Medicinal Natural Products. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic)

Medicinal Natural Products

A Biosynthetic Approach Second Edition Medicinal Natural Products

A Biosynthetic Approach Second Edition

Paul M Dewick School of Pharmaceutical Sciences University of Nottingham, UK Copyright  2002 by John Wiley & Sons Ltd Baffins Lane, Chichester, West Sussex, PO19 1UD, England National 01243 779777 International (+44) 1243 779777 e-mail (for orders and customer service enquiries): [email protected] Visit our Home Page on http://www.wiley.co.uk or http://www.wiley.com All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London, W1P 9HE, UK, without the permission in writing of the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the publication. Neither the authors nor John Wiley & Sons Ltd accept any responsibility or liability for loss or damage occasioned to any person or property through using the material, instructions, methods or ideas contained herein, or acting or refraining from acting as a result of such use. The authors and Publisher expressly disclaim all implied warranties, including merchantability of fitness for any particular purpose. Designations used by companies to distinguish their products are often claimed as trademarks. In all instances where John Wiley & Sons is aware of a claim, the product names appear in initial capital or capital letters. Readers, however, should contact the appropriate companies for more complete information regarding trademarks and registration. Other Wiley Editorial Offices John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, USA Wiley-VCH Verlag GmbH, Pappelallee 3, D-69469 Weinheim, Germany John Wiley, Australia, Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Canada) Ltd, 22 Worcester Road, Rexdale, Ontario, M9W 1L1, Canada John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809

Library of Congress Cataloguing in Publication Data Dewick, Paul M. Medicinal natural products : a biosynthetic approach / Paul M. Dewick. – 2nd ed. p. cm. Includes bibliographical references and index. ISBN 0 471 49640 5 (cased) – ISBN 0 471 49641 3 (pbk.) 1. Natural products. 2. Biosynthesis. 3. Pharmacognosy. I. Title.

RS160 .D48 2001 615
.321 – dc21 2001026766

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0 471 49640 5 (cased) ISBN 0 471 49641 3 (pbk.) Typeset by Laserwords Private Ltd, Chennai, India Printed and bound in Great Britain by Antony Rowe Ltd, Chippenham, Wilts This book is printed on acid-free paper responsibly manufactured from sustainable forestry, in which at least two trees are planted for each one used for paper production. Dedicated to the many hundreds of pharmacy students I have taught over the years, who, unknowingly, have encouraged me to write this text CONTENTS

1 ABOUT THIS BOOK, AND HOW TO USE IT ...... 1 The Subject ...... 1 The Aim ...... 1 The Approach ...... 2 The Topics ...... 2 Be Selective ...... 2 To Learn, or to Understand? ...... 3 Conventions Regarding Acids, Bases, and Ions ...... 3 Nomenclature ...... 3 Some Common Abbreviations ...... 4 Further Reading ...... 4

2 SECONDARY METABOLISM: THE BUILDING BLOCKS AND CONSTRUCTION MECHANISMS ...... 7 Primary and Secondary Metabolism ...... 7 The Building Blocks ...... 8 The Construction Mechanisms ...... 12 Alkylation Reactions: Nucleophilic Substitution ...... 12 Alkylation Reactions: Electrophilic Addition ...... 13 Wagner–Meerwein Rearrangements ...... 15 Aldol and Claisen Reactions ...... 15 Schiff Base Formation and the Mannich Reaction ...... 18 Transamination ...... 20 Decarboxylation Reactions ...... 20 Oxidation and Reduction Reactions ...... 24 Dehydrogenases ...... 24 Oxidases ...... 25 Mono-oxygenases ...... 25 Dioxygenases ...... 27 Amine Oxidases ...... 28 Baeyer–Villiger Oxidations ...... 28 Phenolic Oxidative Coupling ...... 28 Glycosylation Reactions ...... 29 Some Vitamins Associated with the Construction Mechanisms ...... 30 Vitamin B1 , Vitamin B2 , Vitamin B5 , Vitamin B6 , Vitamin B12 , Vitamin H Further Reading ...... 33

3 THE ACETATE PATHWAY: FATTY ACIDS AND POLYKETIDES ...... 35 Saturated Fatty Acids ...... 36 Unsaturated Fatty Acids ...... 40 Fixed Oils and Fats, Evening Primrose Oil Acetylenic Fatty Acids ...... 47 Branched-Chain Fatty Acids ...... 48 Echinacea viii CONTENTS

Prostaglandins ...... 51 Prostaglandins, Isoprostanes Thromboxanes ...... 58 Thromboxanes Leukotrienes ...... 58 Leukotrienes Aromatic Polyketides ...... 60 Cyclization: Simple Phenols ...... 61 Structural Modifications: Anthraquinones ...... 63 Senna, Cascara, Frangula, Allied Drugs, Hypericum/St John’s Wort C-Alkylation Reactions ...... 72 Mycophenolic Acid, Khellin and Cromoglicate Phenolic Oxidative Coupling ...... 76 Griseofulvin Oxidative Cleavage of Aromatic Rings ...... 78 Starter Groups Other Than Acetate ...... 79 Poison Ivy and Poison Oak, Aflatoxins, Cannabis, Tetracyclines, Anthracycline Antibiotics Macrolides and Polyethers ...... 92 Extender Groups other than Malonate ...... 92 Macrolide Antibiotics, Avermectins, Polyene Antifungals, Tacrolimus and Sirolimus, Ansa Macrolides Cyclization Through Diels–Alder Reactions ...... 111 Mevastatin and other Statins Genetic Manipulation of the Acetate Pathway ...... 114 Further Reading ...... 117

4 THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND PHENYLPROPANOIDS ...... 121 Aromatic Amino Acids and Simple Benzoic Acids ...... 121 Folic Acid (Vitamin B9 ), Chloramphenicol Cinnamic Acids ...... 130 Lignans and Lignin ...... 132 Podophyllum Phenylpropenes ...... 135 Volatile Oils Containing Principally Aromatic Compounds Benzoic Acids from C6C3 Compounds ...... 141 Coumarins ...... 142 Dicoumarol and Warfarin, Psoralens Styrylpyrones ...... 147 Kava Flavonoids and Stilbenes ...... 149 Flavonolignans ...... 151 Silybum marianum Isoflavonoids ...... 154 Phyto-oestrogens, Derris and Lonchocarpus Terpenoid Quinones ...... 158 Vitamin E, Vitamin K Further Reading ...... 164 CONTENTS ix

5 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS: TERPENOIDS AND STEROIDS ...... 167 Hemiterpenes (C5) ...... 172 Monoterpenes (C10) ...... 172 Volatile Oils Containing Principally Terpenoid Compounds Irregular Monoterpenes ...... 186 Pyrethrins Iridoids (C10) ...... 187 Valerian Sesquiterpenes (C15) ...... 191 Feverfew, Chamomile and Matricaria, Artemisia annua and Artemisinin, Gossypol, Trichothecenes Diterpenes (C20) ...... 203 Taxus brevifolia and Taxol (Paclitaxel), Ginkgo biloba, Forskolin Sesterterpenes (C25) ...... 212 Triterpenes (C30) ...... 212 Triterpenoid Saponins ...... 219 Liquorice, Quillaia, Ginseng Modified Triterpenoids ...... 225 Tetraterpenes (C40) ...... 226 Vitamin A Higher Terpenoids ...... 231 Steroids ...... 232 Stereochemistry ...... 232 Cholesterol ...... 233 Cholesterol Steroidal Saponins ...... 237 Dioscorea, Fenugreek, Sisal, Sarsaparilla, Yucca Cardioactive Glycosides ...... 241 Digitalis purpurea, Digitalis lanata, Strophanthus, Convallaria, Squill, Toxic Plants Phytosterols ...... 252 Soya Bean Sterols, Fusidic Acid Vitamin D ...... 257 Vitamin D Bile Acids ...... 260 Bile Acids Adrenocortical Hormones/Corticosteroids ...... 262 Semi-Synthesis of Corticosteroids ...... 264 Corticosteroid Drugs Progestogens ...... 267 Progestogen Drugs Oestrogens ...... Oestrogen Drugs, Aromatase Inhibitors, Oestrogen Receptor Antagonists Androgens ...... 282 Androgen Drugs Further Reading ...... 285 x CONTENTS

6 ALKALOIDS ...... 291 Alkaloids Derived from Ornithine ...... 292 Pyrrolidine and Tropane Alkaloids ...... 292 Belladonna, Stramonium, Hyosycamus, Duboisia, Allied Drugs, Hyoscyamine, Hyoscine and Atropine, Coca Pyrrolizidine Alkaloids ...... 305 Alkaloids Derived from Lysine ...... 307 Piperidine Alkaloids ...... 307 Lobelia ...... 308 Quinolizidine Alkaloids ...... 309 Indolizidine Alkaloids ...... 310 Alkaloids Derived from Nicotinic Acid ...... 311 Pyridine Alkaloids ...... 311 Vitamin B3 , Tobacco, Areca Alkaloids Derived from Tyrosine ...... 315 Phenylethylamines and Simple Tetrahydroisoquinoline Alkaloids ...... 315 Catecholamines, Areca, Lophophora, Curare Modified Benzyltetrahydroisoquinoline Alkaloids ...... 327 Opium ...... 329 Phenethylisoquinoline Alkaloids ...... 341 Colchicum Terpenoid Tetrahydroisoquinoline Alkaloids ...... 343 Ipecacuanha Amaryllidaceae Alkaloids ...... 345 Galanthamine Alkaloids Derived from Tryptophan ...... 346 Simple Indole Alkaloids ...... 347 5-Hydroxytryptamine (Serotonin), Psilocybe Simple β-Carboline Alkaloids ...... 349 Terpenoid Indole Alkaloids ...... 350 Rauwolfia, Catharanthus, Iboga, Nux-vomica, Ellipticine Quinoline Alkaloids ...... 359 Cinchona, Camptothecin Pyrroloindole Alkaloids ...... 365 Physostigma Ergot Alkaloids ...... 368 Ergot, Morning Glories Alkaloids Derived from Anthranilic Acid ...... 376 Quinazoline Alkaloids ...... 377 Quinoline and Acridine Alkaloids ...... 377 Alkaloids Derived from Histidine ...... 379 Imidazole Alkaloids ...... 379 Pilocarpus Alkaloids Derived by Amination Reactions ...... 381 Acetate-derived Alkaloids ...... 381 Conium maculatum Phenylalanine-derived Alkaloids ...... 382 Ephedra, Khat Terpenoid Alkaloids ...... 385 Aconite CONTENTS xi

Steroidal Alkaloids ...... 388 Solanum Alkaloids Purine Alkaloids ...... 394 Caffeine, Theobromine, and Theophylline, Coffee, Tea, Cola, Cocoa, Mat´eTea, Guarana Saxitoxin and Tetrodotoxin ...... 396 Saxitoxin, Tetrodotoxin Further Reading ...... 398

7 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES ...... 405 Peptides and Proteins ...... 405 Ribosomal Peptide Biosynthesis ...... 407 Peptide Hormones ...... 410 Thyroid Hormones ...... 410 Thyroxine, Calcitonin Hypothalamic Hormones ...... 411 Thyrotrophin-Releasing Hormone, Luteinizing Hormone-Releasing Hormone, Growth Hormone-Releasing Hormone/Factor, Somatostatin Anterior Pituitary Hormones ...... 413 Corticotropin, Growth Hormone, Prolactin, Gonadotrophins Posterior Pituitary Hormones ...... 413 Oxytocin, Vasopressin Pancreatic Hormones ...... 416 Insulin, Glucagon Interferons ...... 417 Interferons Opioid Peptides ...... 417 Enzymes ...... 419 Nonribosomal Peptide Biosynthesis ...... 421 Peptide Antibiotics ...... 423 Cycloserine, Polymyxins, Bacitracins, Tyrothricin and Gramicidins, Capreomycin, Vancomycin and Teicoplanin, Bleomycin, Cyclosporins, Streptogramins, Dactinomycin Peptide Toxins ...... 433 Death Cap (Amanita phalloides), Ricin, Botulinum Toxin, Microcystins, Snake Venoms Modified Peptides: Penicillins, Cephalosporins, and Other β-Lactams ...... 422 Penicillins ...... 422 Penicillins Cephalosporins ...... 444 Cephalosporins, Cephamycins, Carbacephems Other β-Lactams ...... 450 Clavulanic Acid, Carbapenems, Monobactams Cyanogenic Glycosides ...... 455 Glucosinolates ...... 457 Cysteine Sulphoxides ...... 460 Garlic Further Reading ...... 461 xii CONTENTS

8 CARBOHYDRATES ...... 465 Monosaccharides ...... 465 Oligosaccharides ...... 469 Monosaccharides and Disaccharides, Vitamin C Polysaccharides ...... 473 Polysaccharides ...... Aminosugars ...... 477 Aminoglycosides ...... 478 Aminoglycoside Antibiotics, Streptamine-containing Antibiotics, 2-Deoxystreptamine-containing Antibiotics, Acarbose, Lincomycin and Clindamycin Further Reading ...... 486

Index ...... 487 Medicinal Natural Products. PaulMDewick Copyright  2002 John Wiley & Sons, Ltd ISBNs:0471496405 (Hardback); 0471496413 (paperback); 0470846275(Electronic)

INDEX

Bold type has been used for major references and monograph material; asterisks indicate structural formulae are presented.

∗ Abamectin 101 Acridine alkaloids, see alkaloids β-Alanine 429 ∗ ∗ Abbreviations 4 Acrinylisothiocyanate 457–8 Albuterol 318 , 319 ∗ amino acids 407 Acronychia baueri 379 Alclometasone 272 ∗ ∗ ∗ Abietadiene 209–10 Acronycine 378 –9 Alcuronium 327, 359–60 ∗ Abietenyl cation 210 ACTH, see corticotropin Aldol reaction 15–18 ∗ Abietic acid 185, 209–10 Actinidia polygama 386 imines 19–20, 309, 313 ∗ Abrin 435 Actinidine 386 reverse 18 ∗ Abrus precatorius 435 Actinomycin D, see dactinomycin Aldosterone 262–3 , 264, 271 ∗ Absinthe 176 Actinoplanes 484 Alfacalcidol 259 , 259 ∗ Acacia 457 A. teichomyceticus 426 Alfentanil 334 , 335 ∗ A. senegal 476 ACV 437–8 Algae, brown 226, 253 Acacia 476 Acyl carrier protein (ACP) 36, 115 Alginate, calcium 476 Acanthopanax senticosus 223 Adaptogen 223 Alginate, sodium 476 ∗ ∗ Acarbose 484 , 484 Adenosine monophosphate (AMP) 393–4 Alginic acid 475 , 476 ∗ ∗ Acebutolol 318 , 319 S-Adenosylmethionine (SAM) 12–13 , Alicyclobacillus acidocaldarius 218 ∗ Acenocoumarol 144 14 Alimentary toxic aleukia 203 ∗ Acer saccharum 471 Adenylate cyclase 212 Alizarin 164 Acetate, alkaloids derived from 381–2 Adenylic acid 480 Alkaloids Acetate pathway, genetic manipulation Adhatoda vasica 377 acetate-derived 381–2 ∗ 114–17 Adrenaline 129, 316 , 317, 394 acridine 377–9 Acetyl-CoA Adrenergic receptors 317 acridine 81 as building block 8–10 Adrenocorticotrophin, see corticotropin Amaryllidaceae 345–6 in aldol and Claisen reactions 15–18 Adrenoylethanolamide 89 aporphine 337–9 ∗ Acetyl-intermedine 306 Adriamycin, see doxorubicin numbering 337 ∗ Acetyl-lycopsamine 306 Aescin 220 benzophenanthridine 339 ∗ ∗ N-Acetylanthranilic acid 377 Aesculetin 142–3 benzyltetrahydroisoquinoline 323–4 ∗ ∗ ∗ Acetylcholine 299 , 315 , 326 , 367, Aesculus hippocastrum 220 numbering 322 ∗ ∗ 368 , 380 , 435, 436 Aflatoxins 82–5, 84 benzyltetrahydroisoquinoline, modified ∗ ∗ Acetylcholine receptors Aflatoxin B 82–3 ,84 327–41 1 ∗ muscarinic 299, 316, 380 Aflatoxin B 84 β-carboline 349–50 2 ∗ ∗ nicotinic 299, 304, 308, 315, 325, 327, Aflatoxin G 82–3 ,84 –5 numbering 349 1 ∗ 436 Aflatoxin G 84 derived by amination reactions 381–93 ∗ 2 ∗ Acetyldiginatin 247 –8 Aflatoxin M 84 derived from anthranilic acid 376–9 ∗ 1 ∗ Acetyldigitoxin 247 –9 Afzalechin 150 derived from histidine 379–80 ∗ Acetyldigoxin 247 –9 Agar 476 derived from lysine 307–11 ∗ Acetylgitaloxin 247 –8 Agaricus campestris 433 derived from nicotinic acid 311–15 ∗ Acetylgitoxin 247 –8 Agaropectin 476 derived from ornithine 292–307 ∗ ∗ N-Acetylglucosamine 428 , 473–4 Agarose 476 derived from phenylalanine 382–5 ∗ N-Acetylmuramic acid 473–4 Agave 237 derived from tryptophan 346–71 ∗ Acevaltrate 190–1 A. sisalana 238, 240, 264 derived from tyrosine 315–46 ∗ ∗ Acitretin 231 Agmatine 292 ergot 368–71 ∗ ∗ Aclacinomycin A 94 Agroclavine 369 –70, 374 numbering 368 ∗ ∗ ∗ ∗ Aclarubicin 94 , 94 Ajmalicine 350 –1 , 353, 354 , 357 table 373 ∗ Aconine 387 Ajmaline 353, 354, 358 furoquinoline 378–9 ∗ Aconite 387, 387 Ajoene 461 homoaporphine 341 ∗ ∗ Aconitine 387 Aklanonic acid 93 homomorphinan 342 ∗ Aconitum 387 Aklaviketone 93 imidazole 379–80 ∗ A. napellus 387 Aklavinone 93 –4 indole, simple 347–9 ∗ Acremonium chrysogenum 445 Akuammicine 350 , 357 indole, terpenoid 350–71 ∗ A. fusioides 257 Alanine 406 bis-indole 356 488 INDEX

∗ Alkaloids (continued) Alprostadil 55,56 Androgens 282–3, 283–5 indolizidine 310–11 Alteplase 420 characteristic features 282 ∗ numbering 311 Alternaria tenuis 63 Androstadienedione 279–80 ∗ ∗ morphinan 327–9 Alternariol 63 Androstane 233–4 ∗ numbering 328 Amanita muscaria 299 Androstenedione 277 ∗ ∗ ∗ norditerpenoid 387 A. phalloides 153, 433–4 Androstenolone 273 –4, 279–80 , 283 ∗ ∗ phenethylisoquinoline 341–2 α-Amanitin 153, 433 Anethole 138 –40 phenylalanine-derived 382–5 Amaranthus cruentus 213 Anethum graveolens 180 ∗ phenylethylamine 315–16 Amaryllidaceae alkaloids, see alkaloids Angelic acid 180, 194 , 197, 225 ∗ phthalideisoquinoline 331, 339 Amaryllis 345 Angelicin 145 ∗ piperidine 307–9 Amatoxins 433 Anhalamine 320 –1 ∗ protoberberine 339–41 Amethocaine, see tetracaine Anhalonidine 320 –1 ∗ ∗ numbering 339 Amfetamine 383 , 384–5 Anhalonine 320 –1 ∗ ∗ protopine 339 Amikacin 481–3 , 482 Anhydrochlortetracycline 90 ,92 ∗ purine 394 Amine oxidases 28 Anhydrovinblastine 355 –7 pyridine 311–14 Amino acids Anise, see aniseed pyridocarbazole 359–60 abbreviations 407 star, see star anise ∗ pyrrolidine 292–304 D-amino acids 405 Aniseed 138 ∗ pyrrolizidine 305–6 L-amino acids 405 oil 139 numbering 305 table 406 Anistreplase 420 pyrroloindole 365–6 Amino sugars 477–8 Annatto 231 ∗ numbering 366 Aminoadipic acid 437–8 Ansa macrolides 105–6, 107–8 ∗ pyrroloquinoline 364–5 p-Aminobenzoic acid (PABA) 124 , 126, Anser anser 49 ∗ ∗ quinazoline 377 129, 131 Antamanide 433 –4 ∗ quinoline 81, 359–65, 377–9 α-Aminobutyric acid 372–3, 429, 430 Anthemis nobilis 180, 196 quinolizidine 309–10 γ-Aminobutyric acid 191 Anthocercis littorea 295 ∗ numbering 310 9-Aminocamptothecin 365 Anthocyanidins 150 ∗ steroidal 240–1, 388–93 7-Aminocephalosporanic acid 446 Anthracyclines 92, 93–94 ∗ ∗ ∗ terpenoid 385–8 Aminocyclitol 478 Anthranilic acid 81, 124 , 126–8 , 312 , ∗ ∗ terpenoid tetrahydroisoquinoline 343–4 Aminodehydroshikimic acid 105–6 376–7 tetrahydroisoquinoline, simple 320–4 Aminoglycoside antibiotics 478–80, alkaloids derived from 376–9 numbering 320 480–3 Anthraquinones 163 tropane 292–304 Aminoglycosides 478–85 biosynthesis 63–5 ∗ numbering 292 3-Amino-5-hydroxybenzoic acid 105–6 Antibiotics ∗ ∗ ∗ ∗ Valerian 190 , 386 6-Aminopenicillanic acid 438 , 440 , 444 aminoglycoside 478–80, 480–3 ∗ Alkylamides 49–50 p-Aminophenylalanine 432 anthracyclines 92, 93–4 Alkylation Aminophylline 394 cephalosporins 445–50 electrophilic 13–15 Ammi majus 75, 146 β-lactams 437–55 nucleophilic 12–13 A. visnaga 74, 75 macrolide 94–8, 98–100, 107–8 ∗ ∗ ∗ Allicin 459 –60, 461 Amorphadiene 197–8 penicillins 437–44 ∗ ∗ Alliin 459 –60 Amoxicillin 440, 441 , 452 peptide 422–32 Alliinase 459 Amoxycillin, see amoxicillin tetracycline 89–92, 90–2 Allium 460 AMP, see adenosine monophosphate Anticodon 407 A. cepa 460 Amphetamine, see amfetamine Antihistamines 379 A. sativum 460 Amphotericin 103, 254 Antithrombin 477 A. schoenoprasum 460 Amphotericin A 102 Aphanizomenon 397 ∗ Allopurinol 343 Amphotericin B 102 A. flos-aquae 304 ∗ ∗ ∗ S-Allyl cysteine 459 Ampicillin 440, 441 Apigenin 150 ∗ ∗ S-Allyl cysteine sulphoxide 459 –60 Amycolatopsis mediterranei 105, 107, Apiose 68 ∗ Allyl sulphenic acid 459 108 Apium graveolens 147 ∗ Allylisothiocyanate 457–8 A. orientalis 426 Apocarotenoids 230 ∗ ∗ Allylphenols 138 Amygdalin 455–6 Apoeuphol 225 ∗ ∗ Almond Amylopectin 473–4 , 475 Apomorphine 332 , 333 ∗ bitter 455 Amylose 473–4 , 475 Aporphine alkaloids, see alkaloids ∗ ∗ oil 41 α-Amyrin 218–19 Apotirucallol 225 ∗ sweet 456 β-Amyrin 218–19 , 220 APSAC, see anistreplase Aloe barbadensis 70 Anabaena flos-aquae 304, 435 Aquocobalamin, see vitamin B ∗ ∗ 12 A. ferox 70 Anabasine 312–14 , 315 Arabinose 469 ∗ ∗ A. perryi 70 Anacardic acid 81 Arachidic acid 38 ,41 ∗ ∗ ∗ ∗ A. vera 70 Anacardium occidentale 82 Arachidonic acid 38 ,45 ,52 –3 , ∗ ∗ ∗ ∗ ∗ Aloe-emodin 64 ,66 , 69, 164 Anaferine 307 57–8 ,60 , 268 ∗ ∗ ∗ Aloe-emodin anthrone 66 ,68 ,70 Anandamide 87 –8 Arachidonoylethanolamide, see ∗ Aloes 65, 70 Anatabine 313–14 anandamide ∗ ∗ ∗ Aloin A 68 –9 Anatoxin-a 304 Arachidonoylglycerol 87 ,89 ∗ ∗ Aloin B 68 –9 Androcymbine 342 Arachis hypogaea 41, 82 ∗ Aloinoside A 68 ,70 Androcymbium melanthioides 342 Arachis oil 41 ∗ Aloinoside B 68 ,70 Androgen antagonist 284 Areca 315–16 INDEX 489

∗ Areca catechu 314, 315 Avenasterol 255 Bhang, see cannabis ∗ ∗ Arecaidine 316 Avermectins 97, 100–1, 101, 117 Bicuculline 341 ∗ ∗ Arecoline 314, 315 –16 Avermectin B 100–1 Biflavonoids 211 ∗ ∗ ∗ 1a ∗ Arginine 292 , 305, 388, 396–7 , 406 , Avermectin B 101 Bile acids 260–1, 261–2 ∗ ∗ 1b ∗ 451 , 479 Avermectin B 101 characteristic features 260 2a∗ ∗ Arginine vasopressin, see argipressin Averufin 82–3 Bilobalide 209, 211 ∗ ∗ Argipressin 415 , 416 Azadirachta indica 226 Bioresmethrin 187 , 188 ∗ ∗ Aristolochia 339 Azadirachtin 225 –6 Biotin (vitamin H) 31 , 33 ∗ ∗ A. serpentina 339 Azithromycin 99 , 99 Biotin 17 ∗ ∗ Aristolochic acid 338 –9 Azlocillin 442 , 443 in carboxylation of acetyl-CoA 17 ∗ Armoracia rusticana 458 Aztreonam 453 , 454 ∗ Bipolaris 84 Arogenic acid 128–9 Bis-indole alkaloids, see alkaloids ∗ Aromatase 277 Bisabolene 167, 169 ∗ ∗ Aromatase inhibitors 277, 282 Bacampicillin 441 , 443 α-Bisabolene 194 ∗ ∗ Arrowroot 475 Baccatin III 206 β-Bisabolene 181, 194 ∗ ∗ Artabotrys uncinatus 200 Baccharenyl cation 218–19 γ-Bisabolene 192, 194 ∗ Arteannuic acid 199 Baccharis 203 α-Bisabolol 182, 192, 194 , 197 ∗ ∗ Arteannuin B 199 Bacillus 477 α-Bisabolol oxide A 182, 192, 194 , 197 ∗ ∗ Arteether 199 B. brevis 424 α-Bisabolol oxide B 182, 192, 194 , 197 ∗ ∗ ∗ ∗ Artelinic acid 199 B. colistinus 423 Bisabolyl cation 193 –4 , 202 ∗ ∗ Artemether 199 , 199 B. polymyxa 423 Bisoprolol 318 , 319 Artemisia absinthium 176 B. subtilis 424 Bixa orellana 231 A. annua 197–8, 198–200 Bacitracin 424 ∗ ∗ Bixin 230 –1 A. cinia 196 Bacitracin A 424 ∗ Bleomycin 427–9 Artemisinic acid 197–8 , 199 Bacitracins 424 ∗ ∗ ∗ Bleomycin A2 428, 429 Artemisinin 197, 198 –9 , 198–200 Baeyer-Villiger oxidation 28,82–3,85 ∗ ∗ Bleomycin B2 428, 429 Artemisitene 199 Balanocarpus heimii 217 ∗ ∗ ∗ Bleomycinic acid 428, 429 Artesunic acid 199 , 199 Baldrinal 190–1 ∗ Borage 46 Arthrobacter simplex 268 Bambuterol 318 ∗ oil 41, 46 Articaine 303, 304 Banisteriopsis caapi 350 ∗ ∗ Borago officinalis 41, 46 Ascladiol 79 ∗ Barbaloin 68 –9, 70 Bornane 174 Asclepias syriaca 246 Barbasco 239 ∗ Borneol 174, 176 , 178, 183 Ascophyllum species 476 Barley 316, 349 ∗ Bornyl acetate 183 Ascorbic acid (vitamin C) 26, 28, 230, Barringtogenol 220–1 ∗ ∗ ∗ ∗ Bornyl cation 174, 176 472–3, 472 –3 Bebeerine 325 ∗ Bos taurus 38, 44 Ash, manna 471 Beclometasone dipropionate 270 , 271 Botulinum toxin 435 mountain 471 Beclomethasone, see beclometasone ∗ Botulinum toxin A 435 Ashbya gossypii 31 Behenic acid 38 ,41 Botulism 435 Asparaginase 421 Belladonna 295–6 ∗ ∗ antitoxin 435 Asparagine 406 , 421 Benzaldehyde cyanohydrin 456 ∗ ∗ ∗ Brassica napus Aspartic acid 312 , 406 Benzatropine 300 , 301 43, 458 ∗ Aspergillus species 63, 74, 84, 370 Benzocaine 303, 304 B. nigra 457, 458 ∗ B. oleracea italica 460 A. flaviceps 72 Benzoic acid 141–2 ∗ A. flavus 82, 84 Benzoic acids 121–4, 141–2 Brevetoxin A 109–10 A. parasiticus 76, 84 Benzophenanthridine alkaloids, see Broad bean 48 Broccoli 460 A. terreus 112, 113 alkaloids ∗ Brodifenacoum 144 , 145 A. versicolor 84 Benzoylaconine 387 ∗ Aspidosperma 351 Benztropine, see benzatropine Bromocryptine 372 , 375 ∗ Aspidosperma-type alkaloids 350 –1, Benzyl alcohol 395 Brompton’s cocktail 303 ∗ ∗ 353, 357–8 Benzylglucosinolate 458 Broom 309 ∗ ∗ ∗ ∗ ∗ ∗ Aspirin 55, 142 Benzylpenicillin 437, 438 –9 , 440 –1 Brucine 358–9 ∗ Astaxanthin 226, 228 Benzyltetrahydroisoquinoline alkaloids, see Brugmansia 296 Astragalus 311, 476 alkaloids Buckthorn, alder 70 ∗ ∗ A. gummifer 476 Berberine 339 –40 Buckwheat 151 ∗ ∗ Atenolol 318 , 319 Berberine bridge 339 Budenoside 272 ∗ Atisine 387 –8 Berberis 339 Bufa 243 ∗ Atorvastatin 113 , 113 Bergamot oil 146, 179 Bufadienolides 243 ∗ ∗ ∗ Atracurium 326 , 327, 367 Bergapten 145 –6, 147, 179 Bufalin 243–4 ∗ ∗ Atrochrysone 65–6 Bergaptol 145 Bulbocodium 343 ∗ ∗ Atrochrysone carboxylic acid 65–6 Beta vulgaris 471 Bupivacaine 303, 304 ∗ Atropa 301 Beta-blockers 317–19 Buprenorphine 336 , 336 ∗ ∗ A. belladonna 295 Betamethasone 270 , 270 Buserelin 412 , 413 ∗ ∗ ∗ Atropine 297–301, 298 , 367, 380 Betamethasone dipropionate 270 , 271 Butanoylcastanospermine 311 ∗ ∗ Atroscine 298 Betamethasone valerate 270 , 270 Butenyl-dimethyl-threonine 429–30 ∗ Attar of rose, see rose oil Betaxolol 318 , 319 Butterfat 40–1, 48 ∗ Autumn crocus 343 Betel nuts 314–15 Butyric acid 38 ,41 ∗ ∗ ∗ Autumnaline 341 –2 Betula lenta 140 Butyrospermol 225 490 INDEX

∗ ∗ ∗ Cabergoline 372 , 375 Carbenoxolone sodium 221 , 222 Cefamandole 446, 447 ∗ ∗ Cacao, see cocoa Carbidopa 317 , 317 Cefazolin 446, 447 ∗ ∗ ∗ ∗ Cadaverine 307 , 309–10 , 314 β-Carboline alkaloids, see alkaloids Cefixime 448 , 450 ∗ ∗ ∗ α-Cadinene 196–7 Carboprost 55,56 Cefodizime 446, 448 ∗ ∗ δ-Cadinene 200–1 Carboxymethylcellulose 476 Cefotaxime 446, 448 ∗ ∗ ∗ ∗ Cadinyl cation 193 , 197 , 200–1 Cardamom oil 179 Cefoxitin 449 , 450 ∗ ∗ Caffeic acid 49, 131 –2, 395 Cardenolides 243 Cefpirome 448 ∗ ∗ Caffeine 375, 393 –4, 394–5, 396 Cardiac glycosides, see cardioactive Cefpodoxime-proxetil 449 , 450 ∗ Calabar beans 366 glycosides Cefprozil 447 ∗ ∗ Calcidiol 258 –9 Cardioactive glycosides 241–6 Cefradine 446, 447 ∗ Calcineurin 104 characteristic features 243 Ceftazidime 446, 448 ∗ ∗ Calcipotriol 259 , 260 numbering 243 Ceftibuten 448 , 450 ∗ ∗ Calcitonin 411 Carene 175–6 Ceftizoxime 446, 448 ∗ ∗ Calcitonin, human 411 Carica papaya 420 Ceftriaxone 446, 448 ∗ ∗ ∗ Calcitonin, salmon 411 , 411 α-Carotene 226, 228 , 229–30 Cefuroxime 446, 447 ∗ ∗ ∗ ∗ Calcitriol 258 , 259 β-Carotene 226, 228 –9 , 230 Cefuroxime-axetil 449 , 450 ∗ Calcium gluconate 471 γ-Carotene 226, 228 , 230 Celery 147 ∗ ∗ Calcotonin, porcine 411 δ-Carotene 226, 228 Celiprolol 318 ∗ Calstegines 301 Carotenoids 168, 226–30 Cellulose 473–4 , 476 ∗ Calvin cycle 465 Carotyl cation 193 Cellulose acetate phthalate 476 ∗ Calystegia sepium 301 Carrot 226 Cephaeline 343–4 ∗ ∗ Calystegin A 301 Carteolol 318 , 319 Cephaelis acuminata 344 3 ∗ Calystegin B2 301 Carticaine, see articaine C. ipecacuanha 343, 344 Camellia sinensis 395 Carum carvi 177, 179 Cephalexin, see cefalexin ∗ ∗ Camomile, see chamomile Carvacrol 178 , 185–6 Cephalomannine 206 ∗ ∗ ∗ ∗ Campestane 233–4 Carvedilol 318 Cephalosporin C 444–5 , 446 ∗ ∗ Campestanol 256 Carveol 178 Cephalosporin N 445 ∗ ∗ Campesterol 253 , 256 Carvone 177–8 , 179–80, 185–6 Cephalosporinase 446, 451 ∗ ∗ Camphane 174 β-Caryophyllene 140, 184, 198, 200 Cephalosporins 444–5, 445–50 ∗ ∗ ∗ Camphene 176 Caryophyllyl cation 193 , 198, 200 table 447–9 ∗ ∗ Camphor 175–6 , 177–80, 184, 195 Casbene 207–8 Cephalosporium 257, 444 Camphor oil 179 Cascara 65, 69–70 C. acremonium 445 ∗ Camptotheca acuminata 364, 365 Cascarosides 65–6 ,69 Cephalothin, see cefalotin ∗ ∗ Camptothecin 364 , 365 Cascaroside A 65, 68 –9 Cephamandole, see cefamandole ∗ ∗ Cannabichromene 87 Cascaroside B 68 –9 Cephamycins 445, 450 ∗ ∗ ∗ ∗ Cannabidiol (CBD) 85 –6 Cascaroside C 68 –9 Cephamycin C 445 , 449 –50, 452 ∗ Cannabidiol 87–8 Cascaroside D 68 –9 Cephazolin, see cefazolin ∗ ∗ Cannabidiolic acid 85 –6 Cashew nuts 82 Cephem 450 Cannabidiolic acid 87 Cassava 456 Cephradine, see cefradine ∗ ∗ Cannabigerol 87 Cassia acutifolia 67 Cerivastatin 113 , 113 ∗ ∗ Cannabigerolic acid 85 –6 C. angustifolia 67 Cerotic acid 38 Cannabinoids 85, 87 C. senna 67 Certoparin 477 ∗ Cannabinol (CBN) 85 –6 Cassia oil 139 Chalcones 150 ∗ Cannabinol 87 Castanospermine 310–11 Chamaemelum nobile 180, 196 ∗ ∗ Cannabinolic acid 85 –6 Castanospermum australe 310 Chamazulene 180, 182, 196 –7 ∗ Cannabis 86–9 Castor oil 41 Chamazulene carboxylic acid 196 oil, see cannabis plant 207, 434 Chamomile 196–7 resin, see cannabis seed 314 German 196 Cannabis sativa 85, 86 Catalpa 163 Roman 196 ∗ ∗ Capreomycidine 425 Catalponone 163–4 oil 197 ∗ Capreomycin 425 Catechin 150 oil, German, see matricaria oil ∗ Capreomycin IA 425 Catechins 150–1, 395 oil, Roman 180 ∗ Capreomycin IB 425 Catecholamines 129–30, 316, 317–19, Chamomilla recutica 182, 196 ∗ ∗ ∗ Capric acid 38 , 41–3, 381 384 Chanoclavine-I 369 –70, 374 ∗ ∗ Caproic acid 38 ,41 Catha edulis 383, 384 Chanoclavine-I aldehyde 369 –70 ∗ ∗ ∗ ∗ Caprylic acid 38 , 42–3 Catharanthine 350 , 353–4 , 355 Charas, see cannabis ∗ ∗ Capsaicin 385 Catharanthine N-oxide 355 , 357 Chaulmoogra oil 50–1 ∗ ∗ Capsanthin 226, 228 –9 Catharanthus roseus 353, 356–7 Chaulmoogric acid 50–1 ∗ ∗ Capsicum annuum 226, 385 Cathenamine 351 Chelidonine 339–40 ∗ ∗ Carane 174 –5 Cathine 382 –3, 384 Chelodonium majus 339 ∗ ∗ ∗ Caraway 177 Cathinone 382 –3 , 384 Chenodeoxycholic acid 260–1 , 261 oil 179 CBD, see cannabidiol Cherry, black 456 Carbacephems 450 CBN, see cannabinol Chestnut, Moreton Bay 310 ∗ ∗ Carbapenem 450 Cefaclor 446, 447 Chikusetsusaponins 223 ∗ ∗ Carbapenems 451, 454 Cefadroxil 446, 447 Chimonanthine 366 ∗ ∗ Carbaryl 366 , 368 Cefalexin 446, 447 Chimonanthus fragrans 366 ∗ ∗ ∗ Carbenicillin 441 , 443 Cefalotin 446, 447 Chitin 473–4 INDEX 491

∗ Chitosan 473 Cinerolone 186–7 , 188 Cocoa butter, see theobroma oil ∗ Chives 460 Cinerulose 94 Coconut oil 40, 42 ∗ Chloramphenicol 130–1 , 130 Cinnabar moth 306 fractionated 42 ∗ ∗ Chlorogenic acid 49, 132 , 395–6 Cinnamaldehyde 135, 138 –9 Cocos nucifera 42 ∗ ∗ ∗ Chloromycetin, see chloramphenicol Cinnamic acid 130–1 , 142–3 , 302 , Cod-liver oil 40, 42, 230, 259 ∗ ∗ ∗ Chlorophylls 203 308 Codeine 327–8 , 329–30, 331, 332 , ∗ ∗ Chlorophyll a 203–4 Cinnamic acids 130–2 335 , 337 ∗ ∗ Chloroquine 198, 200, 363 , 363 Cinnamomum camphora 179 Codeinone 328 –9 ∗ ∗ ∗ Chlortetracycline 89 –90 , 90,92 C. cassia 139 Codeinone 335 Chocolate 394, 396 C. zeylanicum 137, 139, 200 Codon 407 ∗ ∗ Cholane 233–4 Cinnamon 137, 200 Coenzyme A 16 ∗ Cholecalciferol, see colecalciferol bark oil 139 Coenzyme B 32 ∗ 12 Cholestane 233–4 leaf oil 139 Coenzyme Q, see ubiquinones ∗ ∗ Cholesterol 103, 112, 217, 232–3 , Cinnamoylcocaine 301 –2 Coffea 395 ∗ 236–7, Cinnamoylglucose 132 C. arabica 395 ∗ biosynthesis 233–6 Cinnamyl acetate 137–8 , 139 C. canephora 395 HDL 237 Cinnamylcocaine, see cinnamoylcocaine C. liberica 395 ∗ ∗ in bile acid biosynthesis 260–1 Cisatracurium 326 , 327 Coffee 313, 394, 395 ∗ in cardiacative glycoside biosynthesis Citral 174 , 181–2 decaffeinated 395 ∗ ∗ 243–4 Citrinin 74 Cola 394, 395–6 ∗ in corticosteroid biosynthesis 263 –4 Citronella oil 180 Cola 395 ∗ in semi-synthesis of hormones Citronellal 174 , 180 C. acuminata 395 ∗ ∗ 279–80 Citronellol 174 , 180, 184 C. nitida 395 ∗ ∗ in steroidal alkaloid biosynthesis Citrostadienol 255 Colchicine 342 , 343 ∗ 388–90 Citrus aurantium 151 Colchicum 343 in steroidal saponin biosynthesis C. aurantium ssp. amara 182, 183 Colchicum 342 ∗ 237–8 C. aurantium ssp. bergamia 146, 179 C. autumnale 342, 343 ∗ LDL 237, 256 C. limon 181 Colecalciferol 257–8 , 259 Cholestyramine, see colestyramine C. paradisi 151 Coleonol 212 ∗ ∗ Cholic acid 260 –1 C. sinensis 182 Colestipol 261 ∗ Choline theophyllinate 394 Cladinose 96–7 Colestyramine 261 ∗ Cholinesterase 346, 367–8, 436 Cladinose 98–9 Coleus forskohlii 212 Chondrodendron 359 Cladonia 76 Colistin 423 ∗ C. tomentosum 324 Claisen reaction 15–18 Colistin A 423 ∗ Chorionic gonadotrophin 415 reverse 18 Colistin B 423 ∗ ∗ ∗ Chorismic acid 122–3 , 124 , 127 , Claisen rearrangement 128 Colophony 185 ∗ ∗ ∗ ∗ 129 , 158 –9, 162 Clam, Alaskan butter 397 Columbamine 339–40 ∗ Chromobacterium violaceum 454 Clarithromycin 99 , 99 Comfrey 306 ∗ ∗ Chrysaloin 68 –9 Clavam 450 Compactin, see mevastatin ∗ ∗ Chrysanthemic acid 186–7 , 188 Clavaminic acid 451 Compound Benzoin Tincture 70 ∗ Chrysanthemum cinerariaefolium 186 Claviceps 368, 370 Conessine 392 –3 ∗ C. parthenium 195 C. paspali 374 γ-Coniceine 381 –2 ∗ ∗ ∗ ∗ ∗ Chrysophanol 64 ,66 , 69–70 C. purpurea 370, 371, 374 Coniferyl alcohol 131 –2, 133 –4 , ∗ ∗ Chrysophanol anthrone 66 Clavine alkaloids 374, 376 151, 153 ∗ ∗ ∗ Chymotrypsin 420 Clavulanic acid 443, 450–1 , 452, 452 Coniine 381 –2 ∗ Cichoric acid 49–50 Clidamycin phosphate 485 Conium maculatum 381, 382 ∗ ∗ Ciclosporin 104, 429, 430 Clindamycin 485 , 485 Conjugated equine oestrogens, see ∗ Cicuta virosa 47 Clobetasol propionate 270 , 271 oestrogens ∗ ∗ Cicutoxin 47, 49 Clobetasone butyrate 270 , 271 Conocybe 348 ∗ Ciguatera 109 Clomifene 281 , 282 Contraceptives ∗ Ciguatoxin 109–10 Clomiphene, see clomifene emergency 276 ∗ Cilastatin 453 –4 Clostridium botulinum 435 oral 275, 279 ∗ Cinchocaine 303, 304 Clove 137, 200 Convallaria 245 Cinchona 362–4 oil 140 C. majalis 251, 252 ∗ Cinchona 359, 362 Clover 154, 278 Convallatoxin 251 ∗ ∗ C. calisaya 362 red 157 Copalyl diphosphate 207–8 , 209 C. ledgeriana 362 sweet 143, 144 Coriander oil 180 ∗ C. succirubra 362 Cloxacillin 442 , 443 Coriandrum sativum 180 ∗ Cinchonamine 361 –2 Co-amoxyclav 452 Corn oil, see maize oil ∗ Cinchonidine 359, 361 –2, 364 Co-dergocrine 375 Corrin ring 32 ∗ ∗ Cinchonine 361 –2, 364 Cobalamins, see vitamin B Cortexolone 266–7 ∗ 12 Cinchoninone 361 Cobra 436, 437 Cortexone, see desoxycorticosterone Cinchotannic acid 362 Coca 301, 302–4 Corticosteroids 55, 262–7, 268–72 ∗ Cineole 175 , 178–80, 184–5 Coca-Cola 303, 396 semi-synthesis 264–7 ∗ ∗ ∗ ∗ ∗ Cinerins 186–7 Cocaine 12 , 293 –5, 301–2, 303 Corticosterone 262 –3 ∗ ∗ Cinerin I 187 –8 Coclaurine 322 –3 Corticotrophin, see corticotropin ∗ Cinerin II 187 –8 Cocoa 394, 396 Corticotropin 411, 413, 414 492 INDEX

∗ ∗ ∗ ∗ Corticotropin-releasing hormone, see Cypermethrin 187 , 188 Dehydrogeissoschizine 351 –2 , 354 , ∗ ∗ hormone Cyproterone acetate 284 , 284 357–8 ∗ Cortisol, see hydrocortisone Cysteine 406 Dehydrogenases 24–5 ∗ ∗ ∗ Cortisone 262 , 264, 268 , 268 Cysteine sulphoxides 460 Dehydrogriseofulvin 78 ∗ ∗ Cortisone acetate 268 Cytisine 309–10 Dehydromatricaria acid 47–8 ∗ ∗ Corydalis 322, 341 Cytisus 309 Dehydropregnenolone acetate 266 –7 , ∗ ∗ Corynanthe-type alkaloids 350 –1, 353, C. scoparius 309 273 –4 ∗ ∗ 357–8 Cytochrome P–450 26, 27–9 Dehydroquinic acid 122 ∗ ∗ Corynantheal 361 –2 Dehydroretinol (Vitamin A ) 229 , ∗ 2 Corynebacterium dipheriae 49 230–1 ∗ ∗ ∗ Corynomycolic acid 49–50 Dactinomycin 432, 432 Dehydroshikimic acid 122 Cotton 200 Dactylocyclines 91 Dehydrositosterol 259 Cottonseed oil 42, 50, 200 ∗ Dehydrostigmasterol 259 ∗ Dactylocycline-A 92 Coumachlor 144 , 145 ∗ Delphinium 387 ∗ ∗ Dactylocycline-B 92 ∗ 2-Coumaric acid 141–2 , 143 ∗ Demeclocycline 89 , 90 ∗ ∗ ∗ Dactylocycline-C 92 ∗ 4-Coumaric acid 130–1 , 132, 141 –3 , ∗ Demecolcine 342 –3 ∗ Dactylocycline-E 92 ∗ 158 –9 Demethoxyyangonin 148 ∗ Dactylosporangium 91 ∗ Coumarin 142–3 Demethylepipodophyllotoxin 136 Daffodil 345, 346 ∗ Coumarinolignans 152 N-Demethylgalanthamine 346 Dagga, see cannabis ∗ Coumarins 142–6 ∗ ∗ Demethylmunduserone 155–6 ∗ Daidzein 154–5 , 157, 278 ∗ Coumatetralyl 144 , 145 Demethylmycophenolic acid 73 Dalbaheptides 426 ∗ Coumestans 154 ∗ Demethylpodophyllotoxin 136 ∗ ∗ Dalfopristin 431, 431 ∗ Coumestrol 154–5 , 278 Demethylsuberosin 145 Dalteparin 477 Cow 38, 44 Dendroaspis 436 Dammar resin 217 COX, see cyclooxygenase ∗ Deoxybarbaloin, see chrysaloin Dammarenediols 217, 219 ∗ Crack cocaine 303 ∗ ∗ Deoxycholic acid 260–1 , 264 ∗ Dammarenyl cation 217–18 , 219 ∗ Crepenynic acid 47–8 ∗ Deoxycortisol 263 Danazol 284 , 285 ∗ CRH, see hormone, corticotropin-releasing Deoxyerythronolide 96–7 ∗ Dandelion 218 Crinine 345–6 Deoxyerythronolide synthase 115 Danthron, see dantron ∗ Crisantaspase 421 ∗ 2-Deoxyfucose 94 ∗ 70 ∗ Crocetin 230 –1 Dantron ,71 Deoxylapachol 163–4 ∗ Crocus sativus 231 Date palm 276 Deoxyloganin 189 ∗ ∗ Cromoglicate 75–6,75 Datura 295, 296, 301 Deoxymannojirimycin 477–8 ∗ Cromoglycate, see cromoglicate D. metel 296 Deoxynivalenol 202 –3 ∗ Crotalaria 305 D. meteloides 295 Deoxynojirimycin 477–8 ∗ Cucurbitacins 217 D. sanguinea 296 Deoxypumiloside 364 –5 ∗ Cucurbitacin E 217 D. stramonium 295, 296 Deoxyribonucleic acid, see DNA ∗ ∗ ∗ Curare 324, 324–7, 359, 367 Daucus carota 226 Deoxystreptamine 478–9 , 481 , 482 , ∗ Daunomycin, see daunorubicin ∗ Curine 325 ∗ 483 Daunorubicin 92–3 , 94 Curvularia lunata 266 ∗ Deoxyxylulose phosphate (DXP) 168, ∗ Daunosamine 92–3 ∗ Cuscohygrine 293 , 295–6, 302 ∗ ∗ 170–1 ∗ 10-Deacetylbaccatin III 205 –6 , 207 Cyanidin 150 ∗ as building block 8–10 10-Deacetylcephalomannine 206 Cyanocobalamin, see vitamin B12 ∗ Depside 63 Cyanogenic glycosides 455–7 Deacetylcolchicine 342 Depsipeptides 430 ∗ ∗ Cycloartane 233–4 N-Deacetylipecoside 343–4 Dermocybe 67 ∗ ∗ Cycloartenol 215 , 216 –17, 234, 253, 10-Deacetyltaxol 206 Derris 157 ∗ 255 Deadly nightshade, see belladonna Derris elliptica 156, 157 ∗ Cycloeucalenol 255 Death cap 153, 433–4 D. malaccensis 157 ∗ ∗ ∗ Cyclohexanecarboxylic acid 103–4 Decalins 232–3 Desacetyl-lanatoside C 248–9 , 250 ∗ ∗ Cyclooxygenase (COX) 52, 55, 197 Decamethonium 326 –7, 326 Desacetylcephalosporin C 445 ∗ ∗ Cyclopamine 390–1 Decanal 182–3 Deserpidine 351–2 , 353 ∗ ∗ Cyclopentolate 300 , 300 Decarboxylation reactions 20–4 Deslanoside 249 , 250 ∗ Cycloposine 391 of α-keto acids 21–4 Desmopressin 415 , 416 ∗ Cyclopropane ring formation 14–15, 50, of β-keto acids 20, 22 Desogestrel 275 , 275–6 ∗ ∗ ∗ 186, 207, 213, 217, 227 Deflazacort 271, 272 Desosamine 96–7 , 98–9 ∗ ∗ ∗ Cycloserine 422, 423 Deguelin 155–6 , 157 Desoximetasone 272 ∗ ∗ Cyclosporins 429–30 Dehydroabietadiene 209–10 Desoxycorticosterone 262–3 ∗ ∗ Cyclosporin 74, 429 Dehydroalanine 425 , 459 Desoxymethasone, see desoximetasone ∗ ∗ ∗ Cyclosporin A 104, 429–30 Dehydrocampesterol 259 Desoxypodophyllotoxin 134 , 136 ∗ ∗ Cylindrocarpon lucidum 429 Dehydrochlortetracycline 90 ,92 Dexamethasone 270 , 270 ∗ ∗ Cymarin 250–1 Dehydrocholesterol 257–8 , 259 Dexamfetamine 383–4 ∗ ∗ Cymarose 245 , 251 Dehydrocholic acid 261, 262 Dexamphetamine, see dexamfetamine ∗ ∗ Cymbopogon citratus 182 Dehydrocrepenynic acid 47–8 Dextromethorphan 334 , 334 ∗ ∗ C. nardus 180 Dehydrodiconiferyl alcohol 132–3 Dextromoramide 334 , 335 ∗ ∗ C. winterianus 180 2,3-Dehydroemetine 344, 345 Dextropropoxyphene 334 , 335 ∗ p-Cymene 178 , 185–6 Dehydroepiandrosterone (DHEA) 284, Dextrose 471 ∗ Cynoglossum 305 277 DHA, see docosahexaenoic acid INDEX 493

∗ ∗ ∗ DHEA, see dehydroepiandrosterone Dihydronicotinic acid 313 –14 Dopexamine 317 , 317 ∗ ∗ Dhurrin 457 Dihydropteroic acid 125 –6 Dover’s powder 330, 344 ∗ ∗ ∗ ∗ Diacetoxyscirpenol 203 Dihydropyridine 313 –14 Doxorubicin 92–3 , 93 ∗ ∗ Diacetylrhein 70 Dihydroquercetin 150 Doxycycline 89 , 91 ∗ ∗ Diallyl disulphide 461 Dihydrosterculic acid 50–1 DPA, see docosapentaenoic acid ∗ ∗ Diallyl trisulphide 461 Dihydrotachysterol 259 , 259 Drimia maritima 252 Diamine oxidases 29 Dihydrotestosterone 284 Dronabinol 88 ∗ ∗ α,γ-Diaminobutyric acid 423 3,4-Dihydroxybenzaldehyde 345–6 Duboisia 295, 297, 301 ∗ ∗ ∗ Diaminopropionic acid 425 2,3-Dihydroxybenzoic acid 124 –5 D. hopwoodi 297 ∗ Diamorphine 332 , 332–3 3,4-Dihydroxybenzoic acid, see D. leichhardtii 297 Dianthrone 65 protocatechuic acid D. myoporoides 297 ∗ Dicentra 341 2,4-Dihydroxycinnamic acid 142–3 DXP, see deoxyxylulose phosphate ∗ ∗ ∗ Dicoumarol 143–4 , 144–5, 163 Dihydroxynaphthoic acid 161–2 , 163 Dydrogesterone 275 ∗ Dictamnine 378 –9 3,4-Dihydroxyphenylalanine, see DOPA Dynorphins 418 ∗ ∗ Dictamnus albus 379 3,5-Dihydroxyphenylglycine 426–7 , Dynorphin A 419 ∗ ∗ Didrovaltrate 190–1 428 Diels-Alder reaction 111, 336 1α,25-Dihydroxyvitamin D , see calcitriol ∗ ∗ 3 Eburicol 254–5 Dienestrol 281 , 281 Dill oil 180 ∗ Ecgonine 295, 302 Dienoestrol, see dienestrol Dimethylallyl cation 170 ∗ ∗ Echinacea 48, 49 Diethylstilbestrol 278 , 281 , 281 Dimethylallyl diphosphate (DMAPP) ∗ ∗ ∗ ∗ Echinacea angustifolia 49 Difenacoum 144 , 145 12–13, 167–8 , 170 , 171 –2 ∗ ∗ E. pallida 49 Diflucortolone 272 Dimethylfecosterol 255 ∗ E. purpurea 48–9 Diginatigenin 247 , 249 Dinophysis 108 ∗ Ecstasy 383, 385 Diginatin 248 Dinoprost 55,56 ∗ herbal 384 Digitalis 245, 252 Dinoprostone 55,56 ∗ ∗ ∗ Edrophonium 366 , 368 D. dubia 250 Dioscin 237 , 239 Eformoterol, see formoterol D. ferruginea 250 Dioscorea 237, 238, 239,282, 264 Eicosanoids 52 ∗ D. grandiflora 250 D. alata 239 Eicosapentaenoic acid (EPA) 38 , 40, 42, ∗ ∗ D. lanata 249–50 D. cayenensis 239 45 –6, 52 , 57, 59–60 ∗ D. lutea 250 D. collettii 239 Eicosatetraenoic acid 45 D. mertonensis 250 D. composita 239 Eicosatrienoic acid 60 D. nervosa 250 D. deltoidea 239 Elaeis guineensis 43 D. purpurea 241, 243, 246 D. esculenta 239 Elder 457 ∗ D. subalpina 250 D. floribunda 239 Eleagnine 349 –50 D. thaspi 250 D. mexicana 239 Eleagnus angustifolia 350 ∗ Digitalis glycosides, partial hydrolysis D. nipponica 239 Elemicin 138 , 140 248 D. pathaica 239 ∗ ∗ Elephantopin 194 Digitalose 245 D. rotundata 239 ∗ ∗ ∗ Elephantopus elatus 194 Digitoxigenin 243 –4 , 246–7 , 249 D. sylvatica 239 ∗ Eleutherococcus senticosus 223 Digitoxin 246, 247 –9 D. trifida 239 Eleutherosides 223 ∗ ∗ ∗ ∗ ∗ Digitoxose 245 , 247 Diosgenin 237 –8 , 239–40, 264, Eleutheroside B 223–4 ∗ ∗ ∗ Digoxigenin 244 , 247 , 249 266 –7, 279–80, 282–3 ∗ ∗ Eleutheroside E 223–4 Digoxin 248–9 , 249 Dioxygenases 27–8,78 Elletaria cardamomum 179 ∗ Digoxin-specific antibody 250 2-oxoglutarate-dependent 27–8 Ellipticine 359–60 , 360 ∗ ∗ ∗ Dihomo-γ-linoleic acid 45 ,52 ,57 Diphylleia 137 Elliptinium acetate 360 , 360 ∗ ∗ Dihomo-γ-linolenoylethanolamide 89 Dipipanone 334 , 335 Elymoclavine 369 –70, 374 ∗ ∗ Dihydroarteannuin B 198 Disaccharides 471–3 Emetine 343–4 , 344 ∗ ∗ ∗ ∗ Dihydroartemisinic acid 197–8 Distigmine 366 , 367 Emodin 64 ,66 , 70, 164 ∗ ∗ Dihydroartemisinin 199 Disulphide bridge 409 Emodin anthranol 67 ∗ ∗ Dihydrochalcones 151 Diterpenes 167–8, 203–12 Emodin anthrone 65–6 ,67 ∗ ∗ ∗ Dihydroclavaminic acid 451 Dithranol 70,71 Emodin dianthrone 67 ∗ ∗ ∗ Dihydrocodeine 332 , 332 DMAPP, see dimethylallyl diphosphate Endocrocin 63–4 ,66 ∗ ∗ Dihydroequilin 281 DNA 407, 409 Endocrocin anthrone 65–6 ∗ ∗ Dihydroergocornine 375 Dobutamine 317 , 317 β-Endorphin 418, 419 ∗ Dihydroergocristine 375 Docetaxel 206 , 207 Endorphins 333, 418 ∗ Dihydroergocyptine 375 Docosahexaenoic acid (DHA) 38 , 40, Enkephalins 333, 418 ∗ ∗ ∗ Dihydroergosterol 259 42, 45 –6 Leu-Enkephalin 333 , 418–19 ∗ ∗ ∗ Dihydroergotamine 375 Docosapentaenoic acid (DPA) 38 , Met-Enkephalin 333 , 418–19 ∗ Dihydroergotoxine 375 45 –6 Enolpyruvylshikimic acid phosphate ∗ Dihydroflavonol 151 Dolichothele sphaerica 379 (EPSP) 123 ∗ Dihydrofolate reductase 125–6 Dolichotheline 379 Enoxaparin 477 ∗ ∗ ∗ ∗ ∗ Dihydrofolic acid 125 –6 DOPA 129–30 , 316 , 317, 322 –3, Enterobactin 124–5 ∗ ∗ ∗ Dihydrokaempferol 150 485 Enterodiol 134 –5, 157 ∗ ∗ ∗ Dihydrokawain 148 DOPAchrome 130 Enterolactone 134 –5, 157 ∗ ∗ ∗ Dihydrolanosterol 236 Dopamine 316 , 317, 322 , 341 , Enzymes 419–21 ∗ ∗ ∗ Dihydromethysticin 148 343–4 , 374–5 fibrinolytic 420 ∗ ∗ Dihydromonacolin L 112 DOPAquinone 129–30 hydrolytic 420 494 INDEX

Enzymes 419–21 (continued) E. chrysanthemi 421 branched chain, biosynthesis 48–51 in snake venoms 436 Erythromycin 98–9, 115 essential 46, 55 ∗ ∗ table 420–1 Erythromycin A 96, 99 –100 ω–3 46 ∗ EPA, see eicosapentaenoic acid Erythromycin B 99 β-oxidation 18 ∗ Ephedra 382, 383–4 Erythromycin C 99 saturated, biosynthesis 36–8 ∗ ∗ E. equisetina 383 Erythronolide 97 saturated, table 38 ∗ ∗ E. geriardiana 383 Erythrose 4-phosphate 121–2 , 466–7 unsaturated, biosynthesis 40, 45–7 ∗ E. intermedia 383, 384 Erythroxylum 301, 302 unsaturated, table 38 E. major 383 E. truxillense 302 ω–3 46 ∗ E. sinica 383 E. coca 295, 301, 302 Fecosterol 254–5 ∗ ∗ ∗ Ephedrine 317, 382 –3, 384 , 384 Escherichia coli 121,124, 407, 409, 417, Fenchane 174 , 176 ∗ ∗ Epibatidine 304–5 419, 434, 440 Fenchol 176 ∗ ∗ ∗ Epicatechin 151 Eseramine 366 –7 Fenchone 140, 176 ∗ Epinephrine, see adrenaline Eserine, see physostigmine Fenchyl cation 176 ∗ Epipedobates tricolor 304 Eseroline 367 Fennel 138 ∗ ∗ Epirubicin 93 , 94 Esmolol 318 , 319 oil 140 ∗ ∗ Episterol 255 Essential fatty acids 46, 55 Fenoterol 318 ∗ ∗ ∗ ∗ Epoprostenol 56,56 Estradiol 155–6, 276 –7 , 279, 281 Fentanyl 334 , 335 Epothilones 103 Estradiol valerate 279 Fenugreek 238, 240 ∗ ∗ ∗ ∗ ∗ Epothilone A 103, 105 Estragole 138 , 139–40 Ferulic acid 131 –2, 141 , 385 ∗ ∗ Epothilone B 103, 105 Estramustine 281 , 281 Feverfew 192, 195–6 ∗ ∗ EPSP, see enolpyruvylshikimic acid Estrane 233–4 Finasteride 284 , 284 phosphate Estriol 276 Fish oils 40 ∗ ∗ Eptastigmine 366 , 367 Estriol 279 FK-506 102, 104 , 429 ∗ Equilin 281 Estrogens, see oestrogens Flavanones 150 ∗ ∗ ∗ Eremothecium ashbyii 31 Estrone 274, 276 –7 , 278, 279, 280 –1 Flavin adenine dinucleotide, see FAD ∗ ∗ Ergine 368 , 370, 374, 376 Estropipate 281 , 281 Flavin mononucleotide, see FMN ∗ Erginine 376 Ethinylestradiol 275–6, 279, 280 Flavones 150 Ergobasine, see ergometrine Ethinyloestradiol, see ethinylestradiol Flavonoids 80, 149–51 ∗ ∗ Ergobine 373 Ethisterone 273 –4 Flavonolignans 151–2 ∗ ∗ Ergobutine 373 Ethylidenelophenol 255 Flavonols 150, 211 ∗ Ergobutyrine 373 Ethylmalonyl-CoA, as chain extender 97 Flax 457 ∗ Ergocalciferol 258 , 259, 260 Ethynodiol, see etynodiol Flaxseed 157 ∗ ∗ ∗ Ergocornine 372 –3 Etonogestrel 275 oil, see linseed oil ∗ ∗ ∗ ∗ Ergocristine 372 –3 Etopophos 136 , 137 Flecainide 303, 304 ∗ ∗ ∗ ∗ α-Ergocryptine 372 –3 Etoposide 136 , 137 Floramultine 341 ∗ ∗ ∗ β-Ergocryptine 373 Etorphine 336 , 336 Flucloxacillin 442 , 443 ∗ ∗ ∗ Ergoline 368 Etynodiol diacetate 275 Fludrocortisone acetate 269, 270 ∗ ∗ ∗ Ergometrine 368 , 370–2 , 374, 375, Eucalyptol 180 Fludroxycortide 272 ∗ 376 Eucalyptus citriodora 180 Flumetasone 272 Ergometrinine 374 E. globulus 180 Flumethasone, see flumetasone ∗ ∗ Ergonine 373 E. polybractea 180 Flunisolide 272 ∗ Ergonovine, see ergometrine E. smithii 180 Fluocinolone acetonide 272 ∗ ∗ Ergoptine 373 Eucalyptus oil 180 Fluocinonide 272 ∗ ∗ β-Ergoptine 373 lemon-scented 180 Fluocortolone 272 ∗ ∗ Ergosine 373 Eudesmane 196 Fluorometholone 270 , 271 ∗ ∗ ∗ β-Ergosine 373 Eudesmyl cation 193 , 196 Fluoroprednisolone 270–1 ∗ Ergostane 233–4 Eugenia caryophyllus 140 Fluoxetine 71, 347 ∗ ∗ ∗ ∗ Ergosterol 103, 253 , 254–5 , 258 –9, Eugenol 137–8 , 139–40 Flurandrenolone, see fludroxycortide ∗ 266 Eugenyl acetate 127, 140 Fluticasone propionate 270 , 271 ∗ ∗ ∗ ∗ Ergostine 373 Euphol 217–18 , 225 Fluvastatin 113 , 113 Euphorbia 207, 217 ∗ Ergot 368, 371–6 FMN, FMNH2 25 ,31 Ergot alkaloids, see alkaloids Evening primrose 46 Foeniculum vulgare 138, 140 ∗ ∗ ∗ ∗ oil 42, 46 ∗ Ergotamine 368 , 370 , 372 , 373 –4, Folic acid (vitamin B9) 124–5 , 375 126–127 ∗ ∗ Ergotamine group alkaloids 372–3 FAD, FADH2 28, 31, 25 Folinic acid 126 , 127 Ergotaminine 374 Fagopyrum esculentum 151 Follicle-stimulating hormone, see hormone Ergotism 371 Falcaria vulgaris 47 Follitropin alfa 415 ∗ Ergotoxine 373, 375 Falcarinol 47, 49 Follitropin beta 415 ∗ ∗ Ergotoxine group alkaloids 372–4 Farnesol 167, 169 Formestane 281 , 282 ∗ ∗ ∗ Ergovaline 373 Farnesyl cation 193 Formononetin 154–5 , 157 ∗ ∗ Ergoxine group alkaloids 372–3 Farnesyl diphosphate (FPP) 191–2 Formoterol 318 ∗ ∗ Eriodictyol 150 Fats 39–40 N-Formyldemecolcine 342 ∗ Erucamide 47 table 41–4 N-Formylkynurenine 312 ∗ Erucic acid 38 , 40, 42–3 Fatty acid synthase 36–8 Forosamine 98 ∗ Ervatmia heyneana 365 Fatty acids Forskolin 210, 212 , 212 ∗ Erwinia 440 acetylenic, biosynthesis 47–8 Fosfestrol 281 , 281 INDEX 495

∗ Fosmidomycin 172 Gentibiose 231 Glucogitaloxin 247 –8 ∗ ∗ Foxglove, Grecian 249 Gentiopicroside 189–90 , 386 –7 Gluconasturtiin 458 ∗ red 246 Gentisaldehyde 79 Gluconic acid 471 ∗ ∗ FPP, see farnesyl diphosphate Gentisyl alcohol 79 Gluconic acid 6-phosphate 467–8 ∗ ∗ Framycetin, see neomycin B Geranial 174 , 181–2 Gluconolactone 6-phosphate 467–8 ∗ ∗ ∗ Frangula 65, 70 Geraniol 167, 169 , 174 , 180, 184, Glucoraphanin 459 –60 ∗ ∗ ∗ ∗ ∗ ∗ Frangulin A 68 ,70 189 , 350 , 386 , 395 Glucosamine 428 , 468 , 474 ∗ ∗ Frangulin B 68 ,70 Geranyl acetate 180 Glucoscilliroside 252 ∗ ∗ ∗ Fraxinus ornus 471 Geranyl cation 173 Glucose 468 –9 , 471 ∗ ∗ Frog, poison 304 Geranyl diphosphate (GPP) 172–3 cyclic hemiacetal forms 468–9 ∗ ∗ ∗ ∗ Fructose 468 –9 , 471, 478 Geranylfarnesyl diphosphate (GFPP) Glucose 1,6-diphosphate 466 ∗ ∗ ∗ cyclic hemiketal forms 468–9 212–13 Glucose 1-phosphate 465–6 ∗ ∗ ∗ ∗ ∗ Fructose 6-phosphate 466–7 Geranylgeraniol 167, 169 Glucose 6-phosphate 465–6 , 467 –8 FSH, see hormone, follicle-stimulating Geranylgeranyl diphosphate (GGPP) β-Glucosidase 455 ∗ ∗ ∗ ∗ ∗ ∗ ∗ Fucosterol 253 203–4 , 205 , 208 , 212 , 226–7 α-Glucosides, formation 470 ∗ ∗ Fucoxanthin 226, 228 –9 Germacrane 192 β-Glucosides, formation 470 Fucus 226, 253 Germacranolide 192, 194 Glucosinolates 457–60 ∗ ∗ ∗ Fugu 398 Germacrene A 195 Glucuronic acid 220–1 , 472 ∗ ∗ ∗ ∗ Furochromones 74 Germacryl cation 192–3 , 195 , 196–7 Glutamic acid 292 , 406 , 453 ∗ Furocoumarins 67, 145–6, 179 Gestodene 275 , 276 in transamination 20–1 ∗ angular 145 Gestogens, see progestogens Glutamine 406 ∗ linear 145 Gestonorone caproate 274, 275 –6 Glutathione 60 ∗ ∗ Furoquinoline alkaloids, see alkaloids Gestrinone 284 , 285 Glyceraldehyde 3-phosphate 170, 312 , ∗ ∗ Fusarium 94, 202, 203, 440 Gestronol, see gestonorone 451 , 465–6 , 467 F. sporotrichiodes 203 GFPP, see geranylfarnesyl diphosphate ∗ ∗ ∗ Glycine 260 , 406 Fusidic acid 257 , 257 GGPP, see geranylgeranyl diphosphate Glycine max 44, 155, 157, 254, 256, 264 ∗ Fusidium coccineum 257 GH, see hormone, growth Glycogen 473–4 GHRH, see hormone, growth Glycoproteins 409 ∗ GABA, see γ-Aminobutyric acid hormone-releasing Glycopyrronium 300 , 300 ∗ Gadoleic acid 38 ,43 GHRIH, see hormone, growth Glycosaminoglycans 475, 477 Gadus morrhua 42 hormone-release inhibiting Glycosides, cardioactive, see cardioactive ∗ Galactosamine 468 Gibberella zeae 94 glycosides ∗ Galactose 469 Gin 181, 197 C-Glycosides, formation 29–30 ∗ Galacturonic acid 220, 475 Ginger 192 O-Glycosides, formation 29–30 Galantamine, see galanthamine oil 181 hydrolysis 29–30 ∗ Galanthamine 345–6 , 346 Gingerols 181 S-Glycosides 29–30 Galanthus 345, 346 Ginkgo biloba 209, 211 Glycosylation reactions 29–30 ∗ Ginkgolides 209–10 Glycyrrhetic acid 220–1 , 222 Galium 164 ∗ ∗ Ginkgolide A 211 Glycyrrhetinic acid, see glycyrrhetic acid Gallic acid 122 ∗ Ginkgolide B 211 Glycyrrhiza glabra 220, 221 Gallotannins, see tannins ∗ Ginkgolide C 211 G. glabra var.glandulifera 221 Gallstones 236, 260, 261 ∗ Ginkgolide J 211 G. glabra var.typica 221 Gambierdiscus toxicus 110 ∗ Gamolenic acid 46 Ginkgolide M 211 G. uralensis 222 ∗ Ganja, see cannabis Ginseng 48, 211, 217, 222–4 Glycyrrhizic acid 220–1 , 222 Garlic 460, 460–1 Russian 223 Glycyrrhizin 220, 222 oil 461 Siberian 223 Glycyrrhizinic acid, see glycyrrhizic acid ∗ ∗ Garosamine 481 –2 Ginsenosides 223–4 Glyoxylic acid 320–1 ∗ ∗ ∗ Gastrin, human 409 Ginsenoside R − 223–4 Glyphosate 123 b 1 ∗ Gaultheria procumbens 140, 141 Ginsenoside R − 224 GMP, see guanosine monophosphate b 2 ∗ Gee’s linctus 252, 330 Ginsenoside Rc 224 GnRH, see hormone, ∗ Gelidium 476 Ginsenoside Rd 223–4 gonadotrophin-releasing ∗ ∗ ∗ Gemeprost 55,56 Ginsenoside Re 223–4 Goitrin 458–9 ∗ ∗ Genes 114 Ginsenoside Rf 224 Gonadorelin 412 , 413 ∗ ∗ Geneserine 367 Ginsenoside R − 223–4 Gonadotrophins 413, 415 g 1 ∗ Genetic manipulation, acetate pathway Ginsenoside R − 224 chorionic 415 g 2 ∗ 114–17 Gitaloxigenin 246–7 , 249 human menopausal 415 ∗ ∗ ∗ Genistein 154–5 , 157, 278 Gitaloxin 247 –8 Gonyaulax 397 ∗ ∗ Gentamicin 481, 482 Gitoxigenin 244 , 246–7 , 249 Goose 49 ∗ ∗ Gentamicin A 482 Gitoxin 246–7 , 248 Goserelin 412 , 413 Gentamicin B 482 Gloriosa 343 Gossypium 50, 200, 201 ∗ Gentamicin C 482 Glucagon 416 , 417, 418 G. arboreum 201 ∗ ∗ Gentamicin C 478, 481 –2 Glucitol 468 G. barbadense 201 1 ∗ ∗ ∗ Gentamicin C 481 –2 Glucobrassicin 458 –9 G. herbaceum 201 1a ∗ Gentamicin C 481 –2 Glucocorticoids 262, 268 G. hirsutum 42, 201 2 ∗ Gentian 190 characteristic features 262 Gossypol 42, 200–1 , 201 ∗ Gentiana lutea 190, 387 Glucofrangulin A 68 ,70 GPP, see geranyl diphosphate ∗ ∗ Gentianine 190, 386 –7 Glucofrangulin B 68 ,70 Gracilaria 476 496 INDEX

Gram stain 475 poison 381, 382 Horsechestnut 220 Gramicidins 424 water 47 Horseradish 458 Gramicidin D 424 Hemlock water dropwort 47 HRT, see hormone replacement therapy Gramicidin S 424 Hemp, Indian 85 5-HT, see 5-hydroxytryptamine ∗ Gramicidin S 425 Hemp, sisal, see sisal Human chorionic gonadotrophin (HCG) 1 ∗ Ile-Gramicidin A 425 Henbane, see hyoscyamus 415 ∗ Ile-Gramicidin B 425 Henbane, Egyptian 296 Human growth hormone, see hormone, ∗ ∗ Ile-Gramicidin C 425 Heparin 163, 475 , 477 growth ∗ ∗ Val-Gramicidin A 424–5 Heracleum mantegazzianum 147 Humulene 198, 200 ∗ ∗ Val-Gramicidin B 425 Heroin 303, 332 , 332–3, 337 Humulus lupulus 200 ∗ ∗ ∗ ∗ Val-Gramicidin C 425 Hesperetin 152 Humulyl cation 192–3 , 198, 200 ∗ ∗ Gramine 348 –9 Hesperidin 151–2 Hyaluronidase 420, 436 ∗ ∗ Gramisterol 254–5 Hesperidin dihydrochalcone 152 ∗ ∗ Hydnocarpic acid 50–1 Grapefruit 151 Heterodendrin 457 Hydnocarpus wightiana 50 ∗ ∗ Griseofulvin 76–8, 77 Hevea brasiliensis 232 Hydrastine 339–40 , 341 ∗ Griseophenone B 77–8 High density lipoprotein (HDL) 237 Hydrastis 339 ∗ Griseophenone C 77–8 Hippoglossus vulgaris 42 H. canadensis 339 ∗ ∗ ∗ Groundnut oil, see arachis oil Histamine 379 Hydrocortisone 222, 262 –3 , 264, ∗ ∗ ∗ ∗ ∗ Groundsel 306 Histidine 379 –80 , 406 266–7 , 268 , 268 alkaloids derived from 379–80 ∗ Growth hormone, see hormone ∗ Hydromorphone 332 , 332 Growth hormone-release inhibiting HMG-CoA 169–70 Hydroxocobalamin, see vitamin B ∗ 12 ∗ hormone, see hormone HMG-CoA reductase 112–14, 169, 172 3-Hydroxyanthranilic acid 312 , 432 ∗ Growth hormone-releasing hormone, see Hogweed, giant 147 4-Hydroxybenzaldehyde 141 ∗ ∗ ∗ hormone Holaphyllamine 392 –3 4-Hydroxybenzoic acid 123–4 , 141 , ∗ ∗ Guaiacylglycerol coniferyl ether 132–3 Holarrhena antidysenterica 393 158 –9 H. floribunda 393 Guaiane 195–6 ∗ 10-Hydroxycamptothecin 365 ∗ Guaianolide 194, 196 Homatropine 300 , 300 Hydroxychloroquine 363 , 363 ∗ Homoaporphine alkaloids, see alkaloids ∗ Guaiyl cation 193 , 196 ∗ 26-Hydroxycholesterol 388, 390 Homobaldrinal 190–1 ∗ Guanosine monophosphate (GMP) 393–4 ∗ 4-Hydroxycinnamyl alcohol 131 –2 Homogentisic acid 159–60 ∗ Guanosine triphosphate (GTP) 74 ∗ 4-Hydroxycoumarin 143–4 Homomethionine 458–9 ∗ Guarana 396 4-Hydroxydihydrocinnamaldehyde 341 Homomorphinan alkaloids, see alkaloids ∗ Guaranine 396 ∗ 6-Hydroxyhyoscyamine 294 –5 ∗ Homophenylalanine 458–9 ∗ Gulonic acid 472 ∗ Hydroxykynurenine 312 ∗ Homospermidine 305 Gulonolactone 472 Hydroxylases 26 ∗ Honesty oil 42 Gulose 429 Hydroxylation pattern Honey 471 Gum arabic, see acacia anthraquinones 164 ∗ Hopanoids 218 Gutta percha 232 ∗ meta 62 ∗ Hopanol 218, 220 Guvacine 316 ortho ∗ Hops 200 123 Guvacoline 316 ∗ Hydroxymethylcellulose 476 Gymnodimium breve 109 Hordenine 316 β β Hordeum vulgare 316, 349 -Hydroxy- -methylglutaryl-CoA, see Gymnothorax javanicus 109 HMG-CoA Hormone ∗ 4-Hydroxyphenylacetaldehyde 322 –3 antidiuretic (ADH), see vasopressin ∗ N-Hydroxyphenylalanine 456 Haem ring 32 corticotropin-releasing (CRH) ∗ ∗ ∗ 4-Hydroxyphenylglycine 426–7 , 428 , Haemin 200 411–12 ∗ 454–5 Haemoglobin 200, 204, 363 follicle-stimulating (FSH) 415 ∗ Haemozoin 200 4-Hydroxyphenylpyruvic acid 128–9 , ∗ gonadotrophin-releasing (GnRH) 412 ∗ ∗ Halcinonide 272 growth (GH) 413, 414 159–60 , 322 –3 α ∗ Halibut-liver oil 42, 230, 259 growth hormone-release inhibiting 11 -Hydroxyprogesterone 264, 265 ∗ ∗ α ∗ Halofantrine 363 , 363 (GHRIH) 411–12 , 413 17 -Hydroxyprogesterone 263 , 274, ∗ ∗ Harmaline 349 growth hormone-releasing (GHRH) 277 ∗ ∗ Harman 349 –50 413 Hydroxyprogesterone caproate 274, 275 ∗ ∗ Harmine 349 –50 Hydroxyproline 406 luteinizing (LH) 415 ∗ Hashish, see cannabis luteinizing hormone-releasing (LH-RH) 5-Hydroxytryptamine (5-HT) 347 , ∗ ∗ ∗ Hecogenin 238 , 240, 264 –5 ∗ 347–8, 374–5 411–12 , 412 ∗ Hedera helix 47 replacement therapy (HRT) 155, 157, 5-Hydroxytryptophan 347 Helianthus annuus 44 239, 276, 283, 279 1α-Hydroxyvitamin D2 259 Heliotropium 305 thyrotrophin-releasing (TRH) 1α-Hydroxyvitamin D3 259 ∗ H. indicum 306 411–12 , 412 25-Hydroxyvitamin D , see calcidiol ∗ 3 Helleborus 246, 252 Hormones Hygrine 293 –4, 302 ∗ H. niger 243 adrenocortical, see corticosteroids Hygroline 301 –2 ∗ ∗ ∗ Hellebrigenin 243 , 244 –5 anterior pituitory 413 Hyoscine 294 –5, 296, 297–301, ∗ ∗ Helminthosporium maydis 212 hypothalamic 411–12 299 –300 ∗ Hemachatus 437 pancreatic 416–17 Hyoscine butylbromide 300 , 300 ∗ ∗ ∗ Hemigossypol 200–1 peptide 410–19 Hyoscyamine 293 –4 , 295–6, ∗ Hemiterpenes 167–8, 172 posterior pituitory 413, 415 297–301, 298 , 367 Hemlock 382 thyroid 410–11 Hyoscyamus 296 INDEX 497

∗ ∗ Hyoscyamus 301 Isocamphyl cation 176 Khellol 75 –6 ∗ H. muticus 296 Isochondrodendrine 325 Kief, see cannabis ∗ ∗ H. niger 295, 296 Isochorismic acid 124 , 159, 162 Kola, see cola ∗ Hyperforin 71 –2 Isoflavones 154 Kreysigia multiflora 342 ∗ ∗ ∗ Hypericin 65, 67 ,71 –2 Isoflavonoids 154–6, 278, 282 Kreysigine 341 ∗ ∗ Hypericism 72 Isoleucine 306, 406 , 457 Kynurenine 312 , 432 ∗ Hypericum 71–2 Isoliquiritigenin 149 –50 ∗ Hypericum perforatum 67, 71 Isoliquiritin 221 –2 ∗ ∗ Hypothalamic hormones, see hormones Isolysergic acid 372 , 374 Labdadienyl diphosphate 207–8 , ∗ ∗ Isomenthol 178 209–10 ∗ ∗ Isomenthone 178 Labetalol 318 ∗ ∗ Iboga 357 Isopenicillin N 437–8 , 445 Laburnum 309 ∗ Iboga-type alkaloids 350 –1, 353–4, Isopentenyl diphosphate (IPP) 14, β-Lactamase 443–4, 445–6, 450, 452, ∗ ∗ ∗ ∗ 358 167–8 , 170 , 171 –2 454 ∗ Ibogaine 354, 357–8 Isophane insulin 417 β-Lactams 437–55 ∗ ∗ Ibuprofen 55 Isopilocarpine 379 –80 Lactose 330, 470 , 471 ∗ ∗ Idarubicin 94 , 94 Isopilosine 380 Lactulose 470 , 471 ∗ ∗ Iduronic acid 475 Isopimpinellin 145 –6 Laminaria 476 ∗ Ilex paraguensis 396 Isopiperitenol 178 Lanatosides ∗ ∗ Illicium 121 Isopiperitenone 178 Lanatoside A 247 –9 ∗ ∗ I. verum 138, 139 Isoprenaline 318 , 319 Lanatoside B 247 –9 ∗ ∗ ∗ ∗ ∗ Iloprost 56,56 Isoprene 167–8 , 172 Lanatoside C 247 –9 , 250 ∗ ∗ Imidazole alkaloids, see alkaloids unit 10, 167, 168 Lanatoside D 247 –9 ∗ Imines, see Schiff base Isoprostanes 57–8 Lanatoside E 247 –9 ∗ ∗ Imipenem 453 , 454 Isoproterenol 318 , 319 Lanolin 236 ∗ IMP, see inosine monophosphate Isopulegone 178 Lanolin alcohols 236 ∗ ∗ ∗ Indicine-N-oxide 306 Isosilybin 152–4 Lanostane 233–4 ∗ ∗ ∗ ∗ Indole 128 Isothebaine 337–8 Lanosterol 215 , 216 –17, 233, 236, ∗ Indole alkaloids, see alkaloids Isothiocyanates 457 253–5 ∗ ∗ Indolizidine alkaloids, see alkaloids Isotretinoin 231 , 231 Lanreotide 412 , 413 ∗ Indometacin 55 Isotrichodermol 202 Lard 43 ∗ ∗ Inosine monophosphate (IMP) 74, Isotrichotriol 202 Lasalocid A 106, 108 ∗ ∗ ∗ ∗ 393 –4 Isovaleric acid 180, 190, 194 , 197, 379 Latanoprost 56 , 56 ∗ ∗ myo-Inositol 478–9 Isovaltrate 190–1 Laudanum 330 ∗ ∗ myo-Inositol phosphate 479 Ivermectin 101 , 101 Laurel, cherry 456 ∗ Insulin 416–17, 417–418 Lauric acid 38 , 40–3 ∗ aspart 416 , 418 Lavandula angustifolia 181 ∗ bovine 416 , 417 Jaborandi 380 L. officinalis 181 glargine 418 Jamestown weed, see stramonium Lavender oil 181 ∗ ∗ ∗ human 416 , 417 Jasmolins 186–7 Lawsone 163–4 ∗ ∗ isophane 418 Jasmolin I 187 –8 Lecanoric acid 63 ∗ ∗ lispro 416 , 418 Jasmolin II 187 –8 Lectin 434 ∗ ∗ porcine 416 , 417 Jasmolone 186–7 , 188 Lemon oil 181 ∗ Interferons 417, 418–19 Jasmonic acid 187 terpeneless 181 alfa 419 Jequirity seeds 435 Lemon-grass oil 182 ∗ beta 419 Jervine 390–1 , 392 Leptodontium elatius 111 gamma 419 Jimson weed, see stramonium Leucanthemum parthenium 195 ∗ ∗ ∗ Inulin 473–4 Juniper 197 Leucine 322, 379 , 406 , 457 ∗ Invert sugar 471 oil 181 Leucocyanidin 150 Ipecac, see ipecacuanha Juniperus communis 181,197 Leucojum 346 Ipecacuanha 330, 343, 344–5 Justicia adhatoda 377 L. aestivum 346 ∗ emetic mixture 345 Leuconolide A 98 ∗ 1 ∗ Ipecoside 343–4 Leucopelargonidin 150 ∗ ∗ Ipomoea 370 Kaempferol 150 –1, 211 Leucovorin, see folinic acid I. violacea 376 Kanamycins 481, 482 Leukotrienes 59 ∗ IPP, see isopentenyl diphosphate Kanamycin A 482–3 biosynthesis 58–60 ∗ ∗ Ipratropium bromide 300 , 301 Kanamycin B 483 Leukotriene A (LTA )60 ∗ 3 3 ∗ Iridane 187–8 Karaya gum 477 Leukotriene A (LTA )60 ∗ ∗ ∗ ∗ 4 4 Iridodial 188–9 , 386 ent-Kaurene 208–9 , 387–8 Leukotriene A (LTA )60 ∗ 5 5 ∗ Iridoids 168, 187–90 ent-Kaurenoic acid 209 Leukotriene B (LTB ) 59–60 ∗ 4 4 ∗ Iridotrial 188–9 Kava 147, 148 Leukotriene C (LTC ) 59–60 ∗ 4 4 ∗ Irinotecan 365 , 365 Kavalactones 148 Leukotriene D (LTD ) 59–60 ∗ 4 4 ∗ Islandicin 64 Kavapyrones 148 Leukotriene E (LTE ) 59–60 ∗ ∗ 4∗ 4 Isoandrocymbine 342 Kawain 148 Leuprorelin 412 , 413 ∗ Isoboldine 337 Ketoconazole 254 Leurocristine, see vincristine ∗ Isobutyric acid 180, 194 , 197 Khat 383, 384–5 Leurosidine 356 ∗ ∗ Isocamphane 174 Khellin 74–5 , 75–6 Leurosine 356 498 INDEX

∗ ∗ Levobunolol 318 , 319 Lupanine 309–10 Marker degradation 264, 266, 273–4, ∗ Levodopa, see DOPA Lupenyl cation 218–19 279–80, 283, 391 ∗ ∗ ∗ Levofolinic acid 127 Lupeol 218–19 Marmesin 145 –6 ∗ Levoleucovorin 127 Lupin 218 Matairesinol 134 –5, 157 ∗ Levomethorphan 334 bitter 310 Matricaria 192, 196, 196–7 ∗ Levonorgestrel 275 –6, 275 sweet 310 oil 182, 197 ∗ Levopropoxyphene 334 , 335 Lupin alkaloids 309 Matricaria chamomilla 182, 192, 196 ∗ ∗ Levothyroxine, see thyroxine Lupinine 309–10 Matricin 196 LH, see hormone, luteinizing Lupinus 309 May apple 135 ∗ LH-RH, see hormone, luteinizing L. luteus 218, 309 Maytansine 106 ∗ hormone-releasing Lutein 226, 228 –9 Maytansinol 106 Liberty cap 348 Luteinizing hormone, see hormone Maytenus serrata 106 Licorice, see liquorice MDMA 383, 385 ∗ Luteinizing hormone-releasing hormone, Lidocaine 303, 304 see hormone Meadow saffron 343 ∗ Lignans 132–5, 157 Luteolin 150 Mecillinam 443 ∗ Lignin 132 Lutjanus bohar 109 Meconic acid 330 ∗ Lignocaine, see lidocaine Lycopene 226–7 Medicago sativa 154, 246 ∗ ∗ Lignoceric acid 38 Lycopersicon esculente 226, 241, 388 Medicarpin 154–5 Lily of the valley 251 ∗ ∗ ∗ ∗ ∗ Lycorine 345–6 Medigoxin 249 , 250 Limonene 174–5 , 177 –8 , 179, 181–6 Lycorus radiata 345 Medroxyprogesterone acetate 274, ∗ ∗ Limonoids 225–6 Lymecycline 89 , 91 275 –6 ∗ ∗ ∗ Linalool 174 , 179–81, 183, 185, 395 Lypressin 415 , 416 Mefloquine 363 , 363 Linalyl acetate 179, 181, 183 ∗ ∗ ∗ ∗ ∗ ∗ Lysergic acid 12 , 368 –9 , 370–2 , Megestrol acetate 274, 275 –6 Linalyl diphosphate (LPP) 172–3 ∗ 374 Melaleuca alternifolia 176, 185 Linamarin 456–7 α ∗ ∗ Lysergic acid -hydroxyethylamide 372 , Melanins 129, 146 Lincomycin 484–5 , 485 374, 376 Melicope fareana 379 ∗ ∗ ∗ ∗ Linoleic acid 38 , 41–5 ,48 , 253, 396 Lysergic acid diethylamide, see LSD Melicopicine 378 –9 α ∗ ∗ -Linolenic acid 38 ,43–4,45 –6, 186, Lysergide, see LSD Melilotus 143 253 ∗ ∗ ∗ ∗ ∗ Lysine 307 , 406 , 437–8 M. officinalis 144 γ ∗ -Linolenic acid 35 , 41–2, 45 –6 alkaloids derived from 307–11 Melissic acid 38 43 Linseed oil as building block 9–10 Meloxicam 55 Linum usitatissimum 43, 157, 457 ∗ Lysine vasopressin, see lypressin Menadiol phosphate 163 Liothyronine 411 ∗ ∗ ∗ Lysuride, see lisuride Menaquinones 158 –9, 162 –3, 204, Lipoic acid, in decarboxylation 23 –24 ∗ ∗ 232 Liquiritigenin 149 –50, 154–5 ∗ ∗ Menaquinone-1 163–4 Liquiritin 221 –2 Menotrophin 415 Liquorice 220, 221–2 Ma Huang 383 Mentha 178 deglycyrrhizinized 222 Macrocystis 476 ∗ M.x piperita 177, 178, 183 Lisuride 372 , 375 Macrolides 94–8, 98–100 ∗ M. pulegium 186 Lithocholic acid 260–1 ansa 105–6, 107–8 ∗ polyene 101–2 M. spicata 177, 185, 186 Littorine 294 –5, 298 ∗ ∗ Madder 164 Menthane 174 Lobelanine 308 ∗ Magic mushrooms 347–8 Menthofuran 178 , 183, 186 Lobelia 308 ∗ ∗ Lobelia inflata 308 Mahonia 339 Menthol 167, 169 , 178 , 183 ∗ ∗ Lobeline 308 , 308 Maize 475 Menthone 178 ,183, 186 ∗ ∗ oil 43, 160 Menthyl acetate 183 Loganin 188–9 , 190, 350 –1, 359 ∗ ∗ Malonyl-CoA Menthyl cation 173–4, 175 –6 Lonchocarpus 157 ∗ Lonchocarpus 156 as building block 10 Mepacrine 363 , 363 formation 17 Meperidine, see pethidine L. urucu 157 ∗ in Claisen reaction 17–18 Mepivacaine 303, 304 L. utilis 157 ∗ ∗ ∗ Maltose 470 , 484 Meptazinol 334 , 335 Lophocerine 320 ∗ Lophophora 320 Malvalic acid 42, 50–1 Merendera 343 Mamba 436 Merilliodendron megacarpum 365 Lophophora schotti 320 ∗ ∗ L. williamsii 316, 320 Mandelonitrile 455–6 Meropenem 453 , 454 ∗ Loracarbef 449 , 450 Mandragora 301 Meroterpenoids 168 ∗ M. officinarum 297 Mescal buttons 320 Lotaustralin 456–7 ∗ ∗ ∗ Mandrake, American 135 Mescaline 316 , 320 Lovastatin 111–12 , 112, 113 , 237 ∗ European 297 Mesterolone 283, 284 Low density lipoprotein (LDL) 237 ∗ LPP, see linalyl diphosphate Manihot esculenta 456 Mestranol 279, 280 ∗ ∗ LSD 372 , 376 Mannich reaction 18–19 Metaproterenol 318 , 319 ∗ ∗ Lucerne 154, 246, 278 Mannitol 468 , 471 Metaraminol 317 , 317 ∗ ∗ ∗ Lucidin 164 Mannojirimycin 477–8 Meteloidine 295 ∗ ∗ ∗ ∗ Lumiestrone 278 Mannose 468 –9 Methacycline 89 , 91 ∗ Lumisterol 259 Mannuronic acid 475 Methadone 334, 335 ∗ Lunaria annua 42 Mappia foetida 365 Methandienone 283 , 284 Lunularia cruciata 81 Maranta arudinacea 475 Methandrostenolone 284 ∗ ∗ Lunularic acid 80 –1 Marihuana, see cannabis Methicillin 442 , 443 INDEX 499

∗ Methicillin-resistant Staphylococcus Mineralocorticoids 262, 268 Nandrolone 284 , 284 aureus, see MRSA characteristic features 263 Naphthoquinones 163 ∗ ∗ ∗ ∗ Methionine 406 , 458–9 Minocycline 89 , 91 Naratripan 347, 348 ∗ ∗ ∗ as building block 9 Misoprostol 56 , 56 Narceine 330 , 339–40 ∗ Methotrexate 127 , 127 Mistletoe 435 Narcissus 345 ∗ ∗ Methoxamine 317 , 317 Mitoxantrone 94 , 94 N. pseudonarcissus 346 Methoxsalen 146–7 Mitozantrone, see mitoxantrone Narcotine, see noscapine ∗ ∗ ∗ ∗ ∗ 2-Methoxycinnamaldehyde 139 Mivacurium 326 , 327 Naringenin 80 , 149 –50 , 152 , ∗ ∗ ∗ 9-Methoxyellipticine 360 MMDA 383 , 385 154–5 ∗ ∗ ∗ S-Methyl cysteine sulphoxide 460 Mometasone 272 Naringenin-chalcone 149 ∗ ∗ ∗ Methyl salicylate 140, 141–2 Monacolin K, see lovastatin Naringin 12 , 151–2 ∗ Methyl vitamin B , see vitamin B Monascus ruber 112 Naringin dihydrochalcone 152 12 ∗ 12 ∗ O-Methylandrocymbine 342 Monensin A 106, 108–9 Nasturtium 458 ∗ Methylcellulose 476 Monkshood 387 Nebivolol 318 , 319 ∗ Methylchavicol 138 Mono-oxygenases 25–7 Nebramycin factor 6, see tobramycin ∗ Methylcobalamin, see vitamin B Monoamine oxidases 28, 347 Nebrosamine 483 ∗ ∗ 12 ∗ N-Methylcoclaurine 322 –3 , 324 –5 inhibitors 319 Necic acids 305 ∗ ∗ N-Methylconiine 382 Monobactams 450 , 453, 454 Nedocromil 75 –6 ∗ Methyldopa 317 , 317 Monosaccharides 465–9, 471–3 Neem 226 ∗ ∗ ∗ Methylecgonine 293 , 295, 301 –2 Monoterpenes 167–8 Neohesperidin 151–2 ∗ ∗ Methylenecycloartanol 255 biosynthesis 172–8, 186 Neohesperidose 152 ∗ Methylenedihydrolanosterol 254 irregular 186–7 Neoisomenthol 178 ∗ Methylenedioxy group, formation 27 Montanic acid 38 Neolignans 135 ∗ ∗ Methylenelophenol 254–5 Montelukast 59 Neomenthol 178 ∗ Methylerythritol phosphate 171 Montezuma speciosissima 201 Neomycin 481, 483 ∗ pathway 169 Moray eel 109 Neomycin B 483 ∗ ∗ N-Methylglucosamine 479–80 Morning glories 370, 376 Neomycin C 483 ∗ N-Methylglycine, see sarcosine Morphinan alkaloids, see alkaloids Neopatulin 79 ∗ ∗ Methylmalonyl-CoA, as chain extender Morphine 327–8 , 329–30, 331, Neopinone 328 –9 ∗ ∗ ∗ ∗ 49–50, 92, 95 332 –3 , 334 , 337, 419 Neosamine B 483 ∗ ∗ N-Methylnarwedine 346 antagonist 336 Neostigmine 299, 325, 366 , 367  ∗ ∗ ∗ 4 -O-Methylnorbelladine 345–6 Morphine O-glucuronide 330 –1 Neotigogenin 240 –1 ∗ ∗ 5-Methylorsellinic acid 72–3 Morphinone 328 –9 Nepeta cataria 188 ∗ ∗ N-Methylpelletierine 307 Morus 477 Nepetalactone 188 ∗ ∗ Methylphloracetophenone 76–7 MRSA 426, 431, 443, 454 Neral 174 , 181–2 ∗ Methylprednisolone 270 , 270 Mucopolysaccharides 475 Nerium oleander 252 ∗ ∗ ∗ ∗ 6-Methylpretetramide 90 ,92 Muscarine 299 , 380 Nerol 174 ∗ O-Methylpsychotrine 344, 345 Mushroom, common 433 Neroli oil, see orange flower oil ∗ ∗ N-Methylputrescine 292 Mustard, black 457 Nerolidyl cation 193 ∗ ∗ ∗ 6-Methylsalicylic acid 62 ,79 white 457 Nerolidyl diphosphate 191–2 , 202 ∗ ∗ 17α-Methyltestosterone 283 MVA, see mevalonic acid Nervonic acid 38 ,42 ∗ ∗ ∗ 7-Methylxanthine 393 Mycaminose 98 Neryl cation 173 ∗ ∗ ∗ 7-Methylxanthosine 393 –4 Mycarose 98 Neryl diphosphate (NPP) 172–3 ∗ ∗ ∗ Methysergide 372 , 376 Mycinose 98 Netilmicin 481–2 ∗ Methysticin 148 Mycobacterium phlei 279 Niacin, see nicotinic acid ∗ Metildigoxin 249 , 250 M. tuberculosis 49 Nicotiana rustica 315 ∗ ∗ Metipranolol 318 , 319 Mycophenolate mofetil 73 N. tabacum 308, 311, 314 ∗ ∗ ∗ Metoprolol 318 , 319 Mycophenolic acid 72–3 , 73–4 Nicotinamide 313, 315 ∗ ∗ Mevaldic acid 112–13, 170 Mycosamine 102 Nicotinamide adenine dinucleotide, see ∗ + Mevalonate-independent pathway 169 β-Myrcene 174 , 178, 181–2 NAD ,NADH ∗ ∗ Mevalonic acid (MVA) 168–70 Myristic acid 38 , 41–4 Nicotinamide adenine dinucleotide + as building block 8–10 Myristica fragrans 138, 140, 385 phosphate, see NADP ,NADPH ∗ ∗ ∗ ∗ Mevastatin 112–13, 113 Myristicin 138 , 140, 385 Nicotine 297, 299, 311, 313 –14, 315 ∗ ∗ Mevinolin, see lovastatin Myrosinase 457 Nicotinic acid (vitamin B ) 312 –13 , ∗ 3 Mexiletene 303, 304 Myrothecium 203 313, 395 Miconazole 254 alkaloids derived from 311–15 ∗ Microcystins 435–6 Nicoumalone 144 , 144 ∗ ∗ Microcystin-LR 436 Nabilone 87 , 88 NIH shift 26 + ∗ Microcystis aeruginosa 435 NAD , NADH 24 –5, 312–13 Nitritocobalamin, see vitamin B ∗ 12 M. viridis 435 Nadolol 318 , 319 Nocardia 106 + ∗ Micromonospora 481 NADP , NADPH 24 –5, 312–13 N. mediterranei 107 ∗ M. inyoensis 484 Nafarelin 412 , 413 N. uniformis 453 M. purpurea 478, 482 Naja 436, 437 Nocardicins 453 ∗ ∗ ∗ ∗ Mifepristone 275 , 276 Nalbuphine 335 , 336 Nocardicin A 453 , 455 ∗ ∗ Mikamycin A 431 Nalorphine 332 , 333 Nocardicin E 455 ∗ ∗ Mikamycin B 431 Naloxone 333, 335 , 336 Nocardicin G 455 ∗ ∗ Milkweed, common 246 Naltrexone 335 , 336 Nojirimycin 477–8 500 INDEX

∗ ∗ Non-ribosomal peptide synthase (NRPS) Oleanolic acid 223–4 Oxytocin 375, 413, 415 , 415 ∗ 421 Oleanyl cation 218–19 Oxytropis 311 ∗ ∗ ∗ ∗ Non-steroidal anti-inflammatory drugs Oleic acid 38 ,40–4,45 ,47 –8 , 253, (NSAID) 55 396 ∗ ∗ Noradrenaline 129, 316 , 317, 374–5 , Oligosaccharides 469–71 PABA, see p-Aminobenzoic acid ∗ ∗ 384 Olivanic acids 451, 453 Paclitaxel 205 , 205–7 ∗ Noratropine 300 Olive oil 40, 43 PAF, see platelet-activating factor ∗ ∗ Norbelladine 345–6 Olivetolic acid 85 –6 PAL, see phenylalanine ammonia lyase ∗ Norbufalin 245 –6 Ololiuqui 376 Palaquium gutta 232 ∗ Norcoclaurine 322 –3 Onchorhynchus 418, 477 Palm kernel oil 40, 43 ∗ Norephedrine 382 –3, 384 Onion 460 fractionated 43 ∗ Norepinephrine, see noradrenaline lachrymatory factor 460 Palmitic acid 38 , 41–4, 230, 253, 396 ∗ ∗ ∗ ∗ Norethisterone 274 –6 Ophiobolene 212–13 Palmitoleic acid 38 , 41–4, 81 ∗ ∗ Norethisterone acetate 274 , 274 Ophiobolin A 212–13 Panaeolus 347, 348 ∗ ∗ Norgestimate 275 Ophiobolin F 213 Panax ginseng 48, 217, 222 ∗ Norgestrel 274, 275 Ophiorrhiza mungos 365 P. notoginseng 223 C-Nor-D-homosteroids 390–1 Opioid peptides, see peptides P. quinquefolium 222 ∗ Norhyoscine 301 Opioids 332 Panaxadiol 223–4 ∗ ∗ Normorphine 332 Opium 327, 329–37 Panaxatriol 223–4 ∗ Nornicotine 297, 313 –15 powder 330, 344 Panaxosides 223–4 ∗ ∗ ∗ Norpseudoephedrine 382 –3 , 384 Opsin 230 Panaxynol 48–9 ∗ ∗ N-Nor-reticuline 322 Orange Panaxytriol 48–9 Norsolorinic acid 82 bitter 151 Pancreatin 420 ∗ ∗ Norsteroids 233–4 flower oil 183 Pancuronium 326 , 327 ∗ ∗ Nortestosterone 274, 284 oil, bitter 182 Pantotheine 36–7, 371, 421–2  ∗ 8 -Norvinblastine 356 oil, sweet 182 Pantothenic acid (vitamin B )31, 31, ∗ ∗ ∗ ∗ 5 Noscapine 330 , 331, 339, 341 oil, terpeneless 182–3 36–7 , 421–2 ∗ Nothapodytes foetida 365 Orciprenaline 318 , 319 PAPA, see p-Aminophenylalanine ∗ NPP, see neryl diphosphate Orientaline 337–8 , 339 Papain 420 ∗ NRPS, see non-ribosomal peptide synthase Orientalinol 338 Papaver 330 ∗ NSAID, see non-steroidal Orientalinone 338 P. bracteatum 337 ∗ anti-inflammatory drugs Oripavine 328 –9 P. orientale 337, 338 ∗ ∗ Nutmeg 138, 385 Ornithine 292 , 377, 406 P. pseudoorientale 337 oil 140 alkaloids derived from 292–307 P. somniferum 328, 329, 337, 339 Nux-vomica 359 as building block 9–10 Papaveretum 330, 332 ∗ ∗ ∗ ∗ ∗ ∗ Nystatin 103, 254 Orsellinic acid 12 ,61 ,63 , 79–80 Papaverine 12 , 322 –3, 330, 331, 332 ∗ Nystatin A1 102 –3 Oryza sativa 475 Papaya 420 Nystatin A 103 OSB, see o-Succinylbenzoic acid PAPS 457 2 ∗ Nystatin A3 103 Oscine 298–9 Paracetamol 153 Otto of rose, see rose oil Paregoric 330 ∗ ∗ Ouabain 250, 251 Parillin 242 ∗ ∗ Obtusafoliol 254–5 Ouabigenin 250–1 Parsley 147 ∗ Ochrosia 360 Ovis aries 44 Parthenin 194 O. elliptica 359, 360 Oxalic acid 71 Parthenium hysterophorus 194 ∗ ∗ Octanal 183 Oxidases 25 Parthenolide 12 , 192, 194–5 ∗ ∗ Octreotide 412 , 413 β-Oxidation 18, 141, 260 Paspalic acid 369 –70, 374 ∗ Oenanthe crocata 47 Oxidation reactions 24–8 Patulin 78–9 ∗ Oenanthotoxin 47, 49 Oxidative cleavage of aromatic rings 27, Paullinia cupana 396 Oenothera biennis 42, 46 78–9, 485 Pausinystalia yohimbe 351 Oestradiol, see estradiol Oxidative coupling, see phenolic oxidative Pea 154 Oestriol, see estriol coupling Peanut 82 ∗ Oestrogens 273, 276–8, 279–82 Oxitropium bromide 300 , 301 oil, see arachis oil ∗ characteristic features 276 2-Oxoglutarate-dependent dioxygenases Pectin 475 ∗ conjugated equine 280 27–8 Peganine 377 ∗ receptor antagonists 282 2-Oxoglutaric acid 159, 162 Peganum harmala 350, 377 ∗ Oestrone, see estrone in transamination 20–1 Pelargonidin 150 ∗ Oils 39, 40 5-Oxoproline, see pyroglutamic acid Pelletierine 307 ∗ ∗ fixed, table 41–4 Oxprenolol 318 α-Peltatin 136 ∗ ∗ volatile, aromatic, table 139–40 Oxybuprocaine 303, 304 β-Peltatin 136 ∗ ∗ volatile, terpenoid, table 179–85 Oxycodone 335 , 336 Penam 450 ∗ ∗ Okadaic acid 108–9 Oxygenation pattern Penem 450 ∗ Olea europaea 43 anthraquinones 164 Penicillenic acid 437, 439 ∗ Oleamide 47 meta 62 Penicillic acid 79–80 Oleander 252 ortho 123 Penicillins 437, 437–44 ∗ ∗ Oleandolide 97 Oxymetholone 284 , 284 binding proteins 443–4 ∗ ∗ Oleandomycin 96–7 , 100 Oxymorphone 335 , 336 Penicillin G, see benzylpenicillin ∗ ∗ ∗ ∗ Oleandrose 97 –8, 101 Oxytetracycline 89 , 90–1 Penicillin N 445 INDEX 501

∗ ∗ ∗ Penicillin V, see Phenylalanine 121, 380 , 382 , 406 , Pinus 177, 183, 185, 381 ∗ ∗ phenoxymethylpenicillin 456 –7, 458–9 P. mugo ssp. pumilio 183 table 441–2 alkaloids derived from 382–5 P. palustris 183, 185 ∗ ∗ Penicillinase 443, 445 as building block 9–10 Pinyl cation 174–6 , 177 ∗ ∗ Penicillium 63, 64, 370 biosynthesis 128–9 Pipecolic acid 103, 310–11 , 431 –2 P. baarnense 79 Phenylalanine ammonia lyase (PAL) 130 Piper betle 315 ∗ P. brevicompactum 72, 73, 112 Phenylephrine 317 , 317 P. methysticum 147, 148 P. chrysogenum 437, 440 2-Phenylethanol 184, 395 P. nigrum 308 ∗ P. citrinum 74, 112 Phenylethylamines, see alkaloids Piperacillin 441 , 443, 452 ∗ ∗ P. cyclopium 79 Phenyl-lactic acid 293–4 , 295, 298 Piperic acid 309 P. griseofulvum 76, 77 Phenylpropenes 135–8 ∆1 ∗ ∗ ∗ ∗ -Piperideine 307 –9 P. islandicum 64 Phenylpyruvic acid 128–9 , 294 ∗ ∗ Piperideine-6-carboxylic acid 437–8 P. janczewski 77 Phloracetophenone 61 –2 ∗ ∗ Piperidine 309 P. nigrum 77 Pholcodine 332 , 332 Piperidine alkaloids, see alkaloids ∗ ∗ P. patulum 62, 77, 78 Phorbol 207–8 Piperine 308–9 ∗ Phosphatidic acid 39 ∗ Penicilloic acid 438–9 , 444 ∗ Piperitenone 178 ∗ Phosphatidylcholine 39 ∗ Penillic acid 437, 439 ∗ Piperitone 178 , 186 Phosphatidylethanolamine 39 ∗ Pennyroyal oil 186 ∗ Pisatin 154–5 ∗ Phosphatidyl-myo-inositol 39 Pentagalloylglucose 122–3 ∗ Pisum sativum 154 ∗ Phosphatidylserine 39 ∗ Pentazocine 334 , 334 Pivampicillin 441 , 443 Phosphoadenosine-phosphosulphonate, see ∗ PEP, see phosphoenolpyruvate Pivmecillinam 442 , 443 ∗ PAPS Peplomycin 428, 429 ∗ Phosphoenolpyruvate (PEP) 121–2 , Plant stanol esters 237, 256 Pepper 226 ∗ Plasminogen activator, recombinant betel 315 123 3-Phosphoglyceraldehyde, see tissue-type (rt-PA) 420 black 308 rt-PA, see plasminogen activator chilli 385 glyceraldehyde 3-phosphate Phospholipids 39 Plasminogen–streptokinase, acylated Peppermint 177–8 activator complex (APSAC) 420 183 Phosphoproteins 409 oil Plasmodium falciparum 198, 200, 362, Pepsi-Cola 396 Photosynthesis 226, 465 Phthalideisoquinoline alkaloids, see 363 Pepsin 420 ∗ ∗ alkaloids Plastoquinones 158 –9, 160 , 232 Peptidase 409 ∗ ∗ Phylloquinones (vitamin K ) 158 –9, Platelet-activating factor (PAF) 39 , 211 Peptides 405–7 ∗ 1 ∗ 163, 204 Platydesmine 378 antibiotics 422–32 ∗ Phyllostine 79 Plexaura homomalla 54 biosynthesis, non-ribosomal 371, ∗ ∗ Physcion 64 ,66 ,70 PLP, see pyridoxal phosphate 421–2 Physostigma venenosum 366, 368, 366–8 Podophyllin 135 biosynthesis, ribosomal 407–8 ∗ ∗ ∗ ∗ ∗ Physostigmine 299, 325, 365–6 , 367 , Podophyllotoxin 12 , 134 –5, 136 , 136 hormones, see hormones ∗ 367, 380 Podophyllotoxone 136 opioid 417 ∗ Physostigmine N-oxide 367 Podophyllum 135–7 toxins 433–7 ∗ ∗ Physovenine 366 –7 Peptidoglycan 426, 444, 473–4 resin 135–6 Phyto-oestrogens 135, 154, 156–7, 282 Podophyllum biosynthesis 444 ∗ ∗ 135, 137 ∗ Phytoene 167, 169 , 226–7 ∗ P. emodi 135 Pergolide 372 , 375 Phytol 203–4 Periwinkle, Madagascar 353, 356 ∗ P. hexandrum 135 ∗ Phytomenadione 158 , 163 Permethrin 187 , 188 P. peltatum 135 Phytosterols 252–7 Poison alder 82 Peroxidases 25 Phytyl diphosphate 159 Pestalotiopsis microspora 207 ∗ dogwood 82 ∗ Picolinic acid 312 , 432 Pethidine 334 , 334 ∗ hemlock 381, 382 Picropodophyllin 137 ivy 81, 82 Petroselinum crispum 147 Pictet-Spengler synthesis 321, 323, 349 ∗ ivy, oakleaf 82 Peucenin 75 Pig 43 ∗ ∗ oak 81, 82 Peyote 320 Pilocarpine 367, 379 –80 , 380 oak, Pacific 82 Phalloidin 153 Pilocarpus 380 ∗ sumach 82 Phalloin 433 Pilocarpus jaborandi 380 Phallotoxins 433 Polyene macrolides 101–2, 102–3 ∗ P. microphyllus 380 α-Phellandrene 175 –6, 183 Polyethers 106–11 ∗ P. pennatifolius 380 β-Phellandrene 175 –6, 181, 183 ∗ Polyketide cyclase 62 ∗ Pilosine 379 –80 Phellandryl cation 175 –6 Pimpinella anisum 138, 139 Polyketide synthase 62, 96, 114–17 ∗ ∗ Phenazocine 334 , 334 Pinane 174 Polyketides, aromatic 60–92 ∗ Phenethylisoquinoline alkaloids, see Pindolol 318 Polymyxins 423 alkaloids Polymyxin B 423 Pine 177 ∗ Phenolic acids, decarboxylation 20–2 Polymyxin B 423 oil 183 1 ∗ Phenolic oxidative coupling 28–9 oil, pumilio 183 Polymyxin B2 423 ∗ ∗ ∗ Phenothrin 187 , 188 α-Pinene 140, 175–6 , 177 –8, 180–1, Polymyxin E 423 ∗ ∗ Phenoxyacetic acid 439–40 183–5 Polymyxin E1 423 ∗ ∗ ∗ ∗ Phenoxymethylpenicillin 439, 440 –1 β-Pinene 140, 175–6 , 178, 181, 183–5 Polymyxin E2 423 ∗ Phenylacetic acid 437 Pinidine 381 Polysaccharides 473–5, 475–7 ∗ ∗ ∗ Phenylacetonitrile 456 Pinoresinol 133 –4 , 135 Pomegranate 276, 307 502 INDEX

∗ ∗ Poppy Prostanoic acid 52, 54 Pyridoxal phosphate (PLP) 20–1 , 32, opium 328, 329 Prostanoids 52 172 seed 329 Protamine 418, 477 in decarboxylation 20, 22 straw 329 Protein kinase C 207 in transamination 20–1 ∗ ∗ Poriferastane 233–4 Proteins 405–7 Pyridoxamine 31 ∗ Porphyrin 32, 203 ribosome-inactivating 434 Pyridoxamine phosphate 20–1 ,32 ∗ Potato 388, 475 Prothrombin 163 Pyridoxine 31 –2, 32 ∗ ∗ Pravastatin 112, 113 Protirelin 412 , 412 Pyridoxine phosphate 32 ∗ ∗ ∗ ∗ Preakuammicine 353–4 , 357–8 Protoaescigenin 220–1 Pyridoxol 31 ∗ Precholecalciferol 257–8 Protoberberine alkaloids, see alkaloids Pyridoxol phosphate 32 ∗ ∗ ∗ Prednisolone 267–8 , 268 Protocatechuic acid 122 Pyrimethamine 127 , 127 ∗ ∗ ∗ Prednisone 267–8 , 268 Protodioscin 238–9 Pyroglutamic acid 406 , 409 ∗ ∗ ∗ Pre-ergocalciferol 258 Protohypericin 65, 67 ,71 Pyrrolidine alkaloids, see alkaloids ∗ ∗ Pregnane 233–4 Protopanaxadiol 223–4 Pyrrolizidine alkaloids, see alkaloids ∗ ∗ ∗ ∗ Pregnenolone 243–4 , 263 , 266 , Protopanaxatriol 223–4 Pyrroloindole alkaloids, see alkaloids ∗ ∗ ∗ 276–7 , 392 –3 Protopine 339–40 Pyrroloquinoline alkaloids, see alkaloids Prenyl group 168 Protopine alkaloids, see alkaloids Pyruvic acid 170 ∗ ∗ ∗ Prephenic acid 127 –8, 129 Protopseudohypericin 71 ∗ ∗ ∗ ∗ ∗ Prephytoene diphosphate 226–7 Protosteryl cation 215 –16 , 217 , 257 ∗ Preproinsulin 416–17 Protoveratrine A 391 Qinghao, see Artemisia annua ∗ ∗ ∗ Presqualene diphosphate 111 , 213–14 Protoveratrine B 391 Qinghao acid 199 ∗ Prilocaine 303, 304 Protoverine 391–2 Qinghaosu, see artemisinin ∗ Primaquine 363 , 363 Provitamins 230 Qinghaosu-II, see arteannuin B ∗ Primary metabolism 7–8 Proxymetacaine 303, 304 Quassia amara 226 ∗ Prisinamycin IA 431 Prozac 71, 347 Quassin 225 –6 ∗ ∗ Prisinamycin IIA 431 Prunasin 455–6 , 457 Quassinoids 225–6 ∗ ∗ ∗ ∗ Proansamycin X 105–6 Prunus 455 Quercetin 150 –1, 152 , 211 ∗ Procainamide 303, 304 P. amygdalus var. amara 41, 455 Quillaia 220, 222 ∗ ∗ Procaine 303, 304 P. amygdalus var. dulcis 41, 456 Quillaic acid 220–1 , 222 ∗ Proclavaminic acid 451 P. laurocerasus 456 Quillaja 222 Prodynorphin 419 P. serotina 456 Q. saponaria 220, 222 ∗ Proenkephalin A 418 Pseudoephedrine 382 –3, 384 Quinazoline alkaloids, see alkaloids ∗ Proenkephalin B 419 Pseudoguaianolide 194 Quinic acid 122 , 139, 362, 395 ∗ ∗ ∗ ∗ Progesterone 239, 243–4 , 263 –4, Pseudohypericin 71 –2 Quinidine 359, 361 –2, 364 ∗ ∗ ∗ ∗ ∗ ∗ 265 –6 , 267 , 271–3 , 273, 277, Pseudopelletierine 307 Quinine 359, 361 –2, 363–4 ∗ 282 Psilocin 347 –8 Quinoline alkaloids, see alkaloids ∗ Progestins, see progestogens Psilocybe 347, 348 Quinolinic acid 312 –13 Progestogens 272–3, 273–6 P. mexicana 348 Quinolizidine alkaloids, see alkaloids antagonist 276 P. semilanceata 348 Quinones, terpenoid 158–60 ∗ ∗ characteristic features 273 Psilocybin 347 –8 Quinupristin 431, 431 ∗ ∗ ∗ Progoitrin 458–9 Psoralen 145 –6 , 147 Proinsulin 416–17 Psoralens 146 ∗ Prolactin 375, 413, 414 Psychotrine 344–5 Ragwort 306 ∗ ∗ Proline 406 Pterocarpans 154 Rapamycin 103–4 , 104–5, 117 Pro-opiomelanocortin 418 Puffer fish 397–8 Rapeseed 458 ∗ Proparacaine, see proxymetacaine Pulegone 178 , 186 oil 43, 256 ∗ S-Propenyl cysteine sulphoxide 460 Pumilio pine oil, see pine oil Raubasine 353 ∗ Propenylphenols 138 Pumiloside 364 –5 Rauvolfia, see Rauwolfia Propionibacterium 33 Punica granatum 307 Rauwolfia 352, 391 ∗ Propranolol 318 , 318–19 Purine alkaloids, see alkaloids Rauwolfia 351 ∗ ∗ S-Propyl cysteine sulphoxide 460 Purpureaglycoside A 246–7 , 248 R. canescens 352, 353 ∗ ∗ Proscillaridin A 252 Purpureaglycoside B 246–7 , 248 R. serpentina 351, 352, 353, 354, 357 ∗ Prostacyclin, see prostaglandin I Purpurosamine 481 R. tetraphylla 352 2 ∗ ∗ ∗ ∗ Prostaglandins 45–6, 54–7, 222, 268 Putrescine 292 , 304 –5 , 313 R. vomitoria 352, 353 biosynthesis 51–4 PUVA 147 Red snapper 109 nomenclature 52, 54 Pyrenacantha klaineana 365 Reduction reactions 24–5 ∗ ∗ ∗ Prostaglandin D (PGD ) 52–3 Pyrethric acid 186–7 , 188 Remifentanil 334 , 335 2 2 ∗ ∗ ∗ Prostaglandin E (PGE )52, 55–6 Pyrethrins 186–7 , 188 Remija pendunculata 364 1 1 ∗ ∗ ∗ ∗ Prostaglandin E (PGE )52–3 , Pyrethrin I 187 –8 Reproterol 319 ∗ 2 2 ∗ ∗ 55–6 Pyrethrin II 187 –8 Rescinnamine 351–2 , 353 ∗ ∗ ∗ Prostaglandin E (PGE )52 Pyrethrolone 186–7 , 188 Reserpine 351–2 , 353 3 3 ∗ ∗ ∗ Prostaglandin F α (PGF α) 52–3 , Pyrethrum 186, 188 Resveratrol 80 –1, 149 ∗ 2 2 55–6 Pyrethrum parthenium 195 Reteplase 420 ∗ ∗ ∗ ∗ Prostaglandin G (PGG ) 52–3 Pyridine alkaloids, see alkaloids Reticuline 322 –3, 327–8 , 329, 337 , 2 2 ∗ ∗ ∗ ∗ Prostaglandin H (PGH ) 52–3 ,59 Pyridocarbazole alkaloids, see alkaloids 339 –40 2 2 ∗ ∗ ∗ ∗ Prostaglandin I (PGI ) 52–3 , Pyridostigmine 366 , 367 Retinal 229 –0, 231 ∗ ∗ 2 2 ∗ ∗ 56 –7 Pyridoxal 31 Retinoic acid 231 INDEX 503

Retinoids 230–1 Rotenoids 155 Scilla maritima 252 ∗ ∗ ∗ Retinol (Vitamin A ) 227, 229 –30, Rotenone 155 , 157 Scillaren A 252 ∗ 1 ∗ ∗ 231 Rotenonic acid 155–6 Scillarenin 252 ∗ ∗ ∗ Retronecine 305 Rubber 232 Scilliroside 252 ∗ ∗ Rhamnose 469 Rubber tree 232 Scillirosidin 252 ∗ Rhamnus 64 Rubia tinctorum 164 Sclarin 212–13 ∗ R. frangula 70 Rubreserine 367 Sclerotinia sclerotiorum 147 ∗ R. purshianus 69 Rue 147, 151 Scopine 297–8, 299 ∗ ∗ Rhein 64 ,66 Rumex 64 Scopolamine, see hyoscine ∗ ∗ Rhein anthrone 68 –9 Ruta graveolens 147, 151, 379 Scopoletin 142–3 ∗ Rheum 70 Rutacridone 378 –9 Scopolia 297 ∗ R. officinale 70 Rutin 151–2 Scopolia 301 ∗ R. palmatum 70 Rutinose 152 S. carniolica 297 ∗ R. rhaponticum 71 Rye 369, 371, 374 Scopolin 143 ∗ Rhizoctonia leguminicola 311 Scoulerine 339–40 Rhizopus 370 Sea whip 54 ∗ R. arrhizus 264, 265 Sabinene 140, 176 , 181 Secale cereale 371 ∗ R. nigricans 264, 265 Saccharomyces 384 Secoiridoids 188 –9, 343, 350 ∗ ∗ ε-Rhodomycinone 93 S. cerevisiae 258, 418 Secoisolariciresinol 134 –5, 157 ∗ ∗ ∗ ∗ Rhodopsin 230–1 Saccharopolyspora erythraea 96, 98 Secologanin 189 –90 , 343–4 , ∗ ∗ ∗ ∗ Rhodosamine 94 Saccharum officinarum 471 350 –1 , 386 Rhubarb 65, 70 Safflower oil 160 Secondary metabolism 8 Rhus diversiloba 82 Saffron 231 Secosteroids 258 ∗ R. radicans 81, 82 Safrole 138 , 179 numbering 258 R. toxicodendron 81, 82 Sage 177 Sedamine 308 ∗ R. vernix 82 oil 184 Sedoheptulose 7-phosphate 466–7 , ∗ ∗ ∗ Riboflavin (vitamin B )31, 31 Salbutamol 318 , 319 484 2 ∗ Ribonucleic acid, see RNA Salcatonin 411 , 411 Sedum acre 308 ∗ ∗ ∗ Ribose 469 Salicin 142 Senecic acid 305 ∗ ∗ Ribose 5-phosphate 465–6 , 467 Salicyladehyde 142 Senecio 305 ∗ ∗ Ribosome 407–8 Salicylic acid 124 , 141–2 S. jacobaea 306 Ribosome-inactivating proteins 434 Salix 142 S. vulgaris 306 ∗ ∗ ∗ Ribulose 5-phosphate 465–6 Salmeterol 319 Senecionine 305 Rice 475 Salmo species 418, 477 Senna 65, 67–9 ∗ Ricin 41, 314, 434 Salmon 418, 477 Sennidin A 68 ∗ ∗ Ricin A 434 Salsolinol 322 Sennidin B 68 ∗ ∗ Ricin B 434 Salutaridine 328 Sennidin C 68 ∗ ∗ ∗ Ricinine 314–15 Salutaridinol 328 Sennidin D 68 ∗ Ricinoleic acid 41, 47 , 434 Salvia officinalis 177, 184 Sennosides 65 ∗ Ricinus communis 41, 47, 207, 314, 434 SAM, see S-Adenosylmethionine Sennoside A 67, 68 –9 ∗ ∗ Rifabutin 107 , 108 Sambucus nigra 457 Sennoside B 67, 68 –9 ∗ ∗ Rifampicin 107 , 108 Sambunigrin 457 Sennoside C 68 ∗ Rifamycin 108 Sandalwood oil 184 Sennoside D 68 ∗ ∗ ∗ ∗ ∗ Rifamycin B 105–6 , 107 –8 Sandaracopimaradiene 209–10 Serine 406 , 454, 455 ∗ ∗ ∗ Rifamycin O 107 –8 Sandaracopimaradienyl cation 210 Sermorelin 412 , 413 ∗ Rifamycin S 107 –8 Sandersonia 343 Serotonin, see 5-hydroxytryptamine ∗ ∗ Rifamycin SV 107 –8 α-Santalol 184 Serpentine 352 ∗ Rifamycin W 106 β-Santalol 184 Sesame oil 44 ∗ Rimexolone 270 , 271 Santalum album 184 Sesamum indicum 44 ∗ ∗ Ringhal 437 α-Santonin 194 –6 β-Sesquiphellandrene 181, 192, 194 ∗ Rivastigmine 366 , 367 Sapogenin 220, 237 Sesquiterpenes 167–8, 191–203 Rivea 370 Saponaria officinalis 220 Sesterterpenes 167–8, 212 R. corymbosa 376 Saponins Shark 212 ∗ Rizatripan 347, 348 steroidal 237–41 dogfish 393 mRNA 407 steroidal, characteristic features 237 Sheep 44 tRNA 407 triterpenoid 219–25 Shellfish poisoning ∗ ∗ ∗ Rocuronium 326 , 327 Sarcosine 406 , 430 , 432 diarrhoeic 109 ∗ Rofecoxib 55 Sarmentogenin 264 neurotoxic 109 ∗ Ropivacaine 303, 304 Sarsaparilla 220, 241, 242 paralytic 397 ∗ ∗ ∗ ∗ Rosa alba 184 Sarsasapogenin 240 –1, 242 Shikimic acid 121–2 , 123 , 139 R. centifolia 184 Sassafras 138 as building block 8 ∗ R. damascena 184 Sassafras albidum 138 Silybin 151, 153 , 433 R. gallica 184 Saxidomus giganteus 397 Silybum marianum 151, 153 ∗ ∗ Rose oil 184 Saxitoxin 396–7, 397 , 397–8 Silychristin 152–4 ∗ Rosemary oil 184 Schiff base Silydianin 152–4 Rosin 185, 209 formation 18–19 Silymarin 153 ∗ Rosmarinus officinalis 184 hydrolysis 18–19 Simvastatin 112, 113 504 INDEX

∗ ∗ Sinalbin 457–8 Spiramycin II 98 , 100 Streptomyces 481 ∗ ∗ Sinapic acid 131 –2 Spiramycin III 98 , 100 S. albogriseolus 357 ∗ Sinapine 132 Spiroketal 237 S. ambofaciens 97, 100 ∗ Sinapis alba 457 Spironolactone 271, 272 S. antibioticus 96, 100, 432 ∗ Sinapyl alcohol 131 –2 Sporomiella intermedia 111 S. aureofaciens 89, 90 ∗ ∗ ∗ ∗ Sinigrin 457–8 , 459 SQ 26,180 453 , 455 S. avermectilis 101 ∗ ∗ Sirolimus 103–4 , 104–5 Squalamine 392 –3 S. capreolus 425 ∗ ∗ ∗ Sisal 238, 240, 264 Squalene 167, 169 , 212, 214 –15 , S. carbophilus 112 ∗ ∗ Sisomycin 482 220 S. cattleya 451, 454 ∗ ∗ Sitostanol 256 Squalene oxide 214–15 , 217 S. cinnamonensis 106 ∗ ∗ Sitosterol 44, 253 –4, 256–7, 279–80 Squalene synthase 214 S. clavuligerus 450, 452 Skimmia japonica 379 Squalestatins 110 S. coeruleorubicus 92, 94 ∗ ∗ Skimmianine 378 –9 Squalestatin S1 111 S. erythreus 98 ∗ β-Skytanthine 386 Squalus 212 S. fradiae 97, 100, 483 Skytanthus acutus 386 S. acanthias 393 S. galilaeus 94 Slow releasing substance of anaphylaxis Squill, red 252 S. griseus 33, 478, 481 (SRSA) 59 white 252 S. hygroscopicus 105 ∗ Smilagenin 240 –1, 242 St Anthony’s Fire 371 S. kanamyceticus 482 Smilax 237, 241, 242 St John’s Wort 67, 71–2 S. lasaliensis 106 S. aristolochiaefolia 242 Stanols 256 S. lavendulae 172 S. febrifuga 242 Stanol esters, see plant stanol esters S. lincolnensis 484, 485 ∗ S. regelii 242 Stanozolol 284 , 284 S. mediterranei 107 Snake venoms 436–7 Staphylococcus aureus 474 S. nodosus 102, 103 Snakeroot 339, 352 methicillin-resistant, see MRSA S. noursei 102, 103 Snowdrop 345, 346 Star anise 138 S. olivaceus 451 Snowflake 346 oil 139 S. orchidaceus 422 Soapbark 220 Starch 473, 475 S. orientalis 426 Soapwort 220 soluble 476 S. parvullus 432 ∗ Sodium glycocholate 260 Starflower oil, see borage oil S. peuceticus 92, 94 ∗ Sodium taurocholate 260 Starter groups, in acetate pathway 79–92 S. peuceticus var. caesius 93 Solanesyl diphosphate 159 Statins 112–113 S. rimosus 90 ∗ ∗ ∗ ∗ Solanidine 388–9 , 390 Stearic acid 38 , 41–4, 45 , 396 S. roseochromogenus 271 ∗ ∗ α-Solanine 388–9 Stearidonic acid 45 S. spectabilis 481 ∗ ∗ ∗ β-Solanine 389 Stemmadenine 353–4 , 359–60 S. subrutilis 477 ∗ γ-Solanine 389 Stephania 338 S. tenebrarius 483 Solanum 240, 301, 388 S. tetrandra 323 S. tsukubaensis 102, 104 ∗ alkaloids 389, 391 Stephanine 338 S. venezuelae 130 S. aviculare 391 Sterculia gum 477 S. verticillus 427 S. khasianum 391 Sterculia 477 S. virginiae 430 ∗ S. laciniatum 391 S. foetida 50 Streptomycin 478, 480 , 481 S. marginatum 391 S. urens 477 Streptose 479 ∗ ∗ S. tuberosum 388, 475 Sterculic acid 42, 50–1 Strictosamide 364 –5 ∗ ∗ ∗ ∗ ∗ ∗ Solasodine 240–1 , 266, 388–9 , Sterigmatocystin 82–3 ,84 Strictosidine 351 , 354 , 361 –2, ∗ ∗ 390 –1 Steroidal alkaloids, see alkaloids 364 –5 ∗ ∗ ∗ Solasonine 240–1 , 388–9 Steroidal saponins, see saponins Strophanthidin 250–1 , 274 ∗ ∗ Somatostatin 411–12 , 413 Steroids 168, 232–85 G-Strophanthin 250–1 Somatotrophin 414 nomenclature 233 K-Strophanthin 250 ∗ Somatropin 414 numbering 233–4 K-Strophanthin-β 250–1 ∗ Sorangium cellulosum 103 stereochemistry 232–3 K-Strophanthoside 250–1 ∗ Sorbitol 468 , 471 Stevia rebaudiana 209 Strophanthus 250–1 ∗ Sorbus aucuparia 471 Steviol 209 Strophanthus 241, 245 ∗ Sorghum 457 Stevioside 209 S. gratus 250 ∗ Sorghum bicolor 457 Stigmastane 233–4 S. komb´e 250 ∗ ∗ Sotalol 319 , 319 Stigmasterol 44, 253 –4, 256–7, 264, S. sarmentosus 264 ∗ Soy sauce 256 266–7 , 279 Stropharia 348 ∗ ∗ Soya bean 155, 157, 254, 256, 264, 278, Stilbenes 80, 149–50 Strychnine 358 –9 , 359 279 Stilboestrol, see diethylstilbestrol Strychnos 357 oil 44, 160, 460 Stramonium 296 S. ignatii 359 ∗ sterols 256 Streptamine 478–9 S. nux-vomica 358, 359, 359 ∗ ∗ Soybean, see soya bean Streptidine 478–9 , 480 S. toxifera 324, 325, 359 ∗ Sparteine 309–10 Streptocarpus 163 Strychnos-type alkaloids 353, 357 Spearmint 177 Streptococcus haemolyticus 420, 421 Styrylpyrones 147–8 oil 185, 186 Streptodornase 421 o-Succinylbenzoic acid (OSB) 159, ∗ ∗ ∗ Spectinomycin 481, 482 Streptogramins 430–2 162 –4 ∗ ∗ ∗ Spermidine 392, 394 Streptogramin A 431 Sucrose 470 –1 , 471 ∗ Spiramycins 100 Streptogramin B 431 Suet 44 ∗ Spiramycin I 97–8 , 100 Streptokinase 420 Sugar beet 471 INDEX 505

∗ cane 471 Temocillin 442 , 443 Thiocyanates 457 ∗ maple 471 Teniposide 136 , 137 Thiosulphinates 460 Sugars, α-andβ- 468–9 Tequila 240 Thistle, milk 152, 153 ∗ D- and L- 469 Terbutaline 319 , 319 Thornapple, see stramonium ∗ ∗ ∗ ∗ Sulbactam 452, 452 Terlipressin 415 , 416 Threonine 380 , 406 ∗ Sulphanilamide 125 , 126 Terpenoid indole alkaloids, see alkaloids Thrombin 477 ∗ Sulphoraphane 459 –60 Terpenoid tetrahydroisoquinoline alkaloids, Thromboxanes 59 ∗ Sumatripan 347, 348 see alkaloids biosynthesis 58–9 ∗ Sunflower oil 44 Terpenophenolics 85, 87 Thromboxane A2 (TXA2) 58–9 ∗ Sus scrofa 43 α-Terpinene 176 , 185 Thromboxane A3 (TXA3)59 ∗ ∗ ∗ Suxamethonium 326 –7, 326 γ-Terpinene 176 , 179–81, 185 Thromboxane B2 (TXB2) 58–9 ∗ Swainsona 311 Terpinen-4-ol 140, 176 , 185 Thromboxane B3 (TXB3)59 ∗ ∗ S. canescens 310 Terpinen-4-yl cation 176 Thujane 174 , 176 ∗ ∗ ∗ Swainsonine 310–11 α-Terpineol 174–5 , 178, 183, 185 Thujone 176 , 184 ∗ Symphytum 305 α-Terpinyl acetate 179 Thujyl cation 176 S. officinale 306 α-Terpinyl cation, see menthyl cation Thyme 186 ∗ ∗ ∗ ∗ Syringaresinol 223–4 Testosterone 274, 277 , 282 –3 , 283, oil 185 ∗ ∗ Syringin 223–4 284 Thymol 178 , 185–6 Syzygium aromaticum 137, 140, 200 Testosterone undecanoate 283 Thymus vulgaris 185, 186 ∗ Tetracaine 303, 304 Thyroglobulin 411 ∗ Tetracosactide 414 , 414 Thyroid hormones, see hormones ∗ T-2 toxin 203 Tetracosactrin, see tetracosactide Thyrotrophin-releasing hormone, see ∗ Tabernanthe iboga 354, 357 Tetracycline 89 , 90–1 hormone ∗ ∗ ∗ ∗ Tabersonine 350 , 353–4 , 357–8 Tetracyclines 89–92, 90–2 Thyroxine 410 , 410–11, 458 ∗ ∗ Tacalcitol 259 , 260 table 89 Tibolone 281 , 281 ∗ ∗ ∗ Tacrolimus 102, 104 , 104–5, 429 Tetrahydrocannabinol (THC) 85 –6, Ticarcillin 441 , 443, 452 ∗ Tall oil 256 87–8 Tiglic acid 180, 194 , 197, 225, 295 ∗ ∗ ∗ Tallow, see suet Tetrahydrocannabinolic acid 12 ,85 –6, Tigogenin 240 –1 ∗ ∗ Tamoxifen 281 , 282 87 Timolol 319 , 319 ∗ Tanacetum cinerariifolium 188 Tetrahydrocannabinolic acid B 87 Tinzaparin 477 ∗ ∗ T. parthenium 192, 195 Tetrahydrocannabivarin 87 Tirucallol 225 ∗ T. vulgare 177 Tetrahydrocolumbamine 339–40 Toad 243, 246 ∗ Tannins 362, 395–6 Tetrahydrofolic acid 125 –6 Tobacco 308, 311, 314–15 condensed 151 as C carrier 126 Indian, see lobelia 1 ∗ ∗ gallotannins 122 Tetrahydroharmine 349 –50 Tobramycin 481, 483 , 483 ∗ Tansy 177 Tetrahydroisoquinoline alkaloids, see Tocainide 303, 304 Tapioca 456 alkaloids Tocopherols (vitamin E) 159, 160–1, ∗ Taraxacum officinale 218 Tetrahydronicotinic acid 316 204, 230 ∗ ∗ ∗ ∗ ∗ Taraxasterol 218–19 Tetrahymanol 218, 220 α-Tocopherol 158 –9, 160 –1 ∗ ∗ ∗ Taraxasteryl cation 218–19 Tetrahymena pyriformis 218 β-Tocopherol 158 , 160–1 ∗ ∗ ∗ ∗ ∗ Taurine 260 Tetramethrin 187 , 188 γ-Tocopherol 158 –9, 160 –1 ∗ ∗ ∗ ∗ ∗ Taxadiene 167, 169 , 205 Tetramethyldecanoic acid 49–50 δ-Tocopherol 158 , 160–1 ∗ ∗ Taxane 206 Tetrandrine 323 –4 α-Tocopherolquinone 161 ∗ Taxifolin 151, 153 Tetranortriterpenoids 225 α-Tocopheryl acetate 160 Taxine 205 Tetraodon 397, 398 Tolypocladium inflatum 429 ∗ ∗ ∗ Taxol 105, 205 , 205–7 Tetraterpenes 167–8, 226–30 Tomatidine 241 , 388–9 ∗ ∗ ∗ ∗ Taxomyces adreanae 207 Tetrodotoxin 396–7, 397 , 398 Tomatine 240–1 , 388–9 , 390 ∗ Taxotere 206 , 207 Thapsia garganica 196 Tomato 226, 227, 241, 388 ∗ Taxus 206 Thapsigargin 194 , 196 Tonic water 363–4 T. baccata 205, 206 THC acid, see tetrahydrocannabinolic acid Topoisomerase 137, 365 ∗ T. brevifolia 205, 205–7 THC, see tetrahydrocannabinol Topotecan 365 , 365 ∗ T. wallachiana 207 Thea sinensis 395 Toremifene 281 , 282 ∗ ∗ Tazobactam 452, 452 Theaflavin 151 , 395 Totaquine 364 Tea 151, 394, 395 Thearubigin 395 Toxicodendron radicans 82 ∗ ∗ ∗ Abyssinian 384 Thebaine 327–8 , 330, 331, 335 –6 , T. toxicaria 82 black 395 336 Toxiferines 327, 359 ∗ green 395 Theobroma cacao 44, 396 C-Toxiferine 325, 327, 359–60 mate´ 396 Theobroma oil 40, 44, 396 Toxiferine-1, see C-Toxiferine ∗ Oolong 395 Theobromine 393 –4, 394–5, 396 TPP, see thiamine diphosphate ∗ Paraguay 396 Theophylline 393 –4, 394–5, 396 Tragacanth 476 ∗ Tea tree oil 176, 185 Thespia populnea 201 Tramadol 334 , 335 ∗ Teicoplanin 426–7 Thiamine (vitamin B ) 30,31 , 172 Transaldolase 466–7 ∗ 1 Teicoplanin T-A2-1 428 Thiamine diphosphate (TPP) 31, 159, Transamination 20–1, 468 ∗ Teicoplanin T-A2-2 428 170, 383–4, 466 Transketolase 466–7 ∗ ∗ ∗ Teicoplanin T-A2-3 428 in decarboxylation 21, 23 –4 Tretinoin 231 , 231 ∗ ∗ Teicoplanin T-A2-4 428 Thienamycin 451, 453 –4 TRH, see hormone, thyrotrophin-releasing ∗ ∗ ∗ Teicoplanin T-A2-5 428 Thioacrolein 461 Tri-iodothyronine 410 , 411 506 INDEX

∗ ∗ ∗ Triamcinolone 270, 271 Ubiquinone 157, 158 –9, 231 Vinyldithiin 461 ∗ ∗ ∗ ∗ ∗ ∗ Triamcinolone acetonide 270, 271 UDPglucose 29–30 , 468 , 470 –1 Violaxanthin 226, 228 –9 ∗ Trichocereus pachanoi 320 UDPglucuronic acid 468 Virginiamycin M1 431 ∗ ∗ ∗ Trichoderma 203 Umbelliferone 142–3 , 145 Visamminol 75 ∗ ∗ Trichodiene 202 Undecenoic acid 47 Viscum album 435 Trichosanthes kirilowii 435 Urginea 246 Viscumin 435 Trichosanthin 435 U. maritima 252 Vision 230 ∗ Trichothecenes 202, 203 Uric acid 343 Visnadin 75 –6 ∗ Trichothecium 202, 203 Uridine diphosphoglucose, see UDPglucose Visnagin 74–5 ,76 Trifolium 154 Urofollitrophin, see urofollitropin Vitamin A 42, 227, 230–1 T. pratense 157 Urofollitropin 415 Vitamin A1, see retinol Triglycerides 39 Urokinase 420 Vitamin A2, see dehydroretinol Trigonella foenum-graecum 238, 240 Uronic acids 220, 222, 467, 475 Vitamin B , see thiamine ∗ 1 ∗ Trigonelline 313, 395 Ursodeoxycholic acid 261, 262 Vitamin B 32 , 32–3 ∗ 12 3,4,5-Trihydroxybenzoic acid, see gallic Urushiols 81 –2 Vitamin B2, see riboflavin acid Usnea 76 Vitamin B , see nicotinic acid ∗ 3 Trillium 237 Usnic acid 76–7 Vitamin B , see pantothenic acid ∗ 5 Trilostane 271, 272 Vitamin B6 32 ∗ ∗ Trimethoprim 127 , 127 Vaccenic acid 38 Vitamin B9, see folic acid ∗ Triptorelin 412 , 413 Valepotriates 190 Vitamin C, see ascorbic acid ∗ Triterpenes 167–8, 212–26 Valeranone 190 Vitamin D 42, 257–8, 259–60 ∗ Triterpenoid, numbering 221 Valerenic acid 190 Vitamin D1 259 Triterpenoid saponins 219–25 Valerian 190, 190–1, 386 Vitamin D2, see ergocalciferol Triticale 374 alkaloids, see alkaloids Vitamin D3, see colecalciferol Triticosecale 374 Valeriana edulis 190 Vitamin D4 259 Triticum aestivum 374, 475 V. mexicana 190 Vitamin D5 259 Troleandomycin 100 V. officinalis 190, 386 Vitamin D6 259 ∗ Tropacocaine 301 –2 V. wallichi 190 Vitamin D7 259 ∗ Tropaeolium majus 458 Valienamine 484 Vitamin E, see tocopherols ∗ ∗ Tropane alkaloids, see alkaloids Valine 385 , 406 , 457 Vitamin H, see biotin ∗ ∗ Tropic acid 294 , 297 Valtrate 190–1 Vitamin K 144, 163 ∗ ∗ Tropicamide 300 , 300 Vancomycin 426–7, 427 , 485 Vitamin K1, see phylloquinones ∗ ∗ Tropine 293 –4 , 297–8 ∗ Vitamin K , see menaquinones Vancosamine 427 2 ∗ ϕ-Tropine 302 Vanilla 141 Vitamin K epoxide 163 ∗ Tropinone 293 –4 Vanilla planiflora 141 Vitamin P 151 ∗ ∗ α-Truxillic acid 302 Vanillic acid 141 Vomitoxin 202 ∗ ∗ ∗ α-Truxilline 301 –2 Vanillin 141 , 385 ∗ ∗ β-Truxilline 301 –2 Vanillylamine 385 Wagner–Meerwein rearrangement 15 ∗ ∗ ∗ β-Truxinic acid 302 Vasicine 377 Warfarin 143–4 , 144–5, 163 Trypsin 420 Vasopressin 413, 416 Waxes 40 ∗ ∗ ∗ ∗ ∗ Tryptamine 347 , 349 , 351 , 366 Vecuronium 326 , 327 Wheat 374, 475 ∗ ∗ ∗ Tryptophan 121, 128 , 312 , 346–7 , Venoms, snake 436–7 oil 160 ∗ ∗ ∗ ∗ 348 , 369 , 406 , 458 Veratrum 389 Wieland–Gumlich aldehyde 358 –9, ∗ alkaloids derived from 346–71 V. album 391 360 as building block 9–10 V. californicum 390 Willow 142 biosynthesis 126–8 V. viride 391 Wine 150–1 ∗ Tsuga 382 Verrucarin A 203 Wintergreen oil 140, 141 ∗ Tuberculostearic acid 49–50 Versicolorins 84 Wolfsbane 387 ∗ ∗ ∗ Tubocurarine 324 –5 , 325, 367 Versicolorin A 82–3 Wool alcohols 236 ∗ Tubulin 136, 207, 331, 343, 356 Versicolorin B 82–3 fat 48, 279 ∗ ∗ Tulobuterol 319 Versiconal acetate 82–3 Wormseed 196 ∗ Turpentine 185, 209 Verticillene 205 Wormwood, annual 198 ∗ oil 185, 209 Verticillyl cation 205 oil 176 ∗ Tylonolide 98 Very Fast Death Factor 304 sweet 198 ∗ ∗ Tylosin 97–8 , 100 Vibrio 398 Wyerone 48–9 ∗ ∗ ∗ Tyramine 315–16 , 319, 322 , 345–6 , Vicia faba 48 ∗ ∗ ∗ 386 Vinblastine 354–5 , 356–7 Xamoterol 319 ∗ Tyria jacobaeae 306 Vinca minor 354 Xanthine 393 Tyrocidines 424 V. rosea, see Catharanthus roseus Xanthophylls 226 ∗ ∗ Tyrocidine A 425 Vincaleukoblastine, see vinblastine Xanthosine 393 ∗ ∗ Tyrocidine B 425 Vincamine 354, 357–8 Xanthosine monophosphate (XMP) 74, ∗ ∗ ∗ Tyrocidine C 425 Vincristine 354–5 , 356–7 393 –4 ∗ ∗ ∗ ∗ ∗ Tyrosine 121, 315–16 , 406 , 457 Vindesine 355 , 356 Xanthotoxin 145 –6, 147 ∗ ∗ ∗ alkaloids derived from 315–46 Vindoline 353–4 , 355 Xanthotoxol 145 as building block 9–10 Vinleurosine 356 XMP, see xanthosine monophosphate ∗ ∗ biosynthesis 128–9 Vinorelbine 355 , 356, 357 Xylose 469 ∗ Tyrothricin 424 Vinrosidine 356 Xylulose 5-phosphate 465–6 INDEX 507

Yam 282 Himalayan 207 Zafirlukast 59 Mexican 238, 239, 264 Pacific 205 Zaragozic acids 110 ∗ ∗ Yamogenin 237–8 , 239–40 Yingzhao 200 Zaragozic acid A 110–11 ∗ ∗ Yangonin 147–8 Yingzhaosu A 199 –200 Zea mays 43, 475 ∗ ∗ ∗ ∗ Yatein 134 –5 Yingzhaosu C 199 –200 Zearalenone 94–95 ,96 ∗ Yeast 257, 283 Yohimbe 351 Zeathanthin 226, 228 –9 ∗ baker’s 418 Yohimbine 351–2 Zingiber officinale 181, 192 ∗ brewer’s 384 Yucca 237, 242 Zingiberene 181, 192, 194 ∗ Yew, common 205 Y. brevifolia 241, 242 Zolmitripan 348 , 348 Medicinal Natural Products. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback);; 0470846275 (Electronic) 1 ABOUT THIS BOOK, AND HOW TO USE IT

An introductory chapter briefly describing the subject, the aim, the approach and the topics. Students are offered advice on how to select material for study, and encouraged to understand the information rather than learn the factual material. General information on ionization in biochemicals and on nomenclature is given, together with a list of common abbreviations.

THE SUBJECT derived agents were never surpassed and remain as valued medicines to this day. Natural drugs This book has been written primarily for pharmacy derived from microorganisms have a much shorter undergraduates to provide a modern text to com- history, and their major impact on medicine goes plement lecture courses dealing with pharmacog- back only about 60 years to the introduction nosy and the use of natural products in medicine. of the antibiotic penicillin. Microbially produced Nevertheless, it should be of value in most antibiotics now account for a very high proportion other courses where the study of natural prod- of the drugs commonly prescribed. There is ucts is included, although the examples chosen are currently a renewed interest in pharmacologically predominantly those possessing pharmacological active natural products, be they from plants, activity. microorganisms, or animals, in the continued For centuries, drugs were entirely of natural search for new drugs, particularly for disease states origin and composed of herbs, animal products, where our present range of drugs is less effective and inorganic materials. Early remedies may have than we would wish. Herbal remedies are also combined these ingredients with witchcraft, mysti- enjoying a revival as many sufferers turn away cism, astrology, or religion, but it is certain that from modern drugs and embrace ‘complementary those treatments that were effective were sub- medicine’. sequently recorded and documented, leading to the early Herbals. The science of pharmacog- THE AIM nosy – the knowledge of drugs – grew from these records to provide a disciplined, scientific descrip- Many modern university pharmacy courses include tion of natural materials used in medicine. Herbs a pharmacognosy component covering a study of formed the bulk of these remedies. As chemical plant-derived drugs, and traditionally this area of techniques improved, the active constituents were natural products has been taught separately from isolated from plants, were structurally character- the microbially derived antibiotics, or the animal- ized, and, in due course, many were synthesized related steroidal and prostanoid drugs. These topics in the laboratory. Sometimes, more active, better usually form part of a pharmaceutical chem- tolerated drugs were produced by chemical modi- istry course. The traditional boundaries may still fications (semi-synthesis), or by total synthesis of remain, despite a general change in pharma- analogues of the active principles. cognosy teaching from a descriptive study to a Gradually synthetic compounds superseded phytochemical-based approach, a trend towards many of the old plant drugs, though certain plant- integrating pharmacognosy within pharmaceutical 2 ABOUT THIS BOOK, AND HOW TO USE IT chemistry, and the general adoption of modular should be familiar, having been met previously in course structures. A chemistry-based teaching pro- courses dealing with the fundamentals of organic gramme encompassing all types of natural product chemistry and biochemistry. These principles are of medicinal importance, semi-synthetic deriva- then seen in action as representative natural prod- tives, and synthetic analogues based on natural uct structures are described in the following chap- product templates, is a logical development, and ters. These are subdivided initially into areas of one we have practised at Nottingham for several metabolism fed by the acetate, shikimate, meval- years. This coursebook provides a suitable text for onate and deoxyxylulose phosphate pathways. such a programme, and attempts to break down the Remaining chapters then cover alkaloids, peptides artificial divisions. and proteins, and carbohydrates. The book tries to include a high proportion of those natural products THE APPROACH currently used in medicine, the major drugs that are derived from natural materials by semi-synthesis, This book establishes a groundwork in natural and those drugs which are structural analogues. product chemistry/phytochemistry by considering Some of the compounds mentioned may have a biosynthesis – the metabolic sequences leading significant biological activity which is of inter- to various selected classes of natural products. est, but not medicinally useful. The book is also This allows application of fundamental chemical designed to be forward looking and gives informa- principles and shows the relationships between tion on possible leads to new drugs. A selection the diverse structures encountered in nature, of supplementary reading references is provided at thus giving a rationale for natural products the end of each chapter; these are limited as far as and replacing the traditional descriptive approach possible to recent review articles in easily acces- with one based more on deductive reasoning. sible journals rather than books, and have been Subdivision of the topics is predominantly via chosen as student friendly. biosynthesis, not class or activity, and this provides a logical sequence of structural types and avoids a BE SELECTIVE catalogue effect. There is extensive use of chemical schemes and mechanism, with detailed mechanistic Coverage is extensive to allow maximum flexibil- explanations being annotated to the schemes, as ity for courses in different institutions, but not all well as outline discussions in the text. Extensive the material will be required for any one course. cross-referencing is used to emphasize links and However, because of the many subdivisions and similarities. As important classes of compounds or the highlighted keywords, it should be relatively drugs (indicated by an asterisk) are reached, more easy to find and select the material appropriate to detailed information is then provided in the form a particular course. On the other hand, the detail of short separate monographs in boxes, which can given in monographs is purposely limited to ensure be studied or omitted as required, in the latter students are provided with enough factual infor- case allowing the main theme to continue. The mation, but are not faced with the need to assess monograph information covers sources, production whether or not the material is relevant. Even so, methods, principal components, drug use, mode these monographs will undoubtedly contain data of action, semi-synthetic derivatives, synthetic which exceed the scope of any individual course. It analogues, etc, as appropriate. Those materials is thus necessary to apply selectivity, and portions currently employed as drugs are emphasized in the of the book will be surplus to immediate require- monographs by the use of bold type. ments. The book is designed to be user friendly, suitable for modular courses and student-centred THE TOPICS learning exercises, and a starting point for later project and dissertation work. The information pre- A preliminary chapter is used to outline the main sented is as up to date as possible, but undoubtedly building blocks and the basic construction mech- new research will be published that modifies or anisms employed in the biosynthesis of natural even contradicts some of the statements made. The products. Many of these fundamental principles reader is asked always to be critical and to maintain ABOUT THIS BOOK, AND HOW TO USE IT 3 a degree of flexibility when reading the scientific acids are shown in unionized form, although they literature, and to appreciate that science is always will typically exist as zwitterions: changing.

RCO2H RCO2 TO LEARN, OR TO UNDERSTAND? H NH2 H NH3 The primary aim of the book is not to rely just on factual information, but to impart an understand- Ionized and unionized forms of many com- ing of natural product structures and the way they pounds are regarded as synonymous in the text, are put together by living organisms. Rationaliza- thus acetate/acetic acid, shikimate/shikimic acid, tion based on mechanistic reasoning is paramount. and mevalonate/mevalonic acid may be used The sequences themselves are not important; the according to the author’s whim and context, and mechanistic explanations for the processes used are have no especial relevance. the essence. Students should concentrate on under- standing the broad features of the sequences, and absorb sufficient information to be able to predict NOMENCLATURE how and why intermediates might be elaborated and transformed. The mechanistic explanations appen- Natural product structures are usually quite com- ded to the schemes should reinforce this approach. plex, some exceedingly so, and fully systematic Anyone who commits to memory a sequence of nomenclature becomes impracticable. Names are reactions for examination purposes has missed the thus typically based on so-called trivial nomencla- point. Of course, passing exams is probably the ture, in which the discoverer of the natural product main reason why students are prompted to read this exerts his or her right to name the compound. book, and the retention of some factual information The organism in which the compound is found is will be essential. There is no alternative to mem- frequently chosen to supply the root name, e.g. ory for some of the material covered in the mono- podophyllotoxin and peltatins from Podophyllum graphs, but wherever possible, information should peltatum.Namesuffixes might be -in to indicate ‘a be reduced to a concept that can be deduced, rather constituent of’, -oside to show the compound is a than remembered. The approach used here should sugar derivative, -genin for the aglycone released help students to develop such deductive skills. by hydrolysis of the sugar derivative, -toxin for a poisonous constituent, or may reflect chemical functionality, such as -one or -ol. Traditionally -ine CONVENTIONS REGARDING ACIDS, is always used for alkaloids (amines). Structurally BASES, AND IONS related compounds are then named as derivatives of the original, using standard prefixes, such as In many structures, the abbreviation P is used hydroxy-, methoxy-, methyl-, dihydro-, homo-, to represent the phosphate group and PP the etc for added substituents, or deoxy-, demethyl-, diphosphate (or pyrophosphate) group: demethoxy-, dehydro-, nor-, etc for removed sub- stituents, positioning being indicated from system- O O O atic numbering of the carbon chains or rings. PO POPO Some groups of compounds, such as steroids, O O O fatty acids, and prostaglandins, are named semi- P (phosphate) PP (diphosphate) systematically from an accepted root name. In this book, almost all structures depicted in the figures At physiological pHs, these groups will be ion- carry a name; this is primarily to help identification ized as shown, but in schemes where structures and, for the student, structural features are more are given in full, the unionized acids are usually pertinent than the names used. It will soon become depicted. This is done primarily to simplify struc- apparent that drug names chosen by pharmaceuti- tures, to eliminate the need for counter-ions, and cal manufacturers are quite random, and have no to avoid mechanistic confusion. Likewise, amino particular relationship to the chemical structure. 4 ABOUT THIS BOOK, AND HOW TO USE IT

We are also currently experiencing a transitional NAD+ nicotinamide adenine dinucleotide period during which many established drug names NADH nicotinamide adenine dinucleotide are being changed to recommended international (reduced) non-proprietary names (rINN); both names are NADP+ nicotinamide adenine dinucleotide included here, the rINN preceding the older name. phosphate NADPH nicotinamide adenine dinucleotide SOME COMMON ABBREVIATIONS phosphate (reduced) ACP acyl carrier protein O oxidation – in schemes ADP adenosine diphosphate P phosphate – in text Api apiose P phosphate – in structures Ara arabinose PEP phosphoenolpyruvate ATP adenosine triphosphate PG prostaglandin B: general base PLP pyridoxal 5 -phosphate CoA coenzyme A as part of a thioester, PP diphosphate e.g. acetyl-CoA (CH3COSCoA) (pyrophosphate) – in text CDP cytidine diphosphate PP diphosphate CTP cytidine triphosphate (pyrophosphate) – in structures Dig digitoxose Rha rhamnose DMAPP dimethylallyl diphosphate (dimethylallyl pyrophosphate) Rib ribose Enz enzyme SAM S-adenosyl methionine FAD flavin adenine dinucleotide TDPGlc thymidine diphosphoglucose TPP thiamine diphosphate (thiamine FADH2 flavin adenine dinucleotide (reduced) pyrophosphate) FMN flavin mononucleotide TX thromboxane UDP uridine diphosphate FMNH2 flavin mononucleotide (reduced) FPP farnesyl diphosphate (farnesyl UDPGlc uridine diphosphoglucose pyrophosphate) UTP uridine triphosphate Fru fructose W–M Wagner–Meerwein rearrangement Gal galactose Xyl xylose GFPP geranylfarnesyl diphosphate hν electromagnetic radiation; usually (geranylfarnesyl UV or visible pyrophosphate) ∆ heat GGPP geranylgeranyl diphosphate (geranylgeranyl pyrophosphate) FURTHER READING Glc glucose GPP geranyl diphosphate (geranyl Pharmacognosy, Phytochemistry, pyrophosphate) Natural Drugs HA general acid HMG-CoA β-hydroxy-β-methylglutaryl Books coenzyme A HSCoA coenzyme A Bruneton J (1999) Pharmacognosy. Lavoisier, Andover. Evans WC (1996) Trease and Evans’ Pharmacognosy. IPP isopentenyl diphosphate Saunders, London. (isopentenyl pyrophosphate) Newall CA, Anderson LA and Phillipson JD (1996) LT leukotriene Herbal Medicines. A Guide for Health-care Profes- Mann mannose sionals. Pharmaceutical, London. FURTHER READING 5

Robbers JE, Speedie MK and Tyler VE (1996) Pharma- Cordell GA (2000) Biodiversity and drug discovery – a cognosy and Pharmacobiotechnology. Williams and symbiotic relationship. Phytochemistry 55, 463–480. Wilkins, Baltimore, MD. Cragg G and Newman D (2001) Nature’s bounty. Chem Robbers JE and Tyler VE (1999) Tyler’s Herbs of Brit 37 (1), 22–26. Choice. The Therapeutic Use of Phytomedicinals. Cragg GM, Newman DJ and Snader KM (1997) Natural Haworth Herbal, New York. products in drug discovery and development. JNat Samuelsson G (1999) Drugs of Natural Origin.Swedish Prod 60,52–60. Pharmaceutical, Stockholm. Newman DJ, Cragg GM and Snader KM (2000) The Schultz V, Hansel R and Tyler VE (1998) Rational Phy- influence of natural products upon drug discovery. totherapy. A Physician’s Guide to Herbal Medicine. Nat Prod Rep 17, 215–234. Springer, Berlin. Shu Y-Z (1998) Recent natural product based drug development: a pharmaceutical industry perspective. JNatProd 61, 1053–1071. Reviews Sneader W (1990) Chronology of drug introductions. Comprehensive Medicinal Chemistry. Vo l 1 . Perg a- Briskin DP (2000) Medicinal plants and phytomedicines. mon, Oxford, 7–80. Linking plant biochemistry and physiology to human Tyler VE (1999) Phytomedicines: back to the future. J health. Plant Physiol 124, 507–514. Nat Prod 62, 1589–1592. MedicinalNaturalProducts. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback);; 0470846275 (Electronic) 2 SECONDARY METABOLISM: THE BUILDING BLOCKS AND CONSTRUCTION MECHANISMS

Distinctions between primary and secondary are defined, and the basic building blocks used in the biosynthesis of secondary natural products are introduced. The chemistry underlying how these building blocks are assembled in nature is described, subdivided according to chemical mechanism, including alkylation reactions, Wagner–Meerwein rearrangements, aldol and Claisen reactions, Schiff base formation and Mannich reactions, transaminations, decarboxylations, oxidation and reduction reactions, phenolic oxidative coupling, and glycosylations.

PRIMARY AND SECONDARY others are then required to synthesize specialized METABOLISM molecules from the basic compounds so obtained. Despite the extremely varied characteristics of living organisms, the pathways for generally mod- All organisms need to transform and interconvert a ifying and synthesizing carbohydrates, proteins, vast number of organic compounds to enable them fats, and nucleic acids are found to be essentially to live, grow, and reproduce. They need to pro- the same in all organisms, apart from minor vide themselves with energy in the form of ATP, variations. These processes demonstrate the fun- and a supply of building blocks to construct their damental unity of all living matter, and are col- own tissues. An integrated network of enzyme- lectively described as primary metabolism, with mediated and carefully regulated chemical reac- the compounds involved in the pathways being tions is used for this purpose, collectively referred termed primary metabolites. Thus degradation to as intermediary metabolism, and the pathways of carbohydrates and sugars generally proceeds involved are termed metabolic pathways.Someof via the well characterized pathways known as the crucially important molecules of life are car- glycolysis and the Krebs/citric acid/tricarboxylic bohydrates, proteins, fats, and nucleic acids. Apart acid cycle, which release energy from the organic from fats, these are polymeric materials. Carbohy- compounds by oxidative reactions. Oxidation of drates are composed of sugar units, whilst proteins fatty acids from fats by the sequence called β- are made up from amino acids, and nucleic acids oxidation also provides energy. Aerobic organisms are based on nucleotides. Organisms vary widely are able to optimize these processes by adding on in their capacity to synthesize and transform chem- a further process, oxidative phosphorylation. This icals. For instance, plants are very efficient at syn- improves the efficiency of oxidation by incorpo- thesizing organic compounds via photosynthesis rating a more general process applicable to the from inorganic materials found in the environ- oxidation of a wide variety of substrates rather than ment, whilst other organisms such as animals and having to provide specific processes for each indi- microorganisms rely on obtaining their raw mate- vidual substrate. Proteins taken in via the diet pro- rials in their diet, e.g. by consuming plants. Thus, vide amino acids, but the proportions of each will many of the metabolic pathways are concerned almost certainly vary from the organism’s require- with degrading materials taken in as food, whilst ments. Metabolic pathways are thus available to 8 SECONDARY METABOLISM interconvert amino acids, or degrade those not is merely a convenience and there is considerable required and thus provide a further source of overlap. energy. Most organisms can synthesize only a pro- portion of the amino acids they actually require for THE BUILDING BLOCKS protein synthesis. Those structures not synthesized, so-called essential amino acids, must be obtained The building blocks for secondary metabolites are from external sources. derived from primary metabolism as indicated in In contrast to these primary metabolic pathways, Figure 2.1. This scheme outlines how metabolites which synthesize, degrade, and generally inter- from the fundamental processes of photosynthe- convert compounds commonly encountered in all sis, glycolysis, and the Krebs cycle are tapped organisms, there also exists an area of metabolism off from energy-generating processes to provide concerned with compounds which have a much biosynthetic intermediates. The number of build- more limited distribution in nature. Such com- ing blocks needed is surprisingly few, and as with pounds, called secondary metabolites, are found any child’s construction set a vast array of objects in only specific organisms, or groups of organ- can be built up from a limited number of basic isms, and are an expression of the individuality building blocks. By far the most important building of species. Secondary metabolites are not neces- blocks employed in the biosynthesis of secondary sarily produced under all conditions, and in the metabolites are derived from the intermediates vast majority of cases the function of these com- acetyl coenzyme A (acetyl-CoA), shikimic acid, pounds and their benefit to the organism is not yet mevalonic acid, and 1-deoxyxylulose 5-phosphate. known. Some are undoubtedly produced for easily These are utilized respectively in the acetate, appreciated reasons, e.g. as toxic materials provid- shikimate, mevalonate,anddeoxyxylulose phos- ing defence against predators, as volatile attractants phate pathways, which form the basis of succeed- towards the same or other species, or as colouring ing chapters. Acetyl-CoA is formed by oxidative agents to attract or warn other species, but it is decarboxylation of the glycolytic pathway product β logical to assume that all do play some vital role pyruvic acid. It is also produced by the -oxidation for the well-being of the producer. It is this area of fatty acids, effectively reversing the process of secondary metabolism that provides most of by which fatty acids are themselves synthesized the pharmacologically active natural products. It is from acetyl-CoA. Important secondary metabolites formed from the acetate pathway include phenols, thus fairly obvious that the human diet could be prostaglandins, and macrolide antibiotics, together both unpalatable and remarkably dangerous if all with various fatty acids and derivatives at the plants, animals, and fungi produced the same range primary/secondary metabolism interface. Shikimic of compounds. acid is produced from a combination of phos- The above generalizations distinguishing pri- phoenolpyruvate, a glycolytic pathway intermedi- mary and secondary metabolites unfortunately ate, and erythrose 4-phosphate from the pentose leave a ‘grey area’ at the boundary, so that some phosphate pathway. The reactions of the pentose groups of natural products could be assigned to phosphate cycle may be employed for the degra- either division. Fatty acids and sugars provide good dation of glucose, but they also feature in the syn- examples, in that most are best described as pri- thesis of sugars by photosynthesis. The shikimate mary metabolites, whilst some representatives are pathway leads to a variety of phenols, cinnamic extremely rare and found only in a handful of acid derivatives, lignans, and alkaloids. Meval- species. Likewise, steroid biosynthesis produces a onic acid is itself formed from three molecules of range of widely distributed fundamental structures, acetyl-CoA, but the mevalonate pathway channels yet some steroids, many of them with pronounced acetate into a different series of compounds than pharmacological activity, are restricted to certain does the acetate pathway. Deoxyxylulose phos- organisms. Hopefully, the blurring of the bound- phate arises from a combination of two glycolytic aries will not cause confusion; the subdivision pathway intermediates, namely pyruvic acid and into primary metabolism (≡ biochemistry) or sec- glyceraldehyde 3-phosphate. The mevalonate and ondary metabolism (≡ natural products chemistry) deoxyxylulose phosphate pathways are together THE BUILDING BLOCKS 9

GLYCOLYSIS OH OH PENTOSE PHOSPHATE OH OH CYCLE OH O O PO HO PO O PHOTOSYNTHESIS OH OH OH OH OH erythrose 4-P D-glucose glucose 6-P

OHC OH CO2H CO2H NH PO 2 NH glycine glyceraldehyde 3-P 2 L-phenylalanine CO2H CO2H CO H CO H HO2C OH 2 HO 2 HS NH HO 2 NH2 NH2 PO HO OH L-cysteine L-serine L-tyrosine 3-phosphoglyceric acid OH CO2H SHIKIMIC ACID NH2 CO H HO2COP 2 NH L-tryptophan NH2 phosphoenolpyruvate L-valine OH

CO2H HO2CO OP NH2 O OH L-alanine pyruvic acid DEOXYXYLULOSE 5-P HO CO2H CoAS O HO2C NH2 OH L-leucine ACETYL-CoA MEVALONIC ACID

KREBS CYCLE CO H CO H 2 2 CO2H HO2CCO2H HO2CCO2H HO2C HO2C NH2 NH2 O O NH2 L-isoleucine L-aspartic acid oxaloacetic acid 2-oxoglutaric acid L-glutamic acid NH SCOH 2 H2NCO2H CO2H CO2H H2N N H2N NH 2 NH2 H NH2 NH2 L-methionine L-lysine L-arginine L-ornithine

Figure 2.1 responsible for the biosynthesis of a vast array of and tryptophan are themselves products from terpenoid and steroid metabolites. the shikimate pathway. Ornithine, a non-protein In addition to acetyl-CoA, shikimic acid, meval- amino acid, along with its homologue lysine,are onic acid, and deoxyxylulose phosphate, other important alkaloid precursors having their origins building blocks based on amino acids are fre- in Krebs cycle intermediates. quently employed in natural product synthesis. Of special significance is the appreciation that Peptides, proteins, alkaloids, and many antibiotics secondary metabolites can be synthesized by com- are derived from amino acids, and the origins bining several building blocks of the same type, of the most important amino acid components of or by using a mixture of different building blocks. these are briefly indicated in Figure 2.1. Interme- This expands structural diversity, and consequently diates from the glycolytic pathway and the Krebs makes subdivisions based entirely on biosynthetic cycle are used in constructing many of them, but pathways rather more difficult. A typical natural the aromatic amino acids phenylalanine, tyrosine, product might be produced by combining elements 10 SECONDARY METABOLISM from the acetate, shikimate, and deoxyxylulose is the product from three acetyl-CoA molecules, phosphate pathways. Many secondary metabolites but only five of mevalonate’s six carbons are also contain one or more sugar units in their used, the carboxyl group being lost. The alter- structure, either simple primary metabolites such native precursor deoxyxylulose phosphate, a as glucose or ribose, or alternatively substantially straight-chain sugar derivative, undergoes a modified and unusual sugars. To appreciate how skeletal rearrangement to form the branched- a natural product is elaborated, it is of value chain isoprene unit. to be able to dissect its structure into the basic • C C : This refers to a phenylpropyl unit and building blocks from which it is made up, and 6 3 is obtained from the carbon skeleton of either to propose how these are mechanistically joined L-phenylalanine or L-tyrosine,twoofthe together. With a little experience and practice, this shikimate-derived aromatic amino acids. This, becomes a relatively simple process, and it allows of course, requires loss of the amino group. The the molecule to be rationalized, thus exposing logi- C side-chain may be saturated or unsaturated, cal relationships between apparently quite different 3 and may be oxygenated. Sometimes the side- structures. In this way, similarities become much chain is cleaved, removing one or two carbons. more meaningful than differences, and an under- Thus, C C and C C units represent modified standing of biosynthetic pathways allows rational 6 2 6 1 shortened forms of the C C system. connecting links to be established. This forms the 6 3 basic approach in this book. • C6C2N: Again, this building block is formed Relatively few building blocks are routinely from either L-phenylalanine or L-tyrosine,L- employed, and the following list, though not com- tyrosine being by far the more common. In the prehensive, includes those most frequently encoun- elaboration of this unit, the carboxyl carbon of tered in producing the carbon and nitrogen skeleton the amino acid is removed. of a natural product. As we shall see, oxygen • indole.C2N: The third of the aromatic amino atoms can be introduced and removed by a vari- acids is L-tryptophan. This indole-containing ety of processes, and so are not considered in the system can undergo decarboxylation in a sim- initial analysis, except as a pointer to an acetate ilar way to L-phenylalanine and L-tyrosine so (see page 62) or shikimate (see page 123) origin. providing the remainder of the skeleton as an The structural features of these building blocks are indole.C2N unit. shown in Figure 2.2. • C4N: The C4N unit is usually found as a hetero- • C1: The simplest of the building blocks is com- cyclic pyrrolidine system and is produced from posed of a single carbon atom, usually in the the non-protein amino acid L-ornithine.In form of a methyl group, and most frequently it marked contrast to the C6C2N and indole.C2N is attached to oxygen or nitrogen, but occasion- units described above, ornithine supplies not its ally to carbon. It is derived from the S -methyl α-amino nitrogen, but the δ-amino nitrogen. The of L-methionine. The methylenedioxy group carboxylic acid function and the α-amino nitro- (OCH2O) is also an example of a C1 unit. gen are both lost. • C2: A two-carbon unit may be supplied by • C5N: This is produced in exactly the same way acetyl-CoA. This could be a simple acetyl as the C4N unit, but using L-lysine as precursor. group, as in an ester, but more frequently it The ε-amino nitrogen is retained, and the unit forms part of a long alkyl chain (as in a fatty tends to be found as a piperidine ring system. acid) or may be part of an aromatic system (e.g. These eight building blocks will form the basis of phenols). Of particular relevance is that in the many of the natural product structures discussed in latter examples, acetyl-CoA is first converted the following chapters. Simple examples of how into the more reactive malonyl-CoA before its compounds can be visualized as a combination of incorporation. building blocks are shown in Figure 2.3. At this • C5: The branched-chain C5 ‘isoprene’ unit is a stage, it is inappropriate to justify why a particular feature of compounds formed from mevalonate combination of units is used, but this aspect or deoxyxylulose phosphate. Mevalonate itself should become clear as the pathways are described. THE BUILDING BLOCKS 11

The building blocks

SCO2H H3C X CH3 (X = O, N, C) NH2 C L-Met 1

CO2H SCoA SCoA CC

O O C2

acetyl-CoA malonyl-CoA

SCoA HO 3 x HO2C O OH acetyl-CoA mevalonic acid isoprene unit OH HO C5 OP OP O OH OH OH deoxyxylulose methylerythritol phosphate phosphate

CO2H

NH2

L-Phe C6C2

CO2H C6C3 NH2 HO L-Tyr C6C1

CO2H CO2H NH2 NH2 N L-Phe N NH N CO2H C C N 6 2 L-Trp indole.C2N NH2 HO

L-Tyr

CO2H H2N N NH2 C4N N L-Orn

H N CO H 2 2 N

NH2 L-Lys C5N N

Figure 2.2 12 SECONDARY METABOLISM

OH OH HO O O O Glucose O CO H 2 O O O Rhamnose OH O orsellinic acid O OH O parthenolide naringin 4 x C2 3 x C MeO OMe 5 C6C3 + 3 x C2 + sugars OMe podophyllotoxin MeO HO C Me 2 x C C + 4 x C 2 N 6 3 1 N OH MeO CO2Me CO2H MeO N Me O

O MeO NH O tetrahydrocannabinolic acid papaverine lysergic acid cocaine + + + + 6 x C2 2 x C5 C6C2N + (C6C2) + 4 x C1 indole.C2N + C5 + C1 C4N 2 x C2 (C6C1) 2 x C1

C6C3 C6C3

Figure 2.3

A word of warning is also necessary. Some natural discussed in terms of chemical analogies. Enzymes products have been produced by processes in have the power to effect these transformations more which a fundamental rearrangement of the carbon efficiently and more rapidly than the chemical anal- skeleton has occurred. This is especially common ogy, and also under very much milder conditions. with structures derived from isoprene units, and it Where relevant, they also carry out reactions in a obviously disguises some of the original building stereospecific manner. Some of the important reac- blocks from immediate recognition. The same is tions frequently encountered are now described. true if one or more carbon atoms are removed by oxidation reactions. Alkylation Reactions: Nucleophilic Substitution THE CONSTRUCTION MECHANISMS The C1 methyl building unit is supplied from L- Natural product molecules are biosynthesized by methionine and is introduced by a nucleophilic a sequence of reactions which, with very few substitution reaction. In nature, the leaving group exceptions, are catalysed by enzymes. Enzymes are is enhanced by converting L-methionine into S - protein molecules which facilitate chemical mod- adenosylmethionine (SAM) [Figure 2.4(a)]. This ification of substrates by virtue of their specific gives a positively charged sulphur and facilitates binding properties conferred by the particular com- the nucleophilic substitution (SN2) type mech- bination of functional groups in the constituent anism [Figure 2.4(b)]. Thus, O-methyl and N - amino acids. In many cases, a suitable cofactor, e.g. methyl linkages may be obtained using hydroxyl NAD+, PLP, HSCoA (see below), as well as the and amino functions as nucleophiles. The genera- substrate, may also be bound to participate in the tion of C -methyl linkages requires the participation transformation. Although enzymes catalyse some of nucleophilic carbon. Positions ortho or para to fairly elaborate and sometimes unexpected changes, a phenol group, or positions adjacent to one or it is generally possible to account for the reactions more carbonyl groups, are thus candidates for C - using sound chemical principles and mechanisms. methylation [Figure 2.4(c)]. As we explore the pathways to a wide variety of AC5 isoprene unit in the form of dimethylallyl natural products, the reactions will generally be diphosphate (DMAPP) may also act as an THE CONSTRUCTION MECHANISMS 13

Alkylation reactions: nucleophilic substitution (a) formation of SAM NH2

N N H C H3C 3 S ATP P, PP S CH2 N N O

H N HO OH H2N 2 CO H CO2H 2 L-Met S-adenosylmethionine (SAM)

(b) O- and N-alkylation using SAM neutral molecule is Ad good leaving group S 2 reaction Ad SCO2H N OH H3C OCH3 SCO2H NH H or NH2 2 SAM NH2 – H S-adenosylhomocysteine

OCH3 or NH CH3 (c) C-alkylation using SAM

Ad ortho (and para) Ad carbonyl groups O H positions are SCOH increase acidity and SCO2H 2 H C activated by OH H3C allow formation of 3 NH2 enolate anion NH2 OH O

(d) O-alkylation using DMAPP

H OH SN2 reaction O – H O PPO dimethylallyl diphosphate diphosphate is (DMAPP) good leaving or group OH SN1 reaction PPO resonance stabilized allylic carbocation

Figure 2.4

alkylating agent, and a similar SN2 nucleophilic Alkylation Reactions: Electrophilic displacement can be proposed, the diphosphate Addition making a good leaving group [Figure 2.4(d)]. In some cases, there is evidence that DMAPP may As indicated above, the C5 isoprene unit in the ionize first to the resonance-stabilized allylic car- form of dimethylallyl diphosphate (DMAPP) bocation and thus an SN1 process operates instead. can be used to alkylate a nucleophile. In the C -Alkylation at activated positions using DMAPP elaboration of terpenoids and steroids, two or more is analogous to the C -methylation process above. C5 units are joined together, and the reactions 14 SECONDARY METABOLISM are rationalized in terms of carbocation chem- of mechanism can be responsible for cyclization istry, including electrophilic addition of carbo- reactions [Figure 2.5(a)]. cations on to alkenes. DMAPP may ionize to The initial carbocation may be generated by generate a resonance-stabilized allylic carboca- a number of mechanisms, important examples tion as shown in Figure 2.4(d), and this can then being loss of a leaving group, especially diphos- react with an alkene [e.g. isopentenyl diphos- phate (i.e. SN1 type ionization), protonation of phate (IPP)] as depicted in Figure 2.5(a). The an alkene, and protonation/ring opening of epox- resultant carbocation may then lose a proton ides [Figure 2.5(b)]. S -Adenosylmethionine may to give the uncharged product geranyl diphos- also alkylate alkenes by an electrophilic addition phate (GPP). Where the alkene and carbocation mechanism, adding a C1 unit, and generating an functions reside in the same molecule, this type intermediate carbocation.

Alkylation reactions: electrophilic addition

(a) inter- and intra-molecular additions electrophilic addition of cation on to alkene

– H OPP OPP OPP isopentenyl diphosphate H geranyl diphosphate (IPP) (GPP)

intramolecular addition: cyclization

(b) generation of carbocation H O OH L H H loss of leaving group protonation of alkene protonation and ring opening of epoxide

Ad

SCO2H H3C SAM NH2 CH3

methylation of alkene via SAM

(c) discharge of carbocation

H H2O H H HO H H H

loss of proton cyclization / loss of proton quenching with nucleophile (water)

Figure 2.5 THE CONSTRUCTION MECHANISMS 15

The final carbocation may be discharged by and the usual objective in these rearrangements loss of a proton (giving an alkene or sometimes a is to achieve tertiary status at the positive cen- cyclopropane ring) or by quenching with a suitable tre. However, a tertiary to secondary transition nucleophile, especially water [Figure 2.5(c)]. might be favoured if the rearrangement allows a significant release of ring strain. These gen- eral concepts are occasionally ignored by nature, Wagner–Meerwein Rearrangements but it must be remembered that the reactions are enzyme-mediated and carbocations may not exist A wide range of structures encountered in nat- as discrete species in the transformations. An inter- ural terpenoid and steroid derivatives can only esting feature of some biosynthetic pathways, e.g. be rationalized as originating from C5 isoprene that leading to steroids, is a remarkable series of units if some fundamental rearrangement process concerted 1,2-migrations rationalized via carboca- has occurred during biosynthesis. These rearrange- tion chemistry, but entirely a consequence of the ments have, in many cases, been confirmed experi- enzyme’s participation (Figure 2.6). mentally, and are almost always consistent with the participation of carbocation intermediates. Rear- rangements in chemical reactions involving car- Aldol and Claisen Reactions bocation intermediates, e.g. SN1 and E1 reactions, are not uncommon, and typically consist of 1,2- The aldol and Claisen reactions both achieve shifts of hydride, methyl, or alkyl groups. Occa- carbon–carbon bond formation and in typical base- sionally, 1,3- or longer shifts are encountered. catalysed chemical reactions depend on the gener- These shifts, termed Wagner–Meerwein rear- ation of a resonance-stabilized enolate anion from rangements, are readily rationalized in terms of a suitable carbonyl system (Figure 2.7). Whether generating a more stable carbocation, or relax- an aldol-type or Claisen-type product is formed ing ring strain (Figure 2.6). Thus, tertiary carbo- depends on the nature of X and its potential as cations are favoured over secondary carbocations, a leaving group. Thus, chemically, two molecules

Wagner–Meerwein rearrangements

1,2-hydride 1,2-methyl H H shift H H shift H H

secondary tertiary secondary tertiary carbocation carbocation carbocation carbocation

1,3-hydride 1,2-alkyl shift shift

H HHH tertiary carbocation, secondary carbocation, but strained but reduced ring strain in 4-membered ring 5-membered ring tertiary resonance-stabilized allylic carbocation cation

H a series of concerted 1,2 hydride and methyl shifts H

H H

Figure 2.6 16 SECONDARY METABOLISM

Aldol and Claisen reactions O O O R CH C R CH C R CH C X H X X resonance-stabilized B enolate anion

loss of leaving O O group O

R CH2 C R CH2 C X R CH2 C O O X CH C nucleophilic addition R CH C O on to carbonyl − X R X R CH C + Claisen-type product H X R = H, X = OEt, ethyl acetoacetate R = X = H, acetaldehyde R = H, X = OEt, ethyl acetate OH if no suitable leaving group, R CH2 C X protonation occurs O R CH C X aldol-type product R = X = H, aldol

Figure 2.7

NH2 H3C SCoA H2C SCoA H + O O N N O O OO acetyl-CoA HS N N O P O P O CH2 N N H H O OH OH OH cysteamine pantothenic acid PO OH pantotheine Coenzyme A HSCoA

resonance decreases acidity resonance of this type is less α of -hydrogens favourable in the sulphur ester

O O O O C R C R C R C R H C O 3 H3C O H3C S H3C S ester thioester

Figure 2.8 of acetaldehyde yield aldol, whilst two molecules In most cases, the biological reactions involve of ethyl acetate can give ethyl acetoacetate. These coenzyme A esters, e.g. acetyl-CoA (Figure 2.8). processes are vitally important in biochemistry for This is a thioester of acetic acid, and it has sig- the elaboration of both secondary and primary nificant advantages over oxygen esters, e.g. ethyl metabolites, but the enzyme catalysis obviates the acetate, in that the α-methylene hydrogens are now need for strong bases, and probably means the eno- more acidic, comparable in fact to those in the late anion has little more than transitory existence. equivalent ketone, thus increasing the likelihood of Nevertheless, the reactions do appear to parallel generating the enolate anion. This is explained in enolate anion chemistry, and are frequently respon- terms of electron delocalization in the ester func- sible for joining together of C2 acetate groups. tion (Figure 2.8). This type of delocalization is THE CONSTRUCTION MECHANISMS 17 more prominent in the oxygen ester than in the (gluconeogenesis). The conversion of acetyl-CoA sulphur ester, due to oxygen’s smaller size and thus into malonyl-CoA means the α-hydrogens are closer proximity of the lone pair for overlap with now flanked by two carbonyl groups, and have carbon’s orbitals. Furthermore, the thioester has a increased acidity. Thus, a more favourable nucle- much more favourable leaving group than the oxy- ophile is provided for the Claisen reaction. No acy- gen ester, and the combined effect is to increase lated malonic acid derivatives are produced, and the reactivity for both the aldol and Claisen-type the carboxyl group introduced into malonyl-CoA is reactions. simultaneously lost by a decarboxylation reaction Claisen reactions involving acetyl-CoA are during the Claisen condensation (Figure 2.9). An made even more favourable by first converting alternative rationalization is that decarboxylation acetyl-CoA into malonyl-CoA by a carboxylation of the malonyl ester is used to generate the acetyl reaction with CO2 using ATP and the coenzyme enolate anion without any requirement for a strong biotin (Figure 2.9). ATP and CO2 (as bicarbonate, base. Thus, the product formed from acetyl-CoA − HCO3 ) form the mixed anhydride, which car- as electrophile and malonyl-CoA as nucleophile boxylates the coenzyme in a biotin–enzyme com- is acetoacetyl-CoA, which is actually the same as plex. Fixation of carbon dioxide by biotin–enzyme in the condensation of two molecules of acetyl- complexes is not unique to acetyl-CoA, and CoA. Accordingly, the role of the carboxylation another important example occurs in the gener- step is clear cut: the carboxyl activates the α- ation of oxaloacetate from pyruvate in the syn- carbon to facilitate the Claisen condensation, and it thesis of glucose from non-carbohydrate sources is immediately removed on completion of this task.

ATP HCO O 3 nucleophilic attack CoAS C CH2 on to carbonyl; loss O acetyl-CoA O of biotin−enzyme as (enolate) O leaving group O O HN NH C N NH ADP HO P O C OH H H HO H H OH CO Enz CO Enz mixed anhydride S S biotin-enzyme N1-carboxybiotin−enzyme nucleophilic attack on to mixed anhydride

O − CoAS C CH2 + biotin enzyme

CO2H malonyl-CoA

loss of CoAS O O as leaving group CH3 C SCoA O O CH3 C SCoA O O CH3 C CH2 C SCoA

H2CCSCoA CH2 C SCoA acetoacetyl-CoA CO2 C OOH nucleophilic attack on carbonyl but with simultaneous loss of CO2

Figure 2.9 18 SECONDARY METABOLISM

O O O NaOEt H CH3 C OEt CH2 CO2Et CH3 C CH2 CO2Et CH3 C CH2 CO2H EtOH

enolate anion from ethyl acetoacetate acetoacetic acid ethyl acetate ∆

– CO2 O

O CO2Et O CO Et O C O 2 H thermal NaOEt H CH3 C OEt CH CO2Et CH3 C CH CO2Et CH3 C CH decarboxylation EtOH CO enolate anion from acylated diethyl diethyl malonate malonate HO gem-diacid

Figure 2.10

β-Oxidation of fatty acids

dehydrogenation; stereospecific dehydrogenation; hydrogen atoms hydration of hydrogen atoms + passed to FAD double bond passed to NAD + FAD FADH2 H2O NAD NADH O O O HO H O O E R SCoA R SCoA R SCoA S R SCoA fatty acyl-CoA HSCoA (chain length C2n) reverse Claisen reaction O O

R SCoA CH3 SCoA fatty acyl-CoA acetyl-CoA (chain length C2n−2)

Figure 2.11

By analogy, the chemical Claisen condensation in which a C2 unit as acetyl-CoA is cleaved off using the enolate anion from diethyl malonate in from a fatty acid chain, leaving it two carbons Figure 2.10 proceeds much more favourably than shorter in length. that using the enolate from ethyl acetate. The same acetoacetic acid product can be formed in the mal- onate condensation by hydrolysis of the acylated Schiff Base Formation and the malonate intermediate and decarboxylation of the Mannich Reaction gem-diacid. Formation of C−N bonds is frequently achieved by Both the reverse aldol and reverse Claisen condensation reactions between amines and alde- reactions may be encountered in the modification hydes or ketones. A typical nucleophilic addition of natural product molecules. Such reactions is followed by elimination of water to give an remove fragments from the basic skeleton already imine or Schiff base [Figure 2.12(a)]. Of almost generated, but may extend the diversity of struc- equal importance is the reversal of this process, tures. The reverse Claisen reaction is a promi- i.e. the hydrolysis of imines to amines and alde- nent feature of the β-oxidation sequence for the hydes/ketones [Figure 2.12(b)]. The imine so pro- catabolic degradation of fatty acids (Figure 2.11), duced, or more likely its protonated form the THE CONSTRUCTION MECHANISMS 19

(a) Schiff base formation equilibrium between protonated species; proton nucleophilic attack on may be on N or O to carbonyl elimination H – H , + H of water RNH2 COH RNCOH RNCOH2 RNC primary H H imine or amine Schiff base

R R R R 1 1 NH COH R N COH R N COH2 NC 1 R H R1 secondary H amine quaternary Schiff base

(b) Schiff base hydrolysis

nucleophilic attack on to imine or loss of amine leaving group; protonated imine formation of carbonyl H H

RNC RNC RNH2 C OH OH H2O H

(c) Mannich reaction

nucleophilic addition on to iminium ion R R NC C NC C H H iminium ion carbanion-type nucleophile, e.g. enolate anion

Figure 2.12 iminium ion, can then act as an electrophile in from nucleophilic addition to a carbonyl group. a Mannich reaction [Figure 2.12(c)]. The nucle- Indeed, the imine/iminium ion is merely acting ophile might be provided by an enolate anion, or as the nitrogen analogue of a carbonyl/protonated in many examples by a suitably activated centre carbonyl. To take this analogy further, protons in an aromatic ring system. The Mannich reaction on carbon adjacent to an imine group will be is encountered throughout alkaloid biosynthesis, acidic, as are those α to a carbonyl group, and the and in its most general form involves combination isomerization to the enamine shown in Figure 2.13 of an amine (primary or secondary), an aldehyde is analogous to keto–enol tautomerism. Just as or ketone, and a nucleophilic carbon. Secondary two carbonyl compounds can react via an aldol amines will react with the carbonyl compound reaction, so can two imine systems, and this is to give an iminium ion (quaternary Schiff base) indicated in Figure 2.13. Often aldehyde/ketone directly, and the additional protonation step is thus substrates in enzymic reactions become covalently not necessary. bonded to the enzyme through imine linkages; in It should be appreciated that the Mannich-like so doing they lose none of the carbonyl activation addition reaction in Figure 2.12(c) is little different as a consequence of the new form of bonding. 20 SECONDARY METABOLISM

H R CH2 R CH R CH H imine−enamine tautomerism NC NC NC H H imine enamine

R R CH C NHR R CH C N NC aldol-type reaction between NC H two imine systems behaving H H as enamine−iminium ion pair enamine protonated aldol-type addition imine product

Figure 2.13

Transamination cleaved off via the reverse aldol or reverse Claisen reactions mentioned above, by far the most com- Transamination is the exchange of the amino mon degradative modification is loss of one carbon group from an amino acid to a keto acid, and atom by a decarboxylation reaction. Decarboxy- provides the most common process for the intro- lation is a particular feature of the biosynthetic duction of nitrogen into amino acids, and for utilization of amino acids, and it has already the removal of nitrogen from them. The cou- been indicated that several of the basic building ple glutamic acid/2-oxoglutaric acid are the blocks, e.g. C6C2N, indole.C2N, are derived from usual donor/acceptor molecules for the amino an amino acid via loss of the carboxyl group. This group. Reductive amination of the Krebs cycle decarboxylation of amino acids is also a pyridoxal intermediate 2-oxoglutaric acid to glutamic acid phosphate-dependent reaction (compare transam- (Figure 2.14) is responsible for the initial incorpo- ination) and is represented as in Figure 2.15(a). ration of nitrogen, a reaction which involves imine This similarly depends on Schiff base formation formation and subsequent reduction. Transamina- and shares features of the transamination sequence tion then allows the amino group to be transferred of Figure 2.14. Decarboxylation is facilitated in the from glutamic acid to a suitable keto acid, or in the same way as loss of the α-hydrogen was facilitated reverse mode from an amino acid to 2-oxoglutaric for the transamination sequence. After protonation acid. This reaction is dependent on the coenzyme of the original α-carbon, the amine is released from pyridoxal phosphate (PLP) and features a Schiff the coenzyme by hydrolysis of the Schiff base base/imine intermediate (aldimine) with the alde- function. α hyde group of PLP (Figure 2.14). The -hydrogen β-Keto acids are thermally labile and rapidly of the original amino acid is now made consider- decarboxylated in vitro via a cyclic mechanism ably more acidic and is removed, leading to the which proceeds through the enol form of the ketimine by a reprotonation process which also final ketone [Figure 2.15(b)]. Similar reactions are restores the aromaticity in the pyridine ring. The found in nature, though whether cyclic processes keto acid is then liberated by hydrolysis of the are necessary is not clear. ortho-Phenolic acids also Schiff base function, which generates pyridoxam- decarboxylate readily in vitro and in vivo, and it is ine phosphate. The remainder of the sequence is again possible to invoke a cyclic β-ketoacidtau- now a reversal of this process, and transfers the tomer of the substrate. The corresponding decar- amine function from pyridoxamine phosphate to boxylation of para-phenolic acids cannot have a another keto acid. cyclic transition state, but the carbonyl group in the proposed keto tautomer activates the system for Decarboxylation Reactions decarboxylation. The acetate pathway frequently yields structures containing phenol and carboxylic Many pathways to natural products involve steps acid functions, and decarboxylation reactions may which remove portions of the carbon skeleton. thus feature as further modifications. Although the Although two or more carbon atoms may be carboxyl group may originate by hydrolysis of the THE CONSTRUCTION MECHANISMS 21

Transamination reductive amination

NH3 H2O HO2C NAD(P)H NAD(P)+ HO2C O NH2

glutamate dehydrogenase HO2C HO2C

2-oxoglutaric acid glutamic acid L-Glu

HO2C HO2C NH2 R transaminase O R O NH2 HO2C HO2C HO2C HO2C glutamic acid keto acid2-oxoglutaric amino acid acid RCOH 2 RCO2H

H NH2 O NH CHO 2 OH OH PO PO

N CH3 N CH3 pyridoxal P pyridoxamine P (PLP) formation of imine hydrolysis of from aldehyde and imine to keto amino acid acid and amine

R CO2H R CO2H R CO2H α -hydrogen H restoring N H N N is now acidic aromaticity OH OH OH PO PO PO

N CH3 N CH3 N CH3 H H aldimine ketimine

Figure 2.14 thioester portion of the acetyl-CoA precursor, there into the Krebs cycle. Both types of reaction are also occasions when a methyl group can be depend upon thiamine diphosphate (TPP). TPP sequentially oxidized to a carboxyl, which then is a coenzyme containing a thiazole ring, which subsequently suffers decarboxylation. has an acidic hydrogen and is thus capable of Decarboxylation of α-keto acids is a fea- yielding the carbanion. This acts as a nucle- ture of primary metabolism, e.g. pyruvic acid ophile towards carbonyl groups. Decarboxylation → acetaldehyde in glycolysis, and pyruvic acid of pyruvic acid to acetaldehyde is depicted as → acetyl-CoA, an example of overall oxidative in Figure 2.15(c), which process also regenerates decarboxylation prior to entry of acetyl-CoA the carbanion. In the oxidation step of oxidative 22 SECONDARY METABOLISM

Decarboxylation reactions

(a) amino acids

RCO2H R

H NH2 NH2

CHO CHO OH OH PO PO

N CH3 N CH3 pyridoxal P pyridoxal P (PLP) (PLP)

formation of hydrolysis of imine imine to aldehyde and amine O H R H R H R O H H H N N restoring N decarboxylation aromaticity OH OH OH PO PO PO

N CH3 N CH3 N CH3 H H

(b) β-keto acids

keto-enol H 6-membered .... tautomerism O O OH O H-bonded system CO2 C C C C O R CH2 R CH3 R CH2 β-keto acid intermediate enol 6-membered H-bonded system OH O .... OH H O CO2 CO2H C H phenolic acid O (i.e. enol tautomer) keto tautomer ≡ β-keto acid

H HO O HO

O CO CO H C H 2 2 H O keto tautomer

Figure 2.15 THE CONSTRUCTION MECHANISMS 23

(c) α-keto acids

NH2 nucleophilic attack of N N S carbanion on to carbonyl: decarboxylation of aldol-type reaction β-iminium acid N OPP H O acidic O thiamine diphosphate hydrogen C HO (TPP) B H C CO H H3C C H H 3 2 C O R1 R1 R1 N S N S N S

R2 R2 R2 TPP TPP anion

O H C reverse O − H3C H enamine imine H C OH H3C H aldol-type C 3 tautomerism C H reaction 1 R1 R R1 TPP anion N S N S N S regenerated 2 R2 R R2 O iminium ion enamine S S OH lipoic acid

H

H3C OH S R3 C S 1 R 3 N S S R enzyme-bound S lipoic acid R2 enzyme-bound enamine enamine attacks S of acetyl group lipoic acid lipoic acid fragment displaced by with S–S bond fission coenzyme A O FAD O HSCoA H C HS 3 HS 3 O R C R H3C SCoA H3C S H3C S C acetyl-CoA HS R3 HS 1 R R1 N S N S original lipoic acid fragment 2 R R2 has become reduced to dithiol; oxidation regenerates the regeneration of TPP enzyme-bound lipoic acid carbanion leaves acetyl group attached to dihydrolipoic acid

Figure 2.15 (continued) 24 SECONDARY METABOLISM decarboxylation, the enzyme-bound disulphide- coenzyme acceptor. The coenzyme system in- containing coenzyme lipoic acid is also involved. volved can generally be related to the functional The intermediate enamine in Figure 2.15(c), in- group being oxidized in the substrate. Thus if the stead of accepting a proton, is used to attack a oxidation process is sulphur in the lipoic acid moiety with subsequent S−S bond fission, thereby effectively reducing the CH OH C O lipoic acid fragment. This allows regeneration of the TPP carbanion, and the acetyl group is bound to the dihydrolipoic acid. This acetyl group is then then a pyridine nucleotide, nicotinamide adenine released as acetyl-CoA by displacement with the dinucleotide (NAD+)ornicotinamide adenine thiol coenzyme A. The bound dihydrolipoic acid dinucleotide phosphate (NADP+), tends to be fragment is then reoxidized to restore its function. utilized as hydrogen acceptor. One hydrogen from An exactly equivalent reaction is encountered in the substrate (that bonded to carbon) is transferred the Krebs cycle in the conversion of 2-oxoglutaric as hydride to the coenzyme, and the other, as acid into succinyl-CoA. a proton, is passed to the medium (Figure 2.16). NAD(P)+ may also be used in the oxidations

Oxidation and Reduction Reactions H Changes to the oxidation state of a molecule are CO CO2H frequently carried out as a secondary metabolite is synthesized or modified. The processes are not always completely understood, but the following CH NH2 CNH general features are recognized. The processes may be classified according to the type of enzyme The reverse reaction, i.e. reduction, is also involved and their mechanism of action. indicated in Figure 2.16, and may be compared with the chemical reduction process using complex Dehydrogenases metal hydrides, e.g. LiAlH4 or NaBH4, namely nu- cleophilic addition of hydride and subsequent pro- Dehydrogenases remove two hydrogen atoms tonation. The reduced forms NADH and NADPH from the substrate, passing them to a suitable are conveniently regarded as hydride-donating

Dehydrogenases: NAD+ and NADP+

NH2 CONH N 2 N O O adenine nicotinamide

N NPCH2 O OPOCH2 N ≡ ribose P P ribose O O OH OH RO OH P HO OH R = H, NAD+ R = P, NADP+ C H C H H O O H H

CONH2 CONH2

N N R R NAD+ NADH NADP+ NADPH

Figure 2.16 THE CONSTRUCTION MECHANISMS 25

Dehydrogenases: FAD and FMN O

H3C N NH NH2 N H3C N N O adenine flavin N O O OH ≡ riboseP P ribitol N NPCH2 O OPO O OH OH OH OH HO OH FMN FAD H H C C C C H O H O

H3C N H3C N NH NH

H3C N N O H3C N N O R H R H

FAD FADH2 FMN FMNH2

Figure 2.17 reducing agents. In practice, NADPH is generally of oxidative phosphorylation, so that the hydrogen employed in reductive processes, whilst NAD+ is atoms eventually pass to oxygen giving water. used in oxidations. Should the oxidative process be the conversion Oxidases CH CH CH CH 2 2 Oxidases also remove hydrogen from a substrate, but pass these atoms to molecular oxygen or to the coenzyme used as acceptor is usually a flavin hydrogen peroxide, in both cases forming water. nucleotide, flavin adenine dinucleotide (FAD)or Oxidases using hydrogen peroxide are termed per- flavin mononucleotide (FMN). These entities are oxidases. Mechanisms of action vary and need bound to the enzyme in the form of a flavoprotein, not be considered here. Important transforma- and take up two hydrogen atoms, represented tions in secondary metabolism include the oxi- in Figure 2.17 as being derived by addition of dation of ortho-andpara-quinols to quinones hydride from the substrate and a proton from the (Figure 2.18), and the peroxidase-induced phenolic medium. Alternative mechanisms have also been oxidative coupling processes (see page 28). proposed, however. Reductive sequences involving flavoproteins may be represented as the reverse reaction in Figure 2.17. NADPH may also be Mono-oxygenases employed as a coenzyme in the reduction of a carbon–carbon double bond. Oxygenases catalyse the direct addition of oxygen These oxidation reactions employing pyridine from molecular oxygen to the substrate. They are nucleotides and flavoproteins are especially impor- subdivided into mono- and di-oxygenases accord- tant in primary metabolism in liberating energy ing to whether just one or both of the oxygen atoms from fuel molecules in the form of ATP. The are introduced into the substrate. With mono- reduced coenzymes formed in the process are nor- oxygenases, the second oxygen atom from O2 mally reoxidized via the electron transport chain is reduced to water by an appropriate hydrogen 26 SECONDARY METABOLISM

Oxidases cleavage of an oxygen atom. Many such sys- tems have been identified, capable of hydroxy- OH O lating aliphatic or aromatic systems, as well as + + 1/2 O2 H2O producing epoxides from alkenes (Figure 2.19). OH O In most cases, NADPH features as hydrogen ortho-quinol ortho-quinone donor. Aromatic hydroxylation catalysed by mono- OH O oxygenases (including cytochrome P-450 systems) probably involves arene oxide (epoxide) interme- + + 1/2 O2 H2O diates (Figure 2.20). An interesting consequence of this mechanism is that when the epoxide opens OH O up, the hydrogen atom originally attached to the para-quinol para-quinone position which becomes hydroxylated can migrate to the adjacent carbon on the ring. A high pro- Figure 2.18 portion of these hydrogen atoms is subsequently retained in the product, even though enolization donor, e.g. NADH, NADPH, or ascorbic acid allows some loss of this hydrogen. This migration (vitamin C). In this respect they may also be is known as the NIH shift, having been origi- considered to behave as oxidases, and the term nally observed at the National Institute of Health, ‘mixed-function oxidase’ is also used for these Bethesda, MD, USA. enzymes. Especially important examples of these enzymes are the cytochrome P-450-dependent Mono-oxygenases mono-oxygenases. These are frequently involved O2 in biological hydroxylations, either in biosyn- CH COH thesis, or in the mammalian detoxification and NADPH metabolism of foreign compounds such as drugs, O2 and such enzymes are thus termed ‘hydroxylases’. H OH Cytochrome P-450 is named after its intense NADPH absorption band at 450 nm when exposed to CO, O which is a powerful inhibitor of these enzymes. O2 It contains an iron–porphyrin complex (haem), NADPH which is bound to the enzyme, and a redox change involving the Fe atom allows binding and the Figure 2.19

NIH shift hydride migration R R R NIH shift R

H H H H H O H H H O O – H arene oxide – H enolization

R R

loss of retention of labelled labelled hydrogen H H hydrogen OH OH

Figure 2.20 THE CONSTRUCTION MECHANISMS 27

The oxidative cyclization of an ortho-hydroxy- aromatic rings. Cyclic peroxides (dioxetanes) are methoxy-substituted aromatic system giving a likely to be intermediates (Figure 2.22). Oxidative methylenedioxy group is also known to involve cleavage of aromatic rings typically employs cat- a cytochrome P-450-dependent mono-oxygenase. echol (1,2-dihydroxy) or quinol (1,4-dihydroxy) This enzyme hydroxylates the methyl to yield substrates, and in the case of catechols, cleavage a formaldehyde hemiacetal intermediate, which may be between or adjacent to the two hydrox- can cyclize to the methylenedioxy bridge (the yls, giving products containing aldehyde and/or acetal of formaldehyde) by an ionic mechanism carboxylic acid functionalities (Figure 2.22). (Figure 2.21). Some dioxygenases utilize two acceptor sub- strates and incorporate one oxygen atom into Dioxygenases each. Thus, 2-oxoglutarate-dependent dioxyge- nases hydroxylate one substrate, whilst also trans- Dioxygenases introduce both atoms from molecu- forming 2-oxoglutarate into succinate with the lar oxygen into the substrate, and are frequently release of CO2 (Figure 2.23). 2-Oxoglutarate- involved in the cleavage of bonds, including dependent dioxygenases also require as cofactors

Methylenedioxy groups nucleophilic attack on to carbonyl equivalent H

O O OH O O O CH3 2 CH2 CH2 NADPH OH OH O O H ortho-hydroxy- formaldehyde methylenedioxy methoxy derivative hemiacetal derivative

Figure 2.21

Dioxygenases

O O + O2 O O

dioxetane

O CH O + O2 O CH O

OH cleavage between OH hydroxyls O2 O CO2H O CO H OH 2 OH

cleavage adjacent to O hydroxyls 2 O CHO O CO H OH 2 OH OH OH OH

Figure 2.22 28 SECONDARY METABOLISM

2-Oxoglutarate-dependent dioxygenases Amine oxidases

HO C FAD, O NH 2 O2 CO2 2 3 O CO2H RCH2NH2 RCHO RH ROH monoamine 2-oxoglutarate- oxidase dependent HO2C dioxygenase HO2C O2, H2O NH3, H2O2 2-oxoglutaric acid succinic acid CHO H2NNH2 H N n 2 n diamine oxidase Figure 2.23 Figure 2.24 Fe2+ to generate an enzyme-bound iron–oxygen complex, and ascorbic acid (vitamin C) to subse- Baeyer–Villiger Oxidations quently reduce this complex. The chemical oxidation of ketones by peracids, the Baeyer–Villiger oxidation, yields an ester, and Amine Oxidases the process is known to involve migration of an alkyl group from the ketone (Figure 2.25). For In addition to the oxidizing enzymes outlined comparable ketone → ester conversions known above, those which transform an amine into an to occur in biochemistry, cytochrome-P-450- or aldehyde, the amine oxidases, are frequently FAD-dependent enzymes requiring NADPH and involved in metabolic pathways. These include O2 appear to be involved. This leads to formation monoamine oxidases and diamine oxidases. of a peroxy–enzyme complex and a mechanism Monoamine oxidases utilize a flavin nucleotide, similar to that for the chemical Baeyer–Villiger typically FAD, and molecular oxygen, and involve oxidation may thus operate. The oxygen atom initial dehydrogenation to an imine, followed introduced thus originates from O2. by hydrolysis to the aldehyde and ammonia (Figure 2.24). Diamine oxidases require a diamine substrate, and oxidize at one amino group using Phenolic Oxidative Coupling molecular oxygen to give the corresponding aldehyde. Hydrogen peroxide and ammonia are Many natural products are produced by the the other products formed. The aminoaldehyde so coupling of two or more phenolic systems, in formed then has the potential to be transformed a process readily rationalized by means of free into a cyclic imine via Schiff base formation. radical reactions. The reactions can be brought

Baeyer−Villiger oxidations

carbonyl reforms: alkyl nucleophilic attack of group migrates from carbon peracid on to carbonyl to adjacent oxygen

− H Baeyer Villiger H O O oxidation O HO

R1 R2 O O O O O O R2 peracid HO 1 ketone R2 R O R1 ester

O O O2, NADPH O OEnz HO R2 2 1 R1 2 (HOO–Enz) R R O R R1

Figure 2.25 THE CONSTRUCTION MECHANISMS 29

Phenolic oxidative coupling

OH O O O – H – e resonance-stabilized free radical phenol x 2 coupling of two radicals x 2 O O O O O H H H O H H H H O keto tautomers O bis-dienone bis-dienone bis-dienone enolization

OH OH OH OH OH O enol tautomers OH HO ether linkage ortho–ortho coupling ortho–para coupling para–para coupling

Figure 2.26 about by oxidase enzymes, including peroxidase sugar units to a suitable atom of an aglycone and laccase systems, known to be radical to give a glycoside, or to another sugar giving generators. Other enzymes catalysing phenolic a polysaccharide. Linkages tend to be through oxidative coupling have been characterized as oxygen, although they are not restricted to cytochrome P-450-dependent proteins, requiring oxygen, since S -, N -, and C -glycosides are NADPH and O2 cofactors, but no oxygen is well known. The agent for glycosylation is incorporated into the substrate. A one-electron a uridine diphosphosugar,e.g.UDPglucose. oxidation of a phenol gives the free radical, and This is synthesized from glucose 1-phosphate the unpaired electron can then be delocalized and UTP, and then the glucosylation process via resonance forms in which the free electron can be envisaged as a simple SN2 nucleophilic is dispersed to positions ortho and para to the displacement reaction [Figure 2.27(a)]. Since original oxygen function (Figure 2.26). Coupling UDPglucose has its leaving group in the α- of two of these mesomeric structures gives a range configuration, the product has the β-configuration, of dimeric systems as exemplified in Figure 2.26. as is most commonly found in natural glucosides. The final products indicated are then derived by Note, however, that many important carbohydrates, enolization, which restores aromaticity to the rings. e.g. sucrose and starch, possess α-linkages, Thus, carbon–carbon bonds involving positions and these appear to originate via double SN2 ortho or para to the original phenols, or ether processes (see page 470). Other UDPsugars, e.g. linkages, may be formed. The reactive dienone UDPgalactose or UDPxylose, are utilized in systems formed as intermediates may in some the synthesis of glycosides containing different cases be attacked by other nucleophilic groupings, sugar units. extending the range of structures ultimately The hydrolysis of glycosides is achieved by spe- derived from this basic reaction sequence. cific hydrolytic enzymes, e.g. β-glucosidase for β- glucosides and β-galactosidase for β-galactosides. Glycosylation Reactions These enzymes mimic the readily achieved acid- catalysed processes [Figure 2.27(b)]. Under acidic The widespread occurrence of glycosides and conditions, the α-andβ-anomeric hemiacetal polysaccharides requires processes for attaching forms can also equilibrate via the open chain 30 SECONDARY METABOLISM

Glycosylation reactions (a) O-glucosylation SN2 reaction OH ROH O HO O OH HO H OH O O HO O HO O + UTP O N HO + UDP HO H O P O P O CH2 HO OR O HO OH OH HO OP H glucose 1-P HO OH O-β-D-glucoside UDPglucose

(b) hydrolysis of O-glucosides

OH OH OH OH O H O H O O HO HO HO OH2 a HO HO OR HO OR HO b HO H HO HO HO H HO H H a OH β OH O- -D-glucoside 2 α-D-glucose b

OH H OH O OH HO HO HO OH HO OH HO HO H H β-D-glucose (c) C-glucosylation HO OH OH HO HO O HO O HO H OH HO HO HO OPPU H OH UDPglucose C-β-D-glucoside

Figure 2.27 sugar. Of particular importance is that although a suitable nucleophilic carbon is available, e.g. O-, N -, and S -glycosides may be hydrolysed aromatic systems activated by phenol groups by acid, C -glycosides are stable to acid. C - [Figure 2.27(c)]. The resultant C -glycoside thus Glycosides are produced in a similar manner to contains a new carbon–carbon linkage, and the C -alkylation process described above, where cleavage would require oxidation, not hydrolysis.

SOME VITAMINS ASSOCIATED WITH THE CONSTRUCTION MECHANISMS Vitamin B1

Vitamin B1 (thiamine) (Figure 2.28) is a water-soluble vitamin with a pyrimidinylmethylthia- zolium structure. It is widely available in the diet, with cereals, beans, nuts, eggs, yeast, and vegetables providing sources. Wheat germ and yeast have very high levels. Dietary deficiency leads to beriberi, characterized by neurological disorders, loss of appetite, fatigue,

(Continues) THE CONSTRUCTION MECHANISMS 31

(Continued)

NH2 O N O N N NH S HO2C OH N N N N O H OH OH OH thiamine pantothenic acid (vitamin B1) riboflavin OH (vitamin B5) (vitamin B2) OH OH

OH NH2 O CHO 5′ HO HO HO HN NH OH OH OH H H N N N S CO2H pyridoxine pyridoxal pyridoxamine (pyridoxol) biotin (vitamin H)

(vitamin B6)

Figure 2.28 and muscular weakness. Thiamine is produced synthetically, and foods such as cereals are often enriched. The vitamin is stable in acid solution, but decomposes above pH 5, and is also partially decomposed during normal cooking. As thiamine diphosphate, vitamin B1 is a coenzyme for pyruvate dehydrogenase which catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA (see page 21), and also for transketolase which transfers a two- carbon fragment between carbohydrates in the pentose phosphate pathway (see page 446). Accordingly, this is a very important component in carbohydrate metabolism.

Vitamin B2

Vitamin B2 (riboflavin) (Figure 2.28) is a water-soluble vitamin having an isoalloxazine ring linked to D-ribitol. It is widely available in foods, including liver, kidney, dairy products, eggs, meat, and fresh vegetables. Yeast is a particularly rich source. It is stable in acid solution, not decomposed during cooking, but is sensitive to light. Riboflavin may be produced synthetically, or by fermentation using the yeastlike fungi Eremothecium ashbyii and Ashbya gossypii. Dietary deficiency is uncommon, but manifests itself by skin problems and eye disturbances. Riboflavin is a component of FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), coenzymes which play a major role in oxidation–reduction reactions (see page 25). Many key enzymes containing riboflavin (flavoproteins) are involved in metabolic pathways. Since riboflavin contains ribitol and not ribose in its structure, FAD and FMN are not strictly nucleotides, though this nomenclature is commonly accepted and used.

Vitamin B5

Vitamin B5 (pantothenic acid) (Figure 2.28) is a very widely distributed water-soluble vitamin, though yeast, liver, and cereals provide rich sources. Even though animals must obtain the vitamin through the diet, pantothenic acid deficiency is rare, since most foods provide

(Continues) 32 SECONDARY METABOLISM

(Continued)

adequate quantities. Its importance in metabolism is as part of the structure of coenzyme A (see page 16), the carrier molecule essential for carbohydrate, fat, and protein metabolism. Pantothenic acid is specifically implicated in enzymes responsible for the biosynthesis of fatty acids (see page 36), polyketides (page 62) and some peptides (page 421).

Vitamin B6

Vitamin B6 covers the three pyridine derivatives pyridoxine (pyridoxol), pyridoxal, and pyridoxamine, and also their 5-phosphates (Figure 2.28). These are water-soluble vitamins, pyridoxine predominating in plant materials, whilst pyridoxal and pyridoxamine are the main forms in animal tissues. Meat, salmon, nuts, potatoes, bananas, and cereals are good sources. A high proportion of the vitamin activity can be lost during cooking, but a normal diet provides an adequate supply. Vitamin B6 deficiency is usually the result of malabsorption, or may be induced by some drug treatments where the drug may act as an antagonist or increase its renal excretion as a side-effect. Symptoms of deficiency are similar to those of niacin (vitamin B3) and riboflavin (vitamin B2) deficiencies, and include eye, mouth, and nose lesions, and neurological changes. Synthetic pyridoxine is used for supplementation. Pyridoxal 5-phosphate is a coenzyme for a large number of enzymes, particularly those involved in amino acid metabolism, e.g. in transamination (see page 20) and decarboxylation (see page 20). The production of the neurotransmitter γ-aminobutyric acid (GABA) from glutamic acid is an important pyridoxal-dependent reaction.

Vitamin B12

Vitamin B12 (cobalamins) (Figure 2.29) are extremely complex structures based on a corrin ring, which, although similar to the porphyrin ring found in haem, chlorophyll, and cytochromes,

R = CN, cyanocobalamin (vitamin B12) H NOC 2 R = OH, hydroxocobalamin (vitamin B12a) CONH2 R = H2O, aquocobalamin (vitamin B12b) H2NOC R = NO2, nitritocobalamin (vitamin B12c) N R N CONH2 R = Me, methylcobalamin (methyl vitamin B12) Co+ H NH2 N N H NOC 2 N N N CONH2 R = N O O 5′-deoxyadenosylcobalamin NH N OH HO OH (coenzyme B12) O PO HO N O O HO N N NH N

NH N N HN

corrin ring system porphyrin ring system

Figure 2.29

(Continues) FURTHER READING 33

(Continued)

has two of the pyrrole rings directly bonded. The central metal atom is cobalt; haem and cytochromes have iron, whilst chlorophyll has magnesium. Four of the six coordinations are provided by the corrin ring nitrogens, and a fifth by a dimethylbenzimidazole moiety. The sixth is variable, being cyano in cyanocobalamin (vitamin B12), hydroxyl in hydroxocobalamin (vitamin B12a), or other anions may feature. Cyanocobalamin is actually an artefact formed as a result of the use of cyanide in the purification procedures. The physiologically active coenzyme form of the vitamin is 5 -deoxyadenosylcobalamin (coenzyme B12). Vitamin B12 appears to be entirely of microbial origin, with intestinal flora contributing towards human dietary needs. The vitamin is then stored in the liver, and animal liver extract has been a traditional source. Commercial supplies are currently obtained by semi-synthesis from the total cobalamin extract of Streptomyces griseus, Propionibacterium species, or other bacterial cultures. This material can be converted into cyanocobalamin or hydroxocobalamin. The cobalamins are stable when protected against light. Foods with a high vitamin B12 content include liver, kidney, meat, and seafood. Vegetables are a poor dietary source, and strict vegetarians may therefore risk deficiencies. Insufficient vitamin B12 leads to pernicious anaemia, a disease that results in nervous disturbances and low production of red blood cells, though this is mostly due to lack of the gastric glycoprotein (intrinsic factor) which complexes with the vitamin to facilitate its absorption. Traditionally, daily consumption of raw liver was used to counteract the problem. Cyanocobalamin, or preferably hydroxocobalamin which has a longer lifetime in the body, may be administered orally or by injection to counteract deficiencies. Both agents are converted into coenzyme B12 in the body. Coenzyme B12 is a cofactor for a number of metabolic rearrangements, such as the conversion of methylmalonyl-CoA into succinyl-CoA in the oxidation of fatty acids with an odd number of carbon atoms, and for methylations, such as in the biosynthesis of methionine.

Vitamin H Vitamin H (biotin) (Figure 2.28) is a water-soluble vitamin found in eggs, liver, kidney, yeast, cereals, and milk, and is also produced by intestinal microflora so that dietary deficiency is rare. Deficiency can be triggered by a diet rich in raw egg white, in which a protein, avidin, binds biotin so tightly so that it is effectively unavailable for metabolic use. This affinity disappears by cooking and hence denaturing the avidin. Biotin deficiency leads to dermatitis and hair loss. The vitamin functions as a carboxyl carrier, binding CO2 via a carbamate link, then donating this in carboxylase reactions, e.g. carboxylation of acetyl-CoA to malonyl-CoA (see page 17), of propionyl-CoA to methylmalonyl-CoA (see page 92), and of pyruvate to oxaloacetate during gluconeogenesis.

FURTHER READING Vitamins Natural Products, Biosynthesis Battersby AR (2000) Tetrapyrroles: The pigments of life. Nat Prod Rep 17, 507–526. Mann J (1994) Chemical Aspects of Biosynthesis.Ox- Burdick D (1998) Vitamins [pyridoxine (B6)]. Kirk– ford Chemistry Primers, Oxford. Othmer Encyclopedia of Chemical Technology,4th Mann J, Davidson RS, Hobbs JB, Banthorpe DV and edn, Vol 25. Wiley, New York, pp 116–132. Harborne JB (1994) Natural Products: Their Chem- Burdick D (1998) Vitamins [thiamine (B1)]. Kirk– istry and Biological Significance. Longman, Harlow. Othmer Encyclopedia of Chemical Technology,4th Torssell KBG (1997) Natural Product Chemistry. A edn, Vol 25. Wiley, New York, pp 152–171. Mechanistic, Biosynthetic and Ecological Approach. Kingston R (1999) Supplementary benefits? Chem Brit Apotekarsocieteten, Stockholm. 35 (7), 29–32. 34 SECONDARY METABOLISM

Outten RA (1998) Vitamins (biotin). Kirk–Othmer En- Scott JW (1998) Vitamins (vitamin B12). Kirk–Othmer cyclopedia of Chemical Technology, 4th edn, Vol 25. Encyclopedia of Chemical Technology, 4th edn, Vol Wiley, New York, pp 48–64. 25. Wiley, New York, pp 192–217. Rawalpally TR (1998) Vitamins (pantothenic acid). Yoneda F (1998) Vitamins [riboflavin (B2)]. Kirk– Kirk–Othmer Encyclopedia of Chemical Technology, Othmer Encyclopedia of Chemical Technology,4th 4th edn, Vol 25. Wiley, New York, pp 99–116. edn, Vol 25. Wiley, New York, pp 132–152. Medicinal Natural Products. PaulMDewick Copyright  2002 John Wiley & Sons, Ltd ISBNs:0471496405 (Hardback); 0471496413 (paperback); 0470846275(Electronic) 3 THE ACETATE PATHWAY: FATTY ACIDS AND POLYKETIDES

Polyketides, metabolites built primarily from combinations of acetate units, are described. The biosynthesis of saturated and unsaturated fatty acids is covered, together with prostaglandins, thromboxanes, and leukotrienes. Cyclization of polyketides to give aromatic structures is then rationalized in terms of aldol and Claisen reactions. More complex structures formed via pathways involving alkylation reactions, phenolic oxidative coupling, oxidative cleavage of aromatic rings, and employing starter groups other than acetate are developed. The use of extender groups other than malonate gives rise to macrolides and polyethers, whilst further cyclization of polyketide structures may be achieved through Diels–Alder reactions. The application of genetic engineering to modify products from the acetate pathway is discussed. Monograph topics giving more detailed information on medicinal agents include fixed oils and fats, evening primrose oil, echinacea, prostaglandins and isoprostanes, thromboxanes, leukotrienes, senna, cascara, frangula and allied drugs, St John’s wort, mycophenolic acid, khellin and cromoglicate, griseofulvin, poison ivy and poison oak, aflatoxins, cannabis, tetracyclines, anthracycline antibiotics, macrolide antibiotics, avermectins, polyene antifungals, tacrolimus and sirolimus, ansa macrolides, mevastatin and other statins.

Polyketides constitute a large class of natural reactions was operating. It is now known that fatty products grouped together on purely biosynthetic acid biosynthesis involves initial carboxylation of grounds. Their diverse structures can be explained acetyl-CoA to malonyl-CoA, a reaction involving β − as being derived from poly- -keto chains, formed ATP, CO 2 (as bicarbonate, HCO3 ), and the coen- by coupling of acetic acid (C2) units via conden- zyme biotin as the carrier of CO2 (see page 17). sation reactions, The conversion of acetyl-CoA into malonyl- CoA increases the acidity of the α-hydrogens, and i.e. nCH CO H −−−→−[CH CO] − 3 2 2 n thus provides a better nucleophile for the Claisen Included in such compounds are the fatty condensation. In the biosynthetic sequence, no acy- acids, polyacetylenes, prostaglandins, macrolide lated malonic acid derivatives are produced, and antibiotics and many aromatic compounds, e.g. no label from [14C]bicarbonate is incorporated, so anthraquinones and tetracyclines. the carboxyl group introduced into malonyl-CoA The formation of the poly-β-keto chain could is simultaneously lost by a decarboxylation reac- be envisaged as a series of Claisen reactions, tion during the Claisen condensation (Figure 3.1). the reverse of which are involved in the β- Accordingly, the carboxylation step helps to acti- oxidation sequence for the metabolism of fatty vate the α-carbon and facilitate Claisen condensa- acids (see page 18). Thus, two molecules of acetyl- tion, and the carboxyl is immediately removed on CoA could participate in a Claisen condensation completion of this task. An alternative rationaliza- giving acetoacetyl-CoA, and this reaction could tion is that decarboxylation of the malonyl ester is be repeated to generate a poly-β-keto ester of used to generate the acetyl enolate anion without appropriate chain length (Figure 3.1). However, any requirement for a strong base. a study of the enzymes involved in fatty acid The pathways to fatty acids and aromatic biosynthesis showed this simple rationalization polyketides branch early. The chain extension could not be correct, and a more complex series of process of Figure 3.1 continues for aromatics, 36 THE ACETATE PATHWAY

CH3COSCoA CH3COSCoA O O O O CH C SCoA CH C CH C SCoA 3 3 2 CH3CO CH2 CCHn 2COSCoA Claisen acetyl-CoA acetoacetyl-CoA Claisen β reaction reaction poly- -keto ester

O Claisen O reaction CH3 C SCoA O O CH3 C SCoA acetyl-CoA O O CH3 C CH2 C SCoA

H2CCSCoA CH2 C SCoA acetoacetyl-CoA CO 2 malonyl-CoA repeat of Claisen C OOH reaction CO2 nucleophilic attack on malonyl-CoA carbonyl with simultaneous O loss of CO2 CH3CO CH2 CCHn 2COSCoA poly-β-keto ester

Figure 3.1 generating a highly reactive poly-β-keto chain, acyl-ACP; R=H) which is two carbons longer than which has to be stabilized by association with the starting material. This can feed back into the groups on the enzyme surface until chain assem- system, condensing again with malonyl-ACP, and bly is complete and cyclization reactions occur. going through successive reduction, dehydration, However, for fatty acids, the carbonyl groups are and reduction steps, gradually increasing the chain reduced before attachment of the next malonate length by two carbons for each cycle, until the group. Partial reduction processes, leading to a required chain length is obtained. At that point, mixture of methylenes, hydroxyls, and carbonyls, the fatty acyl chain can be released as a fatty are characteristic of macrolides (see page 92). acyl-CoA or as the free acid. The chain length actually elaborated is probably controlled by the specificity of the thioesterase enzymes that subse- SATURATED FATTY ACIDS quently catalyse release from the enzyme. The fatty acid synthase protein is known to con- The processes of fatty acid biosynthesis are tain an acyl carrier protein binding site, and also well studied and are known to be catalysed an active site cysteine residue in the β-ketoacyl by the enzyme fatty acid synthase.Inani- synthase domain. Acetyl and malonyl groups are mals, this is a multifunctional protein contain- successively transferred from coenzyme A esters ing all of the catalytic activities required, whilst and attached to the thiol groups of Cys and ACP bacteria and plants utilize an assembly of separa- (Figure 3.3). The Claisen condensation occurs, and ble enzymes. Acetyl-CoA and malonyl-CoA them- the processes of reduction, dehydration, and reduc- selves are not involved in the condensation step: tion then occur whilst the growing chain is attached they are converted into enzyme-bound thioesters, to ACP. The ACP carries a phosphopantatheine the malonyl ester by means of an acyl carrier group exactly analogous to that in coenzyme A, protein (ACP) (Figure 3.2). The Claisen reac- and this provides a long flexible arm, enabling tion follows giving acetoacetyl-ACP (β-keto acyl- the growing fatty acid chain to reach the active ACP; R=H), which is reduced stereospecifically site of each enzyme in the complex, allowing to the corresponding β-hydroxy ester, consuming the different chemical reactions to be performed NADPH in the reaction. Then follows elimina- without releasing intermediates from the enzyme tion of water giving the E (trans) α,β-unsaturated (compare polyketide synthesis page 62 and pep- ester. Reduction of the double bond again utilizes tide synthesis, page 421). Then the chain is trans- NADPH and generates a saturated acyl-ACP (fatty ferred to the thiol of Cys, and the process can SATURATED FATTY ACIDS 37

acyl carrier CH3CO SCoA protein (ACP) acetyl-CoA CO2H CO2H

CH2CO SCoA CH2CO S ACP malonyl-CoA malonyl-ACP

Claisen RCH2CO S Enz reaction acyl-enzyme thioester HOH NADPH

RCH2 CH2CO S ACP RCH2COCH2CO S ACP R each turn of the cycle β β-keto acyl-ACP -hydroxy acyl-ACP stereospecific extends the chain length of reduction of carbonyl E2 elimination – H2O the acyl group by two of H2O carbons E NADPH CO S ACP RCH2CH2CH2CO S ACP RCH2 reduction of fatty acyl-ACP α,β-unsaturated acyl-ACP double bond

HSCoA H2O

RCH2CH2CH2CO SCoA RCH2CH2CH2CO2H fatty acyl-CoA fatty acid

Figure 3.2

O OO SH malonyl-CoA acetyl-CoA S CH3 H SH SO Cys Cys ACP (acetyl/malonyl transacylases) ACP fatty acid synthase Claisen reaction O (β-ketoacyl synthase) O OO S reduction SH dehydration SH SH transfer S reduction S Cys Cys Cys ACP ACP (β-ketoacyl reductase; ACP β-hydroxyacyl dehydratase; enoyl reductase)

OO O R S growing fatty N N O POCH2 Ser ACP acyl chain H H O OH OH pantotheine bound to serine cysteamine pantothenic acid group of ACP through phosphate phosphopantotheine

Figure 3.3 continue. Making the process even more efficient, not correspond with the order in which they are animal fatty acid synthase is a dimeric protein employed. containing two catalytic centres and is able to Thus, combination of one acetate starter unit generate two growing chains. The monomeric with seven malonates would give the C16 fatty subunits are also arranged head to tail so that acid, palmitic acid, and with eight malonates the the acyl group of one unit actually picks up a C18 fatty acid, stearic acid. Note that the two malonyl extender from the other unit (Figure 3.4). carbons at the head of the chain (methyl end) Note that the sequence of enzyme activities along are provided by acetate, not malonate, whilst the the protein chain of the enzyme complex does remainder are derived from malonate, which itself 38 THE ACETATE PATHWAY

dotted lines indicate two sites for fatty KS MAT DH ER KR ACP TE subunit-1 acid synthesis, utilizing enzyme activities from both subunits SH SH ACP: acyl carrier protein SH SH DH: dehydratase ER: enoylreductase subunit-2 TE ACP KR ER DH MAT KS KR: β-ketoacylreductase KS: β-ketoacylsynthase dimeric fatty acid synthase MAT: malonyl/acetyltransferase TE: thioesterase

Figure 3.4

Table 3.1 Common naturally occurring fatty acids Saturated butyric CH3(CH2)2CO2H 4:0 stearic CH3(CH2)16CO2H 18:0 ∗ caproic CH3(CH2)4CO2H 6:0 arachidic CH3(CH2)18CO2H 20:0 ∗ caprylic CH3(CH2)6CO2H 8:0 behenic CH3(CH2)20CO2H 22:0 ∗ capric CH3(CH2)8CO2H 10:0 lignoceric CH3(CH2)22CO2H 24:0 lauric CH3(CH2)10CO2H 12:0 cerotic CH3(CH2)24CO2H 26:0 myristic CH3(CH2)12CO2H 14:0 montanic CH3(CH2)26CO2H 28:0 palmitic CH3(CH2)14CO2H 16:0 melissic CH3(CH2)28CO2H 30:0 ∗To avoid confusion, systematic nomenclature (hexanoic, octanoic, decanoic) is recommended Unsaturated = palmitoleic CH3(CH2)5CH CH(CH2)7CO2H 16:1 (9c) = oleic CH3(CH2)7CH CH(CH2)7CO2H 18:1 (9c) = cis-vaccenic CH3(CH2)5CH CH(CH2)9CO2H 18:1 (11c) = = linoleic CH3(CH2)4CH CHCH2CH CH(CH2)7CO2H 18:2 (9c,12c) = = = α-linolenic CH3CH2CH CHCH2CH CHCH2CH CH(CH2)7CO2H 18:3 (9c,12c,15c) = = = γ-linolenic CH3(CH2)4CH CHCH2CH CHCH2CH CH(CH2)4CO2H 18:3 (6c,9c,12c) = gadoleic CH3(CH2)9CH CH(CH2)7CO2H 20:1 (9c) = = = = arachidonic CH3(CH2)4CH CHCH2CH CHCH2CH CHCH2CH CH(CH2)3CO2H 20:4 (5c,8c,11c,14c) = = = = = eicosapentaenoic CH3CH2CH CHCH2CH CHCH2CH CHCH2CH CHCH2CH CH(CH2)3CO2H (EPA) 20:5 (5c,8c,11c,14c,17c) = erucic CH3(CH2)7CH CH(CH2)11CO2H 22:1 (13c) = = = = = docosapentaenoic CH3CH2CH CHCH2CH CHCH2CH CHCH2CH CHCH2CH CH(CH2)5CO2H (DPA) 22:5 (7c,10c,13c,16c,19c) ======docosahexaenoic CH3CH2CH CHCH2CH CHCH2CH CHCH2CH CHCH2CH CHCH2CH CH(CH2)2CO2H (DHA) 22:6 (4c,7c,10c,13c,16c,19c) = nervonic CH3(CH2)7CH CH(CH2)13CO2H 24:1 (15c) all double bonds are Z (cis) Number of carbon atoms

Position of double bonds

Abbreviations: 18:2 (9c,12c) Stereochemistry of double bonds (c = cis/Z; t = trans/E) Number of double bonds is produced by carboxylation of acetate. This of carbon atoms. Natural fatty acids may contain means that all carbons in the fatty acid originate from four to 30, or even more, carbon atoms, the from acetate, but malonate will only provide the C2 most abundant being those with 16 or 18 carbons. chain extension units and not the C2 starter group. Some naturally occurring fatty acids are shown The linear combination of acetate C2 units as in in Table 3.1. The rarer fatty acids containing an Figure 3.2 explains why the common fatty acids odd number of carbon atoms typically originate are straight chained and possess an even number from incorporation of a different starter unit, e.g. SATURATED FATTY ACIDS 39 propionic acid, or can arise by loss of one carbon acyl-CoA residues, the phosphate being removed from an even-numbered acid. prior to the last esterification (Figure 3.5). The di- Fatty acids are mainly found in ester combi- acyl ester of glycerol 3-phosphate is also known nation with glycerol in the form of triglycerides as a phosphatidic acid, and is the basis of phos- (Figure 3.5). These materials are called fats or pholipid structures. In these structures, the phos- oils, depending on whether they are solid or liquid phate is also esterified with an alcohol, which at room temperature. If all three esterifying acids is usually choline, ethanolamine, serine, or myo- are the same, the triglyceride is termed simple, inositol, e.g. phosphatidyl choline (Figure 3.6). whereas a mixed triglyceride is produced if two or Phospholipids are important structural compo- more of the fatty acids are different. Most natural nents of cell membranes, and because of the polar fats and oils are composed largely of mixed triglyc- and non-polar regions in their structure, they have erides. In this case, isomers can exist, including detergent-like properties. They are also able to potential optical isomers, since if the primary alco- form liposomes, which have considerable potential hols are esterified with different fatty acids the as drug delivery systems. A particularly important central carbon of glycerol will become chiral. In natural phospholipid is platelet-activating factor practice, only one of each pair of enantiomers is (PAF) (Figure 3.6), which resembles a phos- formed in nature. Triglycerides are produced from phatidylcholine, though this compound possesses glycerol 3-phosphate by esterification with fatty an ether linkage to a long chain fatty alcohol,

R1CO SCoA R2CO SCoA OH OCOR1 OCOR1 HO HO R2COO OP esterification OP esterification OP glycerol 3-Pwith first 1-acylglycerol 3-Pwith second 1,2-diacylglycerol 3-P fatty acyl-CoA fatty acyl-CoA (phosphatidic acid)

H2O hydrolysis of phosphate R3CO SCoA OCOR1 OCOR1 R2COO R2COO 3 OCOR esterification OH triacylglycerol with third 1,2-diacylglycerol (triglyceride) fatty acyl-CoA

Figure 3.5

OCOR1 3 R = CH2CH2NMe3 phosphatidylcholine R2COO O O P OR3 CH2CH2NH3 phosphatidylethanolamine OH

CH2 CHCO2H phosphatidylserine

NH2 HO OH phosphatidyl-myo-inositol OH OH OH OR MeCOO O e.g. R = (CH2)15CH3 O P OCH2CH2NMe3 OH platelet-activating factor (PAF)

Figure 3.6 40 THE ACETATE PATHWAY usually hexadecanol, rather than an ester linkage. aids are listed in Table 3.2. Typical fatty acid anal- The central hydroxyl of glycerol is esterified, but yses are shown, though it must be appreciated that to acetic acid rather than to a long chain fatty these figures can vary quite widely. For instance, acid. PAF functions at nanomolar concentrations, plant oils show significant variation according to activates blood platelets and contributes to diverse the climatic conditions under which the plant was biological effects, including thrombosis, inflamma- grown. In colder climates, a higher proportion of tory reactions, allergies, and tissue rejection. Long polyunsaturated fatty acids is produced, so that chain alcohols are reduction products from fatty the plant can maintain the fluidity of its storage acids and also feature in natural waxes.These fats and membranes. The melting points of these are complex mixtures of esters of long chain fatty materials depend on the relative proportions of the acids, usually C20−C24, with long chain monohy- various fatty acids, reflecting primarily the chain dric alcohols or sterols. length and the amount of unsaturation in the chain. Saturation, and increasing chain length in the fatty UNSATURATED FATTY ACIDS acids gives a more solid fat at room temperature. Thus, butterfat and cocoa butter (theobroma oil) Animal fats contain a high proportion of glyc- contain a relatively high proportion of saturated erides of saturated fatty acids and tend to be solids, fatty acids and are solids. Palm kernel and coconut whilst those from plants and fish contain predom- oils are both semi-solids having a high concen- inantly unsaturated fatty acid esters and tend to tration of the saturated C12 acid lauric acid.A be liquids. Some of the common naturally occur- characteristic feature of olive oil is its very high ring unsaturated fatty acids are also included in oleic acid (18:1) content, whilst rapeseed oil pos- Table 3.1. A convenient shorthand representation sesses high concentrations of long chain C20 and for fatty acids indicating chain length with number, C22 fatty acids, e.g. erucic acid (22:1). Typical position and stereochemistry of double bonds is fatty acids in fish oils have high unsaturation and also presented in Table 3.1. A less systematic num- also long chain lengths, e.g. eicosapentaenoic acid bering starting from the methyl (the ω end) may (EPA) (20:5) and docosahexaenoic acid (DHA) also be encountered. Major groups of fatty acids (22:6) in cod liver oil. are designated ω-3 (omega-3), ω-6 (omega-6), ω- Unsaturated fatty acids can arise by more than 9 (omega-9), etc (or sometimes n-3, n-6, n-9), if one biosynthetic route, but in most organisms the there is a double bond that number of carbons from common mechanism is by desaturation of the cor- the methyl terminus. This has some value in relat- responding alkanoic acid, with further desatura- ing structures when an unsaturated fatty acid is tion in subsequent steps. Most eukaryotic organ- 9 biosynthetically elongated from the carboxyl end isms possess a ∆ -desaturase that introduces a cis as during prostaglandin biosynthesis (see page 45). double bond into a saturated fatty acid, requir- Double bonds at position 9 are common, but unsat- ing O2 and NADPH or NADH cofactors. The uration can occur at other positions in the chain. mechanism of desaturation does not involve any Polyunsaturated fatty acids tend to have their dou- intermediates hydroxylated at C-9 or C-10, and ble bonds in a non-conjugated array as a repeating the requirement for O2 is as an acceptor at the = unit −(CH CHCH2)n−. In virtually all cases, the end of an electron transport chain. A stearoyl stereochemistry of the double bond is Z (cis), (C18) thioester is the usual substrate giving an introducing a ‘bend’ into the alkyl chain. This oleoyl derivative (Figure 3.7), coenzyme A esters interferes with the close association and aggre- being utilized by animal and fungal enzymes, gation of molecules that is possible in saturated and ACP esters by plant systems. The position structures, and helps to maintain the fluidity in oils of further desaturation then depends very much and cellular membranes. on the organism. Non-mammalian enzymes tend Fats and oils represent long term stores of to introduce additional double bonds between energy for most organisms, being subjected to the existing double bond and the methyl termi- oxidative metabolism as required. Major oils which nus,e.g.oleate→ linoleate → α-linolenate. Ani- are produced commercially for use as foods, toi- mals always introduce new double bonds towards letries, medicinals, or pharmaceutical formulation the carboxyl group. Thus oleate is desaturated to Table 3.2 Fixedoilsandfats The term fat or oil has no precise significance, and merely describes whether the material is a solid (fat) or liquid (oil) at room temperature. Most commercial oils are obtained from plant sources, particularly seeds and fruits, and the oil is extracted by cold or hot expression, or less commonly by solvent extraction with hexane. The crude oil is then refined by filtration, steaming, neutralization to remove free acids, washing, and bleaching as appropriate. Many food oils are then partially hydrogenated to produce semi-solid fats. Animal fats and fish oils are usually extracted by steaming, the higher temperature deactivating enzymes that would otherwise begin to hydrolyse the glycerides. Oils and fats feature as important food components and cooking oils, some 80% of commercial production being used as human food, whilst animal feeds account for another 6%. Most of the remaining production is used as the basis of soaps, detergents, and pharmaceutical creams and ointments. A number of oils are used as diluents (carrier or base oils) for the volatile oils employed in aromatherapy. Oil Source Part used Oil content† Typical fatty acid Uses, notes (%) composition† (%) Almond Prunus amygdalus seed 40–55 oleic (62–86), linoleic (7–30), emollient base, toiletries, carrier oil var. dulcis,orvar. palmitic (4–9), stearic (1–2) (aromatherapy) amara (Rosaceae) Arachis Arachis hypogaea seed 45–55 oleic (35–72), linoleic (13–43), food oil, emollient base (groundnut, (Leguminosae/ palmitic (7–16), stearic (1–7), peanut) Fabaceae) behenic (1–5), arachidic (1–3) Borage Borago officinalis seed 28–35 linoleic (38), γ-linolenic (23–26), dietary supplement for γ-linolenic (Boraginaceae) oleic (16), palmitic (11) acid content (see page 46) Butterfat cow milk 2–5 palmitic (29), oleic (28), stearic (13), food Bos taurus myristic (12), butyric (4), lauric (3), (Bovidae) caproic (2), capric (2), palmitoleic (2) Castor Ricinus communis seed 35–55 ricinoleic (80–90), oleic (4–9), emollient base, purgative, soap (Euphorbiaceae) linoleic (2–7), palmitic (2–3), manufacture stearic (2–3) Castor seeds contain the highly toxic, but heat-labile protein ricin (see page 434) (Continued overleaf ) Table 3.2 (Continued) Oil Source Part used Oil content† Typical fatty acid Uses, notes (%) composition† (%) Coconut Cocos nucifera seed kernel 65–68 lauric (43–53), myristic (15–21), soaps, shampoos (Palmae/ palmitic (7–11), caprylic (5–10), Fractionated coconut oil containing Arecaceae) capric (5–10), oleic (6–8), only short to medium length fatty stearic (2–4) acids (mainly caprylic and capric) is a dietary supplement Cod-liver cod fresh liver 50 oleic (24), DHA (14), palmitic (11), dietary supplement due to presence of Gadus morrhua EPA (6), palmitoleic (7), stearic (4), EPA and DHA, plus vitamins A (see (Gadidae) myristic (3) page 230) and D (see page 259); halibut-liver oil from halibut Hippoglossus vulgaris (Pleurnectideae) has similar properties andisusedinthesameway Cottonseed Gossypium seed 15–36 linoleic (33–58), palmitic (17–29), solvent for injections, soaps hirsutum oleic (13–44), stearic (1–4) Cotton seeds also contain 1.1–1.3% (Malvaceae) gossypol (see page 200) and small amounts of cyclopropenoid fatty acids, e.g. sterculic and malvalic acids (see page 50) Evening primrose Oenothera biennis seed 24 linoleic (65–80), γ-linolenic (7–14), dietary supplement for γ-linolenic (Onagraceae) oleic (9), palmitic (7) acid content (see page 46) Honesty Lunaria annua seed 30–40 erucic (43), nervonic (25), oleic (24) nervonic acid is being investigated (Cruciferae/ for the treatment of multiple Brassicaceae) sclerosis; the disease is characterized by a deficiency in nervonic acid Lard pig abdominal oleic (45), palmitic (25), stearic (12), foods Sus scrofa fat linoleic (10), palmitoleic (3) (Suidae) Linseed (flaxseed) Linum seed 35–44 α-linolenic (30–60), oleic (39), liniments, dietary supplement for usitatissimum linoleic (15), palmitic (7), stearic (4) α-linolenic acid content (Linaceae) Formerly the basis of paints, reacting with oxygen, polymerizing, anddryingtoahardfilm Maize (corn) Zea mays embryo 33–39 linoleic (34–62), oleic (19–50), food oil, dietary supplement, solvent (Graminae/ palmitic (8–19), stearic (0–4) for injections Poaceae) Olive Olea europaea fruits 15–40 oleic (56–85), palmitic (8–20), food oil, emollient base (Oleaceae) linoleic (4–20), stearic (1–4) Palm kernel Elaeis guineensis kernel 45–50 lauric (40–52), myristic (14–18), soaps (Palmae/ oleic (9–16), palmitoleic (6–10), Fractionated palm oil is a solid Arecaceae) caprylic (3–6), capric (3–5), obtained by fractionation and stearic (1–4), linoleic (1–3) hydrogenation and is used as a suppository base Rapeseed Brassica napus seed 40–50 erucic (30–60), oleic (9–25), linoleic food oil, using varieties producing (Cruciferae/ (11–25), gadoleic (5–15), α-linolenic lower levels of erucic acid where the Brassicaceae) (5–12), palmitic (0–5) main components are now oleic (48–60%), linoleic (18–30%), α-linolenic (6–14%), and palmitic (3–6%) acids Erucic acid is used as a plasticizer in PVC clingfilm (Continued overleaf ) Table 3.2 (Continued) Oil Source Part used Oil content† Typical fatty acid Uses, notes (%) composition† (%) Sesame Sesamum indicum seed 44–54 oleic (35–50), linoleic (35–50), food oil, soaps, solvent for injections, (Pedaliaceae) palmitic (7–12), stearic (4–6) carrier oil (aromatherapy) Soya (soybean) Glycine max seed 18–20 linoleic (44–62%), oleic (19–30), food oil, dietary supplement, carrier (Leguminosae/ palmitic (7–14), α-linolenic (4–11), oil (aromatherapy) Fabaceae) stearic (1–5) Soya oil contains substantial amounts of the sterols sitosterol and stigmasterol (see page 256) Suet (mutton sheep abdominal fat stearic (32), oleic (31), palmitic (27), foods tallow) Ovis aries myristic (6) (Bovidae) Suet (beef tallow) cow abdominal fat oleic (48), palmitic (27), palmitoleic foods Bos taurus (11), stearic (7), myristic (3) (Bovidae) Sunflower Helianthus annuus seed 22–36 linoleic (50–70), oleic (20–40), food oil, carrier oil (aromatherapy) (Compositae/ palmitic (3–10), stearic (1–10) Asteraceae) Theobroma Theobroma cacao kernel 35–50 oleic (35), stearic (35), palmitic (26), suppository base, chocolate (Sterculiaceae) linoleic (3) manufacture Theobroma oil (cocoa butter) is a solid

†The oil yields and fatty acid compositions given in the table are typical values, and can vary widely. The quality of an oil is determined principally by its fatty acid analysis. Structures of the fatty acids are shown in Table 3.1 (see page 38). UNSATURATED FATTY ACIDS 45

CO SR R = SCoA in animals/fungi R = ACP in plants

stearic 18:0 desaturation towards methyl terminus plants plants fungi CO SR fungi CO SR CO SR

oleic linoleic α-linolenic 18:1 (9c) 18:2 (9c,12c) 18:3 (9c,12c,15c) desaturation animals towards carboxyl animals animals terminus

CO SR CO SR CO SR

18:2 (6c,9c) γ-linolenic stearidonic 18:3 (6c,9c,12c) 18:4 (6c,9c,12c,15c) chain extension by Claisen reaction with malonate; chain + C2 (malonate) + C2 (malonate) length increased by two carbons CO SR CO SR

prostaglandins dihomo-γ-linolenic eicosatetraenoic 1-series 20:3 (8c,11c,14c) 20:4 (8c,11c,14c,17c)

CO SR CO SR

prostaglandins arachidonic eicosapentaenoic (EPA) 2-series 20:4 (5c,8c,11c,14c) 20:5 (5c,8c,11c,14c,17c)

Note: the names given prostaglandins are for the appropriate 3-series + C2 (malonate) fatty acid; the structures shown are actually the CO SR thioesters involved in CO SR the conversions

docosahexaenoic (DHA) docosapentaenoic (DPA) 22:6 (4c,7c,10c,13c,16c,19c) 22:5 (7c,10c,13c,16c,19c)

Figure 3.7

∆6,9-octadecadienoate rather than linoleate. How- prostaglandins in the ‘one’ and ‘two’ series ever, animals need linoleate for the biosynthesis respectively (see page 52). Accordingly, linoleic of dihomo-γ-linolenate (∆8,11,14-eicosatrienoate) acid must be obtained from plant material in the and arachidonate (∆5,8,11,14-eicosatetraenoate), diet, and it is desaturated towards the carboxyl C20 polyunsaturated fatty acid precursors of to yield γ-linolenate, which is then used as the 46 THE ACETATE PATHWAY

substrate for further chain extension, adding a oils present in fish are rich in the later metabolites C2 unit from malonate, and producing dihomo- derived from α-linolenic acid, e.g. EPA and DHA, γ-linolenate. Arachidonate derives from this by and are also beneficial to health. Since these fatty additional desaturation, again towards the carboxyl acids all have a double bond three carbons from end of the chain (Figure 3.7). α-Linolenate is the methyl end of the chain, they are grouped similarly a precursor on the way to ∆5,8,11,14,17- together under the term ω-3 fatty acids (omega- eicosapentaenoate (EPA), required for the syn- 3 fatty acids). Regular consumption of fish oils thesis of prostaglandins of the ‘three’ series, and is claimed to reduce the risk of heart attacks and it is also obtained from the diet. A similar chain atherosclerosis. extension process using further molecules of mal- Although most plant-derived oils contain high onate is encountered in the sequence from α- amounts of unsaturated fatty acid glycerides, linolenate in animal systems (Figure 3.7). Chain including those of linoleic and α-linolenic acids, extension/dehydrogenations lead to formation of the conversion of linoleate into γ-linolenate can eicosapentaenoate (EPA) with further elaborations be blocked or inhibited in certain conditions in producing docosapentaenoate (DPA)andthen humans. This restricts synthesis of prostaglandins. docosahexaenoate (DHA). DHA is a component In such cases, the use of food supplements, of lipids in sperm, the retina, and the brain. It is e.g. evening primrose oil∗ from Oenothera bien- thought to be important for brain development, and nis (Onagraceae), which are rich in γ-linolenic deficiency is associated with abnormalities in brain esters, can be valuable and help in the disorder. function. Linoleate and α-linolenate are referred to Many plants in the Boraginaceae, e.g. borage as ‘essential fatty acids’ (EFAs) since they and (Borago afficinalis), also accumulate significant their metabolites are required in the diet for nor- amounts of γ-linolenic acid glycerides, as does mal good health. Some food sources such as the evening primrose, indicating their unusual ability

Evening Primrose Oil

Evening primrose oil is extracted from the seeds of selected strains of the evening primrose (Oenothera biennis; Onagraceae), a biennial plant native to North America, which is now widely cultivated in temperate countries. The seeds contain about 24% fixed oil, which has a high content of glycerides of the unsaturated fatty acids linoleic acid (65–80%) and γ-linolenic acid (gamolenic acid) (7–14%). Because of this high γ-linolenic acid content, evening primrose oil is widely used as a dietary supplement, providing additional quantities of this essential fatty acid, which is a precursor in the biosynthesis of prostaglandins, which regulate many bodily functions (see page 54). Genetic and a number of other factors may inhibit the desaturation of linoleic acid into γ-linolenic acid. Ageing, diabetes, excessive alcohol intake, catecholamines, and zinc deficiency have all been linked to inhibition of the desaturase enzyme. The conversion may also be inhibited if there is a high proportion of fatty acids in the diet, which compete for the desaturase enzyme, including saturated and trans-unsaturated fatty acids. The latter group may be formed during the partial hydrogenation of polyunsaturated fatty acids which is commonly practised during food oil processing to produce semi-solid fats. Evening primrose oil appears to be valuable in the treatment of premenstrual tension, multiple sclerosis, breast pain (mastalgia), and perhaps also in eczema. There is potential for further applications, e.g. in diabetes, alcoholism, and cardiovascular disease. In evening primrose, γ-linolenic acid is usually present in the form of a dilinoleoylmono-γ-linolenylglycerol. This triglyceride is also being explored as a drug material for the treatment of diabetes-related neuropathy and retinopathy. γ-Linolenic acid is also found in the fixed oil of other plants, e.g. blackcurrant, comfrey, and borage, and in human milk. Borage oil (starflower oil) from the seeds of Borago officinalis (Boraginaceae) is used in the same way as evening primrose oil. It contains higher concentrations of γ-linolenic acid (23–26%), but rather less linoleic acid. ACETYLENIC FATTY ACIDS 47

CO2H oleic acid

O2 / NADPH pyrolysis

CO2H CO2H OH undecenoic acid ricinoleic acid

Figure 3.8 to desaturate linoleic esters towards the carboxyl Basidiomycetes. These compounds tend to be terminus, rather than towards the methyl terminus highly unstable and some are even explosive if as is more common in plants. Arachidonic acid sufficient amounts are accumulated. Since only itself has not been found in higher plants, but does very small amounts are present in plants, this occur in some algae, mosses, and ferns. does not present any widespread hazard. Whilst Ricinoleic acid (Figure 3.8) is the major fatty fatty acids containing several double bonds usually acid found in castor oil from seeds of the cas- have these in a non-conjugated array, molecules tor oil plant (Ricinus communis; Euphorbiaceae), containing triple bonds tend to possess conjugated and is the 12-hydroxy derivative of oleic acid. unsaturation. This gives the compounds intense It is formed by direct hydroxylation of oleic and highly characteristic UV spectra which aids acid (usually esterified as part of a phospholipid) their detection and isolation. by the action of an O2- and NADPH-dependent The processes of desaturation are exemplified mixed function oxidase, but this is not of the in Figure 3.9, in which oleic acid (probably as a cytochrome P-450 type. Castor oil has a long his- thiol ester) features as a precursor of crepenynic tory of use as a domestic purgative, but it is now acid and dehydrocrepenynic acid. The acetylenic mainly employed as a cream base. Undecenoic bond is now indicated by a in the semi-systematic acid (∆9-undecenoic acid) can be obtained from shorthand nomenclature. Chain shortening by β- ricinoleic acid by thermal degradation, and as the oxidation (see page 18) is often a feature of these zinc salt or in ester form is used in fungistatic pathways, and formation of the C10 acetylenic preparations. acid dehydromatricaria acid proceeds through Primary amides of unsaturated fatty acids have C18 intermediates, losing eight carbons, presum- been characterized in humans and other mam- ably via four β-oxidations. In the latter part mals, and although their biological role is not fully of the pathway, the Z -double bond from oleic understood, they may represent a group of impor- acid moves into conjugation with the polyacety- tant signalling molecules. Oleamide,thesimple lene chain via an allylic isomerization, giving amide of oleic acid, has been shown to be a sleep- the more favoured E-configuration. Some note- inducing lipid, and the amide of erucic acid, eru- worthy acetylenic structures (though they are no camide, stimulates the growth of blood vessels. longer acids and components of fats) are given in Figure 3.10. Cicutoxin from the water hem- lock (Cicuta virosa; Umbelliferae/Apiaceae) and ACETYLENIC FATTY ACIDS oenanthotoxin from the hemlock water drop- wort (Oenanthe crocata; Umbelliferae/Apiaceae) Many unsaturated compounds found in nature are extremely toxic to mammals, causing persis- contain one or more acetylenic bonds, and these are tent vomiting and convulsions, leading to respi- predominantly produced by further desaturation of ratory paralysis. Ingestion of the roots of these olefinic systems in fatty acid-derived molecules. plants may frequently lead to fatal poisoning. They are surprisingly widespread in nature, and Falcarinol is a constituent of Falcaria vulgaris are found in many organisms, but are especially (Umbelliferae/Apiaceae), Oenanthe crocata, Hed- common in plants of the Compositae/Asteraceae, era helix (Araliaceae), and several other plants, the Umbelliferae/Apiaceae, and fungi of the group and is known to cause contact dermatitis in certain 48 THE ACETATE PATHWAY

CO2H

oleic acid 18:1 (9c)

CO2H

linoleic acid 18:2 (9c,12c)

CO2H

crepenynic acid 18:2 (9c,12a)

CO2H

dehydrocrepenynic acid 18:3 (9c,12a,14c)

CO2H

18:3 (9c,12a,14a)

CO2H

18:4 (9c,12a,14a,16a) H

β-oxidations

H allylic isomerization CO2H

dehydromatricaria acid 10:4 (2t,4a,6a,8a)

Figure 3.9 individuals when the plants are handled. Falcarinol treatment for the common cold. At least some of its (sometimes called panaxynol) and the structurally activity arises from a series of alkylamides, amides related panaxytriol are also characteristic poly- of polyunsaturated acids with isobutylamine. These acetylene components of ginseng (Panax ginseng; acids are predominantly C11 and C12 diene-diynes Araliaceae) (see page 222). Wyerone from the (Figure 3.11). broad bean (Vicia faba; Leguminosae/Fabaceae) has antifungal properties, and its structure exempli- fies how the original straight chain may be cross- BRANCHED-CHAIN FATTY ACIDS linked to produce a ring system. The furan ring may originate from a conjugated diyne. Whilst straight-chain fatty acids are the most com- The herbal preparation echinacea∗ is derived mon, branched-chain acids have been found to from the roots of Echinacea purpurea (Compositae/ occur in mammalian systems, e.g. in wool fat and Asteraceae) and is used for its immunostimu- butter fat. They are also characteristic fatty acid lant properties, particularly as a prophylactic and constituents of the lipid part of cell walls in some BRANCHED-CHAIN FATTY ACIDS 49

OH pathogenic bacteria. Several mechanisms appear to OH operate in their formation. Thus, the structure of corynomycolic acid from Corynebacterium diph- cicutoxin theriae can be rationalized from a combination of two palmitic acid units (Figure 3.12). Methyl side- OH OH chains can be introduced by using methylmalonyl- CoA instead of malonyl-CoA as the chain extending agent (Figure 3.13). Methylmalonyl- oenanthotoxin CoA arises by biotin-dependent carboxylation of propionyl-CoA in exactly the same way as malonyl-CoA was formed (see page 17). OH falcarinol (panaxynol) 2,4,6,8-Tetramethyldecanoic acid found in the preen gland wax of the goose (Anser anser) OH is produced from an acetyl-CoA starter, and four methylmalonyl-CoA chain extender units. The incorporation of propionate as well as OH OH acetate is also a feature of many microbial panaxytriol antibiotic structures (see page 17). However, in other examples, methyl side-chains can be produced by a C-alkylation mechanism using O CO2Me S-adenosylmethionine (SAM). Tuberculostearic O acid (Figure 3.14) found in Mycobacterium tuber- wyerone culosis, the bacterium causing tuberculosis, is Figure 3.10 derived from oleic acid by alkylation on C-10,

Echinacea Echinacea consists of the dried roots of Echinacea purpurea, E. angustifolia,orE. pallida (Compositae/Asteraceae), herbaceous perennial plants indigenous to North America, and widely cultivated for their large daisy-like flowers, which are usually purple or pink. Herbal preparations containing the dried root, or extracts derived from it, are promoted as immunostimulants, particularly as prophylactics and treatments for bacterial and viral infections, e.g. the common cold. Tests have validated stimulation of the immune response, though the origins of this activity cannot be ascribed to any specific substance. Activity has variously been assigned to lipophilic alkylamides, polar caffeic acid derivatives, high molecular weight polysaccharide material, or to a combination of these. Compounds in each group have been demonstrated to possess some pertinent activity, e.g. immunostimulatory, anti-inflammatory, antibacterial or antiviral effects. The alkylamides comprise a complex mixture of unsaturated fatty acids as amides with 2-methylpropanamine (isobutylamine) or 2-methylbutanamine, amines which are probably decarboxylation products from valine and isoleucine respectively. The acid portions are predominantly C11 and C12 diene-diynes or tetraenes (Figure 3.11). These compounds are found throughout the plant though relative proportions of individual components vary considerably. The root of E. purpurea contains at least 12 alkylamides (about 0.6%), of which C12 diene-diynes predominate; levels of these compounds fall significantly during drying and storage. Caffeic acid derivatives present include caffeic acid (see page 132), chlorogenic acid (5-O-caffeoylquinic acid, see page 132), 2-O- caffeoyltartaric acid, and cichoric acid (2,3-di-O-caffeoyltartaric acid) (Figure 3.11). Cichoric acid is a major component (0.6–2.1%) in E. purpurea, but only minor in the other species.

(Continues) 50 THE ACETATE PATHWAY

(Continued)

O O E Z E N R N H O R = H or Me H Z O N E Z H R N tetraene alkylamides H R = H or Me OH O O CO2H Z E HO O N O OH H HO2C O diene-diyne alkylamides HO cichoric acid

Figure 3.11

O OH

SCoA

SCoA CO2H corynomycolic acid O

Figure 3.12

CO2H CO2, ATP SCoA biotin SCoA

O O propionyl-CoA methylmalonyl-CoA

SCoA CO2H

O 2,4,6,8-tetramethyldecanoic acid

Figure 3.13 initiated by the double bond electrons. A postulated (Malvaceae). Malvalic acid is produced from ster- carbocation intermediate could then be discharged culic acid by chain shortening from the carboxyl by accepting hydride from NADPH giving tuber- end (Figure 3.14). Sterculic acid is an inhibitor of culostearic acid. Alternatively, loss of a proton the ∆9-desaturase which converts stearic acid into via cyclopropane ring formation could occur giv- oleic acid and is potentially harmful to humans in ing dihydrosterculic acid. This is known to be that it can alter membrane permeability and inhibit dehydrogenated to sterculic acid, an unusual fatty reproduction. acid containing a highly strained cyclopropene Chaulmoogric and hydnocarpic acids (Figure ring. Sterculic acid is present in the seed oil from 3.15) are cyclopentenyl fatty acids found in chaul- Sterculia foetida (Sterculiaceae) and with similar moogra oil expressed from seeds of Hydnocar- cyclopropene acids, e.g. malvalic acid, is present pus wightiana (Flacourtiaceae). These acids are in edible cottonseed oil from Gossypium species known to arise by malonate chain extension of the PROSTAGLANDINS 51

Ad SAM electrophilic addition: H3CS SAM as alkylating agent R O

SCoA oleoyl-CoA

cation discharged by formation of cyclopropane ring H and loss of proton H2C O CO2H SCoA carbocation intermediate dihydrosterculic acid

cation discharged by NADPH addition of hydride desaturation from NADPH

CO2H CO2H tuberculostearic acid sterculic acid

α-oxidation

CO2H malvalic acid

Figure 3.14

CO2H malonyl-CoA COSCoA hydnocarpic acid

2-cyclopentenyl- CO2H carboxyl-CoA chaulmoogric acid

Figure 3.15 coenzyme A ester of 2-cyclopentenyl carboxylic PROSTAGLANDINS acid as an alternative starter unit to acetate, ∗ demonstrating a further approach to unusual fatty The prostaglandins are a group of modified C20 acids. Chaulmoogra oil provided for many years fatty acids first isolated from human semen and ini- the only treatment for the relief of leprosy, these tially assumed to be secreted by the prostate gland. two acids being strongly bactericidal towards They are now known to occur widely in animal tis- the leprosy infective agent Mycobacterium lep- sues, but only in tiny amounts, and they have been rae.Purified salts and esters of hydnocarpic and found to exert a wide variety of pharmacological chaulmoogric acids were subsequently employed, effects on humans and animals. They are active until they were then themselves replaced by more at very low, hormone-like concentrations and can effective synthetic agents. regulate blood pressure, contractions of smooth 52 THE ACETATE PATHWAY muscle, gastric secretion, and platelet aggregation. acyclic peroxide group in PGG2 is then cleaved by Their potential for drug use is extremely high, but a peroxidase to yield prostaglandin H2 (PGH2), it has proved difficult to separate the various bio- which occupies a central role and can be modi- logical activities into individual agents. fied in several different ways. These modifications The basic prostaglandin skeleton is that of a can be rationally accommodated by initial cleavage cyclized C20 fatty acid containing a cyclopen- of the cyclic peroxide to the diradical; alternative tane ring, a C7 side-chain with the car- ionic mechanisms may also be proposed. Quench- boxyl function, and a C8 side-chain with the ing of the free radicals by abstraction of hydrogen methyl terminus. Prostaglandins are biosynthe- atoms gives rise to prostaglandin F2α (PGF2α), sized from three essential fatty acids, ∆8,11,14- whilst capture and loss of hydrogen atoms eicosatrienoic acid (dihomo-γ-linolenic acid), would provide either prostaglandin E2 (PGE2) 5,8,11,14 ∆ -eicosatetraenoic acid (arachidonic or prostaglandin D2 (PGD2). The bicyclic sys- 5,8,11,14,17 acid), and ∆ -eicosapentaenoic acid, tem in prostaglandin I2 (PGI2; prostacyclin)is which yield prostaglandins of the 1-, 2-, and 3- envisaged as arising by involvement of a side- series, respectively (Figure 3.16) (see below for chain double bond, then loss of a hydrogen principles of nomenclature). The three precur- atom. Prostaglandin structures representative of the sors lead to products of similar structure, but 1-series, e.g. PGE1, or of the 3-series, e.g. PGE3, with varying levels of unsaturation in the two can be formed in a similar way from the appropri- side-chains. Some of the structures elaborated ate fatty acid precursor (Figure 3.16). from arachidonic acid are shown in Figure 3.17. The basic skeleton of the prostaglandins is In the first reaction, arachidonic acid is con- termed prostanoic acid, and derivatives of this verted into prostaglandin G2 (PGG2) by an oxy- system are collectively known as prostanoids. genase (cyclooxygenase; COX) enzyme, which The term eicosanoids is also used to encompass incorporates two molecules of oxygen, liberating prostaglandins, thromboxanes, and leukotrienes, a compound with both cyclic and acyclic perox- which are all derived from C20 fatty acids ide functions. In arachidonic acid the methylene (eicosanoic acids). Semi-systematic nomenclature group flanked by two double bonds is susceptible of prostaglandins is based on the substitution to oxidation, probably via a free radical process. pattern in the five-membered ring, denoted by This may lead to incorporation of oxygen giving a letter suffix (Figure 3.18), and the number of the proposed free radical intermediate. Formation double bonds in the side-chains is given by a of PGG2 is then depicted as a concerted cyclization numerical subscript. Greek letters α and β are reaction, initiated by the peroxide radical, in which used to indicate the configuration at C-9, α a second oxygen molecule is incorporated. The indicating the substituent is below the plane (as

8 8 5 8 5 CO2H CO2H CO2H

11 14 11 14 11 14 17 dihomo-γ-linolenic (∆8,11,14) arachidonic (∆5,8,11,14) eicosapentaenoic (∆5,8,11,14,17)

O O O CO2H CO2H CO2H

HO OH HO OH HO OH

PGE1 PGE2 PGE3

Figure 3.16 PROSTAGLANDINS 53

concerted formation of cyclic peroxide, cyclopentane methylene flanked by double bonds is ring, and acyclic peroxide by addition of second susceptible to free radical oxidation; molecule of O2; mechanistically, this is analogous to the free radical reaction allows addition of first step but exploits the unsaturation; the peroxide O2 and formation of peroxide radical radical finally abstracts a H atom cyclooxygenase CO2H CO2H (COX) H O

O O O arachidonic acid OO

cleavage of acyclic peroxide

O peroxidase O CO2H CO2H

O O OH cyclic OOH peroxide acyclic peroxide PGH2 PGG2 radical cleavage of cyclic peroxide O

CO2H

O CO2H – H , + H HO OH

PGE2 O OH HO + 2H CO2H HO

CO2H O OH

PGD2

HO OH

PGF2α

CO2H CO2H

O H O O CO2H

O HO H OH HO OH OH

PGI2

Figure 3.17

found in natural prostaglandins), and β indicating abbreviated to PG. Prostaglandins A, B, and C the substituent is above the plane (as in some are inactive degradation products from the natural synthetic analogues). ‘Prostaglandin’ is usually prostaglandins. 54 THE ACETATE PATHWAY

R1 R2 1 9 7 5 3 CO H 8 6 4 2 CO2H 1-series 2 10 OH 12 14 16 18 20 11 13 15 17 19 2-series CO2H prostanoic acid OH 3-series CO2H OH

O O O HO O HO CO2H R1 R1 R1 R1 R1 R1

R2 R2 R2 R2 R2 R2 O HO HO PGA PGB PGC PGD PGE PGF O O R1 O R1

O O R2 R2 HO PGG OOH PGH PGI

Figure 3.18

Prostaglandins

Prostaglandins occur in nearly all mammalian tissues, but only at very low concentrations. PGE1 and PGF1α were initially isolated from sheep seminal plasma, but these compounds and PGD2,PGE2,andPGF2α are widely distributed. Animal sources cannot supply sufficient amounts for drug usage. The soft coral Plexaura homomalla (sea whip) from the Caribbean has been identified as having very high (2–3%) levels of prostaglandin esters, predominantly the C-15 epimer of PGA2 (1–2%) with related structures. Prostaglandins of the A-, E-, and F-types are widely distributed in soft corals, especially Plexaura, but these are unlikely to provide a satisfactory and renewable natural source. Considerable effort has been exerted on the total synthesis of prostaglandins and their interconversions, and the high level of success achieved has opened up the availability of compounds for pharmacological testing and subsequent drug use. Synthetic analogues have also been developed to modify or optimize biological activity. The studies have demonstrated that biological activity is effectively confined to the natural enantiomers; the unnatural enantiomer of PGE1 had only 0.1% of the activity of the natural isomer. The prostaglandins display a wide range of pharmacological activities, including contrac- tion and relaxation of smooth muscle of the uterus, the cardiovascular system, the intestinal tract, and of bronchial tissue. They may also inhibit gastric acid secretion, control blood pressure and suppress blood platelet aggregation. Some of these effects are consistent with the prostaglandins acting as second messengers, modulating transmission of hormone stimulation and thus metabolic response. Some prostaglandins in the A and J series have demonstrated potent antitumour properties. Since the prostaglandins control many important physiological processes in animal tissues, their drug potential is high, but the chances of pre- cipitating unwanted side-effects are also high, and this has so far limited their therapeutic use.

(Continues) PROSTAGLANDINS 55

(Continued)

There is, however, much additional scope for controlling the production of natural prostaglandins in body tissues by means of specific inhibitors. Indeed it has been found that some established non-steroidal anti-inflammatory drugs (NSAIDs), e.g. aspirin, indometacin, and ibuprofen, inhibit early steps in the prostaglandin biosynthetic pathway that transform the unsaturated fatty acids into cyclic peroxides. Thus aspirin is known to irreversibly inactivate the cyclooxygenase activity (arachidonic acid → PGG2), though not the peroxidase activity (PGG2 → PGH2), by selective acetylation of a serine residue of the enzyme; ibuprofen and indometacin compete with arachidonic acid at the active site and are reversible inhibitors of the cyclooxygenase. A recent discovery is that two forms of the cyclooxygenase enzyme exist, designated COX-1 and COX-2. COX-1 is expressed constitutively in most tissues and cells and is thought to control synthesis of those prostaglandins important for normal cellular functions such as gastrointestinal integrity and vascular homeostasis. COX-2 is not normally present, but is inducible in certain cells in response to inflammatory stimuli, resulting in enhanced prostaglandin release in the CNS and inflammatory cells with the characteristic inflammatory response. Current NSAIDs do not discriminate between the two COX enzymes, and so this leads to both therapeutic effects via inhibition of COX-2, and adverse effects such as gastrointestinal problems, ulcers, and bleeding via inhibition of COX-1. Because of differences in the nature of the active sites of the two enzymes, it has now been possible to develop agents that can inhibit COX-2 rather than COX-1 as potential new anti-inflammatory drugs. The first of these, meloxicam and rofecoxib, have recently been introduced for relief of pain and inflammation in osteoarthritis. The anti-inflammatory activity of corticosteroids correlates with their preventing the release of arachidonic acid from storage phospholipids, but expression of COX-2 is also inhibited by glucocorticoids. The role of essential fatty acids (see page 46) such as linoleic and γ-linolenic acids, obtained from plant ingredients in the diet, can now be readily appreciated. Without a source of arachidonic acid, or compounds which can be converted into arachidonic acid, synthesis of prostaglandins would be compromised, and this would seriously affect many normal metabolic processes. A steady supply of prostaglandin precursors is required since prostaglandins are continuously being synthesized and then deactivated. Prostaglandins are rapidly degraded by processes which include oxidation of the 15-hydroxyl to a ketone, reduction of the 13,14-double bond, and oxidative degradation of both side-chains. A major area of application of prostaglandins as drugs is in obstetrics, where they are used to induce abortions during the early to middle stages of pregnancy, or to induce labour at term. PGE2 (dinoprostone) (Figure 3.19) is used in both capacities, whilst PGF2α (dinoprost) is now less commonly prescribed and restricted to abortions. PGF2α is rapidly metabolized in body tissues (half-life less than 10 minutes), and the modified version 15-methyl PGF2α (carboprost) has been developed to reduce deactivation by blocking oxidation at position 15. Carboprost is produced by oxidizing the 15-hydroxyl in a suitably-protected PGF2α,then alkylating the 15-carbonyl with a Grignard reagent. Carboprost is effective at much reduced dosage compared with dinoprost, and is of value in augmenting labour at term, especially in cases where ergometrine (see page 375) or oxytocin (see page 415) are ineffective. Gemeprost is another unnatural structure and is used to soften and dilate the cervix in early abortions. These agents are usually administered vaginally. PGE1 (alprostadil) differs from PGE2 by having unsaturation in only one side-chain. Though having effects on uterine muscle, it also has vasodilator properties, and these are exploited for maintaining new-born infants with congenital heart defects, facilitating blood oxygenation prior to corrective surgery. The very rapid metabolism of PGE1 means this drug must be

(Continues) 56 THE ACETATE PATHWAY

(Continued)

HO HO O

CO2H CO2H CO2H 15

HO OH HO Me OH HO OH dinoprost carboprost dinoprostone

(PGF2α) (15-methyl PGF2α) (PGE2)

O O O CO2H CO2Me CO2H

RS HO OH HO OH HO HO 16 alprostadil gemeprost Me (PGE1) misoprostol

CO2H CO2H

HO

i CO2Pr

O HO HO

RS

HO OH HO OH latanoprost epoprostenol / prostacyclin iloprost (PGI2)

Figure 3.19

delivered by continuous intravenous infusion. Alprostadil is also of value in male impotence, self-injectable preparations being used to achieve erection of the penis. An interesting modifi- cation to the structure of PGE1 is found in the analogue misoprostol. This compound has had the oxygenation removed from position 15, transferred to position 16, plus alkylation at posi- tion 16 to reduce metabolism (compare 15-methyl PGF2α above). These modifications result in an orally active drug which inhibits gastric secretion effectively and can be used to promote healing of gastric and duodenal ulcers. In combination with non-specific NSAIDs, it can sig- nificantly lower the incidence of gastrointestinal side-effects such as ulceration and bleeding. PGI2 (epoprostenol, prostacyclin) reduces blood pressure and also inhibits platelet aggregation by reducing calcium concentrations. It is employed to inhibit blood clotting during renal dialysis, but its very low half-life (about 3 minutes) again necessitates continuous intravenous administration. The tetrahydrofuran ring is part of an enol ether and is readily opened by hydration, leading to 6-ketoprostaglandin F1α (Figure 3.20). Iloprost (Figure 3.19) is a stable carbocyclic analogue of potential use in the treatment of thrombotic diseases. Latanoprost (Figure 3.19) is a recently introduced prostaglandin analogue which increases the outflow of aqueous humour from the eye. It is thus used to reduce intraocular pressure in the treatment of the eye disease glaucoma.

(Continues) PROSTAGLANDINS 57

(Continued)

CO2H

hydrolysis of enol ether H HO O CO2H O O H O HO H

HO OH HO HO HO OH

PGI2 6-keto PGF1α

Figure 3.20

Isoprostanes

Isoprostanes represent a new class of prostaglandin-like compounds produced in vivo in humans and animals by non-enzymic free-radical-mediated oxidation of membrane-bound polyunsaturated fatty acids. An isomer of PGF2α in which the two alkyl substituents on the five-membered ring were arranged cis rather than trans was detected in human urine and was the first of these compounds to be characterized. This compound was initially termed 8-iso-PGF2α,or8-epi-PGF2α, though as many more variants in the isoprostane series were discovered it is now termed iPF2α-III (Figure 3.21). The last figure refers to the compound being of type III, with eight types being differentiated by the nature of the non-carboxylic chain. Compounds may be formed from linolenic acid and γ-linolenic acid, as well as from arachidonic, eicosapentaenoic, and dihomo-γ-linolenic acids. Structural characteristics of the four classes of isoprostanes derived from arachidonic acid are shown in Figure 3.22; the letter code as in prostaglandin nomenclature is used to define the ring substitution pattern. The four types of isoprostane shown in Figure 3.22 can be viewed as arising by a free radical mechanism which resembles the enzyme-mediated formation of prostaglandins shown in Figure 3.17. The varying side-chain substituents arise by utilizing different double bonds from the several available in the cyclization mechanism, and incorporating an oxygen atom from molecular oxygen at different positions. Many variants are produced because chemical processes rather than enzyme-controlled processes are employed. Free-radical-derived isomers of leukotrienes and thromboxanes have also been reported.

HO HO HO 8 CO2H CO2H CO2H

12 HO OH HO OH HO OH α PGF2 iPF2α-III 8,12-iso-iPF2α-III (8-iso-PGF2α; 9-epi-PGF2α)

Figure 3.21

(Continues) 58 THE ACETATE PATHWAY

(Continued)

O O CO2H CO2H H H O O O arachidonic acid CO2H arachidonic acid CO2H O H H

O arachidonic acid arachidonic acid CO2H O CO2H O O

OO OO O O O O OO CO2H OO CO2H O O

O O CO2H CO2H

O O OH O O OH OOH O OH O CO2H O CO2H

O O HO HO CO2H CO2H

OH OH HO OH HO HO CO2H HO OH CO2H Type III Type V

HO Type IV HO Type VI

Figure 3.22

Interest in these isoprostanoid derivatives stems partly from the finding that certain compounds possess biological activity, probably via interaction with receptors for prostaglandins. For example, iPF2α-III is a potent vasoconstrictor and also aggregates platelets, whilst 8,12-iso-iPF2α-III (Figure 3.21) possesses activity similar to PGF2α. Another potential application relates to their origin via free radical peroxidation of unsaturated fatty acids. Free radicals are implicated in inflammatory and degenerative diseases such as atherosclerosis, cancer, and Alzheimer’s disease. Isoprostane analysis of urine or serum may thus allow non-invasive monitoring of oxidative damage as a insight into these disease states.

THROMBOXANES reacts to yield the hemiacetal thromboxane B2 (TXB2). An intriguing side-branch from the prostaglandin ∗ pathway leads to thromboxanes (Figure 3.23). LEUKOTRIENES The peroxide and cyclopentane ring functions of PGH2 are cleaved and restructured to form Yet another variant for the metabolism of arachi- ∗ thromboxane A2 (TXA2), which contains a donic acid is the formation of leukotrienes ,a highly strained four-membered oxetane ring. series of fatty acid derivatives with a conjugated TXA2 is highly unstable, and reacts readily triene functionality, and first isolated from leuko- with nucleophiles. In an aqueous environment, it cytes. In a representative pathway (others have THROMBOXANES, LEUKOTRIENES 59

O O O CO2H CO2H CO2H

O O OH OH O OH PGH2 highly strained acetal ring HO system is susceptible to nucleophilic attack CO H 2 CO2H O HO O H2O O hemiacetal OH acetal OH TXB2 TXA2

Figure 3.23

Thromboxanes

The thromboxanes were isolated from blood platelets, and whilst TXA2 showed high biological activity TXB2 was only weakly active. TXA2 causes blood platelets to aggregate to form a clot or thrombus, by increasing cytoplasmic calcium concentrations and thus deforming the platelets which then fuse together. It has the opposite effect to PGI2, and presumably the development of thrombosis reflects an imbalance in the two activities. Both compounds are produced from the same precursor, PGH2, which is converted in the blood platelets to TXA2, and in the blood vessel wall to PGI2. Thromboxanes A3 and B3 have also been isolated from blood platelets, are structurally analogous to prostaglandins in the 3-series, and are 5,8,11,14,17 derived from ∆ -eicosapentaenoic acid. TXA3 is not strongly aggregatory towards blood platelets. The highly unstable nature of the biologically active thromboxanes has made their synthesis difficult, and drug use of natural structures will probably be impracticable. It is likely that most efforts will be directed towards thromboxane antagonists to help reduce blood platelet aggregation in thrombosis patients. The value of aspirin in preventing cardiovascular disease is now known to be related to inhibition of thromboxane A2 biosynthesis in platelets.

Leukotrienes The leukotrienes are involved in allergic responses and inflammatory processes. An antigen–antibody reaction can result in the release of compounds such as histamine (see page 379) or materials termed slow reacting substance of anaphylaxis (SRSA). These substances are then mediators of hypersensitive reactions such as hay fever and asthma. Structural studies have identified SRSA as a mixture of LTC4, LTD4 and LTE4. These cysteine- containing leukotrienes are powerful bronchoconstrictors and vasoconstrictors, and induce mucus secretion, the typical symptoms of asthma. LTE4 is some 10–100-fold less active than LTD4, so that degradation of the peptide side-chain represents a means of eliminating leukotriene function. LTB4 appears to facilitate migration of leukocytes in inflammation, and is implicated in the pathology of psoriasis, inflammatory bowel disease, and arthritis. The biological effects of leukotrienes are being actively researched to define the cellular processes involved. This may lead to the development of agents to control allergic and inflammatory reactions. Drugs inhibiting the formation of LTC4 and LTB4 are in clinical trials, whilst montelukast and zafirlukast have been introduced as orally active leukotriene (LTD4) receptor antagonists for the prophylaxis of asthma. 60 THE ACETATE PATHWAY

this reaction is analogous to OO that initiating prostaglandin formation (Figure 3.17) OOH cleavage of peroxide CO H 2 CO2H allows formation of H H epoxide; overall a dehydration reaction arachidonic acid hydroperoxide conjugate nucleophilic attack of H2O on to epoxide H2O O HO CO2H CO2H OH

LTB4 LTA4 nucleophilic attack of glutathione γ thiol on to epoxide ( -glutamylcysteinylglycine) OH OH CO H CO2H – Glu 2

partial hydrolysis of S S glutathione portion O H H HO C N CO H NCO2H 2 N 2 H2N NH H O LTD4 O 2 Glu Cys Gly – Gly LTC4 OH

CO2H

S

H2N CO2H LTE4

Figure 3.24 been characterized) (Figure 3.24), arachidonic also known, and these are derived from ∆5,8,11- acid is converted into a hydroperoxide, the point eicosatrienoic acid and ∆5,8,11,14,17-eicosapen- of oxygenation being C-5, rather than C-11 as taenoic acid respectively. The subscript numeral in the prostaglandin pathway (Figure 3.17). This indicates the total number of double bonds in the compound loses water via formation of an epoxide leukotriene chain. ring, giving leukotriene A4 (LTA 4). This unsta- ble allylic epoxide may hydrolyse by conjugate AROMATIC POLYKETIDES addition giving leukotriene B4 (LTB 4), or alter- natively the epoxide may be attacked directly by For fatty acid biosynthesis, reduction after each a nucleophile, in this case the sulphur atom of the condensation step affords a growing hydrocarbon tripeptide glutathione (γ-glutamylcysteinylglycine) chain. In the absence of this reduction process, (Figure 3.24). The adduct produced in the latter the growing poly-β-keto chain needs to be sta- reaction is termed leukotriene C4 (LTC4). Partial bilized on the enzyme surface until the chain hydrolysis in the tripeptide fragment then leads length is complete, at which point cyclization or to leukotriene D4 (LTD4)andleukotriene E4 other reactions can occur. The poly-β-keto ester is (LTE 4). Analogues, e.g. LTA3 and LTA5,are very reactive, and there are various possibilities AROMATIC POLYKETIDES 61 for undergoing intramolecular Claisen or aldol malonate chain extension units) is capable of being reactions, dictated of course by the nature of folded in at least two ways, A and B (Figure 3.25). the enzyme and how the substrate is folded. For A, ionization of the α-methylene allows aldol Methylenes flanked by two carbonyls are activated, addition on to the carbonyl six carbons distant allowing formation of carbanions/enolates and sub- along the chain, giving the tertiary alcohol. Dehy- sequent reaction with ketone or ester carbonyl dration occurs as in most chemical aldol reac- groups, with a natural tendency to form strain-free tions, giving the alkene, and enolization follows to six-membered rings. attain the stability conferred by the aromatic ring. The thioester bond (to coenzyme A or ACP) is Cyclization: Simple Phenols then hydrolysed to produce orsellinic acid. Alter- natively, folding of the polyketo ester as in B The polyketo ester (Figure 3.25), formed from four allows a Claisen reaction to occur, which, although acetate units (one acetate starter group and three mechanistically analogous to the aldol reaction, is

CO2H SCoA SCoA SEnz + 3 x O O OOOO acetyl-CoA malonyl-CoA poly-β-keto ester folding folding A B

O O O

O H SEnz O aldol addition on to carbonyl O O O SEnz

aldol reaction Claisen reaction

OH O O O

SEnz dehydration favoured H O SEnz by formation of OO O re-formation of carbonyl conjugated system possible by expulsion of leaving group

O O O

SEnz

OO OO

enolization enolization hydrolysis

enolization favoured HO OH O by formation of aromatic ring enolization favoured by formation of CO2H aromatic ring OH HO OH

orsellinic acid phloracetophenone

Figure 3.25 62 THE ACETATE PATHWAY terminated by expulsion of the thiol leaving group, and three molecules of malonyl-CoA, and the and direct release from the enzyme. Enolization of ‘missing’ oxygen function is removed during the the cyclohexatrione produces phloracetophenone. biosynthesis. Orsellinic acid is not itself deoxy- As with fatty acid synthases, the whole sequence genated to 6-methylsalicylic acid. The enzyme of reactions is carried out by an enzyme complex 6-methylsalicylic acid synthase requires NADPH which converts acetyl-CoA and malonyl-CoA into as cofactor, and removes the oxygen function by the final product without giving any detectable free reduction of a ketone to an alcohol, followed by intermediates. These enzyme complexes combine a dehydration step (Figure 3.26). Whilst on paper polyketide synthase and polyketide cyclase activ- this could be carried out on an eight-carbon inter- ities and share many structural similarities with mediate involved in orsellinic acid biosynthesis fatty acid synthases, including an acyl carrier pro- (Figure 3.25), there is evidence that the reduc- tein with a phosphopantatheine group, a reactive tion/dehydration actually occurs on a six-carbon cysteine residue, and an analogous β-ketoacyl syn- intermediate as the chain is growing (compare fatty thase activity. acid biosynthesis, page 36), prior to the final chain A distinctive feature of an aromatic ring system extension (Figure 3.26). Aldol condensation, eno- derived through the acetate pathway is that several lization, and release from the enzyme then gen- of the carbonyl oxygens of the poly-β-keto sys- erate 6-methylsalicylic acid. Important evidence tem are retained in the final product. These end for reduction occurring at the C6 stage as shown up on alternate carbons around the ring system. in Figure 3.26 comes from the formation of tri- Of course, one or more might be used in form- acetic acid lactone if NADPH is omitted from the ing a carbon–carbon bond, as in orsellinic acid. enzymic incubation. Nevertheless, this oxygenation on alternate car- The folding of a polyketide chain can be estab- bon atoms, a meta oxygenation pattern, is usually lished by labelling studies, feeding carbon-labelled easily recognizable, and points to the biosynthetic sodium acetate to the appropriate organism and origin of the molecule. This meta oxygenation pat- establishing the position of labelling in the final tern contrasts to that seen on aromatic rings formed product by chemical degradation and counting (for via the shikimate pathway (see Chapter 4). the radioactive isotope 14C), or by NMR spec- 6-methylsalicylic acid (Figure 3.26) is a meta- trometry (for the stable isotope 13C). 13CNMR bolite of Penicillium patulum, and differs from spectrometry is also valuable in establishing the orsellinic acid by the absence of a phenol group location of intact C2 units derived from feed- 13 at position 4. It is also derived from acetyl-CoA ing C2-labelled acetate. This is exemplified in

SEnz

OOO enolization then ester NADPH formation HO O HO NADPH reduction O O O of carbonyl to SEnz SEnz alcohol O O O triacetic acid lactone

– H2O enolization dehydration favoured aldol reaction malonyl-CoA hydrolysis by formation of – H2O conjugated system O O SEnz SEnz SEnz chain extension CO2H O follows after O O OO OH dehydration / removal 6-methylsalicylic of oxygen function acid

Figure 3.26 AROMATIC POLYKETIDES 63

O OH

* * * * * * * * * O 7 x H3CCO2H O O OH ** * * * * O O O SEnz HOO O

C14 poly-β-keto chain alternariol refers to intact acetate C2 unit 2 x aldol lactone – H2O formation

O OH enolization

O OH COSEnz COSEnz O O HO OH

Figure 3.27

OH OH CO2H CO2H O

CO2H HO O

HO OH HO OH

orsellinic acid lecanoric acid

Figure 3.28

Figure 3.27, where alternariol, a metabolite from formed from two phenolic acids) found in lichens the mould Alternaria tenuis, can be established and produced from two orsellinic acid molecules to be derived from a single C14 polyketide chain, (Figure 3.28). folded as shown, and then cyclized. Whilst the pre- cise sequence of reactions involved is not known, paper chemistry allows us to formulate the essen- Structural Modifications: tial features. Two aldol condensations followed by Anthraquinones enolization in both rings would give a biphenyl, A number of natural anthraquinone derivatives are and lactonization would then lead to alternariol. also excellent examples of acetate-derived struc- The oxygenation pattern in alternariol shows alter- tures. Endocrocin (Figure 3.29) found in species nate oxygens on both aromatic rings, and an acetate of Penicillium and Aspergillus fungi is formed by origin is readily surmised, even though some folding a polyketide containing eight C2 units to oxygens have been used in ring formation pro- form the periphery of the carbon skeleton. Three cesses. The lone methyl ‘start-of-chain’ is also usu- aldol-type condensations would give a hypothetical ally very obvious in acetate-derived compounds, intermediate 1, and, except for a crucial carbonyl though the carboxyl ‘end-of-chain’ can often react oxygen in the centre ring, endocrocin results by with convenient hydroxyl functions, which may enolization reactions, one of which involves the = have arisen through enolization, and lactone or vinylogous enolization −CH2−CH CH−CO−→ ester functions are thus reasonably common. For −CH=CH−CH=C(OH)−. The additional car- example, lecanoric acid is a depside (an ester bonyl oxygen must be introduced at some stage 64 THE ACETATE PATHWAY

O

O O O SEnz aldol reactions NADPH reduces – H2O OOOO carbonyl NADPH NADPH

O – H O – H2O OO 2 O

O HO HO

CO2H CO2H CO2H OOO OOO OOO hypothetical intermediate 1 hypothetical intermediate 2 hypothetical intermediate 2 –H O –H O enolizations 2 2 enolization enolization oxidation oxidation 2 x oxidation – CO O O – CO2 O OH 2 HO

CO2H OH O OH OH O OH OH O OH endocrocin chrysophanol islandicin decarboxylation oxidation of methyl facilitated by – CO2 O ortho-hydroxyl to alcohol O O O

HO CO2H OH O

oxidation of alcohol OH O OH OH O OH to acid OH O OH emodin aloe-emodin rhein O-methylation SAM of phenol O MeO

OH O OH physcion

Figure 3.29 during the biosynthesis by an oxidative process, One hydroxyl is missing, and a new hydroxyl has for which we have little information. Emodin, been incorporated adjacent to the methyl. With- a metabolite of some Penicillium species, but out any evidence for the sequence of such reac- also found in higher plants, e.g. Rhamnus and tions, the structure of intermediate 2 shows the Rumex species, would appear to be formed from result of three aldol condensations and reduction endocrocin by a simple decarboxylation reac- of a carbonyl. A dehydration reaction, two oxi- tion. This is facilitated by the adjacent phenol dations, and a decarboxylation are necessary to function (see page 20). O-Methylation of emodin attain the islandicin structure. In chrysophanol, would then lead to physcion. Islandicin is another aloe-emodin,andrhein, the same oxygen func- anthraquinone pigment produced by Penicillium tion is lost by reduction as in islandicin, and islandicum, and differs from emodin in two ways. decarboxylation also occurs. The three compounds AROMATIC POLYKETIDES 65 are interrelated by a sequential oxidation of the the speculative pathway of Figure 3.29, but the methyl in chrysophanol to a hydroxymethyl in sequence of reactions is altered. Decarboxylation aloe-emodin, and a carboxyl in rhein. appears to take place before aromatization of the These structural modifications undergone by the last-formed ring system, and tetrahydroanthracene basic polyketide are conveniently considered under intermediates such as atrochrysone carboxylic acid two main headings, according to the timing of and atrochrysone are involved. These dehydrate to the steps in the synthetic sequence. Thus, ‘miss- the anthrones endocrocin anthrone and emodin ing’ oxygen functions appear to be reduced out anthrone, respectively, prior to introduction of the well before the folded and cyclized polyketide extra carbonyl oxygen as a last transformation in is detached from the enzyme, and are mediated the production of anthraquinones. This oxygen is by a reductase component of the enzyme com- derived from O2. plex during chain elongation before the cycliza- Note that many other natural anthraquinone tion reaction. On the other hand, reactions like structures are not formed via the acetate pathway, the decarboxylation, O-methylation, and sequen- but by a more elaborate sequence involving shiki- tial oxidation of a methyl to a carboxyl are rep- mate and an isoprene unit (see page 158). Such resentative of transformations occurring after the structures do not contain the characteristic meta cyclization reaction. It is often possible to demon- oxygenation pattern, and often have oxygenation strate these later conversions by the isolation of in only one aromatic ring (see page 164). enzymes catalysing the individual steps. Most of Emodin, physcion, chrysophanol, aloe-emodin, the secondary transformations are easily rational- and rhein form the basis of a range of purga- ized by careful consideration of the reactivity con- tive anthraquinone derivatives found in long- ferred on the molecule by the alternating and established laxatives such as Senna∗, Cascara∗, usually phenolic oxygenation pattern. These oxy- Frangula∗, Rhubarb∗, and Aloes∗. The free anthra- gens activate adjacent sites creating nucleophilic quinones themselves have little therapeutic activity centres. Introduction of additional hydroxyl groups and need to be in the form of water-soluble ortho or para to an existing phenol will be facil- glycosides to exert their action. Although sim- itated (see page 26), allowing the extra hydroxyl ple anthraquinone O-glycosides are present in the of islandicin to be inserted, for example. Ortho-or drugs, the major purgative action arises from para-diphenols are themselves susceptible to fur- compounds such as cascarosides, e.g. cascaro- ther oxidation in certain circumstances, and may side A (Figure 3.33), which are both O-and give rise to o-andp-quinones (see page 25). The C-glycosides, and sennosides, e.g. sennoside A quinone system in anthraquinones is built up by (Figure 3.33), which are dianthrone O-glycosides. an oxidation of the central cyclohexadienone ring, These types of derivative are likely to be produced again at a nucleophilic centre activated by the from intermediate anthrone structures. This could enone system. Methyls on an aromatic ring are also act as substrate for both O-andC-glucosylation, activated towards oxidation, facilitating the chryso- employing the glucose donor UDPglucose (see phanol → aloe-emodin oxidation, for example. page 29), and would generate a cascaroside struc- Decarboxylation, e.g. endocrocin → emodin, is ture (Figure 3.31). Alternatively, a one-electron readily achieved in the presence of an ortho phenol oxidation allows oxidative coupling (see page 28) function, though a para phenol can also facilitate of two anthrone systems to give a dianthrone this (see page 20). (Figure 3.32). This can be formulated as direct oxi- It is now appreciated that the assembly of dation at the benzylic −CH2−, or via the anthra- the anthraquinone skeleton (and related polycyclic nol, which is the phenolic tautomer of the anthrone structures) is achieved in a step-wise sequence. (Figure 3.32). Glycosylation of the dianthrone sys- After the polyketide chain is folded, the ring tem would then give a sennoside-like product. at the centre of the fold is formed first, fol- However, further oxidative steps can create a lowed in turn by the next two rings. The path- dehydrodianthrone, and then allow coupling of the way outlined for the biosynthesis of endocrocin aromatic rings through protohypericin to give a and emodin is shown in Figure 3.30. Mechanis- naphthodianthrone, e.g. hypericin (Figure 3.32). tically, there is little difference between this and The reactions of Figure 3.32 can be achieved 66 THE ACETATE PATHWAY

O

O O O SEnz

aldol OOOO – H2O + NADPH enolization

HO O O O O SEnz SEnz

OH O O O OH O O O aldol – H2O enolization O HO O O SEnz CO2H OH OH O O OH O OH OH O OH chrysophanol anthrone chrysophanol

aldol O O O OH HO CO H OH 2 SEnz O

OH OH O O OH O OH OH O OH aloe-emodin rhein hydrolysis from enzyme O OH HO HO HO – H2O O

CO2H CO2H CO2H OH OH O OH O OH OH O OH atrochrysone carboxylic acid endocrocin anthrone endocrocin

– CO2 O O OH HO HO HO MeO – H2O O SAM

OH OH O OH O OH OH O OH OH O OH atrochyrsone emodin anthrone emodin physcion

Figure 3.30

OH O OH GlcO O OH

2 x UDPGlc OH OH HO aloe-emodin anthrone H OH O H OH cascaroside OH

Figure 3.31 AROMATIC POLYKETIDES 67

radical OH O OH OH O OH coupling OH O OH

– H , – 1e x 2

HO HO HO emodin anthrone HO tautomerism

OH OH OH OH O OH OH O OH – H , – 1e emodin dianthrone

HO HO O emodin anthranol

OH O OH OH O OH OH O OH

HO O HO O HO HO HO HO

OH O OH OH O OH OH O OH hypericin protohypericin

Figure 3.32 chemically by passing air into an alkaline solution hypericin can act as a photosensitizing agent in a of emodin anthrone. Hypericin is found in cul- similar manner to furocoumarins (see page 146). tures of Dermocybe fungi, and is also a constituent Thus ingestion of hypericin results in an increased of St John’sWort,Hypericum perforatum (Gut- absorption of UV light and can lead to dermatitis tiferae/Hypericaceae), which is a popular herbal and burning. Hypericin is also being investigated medicine in the treatment of depression. The for its antiviral activities, in particular for its poten- naphthodianthrones have no purgative action, but tial activity against HIV.

Senna Senna leaf and fruit are obtained from Cassia angustifolia (Leguminosae/Fabaceae), known as Tinnevelly senna, or less commonly from Cassia senna (syn C. acutifolia), which is described as Alexandrian senna. The plants are low, branching shrubs, C. angustifolia being cultivated inIndiaandPakistan,andC. senna being produced in the Sudan, much of it from wild plants. Tinnevelly senna is cultivated in wetter conditions than Alexandrian senna, which gives more luxuriant growth. Early harvests provide leaf material whilst later on, both leaf and fruit (senna pods) are obtained, a mixture which is separated by sieving (Alexandrian) or hand picking after drying (Tinnevelly). There are no significant differences in the chemical constituents of the two sennas, or between leaf and fruit drug. However, amounts of the active constituents do vary, and appear to be a consequence of cultivation conditions and the time of harvesting of the plant material. The active constituents in both senna leaf and fruit are dianthrone glycosides, principally sennosides A and B (Figure 3.33). These compounds are both di-O-glucosides of rhein

(Continues) 68 THE ACETATE PATHWAY

(Continued)

RO O OH RO O OH RO O OH RO O OH

R R RR CO2H CO2H CO2H CO2H HH HH HH HH CO2H CO2H R S R OH S OH

RO O OH RO O OH RO O OH RO O OH R = Glc, sennoside A R = Glc, sennoside B R = Glc, sennoside C R = Glc, sennoside D R = H, sennidin A R = H, sennidin B R = H, sennidin C R = H, sennidin D

HO O OH GlcO O OH GlcO O OH

10 OH 10 R 2 R1 R2 R1 R 1 = 2 = 1 = 2 = R = CO2H, rhein anthrone R H, R Glc, cascaroside A (10S) R H, R Glc, cascaroside C (10S) 1 2 1 2 R = CH2OH, aloe-emodin anthrone R = Glc, R = H, cascaroside B (10R) R = Glc, R = H, cascaroside D (10R)

HO O OH HO O OH HO OH OH HO O OH

RS OH OH OH RS

H Glc R1 R2 Glc H Glc 1 2 barbaloin R = H, R = Glc, aloin A (10S) barbaloin chrysaloin R1 = Glc, R2 = H, aloin B (10R) (anthranol tautomer) (deoxybarbaloin)

HOO OH GlcO O OH HO O OH

HO OH O ORha RO RO OH H Glc O O HO aloinoside A (10S) R = Rha, glucofrangulin A R = Rha, frangulin A β-D-apiose aloinoside B (10R) R = Api, glucofrangulin B R = Api, frangulin B (Api)

Figure 3.33

dianthrone (sennidins A and B), and liberate these aglycones on acid hydrolysis, or the anthraquinone rhein (Figure 3.30) on oxidative hydrolysis (e.g. aq HNO3 or H2O2/HCl). Sennidins A and B are optical isomers: sennidin A is dextrorotatory (+) whilst sennidin B is the optically inactive meso form. Minor constituents include sennosides C and D (Figure 3.33), which are also a pair of optical isomers, di-O-glucosides of heterodianthrones sennidins C and D. Sennidin C is dextrorotatory, whilst sennidin D is optically inactive, approximating to a meso form in that the modest change in substituent does not noticeably affect the optical rotation. Oxidative hydrolysis of sennosides C and D would produce the anthraquinones rhein and aloe-emodin (Figure 3.30). Traces of other anthraquinone glycoside derivatives are also present in the plant material. Much of the sennoside content of the dried leaf appears to be formed by enzymic oxidation of anthrone glycosides during the drying process. Fresh leaves

(Continues) AROMATIC POLYKETIDES 69

(Continued) and fruits also seem to contain primary glycosides which are more potent than sennosides A and B, and which appear to be partially hydrolysed to sennosides A and B (the secondary glycosides) by enzymic activity during collection and drying. The primary glycosides contain additional glucose residues. Senna leaf suitable for medicinal use should contain not less than 2.5% dianthrone glycosides calculated in terms of sennoside B. The sennoside content of Tinnevelly fruits is between 1.2 and 2.5%, that of Alexandrian fruits being 2.5–4.5%. Senna preparations, in the form of powdered leaf, powdered fruit, or extracts, are typically standardized to a given sennoside content. Non-standardized preparations have unpredictable action and should be avoided. Senna is a stimulant laxative and acts on the wall of the large intestine, increasing peristaltic movement. After oral administration, the sennosides are transformed by intestinal flora into rhein anthrone (Figure 3.33), which appears to be the ultimate purgative principle. The glycoside residues in the active constituents are necessary for water-solubility and subsequent transportation to the site of action. Although purgative action is provided by the aglycones, including anthraquinones, these materials are conjugated and excreted in the urine after oral administration rather than being transported to the colon. Senna is a purgative drug suitable for either habitual constipation, or for occasional use, and is widely prescribed.

Cascara Cascara is the dried bark of the cascara buckthorn Rhamnus purshianus (Rhamnaceae), a small tree native to the forests of the Pacific coast of North America. Most of the drug material is gathered from wild trees in Oregon, Washington, and British Columbia. Trees are felled and the bark is stripped from the trunk and branches, then dried. The fresh bark is unsuitable for drug use, causing griping and nausea, and thus the bark is stored for at least a year before being processed. During this time, enzymic hydrolysis and oxidation modify the anthraquinone-based constituents and thus the cathartic activity. Cascara preparations are mainly formulated from extracts of the bark. Cascara bark contains about 6–9% of anthracene derivatives, 80–90% of which are anthrone C-glycosides. The major constituents are cascarosides A and B (Figure 3.33), which contain both O-andC-glucoside linkages, and represent a pair of optical isomers differing only in the stereochemistry of the C-glucoside bond. These have a substitution pattern analogous to aloe-emodin (Figure 3.30) and oxidative hydrolysis (e.g. aq HNO3 or H2O2/HCl) liberates aloe-emodin. Acid hydrolysis does not cleave the C-glucose linkage, and instead generates barbaloin (Figure 3.33), a mixture of two diastereoisomeric forms, which have been named aloin A and aloin B. It is likely that during any chemical manipulation, the two forms may interconvert via the anthranol tautomer (Figure 3.33). Similar components in the bark, though usually present in smaller amounts than cascarosides A and B, are cascarosides C and D (Figure 3.33). These are also a pair of diastereoisomers, and have a substitution pattern analogous to chrysophanol (Figure 3.30). Hydrolysis of the O-glucose linkage yields chrysaloin, sometimes referred to as deoxybarbaloin. Barbaloin and chrysaloin are also found in the bark, and are thought to be breakdown products formed by enzymic hydrolysis of the cascarosides. Other compounds identified in the bark include simple anthraquinones and their O-glycosides, and some dianthrone derivatives. The principal purgative activity originates from the cascarosides, the C-glycosides barbaloin and chrysaloin being less active when taken orally. As with the sennosides, the actual purgative

(Continues) 70 THE ACETATE PATHWAY

(Continued)

agent is produced by the action of intestinal flora, and the cascarosides are trans- formed into aloe-emodin anthrone (Figure 3.33). Cascara has a similar pharmaco- logical action to senna, i.e. it stimulates peristalsis of the large intestine, and has found major use in the correction of habitual constipation. It has a stronger effect than senna, however, and its routine usage is not now recommended.

Frangula

The bark of the alder buckthorn, Rhamnus frangula (Rhamnaceae) is used in a similar way to cascara, and is preferred to cascara in many European countries, though not in the UK. Rhamnus frangula is a small tree of European origin, and supplies of the bark come from South-Eastern Europe. The bark is also stored for a year before use. Frangula bark contains about 6% anthracene derivatives, mainly anthraquinone O-glycosides. These are derivatives of emodin (Figure 3.30) and comprise glucofrangulins A and B, and frangulins A and B (Figure 3.33). Free aglycones emodin, physcion and chrysophanol are also present.

Allied Drugs

Aloes and rhubarb have found considerable use as purgatives in the past, but they both have a rather drastic action and their use for this purpose has largely been abandoned. Aloes consists of the dried juice from the leaves of various Aloe species (Liliaceae/ Aloeaceae), including A. ferox (Cape aloes), A. barbadensis (Curacao aloes), and A. perryi (Socotrine aloes). The dark brown–black solid extract is extremely bitter, and contains 10–30% anthracene derivatives, the main component of which is barbaloin (Figure 3.33). Aloinosides A and B (Figure 3.33) are present in some varieties. Large amounts of resinous material form the bulk of the extract. Aloes is still used as a pharmaceutical aid in Compound Benzoin Tincture. The fresh mucilaginous gel obtained from Aloe species, particularly Aloe vera (= A. barbadensis), is held to assist wound healing, and is also widely used in skin cosmetics for its moisturizing and emollient properties. This material, mainly carbohydrate in nature (pectins and glucomannans), does not contain anthraquinone derivatives. Rhubarb consists of the dried rhizome and root of Rheum officinale, R. palmatum,and other Rheum species (Polygonaceae). This contains 3–7.5% anthracene derivatives, mainly in the form of mono- and di-O-glucosides of rhein, physcion, and chrysophanol. Aglycones, especially rhein, are also present, and dianthrone derivatives have also been characterized. A high proportion of tannin-like materials gives rhubarb astringent as well as purgative properties. The common rhubarb cultivated for culinary use is Rheum rhaponticum, a species containing similar anthraquinone derivatives to the drug material, but which was not officially acceptable. In common with other Rheum species, this plant is considered poisonous due to the high concentration of oxalic acid present in the leaf (though not in the stem, which is edible). Toxic effects result from hypocalcaemia caused by removal of calcium from the bloodstream by formation of the insoluble calcium oxalate. Dantron (danthron; 1,8-dihydroxyanthraquinone) (Figure 3.34) is known as a natural product, but for drug use is produced synthetically. It is prescribed to relieve constipation in geriatric and terminally ill patients. Dithranol (1,8-dihydroxyanthrone) is used as topical agent to treat troublesome cases of psoriasis. Diacetylrhein is marketed in some countries for the treatment of osteoarthritis.

(Continues) AROMATIC POLYKETIDES 71

(Continued)

HO O OH HO O OH

O dithranol dantron (danthron)

Figure 3.34

Hypericum/St John’s Wort

The dried flowering tops of St John’s Wort (Hypericum perforatum; Guttiferae/Hypericaceae) have been used as a herbal remedy for many years, an extract in vegetable oil being used for its antiseptic and wound healing properties. St John’s Wort is now a major crop marketed as an antidepressant, that is claimed to be as effective in its action as the widely prescribed antidepressants of the selective serotonin re-uptake inhibitor (SSRI) class such as fluoxetine (Prozac ), and with fewer side-effects. There is considerable clinical evidence that extracts of St John’s Wort are effective in treating mild to moderate depression and improving mood. However, to avoid potentially dangerous side-effects, St John’s Wort should not be used at the same time as prescription antidepressants. St John’s Wort is a small to medium height herbaceous perennial plant with numerous yellow flowers characteristic of this genus. It is widespread throughout Europe, where it is generally considered a weed, and has also become naturalized in North America. The tops, including flowers at varying stages of development, which contain considerable amounts of the active principles, are harvested and dried in late summer. The dried herb contains significant amounts of phenolic derivatives, including 4–5% of flavonoids, though the antidepressant activity is considered to derive principally from naphthodianthrone structures such as hypericin (about 0.1%) and pseudohypericin (about 0.2%), and a prenylated phloroglucinol derivative hyperforin (Figure 3.35). The fresh plant also contains significant levels of protohypericin and protopseudohypericin, which are converted into hypericin and pseudophypericin during drying and processing, as a result of irradiation with visible light. Hyperforin is a major lipophilic constituent in the leaves and flowers (2–3%),

OH O OH OH O OH HO O O OH

R R HO HO HO HO OO Note: hyperforin is a mixture of tautomeric forms

OH O OH OH O OH R = H, hypericin R = H, protohypericin R = OH, pseudohypericin R = OH, protopseudohypericin hyperforin

Figure 3.35

(Continues) 72 THE ACETATE PATHWAY

(Continued)

and is now thought to be the major contributor to the antidepressant activity, as well as to the antibacterial properties of the oil extract. Studies show clinical effects of St John’s Wort on depression correlate well with hyperforin content. Standardized aqueous ethanolic extracts containing 0.15% hypericin and 5% hyperforin are usually employed. The aqueous solubility of hypericin and pseudohypericin is markedly increased by the presence of flavonoid derivatives in the crude extract, particularly procyanidin B2,adimerof epicatechin (see page 151). Hypericum extracts have been demonstrated to increase levels of serotonin, noradrenaline, and dopamine, which may be responsible for the antidepressant activity. Hypericin also possesses extremely high toxicity towards certain viruses, a property that requires light and may arise via photo-excitation of the polycyclic quinone system. It is currently under investigation as an antiviral agent against HIV and hepatitis C. Antiviral activity appears to arise from an inhibition of various protein kinases, including those of the protein kinase C family. Hypericin and pseudohypericin are potent photosensitizers initiating photochemical reactions, and are held responsible for hypericism, a photodermatosis seen in cattle after consuming Hypericum plants present in pasture. Patients using St John’s Wort as an antidepressant should also be warned to avoid over- exposure to sunlight. There is also considerable evidence that St John’s Wort interacts with a number of prescription drugs including the anticoagulant warfarin, the cardiac glycoside digoxin, the bronchodilator theophylline, the HIV protease inhibitor indinavir, the immunosuppressive drug cyclosporin, and oral contraceptives. In some cases, it is known to promote the cytochrome P-450-dependent metabolism of the co-administered drugs.

C-Alkylation Reactions that, while C-alkylation with dimethylallyl and higher diphosphates is mediated after the initial A common feature of many natural products con- polyketide cyclization product is liberated from taining phenolic rings is the introduction of alkyl the enzyme, there are several examples where groups at nucleophilic sites. Obviously, the phe- C-methylation undoubtedly occurs before release nol groups themselves are nucleophilic, and with a of any aromatic compound from the enzyme. suitable alkylating agent, O-alkyl derivatives may 5-methylorsellinic acid (Figure 3.36) is a sim- be formed (see page 12), e.g. the O-methylation ple C-methylated analogue of orsellinic acid found of emodin to physcion (Figure 3.30). However, a in Aspergillus flaviceps, and the extra methyl is phenol group also activates the ring carbons at derived from SAM. However, orsellinic acid is the ortho and para positions, so that these posi- not a precursor of 5-methylorsellinic acid and it tions similarly become susceptible to alkylation, is proposed that the poly-β-keto ester is therefore leading to C-alkyl derivatives. The meta oxygena- methylated as part of the series of reactions catal- tion pattern, which is a characteristic feature of ysed by the synthase complex (Figure 3.36). Sim- acetate-derived phenolics, has the effect of increas- ilarly, 5-methylorsellinic acid, but not orsellinic ing this nucleophilicity considerably, and the pro- acid is a precursor of mycophenolic acid∗ in cess of C-alkylation is very much facilitated (see Penicillium brevicompactum (Figure 3.36). How- page 12). Suitable natural alkylating agents are ever, C-alkylation by farnesyl diphosphate (see S-adenosylmethionine (SAM), and dimethylallyl page 191) proceeds after the aromatization step, diphosphate (DMAPP). Other polyprenyl diphos- and a phthalide intermediate is the substrate phate esters may also be encountered in biological involved. The phthalide is a lactone derived from alkylation reactions (e.g. see vitamin K, page 159). 5-methylorsellinic acid by hydroxylation of its A minor inconsistency has been discovered, in starter methyl group and reaction with the end- AROMATIC POLYKETIDES 73

Ad H3CS R C-methylation O O SEnz SAM O SEnz O OOOO SEnz O O O O Ad aldol reaction, aromatization H3CS R O HO HO

O SEnz CO2H CO2H OH O O OH orsellinic acid 5-methylorsellinic acid oxidation of methyl to CH2OH, then O farnesyl PP lactonization HO C-alkylation HO O FPP O

OH O OH O phthalide intermediate Ad S CH3 O-methylation R H CO HO SAM 3 O O HO2C HO2C O OH O OH demethylmycophenolic acid mycophenolic acid

Figure 3.36

Mycophenolic Acid

Mycophenolic acid (Figure 3.36) is produced by fermentation cultures of the fungus Penicillium brevicompactum. It has been known for many years to have antibacterial, antifungal, antiviral, and antitumour properties. It has recently been introduced into medicine as an immunosuppressant drug, to reduce the incidence of rejection of transplanted organs, particularly kidney and heart transplants. It is formulated as the N-morpholinoethyl ester mycophenolate mofetil (Figure 3.37), which is metabolized after ingestion to mycophenolic

H3CO O O O N O OH O mycophenolate mofetil

Figure 3.37

(Continues) 74 THE ACETATE PATHWAY

(Continued)

acid, and is usually administered in combination with cyclosporin (see page 429). The drug is a specific inhibitor of mammalian inosine monophosphate dehydrogenase and has an antiproliferative activity on cells due to inhibition of guanosine nucleotide biosynthesis. This enzyme catalyses the NAD+-dependent oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), a key transformation in the synthesis of guanosine triphosphate (GTP) (see also caffeine biosynthesis, page 394). Rapidly growing cells have increased levels of the enzyme, so this forms an attractive target for anticancer, antiviral, and immunosuppressive therapy.

aldol reaction, aromatization O O 3 x Me HO OO SEnz OO O SEnz CHO reduction O O O O OH reduction dehydration leads to quinone formation O HO HO OH O O H HO C 2 formation of OH O OH OH OH hemiacetal H citrinin O H

Figure 3.38 of-chain carboxyl. The chain length of the far- simpler aromatic ring. The methyls are probably nesyl alkyl group is then shortened by oxidation introduced into the polyketide prior to release of of a double bond giving demethylmycophenolic the first aromatic intermediate, which could well acid, which is then O-methylated, again involv- be an aldehyde rather than the corresponding acid ing SAM, to produce mycophenolic acid. Note that if a reductase component also forms part of the the O-methylation step only occurs after the C- synthase complex. The hemiacetal can be produced alkylations, so that the full activating benefitof after reduction of the side-chain carbonyl, and then two meta positioned phenols can be utilized for in the later stages, oxidation of one methyl to a the C-alkylation. carboxyl will follow. The quinonemethide system Three C-methyl substituents are inserted in citrinin is simply the result of a dehydration into the acetate-derived skeleton of citrinin reaction on the hemiacetal (Figure 3.38). (Figure 3.38), an antimicrobial metabolite from Khellin∗ and visnagin (Figure 3.39) are Penicillium citrinum and several Aspergillus furochromones found in the fruits of Ammi visnaga species, which also displays potentially dangerous (Umbelliferae/Apiaceae), and the active principles carcinogenic and nephrotoxic (kidney-damaging) of a crude plant drug which has a long history of activity. One of these introduced methyls has use as an antiasthmatic agent. Figure 3.39 presents undergone oxidation to a carboxyl, adding to the sequence of steps utilized in the biosynthesis the difficulties in immediately recognizing the of these compounds, fully consistent with the biosynthetic origins of this compound which biosynthetic rationale developed above. The two contains a quinonemethide system rather than the carbons C-2 and C-3 forming part of the furan AROMATIC POLYKETIDES 75

Claisen reaction, heterocyclic ring formation via Michael-type nucleophilic attack aromatization of OH on to enol tautomer followed by loss of leaving group H O SEnz OH OH OH OH O HO O HO HO O O

OO OH O OH O OH O cyclization of hydroxyl on to oxidative cleavage of side-chain: dimethylallyl group: see C-alkylation see furocoumarins Figure 4.35 furocoumarins Figures 4.33 and 4.34 with DMAPP HO O O O O O HO O HO

OH O OH O OH O OH O visamminol peucenin 5,7-dihydroxy- hydroxylation OPP SAM 2-methylchromone DMAPP OH OMe ′ 1 8 1 O O O 2 O O SAM O 2′ 4 3 ′ 3 5 OMe O OH O OMe O visnagin khellin

Figure 3.39

Khellin and Cromoglicate

The dried ripe fruits of Ammi visnaga (Umbelliferae/Apiaceae) have a long history of use in the Middle East as an antispasmodic and for the treatment of angina pectoris. The drug contains small amounts of coumarin derivatives, e.g. visnadin (Figure 3.40) (compare Ammi majus, a rich source of furocoumarins, page 146), but the major constituents (2–4%) are

OMe O O OR O O O

OAc OMe O OO R = H, khellol R = Glc, khellol glucoside Et

O2C O OCO2 visnadin O2C N OCO2

OH 2Na 2Na O OOO O O cromoglicate (cromoglycate) nedocromil

Figure 3.40

(Continues) 76 THE ACETATE PATHWAY

(Continued)

furochromones, including khellin and visnagin (Figure 3.34), and khellol and khellol glucoside (Figure 3.40). Both khellin and visnadin are coronary vasodilators and spasmolytic agents, with visnadin actually being the more potent agent. Khellin has been used in the treatment of angina pectoris and bronchial asthma. The synthetic analogue cromoglicate (cromoglycate) (Figure 3.40) is a most effective and widely used agent for the treatment and prophylaxis of asthma, hay fever, and allergic rhinitis. Cromoglicate contains two chromone systems containing polar carboxylic acid functions, joined by a glycerol linker. The mode of action is not fully established. It was believed to prevent the release of bronchospasm mediators by stabilizing mast cell membranes, but an alternative suggestion is that it may act by inhibiting the effect of sensory nerve activation, thus interfering with bronchoconstriction. It is poorly absorbed orally and is thus administered as inhalation or nasal spray. Eyedrops for relief of allergic conjunctivitis are also available. The more potent nedocromil (Figure 3.40) has also been introduced. ring originate by metabolism of a five-carbon methylation of phloracetophenone (Figure 3.41). dimethylallyl substituent attached to C-6 (for a full The two molecules are joined together by an oxida- discussion, see furocoumarins, page 145). The 8- tive coupling mechanism which can be rational- methoxy group in khellin is absent from visnagin, ized via the one-electron oxidation of a phenol so must be introduced late in the sequence. group in methylphloracetophenone giving free rad- The key intermediate is thus 5,7-dihydroxy-2- ical A, for which resonance forms B and C can methylchromone. On inspection, this has the be written. Coupling of B and C occurs. Only alternate acetate-derived oxygenation pattern and the left-hand ring can subsequently be restored to a methyl chain starter, so is formed from a aromaticity by keto–enol tautomerism, this state poly-β-keto chain through Claisen condensation being denied to the right-hand ring because cou- then heterocyclic ring formation by an overall pling occurred on to the methyl-containing posi- dehydration reaction. After formation of the tion para to the original phenol. Instead, a het- furan ring via the C-dimethylallyl derivative erocyclic ring is formed by attack of the phenol peucenin and then visamminol, visnagin can on to the enone system (see khellin, above). The be obtained by O-methylation. Alternatively, outcome of this reaction is enzyme controlled, further hydroxylation para to the free phenol, since two equivalent phenol groups are present as followed by two methylations, yields khellin. potential nucleophiles, and two equivalent enone The antiasthmatic properties of khellin have been systems are also available. Therefore, four differ- exploited by developing the more polar, water- ent products could be formed, but only one is soluble derivative cromoglicate∗. actually produced. Loss of water then leads to usnic acid. Phenolic oxidative coupling is widely encoun- Phenolic Oxidative Coupling tered in natural product biosynthesis, and many other examples are described in subsequent C-Methylation also features in the biosynthesis of sections. A further acetate-derived metabolite usnic acid (Figure 3.41), an antibacterial metabo- formed as a result of oxidative coupling is the lite found in many lichens, e.g. Usnea and Clado- antifungal agent griseofulvin∗ (Figure 3.42) syn- nia species, which are symbiotic combinations of thesized by cultures of Penicillium griseofulvin. alga and fungus. However, the principal struc- The sequence of events leading to griseofulvin has tural modification encountered involves phenolic now been established in detail, and the pathway oxidative coupling (see page 28). Two molecules also includes O-methylation steps and the intro- of methylphloracetophenone are incorporated, duction of a halogen (chlorine) atom at one of the and these are known to derive from a pre- nucleophilic sites, which is represented as involv- aromatization methylation reaction and not by ing the electrophile Cl+ (Figure 3.42). AROMATIC POLYKETIDES 77

C-methylation prior Claisen reaction, O O to cyclization O O aromatization OH O SEnz SAM O O OOOO O SEnz O SEnz HO OH methylphloracetophenone one-electron oxidation to – H , – 1e free radical

OH O OH O OH O resonance forms of free radical HO O HO O HO O C B A

radical coupling keto–enol tautomerism can O restore aromaticity only in OH HO the left-hand ring OH HO O H HO O HO O O HO OH O HO nucleophilic attack on O to enone system O O OH HO OH HO

HO O HO O O O OH O O H (+)-usnic acid

Figure 3.41

Griseofulvin Griseofulvin is an antifungal agent produced by cultures of Penicillium griseofulvum and a number of other Penicillium species, including P. janczewski, P. nigrum,andP. patulum. Griseofulvin is the drug of choice for widespread or intractable dermatophyte infections, but is ineffective when applied topically. However, it is well absorbed from the gut and selectively concentrated into keratin, so may be used orally to control dermatophytes such as Epidermophyton, Microsporium,andTrichophyton. Treatment for some conditions, e.g. infections in fingernails, may have to be continued for several months, but the drug is generally free of side-effects. The antifungal action appears to be through disruption of the mitotic spindle, thus inhibiting fungal mitosis.

Initial inspection of the structure of griseofulvin Claisen (left-hand ring) and aldol (right-hand shows the alternate oxygenation pattern, and ring) reactions to occur giving a benzophenone also a methyl group which identifies the start intermediate. Two selective methylations lead of the polyketide chain. Cyclization of the C14 to griseophenone C, which is the substrate poly-β-keto chain folded as shown allows both for chlorination to griseophenone B; both these 78 THE ACETATE PATHWAY

O O O OH OOH OH O OMe 2 x SAM O O HO OH MeO OH O O OH OH SEnz benzophenone intermediate griseophenone C Cl Claisen aldol halogenation at activated site ortho to phenol group

phenolic oxidation in OH O OMe OH O OMe both rings OH O OMe

O

MeO O O MeO O O MeO OH OH Cl Cl Cl resonance forms stereospecific griseophenone B radical coupling stereospecific reduction OMe of double bond OMe OH O OMe O OMe O OMe SAM O O O O MeO O MeO O MeO Cl Cl Cl dehydrogriseofulvin griseofulvin

Figure 3.42 compounds appear as minor metabolites in compounds in which the characteristic acetate- P. griseofulvin cultures. One-electron oxidations derived features are probably no longer apparent. on a phenolic group in each ring give a diradical Shikimate-derived aromatic rings can suffer similar and its mesomer, the latter allowing radical oxidative cleavage reactions. coupling to the basic grisan skeleton. Griseofulvin Patulin is an excellent example of an acetate- is then the result of methylation of the remaining derived structure synthesized from an aromatic phenol group and stereospecific reduction of the substrate via oxidative cleavage and subsequent double bond in dehydrogriseofulvin. modifications (Figure 3.44). Patulin is a potent carcinogen produced by Penicillium patulum,a common contaminant on apples. If mould-infected Oxidative Cleavage of Aromatic Rings apples find their way into food products, e.g. Perhaps the most drastic modification which can happen to an aromatic ring is ring cleavage brought OH cleavage between OH O2 hydroxyls about by oxidative enzymes called dioxygenases O CO2H (see page 27). These enzymes typically use cat- O CO H OH 2 echol (1,2-dihydroxy) or quinol (1,4-dihydroxy) OH substrates, require molecular oxygen and Fe2+ diacid cleavage adjacent to cofactors, and incorporate both the oxygen atoms hydroxyls O2 into the ring-cleaved product. In the case of cat- O CHO O CO H echols, cleavage may be between or adjacent OH 2 to the two hydroxyls, giving products contain- OH OH OH OH ing aldehyde and/or carboxylic acid functionalities aldehyde-acid (Figure 3.43). These groups are then able to react with other substituents in the molecule creating Figure 3.43 AROMATIC POLYKETIDES 79

hydroxylation at decarboxylation facilitated oxidation activated position by ortho hydroxyl of methyl to alcohol para to phenol OH O – CO2 OH OH O SEnz CO2H OO OH OH OH OH 6-methylsalicylic m-cresol m-hydroxybenzyl gentisyl alcohol acid alcohol oxidative cleavage of 1,4-quinol O neopatulin to patulin conversion involves change of functionality in hemiacetal ring via reduction and oxidation formation of lactone and cyclic hemiacetal O O O O HO O OH O O O O HO OH ≡

O OH OH O OH OH HO O O O OH O patulin ascladiol neopatulin OH

O OH O O * CHO OH * * O * * * * H3CCO2H * CO2H OH O OH OH O 6-methylsalicylic patulin gentisaldehyde phyllostine acid

Figure 3.44 apple juice, fruit pies, etc, then these products may Penicillic acid (Figure 3.45), another micro- contain unacceptable and dangerous levels of pat- bially produced food contaminant with carcino- ulin. Such food materials are routinely screened genic properties, is synthesized by cultures of for patulin content, with a tolerance level set at Penicillium cyclopium and P. baarnense,andalso 50 µgkg−1. Patulin is derived from acetate via 6- features oxidative ring fission of an aromatic com- methylsalicylic acid (Figure 3.26). Decarboxyla- pound. This time orsellinic acid (Figure 3.25) is a tion and hydroxylation reactions then lead to genti- precursor, and ring fission appears to proceed via syl alcohol (Figure 3.44), which may suffer oxida- a quinone, which is the result of decarboxylation, tive cleavage as shown. Cleavage of the aromatic oxidation, and methylation reactions. Figure 3.45 ring would generate aldehyde and carboxylic acid also represents an over-simplistic rationalization of functions. By rotating the molecule around the car- the ring fission process. bon–carbon single bond as shown, it is easy to see that neopatulin can result by formation of hemi- Starter Groups Other Than Acetate acetal and lactone groups. The reversal of func- tionality in the hemiacetal ring to produce patulin In the examples so far discussed, the basic car- is achieved by reduction and oxidation reactions bon skeleton has been derived from an acetate involving aldehyde and alcohol components of the starter group, with malonate acting as the chain hemiacetal. The sequence shown in Figure 3.44 extender. The molecule has then, in some cases, has been deliberately simplified to rationalize the been made more elaborate by the inclusion of other oxidative cleavage. The true sequence involves carbon atoms, principally via alkylation reactions. gentisaldehyde and the epoxyquinone phyllostine However, the range of natural product structures as intermediates between gentisyl alcohol and that are at least partly derived from acetate is neopatulin. increased enormously by altering the nature of the 80 THE ACETATE PATHWAY

oxidative cleavage OH OMe of 1,4-quinone OMe CO2H O O O

HO HO2C O HO orsellinic acid reduction

hemiketal OMe OMe OMe formation O H O – H2O HO C O O O 2 OH HO HO penicillic acid

Figure 3.45

OH OH OH O SEnz 3 x malonyl-CoA O O ≡ CoAS O SEnz O O O 4-hydroxycinnamoyl-CoA O O aldol Claisen OH OH Michael-type nucleophilic addition on to HO OH HO α,β-unsaturated ketone

CO2H OH O OH

– CO2 OH OH OH

HO O HO

CO2H OH OH O OH lunularic acid naringenin resveratrol (a flavonoid) (a stilbene)

Figure 3.46 starter group from acetate to a different carboxylate page 130) has acted as a starter group. Thus, if system, as its coenzyme A ester, with malonyl- 4-hydroxycinnamoyl-CoA (Figure 3.46) is chain CoA again providing the chain extender. There extended with three malonyl-CoA units, the poly- is less detailed knowledge here about the precise β-keto chain can then be folded in two ways, nature of how substrates are bound to the enzyme, allowing aldol or Claisen-type cyclizations to and whether coenzyme A esters are initially trans- occur, respectively. The six-membered hetero- formed into thio esters of the ACP type. cyclic ring characteristic of most flavonoids, e.g. Flavonoids and stilbenes are simple examples naringenin, is formed by nucleophilic attack of of molecules in which a suitable cinnamoyl-CoA a phenol group from the acetate-derived ring on C6C3 precursor from the shikimate pathway (see to the α,β-unsaturated ketone. Stilbenes, such as AROMATIC POLYKETIDES 81 resveratrol, incorporate the carbonyl carbon of the biosynthesis and aromatic polyketide biosynthesis cinnamoyl unit into the aromatic ring, and typically are distinguished by the sequential reductions as lose the end-of-chain carboxyl by a decarboxy- the chain length increases in the former, and by lation reaction. Although some related structures, the stabilization of a reactive poly-β-keto chain in e.g. lunularic acid from the liverwort Lunularia the latter, with little or no reduction involved. It cruciata, still contain this carboxyl, in general it is thus interesting to see natural product structures is lost in a pre-cyclization modification, and inter- containing both types of acetate–malonate-derived mediates of the type shown in brackets are not chains. In plants of the Anacardiaceae, e.g. poison produced. Flavonoids and stilbenes are discussed ivy∗ (Rhus radicans) and poison oak∗ (Rhus toxi- in more detail in Chapter 4 (see page 149). codendron), contact allergens called urushiols are Anthranilic acid (2-aminobenzoic acid) (see encountered, which derive from just such a path- page 126) is another shikimate-derived compound way. Thus, palmitoleoyl-CoA (∆9-hexadecenoyl- which, as its CoA ester anthraniloyl-CoA, can CoA) can act as starter group for extension by three act as a starter unit for malonate chain exten- malonyl-CoA units, with a reduction step dur- sion. Aromatization of the acetate-derived por- ing chain extension (Figure 3.48). Aldol cycliza- tion then leads to quinoline or acridine alkaloids, tion then gives anacardic acid, which is likely according to the number of acetate units incorpo- to be the precursor of urushiol by decarboxyla- rated (Figure 3.47). These products are similarly tion/hydroxylation. It is likely that different fatty discussed elsewhere, under alkaloids (Chapter 6, acyl-CoAs can participate in this sequence, since page 376). urushiols from poison ivy can contain up to three Fatty acyl-CoA esters are similarly capable double bonds in the C15 side-chain, whilst those of participating as starter groups. Fatty acid from poison oak also have variable unsaturation

O OH

2 x malonyl-CoA O O NH2 N O SCoA H SCoA quinoline alkaloid

O NH2 O OOH anthraniloyl-CoA 3 x O malonyl-CoA NH2 N OH CoAS O H acridine alkaloid

Figure 3.47

3 x malonyl-CoA reduction

SCoA

O O palmitoleoyl-CoA EnzS

O O

– CO2 aldol hydroxylation

HO HO2C urushiol OH anacardic acid OH

Figure 3.48 82 THE ACETATE PATHWAY

Poison Ivy and Poison Oak

Poison ivy (Rhus radicans or Toxicodendron radicans; Anacardiaceae) is a woody vine with three-lobed leaves that is common in the USA. The plant may be climbing, shrubby, or may trail over the ground. It presents a considerable hazard to humans should the sap, which exudes from damaged leaves or stems, come into contact with the skin. The sap sensitizes most individuals, producing delayed contact dermatitis after a subsequent encounter. This results in watery blisters that break open, the fluid quickly infecting other parts of the skin. The allergens may be transmitted from one person to another on the hands, on clothing, or by animals. The active principles are urushiols, a mixture of alkenyl polyphenols. In poison ivy, these are mainly pentadecylcatechols with varying degrees of unsaturation (∆8, ∆8,11, 8,11,14 ∆ ) in the side-chain. Small amounts of C17 side-chain analogues are present. These catechols become oxidized to an ortho-quinone, which is then attacked by nucleophilic groups in proteins to yield an antigenic complex. Poison oak (Rhus toxicodendron or Toxicodendron toxicaria: Anacardiaceae) is nearly always found as a low-growing shrub, and has lobed leaflets similar to those of oak. It is also common throughout North America. There appears considerable confusion over nomenclature, and Rhus radicans may also be termed poison oak, and R. toxicodendron oakleaf poison ivy. Poison oak contains similar urushiol structures in its sap as poison ivy, though heptadecylcatechols (i.e. C17 side-chains) predominate over pentadecylcatechols (C15 side-chains). Related species of Rhus, e.g. R. diversiloba (Pacific poison oak) and R. vernix (poison sumach, poison alder, poison dogwood) are also allergenic with similar active constituents. The allergen-containing species of Rhus have been reclassified under the genus Toxicodendron, though this nomenclature is not commonly employed. Dilute purified extracts containing urushiols may be employed to stimulate antibody production and thus build up immunity to the allergens.

in a C17 side-chain. Large quantities of anacardic ring, e.g. aflatoxin B1 and aflatoxin G1,andtheir acids containing C15 side-chains with one, two, and remarkably complex biosynthetic origin begins three double bonds are also found in the shells with a poly-β-keto chain derived from a hexanoyl- of cashew nuts (Anacardium occidentale; Anacar- CoA starter and seven malonyl-CoA extender diaceae). units (Figure 3.49). This gives an anthraquinone A saturated C6 hexanoate starter unit is used norsolorinic acid by now-familiar condensation in the formation of the aflatoxins∗, a group of reactions, but the folding of the chain is rather dif- highly toxic metabolites produced by Aspergillus ferent from that seen with simpler anthraquinones flavus, and probably responsible for the high inci- (see page 64). The six-carbon side-chain of nor- dence of liver cancer in some parts of Africa. solorinic acid is cyclized to give, in several steps, These compounds were first detected following the ketal averufin. Versiconal acetate is another the deaths of young turkeys fed on mould- known intermediate, and its formation involves a contaminated peanuts (Arachis hypogaea; Legu- Baeyer–Villiger oxidation (see page 28), resulting minosae/Fabaceae). Peanuts still remain one of the principally in transfer of a two-carbon fragment crops most likely to represent a potential risk to (the terminal ethyl of hexanoate) to become an human health because of contamination with fun- ester function. These two carbons can then be lost gal toxins. These and other food materials must be in formation of versicolorin B, now containing routinely screened to ensure levels of aflatoxins do the tetrahydrobisfuran moiety, oxidized in versi- not exceed certain set limits. The aflatoxin struc- colorin A to a dihydrobisfuran system. Sterig- tures contain a bisfuran unit fused to an aromatic matocystin is derived from versicolorin A by AROMATIC POLYKETIDES 83

aldol and Claisen reactions; O O SEnz aromatizations; oxidation O OH O OH O O 7 x malonyl-CoA O OO CoAS O O HO OH hexanoyl-CoA O norsolorinic acid rearrangement - details not known; several steps: cyclizations to ketal substrate shown hydrolysed back achieve bondings indicated hemiacetal to ketone/alcohols gives ketal, probably via formation H OH O OH O O O O O OH O OH H OH Baeyer–Villiger oxidation several steps including OH O OH Baeyer–Villiger oxidation OH O OH H H ketone → ester O O OH O (ester) HO O HO O O O H (ketal) versiconal acetate OH averufin acetyl carbons lost by hydrolysis; formation O of new furan ring dihydro- OH O OH OH O OH bisfuran OH O OH H H H O O O O HO O H HO O H HO HO O H tetrahydro- CO2H O bisfuran O methylation versicolorin B versicolorin A reduction Baeyer–Villiger oxidation decarboxylation ketone → ester allows several steps: rotation hydrolysis and ring cleavage of left-hand ring system; formation of ether linkage to give xanthone oxidative cleavage of aromatic ring with loss of one carbon; O O O O Baeyer–Villiger recyclization as indicated O O oxidation O HO O H H H O O O O MeO O H MeO O H MeO O H

aflatoxin G1 aflatoxin B1 sterigmatocystin (xanthone)

Figure 3.49 oxidative cleavage of the anthraquinone system and, quite unusually, another phenol group is involving a second Baeyer–Villiger oxidation, and lost (contrast loss of oxygen functions via reduc- recyclization through phenol groups to give a tion/dehydration prior to cyclization, see page 62). xanthone skeleton. Rotation of an intermediate Aflatoxin B1 formation requires oxidative cleav- leads to the angular product as opposed to a age of an aromatic ring in sterigmatocystin, loss linear product. One phenol group is methylated, of one carbon and recyclization exploiting the 84 THE ACETATE PATHWAY

Aflatoxins Aflatoxins are potent mycotoxins produced by the fungi Aspergillus flavus and A. parasiticus. Four main naturally occurring aflatoxins, aflatoxins B1,B2,G1,andG2 (Figure 3.50), are recognized, but these can be metabolized by microorganisms and animals to other aflatoxin structures, which are also toxic. Aflatoxin B1 is the most commonly encountered member of the group, and is also the most acutely toxic and carcinogenic example. Aflatoxin B2 is a dihydro derivative of aflatoxin B1, whilst aflatoxins G1 and G2 are an analogous pair with a six-membered lactone rather than a five-membered cyclopentenone ring. These toxins are most commonly associated with peanuts (groundnuts), maize, rice, pistachio nuts, and Brazil nuts, though other crops can be affected, and, although found world-wide, they are partic- ularly prevalent in tropical and subtropical regions. Aflatoxin M1 (Figure 3.50) is a hydroxy derivative of aflatoxin B1 and equally toxic. It may occur in cow’s milk as a result of mammalian metabolism of aflatoxin B1 originally contaminating the animal’s food. Because these com- pounds fluoresce strongly under UV light, they are relatively easily detected and monitored. The aflatoxins primarily affect the liver, causing enlargement, fat deposition, and necrosis, at the same time causing cells of the bile duct to proliferate, with death resulting from irreversible loss of liver function. In the case of aflatoxin B1, this appears to be initiated by cytochrome P- 450-dependent metabolism in the body to the epoxide (Figure 3.50). The epoxide intercalates with DNA, and in so doing becomes orientated towards nucleophilic attack from guanine residues. This leads to inhibition of DNA replication and of RNA synthesis, and initiates mutagenic activity. Aflatoxins are also known to cause hepatic carcinomas, this varying with the species of animal. The above normal incidence of liver cancer in parts of Africa and Asia has been suggested to be linked to the increased amounts of aflatoxins found in foodstuffs, and a tolerance level of 30 ppb has been recommended. Acute hepatitis may result from food containing aflatoxin B1 at levels of the order of 0.1 ppm, and levels of more than 1 ppm are frequently encountered. The biosynthesis of aflatoxins proceeds through intermediates sterigmatocystin and versicolorin (see Figure 3.49). Toxins related to these structures but differing in aromatic substituents are also produced by various fungi. The sterigmatocystins are synthesized by species of Aspergillus and Bipolaris, and contain a reduced bifuran fused to a xanthone, whilst the versicolorins from Aspergillus versicolor contain the same type of reduced bisfuran system but fused to an anthraquinone. Like the aflatoxins, the sterigmatocystins are acutely toxic and carcinogenic. The versicolorins are less toxic though still carcinogenic.

O O O O O O O O O H H HO O O O MeO O H MeO O H MeO O H N N NH2 aflatoxin B1 aflatoxin B2 aflatoxin M1 NH O O O O O O N O O O O O O O H H H guanine residue in DNA O O O MeO O H MeO O H MeO O H

aflatoxin G1 aflatoxin G2 aflatoxin B1-epoxide

Figure 3.50 AROMATIC POLYKETIDES 85 carbonyl functionality. Aflatoxin G1 is derived The principal psychoactive component is tetrahy- by further modification of aflatoxin B1,cleav- drocannabinol (THC) (Figure 3.51), whilst struc- ing the cyclopentenone ring and forming a lac- turally similar compounds such as cannabinol tone, perhaps via a further Baeyer–Villiger reac- (CBN)andcannabidiol (CBD), present in simi- tion. lar or larger amounts, are effectively inactive. In Hexanoate is also likely to feature as a starter recent years, the beneficial effects of cannabis∗, unit in the formation of the cannabinoids,a and especially THC, in alleviating nausea and group of terpenophenolics found in Indian hemp vomiting in cancer patients undergoing chemother- (Cannabis sativa; Cannabaceae). This plant, and apy, and in the treatment of glaucoma and multi- preparations from it, known under a variety of ple sclerosis, has led to a study of cannabinoid names including hashish, marihuana, pot, bhang, analogues for potentially useful medicinal activity. charas, and dagga, have been used for centuries for All the cannabinoid structures contain a monoter- the pleasurable sensations and mild euphoria expe- pene C10 unit attached to a phenolic ring hav- rienced after its consumption, usually by smoking. ing a C5 alkyl chain. The aromatic ring/C5 chain

OO OH

O CO2H 3 x malonyl-CoA SEnz aldol O CoAS HO hexanoyl-CoA O olivetolic acid

OPP C-alkylation geranyl PP oxidation to allylic cation; allows change in configuration around electrophilic cyclization double bond (see Figure 3.52) OH on to carbocation E OH OH CO2H

CO2H CO2H a HO H cannabigerolic acid b OH OH nucleophilic attack of − b a H hydroxyl on to carbocation sequential oxidation of cyclohexene gives OH OH aromatic ring OH

CO2H CO2H CO2H

O O OH cannabidiolic acid tetrahydrocannabinolic acid cannabinolic acid decarboxylations − −CO2 facilitated by ortho CO2 −CO2 hydroxyl OH OH OH

O O OH cannabidiol tetrahydrocannabinol cannabinol CBD THC CBN

Figure 3.51 86 THE ACETATE PATHWAY

is likely to originate from hexanoate and mal- and compare terpenoid cyclization mechanisms, onate, cyclization to a polyketide giving olive- page 173). Cannabidiolic acid is the result of tolic acid, from which cannabigerolic acid can proton loss, whilst tetrahydrocannabinolic acid be obtained by C-alkylation with the monoterpene is the product from heterocyclic ring forma- unit geranyl diphosphate (Figure 3.51). Cycliza- tion. CBD and THC are then the respective tion in the monoterpene unit necessitates a change decarboxylation products from these two com- in configuration of the double bond, and this may pounds. The aromatic terpenoid derived ring in be rationalized as involving the allylic cation, cannabinolic acid and cannabinol can arise which will then also allow electrophilic cyclization via a dehydrogenation process (compare thymol, to proceed (for further detail see Figure 3.52, page 186).

E E Z O

Figure 3.52

Cannabis Indian hemp, Cannabis sativa (Cannabaceae) is an annual herb indigenous to Central and Western Asia, cultivated widely in India and many tropical and temperate regions for its fibre (hemp) and seed (for seed oil). The plant is also grown for its narcotic and mild intoxicant properties, and in most countries of the world its possession and consumption is illegal. Over many years, cannabis plants have been selected for either fibre production or drug use, the former resulting in tall plants with little pharmacological activity, whilst the latter tend to be short, bushy plants. Individual plants are almost always male or female, though the sex is not distinguishable until maturity and flowering. Seeds will produce plants of both sexes in roughly equal proportions. The active principles are secreted as a resin by glandular hairs, which are more numerous in the upper parts of female plants, and resin is produced from the time flowers first appear until the seeds reach maturity. However, all parts of the plant, both male and female, contain cannabinoids. In a typical plant, the concentration of cannabinoids increases in the following order: large leaves, small leaves, flowers, and bracts (which surround the ovaries), with stems containing very little. Material for drug use (ganja) is obtained by collecting the flowering tops (with little leaf) from female plants, though lower quality material (bhang) consisting of leaf from both female and male plants may be employed. By rubbing the flowering tops, the resin secreted by the glandular hairs can be released and subsequently scraped off to provide cannabis resin (charas) as an amorphous brown solid or semi-solid. A potent form of cannabis, called cannabis oil, is produced by alcoholic extraction of cannabis resin. A wide variety of names are used for cannabis products according to their nature and the geographical area. In addition to the Indian words above, the names hashish (Arabia), marihuana (Europe, USA), kief and dagga (Africa) are frequently used. The term ‘assassin’ is a corruption of ‘hashishin’, a group of 13th century murderous Persians who were said to have been rewarded for their activities with hashish. The names grass, dope, pot, hash, weed, and wacky backy are more likely to be in current usage.

(Continues) AROMATIC POLYKETIDES 87

(Continued)

7 OH OH 1 9 6 2 8 10 ′ 3 2 10a 1 5 4 1′ 7 6a 3′ 2 HO O 10 8 6 4′ 3 6′ 9 O ′ O 5 5 4 cannabigerol cannabichromene numbered as systematic numbering OH substituted terpene O O OH OH N O H

anandamide 2-arachidonoylglycerol O

OH OH OH

O O O

CO2H tetrahydrocannabivarin nabilone tetrahydrocannabinolic acid B

Figure 3.53

The quantity of resin produced by the flowering tops of high quality Indian cannabis is about 15–20%. The amount produced by various plants is dependent on several features, however, and this will markedly alter biological properties. Thus, in general, plants grown in a tropical climate produce more resin than those grown in a temperate climate. The tall fibre-producing plants are typically low resin producers, even in tropical zones. However, the most important factor is the genetic strain of the plant, and the resin produced may contain high levels of psychoactive compounds, or mainly inactive constituents. The quality of any cannabis drug is potentially highly variable. The major constituents in cannabis are termed cannabinoids, a group of more than 60 structurally related terpenophenolics. The principal psychoactive agent is tetrahydrocannabinol (THC) (Figure 3.51). This is variously referred to as ∆1-THC or ∆9- THC according to whether the numbering is based on the terpene portion, or as a systematic dibenzopyran (Figure 3.53). Both systems are currently in use. Also found, often in rather similar amounts, are cannabinol (CBN) and cannabidiol (CBD) (Figure 3.51), which have negligible psychoactive properties. These compounds predominate in the inactive resins. Many other cannabinoid structures have been characterized, including cannabigerol and cannabichromene (Figure 3.53). A range of cannabinoid acids, e.g. cannabidiolic acid, tetrahydrocannabinolic acid, and tetrahydrocannabinolic acid B (Figure 3.53) are also present, as are some analogues of the other compounds mentioned, where a propyl side-chain replaces the pentyl group, e.g. tetrahydrocannabivarin (Figure 3.53). The latter compounds presumably arise from the use of butyrate rather than hexanoate as starter unit in the biosynthetic sequence.

(Continues) 88 THE ACETATE PATHWAY

(Continued)

The THC content of high quality cannabis might be in the range 0.5–1% for large leaves, 1–3% for small leaves, 3–7% for flowering tops, 5–10% for bracts, 14–25% for resin, and up to 60% in cannabis oil. Higher amounts of THC are produced in selected strains known as skunk cannabis, so named because of their powerful smell; flowering tops from skunk varieties might contain 10–15% THC. The THC content in cannabis products tends to deteriorate on storage, an effect accelerated by heat and light. Cannabis leaf and resin stored under ordinary conditions rapidly lose their activity and can be essentially inactive after about 2 years. A major change which occurs is oxidation in the cyclohexene ring resulting in conversion of THC into CBN. THC is more potent when smoked than when taken orally, its volatility allowing rapid absorption and immediate effects, so smoking has become the normal means of using cannabis. Any cannabinoid acids will almost certainly be decarboxylated upon heating, and thus the smoking process will also effectively increase somewhat the levels of active cannabinoids available, e.g. THC acid → THC (Figure 3.51). The smoking of cannabis produces a mild euphoria similar to alcohol intoxication, inducing relaxation, contentment, and a sense of well-being, with some changes in perception of sound and colour. However, this is accompanied by a reduced ability to concentrate and do complicated tasks, and a loss of short-term memory. Users claim cannabis is much preferable to alcohol or tobacco, insisting it does not cause dependence, withdrawal symptoms, or lead to the use of other drugs, and they campaign vociferously for its legalization. However, psychological dependence does occur, and cannabis can lead to hallucinations, depression, anxiety, and panic, with the additional risk of bronchitis and lung cancer if the product is smoked. Cannabis has been used medicinally, especially as a mild analgesic and tranquillzer, but more effective and reliable agents replaced it, and even controlled prescribing was discontinued. In recent times, cannabis has been shown to have valuable anti-emetic properties, which help to reduce the side-effects of nausea and vomiting caused by cancer chemotherapeutic agents. This activity stems from THC, and has resulted in some use of THC (dronabinol) and the prescribing of cannabis for a small number of patients. A synthetic THC analogue, nabilone (Figure 3.53), has been developed as an anti-emetic drug for reducing cytotoxic-induced vomiting. Some of the psychoactive properties of THC, e.g. euphoria, mild hallucinations, and visual disturbances, may be experienced as side-effects of nabilone treatment. Cannabis has also been shown to possess properties which may be of value in other medical conditions. There is now ample evidence that cannabis can give relief to patients suffering from chronic pain, multiple sclerosis, glaucoma, asthma, migraine, epilepsy, and other conditions. Many sufferers who cannot seem to benefit from any of the current range of drugs are obtaining relief from their symptoms by using cannabis, but are breaking the law to obtain this medication. Current thinking is that cannabis offers a number of beneficial pharmacological responses and that there should be legal prescribing of cannabinoids or derivatives. Clinical trials have already confirmed the value of cannabis and/or THC taken orally for the relief of chronic pain and the painful spasms characteristic of multiple sclerosis, and in reducing intraocular pressure in glaucoma sufferers. In general, cannabis is only able to alleviate the symptoms of these diseases, and does not provide a cure. The non-psychoactive CBD has been shown to have anti-inflammatory properties potentially useful in arthritis treatment. Recently, the ethanolamide of arachidonic acid (anandamide; ananda is the Sanskrit word for bliss) (Figure 3.53) has been isolated from animal brain tissue, and has been shown

(Continues) AROMATIC POLYKETIDES 89

(Continued)

to mimic several of the pharmacological properties of THC. This appears to be a natural ligand which interacts with central receptors (CB1) to which cannabinoids also bind. Two other polyunsaturated fatty acid ethanolamides, namely dihomo-γ-linolenoyl- (20:3) and adrenoyl- (22:4) ethanolamides have also been isolated from mammalian brain, and shown to have THC-like properties. Another type of cannabinoid receptor (CB2), expressed mainly in the immune system, has been identified; its natural ligand is 2-arachidonoylglycerol (Figure 3.53). Since this compound also interacts with the anandamide receptor, and levels of 2-arachidonoylglycerol in the brain are some 800 times higher than those of anandamide, it is now thought to be the physiological ligand for both receptors, rather than anandamide. The identification of these endogenous materials may open up other ways of exploiting some of the desirable pharmacological features of cannabis.

Table 3.3 Tetracyclines

4 1 R R3 R2 R NMe2 7 H H 3 OH 8 6 5 4 D C A B 5 9 NHR 2 10 11 12 OH 1 OH O OH O O

R1 R2 R3 R4 R5 56α 6β 7  tetracycline H Me OH H H  chlortetracycline H Me OH Cl H   natural oxytetracycline OH Me OH H H  demeclocycline H H OH H H ------ methacycline OH =CH HH 2   doxycycline OH Me H H H   semi- minocycline H H H NMe2 H   synthetic H  N CO2H  lymecycline H Me OH H

NH2

The tetracyclines∗ (Table 3.3) are a group of causing accumulation of mutant metabolites or broad spectrum, orally active antibiotics produced production of abnormal tetracyclines. This organ- by species of Streptomyces, and several natural and ism typically produces chlortetracycline, whilst semi-synthetic members are used clinically. They the parent compound tetracycline (Table 3.3) is contain a linear tetracyclic skeleton of polyketide in fact an aberrant product synthesized in mutants origin in which the starter group is malonamyl- blocked in the chlorination step. The use of CoA (Figure 3.54), i.e. the coenzyme A ester of mutants with genetic blocks has also enabled the malonate semi-amide. Thus, in contrast to most shikimate pathway (Chapter 4) to be delineated. In acetate-derived compounds, malonate supplies all that case, since a primary metabolic pathway was carbon atoms of the tetracycline skeleton, the affected, mutants tended to accumulate intermedi- starter group as well as the chain extenders. The ates and could not grow unless later components main features of the pathway (Figure 3.54) were of the pathway were supplied. With the tetracy- deduced from extensive studies of mutant strains clines, a secondary metabolic pathway is involved, of Streptomyces aureofaciens with genetic blocks and the relatively broad specificity of some of the 90 THE ACETATE PATHWAY

reduction C-methylation

SEnz 8 x malonyl-CoA SEnz CoAS NH2 OOOO O NH NH OO 2 2 malonamyl-CoA OOOOO OH O OH O O enzyme-bound anthrone

a oxidation of 1,4-quinol hydroxylation at activated O to quinone OH centre para to phenol OH OH OH

NH2 NH2 NH2

OH OH OH O O OH OH OH OH O OH OH OH OH O 6-methylpretetramide tautomerism to keto form, followed by hydration of double bond H2O chlorination at activated position para to phenol O Cl O Cl NH H H transamination H 2 OH b OH OH NH3 NH 2 Cl NH2 NH2 OH OH OH OH OH O O O OH OH O O O OH OH O O O

reduction of double bond of hydroxylation; consider the α,β-unsaturated ketone; enol form is tautomeric SAM then favoured due to conjugation CH–CH=CH–C=O form

Cl HO NMe2 Cl HO NMe2 Cl NMe2 H H H H OH O2 OH NADPH NADPH OH

NH2 NH2 NH2 OH OH OH OH O OH O O OH O O O O OH OH O O O chlortetracycline dehydrochlortetracycline anhydrochlortetracycline

Figure 3.54

Tetracyclines

The tetracyclines (Table 3.3) are a group of broad spectrum, orally active antibiotics produced by cultures of Streptomyces species. Chlortetracycline isolated from Streptomyces aureofaciens was the first of the group to be discovered, closely followed by oxytetracycline from cultures of S. rimosus. Tetracycline was found as a minor antibiotic in S. aureofaciens, but may be produced in quantity by utilizing a mutant strain blocked in the chlorination step b (Figure 3.54). Similarly, the early C-6 methylation step (included in a) can also be blocked, and such mutants accumulate 6-demethyltetracyclines, e.g. demeclocycline (demethylchlorotetracycline). These reactions can also be inhibited in the normal strain of S. aureofaciens by supplying cultures with either aminopterin (which inhibits C-6 methylation) or mercaptothiazole (which inhibits C-7 chlorination). Oxytetracycline from S. rimosus lacks

(Continues) AROMATIC POLYKETIDES 91

(Continued) the chlorine substituent, but has an additional 5α-hydroxyl group, probably introduced at a late stage. Only minor alterations can be made to the basic tetracycline structure to modify the antibiotic activity, and these are at positions 5, 6, and 7. Other functionalities in the molecule are all essential to retain activity. Semi-synthetic tetracyclines used clinically include methacycline, obtained by a dehydration reaction from oxytetracycline, and doxycycline,via reduction of the 6-methylene in methacycline. Minocycline contains a 7-dimethylamino group and is produced by a sequence involving aromatic nitration. Lymecycline is an example of an antibiotic developed by chemical modification of the primary amide function at C-2. Having both amino and phenolic functions, tetracyclines are amphoteric compounds, and are more stable in acid than under alkaline conditions. They are thus suitable for oral adminis- tration, and are absorbed satisfactorily. However, because of the sequence of phenol and car- bonyl substituents in the structures, they act as chelators and complex with metal ions, espe- cially calcium, aluminium, iron, and magnesium. Accordingly, they should not be administered with foods such as milk and dairy products (which have a high calcium content), aluminium- and magnesium-based antacid preparations, iron supplements, etc, otherwise erratic and unsatisfactory absorption will occur. A useful feature of doxycycline and minocycline is that their absorptions are much less affected by metal ions. Chelation of tetracyclines with calcium also precludes their use in children developing their adult teeth, and in pregnant women, since the tetracyclines become deposited in the growing teeth and bone. In children, this would cause unsightly and permanent staining of teeth with the chelated yellow tetracycline. Although the tetracycline antibiotics have a broad spectrum of activity spanning Gram- negative and Gram-positive bacteria, their value has decreased as bacterial resistance has developed in pathogens such as Pneumococcus, Staphylococcus, Streptococcus,andE. coli. These organisms appear to have evolved mechanisms of resistance involving decreased cell permeability; a membrane-embedded transport protein exports the tetracycline out of the cell before it can exert its effect. Nevertheless, tetracyclines are the antibiotics of choice for infections caused by Chlamydia, Mycoplasma, Brucella,andRickettsia, and are valuable in chronic bronchitis due to activity against Haemophilus influenzae. They are also used systemically to treat severe cases of acne, helping to reduce the frequency of lesions by their effect on skin flora. There is little significant difference in the antimicrobial properties of the various agents, except for minocycline, which has a broader spectrum of activity, and being active against Neisseria meningitidis is useful for prophylaxis of meningitis. The individual tetracyclines do have varying bioavailabilities, however, which may influence the choice of agent. Tetracycline and oxytetracycline are probably the most commonly prescribed agents. Tetracyclines are formulated for oral application or injection, as ear and eye drops, and for topical use on the skin. Doxycycline also finds use as a prophylactic against malaria in areas where there is widespread resistance to chloroquine and mefloquine (see page 363). Their antimicrobial activity arises by inhibition of protein synthesis. This is achieved by interfering with the binding of aminoacyl-tRNA to acceptor sites on the ribosome by disrupting the codon–anticodon interaction (see page 407). Evidence points to a single strong binding site on the smaller 30S subunit of the ribosome. Although tetracyclines can also bind to mammalian ribosomes, there appears to be preferential penetration into bacterial cells, and there are few major side-effects from using these antibiotics. A series of tetracycline derivatives has recently been isolated from species of Dactylosporangium. These compounds, the dactylocyclines (Figure 3.55), are glyco- sides and have the opposite configuration at C-6 to the natural tetracyclines. Importantly, these compounds are active towards tetracycline-resistant bacteria.

(Continues) 92 THE ACETATE PATHWAY

(Continued)

O OMe R

O OH NMe2 H HO MeO OH

CONH2 OH OH O OH O R = NHOH, dactylocycline-A R = NO2, dactylocycline-B R = NHOAc, dactylocycline-C R = OH, dactylocycline-E

Figure 3.55 enzymes concerned allows many of the later steps the anthracyclines the starter group is propionate to proceed even if one step, e.g. the chlorination, rather than malonamide, and labelling studies have is not achievable. This has also proved valuable demonstrated a rather different folding of the poly- for production of some of the clinical tetracycline β-keto chain (Figure 3.56). As a result, the end- antibiotics. of-chain carboxyl is ultimately lost through decar- One of the early intermediates in the path- boxylation. This carboxyl is actually retained for way to chlortetracycline is 6-methylpretetramide a considerable portion of the pathway, and is even (Figure 3.54). This arises from the poly-β-keto protected against decarboxylation by methylation ester via an enzyme-bound anthrone (compare to the ester, until no longer required. Most of Figure 3.30). Reduction of one carbonyl will occur the modifications which occur during the biosyn- during chain extension, whilst the methylation must thetic pathway are easily predictable. Thus, the be a later modification. Hydroxylation in ring A fol- anthraquinone portion is likely to be formed first, lowed by oxidation gives a quinone, the substrate for then the fourth ring can be elaborated by a hydration at the A/B ring fusion. The product now aldol reaction (Figure 3.56). A feature of note in features the keto tautomer in ring B, since its aro- molecules such as doxorubicin and daunorubicin is maticity has been destroyed. Chlorination of ring D the amino sugar L-daunosamine which originates at the nucleophilic site para to the phenol follows, from TDPglucose (thymidine diphosphoglucose; and an amine group is then introduced stereospecif- compare UDPglucose, page 29) and is introduced ically into ring A by a transamination reaction. This in the latter stages of the sequence. Hydroxylation amino function is then di-N-methylated using SAM of daunorubicin to doxorubicin is the very last step. as the methylating agent yielding anhydrochlorte- Doxorubicin and daunorubicin are used as antitu- tracycline. In the last two steps, C-6 is hydroxylated mour drugs rather than antimicrobial agents. They act primarily at the DNA level and so also have via an O2-, NADPH-, and flavin-dependent oxyge- nase giving the enone dehydrochlortetracycline, cytotoxic properties. Doxorubicin in particular is and NADPH reduction of the C-5a/11a double bond a highly successful and widely used antitumour generates chlortetracycline. agent, employed in the treatment of leukaemias, A number of anthracycline antibiotics∗,e.g. lymphomas, and a variety of solid tumours. doxorubicin (Figure 3.56) from Streptomyces peuceticus and daunorubicin from S. coeruleoru- MACROLIDES AND POLYETHERS bicus, have structurally similar tetracyclic skele- Extender Groups other than Malonate tons and would appear to be related to the tetracyclines. There are similarities in that the The use of propionate as a starter group as in molecules are essentially acetate derived, but for the formation of the anthracyclines is perhaps MACROLIDES AND POLYETHERS 93

reduction O SEnz O CO2H 9 x malonyl-CoA O O CoAS OOO O propionyl-CoA OOOO OH O OH O aklanonic acid SAM aldol 11-hydroxylation reduction of C=O O OH CO2Me O CO2Me O CO2Me O2 NADPH 11 NADPH OH OH OH

OH O OH OH OH O OH OH OH O OH O ε-rhodomycinone aklavinone aklaviketone TDP-L-daunosamine TDP-D-glucose (i) glycosylation of 7-hydroxyl (ii) hydrolysis of ester (iii) decarboxylation of 10-acid (iv) oxidation to 13-ketone (v) methylation of 4-hydroxyl O OH O O OH O O 13 2 NADPH OH 11 10 14 OH OH 4 7 hydroxylation OMe O OH O at C-14 OMe O OH O O

O O O 4′ L-daunosamine HO 4′ NH2 NH2 NH2 HO HO daunorubicin doxorubicin [epimer at 4′ = epirubicin (daunomycin) (adriamycin) (4′-epidoxorubicin)]

Figure 3.56

Anthracycline Antibiotics

Doxorubicin (adriamycin) (Figure 3.56) is produced by cultures of Streptomyces peucetius var caesius and is one of the most successful and widely used antitumour drugs. The organism is a variant of S. peucetius, a producer of daunorubicin (see below), in which mutagen treatment resulted in expression of a latent hydroxylase enzyme and thus synthesis of doxorubicin by 14-hydroxylation of daunorubicin. Doxorubicin has one of the largest spectra of antitumour activity shown by antitumour drugs and is used to treat acute leukaemias, lymphomas, and a variety of solid tumours. It is administered by intravenous injection and largely excreted in the bile. It inhibits the synthesis of RNA copies of DNA by intercalation of the planar molecule between base pairs on the DNA helix. The sugar unit provides further binding strength and also plays a major role in sequence-recognition for the binding. Doxorubicin also exerts some of its cytotoxic effects by inhibition of the enzyme topoisomerase II, which is responsible for cleaving and resealing of double-stranded DNA during replication (see page 137). Common toxic effects include nausea and vomiting, bone marrow suppression, hair loss, and local tissue necrosis, with cardiotoxicity at higher dosage. Daunorubicin (Figure 3.56) is produced by Streptomyces coeruleorubidus and S. peucetius,

(Continues) 94 THE ACETATE PATHWAY

(Continued)

O CO2Me O OH O

OH OH aklavinone OH O OH O OOHO

O O H L-rhodosamine NMe2 N NH2 O OH OHN OH HO

O idarubicin L-2-deoxyfucose OH L-cinerulose O OH O HN OH O N aclacinomycin A mitoxantrone H O (aclarubicin) (mitozantrone)

Figure 3.57

and, though similar to doxorubicin in its biological and chemical properties, it is no longer used therapeutically to any extent. It has a much less favourable therapeutic index than doxorubicin, and the markedly different effectiveness as an antitumour drug is not fully understood, though differences in metabolic degradation may be responsible. Epirubicin (Figure 3.56), the 4- epimer of doxorubicin, is particularly effective in the treatment of breast cancer, producing lower side-effects than doxorubicin. The antileukaemics aclarubicin from Streptomyces galilaeus, a complex glycoside of aklavinone (Figure 3.56), and the semi-synthetic idarubicin are shown in Figure 3.57. These compounds are structurally related to doxorubicin but can show increased activity with less cardiotoxicity. The principal disadvantage of all of these agents is their severe cardiotoxicity which arises through inhibition of cardiac Na+,K+-ATPase. Mitoxantrone (mitozantrone) (Figure 3.57) is a synthetic analogue of the anthracyclinones in which the non-aromatic ring and the aminosugar have both been replaced with aminoalkyl side-chains. This agent has reduced toxicity compared with doxorubicin, and is effective in the treatment of solid tumours and leukaemias. less common than incorporating it as a chain employ propionate by the latter route. The incorpo- extender via methylmalonyl-CoA. We have already ration of propionate by methylmalonate extender encountered this process in the formation of some units can frequently be interrupted and normal branched-chain fatty acids with methyl substituents malonate extenders are added, thus giving an irreg- on the basic chain (see page 49). Of course, ular sequence of methyl side-chains. methyl groups can also be added to a fatty acid The macrolide antibiotics∗ provide us with chain via SAM (see page 49), and there are also excellent examples of natural products conforming many examples for the methylation of poly-β-keto to the acetate pathway, but composed princi- chains, several of which have already been dis- pally of propionate units, or mixtures of propi- cussed. Accordingly, methylation using SAM, and onate and acetate units. The macrolides are a incorporation of propionate via methylmalonyl- large family of compounds, many with antibiotic CoA, provide two different ways of synthesiz- activity, characterized by a macrocyclic lac- ing a methylated polyketide (Figure 3.58). The tone ring, typically 12, 14, or 16 membered, former process is the more common in fungi, reflecting the number of units utilized. Zear- whilst Actinomycetes (e.g. Streptomyces)tendto alenone (Figure 3.59), a toxin produced by the MACROLIDES AND POLYETHERS 95

Methylation using SAM Ad Me S CO2H R SCoA + 3 x SCoA SEnz SEnz

O O OOOO OOOO malonyl-CoA

Incorporation of propionate via methylmalonyl-CoA SCoA

O propionyl-CoA

CO2, ATP biotin

CO2H SCoA + 3x SCoA SEnz

O O OOOO methylmalonyl-CoA

Figure 3.58

Fate of carbonyls: reduction O SEnz CO2H no reduction; reduction SCoA O + 8 x SCoA cyclization to O O dehydration aromatic ring reduction O O O O O no reduction O O see Figure 3.60 O CCH reduction reduction 2 reduction dehydration dehydration (ketoreductase) reduction O SEnz OH OH O aldol reaction, CH CH2 aromatization, O dehydration O lactone formation O HO (dehydratase) HO O CH CH reduction O O (enoylreductase) zearalenone enzyme-bound partially-reduced intermediate CH2 CH2

Figure 3.59 fungus Gibberella zeae and several Fusarium tetracyclines, page 89). Instead, appropriate reduc- species, has a relatively simple structure which tions, dehydrations, etc, involving the β-carbonyl is derived entirely from acetate–malonate units. group are achieved during the chain extension pro- It could be envisaged as a cyclization prod- cess as in the fatty acid pathway (see page 36), uct from a poly-β-keto ester, requiring a vari- and before further malonyl-CoA extender units ety of reduction processes and formation of an are added (Figure 3.60). In contrast to fatty acid aromatic ring by aldol condensation near the biosynthesis, where there is total reduction of each carboxyl terminus (Figure 3.59). However, the carbonyl group before further chain extension, poly-β-keto ester shown in Figure 3.59 would macrolide biosynthesis frequently involves par- not be produced, since its reactivity might tend tial reduction, with the enzymic machinery being to favour formation of a polycyclic aromatic accurately controlled to leave the units at the system (compare anthraquinones, page 63, and right oxidation level before further chain extension 96 THE ACETATE PATHWAY

SEnz SEnz SEnz SEnz SEnz SEnz SEnz SEnz SEnz O O O O O O O O O

HO O O O O

HO O O O

HO O O

HO O

HO O

HO O OH O

O HO

HO HO

O zearalenone

Figure 3.60 occurs. This then provides an enzyme-bound inter- restricted to this group of natural products. In mediate, which leads on to the final product erythromycin A, the sugars are L-cladinose and (Figure 3.59). As a result, zearalenone is a remark- D-desosamine. Chain extension and appropriate able example of an acetate-derived metabolite con- reduction processes lead to an enzyme-bound taining all types of oxidation level seen during the polyketide in which one carbonyl group has suf- fatty acid extension cycle, i.e. carbonyl, secondary fered total reduction, four have been reduced to alcohol (eventually forming part of the lactone), alcohols, whilst one carbonyl is not reduced, and alkene, and methylene, as well as having a portion remains throughout the sequence. These processes which has cyclized to an aromatic ring because no ultimately lead to release of the modified polyke- reduction processes occurred in that fragment of tide as the macrolide ester deoxyerythronolide, the chain. There is now extensive genetic evidence a demonstrated intermediate in the pathway to from a variety of polyketide synthase systems to erythromycins (Figure 3.61; see also page 115). show that macrolide assembly is accomplished on The stereochemistry in the chain is controlled a biological production line of multifunctional pro- by the condensation and reduction steps during teins organised as discrete modules, in which the chain extension, but a reassuring feature is that developing polyketide chain attached to an acyl there appears to be a considerable degree of carrier protein is modified according to the appro- stereochemical uniformity throughout the known priate enzyme activities encoded genetically, and macrolide antibiotics. In the later stages of the is then passed on to another ACP prior to the next biosynthesis of erythromycin, hydroxylations at condensation and modification (see page 115 for carbons 6 and 12, and addition of sugar units, are more details). achieved. Erythromycin A (Figure 3.61) from Saccha- A combination of propionate and acetate units ropolyspora erythraea is a valuable antibacte- is used to produce the 14-membered macrocyclic rial drug and contains a 14-membered macrocy- ring of oleandomycin (Figure 3.62) from Strep- cle composed entirely of propionate units, both tomyces antibioticus, but otherwise many of the as starter and extension units, the latter via structural features and the stereochemistry of ole- methylmalonyl-CoA. In common with many anti- andomycin resemble those of erythromycin A. One bacterial macrolides, sugar units, including amino acetate provides the starter unit, whilst seven propi- sugars, are attached through glycoside linkages. onates, via methylmalonyl-CoA, supply the exten- These unusual 6-deoxy sugars are frequently sion units (Figure 3.62). One methyl group derived MACROLIDES AND POLYETHERS 97

Fate of carbonyls: not reduced O reduced reduced CO2H dehydrated SCoA + SCoA reduced 6 x reduced OH O O OH OH SEnz O reduced O O OH 9 8 HO10 OH reduced 11 7

12 OH 6 NMe2 OH 13 5 HO O 4 O O OOH 1 3 2 D-desosamine O O OMe O OH erythronolide O OH deoxyerythronolide L-cladinose erythromycin A

Figure 3.61

O O SCoA O O OH OH NMe + 2 HO O CO H OH OH O O 2 SEnz D-desosamine 6 x SCoA O O O OH O oleandolide OMe O OH L-oleandrose oleandomycin

Figure 3.62 from propionate has been modified to give an the result of carbonyl reductions being followed epoxide function. The sugar units in oleandomycin by dehydration during chain assembly. Tylosin are L-oleandrose and D-desosamine. Spiramycin I (Figure 3.64) from Streptomyces fradiae has many (Figure 3.63) from Streptomyces ambofaciens has structural resemblances to the spiramycins, but a 16-membered lactone ring, and is built up from can be analysed as a propionate starter with a combination of six acetate units (one as starter), chain extension from two malonyl-CoA, four one propionate extender, together with a further methylmalonyl-CoA, and one ethylmalonyl-CoA. variant, butyrate as chain extender. Butyrate will The avermectins∗ (Figure 3.67) have no anti- be incorporated via ethylmalonyl-CoA and yield bacterial activity, but possess anthelmintic, insec- an extension unit having an ethyl side-chain. This ticidal, and acaricidal properties, and these are is outlined in Figure 3.63. In due course, this exploited in human and veterinary medicine. The ethyl group is oxidized generating an aldehyde. avermectins are also 16-membered macrolides, but Spiramycin I also contains a conjugated diene, their structures are made up from a much longer 98 THE ACETATE PATHWAY

CO2HCOCO2H 2H SCoA + 5 x SCoA ++SCoA SCoA

O O O O ethylmalonyl-CoA

O Me2N OH O D-forosamine O

NMe2 MeO HO OOH O OH O O O D-mycaminose OH OOR O OH EnzS OH R = H, leuconolide A1 L-mycarose R = H, spiramycin I R = COCH3, spiramycin II R = COCH2CH3, spiramycin III

Figure 3.63

CO2H CO2H CO2H SCoA + 2 x SCoA + 4 x SCoA + SCoA O O O O O O O

NMe2 HO O HO OOH O O O O OMe O OH D-mycaminose OMe OOH O OH OH OH D-mycinose EnzS tylonolide L-mycarose tylosin

Figure 3.64

Macrolide Antibiotics The macrolide antibiotics are macrocyclic lactones with a ring size typically 12–16 atoms, and with extensive branching through methyl substituents. Two or more sugar units are attached through glycoside linkages, and these sugars tend to be unusual 6-deoxy structures often restricted to this class of compounds. Examples include L-cladinose, L-mycarose, D-mycinose, and L-oleandrose. At least one sugar is an amino sugar, e.g. D-desosamine, D-forosamine, and D-mycaminose. These antibiotics have a narrow spectrum of antibacterial activity, principally against Gram-positive microorganisms. Their antibacterial spectrum resembles, but is not identical to, that of the penicillins, so they provide a valuable alternative for patients allergic to the penicillins. Erythromycin is the principal macrolide antibacterial currently used in medicine. The erythromycins (Figure 3.65) are macrolide antibiotics produced by cultures of Saccharopolyspora erythraea (formerly Streptomyces erythreus). The commercial product

(Continues) MACROLIDES AND POLYETHERS 99

(Continued)

O O Me N 1 R OH D-desosamine HO OMe HO OH D-desosamine OH NMe2 OH NMe2 OH NMe2 HO HO HO O O O O O O O O O

2 O OOR O O OMe O O OMe

OH O O OH O OH 1 2 R = OH, R = Me, erythromycin A clarithromycin L-cladinose azithromycin R1 = H, R2 = Me, erythromycin B (6-O-methyl erythromycin A) R1 = OH, R2 = H, erythromycin C

Figure 3.65 erythromycin is a mixture containing principally erythromycin A, plus small amounts of erythromycins B and C (Figure 3.65). Erythromycin activity is predominantly against Gram- positive bacteria, and the antibiotic is prescribed for penicillin-allergic patients. It is also used against penicillin-resistant Staphylococcus strains, in the treatment of respiratory tract infections, and systemically for skin conditions such as acne. It is the antibiotic of choice for infections of Legionella pneumophila, the cause of legionnaire’s disease. Erythromycin exerts its antibacterial action by inhibiting protein biosynthesis in sensitive organisms. It binds reversibly to the larger 50S subunit of bacterial ribosomes and blocks the translocation step in which the growing peptidyl-tRNA moves from the aminoacyl acceptor site to the peptidyl donor site on the ribosome (see page 408). The antibiotic is a relatively safe drug with few serious side-effects. Nausea and vomiting may occur, and if high doses are prescribed, a temporary loss of hearing might be experienced. Hepatotoxicity may also occur at high dosage. Erythromycin is unstable under acidic conditions, undergoing degradation to inactive compounds by a process initiated by the 6-hydroxyl attacking the 9-carbonyl to form a hemiketal. Dehydration then follows (Figure 3.66). The 14-membered ring in erythromycin A adopts a modified version of the diamond lattice chairlike conformation shown in Figure 3.66. Studies have indicated that carbon 6 is displaced from this conformation to reduce the 1,3-diaxial interactions at C-4 and C-6, and the two relatively large sugar units attached to the hydroxyls at C-3 and C-5 also distort the ring system further. The distortion of the chairlike conformation brings the 6-hydroxyl sufficiently close to react with the 9-carbonyl. A similar reaction may occur between the C-12 hydroxyl and the 9-carbonyl. Thus, to protect oral preparations of erythromycin against gastric acid, they are formulated as enteric-coated tablets, or as insoluble esters (e.g. ethyl succinate esters), which are then hydrolysed in the intestine. Esterification typically involves the hydroxyl of the amino sugar desosamine. To reduce this acid instability, semi-synthetic analogues of erythromycin have also been developed. Clarithromycin (Figure 3.65) is a 6-O-methyl derivative of erythromycin A; this modification blocks hemiketal formation as in Figure 3.66. Azithromycin (Figure 3.65) is a ring-expanded aza-macrolide in which the carbonyl function has been reduced. In both analogues, the changes enhance activity compared with that of erythromycin. Bacterial resistance to erythromycin has become significant and has limited its therapeutic use against many strains of Staphylococcus. Several mechanisms of resistance have been

(Continues) 100 THE ACETATE PATHWAY

(Continued)

O acid-catalysed formation 9 OH of hemiacetal HO OH OH H 12 OH 6 NMe2 OH HO O O O O

O O OMe

O OH erythromycin A H2O H O dehydration OH – H2O OH 7 5 9 6 OSugar O O 8 10 ...... 4 11 ...... 3 OSugar HO ...... 13 O 1 12 O 2

diamond-lattice conformation

Figure 3.66

implicated, one of which is a change in permeability of the bacterial cell wall. Differences in permeability also appear to explain the relative insensitivity of Gram-negative bacteria to erythromycin when compared to Gram-positive bacteria. Resistant bacteria may also modify the chemical nature of the binding site on the ribosome, thus preventing antibiotic binding, and some organisms are now known to metabolize the macrolide ring to yield inactive products. Oleandomycin (Figure 3.62) is produced by fermentation cultures of Streptomyces antibioticus and has been used medicinally as its triacetyl ester troleandomycin against Gram-positive bacterial infections. The spiramycins (Figure 3.63) are macrolides produced by cultures of Streptomyces ambofaciens. The commercial antibiotic is a mixture containing principally spiramycin I, together with smaller amounts (10–15% each) of the acetyl ester spiramycin II and the propionyl ester spiramycin III. This antibiotic has recently been introduced into medicine for the treatment of toxoplasmosis, infections caused by the protozoan Toxoplasma gondii. Tylosin (Figure 3.64) is an important veterinary antibiotic. It is produced by Streptomyces fradiae, and is used to control chronic respiratory diseases caused by Mycoplasma galliseptum in poultry, and to treat Gram-positive infections in pigs. polyketide chain, which is also used to form Both malonyl-CoA and methylmalonyl-CoA are oxygen heterocycles fused to the macrolide. Aver- then utilized as extender units. The heterocyclic mectin B1a exemplifies a typical structure and the rings are easily accounted for: the spiro sys- basic carbon skeleton required to produce this can tem is merely a ketal, though the tetrahydrofu- be postulated as in Figure 3.67. The starter unit in ran ring requires further hydroxylations of the this case would be 2-methylbutyryl-CoA, which is basic skeleton for its construction. Avermectins derived from the amino acid L-isoleucine (compare are usually isolated as a mixture in which the necic acids, page 305, and tiglic acid, page 197). main a component has a 2-methylpropyl group MACROLIDES AND POLYETHERS 101

CO H OH CO2H 2 HO SCoA SCoA O + 7 x SCoA + 5 x HO

O O O HO O SEnz

O CO2H spiro-ketal 23 NH2 22 O L-Ile O H O OH O 25 O O O H OH 23 MeO 22 O O O HO L-oleandrose avermectin B HO 1a MeO aglycone O H L-oleandrose 5 O avermectin B2a H OH 23 22 avermectin B1a

23 O 22 H CO2H SCoA 25 O ivermectin NH2 O H L-Val avermectin B1b

Figure 3.67

Avermectins The avermectins (Figure 3.67) are a group of macrolides with strong anthelmintic, insecticidal, and acaricidal properties, but with low toxicity to animals and humans. They are produced by cultures of Streptomyces avermectilis. Some eight closely related structures have been identi- fied, with avermectins B1a and B2a being the most active antiparasitic compounds. Abamectin (a mixture of about 85% avermectin B1a and about 15% avermectin B1b) is used on agricultural crops to control mites and insects. Ivermectin (Figure 3.67) is a semi-synthetic 22,23-dihydro derivative of avermectin B1a and was first used in veterinary practice against insects, ticks, mites, and roundworms. Although it is a broad spectrum nematocide against roundworms, it is inactive against tapeworms and flatworms, or against bacteria and fungi. It is an extremely potent agent, and is effective at very low dosages. It has now been introduced for use against filarial and several other worm parasites in humans. Avermectins act by blocking neuromus- cular transmission in sensitive organisms by acting on GABA (γ-aminobutyric acid) receptors.

(derived from isoleucine) at C-25, whilst the minor Even larger macrolides are encountered in the b component has an isopropyl group instead, e.g. polyene macrolides∗, most of which have anti- avermectin B1b. In this case, the starter group is fungal properties, but not antibacterial activity. 2-methylpropionyl-CoA, derived from the amino The macrolide ring size ranges from 26 to 38 acid L-valine. The A-series of avermectins are the atoms, and this also accommodates a conjugated 5-methoxy analogues of the B-series. polyene of up to seven E double bonds. Relatively 102 THE ACETATE PATHWAY few methyl groups are attached to the ring, and of which is introduced by hydroxylation, and thus malonyl-CoA is utilized more frequently than the two areas of conjugation in nystatin A1 are methylmalonyl-CoA as chain extender. Typical extended into a heptaene system in amphotericin examples are amphotericin B (Figure 3.68) from B. Both compounds are glycosylated with the Streptomyces nodosus and nystatin A1 from Strep- amino sugar D-mycosamine, and both are car- tomyces noursei. These have very similar struc- boxylic acids, a result of oxidation of a propionate- tures and are derived from the same basic precur- derived methyl group. sors (Figure 3.68). The ring size is contracted due An unusual and clinically significant macrolide to cross-linking by formation of a hemiketal. They isolated from Streptomyces tsukubaensis is FK-506 have slightly different hydroxylation patterns, part (tacrolimus)∗ (Figure 3.69), which contains a

CO2H CO2H SCoA + 15 x SCoA + 3 x SCoA O O O O

EnzS OH OH O HO OOHOHOHOH HO O

O OH EnzS OH OH OOHOHOH OHO hemiketal HO HO

OH O OH OH O OH hemiketal HO OOHOH OH OH O CO H 28 2 OH OH 29 HO O OH HO O O H2N HO OOHOHOH OH O CO H D-mycosamine 2 amphotericin B HO HO O O H2N D-mycosamine nystatin A1

Figure 3.68

Polyene Antifungals

The polyene antifungals are a group of macrocyclic lactones with a very large 26–38- membered ring. They are characterized by the presence of a series of conjugated E double bonds and are classified according to the longest conjugated chain present. Medicinally important ones include the heptaene amphotericin B, and the tetraene nystatin. There are relatively few methyl branches in the macrocyclic chain. The polyenes have no antibacterial

(Continues) MACROLIDES AND POLYETHERS 103

(Continued)

activity but are useful antifungal agents. Their activity is a result of binding to sterols in the eukaryotic cell membrane, which action explains the lack of antibacterial activity because bacterial cells do not contain sterol components. Fungal cells are also attacked rather than mammalian cells, since the antibiotics bind much more strongly to ergosterol, the major fungal sterol (see page 253), than to cholesterol, the main animal sterol component (see page 236). This binding modifies the cell wall permeability and leads to formation of transmembrane pores allowing K+ ions, sugars, and proteins to be lost from the microorganism. Though binding to cholesterol is less than to ergosterol, it is responsible for the observed toxic side-effects of these agents on humans. The polyenes are relatively unstable, undergoing light-catalysed decomposition, and are effectively insoluble in water. This insolubility actually protects the antibiotic from gastric decomposition, allowing oral treatment of infections in the intestinal tract. Amphotericin is an antifungal polyene produced by cultures of Streptomyces nodosus and contains principally the heptaene amphotericin B (Figure 3.68) together with structurally related compounds, e.g. the tetraene amphotericin A (about 10%), which is the 28,29- dihydro analogue of amphotericin B. Amphotericin A is much less active than amphotericin B. Amphotericin is active against most fungi and yeasts, but it is not absorbed from the gut, so oral administration is restricted to the treatment of intestinal candidiasis. It is administered intravenously for treating potentially life-threatening systemic fungal infections. However, it then becomes highly protein bound resulting in poor penetration and slow elimination from the body. After parenteral administration, toxic side-effects, including nephrotoxicity, are relatively common. Close supervision and monitoring of the patient is thus necessary, especially since the treatment may need to be prolonged. A liposome-encapsulated formulation of amphotericin has been shown to be much less toxic and may prove a significant advance. Candida infections in the mouth or on the skin may be treated with appropriate formulations. Nystatin is a mixture of tetraene antifungals produced by cultures of Streptomyces noursei. The principal component is nystatin A1 (Figure 3.68), but the commercial material also contains nystatin A2 and A3, which have additional glycoside residues. Nystatin is too toxic for intravenous use, but has value for oral treatment of intestinal candidiasis, as lozenges for oral infections, and as creams for topical control of Candida species.

23-membered macrolactone that also incorporates again utilized in its formation, whilst the rest of the an N-heterocyclic ring. This compound is known to skeleton is supplied by simple acetate and propi- be derived from acetate and propionate, the frag- onate residues (Figure 3.69). ments of which can readily be identified in the Attracting considerable interest at the present main chain. The starter unit is cyclohexanecar- time are the epothilones (Figure 3.70), a group boxylic acid, a reduction product from shikimate, of macrolides produced by cultures of the bac- and the piperidine ring and adjacent carbonyl are terium Sorangium cellulosum. These compounds incorporated as pipecolic acid (see page 310) via employ an unusual starter unit containing a thi- an amide linkage on to the end of the growing azole ring, which is almost certainly constructed chain. An unusual pentanoic acid unit is also incor- from the amino acid cysteine and an acetate unit porated to provide the propenyl side-chain. FK-506 (see also thiazole rings in bleomycin, page 429). is a particularly effective immunosuppressant, and The macrolide ring also contains an extra methyl is proving valuable in organ transplant surgery. group at C-4, the result of methylation after or Although rapamycin (sirolimus)∗ contains a very during polyketide chain assembly. The other inter- large 31-membered macrocycle, several portions esting feature is that this bacterium produces of the structure are identical to those of FK-506. epothilone A and epothilone B in the ratio of Cyclohexanecarboxylic acid and pipecolic acid are about 2:1. These compounds differ in the nature 104 THE ACETATE PATHWAY

CO2H CO2H CO2H CO2H SCoA SCoA SCoA + 4 x SCoA + 5 x + + NH O O O O cyclohexanecarbonyl-CoA propylmalonyl-CoA pipecolic acid (from shikimic acid) HO HO

22 O O MeO MeO H OH OH O OOH O SEnz H

N 17 N O O OH OH O O O OMe H OMe tacrolimus (FK506)

OMe HO O HO O

OH O OH O MeO MeO H OH O O O SEnz H N N O HO O MeO OH OH O O O H OMe rapamycin (sirolimus)

Figure 3.69

Tacrolimus and Sirolimus Tacrolimus (FK-506) (Figure 3.69) is a macrolide immunosuppressant isolated from cultures of Streptomyces tsukubaensis. It is used in liver and kidney transplant surgery. Despite the significant structural differences between tacrolimus and the cyclic peptide cyclosporin A (ciclosporin; see page 429), these two agents have a similar mode of action. They both inhibit T-cell activation in the immunosuppressive mechanism by binding first to a receptor protein giving a complex, which then inhibits a phosphatase enzyme called calcineurin. The resultant aberrant phosphorylation reactions prevent appropriate gene transcription and subsequent T-cell activation. Structural similarities between the region C-17 to C-22 and fragments of the cyclosporin A peptide chain have been postulated to account for this binding. Tacrolimus is

(Continues) MACROLIDES AND POLYETHERS 105

(Continued)

up to 100 times more potent than cyclosporin A, but produces similar side-effects including neurotoxicity and nephrotoxicity. Rapamycin (sirolimus) (Figure 3.69) is produced by cultures of Streptomyces hygroscop- icus and is also being investigated as an immunosuppressant drug. Although tacrolimus and rapamycin possess a common structural unit, and both inhibit T-cell activation, they appear to achieve this by somewhat different mechanisms. The first-formed rapamycin–receptor protein binds not to calcineurin, but to a different protein. Rapamycin suppresses lympho- cyte production. Rapamycin also possesses pronounced antifungal activity, but is not active against bacteria.

HS HS S O O CO2H CO2H + + SCoA N SCoA 3 x SCoA 5 x SCoA H2N CO2H N CO2H H O O O L-Cys

R O 12 S S OH N OH N Me O OH 4 SEnz O OH O O OH O R = Me, epothilone B R = H, epothilone A

Figure 3.70 of the substituent at C-12, which is hydrogen in macrolides∗. These are actually lactams rather epothilone A but a methyl group in epothilone B. than lactones, and the nitrogen atom originates Genetic evidence shows that the polyketide syn- from 3-amino-5-hydroxybenzoic acid,whichacts thase enzyme can accept either malonyl-CoA or as the starter unit for chain extension with methylmalonyl-CoA extender units for this posi- malonyl-CoA or methylmalonyl-CoA. 3-Amino- tion. Thus, epothilone B is constructed from three 5-hydroxybenzoic acid (Figure 3.71) is a simple malonate and five methylmalonate extender units phenolic acid derivative produced by an unusual as shown in Figure 3.70, whilst epothiolone A variant of the shikimate pathway (see Chapter 4), requires four units of each type. The epothilones in which aminoDAHP is formed in the initial display marked antitumour properties with a mode step, and then the pathway continues with of action paralleling that of the highly success- amino analogues. This proceeds through to ful anticancer drug taxol (see page 205). How- aminodehydroshikimic acid which yields 3-amino- ever, the epothilones have a much higher potency 5-hydroxybenzoic acid on dehydration. In the (2000–5000 times) and are active against cell lines biosynthesis of rifamycin B (Figure 3.71) in which are resistant to taxol and other drugs. There Amycolatopsis mediterranei, this starter unit, appears to be considerable potential for developing plus two malonyl-CoA and eight methylmalonyl- the epothilones or analogues into valuable anti- CoA extenders, are employed to fabricate cancer drugs. proansamycin X as the first product released A further group of macrolides in which non- from the enzyme. The enzyme-bound intermediate adjacent positions on an aromatic ring are bridged shown in Figure 3.71 is not strictly correct, in by the long aliphatic chain is termed ansa that the naphthoquinone ring system is now 106 THE ACETATE PATHWAY

PEP CO H 2 CO2H CO2H NH3 CO2H PO PO O – H O PO 2

HO NH2 O NH2 HO O HO NH2 OH OH OH aminoDAHP aminodehydroshikimic 3-amino-5-hydroxy- D-erythrose 4-P acid benzoic acid HSCoA

HO O SCoA CO H CO H HO OH OH 2 2 + 2 x SCoA + 8 x SCoA O EnzS NH2 HO NH2 O O O O OH O

HO HO O O OH OH HO OH OH HO OH OH MeO O H OH O H OH H OH OH H H N N N

O HO O O HO HO O O O O CO H O carbon O 2 O O O lost proansamycin X rifamycin W rifamycin B

Figure 3.71 known to be constructed during, not after, chain O Me assembly. Rifamycin W and then the antibiotic N Me O rifamycin B are the result of further modifications Cl O N O O including cleavage of the double bond, loss of one MeO carbon, then formation of the ketal. Maytansine (Figure 3.72) is a plant-derived ansa macrolide O from Maytenus serrata (Celastraceae), though other esters of the parent alcohol, maytansinol, N O H are produced by species of the fungus Nocardia. MeO OH Maytansine has been extensively investigated for maytansine its potential antitumour activity. The macrolide systems described above are pro- Figure 3.72 duced by formation of an intramolecular ester or amide linkage, utilizing appropriate functionali- are lasalocid A (Figure 3.74) from Streptomyces ties in the growing polyketide chain. Macrolide lasaliensis and monensin A (Figure 3.75) from formation does not always occur, and simi- Streptomyces cinnamonensis, representatives of a lar acetate–propionate precursors might also be large group of compounds called polyether antibi- expected to yield molecules which are essentially otics. These, and other examples, are of value in linear in nature. Good examples of such molecules veterinary medicine, being effective in preventing MACROLIDES AND POLYETHERS 107

Ansa Macrolides Ansamycins are a class of macrocyclic compounds in which non-adjacent positions on an aromatic ring system are spanned by the long aliphatic bridge (Latin: ansa = handle). The aromatic portion may be a substituted naphthalene or naphthaquinone, or alternatively a substituted benzene ring. The macrocycle in the ansamycins is closed by an amide rather than an ester linkage, i.e. ansamycins are lactams. The only ansamycins currently used therapeutically are semi-synthetic naphthalene-based macrocycles produced from rifamycin B. The rifamycins are ansamycin antibiotics produced by cultures of Amycolatopsis mediterranei (formerly Nocardia mediterranei or Streptomyces mediterranei). The crude antibiotic mixture was found to contain five closely related substances rifamycins A–E, but if the organism was cultured in the presence of sodium diethyl barbiturate (barbitone or barbital), the product was almost entirely rifamycin B (Figure 3.71). Rifamycin B has essentially no antibacterial activity, but on standing in aqueous solution in the presence of air, it is readily transformed by oxidation and intramolecular nucleophilic addition into rifamycin O, which

OH O OH OH H H OH O H N O2 N N HONO O O or O O O OH O electrochemical O O O OCO2H oxidation O O O O O O O O rifamycin B nucleophilic attack on rifamycin O to quinone system hydrolysis of H ketal O OH O H2NMeNN OH OH H O H MeO OH OH N N HCHO reduction OH OH H O O N Mannich O ascorbic O reaction O OH acid O O O O O O rifamycin SV rifamycin S O OH N O N conjugate N NH3 rifampicin Me O addition on to quinone O MeO OH OH OH OH H OH O H N N O O O NH2 O NH O OH O N O O 3-amino-rifamycin SV N rifabutin

Figure 3.73

(Continues) 108 THE ACETATE PATHWAY

(Continued)

under acidic conditions then hydrolyses and gives rifamycin S, a highly active antibacterial agent (Figure 3.73). Chemical reduction of rifamycin S using ascorbic acid (vitamin C) converts the quinone into a quinol and provides a further antibacterial, rifamycin SV. Rifamycins O, S, and SV can all be obtained by fermentation using appropriate strains of A. mediterranei. Rifamycin SV is actually the immediate biosynthetic precursor of rifamycin B under normal conditions, so this conversion can be genetically blocked and lead to accumulation of rifamycin SV. Several other rifamycin analogues have also been characterized. Rifamycin O is usually produced by chemical or electrochemical oxidation of rifamycin B, and converted into rifamycin SV as in Figure 3.73. The most useful rifamycin employed clinically is rifampicin (Figure 3.73), a semi- synthetic derivative produced from rifamycin SV via a Mannich reaction (see page 18) using formaldehyde and N-amino-N-methylpiperazine. Rifampicin has a wide antibacterial spectrum, with high activity towards Gram-positive bacteria and a lower activity towards Gram-negative organisms. Its most valuable activity is towards Mycobacterium tuberculosis and rifampicin is a key agent in the treatment of tuberculosis, usually in combination with at least one other drug to reduce the chances for development of resistant bacterial strains. It is also useful in control of meningococcal meningitis and leprosy. Rifampicin’s antibacterial activity arises from inhibition of RNA synthesis by binding to DNA-dependent RNA polymerase. RNA polymerase from mammalian cells does not contain the peptide sequence to which rifampicin binds, so RNA synthesis is not affected. In contrast to the natural rifamycins which tend to have poor absorption properties, rifampicin is absorbed satisfactorily after oral administration, and is also relatively free of toxic side-effects. The most serious side-effect is disturbance of liver function. A trivial, but to the patient potentially worrying, side-effect is discoloration of body fluids, including urine, saliva, sweat, and tears, to a red–orange colour, a consequence of the naphthalene/naphthoquinone chromophore in the rifamycins. Rifamycin, the sodium salt of rifamycin SV (Figure 3.73), has also been used clinically in the treatment of Gram-positive infections, and particularly against tuberculosis. Rifabutin (Figure 3.73) is a newly introduced derivative, synthesized via 3-amino-rifamycin SV, which also has good activity against the Mycobacterium avium complex frequently encountered in patients with AIDS.

CO H increase in osmotic pressure. Current thinking is 2 that these ring systems arise via a cascade cycliza- HO tion mechanism, probably involving epoxide inter- O OH OH O O mediates. Thus, in the biosynthesis of monensin A (Figure 3.75), chain assembly from acetate, mal- lasalocid A onate, methylmalonate, and ethylmalonate precur- sors could produce the triene shown. If the triepox- Figure 3.74 ide is then formed, a concerted stereospecific cyclization sequence initiated by a hydroxyl and and controlling coccidiae and also having the involving carbonyls and epoxides could proceed ability to improve the efficiency of food con- as indicated. version in ruminants. The polyether antibiotics Even more remarkable polyether structures are are characterized by the presence of a number found in some toxins produced by marine dinoflag- of tetrahydrofuran and/or tetrahydropyran rings ellates, which are in turn taken up by shellfish and along the basic chain. The polyether acts as pass on their toxicity to the shellfish. Okadaic an ionophore, increasing influx of sodium ions acid (Figure 3.76) and related polyether struc- into the parasite, causing a resultant and fatal tures from Dinophysis species are responsible for MACROLIDES AND POLYETHERS 109

CO2H CO2H CO2H SCoA + 4 x SCoA + 7 x SCoA + SCoA O O O O

epoxidations HO HO

O O OOO OH OH HO O HO O EnzS EnzS concerted cyclization O O sequence

HO

O O O O O MeO HO OH

HO2C monensin A

Figure 3.75

OH O HO2C O O O O OH OH OO OH okadaic acid

Figure 3.76 diarrhoeic shellfish poisoning in mussels, causing concerted sequence of epoxide ring openings leads severe diarrhoea in consumers of contaminated to the extended polyether structure (Figure 3.77). shellfish in many parts of the world. Brevetoxin A The carbon skeleton does not conform to a sim- (Figure 3.77) is an example of the toxins asso- ple polyketide chain, and biosynthetic studies have ciated with ‘red tide’ blooms of dinoflagellates, shown that fragments from the citric acid cycle and which affect fishing and also tourism especially in a four-carbon starter unit from mevalonate are also Florida and the Gulf of Mexico. The red tide toxins involved, and that some of the methyls originate are derived from Gymnodimium breve and are the from methionine. Ciguatoxin (Figure 3.78) is one causative agents of neurotoxic shellfish poisoning, of the most complex examples of a polyether struc- leading to neurological disorders as well as gas- ture found in nature. This is found in the moray eel trointestinal troubles. The toxins are known to bind (Gymnothorax javanicus) and in a variety of coral to sodium channels, keeping them in an open state. reef fish, such as red snapper (Lutjanus bohar). Fatalities among marine life, e.g. fish, dolphins, Ciguatoxin is remarkably toxic even at microgram whales, and in humans, are associated with these levels, causing widespread food poisoning (ciguat- toxins synthesized by organisms at the base of the era) in tropical and subtropical regions, charac- marine food chain. These compounds are postu- terized by vomiting, diarrhoea, and neurological lated to be produced from a polyunsaturated fatty problems. Most sufferers slowly recover, and few acid by epoxidation of the double bonds, and then a cases are fatal, due principally to the very low 110 THE ACETATE PATHWAY

HO

O

EnzS

HO H O epoxidations O O O O O O O O O O O

O HO concerted cyclization sequence O O O O O O O

O O O O

O brevetoxin-A

Figure 3.77

O OH HO O O O O O O O OH O O O O O OH

HO OH ciguatoxin

Figure 3.78 levels of toxin actually present in the fish. A formed by an epoxide-initiated process resembling dinoflagellate Gambierdiscus toxicus is ultimately the polyether derivatives just described. Thus, responsible for polyether production, synthesizing zaragozic acid A (Figure 3.79) is known to be a less toxic analogue, which is passed through the constructed from two acetate-derived chains and food chain and eventually modified into the very aC4 unit such as the Krebs cycle intermediate toxic ciguatoxin by the fish. oxaloacetate (see Figure 2.1). One chain has a The zaragozic acids (squalestatins) are not benzoyl-CoA starter (from the shikimate pathway, macrolides, but they are primarily acetate derived, see page 141), and both contain two methionine- and the central ring system is suggested to be derived side-chain substituents (Figure 3.79). The CYCLIZATION THROUGH DIELS–ALDER REACTIONS 111

O OH OAc O O H3C COSCoA HO2C O CoAS HO C H • Met 2 O CO2H OH HO C 2 zaragozic acid A CO2H (squalestatin S1) succinic acid

OPP presqualene PP

Figure 3.79

concerted aldol formation of O O H nucleophilic HO reaction epoxide functions O reactions HO2C HO C O 2 HO2C O HO2C O O O HO2C HO C O HO2C 2 HO2C HO CO2H CO2H HO H2O CO2H H OCO2H oxaloacetic acid

Figure 3.80 heterocyclic ring system can be envisaged as the acetate pathway, but experimental evidence arising via nucleophilic attack on to oxaloacetic supports cyclization processes different from the acid, formation of a diepoxide, then a concerted aldol and Claisen reactions seen in the biosynthe- sequence of reactions as indicated (Figure 3.80). sis of aromatic compounds. They can, however, be The zaragozic acids are produced by a number rationalized in terms of an enzymic Diels–Alder of fungi, including Sporomiella intermedia and reaction, represented as the electrocyclic sequence Leptodontium elatius, and are attracting consider- shown in Figure 3.81. Thus, lovastatin can be able interest since they are capable of reducing formulated as arising from two polyketide chains blood cholesterol levels in animals by acting as with C-methylation as outlined in Figure 3.82, potent inhibitors of the enzyme squalene synthase with relatively few of the oxygen functions being (see page 212). This is achieved by mimicking retained in the final product. Accordingly, it is the steroid precursor presqualene PP (Figure 3.79) possible that lovastatin is formed by cycliza- and irreversibly inactivating the enzyme. They thus tion of the trienoic acid (Figure 3.82), which is have considerable medical potential for reducing likely to arise by a variant of the macrolide the incidence of coronary-related deaths (compare the statins, below).

CYCLIZATION THROUGH DIELS–ALDER REACTIONS diene dienophile Diels−Alder reaction A number of cyclic structures, typically containing cyclohexane rings, are known to be formed via Figure 3.81 112 THE ACETATE PATHWAY

O SEnz HO O

O O O O O

SEnz O O H O Me O O lovastatin Me OO O O SAM O

SEnz SEnz

SCoA HO SEnz HO HO CO2H CO2H O O OH Diels–Alder OH OH CO2H + 8 x SCoA HO H H O + SAM H dihydromonacolin L

Figure 3.82

Mevastatin and other Statins Mevastatin (formerly compactin) (Figure 3.83) is produced by cultures of Penicillium citrinum and P. brevicompactum, and was shown to be a reversible competitive inhibitor of HMG- CoA reductase, dramatically lowering sterol biosynthesis in mammalian cell cultures and animals, and reducing total and low density lipoprotein cholesterol levels (see page 236). Mevastatin in its ring-opened form (Figure 3.84) mimics the half-reduced substrate mevaldate hemithioacetal during the two-stage reduction of HMG-CoA to mevalonate (see page 170), and the affinity of this agent towards HMG-CoA reductase is 10 000-fold more than the normal substrate. High blood cholesterol levels contribute to the incidence of coronary heart disease (see page 236), so mevastatin, or analogues, are of potential value in treating high risk coronary patients, and some agents are already in use. Although lowering of cholesterol levels reduces the risk of heart attacks, there is evidence that the beneficial effects of statins may extend beyond simply cholesterol reduction. Lovastatin (formerly called mevinolin or monacolin K) (Figure 3.83) is produced by Monascus ruber and Aspergillus terreus and is slightly more active than mevastatin, but has been superseded by more active agents. Simvastatin is obtained from lovastatin by ester hydrolysis and then re-esterification, and is two to three times as potent as lovastatin. Pravastatin is prepared from mevastatin by microbiological hydroxylation using Streptomyces carbophilus and is consequently more hydrophilic than the other drugs, with an activity similar to lovastatin. Lovastatin and simvastatin are both lactones, and are inactive until metabolized

(Continues) CYCLIZATION THROUGH DIELS–ALDER REACTIONS 113

(Continued)

HO O HO O HO CO2H O O OH O O O

O O O H H H

R HO R = H, mevastatin simvastatin pravastatin R = Me, lovastatin (mevinolin; monacolin K) HO CO H HO HO 2 CO2H CO2H OH OH OH F F F N

N H MeO N N O

cerivastatin fluvastatin atorvastatin

Figure 3.83

HO HO HO HO CO2H CO2H NADPH CO2H CO2H OH O OH O O H H mevaldic acid CoAS CoAS O H HMG-CoA mevaldic acid NADPH hemithioacetal HO CO2H mevastatin OH (opened lactone form) mevalonic acid

Figure 3.84

in the liver to the open-ring hydroxy acids typified by pravastatin. Other agents currently in use are synthetic, though they feature the same dihydroxycarboxylic acid side-chain as in pravastatin. Atorvastatin, cerivastatin,andfluvastatin have all been introduced recently. biosynthetic processes, though C-methylation must produced. The ester side-chain is derived as a occur during chain assembly whilst activating separate unit from two acetates with a methyl from carbonyl groups are available. The Diels–Alder methionine, again with C-methylation preceding reaction can then account for formation of the reduction processes. Lovastatin was isolated from decalin system and further reactions will allow cultures of Aspergillus terreus and was found to be the other functional groups in lovastatin to be a potent inhibitor of hydroxymethylglutaryl-CoA 114 THE ACETATE PATHWAY

(HMG-CoA) reductase, a rate-limiting enzyme Rapid advances in genetic engineering have in the mevalonate pathway (see page 169). Ana- now opened up tremendous scope for manipu- logues of lovastatin (statins∗) (Figure 3.83) find lating the processes of biosynthesis by provid- drug use as HMG-CoA reductase inhibitors, thus ing an organism with, or depriving it of, specific lowering blood cholesterol levels in patients. enzymes. The genes encoding a particular pro- tein (see page 407) can now be identified, syn- thesized, and inserted into a suitable organism for GENETIC MANIPULATION OF THE expression; to avoid complications with the nor- ACETATE PATHWAY mal biosynthetic machinery, this is usually dif- ferent from the source organism. Specific genes With only a few exceptions, the transformations in can be damaged or deleted to prevent a particu- any particular biosynthetic pathway are catalysed lar enzyme being expressed. Genes from different by enzymes. These proteins facilitate the chemi- organisms can be combined and expressed together cal modification of substrates by virtue of binding so that an organism synthesizes abnormal com- properties conferred by a particular combination of binations of enzyme activities, allowing produc- functional groups in the constituent amino acids. tion of modified products. Although the general As a result, enzymes tend to demonstrate quite approaches for genetic manipulation are essentially remarkable specificity towards their substrates, and the same for all types of organism and/or natu- usually catalyse only a single transformation. This ral product, it has proved possible to make best specificity means enzymes do not accept alterna- progress using the simpler organisms, especially tive substrates, or, if they do, they convert a limited bacteria, and in particular there have been some range of structurally similar substrates and usu- substantial achievements in the area of acetate- ally much less efficiently. Any particular organism derived structures. Accordingly, some results from thus synthesizes a range of secondary metabolites this group of compounds are used to exemplify dictated largely by its enzyme complement and how genetic manipulation may provide an extra the supply of substrate molecules. Occasionally, dimension in the search for new medicinal agents. where enzymes do possess broader substrate speci- However, it is important that an organism is not ficities, it is possible to manipulate an organism’s viewed merely as a sackful of freely diffusible and secondary metabolite pattern by supplying an alter- always available enzymes; biosynthetic pathways native, but acceptable, substrate. A good example are under sophisticated controls in which there may of this approach is in the directed biosynthesis be restricted availability or localization of enzymes of modified penicillins by the use of phenylacetic and/or substrates (see the different localizations of acid analogues in cultures of Penicillium chryso- the mevalonate and deoxyxylulose phosphate path- genum (see page 437), but its scope is generally ways to terpenoids in plants, page 172). Enzymes very limited. It has also been possible, particularly involved in the biosynthesis of many important with microorganisms, to select natural mutants, secondary metabolites are often grouped together or to generate mutants artificially, where the new as enzyme complexes, or may form part of a mul- strain synthesizes modified or substantially differ- tifunctional protein. ent products. For example, mutant strains of Strep- A detailed study of amino acid sequences tomyces aureofaciens synthesize tetracycline or and mechanistic similarities in various polyketide demeclocycline rather than chlortetracycline (see synthase (PKS) enzymes has led to two main page 90). Such mutants are usually deficient in a types being distinguished. Type I enzymes con- single enzyme and are thus unable to carry out a sist of one or more large multifunctional pro- single transformation, but the broader specificity teins that possess a distinct active site for every of later enzymes in the sequence means subse- enzyme-catalysed step. On the other hand, Type II quent modifications may still occur. However, as enzymes are multienzyme complexes that carry exemplified throughout this book, the vast bulk out a single set of repeating activities. Like of modified natural products of medicinal impor- fatty acid synthases, PKSs catalyse the condensa- tance are currently obtained by chemical synthesis tion of coenzyme A esters of simple carboxylic or semi-synthesis. acids. However, the variability at each step in GENETIC MANIPULATION OF THE ACETATE PATHWAY 115 the biosynthetic pathway gives rise to much more contains the activities needed for one cycle of structural diversity than encountered with fatty chain extension. A minimal module contains a β- acids. The usual starter units employed are acetyl- ketoacyl synthase (KS), an acyltransferase (AT), CoA or propionyl-CoA, whilst malonyl-CoA or and an acyl carrier protein (ACP), that together methylmalonyl-CoA are the main extender units. would catalyse a two-carbon chain extension. The At each cycle of chain extension, Type I PKSs specificity of the AT for either malonyl-CoA or an may retain the β-ketone, or modify it to a hydroxyl, alkyl-malonyl-CoA determines which two-carbon methenyl, or methylene, according to the presence chain extender is used. The starter unit used is of ketoreductase, dehydratase, or enoylreductase similarly determined by the specificity of the AT activities (see page 95). The enzyme activities for in a loading domain in the first module. After each extension cycle with its subsequent mod- each condensation reaction, the oxidation state of ification is considered a ‘module’. The linear the β-carbon is determined by the presence of sequence of modules in the enzyme corresponds a β-ketoacyl reductase (KR), a KR + a dehy- to the generated sequence of extender units in the dratase (DH), or a KR + DH + an enoylreductase polyketide product. The β-ketone groups are pre- (ER) in the appropriate module. The sequence is dominantly left intact by Type II PKSs, and the finally terminated by a thioesterase (TE) activity highly reactive polyketide backbone undergoes fur- which releases the polyketide from the enzyme and ther enzyme-catalysed intramolecular cyclization allows cyclization. Thus in DEBS, module 3 lacks reactions, which are responsible for generating a any β-carbon modifying domains, modules 1, 2, 5, range of aromatic structures (see page 61). and 6 contain KR domains and are responsible for 6-Deoxyerythronolide B synthase (DEBS) is hydroxy substituents, whereas module 4 contains a modular Type I PKS involved in erythromycin the complete KR, DH, and ER set, and results in biosynthesis (see page 96) and its structure and complete reduction to a methylene. Overall, the function are illustrated in Figure 3.85. The enzyme AT specificity and the catalytic domains on each contains three subunits (DEBS-1, 2, and 3), each module determine the structure and stereochem- encoded by a gene (eryA-I, II, and III). It has a istry of each two-carbon extension unit, the order linear organization of six modules, each of which of the modules specifies the sequence of the units,

eryAI eryAII eryAIII genes

DEBS 1 DEBS 2 DEBS 3 proteins Module 1 Module 3 Module 5 release load Module 2 Module 4 Module 6 modules

AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KR ACP KS AT KR ACP TE enzyme activities

S S S S S S S O O O O O O O O

HO HO O HO HO

HO HO O HO OH OOH HO HO O SCoA O O OH HO HO O CO H 2 SCoA deoxyerythronolide O HO HO CO2H SCoA O HO CO2H SCoA O ACP: acyl carrier protein CO2H SCoA AT: acyltransferase O DH: dehydratase CO2H SCoA ER: enoyl reductase O KR: β-ketoacyl reductase CO2H KS: β-ketoacyl synthase TE: thioesterase

Figure 3.85 Module 1 release each module programmes the nature of the module 2 O module 3 load Module 2 OH extender unit added (via AT) , and the oxidation state of the β-carbon in the AT ACP KS AT KR ACP KS AT KR ACP TE preceding unit (via KR, DH, ER) module 1 OH module 4 OO DEBS 1 plus TE domain OOH loading Module 1 Module 3 O load Module 2 release O OH module 5 OH module 6 AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP TE O O truncated DEBS plus TE domain H O

Module 1 Module 3 Module 5 release load Module 2 Module 4 Module 6 OH AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KR ACP KS AT KR ACP TE OOH loading domain from H avermectin PKS specifies O OH isobutyryl-CoA

O

Module 1 Module 3 Module 5 release load Module 2 Module 4 Module 6

OH AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KR ACP KS AT KR ACP TE OOH acyltransferase domain from H rapamycin PKS specifies malonyl-CoA O OH O

Module 1 Module 3 Module 5 release load Module 2 Module 4 Module 6 OH AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KRX ACP KS AT KR ACP TE O O deletion of ketoreductase H β domain prevents -carbon O OH processing

Figure 3.86 FURTHER READING 117 and the number of modules determines the size Non-ribosomal peptide synthases (see page 421) of the polyketide chain. The vast structural diver- are also modular and lend themselves to similar sity of natural polyketides arises from combinato- genetic manipulation as the Type I PKSs. The rial possibilities of arranging modules containing production of modified aromatic polyketides by the various catalytic domains, the sequence and genetically engineered Type II PKSs is not quite number of modules, and the post-PKS enzymes so ‘obvious’ as with the modular Type I enzymes, which subsequently modify the first-formed prod- but significant progress has been made in many uct, e.g. 6-deoxyerythronolide B → erythromycin systems. Each Type II PKS contains a minimal set (see page 96). Genetic engineering now offers vast of three protein subunits, two β-ketoacyl synthase opportunities for rational modification of the resul- (KS) subunits and an ACP to which the growing tant polyketide structure. chain is attached. Additional subunits, including A few representative examples of successful KRs, cyclases (CYC), and aromatases (ARO), are experiments leading to engineered polyketides are responsible for modification of the nascent chain shown in Figure 3.86. Reducing the size of the to form the final cyclized structure. Novel polyke- gene sequence so that it encodes fewer modules tides have been generated by manipulating Type results in the formation of smaller polyketides, II PKSs, exchanging KS, CYC, and ARO sub- characterized by the corresponding loss of exten- units among different systems. However, because der units; in these examples the gene encoding of the highly reactive nature of poly-β-keto chains, the chain terminating thioesterase also has to be the cyclizations that occur with the modified gene attached to complete the biosynthetic sequence. product frequently vary from those in the original Replacing the loading domain of DEBS with that compound. Compared with Type I PKSs, the for- from another PKS, e.g. that producing avermectin mation of new products with predictable molecular (see page 97), alters the specificity of the enzyme structure has proven less controllable. for the starter unit. The loading module of the The polyketide synthases responsible for chain avermectin-producing PKS actually has a much extension of cinnamoyl-CoA starter units leading broader specificity than that for DEBS; Figure 3.86 to flavonoids and stilbenes, and of anthraniloyl- shows the utilization of isobutyryl-CoA as fea- CoA leading to quinoline and acridine alkaloids tures in the natural biosynthesis of avermectin (see page 377) do not fall into either of the above categories and have now been termed Type B1b. Other examples include the replacement of an AT domain (in DEBS specifying a methylmalonyl III PKSs. These enzymes differ from the other extender) with a malonyl-specific AT domain from examples in that they are homodimeric proteins, they utilize coenzyme A esters rather than acyl car- the rapamycin-producing PKS (see page 103), and rier proteins, and they employ a single active site to deletion of a KR domain, thus stopping any β- perform a series of decarboxylation, condensation, carbon processing for that module with consequent cyclization, and aromatization reactions. retention of a carbonyl group. Not all experiments in gene modification are successful, and even when they are yields can be disappointingly lower than FURTHER READING in the natural system. There is always a funda- mental requirement that enzymes catalysing steps Biosynthesis after the point of modification need to have suffi- Rawlings BJ (1999) Biosynthesis of polyketides (other ciently broad substrate specificities to accept and than actinomycete macrolides). Nat Prod Rep 16, process the abnormal compounds being synthe- 425–484. Earlier review: (1997) 14, 523–556. sized; this becomes more unlikely where two or more genetic changes have been made. Neverthe- less, multiple modifications have been successful, Fatty Acids and Fats and it has also been possible to exploit changes in Gunstone F (1996) Fatty Acid and Lipid Chemistry. a combinatorial fashion using different expression Blackie, Glasgow. vectors for the individual subunits, thus creating a Harwood JL (1996) Recent advances in the biosynthesis library of polyketides, which may then be screened of plant fatty acids. Biochim Biophys Acta 1301, for potential biological activity. 7–56. 118 THE ACETATE PATHWAY

Hasenhuettl GL (1993) Fats and fatty oils. Kirk–Othmer Collins PW (1996) Prostaglandins. Kirk–Othmer Ency- Encyclopedia of Chemical Technology, 4th edn, clopedia of Chemical Technology, 4th edn, Vol 20. Vol 10. Wiley, New York, 252–267. Wiley, New York, 302–351. Kawaguchi A and Iwamoto-Kihara A (1999) Biosynthe- Collins PW and Djuric SW (1993) Synthesis of ther- sis and degradation of fatty acids. Comprehensive apeutically useful prostaglandins and prostacyclin Natural Products Chemistry, Vol 1. Elsevier, Ams- analogs. Chem Rev 93, 1533–1564. terdam, pp 23–59. Hamanaka N (1999) Eicosanoids in mammals. Compre- Lie Ken Jie MSF and Pasha MK (1998) Fatty acids, hensive Natural Products Chemistry, Vol 1. Elsevier, fatty acid analogues and their derivatives. Nat Prod Amsterdam, pp 159–206. Rep 15, 607–629. Earlier review: Lie Ken Jie MSF, Jackson WT and Fleisch JH (1996) Development of Pasha MK and Syed-Rahmatullah MSK (1997) 14, novel anti-inflammatory agents: a pharmacologic 163–189. perspective on leukotrienes and their receptors. Prog Mason P (2000) Nutrition: fish oils – an update. Pharm Drug Res 46, 115–168. J 265, 720–724. Lawson JA, Rokach J and FitzGerald GA (1999) Iso- Ohlrogge J and Browse J (1995) Lipid biosynthesis. prostanes: formation, analysis and use as indices Plant Cell 7, 957–970. of lipid peroxidation in vivo. J Biol Chem 274, Rawlings BJ (1998) Biosynthesis of fatty acids and related metabolites. Nat Prod Rep 15, 275–308. 24 441–24 444. Earlier review: (1997), 14, 335–358. Marnett LJ, Goodwin DC, Rowlinson SW, Kalgutkar Shanklin J and Cahoon EB (1998) Desaturation and AS and Landino LM (1999) Structure, function, and related modifications of fatty acids. Annu Rev Plant inhibition of prostaglandin endoperoxide synthases. Physiol Plant Mol Biol 49, 611–641. Comprehensive Natural Products Chemistry, Vol 5. Wolf WJ (1997) Soybeans and other oilseeds. Kirk– Elsevier, Amsterdam, pp 225–261. Othmer Encyclopedia of Chemical Technology,4th Marnett LJ and Kalgutker AS (1999) Cyclooxygenase edn, Vol 22. Wiley, New York, pp 591–619. 2 inhibitors: discovery, selectivity and the future. Trends in Pharmacological Sciences 20, 465–469. Marnett LJ, Rowlinson SW, Goodwin DC, Kalgutkar AS Platelet-Activating Factor and Lanzo CA (1999) Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and Prescott SM, Zimmermann GA and McIntyre TM inhibition. JBiolChem274, 22 903–22 906. (1990) Platelet-activating factor. JBiolChem265, Masferrer JL and Needleman P (2000) Anti-inflam- 17 382–17 384. matories for cardiovascular disease. Proc Natl Acad Snyder F (1995) Platelet-activating factor: the biosyn- Sci USA 97, 12 400–12 401. thetic and catabolic enzymes. Biochem J 305, Mueller MJ (1998) Radically novel prostaglandins in 689–705. animals and plants: the isoprostanes. Chem Biol 5, R323–333. Echinacea Peters-Golden M and Brock TG (2001) Intracellular compartmentalization of leukotriene synthesis: unex- Houghton P (1994) Herbal products: echinacea. Pharm pected nuclear secrets. FEBS Lett 487, 323–326. J 253, 342–343. Smith WL, Garavito RM and DeWitt DL (1996) Prosta- Perry NB, van Klink JW, Burgess EJ and Par- glandin endoperoxide H synthases (cyclooxygena- menter GA (1997) Alkamide levels in Echinacea ses)-1 and -2. JBiolChem271, 33 157–33 160. purpurea: a rapid analytical method revealing dif- Talley JJ (1999) Selective inhibitors of cyclooxygenase- ferences among roots, rhizomes, stems, leaves and 2 (COX-2). Prog Med Chem 36, 201–234. flowers. Planta Med 63,58–62. Ullrich V and Brugger R (1994) Prostacyclin and throm- boxane synthase: new aspects of hemethiolate catal- Prostaglandins, Thromboxanes, ysis. Angew Chem Int Ed Engl 33, 1911–1919. Versteeg HH, van Bergen en Henegouwen PMP, van Leukotrienes Deventer SJH and Peppelenbosch MP (1999) Cyclo- Beuck M (1999) Nonsteroidal antiinflammatory drugs: a oxygenase-dependent signalling: molecular events new generation of cyclooxygenase inhibitors. Angew and consequences. FEBS Lett 445,1–5. Chem Int Ed 38, 631–633. Yamamoto S (1999) Biosynthesis and metabolism of Clissold D and Thickitt C (1994) Recent eicosanoid eicosanoids. Comprehensive Natural Products Chem- chemistry. Nat Prod Rep 11, 621–637. istry, Vol 1. Elsevier, Amsterdam, pp 255–271. FURTHER READING 119

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Hlavka JJ, Ellestad GA and Chopra I (1992) Antibiotics Khellin/Cromoglicate (tetracyclines). Kirk–Othmer Encyclopedia of Chem- ical Technology, 4th edn, Vol 3. Wiley, New York, Bernstein PR (1992) Antiasthmatic agents. Kirk– 331–346. Othmer Encyclopedia of Chemical Technology,4th edn, Vol 2. Wiley, New York, 830–854. Anthracyclines

Griseofulvin Fujii I and Ebizuka Y (1997) Anthracycline biosyn- thesis in Streptomyces galilaeus. Chem Rev 97, Cauwenbergh G (1992) Antiparasitic agents (antimy- 2511–2523. cotics). Kirk–Othmer Encyclopedia of Chemical Hutchinson CR (1997) Biosynthetic studies of daunoru- Technology, 4th edn, Vol 3. Wiley, New York, bicin and tetracenomycin C. Chem Rev 97, 473–489. 2525–2535. Lown JW (1993) Discovery and development of anthra- Aflatoxins cycline antitumour antibiotics. Chem Soc Rev 22, 165–176. Minto RE and Townsend CA (1997) Enzymology and molecular biology of aflatoxin biosynthesis. Chem Rev 97, 2537–2555. Macrolides Townsend CA and Minto RE (1999) Biosynthesis of aflatoxins. Comprehensive Natural Products Chem- Kirst HA (1992) Antibiotics (macrolides). Kirk–Othmer istry, Vol 1. Elsevier, Amsterdam, pp 443–471. Encyclopedia of Chemical Technology, 4th edn, Vol 3. Wiley, New York, 169–213. Kirst HA (1993) Semi-synthetic derivatives of ery- Cannabinoids thromycin. Prog Med Chem 30,57–88. Nicolau KC, Roschangar F and Vourloumis D (1998) Devane WA (1994) New dawn of cannabinoid phar- Chemical biology of epothilones. Angew Chem Int macology. Trends in Pharmacological Sciences 15, Ed 37, 2014–2045. 40–41. Pieper R, Kao C, Khosla C, Luo G and Cane DE (1996) Mechoulam R and Ben-Shabat S (1999) From gan-zi- Specificity and versatility in erythromycin biosynthe- gun-nu to anandamide and 2-arachidonoylglycerol: sis. Chem Soc Rev 25, 297–302. the ongoing story of cannabis. Nat Prod Rep 16, Staunton J and Wilkinson B (1997) Biosynthesis of 131–143. erythromycin and rapamycin. Chem Rev 97, Metchoulam R, Hanus L and Fride E (1998) Towards 2611–2629. cannabinoid drugs – revisited. Prog Med Chem 35, Staunton J and Wilkinson B (1999) Biosynthesis of ery- 199–243. thromycin and related macrolides. Comprehensive O’Driscoll C (2000) High hopes for cannabis. Chem Brit Natural Products Chemistry, Vol 1. Elsevier, Ams- 36 (5), 27. terdam, pp 495–532. Piomelli D, Giuffrida A, Calignano A and de Fon- seca FR (2000) The endocannabinoid system as a target for therapeutic drugs. Trends in Pharmacolog- Polyene Antifungals ical Sciences 21, 218–224. Seth R and Sinha S (1991) Chemistry and pharmacol- Cauwenbergh G (1992) Antiparasitic agents (antimy- ogy of cannabis. Prog Drug Res 36, 71-115. cotics). Kirk–Othmer Encyclopedia of Chemical 120 THE ACETATE PATHWAY

Technology, 4th edn, Vol 3. Wiley, New York, Zaragozic Acids (Squalestatins) 473–489. Hoeprich PD (1995) Antifungal chemotherapy. Prog Bergstrom JD, Dufresne C, Bills GF, Nallin-Omstead M Drug Res 44,87–127. and Byrne K (1995) Discovery, biosynthesis and mechanism of action of the zaragozic acids: potent Avermectins inhibitors of squalene synthase. Annu Rev Microbiol 49, 607–639. Campbell WC (1991) Ivermectin as an antiparasitic Nadin A and Nicolaou KC (1996) Chemistry and biol- agent for use in humans. Annu Rev Microbiol 45, ogy of the zaragozic acids (squalestatins). Angew 445–474. Chem Int Ed Engl 35, 1623–1656. Davies HG and Green RH (1991) Avermectins and Watson NS and Procopiou PA (1996) Squalene synthase milbemycins. Chem Soc Rev 20, 211–269; 271–339. inhibitors: their potential as hypocholesterolaemic Fisher MH and Mrozik H (1992) Antiparasitic agents agents. Prog Med Chem 33, 331–378. (avermectins). Kirk–Othmer Encyclopedia of Chem- ical Technology, 4th edn, Vol 3. Wiley, New York, 526–540. Statins Ikeda H and Omura S (1997) Avermectin biosynthesis. Chem Rev 97, 2591–2609. Cervoni P, Crandall DL and Chan PS (1993) Car- diovascular agents – atherosclerosis and antiathero- Ansa Macrolides sclerosis agents. Kirk–Othmer Encyclopedia of Chemical Technology, 4th edn, Vol 5. Wiley, New Antosz FJ (1992) Antibiotics (ansamacrolides). Kirk– York, 257–261. Othmer Encyclopedia of Chemical Technology,4th Endo A and Hasumi K (1993) HMG-CoA reductase edn, Vol 2. Wiley, New York, 926-961. inhibitors. Nat Prod Rep 10, 541–550. Gopinath L (1996) Cholesterol drug dilemma. Chem Tacrolimus Brit 32 (11), 38–41. Laschat S (1996) Pericyclic reactions in biological sys- Clardy J (1995) The chemistry of signal transduction. tems – does nature know about the Diels–Alder Proc Natl Acad Sci USA 92,56–61. reaction? Angew Chem Int Ed Engl 35, 289–291. Rosen MK and Schreiber SL (1992) Natural products as Yalpani M (1996) Cholesterol-lowering drugs. Chem probes of cellular function: studies of immunophilins. Ind,85–89. Angew Chem Int Ed Engl 31, 384–400. Wong S (1995) Immunotherapeutic agents. Kirk– Othmer Encyclopedia of Chemical Technology,4th Polyketide Synthases/Genetic edn, Vol 14. Wiley, New York, 64–86. Manipulation

Polyethers Cane DE, Walsh CT and Khosla C (1998) Harnessing the biosynthetic code: combinations, permutations, Crandall LW and Hamill RL (1992) Antibiotics (poly- and mutations. Science 262,63–68. ethers). Kirk–Othmer Encyclopedia of Chemical Hutchinson CR (1999) Microbial polyketide synthases: Technology, 4th edn, Vol 3. Wiley, New York, more and more prolific. Proc Natl Acad Sci USA 96, 306–331. 3336–3338. Dutton CJ, Banks BJ and Cooper CB (1995) Polyether Katz L (1997) Manipulation of modular polyketide ionophores. Nat Prod Rep 12, 165–181. synthases. Chem Rev 97, 2557–2575. Riddell FG (1992) Ionophoric antibiotics. Chem Brit, Khosla C (1997) Harnessing the potential of modular 533–537. polyketide synthases. Chem Rev 97, 2577–2590. Robinson JA (1991) Chemical and biochemical aspects Khosla C, Gokhale RS, Jacobsen JR and Cane DE of polyether-ionophore antibiotic biosynthesis. Prog (1999) Tolerance and specificity of polyketide Chem Org Nat Prod 58,1–81. synthases. Annu Rev Biochem 68, 219–253. Shimizu Y (1993) Microalgal metabolites. Chem Rev Richardson M and Khosla C (1999) Structure, function, 93, 1685–1698. and engineering of bacterial polyketide synthases. Yasumoto T and Murata M (1993) Marine toxins. Chem Comprehensive Natural Products Chemistry, Vol 1. Rev 93, 1897–1909. Elsevier, Amsterdam, pp 473–494. Medicinal Natural Products. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 4 THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND PHENYLPROPANOIDS

Shikimic acid and its role in the formation of aromatic amino acids, benzoic acids, and cinnamic acids is described, along with further modifications leading to lignans and lignin, phenylpropenes, and coumarins. Combinations of the shikimate pathway and the acetate pathway are responsible for the biosynthesis of styrylpyrones, flavonoids and stilbenes, flavonolignans, and isoflavonoids. Terpenoid quinones are formed by a combination of the shikimate pathway with the terpenoid pathway. Monograph topics giving more detailed information on medicinal agents include folic acid, chloramphenicol, podophyllum, volatile oils, dicoumarol and warfarin, psoralens, kava, Silybum marianum, phyto-oestrogens, derris and lonchocarpus, vitamin E, and vitamin K.

The shikimate pathway provides an alternative glycolysis) and D-erythrose 4-phosphate (from the route to aromatic compounds, particularly the aro- pentose phosphate cycle) to the aromatic amino matic amino acids L-phenylalanine,L-tyrosine acids was broadly outlined. Phenylalanine and and L-tryptophan. This pathway is employed by tyrosine form the basis of C6C3 phenylpropane microorganisms and plants, but not by animals, units found in many natural products, e.g. cin- and accordingly the aromatic amino acids fea- namic acids, coumarins, lignans, and flavonoids, ture among those essential amino acids for man and along with tryptophan are precursors of a wide which have to be obtained in the diet. A cen- range of alkaloid structures. In addition, it is found tral intermediate in the pathway is shikimic acid that many simple benzoic acid derivatives, e.g. (Figure 4.1), a compound which had been iso- gallic acid (Figure 4.1) and p-aminobenzoic acid lated from plants of Illicium species (Japanese (4-aminobenzoic acid) (Figure 4.4) are produced ‘shikimi’) many years before its role in metabolism via branchpoints in the shikimate pathway. had been discovered. Most of the intermediates in the pathway were identified by a careful study of AROMATIC AMINO ACIDS AND aseriesofEscherichia coli mutants prepared by SIMPLE BENZOIC ACIDS UV irradiation. Their nutritional requirements for growth, and any by-products formed, were then The shikimate pathway begins with a coupling characterized. A mutant strain capable of growth of phosphoenolpyruvate (PEP) and D-erythrose usually differs from its parent in only a single 4-phosphate to give the seven-carbon 3-deoxy- gene, and the usual effect is the impaired syn- D-arabino-heptulosonic acid 7-phosphate (DAHP) thesis of a single enzyme. Typically, a mutant (Figure 4.1). This reaction, shown here as an blocked in the transformation of compound A into aldol-type condensation, is known to be mecha- compound B will require B for growth whilst nistically more complex in the enzyme-catalysed accumulating A in its culture medium. In this version; several of the other transformations in the way, the pathway from phosphoenolpyruvate (from pathway have also been found to be surprisingly 122 THE SHIKIMATE PATHWAY

formally an elimination; it actually involves oxidation of the hydroxyl adjacent to the proton lost and therefore requires NAD+ PEP CO H 2 aldol-type cofactor; the carbonyl is subsequently reduced back to an alcohol reaction CO H P O 2 CO2H H PO PO O H – HOP O H NAD+ HO O H HO OH HO OH OH OH OH D-erythrose 4-P DAHP aldol-type reaction

CO2H CO2H HO CO2H HO CO2H

NADPH – H2O NADH

HO OH O OH O OH HO OH OH OH OH OH shikimic acid 3-dehydroshikimic 3-dehydroquinic quinic acid acid acid

– 2H – H O oxidation and dehydration and 2 enolization enolization CO2H CO2H

HO HO OH OH OH protocatechuic gallic acid acid

Figure 4.1 complex. Elimination of phosphoric acid from gallic acid (3,4,5-trihydroxybenzoic acid) can be DAHP followed by an intramolecular aldol reac- formed by branchpoint reactions from 3-dehy- tion generates the first carbocyclic intermediate 3- droshikimic acid, which involve dehydration and dehydroquinic acid. However, this also represents enolization, or, in the case of gallic acid, dehy- an oversimplification. The elimination of phos- drogenation and enolization. Gallic acid features phoric acid actually follows an NAD+-dependent as a component of many tannin materials (gal- oxidation of the central hydroxyl, and this is then lotannins), e.g. pentagalloylglucose (Figure 4.2), re-formed in an NADH-dependent reduction reac- found in plants, materials which have been used tion on the intermediate carbonyl compound prior for thousands of years in the tanning of animal to the aldol reaction occurring. All these changes hides to make leather, due to their ability to cross- occur in the presence of a single enzyme. Reduc- link protein molecules. Tannins also contribute to tion of 3-dehydroquinic acid leads to quinic acid, the astringency of foods and beverages, especially a fairly common natural product found in the tea, coffee and wines (see also condensed tannins, free form, as esters, or in combination with alka- page 151). loids such as quinine (see page 362). Shikimic A very important branchpoint compound in the acid itself is formed from 3-dehydroquinic acid shikimate pathway is chorismic acid (Figure 4.3), via 3-dehydroshikimic acid by dehydration and which has incorporated a further molecule of reduction steps. The simple phenolic acids pro- PEP as an enol ether side-chain. PEP combines tocatechuic acid (3,4-dihydroxybenzoic acid) and with shikimic acid 3-phosphate produced in a AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 123

OH enzyme, and is believed to bind to the PEP bind- OH OH ing site on the enzyme. Glyphosate finds con- HO OH siderable use as a broad spectrum herbicide, a O OH plant’s subsequent inability to synthesize aromatic OH O amino acids causing its death. The transformation OH of EPSP to chorismic acid (Figure 4.3) involves a O O O O O O 1,4-elimination of phosphoric acid, though this is OO OH probably not a concerted elimination. O 4-hydroxybenzoic acid (Figure 4.4) is pro- duced in bacteria from chorismic acid by HO OH HO OH an elimination reaction, losing the recently OH introduced enolpyruvic acid side-chain. However, OH pentagalloylglucose in plants, this phenolic acid is formed by a branch much further on in the pathway via side-chain degradation of cinnamic acids (see CO H CO H 2 2 page 141). The three phenolic acids so far encoun- HN O O tered, 4-hydroxybenzoic, protocatechuic, and gallic O P P OH OH acids, demonstrate some of the hydroxylation OH OH patterns characteristic of shikimic acid-derived glyphosate PEP metabolites, i.e. a single hydroxy para to the side- chain function, dihydroxy groups arranged ortho to Figure 4.2 each other, typically 3,4- to the side-chain, and tri- hydroxy groups also ortho to each other and 3,4,5- simple ATP-dependent phosphorylation reaction. to the side-chain. The single para-hydroxylation This combines with PEP via an addition–elimi- and the ortho-polyhydroxylation patterns contrast nation reaction giving 3-enolpyruvylshikimic acid with the typical meta-hydroxylation patterns char- 3-phosphate (EPSP). This reaction is catalysed acteristic of phenols derived via the acetate path- by the enzyme EPSP synthase. The synthetic way (see page 62), and in most cases allow the N-(phosphonomethyl)glycine derivative glypho- biosynthetic origin (acetate or shikimate) of an aro- sate (Figure 4.2) is a powerful inhibitor of this matic ring to be deduced by inspection.

nucleophilic attack on to protonated double bond of PEP H PEP CO H CO2H CO2H 2 H PO CO2H HH ATP

HO OH PO OH EPSP synthase PO OCO2H OH OH OH OP shikimic acid shikimic acid 3-P 1,2-elimination of – HOP phosphoric acid 1,4-elimination of phosphoric acid CO2H CO2H

L-Phe – HOP prephenic acid L-Tyr OCO2H PO OCO2H OH OH chorismic acid EPSP

Figure 4.3 124 THE SHIKIMATE PATHWAY

elimination of pyruvic acid elimination of pyruvic acid (formally as enolpyruvic acid) (formally as enolpyruvic acid) generates aromatic ring isomerization via generates aromatic ring CO H S 2′ reaction 2 CO2H N CO2H CO2H OH2 OH OH

OCO2H OCO2H OH OH isochorismic acid salicylic acid 4-hydroxybenzoic H chorismic acid acid hydrolysis of L-Gln L-Gln enol ether amination using side-chain oxidation of 3-hydroxyl to ammonia (generated ketone, then enolization from glutamine) as CO2H CO H CO2H nucleophile 2 CO2H NH2 OH NAD+ OH

OCOH 2 OCO2H OH OH NH 2 2,3-dihydroxybenzoic 2-amino-2-deoxy- 4-amino-4-deoxy- acid chorismic acid isochorismic acid

elimination of pyruvic acid

CO2H CO2H

NH2 L-Trp

NH2 anthranilic acid p-aminobenzoic acid (PABA)

Figure 4.4

2,3-dihydroxybenzoic acid,andsalicylic acid available sufficient concentrations of essential (2-hydroxybenzoic acid) (in microorganisms, but iron. Enterobactin comprises three molecules of not in plants, see page 141), are derived from 2,3-dihydroxybenzoic acid and three of the amino chorismic acid via its isomer isochorismic acid L-serine, in cyclic triester form. acid (Figure 4.4). The isomerization involves Simple amino analogues of the phenolic  an SN2 -type of reaction, an incoming water acids are produced from chorismic acid by nucleophile attacking the diene system and related transformations in which ammonia, gen- displacing the hydroxyl. Salicyclic acid arises by erated from glutamine, acts as a nucleophile an elimination reaction analogous to that producing (Figure 4.4). Chorismic acid can be aminated at 4-hydroxybenzoic acid from chorismic acid. In C-4 to give 4-amino-4-deoxychorismic acid and the formation of 2,3-dihydroxybenzoic acid, the then p-aminobenzoic (4-aminobenzoic) acid, or at side-chain of isochorismic acid is first lost by C-2 to give the isochorismic acid analogue which hydrolysis, then dehydrogenation of the 3-hydroxy will yield 2-aminobenzoic (anthranilic) acid. Ami- to a 3-keto allows enolization and formation nation at C-4 has been found to occur with reten- of the aromatic ring. 2,3-Dihydroxybenzoic tion of configuration, so perhaps a double inver- acid is a component of the powerful iron sion mechanism is involved. p-Aminobenzoic acid chelator (siderophore) enterobactin (Figure 4.5) (PABA) forms part of the structure of folic acid ∗ found in Escherichia coli and many other (vitamin B9) (Figure 4.6). The folic acid struc- Gram-negative bacteria. Such compounds play an ture is built up (Figure 4.6) from a dihydropterin important role in bacterial growth by making diphosphate which reacts with p-aminobenzoic AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 125

OH OO O activation to AMP derivative, compare peptide formation, O Figure 7.15 OH O O CO H COAMP 2 L-Ser O NH O NH O HN HN O OH OH ATP O OH HO HO 3+ OH OH O O O Fe O HO 2,3-dihydroxy- OH HO benzoic acid O OH HN N enterobactin as iron chelator H HO O O HO OH enterobactin

Figure 4.5

H2N

SO2NH2 H2N sulphanilamide (acts as antimetabolite of PABA and is enzyme inhibitor) CO2H PABA H H H H2N N N H2N N N H2N N N ATP H N OH N OPP N N N N N SN2 reaction OH OH OH CO2H hydroxymethyl- hydroxymethyl- dihydropteroic acid dihydropterin dihydropterin PP L-Glu ATP H H reduction H2N N N H2N N N H NADPH H N N N N N H N H H dihydrofolate OH N CO2H reductase OH N CO2H (DHFR) O CO H O CO H tetrahydrofolic acid 2 dihydrofolic acid 2 (FH4) (FH2) dihydrofolate NADPH reductase reduction (DHFR) the pteridine system is sometimes H2N N N drawn as the tautomeric amide form: H N N H N N N 2 N H OH N CO H HN 2 N O CO2H O p-amino- benzoic acid a pteridine (PABA) L-Glu

folic acid

Figure 4.6 126 THE SHIKIMATE PATHWAY acid to give dihydropteroic acid, an enzymic regeneration of tetrahydrofolic acid, and forms an step for which the sulphonamide antibiotics are important site of action for some antibacterial, anti- inhibitors. Dihydrofolic acid is produced from the malarial, and anticancer drugs. dihydropteroic acid by incorporating glutamic acid, Anthranilic acid (Figure 4.4) is an intermediate and reduction yields tetrahydrofolic acid.This in the biosynthetic pathway to the indole-containing reduction step is also necessary for the continual aromatic amino acid L-tryptophan (Figure 4.10).

Folic Acid (Vitamin B9)

Folic acid (vitamin B9) (Figure 4.6) is a conjugate of a pteridine unit, p-aminobenzoic acid, and glutamic acid. It is found in yeast, liver, and green vegetables, though cooking may destroy up to 90% of the vitamin. Deficiency gives rise to anaemia, and supplementation is often necessary during pregnancy. Otherwise, deficiency is not normally encountered unless there is malabsorption, or chronic disease. Folic acid used for supplementation is usually synthetic, and it becomes sequentially reduced in the body by the enzyme dihydrofolate reductase to give dihydrofolic acid and then tetrahydrofolic acid (Figure 4.6). Tetrahydrofolic acid then functions as a carrier of one-carbon groups, which may be in the form of methyl, methylene, methenyl, or formyl groups, by the reactions outlined in Figure 4.7. These groups are involved in amino acid and nucleotide metabolism. Thus a methyl group is transferred in the regeneration of methionine from homocysteine, purine biosynthesis involves methenyl and formyl transfer, and pyrimidine biosynthesis utilizes methylene transfer. Tetrahydrofolate derivatives also serve as acceptors of one-carbon units in degradative pathways. Mammals must obtain their tetrahydrofolate requirements from their diet, but microorgan- isms are able to synthesize this material. This offers scope for selective action and led to the use of sulphanilamide and other antibacterial sulpha drugs, compounds which competitively inhibit dihydropteroate synthase, the biosynthetic enzyme incorporating p-aminobenzoic acid into the structure. These sulpha drugs thus act as antimetabolites of p-aminobenzoate. Spe- cific dihydrofolate reductase inhibitors have also become especially useful as antibacterials,

H HCO2H H H ATP ADP H2N N N H2N N N H2N N N

N 5 N N 6 N N N H 10 H OH HN OH N OH HN H HO 10 O 5 FH4 N -formyl-FH4 N -formyl-FH4 L-Ser ATP (folinic acid) Gly ADP

H2O H H H + H2N N N H2N N N NADP NADPH H2N N N

N N N N N N + NADH OHMe HN NAD OH N OH N

5 5 10 5 10 N -methyl-FH4 N ,N -methylene-FH4 N ,N -methenyl-FH4

Figure 4.7

(Continues) AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 127

(Continued)

OMe H2N N N H2N N Me H2N N OMe N N N N H N OMe NH2 N CO2H NH2 Cl NH2 O CO2H trimethoprim pyrimethamine methotrexate

Figure 4.8

H L-Ser Gly H O H2N N N H2N N N HN N N N N H O N OH HN OH N deoxyribose-P 5 10 FH4 N ,N -methylene-FH4 dUMP

NADPH H O H2N N N HN N N O N OH HN deoxyribose-P

FH2 dTMP

Figure 4.9

e.g. trimethoprim (Figure 4.8), and antimalarial drugs, e.g. pyrimethamine, relying on the differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate (Figure 4.8), primarily block pyrimidine biosynthesis, but are less selective than the antimicrobial agents, and rely on a stronger binding to the enzyme than the natural substrate has. Regeneration of tetrahydrofolate from dihydrofolate is vital for DNA synthesis in rapidly proliferating cells. The methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) requires N5,N10-methylenetetrahydrofolate as the methyl donor, which is thereby transformed into dihydrofolate (Figure 4.9). N5-Formyl-tetrahydrofolic acid (folinic acid, leucovorin) (Figure 4.7) is used to counteract the folate-antagonist action of anticancer agents like methotrexate. The natural 6S isomer is termed levofolinic acid (levoleucovorin); folinic acid in drug use is usually a mixture of the 6R and 6S isomers.

In a sequence of complex reactions, which will side-chain of L-tryptophan. Although a precursor of not be considered in detail, the indole ring sys- L-tryptophan, anthranilic acid may also be produced tem is formed by incorporating two carbons from by metabolism of tryptophan. Both compounds fea- phosphoribosyl diphosphate, with loss of the orig- ture as building blocks for a variety of alkaloid inal anthranilate carboxyl. The remaining ribosyl structures (see Chapter 6). carbons are then removed by a reverse aldol reac- Returning to the main course of the shikimate tion, to be replaced on a bound form of indole pathway, a singular rearrangement process occurs by those from L-serine, which then becomes the transforming chorismic acid into prephenic acid 128 THE SHIKIMATE PATHWAY

OH CO2H CO2H CO2H SN2 reaction HO OP H NH2 NH CH OPP OH CH2OP 2 N H imine−enamine anthranilic O O acid OH OH HO OH tautomerism PPO H phosphoribosyl PP phosphoribosyl anthranilic acid OH CO2H HO OP OH N H enol−keto

CO2H tautomerism HO H CO2H NH2 reverse aldol HO OP L-Ser OOHO reaction – CO2 NH OH – H2O O 2 OP PLP OH N N N N H H H H L-Trp indole indole-3-glycerol P (enzyme-bound)

Figure 4.10

HO2C HO C Claisen 2 CO2H O rearrangement O CO2H O CO2H OH HO2C OH OH chorismic acid chorismic acid prephenic acid (pseudoequatorial (pseudoaxial conformer) conformer)

Figure 4.11

(Figure 4.11). This reaction, a Claisen rearrange- transamination, and in the case of tyrosine ment, transfers the PEP-derived side-chain so that biosynthesis an oxidation, but the order in which it becomes directly bonded to the carbocycle, and these reactions occur differentiates the routes. so builds up the basic carbon skeleton of phenylala- Decarboxylative aromatization of prephenic acid nine and tyrosine. The reaction is catalysed in nature yields phenylpyruvic acid, and PLP-dependent by the enzyme chorismate mutase, and, although it transamination leads to L-phenylalanine. In the can also occur thermally, the rate increases some presence of an NAD+-dependent dehydrogenase 106-fold in the presence of the enzyme. The enzyme enzyme, decarboxylative aromatization occurs achieves this by binding the pseudoaxial conformer with retention of the hydroxyl function, though of chorismic acid, allowing a transition state with as yet there is no evidence that any inter- chairlike geometry to develop. mediate carbonyl analogue of prephenic acid Pathways to the aromatic amino acids L-pheny- is involved. Transamination of the resultant lalanine and L-tyrosine via prephenic acid may 4-hydroxyphenylpyruvic acid subsequently gives vary according to the organism, and often more L-tyrosine. L-Arogenic acid is the result of than one route may operate in a particular species transamination of prephenic acid occurring prior according to the enzyme activities that are avail- to the decarboxylative aromatization, and can able (Figure 4.12). In essence, only three reac- be transformed into both L-phenylalanine and tions are involved, decarboxylative aromatization, L-tyrosine depending on the absence or presence AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 129 of a suitable enzymic dehydrogenase activity. by oxidation reactions into a heterogeneous In some organisms, broad activity enzymes polymer melanin, the main pigment in mammalian are known to be capable of accepting both skin, hair, and eyes. In this material, the indole prephenic acid and arogenic acid as substrates. system is not formed from tryptophan, but arises In microorganisms and plants, L-phenylalanine from DOPA by cyclization of DOPAquinone, the and L-tyrosine tend to be synthesized separately nitrogen of the side-chain then attacking the ortho- as in Figure 4.12, but in animals, which lack quinone (Figure 4.13). the shikimate pathway, direct hydroxylation of Some organisms are capable of synthesizing an L-phenylalanine to L-tyrosine, and of L-tyrosine unusual variant of L-phenylalanine, the aminated to L-DOPA (dihydroxyphenylalanine), may be derivative L-p-aminophenylalanine (L-PAPA) (Fig- achieved (Figure 4.13). These reactions are catal- ure 4.14). This is known to occur by a series of ysed by tetrahydropterin-dependent hydroxylase reactions paralleling those in Figure 4.12, but uti- enzymes, the hydroxyl oxygen being derived lizing the PABA precursor 4-amino-4-deoxychori- from molecular oxygen. L-DOPA is a precursor smic acid (Figure 4.4) instead of chorismic acid. of the catecholamines, e.g. the neurotransmitter Thus, amino derivatives of prephenic acid and py- noradrenaline and the hormone adrenaline (see ruvic acid are elaborated. One important metabolite page 316). Tyrosine and DOPA are also converted known to be formed from L-PAPA is the antibiotic

transamination: keto acid amino acid CO2H CO2H

O NH2

PLP

phenylpyruvic acid L-Phe decarboxylation, decarboxylation, aromatization and aromatization and loss of leaving group loss of leaving group

O CO2H O CO2H CO2H H C H C O O O PLP NH2

OCO2H OH H OH OH chorismic acid prephenic acid L-arogenic acid

+ oxidation means OH is an additional oxidation step (of NAD + NAD retained on decarboxylation alcohol to ketone) means OH is and aromatization; no discrete retained on decarboxylation ketone intermediate is formed and aromatization; no discrete CO2H CO2H ketone intermediate is formed O NH2

PLP

OH OH 4-hydroxyphenyl- L-Tyr pyruvic acid

Figure 4.12 130 THE SHIKIMATE PATHWAY

O2 O2 tetrahydro- tetrahydro- CO H CO H HO CO2H 2 biopterin 2 biopterin CATECHOLAMINES noradrenaline, NH NH2 NH2 2 HO HO adrenaline L-Phe L-Tyr L-DOPA

nucleophilic attack

O HO on to enone O CO2H O MELANINS CO2H CO2H NH2 HO N HO N O H DOPAchrome DOPAquinone

Figure 4.13

chloramphenicol∗, produced by cultures of Strep- natural products. In plants, a frequent first step is tomyces venezuelae. The late stages of the path- the elimination of ammonia from the side-chain way (Figure 4.14) have been formulated to involve to generate the appropriate trans (E) cinnamic hydroxylation and N-acylation in the side-chain, acid. In the case of phenylalanine, this would the latter reaction probably requiring a coenzyme give cinnamic acid, whilst tyrosine could yield A ester of dichloroacetic acid. Following reduction 4-coumaric acid (p-coumaric acid) (Figure 4.15). of the carboxyl group, the final reaction is oxida- All plants appear to have the ability to deami- tion of the 4-amino group to a nitro, a fairly rare nate phenylalanine via the enzyme phenylalanine substituent in natural product structures. ammonia lyase (PAL), but the corresponding trans- formation of tyrosine is more restricted, being CINNAMIC ACIDS mainly limited to members of the grass family (the Graminae/Poaceae). Whether a separate enzyme L-Phenylalanine and L-tyrosine, as C6C3 build- tyrosine ammonia lyase (TAL) exists, or whether ing blocks, are precursors for a wide range of grasses merely have a broad specificity PAL also

Chloramphenicol

Chloramphenicol (chloromycetin) (Figure 4.14) was initially isolated from cultures of Streptomyces venezuelae, but is now obtained for drug use by chemical synthesis. It was one of the first broad spectrum antibiotics to be developed, and exerts its antibacterial action by inhibiting protein biosynthesis. It binds reversibly to the 50S subunit of the bacterial ribosome, and in so doing disrupts peptidyl transferase, the enzyme that catalyses peptide bond formation (see page 408). This reversible binding means that bacterial cells not destroyed may resume protein biosynthesis when no longer exposed to the antibiotic. Some microorganisms have developed resistance to chloramphenicol by an inactivation process involving enzymic acetylation of the primary alcohol group in the antibiotic. The acetate binds only very weakly to the ribosomes, so has little antibiotic activity. The value of chloramphenicol as an antibacterial agent has been severely limited by some serious side-effects. It can cause blood disorders including irreversible aplastic anaemia in certain individuals, and these can lead to leukaemia and perhaps prove fatal. Nevertheless, it is still the drug of choice for some life-threatening infections such as typhoid fever and bacterial meningitis. The blood constitution must be monitored regularly during treatment to detect any abnormalities or adverse changes. The drug is orally active, but may also be injected. Eye-drops are useful for the treatment of bacterial conjunctivitis. CINNAMIC ACIDS 131

decarboxylation and aromatization; amino group is retained via an additional oxidation step (amine → imine)

O CO2H CO2H

CO2H HO C O Claisen 2 rearrangement

OCO2H NH NH2 2 NH2 4-amino-4-deoxy- transamination chorismic acid PLP hydroxylation CH2OH CO2H N-acylation CO2H HO HO NHCOCHCl2 NHCOCHCl2 CHCl2COSCoA NH2

reduction of CO2H to CH2OH; oxidation of NH NO2 NH2 to NO2 2 NH2 chloramphenicol L-p-aminophenylalanine L-PAPA

Figure 4.14

E2 elimination of ammonia CO2H CO2H

NH2

PAL

L-Phe cinnamic acid O 2 hydroxylation NADPH sequence of hydroxylation and methylation reactions

CO2H CO2H CO2H CO2H CO2H CO2H

NH2 O O2 2 SAM NADPH SAM NADPH

HO MeO MeO OH MeO OMe OH OH OH OH OH OH L-Tyr 4-coumaric acid caffeic acid ferulic acid sinapic acid (p-coumaric acid)

CH2OH CH2OH CH2OH

MeO MeO OMe OH OH OH 4-hydroxycinnamyl alcohol coniferyl alcohol sinapyl alcohol (p-coumaryl alcohol)

POLYMERS x 2 x n

LIGNANS LIGNIN

Figure 4.15 132 THE SHIKIMATE PATHWAY

HO Pre-eminent amongst these, certainly as far as O O CO2H nature is concerned, is the plant polymer lignin, OH OH a strengthening material for the plant cell wall which acts as a matrix for cellulose microfibrils (see page 473). Lignin represents a vast reservoir of aromatic materials, mainly untapped because OH of the difficulties associated with release of these OH metabolites. The action of wood-rotting fungi chlorogenic acid offers the most effective way of making these use- (5-O-caffeoylquinic acid) ful products more accessible. Lignin is formed OH by phenolic oxidative coupling of hydroxycin- namyl alcohol monomers, brought about by perox- HO O HO O idase enzymes (see page 28). The most important OH of these monomers are 4-hydroxycinnamyl alco- O hol (p-coumaryl alcohol), coniferyl alcohol,and 1-O-cinnamoylglucose sinapyl alcohol (Figure 4.15), though the mono- mers used vary according to the plant type. OMe Gymnosperms polymerize mainly coniferyl alco- OH hol, dicotyledonous plants coniferyl alcohol and O sinapyl alcohol, whilst monocotyledons use all Me3N OMe three alcohols. The alcohols are derived by reduc- O tion of cinnamic acids via coenzyme A esters and sinapine aldehydes (Figure 4.17), though the substitution patterns are not necessarily elaborated completely Figure 4.16 at the cinnamic acid stage, and coenzyme A esters and aldehydes may also be substrates for aromatic capable of deaminating tyrosine, is still debated. hydroxylation and methylation. Formation of the Those species that do not transform tyrosine syn- coenzyme A ester facilitates the first reduction step thesize 4-coumaric acid by direct hydroxylation of − by introducing a better leaving group (CoAS ) cinnamic acid, in a cytochrome P-450-dependent for the NADPH-dependent reaction. The second reaction, and tyrosine is often channelled instead reduction step, aldehyde to alcohol, utilizes a fur- into other secondary metabolites, e.g. alkaloids. ther molecule of NADPH and is reversible. The Other cinnamic acids are obtained by further peroxidase enzyme then achieves one-electron oxi- hydroxylation and methylation reactions, sequen- dation of the phenol group. One-electron oxidation tially building up substitution patterns typical of of a simple phenol allows delocalization of the shikimate pathway metabolites, i.e. an ortho oxy- unpaired electron, giving resonance forms in genation pattern (see page 123). Some of the more which the free electron resides at positions ortho common natural cinnamic acids are 4-coumaric, para caffeic, ferulic,andsinapic acids (Figure 4.15). and to the oxygen function (see page 29). These can be found in plants in free form and in With cinnamic acid derivatives, conjugation allows a range of esterified forms, e.g. with quinic acid the unpaired electron to be delocalized also into as in chlorogenic acid (5-O-caffeoylquinic acid) the side-chain (Figure 4.18). Radical pairing of (see coffee, page 395), with glucose as in 1-O- resonance structures can then provide a range of cinnamoylglucose, and with choline as in sinapine dimeric systems containing reactive quinoneme- (Figure 4.16). thides, which are susceptible to nucleophilic attack from hydroxyl groups in the same system, or by external water molecules. Thus, coniferyl LIGNANS AND LIGNIN alcohol monomers can couple, generating linkages as exemplified by guaiacylglycerol β-coniferyl The cinnamic acids also feature in the pathways to ether (β-arylether linkage), dehydrodiconi- other metabolites based on C6C3 building blocks. feryl alcohol (phenylcoumaran linkage), and LIGNANS AND LIGNIN 133

CO2H COSCoA CHO CH2OH

HSCoA NADPH NADPH

R1 R2 R1 R2 R1 R2 R1 R2 OH OH OH OH

Figure 4.17

OH OH OH OH OH one-electron oxidation

– H+ – e MeO MeO MeO MeO MeO OH O O O O coniferyl alcohol A BCD resonance forms of free radical

A + DB + DD + D radical radical radical pairing pairing pairing OH OH O H OMe

OH HO H HO OMe OMe O O HO enolization H2O O MeO nucleophilic attack of hydroxyls on to OMe OH O O H H quinonemethides OMe nucleophilic attack OH of water on to HO quinonemethide O OH OMe OMe OH MeO O H O OMe HO OH pinoresinol HO OMe nucleophilic attack (resinol linkage) O of hydroxyl on to quinonemethide HO HO

OH OMe OMe O guaiacylglycerol dehydrodiconiferyl alcohol β-coniferyl ether HO β (phenylcoumaran linkage) ( -arylether linkage) OMe

Figure 4.18 134 THE SHIKIMATE PATHWAY

H H H H MeO MeO OH O H H HO HO

OH OH this step probably involves ring OH phenolic oxidative opening to the quinonemethide coupling O followed by reduction OH OMe OMe H H NADPH H H MeO MeO O HO MeO OH HO HO coniferyl alcohol (+)-pinoresinol (–)-secoisolariciresinol

modification of oxidation of one CH2OH ≡ aromatic substitution to CO2H, then lactone ring patterns formation MeO MeO O OH O O OH O HO NAD+ HO O O

MeO OMe MeO MeO OMe OH OH yatein matairesinol (–)-secoisolariciresinol

OH hydroxylation O O O O O O O O O nucleophilic O O O attack on to quinonemethide system MeO OMe MeO OMe MeO OMe OMe OMe OMe

desoxypodophyllotoxin podophyllotoxin

Figure 4.19

MeO HO HO OGlc OH O OGlc intestinal OH HO bacteria O

MeO HO HO OH enterodiol enterolactone secoisolariciresinol diglucoside

Figure 4.20 LIGNANS AND LIGNIN 135 pinoresinol (resinol linkage). These dimers can is known to arise by reductive opening of the react further by similar mechanisms to produce a furan rings of pinoresinol, followed by oxida- lignin polymer containing a heterogeneous series tion of a primary alcohol to the acid and then of inter-molecular bondings as seen in the var- lactonization. The substitution pattern in the two ious dimers. In contrast to most other natural aromatic rings is built up further during the path- polymeric materials, lignin appears to be devoid way, i.e. matairesinol → yatein, and does not of ordered repeating units, though some 50–70% arise by initial coupling of two different cinnamyl of the linkages are of the β-arylether type. The alcohol residues. The methylenedioxy ring sys- dimeric materials are also found in nature and are tem, as found in many shikimate-derived natural called lignans. Some authorities like to restrict products, is formed by an oxidative reaction on the term lignan specifically to molecules in which an ortho-hydroxymethoxy pattern (see page 27). the two phenylpropane units are coupled at the Podophyllotoxin and related lignans are found central carbon of the side-chain, e.g. pinoresinol, in the roots of Podophyllum∗ species (Berberi- whilst compounds containing other types of cou- daceae), and have clinically useful cytotoxic and pling, e.g. as in guaiacylglycerol β-coniferyl ether anticancer activity. The lignans enterolactone and dehydrodiconiferyl alcohol, are then referred and enterodiol (Figure 4.20) were discovered in to as neolignans. Lignan/neolignan formation human urine, but were subsequently shown to and lignin biosynthesis are catalysed by differ- be derived from dietary plant lignans, especially ent enzymes, and a consequence is that natural secoisolariciresinol diglucoside, by the action of lignans/neolignans are normally enantiomerically intestinal microflora. Enterolactone and enterodiol pure because they arise from stereochemically have oestrogenic activity and have been impli- controlled coupling. The control mechanisms for cated as contributing to lower levels of breast lignin biosynthesis are less well defined, but the cancer amongst vegetarians (see phyto-oestrogens, enzymes appear to generate products lacking opti- page 156). cal activity. Further cyclization and other modifications can create a wide range of lignans of very differ- PHENYLPROPENES ent structural types. One of the most important of the natural lignans having useful biologi- The reductive sequence from an appropriate cin- cal activity is the aryltetralin lactone podophyl- namic acid to the corresponding cinnamyl alcohol lotoxin (Figure 4.19), which is derived from is not restricted to lignin and lignan biosynthesis, coniferyl alcohol via the dibenzylbutyrolactones and is utilized for the production of various matairesinol and yatein, cyclization probably phenylpropene derivatives. Thus cinnamaldehyde occurring as shown in Figure 4.19. Matairesinol (Figure 4.23) is the principal component in the

Podophyllum

Podophyllum consists of the dried rhizome and roots of Podophyllum hexandrum (P. emodi) or P. peltatum (Berberidaceae). Podophyllum hexandrum is found in India, China, and the Himalayas and yields Indian podophyllum, whilst P. peltatum (May apple or American mandrake) comes from North America and is the source of American podophyllum. Plants are collected from the wild. Both plants are large-leafed perennial herbs with edible fruits, though other parts of the plant are toxic. The roots contain cytotoxic lignans and their glucosides, P. hexandrum containing about 5%, and P. peltatum about 1%. A concentrated form of the active principles is obtained by pouring an ethanolic extract of the root into water, and drying the precipitated podophyllum resin or ‘podophyllin’. Indian podophyllum yields about 6–12% of resin containing 50–60% lignans, and American podophyllum 2–8% of resin containing 14–18% lignans.

(Continues) 136 THE SHIKIMATE PATHWAY

(Continued)

OH OH O O O O O O O O O O O O O O O O O

MeO4′ OMe MeO OMe MeO OMe MeO OMe OR OR OMe OMe R = Me, podophyllotoxin R = Me, β-peltatin desoxypodophyllotoxin podophyllotoxone R = H, 4′-demethylpodophyllotoxin R = H, α-peltatin

O O O O O S O HO O HO O OH OH OH O O O O O O O O O O O O

MeO OMe MeO4′ OMe MeO OMe OH OR OH 4′-demethylepipodophyllotoxin R = H, etoposide teniposide R = P, etopophos

Figure 4.21

The lignan constituents of the two roots are the same, but the proportions are markedly different. The Indian root contains chiefly podophyllotoxin (Figure 4.21) (about 4%) and 4- demethylpodophyllotoxin (about 0.45%). The main components in the American root are podophyllotoxin (about 0.25%), β-peltatin (about 0.33%) and α-peltatin (about 0.25%). Desoxypodophyllotoxin and podophyllotoxone are also present in both plants, as are the glucosides of podophyllotoxin, 4-demethylpodophyllotoxin, and the peltatins, though preparation of the resin results in considerable losses of the water-soluble glucosides. Podophyllum resin has long been used as a purgative, but the discovery of the cytotoxic properties of podophyllotoxin and related compounds has now made podophyllum a commercially important drug plant. Preparations of podophyllum resin (the Indian resin is preferred) are effective treatments for warts, and pure podophyllotoxin is available as a paint for venereal warts, a condition which can be sexually transmitted. The antimitotic effect of podophyllotoxin and the other lignans is by binding to the protein tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules (compare vincristine, page 356, and colchicine, page 343). During mitosis, the chromosomes separate with the assistance of these microtubules, and after cell division the microtubules are transformed back to tubulin. Podophyllotoxin and other Podophyllum lignans were found to be unsuitable for clinical use as anticancer agents due to toxic side-effects, but the semi-synthetic derivatives etoposide and teniposide (Figure 4.21), which are manufactured from natural podophyllotoxin, have proved excellent antitumour agents. They were developed as modified forms (acetals) of the natural

(Continues) PHENYLPROPENES 137

(Continued)

base removes acidic reformation of keto form results proton α to carbonyl and OH generates enolate anion OH in change of stereochemistry OH O O O O O O O NaOAc O O H H O HB O O B MeO OMe MeO OMe MeO OMe OMe OMe OMe podophyllotoxin picropodophyllin

Figure 4.22

4-demethylpodophyllotoxin glucoside. Attempted synthesis of the glucoside inverted the stereochemistry at the sugar–aglycone linkage, and these agents are thus derivatives of 4- demethylepipodophyllotoxin (Figure 4.21). Etoposide is a very effective anticancer agent, and is used in the treatment of small cell lung cancer, testicular cancer and lymphomas, usually in combination therapies with other anticancer drugs. It may be given orally or intravenously. The water-soluble pro-drug etopophos (etoposide 4-phosphate) is also available. Teniposide has similar anticancer properties, and, though not as widely used as etoposide, has value in paediatric neuroblastoma. Remarkably, the 4-demethylepipodophyllotoxin series of lignans do not act via a tubulin- binding mechanism as does podophyllotoxin. Instead, these drugs inhibit the enzyme topoisomerase II, thus preventing DNA synthesis and replication. Topoisomerases are responsible for cleavage and resealing of the DNA strands during the replication process, and are classified as type I or II according to their ability to cleave one or both strands. Camptothecin (see page 365) is an inhibitor of topoisomerase I. Etoposide is believed to inhibit strand-rejoining ability by stabilizing the topoisomerase II–DNA complex in a cleavage state, leading to double-strand breaks and cell death. Development of other topoisomerase inhibitors based on podophyllotoxin-related lignans is an active area of research. Biological activity in this series of compounds is very dependent on the presence of the trans-fused five- membered lactone ring, this type of fusion producing a highly-strained system. Ring strain is markedly reduced in the corresponding cis-fused system, and the natural compounds are easily and rapidly converted into these cis-fused lactones by treatment with very mild bases, via enol tautomers or enolate anions (Figure 4.22). Picropodophyllin is almost devoid of cytotoxic properties. Podophyllotoxin is also found in significant amounts in the roots of other Podophyllum species, and in closely related genera such as Diphylleia (Berberidaceae).

oil from the bark of cinnamon (Cinnamomum contains large amounts of eugenol (Figure 4.23) zeylanicum; Lauraceae), widely used as a spice and much smaller amounts of cinnamaldehyde. and flavouring. Fresh bark is known to contain Eugenol is also the principal constituent in oil high levels of cinnamyl acetate, and cinnamalde- from cloves (Syzygium aromaticum; Myrtaceae), hyde is released from this by fermentation pro- used for many years as a dental anaesthetic, as cesses which are part of commercial preparation of well as for flavouring. The side-chain of eugenol the bark, presumably by enzymic hydrolysis and is derived from that of the cinnamyl alcohols by participation of the reversible aldehyde–alcohol reduction, but differs in the location of the double oxidoreductase. Cinnamon leaf, on the other hand, bond. This change is accounted for by resonance 138 THE SHIKIMATE PATHWAY

OCOCH3 CHO

MeO MeO O MeO OMe cinnamaldehyde cinnamyl acetate OMe OMe OH O OMe anethole estragole eugenol myristicin elemicin (methylchavicol)

Figure 4.23

loss of hydroxyl as resonance-stabilized leaving group allylic cation H = Ar OH Ar Ar Ar etc cinnamyl alcohol H H OMe OMe OH NADPH NADPH

Ar Ar propenylphenol allylphenol

Figure 4.24 forms of the allylic cation (Figure 4.24), and addi- have been shown to be weak carcinogens in tion of hydride (from NADPH) can generate either laboratory tests on animals. In the case of saf- allylphenols, e.g. eugenol, or propenylphenols, e.g. role (Figure 4.25), the main component of sas- anethole (Figure 4.23). Loss of hydroxyl from safras oil, this has been shown to arise from a cinnamyl alcohol may be facilitated by proto- hydroxylation in the side-chain followed by sul- nation, or perhaps even phosphorylation, though phation, giving an agent which binds to cel- there is no evidence for the latter. Myristicin lular macromolecules. Further data on volatile (Figure 4.23) from nutmeg (Myristica fragrans; oils containing aromatic constituents isolated from Myristicaceae) is a further example of an allylphe- these and other plant materials are given in nol found in flavouring materials. Myristicin also Table 4.1. Volatile oils in which the main compo- has a history of being employed as a mild hallu- nents are terpenoid in nature are listed in Table 5.1, cinogen via ingestion of ground nutmeg. Myris- page 177. ticin is probably metabolized in the body via an amination reaction to give an amfetamine- like derivative (see page 385). Anethole is the main component in oils from aniseed (Pimpinella HO HO3SO anisum; Umbelliferae/Apiaceae), star anise (Illi- cium verum; Illiciaceae), and fennel (Foeniculum vulgare; Umbelliferae/Apiaceae). The propenyl O O O components of flavouring materials such as cin- O O O namon, star anise, nutmeg, and sassafras (Sas- safrole safras albidum; Lauraceae) have reduced their commercial use somewhat since these constituents Figure 4.25 Table 4.1 Volatile oils containing principally aromatic compounds Volatile or essential oils are usually obtained from the appropriate plant material by steam distillation, though if certain components are unstableatthese temperatures, other less harsh techniques such as expression or solvent extraction may be employed. These oils, which typically contain a complex mixture of low boiling components, are widely used in flavouring, perfumery, and aromatherapy. Only a small number of oils have useful therapeutic properties, e.g. clove and dill, though a wide range of oils is now exploited for aromatherapy. Most of those employed in medicines are simply added for flavouring purposes. Some of the materials are commercially important as sources of chemicals used industrially, e.g. turpentine. For convenience, the major oils listed are divided into two groups. Those which contain principally chemicals which are aromatic in nature and which are derived by the shikimate pathway are given in Table 4.1 below. Those oils which are composed predominantly of terpenoid compounds are listed in Table 5.1 on page 177, since they are derived via the deoxyxylulose phosphate pathway. It must be appreciated that many oils may contain aromatic and terpenoid components, but usually one group predominates. The oil yields, and the exact composition of any sample of oil will be variable, depending on the particular plant material used in its preparation. The quality of an oil and its commercial value is dependent on the proportion of the various components. Oil Plant source Plant part Oil Major constituents with Uses, notes used content (%) typical (%) composition Aniseed Pimpinella anisum ripe fruit 2–3 anethole (80–90) flavour, carminative, (Anise) (Umbelliferae/ estragole (1–6) aromatherapy Apiaceae) Star anise Illicium verum ripe fruit 5–8 anethole (80–90) flavour, carminative (Illiciaceae) estragole (1–6) fruits contain substantial amounts of shikimic and quinic acids Cassia Cinnamomum cassia dried bark, 1–2 cinnamaldehyde (70–90) flavour, carminative (Lauraceae) or leaves 2-methoxycinnamal- and twigs dehyde (12) known as cinnamon oil in USA Cinnamon bark Cinnamomum dried bark 1–2 cinnamaldehyde (70–80) flavour, carminative, zeylanicum eugenol (1–13) aromatherapy (Lauraceae) cinnamyl acetate (3–4) Cinnamon leaf Cinnamomum leaf 0.5–0.7 eugenol (70–95) flavour zeylanicum (Lauraceae) (Continued overleaf ) Table 4.1 (Continued) Oil Plant source Plant part Oil Major constituents with Uses, notes used content typical (%) composition (%) Clove Syzygium aromaticum dried flower 15–20 eugenol (75–90) flavour, aromatherapy, antiseptic (Eugenia caryophyllus) buds eugenyl acetate (10–15) (Myrtaceae) β-caryophyllene (3) Fennel Foeniculum vulgare ripe fruit 2–5 anethole (50–70) flavour, carminative, aromatherapy (Umbelliferae/ fenchone (10–20) Apiaceae) estragole (3–20) Nutmeg Myristica fragrans seed 5–16 sabinene (17–28) flavour, carminative, aromatherapy (Myristicaceae) α-pinene (14–22) β-pinene (9–15) although the main constituents are terpinen-4-ol (6–9) terpenoids, most of the flavour myristicin (4–8) comes from the minor aromatic elemicin (2) constituents, myristicin, elemicin, etc myristicin is hallucinogenic (see page 385) Wintergreen Gaultheria procumbens leaves 0.7–1.5 methyl salicylate (98%) flavour, antiseptic, antirheumatic (Ericacae) or Betula lenta bark 0.2–0.6 prior to distillation, plant material (Betulaceae) is macerated with water to allow enzymic hydrolysis of glycosides methyl salicylate is now produced synthetically BENZOIC ACIDS FROM C6C3 COMPOUNDS 141

BENZOIC ACIDS FROM C6C3 Coenzyme A esters are not involved, and though COMPOUNDS a similar hydration of the double bond occurs, chain shortening features a reverse aldol reac- tion, generating the appropriate aromatic alde- Some of the simple hydroxybenzoic acids (C C 6 1 hyde. The corresponding acid is then formed compounds) such as 4-hydroxybenzoic acid and via an NAD+-dependent oxidation step. Thus, gallic acid can be formed directly from inter- aromatic aldehydes such as vanillin,themain mediates early in the shikimate pathway, e.g. flavour compound in vanilla (pods of the orchid 3-dehydroshikimic acid or chorismic acid (see Vanilla planiflora; Orchidaceae) would be formed page 121), but alternative routes exist in which from the correspondingly substituted cinnamic acid cinnamic acid derivatives (C6C3 compounds) are without proceeding through intermediate benzoic cleaved at the double bond and lose two car- acids or esters. Whilst the substitution pattern bons from the side-chain. Thus, 4-coumaric acid in these C6C1 derivatives is generally built up may act as a precursor of 4-hydroxybenzoic at the C6C3 cinnamic acid stage, prior to chain acid, and ferulic acid may give vanillic acid shortening, there exists the possibility of further (4-hydroxy-3-methoxybenzoic acid) (Figure 4.26). hydroxylations and/or methylations occurring at A sequence analogous to that involved in the the C6C1 level, and this is known in certain β-oxidation of fatty acids (see page 18) is possi- examples. Salicylic acid (Figure 4.27) is synthe- ble, so that the double bond in the coenzyme A sized in microorganisms directly from isochorismic ester would be hydrated, the hydroxyl group oxi- acid (see page 124), but can arise in plants by two dized to a ketone, and the β-ketoester would then other mechanisms. It can be produced by hydrox- lose acetyl-CoA by a reverse Claisen reaction, giv- ylation of benzoic acid, or by side-chain cleavage ing the coenzyme A ester of 4-hydroxybenzoic of 2-coumaric acid, which itself is formed by an acid. Whilst this sequence has been generally ortho-hydroxylation of cinnamic acid. Methyl sal- accepted, newer evidence supports another side- icylate is the principal component of oil of win- chain cleavage mechanism, which is different from tergreen from Gaultheria procumbens (Ericaceae), the fatty acid β-oxidation pathway (Figure 4.26). used for many years for pain relief. It is derived by

CO2H COSCoA COSCoA COSCoA HO O

HSCoA ATP H2O NAD+ β R R R R -oxidation pathway, as in fatty acid metabolism OH OH OH OH (Figure 2.11) R = H, 4-coumaric acid R = H, 4-coumaroyl-CoA R = OMe, ferulic acid R = OMe, feruloyl-CoA HSCoA reverse H2O Claisen CH3COSCoA

CO2H HO CHO CO2H COSCoA reverse aldol NAD+

R R R R OH OH OH OH R = H, 4-hydroxy- R = H, 4-hydroxy- benzaldehyde benzoic acid R = OMe, vanillin R = OMe, vanillic acid

Figure 4.26 142 THE SHIKIMATE PATHWAY

hydroxylation methylation CO2H CO2H CO2Me CO2H

OH SAM OH OCOCH3

benzoic acid salicylic acid methyl salicylate aspirin (acetylsalicyclic acid) side-chain cleavage

CO2HCO2H

hydroxylation side-chain CHO glucosylation CHO reduction CH OH cleavage 2 OH OH UDPGlc OGlc OGlc

2-coumaric acid salicylaldehyde salicin

Figure 4.27

SAM-dependent methylation of salicylic acid. The the trans (E) to the less stable cis (Z) form. Whilst salicyl alcohol derivative salicin, found in many trans–cis isomerization would be unfavourable species of willow (Salix species; Salicaceae), is not in the case of a single isolated double bond, in derived from salicylic acid, but probably via gluco- the cinnamic acids the fully conjugated system sylation of salicylaldehyde and then reduction of allows this process to occur quite readily, and the carbonyl (Figure 4.27). Salicin is responsible UV irradiation, e.g. daylight, is sufficient to pro- for the analgesic and antipyretic effects of wil- duce equilibrium mixtures which can be separated low barks, widely used for centuries, and the tem- (Figure 4.29). The absorption of energy promotes plate for synthesis of acetylsalicylic acid (aspirin) an electron from the π-orbital to a higher energy (Figure 4.27) as a more effective analogue. state, the π∗-orbital, thus temporarily destroying the double bond character and allowing rotation. Loss of the absorbed energy then results in re- COUMARINS formation of the double bond, but in the cis- configuration. In conjugated systems, the π–π∗ The hydroxylation of cinnamic acids ortho to the energy difference is considerably less than with a side-chain as seen in the biosynthesis of salicylic non-conjugated double bond. Chemical lactoniza- acid is a crucial step in the formation of a group of tion can occur on treatment with acid. Both the cinnamic acid lactone derivatives, the coumarins. trans–cis isomerization and the lactonization are Whilst the direct hydroxylation of the aromatic enzyme-mediated in nature, and light is not nec- ring of the cinnamic acids is common, hydrox- essary for coumarin biosynthesis. Thus, cinnamic ylation generally involves initially the 4-position acid and 4-coumaric acid give rise to the coumarins para to the side-chain, and subsequent hydrox- coumarin and umbelliferone (Figure 4.28). Other ylations then proceed ortho to this substituent coumarins with additional oxygen substituents on (see page 132). In contrast, for the coumarins, the aromatic ring, e.g. aesculetin and scopoletin, hydroxylation of cinnamic acid or 4-coumaric acid appear to be derived by modification of umbellif- can occur ortho to the side-chain (Figure 4.28). erone, rather than by a general cinnamic acid to In the latter case, the 2,4-dihydroxycinnamic acid coumarin pathway. This indicates that the hydrox- produced confusingly seems to possess the meta ylation meta to the existing hydroxyl, discussed hydroxylation pattern characteristic of phenols above, is a rather uncommon occurrence. derived via the acetate pathway. Recognition of Coumarins are widely distributed in plants, the C6C3 skeleton should help to avoid this confu- and are commonly found in families such as the sion. The two 2-hydroxycinnamic acids then suffer Umbelliferae/Apiaceae and Rutaceae, both in the a change in configuration in the side-chain, from free form and as glycosides. Coumarin itself is COUMARINS 143

trans-cis isomerization lactone formation CO H 2 CO2H

CO2H OH OH OO cinnamic acid 2-coumaric acid coumarin

CO2H CO2H

CO2H HO HO OH HO OH HO OO 4-coumaric acid 2,4-dihydroxy- umbelliferone cinnamic acid

MeO MeO HO

GlcO OO HO OO HO OO scopolin scopoletin aesculetin

Figure 4.28

E Z CO2H hν H

CO2H OH OH OH OO coumarin

Figure 4.29

CO2H hydrolysis and lactonization through damage to the plant tissues during harvesting and process- OGlc ing (Figure 4.30). If sweet clover is allowed to (E)-2-coumaric acid ferment, 4-hydroxycoumarin is produced by the glucoside action of microorganisms on 2-coumaric acid (Figure 4.31) and this can react with formalde- enzymic hydrolysis hyde, which is usually present due to micro- bial degradative reactions, combining to give CO2H ∗ O OO dicoumarol. Dicoumarol is a compound with pro- Glc coumarin nounced blood anticoagulant properties, which can (Z)-2-coumaric acid cause the deaths of livestock by internal bleeding, glucoside and is the forerunner of the warfarin∗ group of medicinal anticoagulants. Figure 4.30 Many other natural coumarins have a more complex carbon framework and incorporate found in sweet clover (Melilotus species; Legu- extra carbons derived from an isoprene unit minosae/Fabaceae) and contributes to the smell (Figure 4.33). The aromatic ring in umbelliferone of new-mown hay, though there is evidence is activated at positions ortho to the hydroxyl that the plants actually contain the glucosides of group and can thus be alkylated by a suitable (E)- and (Z)-2-coumaric acid (Figure 4.30), and alkylating agent, in this case dimethylallyl coumarin is only liberated as a result of enzymic diphosphate. The newly introduced dimethylallyl 144 THE SHIKIMATE PATHWAY

aldol reaction; it may help to lactone formation consider the diketo tautomer and enolization H O OH O OH OH COSCoA COSCoA HH dehydration follows OH OH O O O O

4-hydroxycoumarin nucleophilic – H2O attack on to the HCHO enone system

H OH OH O OH

OOO O O O O O dicoumarol

Figure 4.31

Dicoumarol and Warfarin The cause of fatal haemorrhages in animals fed spoiled sweet clover (Melilotus officinalis; Leguminosae/Fabaceae) was traced to dicoumarol (bishydroxycoumarin) (Figure 4.31). This agent interferes with the effects of vitamin K in blood coagulation (see page 163), the blood loses its ability to clot, and thus minor injuries can lead to severe internal bleeding. Synthetic dicoumarol has been used as an oral blood anticoagulant in the treatment of thrombosis, where the risk of blood clots becomes life threatening. It has been superseded by salts of warfarin and acenocoumarol (nicoumalone) (Figure 4.32), which are synthetic developments from the natural product. An overdose of warfarin may be countered by injection of vitamin K1. Warfarin was initially developed as a rodenticide, and has been widely employed for many years as the first choice agent, particularly for destruction of rats. After

OH O OH OH RS RS RS RS NO Cl O O 2 O O O O warfarin coumatetralyl R = H, difenacoum R = Br, brodifenacoum R OH O OH O RS RS

O O O O acenocoumarol coumachlor (nicoumalone)

Figure 4.32

(Continues) COUMARINS 145

(Continued)

consumption of warfarin-treated bait, rats die from internal haemorrhage. Other coumarin derivatives employed as rodenticides include coumachlor and coumatetralyl (Figure 4.32). In an increasing number of cases, rodents are becoming resistant to warfarin, an ability which has been traced to elevated production of vitamin K by their intestinal microflora. Modified structures defenacoum and brodifenacoum have been found to be more potent than warfarin, and are also effective against rodents that have become resistant to warfarin. group in demethylsuberosin is then able to enzyme then cleaves off the hydroxyisopropyl cyclize with the phenol group giving marmesin. fragment (as acetone) from marmesin giving This transformation is catalysed by a cytochrome the furocoumarin psoralen (Figure 4.35). This P-450-dependent mono-oxygenase, and requires does not involve any hydroxylated intermediate, cofactors NADPH and molecular oxygen. For and cleavage is believed to be initiated by a many years, the cyclization had been postulated radical abstraction process. Psoralen can act as a to involve an intermediate epoxide, so that precursor for the further substituted furocoumarins nucleophilic attack of the phenol on to the bergapten, xanthotoxin,andisopimpinellin epoxide group might lead to formation of either (Figure 4.33), such modifications occurring late five-membered furan or six-membered pyran in the biosynthetic sequence rather than at heterocycles as commonly encountered in natural the cinnamic acid stage. Psoralen, bergapten, products (Figure 4.34). Although the reactions of etc are termed ‘linear’ furocoumarins. ‘Angular’ Figure 4.34 offer a convenient rationalization for furocoumarins, e.g. angelicin (Figure 4.33), can cyclization, epoxide intermediates have not been arise by a similar sequence of reactions, but demonstrated in any of the enzymic systems so far these involve dimethylallylation at the alternative investigated, and therefore some direct oxidative position ortho to the phenol. An isoprene-derived cyclization mechanism must operate. A second furan ring system has already been noted in the cytochrome P-450-dependent mono-oxygenase formation of khellin (see page 74), though the

C-alkylation at activated position OPP ortho to phenol O2 NADPH DMAPP HO HO OO HO OO O OO umbelliferone demethylsuberosin marmesin

O2 NADPH OMe OH O2 SAM NADPH

O OO O OO O OO bergapten bergaptol psoralen O2 (linear furocoumarin) OMe hydroxylation NADPH methylation SAM

O O O OO OO OO O OO OMe OMe OH isopimpinellin xanthotoxin xanthotoxol angelicin (angular furocoumarin)

Figure 4.33 146 THE SHIKIMATE PATHWAY

H O – H2O HO HO O O

nucleophilic attack 5-membered furan ring H on to epoxide

O HO – H2O

HO O O 6-membered pyran ring

Figure 4.34

oxidation leading to radical cleavage of Enz Fe Enz Fe O side-chain carbons H H OH O2 NADPH HO HO HO O OO O OO O OO marmesin psoralen – H O side-chain carbons 2 O HO OH released as acetone

Figure 4.35 aromatic ring to which it was fused was in that towards UV light, resulting in sunburn or serious case a product of the acetate pathway. Linear blistering. Used medicinally, this effect may be furocoumarins (psoralens)∗ can be troublesome to valuable in promoting skin pigmentation and humans since they can cause photosensitization treating psoriasis.

Psoralens Psoralens are linear furocoumarins which are widely distributed in plants, but are particularly abundant in the Umbelliferae/Apiaceae and Rutaceae. The most common examples are psoralen, bergapten, xanthotoxin, and isopimpinellin (Figure 4.33). Plants containing psoralens have been used internally and externally to promote skin pigmentation and sun-tanning. Bergamot oil obtained from the peel of Citrus aurantium ssp. bergamia (Rutaceae) (see page 179) can contain up to 5% bergapten, and is frequently used in external suntan preparations. The psoralen, because of its extended chromophore, absorbs in the near UV and allows this radiation to stimulate formation of melanin pigments (see page 129). Methoxsalen (xanthotoxin; 8-methoxypsoralen) (Figure 4.36), a constituent of the fruits of Ammi majus (Umbelliferae/Apiaceae), is used medically to facilitate skin repigmentation where severe blemishes exist (vitiligo). An oral dose of methoxsalen is followed by long wave UV irradiation, though such treatments must be very carefully regulated to

(Continues) STYRYLPYRONES 147

(Continued)

H O N O N

O O O O O O O hν O hν O HN HN HN

O N O OMe O N O OMe O N O OMe xanthotoxin (methoxsalen) thymine in DNA psoralen−DNA adduct psoralen−DNA di-adduct

Figure 4.36

minimize the risk of burning, cataract formation, and the possibility of causing skin cancer. The treatment is often referred to as PUVA (psoralen + UV-A). PUVA is also of value in the treatment of psoriasis, a widespread condition characterized by proliferation of skin cells. Similarly, methoxsalen is taken orally, prior to UV treatment. Reaction with psoralens inhibits DNA replication and reduces the rate of cell division. Because of their planar nature, psoralens intercalate into DNA, and this enables a UV-initiated cycloaddition reaction between pyrimidine bases (primarily thymine) in DNA and the furan ring of psoralens (Figure 4.36). In some cases, di-adducts can form involving further cycloaddition via the pyrone ring, thus cross-linking the nucleic acid. A troublesome extension of these effects can arise from the handling of plants that contain significant levels of furocoumarins. Celery (Apium graveolens; Umbelliferae/Apiaceae) is normally free of such compounds, but fungal infection with the natural parasite Sclerotinia sclerotiorum induces the synthesis of furocoumarins (xanthotoxin and others) as a response to the infections. Some field workers handling these infected plants have become very sensitive to UV light and suffer from a form of sunburn termed photophytodermatitis. Infected parsley (Petroselinum crispum) can give similar effects. Handling of rue (Ruta graveolens; Rutaceae) or giant hogweed (Heracleum mantegazzianum; Umbelliferae/Apiaceae), which naturally contain significant amounts of psoralen, bergapten, and xanthotoxin, can cause similar unpleasant reactions, or more commonly rapid blistering by direct contact with the sap. The giant hogweed can be particularly dangerous. Individuals vary in their sensitivity towards furocoumarins; some are unaffected whilst others tend to become sensitized by an initial exposure and then develop the allergic response on subsequent exposures.

STYRYLPYRONES plus one or two C2 units from malonyl-CoA. The short poly-β-keto chain frequently cyclizes to form a lactone derivative (compare triacetic acid lac- Cinnamic acids, as their coenzyme A esters, may tone, page 62). Thus, Figure 4.37 shows the pro- also function as starter units for chain exten- posed derivation of yangonin via cyclization of the sion with malonyl-CoA units, thus combining di-enol tautomer of the polyketide formed from elements of the shikimate and acetate pathways 4-hydroxycinnamoyl-CoA and two malonyl-CoA (see page 80). Most commonly, three C2 units extender units. Two methylation reactions com- are added via malonate giving rise to flavonoids plete the sequence. Yangonin and a series of related and stilbenes, as described in the next section structures form the active principles of kava root (page 149). However, there are several examples (Piper methysticum; Piperaceae), a herbal remedy of products formed from a cinnamoyl-CoA starter popular for its anxiolytic activity. 148 THE SHIKIMATE PATHWAY

chain extension; acetate pathway with O a cinnamoyl-CoA starter group OH di-enol tautomer O 2 x malonyl-CoA

SCoA O O ≡ OH O SCoA SCoA HO HO HO 4-hydroxycinnamoyl-CoA OMe lactone OH formation

SAM O O O O

MeO HO yangonin

Figure 4.37

Kava Aqueous extracts from the root and rhizome of Piper methysticum (Piperaceae) have long been consumed as an intoxicating beverage by the peoples of Pacific islands comprising Polynesia, Melanesia and Micronesia, and the name kava or kava-kava referred to this drink. In herbal medicine, the dried root and rhizome is now described as kava, and it is used for the treatment of anxiety, nervous tension, agitation and insomnia. The pharmacological activity is associated with a group of styrylpyrone derivatives termed kavapyrones or kavalactones, good quality roots containing 5–8% kavapyrones. At least 18 kavapyrones have been characterized, the six major ones being the enolides kawain, methysticin, and their dihydro derivatives reduced in the cinnamoyl side-chain, and the dienolides yangonin and demethoxyyangonin (Figure 4.38). Compared with the dienolides, the enolides have a reduced pyrone ring and a chiral centre. Clinical trials have indicated kava extracts to be effective as an anxiolytic, the kavapyrones also displaying anticonvulsive, analgesic, and central muscle relaxing action. Several of these compounds have been shown to have an effect on neurotransmitter systems including those involving glutamate, GABA, dopamine, and serotonin.

OMe OMe OMe OMe

O O OO OO OO OO H H H H O O kawaindihydrokawain methysticin dihydromethysticin OMe OMe

O O OO

MeO yangonin demethoxyyangonin

Figure 4.38 FLAVONOIDS AND STILBENES 149

FLAVONOIDS AND STILBENES Both structures nicely illustrate the different char- acteristic oxygenation patterns in aromatic rings Flavonoids and stilbenes are products from a derived from the acetate or shikimate pathways. cinnamoyl-CoA starter unit, with chain exten- With the stilbenes, it is noted that the terminal sion using three molecules of malonyl-CoA. ester function is no longer present, and there- This initially gives a polyketide (Figure 4.39), fore hydrolysis and decarboxylation have also which, according to the nature of the enzyme taken place during this transformation. No inter- responsible, can be folded in two different mediates, e.g. carboxylated stilbenes, have been ways. These allow aldol or Claisen-like reac- detected, and the transformation from cinnamoyl- tions to occur, generating aromatic rings as CoA/malonyl-CoA to stilbene is catalysed by the already seen in Chapter 3 (see page 80). Enzymes single enzyme. Resveratrol has assumed greater stilbene synthase and chalcone synthase cou- relevance in recent years as a constituent of ple a cinnamoyl-CoA unit with three malonyl- grapes and wine, as well as other food products, CoA units giving stilbenes, e.g. resveratrol or with antioxidant, anti-inflammatory, anti-platelet, chalcones, e.g. naringenin-chalcone respectively. and cancer preventative properties. Coupled with

OH

CoAS

O 4-hydroxycinnamoyl-CoA chain extension; acetate pathway with 3 x malonyl-CoA a cinnamoyl-CoA starter group OH OH OH O SCoA O SCoA O NADPH ≡ O O O SCoA (reductase) O O OH O O O Claisen aldol Claisen (stilbene synthase) (chalcone synthase) CO2 OH OH OH

HO HO OH HO OH

OH OH O O resveratrol naringenin-chalcone isoliquiritigenin (a stilbene) (a chalcone) Michael-type nucleophilic (a chalcone) attack of OH on to α,β-unsaturated ketone

OH OH

HO O HO O

OH O O naringenin liquiritigenin (a flavanone) (a flavanone)

Figure 4.39 150 THE SHIKIMATE PATHWAY the cardiovascular benefits of moderate amounts has a resorcinol oxygenation pattern rather than of alcohol, and the beneficial antioxidant effects the phloroglucinol system. This modification has of flavonoids (see page 151), red wine has now been tracked down to the action of a reductase emerged as an unlikely but most acceptable medic- enzyme concomitant with the chalcone synthase, inal agent. and thus isoliquiritigenin is produced rather than Chalcones act as precursors for a vast range naringenin-chalcone. Flavanones can then give of flavonoid derivatives found throughout the rise to many variants on this basic skeleton, e.g. plant kingdom. Most contain a six-membered flavones, flavonols, anthocyanidins,andcate- heterocyclic ring, formed by Michael-type nucle- chins (Figure 4.40). Modifications to the hydroxy- ophilic attack of a phenol group on to the unsatu- lation patterns in the two aromatic rings may occur, rated ketone giving a flavanone,e.g.naringenin generally at the flavanone or dihydroflavonol stage, (Figure 4.39). This isomerization can occur chem- and methylation, glycosylation, and dimethylal- ically, acid conditions favouring the flavanone lylation are also possible, increasing the range and basic conditions the chalcone, but in nature of compounds enormously. A high proportion of the reaction is enzyme catalysed and stereospe- flavonoids occur naturally as water-soluble gly- cific, resulting in formation of a single fla- cosides. Considerable quantities of flavonoids are vanone enantiomer. Many flavonoid structures, consumed daily in our vegetable diet, so adverse e.g. liquiritigenin, have lost one of the hydroxyl biological effects on man are not particularly groups, so that the acetate-derived aromatic ring intense. Indeed, there is growing belief that some

OH OH OH

HO O HO O HO O R O2 R O2 R 2-oxoglutarate 2-oxoglutarate OH OH OH O OH O OH O R = H, naringenin R = H, dihydrokaempferol R = H, kaempferol R = OH, eriodictyol R = OH, dihydroquercetin R = OH, quercetin (flavanones) (dihydroflavonols) (flavonols) O2 2-oxoglutarate NADPH OH OH OH HO O HO O HO R HO O R O R

OH OH OH O OH OH OH OH R = H, apigenin R = H, leucopelargonidin R = OH, luteolin R = OH, leucocyanidin (flavones) (flavandiols; leucoanthocyanidins) – 2 H2O

NADPH OH OH

HO O HO O R R

OH OH OH OH R = H, afzalechin R = H, pelargonidin R = OH, (+)-catechin R = OH, cyanidin (catechins) (anthocyanidins)

Figure 4.40 FLAVONOIDS AND STILBENES 151

OH

O HO O HO HO OH OH OH OH OH HO O HO O OH OH O OH OH OH OH OH HO O OH HO OH theaflavin OH OH epicatechin trimer

Figure 4.41

flavonoids are particularly beneficial, acting as peels have been included in dietary supplements as antioxidants and giving protection against cardio- vitamin P, and claimed to be of benefit in treating vascular disease, certain forms of cancer, and, conditions characterized by capillary bleeding, but it is claimed, age-related degeneration of cell their therapeutic efficacy is far from conclusive. components. Their polyphenolic nature enables Neohesperidin (Figure 4.42) from bitter orange them to scavenge injurious free radicals such as (Citrus aurantium; Rutaceae) and naringin from superoxide and hydroxyl radicals. Quercetin in grapefruit peel (Citrus paradisi) are intensely particular is almost always present in substan- bitter flavanone glycosides. It has been found tial amounts in plant tissues, and is a power- that conversion of these compounds into dihy- ful antioxidant, chelating metals, scavenging free drochalcones by hydrogenation in alkaline solu- radicals, and preventing oxidation of low density tion (Figure 4.43) produces a remarkable change lipoprotein. Flavonoids in red wine (quercetin, to their taste, and the products are now intensely kaempferol, and anthocyanidins) and in tea (cate- sweet, being some 300–1000 times as sweet as chins and catechin gallate esters) are also demon- sucrose. These and other dihydrochalcones have strated to be effective antioxidants. Flavonoids been investigated as non-sugar sweetening agents. contribute to plant colours, yellows from chal- cones and flavonols, and reds, blues, and violets FLAVONOLIGNANS from anthocyanidins. Even the colourless mate- rials, e.g. flavones, absorb strongly in the UV An interesting combination of flavonoid and lig- and are detectable by insects, probably aiding nan structures is found in a group of compounds flower pollination. Catechins form small poly- called flavonolignans. They arise by oxidative mers (oligomers), the condensed tannins,e.g.the coupling processes between a flavonoid and a epicatechin trimer (Figure 4.41) which contribute phenylpropanoid, usually coniferyl alcohol. Thus, astringency to our foods and drinks, as do the sim- the dihydroflavonol taxifolin through one-electron pler gallotannins (see page 122), and are commer- oxidation may provide a free radical, which may cially important for tanning leather. Theaflavins, combine with the free radical generated from antioxidants found in fermented tea (see page 395), coniferyl alcohol (Figure 4.44). This would lead are dimeric catechin structures in which oxida- to an adduct, which could cyclize by attack of tive processes have led to formation of a seven- the phenol nucleophile on to the quinone methide membered tropolone ring. system provided by coniferyl alcohol. The prod- The flavonol glycoside rutin (Figure 4.42) uct would be silybin, found in Silybum marianum from buckwheat (Fagopyrum esculentum; Polygo- (Compositae/Asteraceae) as a mixture of two trans naceae) and rue (Ruta graveolens; Rutaceae), and diastereoisomers, reflecting a lack of stereospeci- the flavanone glycoside hesperidin from Citrus ficity for the original radical coupling. In addition, 152 THE SHIKIMATE PATHWAY

OH HO HO O L-Rha O OMe OH HO O O O HO OH HO O OH OH D-Glc O-Glc-Rha OH O rutinose OH O = rhamnosyl(α1→6)glucose hesperetinquercetin rutinose

hesperidin rutin

OH OMe HO O D-Glc O O HO OH OH O Rha-Glc-O O O HO OH O HO L-Rha OH OH O neohesperidose = rhamnosyl(α1→2)glucose hesperetin neohesperidose naringenin

neohesperidin naringin

Figure 4.42

R1 R1 R1

Rha-Glc-O O Rha-Glc-O OH Rha-Glc-O OH R2 R2 R2

OH H2 / catalyst H OH O OH O OH O OH R1 = OH, R2 = H, R1 = OH, R2 = H, naringin naringin dihydrochalcone R1 = OMe, R2 = OH, neohesperidin R1 = OMe, R2 = OH, neohesperidin dihydrochalcone

Figure 4.43 the regioisomer isosilybin (Figure 4.45), again a of an enolate nucleophile on to the quinoneme- mixture of trans diastereoisomers, is also found in thide. Hemiketal formation finishes the process. Silybum. Silychristin (Figure 4.45) demonstrates The flavonolignans from Silybum∗ (milk thistle) a further structural variant which can be seen to have valuable antihepatotoxic properties, and can originate from a mesomer of the taxifolin-derived provide protection against liver-damaging agents. free radical, in which the unpaired electron is Coumarinolignans, which are products arising by a localized on the carbon ortho to the original 4- similar oxidative coupling mechanism which com- hydroxyl function. The more complex structure bines a coumarin with a cinnamyl alcohol, may in silydianin is accounted for by the mechanism be found in other plants. The benzodioxane ring shown in Figure 4.46, in which the initial coupling as seen in silybin and isosilybin is a characteristic product cyclizes further by intramolecular attack feature of many such compounds. FLAVONOLIGNANS 153

OH OH one-electron one-electron oxidation OH oxidation O

HO O HO O OH – H OH – H – e – e OH OH OMe OMe OH O OH O O OH taxifolin coniferyl alcohol OH radical coupling O OH HO O OMe O O

OH OH HO O OMe OH OH O + OH OH O H O OH O nucleophilic attack of OH HO O OMe O on to quinonemethide (two stereochemistries possible) OH OH OH O silybin (diastereoisomeric pair)

Figure 4.44

Silybum marianum

Silybum marianum (Compositae/Asteraceae) is a biennial thistle-like plant (milk thistle) common in the Mediterranean area of Europe. The seeds yield 1.5–3% of flavonolignans collectively termed silymarin. This mixture contains mainly silybin (Figure 4.44), together with silychristin (Figure 4.45), silydianin (Figure 4.46), and small amounts of isosilybin (Figure 4.45). Both silybin and isosilybin are equimolar mixtures of two trans diastereoisomers. Silybum marianum is widely used in traditional European medicine, the fruits being used to treat a variety of hepatic and other disorders. Silymarin has been shown to protect animal livers against the damaging effects of carbon tetrachloride, thioacetamide, drugs such as paracetamol, and the toxins α-amanitin and phalloin found in the death cap fungus (Amanita phalloides) (see page 433). Silymarin may be used in many cases of liver disease and injury, though it still remains peripheral to mainstream medicine. It can offer particular benefit in the treatment of poisoning by the death cap fungus. These agents appear to have two main modes of action. They act on the cellular membrane of hepatocytes inhibiting absorption of toxins, and secondly, because of their phenolic nature, they can act as antioxidants and scavengers for free radicals which cause liver damage originating from liver detoxification of foreign chemicals. Derivatives of silybin with improved water-solubility and/or bioavailability have been developed, e.g. the bis-hemisuccinate and a phosphatidylcholine complex. 154 THE SHIKIMATE PATHWAY

OH OH O O OMe O HO O HO O O OMe OH OH HO OH OH O OH O + OH OH O OMe O HO O OH O HO O OH OMe OH OH OH OH O isosilybin OH O (diastereoisomeric pair) silychristin

Figure 4.45

(i) nucleophilic attack of enolate on to quinonemethide MeO (ii) hemiketal formation OH HO MeO radical O (ii) OH coupling O H O H MeO O O H HO O HO O OH OH OH HO O (i) O OH OH OH OH O OH O OH O silydianin

Figure 4.46

ISOFLAVONOIDS of different isoflavonoids have been identified, and structural complexity is brought about by The isoflavonoids form a quite distinct subclass hydroxylation and alkylation reactions, varying the of flavonoid compound, being structural vari- oxidation level of the heterocyclic ring, or forming ants in which the shikimate-derived aromatic additional heterocyclic rings. Some of the many ring has migrated to the adjacent carbon of variants are shown in Figure 4.48. Pterocarpans, the heterocycle. This rearrangement process is e.g. medicarpin from lucerne (Medicago sativa), brought about by a cytochrome P-450-dependent and pisatin from pea (Pisum sativum), have anti- enzyme requiring NADPH and O2 cofactors, fungal activity and form part of these plants’ which transforms the flavanones liquiritigenin natural defence mechanism against fungal attack. or naringenin into the isoflavones daidzein or Simple isoflavones such as daidzein and coumes- genistein respectively via intermediate hydrox- tans such as coumestrol from lucerne and clovers yisoflavanones (Figure 4.47). A radical mecha- (Trifolium species), have sufficient oestrogenic nism has been proposed. This rearrangement is activity to seriously affect the reproduction of graz- quite rare in nature, and isoflavonoids are almost ing animals, and are termed phyto-oestrogens∗. entirely restricted to the plant family the Legu- These planar molecules undoubtedly mimic the minosae/Fabaceae. Nevertheless, many hundreds shape and polarity of the steroid hormone estradiol ISOFLAVONOIDS 155

oxidation to 1,2-aryl OH free radical OH migration O OH HO O 2 HO O HO O NADPH Fe Enz H H O R O R O R O Fe Enz OH R = H, liquiritigenin R = OH, naringenin

(flavonoid) HO 7 O HO O OH – H2O

R O 4′ R O OH OH R = H, daidzein R = OH, genistein (isoflavonoid)

Figure 4.47

HO O HO O HO O H

H O O O OH OMe OMe daidzein formononetin medicarpin (an isoflavone) (an isoflavone) (a pterocarpan)

H HO O O MeO O H O O OH O O H O O OH H O O coumestrol OMe pisatin (a coumestan) rotenone OMe (a pterocarpan) (a rotenoid)

Figure 4.48

(see page 276). The consumption of legume fod- (see page 279). The rotenoids take their name der crops by animals must therefore be restricted, from the first known example rotenone,andare or low isoflavonoid producing strains have to be formed by ring cyclization of a methoxyisoflavone selected. Isoflavonoids in the human diet, e.g. (Figure 4.49). Rotenone itself contains a C5 iso- from soya (Glycine max) products, are believed to prene unit (as do virtually all the natural rotenoids) give some protection against oestrogen-dependent introduced via dimethylallylation of demethyl- cancers such as breast cancer, by restricting the munduserone. The isopropenylfurano system of availability of the natural hormone. In addition, rotenone, and the dimethylpyrano of deguelin, they can feature as dietary oestrogen supplements are formed via rotenonic acid (Figure 4.49) with- in the reduction of menopausal symptoms, in out any detectable epoxide or hydroxy intermedi- a similar way to hormone replacement therapy ates (compare furocoumarins, page 145). Rotenone 156 THE SHIKIMATE PATHWAY

oxidation of OMe group (hydroxylation and loss of hydroxide, compare H HO O Figure 2.21) HO O HO O OMe O O

O O O H OMe OMe OMe OMe OMe OMe H

H C-alkylation at addition of O O activated position hydride O cyclization to 5-membered ring ortho to phenol (reduction)

H O OPP OMe H H HO O DMAPP HO O rotenone OMe O O

H H O O H O O O OMe cyclization to OMe 6-membered ring OMe OMe H rotenonic acid demethylmunduserone O OMe deguelin OMe

Figure 4.49 and other rotenoids are powerful insecticidal and Rotenone thus provides an excellent biodegradable piscicidal (fish poison) agents, interfering with insecticide, and is used as such either in pure or ∗ oxidative phosphorylation. They are relatively powdered plant form. Roots of Derris elliptica ∗ harmless to mammals unless they enter the blood or Lonchocarpus species are rich sources of stream, being metabolized rapidly upon ingestion. rotenone.

Phyto-oestrogens

Phyto-oestrogen (phytoestrogen) is a term applied to non-steroidal plant materials displaying oestrogenic properties. Pre-eminent amongst these are isoflavonoids. These planar molecules mimic the shape and polarity of the steroid hormone estradiol (see page 279), and are able to bind to an oestrogen receptor, though their activity is less than that of estradiol. In some tissues, they stimulate an oestrogenic response, whilst in others they can antagonize the effect of oestrogens. Such materials taken as part of the diet therefore influence overall oestrogenic activity in the body by adding their effects to normal levels of steroidal oestrogens (see page 282). Foods rich in isoflavonoids are valuable in countering some of the side-effects of the menopause in women, such as hot flushes, tiredness, and mood swings. In addition, there is mounting evidence that phyto-oestrogens also provide a range of other beneficial effects, helping to prevent heart attacks and other cardiovascular diseases, protecting against osteoporosis, lessening the risk of breast and uterine cancer, and in addition displaying significant antioxidant activity which may reduce the risk of Alzheimer’s disease. Whilst some of these benefits may be obtained by the use of steroidal

(Continues) ISOFLAVONOIDS 157

(Continued) oestrogens, particularly via hormone replacement therapy (HRT; see page 279), phyto- oestrogens offer a dietary alternative. The main food source of isoflavonoids is the soya bean (Glycine max; Legumi- nosae/Fabaceae) (see also page 256), which contains significant levels of the isoflavones daidzein, and genistein (Figure 4.47), in free form and as their 7-O-glucosides. Total isoflavone levels fall in the range 0.1–0.4%, according to variety. Soya products such as soya milk, soya flour, tofu, and soya-based textured vegetable protein may all be used in the diet for their isoflavonoid content. Breads in which wheat flour is replaced by soya flour are also popular. Extracts from red clover (Trifolium pratense; Leguminosae/Fabaceae) are also used as a dietary supplement. Red clover isoflavones are predominantly formononetin (Figure 4.48) and daidzein, together with their 7-O- glucosides. The lignans enterodiol and enterolactone (see page 135) are also regarded as phyto- oestrogens. These compounds are produced by the action of intestinal microflora on lignans such as secoisolariciresinol or matairesinol ingested in the diet. A particularly important precursor is secoisolariciresinol diglucoside from flaxseed (Linum usitatissimum; Linaceae), and flaxseed may be incorporated into foodstuffs along with soya products. Enterolactone and enterodiol were first detected in human urine, and their origins were traced back to dietary fibre-rich foods. Levels in the urine were much higher in vegetarians, and have been related to a lower incidence of breast cancer in vegetarians.

Derris and Lonchocarpus

Species of Derris (e.g. D. elliptica, D. malaccensis)andLonchocarpus (e.g. L. utilis, L. urucu) (Leguminosae/Fabaceae) have provided useful insecticides for many years. Roots of these plants have been employed as a dusting powder, or extracts have been formulated for sprays. Derris plants are small shrubs cultivated in Malaysia and Indonesia, whilst Lonchocarpus includes shrubs and trees, with commercial material coming from Peru and Brazil. The insecticidal principles are usually supplied as a black, resinous extract. Both Derris and Lonchocarpus roots contain 3–10% of rotenone (Figure 4.49) and smaller amounts of other rotenoids, e.g. deguelin (Figure 4.49). The resin may contain rotenone (about 45%) and deguelin (about 20%). Rotenone and other rotenoids interfere with oxidative phosphorylation, blocking transfer of electrons to ubiquinone (see page 159) by complexing with NADH:ubiquinone oxidoreductase of the respiratory electron transport chain. However, they are relatively innocuous to mammals unless they enter the blood stream, being metabolized rapidly upon ingestion. Insects and also fish seem to lack this rapid detoxification. The fish poison effect has been exploited for centuries in a number of tropical countries, allowing lazy fishing by the scattering of powdered plant material on the water. The dead fish were collected, and when subsequently eaten produced no ill effects on the consumers. More recently, rotenoids have been used in fish management programmes to eradicate undesirable fish species prior to restocking with other species. As insecticides, the rotenoids still find modest use, and are valuable for their selectivity and rapid biodegradability. However, they are perhaps inactivated too rapidly in the presence of light and air to compete effectively with other insecticides such as the modern pyrethrin derivatives (see page 188). 158 THE SHIKIMATE PATHWAY

TERPENOID QUINONES ortho-quinones and quinols (1,4-dihydroxyben- zenes) yielding para-quinones (see page 25). Quinones are potentially derivable by oxi- Accordingly, quinones can be formed from phe- dation of suitable phenolic compounds, cat- nolic systems generated by either the acetate or echols (1,2-dihydroxybenzenes) giving rise to shikimate pathways, provided a catechol or quinol

O O OH = − MeO n 1 12 n = 3−10 R1 R2

MeO n H n H O O O H ubiquinone-n plastoquinone-n 3 (coenzyme Qn) R1 = R2 = Me, α-tocopherol O O R1 = H, R2 = Me, β-tocopherol = − n 1 13 R1 = Me, R2 = H, γ-tocopherol R1 = R2 = H, δ -tocopherol H 3 nH (vitamin E) O O phylloquinone menaquinone-n (vitamin K1; phytomenadione) (vitamin K2)

Figure 4.50

CO2H

plants / H animals nOPP CO2H CO2H

OH 4-coumaric acid C-alkylation with a n H bacteria polyisoprenyl PP OH OH CO2H 4-hydroxybenzoic 1. hydroxylation CH3COCO2H acid 2. O-methylation O2 3. decarboxylation SAM OCO2H or: 3, 1, 2 OH chorismic acid MeO n H OH

1. C-methylation O2 2. hydroxylation oxidation O 3. O-methylation O to quinone MeO SAM O2 SAM

MeO H MeO n n H O O ubiquinone-n

Figure 4.51 TERPENOID QUINONES 159 system has been elaborated, and many examples suffers decarboxylation, and the product is thus an are found in nature. A range of quinone deriva- alkyl methyl p-quinol derivative. Further aromatic tives and related structures containing a terpenoid methylation (via S-adenosylmethionine) and oxida- fragment as well as a shikimate-derived portion tion of the p-quinol to a quinone follow to yield the are also widely distributed. Many of these have plastoquinone. Thus, only one of the two methyl important biochemical functions in electron trans- groups on the quinone ring of the plastoquinone is port systems for respiration or photosynthesis, and derived from SAM. Plastoquinones are involved in some examples are shown in Figure 4.50. the photosynthetic electron transport chain in plants. Ubiquinones (coenzyme Q) (Figure 4.50) are Tocopherols are also frequently found in the found in almost all organisms and function as elec- chloroplasts and constitute members of the vita- tron carriers for the electron transport chain in min E∗ group. Their biosynthesis shares many of mitochondria. The length of the terpenoid chain the features of plastoquinone biosynthesis, with is variable (n = 1−12), and dependent on species, an additional cyclization reaction involving the but most organisms synthesize a range of com- p-quinol and the terpenoid side-chain to give a pounds, of which those where n = 7−10 usually chroman ring (Figure 4.52). Thus, the tocopherols, predominate. The human redox carrier is coenzyme e.g. α-tocopherol and γ-tocopherol, are not in Q10. They are derived from 4-hydroxybenzoic fact quinones, but are indeed structurally related to acid (Figure 4.51), though the origin of this plastoquinones. The isoprenoid side-chain added, compound varies according to organism (see from phytyl diphosphate, contains only four iso- pages 123, 141). Thus, bacteria are known to prene units, and three of the expected double bonds transform chorismic acid by enzymic elimination have suffered reduction. Again, decarboxylation of pyruvic acid, whereas plants and animals uti- of homogentisic acid cooccurs with the alkylation lize a route from phenylalanine or tyrosine via reaction. C-Methylation steps using SAM, and the 4-hydroxycinnamic acid (Figure 4.51). 4-Hydro- cyclization of the p-quinol to γ-tocopherol, have xybenzoic acid is the substrate for C-alkylation been established as in Figure 4.52. Note once again ortho to the phenol group with a polyisoprenyl that one of the nuclear methyls is homogentisate- diphosphate of appropriate chain length (see derived, whilst the others are supplied by SAM. page 231). The product then undergoes further The phylloquinones (vitamin K1)andmena- elaboration, the exact sequence of modifications, quinones (vitamin K2) are shikimate-derived na- i.e. hydroxylation, O-methylation, and decarboxy- phthoquinone derivatives found in plants and ∗ lation, varying in eukaryotes and prokaryotes. algae (vitamin K1 ) or bacteria and fungi (vita- Quinone formation follows in an O2-dependent min K2). The most common phylloquinone struc- combined hydroxylation–oxidation process, and ture (Figure 4.50) has a diterpenoid side-chain, ubiquinone production then involves further hydro- whereas the range of menaquinone structures tends xylation, and O-andC-methylation reactions. to be rather wider with 1–13 isoprene units. Plastoquinones (Figure 4.50) bear considerable These quinones are derived from chorismic acid structural similarity to ubiquinones, but are not via its isomer isochorismic acid (Figure 4.55). derived from 4-hydroxybenzoic acid. Instead, they Additional carbons for the naphthoquinone skele- are produced from homogentisic acid, a pheny- ton are provided by 2-oxoglutaric acid, which is lacetic acid derivative formed from 4-hydroxyphe- incorporated by a mechanism involving the coen- nylpyruvic acid by a complex reaction involving zyme thiamine diphosphate (TPP). 2-Oxoglutaric decarboxylation, O2-dependent hydroxylation, and acid is decarboxylated in the presence of TPP subsequent migration of the −CH2CO2Hside- to give the TPP anion of succinic semialdehyde, chain to the adjacent position on the aromatic ring which attacks isochorismic acid in a Michael-type (Figure 4.52). C-Alkylation of homogentisic acid reaction. Loss of the thiamine cofactor, elimina- ortho to a phenol group follows, and involves tion of pyruvic acid, and then dehydration yield a polyisoprenyl diphosphate with n = 3−10, but the intermediate o-succinylbenzoic acid (OSB). most commonly with n = 9, i.e. solanesyl diphos- This is activated by formation of a coenzyme A phate. However, during the alkylation reaction, ester, and a Dieckmann-like condensation allows the −CH2CO2H side-chain of homogentisic acid ring formation. The dihydroxynaphthoic acid is the 160 THE SHIKIMATE PATHWAY

a complex sequence involving C-alkylation ortho to phenol; hydroxylation, migration of H side-chain, and decarboxylation also decarboxylation PPO n n = 9, solanesyl PP HO O HO O2 HO H n OH OH CO2 CO O CO2H HO2C 2 4-hydroxyphenyl- homogentisic acid SAM pyruvic acid C-methylation H ortho to phenol PPO 3 HO H CO2 phytyl PP n

OH HO

H oxidation of O O quinol to quinone H 3

C-methylation SAM ortho to phenol O H n

HO H O

H O H 3 plastoquinone-n cyclization to 6-membered ring via protonation of double bond

HO HO SAM H H O O 3 C-methylation 3 ortho to phenol γ-tocopherol α-tocopherol

Figure 4.52

Vitamin E Vitamin E refers to a group of fat-soluble vitamins, the tocopherols, e.g. α-, β-, γ-, and δ-tocopherols (Figure 4.53), which are widely distributed in plants, with high levels in cereal seeds such as wheat, barley, and rye. Wheat germ oil is a particularly good source. The proportions of the individual tocopherols vary widely in different seed oils, e.g. principally β- in wheat oil, γ- in corn oil, α- in safflower oil, and γ-andδ-insoybeanoil.VitaminE deficiency is virtually unknown, with most of the dietary intake coming from food oils and margarine, though much can be lost during processing and cooking. Rats deprived of the vitamin display reproductive abnormalities. α-Tocopherol has the highest activity (100%), with the relative activities of β-, γ-, and δ-tocopherols being 50%, 10%, and 3% respectively. α-Tocopheryl acetate is the main commercial form used for food supplementation and

(Continues) TERPENOID QUINONES 161

(Continued)

HO

O α-tocopherol

HO HO HO

O O O

β-tocopherol γ-tocopherol δ-tocopherol

Figure 4.53

initiation of free radical reaction by peroxy radical resonance-stabilized quenching of second free radical peroxy radical HO O O O ROO ROO

O O O O OOR α loss of -tocopherol peroxide leaving group OH hydrolysis of O hemiketal O O H2O

O O O OH α-tocopherolquinone

Figure 4.54

for medicinal purposes. The vitamin is known to provide valuable antioxidant properties, probably preventing the destruction by free radical reactions of vitamin A and unsaturated fatty acids in biological membranes. It is used commercially to retard rancidity in fatty materials in food manufacturing, and there are also claims that it can reduce the effects of ageing and help to prevent heart disease. Its antioxidant effect is likely to arise by reacting with peroxyl radicals, generating by one-electron phenolic oxidation a resonance-stabilized free radical that does not propagate the free radical reaction, but instead mops up further peroxyl radicals (Figure 4.54). In due course, the tocopheryl peroxide is hydrolysed to the tocopherolquinone. more favoured aromatic tautomer from the hydrol- 1,4-dihydroxynaphthoic acid to the isoprenylated ysis of the coenzyme A ester. This compound is naphthoquinone appears to be catalysed by a single now the substrate for alkylation and methylation enzyme, and can be rationalized by the mech- as seen with ubiquinones and plastoquinones. anism in Figure 4.56. This involves alkylation However, the terpenoid fragment is found to (shown in Figure 4.56 using the diketo tautomer), replace the carboxyl group, and the decarboxylated decarboxylation of the resultant β-keto acid, and analogue is not involved. The transformation of finally an oxidation to the p-quinone. 162 THE SHIKIMATE PATHWAY

Michael-type HO C HO2C HO C addition 2 H2O 2 OH O OH CO H O 2 CO H O CO2H O C 2 OH H HO HO CO2H H OTPP chorismic acid isochorismic acid TPP succinic semi-aldehyde -TPP anion TPP-dependent decarboxylation of CO2 α-keto acid to aldehyde; nucleophilic H TPP addition of TPP anion on to aldehyde HO C then allows removal of aldehydic 2 OH HO2CCO2H proton which has become acidic CO2H O CO2H O H 2-oxoglutaric acid O 1,4-elimination of pyruvic acid CH COCO H Claisen-like condensation 3 2 (Dieckmann reaction) O O HSCoA H OH OH H C ATP CO2H CO2H COSCoA COSCoA CO2H CO2H

dehydration to O O O O hydrolysis of thioester; o-succinyl form aromatic ring enolization to more benzoic acid stable tautomer (OSB) HSCoA H OH PP O n O SAM O CO2H H H n n

C-methylation CO2 OH C-alkylation with O O 1,4-dihydroxy- concomitant menaquinone-n naphthoic acid decarboxylation (vitamin K2)

Figure 4.55

R PPO H decarboxylation O O O of β-keto acid CO H 2 O R R O O

CO2 O O R R O

O O

Figure 4.56 TERPENOID QUINONES 163

Vitamin K

Vitamin K comprises a number of fat-soluble naphthoquinone derivatives, with vitamin K1 (phylloquinone) (Figure 4.50) being of plant origin whilst the vitamins K2 (menaquinones) are produced by microorganisms. Dietary vitamin K1 is obtained from almost any green vegetable, whilst a significant amount of vitamin K2 is produced by the intestinal microflora. As a result, vitamin K deficiency is rare. Deficiencies are usually the result of malabsorption of the vitamin, which is lipid soluble. Vitamin K1 (phytomenadione) or the water-soluble menadiol phosphate (Figure 4.57) may be employed as supplements. Menadiol is oxidized in the body to the quinone, which is then alkylated, e.g. with geranylgeranyl diphosphate, to yield a metabolically active product. Vitamin K is involved in normal blood clotting processes, and a deficiency would lead to haemorrhage. Blood clotting requires the carboxylation of glutamate residues in the protein prothrombin, generating bidentate ligands that allow the protein to bind to other factors. This carboxylation requires carbon dioxide, molecular oxygen, and the reduced quinol form of vitamin K (Figure 4.57). During the carboxylation, the reduced vitamin K suffers epoxidation, and vitamin K is subsequently regenerated by reduction. Anticoagulants such as dicoumarol and warfarin (see page 144) inhibit this last reduction step. However, the polysaccharide anticoagulant heparin (see page 477) does not interfere with vitamin K metabolism, but acts by complexing with blood clotting enzymes.

O OH O H N N H

O OH CO2H vitamin K vitamin K (quinol) prothrombin O2, CO2 OP O O reductase H N N O H CO2H

OP O CO2H menadiol phosphate vitamin K epoxide

Figure 4.57

OSB, and 1,4-dihydroxynaphthoic acid, or its Streptocarpus (Gesneriaceae), e.g. catalponone diketo tautomer, have been implicated in the (compare Figure 4.56), and this can be transformed biosynthesis of a wide range of plant naphtho- to deoxylapachol and then menaquinone-1 (Fig- quinones and anthraquinones. There are parallels ure 4.58). Lawsone is formed by an oxidative with the later stages of the menaquinone sequence sequence in which hydroxyl replaces the carboxyl. shown in Figure 4.55, or differences according to A further interesting elaboration is the synthesis the plant species concerned. Some of these path- of an anthraquinone skeleton by effectively cycliz- ways are illustrated in Figure 4.58. Replacement ing a dimethylallyl substituent on to the naph- of the carboxyl function by an isoprenyl sub- thaquinone system. Rather little is known about stituent is found to proceed via a disubstituted how this process is achieved but many examples intermediate in Catalpa (Bignoniaceae) and are known from the results of labelling studies. 164 THE SHIKIMATE PATHWAY

O O O O

CO2H CO2H

O O O O catalponone deoxylapachol menaquinone-1

O O O OH

O O O OH lawsone

OH OH O OH O OH CO H CO2H OH 2 OH

OH OH OH O O 1,4-dihydroxy- lucidin alizarin naphthoic acid

Figure 4.58

Some of these structures retain the methyl from acid at the non-carboxylated carbon. Obviously, the isoprenyl substituent, whilst in others this has this is also a nucleophilic site and alkylation been removed, e.g. alizarin from madder (Rubia here is mechanistically sound. Again, cyclization tinctorum; Rubiaceae), presumably via an oxi- of the dimethylallyl to produce an anthraquinone dation–decarboxylation sequence. Hydroxylation, can occur, and the potently mutagenic lucidin particularly in the terpenoid-derived ring, is also a from Galium species (Rubiaceae) is a typical frequent feature. example. The hydroxylation patterns seen in the Some other quinone derivatives, although anthraquinones in Figure 4.58 should be com- formed from the same pathway, are produced pared with those noted earlier in acetate/malonate- by dimethylallylation of 1,4-dihydroxynaphthoic derived structures (see page 63). Remnants of the alternate oxygenation pattern are usually acetate / malonate very evident in acetate-derived anthraquinones O O (Figure 4.59), whereas such a pattern cannot HO OH easily be incorporated into typical shikimate/2- oxoglutarate/isoprenoid structures. Oxygen sub- stituents are not usually present in positions fitting OH OOH OH OOH the polyketide hypothesis. emodin aloe-emodin

Shikimate / 2-oxoglutarate / isoprenoid O OH O OH FURTHER READING OH OH Shikimate Pathway OH O O Abell C (1999) Enzymology and molecular biology alizarin lucidin of the shikimate pathway. Comprehensive Natural Products Chemistry, Vol 1. Elsevier, Amsterdam, Figure 4.59 pp 573–607. FURTHER READING 165

Floss HG (1997) Natural products derived from unusual Lignin variants of the shikimate pathway. Nat Prod Rep 14, 433–452. Lewis NG and Yamamoto E (1990) Lignin: occurrence, Haslam E (1996) Aspects of the enzymology of the biogenesis and biodegradation. Annu Rev Plant Phys- shikimate pathway. Prog Chem Org Nat Prod 69, iol Plant Mol Biol 41, 455–496. 157–240. Herrmann KM and Weaver LM (1999) The shikimate pathway. Annu Rev Plant Physiol Plant Mol Biol 50, Lignans 473–503. Knaggs AR (2000) The biosynthesis of shikimate meta- Canel C, Moraes RM, Dayan FE and Ferreira D (2000) bolites. Nat Prod Rep 17, 269–292. Earlier reviews: Molecules of interest: podophyllotoxin. Phytochem- 1999, 16, 525–560; Dewick PM (1998) 15,17–58. istry 54, 115–120. Davin LB and Lewis NG (2000) Dirigent proteins and dirigent sites explain the mystery of specificity of radical precursor coupling in lignan and lignin Folic acid biosynthesis. Plant Physiol 123, 453–461. Cossins EA and Chen L (1997) Folates and one-carbon Lewis NG and Davin LB (1999) Lignans: biosynthe- metabolism in plants. Phytochemistry 45, 437–452. sis and function. Comprehensive Natural Products Maden BEH (2000) Tetrahydrofolate and tetrahydro- Chemistry, Vol 1. Elsevier, Amsterdam, pp 639–712. methanopterin compared: Functionally distinct car- Stahelin¨ HF and von Wartburg A (1991) The chemical riers in C metabolism. Biochem J 350, 609–629 and biological route from podophyllotoxin glucoside 1 to etoposide. Cancer Res 51,5–15. (see errata 352, 935–936). Ward RS (1999) Lignans, neolignans and related com- Mason P (1999) Nutrition: folic acid – new roles for a pounds. Nat Prod Rep 16,75–96. Earlier reviews: well known vitamin. Pharm J 263, 673–677. 1997, 14,43–74; 1995, 12, 183–205. Pufulete M (1999) Eat your greens. Chem Brit 35 (6), 26–28. Rawalpally TR (1998) Vitamins (folic acid). Kirk–Oth- Coumarins mer Encyclopedia of Chemical Technology, 4th edn, Vol 25. Wiley, New York, pp 64–82. Bell WR (1992) Blood, coagulants and anticoagulants. Young DW (1994) Studies on thymidylate synthase Kirk–Othmer Encyclopedia of Chemical Technology, and dihydrofolate reductase – two enzymes involved 4th edn, Vol 4. Wiley, New York, pp 333–360. in the synthesis of thymidine. Chem Soc Rev 23, Estevez-Braun´ A and Gonzalez´ AG (1997) Coumarins. 119–128. Nat Prod Rep 14, 465–475. Earlier review: Mur- ray RDH (1995) 12, 477–505. Matern U, Luer¨ P and Kreusch D (1999) Biosynthe- Chloramphenicol sis of coumarins. Comprehensive Natural Products Chemistry, Vol 1. Elsevier, Amsterdam, pp 623–637. Nagabhushan T, Miller GH and Varma KJ (1992) Anti- Murray RDH (1997) Naturally occurring plant couma- biotics (chloramphenicol and analogues). Kirk–Oth- rins. Prog Chem Org Nat Prod 72,1–119. mer Encyclopedia of Chemical Technology, 4th edn, Vol 2. Wiley, New York, pp 961–978. Styrylpyrones

Haberlein¨ H, Boonen G and Beck M-A (1997) Piper Melanins methysticum: enantiomeric separation of kavapyrones by high performance liquid chromatography. Planta Prota G (1995) The chemistry of melanins and melano- Med 63,63–65. genesis. Prog Chem Org Nat Prod 64,93–148.

Flavonoids Volatile Oils Das A, Wang JH and Lien EJ (1994) Carcinogenicity, Mookherjee BD and Wilson RA (1996) Oils, essential. mutagenicity and cancer preventing activities of Kirk–Othmer Encyclopedia of Chemical Technology, flavonoids: a structure–system–activity relationship 4th edn, Vol 17. Wiley, New York, pp 603–674. (SSAR) analysis. Prog Drug Res 42, 133–166. 166 THE SHIKIMATE PATHWAY

Forkmann G and Heller W (1999) Biosynthesis of Ferreira D and Li X-C (2000) Oligomeric proantho- flavonoids. Comprehensive Natural Products Chem- cyanidins: naturally occurring O-heterocycles. Nat istry, Vol 1. Elsevier, Amsterdam, pp 713–748. Prod Rep 17, 193–212. Earlier review: Ferreira D Gordon MH (1996) Dietary antioxidants in disease pre- and Bekker R (1996) 13, 411–433. vention. Nat Prod Rep 13, 265–273. Ferreira D, Nel RJJ and Bekker R (1999) Condensed Harborne JB and Williams CA (1998) Anthocyanins tannins. Comprehensive Natural Products Chemistry, and other flavonoids. Nat Prod Rep 15, 631–652. Vol 3. Elsevier, Amsterdam, pp 747–797. Earlier review: 1995, 12, 639–657. Gross GG (1999) Biosynthesis of hydrolyzable tannins. Harborne JB and Williams CA (2000) Advances in Comprehensive Natural Products Chemistry, Vol 3. flavonoid research since 1992. Phytochemistry 55, Elsevier, Amsterdam, pp 799–826. 481–504. Haslam E (1996) Natural polyphenols (vegetable tan- Waterhouse AL (1995) Wine and heart disease. Chem nins) as drugs: possible modes of action. J Nat Prod Ind 338–341. 59, 205–215. Haslam E and Cai Y (1994) Plant polyphenols (veg- Isoflavonoids etable tannins): gallic acid metabolism. Nat Prod Rep 11,41–66. Crombie L and Whiting DA (1998) Biosynthesis in Okuda T, Yoshida T and Hatano T (1995) Hydrolyzable the rotenoid group of natural products: applica- tannins and related polyphenols. Prog Chem Org Nat tions of isotope methodology. Phytochemistry, 49, Prod 66,1–117. 1479–1507. Davis SR, Dalais FS, Simpson ER and Murkies AL (1999) Phytoestrogens in health and disease. Rec Prog Hormone Res 54, 185–210. Vitamin E Dixon RA (1999) Isoflavonoids: biochemistry, molecu- lar biology, and biological function. Comprehensive Casani R (1998) Vitamins (vitamin E). Kirk–Othmer Natural Products Chemistry, Vol 1. Elsevier, Ams- Encyclopedia of Chemical Technology, 4th edn, terdam, pp 773–823. Vol 25. Wiley, New York, pp 256–268. Donnelly DMX and Boland GM (1998) Isoflavonoids Gordon MH (1996) Dietary antioxidants in disease pre- and related compounds. Nat Prod Rep 15, 241–260. vention. Nat Prod Rep 13, 265–273. Chem Earlier review: 1995, 12, 321–338. Scott G (1995) Antioxidants – the modern elixir? Mason P (2001) Nutrition: isoflavones. Pharm J 266, Brit 879–882. 16–19. Metcalf RL (1995) Insect control technology. Kirk– Othmer Encyclopedia of Chemical Technology,4th Vitamin K edn, Vol 14. Wiley, New York, pp 524–602. Dowd P, Hershline R, Ham SW and Naganathan S (1994) Mechanism of action of vitamin K. Nat Prod Tannins Rep 11, 251–264. Ferreira D, Brandt EV, Coetzee J and Malan E (1999) Van Arnum SD (1998) Vitamins (vitamin K). Kirk– Condensed tannins. Prog Chem Org Nat Prod 77, Othmer Encyclopedia of Chemical Technology,4th 21–67. edn, Vol 25. Wiley, New York, pp 269–283. MedicinalNaturalProducts. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 5 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS: TERPENOIDS AND STEROIDS

The two pathways leading to terpenoids are described: the mevalonate pathway and the recently discovered mevalonate-independent pathway via deoxyxylulose phosphate. Terpenoids may be classified according to the number of isoprenoid units incorporated, and hemiterpenes, monoterpenes and the variants irregular monoterpenes and iridoids, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenes, and higher terpenoids are described in turn, representing groups with increasing numbers of isoprene units. Structures are rationalized through extensive use of carbocation mechanisms and subsequent Wagner–Meerwein rearrangements. Steroids as examples of modified triterpenoids are discussed in detail, including stereochemistry and molecular shape. There follows specific consideration of cholesterol, steroidal saponins, cardioactive glycosides, phytosterols, vitamin D, bile acids, corticosteroids and their semi-synthesis, progestogens, oestrogens, and androgens. Monograph topics giving more detailed information on medicinal agents include volatile oils, pyrethrins, valerian, feverfew, chamomile and matricaria, Artemisia annua and artemisinin, gossypol, trichothecenes, Taxus brevifolia and taxol, Ginkgo biloba, forskolin, liquorice, quillaia, ginseng, vitamin A, cholesterol, dioscorea, fenugreek, sisal, sarsaparilla, yucca, Digitalis purpurea, Digitalis lanata, strophanthus, convallaria, squill, soya bean sterols, fusidic acid, vitamin D, bile acids, corticosteroid drugs, progestogen drugs, oestrogen drugs, aromatase inhibitors, oestrogen receptor antagonists, and androgen drugs.

The terpenoids form a large and structurally these compounds, and the biochemically active diverse family of natural products derived from isoprene units were identified as the diphosphate C5 isoprene units (Figure 5.1) joined in a head- (pyrophosphate) esters dimethylallyl diphosphate to-tail fashion. Typical structures contain carbon (DMAPP)andisopentenyl diphosphate (IPP) skeletons represented by (C5)n, and are classi- (Figure 5.2). Relatively few of the natural ter- fied as hemiterpenes (C5), monoterpenes (C10), penoids conform exactly to the simple concept sesquiterpenes (C15), diterpenes (C20), sesterter- of a linear head-to-tail combination of isoprene penes (C25), triterpenes (C30)andtetraterpenes units as seen with geraniol (C10), farnesol (C15), (C40) (Figure 5.2). Higher polymers are encoun- and geranylgeraniol (C20) (Figure 5.3). Squa- tered in materials such as rubber. Isoprene itself lene (C30)andphytoene (C40), although formed (Figure 5.1) had been characterized as a decompo- entirely of isoprene units, display a tail-to-tail link- sition product from various natural cyclic hydro- age at the centre of the molecules. Most terpenoids carbons, and was suggested as the fundamental are modified further by cyclization reactions, but building block for these compounds, also referred the head-to-tail arrangement of the units can usu- to as ‘isoprenoids’. Isoprene is produced natu- ally still be recognized, e.g. menthol, bisabo- rally but is not involved in the formation of lene,andtaxadiene. The linear arrangement of 168 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

single C5 unit, usually a dimethylallyl substituent, and molecules containing these isolated isoprene units are sometimes referred to as ‘meroter- C isoprene unit isoprene 5 penoids’. Some examples include furocoumarins Figure 5.1 (see page 145), rotenoids (see page 155), and ergot alkaloids (see page 368). One should also note that the term ‘prenyl’ is in general use isoprene units can be more difficult to appreciate to indicate the dimethylallyl substituent. Even in many other structures when rearrangement reac- macromolecules like proteins can be modified by tions have taken place, e.g. steroids, where in addi- attaching terpenoid chains. Cysteine residues are tion, several carbons have been lost. Nevertheless, alkylated with farnesyl or geranylgeranyl groups, such compounds are formed via regular terpenoid thereby increasing the lipophilicity of the protein precursors. and its ability to associate with membranes. Many other natural products contain terpenoid The biochemical isoprene units may be derived elements in their molecules, in combination with by two pathways, by way of intermediates meval- carbon skeletons derived from other sources, such onic acid (MVA) (Figure 5.4) or 1-deoxy-D- as the acetate and shikimate pathways. Many xylulose 5-phosphate (deoxyxylulose phosphate; alkaloids, phenolics, and vitamins discussed in DXP) (Figure 5.6). Mevalonic acid, itself a prod- other chapters are examples of this. A particularly uct of acetate metabolism, had been established as common terpenoid fragment in such cases is a a precursor of the animal sterol cholesterol, and

Mevalonic acid Deoxyxylulose phosphate

Hemiterpenes (C ) OPP OPP 5 dimethylallyl PP isopentenyl PP (DMAPP) (C5) (IPP) (C5)

Monoterpenes (C ) C10 10 Iridoids IPP

C15 Sesquiterpenes (C15)

IPP x 2

C20 Diterpenes (C20)

IPP x 2

C25 Sesterterpenes (C25)

C30 Triterpenoids (C30)

Steroids (C18−C30)

C 40 Tetraterpenes (C40) Carotenoids

Figure 5.2 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS 169

OH geraniol

OH farnesol

OH geranylgeraniol

squalene

phytoene

OH

menthol bisabolene taxadiene

Figure 5.3 the steps leading to and from mevalonic acid were pathway, an acetoacetic acid thioester (bound to gradually detailed in a series of painstakingly exe- the acyl carrier protein) would have been formed cuted experiments. For many years, the early parts using the more nucleophilic thioester of malonic of the mevalonate pathway were believed to be acid. The mevalonate pathway does not use mal- common to the whole range of natural terpenoid onyl derivatives and it thus diverges from the derivatives, but it has since been discovered that an acetate pathway at the very first step. In the alternative pathway to IPP and DMAPP exists, via second step, it should be noted that, on purely deoxyxylulose phosphate, and that this pathway is chemical grounds, acetoacetyl-CoA is the more probably more widely utilized in nature than is the acidic substrate, and might be expected to act as mevalonate pathway. This pathway is also referred the nucleophile rather than the third acetyl-CoA to as the mevalonate-independent pathway or the molecule. The enzyme thus achieves what is a methylerythritol phosphate pathway. less favourable reaction. The conversion of HMG- Three molecules of acetyl-coenzyme A are CoA into (3R)-MVA involves a two-step reduc- used to form mevalonic acid. Two molecules tion of the thioester group to a primary alcohol, combine initially in a Claisen condensation to and provides an essentially irreversible and rate- give acetoacetyl-CoA, and a third is incor- limiting transformation. Drug-mediated inhibition porated via a stereospecific aldol addition of this enzyme (HMG-CoA reductase)canbe giving the branched-chain ester β-hydroxy-β- used to regulate the biosynthesis of mevalonate and methylglutaryl-CoA (HMG-CoA) (Figure 5.4). ultimately of the steroid cholesterol (see statins, This third acetyl-CoA molecule appears to be page 112). bound to the enzyme via a thiol group, and this The six-carbon compound MVA is transformed linkage is subsequently hydrolysed to form the into the five-carbon phosphorylated isoprene free acid group of HMG-CoA. In the acetate units in a series of reactions, beginning with 170 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

stereospecific aldol reaction; Claisen H O also involves hydrolysis of H reaction OO acetyl−enzyme linkage OH O SCoA + EnzSH HO2C SCoA SCoA SCoA acetoacetyl-CoA HMG-CoA SEnz O EnzSH acetyl-CoA O enzyme-bound HMG-CoA NADPH acetyl group reductase reduction of aldehyde reduction of thio to alcohol ester to aldehyde 6 via hemithioacetal 1 OH NADPH OH OH OH 5 HO2C HO2C HO2C 3 OH O SCoA 2 4 H mevalonic acid mevaldic acid mevaldic acid (MVA) hemithioacetal sequential phosphorylation of the 2 x ATP primary alcohol to a stereospecific allylic diphosphate O isomerization; equilibrium HO POADP favours DMAPP ATP 5 O OH OH –CO2 H 3 1 isomerase 2 H OPP O OPP 4 OPP HR HS ATP facilitates the decarboxylation−elimination; the isopentenyl PP dimethylallyl PP anticipated phosphorylation of the (IPP) (DMAPP) tertiary alcohol to make a better leaving group is apparently not involved

Figure 5.4 phosphorylation of the primary alcohol group. Two is more likely to act as a nucleophile, especially different ATP-dependent enzymes are involved, towards the electrophilic DMAPP. These differing resulting in mevalonic acid diphosphate, and reactivities are the basis of terpenoid biosynthesis, decarboxylation/dehydration then follow to give and carbocations feature strongly in mechanistic IPP. Whilst a third molecule of ATP is required rationalizations of the pathways. for this last transformation, there is no evidence for 1-Deoxy-D-xylulose 5-phosphate is formed phosphorylation of the tertiary hydroxyl, though from the glycolytic pathway intermediates pyruvic this would convert the hydroxyl into a better acid and glyceraldehyde 3-phosphate with the loss leaving group. Perhaps ATP assists the loss of of the pyruvate carboxyl (Figure 5.6). Thiamine the hydroxyl as shown in Figure 5.4. IPP is diphosphate-mediated decarboxylation of pyruvate isomerized to the other isoprene unit, DMAPP, by an isomerase enzyme which stereospecifically removes the pro-R proton (HR) from C-2, and OPP incorporates a proton from water on to C-4. Whilst Note: when using this the isomerization is reversible, the equilibrium lies DMAPP representation of the heavily on the side of DMAPP. This conversion S 1 reaction allylic cation, do not N forget the double bond generates a reactive electrophile and therefore a good alkylating agent. DMAPP possesses a good leaving group, the diphosphate, and can yield via an SN1 process an allylic carbocation which is resonance-stabilized allylic cation stabilized by charge delocalization (Figure 5.5). In contrast, IPP with its terminal double bond Figure 5.5 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS 171

(compare page 21) produces an acetaldehyde with a reduction. The expected aldehyde product equivalent bound in the form of an enamine, which from the rearrangement step is not detectable, and reacts as a nucleophile in an addition reaction the single enzyme catalyses the rearrangement and with the glyceraldehyde 3-phosphate. Subsequent reduction reactions without release of any inter- release from the TPP carrier generates deoxy- mediate. Analogous rearrangements are seen in the xylulose phosphate, which is transformed into biosynthesis of the amino acids valine, leucine, and 2-C-methyl-D-erythritol 4-phosphate by a rear- isoleucine. The methylerythritol phosphate con- rangement reaction, conveniently rationalized as tains the branched-chain system equivalent to the a pinacol-like rearrangement (Figure 5.6), coupled isoprene unit, but the complete sequence of steps

nucleophilic attack of H CO2H enamine on to aldehyde H O OH OH O O H3C OH OP H3C OP CO2 C C OP pyruvic acid R1 OH R1 OH O OH 1-deoxy-D-xylulose 5-P thiamine PP N S D-glyceraldehyde N S (TPP) see Figure 2.15 3-P R2 R2 R1 N S TPP anion TPP/pyruvate-derived regenerated enamine R2 OH H pinacol-like rearrangement O N P favoured by stability of reduction of aldehyde to alcohol; the resultant oxonium ion R O fosmidomycin aldehyde intermediate remains H OH O enzyme-bound O OH OH NADPH ≡ OP OP OP OP HO O OH OH O OH OH OH 1-deoxy- C H D-xylulose 5-P 2- -methyl- D-erythritol 4-P nucleophilic attack of CTP phosphate hydroxyl on compare formation of NH NH O diphosphate; formation of 2 UDPglucose, Figure 2.27 2 phosphoanhydride O P OH N N OH OH O O O O N O ATP N O OOP O P CH2 OOP O P CH2 O O OH OH OH OH OH OH OH OH

2-phospho-4-(CDP)- HO OH 4-(CDP)-2-C-methyl- HO OH 2-C-methyl-D-erythritol D-erythritol

steps to be determined OOH O O P O NADPH – H O NADPH P 2 O OH OPP OPP OPP OPP OH H OH OH O OH OH OH OH 2-C-methyl-D-erythritol- – H O 2,4-cyclophosphate intramolecular 2 elimination; then enol–keto tautomerism isomerase ? OPP OPP dimethylallyl PP there is evidence that DMAPP isopentenyl PP (DMAPP) may be formed independently, and (IPP) not via isomerization of IPP

Figure 5.6 172 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS leading to the intermediate isopentenyl phosphate not affect the formation of terpenoids derived via has yet to be elucidated. Reaction of methylery- deoxyxylulose phosphate. Equally, it is possible thritol phosphate with cytidine triphosphate (CTP) to inhibit terpenoid production without affecting produces a cytidine diphospho derivative (com- steroid formation by the use of deoxyxyxlulose pare uridine diphosphoglucose in glucosylation, phosphate pathway inhibitors, such as the antibi- page 29), which is then phosphorylated via ATP. otic fosmidomycin from Streptomyces lavendulae. The resultant 2-phosphate is converted into a cyclic This acts as an analogue of the rearrangement phosphoanhydride with loss of cytidine phosphate. intermediate (Figure 5.6). Regulation of choles- This cyclophosphate, by steps not yet known (a terol production in humans is an important health possible sequence is proposed in Figure 5.6), leads concern (see page 236). to IPP, and links the deoxyxylulose pathway with the mevalonate pathway. DMAPP may then be derived by isomerism of IPP, or may be produced HEMITERPENES (C5) independently; this also remains to be clarified. IPP and DMAPP are reactive hemiterpene interme- Deoxyxylulose phosphate also plays an important diates in the pathways leading to more complex role as a precursor of thiamine (vitamin B , 1 terpenoid structures. They are also used as alky- page 30) and pyridoxol phosphate (vitamin B , 6 lating agents in the formation of meroterpenoids page 33). as indicated above, but examples of these struc- Whether the mevalonate pathway or the tures are discussed under the section appropriate deoxyxylulose phosphate pathway supplies iso- to the major substructure, e.g. alkaloids, shiki- prene units for the biosynthesis of a particular mate, acetate. Relatively few true hemiterpenes are terpenoid has to established experimentally. Ani- produced in nature, with isoprene, a volatile com- mals appear to lack the deoxyxylulose phosphate pound which is released by many species of plants, pathway, so utilize the mevalonate pathway exclu- especially trees, being the notable example. Iso- sively. Many other organisms, including plants, are prene is formed by loss of a proton from the allylic equipped to employ both pathways, often concur- cation (Figure 5.7). rently. In plants, the two pathways appear to be compartmentalized, so that the mevalonate path- – H way enzymes are localized in the cytosol, whereas H the deoxyxylulose phosphate pathway enzymes are found in chloroplasts. Accordingly, triterpenoids isoprene and steroids (cytosolic products) are formed by the mevalonate pathway, whilst most other ter- Figure 5.7 penoids are formed in the chloroplasts and are deoxyxylulose phosphate derived. Of course there MONOTERPENES (C10) are exceptions. There are also examples where the two pathways can supply different portions of a Combination of DMAPP and IPP via the enzyme molecule, or where there is exchange of late-stage prenyl transferase yields geranyl diphosphate common intermediates between the two pathways (GPP) (Figure 5.8). This is believed to involve resulting in a contribution of isoprene units from ionization of DMAPP to the allylic cation, addition each pathway. In the following part of this chapter, to the double bond of IPP, followed by loss of these complications will not be considered further, a proton. Stereochemically, the proton lost (HR) and in most cases there is no need to consider the is analogous to that lost on the isomerization of precise source of the isoprene units. The only area IPP to DMAPP. This produces a monoterpene of special pharmacological interest where the early diphosphate, geranyl PP, in which the new double pathway is of particular concern is steroid biosyn- bond is trans (E). Linalyl PP and neryl PP are thesis, which appears to be from mevalonate in isomers of geranyl PP, and are likely to be formed the vast majority of organisms. Thus, inhibitors from geranyl PP by ionization to the allylic cation, of the mevalonate pathway enzyme HMG-CoA which can thus allow a change in attachment of the reductase will reduce steroid production, but will diphosphate group (to the tertiary carbon in linalyl MONOTERPENES 173

electrophilic addition giving tertiary cation

OPP OPP OPP DMAPP HR HS HR HS stereospecific loss of proton E OPP ≡ OPP geranyl PP (GPP)

Figure 5.8

single bond in LPP allows rotation OPP OPP OPP E Z OPP ≡ OPP OPP

geranyl PP linalyl PP neryl PP (GPP) (LPP) (NPP)

resonance-stabilized allylic cation resonance-stabilized allylic cation (geranyl cation) (neryl cation)

Figure 5.9

PP) or a change in stereochemistry at the double of these would seem more immediate precursors bond (to Z in neryl PP) (Figure 5.9). These three of the monocyclic menthane system, formation compounds, by relatively modest changes, can give of which could be represented as shown in rise to a range of linear monoterpenes found as Figure 5.12, generating a carbocation (termed components of volatile oils used in flavouring and menthyl or α-terpinyl) having the menthane perfumery (Figure 5.10). The resulting compounds skeleton. It has been found that monoterpene may be hydrocarbons, alcohols, aldehydes, or cyclase enzymes are able to accept all three perhaps esters, especially acetates. diphosphates, with linalyl PP being the best The range of monoterpenes encountered is substrate, and it appears they have the ability extended considerably by cyclization reactions, to isomerize the substrates initially as well as and monocyclic or bicyclic systems can be to cyclize them. It is convenient therefore to created. Some of the more important examples consider the species involved in the cyclization as of these ring systems are shown in Figure 5.11. the delocalized allylic cation tightly bound to the Such cyclizations would not be expected to diphosphate anion, and bond formation follows due occur with the precursor geranyl diphosphate, to the proximity of the π-electrons of the double the E stereochemistry of the double bond being bond (Figure 5.12). unfavourable for ring formation (Figure 5.9). In Chapter 2, the possible fates of carbocations Neryl PP or linalyl PP, however, do have were discussed. These include quenching with favourable stereochemistry, and either or both nucleophiles (especially water), loss of a proton, 174 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

O O

O

geranial (Z-citral ) citronellal β-myrcene neral (E-citral) (lemon oil) (citronella oil) (hops) (lemon oil)

OPP OPP

OPP

GPP LPP NPP

OH OH OH

OH

citronellol geraniol linalool nerol (rose oil) (geranium oil) (coriander oil) (rose oil)

Figure 5.10

≡ ≡ ≡

pinane type camphane / bornane type fenchane type menthane type ≡ ≡

isocamphane type carane type thujane type

MONOCYCLIC BICYCLIC

Figure 5.11 cyclization, and the possibility that Wagner– side-chain towards the double bond (via the surface Meerwein rearrangements might occur (see characteristics of the enzyme) would allow a page 15). All feature strongly in terpenoid repeat of the cyclization mechanism, and produce biosynthesis. The newly generated menthyl cation bicyclic bornyl and pinyl cations, according to could be quenched by attack of water, in which which end of the double bond was involved in case the alcohol α-terpineol would be formed, forming the new bonds (Figure 5.14). Borneol or it could lose a proton to give limonene would result from quenching of the bornyl cation (Figure 5.13). Alternatively, folding the cationic with water, and then oxidation of the secondary MONOTERPENES 175

OPP OPP

OPP

GPP LPP NPP

electrophilic addition giving tertiary cation OPP

delocalized allylic cation- menthyl / α-terpinyl cation diphosphate ion-pair

Figure 5.12

H although the menthyl cation is tertiary, the 1,3-shift creates a resonance-stabilized allylic menthyl / α-terpinyl cation cation W–M 1,3-hydride H O 2 – H shift

H – H cyclization α-phellandrene

OH O – H

limonene α-terpineol cineole phellandryl cation

W–M = Wagner–Meerwein rearrangement β-phellandrene

Figure 5.13 alcohol could generate the ketone camphor.As even though, in nature, these cations may not exist an alternative to discharging the positive charge as such discrete species, but rather as tightly bound by adding a nucleophile, loss of a proton would ion pairs with a counter-anion, e.g. diphosphate. generate an alkene. Thus α-pinene and β-pinene The analogy with carbocation chemistry is arise by loss of different protons from the pinyl justified, however, since a high proportion of cation, producing the double bonds as cyclic or natural terpenoids have skeletons which have exocyclic respectively. A less common termination suffered rearrangement processes. Rearrangements step involving loss of a proton is the formation of a of the Wagner–Meerwein type (see page 15), in cyclopropane ring as exemplified by 3-carene and which carbons or hydride migrate to achieve generation of the carane skeleton. enhanced stability for the cation via tertiary The chemistry of terpenoid formation is against secondary character, or by reduction of essentially based on the reactivity of carbocations, ring strain, give a mechanistic rationalization for 176 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS the biosynthetic pathway. The menthyl cation, only changes one tertiary carbocation system for although it is a tertiary, may be converted by another, but allows formation of α-terpinene, a 1,3-hydride shift into a favourable resonance- γ-terpinene,andtheα-terpineol isomer, terpinen- stabilized allylic cation (Figure 5.13). This allows 4-ol. A further cyclization reaction on the the formation of α-andβ-phellandrenes by loss terpinen-4-yl cation generates the thujane skeleton, of a proton from the phellandryl carbocation. e.g. sabinene and thujone. Terpinen-4-ol is the The bicyclic pinyl cation, with a strained four- primary antibacterial component of tea tree oil membered ring, rearranges to the less strained from Melaleuca alternifolia (Myrtaceae); thujone five-membered fenchyl cation (Figure 5.14), a has achieved notoriety as the neurotoxic agent change which presumably more than makes in wormwood oil from Artemisia absinthium up for the unfavourable tertiary to secondary (Compositae/Asteraceae) used in preparation of the carbocation transformation. This produces the drink absinthe, now banned in most countries. fenchane skeleton, exemplified by fenchol and So far, little attention has been given to the fenchone. The isocamphyl tertiary carbocation is stereochemical features of the resultant monoter- formed from the bornyl secondary carbocation pene. Individual enzyme systems present in a by a Wagner–Meerwein rearrangement, and so particular organism will, of course, control the leads to camphene. A hydride shift converting folding of the substrate molecule and thus define the menthyl cation into the terpinen-4-yl cation the stereochemistry of the final product. Most

H 1,2-shift produces H menthyl / α-terpinyl another tertiary cation cation W–M formation of 1,2-hydride cyclopropane – H shift ring

1,2-shift creates pinyl secondary cation, but bornyl 1,2-shift creates cation reduces ring strain cation tertiary cation terpinen-4-yl car-3-ene W–M H O W–M – H 1,2-alkyl 2 1,2-alkyl – H cation – H shift shift – H H2O

OH

α-pinene β-pinene fenchyl borneol isocamphyl OH cation cation α-terpinene γ-terpinene terpinen-4-ol thujyl cation H2O O – H – H

O O O O OH O

+ reduction fenchone fenchol camphor camphene

thujone sabinene

Figure 5.14 MONOTERPENES 177 monoterpenes are optically active, and there due to (+)-carvone whereas (−)-carvone smells of are many examples known where enantiomeric spearmint. (+)-Limonene smells of oranges whilst forms of the same compound can be isolated (−)-limonene resembles the smell of lemons. from different sources, e.g. (+)-camphor in sage The origins of the different enantiomeric forms (Salvia officinalis; Labiatae/Lamiaceae) and (−)- of limonene and α-pinene are illustrated in camphor in tansy (Tanacetum vulgare;Com- Figure 5.15. This shows the precursor geranyl PP positae/Asteraceae), or (+)-carvone in caraway being folded in two mirror image conformations, (Carum carvi; Umbelliferae/Apiaceae) and (−)- leading to formation of the separate enantiomers of carvone in spearmint (Mentha spicata; Labiatae/ linalyl PP. Analogous carbocation reactions will Lamiaceae). There are also examples of com- then explain production of the optically active pounds found in both enantiomeric forms in the monoterpenes. Where a single plant produces both same organism, examples being (+)- and (−)- enantiomers, it appears to contain two separate limonene in peppermint (Mentha x piperita;Labi- enzyme systems each capable of elaborating a sin- atae/Lamiaceae) and (+)- and (−)-α-pinene in gle enantiomer. Furthermore, a single enzyme typ- pine (Pinus species; Pinaceae). The individual ically accepts geranyl PP as substrate, catalyses the enantiomers can produce different biological isomerization to linalyl PP, and converts this into responses, especially towards olfactory receptors in a final product without the release of free inter- the nose. Thus the characteristic caraway odour is mediates. Sometimes, multiple products in varying

GPP can be folded in two different ways, thus allowing generation of enantiomeric LPP molecules

OPP PPO

OPP PPO

GPP GPP

OPP OPP OPP OPP

(–)-(3R)-LPP (+)-(3S)-LPP

– H – H

α-terpinyl α-terpinyl R S cation (+)-( )-limonene (–)-( )-limonene cation

– H – H

pinyl (+)-α-pinene (–)-α-pinene pinyl cation cation

Figure 5.15 178 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS amounts, e.g. limonene, myrcene, α-pinene, and β- In other systems, a particular structure may pinene, are synthesized by a single enzyme, reflect- be found as a mixture of diastereoisomers. Pep- ing the common carbocation chemistry involved permint (Mentha x piperita; Labiatae/Lamiaceae) in these biosyntheses, and suggesting the enzyme typically produces (−)-menthol, with smaller is predominantly providing a suitable environ- amounts of the stereoisomers (+)-neomenthol, ment for the folding and cyclization of the sub- (+)-isomenthol,and(+)-neoisomenthol, cove- strate. Subsequent reactions such as oxidation of ring four of the possible eight stereoisomers an alcohol to a ketone, e.g. borneol to cam- (Figure 5.16). Oils from various Mentha species phor (Figure 5.14), or heterocyclic ring formation also contain significant amounts of ketones, e.g. in the conversion of α-terpineol into cineole (−)-menthone,(+)-isomenthone,(−)-piperitone, (Figure 5.13), require additional enzyme systems. or (+)-pulegone. The metabolic relationship of

oxidation HO O allylic hydroxylation

(–)-trans-carveol (–)-carvone reduction O allylic oxidation isomerization (–)-piperitone (–)-limonene OH O O allylic hydroxylation (–)-trans- (–)-isopiperitenone piperitenone O isopiperitenol reduction OH (+)-piperitone reduction reduction (+)-neomenthol

reduction reduction allylic O OH isomerization

(–)-menthone (–)-menthol O O (levomenthol)

(+)-cis- (+)-pulegone reduction isopulegone reduction O OH ring formation (+)-isomenthone (+)-isomenthol

HO reduction

OH O OH

p-cymene thymol carvacrol (+)-menthofuran (+)-neoisomenthol

Figure 5.16 Table 5.1 Volatile oils containing principally terpenoid compounds Major volatile oils have been divided into two groups. Those oils containing principally chemicals which are terpenoid in nature and which are derivedbythe deoxyxylulose phosphate pathway are given in Table 5.1 below. Oils which are composed predominantly of aromatic compounds which are derived via the shikimate pathway are listed in Table 4.1 on page 139. The introductory remarks to Table 4.1 are also applicable to Table 5.1. Oils Plant source Plant part used Oil content (%) Major constituents with Uses, notes typical (%) composition Bergamot Citrus aurantium ssp. fresh fruit peel 0.5 limonene (42) flavouring, bergamia (Rutaceae) (expression) linalyl acetate (27) aromatherapy, perfumery γ-terpinene (8) linalool (7) also contains the furocoumarin bergapten (upto5%)andmay cause severe photosensitization (see page 146) Camphor oil Cinnamomum camphora wood 1–3 camphor (27–45) soaps (Lauraceae) cineole (4–21) safrole (1–18) Caraway Carum carvi ripe fruit 3–7 (+)-carvone (50–70) flavour, carminative, (Umbelliferae/Apiaceae) limonene (47) aromatherapy Cardamom Elettaria cardamomum ripe fruit 3–7 α-terpinyl acetate (25–35) flavour, carminative, (Zingiberaceae) cineole (25–45) linalool (5) ingredient of curries, pickles (Continued overleaf ) Table 5.1 (Continued) Oils Plant source Plant part used Oil content (%) Major constituents with Uses, notes typical (%) composition Chamomile (Roman Chamaemelum nobile dried flowers 0.4–1.5 aliphatic esters of angelic, flavouring, aromatherapy chamomile) (Anthemis nobilis) tiglic, isovaleric, and blue colour of oil is due (Compositae/Asteraceae) isobutyric acids (75–85) to chamazulene (see small amounts of page 196) monoterpenes Citronella Cymbopogon fresh leaves 0.5–1.2 (+)-citronellal (25–55) perfumery, winterianus geraniol aromatherapy, insect C. nardus (+)-citronellol (10–15) repellent (Graminae/Poaceae) geranyl acetate (8) (20–40) Coriander Coriandrum sativum ripe fruit 0.3–1.8 (+)-linalool (60–75) flavour, carminative (Umbelliferae/Apiaceae) γ-terpinene (5) α-pinene (5) camphor (5) Dill Anethum graveolens ripe fruit 3–4 (+)-carvone (40–65) flavour, carminative (Umbelliferae/Apiaceae) Eucalyptus Eucalyptus globulus fresh leaves 1–3 cineole (= eucalyptol) flavour, antiseptic, E. smithii (70–85) aromatherapy E. polybractea α-pinene (14) (Myrtaceae) Eucalyptus Eucalyptus citriodora fresh leaves 0.8 citronellal (65–85) perfumery (lemon-scented) (Myrtaceae) Ginger Zingiber officinale dried rhizome 1.5–3 zingiberene (34) flavouring (Zingiberaceae) β-sesquiphellandrene (12) the main pungent β-phellandrene (8) principles in ginger β-bisabolene (6) (gingerols) are not volatile Juniper Juniperus communis dried ripe 0.5–2 α-pinene (45–80) flavouring, antiseptic, (Cupressaceae) berries myrcene (10–25) diuretic, aromatherapy limonene (1–10) sabinene (0–15) juniper berries provide the flavouring for gin Lavender Lavandula angustifolia fresh flowering 0.3–1 linalyl acetate (25–45) perfumery, aromatherapy L. officinalis tops linalool (25–38) (Labiatae/Lamiaceae) inhalation produces mild sedation and facilitates sleep Lemon Citrus limon (Rutaceae) dried peel from 0.1–3 (+)-limonene (60–80) flavouring, perfumery, fruit β-pinene (8–12) aromatherapy (expression) γ-terpinene (8–10) citral (= geranial + neral) terpeneless lemon oil is (2–3) obtained by removing much of the terpenes under reduced pressure; this oil is more stable and contains 40–50% citral (Continued overleaf ) Table 5.1 (Continued) Oils Plant source Plant part used Oil content (%) Major constituents with Uses, notes typical (%) composition Lemon-grass Cymbopogon citratus fresh leaves 0.1–0.3 citral (= geranial + neral) perfumery, aromatherapy (Graminae/Poaceae) (50–85) Matricaria (German Matricaria chamomilla dried flowers 0.3–1.5 (−)-α-bisabolol (10–25%) flavouring chamomile) (Chamomilla recutica) bisabolol oxides A and B (Compositae/Asteraceae) (10–25%) dark blue colour of oil is chamazulene (1–15%) due to chamazulene Orange (bitter) Citrus aurantium ssp. dried peel from 0.5–2.5 (+)-limonene (92–94) flavouring, aromatherapy amara (Rutaceae) fruit myrcene (2) (expression) the main flavour and odour comes from the minor oxygenated components terpeneless orange oil is obtained by removing much of the terpenes under reduced pressure; this oil contains about 20% aldehydes, mainly decanal Orange (sweet) Citrus sinensis dried peel from 0.3 (+)-limonene (90–95) flavouring, aromatherapy (Rutaceae) fruit myrcene (2) (expression) the main flavour and odour comes from the minor oxygenated components terpeneless orange oil is obtained by removing much of the terpenes under reduced pressure; this oil contains about 20% aldehydes, mainly octanal and decanal. Orange flower Citrus aurantium ssp. fresh flowers 0.1 linalool (36) flavour, perfumery, (Neroli) amara (Rutaceae) β-pinene (16) aromatherapy limonene (12) linalyl acetate (6) Peppermint Mentha x piperita fresh leaf 1–3 menthol (30–50) flavouring, carminative, (Labiatae/Lamiaceae) menthone (15–32) aromatherapy menthyl acetate (2–10), menthofuran (1–9) Pine Pinus palustris or other needles, twigs α-terpineol (65) antiseptic, disinfectant, Pinus species (Pinaceae) aromatherapy Pumilio pine Pinus mugo ssp. pumilio needles 0.3–0.4 α-andβ-phellandrene (60) inhalant (Pinaceae) α-andβ-pinene (10–20) bornyl acetate (3–10) the minor components bornyl acetate and borneol are mainly responsible for the aroma (Continued overleaf ) Table 5.1 (Continued) Oils Plant source Plant part used Oil content (%) Major constituents with Uses, notes typical (%) composition Rose (attar of rose, Rosa damascena, fresh flowers 0.02–0.03 citronellol (36) perfumery, aromatherapy otto of rose) gallica, R. alba,andR. geraniol (17) R. centifolia (Rosaceae) 2-phenylethanol (3) C14−C23 straight chain hydrocarbons (25) Rosemary Rosmarinus officinalis fresh flowering 1–2 cineole (15–45) perfumery, aromatherapy (Labiatae/Lamiaceae) tops α-pinene (10–25) camphor (10–25) β-pinene (8) Sage Salvia officinalis fresh flowering 0.7–2.5 thujone (40–60) aromatherapy, food (Labiatae/Lamiaceae) tops camphor (5–22) flavouring cineole (5–14) β-caryophyllene (10) limonene (6) Sandalwood Santalum album heartwood 4.5–6.3 sesquiterpenes: perfumery, aromatherapy (Santalaceae) α-santalol (50) β-santalol (21) Spearmint Mentha spicata fresh leaf 1–2 (−)-carvone (50–70) flavouring, carminative, (Labiatae/Lamiaceae) (−)-limonene (2–25) aromatherapy Tea tree Melaleuca alternifolia fresh leaf 1.8 terpinen-4-ol (30–45) antiseptic, aromatherapy (Myrtaceae) γ-terpinene (10–28) α-terpinene (5–13) an effective broad p-cymene (0.5–12) spectrum antiseptic cineole (0.5–10) widely used in creams, α-terpineol (1.5–8) cosmetics, toiletries Thyme Thymus vulgaris fresh flowering 0.5–2.5 thymol (40) antiseptic, aromatherapy, (Labiatae/Lamiaceae) tops p-cymene (30) food flavouring linalool (7) carvacrol (1) Turpentine oil Pinus palustris and distillation of (+)-and(−)-α-pinene counter-irritant, other Pinus species the resin (35:65) (60–70) important source of (Pinaceae) (turpentine) β-pinene (20–25) industrial chemicals secreted from bark residue from distillation is colophony (rosin), composed chieflyof diterpene acids (abietic acids, see page 209) 186 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS these various compounds has been established as in Figure 5.16, which illustrates how the stereo- chemistry at each centre can be established by stereospecific reduction processes on double bonds regular monoterpene skeleton or carbonyl groups. The pathway also exemplifies that oxygen functions can be introduced into the molecule at positions activated by adjacent double bonds (allylic oxidation), as well as being intro- duced by quenching of carbocations with water. Thus limonene is a precursor of carvone (the main irregular monoterpene skeletons constituent of spearmint oil from Mentha spicata) Figure 5.17 as well as menthone and piperitone, initial hydrox- ylation occurring at an alternative allylic site on the fit the regular head-to-tail coupling mechanism, ring. Menthofuran exemplifies a further oxidative e.g. those in Figure 5.17. These structures are modification generating a heterocyclic ring. Both termed irregular monoterpenes and seem to be lim- pulegone and menthofuran are considered hepato- ited almost exclusively to members of the plant toxic. Pulegone is a major constituent of oil of family the Compositae/Asteraceae. Allowing for pennyroyal from Mentha pulegium, which has a possible rearrangements, the two isoprene units folklore history as an abortifacient. Pulegone is appear to have coupled in another manner and metabolized in humans first to menthofuran, and this is borne out by information available on their then to electrophilic metabolites that form adducts biosynthesis, though this is far from fully under- with cellular proteins (compare pyrrolizidine alka- stood. Thus, although DMAPP and IPP are uti- loids, page 305). lized in their biosynthesis, geranyl PP and neryl p-Cymene, and the phenol derivatives thymol PP do not appear to be involved. Pre-eminent and carvacrol (Figure 5.16) found in thyme (Thy- amongst these structures are chrysanthemic acid mus vulgaris ; Labiatae/Lamiaceae), are represen- and pyrethric acid (Figure 5.18), found in ester tatives of a small group of aromatic compounds form as the pyrethrins∗ (pyrethrins, cinerins, and that are produced in nature from isoprene units, jasmolins, Figure 5.18), which are valuable insecti- rather than by the much more common routes cidal components in pyrethrum flowers, the flower to aromatics involving acetate or shikimate (see heads of Chrysanthemum cinerariaefolium (Com- also cannabinol, page 85, and gossypol, page 200). positae/Asteraceae). These cyclopropane structures These compounds all possess the carbon skele- are readily recognizable as derived from two iso- ton typical of monocyclic monoterpenes, and their prene units, and a mechanism for the derivation structural relationship to limonene and the more of chrysanthemic acid is given in Figure 5.19 common oxygenated monoterpenes such as men- (compare this mechanism with that involved in thone or carvone suggests pathways in which addi- the formation of presqualene PP during steroid tional dehydrogenation reactions are involved. biosynthesis, page 214). This invokes two DMAPP Data on volatile oils containing terpenoid con- units joining by a modification of the standard stituents isolated from these and other plant mate- mechanism, with termination achieved by cyclo- rials are given in Table 5.1. Volatile oils in which propane ring formation. Little is known about the main components are aromatic and derived the origins of pyrethrolone, cinerolone,andjas- from the shikimate pathway are listed in Table 4.1, molone (Figure 5.18), the alcohol portions of the page 139. pyrethrins, though it is possible that these are cyclized and modified fatty acid derivatives, the cyclization resembling the biosynthetic pathway IRREGULAR MONOTERPENES to prostaglandins (see page 53). Thus, α-linolenic acid via 12-oxophytodienoic acid could be the A number of natural monoterpene structures con- precursor of jasmolone, with β-oxidation and tain carbon skeletons which, although obviously then decarboxylation accounting for the chain derived from isoprene C5 units, do not seem to shortening (Figure 5.20). Certainly, this type of IRREGULAR MONOTERPENES 187

O CO H CO2H R2 2 R1 O MeO C chrysanthemic acid 2 pyrethric acid O HO HO HO R1 R2

pyrethrin I Me CH=CH2 pyrethrin II CO2Me CH=CH2 O O O cinerin I Me Me pyrethrolone cinerolone jasmolone

cinerin II CO2Me Me jasmolin I Me Et

jasmolin II CO2Me Et O

ON O

O O O O tetramethrin Cl O RS R O bioresmethrin O Cl O

O O phenothrin R = H, permethrin R = CN, cypermethrin

Figure 5.18

electrophilic addition loss of proton via hydrolysis of phosphate ester; giving tertiary cation cyclopropyl ring oxidation of alcohol to acid formation OPP H OPP OPP CO2H

chrysanthemic acid

Figure 5.19

CO H 2 O CO2H CO2H

O O H OO α-linolenic acid OH

HO β-oxidation CO2H etc CO2H ?

O O jasmonic acid 12-oxophytodienoic acidO jasmolone

Figure 5.20 pathway operates in the formation of jasmonic IRIDOIDS (C10) acid (Figure 5.20), which forms part of a gen- eral signalling system in plants, particularly the The iridane skeleton (Figure 5.21) found in synthesis of secondary metabolites in response to iridoids is monoterpenoid in origin and contains wounding or microbial infection. a cyclopentane ring which is usually fused 188 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

Pyrethrins

The pyrethrins are valuable insecticidal components of pyrethrum flowers, Chrysanthemum cinerariaefolium (= Tanacetum cinerariifolium) (Compositae/Asteraceae). The flowers are harvested just before they are fully expanded, and usually processed to an extract. Pyrethrum cultivation is conducted in East Africa, especially Kenya, and more recently in Ecuador and Australia. The natural pyrethrins are used as a constituent of insect sprays for household use and as post-harvest insecticides, having a rapid action on the nervous system of insects, whilst being biodegradable and non-toxic to mammals, though they are toxic to fish and amphibians. This biodegradation, initiated by air and light, means few insects develop resistance to the pyrethrins, but it does limit the lifetime of the insecticide under normal conditions to just a few hours. The flowers may contain 0.7–2% of pyrethrins, representing about 25–50% of the extract. A typical pyrethrin extract contains pyrethrin I (35%), pyrethrin II (32%), cinerin I (10%), cinerin II (14%), jasmolin I (5%), and jasmolin II (4%), which structures represent esters of chrysanthemic acid or pyrethric acid with the alcohols pyrethrolone, cinerolone, and jasmolone (Figure 5.18). Pyrethrin I is the most insecticidal component, with pyrethrin II providing much of the rapid knock-down (paralysing) effect. A wide range of synthetic pyrethroid analogues, e.g. bioresmethrin, tetramethrin, phenothrin, permethrin,and cypermethrin (Figure 5.18), have been developed, which have increased lifetimes up to several days and greater toxicity towards insects. These materials have become widely used household and agricultural insecticides. Tetramethrin, bioresmethrin, and phenothrin are all esters of chrysanthemic acid but with a modified alcohol portion, providing improvements in knock-down effect and in insecticidal activity. Replacement of the terminal methyls of chrysanthemic acid with chlorine atoms, e.g. permethrin, conferred greater stability towards air and light, and opened up the use of pyrethroids in agriculture. Inclusion of a cyano group in the alcohol portion as in cypermethrin improved insecticidal activity several-fold. Modern pyrethroids now have insecticidal activities over a thousand times that of pyrethrin I, whilst maintaining extremely low mammalian toxicity. Permethrin and phenothrin are employed against skin parasites such as head lice.

to a six-membered oxygen heterocycle, e.g. nepetalactone from catmint Nepeta cataria (Labiatae/Lamiaceae), a powerful attractant and stimulant for cats. The iridoid system arises from O O geraniol by a type of folding (Figure 5.22) which iridane iridoid secoiridoid is different from that already encountered with monoterpenoids, and also different is the lack H of phosphorylated intermediates and subsequent O carbocation mechanism in its formation. The H fundamental cyclization to iridodial is formulated O OO as attack of hydride on the dialdehyde, produced nepetalactone iridoid secoiridoid by a series of hydroxylation and oxidation reactions on geraniol. Further oxidation gives Figure 5.21 iridotrial, in which hemiacetal formation then leads to production of the heterocyclic ring. In the aldehyde or in the heterocyclic ring. A large iridotrial, there is an equal chance that the original number of iridoids are found as glycosides, e.g. methyls from the head of geraniol end up as loganin, glycosylation effectively transforming the IRIDOIDS 189 hemiacetal linkage into an acetal. The pathway skeleton still recognizable in loganin to give to loganin involves, in addition, a sequence of secologanin, representative of the secoiridoids reactions in which the remaining aldehyde group (Figure 5.21). This is catalysed by a cytochrome is oxidized to the acid and methylated, giving P-450-dependent mono-oxygenase, and a free deoxyloganin,andthefinal step is a hydroxylation radical mechanism is proposed in Figure 5.22. reaction. Loganin is a key intermediate in the Secologanin now contains a free aldehyde group, biosynthesis of many other iridoid structures, together with further aldehyde and enol groups, and also features in the pathway to a range these latter two fixed as an acetal by the presence of complex terpenoid indole alkaloids (see of the glucose. As we shall see with some of page 350) and tetrahydroisoquinoline alkaloids the complex alkaloids, these functionalities can be (see page 343). Fundamental in this further released again by hydrolysing off the glucose and metabolism is cleavage of the simple monoterpene reopening the hemiacetal linkage. Gentiopicroside

cyclization formulated as initiated by electrophilic addition utilizing the unsaturated carbonyl, terminated by addition of hydride; the Schiff base-assisted mechanism hemiacetal via hydroxylation shown below is more realistic H formation and oxidations H OH H O OH CHO CHO H H H H OH O CHO geraniol iridodial iridodial iridodial O (keto form) (enol form) (hemiacetal form)

(NADPH) H O hemiacetal formation CHO H CHO OH CHO H H O HN HN OHC CHO OHC Enz H Enz iridotrial iridotrial (keto form) (hemiacetal form) formation of alkene Enz Fe oxidation of aldehyde to acid and ring cleavage oxidation glucosylation H O H leading to esterification HO HO radical HO H O2 H H NADPH H H OGlc OGlc OGlc O OGlc H H H H glucosylation has O O O O now transformed MeO2C MeO2C MeO2C MeO2C the hemiacetal loganin deoxyloganin into an acetal Enz Fe OH HO HO OH CHO H H – H O H tryptamine terpenoid OGlc OGlc 2 OGlc indole H H H alkaloids O O O MeO C MeO2C MeO2C 2 secologanin

Figure 5.22 190 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

hydrolysis of ester; formation of enol allylic lactone CHO tautomer isomerization formation OGlc OGlc OGlc OGlc OH O OH O O O O MeO2C HO C HO2C 2 secologanin O gentiopicroside

Figure 5.23

(Figure 5.23) is another example of a secoiridoid, A range of epoxyiridoid esters has been identified and is found in Gentian root (Gentiana lutea; in the drug valerian∗ (Valeriana officinalis; Valeri- Gentianaceae), contributing to the bitter taste of anaceae). These materials, responsible for the seda- this herbal drug. Its relationship to secologanin is tive activity of the crude drug, are termed valepo- suggested in Figure 5.23. The alkaloid gentianine triates. Valtrate (Figure 5.24) is a typical example, (see page 386) is also found in Gentian root, and and illustrates the structural relationship to loganin, represents a nitrogen analogue of the secoiridoids, though these compounds contain additional ester in which the pyran oxygen has been replaced by functions, frequently isovaleryl. The hemiacetal is nitrogen. now fixed as an ester, rather than as a glycoside.

Valerian Valerian root consists of the dried underground parts of Valeriana officinalis (Valerianaceae), a perennial herb found throughout Europe. Drug material comes from wild and cultivated plants, and is carefully dried at low temperature (less than 40 °C) to minimize decomposition of constituents. Valerian preparations are widely used as herbal tranquillizers to relieve nervous tension, anxiety, and insomnia. Valerian was especially popular during the First World War, when it was used to treat shell-shock. The drug does possess mild sedative and tranquillizing properties, but for maximum activity the roots need to be freshly harvested and carefully dried. The major active principles are generally held to be a number of epoxyiridoid esters called valepotriates (0.5–1.6%), the principal component of which is valtrate (about 80%) (Figure 5.24). Minor valepotriates have the same parent iridoid alcohol as valtrate, but differ with respect to esterifying acids, e.g. isovaltrate (Figure 5.24), or are based on the reduced iridoid seen in didrovaltrate, again with various ester functionalities. Acid entities characterized in this group of compounds are mainly isovaleric (3-methylbutyric) and acetic (as in valtrate/isovaltrate/didrovaltrate), though more complex diester groups involving 3-acetoxyisovaleric and isovaleroxyisovaleric acids are encountered. During drying and storage, some of the valepotriate content may decompose by hydrolysis to liberate quantities of isovaleric acid, giving a characteristic odour, and structures such as baldrinal (Figure 5.24) (from valtrate) and homobaldrinal (from isovaltrate). Samples of old or poorly prepared valerian may contain negligible amounts of valepotriates. Standardized mixtures of valepotriates, containing didrovaltrate (80%), valtrate (15%), and acevaltrate (Figure 5.24) (5%), are available in some countries. These materials are usually extracted from the roots of other species of Valeriana, which produce higher amounts of valepotriates than V. officinalis, e.g. V. mexicana contains up to about 8%. Some other species of Valeriana that contain similar valepotriate constituents are used medicinally, including V. wallichi (Indian valerian) and V. edulis (Mexican valerian).

(Continues) SESQUITERPENES 191

(Continued)

O O O O O O

O H O H O H O O O H O O OOO O O O O

O valtrateO didrovaltrateO isovaltrate

O O CHO CHO O H OAc O

O O OOO O O

H O acevaltrateO baldrinalO homobaldrinal

R N

O CO2H OH valeranonevalerenic acid Valerian alkaloids R = H, OH

Figure 5.24

Despite the information given above, many workers believe the sedative activity of valerian cannot be due to the valepotriates, which are very unstable and not water soluble. Some of the sedative activity is said to arise from sesquiterpene derivatives such as valerenic acid (about 0.3%) and those constituting the volatile oil content (0.5–1.3%), e.g. valeranone (Figure 5.24), which have been shown to be physiologically active. GABA (γ-aminobutyric acid) and glutamine have also been identified in aqueous extracts of valerian, and these have been suggested to contribute to the sedative properties. Small amounts of some iridoid-related alkaloids (Figure 5.24) have been isolated from valerian root (see page 386). The valepotriates valtrate and didrovaltrate are reported to be cytotoxic in vitro, and this may restrict future use of valerian. The reactive epoxide group is likely to be responsible for these cytotoxic properties.

SESQUITERPENES (C15) stereochemistry of the double bond nearest the diphosphate can adopt an E configuration (as in Addition of a further C5 IPP unit to geranyl FPP), or a Z configuration via ionization, as found diphosphate in an extension of the prenyl trans- with geranyl/neryl PP (Figure 5.26). In some sys- ferase reaction leads to the fundamental sesquiter- tems, the tertiary diphosphate nerolidyl PP (com- pene precursor, farnesyl diphosphate (FPP) pare linalyl PP, page 172) has been implicated (Figure 5.25). Again, an initial ionization of GPP as a more immediate precursor than farnesyl PP seems likely, and the proton lost from C-2 of IPP (Figure 5.26). This allows different possibilities is stereochemically analogous to that lost in the for folding the carbon chain, dictated of course previous isoprenylation step. FPP can then give by the enzyme involved, and cyclization by elec- rise to linear and cyclic sesquiterpenes. Because trophilic attack on to an appropriate double bond. of the increased chain length and additional dou- As with the monoterpenes, standard reactions ble bond, the number of possible cyclization of carbocations rationally explain most of the modes is also increased, and a huge range of common structural skeletons encountered, and mono-, bi-, and tri-cyclic structures can result. The a representative selection of these is given in 192 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

less volatile than monoterpenes. Simple quench- ing of the bisabolyl cation with water leads OPP to α-bisabolol (Figure 5.28), a major compo- geranyl PP nent of matricaria (German chamomile)∗ flowers (Matricaria chamomilla; Compositae/Asteraceae). So-called bisabolol oxides AandBarealso OPP present, compounds probably derived from bis- allylic cation HR HS abolol by cyclization reactions (Figure 5.28) on electrophilic addition an intermediate epoxide (compare Figure 4.34, giving tertiary cation page 146). Other cyclizations in Figure 5.27 lead to ring OPP systems larger than six carbons, and seven-,

HR HS ten-, and 11-membered rings can be formed stereospecific loss of as shown. The two ten-membered ring sys- proton tems (germacryl and cis-germacryl cations), or the two 11-membered systems (humulyl and OPP cis-humulyl cations), differ only in the stereochem- farnesyl PP istry associated with the double bonds. However, (FPP) this affects further cyclization processes and is Figure 5.25 responsible for extending the variety of natural sesquiterpene derivatives. The germacryl cation, without further cyclization, is a precursor of the Figure 5.27. One of these cyclized systems, the germacrane class of sesquiterpenes, as exemplified bisabolyl cation, is analogous to the monoter- by parthenolide (Figure 5.30), the antimigraine pene menthane system, and further modifica- agent in feverfew∗ (Tanacetum parthenium;Com- tions in the six-membered ring can take place positae/Asteraceae). Parthenolide is actually classi- to give essentially monoterpene variants with fied as a germacranolide, the suffix ‘olide’ referring an extended hydrocarbon substituent, e.g. γ- to the lactone group. Whilst the details of the path- bisabolene (Figure 5.28), which contributes to way are not known, a series of simple oxidative the aroma of ginger (Zingiber officinale;Zin- transformations (Figure 5.30) can produce the α,β- giberaceae) along with the related structures unsaturated lactone and epoxide groupings. such as zingiberene and β-sesquiphellandrene The α,β-unsaturated lactone functionality is (Figure 5.29). Sesquiterpenes will in general be a common feature of many of the biologically

E E OPP E,E-FPP

OPP E Z

nerolidyl PP OPP E,Z-FPP

Figure 5.26 SESQUITERPENES 193

i

H guaiyl cation e.g. matricin, thapsigargin H E germacryl cation ii e.g. parthenolide E

E,E-farnesyl cation eudesmyl cation e.g. α-santonin

humulyl cation e.g. humulene E

caryophyllyl cation e.g. caryophyllene ≡

W–M E 1,3-hydride shift H

nerolidyl cation bisabolyl cation e.g. bisabolene, α-bisabolol e.g. artemisinic acid

a

a E

a carotyl cation W–M Z b b 1,3-hydride shift E,Z-farnesyl cation b H

cis-germacryl cation cadinyl cation e.g. α-cadinene

cis-humulyl cation

Figure 5.27 194 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

H H – H – H

H OH O

(–)-β-bisabolene bisabolyl cation γ-bisabolene (–)-α-bisabolol oxide B – H b H2O OH H H OH H OH H a O b a O (–)-α-bisabolol oxide A

(–)-α-bisabolene (–)-α-bisabolol

Figure 5.28

O

O H H O HO

O O O O O O O α O O O -santonin (–)-zingiberene β (–)- -sesqui- elephantopin parthenin phellandrene

O O

O CO2HCO2H O H O angelic acid tiglic acid O O O OH OH O CO2H CO2H O isovaleric acid isobutyric acid thapsigargin

Figure 5.29 active terpenoids. The activity frequently manifests Michael-type addition of a suitable nucleophile, itself as a toxicity, especially cytotoxicity as e.g. thiols, on to the α,β-unsaturated lactone. Such seen with the germacranolide elephantopin alkylation reactions are believed to explain biolog- (Figure 5.29) from Elephantopus elatus (Com- ical activity, and, indeed, activity is typically lost positae/Asteraceae), or skin allergies, as caused if either the double bond or the carbonyl group is by the pseudoguaianolide (a rearranged guaiano- chemically reduced. In some structures, additional lide) parthenin (Figure 5.29) from Parthenium electrophilic centres offer further scope for alky- hysterophorus (Compositae/Asteraceae), a highly lation reactions. In parthenolide (Figure 5.31), an troublesome weed in India. These compounds can electrophilic epoxide group is also present, allow- be considered as powerful alkylating agents by a ing transannular cyclization and generation of a SESQUITERPENES 195

– H

O O O germacryl cation O O O germacrene A parthenolide

Figure 5.30

transannular cyclization initiated via attack of sulphydryl in nucleophilic attack of sulphydryl on R protonated epoxide; compare protein on to resultant to α,β-unsaturated lactone: S H carbocation Michael reaction formation of guaiyl cation, SR SR Figure 5.27 H H H R HS

O H H H O OH O OH O OH O SR O H O O O parthenolide SR H

HO H O O H SR H O O sesquiterpene lactone O H irreversibly bound to protein

Figure 5.31 second alkylation site. Cytotoxic agents may irre- parthenolide and structurally related compounds in versibly alkylate critical enzymes that control cell feverfew have been demonstrated to relate to alky- division, whilst allergenic compounds may con- lation of thiol groups. jugate with proteins to form antigens which trig- α-Santonin (Figure 5.29) has been identified ger the allergic response. The beneficial effects of as the principal anthelmintic component of

Feverfew Feverfew is a traditional herbal remedy for the relief of arthritis, migraine, toothache, and menstrual difficulties. The plant is a perennial, strongly aromatic herb of the Compositae/Asteraceae family, and has been classified variously as Tanacetum parthenium, Chrysanthemum parthenium, Leucanthemum parthenium,orPyrethrum parthenium,the former name being currently favoured. Studies have confirmed that feverfew is an effective prophylactic treatment in about 70% of migraine sufferers. It reduces the frequency of attacks, the vomiting associated with attacks, and the severity of attacks. The herb has been shown to inhibit blood platelet aggregation, the release of 5-hydroxytryptamine (serotonin) from platelets, the release of histamine from mast cells, and the production of prostaglandins, thromboxanes and leukotrienes. Of a range of sesquiterpene lactones of the germacrane and guianane groups characterized in the leaf material, the principal constituent and major active component is parthenolide (Figure 5.30) (up to about 1% in dried leaves). The powerful pungent odour of the plant arises from the volatile oil constituents, of which the monoterpene camphor (Figure 5.14) is a major constituent. Feverfew may be taken as the fresh leaf, often eaten with bread in the form of a sandwich to minimize the bitter taste, or it can be obtained

(Continues) 196 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

in dosage forms as tablets or capsules of the dried powdered leaf. The parthenolide content of dried leaf deteriorates on storage, and many commercial preparations of feverfew have been shown to contain little parthenolide, or to be well below the stated content. This may be a consequence of complexation with plant thiols via Michael addition. Consumers of fresh leaf can be troubled by sore mouth or mouth ulcers, caused by the sesquiterpenes. Parthenolide is also known to be capable of causing some allergic effects, e.g. contact dermatitis. The proposed mechanism of action of parthenolide via alkylation of thiol groups in proteins is shown in Figure 5.31. various Artemisia species, e.g. wormseed (A. again from matricaria∗ flowers. This compound cinia; Compositae/Asteraceae), and has found degrades on heating, presumably by elimination of considerable use for removal of roundworms, acetic acid and water, and then decarboxylation to although potential toxicity limits its application. the azulene derivative chamazulene, responsible Structurally, α-santonin bears much similarity to for the blue coloration of oil distilled from the parthenolide, and the most marked difference flowers. Thapsigargin (Figure 5.29) from Thapsia lies in the presence of the bicyclic decalin garganica (Umbelliferae/Apiaceae) provides a ring system. This basic skeleton, the eudesmane further example of a guaianolide, esterified with system, is formed from the germacryl cation a variety of acid groups. This compound is of by protonation and cyclization via the eudesmyl considerable pharmacological interest as a tumour cation (Figure 5.27, route ii), whereas protonation promoter, and as a potent activator of cells at the more substituted end of a double bond (anti- involved in the inflammatory response. Markovnikov addition, route i), could generate Another type of decalin-containing sesquiter- the guaiyl cation and guaiane skeleton. This pene is seen in the structures of α-cadinene and latter skeleton is found in matricin (Figure 5.32), amorpha-4,11-diene. α-Cadinene (Figure 5.33) is

elimination of acetic acid and two molecules H2O; consider lactone as hydrolysed to hydroxyacid

∆ – HOAc, OAc – 2 H2O – CO2 H H OH O

HO2C O chamazulene matricin chamazulene carboxylic acid

Figure 5.32

Chamomile and Matricaria Two types of chamomile (camomile) are commonly employed in herbal medicine, Roman chamomile Chamaemelum nobile (formerly Anthemis nobilis) (Compositae/Asteraceae), and German chamomile Matricaria chamomilla (Chamomilla recutica) (Compositae/Asteraceae). German chamomile, an annual plant, is the more important commercially, and is often called matricaria to distinguish it from the perennial Roman chamomile. Both plants are cultivated

(Continues) SESQUITERPENES 197

(Continued)

in various European countries to produce the flowerheads, which are then dried for drug use. Volatile oils obtained by steam distillation or solvent extraction are also available. Roman chamomile is usually taken as an aqueous infusion (chamomile tea) to aid digestion, curb flatulence, etc, but extracts also feature in mouthwashes, shampoos, and many pharmaceutical preparations. It has mild antiseptic and anti-inflammatory properties. The flowerheads yield 0.4–1.5% of volatile oil, which contains over 75% of aliphatic esters of angelic, tiglic, isovaleric, and isobutyric acids (Figure 5.29), products of isoleucine, leucine, and valine metabolism (see pages 100, 295, 306), with small amounts of monoterpenes and sesquiterpenes. Matricaria is also used as a digestive aid, but is mainly employed for its anti-inflammatory and spasmolytic properties. Extracts or the volatile oil find use in creams and ointments to treat inflammatory skin conditions, and as an antibacterial and antifungal agent. Taken internally, matricaria may help in the control of gastric ulcers. The flowers yield 0.5–1.5% volatile oil containing the sesquiterpenes α-bisabolol (10–25%), bisabolol oxides A and B (10–25%) (Figure 5.28), and chamazulene (0–15%) (Figure 5.32). Chamazulene is a thermal decomposition product from matricin, and is responsible for the dark blue coloration of the oil (Roman chamomile oil contains only trace amounts of chamazulene). α-Bisabolol has some anti-inflammatory, antibacterial, and ulcer-protective properties, but chamazulene is probably a major contributor to the anti-inflammatory activity of matricaria preparations. It has been found to block the cyclooxygenase enzyme in prostaglandin biosynthesis (see page 55) and the anti-inflammatory activity may result from the subsequent inhibition of leukotriene formation.

to produce the decalin ring system. In this H H H case, a six-membered ring is most likely formed first giving the bisabolyl cation, and, again, a 1,3-hydride shift is implicated prior to form- H H ing the decalin system (Figure 5.27). Amorpha- 4,11-diene is an intermediate in the pathway cadinyl cation α-cadinene leading to artemisinin in Artemisia annua (Com- positae/Asteraceae) (Figure 5.34). This proceeds Figure 5.33 through artemisinic acid and dihydroartemisinic acid via modest oxidation and reduction pro- cesses. Dihydroartemisinic acid may be converted one of the many terpenoids found in juniper berries chemically into artemisinin by an oxygen-mediated (Juniperus communis; Cupressaceae) used in mak- photochemical oxidation under conditions that ing gin, and this compound is derived from the might normally be present in the plant, suggest- ten-carbon ring-containing cis-germacryl cation. ing that all further transformations may in fact be The double bonds in the cis-germacryl cation non-enzymic. An intermediate in this process also are unfavourably placed for a cyclization reac- found naturally in A. annua is the hydroperoxide tion as observed with the germacryl cation, and of dihydroartemisinic acid. The further modifi- available evidence points to an initial 1,3-shift cations postulated in Figure 5.34 include ring of hydride to the isopropyl side-chain generat- expansion by cleavage of this hydroperoxide ing a new cation, and thus allowing cyclization and a second oxygen-mediated hydroperoxida- (Figure 5.27). Amorpha-4,11-diene (Figure 5.34) tion. The 1,2,4-trioxane system in artemisinin can is structurally related to α-cadinene, but the dif- be viewed more simply as a combination of ferent stereochemistry of ring fusion and site hemiketal, hemiacetal, and lactone functions, and of the second double bond is a consequence the later stages of the pathway merely reflect of a different cyclization mechanism operating their construction. Artemisinin∗ is an important 198 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

photoxidation to H H H hydroperoxide H

H H H H H OO H O HO2C HO2C from bisabolyl cation amorpha-4,11-diene artemisinic acid dihydroartemisinic acid (see Figure 5.27) ν air oxidation to hydrolysis of cleavage of hydroperoxide, O2 / h hydroperoxide ring expansion, and O O hemiketal H H H quenching of carbocation H HO H2O HOO O2 O H H H H H O H O O HO HO2C HO2C HO2C HO2C hemiketal dihydroartemisinic acid formation hydroperoxide formation of hemiacetal, then an enzyme preparation H lactone H has also catalysed this H O O transformation O O HO O ? H H O H O O O HO2C O O artemisinin dihydroarteannuin B (qinghaosu)

Figure 5.34 antimalarial component in Artemisia annua∗,a The 11-carbon ring of the humulyl carboca- Chinese herbal drug. There is currently strong tion may be retained, as in the formation of research effort to produce artemisinin or analogues humulene (Figure 5.36), or modified to give the as new antimalarial drugs, since many of the cur- caryophyllyl cation containing a nine-membered rent drugs have become less satisfactory due to ring fused to a four-membered ring, as in β- resistance (see quinine, page 362). caryophyllene (Figure 5.36). Humulene is found

Artemisia annua and Artemisinin Artemisia annua (Compositae/Asteraceae) is known as qinghao in Chinese traditional medicine, where it has been used for centuries in the treatment of fevers and malaria. The plant is sometimes called annual or sweet wormwood, and is quite widespread, being found in Europe, North and South America, as well as China. Artemisinin (qinghaosu) (Figure 5.34) was subsequently extracted and shown to be responsible for the antimalarial properties, being an effective blood schizontocide in humans infected with malaria, and showing virtually no toxicity. Malaria is caused by protozoa of the genus Plasmodium, especially P. falciparum, entering the blood system from the salivary glands of mosquitoes, and world-wide is responsible for 2–3 million deaths each year. Established antimalarial drugs such as chloroquine (see page 363) are proving less effective in the treatment of malaria due to the appearance of drug-resistant strains of P. falciparum. Artemisinin is currently effective against these drug-resistant strains.

(Continues) SESQUITERPENES 199

(Continued)

Artemisinin is a sesquiterpene lactone containing a rare peroxide linkage which appears essential for activity. Some plants of Artemisia annua have been found to produce as much as 1% artemisinin, but the yield is normally very much less, typically 0.05–0.2%. Apart from one or two low-yielding species, the compound has not been found in any other species of the genus Artemisia (about 400 species). Small amounts (about 0.01%) of the related peroxide structure artemisitene (Figure 5.35) are also present in A. annua, though this has a lower antimalarial activity. The most abundant sesquiterpenes in the plant are artemisinic acid (arteannuic acid, qinghao acid) (typically 0.2–0.8%) (Figure 5.34), and lesser amounts (0.1%) of arteannuin B (qinghaosu-II) (Figure 5.35). Fortunately, arteannuic acid may be converted chemically into artemisinin by a relatively simple and efficient process. Artemisinin may be reduced to the lactol (hemiacetal) dihydroartemisinin (Figure 5.35), and this has been used for the semi- synthesis of a range of analogues, of which the acetals artemether and arteether (Figure 5.35), and the water-soluble sodium salts of artelinic acid and artesunic acid (Figure 5.35), appear very promising antimalarial agents. These materials have increased activity compared with artemisinin and the chances of infection recurring are also reduced. Artemether has rapid action against chloroquinine-resistant P. falciparum malaria, and is currently being used as injection formulations. Arteether has similar activity. Being acetals, artemether and arteether are both extensively decomposed in acidic conditions, but are stable in alkali. The ester artesunic acid is also used in injection form, but is rather unstable in alkaline solution, hydrolysing to dihydroartemisinin. The ether artelinic acid is considerably more stable. These two compounds have a rapid action and particular application in the treatment of potentially fatal cerebral malaria. Dihydroartemisinin is a more active antimalarial than artemisinin and appears to be the main metabolite of these drugs in the body. They rapidly clear the blood of parasites, but do not have a prophylactic effect. Chemically, these agents are quite unlike any other class of current antimalarial agent, and when thoroughly evaluated, they may well become an important group of drugs in the fight against this life-threatening disease.

H H H H O O O O O O O ≡ O O O O O H H O H H O O O O O O

O O O H OH artemisinin artemisitene arteannuin B dihydroartemisinin H O O H O O OH H O O O H O O O OH O O H O O H O H O HO O H O H OR yingzhaosu C HO C O R = Me, artemether HO2C 2 R = Et, arteether artelinic acid artesunic acid yingzhaosu A

Figure 5.35

(Continues) 200 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

The relationship between a peroxide linkage and antimalarial activity is strengthened by the isolation of other sesquiterpene peroxides which have similar levels of activity as artemisinin. Thus, roots of the vine yingzhao (Artabotrys uncinatus; Annonaceae), which is also used as a traditional remedy for malaria, contain the bisabolyl derivatives yingzhaosu A and yingzhaosu C (Figure 5.35), the latter containing an aromatic ring of isoprenoid origin (compare the monoterpenes thymol and carvacrol, page 186). Artemisinin, and other peroxide-containing antimalarial agents, appear to complex with haemin, which is a soluble iron–porphyrin material released from haemoglobin as a result of proteolytic digestion by the malarial parasite. This material is toxic to Plasmodium, so is normally converted into an insoluble non-toxic form haemozoin (malarial pigment) by enzymic polymerization. Agents like chloroquine (see page 363) interfere with the polymerization process. Complexation of haemin with artemisinin by coordination of the peroxide bridge with the iron atom interrupts the detoxification process and leads to the generation of free radical species through homolytic cleavage of the peroxide. The resulting radicals ultimately damage proteins in Plasmodium. in hops (Humulus lupulus; Cannabaceae), and β- cadinyl carbocation via δ-cadinene is involved in caryophyllene is found in a number of plants, e.g. generating the basic aromatic sesquiterpene unit in the oils from cloves (Syzygium aromaticum; hemigossypol, and then dimerization is simply an Myrtaceae) and cinnamon (Cinnamomum zeylan- example of phenolic oxidative coupling ortho to icum; Lauraceae). the phenol groups (Figure 5.37). Gossypol∗ (Figure 5.37) is an interesting and The formation of sesquiterpenes by a carboca- unusual example of a dimeric sesquiterpene in tion mechanism means that there is considerable which loss of hydrogen has led to an aromatic sys- scope for rearrangements of the Wagner– tem (compare the phenolic monoterpenes thymol Meerwein type. So far, only occasional hydride and carvacrol, page 186). This material is found migrations have been invoked in rationalizing in immature flower buds and seeds of the cot- the examples considered. Obviously, fundamen- ton plant (Gossypium species; Malvaceae), though tal skeletal rearrangements will broaden the range originally isolated in small amounts from cotton- of natural sesquiterpenes even further. That such seed oil. It can function as a male infertility agent, processes do occur has been proven beyond and is used in China as a male contraceptive. The doubt by appropriate labelling experiments, and

– H ≡ b a a H humulyl cation humulene b

– H H H H

caryophyllyl cation β-caryophyllene

Figure 5.36 SESQUITERPENES 201

one-electron various oxidative oxidation OH CHO H processes O CHO OH – H OH – e

OH OH H H

cadinyl cation δ-cadinene hemigossypol

radical HO coupling O CHO OH CHO x 2 OH OH HO CHO OH OH OH

gossypol resonance forms of free radical

Figure 5.37

Gossypol

Gossypol occurs in the seeds of cotton (Gossypium species, e.g. G. hirsutum, G. herbaceum, G. arboreum, G. barbadense; Malvaceae) in amounts of 0.1–0.6%. Its contraceptive effects were discovered when subnormal fertility in some Chinese rural communities was traced back to the presence of gossypol in dietary cottonseed oil. Gossypol acts as a male contraceptive, altering sperm maturation, spermatozoid motility, and inactivation of sperm enzymes necessary for fertilization. Extensive clinical trials in China have shown the antifertility effect is reversible after stopping the treatment provided consumption has not been too prolonged. Cases of irreversible infertility have resulted from longer periods of drug use. The molecule is chiral due to restricted rotation, and can thus exist as two atropisomers which do not easily racemize (Figure 5.38). Only the (−)-isomer is pharmacologically active as a contraceptive, whereas most of the toxic symptoms appear to be associated with the (+)-isomer, which also displays antitumour and antiviral activities. Most species of Gossypium (except G. barbadense) produce gossypol where the (+)-isomer predominates over the (−)-isomer, with amounts varying according to species and cultivar. Racemic (±)-gossypol (but neither of the enantiomers) complexes with acetic acid, so that suitable treatment of cotton seed extracts actually separates the racemate from the excess of (+)-isomer. The racemic form can then be resolved. Other plants in the Gossypieae tribe of the Malvaceae also produce gossypol, with the barks of Thespia populnea (3.3%) and Montezuma speciosissima (6.1%) being particularly rich sources. Unfortunately, gossypol from these is almost entirely the inactive (+)-form.

OHC OH HO CHO HO OH OH HO

HO OH OH HO HO CHO OHC OH

(+)-gossypol (–)-gossypol

Figure 5.38 202 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS a single example will be used as illustration. 1,2-methyl shifts then follow, and it is important to The trichothecenes∗ are a group of fungal tox- note that each migrating group attacks the oppo- ins found typically in infected grain foodstuffs. site side of the centre from which the previous Their name comes from the fungal genus Tri- group is departing, i.e. inverting the configuration chothecium, but most of the known structures at these centres. Accordingly, a concerted sequence are derived from cultures of Fusarium species. A of migrations is feasible, such a process being particularly prominent trichothecene contaminant seen more vividly in the formation of triterpenoids is deoxynivalenol (vomitoxin), which is produced and steroids (see page 216). Loss of a proton and from the less substituted trichothecene isotricho- generation of a double bond terminates the pro- dermol by a sequence of oxygenation reactions cess giving trichodiene. Oxygenation of trichodi- (Figure 5.39). The trichothecenes have their ori- ene gives, in several steps, isotrichotriol.Twoof gins in nerolidyl diphosphate, and ring closure the hydroxylations are at activated allylic posi- of the bisabolyl cation derived from it gener- tions; hydroxylation on the five-membered ring ates a new carbocation with a five-membered ring will therefore occur before the epoxidation. Ether (Figure 5.39). At this stage, a series of one hydride formation, involving perhaps protonation, loss of and two methyl migrations occur to give a cation, water and generation of an allylic cation, completes which loses a proton to produce the specifictri- the pathway to the basic trichothecene structure as chothecene precursor trichodiene. These migra- in isotrichodermol. tions are fully backed up by experimental data, Finally, it is worth noting how many of and although not immediately predictable, can be the sesquiterpene derivatives described above are rationalized satisfactorily by consideration of the found in plants belonging to the daisy family, the cation suitably bound to the enzyme surface as Compositae/Asteraceae. Whilst sesquiterpenes are shown in Figure 5.39. The sequence is initiated by no means restricted to this family, the Composi- by a 1,4-hydride shift which is spatially allowed tae/Asteraceae undoubtedly provides a very rich by the relative proximity of the centres. Two source.

electrophilic addition Wagner–Meerwein leads to five-membered rearrangements: 1,4-hydride ring and secondary shift; two 1,2-methyl shifts cation H H

H

bisabolyl cation ≡ trichodiene ≡

H H

sequence of epoxidation and three nucleophilic attack of hydroxyl hydroxylations O on to allylic cation

HH HH OH + H HO OH O OH O OH OH OH O – H2O O O O O O HO OH isotrichodermol allylic cation isotrichotriol deoxynivalenol (DON)

Figure 5.39 DITERPENES 203

Trichothecenes The trichothecenes are a group of sesquiterpene toxins produced by several fungi of the genera Fusarium, Myrothecium, Trichothecium,andTrichoderma, which are parasitic on cereals such as maize, wheat, rye, barley, and rice. About 150 different structures have been identified, with some of these being isolated from plants of the genus Baccharis (Compositae/Asteraceae), where a symbiotic plant–fungus relationship may account for their production. Examples of trichothecene structures commonly encountered as food contam- inants include deoxynivalenol (DON) (Figure 5.39), and diacetoxyscirpenol (DAS), T-2 toxin, and verrucarin A (Figure 5.40). The double bond and the epoxide group in the basic tri- chothecene skeleton are essential for toxicity, and the number of oxygen substituents and ester functions also contribute. Macrocyclic ester functions as seen in verrucarin A tend to produce the most toxic examples. Although these compounds are more toxic when injected, oral toxicity is relatively high, and lethal amounts can easily be consumed because of the nature of the host plants. They are sufficiently toxic to warrant routine analysis of foodstuffs such as wheat and flour, and also flour-derived products, e.g. bread, since they survive pro- cessing and the high temperatures used in baking. DON levels above 1 ppm are considered hazardous for human consumption. It is relevant to note that when mammals ingest these compounds, a degree of de-epoxidation can occur, ascribed to gut microflora, thus providing some detoxification by removing a structural feature necessary for toxicity. As their main mechanism of action, these compounds inhibit protein biosynthesis by binding to the ribosome and inhibiting peptidyl transferase activity (see page 407). They also inhibit DNA biosynthesis. A major human condition known to be caused by trichothecenes is alimentary toxic aleukia (ATA), characterized by destruction of the skin, haemorrhaging, inflammation, sepsis, a decrease in red and white blood corpuscles, bone marrow atrophy, and a high mortality rate. A severe outbreak of ATA was recorded in the former Soviet Union shortly after the Second World War when food shortages necessitated the consumption of grain that had overwintered in the field. This had become badly contaminated with Fusarium sporotrichioides and hence T-2 toxin. It is estimated that tens of thousands died as a result. Many trichothecene derivatives have been tested as potential anticancer agents but have proved too toxic for clinical use.

HH O O HH HH O O OH O OH O O O O O O O AcO OAc AcO OAc O HO diacetoxyscirpenol T-2 toxin O (DAS) verrucarin A

Figure 5.40

DITERPENES (C20) terpenoids (Figure 5.41). One of the simplest and most important of the diterpenes is phytol The diterpenes arise from geranylgeranyl diphos- (Figure 5.42), a reduced form of geranylgeran- phate (GGPP), which is formed by addition of iol, which forms the lipophilic side-chain of the a further IPP molecule to farnesyl diphosphate chlorophylls, e.g. chlorophyll a (Figure 5.42). in the same manner as described for the lower Related haem molecules, porphyrin components 204 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

OPP farnesyl PP (FPP)

OPP allylic cation IPP electrophilic addition gives tertiary cation

OPP

HR HS stereospecific loss of proton

OPP geranylgeranyl PP (GGPP)

Figure 5.41

N N OH 2+ phytol Mg O N N

MeO2C O O O O vitamin K1 (phylloquinone) chlorophyll a

Figure 5.42 of haemoglobin, lack such lipophilic side-chains. group (menaquinones) from bacteria have unsatu- Available evidence suggests that geranylgeranyl rated terpenoid side-chains of variable length. The diphosphate is involved in forming the ester link- phytyl group of phylloquinone is introduced by age, and the three reduction steps necessary to alkylation of dihydroxynaphthoic acid with phytyl form the phytol ester occur after attachment to diphosphate and a similar phytylation of homogen- the chlorophyll molecule. A phytyl substituent tisic acid features in the formation of the E group is also found in vitamin K1 (phylloquinone) vitamins (tocopherols). These compounds are dis- (Figure 5.42), a naphthoquinone derivative found cussed further under shikimate derivatives (see in plants, though other members of the vitamin K page 158). DITERPENES 205

cyclization by electrophilic addition E giving tertiary cation H E

E OPP

GGPP AcO O verticillyl cation generation of cation by H protonation of alkene; – H O HO OH leads to electrophilic O cyclization PhNH O H O H O Ph Ph O AcO H OH paclitaxel (taxol) H L-Phe many steps; oxygenation verticillene O acetyl-CoA of diterpene system and O several OH esterifications O NH O O esterifications 10 O OH OH O HO H H OH HO HO O O O O O O AcO O taxadiene L-Phe O acetyl-CoA paclitaxel L-Phe 10-deacetyl- (taxol) baccatin III

Figure 5.43

Cyclization reactions of GGPP mediated by car- Although these compounds are sometimes classi- bocation formation, plus the potential for Wagner fied as diterpenoid alkaloids, the nitrogen atom is –Meerwein rearrangements, will allow many struc- not incorporated into the diterpene skeleton, as tural variants of diterpenoids to be produced. The exemplified by taxol (paclitaxel)∗ (Figure 5.43) toxic principle ‘taxine’ from common yew (Taxus from Pacificyew(Taxus brevifolia)∗. The side- baccata; Taxaceae) has been shown to be a mixture chains in taxol containing aromatic rings are of at least eleven compounds based on the taxadi- derived from shikimate via phenylalanine. Taxol ene skeleton which can be readily rationalized as is an important new anticancer agent, with a broad in Figure 5.43, employing the same mechanistic spectrum of activity against some cancers which principles as seen with mono- and sesqui-terpenes. do not respond to other agents.

Taxus brevifolia and Taxol (Paclitaxel)

A note on nomenclature: the name taxol was given to a diterpene ester with anticancer properties when it was first isolated in 1971 from Taxus brevifolia. When the compound was subsequently exploited commercially as a drug, Taxol was registered as a trademark. Accordingly, the generic name paclitaxel has been assigned to the compound. The literature now contains an unhappy mixture of the two names, though the original name taxol is most often employed.

(Continues) 206 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

The anticancer drug taxol (Figure 5.43) is extracted from the bark of the Pacific yew, Taxus brevifolia (Taxaceae), a slow growing shrub/tree found in the forests of North-West Canada (British Columbia) and the USA (Washington, Oregon, Montana, Idaho, and North California). Although the plant is not rare, it does not form thick populations, and needs to be mature (about 100 years old) to be large enough for exploitation of its bark. The wood of T. brevifolia is not suitable for timber, and in some areas, plants have been systematically destroyed to allow cultivation of faster-growing commercially exploitable conifers. Harvesting is now strictly regulated, but it is realized that this will not provide a satisfactory long term supply of the drug. The bark from about three mature 100-year-old trees is required to provide one gram of taxol, and a course of treatment may need 2 grams of the drug. Current demand for taxol is in the region of 100–200 kg per annum. All parts of Taxus brevifolia contain a wide range of diterpenoid derivatives termed taxanes, which are structurally related to the toxic constituents found in other Taxus species, e.g. the common yew, Taxus baccata. Over a hundred taxanes have been characterized from various Taxus species, and taxol is a member of a small group of compounds possessing a four- membered oxetane ring and a complex ester side-chain in their structures, both of which are essential for antitumour activity. Taxol is found predominantly in the bark of T. brevifolia,butin relatively low amounts (about 0.01–0.02%). Up to 0.033% of taxol has been recorded in some samples of leaves and twigs, but generally the taxol content is much lower than in the bark. The content of some other taxane derivatives in the bark is considerably higher, e.g. up to 0.2% baccatin III (Figure 5.44). Other taxane derivatives characterized include 10-deacetyltaxol, 10- deacetylbaccatin III, cephalomannine and 10-deacetylcephalomannine. A more satisfactory solution currently exploited for the supply of taxol and derivatives for drug use is to produce these compounds by semi-synthesis from more accessible structurally related materials. Both baccatin III and 10-deacetylbaccatin III (Figure 5.44) have been efficiently transformed into taxol. 10-Deacetylbaccatin III is readily extracted from the leaves and twigs of Taxus baccata, and, although the content is variable, it is generally present at much higher levels (up to 0.2%) than taxol can be found in T. brevifolia. Taxus baccata, the common yew, is widely planted as an ornamental tree in Europe and the USA and is much faster growing than the Pacific yew. Cell cultures of T. baccata also offer excellent potential for production of taxol or 10-deacetylbaccatin III but are not yet economic; taxol yields of up to 0.2% dry weight

O OR OR OH 10 O O NH O 10 O O OH OH O NH O OH HO O O H H H OH OH HO HO HO O O O O O O O O O AcO AcO AcO

R = Ac, baccatin III R = Ac, cephalomannine docetaxel R = H, 10-deacetylbaccatin III R = H, 10-deacetylcephalomannine (taxotere)

Figure 5.44

(Continues) DITERPENES 207

(Continued)

cultured cells have been obtained. The use of microorganisms and enzymes to specifically hydrolyse ester groups from the mixture of structurally related taxanes in crude extracts and thus improve the yields of 10-deacetylbaccatin III has also been reported. There is further optimism for new methods of obtaining taxol by microbial culture. Thus, a fungus, Taxomyces adreanae, isolated from the inner bark of Taxus brevifolia appears to have inherited the necessary genes from the tree (or vice versa) and is able to synthesize taxol and other taxanes in culture, though at only very low levels (20–50 ng l –1). A fungus, Pestalotiopsis microspora, recently isolated from the inner bark of the Himalayan yew (Taxus wallachiana) produces higher levels (60–70 µgl–1), and if this could be optimized further it might form the basis for commercial production. Paclitaxel (Taxol) is being used clinically in the treatment of ovarian and breast cancers, non-small-cell lung cancer, small-cell lung cancer, and cancers of the head and neck. Docetaxel (Taxotere) (Figure 5.44) is a side-chain analogue of taxol, which has also been produced by semi-synthesis from 10-deacetylbaccatin III. It has improved water-solubility compared with taxol, and is being used clinically against ovarian and breast cancers. Taxol acts as an antimitotic by binding to microtubules, promoting their assembly from tubulin, and stabilizing them against depolymerization during cell division. The resultant abnormal tubulin–microtubule equilibrium disrupts the normal mitotic spindle apparatus and blocks cell proliferation. Taxol thus has a different mechanism of action to other antimitotics such as vincristine (see page 356) or podophyllotoxin (see page 136), which bind to the protein tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules. Taxol has also been shown to bind to a second target, a protein which normally blocks the process of apoptosis (cell death). Inhibition of this protein allows apoptosis to proceed.

The latex of some plants in the genus Euphorbia shown in Figure 5.45, is then likely to be the (Euphorbiaceae) is toxic, and can cause poisoning precursor of the phorbol ring system. in humans and animals, skin dermatitis, cell pro- In contrast to the cyclization mechanisms shown liferation, and tumour promotion (co-carcinogen in Figures 5.43 and 5.45, where loss of diphos- activity). Many species of Euphorbia are regarded phate generates the initial carbocation, many of as potentially toxic, and the latex can produce the natural diterpenes have arisen by a different severe irritant effects, especially on mucous mem- mechanism. Carbocation formation is initiated by branes and the eye. Most of the biological effects protonation of the double bond at the head of are due to diterpene esters, e.g. esters of phor- the chain leading to a first cyclization sequence. bol (Figure 5.45), which activate protein kinase Loss of the diphosphate later on also produces a C, an important and widely distributed enzyme carbocation and facilitates further cyclization. The responsible for phosphorylating many biochemi- early part of the sequence resembles that involved cal entities. The permanent activation of protein in hopanoid biosynthesis (see page 218), and to kinase C is thought to lead to the uncontrolled some extent triterpenoid and steroid biosynthesis cancerous growth. The most commonly encoun- (see page 214), though in the latter cases open- tered ester of phorbol is 12-O-myristoylphorbol ing of the epoxide ring of the precursor squa- 13-acetate (Figure 5.45). The origins of phorbol lene oxide is responsible for generation of the are not fully delineated, but may be rationalized cationic intermediates. Protonation of GGPP can as in Figure 5.45. Cyclization of GGPP generates initiate a concerted cyclization sequence, termi- a cation containing a 14-membered ring system. nated by loss of a proton from a methyl, yielding Loss of a proton via cyclopropane ring formation copalyl PP (Figure 5.46, a). The stereochemistry leads to casbene, an antifungal metabolite pro- in this product is controlled by the folding of duced by the castor oil plant, Ricinus communis the substrate on the enzyme surface, though an (Euphorbiaceae). Casbene, via the ring closures alternative folding can lead to labdadienyl PP, 208 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

electrophilic addition formation of cyclopropane gives tertiary cation ring with loss of proton

H OPP H

H

GGPP casbene

O proposed ring closures CH (CH ) O HO 3 2 12 OAc OH esterifications O H H H H H H OH OH various OH O OH O oxygenations HO HO 12-O-myristoylphorbol 13-acetate phorbol

Figure 5.45

protonation of double bond gives tertiary carbocation; this allows concerted series of cyclizations terminating in proton loss H H H H H a OPP

OPP OPP ≡ H

H − GGPP H ( )-copalyl PP

OPP OPP H b OPP ≡ H H H H H H H GGPP labdadienyl PP = (+)-copalyl PP

Figure 5.46 the enantiomeric product having opposite con- one, whilst expanding the five-membered ring to figurations at the newly generated chiral centres give a six-membered ring. The driving force is (Figure 5.46, b). From copalyl PP, a sequence of transformation of a secondary carbocation to give cyclizations and a rearrangement, all catalysed by a a tertiary one, but this also results in the methyl single enzyme, leads to ent-kaurene (Figure 5.47). group no longer being at a bridgehead, and what As shown, this involves loss of the diphosphate appears at first glance to be merely a confusing leaving group enabling carbocation-mediated for- change in stereochemistry. Loss of a proton from mation of the third ring system, and subsequent this methyl generates the exocyclic double bond of production of the fourth ring. Then follows a Wag- ent-kaurene and provides an exit from the carbo- ner–Meerwein migration, effectively contracting cationic system. The prefix ent is used to indicate the original six-membered ring to a five-membered enantiomeric; the most common stereochemistry DITERPENES 209

loss of diphosphate generates cyclization of alkene allylic action; further cyclization on to cation produces of alkene on to cation secondary cation ∗ OPP ∇ secondary cation H H H converted into tertiary cation by 1,2-alkyl H H H migration copalyl PP W–M 1,2-alkyl O shift sequential oxidation of loss of proton ∗ methyl to carboxyl generates alkene H

H H H ∇

H H H CO2H ent-kaurene ent-kaurenoic acid O O2 β → NADPH 2 1 OH OGlc Glc

3 x UDPGlc CO2H H H

H H steviol OH CO2H CO2Glc steviol stevioside

Figure 5.47 is that found in labdadienyl PP (Figure 5.46) and migration to modify the side-chain, and further derivatives, so the kaurene series is termed enan- proton loss to form the diene abietadiene. Abietic tiomeric. acid results from sequential oxidation of the 4α- ent-Kaurene is the precursor of stevioside methyl. Wounding of pine trees leads to an accu- (Figure 5.47) in the plant Stevia rebaudiana (Com- mulation at the wound site of both monoterpenes positae/Asteraceae) by relatively simple hydroxy- and diterpenes, which may be fractionated by dis- lation, oxidation, and glucosylation reactions. Both tillation to give turpentine oil and rosin (Table 5.1). glucosyl ester and glucoside linkages are present The volatile monoterpenes seem to act as a sol- in stevioside, which help to confer an intensely vent to allow deposition of the rosin layer to seal sweet taste to this and related compounds. Stevio- the wound. The diterpenes in rosin have both anti- side is present in the plant leaf in quite large fungal and insecticidal properties. amounts (3–10%), is some 100–300 times as Extensive modification of the labdadienyl diter- sweet as sucrose, and is being used commercially pene skeleton is responsible for generation of as a sweetening agent. the ginkgolides, highly oxidized diterpene trilac- The alternative stereochemistry typified by lab- tones which are the active principles of Ginkgo dadienyl PP can be seen in the structure of abi- biloba∗ (Ginkgoaceae). Several rearrangements, etic acid (Figure 5.48), the major component of ring cleavage, and formation of lactone rings can the rosin fraction of turpentine from pines and broadly explain its origin (Figure 5.49), though other conifers (Table 5.1). Initially, the tricyclic this scheme is highly speculative and likely to system is built up as in the pathway to ent-kaurene be incorrect. Although detailed evidence is lack- (Figure 5.47), via the same mechanism, but gener- ing, it is known that labdadienyl PP is a pre- ating the enantiomeric series of compounds. The cursor, and most probably dehydroabietane also. cation loses a proton to give sandaracopimara- The unusual tert-butyl substituent arises as a diene (Figure 5.48), which undergoes a methyl consequence of the A ring cleavage. Bilobalide 210 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

H loss of proton generates alkene OPP H H H H protonation of alkene allows W–M H H H 1,2-methyl shift labdadienyl PPsandaracopimarenyl cation (–)-sandaracopimaradiene

sequential oxidation of methyl to carboxylic acid loss of proton via alcohol and aldehyde generates diene

H H H H H H H CO2H (–)-abietic acid O (–)-abietadiene abietenyl cation

Figure 5.48

see Figure 5.48 PP O – H H H ginkgolides O H H H labdadienyl PP abietenyl cation dehydroabietane

A ring cleavage AB W–M W–M 1,2-alkyl shift 1,2-alkyl shift

further sequence of loss of carbons as indicated; ginkgolides are usually drawn oxidative reactions lactone formation from residual as if viewed from the other side carboxyl and alcohol functions ring closures O O (1 hemiacetal, 3 lactones) O O OH as indicated O O CO2H O OHC O O O HO2C OH O O ≡ O CO2H O OH O O O O O O ginkgolide e.g. bilobalide

Figure 5.49

(Figure 5.49) contains a related C15-skeleton, and some of the degeneration of the faculties normally is most likely a partially degraded ginkgolide. experienced in old age. Ginkgo is the world’s oldest tree species, and In forskolin (Figure 5.51), the third ring is its leaves are now a currently fashionable health heterocyclic rather than carbocyclic. The basic supplement, taken in the hope that it can delay skeleton of forskolin can be viewed as the result DITERPENES 211

Ginkgo biloba

Ginkgo biloba is a primitive member of the gymnosperms and the only survivor of the Ginkgoaceae, all other species being found only as fossils. It is a small tree native to China, but widely planted as an ornamental, and cultivated for drug use in Korea, France, and the United States. Standardized extracts of the leaves are marketed against cerebral vascular disease and senile dementia. Extracts have been shown to improve peripheral and cerebrovascular circulation. The decline in cognitive function and memory processes in old age can be due to disturbances in brain blood circulation, and thus ginkgo may exert beneficial effects by improving this circulation, and assist with other symptoms such as vertigo, tinnitus, and hearing loss. The active constituents have been characterized as mixtures of terpenoids and flavonoids. The dried leaves contain 0.1–0.25% terpene lactones, comprising five ginkgolides (A, B, C, J, and M) and bilobalide (Figure 5.50). Bilobalide comprises about 30–40% of the mixture, whilst ginkgolide A is the predominant ginkgolide (about 30%). The ginkgolides are diterpenoid in nature, whilst bilobalide is described as sesquiterpenoid. However, bilobalide bears such a structural similarity to the ginkgolides, it is most probably a degraded ginkgolide. The ginkgolides have been shown to have potent and selective antagonistic activity towards platelet-activating factor (PAF, see page 39), which is implicated in many physiological processes. The flavonoid content of the dried leaves is 0.5–1.0%, and consists of a mixture of mono-, di-, and tri-glycosides of the flavonols kaempferol and quercetin (Figure 5.50; see also page 151) and some biflavonoids. These probably also contribute to the activity of ginkgo, and may act as radical scavengers. Extracts of ginkgo for drug use are usually standardized to contain flavonoid glycosides and terpene lactones in a ratio of 24% to 6%, or 27% to 7%. Ginkgo may be combined with ginseng (see page 222) in the treatment of geriatric disorders. Ginkgo and the ginkgolides are undergoing extensive investigation in conditions where there are high PAF levels, e.g. shock, burns, ulceration, and inflammatory skin disease.

O O HO O O R2 HO H O O O OH R3 O O R1 O O O O bilobalide R1 R2 R3 OH ginkgolide A OH H H ginkgolide B OH OH H HO O R ginkgolide C OH OH OH OH ginkgolide J OH H OH OH O ginkgolide M HOHOH R = H, kaempferol R = OH, quercetin

Figure 5.50 212 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

quenching of intermediate loss of diphosphate and formation of sequence of oxidation and cation by H O allylic cation; quenching by attack of esterification reactions 2 hydroxyl gives heterocyclic ring O HO OPP OPP O O H OH H OH OH 2 OAc H H H H OH GGPP forskolin

Figure 5.51 of quenching of the cation by water as opposed to shown in Figure 5.52, and provides no novel fea-  proton loss, followed by SN2 nucleophilic substi- tures except for an experimentally demonstrated tution on to the allylic diphosphate (or nucleophilic 1,5-hydride shift. GFPP arises by a continuation of substitution on to the allylic cation generated by the chain extension process, adding a further IPP loss of diphosphate) (Figure 5.51). A series of unit to GGPP. Ophiobolin A shows a broad spec- oxidative modifications will then lead to forskolin. trum of biological activity against bacteria, fungi, This compound has been isolated from Coleus and nematodes. The most common type of marine forskohlii (Labiatae/Lamiaceae), a plant used in sesterterpenoid is exemplified by sclarin and this Indian traditional medicine and shown to have structure can be envisaged as the result of a con- quite pronounced hypotensive and antispasmodic certed cyclization sequence (Figure 5.53) analo- activities. Forskolin∗ is a valuable pharmacologi- gous to that seen with GGPP in the diterpenoids, cal tool as a potent stimulator of adenylate cyclase and with squalene oxide in the triterpenoids (see activity, and it is being investigated for its cardio- below). vascular and bronchospasmolytic effects.

TRITERPENES (C30) SESTERTERPENES (C25) Triterpenes are not formed by an extension of the Although many examples of this group of nat- now familiar process of adding IPP to the grow- ural terpenoids are now known, they are found ing chain. Instead, two molecules of farnesyl PP principally in fungi and marine organisms, and are joined tail to tail to yield the hydrocarbon span relatively few structural types. The ori- squalene (Figure 5.54), originally isolated from gin of ophiobolene and ophiobolin A in the the liver oil of shark (Squalus sp.). Squalene was plant pathogen Helminthosporium maydis from subsequently found in rat liver and yeast, and these cyclization of geranylfarnesyl PP (GFPP)is systems were used to study its biosynthetic role

Forskolin In a screening programme of Indian medicinal plants, extracts from the roots of Coleus forskohlii (Labiatae/Lamiaceae) were discovered to lower blood pressure and have cardioactive properties. This led to the isolation of the diterpene forskolin (= coleonol) (Figure 5.51) as the active principle in yields of about 0.1%. Forskolin has been shown to exert its effects by direct stimulation of adenylate cyclase, and has become a valuable pharmacological tool in the study of this enzyme and its functions. It has shown promising potential for the treatment of glaucoma, congestive heart failure, hypertension, and bronchial asthma, though drug use is limited by poor water-solubility, and derivatives or analogues will need to be developed. TRITERPENES 213

W−M 1,5-hydride shift generates allylic cation; concerted cyclizations cyclization on to this cation follows, and the initiated by allylic cation cation is eventually quenched by water

H H

OPP H O geranylfarnesyl PP 2 (GFPP) O

H H H O H O H OHC H H H O O O H H H H HO HO ophiobolin A ophiobolene (ophiobolin F)

Figure 5.52

OPP HO AcO O H O H

HH

H H GFPP sclarin

Figure 5.53 as a precursor of triterpenes and steroids; several The formation of presqualene PP is represented seed oils are now recognized as quite rich sources in Figure 5.54 as attack of the 2,3-double bond of squalene, e.g. Amaranthus cruentus (Amaran- of FPP on to the farnesyl cation, analogous to the thaceae). During the coupling process, which on chain extension using IPP (see also the proposal for paper merely requires removal of the two diphos- the origins of irregular monoterpenes, page 186). phate groups, a proton from a C-1 position of The resultant tertiary cation is discharged by loss one molecule of FPP is lost, and a proton from of a proton and formation of the cyclopropane ring, NADPH is inserted. Difficulties with formulating giving presqualene PP. Obviously, to form squa- a plausible mechanism for this unlikely reaction lene, carbons-1 of the two FPP units must eventu- were resolved when an intermediate in the pro- ally be coupled, whilst presqualene PP formation cess, presqualene diphosphate, was isolated from has actually joined C-1 of one molecule to C-2 of rat liver. Its characterization as a cyclopropane the other. To account for the subsequent change derivative immediately ruled out all the hypotheses in bonding of the two FPP units, a further cyclo- current at the time. propane cationic intermediate is proposed. Loss of 214 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

FPP squalene 1′ ′ ′ 3 synthase ∗ 2 PPO FPP 1 ∗ electrophilic addition giving allylic cation tertiary cation

OPP ∗ H H ∗ loss of proton with formation of cyclopropane ring

loss of diphosphate gives ∗ PP H O primary cation ∗ H

∗ ∗ H H 1,3-alkyl shift generates presqualene PP W–M new cyclopropane ring 1,3-alkyl and more favourable shift tertiary cation ∗ ∗ H ∗ ∗ H H H cation quenched by bond cleavage produces alkene and (NADPH) attack of hydride favourable allylic cation

H H ∗ ∗

squalene

Figure 5.54 diphosphate from presqualene PP would give the (Figure 5.55). The cyclizations are carbocation unfavourable primary cation, which via Wagner– mediated and proceed in a step-wise sequence Meerwein rearrangement can generate a tertiary (Figure 5.56). Thus, protonation of the epoxide carbocation and achieve the required C-1–C-1 group will allow opening of this ring and gen- bond. Breaking the original but now redundant eration of the preferred tertiary carbocation, suit- C-1–C-2 bond can give an allylic cation, and the ably placed to allow electrophilic addition to a generation of squalene is completed by supply of double bond, formation of a six-membered ring hydride from NADPH. and production of a new tertiary carbocation. This Cyclization of squalene is via the intermedi- process continues twice more, generating the pre- ate squalene-2,3-oxide (Figure 5.55), produced in ferred tertiary carbocation (Markovnikov addition) a reaction catalysed by a flavoprotein requiring after each ring formation, though the third ring O2 and NADPH cofactors. If squalene oxide is formed is consequently a five-membered one. This suitably positioned and folded on the enzyme sur- is expanded to a six-membered ring via a Wag- face, the polycyclic triterpene structures formed ner–Meerwein 1,2-alkyl shift, resulting in some can be rationalized in terms of a series of cycliza- relief of ring strain, though sacrificing a tertiary tions, followed by a sequence of concerted Wag- carbocation for a secondary one. A further elec- ner–Meerwein migrations of methyls and hydrides trophilic addition generates the tertiary protosteryl TRITERPENES 215

squalene

O2 sequence of W–M 1,2-hydride NADPH and 1,2-methyl shifts

H H H H cyclizations H ≡ H H O HO HO H H squalene oxide protosteryl cation loss of proton leads to animals plants fungi cyclopropane

loss of proton gives alkene H H

H

HO HO H H lanosterol cycloartenol

Figure 5.55

protonation of epoxide electrophilic addition electrophilic addition allows ring opening to gives tertiary cation + gives tertiary cation + tertiary cation 6-membered ring 6-membered ring

H HO HO HO O H H squalene oxide H electrophilic addition gives tertiary cation + 5-membered ring electrophilic addition W–M rearrangement; ring H H gives tertiary cation expansion at expense of H tertiary→ secondary cation

H H H HO HO HO H H H protosteryl cation

Figure 5.56 216 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS cation (Figure 5.56). The stereochemistries in this be produced with these conformational charac- cation are controlled by the type of folding teristics. This cation then undergoes a series of achieved on the enzyme surface, and this probably Wagner–Meerwein 1,2-shifts, firstly migrating a also limits the extent of the cyclization pro- hydride and generating a new cation, migrating the cess. Thus, if the folded squalene oxide approx- next hydride, then a methyl and so on until a pro- imates to a chair–boat–chair–boat conformation ton is lost forming a double bond and thus creat- (Figure 5.57), the transient protosteryl cation will ing lanosterol (Figure 5.57). The stereochemistry

H H O HO H H chair – boat – chair – boat H squalene oxide sequence of W–M 1,2-hydride and 1,2-methyl shifts H H 10 9 2 1 HO 3 HO 4 6 7 H 5 8 H H H H protosteryl cation H sequence of W–M 1,2-hydride and 1,2-methyl shifts H H H H H H 2 10 9 1 HO 3 HO H 4 6 7 5 8 H H H H cyclopropane ring protosteryl cation formation and loss of loss of H-9 creates proton from C-10 methyl double bond H H

H

HO H HO H

H H ≡ ≡

H H

H

HO HO H H cycloartenol lanosterol

Figure 5.57 TRITERPENES 217

first sequence of W–M 1,2-hydride and H H H 1,2-methyl shifts H

H further sequence of W–M HO HO H H 1,2-methyl and 1,2-hydride H protosteryl cation shifts, terminated by loss of proton OH various oxidative H OAc modifications O H H H OH HO O H H

O HO cucurbitacin E

Figure 5.58 of the protosteryl cation in Figure 5.57 shows bond (Figure 5.58), as in the pathway to the how favourable this sequence will be, and empha- cucurbitacins, a group of highly oxygenated triter- sizes that in the ring system, the migrating groups penes encountered in the cucumber/melon/marrow are positioned anti to each other, one group family, the Cucurbitaceae. These compounds are entering whilst the other leaves from the oppo- characteristically bitter tasting, purgative, and ex- site side of the stereocentre. This, of course, tremely cytotoxic. inverts configurations at each appropriate centre. Should squalene oxide be folded on to No anti group is available to migrate to C-9 another type of cyclase enzyme, this time in (steroid numbering), and the reaction terminates a roughly chair–chair–chair–boat conformation by loss of proton H-9. Lanosterol is a typical (Figure 5.59), then an identical carbocation animal triterpenoid, and the precursor for choles- mechanism ensues, and the transient dammarenyl terol and other sterols in animals (see page 233) cation formed now has different stereochemical and fungi (see page 254). In plants, its interme- features to the protosteryl cation. Whilst a diate role is taken by cycloartenol (Figure 5.57), series of Wagner–Meerwein migrations can occur, which contains a cyclopropane ring, generated by there is relatively little to be gained on purely inclusion of carbon from the methyl at C-10. chemical grounds, since these would invert For cycloartenol, H-9 is not lost, but migrates to stereochemistry and destroy the already favourable C-8, and the carbocation so formed is quenched conformation. Instead, the dammarenyl cation by cyclopropane formation and loss of one of typically undergoes further carbocation promoted the methyl protons. For many plant steroids, this cyclizations, without any major changes to the cyclopropane ring has then to be reopened (see ring system already formed. There are occasions page 235). Most natural triterpenoids and steroids in which the migrations do occur, however, and contain a 3-hydroxyl group, the original epoxide euphol from Euphorbia species (Euphorbiaceae) oxygen from squalene oxide. is a stereoisomer of lanosterol (Figure 5.55). An additional feature of the protosteryl cation Should the Wagner–Meerwein rearrangements is that the C-10 methyl and H-5 also share not occur, the dammarenyl cation could be quen- an anti-axial relationship, and are also suscep- ched with water, giving the epimeric dammarene- tible to Wagner–Meerwein rearrangements, so diols, as found in Dammar resin from Bal- that the C-9 cation formed in the cycloartenol anocarpus heimii (Dipterocarpaceae) and ginseng∗ sequence may then initiate further migrations. This (Panax ginseng; Araliaceae) (Figure 5.60). Alter- can be terminated by formation of a 5,6-double natively, the migration shown to give the 218 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

O

chair – chair – chair – boat squalene oxide H H

H ≡ H HO H HO H H dammarenyl cation H sequence of W–M 1,2-hydride and 1,2-methyl shifts terminated by loss of proton H

H ≡

HO HO H H H euphol

Figure 5.59 baccharenyl cation relieves some ring strain by taraxasterol found in dandelion (Taraxacum offic- creating a six-membered ring, despite sacrificing inale; Compositae/Asteraceae). Comparison with a tertiary carbocation for a secondary one. A α-amyrin shows the subtly different stereochem- pentacyclic ring system can now be formed by istry present because the inversions of config- cyclization on to the double bond, giving a new uration caused by hydride migrations have not five-membered ring and the tertiary lupenyl cation. occurred. Where evidence is available, these exten- Although this appears to contradict the reason- sive series of cyclizations and Wagner–Meerwein ing used above for the dammarenyl → baccharenyl rearrangements appear to be catalysed by a sin- transformation, the contribution of the enzyme gle enzyme, which converts squalene oxide into involved must also be considered in each case. A the final product, e.g. lanosterol, cycloartenol, α- five-membered ring is not highly strained as evi- amyrin, or β-amyrin. denced by all the natural examples encountered. Bacterial membranes frequently contain hopa- Loss of a proton from the lupenyl cation gives noids (Figure 5.61), triterpenoid compounds that lupeol, found in lupin (Lupinus luteus; Legumi- appear to take the place of the sterols that are typ- nosae/Fabaceae). Ring expansion in the lupenyl ically found in the membranes of higher organisms, cation by bond migration gives the oleanyl sys- helping to maintain the structural integrity and to tem, and labelling studies have demonstrated this control permeability. Hopanoids arise from squa- ion is discharged by hydride migrations and loss of lene by a similar carbocation cyclization mecha- a proton, giving the widely distributed β-amyrin. nism, but do not involve the initial epoxidation Formation of the isomeric α-amyrin involves to squalene oxide. Instead, the carbocation is pro- first the migration of a methyl in the oleanyl duced by protonation (compare the cyclization of cation, then discharge of the new taraxasteryl GGPP to labdadienyl PP, page 207), and the resul- cation by three hydride migrations and loss of a tant compounds tend to lack the characteristic proton. Loss of a proton from the non-migrated 3-hydroxyl group, e.g. hopan-22-ol from Alicy- methyl in the taraxasteryl cation is an alternative clobacillus acidocaldarius (Figure 5.61). On the way of achieving a neutral molecule, and yields other hand, tetrahymanol from the protozoan TRITERPENES 219

ring expansion at expense of cation quenched by H2O; attack may tertiary → secondary cation occur from either side of cation OH H H

H H2O H H H HO HO H H dammarenyl cation dammarenediols W–M ring expansion at expense of 1,2-alkyl 5-membered ring tertiary → secondary cation shift formation gives tertiary cation HHH

H – H H

H H H HO HO HO H H H baccharenyl cation lupenyl cation lupeol

W–M 1,2-methyl shift allows three 1,2-hydride shifts, then loss two 1,2-hydride shifts → 1,2-alkyl secondary tertiary of proton; note inversions of then loss of proton; shift cation stereochemistry note inversion of H stereochemistry H H H H H W–M H H 1,2-methyl H ≡ H shift H H H H HO HO HO H H H oleanyl cation oleanyl cation taraxasteryl cation W–M – H W–M 1,2-hydride 1,2-hydride shifts shifts

H

H H H

H H H HO HO HO H H H β-amyrin taraxasterol α-amyrin

Figure 5.60

Tetrahymena pyriformis, because of its symme- (Markovnikov addition) becomes a secondary try, might appear to have a 3-hydroxyl group, carbocation/six-membered ring. but this is derived from water, and not molec- ular oxygen as would be the case if squalene Triterpenoid Saponins oxide were involved. As in formation of the proto- steryl cation (page 214), Wagner–Meerwein ring The pentacyclic triterpenoid skeletons exemplified expansions occur during the cyclization mech- by lupeol, α-amyrin, and β-amyrin (Figure 5.60) anisms shown in Figure 5.61 so that the first- are frequently encountered in the form of formed tertiary carbocation/five-membered ring triterpenoid saponin structures. Saponins are 220 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

CD

squalene H further carbocation-mediated cyclizations: cyclization is initiated by protonation of rings C and D are initially formed double bond to give tertiary cation 5-membered via Markovnikov addition; they are then expanded to 6-membered rings

via W–M rearrangements OH2

OH2

OH H H H H H H OH H ≡ H H H HO H H H hopan-22-ol tetrahymanol

Figure 5.61 glycosides which, even at low concentrations, families. Medicinally useful examples are mainly produce a frothing in aqueous solution, because based on the β-amyrin subgroup (Figure 5.62), they have surfactant and soaplike properties. The and many of these possess carboxylic acid groups name comes from the Latin sapo, soap, and plant derived by oxidation of methyl groups, those at materials containing saponins were originally used positions 4 (C-23), 17 (C-28), and 20 (C-30) on for cleansing clothes, e.g. soapwort (Saponaria the aglycone ring system being subject to such oxi- officinalis; Caryophyllaceae) and quillaia or dation. In some structures, less oxidized formyl soapbark (Quillaja saponaria; Rosaceae). These (−CHO) or hydroxymethyl (−CH2OH) groups materials also cause haemolysis, lysing red blood may also be encountered. Positions 11 and 16 cells by increasing the permeability of the plasma may also be oxygenated. Sugar residues are usu- membrane, and thus they are highly toxic when ally attached to the 3-hydroxyl, with one to six injected into the blood stream. Some saponin- monosaccharide units, the most common being containing plant extracts have been used as arrow glucose, galactose, rhamnose, and arabinose, with poisons. However, saponins are relatively harmless uronic acid units (glucuronic acid and galacturonic when taken orally, and some of our valuable food acid) also featuring (see page 467). Thus, quillaia∗ materials, e.g. beans, lentils, soybeans, spinach, bark contains a saponin mixture with quillaic and oats, contain significant amounts. Sarsaparilla acid (Figure 5.62) as the principal aglycone, and (see page 242) is rich in steroidal saponins but the medicinally valuable root of liquorice∗ (Gly- is widely used in the manufacture of non- cyrrhiza glabra; Leguminosae/Fabaceae) contains alcoholic drinks. Toxicity is minimized during glycyrrhizin, a mixture of potassium and cal- ingestion by low absorption, and by hydrolysis. cium salts of glycyrrhizic acid (Figure 5.63), Acid-catalysed hydrolysis of saponins liberates which is composed of the aglycone glycyrrhetic sugar(s) and an aglycone (sapogenin), which can acid and two glucuronic acid units. Seeds of be either triterpenoid or steroidal (see page 237) horsechestnut (Aesculus hippocastrum; Hippocas- in nature. Some plants may contain exceptionally tanaceae), sometimes used in herbal prepara- high amounts of saponins, e.g. about 10% in tions as an anti-inflammatory and anti-bruising quillaia bark. remedy, contain a complex mixture of saponins Triterpenoid saponins are rare in monocotyle- termed aescin, based on the polyhydroxylated dons, but abundant in many dicotyledonous aglycones protoaescigenin and barringtogenol TRITERPENES 221

29 30 O 20 19 21 E 12 18 O 13 17 22 11 25 26 H O 1 9 C 14 D 28 2 16 A 10 B 8 15 H 5 27 O 3 7 4 HO 6 H 24 23 O pentacyclic triterpenoid skeleton potential sites for oxidation (β-amyrin type)

OH

OH HCO2H H OH OH OH H H HO HO H H R CHO R = OH, protoaescigenin quillaic acid R = H, barringtogenol

Figure 5.62

CO2H CO2

O O D-glucuronic acid H H

CO2H O HO O H H 2Na HO O O2C O H H CO2H HO O carbenoxolone sodium HO O OH OH OH D-glucuronic acid glycyrrhetic acid GlcO O GlcO OH glycyrrhizic acid

O O liquiritin isoliquiritin

Figure 5.63

Liquorice

Liquorice (licorice; glycyrrhiza) is the dried unpeeled rhizome and root of the perennial herb Glycyrrhiza glabra (Leguminosae/Fabaceae). A number of different varieties are cultivated commercially, including G. glabra var. typica (Spanish liquorice) in Spain, Italy, and France, and G. glabra var. glandulifera (Russian liquorice) in Russia. Russian liquorice is usually peeled

(Continues) 222 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

before drying. Glycyrrhiza uralensis (Manchurian liquorice) from China is also commercially important. Much of the liquorice is imported in the form of an extract, prepared by extraction with water, then evaporation to give a dark black solid. Most of the liquorice produced is used in confectionery and for flavouring, including tobacco, beers, and stouts. Its pleasant sweet taste and foaming properties are due to saponins. Liquorice root contains about 20% of water soluble extractives, and much of this (typically 3–5% of the root, but up to 12% in some varieties) is comprised of glycyrrhizin, a mixture of the potassium and calcium salts of glycyrrhizic (glycyrrhizinic) acid (Figure 5.63). Glycyrrhizic acid is a diglucuronide of the aglycone glycyrrhetic (glycyrrhetinic) acid. The bright yellow colour of liquorice root is provided by flavonoids (1–1.5%) including liquiritin and isoliquiritin (Figure 5.63), and their corresponding aglycones (see page 150). Considerable amounts (5–15%) of sugars (glucose and sucrose) are also present. Glycyrrhizin is reported to be 50–150 times as sweet as sucrose, and liquorice has thus long been used in pharmacy to mask the taste of bitter drugs. Its surfactant properties have also been exploited in various formulations, as have its demulcent and mild expectorant properties. More recently, some corticosteroid-like activity has been recognized, liquorice extracts displaying mild anti-inflammatory and mineralocorticoid activities. These have been exploited in the treatment of rheumatoid arthritis, Addison’s disease (chronic adrenocortical insufficiency), and various inflammatory conditions. Glycyrrhetic acid has been implicated in these activities, and has been found to inhibit enzymes that catalyse the conversion of prostaglandins and glucocorticoids into inactive metabolites. This results in increased levels of prostaglandins, e.g. PGE2 and PGF2α (see page 54), and of hydrocortisone (see page 268). Perhaps the most important current application is to give systematic relief from peptic ulcers by promoting healing through increased prostaglandin activity. A semi-synthetic derivative of glycyrrhetic acid, the hemisuccinate carbenoxolone sodium (Figure 5.63), is widely prescribed for the treatment of gastric ulcers, and also duodenal ulcers. The mineralocorticoid effects (sodium and water retention) may exacerbate hypertension and cardiac problems. Surprisingly, a deglycyrrhizinized liquorice preparation is also marketed for treatment of peptic ulcers, but its efficiency has been questioned.

Quillaia

Quillaia bark or soapbark is derived from the tree Quillaja saponaria (Rosaceae) and other Quillaja species found in Chile, Peru, and Bolivia. The bark contains up to 10% saponins, a mixture known as ‘commercial saponin’, which is used for its detergent properties. Quillaia’s surfactant properties are occasionally exploited in pharmaceutical preparations where it is used in the form of quillaia tincture as an emulsifying agent, particularly for fats, tars, and volatile oils. The bark contains a mixture of saponins which on hydrolysis liberates quillaic acid (Figure 5.62) as the aglycone, together with sugars, uronic acids, and acids from ester functions.

Ginseng

The roots of the herbaceous plants Panax ginseng (Araliaceae) from China, Korea, and Russia, and related Panax species, e.g. P. quinquefolium (American ginseng) from the USA

(Continues) TRITERPENES 223

(Continued) and Canada, and P. notoginseng (Sanchi-ginseng) from China, have been widely used in China and Russia for the treatment of a number of diseases including anaemia, diabetes, gastritis, insomnia, sexual impotence, and as a general restorative. Interest in the drug has increased considerably in recent years and ginseng is widely available as a health food in the form of powders, extracts, and teas. The dried and usually peeled root provides white ginseng, whereas red ginseng is obtained by steaming the root, this process generating a reddish- brown caramel-like colour, and reputedly enhancing biological activity. Ginseng is classified as an ‘adaptogen’, helping the body to adapt to stress, improving stamina and concentration, and providing a normalizing and restorative effect. It is also widely promoted as an aphrodisiac. The Korean root is highly prized and the most expensive. Long term use of ginseng can lead to symptoms similar to those of corticosteroid poisoning, including hypertension, nervousness, and sleeplessness in some subjects, yet hypotension and tranquillizing effects in others. The benefits of ginseng treatment are by no means confirmed at the pharmacological level, though CNS-stimulating, CNS-sedative, tranquillizing, antifatigue, hypotensive, and hypertensive activities have all been demonstrated. Many of the secondary metabolites present in the root have now been identified. It contains a large number of triterpenoid saponins based on the dammarane subgroup, saponins that have been termed ginsenosides by Japanese investigators, or panaxosides by Russian researchers. These are derivatives of two main aglycones, protopanaxadiol and protopanaxatriol (Figure 5.64), though the aglycones liberated on acid hydrolysis are panaxadiol and panaxatriol respectively. Acid- catalysed cyclization in the side-chain produces an ether ring (Figure 5.64). Sugars are present in the saponins on the 3- and 20-hydroxyls in the diol series, and the 6- and 20-hydroxyls in the triol series. About 30 ginsenosides have been characterized from the different varieties of ginseng, with ginsenoside Rb-1 (Figure 5.64) of the diol series typically being the most abundant constituent. Ginsenoside Rg-1 (Figure 5.64) is usually the major component representative of the triol series. Other variants are shown in Figure 5.64. Particularly in white ginseng, many of the ginsenosides are also present as esters with malonic acid. Steaming to prepare red ginseng causes partial hydrolysis of esters and glycosides. Ginsenosides Rb-1 and Rg-1 appear to be the main representatives in Panax ginseng, ginsenosides Rb-1, Rg-1,andRd in P. notoginseng, and ginsenosides Rb-1, Re,and malonylated Rb-1 in P. quinquefolium. The pentacyclic triterpenoid sapogenin oleanolic acid (Figure 5.65) is also produced by hydrolysis of the total saponins of P. ginseng, and is present in some saponin structures (chikusetsusaponins). The saponin contents of Panax notoginseng (about 12%) and P. quinquefolium (about 6%) are generally higher than that of P. ginseng (1.5–2%). The root of Eleutherococcus senticosus (Acanthopanax senticosus) (Araliaceae) is used as an inexpensive substitute for ginseng, and is known as Russian or Siberian ginseng. This material is held to have similar adaptogenic properties as Panax ginseng and a number of eleutherosides have been isolated. However, the term eleutheroside has been applied to compounds of different chemical classes, and the main active anti-stress constituents appear to be lignan glycosides, e.g. eleutheroside E (≡ syringaresinol diglucoside) (Figure 5.65) (see page 132) and phenylpropane glycosides, e.g. eleutheroside B (≡ syringin). The leaves of Russian ginseng contain a number of saponins based on oleanolic acid, but these are quite different to the ginsenosides/panaxosides found in Panax. Whilst there is sufficient evidence to support the beneficial adaptogen properties for Eleutherococcus senticosus, detailed pharmacological confirmation is not available.

(Continues) 224 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

protonation of double bond gives tertiary cation and allows formation 2 of ether by attack of hydroxyl OGly OH OH HO 20 HO HO H H H H H H

H H 3 H H H 1 Gly O HO HO H H H ginsenoside protopanaxadiol (panaxoside) Gly1 Gly2 both 20R- and 20S-panaxadiol are O β1→2 β1→6 HO 20R formed, with the 20R-epimer H ginsenoside R Glc–Glc––Glc–Glc b-1 predominating; since both isomers H β1→2 β1→6 are produced, this implies an ginsenoside R Glc–Glc––Ara–Glc b-2 equilibration, probably through loss (pyranose) H β1→2 β1→6 of H2O and generation of a HO ginsenoside Rc Glc–Glc––Ara–Glc carbocation at C-20 in H (furanose) protopanaxadiol panaxadiol β1→2

ginsenoside Rd Glc–Glc– Glc

OGly2 OH O HO HO HO 20R H 20 H H H H H

H H H H H

HO 6 HO HO H H H OGly1 OH OH ginsenoside protopanaxatriol panaxatriol (panaxoside) as mixture of 20R and 20S epimers; Gly1 Gly2 see notes on panaxadiol above β1→2

ginsenoside Re Rha–Glc– Glc β1→2

ginsenoside Rf Glc–Glc– H

ginsenoside Rg-1 Glc Glc β1→2

ginsenoside Rg-2 Rha–Glc– H

Figure 5.64

OMe OMe GlcO GlcO O MeO HCO2H HH MeO OMe H O HO OH H OGlc syringin oleanolic acid syringaresinol diglucoside OMe (eleutheroside B) (eleutheroside E)

Figure 5.65 TRITERPENES 225

(Figure 5.62). Several of these hydroxyls are ester- the steroids, and these are so important that ified with aliphatic acids, e.g. acetic, tiglic, and they are considered separately. Other degraded angelic acids. triterpenoids include the limonoids (tetranortriter- penoids), in which four terminal carbons from the side-chain are removed, and the quassinoids, Modified Triterpenoids which have lost ten carbons, including one of The triterpenoid skeletons may be subjected to the C-4 methyls. The quassinoids thus have a a variety of structural modifications as already C20 skeleton which could be misinterpreted as illustrated. However, the particular modifications a diterpene structure. Biosynthetic information is considered in this section are those that lead relatively sparse, but the relationship to precur- to loss of several skeletal carbon atoms. Pre- sors of the euphol type is outlined in Figure 5.66. eminent amongst such degraded triterpenoids are Limonoids are found mainly in plants of the

H H H allylic 20 H isomerization H H

O

H H HO O H H HO HO H H H opening of epoxide ring euphol (20R) butyrospermol generates tertiary cation tirucallol (20S) and initiates 1,2-shift; − W M loss of proton gives 1,2-methyl alkene shift H O H

– 4C limonoids H (tetranortriterpenoids) H HO OH cleavage of 4 carbons from H HO OH H side-chain and formation of furan ring as indicated apoeuphol (20R) apotirucallol (20S) via cleavage of ring C – 10C (including one from C-4) HO O CO2Me O O OH cleavage of 8 carbon side-chain H O O and one from ring D; formation of H OH O lactone ring as indicated quassinoids AcO OH H OMe MeO2C O O O azadirachtin MeO H H O O H

quassin

Figure 5.66 226 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS families Rutaceae, Meliaceae, and Simaroubaceae. plants and fungi, an additional isomerization step is Azadirachtin (Figure 5.66) is probably one of the involved to change the configuration of the central most complex limonoid structures to be encoun- double bond. tered, but is currently of considerable interest. This The extended π-electron system confers colour material has potent insect antifeedant properties to the carotenoids, and accordingly they contribute and is extracted commercially from seeds of the yellow, orange, and red pigmentations to plant Neem tree (Azadirachta indica; Meliaceae) for use tissues. Lycopene is the characteristic carotenoid as an agricultural pesticide to prevent insect dam- pigment in ripe tomato fruit (Lycopersicon escu- age to crops. It is a relatively inexpensive and lente; Solanaceae). The orange colour of carrots ecologically sound pesticide. Quassinoids are pro- (Daucus carota; Umbelliferae/Apiaceae) is caused duced by many plants in the Simaroubaceae fam- by β-carotene (Figure 5.68), though this com- ily, in particular Quassia. Quassin (Figure 5.66) pound is widespread in higher plants. β-Carotene from Q. amara (quassia wood) is a typical and other natural carotenoids (Figure 5.68) are example. They have attracted considerable study widely employed as colouring agents for foods, because of their cytotoxic, antimalarial, and amoe- drinks, confectionery, and drugs. β-Carotene dis- bicidal properties. plays additional cyclization of the chain ends, which can be rationalized by the carbocation mechanism shown in Figure 5.69. Depending on TETRATERPENES (C40) which proton is lost from the cyclized cation, three different cyclic alkene systems can arise The tetraterpenes are represented by only one at the end of the chain, described as β-, γ-, or group of compounds, the carotenoids, though sev- ε-ring systems. α-Carotene (Figure 5.68) has a eral hundred natural structural variants are known. β-ring at one end of the chain, and an ε-type These compounds play a role in photosynthe- at the other, and is representative of carotenoids sis, but they are also found in non-photosynthetic lacking symmetry. γ-Carotene (a precursor of plant tissues, in fungi and bacteria. Formation of β-carotene) and δ-carotene (a precursor of α- the tetraterpene skeleton, e.g. phytoene, involves carotene) illustrate carotenoids where only one tail-to-tail coupling of two molecules of ger- end of the chain has become cyclized. Oxy- anylgeranyl diphosphate (GGPP) in a sequence genated carotenoids (termed xanthophylls) are also essentially analogous to that seen for squalene widely distributed, and the biosynthetic origins and triterpenes (Figure 5.67). A cyclopropyl com- of the oxygenated rings found in some of these, pound, prephytoene diphosphate (compare pres- such as zeathanthin, lutein,andviolaxanthin qualene diphosphate, page 213) is an intermediate (Figure 5.68), all common green leaf carotenoids, in the sequence, and the main difference between are shown in Figure 5.69. The epoxide grouping the tetraterpene and triterpene pathways is how in violaxanthin allows further chemical modifi- the resultant allylic cation is discharged. For squa- cations, such as ring contraction to a cyclopen- lene formation, the allylic cation accepts a hydride tane, exemplified by capsanthin (Figure 5.68), ion from NADPH, but for phytoene biosynthesis, the brilliant red pigment of peppers (Capsicum a proton is lost, generating a double bond in the annuum; Solanaceae), or formation of an allene centre of the molecule, and thus a short conjugated as in fucoxanthin, an abundant carotenoid in chain is developed. In plants and fungi, this new brown algae (Fucus species; Fucaceae). Astaxan- double bond has the Z (cis)configuration, whilst in thin (Figure 5.68) is commonly found in marine bacteria, it is E (trans). This triene system prevents animals and is responsible for the pink/red col- the type of cyclization seen with squalene. Conju- oration of crustaceans, shellfish, and fish such gation is extended then by a sequence of desat- as salmon. These animals are unable to synthe- uration reactions, removing pairs of hydrogens size carotenoids and astaxanthin is produced by alternately from each side of the triene system, modification of plant carotenoids, e.g. β-carotene, giving eventually lycopene (Figure 5.67), which, obtained in the diet. in common with the majority of carotenoids, has Carotenoids function along with chlorophylls the all-trans configuration. This means that in in photosynthesis as accessory light-harvesting TETRATERPENES 227

GGPP

PPO GGPP

electrophilic addition giving allylic cation tertiary cation OPP loss of proton with H H formation of cyclopropane ring

loss of diphosphate H OPP gives primary cation H

H H prephytoene PP W–M 1,3-alkyl shift generates new cyclopropane ring and more favourable H H tertiary cation apart from the final proton H loss, this sequence is exactly analogous to that seen with H presqualene PP (Figure 5.54) H loss of proton bond cleavage produces alkene generates alkene and favourable allylic cation

Z

Z-phytoene sequence of desaturation reactions; in plants and fungi, the central double bond is also isomerized Z → E E

lycopene

Figure 5.67 pigments, effectively extending the range of light and affording protection against some forms of absorbed by the photosynthetic apparatus. They cancer. The most significant dietary carotenoid also serve as important protectants for plants and in this respect is lycopene, with tomatoes and algae against photo-oxidative damage, quenching processed tomato products featuring as the pre- toxic oxygen species. Some herbicides (bleaching dominant source. The extended conjugated system herbicides) act by inhibiting carotenoid biosynthe- allows free radical addition reactions and hydrogen sis, and the unprotected plant is subsequently killed abstraction from positions allylic to this chain. by photo-oxidation. Recent research also suggests The A group of vitamins∗ are important carotenoids are important antioxidant molecules in metabolites of carotenoids. Vitamin A1 (retinol) humans, quenching singlet oxygen and scavenging (Figure 5.70) effectively has a diterpene structure, peroxyl radicals, thus minimizing cell damage but it is derived in mammals by oxidative 228 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

β-carotene

α-carotene OH

HO zeathanthin OH O

O HO violaxanthin OH

HO lutein O OH

HO astaxanthin O OH

O

capsanthin OH

O HO fucoxanthin OAc OH

γ-carotene

δ-carotene

Figure 5.68 TETRATERPENES 229

H H a b H protonation of double bond c H gives tertiary cation; then a electrophilic addition b c

β-ring γ-ring ε-ring e.g. β-carotene e.g. α-carotene

O2 O2 NADPH NADPH H O2 NADPH O H HO HO HO e.g. zeathanthin e.g. lutein opening of epoxide ring and loss of proton O2 NADPH pinacol-like rearrangement generates allene generating ketone O opening of epoxide ring

O OH H HO HO HO OH OH e.g. fucoxanthin e.g. violaxanthin e.g. capsanthin

Figure 5.69

a b

β-carotene excentric cleavage excentric central cleavage generates O2 central can generate one O2 cleavage b two molecules of retinal cleavage a molecule of retinal oxidative chain shortening O O

retinal

NADH desaturation extending conjugation

OH OH

retinol dehydroretinol (vitamin A1) (vitamin A2)

Figure 5.70 230 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

metabolism of a tetraterpenoid, mainly β-carotene, taken in the diet. Cleavage occurs in the HO2C mucosal cells of the intestine, and is catalysed by an O2-dependent dioxygenase, probably via bixin an intermediate peroxide. This can theoretically MeO2C yield two molecules of the intermediate aldehyde, CO2H retinal, which is subsequently reduced to the HO2C alcohol, retinol (Figure 5.70). Although β-carotene crocetin cleaved at the central double bond is capable of giving rise to two molecules of retinol, there Figure 5.71 is evidence that cleavage can also occur at other double bonds, so-called excentric cleavage oil and halibut-liver oil are rich sources used as (Figure 5.70). Further chain shortening then dietary supplements. However, carotenoid sources produces retinal, but only one molecule is are equally important. These need to have at least produced per molecule of β-carotene. Vitamin A2 one non-hydroxylated ring system of the β-type, (dehydroretinol) (Figure 5.70) is an analogue of e.g. β-carotene, α-carotene, and γ-carotene. retinol containing a cyclohexadiene ring system; Cleavage of carotenoid precursors is likely the corresponding aldehyde, and retinal, are also to explain the formation of bixin and crocetin included in the A group of vitamins. Retinol and its (Figure 5.71) and, indeed, these are classified as derivatives are found only in animal products, and apocarotenoids. Large amounts (up to 10%) of these provide some of our dietary needs. Cod-liver the red pigment bixin are found in the seed

Vitamin A

Vitamin A1 (retinol)andvitamin A2 (dehydroretinol) (Figure 5.72) are fat-soluble vitamins found only in animal products, especially eggs, dairy products, and animal livers and kidneys. Fish liver oils, e.g. cod liver oil and halibut liver oil (see Table 3.2) are particularly rich sources. They exist as the free alcohols, or as esters with acetic and palmitic acid. Vitamin A2 has about 40% of the activity of vitamin A1. Carotenoid precursors (provitamins) are widely distributed in plants, and after ingestion, these are subsequently transformed into vitamin A in the liver. Green vegetables and rich plant sources such as carrots help to provide adequate levels. A deficiency of vitamin A leads to vision defects, including impairment at low light levels (night blindness) and a drying and degenerative disease of the cornea. It also is necessary for normal growth of young animals. Retinoids (vitamin A and analogues) are now known to act as signalling molecules which regulate diverse aspects of cell differentiation, embryonic development, growth, and vision. For the processes of vision, retinol needs to be converted first by oxidation into the aldehyde all-trans-retinal, and then by enzymic isomerization to cis-retinal (Figure 5.73). cis-Retinal is then bound to the protein opsin in the retina via a Schiff base linkage to give the red visual pigment rhodopsin, and its sensitivity to light involves isomerization of the cis-retinal portion back to the all-trans form, thus translating the light energy into molecular change, which triggers a nerve impulse to the brain. The absorption of light energy promotes an electron from a π-toaπ∗-orbital, thus temporarily destroying the double bond character and allowing rotation. A similar cis–trans isomerization affecting cinnamic acids was discussed under coumarins (see page 142). All-trans-retinal is then subsequently released for the process to continue. Vitamin A is relatively unstable, and sensitive to oxidation and light. Antioxidant stabilizers such as vitamin E and vitamin C are sometimes added. It is more stable in oils such as the fish liver oils, which are thus good vehicles for administering the vitamin. Synthetic material is also used. Excessive intake of

(Continues) HIGHER TERPENOIDS 231

(Continued)

OH OH

retinol dehydroretinol (vitamin A1) (vitamin A2)

CO2H CO2H

CO2H tretinoin isotretinoin MeO acitretin (retinoic acid) (13-cis-retinoic acid)

Figure 5.72

OH

retinol

NADP+ hydrolytic cleavage of H Schiff base H 11 E opsin O N H retinal enzymic trans–cis absorption of light energy isomerism of hν restores trans configuration of formation of Schiff base 11,12-double bond 11,12-double bond (imine) with amino group on protein 11 Z

H2N opsin

opsin 11-cis-retinal H O rhodopsin H N H

Figure 5.73

vitamin A can lead to toxic effects, including pathological changes in the skin, hair loss, blurred vision, and headaches. The synthetic retinoic acids tretinoin (retinoic acid) and isotretinoin (13-cis-retinoic acid) (Figure 5.72) are retinoids that are used as topical or oral treatments for acne vulgaris, reducing levels of dehydroretinol and modifying skin keratinization. Dehydroretinol levels in the skin become markedly elevated in conditions such as eczema and psoriasis. Acitretin (Figure 5.72) is an aromatic analogue which can give relief in severe cases of psoriasis. All these materials can produce toxic side-effects.

coats of annatto (Bixa orellana; Bixaceae), and HIGHER TERPENOIDS bixin is widely used as a natural food colorant. Crocetin, in the form of esters with gentiobiose Terpenoid fragments containing several iso- [D-Glc(β1 → 6)D-Glc], is the major pigment in prene units are found as alkyl substituents in stigmas of Crocus sativus (Iridaceae), which com- shikimate-derived quinones (see page 158). Thus prise the spice saffron. ubiquinones typically have C40−C50 side-chains, 232 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

modifications, especially to the side-chain, help to create a wide range of biologically important natural products, e.g. sterols, steroidal saponins, n n cardioactive glycosides, bile acids, corticosteroids, rubber gutta and mammalian sex hormones. Because of the 3 5 ≈ 2 4 n ≈ 10 –10 n 10 –10 profound biological activities encountered, many Figure 5.74 natural steroids together with a considerable num- ber of synthetic and semi-synthetic steroidal com- pounds are routinely employed in medicine. The plastoquinones usually C45, and menaquinones markedly different biological activities observed up to C65. The alkylating agents are polyprenyl emanating from compounds containing a common diphosphates, formed simply by an extension of the structural skeleton are in part ascribed to the func- prenyltransferase reaction (see page 172), repeat- tional groups attached to the steroid nucleus, and in edly adding IPP residues. Even longer polyiso- part to the overall shape conferred on this nucleus prene chains are encountered in some natural by the stereochemistry of ring fusions. polymers, especially rubber and gutta percha. Rub- Ring systems containing six-membered or five- ber (Figure 5.74), from the rubber tree Hevea membered rings can be trans-fused as exempli- brasiliensis (Euphorbiaceae), is unusual in pos- fied by trans-decalin or cis-fused as in cis-decalin sessing an extended array of cis (Z) double bonds (Figure 5.76). The trans-fusion produces a flat- rather than the normal trans configuration. Gutta tish molecule when two chair conformations are percha, from Palaquium gutta (Sapotaceae) on the present. The only conformational mobility allowed other hand, has trans (E) double bonds. The cis is to less favourable boat forms. Both bridgehead double bonds in rubber are known to arise by loss hydrogens (or other substituents) are axial to both of the pro-S proton (H ) from C-2 of IPP (con- S of the rings. In contrast, the cis-fused decalin is trast loss of H , which gives a trans double bond) R basically a bent molecule, and is found to be flexi- (Figure 5.75). However, a small (up to C ) trans- 20 ble in that alternative conformers are possible, both allylic diphosphate initiator is actually used for rings still being in chair form. However, this flex- the beginning of the chain before the extended cis ibility will be lost if either ring is then trans-fused chain is elaborated. to a third ring. Bridgehead substituents are axial to one ring, whilst being equatorial to the other, in STEROIDS each conformer. In natural steroids, there are examples of the Stereochemistry A/B ring fusion being trans or cis, or having unsat- uration, either ∆4 or ∆5. In some compounds, The steroids are modified triterpenoids con- notably the oestrogens, ring A can even be aro- taining the tetracyclic ring system of lanos- matic; clearly there can then be no bridgehead terol (Figure 5.55), but lacking the three methyl substituent at C-10 and the normal C-10 methyl groups at C-4 and C-14. Cholesterol (Figure 5.76) (C-19) must therefore be lost. All natural steroids typifies the fundamental structure, but further have a trans B/C fusion, though cis forms can be

loss of HR gives – H R R E trans double bond R OPP OPP R OPP HR HS electrophilic HR HS addition – HS Z loss of H gives cis R S double bond

OPP

Figure 5.75 STEROIDS 233

21 22 24 20 23 25 26 18 H 12 17 19 11 27 13 16 H 1 9 C 14 D 2 10 15 H H A B 8 H 3 HO 5 7 trans-decalin 4 6 H cholesterol steroid numbering H H H H C D A B H H H cis-decalin all-trans

H H H D D C C C D B B H A H A H A B H H H H H

A/B cis A/B cis, C/D cis A ring aromatic

H H

C D C D C D A B A B A B H H H H H H ∆5-unsaturation ∆4-unsaturation ∆5,7-unsaturation

Figure 5.76 made synthetically. The C/D fusion is also usually to customary conventions as in Figure 5.77), or β trans, though there are notable exceptions such as (on the upper face). Ring fusions may be desig- the cardioactive glycosides. Of course, such com- natedbyusingα or β for the appropriate bridge- ments apply equally to some of the triterpenoid head hydrogen, particularly those at positions 5 and structures already considered. However, it is in the 14, which will define the A/B and C/D fusions steroid field that the relationship between stereo- respectively, e.g. 5β-cholestane has the A/B rings chemistry and biological activity is most marked. cis-fused. Since the parent hydrocarbon assumes The overall shapes of some typical steroid skele- that ring fusions are trans, the stereochemistry tons are shown in Figure 5.76. for ring fusions is usually only specified where Systematic steroid nomenclature is based it is cis. Cholesterol is thus cholest-5-en-3β-ol or on a series of parent hydrocarbons, includ- 5-cholesten-3β-ol. The term nor is affixed to indi- ing estrane, androstane, pregnane, cholane, cate loss of a carbon atom, e.g. 19-norsteroids (see cholestane, ergostane, campestane, stigmastane page 274) lack C-19 (the methyl at C-10). and poriferastane (Figure 5.77). The triterpenoid hydrocarbons lanostane and cycloartane are sim- ilarly used in systematic nomenclature and are Cholesterol also included in Figure 5.77. It is usual to add only unsaturation (ene/yne) and the highest prior- In animals, the triterpenoid alcohol lanosterol ity functional group as suffixes to the root name; (Figure 5.55) is converted into cholesterol∗ other groups are added as prefixes. Stereochemistry (Figure 5.76), a process requiring, as well as the of substituents is represented by α (on the lower loss of three methyl groups, reduction of the face of the molecule when it is drawn according side-chain double bond, and generation of a ∆5,6 234 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

H H H H H

H H H H H H H H

H H H H estrane androstane pregnane cholane

1 24 241

24 24

H H H

H H H H H H

H H H cholestane ergostane campestane

242 242 241 241

24 24

H H H H

H H H H H 30 30 H HHH 29 28 29 28 stigmastane poriferastane lanostane cycloartane

Figure 5.77 double bond in place of the ∆8,9 double bond. mechanism (Figure 5.79). This is facilitated by The sequence of these steps is to some extent oxidizing the 3-hydroxyl to a ketone, thus generat- variable, and dependent on the organism involved. ing intermediate β-keto acids. In this sequence, the Accordingly, these individual transformations are enolate is restored to a ketone, in which the remain- considered rather than the overall pathway. ing C-4 methyl takes up the more favourable equa- The methyl at C-14 is usually the one lost first, torial (4α) orientation. and this is removed as formic acid. The reac- The side-chain ∆24 double bond is reduced by tion is catalysed by a cytochrome P-450 mono- an NADPH-dependent reductase, hydride from the oxygenase, which achieves two oxidation reactions coenzyme being added at C-25, with H-24 being to give the 14α-formyl derivative (Figure 5.78), derived from water (Figure 5.80). The ∆8 double and loss of this formyl group giving the ∆8,14 bond is effectively migrated to ∆5 via ∆7 and the diene, most probably via homolytic cleavage of the ∆5,7 diene (Figure 5.81). This sequence involves peroxy adduct as indicated (compare similar per- an allylic isomerization, a dehydrogenation, and a oxy adducts involved in side-chain cleavage from reduction. Newly introduced protons at C-9 and ring D, page 278, and in A ring aromatization, C-8 originate from water, and that at C-7 from page 278). The 14-demethyl sterol is then obtained NADPH. by an NADPH-dependent reduction step, the 15- The role of lanosterol in non-photosynthetic proton being derived from water. organisms (animals, fungi) is taken in photo- Loss of the C-4 methyls occurs sequentially, synthetic organisms (plants, algae) by the cyclo- usually after removal of the 14α-methyl, with both propane triterpenoid cycloartenol (Figure 5.55). carbons being cleaved off via a decarboxylation This cyclopropane feature is found in a number of STEROIDS 235

sequential oxidation of peroxy adduct formation via nucleophilic attack of 14-methyl group to aldehyde peroxy-enzyme on to carbonyl R O R R R 2 O NADPH 2 NADPH 14 D Enz–Fe–OOH 15 OH O OH O homolytic cleavage of OFe–Enz reduction of double peroxy bond bond R R R NADPH R H2O

H H H H H OFe–Enz OH O HCO2H methyl group lost as OFe–Enz formic acid

Figure 5.78

sequential oxidation of oxidation of 3β-alcohol decarboxylation of 4α-methyl group to carboxyl to ketone β-keto acid

O2 + NADPH NAD – CO2 A 4 HO HO H O H H H HO H HO C O 2 C enol–keto tautomerism; O H methyl takes up more repeat of whole favoured equatorial process configuration NADPH H O HO H HO H H H reduction of ketone O back to 3β-alcohol

Figure 5.79

reopen the cyclopropane ring is necessary. This is H NADPH shown in Figure 5.82. The stereochemistry at C- 25 H2O 8(Hβ) is unfavourable for a concerted mechanism 24 H H involving loss of H-8 with cyclopropane ring open- ing. It is suggested therefore that a nucleophilic Figure 5.80 group from the enzyme attacks C-9, opening the cyclopropane ring and incorporating a proton from plant sterols, but the majority of plant steroids con- water. A trans elimination then generates the ∆8 tain the normal methyl at C-10. This means that, double bond. The cyclopropane ring-opening pro- in addition to the lanosterol → cholesterol modi- cess seems specificto4α-monomethyl sterols. In fications outlined above, a further mechanism to plants, removal of the first 4-methyl group (4α; 236 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

allylic dehydrogenation gives reduction of conjugated diene H isomerization double bond 9 O2 H 8 NADPH NADPH H H 5 B 7 H H H H H H H 6 H H H H

Figure 5.81

cyclopropane ring opening initiated by attack of nucleophilic group on enzyme R R R H trans elimination

9 H H

8 Enz Enz HO HO HO 4α-monomethyl 4α-monomethyl cycloartanol derivative lanosterol derivative

Figure 5.82 note the remaining 4β-methyl group then takes ring-opening enzyme means cycloartenol is not up the α-orientation) is also known to precede converted into lanosterol, and lanosterol is thus loss of the 14α-methyl. Accordingly, the sub- absent from virtually all plant tissues. Cholesterol strate shown in Figure 5.82 has both 4α- and 14α- is almost always present in plants, though often in methyl groups. The specificity of the cyclopropane only trace amounts, and is formed via cycloartenol.

Cholesterol Cholesterol (Figure 5.76) is the principal animal sterol and since it is a constituent of cell membranes has been found in all animal tissues. Human gallstones are almost entirely composed of cholesterol precipitated from the bile. Cholesterol is currently available in quantity via the brains and spinal cords of cattle as a by-product of meat production, and these form one source for medicinal steroid semi-synthesis. Large quantities are also extractable from lanolin, the fatty material coating sheep’s wool. This is a complex mixture of esters of long chain fatty acids (including straight-chain, branched-chain, and hydroxy acids) with long chain aliphatic alcohols and sterols. Cholesterol is a major sterol component. Saponification of crude lanolin gives an alcohol fraction (lanolin alcohols or wool alcohols) containing about 34% cholesterol and 38% lanosterol/dihydrolanosterol. Wool alcohols are also used as an ointment base. Although the processes involved are quite complex, there appears to be a clear correlation between human blood cholesterol levels and heart disease. Atherosclerosis is a hardening of the arteries caused by deposition of cholesterol, cholesterol esters, and other lipids in the artery wall, causing a narrowing of the artery and thus an increased risk of forming blood clots (thrombosis). Normally, most of the cholesterol serves a structural element in cell walls, whilst the remainder is transported via the blood and is used for synthesis of steroid hormones, vitamin D (page 259), or bile acids (page 261). Transport of cholesterol is facilitated by formation of lipoprotein carriers, comprising protein and phospholipid shells

(Continues) STEROIDS 237

(Continued)

surrounding a core of cholesterol, in both free and esterified forms. Risk of atherosclerosis increases with increasing levels of low density lipoprotein (LDL) cholesterol, and is reduced with increasing levels of high density lipoprotein (HDL) cholesterol. Blood LDL cholesterol levels are thus a good statistical indicator of the potential risk of a heart attack. The risks can be lessened by avoiding foods rich in cholesterol, e.g. eggs, reducing the intake of foods containing high amounts of saturated fatty acids such as animal fats, and replacing these with vegetable oils and fish that are rich in polyunsaturated fatty acids (see page 40). Blood LDL cholesterol levels may also be reduced by incorporating into the diet plant sterol esters or plant stanol esters, which reduce the absorption of cholesterol (see page 256). In humans, dietary cholesterol is actually a smaller contributor to LDL cholesterol levels than is dietary saturated fat. Cholesterol biosynthesis may also be inhibited by drug therapy using specific inhibitors of the mevalonate pathway, e.g. lovastatin and related compounds (see page 112).

Steroidal Saponins steroidal saponins have the same configuration at the spiro centre C-22, but stereoisomers at C-25 Steroidal saponins have similar biological proper- exist, e.g. yamogenin (Figure 5.84), and often ties to the triterpenoid saponins (see page 219), mixtures of the C-25 stereoisomers cooccur in but are less widely distributed in nature. They are the plant. The sugar moiety is usually at position found in many monocotyledon families, especially 3, and typically contains fewer monosaccharide the Dioscoreaceae (e.g. Dioscorea), the Agavaceae units than found with triterpenoid saponins. One to (e.g. Agave, Yucca) and the Liliaceae (e.g. Smi- three monosaccharide units are most common. The lax, Trillium). Their sapogenins are C27 sterols in three-dimensional shape of diosgenin is indicated which the side-chain of cholesterol has undergone in Figure 5.83. modification to produce a spiroketal, e.g. dioscin The spiroketal function is derived from the (Figure 5.83) from Dioscorea. Acid hydrolysis of cholesterol side-chain by a series of oxygena- dioscin liberates the aglycone diosgenin. All the tion reactions, hydroxylating C-16 and one of the

O H D-Glc O H O OH H 25 α1→4 β1→ H H H O O H 22 HO O H O O diosgenin H HO α1→2 O H H HO 3 OH HO L-Rha O HO diosgenin HO OH L-Rha O dioscin H

Characteristic features of steroidal saponins: O • HO H spiroketal at C-22 H H • common configuration at C-22 H • sugar residues on 3β-hydroxyl diosgenin

Figure 5.83 238 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

ketal whilst the different possible stereochemistries O at C-25 are dictated by whether C-26 or C-27 is H hydroxylated in the earlier step. Glycoside deriva- tives, e.g. protodioscin (Figure 5.86) have been H O H isolated from plants. These are readily hydrolysed, H H and then spontaneously cyclize to the spiroketal HO (Figure 5.86). Allowing homogenized fresh plant yamogenin tissues to stand and autolyse through the action O of endogenous glycosidase enzymes not only O achieves cyclization of such open-chain saponins, H 12 but can hydrolyse off the sugar units at C-3, thus H O yielding the aglycone or sapogenin. This is a stan- H dard approach employed in commercial production H H of steroidal sapogenins, important starting materi- HO als for the semi-synthesis of steroidal drugs. Dios- H hecogenin genin is the principal example and is obtained from ∗ Figure 5.84 Mexican yams (Dioscorea spp.; Dioscoreaceae) . Fenugreek∗ (Trigonella foenum-graecum; Legumi- nosae/Fabaceae) is another potentially useful com- terminal methyls, and then producing a ketone mercial source. Sisal∗ (Agave sisalana; Agavaceae) function at C-22 (Figure 5.85). This proposed is also used commercially, yielding hecogenin intermediate is transformed into the hemiketal and (Figure 5.84), a 12-keto derivative with trans- then the spiroketal. The chirality at C-22 is fixed fused A/B rings, the result of reduction of the ∆5 by the stereospecificity in the formation of the double bond.

22 O 25 26 H O O OH OH 27 H 16 O OH OH H H nucleophilic attack of HO H H hydroxyl on to ketone cholesterol gives hemiketal OH H HO OH H H O O H ketal (spiroketal) H H formation from hemiketal H UDPGlc OGlc O OH 25 H H 22 H H O O H chemical hydrolysis H H H allows spontaneous H formation of spiroketal HO diosgenin / yamogenin

Figure 5.85 STEROIDS 239

OGlc

OH O H H O O H β-glucosidase H H H α1→4 β1→ H H α1→4 β1→ H H L-Rha D-Glc O L-Rha D-Glc O α1→2 α1→2 L-Rha protodioscin L-Rha dioscin

Figure 5.86

Dioscorea About 600 species of Dioscorea (Dioscoreaceae) are known, and a number of these are cultivated for their large starchy tubers, commonly called yams, which are an important food crop in many parts of the world. Important edible species are Dioscorea alata and D. esculenta (S E Asia), D. rotundata and D. cayenensis (W Africa) and D. trifida (America). A number of species accumulate quite high levels of saponins in their tubers, which make them bitter and inedible, but these provide suitable sources of steroidal material for drug manufacture. Dioscoreas are herbaceous, climbing, vinelike plants, the tuber being totally buried, or sometimes protruding from the ground. Tubers weigh anything up to 5 kg, but in some species, tubers have been recorded to reach weights as high as 40–50 kg. Drug material is obtained from both wild and cultivated plants, with plants collected from the wild having been exploited considerably more than cultivated ones. Commercial cultivation is less economic, requiring a 4–5 year growing period, and some form of support for the climbing stems. Much of the world’s production has come from Mexico, where tubers from D. composita (barbasco), D. mexicana,andD. floribunda, mainly harvested from wild plants, are utilized. The saponin content of the tubers varies, usually increasing as tubers become older. Typically, tubers of D. composita may contain 4–6% total saponins, and D. floribunda 6–8%. Other important sources of Dioscorea used commercially now include India (D. deltoidea), South Africa (D. sylvatica)andChina(D. collettii, D. pathaica,and D. nipponica). Sapogenins are isolated by chopping the tubers, allowing them to ferment for several days, then completing the hydrolysis of saponins by heating with aqueous acid. The sapogenins can then be solvent extracted. The principal sapogenin in the species given above is diosgenin (Figure 5.83), with small quantities of the 25β-epimer yamogenin (Figure 5.84). Demand for diosgenin for pharmaceuticals is huge, equivalent to 10 000 tonnes of Dioscorea tuber per annum, and it is estimated that about 60% of all steroidal drugs are derived from diosgenin. Powdered Dioscorea (wild yam) root or extract is also marketed to treat the symptoms of menopause as an alternative to hormone replacement therapy (see page 279). Although there is a belief that this increases levels of progesterone, which is then used as a biosynthetic precursor of other hormones, there is no evidence that diosgenin is metabolized in the human body to progesterone, and any beneficial effects may arise from diosgenin itself.

(Continues) 240 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued) Fenugreek

The seeds of fenugreek (Trigonella foenum-graecum; Leguminosae/Fabaceae) are an important spice material, and are ingredients in curries and other dishes. The plant is an annual, and is grown widely, especially in India, both as a spice and as a forage crop. Seeds can yield, after hydrolysis, 1–2% of sapogenins, principally diosgenin (Figure 5.83) and yamogenin (Figure 5.84). Although yields are considerably lower than from Dioscorea, the ease of cultivation of fenugreek and its rapid growth make the plant a potentially viable crop for steroid production in temperate countries. Field trials of selected high-yielding strains have been conducted.

Sisal Sisal (Agave sisalana; Agavaceae) has long been cultivated for fibre production, being the source of sisal hemp, used for making ropes, sacking and matting. The plant is a large, rosette- forming succulent with long, tough, spine-tipped leaves containing the very strong fibres. The main area of sisal cultivation is East Africa (Tanzania, Kenya), with smaller plantations in other parts of the world. The sapogenin hecogenin (Figure 5.84) was initially produced from the leaf waste (0.1–0.2% hecogenin) after the fibres had been stripped out. The leaf waste was concentrated, allowed to ferment for several days, then treated with steam under pressure to complete hydrolysis of the saponins. Filtration then produced a material containing about 12% hecogenin, plus other sapogenins. This was refined further in the pharmaceutical industry. Other sapogenins present include tigogenin and neotigogenin (Figure 5.87). As the demand for natural fibres declined due to the availability of synthetics, so did the supply of sisal waste and thus hecogenin. In due course, hecogenin became a more valuable commodity than sisal, and efforts were directed specifically towards hecogenin production. This has resulted in the cultivation of Agave hybrids with much improved hecogenin content. The fermented sap of several species of Mexican Agave provides the alcoholic beverage pulque. Distillation of the fermented sap produces tequila.

O O H H O H H O H H H AB H ABH H HO HO H A/B cis, smilagenin H A/B cis, sarsasapogenin A/B trans, tigogenin A/B trans, neotigogenin

Figure 5.87

Some steroidal alkaloids are nitrogen ana- are found in many plants of the genus Solanum logues of steroidal saponins, and display sim- (Solanaceae), and such plants must thus be ilar properties such as surface activity and regarded as potentially toxic. In contrast to the haemolytic activity, but these compounds are toxic oxygen analogues, all compounds have the same when ingested. These types of compound, e.g. stereochemistry at C-25 (methyl always equato- solasonine (Figure 5.88) (aglycone solasodine), rial), whilst isomers at C-22 do exist, e.g. tomatine STEROIDS 241

H 25 N H 22 D-Gal H O OH H OH β1→3 OH O β1→ H H HO O HO O O OH D-Glc α1→2 O O HO solasodine HO L-Rha OH

solasonine 25 D-Glc H NH β1→3 OH 22 O β1→4 O HO O HO H HO O O OH H OH OH O β1→ H H D-Xyl O HO O HO H HO O OH OH β1→2 D-Gal tomatidine D-Glc

tomatine

Figure 5.88

(Figure 5.88) (aglycone tomatidine) from tomato the ∆5 double bond yields the trans-fused system, (Lycopersicon esculente; Solanaceae). The nitro- whereas reduction of a ∆4 double bond gives the gen atom is introduced by a transamination reac- alternative cis-fused system (Figure 5.89). Accord- tion, typically employing an amino acid as donor ingly, to obtain the A/B cis fusion, the ∆5 unsatu- (see page 20). Since the production of medici- ration of cholesterol is changed to ∆4 by oxidation nal steroids from steroidal saponins requires pre- of the 3-hydroxyl and allylic isomerization to liminary degradation to remove the ring systems the conjugated 4-ene-3-one system, and this is containing the original cholesterol side-chain, it followed by reduction of both functional groups is immaterial whether these rings contain oxy- (Figure 5.89) (compare biosynthesis of proges- gen or nitrogen. Thus, plants rich in solasodine or terone, page 243). The sarsaparilla saponins are tomatidine could also be employed for commercial not present in sufficient quantities to be commer- steroid production (see page 391). cially important for steroid production, but quite Smilagenin and sarsasapogenin (Figure 5.87) ∗ large amounts of sarsasapogenin can be extracted found in sarsaparilla (Smilax spp.; Liliaceae/ Smi- from the seeds of Yucca brevifolia∗ (Agavaceae). lacaceae) are reduced forms of diosgenin and yamogenin respectively. These contain cis-fused A/B rings, whilst the corresponding trans-fused Cardioactive Glycosides systems are present in tigogenin and neotigogenin (Figure 5.87) found in Digitalis purpurea along Many of the plants known to contain cardiac or with cardioactive glycosides (see page 246). All cardiotonic glycosides have long been used as four stereoisomers are derived from cholesterol, arrow poisons (e.g. Strophanthus) or as heart drugs and the stereochemistry of the A/B ring fusion (e.g. Digitalis). They are used to strengthen a appears to be controlled by the nature of the sub- weakened heart and allow it to function more effi- strate being reduced. Direct enzymic reduction of ciently, though the dosage must be controlled very 242 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

oxidation of keto–enol enol–keto H hydroxyl to C=O H tautomerism H tautomerism H

H H H H HO H O HO O H H ∆5,6 reduction of H reduction of ∆4,5 reduction of H H C=O H

H H H HO HO O H H H A/B trans A/B cis

Figure 5.89

Sarsaparilla

Sarsaparilla consists of the dried roots of various Smilax species (Liliaceae/Smilacaceae), including S. aristolochiaefolia, S. regelii,andS. febrifuga, known respectively as Mexican, Honduran, and Ecuadorian sarsaparilla. The plants are woody climbers indigenous to Central America. Sarsaparilla has a history of use in the treatment of syphilis, rheumatism, and skin diseases, but is now mainly employed as a flavouring in the manufacture of non-alcoholic drinks. It has some potential as a raw material for the semi-synthesis of medicinal steroids, being a source of sarsasapogenin and smilagenin (Figure 5.87). The roots contain 1.8–2.4% steroidal saponins, including parillin (Figure 5.90).

O OH β1→6 H HO O HO O H O OH D-Glc D-Glc H O β1→ H H α1→4 O HO O H O OH HO HO O HO OH HO O sarsasapogenin L-Rha OH β1→2 D-Glc

parillin

Figure 5.90

Yucca Yucca brevifolia (Agavaceae) has been explored as a potential source of sarsasapogenin for steroid production, especially at times when market prices of diosgenin from Dioscorea became prohibitively expensive. The plant grows extensively in the Mojave desert in California, and high levels of sarsasapogenin (8–13%) are present in the seeds. This means the plants can be harvested regularly without damage. The subsequent stabilization of Dioscorea prices in the 1970s stopped further commercial utilization. STEROIDS 243 carefully since the therapeutic dose is so close to group, followed by incorporation of either two or the toxic dose. The cardioactive effects of Digitalis three carbons for cardenolides or bufadienolides were discovered as a result of its application in respectively (Figure 5.92). the treatment of dropsy, an accumulation of water Shortening of the cholesterol side-chain is in the body tissues. Digitalis alleviated dropsy accomplished by stepwise hydroxylation at C-22 indirectly by its effect on the heart, improving and then C-20, then cleavage of the C-20/22 bond the blood supply to the kidneys and so removing giving pregnenolone, which is then oxidized in excess fluid. ring A giving progesterone (Figure 5.92). This The therapeutic action of cardioactive glyco- can be reduced to give the cis-fused A/B sys- sides depends on the structure of the aglycone, and tem as in 3β-hydroxy-5β-pregnan-20-one (com- on the type and number of sugar units attached. pare Figure 5.89) which is the substrate for 14β- Two types of aglycone are recognized, cardeno- hydroxylation, i.e. inverting the stereochemistry lides,e.g.digitoxigenin from Digitalis purpurea, at this centre. Inversion is atypical for hydroxy- which are C23 compounds, and bufadienolides, lation by mono-oxygenases, which are found to e.g. hellebrigenin from Helleborus niger,which hydroxylate with retention of configuration. What- 8 are C24 structures (Figure 5.91). Stereochemistry is ever the mechanism of this hydroxylation, no ∆ very important for activity, and these compounds or ∆15 double bond intermediates are involved. have cis fusions for both the A/B and C/D rings, Hydroxylation in the side-chain at C-21 follows. 3β- and 14β-hydroxyl groups with the glycoside The lactone ring is created at this stage. An inter- function at C-3, and an α,β-unsaturated lactone mediate malonate ester is involved, and ring forma- grouping at C-17β. This lactone ring is five mem- tion probably occurs via the aldol addition process bered in the cardenolides, and six membered in the shown in Figure 5.93 giving the cardenolide dig- bufadienolides. The hellebrigenin structure shows itoxigenin, the carboxyl carbon of the malonate two other modifications not found in the basic ester being lost by decarboxylation during the pro- steroid skeleton, namely a hydroxyl at the bridge- cess (compare malonate in the acetate pathway). head carbon C-5, and a formyl group at C-10, Alternatively, three carbons from oxaloacetate can being an oxidized form of the normal methyl. The be incorporated by a similar esterification/aldol three-dimensional shape of digitoxigenin is shown reaction sequence. This would produce bufalin in Figure 5.91. These basic structures arise biosyn- (Figure 5.92), a bufadienolide structure found in thetically by metabolism of cholesterol, in which the skin of toads (Bufo spp.), from which this the side-chain is cleaved to a two-carbon acetyl class of compound was originally isolated and

O 24 O O O 23 23 21 21 22 20 20 22 17 17

H 14 OHC H 14

10 3 H OH 3 5 H OH HO HO H OH O O cardenolide - digitoxigenin bufadienolide - hellebrigenin H

Characteristic features of cardiac glycosides: OH H • cis-fused A/B and C/D rings H • 14β-hydroxyl H • unsaturated lactone at C-17β HO digitoxigenin • sugar residues on 3β-hydroxyl

Figure 5.91 244 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

oxidative cleavage between hydroxyls, OH perhaps via peroxide H O stepwise hydroxylation via O OH 22 22-hydroxycholesterol HO 20 O

O H H 2 H O2 NADPH NADPH HH HH H H HO HO HO cholesterol pregnenolone 4,5 reduction of ∆ gives NAD+ O cis-fused A/B rings O enol–keto keto–enol tautomerism tautomerism H H H H NADPH H H H H H H O O HO O H 5β-pregnan-3,20-dione progesterone reduction of 3-ketone to NADPH 3β-hydroxyl OH

O 14β-hydroxylation with O O inversion of side-chain stereochemistry 21-hydroxylation H H H

H H H OH H OH HO HO HO H H H 3β-hydroxy- 3β,14β-dihydroxy- 3β,14β,21-trihydroxy- O 5β-pregnan-20-one 5β-pregnan-20-one 5β-pregnan-20-one

malonyl-CoA HO2C SCoA O see Figure 5.93 oxaloacetyl-CoA O

O O O

H H

H OH HOH HO HO H H digitoxigenin bufalin 5β-hydroxylation and C-19 oxidation 12β-hydroxylation 16β-hydroxylation O

O O O O O

HO 12 19 H HOH OHC H 16

H OH HOH 5 H OH HO HO HO H H OH digoxigeningitoxigenin hellebrigenin

Figure 5.92 STEROIDS 245

aldol reaction with concomitant decarboxylation; compare malonyl-CoA as chain extender in acetate pathway dehydration favoured by O O O formation of conjugated system CO2H CO2H CO2H O O OH CoAS O O O O malonyl-CoA O O – CO2 – H2O O H OH ester formation malonate ester cardenolide

O aldol reaction with O O concomitant O CoAS CO2H O O decarboxylation OH O O O O O oxaloacetyl-CoA – CO2 O CO2H CO2H O O H OH dehydration ester formation favoured by oxaloacetate ester – H2O formation of 1,4-elimination conjugated system O favouring formation of O O conjugated dienone O O O OH reduction O H

bufadienolide

Figure 5.93

HO has subsequently taken the general name. Note 6 6 that in the subsequent formation of hellebrigenin HO O O OH (Figure 5.92), hydroxylation at C-5 occurs with the 2 MeO OH 3 OH expected retention of stereochemistry, and not with OH inversion as seen at C-14. D-digitoxose D-digitalose O

The fundamental pharmacological activity resi- 6 O des in the aglycone portion, but is consider- HO O ably modified by the nature of the sugar at C-3. OH This increases water solubility and binding to 2 OMe heart muscle. The sugar unit may have one to H H D-cymarose four monosaccharides; many, e.g. D-digitoxose and D-digitalose (Figure 5.94), are unique to this H OH HO group of compounds. About 20 different sugars H have been characterized, and with the exception of 19-norbufalin D-glucose, they are 6-deoxy- (e.g. L-rhamnose) or 2,6-dideoxy- (e.g. D-digitoxose) hexoses, some of Figure 5.94 which are 3-methyl ethers (e.g. D-digitalose and D-cymarose (Figure 5.94)). In plants, cardiac gly- families the Apocynaceae (e.g. Strophanthus)∗, cosides are confined to the Angiosperms, but are Liliaceae (e.g. Convallaria)∗, and Scrophulari- found in both monocotyledons and dicotyledons. aceae (e.g. Digitalis)∗ yield medicinal agents. The The cardenolides are more common, and the plant rarer bufadienolides are found in some members of 246 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS the Liliaceae (e.g. Urginea)∗ and Ranunculaceae Endogenous Digitalis-like compounds have also (e.g. Helleborus), as well as toads. Monarch but- been detected, albeit in very small quantities, in terflies and their larvae are known to accumulate mammalian tissues. 19-Norbufalin (Figure 5.94) in their bodies a range of cardenolides, which they is found in human eye lenses, at higher levels if ingest from their food plant, the common milk- these are cataract afflicted, and it is believed to weed (Asclepias syriaca; Asclepiadaceae). This regulate ATPase activity under some physiological makes them unpalatable to predators such as birds. and pathological conditions.

Digitalis purpurea

Digitalis leaf consists of the dried leaf of the red foxglove Digitalis purpurea (Scrophulariaceae). The plant is a biennial herb, common in Europe and North America, which forms a low rosette of leaves in the first year, and its characteristic spike of purple (occasionally white) bell- shaped flowers in the second year. It is potentially very toxic, but is unlikely to be ingested by humans. Digitalis purpurea is cultivated for drug production, principally in Europe, the first year leaves being harvested then rapidly dried at 60°C as soon as possible after collection. This procedure is necessary to inactivate hydrolytic enzymes which would hydrolyse glycoside linkages in the cardioactive glycosides giving rise to less active derivatives. Even so, some partial hydrolysis does occur. Excess heat may also cause dehydration in the aglycone to ∆14-anhydro compounds, which are inactive. Because of the pronounced cardiac effects of digitalis, the variability in the cardiac glycoside content, and also differences in the range of structures present due to the effects of enzymic hydrolysis, the crude leaf drug is usually assayed biologically rather than chemically. Prepared digitalis is a biologically standardized preparation of powdered leaf, its activity being assessed on cardiac muscle of guinea pig or pigeon and compared against a standard preparation. It may be diluted to the required activity by mixing in powdered digitalis of lower potency, or inactive materials such as lucerne (Medicago sativa) or grass. The crude drug is hardly ever used now, having been replaced by the pure isolated glycosides. The cardioactive glycoside content of Digitalis purpurea leaf is 0.15–0.4%, consisting of about 30 different structures. The major components are based on the aglycones digitoxigenin, gitoxigenin, and gitaloxigenin (Figure 5.95), the latter being a formate ester. The glycosides comprise two series of compounds, those with a tetrasaccharide glucose–(digitoxose)s – unit and those with a trisaccharide (digitoxose)3 – unit. The latter group (the secondary glycosides) are produced by partial hydrolysis from the former group (the primary glycosides) during drying by the enzymic action of a β-glucosidase, which removes the terminal glucose. Thus the principal glycosides in the fresh leaves, namely purpureaglycoside A and purpureaglycoside B (Figure 5.95), are partially converted into digitoxin and gitoxin respectively (Figure 5.95), which normally predominate in the dried leaf. These transformations are indicated schematically in Figure 5.96. In the fresh leaf, purpureaglycoside A can constitute about 50% of the glycoside mixture, whilst in the dried leaf, the amounts could be negligible if the plant material is old or poorly stored. The gitaloxigenin-based glycosides are relatively unstable, and the formyl group on the aglycone is readily lost by hydrolysis. Other minor glycosides are present, but neither the fresh nor dried leaf contain any significant quantities of the free aglycones. Glycosides of the gitoxigenin series are less active than the corresponding members of the digitoxigenin-derived series. Digitoxin is the only compound routinely used as a drug, and it is employed in congestive heart failure and treatment of cardiac arrhythmias, particularly atrial fibrillation.

(Continues) STEROIDS 247

(Continued)

O O

OH β → O 1 4 HO β → O 1 4 H R HO O β1→4 O O β → OH 1 H OH O O OH O OH H OH R = H, digitoxigenin D-glucose D-digitoxose D-digitoxose D-digitoxose R = OH, gitoxigenin R = O2CH, gitaloxigenin

R = H, digitoxin R = OH, gitoxin R = O2CH, gitaloxin

R = H, purpureaglycoside A R = OH, purpureaglycoside B O O R = O2CH, glucogitaloxin

OH β → O 1 4 HO β → O 1 4 H R HO O β1→4 16 O O OH β1→ H OH O O O O OH H O OH R = H, digitoxigenin D-glucose 3-acetyl- D-digitoxose D-digitoxose R = OH, gitoxigenin D-digitoxose R = O2CH, gitaloxigenin

R = H, acetyldigitoxin R = OH, acetylgitoxin R = O2CH, acetylgitaloxin

R = H, lanatoside A R = OH, lanatoside B O O R = O2CH, lanatoside E R1 OH 12 O β1→4 HO β1→4 H R2 HO O O 16 β1→4 O O OH β1→ H OH O O O O OH H OH O R1 = OH, R2 = H, digoxigenin D-glucose 3-acetyl- D-digitoxose D-digitoxose 1 2 D-digitoxose R = OH, R = OH, diginatigenin

R1 = OH, R2 = H, acetyldigoxin R1 = OH, R2 = OH, acetyldiginatin

R1 = OH, R2 = H, lanatoside C R1 = OH, R2 = OH, lanatoside D

Figure 5.95

(Continues) 248 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

purpureaglycoside A lanatoside A

Glc–Dig–Dig–Dig–digitoxigenin Glc–Dig–Dig–Dig–digitoxigenin

– Glc Ac – Glc

digitoxin acetyldigitoxin – Ac Dig–Dig–Dig–digitoxigenin Dig–Dig–Dig–digitoxigenin

Ac

purpureaglycoside B lanatoside B

Glc–Dig–Dig–Dig–gitoxigenin Glc–Dig–Dig–Dig–gitoxigenin

– Glc Ac – Glc

gitoxin acetylgitoxin – Ac Dig–Dig–Dig–gitoxigenin Dig–Dig–Dig–gitoxigenin

Ac

desacetyl-lanatoside C lanatoside C – Ac Glc–Dig–Dig–Dig–digoxigenin Glc–Dig–Dig–Dig–digoxigenin

– Glc Ac – Glc

lanatoside D digoxin acetyldigoxin – Ac Glc–Dig–Dig–Dig–diginatigenin Dig–Dig–Dig–digoxigenin Dig–Dig–Dig–digoxigenin Ac Ac – Glc

glucogitaloxin lanatoside E acetyldiginatin

Glc–Dig–Dig–Dig–gitaloxigenin Glc–Dig–Dig–Dig–gitaloxigenin Dig–Dig–Dig–diginatigenin Ac – Glc Ac – Glc – Ac

gitaloxin acetylgitaloxin – Ac diginatin Dig–Dig–Dig–gitaloxigenin Dig–Dig–Dig–gitaloxigenin

Ac Dig–Dig–Dig–diginatigenin

Figure 5.96

(Continues) STEROIDS 249

(Continued) Digitalis lanata

Digitalis lanata (Scrophulariaceae), the Grecian foxglove, is a perennial or biennial herb from Southern and Central Europe, and differs in appearance from the red foxglove by its long narrow smoother leaves, and its smaller flowers of a yellow-brown colour. It is cultivated in Europe, the United States and South America, and is harvested and dried in a similar manner to D. purpurea. It has not featured as a crude drug, but is used exclusively for the isolation of individual cardiac glycosides, principally digoxin and lanatoside C (Figure 5.97). The total cardenolide content of up to 1% is two to three times that found in D. purpurea. The main constituents resemble those of D. purpurea, but contain an acetyl ester function on the third digitoxose, that furthest from the aglycone. This acetyl group makes the compounds easier to isolate from the plant material and makes crystallization easier. Drying of the leaf is similarly accompanied by some partial hydrolysis of the original fresh leaf constituents through enzymic action, and both the terminal glucose and the acetyl group may be hydrolysed off, extending the range of compounds isolated. The D. lanata cardiac glycosides are based on five aglycones, digitoxigenin, gitoxigenin, and gitaloxigenin, as found in D. purpurea, plus digoxigenin and diginatigenin (Figure 5.95), which do not occur in D. purpurea. The primary glycosides containing the acetylated tetrasaccharide unit glucose–acetyldigitoxose–(digitoxose)2 –are called lanatosides. Lanatosides A and C (Figure 5.95) constitute the major components in the fresh leaf (about 50–70%) and are based on the aglycones digitoxigenin and digoxigenin respectively. Lanatosides B, D, and E (Figure 5.95) are minor components derived from gitoxigenin, diginatigenin, and gitaloxigenin respectively. Enzymic hydrolysis of the lanatosides generally involves loss of the terminal glucose prior to removal of the acetyl function, so that compounds like acetyldigitoxin and acetyldigoxin as well as digitoxin and digoxin are present in the dried leaf as decomposition products from lanatosides A and C respectively. These transformations are also indicated in simplified form in Figure 5.96. Digoxin (Figure 5.97) has a rapid action and is more quickly eliminated from the body than digitoxin, and is therefore the most widely used of the cardioactive glycosides. Digoxin is more hydrophilic than digitoxin, binds less strongly to plasma proteins and is mainly eliminated by the kidneys, whereas digitoxin is metabolized more slowly by the liver. It is used in congestive

O O

OH

H R1O O O O H OH O O OR2 O OH H OH R1 = R2 = H, digoxin R1 = Me, R2 = H, medigoxin (metildigoxin) R1 = Glc, R2 = Ac, lanatoside C R1 = Glc, R2 = H, deslanoside (desacetyl-lanatoside C)

Figure 5.97

(Continues) 250 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

heart failure and atrial fibrillation. Lanatoside C and deslanoside (desacetyl-lanatoside C) (Figure 5.97) have also been employed, though not to the same extent. They have very rapid action and are suited for treatment of cardiac emergencies by injection. A semi- synthetic derivative medigoxin or metildigoxin (methyl replacing the glucose in lanatoside C) (Figure 5.97) has also been available, being more active via better bioavailability. The cardioactive glycosides increase the force of contractions in the heart, thus increasing cardiac output and allowing more rest between contractions. The primary effect on the heart appears to be inhibition of Na+/K+-ATPase in the cell membranes of heart muscle, specifically inhibiting the Na+ pump, thereby raising the intracellular Na+ concentration. The resultant decrease in the Na+ gradient across the cell membrane reduces the energy available for transport of Ca2+ out of the cell, leads to an increase in intracellular Ca2+ concentration, and provides the positive ionotropic effect and increased force of contractions. The improved blood circulation also tends to improve kidney function leading to diuresis and loss of oedema fluid often associated with heart disease. However, the diuretic effect, historically important in the treatment of dropsy, is more safely controlled by other diuretic drugs. To treat congestive heart failure, an initial loading dose of the cardioactive glycoside is followed by regular maintenance doses, the amounts administered depending on drug bioavailability and subsequent metabolism or excretion. Because of the extreme toxicity associated with these compounds (the therapeutic level is 50–60% of the toxic dose; a typical daily dose is only about 1 mg) dosage must be controlled very carefully. Bioavailability has sometimes proved erratic and can vary between different manufacturers’ formulations, so patients should not be provided with different preparations during their treatment. Individual patients also excrete the glycosides or metabolize them by hydrolysis to the aglycone at different rates, and ideally these processes should be monitored. Levels of the drug in blood plasma can be measured quite rapidly by radioimmunoassay using a specific antibody. A digoxin-specific antibody is available both for assay and also as a means of reversing life-threatening digoxin overdose. It has also successfully reversed digitoxin overdose, thus demonstrating a somewhat broader specificity. The value of digoxin treatment for heart failure where the heartbeat remains regular has recently been called into question. It still remains a recognized treatment for atrial fibrillation. Many other species of Digitalis, e.g. D. dubia, D. ferruginea, D. grandiflora, D. lutea, D. mertonensis, D. nervosa, D. subalpina,andD. thaspi contain cardioactive glycosides in their leaves, and some have been evaluated and cultivated for drug use.

Strophanthus

Strophanthus is the dried ripe seeds of Strophanthus kombe´ or S. gratus (Apocynaceae), which are tall vines from equatorial Africa. Strophanthus kombe´ has a history of use by African tribes as an arrow poison, and the seeds contain 5–10% cardenolides, a mixture known as K-strophanthin. This has little drug use today, though it was formerly used medicinally as a cardiac stimulant. The main glycoside (about 80%) is K-strophanthoside (Figure 5.98) with smaller amounts of K-strophanthin-β and cymarin, related to K-strophanthoside as shown. These are derivatives of the aglycone strophanthidin. Strophanthus gratus contains 4–8% of ouabain (G-strophanthin) (Figure 5.98), the rhamnoside of ouabigenin. Ouabigenin is rather

(Continues) STEROIDS 251

(Continued)

O O

OH HO O α1→4 HO OH OHC H HO β1→4 O O β1→ HOH HO O O OH O OH OMe D-glucoseD-glucose D-cymarose strophanthidin

cymarin

K-strophanthin-β

K-strophanthoside O O O O

HO HO HO 11 H OHC H 1 10 HOH H OH α1→ O α1→ O OH OH O O HO HO HO HO OH OH L-rhamnose ouabigenin L-rhamnose strophanthidin

ouabain convallatoxin (G-strophanthin)

Figure 5.98 unusual in having additional hydroxylation at 1β and 11α, as well as a hydroxymethyl at C-10. Ouabain is a stable, crystalline material, which is often employed as the biological standard in assays for cardiac activity. It is a potent cardiac glycoside and acts quickly, but wears off rapidly. It is very polar with rapid renal elimination and must be injected because it is so poorly absorbed orally. It has been used for emergency treatment in cases of acute heart failure. It is still official in many pharmacopoeias.

Convallaria The dried roots and tops of lily of the valley, Convallaria majalis (Liliaceae/Convallariaceae), contain cardioactive glycosides (0.2–0.3%) and are used in some European countries rather than digitalis. The effects are similar, but the drug is less cumulative. This plant is widely cultivated as an ornamental, particularly for its intensely perfumed small white flowers, and must be considered potentially toxic. The major glycoside (40–50%) is convallatoxin (Figure 5.98), the rhamnoside of strophanthidin.

(Continues) 252 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued) Squill

Squill (white squill) consists of the dried sliced bulbs of the white variety of Urginea maritima (formerly Scilla maritima; also known as Drimia maritima) (Liliaceae/Hyacinthaceae) which grows on seashores around the Mediterranean. The plant contains bufadienolides (up to 4%), principally scillaren A and proscillaridin A (Figure 5.99). The aglycone of scillaren A is scillarenin, which is unusual in containing a ∆4 double bond and thus lacks the cis A/B ring fusion found in the majority of cardiac glycosides. Squill is not usually used for its cardiac properties, as the glycosides have a short duration of action. Instead, squill is employed for its expectorant action in preparations such as Gee’s linctus. Large doses cause vomiting and a digitalis-like action on the heart. Red squill is a variety of Urginea maritima that contains an anthocyanin pigment (see page 150) and bufadienolides that are different from those of the white squill. The main glycosides are glucoscilliroside and scilliroside (Figure 5.99), glucosides of scillirosidin. This chemical variety should not be present in medicinal squill, and has mainly been employed as a rodenticide. Rodents lack a vomiting reflex and are poisoned by the cardiac effects, whilst in other animals and humans vomiting will occur due to the emetic properties of the drug. The use of red squill as a rodenticide is now considered inhumane.

O O O O

H

H OH OH OH β → α1→ O O 1 4 OH OH β → HO β1→ O 1 4 O H OH HO O HO O HO O OH HO O OH HO OH OAc OH D-glucoseL-rhamnose scillarenin D-glucose D-glucose scillirosidin

proscillaridin A scilliroside

scillaren A glucoscilliroside

Figure 5.99

Toxic Plants Many plants containing cardioactive glycosides are widely grown as ornamentals and must be considered toxic and treated with due care and respect. These include Digitalis species, Convallaria majalis, Helleborus species, and oleander (Nerium oleander; Apocynaceae).

Phytosterols corticosteroids. The main sterols in plants, fungi, and algae are characterized by extra one-carbon or The major sterol found in mammals is the C27 com- two-carbon substituents on the side-chain, attached pound cholesterol, which acts as a precursor for at C-24. These substituent carbons are numbered other steroid structures such as sex hormones and 241 and 242 (Figure 5.77); some older publications STEROIDS 253

242 241 22 24 H 24 H E SR

H H H

H H H H H H HO HO HO stigmasterol sitosterol fucosterol

H

HH H H HO HO ergosterol campesterol

Figure 5.100 may use 28 and 29. The widespread plant sterols are found predominantly in free alcohol form, but campesterol and sitosterol (Figure 5.100) are also as esters with long chain fatty acids (e.g. respectively 24-methyl and 24-ethyl analogues palmitic, oleic, linoleic, and α-linolenic acids), as of cholesterol. Stigmasterol contains additional glycosides, and as fatty acylated glycosides. These unsaturation in the side-chain, a trans-∆22 dou- sterols, termed phytosterols, are structural compo- ble bond, a feature seen in many plant sterols, nents of membranes in plants, algae, and fungi, and but never in mammalian ones. The introduction affect the permeability of these membranes. They of methyl and ethyl groups at C-24 generates also appear to play a role in cell proliferation. a new chiral centre, and the 24-alkyl groups in The source of the extra methyl or ethyl side- campesterol, sitosterol, and stigmasterol are desig- chain carbons in both cases is S-adenosylmethio- nated α. The predominant sterol found in fungi is nine (SAM), and to achieve alkylation the ergosterol (Figure 5.100), which has a β-oriented side-chain must have a ∆24 double bond, i.e. the 24-methyl, as well as a trans-∆22 double bond side-chains seen in lanosterol and cycloartenol. and additional ∆7 unsaturation. The descriptors The precise mechanisms involved have been α and β unfortunately do not relate to similar found to vary according to organism, but some terms for the steroid ring system, but are derived of the demonstrated sequences are given in from consideration of Fischer projections for the Figure 5.101. Methylation of the ∆24 double bond side-chain, substituents to the left being desig- at C-24 via SAM yields a carbocation which nated α and those to the right as β. Systematic RS undergoes a hydride shift and loss of a proton nomenclature is preferred, but note that this defines from C-241 to generate the 24-methylene side- sitosterol as 24R whilst stigmasterol, because of chain. This can be reduced to a 24-methyl its extra double bond, is 24S. The majority of either directly, or after allylic isomerization. plant sterols have a 24α-methyl or 24α-ethyl sub- Alternatively, the 24-methylene derivative acts as stituent, whilst algal sterols tend to have 24β- substrate for a second methylation step with SAM, ethyls, and fungi 24β-methyls. The most abundant producing a carbocation. Discharge of this cation sterol in brown algae (Fucus spp.; Fucaceae) is by proton loss produces a 24-ethylidene side- fucosterol (Figure 5.100), which demonstrates a chain, and reduction or isomerization/reduction further variant, a 24-ethylidene substituent. Such gives a 24-ethyl group. The trans-∆22 double groups can have E-configurations as in fucos- bond is introduced only after alkylation at C-24 terol, or the alternative Z-configuration. Sterols is completed. No stereochemistry is intended in 254 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

electrophilic addition; Ad Ad W–M 1,2-hydride shift C-methylation using SAM followed by loss of proton Me S Me S H 241 R R second electrophilic addition involving 24 H SAM

NADPH allylic isomerization H

NADPH

allylic NADPH dehydrogenation isomerization

dehydrogenation 22 NADPH 22

Figure 5.101

Figure 5.101. It is apparent that stereochemistries less variety is encountered. The most common in the 24-methyl, 24-ethyl, and 24-ethylidene pathway from lanosterol to ergosterol in fungi derivatives could be controlled by the reduction involves initial side-chain alkylation to eburicol processes or by proton loss as appropriate. It is (24-methylenedihydrolanosterol), which is the sub- more plausible for different stereochemistries in strate for 14-demethylation (Figure 5.103). Loss the 24-methyl and 24-ethyl side-chains to arise of the 4-methyls then gives fecosterol, from from reduction of different double bonds, rather which ergosterol arises by further side-chain and than reduction of the same double bond in two ring B modifications. Although the transforma- different ways. In practice, other mechanisms tions are similar to those occurring in the mam- involving a 25(26)-double bond are also found to malian pathway for lanosterol → cholesterol, the operate. initial side-chain alkylation means the intermedi- The substrates for alkylation are found to ates formed are different. Some useful anti-fungal be cycloartenol in plants and algae, and lanos- agents, e.g. ketoconazole and miconazole, are spe- terol in fungi. The second methylation step in cific inhibitors of the 14-demethylation reaction plants and algae usually involves gramisterol (24- in fungi, but do not affect cholesterol biosynthe- methylenelophenol) (Figure 5.102). This indicates sis in humans. Inability to synthesize the essential that the processes of side-chain alkylation and sterol components of their membranes proves fatal the steroid skeleton modifications, i.e. loss of for the fungi. Similarly, 14-demethylation in plants methyls, opening of the cyclopropane ring, and proceeds via obtusifoliol (Figure 5.102) and plants migration of the double bond, tend to run con- are unaffected by azole derivatives developed as currently rather than sequentially. Accordingly, the agricultural fungicides. The antifungal effect of range of plant and algal sterol derivatives includes polyene antibiotics such as amphotericin and nys- products containing side-chain alkylation, reten- tatin depends on their ability to bind strongly to tion of one or more skeletal methyls, and pos- ergosterol in fungal membranes and not to choles- session of a cyclopropane ring, as well as those terol in mammalian cells (see page 102). more abundant examples such as sitosterol and Sitosterol and stigmasterol (Figure 5.100) are stigmasterol based on a cholesterol-type skeleton. produced commercially from soya beans∗ (Glycine Most fungal sterols originate from lanosterol, so max; Leguminosae/Fabaceae) as raw materials STEROIDS 255

α H C-24 alkylation H loss of C-4 methyl H

HO HO HO H H H cycloartenol 24-methylenecycloartanol cycloeucalenol opening of cyclopropane

loss of C-14α methyl and further side-chain allylic isomerization alkylation ∆8 ∆7 HH HH (migration of to ) HO HO HO H H H citrostadienol gramisterol obtusifoliol (24-ethylidenelophenol) (24-methylenelophenol)

loss of loss of C-4 methyl C-4 methyl

HH 24-ethyl sterols HH 24-methyl sterols HO HO H H avenasterol episterol

Figure 5.102

C-24 alkylation C-14 demethylation

H HO HO HO H H H lanosterol eburicol 4,4-dimethylfecosterol (24-methylenedihydrolanosterol) demethylations at C-4

side-chain and ring B modifications HH H HO HO H ergosterol fecosterol

Figure 5.103 256 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

Soya Bean Sterols

Soya beans or soybeans (Glycine max; Leguminosae/Fabaceae) are grown extensively in the United States, China, Japan, and Malaysia as a food plant. They are used as a vegetable, and provide a high protein flour, an important edible oil (Table 3.2), and an acceptable non-dairy soybean milk. The flour is increasingly used as a meat substitute. Soy sauce is obtained from fermented soybeans and is an indispensable ingredient in Chinese cookery. The seeds also contain substantial amounts (about 0.2%) of sterols. These include stigmasterol (about 20%), sitosterol (about 50%) and campesterol (about 20%) (Figure 5.100), the first two of which are used for the semi-synthesis of medicinal steroids. In the seed, about 40% of the sterol content is in the free form, the remainder being combined in the form of glycosides, or as esters with fatty acids. The oil is usually solvent extracted from the dried flaked seed using hexane. The sterols can be isolated from the oil after basic hydrolysis as a by-product of soap manufacture, and form part of the unsaponifiable matter. The efficacy of dietary plant sterols in reducing cholesterol levels in laboratory animals has been known for many years. This has more recently led to the introduction of plant sterol esters as food additives, particularly in margarines, as an aid to reducing blood levels of low density lipoprotein (LDL) cholesterol, known to be a contributory factor in atherosclerosis and the incidence of heart attacks (see page 236). Plant sterol esters are usually obtained by esterifying sitosterol from soya beans with fatty acids to produce a fat-soluble product. Regular consumption of this material (recommended 1.3 g per day) is shown to reduce blood LDL cholesterol levels by 10–15%. The plant sterols are more hydrophobic than cholesterol and have a higher affinity for micelles involved in fat digestion, effectively decreasing intestinal cholesterol absorption. The plant sterols themselves are not absorbed from the GI tract. Of course, the average diet will normally include small amounts of plant sterol esters. Related materials used in a similar way are plant stanol esters. Stanols are obtained by hydrogenation of plant sterols, and will consist mainly of sitostanol (from sitosterol and stigmasterol) and campestanol (from campesterol) (Figure 5.104); these are then esterified with fatty acids. Regular consumption of plant stanol esters (recommended 3.4 g per day) is shown to reduce blood LDL cholesterol levels by an average of 14%. Much of the material used in preparation of plant stanol esters originates from tall oil, a by-product of the wood pulping industry. This contains campesterol, sitosterol, and also sitostanol. The stanols are usually transesterified with rapeseed oil, which is rich in unsaturated fatty acids (see page 43).

H H

H H H H HO HO HH sitostanol campestanol

Figure 5.104 STEROIDS 257

O CO2H H O H H H HO – H O OAc

H H H HO HO HO H H H fusidic acid protosteryl cation

O H – CO2

CO2H OH O HO H O H fusidic acid

Figure 5.105 for the semi-synthesis of medicinal steroids (see Vitamin D pages 266, 279). For many years, only stigmas- 22 ∗ terol was utilized, since the ∆ double bond Vitamin D3 (colecalciferol, cholecalciferol) allowed chemical degradation of the side-chain to is a sterol metabolite formed photochemically be effected with ease. The utilization of sitosterol in animals from 7-dehydrocholesterol by the was not realistic until microbiological processes sun’s irradiation of the skin (Figure 5.106). for removal of the saturated side-chain became 7-Dehydrocholesterol is the immediate ∆5,7 available. diene precursor of cholesterol (see page 234), Fusidic acid∗ (Figure 5.105), an antibacterial and a photochemical reaction allows ring agent from Acremonium fusidioides, has no addi- opening to precholecalciferol. A thermal 1,7- tional side-chain alkylation, but has lost one C-4 hydrogen shift follows to give colecalciferol methyl and undergone hydroxylation and oxida- (vitamin D3). Vitamin D3 is also manufactured tion of a side-chain methyl. Its relationship to the photosynthetically by the same route. Vitamin ∗ protosteryl cation is shown in Figure 5.105. The D2 (ergocalciferol) may be obtained from stereochemistry in fusidic acid is not typical of ergosterol in exactly the same way, and, although most steroids, and ring B adopts a boat confor- found in plants and yeasts, large amounts are mation; the molecular shape is comparable to the obtained semi-synthetically by the sequence shown protosteryl cation (Figure 5.57, page 216). in Figure 5.106, using ergosterol from yeast

Fusidic Acid Fusidic acid (Figure 5.105) is a steroidal antibiotic produced by cultures of the fungus Acremonium fusidioides (formerly Fusidium coccineum). It has also been isolated from several Cephalosporium species. Fusidic acid and its salts are narrow-spectrum antibiotics active against Gram-positive bacteria. It is primarily used, as its sodium salt, in infections caused by penicillin-resistant Staphylococcus species, especially osteomyelitis since fusidic acid concentrates in bone. It is usually administered in combination with another antibiotic to minimize development of resistance. Fusidic acid reversibly inhibits protein biosynthesis at the translocation step by binding to the larger subunit of the ribosome (see page 407). 258 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

1,7-hydrogen shift; this should be considered as an extended version of an allylic isomerization:

H HC CH CH2 H absorption of light energy by π-electron however, there is intramolecular transfer of the proton system allows electrocyclic ring opening rather than employing an external source

H hν H H H H HO HO 7-dehydrocholesterol rotation about C–C precholecalciferol single bond OH

OH OH

9 O O 8 H H H 7 6 kidney liver 5 10 4

1 HO OH HO HO 3 2 calcitriol calcidiol colecalciferol (cholecalciferol) (1α,25-dihydroxyvitamin D3) (25-hydroxyvitamin D3) (vitamin D3)

H hν H H H H HO ergosterol

OH HO ergocalciferol pre-ergocalciferol (vitamin D2)

Figure 5.106

(Saccharomyces cerevisiae). Vitamin D3 is not ring-opened analogue of loganin, page 189). The itself the active form of the vitamin, and in the numbering system for steroids is also retained, and body it is hydroxylated first to calcidiol and vitamin D3 becomes a derivative of 9,10-seco- then to calcitriol (Figure 5.106). Colecalciferol cholestane, namely (5Z,7E)-9,10-secocholesta-5, and calcitriol have also been found in several 7,10(19)-trien-3β-ol, ‘9,10’ indicating the site of plant species. ring cleavage. Note that it is necessary to indi- Systematic nomenclature of vitamin D deriva- cate the configuration of two of the double bonds, tives utilizes the obvious relationship to steroids, and the somewhat confusing β-configuration for and the term seco (ring opened) is incorporated the 3-hydroxy shows it is actually the same as in into the root name (compare secologanin as a cholesterol. STEROIDS 259

Vitamin D

Vitamin D3 (colecalciferol, cholecalciferol) (Figure 5.106) is the main form of the fat- soluble vitamin D found in animals, though vitamin D2 (ergocalciferol) (Figure 5.106) is a constituent of plants and yeasts. Vitamin D3 is obtained in the diet from liver and dairy products such as butter, cream, and milk, whilst large amounts can be found in fish liver oils, e.g. cod liver oil and halibut liver oil (Table 3.2). Further requirements are produced naturally when the sterol 7-dehydrocholesterol is converted into colecalciferol by the effects of UV light on the skin. With a proper diet, and sufficient exposure to sunshine, vitamin D deficiency should not occur. Vitamin D deficiency leads to rickets, an inability to calcify the collagen matrix of growing bone, and is characterized by a lack of rigidity in the bones, particularly in children. In adults, osteoporosis may occur. In most countries, foods such as milk and cereals are usually fortified with vitamin D3, obtained commercially by UV irradiation of 7-dehydrocholesterol which is produced in quantity by semi-synthesis from cholesterol. Vitamin D2 has a similar activity in humans and is manufactured by UV irradiation of yeast, thereby transforming the ergosterol content. Other compounds with vitamin D activity have also been produced: vitamin D4 from 22,23-dihydroergosterol, vitamin D5 from 7-dehydrositosterol, vitamin D6 from 7-dehydrostigmasterol, and vitamin D7 from 7-dehydrocampesterol. Vitamin D1 was an early preparation, later shown to be a mixture of vitamin D2 and a photochemical by-product lumisterol (9β,10α-ergosterol). Vitamin D is unstable to heat, light, and air. Vitamin D3 is not itself the active form of the vitamin, and in the body it is hydroxylated firstly to 25-hydroxyvitamin D3 (calcidiol) (Figure 5.106) by an enzyme in the liver, and then to 1α,25-dihydroxyvitamin D3 (calcitriol) by a kidney enzyme. Calcitriol is then transported to the bones, intestine, and other organs. It stimulates the absorption of calcium and phosphate in the intestine and the mobilization of calcium from bone. Calcitriol and other analogues, e.g. alfacalcidol and dihydrotachysterol (Figure 5.107) are available for use where chronic vitamin D deficiency is due to liver or kidney malfunction. The long term use of calcitriol and alfacalcidol (1α-hydroxyvitamin D3) in the treatment of osteoporosis may lead to toxic effects arising from elevated serum calcium levels. Toxicity is much reduced in the related 1α-hydroxyvitamin D2, and this agent is being investigated further. Vitamin D is also known to have other physiological functions, including a role in immune suppression, hormone secretion, and the differentiation of both normal and malignant cells.

OH OH

H H H H

HO OH HO HO OH HO OH alfacalcidol dihydrotachysterol calcipotriol tacalcitol

Figure 5.107

(Continues) 260 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

Two vitamin D derivatives, calcipotriol and tacalcitol (Figure 5.107) are widely used in the topical treatment of psoriasis, to inhibit the cell proliferation characteristic of this condition. Vitamin D2 is also employed as a rodenticide. High doses are toxic to rats and mice, the vitamin causing fatal hypercalcaemia.

Bile Acids unit as propionyl-CoA. Other essential features of the molecule are introduced earlier. The A/B ring ∗ The bile acids are C24 steroidal acids that occur system is cis-fused, and this is achieved by reduc- in salt form in bile, secreted into the gut to emul- tion of a ∆4 rather than a ∆5 double bond (see sify fats and encourage digestion. They act as page 241). Migration of the double bond is accom- detergents by virtue of their relatively non-polar plished via the 3-ketone, and when this is reduced steroid nucleus and the polar side-chain, which back to a hydroxyl the configuration at C-3 is contains a carboxylic acid group, that is typically changed to 3α.Bothcholic acid and chenodeoxy- bound via an amide linkage to glycine or taurine. cholic acid (Figure 5.110) are formed in the liver, Thus, for example, cholic acid (Figure 5.108) is though the 7α-hydroxyl functions of these com- found as sodium glycocholate and sodium tauro- pounds can be removed by intestinal microflora, cholate. Metabolism to bile acids is also the prin- so that mammalian bile also contains deoxycholic cipal way in which mammals degrade cholesterol acid and lithocholic acid (Figure 5.110). The bile absorbed from the diet. These structures are formed salts are then usually reabsorbed and stored in in the liver from cholesterol by a sequence which the gall bladder, although they are also excreted oxidizes off three carbons from the side-chain as the body’s main means of eliminating excess (Figure 5.109). This is achieved by initial oxida- cholesterol. Inability to remove cholesterol by bile tion of one of the side-chain methyls to an acid, fol- acid synthesis and excretion may contribute to lowedbyaβ-oxidation sequence as seen with fatty atherosclerosis and gallstone disease; gallstones acids (see Figure 2.11), removing the three-carbon often contain more than 70% of cholesterol.

O

HO N CO2 Na H H H NCOH CO H 2 2 HO 2 glycine H H 12 HO OH H H sodium glycocholate

O 3 5 H H SO3H HO 7 OH H2N HO SO3 Na H taurine N H cholic acid H Characteristic features of bile acids: H H • C24 cholane skeleton • cis-fusion of A/B rings HO OH H • C5-carboxylic acid side-chain sodium taurocholate • 3α- and 7α-hydroxyls

Figure 5.108 STEROIDS 261

7α-hydroxylation; oxidation of reduction of 4,5-double bond gives 3-hydroxyl leads to ∆4-3-ketone, cis-fused rings; stereospecific reduction of O then 12α-hydroxylation carbonyl generates 3α-hydroxyl

HO HO 12

H H H O

3 H H H H H H 7 oxidation of HO O OH HO OH methyl to carboxyl O H cholesterol O CO H HO 2 HO COSCoA HO CO2H

H β H H -oxidation HSCoA H H H H H H ATP HO OH HO OH HO OH H H H shortening of side-chain; activation of acid to cholic acid β-oxidation removes coenzyme A ester propionyl-CoA

Figure 5.109

R CO2H R CO2H

H intestinal H microflora H H H H HO OH HO H H R = OH, cholic acid R = OH, deoxycholic acid R = H, chenodeoxycholic acid R = H, lithocholic acid

Figure 5.110

Bile Acids Bile acids are obtained by purification from fresh ox bile taken from carcasses as a by- product of the meat trade. Chenodeoxycholic acid (Figure 5.110) and ursodeoxycholic acid (Figure 5.111) are used to dissolve cholesterol gallstones as an alternative to surgery. By suppressing synthesis of both cholesterol and cholic acid, they contribute to removal of biliary cholesterol and consequently a gradual dissolution of gallstones which may have formed due to supersaturation. Partial or complete dissolution requires treatment over a period of many months, and is not effective for radio-opaque gallstones, which contain appreciable levels of calcium salts. Dehydrocholic acid (Figure 5.111) may be used, after surgery, to improve biliary drainage. Anion-exchange resins such as colestyramine (cholestyramine)andcolestipol are used as cholesterol-lowering drugs to bind bile acids and prevent their reabsorption. This promotes hepatic conversion of cholesterol into bile acids, thus increasing breakdown of low density lipoprotein cholesterol, and is of value in treating high risk coronary patients.

(Continues) 262 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

CO H 2 O CO2H

H H

H H H H HO OH O O H H ursodeoxycholic acid dehydrocholic acid

Figure 5.111

Bile acids are still important as starting materials for the semi-synthesis of other medicinal steroids, being a cheap and readily accessible raw material.

Adrenocortical in inflammatory processes. Mineralocorticoids are Hormones/Corticosteroids concerned with the control of electrolyte balance, active compounds promoting the retention of Na+ A large number of steroid hormones have been iso- and Cl−, and the excretion of K+. lated and characterized from the adrenal glands. Examples of natural glucocorticoids include Since they are produced by the adrenal cortex, hydrocortisone (cortisol)andcorticosterone the outer part of the adrenal glands near the kid- (Figure 5.112), whilst aldosterone and desoxy- neys, they are termed adrenocortical hormones corticosterone (cortexone) (Figure 5.113) typify ∗ or corticosteroids . They contain a pregnane C21 mineralocorticoids. Desoxycorticosterone has also skeleton and fall into two main activity groups, been found in plants. Some common features the glucocorticoids and the mineralocorticoids, of these molecules are the β-CO.CH2OH side- although it is difficult to separate entirely the chain at C-17, and frequently an α-hydroxy also two types of activity in one molecule. Glucocor- at this position. Ring A usually contains a ∆4- ticoids are concerned with the synthesis of carbo- 3-keto functionality. The 11β-hydroxy is essen- hydrate from protein, and deposition of glycogen tial for glucocorticoid activity. In aldosterone, the in the liver. They also play an important role principal mineralocorticoid hormone, the methyl

OH OH OH O O O HO 11 17 OH O OH HO H H H

3 H H H H H H 4 O O O hydrocortisone cortisone corticosterone (cortisol) Characteristic features of glucocorticoids: • C21 pregnane skeleton • 17β-CO.CH2OH side-chain • 11β-hydroxyl • ∆4-3-keto (usually) • 17α-hydroxyl (usually)

Figure 5.112 STEROIDS 263 group (C-18) has been oxidized to an aldehyde, involves side-chain cleavage as seen in the biosyn- and this is able to react with the 11β-hydroxyl, so thesis of cardioactive glycosides (see page 243) that aldosterone exists predominantly in the hemi- and the same sequence of reactions is operative. acetal form (Figure 5.113). This essentially elimi- From progesterone, the formation of desoxycor- nates the glucocorticoid activity. ticosterone, corticosterone,andhydrocortisone The corticosteroids are produced from choles- (cortisol) (Figure 5.114) requires only a series terol via pregnenolone and progesterone.This of hydroxylation steps, catalysed by cytochrome

OH OH OH HO O O O OHC O HO 17 H H H

3 H H H H H H 4 O O O aldosterone aldosterone desoxycorticosterone (hemiacetal form) (cortexone) Characteristic features of mineralocorticoids: • C21 pregnane skeleton • 17β-CO.CH2OH side-chain • ∆4-3-keto (usually)

Figure 5.113

17α-hydroxylation O O O 17 OH H O2 H H O2 H NADPH NAD+ NADPH H H H H H H H H HO HO O O cholesterol pregnenolone progesterone 17α-hydroxyprogesterone O side-chain degradation via 2 21-hydroxylation O2 20,22-dihydroxycholesterol NADPH NADPH (see Figure 5.92) OH OH O O OH 11 11 H H

H H H H O O desoxycorticosterone 11-deoxycortisol

O2 11β-hydroxylation O2 NADPH NADPH OH OH OH sequential oxidation of O 18 O O OHC 18-methyl HO HO HO OH O2 H NADPH H H

H H H H H H O O O aldosterone corticosterone hydrocortisone (cortisol)

Figure 5.114 264 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

P-450-dependent hydroxylases with NADPH and O O O2 cofactors. Thus, positions 17, 21 and 11 may be hydroxylated, and the exact order can in fact vary from that shown in Figure 5.114, accord- HO 11 ing to species. It can be seen that production of H hydrocortisone from cholesterol actually utilizes H OH cytochrome P-450-dependent enzymes in four of HO the five steps. The further oxidation of C-18 to H an aldehyde via the alcohol allows formation of sarmentogenin aldosterone from corticosterone, again involving O O a P-450 system. 12 H

H O H Semi-Synthesis of Corticosteroids H H HO The medicinal use of corticosteroids was stimu- H lated by reports of the dramatic effects of cortisone hecogenin on patients suffering from rheumatoid arthritis in the late 1940s and early 1950s. The cortisone Figure 5.115 employed was isolated from the adrenal glands of cattle, and later was produced semi-synthetically modifications in ring C are shown in Figure 5.116. by a laborious process from deoxycholic acid (see Bromination α to the 12-keto function gener- page 260) isolated from ox bile and necessitating ates the 11α-bromo derivative, which on treatment over 30 chemical steps. Increased demand for cor- with base gives the 12-hydroxy-11-ketone by a tisone and hydrocortisone (cortisol) (it had been base-catalysed keto–enol tautomerism mechanism. shown that cortisone was reduced in the liver to The 12-hydroxyl is then removed by hydride dis- hydrocortisone as the active agent) led to exploita- placement of the acetate using calcium in liquid tion of alternative raw materials, particularly plant ammonia. The new 11-keto sapogenin is sub- sterols and saponins. A major difficulty in any jected to the side-chain degradation used with other semi-synthetic conversion was the need to provide sapogenins, e.g. diosgenin (see Figure 5.119), to the 11β-hydroxyl group which was essential for the 11-ketopregnane (Figure 5.117) which can then glucocorticoid activity. be used for conversion into cortisone, hydrocorti- Sarmentogenin (Figure 5.115) had been iden- sone, and other steroid drugs. tified as a natural 11-hydroxy cardenolide in Stro- Of much greater importance was the discovery phanthus sarmentosus but it was soon appreciated in the mid-1950s that hydroxylation at C-11 could that the amounts present in the seeds, and the lim- be achieved via a microbial fermentation. Pro- ited quantity of plant material available, would gesterone was transformed by Rhizopus arrhizus not allow commercial exploitation of this com- into 11α-hydroxyprogesterone (Figure 5.118) in pound. As an alternative to using a natural yields of up to 85%. More recently, Rhizopus 11-oxygenated substrate, compounds containing a nigricans has been employed, giving even higher 12-oxygen substituent might be used instead, in yields. 11α-Hydroxyprogesterone is then converted that this group activates position 11 and allows into hydrocortisone by chemical means, the 11β chemical modification at the adjacent site. Indeed, configuration being introduced via oxidation to this was a feature of the semi-synthesis of corti- the 11-keto and then a stereospecific reduction sone from deoxycholic acid, which contains a 12α- step. Progesterone could be obtained in good hydroxyl. Hecogenin (Figure 5.115) from sisal yields (about 50%) from diosgenin extracted (Agave sisalana; Agavaceae) (see page 240), a from Mexican yams (Dioscorea species; Dioscore- steroidal sapogenin with a 12-keto function, made aceae) (see page 239) or stigmasterol from soya possible the economic production of cortisone on a beans (Glycine max; Leguminosae/Fabaceae) (see commercial scale. This material is still used in the page 256). Steroidal sapogenins such as diosgenin semi-synthesis of steroidal drugs, and the critical maybedegradedbytheMarker degradation STEROIDS 265

α bromination SN2 displacement O to carbonyl O O OH 12 Br HO HO 11 Br2 OH H H H H base-catalysed HH HH HH HH keto–enol–keto tautomerism; the 11-keto form is SN2 displacement; preferred, probably hydride as nucleophile because in the 12-keto AcO HO form, the carbonyl is 11 O Ca / NH O O adjacent to the 3 Ac2O H H H five-membered D-ring

HH HH H H

Figure 5.116

O side-chain O H degradation O O O H H hydrocortisone hecogenin H (cortisol) H H Marker degradation H H (see Figure 5.119) AcO HO H H 3β-hydroxypregnan-11,18-dione

Figure 5.117

O O HO 11 Rhizopus arrhizus H H or R. nigricans chemically hydrocortisone H H > 85% H H (cortisol) O O progesterone 11α-hydroxyprogesterone

Figure 5.118

(Figure 5.119), which removes the spiroketal por- and stereoselective manner, addition of hydrogen tion, leaving carbons C-20 and C-21 still attached being from the less-hindered α-face to give preg- to contribute to the pregnane system. Initial treat- nenolone acetate. Progesterone is obtained by ment with acetic anhydride produces the diacetate, hydrolysis of the ester function and Oppenauer oxi- by opening the ketal, dehydrating in ring E, and dation to give the preferred α,β-unsaturated ketone acetylating the remaining hydroxyls. The double (see page 241). It is immediately obvious from bond in ring E is then selectively oxidized to give Figure 5.119 that, since the objective is to remove a product, which now contains the unwanted side- the unwanted ring F part of the sapogenin, fea- chain carbons as an ester function, easily removed tures like the stereochemistry at C-25 are irrele- by hydrolysis. Under the conditions used, the prod- vant, and the same general degradation procedure uct is the α,β-unsaturated ketone. Hydrogenation can be used for other sapogenins. It is equally of the double bond is achieved in a regioselective applicable to the nitrogen-containing analogues of 266 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

Marker Degradation of Diosgenin AcO Ac O H AcO

O O

involves opening of spiroketal and acetylation of 26-hydroxyl 21 O followed by loss of proton; AcO 20 F 25 3-hydroxyl is also esterified H H E 22 O O H Ac2O / ∆ H H H H H H H HO AcO selective oxidation of diosgenin 20,22 double bond CrO3

ester hydrolysis; resultant AcO O O alcohol dehydrates to form O conjugated system H H O HOAc / ∆ H H H H AcO AcO

dehydropregnenolone acetate selective catalytic Oppenauer oxidation of 3-hydroxyl hydrogenation of 16,17 H2 / Pd to ketone, with tautomerism to give double bond occurs from conjugated system O O O less-hindered α face i Al(O Pr)3 H H H OH Me2CO H H H H H H AcO HO O

pregnenolone acetate pregnenolone progesterone

Figure 5.119 sapogenins, e.g. solasodine (Figure 5.88). In such first reaction. A similar route can be used for compounds, the stereochemistry at C-22 is also the fungal sterol ergosterol, though an additional quite immaterial. step is required for reduction of the ∆7 dou- Degradation of the sterol stigmasterol to pro- ble bond. gesterone is achieved by the sequence shown in An alternative sequence from diosgenin to Figure 5.120. The double bond in the side-chain hydrocortisone has been devised, making use allows cleavage by ozonolysis, and the resul- of another microbiological hydroxylation, this tant aldehyde is chain shortened via formation of time a direct 11β-hydroxylation of the steroid an enamine with piperidine. This can be selec- ring system (Figure 5.121). The fungus Curvu- tively oxidized to progesterone. In this sequence, laria lunata is able to 11β-hydroxylate cortex- the ring A transformations are carried out as the olone to hydrocortisone in yields of about 60%. STEROIDS 267

Oppenauer oxidation of 3-hydroxyl to ketone, with ozonolysis to cleave tautomerism to give side-chain double bond conjugated system O

Al(OiPr) H 3 H H Me2CO O3 A H H H H H H HO O O stigmasterol enamine formation: Schiff base (iminium) formed first, HN followed by base-catalysed iminium−enamine tautomerism O N

H H CrO3 H H H H O O progesterone selective oxidation of side-chain double bond

Figure 5.120

diosgenin Marker degradation OH OH (Figure 5.119) O O O OH HO OH 11 H chemically H Curvularia lunata H

H H H H 60% H H AcO O O dehydropregnenolone cortexolone hydrocortisone acetate (cortisol)

Figure 5.121

Although a natural corticosteroid, cortexolone may processes are in general more expensive than be obtained in large amounts by chemical transfor- chemical transformations, and are only used com- mation from 16-dehydropregnenolone acetate, an mercially when some significant advantage is intermediate in the Marker degradation of dios- achieved, e.g. replacement of several chemical genin (Figure 5.119). steps. The therapeutic properties of cortisone and Some steroid drugs are produced by total syn- hydrocortisone can be further improved by the thesis, but, in general, the ability of microor- microbial introduction of a 1,2-double bond, giv- ganisms to biotransform steroid substrates has ing prednisone and prednisolone respectively proved invaluable in exploiting inexpensive nat- (Figure 5.122). These agents surpass the parent ural steroids as sources of drug materials. It hormones in antirheumatic and antiallergic activity is now possible via microbial fermentation to with fewer side effects. As with cortisone, pred- hydroxylate the steroid nucleus at virtually any nisone is converted in the body into the active position and with defined stereochemistry. These agent, in this case prednisolone. 268 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

OH OH O O R2 R2 R1 OH R1 OH 1 H Arthrobacter simplex H 2 H H 60–90 % H H O O R1R2 = O, cortisone R1R2 = O, prednisone R1 = OH, R2 = H, hydrocortisone (cortisol) R1 = OH, R2 = H, prednisolone

Figure 5.122

Corticosteroid Drugs

Glucocorticoids are primarily used for their antirheumatic and anti-inflammatory activities. They give valuable relief to sufferers of rheumatoid arthritis and osteoarthritis, and find considerable use for the treatment of inflammatory conditions by suppressing the characteristic development of swelling, redness, heat, and tenderness. They exert their action by interfering with prostaglandin biosynthesis, via production of a peptide that inhibits the phospholipase enzyme responsible for release of arachidonic acid from phospholipids (see page 55). However, these agents merely suppress symptoms and they do not provide a cure for the disease. Long term usage may result in serious side-effects, including adrenal suppression, osteoporosis, ulcers, fluid retention, and increased susceptibility to infections. Because of these problems, steroid drugs are rarely the first choice for inflammatory treatment, and other therapies are usually tried first. Nevertheless corticosteroids are widely used for inflammatory conditions affecting the ears, eyes, and skin, and in the treatment of burns. Some have valuable antiallergic properties helping in reducing the effects of hay fever and asthma. In some disease states, e.g. Addison’s disease, the adrenal cortex is no longer able to produce these hormones, and replacement therapy becomes necessary. The most common genetic deficiency is lack of the 21-hydroxylase enzyme in the biosynthetic pathway, necessary for both hydrocortisone and aldosterone biosynthesis (Figure 5.114). This can then lead to increased synthesis of androgens (see Figure 5.133). Mineralocorticoids are primarily of value in maintaining electrolyte balance where there is adrenal insufficiency. Natural corticosteroid drugs cortisone (as cortisone acetate)andhydrocortisone (cortisol) (Figure 5.112) are valuable in replacement therapies, and hydrocortisone is one of the most widely used agents for topical application in the treatment of inflammatory skin conditions. The early use of natural corticosteroids for anti-inflammatory activity tended to show up some serious side-effects on water, mineral, carbohydrate, protein, and fat metabolism. In particular, the mineralocorticoid activity is usually considered an undesirable effect. In an effort to optimize anti-inflammatory activity, many thousands of chemical modifications to the basic structure were tried. Introduction of a ∆1 double bond modifies the shape of ring A and was found to increase glucocorticoid over mineralocorticoid activity, e.g. prednisone and prednisolone (Figure 5.122). A 9α-fluoro substituent increased all activities, whereas 16α-or16β-methyl groups reduced the mineralocorticoid activity without affecting

(Continues) STEROIDS 269

(Continued)

esterification with tosyl chloride SN2 displacement to generate good leaving group does not occur

11 AcO HO TsCl TsO AcO 9 H H H

H H H H H H

base-catalysed E1 elimination AcO

HO HO HO SOCl2 / Py Br2 HO H H Br H H

H H H H Br H E2 elimination bromination from less-hindered α face

base-catalysed AcO intramolecular SN2 gives epoxide

HO HF H O H

F H H acid-catalysed opening of epoxide (favouring trans-fused ring system)

Figure 5.123

the glucocorticoid activity. The discovery that 9α-fluoro analogues had increased activity arose indirectly from attempts to epimerize 11α-hydroxy compounds into the active 11β-hydroxy derivatives (Figure 5.123). Thus, when an 11α-tosylate ester was treated with acetate, a base-catalysed elimination was observed rather than the hoped-for substitution, which is hindered by the methyl groups (Figure 5.123). This syn elimination suggests an E1 mechanism is involved. The same ∆9(11)-ene can also be obtained by dehydration of the 11β-alcohol by using thionyl chloride. Addition of HOBr to the 9(11)-double bond proceeds via electrophilic attack from the less-hindered α-face, giving the cyclic bromonium ion, and then ring opening by β-attack of hydroxide at C-11. Attack at C-9 is sterically hindered by the methyl at C-10. 9α- Bromocortisol 21-acetate produced in this way was less active as an anti-inflammatory than cortisol 21-acetate by a factor of three, and 9α-iodocortisol acetate was also less active by a factor of ten. Fluorine must be introduced indirectly by the β-epoxide formed by base treatment of the 9α-bromo-10β-hydroxy analogue (Figure 5.123). The resultant 9α-fluorocortisol 21- acetate (fluorohydrocortisone acetate; fludrocortisone acetate) (Figure 5.124) was found to be about 11 times more active than cortisol acetate. However, its mineralocorticoid activity was

(Continues) 270 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

OAc OH OH OH O O O O HO OH HO OH HO OH HO OH H H H H

F H H H F H F H O O O O fludrocortisone acetate dexamethasone betamethasone (9α-fluorocortisol acetate) methylprednisolone OH OCOEt FH2CS O O O HO O HO OCOEt HO OCOEt H H O H F H X H F H O O O betamethasone 17-valerate X = F, betamethasone 17,21-dipropionate F X = Cl, beclometasone (beclomethasone) fluticasone propionate 17,21-dipropionate Cl Cl

O O O O HO OH HO OCOEt O OCOPr HO H H H H

F H F H F H H H O O O O

fluorometholone clobetasol 17-propionate clobetasone 17-butyrate rimexolone

Figure 5.124

also increased some 300-fold, so its anti-inflammatory activity has no clinical relevance, and it is only employed for its mineralocorticoid activity. The introduction of a 9α-fluoro substituent into prednisolone causes powerful Na+ retention. These effects can be reduced (though usually not eliminated entirely) by introducing a substituent at C-16, either a 16α-hydroxy or a 16α/16β-methyl. The 16α-hydroxyl can be introduced microbiologically, e.g. as in the conversion of 9α-fluoroprednisolone into triamcinolone (Figure 5.125). The ketal formed from triamcinolone and acetone, triamcinolone acetonide (Figure 5.125) provides a satisfactory means of administering this anti-inflammatory by topical application in the treatment of skin disorders such as psoriasis. Methylprednisolone (Figure 5.124) is a 6α-methyl derivative of prednisolone showing a modest increase in activity over the parent compound. A 6-methyl group can be supplied by reaction of the Grignard reagent MeMgBr with a suitable 5,6-epoxide derivative. Dexamethasone and betamethasone (Figure 5.124) exemplify respectively 16α- and 16β-methyl derivatives in drugs with little, if any, mineralocorticoid activity. The 16-methyl group is easily introduced by a similar Grignard reaction with an appropriate α,β-unsaturated ∆16-20-ketone. Betamethasone, for topical application, is typically formulated as a C-17 ester with valeric acid (betamethasone 17-valerate), or as

(Continues) STEROIDS 271

(Continued)

OH OH OH O O O O HO OH HO OH HO O H 16 Streptomyces H OH H roseochromogenus Me2CO F H F H F H O O O 9α-fluoroprednisolone triamcinolone triamcinolone acetonide

Figure 5.125

the 17,21-diester with propionic acid (betamethasone 17,21-dipropionate) (Figure 5.124). The 9α-chloro compound beclometasone 17,21-dipropionate (beclomethasone 17,21- dipropionate) is also an important topical agent for eczema and psoriasis, and as an inhalant for the control of asthma. Fluticasone propionate (Figure 5.124) is also used in asthma treatment, and is representative of compounds where the 17-side-chain has been modified to a carbothiate (sulphur ester). Although the anti-inflammatory activity of hydrocortisone is lost if the 21-hydroxyl group is not present, considerable activity is restored when a 9α-fluoro substituent is introduced. Fluorometholone (Figure 5.124) is a corticosteroid that exploits this relationship and is of value in eye conditions. Other agents are derived by replacing the 21-hydroxyl with a halogen, e.g. clobetasol 17-propionate and clobetasone 17- butyrate (Figure 5.124), which are effective topical drugs for severe skin disorders. In rixemolone, a recently introduced anti-inflammatory for ophthalmic use, neither a 21-hydroxy nor a 9α-fluoro substituent is present, but instead there are 17α- and 16α-methyl substituents. Rimexolone has significant advantages in eye conditions over drugs such as dexamethasone, in that it does not significantly raise intraocular pressure. Many other corticosteroids are currently available for drug use. Structures of some of these are given in Figure 5.126, grouped according to the most characteristic structural features, namely 16-methyl, 16-hydroxy, and 21-chloro derivatives. The recently introduced deflazacort (Figure 5.127) is a drug with high glucocorticoid activity, but does not conveniently fit into any of these groups in that it contains an oxazole ring spanning C-16 and C-17. Trilostane (Figure 5.127) is an adrenocortical suppressant, which inhibits synthesis of glucocorticoids and mineralocorticoids and has value in treating Cushing’s syndrome, characterized by a moon-shaped face and caused by excessive glucocorticoids. This drug is an inhibitor of the dehydrogenase–isomerase that transforms pregnenolone into progesterone (Figures 5.92 and 5.114). Spironolactone (Figure 5.127) is an antagonist of the endogenous mineralocorticoid aldosterone and inhibits the sodium-retaining action of aldosterone whilst also decreasing the potassium-secreting effect. Classified as a potassium-sparing diuretic, it is employed in combination with other diuretic drugs to prevent excessive potassium loss. Progesterone (page 273) is also an aldosterone antagonist; the spironolactone structure differs from progesterone in its 7α-thioester substituent, and replacement of the 17β side-chain with a17α-spirolactone.

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(Continued)

OH OH OH OH O O O O HO OH HO HO HO OH H H H H

H H F H F H F H O Cl O O O F F alclometasone desoximetasone diflucortolone flumetasone (flumethasone) (used as 17,21-dipropionate) (desoxymethasone) (used as 21-valerate) [used as pivalate (trimethylacetate)]

OH OH OH OH O O O O O RS O O HO HO HO HO O H H O H O H

H H H H H H H H O O O O F F F fluocortolone budesonide fludroxycortide flunisolide (used as 21-hexanoate) (flurandrenolone)

OH OAc Cl Cl O O O O O O O HO HO HO HO OH O O O H H H H

F H F H F H Cl H O O O O F F mometasone fluocinolone acetonide fluocinonide halcinonide (used as 17-furoate)

Figure 5.126

OAc O O N OH O HO H H O H NC H H HH H H O S O O O O deflazacort trilostane spironolactone

Figure 5.127

Progestogens (Figure 5.128), which is secreted by the corpus luteum following release of an ovum. Progesterone Progestogens∗ (progestins; gestogens) are female is also an intermediate in the biosynthesis of the sex hormones, concerned with preparing the uterus corticosteroids, e.g. hydrocortisone and aldosterone for pregnancy, and then maintaining the necessary (see page 263), and its derivation from cholesterol conditions. There is only one naturally occurring via pregnenolone has already been seen in the for- progestational steroid and that is progesterone mation of cardioactive glycosides (see page 243). STEROIDS 273

O Oestrogens

H The oestrogens (US spelling: estrogens)are female sex hormones produced in the ovaries, and H H 3 4 also in the placenta during pregnancy. They are O responsible for the female sex characteristics, and progesterone together with progesterone control the menstrual Characteristic features of progestogens: cycle. Oestrogens were first isolated from the urine

• C21 pregnane skeleton of pregnant women, in which levels increase some • ∆4-3-keto 50-fold during the pregnancy. In horses, levels rise by as much as 500 times during pregancy. Oestro- Figure 5.128 gens occur both in free form, and as glucuronides

Progestogen Drugs

Quantities of progesterone (Figure 5.128) for drug use are readily available by semi-synthesis using the Marker degradation (see page 264). However, progesterone is poorly absorbed, and it is not suitable for oral use, being rapidly metabolized in the liver. Many semi-synthetic analogues have been produced, and it was thus appreciated that the α,β-unsaturated ketone system in ring A was essential for activity. The side-chain function at C-17 could be modified, and ethisterone (17α-ethynyltestosterone) (Figure 5.129), developed as a potential androgen, was found to be active orally as a progestational agent. This incorporates an ethynyl side-chain at C-17, a feature of several semi-synthetic steroidal hormones used as drugs. This group, referred to as ‘ethinyl’ in drug molecules, is introduced by nucleophilic

′ R OH2 R′ H2O R′ N N NH diosgenin R R O R hydrolysis of amide function Beckmann rearrangement: oxime → amide Marker gives enamine; tautomerism to degradation imine then allows further oxime formation (Figure 5.119) OH O hydrolysis to ketone O N HN O

NH2OH ArSO2Cl / Py H HCl Beckmann H H H rearrangement H H AcO hydrolysis of dehydropregnenolone HO ester at C-3 acetate Oppenauer OH oxidation OH O

19 Al(OiPr) HC C Na H 3 H H Me2CO liq NH3 H H H H H H O HO nucleophilic attack of HO ethisterone acetylide anion on to carbonyl androstenolone from less-hindered α face

Figure 5.129

(Continues) 274 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

O OH OAc 17 H H H H 14 H H

H H H H H H O O O 19-nor-14β,17α-progesterone norethisterone norethisterone acetate

Figure 5.130

attack of acetylide anion on to a C-17 carbonyl (Figure 5.129), attack coming from the α-face, the methyl C-18 hindering approach from the β-face. The substrate androstenolone is readily obtained from the Marker degradation intermediate dehydropregnenolone acetate (Figure 5.119). The oxime (Figure 5.129) is treated with a sulphonyl chloride in pyridine and undergoes a Beckmann rearrangement in which C-17 migrates to the nitrogen giving the amide. This amide is also an enamine and can be hydrolysed to the 17-ketone. Acetylation or other esterification of the 17-hydroxyl in progestogens increases lipid solubility and extends the duration of action by inhibiting metabolic degradation. Examples include norethisterone acetate, medroxyprogesterone acetate, and hydroxyprogesterone caproate, discussed below. Though considerably better than progesterone, the oral activity of ethisterone is still relatively low, and better agents were required. An important modification from ethisterone was the 19-nor analogue, norethisterone and its ester norethisterone acetate (Figure 5.130). Attention was directed to the 19-norsteroids by the observation that 19-nor- 14β,17α-progesterone (Figure 5.130), obtained by degradation of the cardioactive glycoside strophanthidin (see page 250), displayed eight times higher progestational activity than progesterone, despite lacking the methyl C-19, and having the unnatural configurations at the two centres C-14 (C/D rings cis-fused) and C-17. Norethisterone can be synthesized from the oestrogen estrone (see page 279) which already lacks the C-9 methyl, or from androstenolone (Figure 5.129) by a sequence which allows oxidation of C-19 to a carboxyl, which is readily lost by decarboxylation when adjacent to the α,β-unsaturated ketone system. Although ethisterone and norethisterone are structurally C21 pregnane derivatives, they may also be regarded as 17-ethynyl derivatives of testosterone (see page 282), the male sex hormone, and 19-nortestosterone respectively. Many of the commonly used progestogens fall into these two classes. Semi-synthetic analogues of progesterone, still containing the 17-acetyl side-chain, tend to be derivatives of 17α-hydroxyprogesterone, another biosynthetic intermediate on the way to hydrocortisone (Figure 5.114) that also has progesterone-like activity. Examples include hydroxyprogesterone caproate, and gestonorone (gestronol) caproate (Figure 5.131). Medroxyprogesterone acetate (Figure 5.131) contains an additional 6α-methyl, introduced to block potential deactivation by metabolic hydroxylation, and is 100–300 times as potent as ethisterone on oral administration. Megestrol acetate (Figure 5.131) contains a 6-methyl group and an additional ∆6 double bond. Norgestrel (Figure 5.131) is representative of progestogens with an ethyl group replacing the 13-methyl. Although these can be obtained by semi-synthesis from natural 13-methyl compounds, norgestrel is produced by total synthesis as the racemic compound.

(Continues) STEROIDS 275

(Continued)

O O O O O2CC5H11 O2CC5H11 OAc OAc H H H H H

H H H H H H H H

O O O 6 O hydroxyprogesterone gestonorone (gestronol) caproate (hexanoate) caproate (hexanoate) medroxyprogesterone megestrol acetate acetate

OH OH O OH

H H H H H H H

H H H H H H H H O O O (–): levonorgestrel desogestrel dydrogesterone etonogestrel (±): norgestrel Me2N OAc OH OAc OH

H H H H H H H H H H H H H H HO AcO O N O etynodiol (ethynodiol) gestodene norgestimate mifepristone diacetate

Figure 5.131

Since only the laevorotatory enantiomer which has the natural configuration is biologically active, this enantiomer, levonorgestrel, is now replacing the racemic form for drug use. In desogestrel (Figure 5.131), further features are the modification of an 11-oxo function to an 11-methylene, and removal of the 3-ketone. Structures of some other currently available progestogen drugs are shown in Figure 5.131. During pregnancy, the corpus luteum continues to secrete progesterone for the first three months, after which the placenta becomes the supplier of both progesterone and oestrogen. Progesterone prevents further ovulation and relaxes the uterus to prevent the fertilized egg being dislodged. In the absence of pregnancy, a decline in progesterone levels results in shedding of the uterine endometrium and menstruation. Progestogens are useful in many menstrual disorders, and as oral contraceptives either alone at low dosage (progestogen- only contraceptives, e.g. norethisterone, levonorgestrel) or in combination with oestrogens (combined oral contraceptives, e.g. ethinylestradiol + norethisterone, ethinylestradiol + levonorgestrel). The combined oestrogen–progestogen preparation inhibits ovulation, but normal menstruation occurs when the drug is withdrawn for several days each month. The low dosage progestogen-only pill appears to interfere with the endometrial lining to inhibit fertilized egg implantation, and thickens cervical mucus making a barrier to sperm movement. The progestogen-only formulation is less likely to cause thrombosis, a serious side-effect sometimes experienced from the use of oral contraceptives. There appears to be a slightly higher risk of thrombosis in patients using the so-called ‘third generation’ oral

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(Continued)

contraceptive pills containing the newer progestogens desogestrel and gestodene. Current oral contraceptives have a much lower hormone content than the early formulations of the 1960s and 1970s, typically about 10% of the progestogen and 50% of the oestrogen content. Deep muscular injections of medroxyprogesterone or norethisterone esters, and implants of levonorgestrel can be administered to provide long-acting contraception. A high dose of levonorgestrel, alone or in combination with ethinylestradiol, is the drug of choice for emergency contraception after unprotected intercourse, i.e. the ‘morning-after’ pill. Hormone replacement therapy (HRT) in non-hysterectomized women also uses progestogen–oestrogen combinations (see page 279), whilst progestogens such as norethisterone, megestrol acetate, medroxyprogesterone acetate, and gestonorone caproate also find application in the treatment of breast cancers. Mifepristone (Figure 5.131) is a progestogen antagonist used orally as an abortifacient to terminate pregnancy. This drug has a higher affinity for the progesterone receptor than does the natural hormone, and prevents normal responses. This leads to loss of integrity of the uterine endometrial lining, and detachment of the implanted fertilized egg.

at position 3; they are not restricted to females have an aromatic A ring, a consequence of which since small amounts are produced in the male is that C-19, the methyl on C-10, is absent. There testis. The principal and most potent example is is now no carbon side-chain at C-17, and the basic estradiol (also oestradiol, but US spelling has C18 skeleton is termed estrane. been generally adopted), though only low lev- The biosynthetic pathway to estradiol and els are found in urine, and larger amounts of estrone (Figure 5.133) proceeds from choles- the less active metabolites estrone (oestrone)and terol via pregnenolone and bears a resemblance the 16α-hydroxylated derivative estriol (oestriol) to the hydrocortisone pathway (Figure 5.114) are present (Figure 5.132). Estrone has also been in the early 17-hydroxylation step. Indeed, the found in significant quantities in some plant seeds, same cytochrome P-450-dependent enzyme catal- e.g. pomegranate and date palm. These compounds yses 17-hydroxylation of both pregnenolone and

O

H

H H HO OH estrone (oestrone) H OH

H H H OH HO estradiol H H (oestradiol) HO estriol (oestriol) Characteristic features of oestrogens:

• C18 estrane skeleton • aromatic A ring (consequent loss of C-10 methyl) • no side-chain

Figure 5.132 STEROIDS 277

oxidative removal of O 17α-hydroxylation O side-chain (Figure 5.134) O 17 OH

H O2 H O2 H NADPH NADPH H H H H H H HO HO HO pregnenolone 17α-hydroxypregnenolone dehydroepiandrosterone oxidation of 3-hydroxyl NAD+ and tautomerism to sequential oxidation of C-10 conjugated system O methyl to aldehyde O O

O HO H O2 H O2 H NADPH NADPH H H H H H H aromatase aromatase O O O oxidative loss of C-10 androstenedione O2 formyl and aromatization NADPH of ring A (Figure 5.135) NADH/ aromatase NADPH O OH OH

H NADH/ H O2 H NADPH NADPH H H H H H H aromatase HO HO O estrone estradiol testosterone (oestrone) (oestradiol)

Figure 5.133 progesterone, as well as the next step in oestro- testosterone, is a substrate for aromatization in gen biosynthesis, and it plays a significant role ring A, with loss of C-19. This sequence is also in controlling the direction of steroid synthe- catalysed by a single cytochrome P-450-dependent sis. Whilst 17α-hydroxyprogesterone is trans- enzyme, called aromatase, and the reaction pro- formed by 21-hydroxylation for hydrocortisone ceeds via sequential oxidation of the methyl, with biosynthesis, in oestrogen biosynthesis the α- its final elimination as formic acid (Figure 5.135). hydroxyketone function is oxidized from 17α- The mechanism suggested is analogous to that of hydroxypregnenolone, cleaving off the two-carbon the side-chain cleavage reaction. Formation of the side-chain as acetic acid. The product is the aromatic ring is then a result of enolization. As 17-ketone dehydroepiandrosterone, which is the with other steroid hormones, the exact order of most abundant steroid in the blood of young adult some of the steps, including formation of the ∆4- humans, with levels peaking at about 20 years of 3-keto function, 17-hydroxylation, reduction of the age, then declining as the person ages. Apart from 17-keto, and aromatization in ring A, can vary its role as a precursor of hormones, it presumably according to organism, or site of synthesis in the has other physiological functions, though these still body. Since breast tumours require oestrogens for remain to be clarified. A mechanism for the side- growth, the design of aromatase inhibitors∗ has chain cleavage reaction, initiated by attack of an become an important target for anticancer drug enzyme-linked peroxide, is shown in Figure 5.134, research. and is analogous to that proposed for loss of the The aromatic ring makes the oestrogen molecule 14-methyl group during cholesterol biosynthesis almost planar (see page 233) and is essential for (see page 235). Oxidation and tautomerism in rings activity. Changes which remove the aromatic- A/B then give androstenedione (Figure 5.133). ity, e.g. partial reduction, or alter stereochem- Either androstenedione, or its reduction product istry, give analogues with reduced or no activity. 278 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

peroxy adduct formation via homolytic cleavage nucleophilic attack of of peroxy bond 17α-hydroxylation peroxy-enzyme on to carbonyl OH OH O–Fe–Enz OO–Fe–Enz O O O O2 O 17 OH 2 OH OH NADPH NADPH

Enz–Fe–OOH side-chain lost H H H H as acetic acid

CH3CO2H

O O–Fe–Enz O H

H H

Figure 5.134

peroxy adduct formation via homolytic cleavage sequential oxidation of C-10 methyl to aldehyde nucleophilic attack of of peroxy bond peroxy-enzyme on to carbonyl Enz–Fe–O O HO OH OH O 2 O2 O2 NADPH NADPH NADPH 10 Enz–Fe–OOH O O O O

Enz–Fe–O HCO2H Enz–Fe–O O OH HH enolization

HO O O O side-chain lost as formic acid

Figure 5.135

O Thus, exposure of estrone to UV light leads to OH 13 inversion of configuration at C-13 adjacent to the H carbonyl function, and consequently formation of H H a cis-fused C/D ring system. The product, lumie- HO HO strone (Figure 5.136) is no longer biologically lumiestrone diethylstilbestrol active. Some planar non-steroidal structures can (stilboestrol) OH also demonstrate oestrogenic activity as a result OH O R O of a similar shape and relative spacing of oxygen functions. Thus, the synthetic diethylstilbestrol (stilboestrol) (Figure 5.136) has been widely used HO O OOHO as an oestrogenic drug, and coumestrol, daidzein, coumestrol R = H, daidzein and genistein (Figure 5.136) are naturally occur- R = OH, genistein ring isoflavonoids with oestrogenic properties from Figure 5.136 lucerne, clovers, and soya beans, and are termed STEROIDS 279

Oestrogen Drugs

Oestrogens suppress ovulation and with progestogens form the basis of combined oral contraceptives and hormone replacement therapy (HRT). They are also used to supplement natural oestrogen levels where these are insufficient as in some menstrual disorders, and to suppress androgen formation and thus tumour growth of cancers dependent on androgens, e.g. prostate cancers. Oestrogens appear to offer a number of beneficial effects to women, including protection against osteoporosis, heart attacks, and possibly Alzheimer’s disease. However, some cancers, e.g. breast and uterine cancers, are dependent on a supply of oestrogen for growth, especially during the early stages, so high oestrogen levels are detrimental. Steroidal oestrogens for drug use were originally obtained by processing pregnancy urines, but the dramatic increase in demand due to the introduction of oral contraceptives required development of semi-synthetic procedures. Androstenolone formed via the Marker degradation of diosgenin (Figure 5.129) may be transformed to the dione by catalytic reduction of the ∆5 double bond and oxidation of the 3-hydroxyl (Figure 5.137). This then allows production of androstadienedione by dibromination and base-catalysed elimination of HBr. Alternatively, it is now possible to achieve the synthesis of androstadienedione in a single step by a microbiological fermentation of either sitosterol obtained from soya beans (see page 256), or of cholesterol obtained in large quantities from the woolfat of sheep, or from the spinal cord of cattle (see page 236). These materials lack unsaturation in the side-chain and were not amenable to simple chemical oxidation processes, as for example with stigmasterol (see page 266). Their exploitation required the development of suitable biotransformations, and use of Mycobacterium phlei has now achieved this objective (Figure 5.137). The aromatization step to estrone can be carried out in low yields by vapour-phase free-radical-initiated thermolysis, or more recently with considerably better yields using a dissolving-metal reductive thermolysis. In both processes, the methyl at C-10 is lost. This sequence gives estrone, from which estradiol (oestradiol) may be obtained by reduction of the 17-carbonyl. However, by far the most commonly used medicinal estrogen is ethinylestradiol (ethinyloestradiol) (Figure 5.137), which is 12 times as effective as estradiol when administered orally. This analogue can be synthesized from estrone by treatment with potassium acetylide in liquid ammonia, which attacks from the less-hindered α-face (see page 273). The ethynyl substituent prevents oxidation at C-17, as in the metabolism of estradiol to the less active estrone. The phenol group allows synthesis of other derivatives, e.g. the 3-methyl ether mestranol (Figure 5.137), which acts as a pro-drug, being oxidized in the liver to ethinylestradiol. To retain oestrogenic activity, structural modifications appear effectively limited to the addition of the 17α-ethynyl group, and to substituents on the 3-hydroxyl. The ester estradiol valerate (oestradiol valerate) facilitates prolonged action through slower absorption and metabolism. The lower activity metabolites estriol (oestriol) (about 2% activity of estradiol) and estrone (oestrone) (about 33% activity) (Figure 5.132) are sometimes used in hormone replacement therapy (HRT). Oestrogen and progesterone levels decline naturally at menopause when the menstrual cycle ceases. The sudden reduction in oestrogen levels can lead to a number of unpleasant symptoms, including tiredness, hot flushes, vaginal dryness, and mood changes. HRT reduces these symptoms, and delays other long-term consequences of reduced oestrogen levels, including osteoporosis and atherosclerosis. HRT currently provides the best therapy for preventing osteoporosis, a common disease in post-menopausal women.

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(Continued)

diosgenin Marker degradation and removal of side-chain reduction of double bond; oxidation (Figure 5.129) of 3-hydroxyl brominations O O α to ketone O

H i H2 / catalyst H H ii CrO3 Br2 / HOAc Br H H H H H H HO O O base-catalysed H H androstenolone Br eliminations collidine / ∆ O

cholesterol H H Mycobacterium phlei or H H H H HO sitosterol O androstadienedione vapour-phase H radical-initiated thermolysis H H tetralin / ∆ / 600° or HO Li /biphenyl / 66° dissolving metal O reduction OH SN2 alkylation of OH O phenol

H H HC C Na H Me2SO4 liq NH3 H H H H H H MeO HO HO mestranol ethinylestradiol nucleophilic attack of estrone (ethinyloestradiol) acetylide anion on to (oestrone) carbonyl from less-hindered α face

Figure 5.137

Osteoporosis is characterized by a generalized loss of bone mass leading to increased risk of fracture, and a sharp reduction in endogenous oestrogen levels is recognized as a critical factor. Oestrogen and progestogen combinations are used in HRT unless the woman has had a hysterectomy, in which case oestrogen alone is prescribed. Natural oestrogen structures are preferred to synthetic structures such as ethinylestradiol or mestranol. Before the availability of plant-derived semi-synthetic oestrogens, extraction of urine from pregnant women and pregnant horses allowed production of oestrogen mixtures for drug use. Conjugated equine oestrogens are still widely prescribed for HRT and are obtained by extraction from the urine of pregnant mares and subsequent purification, predominantly in Canada and the USA. Mares are about 120 days pregnant when urine collection begins, and collection continues for another 150–160 days. During this period, a mare will produce about 400–500 litres of urine.

(Continues) STEROIDS 281

(Continued)

O OH O OH 17 H H H N H H HH H H + H H HO HO HO3SO N O equilin dihydroequilin estropipate H tibolone O OH OP OH H

H H HO PO HO O diethylstilbestrol fosfestrol dienestrol OH (stilboestrol) (dienoestrol) formestane NMe2 O OH

H O H H Cl N O Me N Cl 2 O Cl R = H, tamoxifen estramustine R R = Cl, toremifene clomifene (clomiphene)

Figure 5.138

Horses are maintained in an almost continuous state of pregnancy, and need to be kept in confined stalls and fitted with a suitable urine collection device, though the use of catheters to collect urine has been discontinued. Animal welfare groups urge women to reject these drug preparations in favour of plant-derived alternatives. Conjugated equine oestrogens provide a profile of natural oestrogens based principally on estrone and equilin (Figure 5.138). It consists of a mixture of oestrogens in the form of sodium salts of their sulphate esters, comprising mainly estrone (50–60%) and equilin (20–30%), with smaller amounts of 17α-dihydroequilin, 17α-estradiol, and 17β-dihydroequilin. The semi- synthetic estropipate is also a conjugated oestrogen, the piperazine salt of estrone sulphate. The structure of tibolone (Figure 5.138) probably resembles that of a progestogen more than it does an oestrogen. Although it does not contain an aromatic A-ring, the 5(10)-double bond ensures a degree of planarity. This agent combines both oestrogenic and progestogenic activity, and also has weak androgenic activity, and has been introduced for treatment of vasomotor symptoms of menopause. Diethylstilbestrol (stilboestrol)anddienestrol (dienoestrol) (Figure 5.138) are the principal non-steroidal oestrogen drugs, used topically via the vagina. Fosfestrol (Figure 5.138) has value in the treatment of prostate cancer, being hydrolysed by the enzyme phosphatase to produce diethylstilbestrol as the active agent. In estramustine (Figure 5.138), estradiol is combined with a cytotoxic alkylating agent of the nitrogen mustard class via a carbamate linkage. This drug has a dual function, a hormonal effect by suppressing androgen (testosterone) formation, and an antimitotic effect from the mustine residue. It is of value in treating prostate cancers.

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(Continued)

Phyto-oestrogens are predominantly isoflavonoid derivatives found in food plants and are used as dietary supplements to provide similar benefits to HRT, especially in countering some of the side-effects of the menopause in women. These compounds are discussed under isoflavonoids (see page 156). Dioscorea (wild yam) root or extract (see page 239) is also marketed to treat the symptoms of menopause as an alternative to HRT. Although there is a belief that this increases levels of progesterone, which is then used as a biosynthetic precursor of other hormones, there is no evidence that diosgenin is metabolized in the human body to progesterone.

Aromatase Inhibitors Formestane (Figure 5.138), the 4-hydroxy derivative of androstenedione, represents the first steroid aromatase inhibitor to be used clinically. It reduces the synthesis of oestrogens and is of value in treating advanced breast cancer in post-menopausal patients.

Oestrogen Receptor Antagonists

Breast cancer is dependent on a supply of oestrogen, and a major success in treating this disease has been the introduction of tamoxifen (Figure 5.138). This drug contains the stilbene skeleton seen in diethylstilbestrol and related oestrogens, but acts as an oestrogen-receptor antagonist rather than as an agonist in breast tissue, and deprives the cells of oestrogen. However, it is an agonist in bone and uterine tissue. The chlorinated analogue toremifene is also available, but is used primarily in post-menopausal women. Oestrogen antagonists can also be used as fertility drugs, occupying oestrogen receptors and interfering with feedback mechanisms and leading to ova release. Clomifene (clomiphene) (Figure 5.138), and to a lesser extent tamoxifen, are used in this way, but can lead to multiple pregnancies.

phyto-oestrogens (see page 156). Dietary natural OH isoflavonoids are believed to give some protection 17 against breast cancers, and are also recommended H to alleviate the symptoms of menopause. H H 3 4 O Androgens testosterone Characteristic features of androgens: The primary male sex hormone, or androgen, • C androstane skeleton is testosterone (Figure 5.139). This is secreted 19 • no side-chain by the testes and is responsible for develop- • ∆4-3-keto ment and maintenance of the male sex char- • 17β-hydroxyl acteristics. Androgens∗ also have a secondary physiological effect, an anabolic activity which Figure 5.139 stimulates growth of bone and muscle, and pro- motes storage of protein. The biosynthetic pathway to testosterone is outlined in Figure 5.133, where lacks any side-chain and has a 17β-hydroxyl as in it can feature as an intermediate in the pathway estradiol, but still contains the methyl C-19 and 4 to oestrogens. Low levels of testosterone are also the ∆ -3-one system in ring A. This C19 skeleton synthesized in females in the ovary. Testosterone is designated androstane. STEROIDS 283

It is particularly worthy of note that the aperson’s normal physiological functions and routes to corticosteroids, progestogens, oestro- characteristics are to be maintained. This balanced gens, and androgens involve common precur- production is regulated primarily by gonadotrophic sors or partial pathways. This means that these and hypothalamic proteins from the pituitary (see processes need to be under very tight control if page 411).

Androgen Drugs

Testosterone can be produced from androstenolone (Figure 5.129) by chemical routes requiring reduction of the 17-carbonyl and oxidation of the 3-hydroxyl, and necessitating appropriate protecting groups. A simple high-yielding process (Figure 5.140) exploits yeast, in which fermentation firstly under aerobic conditions oxidizes the 3-hydroxyl, and then in the absence of air reduces the 17-keto group. Testosterone is not active orally since it is easily metabolized in the liver, and it has to be implanted, or injected in the form of esters. Transdermal administration from impregnated patches has also proved successful, and is now the method of choice for treating male sexual impotence caused by low levels of sex hormones (hypogonadism). Testosterone may also be prescribed for menopausal women as an adjunct to hormone replacement therapy (see page 279) to improve sex drive, and occasionally in the treatment of oestrogen-dependent breast cancer. The ester testosterone undecanoate is orally active, as is mesterolone (Figure 5.141), which features introduction of a1α-methyl group and reduction of the ∆4 double bond. Oral activity may also be attained by adding a 17α-alkyl group to reduce metabolism, e.g. 17α-methyltestosterone (Figure 5.140), such groups being introduced using appropriate Grignard reagents. However, these types of 17α-alkyl derivative are being replaced since they can sometimes cause jaundice as a side-effect. The ratio of androgenic to anabolic activity can vary in different molecules. There have been attempts to produce steroids with low androgenic but high anabolic activity to use for various

diosgenin Marker under aerobic conditions get β under anaerobic conditions get degradation and oxidation of 3 -hydroxyl leading reduction of 17-ketone removal of to ∆4-3-ketone side-chain O O OH (Figure 5.129) H yeast H yeast H O2 no O2 H H H H H H HO O O androstenolone testosterone nucleophilic attack on to MeMgI carbonyl from less-hindered α face OH OH OH

chemical or H H microbiological H

H H H H H H HO O O 17α-methyltestosterone methandienone

Figure 5.140

(Continues) 284 THE MEVALONATE AND DEOXYXYLULOSE PHOSPHATE PATHWAYS

(Continued)

OH OH O OH OAc H H H H H HO H H H H H H H H O O O O H H Cl mesterolone nandrolone oxymetholone (19-nortestosterone) cyproterone acetate H2NNH2 O t OH NH Bu OH OH H H H H HN H H H H N H H H N O N O O H H H stanozolol finasteride danazol gestrinone

Figure 5.141

metabolic and endocrine disorders. Methandrostenolone (methandienone) (Figure 5.140), oxymetholone,andnandrolone (19-nortestosterone) (Figure 5.141) are modifications used in this way. Treatment of oxymetholone with hydrazine leads to formation of a pyrazole ring fused to a saturated ring A as seen in stanozolol (Figure 5.141). However, it is difficult to completely remove the androgenic activity from anabolic steroids. These materials are frequently abused by athletes wishing to promote muscle development and strength. Androgenic activity can affect the sexual characteristics of women, making them more masculine, whilst prolonged use of these drugs can lower fertility in either sex and endanger long term health by increasing the risks of heart and liver disease or cancer. The progestogen cyproterone acetate (Figure 5.141) is a competitive androgen antagonist or anti-androgen, that reduces male libido and fertility, and finds use in the treatment of severe hypersexuality and sexual deviation in the male, as well as in prostate cancer. Finasteride (Figure 5.141) is another anti-androgen, which is of value in prostate conditions. It is a 4-aza-steroid and a specific inhibitor of the 5α-reductase involved in testosterone metabolism. This enzyme converts testosterone into dihydrotestosterone, which is actually a more potent androgen, so that its inhibition helps to reduce prostate tissue growth. Finasteride has also been noted to prevent hair loss in men, and is marketed to treat male-pattern baldness. Dehydroepiandrosterone (DHEA) (Figure 5.133) is a precursor of androgens and oestrogens, and is the most abundant steroid in the blood of young adult humans, with levels peaking at about 20 years of age, then declining as the person ages. Whilst this hormone has a number of demonstrated biological activities, its precise physiological functions remain to be clarified. This material has become popular in the hope that it will maintain youthful vigour and health, countering the normal symptoms of age. These claims are as yet unsubstantiated, but taking large amounts of this androgen and oestrogen precursor can lead to side-effects associated with high levels of these hormones, e.g. increased risk of prostate cancer in men, or of breast cancer in women, who may also develop acne and facial hair. DHEA is not a precursor of glucocorticoids, mineralocorticoids, or of progestogens.

(Continues) FURTHER READING 285

(Continued)

Danazol and gestrinone (Figure 5.141) are inhibitors of pituitary gonadotrophin release, combining weak androgenic activity with antioestrogenic and antiprogestogenic activity. These highly modified structures bear one or more of the features we have already noted in discussions of androgens, progestogens and oestrogens, possibly accounting for their complex activity. These compounds are used particularly to treat endometriosis, where endometrial tissue grows outside the uterus.

FURTHER READING Wise ML and Croteau R (1999) Monoterpene biosyn- thesis. Comprehensive Natural Products Chemistry, Biosynthesis, General Vol 2. Elsevier, Amsterdam, pp 97–153.

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Bile Acids Banting L (1996) Inhibition of aromatase. Prog Med Chem 33, 147–184. Russell DW and Setchell KDR (1992) Bile acid biosyn- Guo JZ, Hahn DW and Wachter MP (1995) Hormones thesis. Biochemistry 31, 4737–4749. (estrogens and antiestrogens). Kirk–Othmer Ency- clopedia of Chemical Technology, 4th edn, Vol 13. Wiley, New York, pp 481–512. Corticosteroids Hahn DW, McGuire JL and Bialy G (1993) Con- traceptives. Kirk–Othmer Encyclopedia of Chemi- Bernstein PR (1992) Antiasthmatic agents. Kirk–Othmer cal Technology, 4th edn, Vol 7. Wiley, New York, Encyclopedia of Chemical Technology, 4th edn, pp 219–250. Vol 2. Wiley, New York, pp 830–854. Cervoni P and Chan PS (1993) Diuretic agents. Kirk– Othmer Encyclopedia of Chemical Technology,4th Androgens edn, Vol 8. Wiley, New York, pp 398–432. Hirschmann R (1991) Medicinal chemistry in the Jarman M, Smith HJ, Nicholls PJ and Simons C (1998) golden age of biology: lessons from steroid and Inhibitors of enzymes of androgen biosynthesis: α peptide research. Angew Chem Int Edn Engl 30, cytochrome P45017α and 5 -steroid reductase. Nat 1278–1301. Prod Rep 15, 495–512. MedicinalNaturalProducts. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 6 ALKALOIDS

Alkaloids are classified according to the amino acid that provides both the nitrogen atom and the funda- mental portion of the alkaloid skeleton, and these are discussed in turn. Ornithine gives rise to pyrrolidine and tropane alkaloids, lysine to piperidine, quinolizidine, and indolizidine alkaloids, and nicotinic acid to pyridine alkaloids. Tyrosine produces phenylethylamines and simple tetrahydroisoquinoline alkaloids, but also many others in which phenolic oxidative coupling plays an important role, such as modified ben- zyltetrahydroisoquinoline, phenethylisoquinoline, terpenoid tetrahydroisoquinoline, and Amaryllidaceae alkaloids. Alkaloids derived from tryptophan are subdivided into simple indole, simple β-carboline, terpenoid indole, quinoline, pyrroloindole, and ergot alkaloids. Anthranilic acid acts as a precursor to quinazoline, quinoline and acridine alkaloids, whilst histidine gives imidazole derivatives. However, many alkaloids are not derived from an amino acid core, but arise by amination of another type of substrate, which may be acetate derived, phenylalanine derived, a terpene or a steroid, and examples are discussed. Purine alkaloids are constructed by pathways that resemble those for purines in nucleic acids. Monograph topics giving more detailed information on medicinal agents include belladonna, stramo- nium, hyoscyamus, duboisia and allied drugs, hyoscyamine, hyoscine and atropine, coca, lobelia, vitamin B3, tobacco, areca, catecholamines, lophophora, curare, opium, colchicum, ipecacuanha, galanthamine, serotonin, psilocybe, rauwolfia, catharanthus, iboga, nux-vomica, ellipticine, cinchona, camptothecin, physostigma, ergot, morning glories, pilocarpus, Conium maculatum, ephedra, khat, aconite, Solanum alkaloids, caffeine, theobromine and theophylline, coffee, tea, cola, cocoa, mate tea, guarana, saxitoxin, and tetrodotoxin.

The alkaloids are organic nitrogenous bases found some examples rapidly expands the number of sub- mainly in plants, but also to a lesser extent in divisions. The nitrogen atoms in alkaloids originate microorganisms and animals. One or more nitrogen from an amino acid, and, in general, the carbon atoms are present, typically as primary, secondary, skeleton of the particular amino acid precursor is or tertiary amines, and this usually confers basicity also largely retained intact in the alkaloid struc- to the alkaloid, facilitating their isolation and purifi- ture, though the carboxylic acid carbon is often cation since water-soluble salts can be formed in lost through decarboxylation. Accordingly, subdi- the presence of mineral acids. The name alkaloid is vision of alkaloids into groups based on amino in fact derived from alkali. However, the degree of acid precursors forms a rational and often illumi- basicity varies greatly, depending on the structure of nating approach to classification. Relatively few the alkaloid molecule, and the presence and location amino acid precursors are actually involved in of other functional groups. Indeed, some alkaloids alkaloid biosynthesis, the principal ones being are essentially neutral. Alkaloids containing quater- ornithine, lysine, nicotinic acid, tyrosine, tryp- nary amines are also found in nature. The biological tophan, anthranilic acid, and histidine. Building activity of many alkaloids is often dependent on the blocks from the acetate, shikimate, or deoxyxylu- amine function being transformed into a quaternary lose phosphate pathways are also frequently incor- system by protonation at physiological pHs. porated into the alkaloid structures. However, a Alkaloids are often classified according to the large group of alkaloids are found to acquire nature of the nitrogen-containing structure, e.g. their nitrogen atoms via transamination reactions, pyrrolidine, piperidine, quinoline, isoquinoline, incorporating only the nitrogen from an amino indole, etc, though the structural complexity of acid, whilst the rest of the molecule may be derived 292 ALKALOIDS from acetate or shikimate, or may be terpenoid Thus ornithine supplies a C4N building block or steroid in origin. The term ‘pseudoalkaloid’ is to the alkaloid, principally as a pyrrolidine ring sometimes used to distinguish this group. system, but also as part of the tropane alkaloids (Figure 6.1). Most of the other amino acid alkaloid ALKALOIDS DERIVED FROM precursors typically supply nitrogen from their solitary α-amino group. However, the reactions of ORNITHINE ornithine are almost exactly paralleled by those of L-Ornithine (Figure 6.1) is a non-protein amino L-lysine, which incorporates a C5N unit containing ε acid forming part of the urea cycle in animals, its -amino group (see page 307). where it is produced from L-arginine in a reaction catalysed by the enzyme arginase. In plants it Pyrrolidine and Tropane Alkaloids is formed mainly from L-glutamate (Figure 6.2). Ornithine contains both δ-andα-amino groups, Simple pyrrolidine-containing alkaloid structures and it is the nitrogen from the former group which are exemplified by hygrine and cuscohygrine, is incorporated into alkaloid structures along with found in those plants of the Solanaceae that the carbon chain, except for the carboxyl group. accumulate medicinally valuable tropane alka- loids such as hyoscyamine or cocaine (see Figure 6.3). The pyrrolidine ring system is formed CO2H initially as a ∆1-pyrrolinium cation (Figure 6.2). NH2 N NH2 PLP-dependent decarboxylation (see page 20) of H L-Orn pyrrolidine C4N ornithine gives putrescine, which is then methy- lated to N-methylputrescine. Oxidative deamina- 8 1 2 tion of N-methylputrescine by the action of a Me N N ≡ diamine oxidase (see page 28) gives the alde- Me 5 4 3 hyde, and Schiff base (imine) formation pro- 7 N ∆1 tropane 6 duces the -methyl- -pyrrolinium cation. Indeed, the aminoaldehyde in aqueous solution is known Figure 6.1 to exist as an equilibrium mixture with the

NH NH – CO2 PLP CO2H H2N N H2N N H H NH2 NH2 L-Arg agmatine hydrolysis of imine functionality in O guanidine system urea cycle arginase H2N N H (animals) NH2 urea N-carbamoylputrescine hydrolysis of urea HO2CCO2H plants CO2H H2N – CO2 H N PLP 2 NH NH 2 2 NH2 L-Glu L-Orn putrescine N-methylation Schiff base SAM formation diamine oxidase

N NH O NH NH2 Me Me Me N-methyl-∆1-pyrrolinium N-methylputrescine cation

Figure 6.2 ALKALOIDS DERIVED FROM ORNITHINE 293

attack of enolate anion O O on to iminium cation: Mannich reaction N H2C SCoA SCoA Me N-methyl- ∆1-pyrrolinium cation

O O O O

N S SCoA N S N R SCoA N R Me Me Me Me hydrolysis hydrolysis – CO (–)-hygrine (+)-hygrine acetyl-CoA 2 acetyl-CoA – CO2

O O O O O O

N S SCoA N R SCoA N SCoA N Me Me Me Me oxidation to generate pyrrolinium N-methyl- O cation; formation of enolate anion in O ∆1-pyrrolinium cation side-chain hydrolysis intermolecular COSCoA Mannich N R reaction Me Me N S O O O CO2H intramolecular N N H Mannich intramolecular Mannich – CO Me Me reaction reaction; concomitant 2 CoAS O decarboxylation COSCoA hydrolysis Me N – CO2 Me N O O O methyl ester formation and SAM N N stereospecific reduction of tropinone NADPH carbonyl to give 3β-alcohol Me Me stereospecific L-Phe cuscohygrine reduction of carbonyl α NADPH CO2Me to give 3 -alcohol L-Phe Me N phenyl-lactic OH acid N Me N Me OH CoAS methylecgonine OH O O ester tropine O formation benzoyl-CoA (–)-hyoscyamine CO2Me Me N O

O cocaine

Figure 6.3

Schiff base. An alternative sequence to putrescine and the sequence appears to involve stepwise starting from arginine also operates concurrently addition of two acetyl-CoA units (Figure 6.3). In as indicated in Figure 6.2. The arginine pathway the first step, the enolate anion from acetyl-CoA also involves decarboxylation, but requires addi- acts as nucleophile towards the pyrrolinium ion tional hydrolysis reactions to cleave the guanidine in a Mannich-like reaction, which could yield portion. products with either R or S stereochemistry. The The extra carbon atoms required for hygrine second addition is then a Claisen condensation formation are derived from acetate via acetyl-CoA, extending the side-chain, and the product is the 294 ALKALOIDS

HO2C H CO2H rearrangement

NH2 OH L-Phe (–)-tropic acid

transamination

CO2H CO2H COSCoA

O OH OH N phenylpyruvic acid phenyl-lactic acid Me

OH N N Me Me tropine rearrangement HCH2OH HOH O mutase + dehydrogenase O O O H H OFe–Enz (–)-hyoscyamine littorine

dehydrogenase enzymic generation of free radical adjacent to aromatic ring (resonance H HOH hydroxylation by O stabilized by Phe) 2-oxoglutarate- RO RO dependent dioxygenase O O cyclopropane ring re-formation of formation via electron – H2O from carbonyl double O carbonyl and 2 HO Fe–Enz bond 2-oxoglutarate OH cyclopropane bond H cleavage HO H HO H RO H RO RO O HO O O

Me Me

N O2 N 2-oxoglutarate HCHOH HCHOH 2 O 2 HO O O

O oxidative formation of O epoxide ring 6β-hydroxyhyoscyamine (–)-hyoscine (scopolamine)

Figure 6.4

2-substituted pyrrolidine, retaining the thioester generates tropinone, and stereospecific reduction group of the second acetyl-CoA. Hygrine and of the carbonyl yields tropine with a 3α- most of the natural tropane alkaloids lack this hydroxyl. Hyoscyamine is the ester of tropine particular carbon atom, which is lost by suitable with (S )-tropic acid (Figure 6.4), which is derived hydrolysis/decarboxylation reactions. The bicyclic from L-phenylalanine. A novel rearrangement structure of the tropane skeleton in hyoscyamine process occurs in the phenylalanine → tropic and cocaine is achieved by a repeat of the acid transformation, in which the carboxyl Mannich-like reaction just observed. This requires group apparently migrates to the adjacent carbon an oxidation step to generate a new ∆1-pyrrolinium (Figure 6.4). Phenylpyruvic acid and phenyl-lactic cation, and removal of a proton α to the carbonyl. acid have been shown to be involved and The intramolecular Mannich reaction on the tropine becomes esterified with phenyl-lactic acid R enantiomer accompanied by decarboxylation (as the coenzyme-A ester) to form littorine ALKALOIDS DERIVED FROM ORNITHINE 295 before the rearrangement occurs. The mechanism Should the carboxyl carbon from the acetoacetyl of this rearrangement has yet to be proven, side-chain not be lost as it was in the formation though a free radical process (Figure 6.4) with of tropine, the subsequent intramolecular Mannich an intermediate cyclopropane-containing radical reaction will generate a tropane skeleton with would accommodate the available data. Further an additional carboxyl substituent (Figure 6.3). modifications to the tropane skeleton then occur However, this event is rare, and is only exemplified on the ester, and not on the free alcohol. These by the formation of ecgonine derivatives such as include hydroxylation to 6β-hydroxyhyoscyamine cocaine in Erythroxylum coca (Erythroxylaceae). and additional oxidation allowing formation of an The pathway is in most aspects analogous to epoxide grouping as in hyoscine (scopolamine). that already described for hyoscyamine, but Both of these reactions are catalysed by must proceed through the S-enantiomer of the a single 2-oxoglutarate-dependent dioxygenase N-methylpyrrolidineacetoacetyl-CoA. The ester (see page 27). Other esterifying acids may be function is then modified from a coenzyme A encountered in tropane alkaloid structures, e.g. thioester to a simple methyl oxygen ester, and tiglic acid in meteloidine (Figure 6.5) from Datura methylecgonine is subsequently obtained from meteloides and phenyl-lactic acid in littorine, the methoxycarbonyltropinone by stereospecific above which is a major alkaloid in Anthocercis reduction of the carbonyl. Note that in this case littorea. Tiglic acid is known to be derived from reduction of the carbonyl occurs from the opposite the amino acid L-isoleucine (see page 197). face to that noted with the tropinone → tropine The structure of cuscohygrine arises by an conversion and thus yields the 3β configuration intermolecular Mannich reaction involving a sec- in ecgonine. Cocaine is a diester of ecgonine, ond N-methyl-∆1-pyrrolinium cation (Figure 6.3). the benzoyl moiety arising from phenylalanine via cinnamic acid and benzoyl-CoA (see page 141). Me The tropane alkaloids (−)-hyoscyamine∗ and N (−)-hyoscine∗ are among the most important HO of the natural alkaloids used in medicine. HO O HO2C They are found in a variety of solanaceous ∗ NH plants, including Atropa belladonna (deadly O 2 ∗ meteloidine L-Ile nightshade), Datura stramonium (thornapple) and other Datura species, Hyoscyamus niger∗ ∗ Figure 6.5 (henbane), and Duboisia species. These alkaloids

Belladonna The deadly nightshade Atropa belladonna (Solanaceae) has a long history as a highly poisonous plant. The generic name is derived from Atropos, in Greek mythology the Fate who cut the thread of life. The berries are particularly dangerous, but all parts of the plant contain toxic alkaloids, and even handling of the plant can lead to toxic effects since the alkaloids are readily absorbed through the skin. Although humans are sensitive to the toxins, some animals, including sheep, pigs, goats, and rabbits, are less susceptible. Cases are known where the consumption of rabbits or birds that have ingested belladonna has led to human poisoning. The plant is a tall perennial herb producing dull-purple bell-shaped flowers followed by conspicuous shiny black fruits, the size of a small cherry. Atropa belladonna is indigenous to Central and Southern Europe, though it is not especially common. It is cultivated for drug use in Europe and the United States. The tops of the plant are harvested two or three times per year and dried to give belladonna herb. Roots from plants some 3–4 years old are less commonly employed as a source of alkaloids.

(Continues) 296 ALKALOIDS

(Continued)

Belladonna herb typically contains 0.3–0.6% of alkaloids, mainly (−)-hyoscyamine (Figure 6.4). Belladonna root has only slightly higher alkaloid content at 0.4–0.8%, again mainly (−)-hyoscyamine. Minor alkaloids including (−)-hyoscine (Figure 6.4) and cuscohygrine (Figure 6.3) are also found in the root, though these are not usually significant in the leaf. The mixed alkaloid extract from belladonna herb is still used as a gastrointestinal sedative, usually in combination with antacids. Root preparations can be used for external pain relief, e.g. in belladonna plasters.

Stramonium Datura stramonium (Solanaceae) is commonly referred to as thornapple on account of its spikey fruit. It is a tall bushy annual plant widely distributed in Europe and North America, and because of its alkaloid content is potentially very toxic. Indeed, a further common name, Jimson or Jamestown weed, originates from the poisoning of early settlers near Jamestown, Virginia. At subtoxic levels, the alkaloids can provide mild sedative action and a feeling of well-being. In the Middle Ages, stramonium was employed to drug victims prior to robbing them. During this event, the victim appeared normal and was cooperative, though afterwards could usually not remember what had happened. For drug use, the plant is cultivated in Europe and South America. The leaves and tops are harvested when the plant is in flower. Stramonium leaf usually contains 0.2–0.45% of alkaloids, principally (−)-hyosycamine and (−)-hyoscine in a ratio of about 2:1. In young plants, (−)-hyoscine can predominate. The generic name Datura is derived from dhat, an Indian poison used by the Thugs. The nar- cotic properties of Datura species, especially D. metel, have been known and valued in India for centuries. The plant material was usually absorbed by smoking. Most species of Datura contain similar tropane alkaloids and are potential sources of medicinal alkaloids. In partic- ular, Datura sanguinea, a perennial of treelike stature with blood-red flowers, is cultivated in Ecuador, and yields leaf material with a high (0.8%) alkaloid content in which the principal com- ponent is (−)-hyoscine. The plants can be harvested several times a year. Datura sanguinea, and several other species of the tree-daturas (now classified as a separate genus Brugmansia) are widely cultivated as ornamentals, especially for conservatories, because of their attractive large tubular flowers. The toxic potential of these plants is not always recognized.

Hyosycamus

Hyoscyamus niger (Solanaceae), or henbane, is a European native with a long history as a medicinal plant. Its inclusion in mediaeval concoctions and its power to induce hallucinations with visions of flight may well have contributed to our imaginary view of witches on broomsticks. The plant has both annual and biennial forms, and is cultivated in Europe and North America for drug use, the tops being collected when the plant is in flower, and then dried rapidly. The alkaloid content of hyoscyamus is relatively low at 0.045–0.14%, but this can be composed of similar proportions of (−)-hyoscine and (−)-hyosycamine. Egyptian henbane, Hyosycamus muticus, has a much higher alkaloid content than H. niger, and although it has mainly been collected from the wild, especially from Egypt, it functions as a major commercial source for alkaloid production. Some commercial cultivation occurs in California. The alkaloid content of the leaf is from 0.35% to 1.4%, of which about 90% is (−)-hyoscyamine.

(Continues) ALKALOIDS DERIVED FROM ORNITHINE 297

(Continued) Duboisia Duboisia is a small genus of trees, containing only three species, found in Australia, again from the family Solanaceae. Two of these, Duboisia myoporoides and D. leichhardtii are grown commercially in Australia for tropane alkaloid production. The small trees are kept as bushes to allow frequent harvesting, with up to 70–80% of the leaves being removed every 7–8 months. The alkaloid content of the leaf is high, up to 3% has been recorded, and it includes (−)-hyoscyamine, (−)-hyoscine, and a number of related structures. The proportion of hyoscyamine to hyoscine varies according to the species used, and the area in which the trees are grown. The hyoscine content is frequently much higher than that of hyoscyamine. Indeed, interest in Duboisia was very much stimulated by the demand for hyoscine as a treatment for motion sickness in military personnel in the Second World War. Even higher levels of alkaloids, and higher proportions of hyoscine, can be obtained from selected D. myoporoides × D. leichhardtii hybrids, which are currently cultivated. The hybrid is superior to either parent, and can yield 1–2.5% hyoscine and 0–1% hyoscyamine. Duboisia leaf is an important commercial source of medicinal tropane alkaloids. The third species of Duboisia, D. hopwoodi, contains little tropane alkaloid content, but produces mainly nicotine and related alkaloids, e.g. nornicotine (see page 313). Leaves of this plant were chewed by aborigines for their stimulating effects.

Allied Drugs

Tropane alkaloids, principally hyoscyamine and hyoscine, are also found in two other medicinal plants, scopolia and mandrake, but these plants find little current use. Scopolia (Scopolia carniolica; Solanaceae) resembles belladonna in appearance, though it is considerably smaller. Both root and leaf materials have been employed medicinally. The European mandrake (Mandragora officinarum; Solanaceae) has a complex history as a hypnotic, a general panacea, and an aphrodisiac. Its collection has been surrounded by much folklore and superstition, in that pulling it from the ground was said to drive its collector mad due to the unearthly shrieks emitted. The roots are frequently forked and are loosely likened to a man or woman. Despite the Doctrine of Signatures, which teaches that the appearance of an object indicates its special properties, from a pharmacological point of view, this plant would be much more efficient as a pain-reliever than as an aphrodisiac.

Hyoscyamine, Hyoscine and Atropine

All the above solanaceous plants contain as main alkaloidal constituents the tropane esters (−)-hyoscyamine and (−)-hyoscine, together with other minor tropane alkaloids. The piperidine ring in the bicyclic tropane system has a chairlike conformation, and there is a ready inversion of configuration at the nitrogen atom so that the N-methyl group can equilibrate between equatorial and axial positions (Figure 6.6). An equatorial methyl is strongly favoured provided there are no substituents on the two-carbon bridge, in which case the axial form may predominate. (−)-Hyoscyamine is the ester of tropine (Figure 6.4) with (−)- (S)-tropic acid, whilst (−)-hyoscine contains scopine (Figure 6.8) esterified with (−)-(S)-tropic acid. The optical activity of both hyoscyamine and hyoscine stems from the chiral centre

(Continues) 298 ALKALOIDS

(Continued)

Me

Me N N

OH OH equatorial methyl axial methyl (favoured in hyoscyamine) (favoured in hyoscine)

Figure 6.6

base-catalysed or heat-initiated double bond of enol and keto–enol tautomerism aromatic ring in conjugation Me N Me N Me N OH HCH2OH H CH2OH O O O

O OH O

(–)-hyoscyamineMe N (+)-hyoscyamine

CH2OH O RS O atropine

Figure 6.7

in the acid portion, (S)-tropic acid. Tropine itself, although containing chiral centres, is a symmetrical molecule and is optically inactive; it can be regarded as a meso structure. The chiral centre in the tropic acid portion is adjacent to a carbonyl and the aromatic ring, and racemization can be achieved under mild conditions by heating or treating with base. This will involve an intermediate enol (or enolate) which is additionally favoured by conjugation with the aromatic ring (Figure 6.7). Indeed, normal base assisted fractionation of plant extracts to isolate the alkaloids can sometimes result in production of significant amounts of racemic alkaloids. The plant material itself generally contains only the enantiomerically pure alkaloids. Hyoscyamine appears to be much more easily racemized than hyoscine. Hydrolysis of the esters using acid or base usually gives racemic tropic acid. Note that littorine (Figure 6.4), in which the chiral centre is not adjacent to the phenyl ring, is not readily racemized, and base hydrolysis gives optically pure phenyl-lactic acid. The racemic form of hyoscyamine is called atropine (Figure 6.7), whilst that of hyoscine is called atroscine. In each case, the biological activity of the (+)-enantiomer is some 20–30 times less than that of the natural (−)-form. Chemical hydrolysis of hyoscine in an attempt to obtain the alcohol scopine is not feasible. Instead, the alcohol oscine is generated because of the proximity of the 3α-hydroxyl group to the reactive epoxide function (Figure 6.8). Probably for traditional reasons, salts of both (−)-hyoscyamine and (±)-hyoscyamine (atropine) are used medicinally, whereas usage of hyoscine is restricted to the

(Continues) ALKALOIDS DERIVED FROM ORNITHINE 299

(Continued)

nucleophilic attack of α Me 3 -hydroxyl on to either hydrolysis Me carbon of epoxide Me N Me N N N HCH2OH H O HO + O O or OH OH HO O O O (–)-hyoscine scopine (±)-oscine

Figure 6.8

OH OH Me H Me H Me Me N N N Me O Me O O Me O O

muscarine acetylcholine hyoscyamine (as conjugate acid)

Figure 6.9 natural laevorotatory form. These alkaloids compete with acetylcholine for the muscarinic site of the parasympathetic nervous system, thus preventing the passage of nerve impulses, and are classified as anticholinergics. Acetylcholine binds to two types of receptor site, described as muscarinic or nicotinic, from the specific triggering of a response by the Amanita muscaria alkaloid muscarine or the tobacco alkaloid nicotine (see page 314) respectively. The structural similarity between acetylcholine and muscarine (Figure 6.9) can readily be appreciated, and hyoscyamine is able to occupy the same receptor site by virtue of the spatial relationship between the nitrogen atom and the ester linkage (Figure 6.9). The side-chain also plays a role in the binding, explaining the difference in activities between the two enantiomeric forms. The agonist properties of hyoscyamine and hyoscine give rise to a number of useful effects, including antispasmodic action on the gastrointestinal tract, antisecretory effect controlling salivary secretions during surgical operations, and as mydriatics to dilate the pupil of the eye. Hyoscine has a depressant action on the central nervous system and finds particular use as a sedative to control motion sickness. One of the side-effects from oral administration of tropane alkaloids is dry mouth (the antisecretory effect) but this can be much reduced by transdermal administration. In motion sickness treatment, hyoscine can be supplied via an impregnated patch worn behind the ear. Hyoscine under its synonym scopolamine is also well known, especially in fiction, as a ‘truth drug’. This combination of sedation, lack of will, and amnesia was first employed in child-birth, giving what was termed ‘twilight sleep’, and may be compared with the mediaeval use of stramonium. The mydriatic use also has a very long history. Indeed, the specific name belladonna for deadly nightshade means ‘beautiful lady’ and refers to the practice of ladies at court who applied the juice of the fruit to the eyes, giving widely dilated pupils and a striking appearance, though at the expense of blurred vision through an inability to focus. Atropine also has useful antidote action in cases of poisoning caused by cholinesterase inhibitors, e.g. physostigmine and neostigmine (see page 366) and organophosphate insecticides.

(Continues) 300 ALKALOIDS

(Continued)

It is valuable to reiterate here that the tropane alkaloid-producing plants are all regarded as very toxic, and that since the alkaloids are rapidly absorbed into the blood stream, even via the skin, first aid must be very prompt. Initial toxicity symptoms include skin flushing with raised body temperature, mouth dryness, dilated pupils, and blurred vision.

Me N Me N

OH NEtCH2OH O N RS O RS O O homatropine tropicamide

HO benzatropine (benztropine) OH O O RS Me2N Me N 2 O O cyclopentolate glycopyrronium

Figure 6.10

Homatropine (Figure 6.10) is a semi-synthetic ester of tropine with racemic mandelic (2- hydroxyphenylacetic) acid and is used as a mydriatic, as are tropicamide and cyclopentolate (Figure 6.10). Tropicamide is an amide of tropic acid, though a pyridine nitrogen is used to mimic that of the tropane. Cyclopentolate is an ester of a tropic acid-like system, but uses a non-quaternized amino alcohol resembling choline. Glycopyrronium (Figure 6.10) has a quaternized nitrogen in a pyrrolidine ring, with an acid moiety similar to that of cyclopentolate. This drug is an antimuscarinic used as a premedicant to dry bronchial and salivary secretions. Hyoscine butylbromide (Figure 6.11) is a gastro-intestinal antispasmodic synthesized from (−)-hyoscine by quaternization of the amine function with butyl bromide. The quaternization of tropane alkaloids by N-alkylation proceeds such that the incoming alkyl group always

Me Me Br N Bu N HCHOH HCHOH O 2 BuBr O 2 O O

O O (–)-hyoscine hyoscine butylbromide

i Pr Br Et Br HN N Me Me N (i) iPrBr CH2OH (ii) MeBr CH2OH HCHOH O 2 O O O RS RS O O O noratropine ipratropium bromide oxitropium bromide

Figure 6.11

(Continues) ALKALOIDS DERIVED FROM ORNITHINE 301

(Continued)

approaches from the equatorial position. The potent bronchodilator ipratropium bromide (Figure 6.11) is thus synthesized from noratropine by successive isopropyl and methyl alkylations whilst oxitropium bromide is produced from norhyoscine by N-ethylation and then N-methylation. Both drugs are used in inhalers for the treatment of chronic bronchitis. Benzatropine (benztropine) (Figure 6.10) is an ether of tropine used as an antimuscarinic drug in the treatment of Parkinson’s disease. It is able to inhibit dopamine reuptake, helping to correct the deficiency which is characteristic of Parkinsonism.

HN HN OH page 477) in the development of drugs with HO OH HO OH activity against the AIDS virus HIV. It is likely that OH OH these alkaloids are produced by a similar pathway to that which yields tropine, but the stereochem- calystegine A3 calystegine B2 istry of reduction of tropinone (or nortropinone) Figure 6.12 yields the 3β-alcohol, and further hydroxylation steps are necessary. Examples of tri-, tetra-, and penta-hydroxy calystegines are currently known. are also responsible for the pronounced toxic Cocaine (Figure 6.3) is a rare alkaloid restricted properties of these plants. to some species of Erythroxylum (Erythroxy- The calystegines (Figure 6.12) are a group of laceae). Erythroxylum coca (coca)∗ is the most recently discovered, water-soluble, polyhydroxy prominent as a source of cocaine, used medicinally nortropane derivatives that are found in the leaves as a local anaesthetic, and as an illicit drug for its and roots of many of the solanaceous plants, euphoric properties. Coca also contains significant including Atropa, Datura, Duboisia, Hyoscyamus, amounts of cinnamoylcocaine (cinnamylcocaine) Mandragora, Scopolia,andSolanum.Theywere (Figure 6.13), where cinnamic acid rather than first isolated from Calystegia sepium (Convolvu- benzoic acid is the esterifying acid, together with laceae). These compounds, e.g. calystegin A3 some typical tropine derivatives without the extra and calystegin B2 (Figure 6.12), are currently of carboxyl, e.g. tropacocaine (Figure 6.13). Tropa- great interest as glycosidase inhibitors and have cocaine still retains the 3β-configuration, showing similar potential as the polyhydroxyindolizidines that the stereospecific carbonyl reduction is the such as castanospermine (see page 310) and same as with the cocaine route, and not as with the aminosugars such as deoxynojirimycin (see the hyoscyamine pathway.

CO2Me CO2Me Me N Me N Me N O O OH

O O cinnamoylcocaine tropacocaine methylecgonine (cinnamylcocaine) CO Me 2 OH CO2Me Me N Me N O N O Me O hygroline O MeO2C O CO2Me O N N Me Me O O

α-truxilline β-truxilline

Figure 6.13 302 ALKALOIDS

Coca Coca leaves are obtained from species of Erythroxylum (Erythroxylaceae), small shrubs native to the Andes region of South America, namely Colombia, Ecuador, Peru, and Bolivia. Plants are cultivated there, and in Indonesia. Two main species provide drug materials, Erythroxylum coca (Bolivian or Huanaco coca) and E. truxillense (Peruvian or Truxillo coca). Cultivated plants are kept small by pruning and a quantity of leaves is harvested from each plant three or more times per year. Coca-leaf chewing has been practised by South American Indians for many years and is an integral part of the native culture pattern. Leaf is mixed with lime, this liberating the principal alkaloid cocaine as the free base, and the combination is then chewed. Cocaine acts as a potent antifatigue agent, and this allows labourers to ignore hunger, fatigue, and cold, enhancing physical activity and endurance. Originally the practice was limited to the Inca high priests and favoured individuals, but became widespread after the Spanish conquest of South America. It is estimated that 25% of the harvest is consumed in this way by the local workers, who may each use about 50 g of leaf per day (≡ 350 mg cocaine). Only a tiny amount (1–2%) of the coca produced is exported for drug manufacture. The rest contributes to illicit trade and the world’s drug problems. Efforts to stem the supply of illicit coca and cocaine have been relatively unsuccessful. Coca leaf contains 0.7–2.5% of alkaloids, the chief component (typically 40–50%) of which is (−)-cocaine (Figure 6.3), a diester of (−)-ecgonine. Note that although tropine is an optically inactive meso structure, ecgonine contains four chiral centres, is no longer symmetrical, and is therefore optically active. Cinnamoylcocaine (cinnamylcocaine), α-truxilline, β-truxilline, and methylecgonine (Figure 6.13) are minor constituents also based on ecgonine. The truxillines contain dibasic acid moieties, α-truxillic and β-truxinic acids, which are cycloaddition products from two cinnamic acid units (Figure 6.14). Other alkaloids present include structures based on ϕ-tropine (the 3β-isomer of tropine) such as tropacocaine (Figure 6.13) and on hygrine, e.g. hygrine, hygroline (Figure 6.13) and cuscohygrine (Figure 6.3). Cuscohygrine typically accounts for 20–30% of the alkaloid content. Illegal production of cocaine is fairly unsophisticated, but can result in material of high quality. The alkaloids are extracted from crushed leaf using alkali (lime) and petrol. The petrol extract is then re-extracted with aqueous acid, and this alkaloid fraction is basified and allowed to stand, yielding the free alkaloid as a paste. Alternatively, the hydrochloride or sulphate salts may be prepared. The coca alkaloids are often diluted with carrier to give a

HO2C HO2C HO2C HO2C

CO2H HO2C CO2H HO2C

cinnamic acid α-truxillic acid cinnamic acid β-truxinic acid

Figure 6.14

(Continues) ALKALOIDS DERIVED FROM ORNITHINE 303

(Continued) preparation with 10–12% of cocaine. The illicit use of cocaine and cocaine hydrochloride is a major problem worldwide. The powder is usually sniffed into the nostrils, where it is rapidly absorbed by the mucosa, giving stimulation and short-lived euphoria through inhibiting reuptake of neurotransmitters dopamine, noradrenaline, and serotonin, so prolonging and augmenting their effects. Regular usage induces depression, dependence, and damage to the nasal membranes. The drug may also be injected intravenously, or the vapour inhaled. For inhalation, the free base or ‘crack’ is employed to increase volatility. The vaporized cocaine is absorbed extremely rapidly and carried to the brain within seconds, speeding up and enhancing the euphoric lift. Taken in this form, cocaine has proved highly addictive and dangerous. Cocaine abuse is currently a greater problem than heroin addiction, and, despite intensive efforts, there is no useful antagonist drug available to treat cocaine craving and addiction. In the 1800s, coca drinks were fashionable, and one in particular, Coca-Cola, became very popular. This was originally based on extracts of coca (providing cocaine) and cola (supplying caffeine) (see page 395), but although the coca content was omitted from 1906 onwards, the name and popularity continue. Medicinally, cocaine is of value as a local anaesthetic for topical application. It is rapidly absorbed by mucous membranes and paralyses peripheral ends of sensory nerves. This is achieved by blocking ion channels in neural membranes. It was widely used in dentistry, but has been replaced by safer drugs, though it still has applications in ophthalmic and ear, nose, and throat surgery. As a constituent of Brompton’s cocktail (cocaine and heroin in sweetened alcohol) it is available to control pain in terminal cancer patients. It increases the overall analgesic effect, and its additional CNS stimulant properties counteract the sedation normally associated with heroin (see page 332). The essential functionalities of cocaine required for activity were eventually assessed to be the aromatic carboxylic acid ester and the basic amino group, separated by a lipophilic hydrocarbon chain. Synthetic drugs developed from the cocaine structure have been introduced to provide safer, less toxic local anaesthetics (Figure 6.15). Procaine, though little used now, was the first major analogue employed. Benzocaine is used topically, but has a short duration of action. Tetracaine (amethocaine), oxybuprocaine,andproxymetacaine are valuable local anaesthetics employed principally in ophthalmic work. The ester function can be replaced by an amide, and this gives better stability towards hydrolysis in aqueous solution or by esterases. Lidocaine (lignocaine) is an example of an amino amide analogue and is perhaps the most widely used local anaesthetic, having rapid action, effective absorption, good stability, and may be used by injection or topically. Other amino amide local anaesthetic structures include prilocaine, with similar properties to lidocaine and very low toxicity, and bupivacaine, which has a long duration of action. Ropivacaine, mepivacaine,andarticaine (carticaine) are some recently introduced amide-type local anaesthetics, the latter two being used predominantly in dentistry. Cinchocaine is often incorporated into preparations to soothe haemorrhoids. Lidocaine, although introduced as a local anaesthetic, was subsequently found to be a potent antiarrhythmic agent, and it now finds further use as an antiarrhythmic drug, for treatment of ventricular arrhythmias especially after myocardial infarction. Other cocaine- related structures also find application in the same way, including tocainide, procainamide, and flecainide (Figure 6.15). Tocainide is a primary amine analogue of lidocaine, whilst procainamide is an amide analogue of procaine. In mexiletene, a congener of lidocaine, the amide group has been replaced by a simple ether linkage.

(Continues) 304 ALKALOIDS

(Continued)

n NH2 NH2 NH Bu NH2

O O EtO O n Et2N Et2N Et2N O Bu O O O O procaine benzocaine tetracaine / amethocaine oxybuprocaine

OPr H H RS H N N RS N O Et2N N N Et2N NH2 n H O Bu O O O proxymetacaine lidocaine / lignocaine bupivacaine prilocaine

H H H CO2Me N RS N RS N N N N N H H S N n Pr O Me O O Et2NOBu ropivacaine mepivacaine articaine O F3C cinchocaine

NH2 O H RS H RS H N N O RS H N H N N 2 Et2N 2 N O CF3 H O O O tocainide procainamide mexiletine flecainide

Figure 6.15

Anatoxin-a (Figure 6.16) is a toxic tropane- the highly coloured skin of the Ecuadorian poison related alkaloid produced by a number of frog Epipedobates tricolor. This compound, called cyanobacteria, e.g. Anabaena flos-aquae and Aph- epibatidine (Figure 6.17), is exciting considerable anizomenon flos-aquae, species which proliferate interest as a lead compound for analgesic drugs. It in lakes and reservoirs during periods of hot, is 200–500 times more potent than morphine (see calm weather. A number of animal deaths have page 331), and does not act by the normal opioid been traced back to consumption of water con- mechanism, but is a specific agonist at nicotinic taining the cyanobacteria, and ingestion of the acetylcholine receptors. Whether or not this struc- highly potent neurotoxin anatoxin-a, which has ture is ornithine derived remains to be established. been termed Very Fast Death Factor. Anatoxin- a is a powerful agonist at nicotinic acetylcholine L-Orn receptors, and has become a useful pharmacolog- ical probe. The ring system may be regarded as O a homotropane, and it has been suggested that acetyl-CoA the pyrrolidine ring originates from ornithine via H2N N HN H2N putrescine and ∆1-pyrroline, in a way similar to the tropane alkaloids (Figure 6.16). The remain- ing carbons may originate from acetate precursors. putrescine∆1-pyrroline anatoxin-a A remarkable compound with a nortropane ring system has been isolated in tiny amounts from Figure 6.16 ALKALOIDS DERIVED FROM ORNITHINE 305

Cl condenses with a second putrescine molecule to give the imine, but this mechanism has been N proven to be incorrect. The pyrrolizidine skele- ton is formed from homospermidine by a sequence HN of oxidative deamination, imine formation, and an intramolecular Mannich reaction, which exploits the enolate anion generated from the aldehyde. epibatidine This latter reaction is analogous to that proposed in formation of the tropane ring system (see Figure 6.17 page 293). A typical simple natural pyrrolizidine structure is that of retronecine (Figure 6.18), Pyrrolizidine Alkaloids which can be derived from the pyrrolizidine alde- hyde by modest oxidative and reductive steps. The Two molecules of ornithine are utilized in for- pyrrolizidine skeleton thus incorporates a C4N unit mation of the bicyclic pyrrolizidine skeleton, the from ornithine, plus a further four carbons from the pathway (Figure 6.18) proceeding via the inter- same amino acid precursor. mediate putrescine. Because plants synthesiz- Pyrrolizidine alkaloids have a wide distribution, ing pyrrolizidine alkaloids appear to lack the but are characteristic of certain genera of the decarboxylase enzyme transforming ornithine into Boraginaceae (e.g. Heliotropium, Cynoglossum, putrescine, ornithine is actually incorporated by and Symphytum), the Compositae/Asteraceae way of arginine (Figure 6.2). Two molecules of (e.g. Senecio) and the Leguminosae/Fabaceae putrescine are condensed in an NAD+-dependent (e.g. Crotalaria). The pyrrolizidine bases rarely oxidative deamination reaction to give the imine, occur in the free form, but are generally found which is then converted into homospermidine by as esters with rare mono- or di-basic acids, the NADH reduction. On paper, one might predict necic acids. Thus, senecionine (Figure 6.18) from that one molecule of putrescine is converted by Senecio species is a diester of retronecine with oxidative deamination into the aldehyde, which senecic acid. Inspection of the ten-carbon skeleton

oxidative deamination NH H N 2 2 NH2 H2N NH2 L-Orn NH2 NAD+ NADH or NH H N 2 2 N L-Arg NH putrescine putrescine homospermidine NH2 aminoaldehyde is oxidative OHC not involved deamination intramolecular oxidative Schiff base Mannich reaction CHO CHO H CHO deamination formation NH2 NH2 CHO N N N N NH

senecic acid is derived from two HO 2 x CO2H molecules of isoleucine O senecic acid 7 1 OH L-Ile NH2 8 HO H O O O 6 2 H retronecine N N 5 4 3 N O N pyrrolizidine pyrrolizidine retronecine N-oxide senecionine

Figure 6.18 306 ALKALOIDS of senecic acid suggests it is potentially derivable oxidases into reactive pyrrole structures, which are from two isoprene units, but experimental evidence potent alkylating agents and react with suitable has demonstrated that it is in fact obtained by cell nucleophiles, e.g. nucleic acids and proteins incorporation of two molecules of the amino (Figure 6.19). N-oxides are not transformed by acid L-isoleucine. Loss of the carboxyl from these oxidases, only the free bases. The presence isoleucine supplies a carbon fragment analogous of pyrrolizidine alkaloids, e.g. acetyl-intermedine to isoprene units (compare tiglic acid in the and acetyl-lycopsamine (Figure 6.20) in medi- tropane alkaloid meteloidine, Figure 6.5). Other cinal comfrey (Symphytum officinale;Boragi- necic acid structures may incorporate fragments naceae) has emphasized potential dangers of using from valine, threonine, leucine, or acetate. It is also this traditional herbal drug as a remedy for inflam- worthy of note that, in general, the pyrrolizidine matory, rheumatic, and gastrointestinal disorders. alkaloids accumulate in the plant as polar N- Prolonged usage may lead to liver damage. Cater- oxides, facilitating their transport, and above all, pillars of the cinnabar moth Tyria jacobaeae feed maintaining them in a non-toxic form. The N- on species of Senecio (e.g. ragwort, S. jacobaea, oxides are easily changed back to the tertiary and groundsel, S. vulgaris) with impunity, building amines by mild reduction, as will occur in the gut up levels of pyrrolizidine alkaloids in their bod- of a herbivore. ies (in the form of non-toxic N-oxides) making Many pyrrolizidine alkaloids are known to pro- them distasteful to predators, and potentially toxic duce pronounced hepatic toxicity and there are should the predator convert the alkaloids into the many recorded cases of livestock poisoning. Poten- free bases. Indicine-N-oxide (Figure 6.20) from tially toxic structures have 1,2-unsaturation in the Heliotropium indicum (Boraginaceae) demon- pyrrolizidine ring and an ester function on the strated significant antileukaemic activity in clinical side-chain. Although themselves non-toxic, these trials but undesirable hepatotoxicity prevented any alkaloids are transformed by mammalian liver further development.

loss of leaving group generates oxidation to conjugated iminium ion which O pyrrole O acts as electrophile O 1 2 R R2 R1 R2 R O Nu O H O liver O O O oxidases O

N N N

irreversible alkylation of nucleophiles in O proteins or nucleic acids Nu R2 Nu Nu Nu Nu O loss of second leaving group generates another N N N electrophilic iminium ion

Figure 6.19

O O O OH OH OH O H O O H O HO H O O H OH O H OH HO N N N acetyl-intermedine acetyl-lycopsamine O indicine-N-oxide

Figure 6.20 ALKALOIDS DERIVED FROM LYSINE 307

The tobacco alkaloids, especially nicotine, are Piperidine Alkaloids derived from nicotinic acid (see page 311) but also contain a pyrrolidine ring system derived from N-Methylpelletierine (Figure 6.22) is an alka- ornithine as a portion of their structure. loidal constituent of the bark of pomegranate (Punica granatum; Punicacae), where it cooc- curs with pelletierine and pseudopelletierine ALKALOIDS DERIVED FROM LYSINE (Figure 6.22), the mixture of alkaloids hav- ing activity against intestinal tapeworms. N- L-Lysine is the homologue of L-ornithine, and Methylpelletierine and pseudopelletierine are it too functions as an alkaloid precursor, using homologues of hygrine and tropinone respec- pathways analogous to those noted for ornithine. tively, and a pathway similar to Figure 6.3 using The extra methylene group in lysine means this the diamine cadaverine (Figure 6.22) may be amino acid participates in forming six-membered proposed. (The rather distinctive names cadav- piperidine rings, just as ornithine provided five- erine and putrescine reflect early isolations membered pyrrolidine rings. As with ornithine, the of these compounds from decomposing animal carboxyl group is lost, the ε-amino nitrogen rather flesh.) In practice, the Mannich reaction involv- than the α-amino nitrogen is retained, and lysine ∆1 thus supplies a C N building block (Figure 6.21). ing the -piperidinium salt utilizes the more 5 nucleophilic acetoacetyl-CoA rather than acetyl- CoA, and the carboxyl carbon from acetoac- etate appears to be lost during the reaction CO2H by suitable hydrolysis/decarboxylation reactions NH2 NH2 N (Figure 6.22). Anaferine (Figure 6.22) is an ana- H L-Lys piperidine C5N logue of cuscohygrine in which a further piperi- dine ring is added via an intermolecular Mannich Figure 6.21 reaction.

oxidative deamination via Schiff base 2 x acetyl-CoA – CO2 diamine oxidase formation PLP CO2H COSCoA NH2 NH2 NH2 NH2 NH2 O N L-Lys cadaverine ∆1-piperideine O H hydrolysis acetoacetyl-CoA decarboxylation O SAM O O COSCoA

N N N N Mannich Me H H H COSCoA reaction O N-methylpelletierine pelletierine ∆1-piperidinium cation intramolecular intermolecular Mannich Mannich reaction N reaction H Me N O O N N H H pseudopelletierine anaferine

Figure 6.22 308 ALKALOIDS

The alkaloids found in the antiasthmatic lobeline and lobelanine from Lobelia inflata, plant Lobelia inflata∗ (Campanulaceae) contain or sedamine from Sedum acre (Crassulaceae), piperidine rings with alternative C6C2 side-chains are products from further N-methylation and/or derived from phenylalanine via cinnamic acid. carbonyl reduction reactions (Figure 6.23). These alkaloids are produced as in Figure 6.23, The simple piperidine alkaloid coniine from in which benzoylacetyl-CoA, an intermediate in poison hemlock is not derived from lysine, but the β-oxidation of cinnamic acid (see page 141) originates by an amination process and is discussed provides the nucleophile for the Mannich reaction. on page 381. Oxidation in the piperidine ring gives a new The pungency of the fruits of black pepper iminium species, and this can react further with (Piper nigrum; Piperaceae), a widely used a second molecule of benzoylacetyl-CoA, again condiment, is mainly due to the piperidine alkaloid via a Mannich reaction. Naturally, because of the piperine (Figure 6.24). In this structure, the piperi- nature of the side-chain, the second intramolecular dine ring forms part of a tertiary amide structure, Mannich reaction, as involved in pseudopelletierine and is incorporated via piperidine itself, the biosynthesis, is not feasible. Alkaloids such as reduction product of ∆1-piperideine (Figure 6.22).

Mannich reaction, hydrolysis and decarboxylation CoASOC oxidation O O

N N N H O H H ∆1-piperidinium benzoylacetyl-CoA Mannich reaction, cation enolate anion reduction hydrolysis and benzoylacetyl-CoA decarboxylation

OH O O

N HO C N 2 H H cinnamic acid sedamine SAM

O OH O O reduction N N Me Me

lobeline lobelanine

Figure 6.23

Lobelia Lobelia or Indian tobacco consists of the dried leaves and tops of Lobelia inflata (Campanulaceae), an annual herb from the USA and Canada. Lobelia contains about 0.2–0.4% of alkaloids, of which the piperidine derivative lobeline (Figure 6.23) is the chief constituent. Minor alkaloids identified include closely related structures, e.g. lobelanine (Figure 6.23). The North American Indians employed lobelia as an alternative or substitute for tobacco (Nicotiana tabacum; Solanaceae), and it is found that lobeline stimulates nicotinic receptor sites in a similar way to nicotine, but with a weaker effect. Lobeline has been employed in preparations intended as smoking deterrents. The crude plant drug has also long been used to relieve asthma and bronchitis, though in large doses it can be quite toxic. ALKALOIDS DERIVED FROM LYSINE 309

Claisen reaction; chain extension reduction/dehydration reactions using malonyl-CoA as in fatty acid biosynthesis O O O OH O malonyl-CoA O O NADPH SCoA SCoA O SCoA O O O reduction N HN – H2O

O ∆1-piperideine piperidine O O O N SCoA

O amide formation O piperine piperoyl-CoA (piperic acid CoA ester)

Figure 6.24

The piperic acid portion is derived from a incorporated into lupinine via cadaverine, but the cinnamoyl-CoA precursor, with chain exten- intermediate corresponding to homospermidine is sion using acetate/malonate (compare flavonoids, excluded. ∆1-Piperideine seems to be an important page 149), and combines as its CoA ester with intermediate after cadaverine and the pathway pro- piperidine. posed (Figure 6.25) invokes coupling of two such molecules. The two tautomers of ∆1-piperideine, as N-analogues of corresponding carbonyl com- Quinolizidine Alkaloids pounds, are able to couple by an aldol-type mech- anism (see page 19). Indeed, this coupling occurs The lupin alkaloids, found in species of Lupinus in solution at physiological pHs, though stereospe- (Leguminosae/Fabaceae), and responsible for the cific coupling to the product shown in Figure 6.25 toxic properties associated with lupins, are charac- would require appropriate enzymic participation. terized by a quinolizidine skeleton (Figure 6.25). Following the coupling, it is suggested that the This bicyclic ring system is closely related to imine system is hydrolysed, the primary amine the ornithine-derived pyrrolizidine system, but is group then oxidized, and formation of the quino- formed from two molecules of lysine. Lupi- lizidine ring is achieved by Schiff base formation. nine from Lupinus luteus is a relatively sim- Lupinine is then synthesized by two reductive ple structure very comparable to the basic ring steps. system of the pyrrolizidine alkaloid retronecine The pathway to sparteine and lupanine (see page 305), but other lupin alkaloids, e.g. undoubtedly requires participation of another lupanine and sparteine (Figure 6.25) contain a molecule of cadaverine or ∆1-piperideine. Exper- tetracyclic bis-quinolizidine ring system, and are imental data are not clear-cut and Figure 6.25 formed by incorporation of a third lysine molecule. merely indicates how incorporation of a further Sparteine is also the major alkaloid in broom piperidine ring might be envisaged. Loss of one (Cytisus scoparius; Leguminosae/Fabaceae). The or other of the outermost rings and oxidation to a alkaloid cytisine, a toxic component of Laburnum pyridone system offers a potential route to cytisine. species (Leguminosae/Fabaceae) contains a mod- Quinolizidine alkaloids are mainly found in ified tricyclic ring system, and comparison with plants of the Leguminosae/Fabaceae family. They the structures of lupanine or sparteine shows its deter or repel feeding of herbivores, and are toxic likely relationship by loss of carbon atoms from to them by a variety of mechanisms. A number the tetracyclic system (Figure 6.25). However, the of plants (Laburnum, Cytisus, Lupinus) containing structural similarity of lupinine and retronecine significant quantities of these alkaloids must be is not fully reflected in the biosynthetic path- regarded as potentially toxic to humans, and are ways. Experimental evidence shows lysine to be known to be responsible for human poisoning. 310 ALKALOIDS

L-Lys aldol-type reaction between enamine and iminium NH2 diamine Schiff base NH H N oxidase formation H 2 NH N 2 CHO H NH NH2 NH2 NH NH hydrolysis of cadaverine imine to aldehyde/amine Schiff base oxidative OH CHO CHO CHO CHO formation deamination H H H H H

NH N N N NH OHC (–)-lupinine NH2 Schiff base HN formation

H further coupling H H N N N

N N N H O (+)-lupanine

O 9 1 10 H cleavage of C4 unit 8 2 N N N 7 3 5 N HN 6 4 H quinolizidine (–)-sparteine (+)-cytisine

Figure 6.25

The widely planted and ornamental laburnum trees they are derived from lysine, their origin devi- offer a particular risk, since all parts, including the ates from the more common lysine-derived struc- pealike seeds, contain dangerously high amounts tures in that L-pipecolic acid is an intermediate of alkaloids. So-called ‘sweet lupins’ are selected in the pathway. Two routes to pipecolic acid are strains with an acceptably low alkaloid content known in nature as indicated in Figure 6.26, and (typically about a quarter of the total alkaloids these differ with respect to whether the nitro- of ‘bitter’ strains), which are grown as a high gen atom originates from the α-ortheε-amino protein crop. group of lysine. For indolizidine alkaloid biosyn- thesis, pipecolic acid is formed via the aldehyde and Schiff base with retention of the α-amino Indolizidine Alkaloids group nitrogen. The indolizidinone may then be produced by incorporating a C2 acetate unit by Indolizidine alkaloids (Figure 6.26) are charac- simple reactions, though no details are known. terized by fused six- and five-membered rings, This compound leads to castanospermine by a with a nitrogen atom at the ring fusion, e.g. sequence of hydroxylations, but is also a branch- swainsonine from Swainsona canescens (Legu- point compound to alkaloids such as swainsonine, minosae/Fabaceae) and castanospermine from which have the opposite configuration at the ring the Moreton Bay chestnut Castanospermum aus- fusion. Involvement of a planar iminium ion could trale (Leguminosae/Fabaceae). In this respect, they account for the change in stereochemistry. Polyhy- appear to be a hybrid between the pyrrolizidine and droxyindolizidines such as swainsonine and cas- quinolizidine alkaloids described above. Although tanospermine have demonstrated activity against ALKALOIDS DERIVED FROM LYSINE 311

CO2H in some organisms, CO2H pipecolic acid is produced L-Lys O by this alternative pathway NH2 N formation of CoA ester, then Claisen reaction with acetyl (or malonyl) CoA HSCoA acetyl-CoA O HO2C HO2C H NH2 NH2 CO2H CO2H

NH2 O N NH NH O SCoA L-Lysα-aminoadipic acid piperideine-6- L-pipecolic acid ring closure δ-semialdehyde carboxylic acid via amide or imine further hydroxylation 8 1 HO steps OH O 9 H OH H H 7 2 HO 6 N 4 N 5 3 N N HO indolizidine castanospermine 1-indolizidinone

reduction of planar system allows change in stereochemistry HO at ring fusion OH OH H OH H H OH

OH OH OH N N N N swainsonine

Figure 6.26

OH exocyclic OH OH HO OH hydrolytic H HO OH cleavage HO OH HO OH HO H O R OH OH N O O O N OH O H 6-O-butanoyl- β-D-glucosideoxonium ion castanospermine castanospermine

Figure 6.27 the AIDS virus HIV, by their ability to inhibit gly- animals, causing severe gastro-intestinal upset and cosidase enzymes involved in glycoprotein biosyn- malnutrition by severely affecting intestinal hydro- thesis. The glycoprotein coating is essential for the lases. Indolizidine alkaloids are found in many proliferation of the AIDS virus. This has stimulated plants in the Leguminosae/Fabaceae (e.g. Swain- considerable research on related structures and sona, Astragalus, Oxytropis) and also in some their mode of action. The ester 6-O-butanoyl- fungi (e.g. Rhizoctonia leguminicola). castanospermine (Figure 6.27) is currently in clin- ical trials as an anti-AIDS agent. There is a strong ALKALOIDS DERIVED FROM similarity between castanospermine and the oxo- nium ion formed by hydrolytic cleavage of a gluco- NICOTINIC ACID side (Figure 6.27) (see page 30), but there appears Pyridine Alkaloids to be little stereochemical relationship with some other sugars, whose hydrolytic enzymes are also The alkaloids found in tobacco∗ (Nicotiana strongly inhibited. These alkaloids are also toxic to tabacum; Solanaceae) include nicotine and 312 ALKALOIDS

the latter rings arising from ornithine and lysine CO2H CONH2 CO2 respectively. The pyridine unit has its origins ∗ in nicotinic acid (vitamin B3) (Figure 6.28), N N N the vitamin sometimes called niacin,which nicotinic acid nicotinamide Me forms an essential component of coenzymes (niacin / vitamin B ) 3 trigonelline such as NAD+ and NADP+ (see page 24). The nicotinic acid component of nicotinamide is synthesized in animals by degradation of N N N H Me H H H H L-tryptophan through the kynurenine pathway N N N and 3-hydroxyanthranilic acid (Figure 6.29) (see nicotine nornicotine anabasine also dactinomycin, page 432), the pyridine ring being formed by oxidative cleavage of the Figure 6.28 benzene ring and subsequent inclusion of the amine nitrogen (Figure 6.29). However, plants anabasine (Figure 6.28). The structures contain such as Nicotiana use a different pathway a pyridine ring together with a pyrrolidine ring employing glyceraldehyde 3-phosphate and L- (in nicotine) or a piperidine unit (in anabasine), aspartic acid precursors (Figure 6.30). The dibasic

oxidative cleavage hydrolysis of CO2H CO2H CO2H CO2H of indole system amide NH2 NH2 NH2 O NH2 O2 2 O O NADPH O N CHO H N NH2 NH2 H L-Trp OH N-formylkynurenine L-kynurenine 3-hydroxykynurenine pyridoxal P-dependent PLP L-Ala PLP reactions allow cleavage L-Ala of side-chain CO2H CO2H

CONH2

NH2 NH2 N NCO2H anthranilic acid OH R picolinic acid 3-hydroxyanthranilic acid + NAD cis–trans isomerization of oxidative Schiff base – CO2 double bond via cleavage of formation generates enamine–imine aromatic ring heterocyclic ring CO2H CO H tautomerism CO2H – CO2 2 CO2H

non-enzymic O O N N CO2H CO2H non-enzymic NH H2N HO C 2 nicotinic acid quinolinic acid 2

Figure 6.29

dehydration and OP dehydrogenation HO CO2H HO CO2H CO2H CO2H – CO2

O H2NCO2H N CO2H N CO2HN 3-phospho- L-Asp quinolinic acid nicotinic acid glyceraldehyde

Figure 6.30 ALKALOIDS DERIVED FROM NICOTINIC ACID 313

Vitamin B3

Vitamin B3 (nicotinic acid, niacin) (Figure 6.28) is a stable water-soluble vitamin widely distributed in foodstuffs, especially meat, liver, fish, wheat germ, and yeast. However, in some foods, e.g. maize, it may be present in a bound form, and is not readily available. Diets based principally on maize may lead to deficiencies. The amino acid tryptophan can be converted in the body into nicotinic acid (Figure 6.29), and may provide a large proportion of the requirements. Nicotinic acid is also produced during the roasting of coffee from the decomposition of the N-methyl derivative trigonelline (Figure 6.28). Nicotinic acid is converted into nicotinamide (Figure 6.28), though this compound also occurs naturally in many foods. The term vitamin B3 is often used for the combined nicotinamide–nicotinic acid complement. In the form of the coenzymes NAD+ and NADP+, nicotinamide plays a vital role in oxidation–reduction reactions (see page 24), and is the most important electron carrier in primary metabolism. Deficiency in nicotinamide leads to pellagra, which manifests itself in diarrhoea, dermatitis, and dementia. Oral lesions and a red tongue may be more noticeable than the other symptoms. Nicotinamide is usually preferred over nicotinic acid for dietary supplements since there is less risk of gastric irritation. Both are produced synthetically. It is common practice to enrich many foods, including bread, flour, corn, and rice products. Nicotinic acid in large doses can lower both cholesterol and triglyceride concentrations by inhibiting their synthesis.

1,4-reduction of pyridine L-Orn to dihydropyridine aldol-type reaction between H enamine and iminium ion H O H CO2H H NADPH – CO2 O H N NH NH2 H N N H 2 H N putrescine (NADPH) nicotinic acid H H Me H 1,2-dihydro- N-methyl- dihydronicotinic pyridine pyrrolinium acid cation hydroxylation of oxidation of non-enzymic N-methyl dihydropyridine back decomposition to pyridine system O2 H N – HCHO N NADPH N NADP+ N H H H H Me H CH OH H Me N N 2 N N H nornicotine nicotine NADP+

Figure 6.31

acid quinolinic acid features in both pathways, This allows an aldol-type reaction with the decarboxylation yielding nicotinic acid. N-methylpyrrolinium cation, and finally dehydro- In the formation of nicotine, a pyrrolidine genation of the dihydropyridine ring back to a ring derived from ornithine, most likely as the pyridine gives nicotine. Nornicotine is derived N-methyl-∆1-pyrrolinium cation (see Figure 6.2) by oxidative demethylation of nicotine. Anaba- is attached to the pyridine ring of nicotinic sine is produced from nicotinic acid and lysine acid, displacing the carboxyl during the sequence via the ∆1-piperidinium cation in an essentially (Figure 6.31). A dihydronicotinic acid interme- analogous manner (Figure 6.32). A subtle anomaly diate is likely to be involved allowing decar- has been exposed in that a further Nicotiana boxylation to the enamine 1,2-dihydropyridine. alkaloid anatabine appears to be derived by 314 ALKALOIDS

combination of two nicotinic acid units, and the nicotinic L-Lys ∆3-piperideine ring is not supplied by lysine acid (Figure 6.33). Nicotinic acid undoubtedly provides the basic H skeleton for some other alkaloids. Ricinine H N NH2 NH2 (Figure 6.35) is a 2-pyridone structure and contains N H H H ∆1-piperidinium cadaverine a nitrile grouping, probably formed by dehydration 1,2-dihydro- cation of a nicotinamide derivative. This alkaloid is pyridine a toxic constituent of castor oil seeds (Ricinus aldol-type reaction between enamine and iminium ion communis; Euphorbiaceae), though the toxicity of the seeds results mainly from the polypeptide ricin (see page 434). Arecoline (Figure 6.36) is found ∗ N in Betel nuts (Areca catechu: Palmae/Arecaceae) H H and is a tetrahydronicotinic acid derivative. Betel N anabasine nuts are chewed in India and Asia for the stimulant effect of arecoline. Figure 6.32

H H nicotinic CO2H – CO2 H acid H H N N H H H dihydronicotinic 1,2-dihydro- acid pyridine enamine–imine tautomerism – CO aldol-type reaction 2 H between enamine and H iminium ion H H N N N H H H N N H H 1,4-dihydro- anatabine H pyridine 1,2-dihydro- pyridine

Figure 6.33

Tobacco Tobacco is the cured and dried leaves of Nicotiana tabacum (Solanaceae), an annual herb indigenous to tropical America, but cultivated widely for smoking. Tobacco leaves may contain from 0.6–9% of (−)-nicotine (Figure 6.28), an oily, volatile liquid alkaloid, together with smaller amounts of structurally related alkaloids, e.g. anabasine and nornicotine (Figure 6.28). In the leaf, the alkaloids are typically present as salts with malic and citric acids. Nicotine in small doses can act as a respiratory stimulant, though in larger doses it causes respiratory depression. Despite the vast array of evidence linking tobacco smoking and cancer, the smoking habit continues throughout the world, and tobacco remains a major crop plant. Tobacco smoke contains a number of highly carcinogenic chemicals formed by incomplete combustion, including benzpyrene, 2-naphthylamine, and 4-aminobiphenyl. Metabolism by the body’s P-450 system leads to further reactive intermediates, which can combine with DNA and cause mutations. Tobacco smoking also contributes to atherosclerosis, chronic

(Continues) ALKALOIDS DERIVED FROM NICOTINIC ACID 315

(Continued)

bronchitis, and emphysema, and is regarded as the single most preventable cause of death in modern society. Nicotine is being used by former smokers who wish to stop the habit. It is available in the form of chewing gum or nasal sprays, or can be absorbed transdermally from nicotine-impregnated patches.

Me Me Me H H N N N N OMe Me O O Me O acetylcholine nicotine arecoline (as conjugate acid) (as conjugate acid)

Figure 6.34

Powdered tobacco leaves have long been used as an insecticide, and nicotine from Nicotiana tabacum or N. rustica has been formulated for agricultural and horticultural use. The free base is considerably more toxic than salts, and soaps were included in the formulations to ensure a basic pH and to provide a surfactant. Other Nicotiana alkaloids, e.g. anabasine and nornicotine, share this insecticidal activity. Although an effective insecticide, nicotine has been replaced by other agents considered to be safer. Nicotine is toxic to man due to its effect on the nervous system, interacting with the nicotinic acetylcholine receptors, though the tight binding observed is only partially accounted for by the structural similarity between acetylcholine and nicotine (Figure 6.34). Recent studies suggest that nicotine can improve memory by stimulating the transmission of nerve impulses, and this finding may account for the lower incidence of Alzheimer’s disease in smokers. Any health benefits conferred by smoking are more than outweighed by the increased risk of heart, lung, and respiratory diseases.

OMe ALKALOIDS DERIVED FROM CONH2 CN TYROSINE nicotinic acid Phenylethylamines and Simple N N O nicotinamide Me Tetrahydroisoquinoline Alkaloids ricinine PLP-dependent decarboxylation of L-tyrosine Figure 6.35 gives the simple phenylethylamine derivative

Areca Areca nuts (betel nuts) are the seeds of Areca catechu (Palmae/Arecaceae), a tall palm cultivated in the Indian and Asian continents. These nuts are mixed with lime, wrapped in leaves of the betel pepper (Piper betle) and then chewed for their stimulant effect, and subsequent feeling of well-being and mild intoxication. The teeth and saliva of chewers stain bright red. The major stimulant alkaloid is arecoline (up to 0.2%) (Figure 6.36), the remainder of the alkaloid content (total about 0.45%) being composed of related reduced pyridine

(Continues) 316 ALKALOIDS

(Continued)

structures, e.g. arecaidine, guvacine (tetrahydronicotinic acid), and guvacoline (Figure 6.36). Arecoline is an agonist for muscarinic acetylcholine receptors (see Figure 6.34), although it possesses a reversed ester profile compared with acetylcholine. Areco- line has been employed in veterinary practice as a vermicide to eradicate worms.

CO2Me CO2H CO2Me CO2H

N N N N Me Me H H arecolinearecaidine guvacoline guvacine

Figure 6.36 tyramine, which on di-N-methylation yields or flight’ hormone released in animals from hordenine, a germination inhibitory alka- the adrenal gland as a result of stress. loid from barley (Hordeum vulgare;Gram- These compounds are synthesized by succes- inae/Poaceae) (Figure 6.37). More commonly, sive β-hydroxylation and N-methylation reactions phenylethylamine derivatives possess 3,4-di- or on dopamine (Figure 6.37). Aromatic hydroxy- 3,4,5-tri-hydroxylation patterns, and are derived lation and O-methylation reactions in the cac- via dopamine (Figure 6.37), the decarboxylation tus Lophophora williamsii∗ (Cactaceae) convert product from L-DOPA (L-dihydroxyphenylal- dopamine into mescaline (Figure 6.37), an alka- anine) (see page 129). Pre-eminent amongst loid with pyschoactive and hallucinogenic prop- these are the catecholamines∗ noradrenaline erties. Note that the sequence of hydroxylations (norepinephrine), a mammalian neurotransmit- and methylations exactly parallel those described ter, and adrenaline (epinephrine), the ‘fight for the cinnamic acids (see page 131).

CO H 2 PLP SAM

NH2 NH2 NMe2 HO HO HO L-Tyr tyramine hordenine O2 tetrahydrobiopterin OH OH

HO CO2H HO O2 HO HO PLP ascorbate SAM

NH2 NH2 NH2 NHMe HO HO HO HO L-DOPA dopaminenoradrenaline adrenaline (norepinephrine) (epinephrine) SAM MeO

NH2 HO

MeO MeO MeO SAM SAM NH 2 NH2 NH2 HO HO MeO OH OMe OMe mescaline

Figure 6.37 ALKALOIDS DERIVED FROM TYROSINE 317

Catecholamines The catecholamines dopamine, noradrenaline (norepinephrine), and adrenaline (epinephrine) are produced in the adrenal glands and nervous tissue and act as neurotransmitters in mammals. Several adrenergic receptors have been identified. α-Receptors are usually excitatory and produce a constricting effect on vascular, uterine, and intestinal muscles. β-Receptors are usually inhibitory on smooth muscle, but stimulatory on heart muscles. Dopamine (Figure 6.37) can act on both vascular α1 and cardiac β1 receptors, but also has its own receptors in several other structures. In Parkinson’s disease, there is a deficiency of dopamine due to neural degeneration, affecting the balance between excitatory and inhibitory transmitters. Treatment with L-DOPA (levodopa) (Figure 6.37) helps to increase the dopamine levels in the brain. Unlike dopamine, DOPA can cross the blood–brain barrier, but needs to be administered with a DOPA-decarboxylase inhibitor, e.g. carbidopa (Figure 6.38), to prevent rapid decarboxylation in the bloodstream. Injections of dopamine or dobutamine (Figure 6.38) are valuable as cardiac stimulants in cases of cardiogenic shock. These agents act on β1 receptors; dopexamine (Figure 6.38) is also used for chronic heart failure but acts on β2 receptors in cardiac muscle. Noradrenaline (norepinephrine) (Figure 6.37) is a powerful peripheral vasoconstrictor predominantly acting on α-adrenergic receptors, and is useful in restoring blood pressure in cases of acute hypotension. The structurally related alkaloid ephedrine (see page 384) may be used in the same way, and synthetic analogues of noradrenaline, e.g. phenylephrine, methoxamine,andmetaraminol (Figure 6.38), have also been developed. Methyldopa is used to treat hypertension; it is a centrally acting agent that becomes decarboxylated and hydroxylated to form the false transmitter α-methylnoradrenaline, which competes with noradrenaline. Adrenaline (epinephrine) (Figure 6.37) is released from the adrenal glands when an animal is confronted with an emergency situation, markedly stimulating glycogen breakdown in muscle, increasing respiration, and triggering catabolic processes that result in energy release. Adrenaline interacts with both α-andβ- receptors, an α-response being vasoconstriction of smooth muscle in the skin. β-Responses include mediation of cardiac muscle contractions and the relaxation of smooth muscle in the bronchioles of the lung. Injection of adrenaline is thus of value in cases of cardiac arrest, or in allergic emergencies such as bronchospasm or severe allergy (anaphylactic shock). It is not effective orally. A wide range of cardioactive β-adrenoceptor blocking agents (beta-blockers) has been developed to selectively bind

HO CO2H HO OH HO

NHNH2 HN HN HO HO RS HO NH carbidopa dobutamine dopexamine

OH OH OH RS HO MeO HO HO CO2H RS NHMe NH2 NH2 NH2 OMe HO phenylephrine methoxamine metaraminol methyldopa

Figure 6.38

(Continues) 318 ALKALOIDS

(Continued)

to β-receptors to control the rate and force of cardiac contractions in the management of hypertension and other heart conditions. The prototype of the beta-blocker drugs is propranolol (Figure 6.39), in which the catechol ring system has been modified to a

OH OH OH RS RS O O O RS O O HN HN HN N H2N H O atenolol propranolol acebutolol

OH OH OH RS H2N O O RSO RS

O HN HN O HN O O H2N O betaxolol bisoprolol O bambuterol OH OH OH O RSO RSO RS O HN HN HN O Et2N N H HN N OMe O H carteolol carvedilol celiprolol O

OH OH H OH O RS HO RS O N RS

HN HN RS HN RS MeO2C HO esmolol OH OH OMe fenoterol formoterol (eformoterol)

OH O OH OH OH HO RS RS O O RS H2N O HN HN HN HN O HO HO RS isoprenaline labetalol (isoproterenol) O levobunolol metipranolol

OH OH OH RS RS O O O RS RS OH HN HN HN RS O MeO F RS metoprolol OH nebivolol F HO nadolol OH O OH OH OH HO RS O RS O RS RS HO HN HN HN HN HO OH N oxprenolol H pindolol salbutamol orciprenaline (albuterol) (metaproterenol)

Figure 6.39 (continues)

(Continues) ALKALOIDS DERIVED FROM TYROSINE 319

(Continued)

OH OH HO RS Me RS N HO N HN N HN O HO O N OH salmeterol reproterol O Me O OH OH RS HO RS N OH O HN HN MeSO2HN N N HN OH S sotalol Cl OH terbutaline OH timolol RS O RS O HN HN HO N N H tulobuterol xamoterol O

Figure 6.39 (continued) naphthalene ether, and a bulky N-alkyl substituent has been incorporated. Many structural variants have been produced and there is now a huge, perhaps bewildering, variety of beta-blockers in regular use, with subtle differences in properties and action affecting the choice of drug for a particular condition or individual patient. These are shown in Figure 6.39. Atenolol, betaxolol, bisoprolol, metoprolol, nebivolol,andto a lesser extent acebutolol, have less effect on the β2 bronchial receptors and are thus relatively cardioselective. Most other agents are non-cardioselective, and could also provoke breathing difficulties. Esmolol and sotalol are used only in the management of arrhythmias. Other β-agonists are valuable as antiasthmatic drugs. Important examples include salbutamol (albuterol)andterbutaline, which are very widely prescribed, principally for administration by inhalation at the onset of an asthma attack, but, as with cardioactive beta-blockers, a wide range of agents is in current use (Figure 6.39). These agents are mainly selective towards the β2-receptors, and supersede the earlier less selective bronchodilator drugs such as isoprenaline (isoproterenol)andorciprenaline (metaproterenol) (Figure 6.39). Topical application of a beta-blocker to the eye reduces intra- ocular pressure by reducing the rate of production of aqueous humour. Some drugs in this class, namely betaxolol, carteolol, levobunolol, metipranolol,andtimolol, are thus useful in treating glaucoma. Propranolol, metoprolol, nadolol,andtimolol also have additional application in the prophylaxis of migraine. Catecholamine neurotransmitters are subsequently inactivated by enzymic methylation of the 3-hydroxyl (via catechol-O-methyltransferase) or by oxidative removal of the amine group via monoamine oxidase. Monoamine oxidase inhibitors are sometimes used to treat depression, and these drugs cause an accumulation of amine neurotransmitters. Under such drug treatment, simple amines such as tyramine in cheese, beans, fish, and yeast extracts are also not metabolized and can cause dangerous potentiation of neurotransmitter activity.

(Continues) 320 ALKALOIDS

(Continued)

Lophophora

Lophophora or peyote consists of the dried sliced tops of Lophophora williamsii (Cactaceae), a small cactus from Mexico and the SW United States. The plant has been used by the Aztecs and since by the Mexican Indians for many years, especially in religious ceremonies to produce hallucinations and establish contact with the gods. The so-called mescal buttons were ingested and this caused unusual and bizarre coloured images. The plant is still used by people seeking drug-induced experiences. The most active of the range of alkaloids found in lophophora (total 8–9% alkaloids in the dried mescal buttons) is mescaline (Figure 6.37), a simple phenylethylamine derivative. Other constituents include anhalamine, anhalonidine, and anhalonine (Figure 6.40). Mescaline has been used as a hallucinogen in experimental psychiatry. The dosage required is quite large (300–500 mg), but the alkaloid can readily be obtained by total synthesis, which is relatively uncomplicated. Mescaline is also found in other species of cactus, e.g. Trichocereus pachanoi,a substantially larger columnar plant that can grow up to 20 feet tall, and found mainly in the Andes.

Closely-related alkaloids cooccurring with The additional carbon atoms, two in the case of mescaline are anhalamine, anhalonine,and anhalonidine and anhalonine, and one for anhala- anhalonidine (Figure 6.40), which are representa- mine, are supplied by pyruvate and glyoxylate tives of simple tetrahydroisoquinoline derivatives. respectively. In each case, a carboxyl group is

5 4 MeO 6 3

7 NH NH2 2 HO 8 1 OH formation of tetrahydroisoquinoline O Schiff base SAM Mannich-like reaction; para position Me CO2H in aromatic ring is nucleophilic due pyruvic acid to mesomeric effect MeO MeO MeO

NH2 N H NH MeO MeO MeO H OH HO Me CO H HO Me CO H O 2 2 formation of favoured oxidative aromatic tautomer HCO2H decarboxylation MeO MeO glyoxylic acid N NH MeO MeO

HO Me HO Me CO2H reduction

MeO MeO MeO SAM NH NH NH MeO MeO MeO RS RS OH HO Me MeO Me anhalamine anhalonidine anhalonine

Figure 6.40 ALKALOIDS DERIVED FROM TYROSINE 321 lost from this additional precursor. The keto acid usually employ keto acids like pyruvate or alde- pyruvate reacts with a suitable phenylethylamine, hyde acids like glyoxylate. Instead, simple alde- in this case the dimethoxy-hydroxy derivative, hydes, e.g. acetaldehyde or formaldehyde, could giving a Schiff base (Figure 6.40). In a Mannich- be used (Figure 6.41, a), giving the same product like mechanism, cyclization occurs to generate directly without the need for a decarboxylation step the isoquinoline system, the mesomeric effect of to convert the intermediate tetrahydroisoquino- an oxygen substituent providing the nucleophilic linecarboxylic acid (Figure 6.41, b). In nature, both site on the aromatic ring. Restoration of aro- routes are in fact found to operate, depending on maticity via proton loss gives the tetrahydroiso- the complexity of the R group. Thus, the keto acid quinoline, overall a biosynthetic equivalent of the (route b) is used for relatively simple substrates Pictet–Spengler synthesis. The carboxyl group is (R = H, Me) whilst more complex precursors (R = then removed, not by a simple decarboxylation, ArCH2,ArCH2CH2, etc) are incorporated via the but via an unusual oxidative decarboxylation first corresponding aldehydes (route a). The stereo- generating the intermediate imine, reduction finally chemistry in the product is thus controlled by leading to anhalonidine with further methylation the condensation/Mannich reactions (route a), or giving anhalonine. Anhalamine is derived from by the final reduction reaction (route b). Occa- the same phenylethylamine precursor utilizing gly- sionally, both types of transformation have been oxylic acid (Figure 6.40). demonstrated in the production of a single com- The chemical synthesis of tetrahydroisoquino- pound, an example being the Lophophora schotti lines by the Pictet–Spengler reaction does not alkaloid lophocerine (Figure 6.42). This requires

(O) aldehyde (O) RCHO NH2 a (O) NH (O) (O) (O) R b keto acid RCOCO2H – CO2 Route a: R = Ar, ArCH2, ArCH2CH2, (O) OGlc

NH O (O) MeO2C (O) R CO2H Route b: R = H, Me

Figure 6.41

PPO H2NCO2H HO O OCO2H

isopentenyl PP MeO L-Leu

NH2 HO Mannich-like reaction

MeO MeO MeO SAM – CO2 NMe NH NH HO HO HO RS HO2C

lophocerine

Figure 6.42 322 ALKALOIDS

CH3CH2OH 5 4 6 3

7 NH 2 CH3CHO 8 1 2′ HO CO2H HO α ′ 3 1′ NH2 NH ′ HO HO 4′ 6 RS 5′ L-DOPA salsolinol benzyltetrahydroisoquinoline

Figure 6.43 Figure 6.44

utilization of a C5 isoprene unit, incorporated via alkaloid salsolinol (Figure 6.43) is found in plants, an aldehyde. However, a second route using the e.g. Corydalis spp. (Papaveraceae), but can also keto acid derived from the amino acid L-leucine be detected in the urine of humans as a product by transamination has also been demonstrated. The from dopamine and acetaldehyde combining via

Mannich-like HO O reaction

NH2 NH HO MeO HO 2 –CO2 HO PLP tyramine dopamine NH NH HO SAM HO H H L-Tyr O CO H 2 CHO PLP –CO2 transamination HO HO (S)-norcoclaurine (S)-coclaurine HO HO 4-hydroxyphenyl- 4-hydroxyphenyl- pyruvic acid acetaldehyde SAM

MeO methylations and MeO MeO dehydrogenations N NH NMe MeO HO HO H H MeO HO

MeO MeO HO papaverine N-nor-reticuline (S)-N-methyl- coclaurine O reduction of planar iminium ion 2 ascorbate allows change in stereochemistry MeO MeO MeO MeO

NMe NMe NMe NMe HO NADPH HO NADP+ HO SAM HO H H H HO HO HO HO

MeO MeO MeO HO (R)-reticuline 1,2-dehydroreticulinium (S)-reticuline (S)-3′-hydroxy- cation N-methylcoclaurine

morphinans aporphines protoberberines benzophenanthridines

Figure 6.45 ALKALOIDS DERIVED FROM TYROSINE 323 a Pictet–Spengler reaction. Acetaldehyde is typi- (S )-reticuline, a pivotal intermediate to other cally formed after ingestion of ethanol. alkaloids, is attained by N-methylation. Sur- Incorporation of a phenylethyl unit into the prisingly, some alkaloids, such as the opium phenylethylamine gives rise to a benzyltetrahy- alkaloids morphine, codeine, and thebaine (see droisoquinoline skeleton (Figure 6.44), which can page 327) are elaborated from (R)-reticuline rather undergo further modifications to produce a wide than the first-formed (S )-isomer. The change range of plant alkaloids, many of which feature in configuration is known to be achieved by as important drug materials. Fundamental changes an oxidation–reduction process and the inter- to the basic skeleton increase the diversity of mediate 1,2-dehydroreticulinium ion, as shown structural types as described under ‘modified ben- in Figure 6.45. Papaverine, a benzylisoquino- zyltetrahydroisoqinolines’. Most examples of ben- line alkaloid found in opium (see page 331), is zyltetrahydroisoquinoline alkaloids and modified formed from N-nor-reticuline by successive O- structures contain ortho di-oxygenation in each methylations and oxidation in the heterocyclic ring aromatic ring, which pattern is potentially deriv- (Figure 6.45). able from the utilization of two DOPA molecules. Structures in which two (or more) benzylte- Although two tyrosine molecules are used in the trahydroisoquinoline units are linked together are biosynthetic pathway, only the phenylethylamine readily explained by a phenolic oxidative cou- fragment of the tetrahydroisoquinoline ring system pling mechanism (see page 28). Thus, tetrandrine is formed via DOPA, the remaining carbons com- (Figure 6.46), a bis-benzyltetrahydroisoquinoline ing from tyrosine via 4-hydroxyphenylpyruvic acid alkaloid isolated from Stephania tetrandra (Menis- and 4-hydroxyphenylacetaldehyde (Figure 6.45). permaceae) is easily recognized as a coupling prod- The product from the Mannich-like reaction uct from two molecules of (S )-N-methylcoclaurine is thus the trihydroxy alkaloid norcoclaurine, (Figure 6.46). The two diradicals, formed by one- formed stereospecifically as the (S )-enantiomer. electron oxidations of a free phenol group in each The tetrahydroxy substitution pattern is built up ring, couple to give ether bridges, and the product by further hydroxylation in the benzyl ring, is then methylated to tetrandrine. The pathway is though O-methylation [giving (S )-coclaurine]and much more likely to follow a stepwise coupling N-methylation steps precede this. Eventually, process requiring two oxidative enzymes rather

one electron oxidations of phenol groups give resonance-stabilized OMe free radicals radical coupling: this is likely to be a stepwise process MeN O MeO H OMe MeO

NMe MeN NMe HO O2 OH O H NADPH O H H

MeO HO OH O NMe (S)-N-methyl- O coclaurine H SAM

OMe MeO O MeN NMe OMe O H H

OMe O tetrandrine

Figure 6.46 324 ALKALOIDS than the combined one suggested in Figure 6.46. the principal active component in the arrow poi- Tetrandrine is currently of interest for its ability son curare∗ from Chondrodendron tomentosum to block calcium channels, and may have applica- (Menispermaceae), can be elaborated by a different tions in the treatment of cardiovascular disorders. By coupling of one molecule each of (S )- and (R)-N- a similar mechanism, tubocurarine (Figure 6.47), methylcoclaurine (Figure 6.47).

one-electron oxidations of phenol groups give resonance-stabilized MeO free radicals radical coupling: this is likely NMe O2 MeO to be a stepwise process HO NADPH H NMe MeO O MeO H NMe NMe HO H H (S)-N-methyl- O OH O OH coclaurine O SAM MeO O

O2 NMe NADPH O O HO H H H H O OH OH MeN MeN Me2N OMe HO OMe OMe (R)-N-methyl- tubocurarine coclaurine

Figure 6.47

Curare Curare is the arrow poison of the South American Indians, and it may contain as many as 30 different plant ingredients, which may vary widely from tribe to tribe according to local custom. Curare is prepared in the rain forests of the Amazon and Orinoco, and represents the crude dried extract from the bark and stems of various plants. The young bark is scraped off, pounded, and the fibrous mass percolated with water in a leaf funnel. The liquor so obtained is then concentrated by evaporation over a fire. Further vegetable material may be added to make the preparation more glutinous so that it will stick to the arrows or darts. The product is dark brown or black, and tarlike. In the 1880s, it was found that the traditional container used for curare was fairly indicative of the main ingredients that had gone into its preparation. Three main types were distinguished. Tube curare was packed in hollow bamboo canes, and its principal ingredient was the climbing plant Chondrodendron tomentosum (Menispermaceae). Calabash curare was packed in gourds, and was derived from Strychnos toxifera (Loganiaceae). Pot curare was almost always derived from a mixture of loganiaceous and menispermaceous plants, and was packed in small earthenware pots. Current supplies of curare are mainly of the menispermaceous type, i.e. derived from Chondrodendron. The potency of curare as an arrow poison is variable and consequently needs testing. A frequently quoted description of this testing is as follows: ‘If a monkey hit by a dart is only able to get from one tree to the next before it falls dead, this is ‘‘one-tree curare’’, the superior grade. ‘‘Two-tree curare’’ is less satisfactory, and ‘‘three-tree curare’’ is so weak that it can be used to bring down live animals that the Indians wish to keep in captivity.’ Thus, the poison

(Continues) ALKALOIDS DERIVED FROM TYROSINE 325

(Continued) does not necessarily cause death; it depends on the potency. Curare is only effective if it enters the bloodstream, and small amounts taken orally give no ill effects provided there are no open sores in the mouth or throat. Curare kills by producing paralysis, a limp relaxation of voluntary muscles. It achieves this by competing with acetylcholine at nicotinic receptor sites (see page 299), thus blocking nerve impulses at the neuromuscular junction. Death occurs because the muscles of respiration cease to operate, and artificial respiration is an effective treatment prior to the effects gradually wearing off through normal metabolism of the drug. Anticholinesterase drugs such as physostigmine and neostigmine are specific antidotes for moderate curare poisoning. Curare thus found medicinal use as a muscle relaxant, especially in surgical operations such as abdominal surgery, tonsillectomy, etc, where tense muscles needed to be relaxed. Curare was also found to be of value in certain neurological conditions, e.g. multiple sclerosis, tetanus, and Parkinson’s disease, to temporarily relax rigid muscles and control convulsions, but was not a curative. However, the potency of curare varied markedly, and supplies were sometimes limited. The alkaloid content of curare is from 4% to 7%. The most important constituent in menispermaceous curare is the bis-benzyltetrahydroisoquinoline alkaloid (+)-tubocurarine (Figure 6.48). This is a monoquaternary ammonium salt, and is water soluble. Other main alkaloids include non-quaternary dimeric structures, e.g. isochondrodendrine and curine (bebeerine) (Figure 6.48), which appear to be derived from two molecules of (R)-N- methylcoclaurine, with the former also displaying a different coupling mode. The constituents in loganiaceous curare (from calabash curare, i.e. Strychnos toxifera) are even more complex, and a series of 12 quaternary dimeric strychnine-like alkaloids has been identified, e.g. C-toxiferine (toxiferine-1) (see page 359). Tubocurarine (Figure 6.48) is still extracted from menispermaceous curare and injected as a muscle relaxant in surgical operations, reducing the need for deep anaesthesia. Artificial respiration is required until the drug has been inactivated (about 30 minutes) or antagonized (e.g. with neostigmine). The limited availability of tubocurarine has led to the development of a series of synthetic analogues, some of which have improved characteristics and are now

MeO MeO MeO

NMe NMe NMe HO H H H O OH O OH O

O O O H H H OH OH OH Me2N MeN MeN

OMe OMe OMe (+)-tubocurarine(–)-curine (+)-(R,R)-isochondrodendrine [(–)-(R,R)-bebeerine]

Figure 6.48

(Continues) 326 ALKALOIDS

(Continued)

preferred over the natural product. Interestingly, the structure of tubocurarine was originally formulated incorrectly as a diquaternary salt, rather than the monoquaternary salt, and analogues were based on the pretext that curare-like effects might be obtained from compounds containing two quaternary nitrogens separated by a polymethylene chain. This was borne out in practice, and the separation was found to be optimal at about ten carbons. Decamethonium (Figure 6.49) was the first synthetic curare-like muscle relaxant, but has since been superseded. In tubocurarine, the two nitrogens are also separated by ten atoms, and at physiological pHs it is likely that both centres will be positively charged. Obviously, the interatomic distance (1.4 nm in tubocurarine) is very dependent on the structure and stereochemistry rather than just the number of atoms separating the centres, but an extended conformation of decamethonium approximates to this distance. Suxamethonium

O O

Me3N Me3N Me3N O O NMe3 O NMe3 acetylcholine decamethonium suxamethonium O MeO OMe Me Me N OON MeO OMe Hofmann E2 elimination favoured by OO electron-withdrawing carbonyl group Me H OMe MeO atracurium NO OMe (mixture of stereoisomers) OMe O MeO OMe Me Me N OON MeO OMe OAc OO O H N OMe MeO N H H OMe cisatracurium OMe HO H OAc OAc rocuronium β Me β H N H N N N β Me Me H H H H AcO α AcO OMe H H MeO pancuronium vecuronium MeO Me O MeO N O OMe MeO ON Me OMe O OMe

OMe mivacurium OMe

Figure 6.49

(Continues) ALKALOIDS DERIVED FROM TYROSINE 327

(Continued)

(Figure 6.49) is an effective agent with a very short duration of action, due to the two ester functions, which are rapidly metabolized by an esterase (a pseudocholinesterase) in the body, and this means the period during which artificial respiration is required is considerably reduced. It also has ten-atom separation between the quaternary nitrogens. Atracurium (Figure 6.49) is a recent development, containing two quaternary nitrogens in benzyltetrahydroisoquinoline structures separated by 13 atoms. In addition to enzymic ester hydrolysis, atrocurium is also degraded in the body by non-enzymic E2 Hofmann elimination (Figure 6.49), which is independent of liver or kidney function. Normally, this elimination would require strongly alkaline conditions and a high temperature, but the presence of the carbonyl group increases the acidity and thus facilitates loss of the proton, and the elimination can proceed readily under physiological conditions, giving atracurium a half life of about 20 minutes. This is particularly valuable where patients have low or atypical pseudocholinesterase enzymes. Atracurium contains four chiral centres (including the quaternary nitrogens) and is supplied as a mixture of stereoisomers; the single isomer cisatracurium has now been introduced. This isomer is more potent than the mixture, has a slightly longer duration of action, and produces fewer cardiovascular side-effects. Mivacurium (Figure 6.49) has similar benzyltetrahydroisoquinoline structures to provide the quaternary centres, but the separation has now increased to 16 atoms. In pancuronium, separation of the two quaternary centres is achieved by a steroidal skeleton. This agent is about five times as potent as tubocurarine. Vecuronium is the equivalent monoquaternary structure, and has the fewest side-effects. Rocuronium is also based on a steroidal skeleton, and provides rapid action with no cardiovascular effects. The toxiferines (see Figure 6.85, page 359) also share the diquaternary character. Alcuronium is a semi-synthetic skeletal muscle relaxant containing the dimeric strychnine-like structure and is produced chemically from C-toxiferine (see page 360). These neuromuscular blocking agents act by occupying nicotinic acetylcholine (Figure 6.49) receptor sites. All the structures have two acetylcholine-like portions, which can interact with the receptor. Where these are built into a rigid framework, e.g. tubocurarine and pancuronium, the molecule probably spans and blocks several receptor sites. Tubocurarine and the heterocyclic analogues are termed non-depolarizing or competitive muscle relaxants. The straight chain structures, e.g. decamethonium and suxamethonium, initially mimic the action of acetylcholine but then persist at the receptor, and are termed depolarizing blocking agents. Thus they trigger a response, a brief contraction of the muscle, which is then followed by a prolonged period of muscular paralysis until the compound is metabolized.

Modified opium∗ alkaloids morphine, codeine,andthe- Benzyltetrahydroisoquinoline baine (Figure 6.50) are derived by this type of Alkaloids coupling, though the subsequent reduction of one aromatic ring to some extent disguises their ben- The concept of phenolic oxidative coupling is a zyltetrahydroisoquinoline origins. (R)-Reticuline crucial theme in modifying the basic benzylte- (Figure 6.45) is firmly established as the precursor trahydroisoquinoline skeleton to many other types of these morphinan alkaloids. of alkaloid. Tetrandrine (Figure 6.46) and tubocu- (R)-Reticuline, rewritten as in Figure 6.50, is rarine (Figure 6.47) represent coupling of two the substrate for one-electron oxidations via the benzyltetrahydroisoquinoline molecules by ether phenol group in each ring, giving the diradi- bridges, but this form of coupling is perhaps cal. Coupling ortho to the phenol group in the less frequent than that involving carbon–carbon tetrahydroisoquinoline, and para to the phenol in bonding between aromatic rings. The principal the benzyl substituent, then yields the dienone 328 ALKALOIDS

one-electron oxidation of phenol groups to give resonance-stabilized free radicals radical MeO MeO MeO MeO coupling O2 NMe NADPH HO HO O HO H ≡ HO NMe NMe NMe H H H MeO MeO MeO MeO O (R)-reticuline OH O salutaridine ′ stereospecific SN2 nucleophilic attack with reduction of carbonyl NADPH acetate as leaving group MeO MeO MeO MeO

O CH3COSCoA HO HO O O NMe NMe NMe NMe H H H H O O MeO MeO MeO H H thebaine OAc esterification provides OH demethylation of thebaine better leaving group salutaridinol via hydroxylation, cleaving demethylation off methyl as formaldehyde

MeO HO HO

demethylation O O O NMe NMe NMe H H H H O MeO O neopinone oripavine morphinone demethylation of codeine; keto–enol tautomerism; stereospecific reduction allylic isomerization probably via oxidation of favoured by conjugation methyl to hydroxymethyl and cleavage of 2 MeO MeO formaldehyde HO 3 1 4 12 11 10 NADPH 15 16 13 O O O 14 9 5 NH NMe NMe NMe 9a H H stereospecific H H H H 6 8 O reduction of HO HO 7 codeinonecarbonyl codeine morphine morphinan

Figure 6.50

salutaridine, found as a minor alkaloid constituent by stereospecific reduction of the carbonyl group. in the opium poppy Papaver somniferum (Papaver- Ring closure to form the ether linkage in the- aceae). Only the original benzyl aromatic ring baine would be the result of nucleophilic attack can be restored to aromaticity, since the tetrahy- of the phenol group on to the dienol system and droisoquinoline fragment is coupled para to the subsequent displacement of the hydroxyl. This phenol function, a position which is already sub- cyclization step can be demonstrated chemically by stituted. The alkaloid thebaine is obtained by treatment of salutaridinol with acid. In vivo, how- way of salutaridinol, formed from salutaridine ever, an additional reaction is used to improve the ALKALOIDS DERIVED FROM TYROSINE 329 nature of the leaving group, and this is achieved by The involvement of these O-demethylation acetylation with acetyl-CoA. The cyclization then reactions is rather unusual; secondary metabolic occurs readily, and without any enzyme participa- pathways tend to increase the complexity of the tion. Subsequent reactions involve conversion of product by adding methyls rather than removing thebaine into morphine by way of codeine, a pro- them. In this pathway, it is convenient to view the cess which modifies the oxidation state of the diene methyl groups in reticuline as protecting groups, ring, but most significantly removes two O-methyl which reduce the possible coupling modes avail- groups. One is present as an enol ether, removal able during the oxidative coupling process, and generating neopinone, which gives codeinone and these groups are then removed towards the end then codeine by allylic isomerization and reduc- of the synthetic sequence. There is also some tion respectively. The last step, demethylation of evidence that the later stages of the pathway in the phenol ether codeine to the phenol morphine, Figure 6.50 are modified in some strains of opium is the type of reaction only achievable in the poppy. In such strains, thebaine is converted by laboratory by the use of powerful and reactive way of oripavine and morphinone, this pathway demethylating agents, e.g. HBr or BBr3. Because removing the phenolic O-methyl before that of of the other functional groups present, chemical the enol ether, i.e. carrying out the same steps conversion of codeine into morphine is not usu- but in a different order. The enzymic transforma- ally a satisfactory process. However, the enzyme- tion of thebaine into morphine, and the conver- mediated conversion in P. somniferum proceeds sion of (R)-reticuline into salutaridinol have also smoothly and efficiently. The enzymic demethy- been observed in mammalian tissues, giving strong lations of both the enol ether and the phenol ether evidence that the trace amounts of morphine and probably involve initial hydroxylation followed related alkaloids which can sometimes be found in by loss of the methyl groups as formaldehyde mammals are actually of endogenous origin rather (Figure 6.50). than dietary.

Opium

Opium is the air-dried milky exudate, or latex, obtained by incising the unripe capsules of the opium poppy Papaver somniferum (Papaveraceae). The plant is an annual herb with large solitary flowers, of white, pink, or dull red-purple colour. For opium production, the ripening capsules, which are just changing colour from blue-green to yellow, are carefully incised with a knife to open the latex tubes, but not to cut through to the interior of the capsule. These latex tubes open into one another, so it is not necessary to incise them all. Cuts are made transversely or longitudinally according to custom. The initially white milky latex quickly oozes out, but rapidly turns brown and coagulates. This material, the raw opium, is then removed early the following morning, being scraped off and moulded into balls or blocks. Typically, these are wrapped in poppy leaves and shade-dried. The blocks may be dusted with various plant materials to prevent cohering. Fresh opium is pale to dark brown and plastic, but it becomes hard and brittle when stored. Opium has been known and used for 4000 years or more. In recent times, attempts have been made at governmental and international levels to control the cultivation of the opium poppy, but with only limited success. In endeavours to reduce drug problems involving opium-derived materials, especially heroin, where extremely large profits can be made from smuggling relatively small amounts of opium, much pharmaceutical production has been replaced by the processing of the bulkier ‘poppy straw’. The entire plant tops are harvested and dried, then extracted for their alkaloid content in the pharmaceutical industry. Poppy straw now accounts for most of the medicinal opium alkaloid production, but there is still

(Continues) 330 ALKALOIDS

(Continued)

considerable trade in illicit opium. In addition to opium, the opium poppy yields seeds, which are used in baking and are also pressed to give poppy seed oil. The remaining seed cake is used as cattle feed, and it is held that these poppy seed products cover all the growing expenses, with opium providing the profit. Poppy seeds do not contain any significant amounts of alkaloids. The main producer of medicinal opium is India, whilst poppy straw is cultivated in Turkey, Russia, and Australia. Opium destined for the black market originates from the Golden Triangle (Burma, Laos, and Thailand), the Golden Crescent (Iran, Pakistan, and Afghanistan), and Mexico. Crude opium has been used since antiquity as an analgesic, sleep-inducer (narcotic), and for the treatment of coughs. It has been formulated in a number of simple preparations for general use, though these are now uncommon. Laudanum, or opium tincture, was once a standard analgesic and narcotic mixture. Paregoric, or camphorated opium tincture, was used in the treatment of severe diarrhoea and dysentery, but is still an ingredient in the cough and cold preparation Gee’s linctus. In Dover’s powder, powdered opium was combined with powdered ipecacuanha (see page 344) to give a popular sedative and diaphoretic (promotes perspiration) to take at the onset of colds and influenza. Opium has traditionally been smoked for pleasure, but habitual use develops a craving for the drug followed by addiction. An unpleasant abstinence syndrome is experienced if the drug is withdrawn. In modern medicine, only the purified opium alkaloids and their derivatives are commonly employed. Indeed, the analgesic preparation ‘papaveretum ’ (see below), which once con- tained the hydrochlorides of total opium alkaloids, is now formulated from selected purified alkaloids, in the proportions likely to be found in opium. Although the ripe poppy capsule can contain up to 0.5% total alkaloids, opium represents a much concentrated form and up to 25% of its mass is composed of alkaloids. Of the many (>40) alkaloids identified, some six represent almost all of the total alkaloid content. Actual amounts vary widely, as shown by the following figures: morphine (Figure 6.50) (4–21%); codeine (Figure 6.50) (0.8–2.5%); thebaine (Figure 6.50) (0.5–2.0%); papaverine (Figure 6.45) (0.5–2.5%); noscapine (narcotine) (Figure 6.51) (4–8%); narceine (Figure 6.51; see also Figure 6.63, page 340) (0.1–2%). A typ- ical commercial sample of opium would probably have a morphine content of about 12%. Powdered opium is standardized to contain 10% of anhydrous morphine, usually by dilution with an approved diluent, e.g. lactose or cocoa husk powder. The alkaloids are largely com- bined in salt form with meconic acid (Figure 6.51), opium containing some 3–5% of this mate- rial. Meconic acid is invariably found in opium, but, apart from its presence in other Papaver species, has not been detected elsewhere. It gives a deep red-coloured complex with ferric chloride, and this has thus been used as a rapid and reasonably specific test for opium. In the

O O O HO 3 NMe OH O 2 NMe CO H O 2 H MeO OMe MeO HO C O CO H O O 2 2 O meconic acid HO2C NMe O H OMe OMe HO 6 H narceine O OMe HO O noscapine OH (narcotine) morphine 6-O-glucuronide

Figure 6.51

(Continues) ALKALOIDS DERIVED FROM TYROSINE 331

(Continued) past, the urine of suspected opium smokers could also be tested in this way. Of the main opium alkaloids, only morphine and narceine display acidic properties, as well as the basic properties due to the tertiary amine. Narceine has a carboxylic acid function, whilst morphine is acidic due to its phenolic hydroxyl. This acidity can be exploited for the preferential extraction of these alkaloids (principally morphine) from an organic solvent by partitioning with aqueous base. Morphine (Figure 6.50) is a powerful analgesic and narcotic, and remains one of the most valuable analgesics for relief of severe pain. It also induces a state of euphoria and mental detachment, together with nausea, vomiting, constipation, tolerance, and addiction. Regular users experience withdrawal symptoms, including agitation, severe abdominal cramps, diarrhoea, nausea, and vomiting, which may last for 10–14 days unless a further dose of morphine is taken. This leads to physical dependence, which is difficult to overcome, so that the major current use of morphine is thus in the relief of terminal pain. Although orally active, it is usually injected to obtain rapid relief of acute pain. The side-effect of constipation is utilized in some anti-diarrhoea preparations, e.g. kaolin and morphine. Morphine is metabolized in the body to glucuronides, which are readily excreted. Whilst morphine 3-O-glucuronide is antagonistic to the analgesic effects of morphine, morphine 6-O-glucuronide (Figure 6.51) is actually a more effective and longer lasting analgesic than morphine, with fewer side-effects. Codeine (Figure 6.50) is the 3-O-methyl ether of morphine, and is the most widely used of the opium alkaloids. Because of the relatively small amounts found in opium, most of the material prescribed is manufactured by semi-synthesis from morphine. Its action is dependent on partial demethylation in the liver to produce morphine, so it produces morphine- like analgesic effects, but little if any euphoria. As an analgesic, codeine has about one-tenth the potency of morphine. Codeine is almost always taken orally and is a component of many compound analgesic preparations. Codeine is a relatively safe non-addictive medium analgesic, but is still too constipating for long-term use. Codeine also has valuable antitussive action, helping to relieve and prevent coughing. It effectively depresses the cough centre, raising the threshold for sensory cough impulses. Thebaine (Figure 6.50) differs structurally from morphine/codeine mainly by its possession of a conjugated diene ring system. It is almost devoid of analgesic activity, but may be used as a morphine antagonist. Its main value is as substrate for the semi-synthesis of other drugs (see below). Papaverine (Figure 6.45) is a benzylisoquinoline alkaloid, and is structurally very different from the morphine, codeine, thebaine group of alkaloids (morphinans). It has little or no analgesic or hypnotic properties put possesses spasmolytic and vasodilator activity. It has been used in some expectorant preparations, and in the treatment of gastrointestinal spasms, but its efficacy was not substantiated. It is sometimes used as an effective treatment for male impotence, being administered by direct injection to achieve erection of the penis. Noscapine (Figure 6.51) is a member of the phthalideisoquinoline alkaloids (see page 339) and provides a further structural variant in the opium alkaloids. Noscapine has good antitussive and cough suppressant activity comparable to that of codeine, but no analgesic or narcotic action. Its original name ‘narcotine’ was changed to reflect this lack of narcotic action. Despite many years of use as a cough suppressant, the finding that noscapine may have teratogenic properties (i.e. may deform a fetus) has resulted in noscapine preparations being deleted. In recent studies, antitumour activity has been noted from noscapine, which binds to tubulin as do podophyllotoxin and colchicine (see pages 136 and 343), thus arresting cells at mitosis. The chemotherapeutic potential of this orally effective agent merits further evaluation.

(Continues) 332 ALKALOIDS

(Continued)

HO HO HO

Br O O O NMe NH N H H H H H H O HO HO hydromorphone normorphine nalorphine (i) H2, catalyst (ii) oxidation (i) BrCN (ii) H MeO HO AcO

Ac2O Me2SO4 Py O O O NMe NMe NMe H H H H H H HO HO AcO codeine morphine heroin ∆ Cl H , (diamorphine) N H2, catalyst O

MeO O HO N O HO O O H NMe NMe NMe H H H H HO HO dihydrocodeine pholcodine apomorphine

Figure 6.52

Papaveretum is a mixture of purified opium alkaloids, as their hydrochlorides, and is now formulated to contain only morphine (85.5%), codeine (7.8%), and papaverine (6.7%). It is used for pain relief during operations. A vast range of semi-synthetic or totally synthetic morphine-like derivatives have been produced. These are collectively referred to as ‘opioids’. Many have similar narcotic and pain-relieving properties as morphine, but are less habit forming. Others possess the cough-relieving activity of codeine, but without the analgesic effect. More than 90% of the morphine extracted from opium (or poppy straw) is currently processed to give other derivatives (Figure 6.52). Most of the codeine is obtained by semi-synthesis from morphine, mono-O-methylation occurring at the acidic phenolic hydroxyl. Similarly, pholcodine (Figure 6.52), an effective and reliable antitussive, can be obtained by alkylation with N-(chloroethyl)morpholine. Dihydrocodeine (Figure 6.52) is a reduced form of codeine with similar analgesic properties, the double bond not being essential for activity. In hydromorphone, the double bond of morphine has been reduced, and in addition the 6-hydroxyl has been oxidized to a ketone. This increases the analgesic effects, but also the side-effects. Diamorphine,orheroin (Figure 6.52) is merely the diacetate of morphine, and is a highly addictive analgesic and hypnotic. The increased lipophilic

(Continues) ALKALOIDS DERIVED FROM TYROSINE 333

(Continued) character results in better transport and absorption, though the active agent is probably the 6-acetate, the 3-acetate group being hydrolysed by esterases in the brain. Heroin was synthesized originally as a cough suppressant, and though most effective in this role has unpleasant addictive properties, with users developing a psychological craving for the drug. It is widely used for terminal care, e.g. cancer sufferers, both as an analgesic and cough suppressant. The euphoria induced by injection of heroin has resulted in much abuse of the drug, and creation of a world-wide major drug problem. The N-methyl group of morphine can be removed by treatment with cyanogen bromide, then hydrolysis. A variety of N-alkyl derivatives, e.g. N-allyl-normorphine (nalorphine) (Figure 6.52) may be produced by use of appropriate alkyl bromides. Nalorphine has some analgesic activity, but was also found to counter the effects of morphine, and is thus a mixed agonist–antagonist. It is sometimes used as a narcotic antagonist, but is principally regarded as the forerunner of pure opiate antagonists such as naloxone (see below). Treatment of morphine with hot acid induces a rearrangement process, resulting in a highly modified structural skeleton, a representative of the aporphine group of alkaloids (see page 337). The product apomorphine (Figure 6.52) has no analgesic properties, but morphine’s side-effects of nausea and vomiting are highly emphasized. Apomorphine is a powerful emetic, and can be injected for emergency treatment of poisoning. This is now regarded as dangerous, but apomorphine is also valuable to control the symptoms of Parkinson’s disease, being a stimulator of D1 and D2 dopamine receptors. Apomorphine’s structure contains a dihydroxyphenylethylamine (dopamine) fragment, conferring potent dopamine agonist properties to this agent. It has been found that a common structural feature required for centrally acting analgesic activity in the opioids is the combination of an aromatic ring, and a piperidine ring which maintain the stereochemistry at the chiral centre as shown in Figure 6.53. The three- dimensional disposition of the nitrogen function to the aromatic ring allows morphine and other analgesics to bind to a pain-reducing receptor in the brain. Several different receptors and groups of receptors are known. The natural agonists include peptides called enkephalins, Met-enkephalin and Leu-enkephalin (Figure 6.53), produced from a larger peptide endorphin (see page 419). The terminal tyrosine residue in the enkephalins is mimicked by portions of

SMe

O H

HO2CN HO2CN H H H H HN O O N OH H O Me N H2N N H H Leu-enkephalin O (Tyr–Gly–Gly–Phe–Leu)

OH OH morphine Met-enkephalin (Tyr–Gly–Gly–Phe–Met)

Figure 6.53

(Continues) 334 ALKALOIDS

(Continued)

HO HO HO HO

O NMe NMe Me N Me N H H H H H H Me Me HO morphine dextromethorphan phenazocine pentazocine

≡ (enantiomer = levomethorphan)

HO Et Et

ON ON O MeO C EtO2C Me 2 N CO2Me Me N N HO N pethidine fentanyl remifentanil morphine (meperidine)

Et

ON O O MeO N N O NN N RS NMe2 NMe2 dextropropoxyphene alfentanil O methadone (enantiomer = levopropoxyphene)

HO MeO

O O N N Me HO RS N N O meptazinol tramadol dipipanone dextromoramide NMe2

Figure 6.54

the morphine structure. The enkephalins themselves are rapidly degraded in the body and are unsuitable for drug use. Some totally synthetic opioid drugs modelled on morphine are shown in Figure 6.54. Removal of the ether bridge and the functionalities in the cyclohexene ring are exemplified in levomethorphan and dextromethorphan. Levomethorphan has analgesic properties, whilst both enantiomers possess the antitussive activity of codeine. In practice, the ‘unnatural’ isomer dextromethorphan is the preferred drug material, being completely non-addictive and possessing no analgesic activity. Pentazocine and phenazocine are examples of morphine- like structures where the ether bridge has been omitted and the cyclohexene ring has been replaced by simple methyl groups. These drugs are good analgesics and are non-addictive, though pentazocine can induce withdrawal symptoms. Even more drastic simplification of the morphine structure is found in pethidine (meperidine), one of the most widely used synthetic opiates. Only the aromatic ring and the piperidine systems are retained. Pethidine is less potent than morphine, but produces prompt, short-acting analgesia, and is also less

(Continues) ALKALOIDS DERIVED FROM TYROSINE 335

(Continued) constipating than morphine. It can be addictive. Fentanyl has a 4-anilino- rather than a 4-phenyl-piperidine structure, and is 50–100 times more active than morphine due to its high lipophilicity and excellent transport properties. Alfentanil and remifentanil are further variants on the fentanyl structure; all three drugs are rapid-acting and used during oper- ative procedures. The piperidine ring system is no longer present in methadone, though this diphenylpropylamine derivative can be drawn in such a way as to mimic the piperidine ring conformation. Methadone is orally active, has similar activity to morphine, but is less euphorigenic and has a longer duration of action. Although it is as potentially addictive as morphine, the withdrawal symptoms are different and much less severe than with other drugs such as heroin, and methadone is widely used for the treatment and rehabilitation of heroin addicts. However, it only replaces one addiction with another, albeit a less dangerous one. Dextropropoxyphene mimics the piperidine ring in a rather similar manner, but this agent has only low analgesic activity, about half that of codeine, and finds application in combination formulations with aspirin or paracetamol. The enantiomeric levopropoxyphene has antitus- sive activity, but no analgesic properties. Dipipanone and dextromoramide are structural variants on methadone, and are used for moderate to severe pain; dipipanone is usually administered in combination with an anti-emetic. Meptazinol is structurally unlike the other opiate analgesics in that it contains a seven-membered nitrogen heterocycle. It is an effective analgesic, and produces relatively few side-effects with a low incidence of respiratory depres- sion. Tramadol is a recent drug claimed to produce analgesia by an opioid mechanism and by enhancement of serotoninergic and adrenergic pathways, with few typical opioid side-effects.

hydrolysis of MeO 1,4-addition MeO MeO hemiketal H O 2 2 H O O O NMe NMe NMe HO HO H HO H H MeO O thebaine MeO

hydrolysis of H2 / Pd enol ether HBr MeO HO demethylation MeO using strong acid

HBr O O O NMe NMe NMe H H HO H HO H O O O codeinone oxymorphone oxycodone stereospecific reduction from NaBH4 less-hindered side MeO HO HO HO

O O O O NMe N N N H H HO H HO H HO H HO O O HO codeine naloxone naltrexone nalbuphine

Figure 6.55

(Continues) 336 ALKALOIDS

(Continued)

Thebaine, for many years regarded as an unwanted by-product from opium, is now utilized for the semi-synthesis of useful new drugs. On treatment with hydrogen peroxide, the conjugated diene undergoes 1,4-addition, and hydrolysis results in formation of a 4- hydroxy cyclohexenone system (Figure 6.55). Reduction and demethylation lead respectively to oxycodone and oxymorphone, which are potent analgesics. The conjugated diene system can also be exploited in a Diels–Alder reaction, building on another ring system (Figure 6.56). Some of these adducts have quite remarkable levels of analgesic activity, but are too powerful for human use. Some, e.g. etorphine (Figure 6.57), are used in veterinary practice to sedate large animals (elephants, rhinos) by means of tranquillizer darts. Etorphine is some 5000–10 000 times more potent than morphine. Buprenorphine (Figure 6.57) is an etorphine analogue with an N-cyclopropylmethyl substituent and tert-butyl instead of N-propyl in the side-chain. This material has both opioid agonist and antagonist properties. Mixed agonist–antagonist properties offer scope for producing analgesia whilst negating the effects of other opioids to which a patient may be addicted. Buprenorphine has a long duration of action, and only low dependence potential, but may precipitate withdrawal symptoms in patients dependent on other opioids. It is now being used as an alternative to methadone in the treatment of opioid dependence. Nalbuphine (Figure 6.55), produced semi-sythetically from thebaine, also displays mixed agonist–antagonist properties, and has similar agonist activity as morphine, but produces less side-effects and has less abuse potential. Naloxone (Figure 6.55) shows hardly any agonist activity but is a potent antagonist, and is used to treat opiate poisoning, including that in children born to heroin addicts. Naltrexone (Figure 6.55)

Grignard reagent attacks Pr O stereospecifically from less-hindered Diels-Alder H O side; demethylation with strong base H OH reaction OMe OMe Me N OMe Me N Me N PrMgI NaOH / ∆ O O O

OMe OMe OH thebaine etorphine

Figure 6.56

HO HO

O O NMe N H H MeO MeO H H HO HO

etorphine buprenorphine

Figure 6.57

(Continues) ALKALOIDS DERIVED FROM TYROSINE 337

(Continued)

also has antagonist activity similar to naloxone. These agents are N-alkyl derivatives related to oxymorphone/oxycodone. Thebaine may also be transformed very efficiently into codeine in about 75% yield (Figure 6.55). The two-stage synthesis involves acid-catalysed hydrolysis of the enol ether function to give codeinone (this being the more favoured tautomer of the first-formed β, γ- unsaturated ketone) followed by selective borohydride reduction of the carbonyl. This opens up possibilities for producing codeine (the most widely used of the opium alkaloids) without using morphine. At present, most of the codeine is synthesized by methylation of morphine. The advantage of using thebaine is that the raw material for the pharmaceutical industry could be shifted away from morphine and opium. This might then help in the battle to eliminate illicit morphine production and its subsequent conversion into heroin. Conversion of thebaine into morphine and heroin is much more difficult and low yielding. Thus, there is interest in cultivating Papaver bracteatum rather than P. somniferum. This plant produces mainly thebaine, no morphine, and only faint traces of codeine. Experiments have shown it has the enzymic activity to convert codeinone into codeine, but it appears to lack enzymes which carry out the late demethylation steps in Figure 6.50. The capsules can produce up to 3% thebaine, but, regrettably, there have been difficulties in making this a commercially viable project, and this idea has not materialized. Other species of Papaver seem to lack the enzyme that reduces salutaridine to salutaridinol (Figure 6.50) and they thus do not synthesize morphine-like alkaloids. Remarkably, there is now considerable evidence that various animals, including humans and other mammals, are also able to synthesize morphine and related alkaloids in small amounts. These compounds have been detected in various tissues, including brain, liver, spleen, adrenal glands and skin, and endogenous morphine may thus play a role in pain relief, combining its effects with those provided by the enkephalin peptides.

A minor constituent of P. somniferum is (Figure 6.58). Some structures, e.g. isothebaine the aporphine alkaloid isoboldine (Figure 6.58). (Figure 6.59) from P. orientale, are not as eas- Other species of poppy, e.g. Papaver orientale ily rationalized. (S )-Orientaline is a precursor and P. pseudoorientale, are known to synthesize of isothebaine (Figure 6.59). This benzyltetrahy- aporphine alkaloids as principal constituents rather droisoquinoline, with a different methylation pat- than morphinan structures. (S )-Isoboldine is read- tern to reticuline, is able to participate in oxidative ily appreciated to be the product of oxidative coupling, but inspection of the structures indi- coupling of (S )-reticuline, coupling ortho to the cates a phenol group is lost in the transformation. phenol group in the tetrahydroisoquinoline, and The pathway (Figure 6.59) involves an unexpected para to the phenol of the benzyl substituent rearrangement process, however. Thus, oxidative

MeO MeO oxidative MeO 3 4 coupling 2 5 NMe NMe NMe 6a NH HO O O HO 1 H H H 6 7 11

MeO MeO MeO 10 8 OH O OH 9 (S)-reticuline (S)-isoboldine aporphine

Figure 6.58 338 ALKALOIDS

oxidative MeO MeO MeO coupling NMe NMe NMe HO O O HO H H H MeO MeO MeO HO O (S)-orientaline O (S)-orientalinone reduction of dienone to dienol MeO MeO

NMe NMe HO HO H H MeO 1,2-aryl migration with loss of MeO leaving group; favoured by formation of aromatic ring H OH (S)-isothebaine orientalinol

Figure 6.59

oxidative MeO MeO MeO coupling NMe NMe NMe HO HO HO H H H MeO MeO MeO 1,2-alkyl migration with loss of leaving group; favoured by HO formation of aromatic ring O H (R)-orientaline OH (R)-orientalinone orientalinol stereoisomer formation of methylenedioxy ring oxidative ring O CO2H cleavage O NMe O NO2 O H

OMe OMe aristolochic acid stephanine

Figure 6.60 coupling ortho–para to the phenol groups gives because normal keto–enol tautomerism is not pos- a dienone orientalinone (compare the structure sible for rearomatization when coupling involves of salutaridine (Figure 6.50)). After reduction of positions already substituted. The process is fully the carbonyl group, a rearrangement occurs, restor- borne out by experimental evidence, including the ing aromaticity and expelling the hydroxyl (orig- subsequent isolation of orientalinone and oriental- inally a phenol group) to produce isothebaine. inol from P. orientale. This type of rearrangement, for which good chem- Stephanine (Figure 6.60) from Stephania ical analogies are available, is a feature of many species (Menispermaceae) is analogous to isothe- other alkaloid biosynthetic pathways, and occurs baine and shares a similar pathway, though from ALKALOIDS DERIVED FROM TYROSINE 339

(R)-orientaline. The different substitution pat- reaction (see page 320). The product from the tern in stephanine compared to isothebaine is enzymic transformation of (S )-reticuline is the a consequence of the intermediate dienol suf- protoberberine alkaloid (S )-scoulerine, the berber- fering migration of the alkyl rather than aryl ine bridge enzyme requiring molecular oxygen group (Figure 6.60). Aristolochic acid is a novel as oxidant and releasing H2O2 as by-product modified aporphine containing a nitro group (Figure 6.62). Its role in the cyclization reaction and is produced from stephanine by oxidative completed, the phenol group in scoulerine is then reactions leading to ring cleavage (Figure 6.60). methylated, and tetrahydrocolumbamine is oxi- Aristolochic acid is present in many species of dized further to give the quaternary isoquinoline Aristolochia (Aristolochiaceae) used in traditional system in columbamine. This appears to involve medicine, e.g. snake-root A. serpentina. However, two separate oxidation steps, both requiring molec- because aristolochic acid is now known to be ular oxygen, though H2O2 and H2O are pro- nephrotoxic and to cause acute kidney failure, the duced in the successive processes. The mechanistic use of Aristolochia species in herbal medicines, sequence through an iminium ion has been sug- especially Chinese remedies, has been banned in gested to account for these observations. Finally, several countries. berberine is produced by transformation of the The alkaloid berberine (Figure 6.61) is found ortho-methoxyphenol to a methylenedioxy group, in many members of the Berberidaceae (e.g. via the O2-, NADPH-, and cytochrome P-450- Berberis, Mahonia), the Ranunculaceae (e.g. dependent enzyme. Hydrastis), and other families. Berberine has The protoberberine skeleton of scoulerine may antiamoebic, antibacterial, and anti-inflammatory be subjected to further modifications, some of properties and plants containing berberine have which are given in Figure 6.63. Cleavage of the long been used in traditional medicine. Its heterocyclic ring systems adjacent to the nitro- tetracyclic skeleton is derived from a benzyl- gen atom as shown give rise to new skeletal tetrahydroisoquinoline system with the incorpo- types: protopine, e.g. protopine from Chelido- ration of an extra carbon atom, supplied from nium majus (Papaveraceae), phthalideisoquino- S-adenosylmethionine via an N-methyl group line, e.g. hydrastine from Hydrastis canadensis (Figure 6.61). This extra skeletal carbon is known (Ranunculaceae), and benzophenanthridine, e.g. as a ‘berberine bridge’. Formation of the berber- chelidonine, also from Chelidonium majus.The ine bridge is readily rationalized as an oxidative non-heterocyclic system seen in the opium alkaloid process in which the N-methyl group is oxidized narceine from Papaver somniferum can be visual- to an iminium ion, and a cyclization to the aro- ized as the result of cleavage of two of these bonds. matic ring occurs by virtue of the phenolic group Some alkaloids of the phthalide type are medici- (Figure 6.62). nally important. Noscapine (Figure 6.64) is one of The oxidative cyclization process is analogous the opium alkaloids and although it lacks any anal- to the formation of a methylenedioxy group (see gesic activity it is an effective cough suppressant page 27), whilst the mechanism of cyclization is (see page 331). Hydrastine is beneficial as a tra- exactly the same as that invoked in formation of ditional remedy in the control of uterine bleeding. a tetrahydroisoquinoline ring, i.e. a Mannich-like Hydrastis also contains berberine, indicating the

4 5 MeO O 3 6 7 N N 2 N HO CH O 8 3 1 14 OH OMe 13 9

12 10 OMe OMe reticuline berberine protoberberine 11

Figure 6.61 340 ALKALOIDS

oxidation of tertiary Mannich-like reaction; position amine to iminium ion ortho to phenol is nucleophilic MeO MeO MeO

O2 H2O2 NMe N N HO HO HO H H H H OH berberine OH O bridge enzyme OMe OMe OMe (S)-reticuline keto–enol tautomerism oxidation of amine MeO to iminium ion MeO MeO H O O N H 2 2 2 N N HO HO SAM HO H H OMe OMe OH H

oxidation leading OMe OMe OMe to formation of (S)-tetrahydrocolumbamine (S)-scoulerine O2 aromatic system

H2O oxidation: formation of MeO methylenedioxy ring O N O2 HO NADPH O N OMe OMe

OMe OMe columbamine berberine

Figure 6.62

MeO c O Me a N b a N HO O O OH O

OMe O scoulerine protopine (protoberberine-type) (protopine-type) b a + b c

O O O O Me O NMe H NMe2 H O N O O CO2H H MeO OMe O O OMe H HO O OMe narceine hydrastine OMe chelidonine (phthalideisoquinoline-type) (benzophenanthridine-type)

Figure 6.63 ALKALOIDS DERIVED FROM TYROSINE 341

O O O

NMe NMe O O O NMe H H H MeO O O O OMe OMe O O OMe O OMe O O hydrastine noscapine bicuculline (narcotine)

Figure 6.64 close biosynthetic relationship of the two types of (Figure 6.65), which contain an extra carbon alkaloid. Bicuculline (Figure 6.64) from species of between the tetrahydroisoquinoline and the pen- Corydalis and Dicentra (Fumariaceae) and its qua- dant aromatic rings. This skeleton is formed in ternary methiodide have been identified as potent a similar way to that in the benzyltetrahydroiso- GABA (γ-aminobutyric acid) antagonists and have quinolines from a phenylethylamine and an alde- found widespread application as pharmacological hyde (Figure 6.65), but a whole C6C3 unit rather probes for convulsants acting at GABA neurore- than a C6C2 fragment functions as the react- ceptors. ing aldehyde. Typically, dopamine (from tyro- sine) and 4-hydroxydihydrocinnamaldehyde (from phenylalanine) are involved in the initial conden- Phenethylisoquinoline Alkaloids sation, and further hydroxylation and methylation steps then build up the substitution pattern to Several genera in the lily family (Liliaceae) are that of autumnaline. Phenolic oxidative coupling found to synthesize analogues of the benzylte- accounts for the occurrence of homoaporphine trahydroisoquinoline alkaloids, e.g. autumnaline alkaloids such as floramultine and kreysigine in

HO Mannich-like HO MeO L-Tyr reaction NH2 HO NH NMe CHO HO HO dopamine H H MeO

L-Phe HO MeO OH OH (S)-autumnaline 4-hydroxydihydro- oxidative cinnamaldehyde coupling MeO MeO

NMe NMe HO SAM HO MeO H MeO H

MeO MeO homoaporphine skeleton MeO HO kreysigine floramultine

Figure 6.65 342 ALKALOIDS

MeO oxidative homomorphinan coupling skeleton NMe HO HO MeO HO NMe NMe H NMe ≡ MeO H MeO H RO H MeO SAM MeO MeO MeO OH O O MeO MeO MeO MeO OH isoandrocymbine R = Me, O-methylandrocymbine (S)-autumnaline (R = H, androcymbine)

cleavage of cyclopropane oxidation to alkene (enamine); generates aromatic 7-membered may proceed through iminium, O ring (tropolone); remaining ring followed by iminium–enamine carbon becomes N-formyl group tautomerism OFe–Enz OFe–Enz MeO CHO MeO MeO NMe NMe O NMe MeO H MeO H MeO H MeO MeO MeO O oxidation leading to formation MeO O O OMe of cyclopropane ring and MeO N-formyldemecolcine generation of free radical hydrolysis of amide O MeO MeO MeO

NHMe O NH2 acetyl-CoA NH MeO H MeO H MeO H MeO MeO MeO oxidative amide O O demethylation O formation OMe OMe OMe demecolcine deacetylcolchicine colchicine

Figure 6.66

Kreysigia multiflora (Liliaceae/Convallariaceae). isolated from Androcymbium melanthioides (Lil- (S )-Autumnaline has also been found to act iaceae/Colchicaceae), thus giving a clue to the as a precursor for colchicine (Figure 6.66), an biosynthetic pathway. Methylation follows giv- alkaloid containing an unusual tropolone ring. ing O-methylandrocymbine, and it is then pro- Colchicine is found in species of Colchicum∗,e.g. posed that enzymic oxidation to an enamine yields Colchicum autumnale (Liliaceae/Colchicaceae), as the substrate for ring modification. Experimental well as many other plants in the Liliaceae. labelling studies are then best explained by forma- Colchicine no longer has its nitrogen atom in tion of a cyclopropane ring followed by ring open- a ring system, and extensive reorganization of ing to generate the 6π electron aromatic tropolone the autumnaline structure is thus necessary. The system, incorporating the original tyrosine ben- seven-membered tropolone ring was shown by zylic carbon into the seven-membered ring, and labelling experiments to originate by ring expan- also breaking the original phenylethylamine side- sion of the tyrosine-derived aromatic ring tak- chain between the carbons. One carbon is left ing in the adjacent benzylic carbon (Figure 6.66). on the nitrogen as a formyl group, and this can Prior to these remarkable rearrangements, oxida- be lost by hydrolysis. Colchicine is produced by tive coupling of autumnaline in the para–para exchanging the N-methyl group for an N-acetyl sense features in the pathway giving the dienone group, by way of an oxidative demethylation fol- isoandrocymbine, which has a homomorphinan lowed by acetylation using acetyl-CoA. Demecol- skeleton (compare salutaridine, Figure 6.50). The cine and deacetylcolchicine are intermediates in isomer androcymbine (Figure 6.66) had been the process. ALKALOIDS DERIVED FROM TYROSINE 343

Colchicum Colchicum seed and corm are obtained from Colchicum autumnale (Liliaceae/Colchicaceae), the autumn crocus or meadow saffron. The plant, though not a crocus, produces crocus-like flowers in the autumn, the leaves not emerging until the spring. It is a native of Europe, is widely cultivated as an ornamental garden plant, and is grown for drug use, mainly in Europe and North Africa. The principal alkaloid is colchicine (Figure 6.66), which occurs to the level of about 0.8% in the seed, and 0.6% in the corm. As an N-acetyl derivative, colchicine does not display any significant basicity, and does not form well-defined salts. Demecolcine (N-deacetyl-N-methylcolchicine) (Figure 6.66) is a minor constituent in both corm and seeds. Extracts of Colchicum autumnale,andlatercolchicine itself, have been used in the treatment of gout, a painful condition in which impaired purine metabolism leads to a build-up of uric acid crystals in the joints. Colchicine is an effective treatment for acute attacks, but it is very toxic, and this restricts its general use. It appears to act primarily as an anti-inflammatory agent, and does not itself affect uric acid metabolism, which needs to be treated with other agents, e.g. a xanthine oxidase inhibitor such as allopurinol. The cytotoxic properties of colchicine and related alkaloid structures from C. autumnale led to their being tested as potential anticancer agents, though they still proved too toxic for medicinal use. Colchicine binds to tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules as do podophyllotoxin (see page 136) and vincristine (see page 356), and is a useful biochemical probe. However, the ability of colchicine to act as a mitotic poison is exploited in plant breeding, since the interference with mitosis results in multiplication of chromosomes in the cell nucleus without the process of cell division. Cell division recommences on cessation of treatment. This allows the generation of mutations (polyploids) and possible new varieties of plant. Colchicine is also found in other species of Colchicum, as well as many other plants in the Liliaceae (e.g. Bulbocodium, Gloriosa, Merendera,andSandersonia), a group of plants now classified as the family Colchicaceae.

Terpenoid Tetrahydroisoquinoline the opposite stereochemistry at C-1 to this biosyn- Alkaloids thetic intermediate. The secologanin fragment con- tains an acetal function, which can be restored to its component aldehyde and alcohol fragments The alkaloids found in ipecacuanha∗, the dried by hydrolysis of the glucosidic bond. The newly rhizome and roots of Cepahaelis ipecacuanha liberated aldehyde can then bond with the sec- (Rubiaceae), have a long history of use in the treat- ondary amine to give the quaternary Schiff base. ment of amoebic dysentery, and provide unusual This intermediate is converted into an aldehyde examples of tetrahydroisoquinoline structures. The by a sequence of reactions: reduction of iminium, principal alkaloids, e.g. emetine and cephaeline reduction of alkene, plus hydrolysis of ester and (Figure 6.67), possess a skeleton with two tetrahy- subsequent decarboxylation, though not necessar- droisoquinoline ring systems, plus a further frag- ily in that order. The decarboxylation step is facil- ment that has its origin in a terpenoid-derived itated by the β-aldehyde function, shown as an molecule. This terpenoid substrate is the secoiri- enol in Figure 6.67. Most of the reactions tak- doid secologanin (see page 189), a compound ing place in the secologanin-derived part of the that also features in the biosynthesis of many structure are also met in discussions of terpenoid complex indole alkaloids (see page 350). Sec- indole alkaloids (see page 350). The resultant alde- ologanin is an aldehyde and can condense with hyde is now able to participate in formation of dopamine to give the tetrahydroisoquinoline alka- a second tetrahydroisoquinoline ring system, by loid N-deacetylisoipecoside (Figure 6.67). Ipeco- reaction with a second dopamine molecule. Methy- side itself is found in ipecacuanha, though it has lation gives cephaeline and emetine. 344 ALKALOIDS

HO Mannich-like HO HO reaction

NH2 NH 1 NAc HO HO HO CHO H H H H dopamine H OGlc OGlc OGlc H H H O O O MeO2C MeO2C MeO2C N-deacetylisoipecoside ipecoside secologanin hydrolysis of glucoside liberates – Glc hemiacetal; further hydrolysis produces aldehyde and alcohol HO HO

NH NH MeO HO CHO ≡ HO H H H H N CHO MeO H H H OH OH MeO C MeO C 2 2 H formation of H quaternary Schiff reduction C=N OMe Me HN base (iminium ion) reduction C=C HO hydrolysis of ester HO decarboxylation OR enol → keto R = Me, emetine N N HO HO R = H, cephaeline H H H repeat of H H Mannich-like OH reaction MeO2C CHO OH NH2

OH dopamine

Figure 6.67

Ipecacuanha

Ipecacuanha or ipecac is derived from the dried rhizome and roots of Cephaelis ipecacuanha or C. acuminata (Rubiaceae). These are low straggling shrubs having horizontal rhizomes with prominently ridged roots. Cephaelis ipecacuanha yields what is termed Rio or Brazilian ipecac, and is cultivated mainly in Brazil, whilst C. acuminata gives Cartagena, Nicaragua, or Panama ipecac, and comes principally from Colombia and Nicaragua. Most of the commercial ipecac now derives from C. acuminata. Ipecac is an age-old remedy of the South American Indians, who used it for the treatment of dysentery. More recently it was mixed with powdered opium to give Dover’s powder (see page 330), where the ipecac content functioned as a diaphoretic. Ipecac contains 2–2.5% of alkaloids, the principal ones being emetine and cephaeline (Figure 6.67). Typically, in C. ipecacuanha the emetine to cephaeline ratio might be about 2:1, whereas in C. acuminata the ratio ranges from about 1:2 to 1:1. Minor alkaloids characterized include psychotrine and O-methylpsychotrine (Figure 6.68), which are dehydro variants of cephaeline and emetine respectively. Both emetine and the synthetic 2,3-dehydroemetine (Figure 6.68) have been useful as anti-amoebics, particularly in the treatment of amoebic dysentery. However, they also cause

(Continues) ALKALOIDS DERIVED FROM TYROSINE 345

(Continued)

MeO MeO

N N MeO MeO H H

H N H OMe HN

OR OMe 2,3-dehydroemetine OR R = Me, O-methylpsychotrine R = H, psychotrine

Figure 6.68

nausea, and this has now made other drugs preferable. The emetic action of the alkaloids is particularly valuable though, and the crude drug extract in the form of ipecacuanha emetic mixture is an important preparation used for drug overdose or poisoning. The emetic mixture is often a standard component in poison antidote kits. Ipecacuanha also has expectorant activity and extracts are still components of a number of compound expectorant preparations. Emetine has more expectorant and less emetic action than cephaeline, and thus the Brazilian drug is preferred for such mixtures. If required, emetine may be obtained in larger amounts by also methylating the cephaeline component of the plant material. Emetine and cephaeline are both potent inhibitors of protein synthesis, inhibiting at the translocation stage. They display antitumour and antiviral as well as antiamoebic activity, but are too toxic for therapeutic use. In recent studies, O-methylpsychotrine has displayed fairly low effects on protein synthesis, but a quite potent ability to curb viral replication through inhibition of HIV-reverse transcriptase. This may give it potential in the treatment of AIDS.

Amaryllidaceae Alkaloids (compare opium alkaloids, page 328), and the sequence is completed by reduction and methylation Various types of alkaloid structure are encountered reactions. For lycorine and crinine, although details in the daffodil family, the Amaryllidaceae, and are not given in Figure 6.69, it is apparent that they can be rationalized better through biosynthesis the nitrogen atom acts as a nucleophile towards than by structural type. The alkaloids arise the dienone system in a similar manner, generating by alternative modes of oxidative coupling of the new heterocyclic ring systems. Alkaloids such precursors related to norbelladine (Figure 6.69), as lycorine, crinine, and galanthamine can undergo which is formed through combination of 3,4- further modifications, which include ring cleavage dihydroxybenzaldehyde with tyramine, these two reactions, generating many more variations than preursors arising from phenylalanine and tyrosine can be considered here. The Amaryllidaceae family respectively. Three structural types of alkaloid includes Amaryllis, Narcissus,andGalanthus, can be related to 4
-O-methylnorbelladine by and the alkaloid content of bulbs from most different alignments of the phenol rings, allowing members makes these toxic. Lycorine was first coupling para–ortho (A), para–para (B), or isolated from Lycorus radiata, but is common ortho–para (C) as shown in Figure 6.69. For and found throughout the family. Galanthamine galanthamine, the dienone formed via oxidative from snowdrops (Galanthus species) is currently coupling (C) undergoes nucleophilic addition from an important drug material of value in treating the phenol group, forming an ether linkage Alzheimer’sdisease. 346 ALKALOIDS

HO H2N L-Phe L-Tyr HO CHO via cinnamic acids OH with side-chain tyramine via Schiff base cleavage and reduction HO MeO H SAM H N N HO HO

norbelladine OH 4′-O-methylnorbelladine OH

HO OH OH HO MeO MeO MeO H H NH N N HO HO A B C oxidative coupling

HO OH O HO O O MeO N O NH O N H nucleophilic attack lycorine crinine of phenol on to dienone OH OH O O O O

MeO SAM MeO NADPH MeO

N NH NH Me galanthamine N-demethylgalanthamine N-demethylnarwedine

Figure 6.69

Galanthamine Galantamine (galanthamine) can be isolated from a number of species of the Amaryllidaceae, including snowdrops (Galanthus species), daffodils (Narcissus pseudonarcissus), and snowflakes (Leucojum species), where typical content varies from about 0.05 to 0.2% in the bulbs. It is currently isolated for drug use from the bulbs of wild Leucojum aestivum and Galanthus species, since commercial synthesis is not economic. Galantamine acts as a competitive cholinesterase inhibitor, and enhances cognitive function in the treatment of Alzheimer’s disease by raising acetylcholine levels in brain areas lacking cholinergic neurones. In common with other treatments for Alzheimer’s disease, it does not cure the condition, but merely slows the rate of cognitive decline.

ALKALOIDS DERIVED FROM the shikimate pathway (Chapter 4) via anthranilic TRYPTOPHAN acid. It acts as a precursor of a wide range of indole alkaloids, but there is also definite proof that major L-Tryptophan is an aromatic amino acid contain- rearrangement reactions can convert the indole ring ing an indole ring system, having its origins in system into a quinoline ring, thus increasing further ALKALOIDS DERIVED FROM TRYPTOPHAN 347

the ability of this amino acid to act as an alkaloid organism involved. 5-Hydroxytryptamine is also precursor (see page 359). found in mammalian tissue, where it acts as a neurotransmitter in the central nervous system. It is formed from tryptophan by hydroxylation and Simple Indole Alkaloids then decarboxylation, paralleling the tyrosine → Tryptamine and its N-methyl and N,N-dimethyl dopamine pathway (see page 316). In the for- derivatives (Figure 6.70) are widely distributed mation of psilocin (Figure 6.70), decarboxyla- in plants, as are simple hydroxylated derivatives tion precedes N-methylation, and hydroxylation such as 5-hydroxytryptamine (serotonin). These occurs last. Phosphorylation of the hydroxyl in are formed (Figure 6.70) by a series of decar- psilocin gives psilocybin. These two compounds boxylation, methylation, and hydroxylation reac- are responsible for the hallucinogenic properties of tions, though the sequences of these reactions are so-called magic mushrooms, which include species found to vary according to final product and/or of Psilocybe∗, Panaeolus,etc.

CO2H

– CO NH2 2 NH2 SAM NHMe SAM NMe2

N N N N H H H H L-Trp tryptamine O O CO H 2 OP OH

HO NH2 HO NH2 NMe2 NMe2 – CO2 N N N N H H H H 5-hydroxy-L-Trp 5-hydroxytryptamine psilocybin psilocin (5-HT; serotonin)

Figure 6.70

5-Hydroxytryptamine (Serotonin)

5-Hydroxytryptamine (5-HT, serotonin) is a monoamine neurotransmitter found in cardiovas- cular tissue, the peripheral nervous system, blood cells, and the central nervous system. It mediates many central and peripheral physiological functions, including contraction of smooth muscle, vasoconstriction, food intake, sleep, pain perception, and memory, a conse- quence of it acting on several distinct receptor types. Although 5-HT may be metabolized by monoamine oxidase, platelets and neurons possess a high affinity 5-HT reuptake mechanism. This mechanism may be inhibited by widely-prescribed antidepressant drugs termed selective serotonin re-uptake inhibitors (SSRIS), e.g. fluoxetine (Prozac), thereby increasing levels of 5-HT in the CNS. Migraine headaches that do not respond to analgesics may be relieved by the use of an agonist of the 5-HT1 receptor, since these receptors are known to mediate vasoconstriction. Though the causes of migraine are not clear, they are characterized by dilation of cerebral blood vessels. 5-HT1 agonists based on the 5-HT structure in current use include the sulphonamide derivative sumatriptan, and the more recent agents naratriptan, rizatriptan,

(Continues) 348 ALKALOIDS

(Continued)

NMe2 Me NMe2 Me2NSO2 N N N N N N H H sumatriptan rizatriptan MeNHSO2 NMe2 O N NH N H H naratriptan O zolmitriptan

Figure 6.71

and zolmitriptan (Figure 6.71). These are of considerable value in treating acute attacks. Several of the ergot alkaloids (page 371) also interact with 5-HT receptors.

Psilocybe The genus Psilocybe constitutes a group of small mushrooms with worldwide distribution. It has achieved notoriety on account of hallucinogenic experiences produced following ingestion of several species, particularly those from Mexico, and has led to the description ‘magic mushrooms’. Over 80 species of Psilocybe have been found to be psychoactive, whereas over 50 species are inactive. More than 30 of the hallucinogenic species have been identified in Mexico, but active species may be found in all areas of the world. Psilocybe mexicana has been used by the Mexican Indians in ancient ceremonies for many years, and its history can be traced back to the Aztecs. In temperate regions, Psilocybe semilanceata, the liberty cap, is a common species with similar activity. All the psychoactive members of the genus are said to stain blue when the fresh tissue, particularly that near the base of the stalk, is damaged, though the converse is not true. Ingestion of the fungus causes visual hallucinations with rapidly changing shapes and colours, and different perceptions of space and time, the effects gradually wearing off and causing no lasting damage or addiction. The active hallucinogens, present at about 0.3%, are the tryptamine derivatives psilocybin and psilocin (Figure 6.70), which are structurally related to the neurotransmitter 5-HT, thus explaining their neurological effects. Psilocybin is probably the main active ingredient, and to produce hallucinations a dose of some 6–20 mg is required. In addition to species of Psilocybe, these compounds may be found in some fungi from other genera, including Conocybe, Panaeolus,andStropharia. Misidentification of fungi can lead to the consumers experiencing possible unwanted toxic effects, especially gastro-intestinal upsets, instead of the desired psychedelic visions.

chain shortening involves retention of nitrogen atom; this may be temporarily bound to PLP CO2H NH2 NMe2 NH2 HO HO

N N N H H H L-Trp gramine

Figure 6.72 ALKALOIDS DERIVED FROM TRYPTOPHAN 349

Gramine (Figure 6.72) is a simple amine found attacking a Schiff base generated from tryptamine in barley (Hordeum vulgare; Graminae/Poaceae) and an aldehyde (or keto acid) (Figure 6.73). Aro- and is derived from tryptophan by a biosynthetic maticity is restored by subsequent loss of the pathway which cleaves off two carbon atoms, yet C-2 proton. (It should be noted that the analo- surprisingly retains the tryptophan nitrogen atom. gous chemical reaction actually involves nucle- Presumably, the nitrogen reacts with a cofactor, ophilic attack from C-3, and then a subsequent e.g. pyridoxal phosphate, and is subsequently rearrangement occurs to give bonding at C-2; transferred back to the indolemethyl group after there is no evidence yet for this type of pro- the chain shortening. cess in biosynthetic pathways.) Extra carbons are supplied by aldehyes or keto acids, accord- Simple β-Carboline Alkaloids ing to the complexity of the substrate (compare tetrahydroisoquinoline alkaloids, page 321). Thus, Alkaloids based on a β-carboline system (Fig- complex β-carbolines, e.g. the terpenoid indole ure 6.73) exemplify the formation of a new six- alkaloid ajmalicine (see page 351), are produced membered heterocyclic ring using the ethylamine by a pathway using an aldehyde such as sec- side-chain of tryptamine in a process analogous to ologanin. Simpler structures employ keto acids, generation of tetrahydroisoquinoline alkaloids (see e.g. harmine (Figure 6.74) incorporates two extra page 320). Position 2 of the indole system is nucle- carbons from pyruvate. In such a case, an acid ophilic due to the adjacent nitrogen, and can partic- is an intermediate, and oxidative decarboxylation ipate in a Mannich/Pictet–Spengler type reaction, gives the dihydro-β-carboline, from which reduced

Schiff base formation Mannich-like reaction; α-carbon can using aldehyde act as nucleophile N NH2 H NH OHC N R N R N H R H H H tryptamine tautomerism to restore aromaticity 4 3 5 N 2 6 N O NH 1 7 N 9 8 H N R N R β-carboline H H

Figure 6.73

Mannich-like reaction oxidative reduction using keto acid decarboxylation NH 2 NH N NH O CO2H RS N CO2H N H N N H H H O elaeagnine

N N NH N O RS MeO N MeO N MeO N N H H H H harmine harmaline tetrahydroharmine harman

Figure 6.74 350 ALKALOIDS tetrahydro-β-carboline structures, e.g. elaeagnine eight plant families, of which the Apocynaceae, from Elaeagnus angustifolia (Elaeagnaceae), or the Loganiaceae, and the Rubiaceae provide the fully aromatic β-carboline structures, e.g. harman best sources. In terms of structural complexity, and harmine from Peganum harmala (Zygophyl- many of these alkaloids are quite outstanding, and laceae) are derived (Figure 6.74). The methoxy it is a tribute to the painstaking experimental stud- substitution in the indole system of harmine is ies of various groups of workers that we are able introduced at some stage in the pathway by suc- to rationalize these structures in terms of their cessive hydroxylation and methylation reactions. biochemical origins. In virtually all structures, a A sequence from 6-hydroxytryptamine is also fea- tryptamine portion can be recognized. The remain- sible. The reported psychoactive properties of the ing fragment is usually a C9 or C10 residue, and plants Peganum harmala and Banisteriopsis caapi three main structural types are discernable. These (Malpighiaceae) is due to the β-carboline alkaloids are termed the Corynanthe type, as in ajmalicine such as harmine, harmaline, and tetrahydroharmine and akuammicine,theAspidosperma type, as in (Figure 6.74). tabersonine,andtheIboga type, exemplified by catharanthine (Figure 6.75). The C9 or C10 frag- ment was shown to be of terpenoid origin, and Terpenoid Indole Alkaloids the secoiridoid secologanin (see page 189) was identified as the terpenoid derivative, which ini- More than 3000 terpenoid indole alkaloids are rec- tially combined with the tryptamine portion of ognized, making this one of the major groups the molecule. Furthermore, the Corynanthe, Aspi- of alkaloids in plants. They are found mainly in dosperma,andIboga groups of alkaloids could

N N

O N N CO2Me H H MeO2C akuammicine ajmalicine HO OHC OH OGlc OGlc

O O MeO2C MeO2C geraniol loganin secologanin Corynanthe type

N

N CO2Me H tabersonine Aspidosperma type N

N CO2Me Iboga H type catharanthine

Figure 6.75 ALKALOIDS DERIVED FROM TRYPTOPHAN 351

hydrolysis of glycoside liberates hemiacetal; further hydrolysis produces aldehyde and Mannich-like alcohol; note rotation about C–C bond reaction CHO OGlc NH2 H H H OGlc NH – Glc NH OHC N O OH O N H N H H H H H H tryptamine H CO2Me CO2Me CO2Me strictosidine secologanin Schiff base allylic isomerization formation favouring conjugation H N N H

OH OH attack of nucleophile on N H N H to α,β-unsaturated H H H H iminium ion CO2Me CO2Me dehydrogeissoschizine (enol form)

N N H NADPH O O N H N H H H H H CO2Me CO2Me cathenamine ajmalicine

Figure 6.76 be related and rationalized in terms of rearrange- the secondary amine function giving a quaternary ments occurring in the terpenoid part of the struc- Schiff base. These reactions are also seen in the tures (Figure 6.75). Secologanin itself contains the pathway to ipecac alkaloids (see page 343). Allylic ten-carbon framework typical of the Corynanthe isomerization, moving the vinyl double bond into group. The Aspidosperma and Iboga groups could conjugation with the iminium generates dehydro- then arise by rearrangement of the Corynanthe geissoschizine, and cyclization to cathenamine fol- skeleton as shown. This is represented by detach- lows. Cathenamine is reduced to ajmalicine in the ment of a three-carbon unit, which is then rejoined presence of NADPH. to the remaining C7 fragment in one of two differ- Carbocyclic variants related to ajmalicine such ent ways. Where C9 terpenoid units are observed, as yohimbine are likely to arise from dehy- the alkaloids normally appear to have lost the car- drogeissoschizine by the mechanism indicated in bon atom indicated in the circle. This corresponds Figure 6.77. Yohimbine is found in Yohimbe bark to the carboxylate function of secologanin and its (Pausinystalia yohimbe; Rubiaceae) and Aspi- loss by hydrolysis/decarboxylation is now under- dosperma bark (Aspidosperma species; Apocy- standable. naceae) and has been used in folk medicine as The origins of loganin and secologanin have an aphrodisiac. It does have some pharmacolog- already been discussed in Chapter 5 (see page 189). ical activity and is known to dilate blood ves- Condensation of secologanin with tryptamine in sels. More important examples containing the a Mannich-like reaction generates the tetrahydro- same carbocyclic ring system are the alkaloids β-carboline system and produces strictosidine found in species of Rauwolfia∗, especially R. ser- (Figure 6.76). Hydrolysis of the glycoside func- pentina (Apocynaceae). Reserpine and deserpi- tion allows opening of the hemiacetal, and expo- dine (Figure 6.78) are trimethoxybenzoyl esters of sure of an aldehyde group, which can react with yohimbine-like alkaloids, whilst rescinnamine is 352 ALKALOIDS

homoallylic isomerization nucleophilic attack on to carbonyl through H N N conjugated system

N H N H H H CHO H H O H CO2Me MeO2C dehydrogeissoschizine (keto form)

N H reduction N

N H N H H H H H OH OH MeO2C MeO2C yohimbine

Figure 6.77

OMe O O OMe N H N H 3 OMe 11 O O OMe R N H MeO N H H H OMe H H OMe OMe OMe MeO2C MeO2C R = OMe, reserpine rescinnamine H R = H, deserpidine N O N H H CO2Me serpentine

Figure 6.78

Rauwolfia Rauwolfia has been used in Africa for hundreds of years, and in India for at least 3000 years. It was used as an antidote to snake-bite, to remove white spots in the eyes, against stomach pains, fever, vomiting, and headache, and to treat insanity. It appeared to be a universal panacea, and was not considered seriously by Western scientists until the late 1940s/early 1950s. Clinical tests showed the drug to have excellent antihypertensive and sedative activity. It was then rapidly and extensively employed in treating high blood pressure and to help mental conditions, relieving anxiety and restlessness, and thus initiating the tranquillizer era. The ‘cure for insanity’ was thus partially justified, and rauwolfia was instrumental in showing that mental disturbance has a chemical basis and may be helped by the administration of drugs. Rauwolfia is the dried rhizome and roots of Rauwolfia (sometimes Rauvolfia) serpentina (Apocynaceae) or snakeroot, a small shrub from India, Pakistan, Burma, and Thailand. Other species used in commerce include R. vomitoria from tropical Africa, a small tree whose leaves after ingestion cause violent vomiting, and R. canescens (= R. tetraphylla) from India and the

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Caribbean. Most of the drug material has been collected from the wild. Rauwolfia serpentina contains a wide range of indole alkaloids, totalling 0.7–2.4%, though only 0.15–0.2% consists of desirable therapeutically active compounds, principally reserpine, rescinnamine, and deserpidine (Figure 6.78). Other alkaloids of note are serpentine (Figure 6.78), ajmalicine (Figure 6.76), and ajmaline (see Figure 6.82). Reserpine and deserpidine are major alkaloids in R. canescens,andR. vomitoria contains large amounts of rescinnamine and reserpine. Reserpine and deserpidine (Figure 6.78) have been widely used as antihypertensives and mild tranquillizers. They act by interfering with catecholamine storage, depleting levels of available neurotransmitters. Prolonged use of the pure alkaloids, reserpine in particular, has been shown to lead to severe depression in some patients, a feature not so prevalent when the powdered root was employed. The complex nature of the alkaloidal mixture means the medicinal action is somewhat different from that of reserpine alone. Accordingly, crude powdered rauwolfia remained an important drug for many years, and selected alkaloid fractions from the crude extract have also been widely used. The alkaloids can be fractionated according to basicity. Thus, serpentine and similar structures are strongly basic, whilst reserpine, rescinnamine, deserpidine and ajmalicine are weak bases. Ajmaline and related compounds have intermediate basicity. The rauwolfia alkaloids are now hardly ever prescribed in the UK, either as antihypertensives or as tranquillizers. Over a period of a few years, they have been rapidly superseded by synthetic alternatives. Reserpine has also been suggested to play a role in the promotion of breast cancers. Both ajmalicine (= raubasine) (Figure 6.76) and ajmaline (Figure 6.82) are used clinically in Europe, though not in the UK. Ajmalicine is employed as an antihypertensive, whilst ajmaline is of value in the treatment of cardiac arrhythmias. Ajmalicine is also extracted commercially from Catharanthus roseus (see page 357). a trimethoxycinnamoyl ester. Both reserpine and of the bond to C-β, and then further fission yields a rescinnamine contain an additional methoxyl sub- hypothetical intermediate, the importance of which stituent on the indole system at position 11, the is that the rearrangeable C3 unit has been cleaved result of hydroxylation and methylation at a late from the rest of the terpenoid carbons. Alkaloids of stage in the pathway. A feature of these alkaloids the Aspidosperma type, e.g. tabersonine and vin- is that they have the opposite stereochemistry at doline,andIboga type, e.g. catharanthine,then position 3 to yohimbine and strictosidine. Rau- arise from this intermediate by different bonding wolfia serpentina also contains significant amounts modes (Figure 6.79). of ajmalicine (Figure 6.76), emphasizing the struc- Many of the experimental studies that have led tural and biosynthetic relationships between the to an understanding of terpenoid indole alkaloid two types of alkaloid. biosynthesis have been carried out using plants of The structural changes involved in converting the Madagascar periwinkle (Catharanthus roseus∗, the Corynanthe type skeleton into those of the Aspi- formerly Vinca rosea; Apocynaceae). Representa- dosperma and Iboga groups are quite complex, tives of all the main classes of these alkaloids and are summarized in Figure 6.79. Early inter- are produced, including ajmalicine (Corynan- mediates are alkaloids such as preakuammicine, the), catharanthine (Iboga), and vindoline (Aspi- which, although clearly of the Corynanthe type, is dosperma). The sequence of alkaloid formation sometimes designated as Strychnos type (compare has been established initially by noting which strychnine, page 358). This is because the Corynan- alkaloids become labelled as a feeding experi- the terpenoid unit, originally attached to the indole ment progresses, and more recently by appropriate α-carbon, is now bonded to the β-carbon, and a new enzymic studies. However, the extensive investi- bonding between the rearrangeable C3 unit and C-α gations of the Catharanthus roseus alkaloids have is in place. Stemmadenine arises through fission also been prompted by the anticancer activity 354 ALKALOIDS

N NH H OGlc b N a b O N H N OH CH2OH H H H N CO Me b 2 CO2Me CO Me preakuammicine strictosidine 2 a dehydrogeissoschizine (enol form)

N H N

O N H H H CH2OH CO2Me N CO2Me ajmalicine H stemmadenine this represents formal cleavage of the C3 unit during the rearrangement process Diels–Alder type N N reaction N

≡ N N CO2Me CO2Me N CO2Me H H H catharanthine hypothetical intermediate

N N OAc H H OH CO Me MeO N H 2 N CO2Me Me H vindoline tabersonine

Figure 6.79 detected in a group of bisindole alkaloids. Two of is then reduced in the dihydropyridinium ring these, vinblastine and vincristine (Figure 6.80), by NADH-dependent 1,4-addition, giving the sub- have been introduced into cancer chemotherapy strate for hydroxylation. Finally, reduction yields and feature as some of the most effective anti- vinblastine. Vincristine, with its N-formyl group cancer agents available. These structures are seen rather than N-methyl on the vindoline fragment, to contain the elements of catharanthine and vin- may be an oxidized product from vinblastine. doline, and, indeed, they are derived by coupling Further variants on the terpenoid indole alka- of these two alkaloids. The pathway is believed loid skeleton (Figure 6.82) are found in ibogaine to involve firstly an oxidative reaction on catha- from Tabernanthe iboga∗, vincamine from Vinca ranthine, catalysed by a peroxidase, generating a minor,andajmaline from Rauwolfia serpentina. peroxide which loses the peroxide as a leaving Ibogaine is simply a C9 Iboga type alkaloid, but group, breaking a carbon–carbon bond as shown is of interest as an experimental drug to treat (Figure 6.81). This intermediate electrophilic ion is heroin addiction. In a number of European coun- attacked by the nucleophilic vindoline, C-5 of the tries, vincamine is used clinically as a vasodilator indole nucleus being suitably activated by the OMe to increase cerebral blood flow in cases of senil- at C-6 and also by the indole nitrogen. The adduct ity, and ajmaline for cardiac arrhythmias. Ajmaline ALKALOIDS DERIVED FROM TRYPTOPHAN 355

N N

N HO HO N N N H H OAc H H OH MeO2C MeO2C OH OH N H CO Me MeO 2 MeO N H CONH2 R Me R = Me, vinblastine vindesine R = CHO, vincristine

N N

N N N N H H OAc H H OAc MeO2C MeO2C OH OH MeO N H CO2Me MeO N H CO2Me Me Me vinorelbine anhydrovinblastine

Figure 6.80

loss of leaving group precipitates ring opening: resembles a reverse Mannich-like reaction OH N N N nucleophilic attack on peroxidase O to conjugated iminium system

N N N CO2Me CO2Me CO2Me H H H catharanthine N m-chloroperbenzoic (CF3CO)2O acid CF3CO O H OAc N OH MeO N H CO2Me Me vindoline (V) N CO2Me H catharanthine N-oxide

N N N H O NADH

OH N N N H H H V FeCl3 V CONH V MeO2C MeO2C 2 MeO2C O2 NADH NaBH4 N

N NaO2C

biosynthetic pathway OH N synthetic route H V MeO2C vinblastine

Figure 6.81 356 ALKALOIDS

Catharanthus The Madagascar periwinkle Catharanthus roseus (= Vinca rosea) (Apocynaceae) is a small herb or shrub originating in Madagascar, but now common in the tropics and widely cultivated as an ornamental for its shiny dark green leaves and pleasant five-lobed flowers. Drug material is now cultivated in many parts of the world, including the USA, Europe, India, Australia, and South America. Because of its folklore usage as a tea for diabetics, the plant was originally investigated for potential hypoglycaemic activity. Although plant extracts had no effects on blood sugar levels in rabbits, the test animals succumbed to bacterial infection due to depleted white blood cell levels (leukopenia). The selective action suggested anticancer potential for the plant, and an exhaustive study of the constituents was initiated. The activity was found in the alkaloid fraction, and more than 150 alkaloids have been characterized in the plant. These are principally terpenoid indole alkaloids, many of which are known in other plants, especially from the same family. Useful antitumour activity was demonstrated in a number of dimeric indole alkaloid structures (more correctly bis-indole alkaloids), including vincaleukoblastine, leurosine, leurosidine, and leurocristine. These compounds became known as vinblastine, vinleurosine, vinrosidine, and vincristine respectively, the vin- prefix being a consequence of the earlier botanical nomenclature Vinca rosea,which was commonly used at that time. The alkaloids vinblastine and vincristine (Figure 6.80) were introduced into cancer chemotherapy and have proved to be extremely valuable drugs. Despite the minor difference in structure between vinblastine and vincristine, a significant difference exists in the spectrum of human cancers that respond to the drugs. Vinblastine (Figure 6.80) is used mainly in the treatment of Hodgkin’s disease, a cancer affecting the lymph glands, spleen, and liver. Vincristine (Figure 6.80) has superior antitumour activity compared to vinblastine but is more neurotoxic. It is clinically more important than vinblastine, and is especially useful in the treatment of childhood leukaemia, giving a high rate of remission. Some other cancer conditions, including lymphomas, small cell lung cancer, and cervical and breast cancers, also respond favourably. The alkaloids need to be injected, and both generally form part of a combination regimen with other anticancer drugs. Vindesine (Figure 6.80) is a semi-synthetic derivative of vinblastine, which has been introduced for the treatment of acute lymphoid leukaemia in children. Vinorelbine (Figure 6.80), an anhydro derivative of 8 -norvinblastine, is a newer semi-synthetic modification obtained from anhydrovinblastine (Figure 6.80), where the indole.C2Nbridgein the catharanthine-derived unit has been shortened by one carbon. It is orally active and has a broader anticancer activity yet with lower neurotoxic side-effects than either vinblastine or vincristine. These compounds all inhibit cell mitosis, acting by binding to the protein tubulin in the mitotic spindle, preventing polymerization into microtubules, a mode of action shared with other natural agents, e.g. colchicine (see page 343) and podophyllotoxin (see page 136). A major problem associated with the clinical use of vinblastine and vincristine is that only very small amounts of these desirable alkaloids are present in the plant. Although the total alkaloid content of the leaf can reach 1% or more, over 500 kg of catharanthus is needed to yield 1 g of vincristine. This yield (0.0002%) is the lowest of any medicinally important alkaloid isolated on a commercial basis. Extraction is both costly and tedious, requiring large quantities

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of raw material and extensive use of chromatographic fractionations. The growing importance of vincristine relative to vinblastine as drugs is not reflected in the plant, which produces a much higher proportion of vinblastine. Fortunately, it is possible to convert vinblastine into vincristine by controlled chromic acid oxidation or via a microbiological N-demethylation using Streptomyces albogriseolus. Considerable effort has been expended on the semi- synthesis of the ‘dimeric’ alkaloids from ‘monomers’ such as catharanthine and vindoline, which are produced in C. roseus in much larger amounts. Efficient, stereospecific coupling has eventually been achieved, and it is now possible to convert catharanthine and vindoline into vinblastine in about 40% yield. The process used is a biomimetic one, virtually identical to the suggested biosynthetic process, and is also included in Figure 6.81. Catharanthine- N-oxide is employed instead of the peroxidase-generated peroxide, and this couples readily in trifluoroacetic anhydride with vindoline in almost quantitative yield. Subsequent reduction, oxidation, and reduction steps then give vinblastine via the same ‘biosynthetic’ intermediates. It is particularly interesting that the most effective reducing agents for the transformation of the dihydropyridinium compound into the tetrahydropyridine were N-substituted 1,4- dihydronicotinamides, simpler analogues of NADH, the natural reducing agent. Excellent yields of anhydrovinblastine (the starting material for vinorelbine production) (Figure 6.80) can also be obtained by electrochemical oxidation of catharanthine/vindoline. These syntheses should improve the supply of these alkaloids and derivatives, and also allow more detailed studies of structure–activity relationships to be undertaken. This group of compounds is still of very high interest, and development programmes for analogues continue. Ajmalicine (see rauwolfia, page 353) is present in the roots of Catharanthus roseus at a level of about 0.4%, and this plant is used as a commercial source in addition to Rauwolfia serpentina.

Iboga The Iboga group of terpenoid indole alkaloids takes its name from Tabernanthe iboga (Apocynaceae), a shrub from the Congo and other parts of equatorial Africa. Extracts from the root bark of this plant have long been used by indigenous people in rituals, to combat fatigue, and as an aphrodisiac. The root bark contains up to 6% indole alkaloids, the principal component of which is ibogaine (Figure 6.82). Ibogaine is a CNS stimulant, and is also psychoactive. In large doses, it can cause paralysis and respiratory arrest. Ibogaine is of interest as a potential drug for relieving heroin craving in drug addicts. Those who use the drug experience hallucinations from the ibogaine, but it is claimed they emerge from this state with a significantly reduced opiate craving. A number of deaths resulting from the unsupervised use of ibogaine has led to its being banned in some countries.

contains a C9 Corynanthe type unit and its rela- Alkaloids like preakuammicine (Figure 6.79) tionship to dehydrogeissoschizine is indicated in and akuammicine (Figure 6.75) contain the C10 Figure 6.82. Vincamine still retains a C10 Aspi- and C9 Corynanthe type terpenoid units respec- dosperma unit, and it originates from tabersonine tively. They are, however, representatives of a by a series of reactions that involve cleavage of subgroup of Corynanthe alkaloids termed the bonds to both α and β positions of the indole Strychnos type because of their structural similar- (Figure 6.82). ity to many of the alkaloids found in Strychnos 358 ALKALOIDS

N MeO

N H C Iboga ibogaine 9

HO N N OH

N N H H H CHO Me dehydrogeissoschizine CO2Me ajmaline C9 Corynanthe (keto form)

N H N

N

N CO2Me HO H CO2Me tabersonine vincamine Aspidosperma

Figure 6.82

N N N O

HO CH2OH CHO N CO2Me N N CHO CO2Me H preakuammicine Wieland–Gumlich aldehyde acetyl-CoA

aldol reaction with N N acetyl-CoA N H H H H H HO H O OH O N H N N H H H H HO O CO2H Wieland–Gumlich aldehyde strychnine (hemiacetal form)

Figure 6.83 species (Loganiaceae), e.g. S. nux-vomica∗, note- of one carbon from a preakuammicine-like struc- worthy examples being the extremely poisonous ture via hydrolysis/decarboxylation and then addi- strychnine (Figure 6.83) and its dimethoxy ana- tion of the extra two carbons by aldol condensation logue brucine (Figure 6.84). The non-tryptamine with the formyl group, complexed as a hemiacetal portion of these compounds contains 11 carbons, in the so-called Wieland–Gumlich aldehyde. The and is constructed from an iridoid-derived C9 unit, subsequent formation of strychnine from this hemi- plus two further carbons supplied from acetate. The acetal is merely construction of ether and amide pathway to strychnine in Figure 6.83 involves loss linkages. ALKALOIDS DERIVED FROM TRYPTOPHAN 359

Nux-vomica Nux-vomica consists of the dried ripe seeds of Strychnos nux-vomica (Loganiaceae), a small tree found in a wide area of East Asia extending from India to Northern Australia. The fruit is a large berry with a hard coat and a pulpy interior containing three to five flattish grey seeds. These seeds contain 1.5–5% of alkaloids, chiefly strychnine (about 1.2%) and brucine (about 1.6%) (Figure 6.82). Strychnine is very toxic, affecting the CNS and causing convulsions. This is a result of binding to receptor sites in the spinal cord that normally accommodate glycine. Fatal poisoning (consumption of about 100 mg by an adult) would lead to asphyxia following contraction of the diaphragm. It has found use as a vermin killer, especially for moles. Its only medicinal use is in very small doses as an appetite stimulant and general tonic, sometimes with iron salts if the patient is anaemic. Brucine is considerably less toxic. Both compounds have been regularly used in synthetic chemistry as optically active bases to achieve optical resolution of racemic acids. Seeds of the related Strychnos ignatii have also served as a commercial source of strychnine and brucine. Of biochemical interest is the presence of quite significant amounts (up to 5%) of the iridoid glycoside loganin (see page 188) in the fruit pulp of Strychnos nux-vomica. This compound is, of course, an intermediate in the biosynthesis of strychnine and other terpenoid indole alkaloids.

N aldehyde-like molecules (Figure 6.85). The pres- H ence of two quaternary nitrogens, separated H by an appropriate distance, is responsible for H O the curare-like activity (compare tubocurarine N H H and synthetic analogues, page 326). Alcuronium (Figure 6.85) is a semi-synthetic skeletal muscle O relaxant produced from C-toxiferine (see curare, strychnine page 327). N Ellipticine∗ (Figure 6.86) contains a pyrido- H carbazole skeleton, which is also likely to be MeO H formed from a tryptamine–terpenoid precursor. H O Although little evidence is available, it is sug- MeO N H H gested that a precursor like stemmadenine may undergo transformations that effectively remove O brucine the two-carbon bridge originally linking the indole and the nitrogen in tryptamine (Figure 6.86). The Figure 6.84 remaining C9 terpenoid fragment now contain- ing the tryptamine nitrogen can then be used The arrow poison curare, when produced from to generate the rest of the skeleton. Ellipticine Ochrosia elliptica Chondrodendron species (Menispermaceae), con- is found in (Apocynaceae) tains principally the bis-benzyltetrahydroisoquino- and related species and has useful anticancer properties. line alkaloid tubocurarine (see page 324). Species of Strychnos, especially S. toxifera, are employed in making loganiaceous curare, and biologically Quinoline Alkaloids active alkaloids isolated from such preparations have been identified as a series of toxiferines, Some of the most remarkable examples of ter- e.g. C-toxiferine (Figure 6.85). The structures penoid indole alkaloid modifications are to be appear remarkably complex, but may be envis- found in the genus Cinchona∗ (Rubiaceae), in aged as a combination of two Wieland–Gumlich the alkaloids quinine, quinidine, cinchonidine, 360 ALKALOIDS

Me via intermolecular N N Schiff base reactions H H N OH OH H H H x 2 chemically H N H N H OH H H N H N N H CHO H H H HO HO Wieland–Gumlich aldehyde H H N N Me C-toxiferine alcuronium

Figure 6.85

H N N NH N

CH2OH N N N CO2Me N H H H H stemmadenine ellipticine

Figure 6.86

Ellipticine Ellipticine (Figure 6.86) and related alkaloids, e.g. 9-methoxyellipticine (Figure 6.87), are found in the bark of Ochrosia elliptica (Apocynaceae) and other Ochrosia species. Clinical trials with these alkaloids and a number of synthetic analogues showed them to be potent inhibitors of several cancerous disorders, but pre-clinical toxicology indicated a number of side-effects, including haemolysis and cardiovascular effects. Ellipticines are planar molecules that intercalate between the base pairs of DNA and cause a partial unwinding of the helical array. There is a some correlation between the degree of unwinding and the biological properties, those showing the largest unwinding inhibiting the greatest number of cancerous cells. Recent research suggests there may be more than one mechanism of action, however. Ellipticine is oxidized in vivo mainly to 9-hydroxyellipticine, which has an increased activity, and it is believed that this may in fact be the active agent. Poor water-solubility of ellipticine and derivatives gave problems in formulation for clinical use, but quaternization of 9-hydroxyellipticine to give the water-soluble 9-hydroxy-2-N-methylellipticinium acetate (elliptinium acetate) (Figure 6.87) has produced a highly active material, of value in some forms of breast cancer, and perhaps also in renal cell cancer. A variety of such quaternized derivatives is being tested, and some water-soluble N-glycosides also show high activity.

Me N N OAc

MeO HO

N N H H 9-methoxyellipticine elliptinium acetate

Figure 6.87 ALKALOIDS DERIVED FROM TRYPTOPHAN 361

H

H H S N R 8 R H OH R 9 OH R N S H H

N N R = OMe, (–)-quinine R = OMe, (+)-quinidine R = H, (–)-cinchonidine R = H, (+)-cinchonine

Figure 6.88

and cinchonine (Figure 6.88), long prized for their antimalarial properties. These structures are NR2 remarkable in that the indole nucleus is no longer present, having been rearranged into a quinoline N N H system (Figure 6.89). The relationship was sus- indole alkaloid quinoline alkaloid pected quite early on, however, since the indole derivative cinchonamine (Figure 6.90) was known Figure 6.89

OH NH H OGlc N O N H H H N H H CO Me strictosidine 2 cleavage of C–N bond (via cinchonamine iminium) then formation of new C–N bond (again via iminium) CHO H N H N N

N H N H N H CHO CHO H H H H H cleavage of indole hydrolysis and MeO2C corynantheal C–N bond decarboxylation

H O 8 O HO N N N H H H R NADPH

CHO N N NH2 R = H, cinchonidine cinchoninone R = OMe, quinine epimerization at C-8 via enol H O HO N N H H R NADPH

N N R = H, cinchonine R = OMe, quinidine

Figure 6.90 362 ALKALOIDS to co-occur with these quinoline alkaloids. An out- could generate new amine and keto functions. The line of the pathway from the Corynanthe-type indole new heterocycle would then be formed by com- alkaloids to cinchonidine is shown in Figure 6.90. bining this amine with the aldehyde produced in The conversion is dependent on the reversible pro- the tryptamine side-chain cleavage. Finally, reduc- cesses by which amines plus aldehydes or ketones, tion of the ketone gives cinchonidine or cincho- imines (Schiff bases), and their reduction products nine. Hydroxylation and methylation at some stage are related in nature. Suitable modification of stric- allows biosynthesis of quinine and quinidine.Qui- tosidine leads to an aldehyde (compare the early nine and quinidine, or cinchonidine and cinchonine, reactions in the ajmalicine pathway (Figure 6.76)). are pairs of diastereoisomers, which have opposite Hydrolysis/decarboxylation would initially remove chiralities at two centres (Figure 6.88). Stereospe- one carbon from the iridoid portion and produce cific reduction of the carbonyl in cinchoninone can corynantheal. An intermediate of the cinchonamine control the stereochemistry adjacent to the quino- type would then result if the tryptamine side-chain line ring (C-9). The stereochemistry at the second were cleaved adjacent to the nitrogen, and if this centre (C-8) is also determined during the reduction nitrogen were then bonded to the acetaldehyde func- step, presumably via the enol form of cinchoninone tion. Ring opening in the indole heterocyclic ring (Figure 6.90).

Cinchona Cinchona bark is the dried bark from the stem and root of species of Cinchona (Rubiaceae), which are large trees indigenous to South America. Trees are cultivated in many parts of the world, including Bolivia, Guatemala, India, Indonesia, Zaire, Tanzania, and Kenya. About a dozen different Cinchona species have been used as commercial sources, but the great variation in alkaloid content, and the range of alkaloids present, has favoured cultivation of three main species, together with varieties, hybrids, and grafts. Cinchona succirubra provides what is called ‘red’ bark (alkaloid content 5–7%), C. ledgeriana gives ‘brown’ bark (alkaloid content 5–14%), and C. calisaya ‘yellow’ bark with an alkaloid content of 4–7%. Selected hybrids can yield up to 17% total alkaloids. Bark is stripped from trees which are about 8–12 years old, the trees being totally uprooted by tractor for the process. A considerable number of alkaloids have been characterized in cinchona bark, four of which account for some 30–60% of the alkaloid content. These are quinine, quinidine, cinchonidine, and cinchonine, quinoline-containing structures representing two pairs of diastereoisomers (Figure 6.88). Quinine and quinidine have opposite configurations at two centres. Cinchonidine and cinchonine are demethoxy analogues, but unfortunately use of the -id- syllable in the nomenclature does not reflect a particular stereochemistry. Quinine is usually the major component (half to two-thirds total alkaloid content) but the proportions of the four alkaloids vary according to species or hybrid. The alkaloids are often present in the bark in salt combination with quinic acid (see page 122) or a tannin material called cinchotannic acid. Cinchotannic acid decomposes due to enzymic oxidation during processing of the bark to yield a red pigment, which is particularly prominent in the ‘red’ bark. Cinchona and its alkaloids, particularly quinine, have been used for many years in the treatment of malaria, a disease caused by protozoa, of which the most troublesome is Plasmodium falciparum. The beneficial effects of cinchona bark were first discovered in South America in the 1630s, and the bark was then brought to Europe by Jesuit missionaries. Religious intolerance initially restricted its universal acceptance, despite the widespread occurrence of malaria in Europe and elsewhere. The name cinchona is a mis-spelling derived from Chinchon. In an often quoted tale, now historically disproved, the Spanish Countess of Chinchon, wife of the viceroy of Peru, was reputedly cured of malaria by the bark. For

(Continues) ALKALOIDS DERIVED FROM TRYPTOPHAN 363

(Continued) many years, the bark was obtained from South America, but cultivation was eventually established by the English in India, and by the Dutch in Java, until just before the Second World War, when almost all the world’s supply came from Java. When this source was cut off by Japan in the Second World War, a range of synthetic antimalarial drugs was hastily produced as an alternative to quinine. Many of these compounds were based on the quinine structure. Of the wide range of compounds produced, chloroquine, primaquine, and mefloquine (Figure 6.91) are important antimalarials. Primaquine is exceptional in having an 8-aminoquinoline structure, whereas chloroquine and mefloquine retain the 4- substituted quinoline as in quinine. The acridine derivative mepacrine (Figure 6.91), though not now used for malaria treatment, is of value in other protozoal infections. Halofantrine (Figure 6.91) dispenses with the heterocyclic ring system completely, and is based on phenanthrene. At one time, synthetic antimalarials had almost entirely superseded natural quinine, but the emergence of Plasmodium falciparum strains resistant to the synthetic drugs, especially the widely used prophylactic chloroquine, has resulted in reintroduction of quinine. Mefloquine is currently active against chloroquine-resistant strains, but, whilst ten times as active as quinine, does produce gastrointestinal upsets and dizziness, and can trigger psychological problems such as depression, panic, or psychosis in some patients. The ability of P. falciparum to develop resistance to modern drugs means malaria still remains a huge health problem, and is probably the major single cause of deaths in the modern world. Chloroquine and its derivative hydroxychloroquine (Figure 6.91), although antimalarials, are also used to suppress the disease process in rheumatoid arthritis. Quinine (Figure 6.88), administered as free base or salts, continues to be used for treatment of multidrug-resistant malaria, though it is not suitable for prophylaxis. The specific mechanism of action is not thoroughly understood, though it is believed to prevent polymerization of toxic haemoglobin breakdown products formed by the parasite (see artemisinin, page 200). Vastly larger amounts of the alkaloid are consumed in beverages, including vermouth and tonic water. It is amusing to realize that gin was originally added to quinine to make the bitter antimalarial more palatable. Typically, the quinine dosage was up to 600 mg three times a

OH RS RS RS NEt2 N NEt2 HN HN HN MeO

Cl N Cl N N Cl hydroxychloroquine chloroquine mepacrine HORS N

HO RS SR N MeO H Cl N F3C HN RS N CF3 NH2 CF3 Cl mefloquine primaquine halofantrine

Figure 6.91

(Continues) 364 ALKALOIDS

(Continued)

day. Quinine in tonic water is now the mixer added to gin, though the amounts of quinine used (about 80 mg l−1) are well below that providing antimalarial protection. Quinine also has a skeletal muscle relaxant effect with a mild curare-like action. It thus finds use in the prevention and treatment of nocturnal leg cramp, a painful condition affecting many individuals, especially the elderly. Quinidine (Figure 6.88) is the principal cinchona alkaloid used therapeutically, and is administered to treat cardiac arrhymias. It inhibits fibrillation, the uncoordinated contraction of muscle fibres in the heart. It is rapidly absorbed by the gastrointestinal tract and overdose can be hazardous, leading to diastolic arrest. Quinidine, cinchonine, and cinchonidine also have antimalarial properties, but these alkaloids are not as effective as quinine. The cardiac effect makes quinidine unsuitable as an antimalarial. However, mixtures of total Cinchona alkaloids, even though low in quinine content, are acceptable antimalarial agents. This mixture, termed totaquine, has served as a substitute for quinine during shortages. Quinine-related alkaloids, especially quinidine, are also found in the bark of Remija pendunculata (Rubiaceae).

Camptothecin∗ (Figure 6.93)fromCamptotheca N acuminata (Nyssaceae) is a further example of N a quinoline-containing structure that is actually N N H derived by modification of an indole system. The β -carboline alkaloid pyrroloquinoline alkaloid main rearrangement process is that the original β- (pyridoindole) carboline 6–5–6 ring system becomes a 6–6–5 Figure 6.92 pyrroloquinoline by ring expansion of the indole

ester hydrolysis, O H OGlc lactam formation NH NH CO2Me N ≡ H H O O O N H N H N H H H H H CO Me H OGlc H OGlc strictosidine 2 strictosamide oxidation of double reduction of carbonyl aldol-type bond to yield two then dehydration to O O condensation carbonyls give pyridine ring O O O N N O N N N N H H H H H O O O H H H

deoxypumiloside H OGlc pumiloside H OGlc H OGlc allylic isomerization

O O N N N N H O O

H OGlc camptothecin HO O

Figure 6.93 ALKALOIDS DERIVED FROM TRYPTOPHAN 365 heterocycle (Figure 6.92). In camptothecin, the Pyrroloindole Alkaloids iridoid portion from strictosidine is effectively still intact, the original ester function being uti- Both C-2 and C-3 of the indole ring can be lized in forming an amide linkage to the sec- regarded as nucleophilic, but reactions involving ondary amine. This occurs relatively early, in C-2 appear to be the most common in alkaloid that strictosamide is an intermediate. Pumiloside biosynthesis. There are examples where the nucle- (also isolated from C. acuminata)anddeoxy- ophilic character of C-3 is exploited, however, pumiloside are potential intermediates. Steps and the rare pyrroloindole skeleton typified by beyond are not yet defined, but involve relatively physostigmine (eserine) (Figure 6.95) is a likely straightforward oxidation and reduction processes case. A suggested pathway to physostigmine is by (Figure 6.93). C-3 methylation of tryptamine, followed by ring

Camptothecin

Camptothecin (Figure 6.93) and derivatives are obtained from the Chinese tree Camptotheca acuminata (Nyssaceae). Seeds yield about 0.3% camptothecin, bark about 0.2%, and leaves up to 0.4%. Camptotheca acuminata is found only in Tibet and West China, but other sources of camptothecin such as Nothapodytes foetida (formerly Mappia foetida) (Icacinaceae), Merilliodendron megacarpum (Icacinaceae), Pyrenacantha klaineana (Icacinaceae), Ophiorrhiza mungos (Rubiaceae), and Ervatmia heyneana (Apocynaceae) have been discovered. In limited clinical trials camptothecin showed broad-spectrum anticancer activity, but toxicity and poor solubility were problems. The natural 10- hydroxycamptothecin (about 0.05% in the bark of C. acuminata) is more active than camptothecin, and is used in China against cancers of the neck and head. Synthetic analogues 9-aminocamptothecin (Figure 6.94) and the water-soluble derivatives topotecan and irinotecan (Figure 6.94) showed good responses in a number of cancers; topotecan and irinotecan are now available for the treatment of ovarian cancer and colorectal cancer, respectively. Irinotecan is a carbamate pro-drug of 10-hydroxy-7-ethylcamptothecin, and is converted into the active drug by liver enzymes. These agents act by inhibition of the enzyme topoisomerase I, which is involved in DNA replication and reassembly, by binding to and stabilizing a covalent DNA–topoisomerase complex (see page 137). Camptothecin has also been shown to have potentially useful activity against pathogenic protozoa such as Trypanosoma brucei and Leishmania donovani, which cause sleeping sickness and leishmaniasis respectively. Again, this is due to topoisomerase I inhibition.

NMe2 N NH2 9 N O 10 O HO O O N N N O N N N O O O

OH O OH O OH O 9-aminocamptothecin topotecan irinotecan

Figure 6.94 366 ALKALOIDS

Ad C-alkylation at C-3 3 4 2 Me S due to nucleophilic nucleophilic attack 3a R character on to iminium ion 5 NH 1 Me Me 6 8a 8 N NH2 7 H NH2 NH pyrroloindole N N N H H H tryptamine H H N MeN Me MeHN O NMe NMe O N H Me N H physostigmine H (eserine) chimonanthine

Figure 6.95 formation involving attack of the primary amine (Figure 6.95) from Chimonanthus fragrans (Caly- function on to the iminium ion (Figure 6.95). Fur- canthaceae), the point of coupling being C-3 of ther substitution is then necessary. Dimers with this the indole, and an analogous radical reaction may ring system are also known, e.g. chimonanthine be proposed. Physostigmine is found in seeds of

Physostigma

Physostigma venenosum (Leguminosae/Fabaceae) is a perennial woody climbing plant found on the banks of streams in West Africa. The seeds are known as Calabar beans (from Calabar, now part of Nigeria) and have an interesting history in the native culture as an ordeal poison. The accused was forced to swallow a potion of the ground seeds, and if the mixture was subsequently vomited, he/she was judged innocent and set free. If the poison took effect, the prisoner suffered progressive paralysis and died from cardiac and respiratory failure. It is said that slow consumption allows the poison to take effect, whilst emesis is induced by a rapid ingestion of the dose.

Me Me Et MeHN O MeHN O N O N O Me NMe2 O CONHMe O N H N H O Me Me eseramine physovenine rivastigmine

Me Me(CH2)6HN O Me2NNMeO 3 Me2N O NMe NMe O N H O O eptastigmine Me neostigmine pyridostigmine O O NHMe O Me HO NMe2Et MeN N O O N NMe Me O distigmine edrophonium carbaryl

Figure 6.96

(Continues) ALKALOIDS DERIVED FROM TRYPTOPHAN 367

(Continued)

oxidation to N-oxide Me Me Me MeHN O MeHN O MeHN O NMe O Me NMe N O O O O O N H N H N H Me Me Me physostigmine physostigmine N-oxide geneserine hydrolysis of H2O carbamate oxidation to quinol, and then to ortho-quinone Me Me HO O O NMe NMe N H O N H Me Me eseroline rubreserine

Figure 6.97

The seeds contain several alkaloids (alkaloid content about 1.5%), the major one (up to 0.3%) being physostigmine (eserine) (Figure 6.95). The unusual pyrroloindole ring system is also present in some of the minor alkaloids, e.g. eseramine (Figure 6.96), whilst physovenine (Figure 6.96) contains an undoubtedly related furanoindole system. Another alkaloid, geneserine (Figure 6.97), is an artefact produced by oxidation of physostigmine, incorporating oxygen into the ring system, probably by formation of an N-oxide and ring expansion. Solutions of physostigmine are not particularly stable in the presence of air and light, especially under alkaline conditions, oxidizing to a red quinone, rubeserine (Figure 6.97). Physostigmine (eserine) is a reversible inhibitor of cholinesterase, preventing normal destruction of acetylcholine and thus enhancing cholinergic activity. Its major use is as a miotic, to contract the pupil of the eye, often to combat the effect of mydriatics such as atropine (see page 297). It also reduces intraocular pressure in the eye by increasing outflow of the aqueous humour, and is a valuable treatment for glaucoma, often in combination with pilocarpine (see page 380). Because it prolongs the effect of endogenous acetylcholine, physostigmine can be used as an antidote to anticholinergic poisons such as hyoscyamine/atropine (see page 297), and it also reverses the effects of competitive muscle relaxants such as curare, tubocurarine, atracurium, etc (see page 324). Anticholinesterase drugs are also of value in the treatment of Alzheimer’s disease, which is characterized by a dramatic decrease in functionality of the central cholinergic system. Use of acetylcholinesterase inhibitors can result in significant memory enhancement in patients, and analogues of physostigmine are presently in use (e.g. rivastigmine) or in advanced clinical trials (e.g. eptastigmine (Figure 6.96)). These analogues have a longer duration of action and less toxicity than physostigmine. The biological activity of physostigmine resides primarily in the carbamate portion, which is transferred to the hydroxyl group of an active site serine in cholinesterase (Figure 6.98). The enzyme is only slowly regenerated by hydrolysis of this group, since resonance contributions reduce the reactivity of the carbonyl in the amide relative to the ester. Accordingly, cholinesterase becomes temporarily inactivated. Synthetic analogues of physostigmine which have been developed retain the carbamate residue, an aromatic ring to achieve binding and to provide a good leaving group, whilst ensuring water-solubility through possession of a quaternary ammonium system. Neostigmine, pyridostigmine,and distigmine (Figure 6.96) are examples of synthetic anticholinesterase drugs used primarily for enhancing neuromuscular transmission in the rare autoimmune condition myasthenia

(Continues) 368 ALKALOIDS

(Continued)

O

NMe3 O O acetylcholine fast OH O hydrolysis OH

Ser Ser Ser cholinesterase resonance stabilization O phenoxide system decreases carbonyl character provides good Ar and slows rate of hydrolysis MeHN O leaving group MeHN O MeHN O slow OH O O hydrolysis OH

Ser Ser Ser Ser cholinesterase

Figure 6.98

gravis, in which muscle weakness is caused by faulty transmission of nerve impulses. Edrophonium is a short-acting competitive blocker of the acetylcholinesterase active site, which is used to help diagnose myasthenia gravis. A number of carbamate insecticides, e.g. carbaryl (Figure 6.96), also depend on inhibition of cholinesterase for their action, insect acetylcholinesterase being more susceptible to such agents than the mammalian enzyme. Physostigmine displays little insecticidal action because of its poor lipid solubility.

Physostigma venenosum∗ (Leguminosae/Fabaceae) Ergot Alkaloids and has played an important role in pharma- cology because of its anticholinesterase activity. Ergot∗ is a fungal disease commonly found on The inherent activity is in fact derived from the many wild and cultivated grasses, and is caused carbamate side-chain rather than the heterocyclic by species of Claviceps. The disease is eventually ring system, and this has led to a range of synthetic characterized by the formation of hard, seedlike materials being developed. ‘ergots’ instead of normal seeds, these structures,

HO N O H O N R 7 6 OH HN HN 8 NH O NMe O H H 9 10 O NMe O NMe 5 4 H H H 11 3 12 2 13 N N 14 H H N N ergoline R = OH, (+)-lysergic acid H H R = NH2, ergine ergometrine ergotamine

Figure 6.99 ALKALOIDS DERIVED FROM TRYPTOPHAN 369 called sclerotia, forming the resting stage of the NH HO C 2 2 NMe fungus. The poisonous properties of ergots in grain, CO2H C5 H especially rye, for human or animal consump- tion have long been recognized, and the causative agents are known to be a group of indole alka- loids, referred to collectively as the ergot alkaloids N N H H or ergolines (Figure 6.99). Under natural condi- Trp D-(+)-lysergic acid tions the alkaloids are elaborated by a combination of fungal and plant metabolism, but they can Figure 6.100

OPP N-methylation

CO2H CO2H CO2H

NH2 NH2 SAM NHMe

N C-alkylation at position 4 N N H which is nucleophilic due to H H L-Trp 4-dimethylallyl- indole nitrogen O L-Trp H O 1,4-elimination of epoxidation H2O to give diene H

CO2H CO2H OH CO2H

NHMe O NHMe NHMe

N N N H H H

≡ Schiff base formation followed by reduction; a H cis–trans isomerization is necessary to suitably position the amine and aldehyde groups O O OHC MeHN HO NHMe NHMe NMe H H H H O – CO 2 H NADPH H H

N opening of epoxide N N N H H H H allows ring closure and chanoclavine-I chanoclavine-I agroclavine decarboxylation aldehyde

allylic isomerization; gives O2 conjugation with aromatic NADPH ring system HO2C HO2C NMe NMe HO NMe H H H H

H O H

N N N H H H D-(+)-lysergic acid paspalic acid elymoclavine

Figure 6.101 370 ALKALOIDS be synthesized in cultures of suitable Claviceps double bond in the dimethylallyl substituent species. Ergoline alkaloids have also been found must become a single bond on two separate in fungi belonging to genera Aspergillus, Rhizo- occasions, allowing rotation to occur as new pus,andPenicillium,aswellasClaviceps,and rings are established. This gives the appearance simple examples are also found in some plants of of cis–trans isomerizations as 4-dimethylallyl- the Convolvulaceae such as Ipomoea and Rivea L-tryptophan is transformed into chanoclavine- (morning glories)∗. Despite their toxicity, some I, and as chanoclavine-I aldehyde cyclizes to of these alkaloids have valuable pharmacologi- agroclavine (Figure 6.101). A suggested sequence cal activities and are used clinically on a routine to account for the first of these is shown. In basis. the later stages, agroclavine is hydroxylated to Medicinally useful alkaloids are derivatives elymoclavine, further oxidation of the primary of (+)-lysergic acid (see Figure 6.99), which alcohol occurs giving paspalic acid,andlysergic is typically bound as an amide with an amino acid then results from a spontaneous allylic alcohol as in ergometrine, or with a small isomerization. polypeptide structure as in ergotamine.The Simple derivatives of lysergic acid require building blocks for lysergic acid are tryptophan the formation of amides; for example, ergine (less the carboxyl group) and an isoprene unit (Figure 6.99) in Rivea and Ipomoea species is (Figure 6.100). Alkylation of tryptophan with lysergic acid amide, whilst ergometrine from dimethylallyl diphosphate gives 4-dimethylallyl-L- Claviceps purpurea is the amide with 2-aminopro- tryptophan, which then undergoes N-methylation panol. The more complex structures containing (Figure 6.101). Formation of the tetracyclic ring peptide fragments, e.g. ergotamine (Figure 6.99), system of lysergic acid is known to proceed are formed by sequentially adding amino acid through chanoclavine-I and agroclavine, though residues to thioester-bound lysergic acid, giving a the mechanistic details are far from clear. linear lysergyl–tripeptide covalently attached to the Labelling studies have established that the enzyme complex (Figure 6.102). Peptide formation

ATP EnzSH Enzyme Enzyme L-Ala, L-Phe, L-Pro the enzyme is known to HO2C NH be comprised of two NMe OS S 2 O S H2N subunits which bind H 1 2 S EnzSH substrates as indicated ATP L-Ala O H N O 3 NMe L-Pro H N activation via AMP H esters, L-Phe sequential formation D-(+)-lysergic acid then attachment to N enzyme H of tripeptide (see Figure 7.15) L-Pro

hydroxylation then lactam formation N HO N N EnzS formation of and release from O O H O hemiketal-like OOH enzyme O L-Phe N product N HN O L-Ala HN O HN O HN O NMe O NMe NMe H O O H H

N H N N ergotamine H H

Figure 6.102 ALKALOIDS DERIVED FROM TRYPTOPHAN 371 involves the same processes seen in the non- various active sites (compare fatty acid biosynthesis, ribosomal biosynthesis of peptides, and involves page 36). The cyclized tripeptide residue is readily ATP-mediated activation of the amino acids prior to rationalized by the formation of a lactam (amide) attachment to the enzyme complex through thioester which releases the product from the enzyme, linkages (see page 421). A phosphopantatheine arm followed by generation of a hemiketal-like linkage is used to enable the growing chain to reach the as shown (Figure 6.102).

Ergot

Medicinal ergot is the dried sclerotium of the fungus Claviceps purpurea (Clavicipitaceae) developed on the ovary of rye, Secale cereale (Graminae/Poaceae). Ergot is a fungal disease of wild and cultivated grasses, and initially affects the flowers. In due course, a dark sclerotium, the resting stage of the fungus, is developed instead of the normal seed. This protrudes from the seed head, the name ergot being derived from the French word argot – a spur. The sclerotia fall to the ground, germinating in the spring and reinfecting grasses or grain crops by means of spores. Two types of spore are recognized: ascospores, which are formed in the early stages and are dispersed by the wind, whilst later on conidiospores are produced, which are insect distributed. The flowers are only susceptible to infection before pollination. Ergots may subsequently be harvested with the grain and contaminate flour or animal feed. The consumption of ergot-infected rye has resulted in the disease ergotism, which has a long, well documented history. There are three broad clinical features of ergot poisoning, which are due to the alkaloids present and the relative proportions of each component:

• Alimentary upsets, e.g. diarrhoea, abdominal pains, and vomiting. • Circulatory changes, e.g. coldness of hands and feet due to a vasoconstrictor effect, a decrease in the diameter of blood vessels, especially those supplying the extremeties. • Neurological symptoms, e.g. headache, vertigo, convulsions, psychotic disturbances, and hallucinations. These effects usually disappear on removal of the source of poisoning, but much more serious problems develop with continued ingestion, or with heavy doses of ergot-contaminated food. The vasoconstrictor effect leads to restricted blood flow in small terminal arteries, death of the tissue, the development of gangrene, and even the shedding of hands, feet, or limbs. Gangrenous ergotism was known as St Anthony’s Fire, the Order of St Anthony traditionally caring for sufferers in the Middle Ages. The neurological effects were usually manifested by severe and painful convulsions. Outbreaks of the disease in both humans and animals were relatively frequent in Europe in the Middle Ages, but once the cause had been established it became relatively simple to avoid contamination. Separation of the ergots from grain, or the use of fungicides during cultivation of the crop, have removed most of the risks, though infection of crops is still common. The ergot sclerotia contain from 0.15–0.5% alkaloids, and more than 50 have been characterized. The medicinally useful compounds are derivatives of (+)-lysergic acid (Figure 6.103), and can be separated into two groups, the water-soluble amino alcohol derivatives (up to about 20% of the total alkaloids), and water-insoluble peptide derivatives (up to 80% of the total alkaloids). Ergometrine (Figure 6.103) (also known as ergonovine in the USA and ergobasine in Switzerland) is an amide of lysergic acid and 2-aminopropanol, and is the only significant member of the first group. The peptide derivatives contain a

(Continues) 372 ALKALOIDS

(Continued)

OH OH OH HN OH HN NEt2 6 8 O NMe O NMe O NMe O NMe O NMe 9 H H H H H 10

N N N N N H H H H H (+)-lysergic acid (+)-isolysergic acid lysergic acid ergometrine lysergic acid diethylamide α-hydroxyethylamide (ergonovine; ergobasine) (lysergide; LSD)

HO N HO N HO N HO N O H O O H O O H O O H O N N N N

HN HN HN HN O O O O O NMe O NMe O NMe O NMe H H H H

N N N N H H H H ergotamine ergocristine ergocornine α-ergocryptine

HO N O H O N NHEt

O NEt2 OH HN O O N NMe2 SMe HN HN O NMe O N NMe NPr O NMe H H H H H

H H

Br N N N N N H H H H Me bromocryptine cabergoline lisuride pergolide methysergide (lysuride)

Figure 6.103

cyclized tripeptide fragment bonded to lysergic acid via an amide linkage. Based on the nature of the three amino acids, these structures can be subdivided into three groups, the ergotamine group, the ergoxine group, and the ergotoxine group (Table 6.1). The amino acids involved are alanine, valine, leucine, isoleucine, phenylalanine, proline, and α-aminobutyric acid, in various combinations (see Figure 6.104). All contain proline in the tripeptide, and one of the amino acids is effectively incorporated into the final structure in the form of an α-hydroxy-α-amino acid. Thus, ergotamine incorporates alanine, phenylalanine, and proline residues in its peptide portion. Hydrolysis gives (+)-lysergic acid, proline, and phenylalanine,

(Continues) ALKALOIDS DERIVED FROM TRYPTOPHAN 373

(Continued)

Table 6.1 Peptide alkaloids in ergot

HO N HO N HO N O H O O H O O H O N N N R R R HN HN HN O O O O NMe O NMe O NMe H H H

N N N H H H ergotamine groupergoxine group ergotoxine group

R = CH2Ph ergotamine ergostine ergocristine α R = CH2CHMe2 ergosine ergoptine -ergocryptine S R = CH(Me)Et [β-ergosine] [β-ergoptine] β-ergocryptine

R = CHMe2 ergovaline ergonine ergocornine

R = Et ergobine ergobutine ergobutyrine

[ ] not yet known in nature together with pyruvic acid and ammonia, the latter hydrolysis products a consequence of the additional hydroxylation involving alanine (Figure 6.104). Hydrolysis of the ergotoxine group of alkaloids results in the proximal valine unit being liberated as dimethylpyruvic acid (not systematic nomenclature) and ammonia, and the ergoxine group similarly yields α-oxobutyric acid from the α-aminobutyric acid fragment. The alkaloid ‘ergotoxine’ was originally thought to be a single compound, but was subsequently shown to be a mixture of alkaloids. The proposed structures β-ergosine and β-ergoptine, which complete the combinations shown in Table 6.1, have not yet been isolated as natural products.

L-Pro

HO N hydrolysis NH = HO2C H R CH2Ph, L-Phe O H O CO H 2 R = CH2CHMe2, L-Leu R′ N OH H2N R = CH(Me)Et, L-Ile R′ R = HN R R CHMe2, L-Val O H2NCO2H R = Et, α-aminobutyric acid

O R = Me, pyruvic acid R′ CO2H R = Et, α-oxobutyric acid R = CHMe2, dimethylpyruvic acid NH3

Figure 6.104

(Continues) 374 ALKALOIDS

(Continued)

Medicinal ergot is cultivated in the Czech Republic, Germany, Hungary, Switzerland, Austria, and Poland. Fields of rye are infected artificially with spore cultures of Claviceps purpurea, either by spraying or by a mechanical process that uses needles dipped in a spore suspension. The ergots are harvested by hand, by machine, or by separation from the ripe grain by flotation in a brine solution. By varying the strain of the fungal cultures, it is possible to maximize alkaloid production (0.4–1.2%), or give alkaloid mixtures in which particular components predominate. Ergots containing principally ergotamine in concentrations of about 0.35% can be cultivated. In recent times, ergot of wheat (Triticum aestivum), and the wheat–rye hybrid triticale (Triticosecale) have been produced commercially. Alternatively, the ergot alkaloids can be produced by culturing the fungus. Initially, cultures of the rye parasite Claviceps purpurea in fermentors did not give the typical alkaloids associated with the sclerotia, e.g. ergometrine and ergotamine. These medicinally useful compounds appear to be produced only in the later stages of development of the fungus. Instead, the cultures produced alkaloids that were not based on lysergic acid, and are now recognized as intermediates in the biosynthesis of lysergic acid, e.g. chanoclavine- I, agroclavine, and elymoclavine (Figure 6.101). Ergot alkaloids that do not yield lysergic acid on hydrolysis have been termed clavine alkaloids. Useful derivatives based on lysergic acid can be obtained by fermentation growth of another fungal species, namely Claviceps paspali. Although some strains are available that produce peptide alkaloids in culture, other strains produce high yields of simple lysergic acid derivatives. These include lysergic acid α-hydroxyethylamide (Figure 6.103), lysergic acid amide (ergine) (Figure 6.99), which is also an acid-catalysed decomposition product from lysergic acid α-hydroxyethylamide, and the ∆8,9-isomer of lysergic acid, paspalic acid (Figure 6.101). Lysergic acid is obtained from the first two by hydrolysis, or from paspalic acid by allylic isomerization. Other alkaloids, e.g. ergometrine and ergotamine, can then be produced semi-synthetically. High yielding fermentation methods have also been developed for direct production of ergotamine and the ergotoxine group of peptide alkaloids. The pharmacologically active ergot alkaloids are based on (+)-lysergic acid (Figure 6.103), but since one of the chiral centres in this compound (and its amide derivatives) is adjacent to a carbonyl, the configuration at this centre can be changed as a result of enolization brought about by heat or base (compare tropane alkaloids, page 298; again note that enolization is favoured by conjugation with the aromatic ring). The new diastereomeric form of (+)- lysergic acid is (+)-isolysergic acid (Figure 6.103), and alkaloids based on this compound are effectively pharmacologically inactive. They are frequently found along with the (+)-lysergic acid derivatives, amounts being significant if old ergot samples are processed, or unsuitable isolation techniques are employed. In the biologically active lysergic acid derivatives, the amide group occupies an 8-equatorial position, whilst in the inactive iso-forms, this group is axial. However, the axial form is actually favoured because this configuration allows hydrogen-bonding from the amide N–H to the hetrocyclic nitrogen at position 6. Derivatives of (+)-isolysergic acid are named by adding the syllable -in- to the corresponding (+)-lysergic acid compound, e.g. ergometrinine, ergotaminine. The ergot alkaloids owe their pharmacological activity to their ability to act at α-adrenergic, dopaminergic and serotonergic receptors. The relationship of the general alkaloid structure to those of noradrenaline, dopamine, and 5-hydroxytryptamine (5-HT, serotonin) is shown in Figure 6.105. The pharmacological response may be complex. It depends on the preferred receptor to which the compound binds, though all may be at least partially involved, and whether the alkaloid is an agonist or antagonist.

(Continues) ALKALOIDS DERIVED FROM TRYPTOPHAN 375

(Continued)

R R HN HN

O NMe NH2 NH2 O NMe NH2 H HO H

HO

N OH OH N N H OH OH H H noradrenaline dopamine 5-hydroxytryptamine (serotonin; 5-HT)

Figure 6.105

Despite the unpleasant effects of ergot as manifested by St Anthony’s Fire, whole ergot preparations have been used since the 16th century to induce uterine contractions during childbirth, and to reduce haemorrhage following the birth. This oxytocic effect (oxytocin is the pituitary hormone that stimulates uterine muscle, see page 415) is still medicinally valuable, but is now achieved through use of the isolated alkaloid ergometrine. The deliberate use of ergot to achieve abortions is dangerous and has led to fatalities. Ergometrine (ergonovine) (Figure 6.103) is used as an oxytocic, and is injected during the final stages of labour and immediately following childbirth, especially if haemorrhage occurs. Bleeding is reduced because of its vasoconstrictor effects, and it is valuable after Caesarian operations. It is sometimes administered in combination with oxytocin itself (see page 415). Ergometrine is also orally active. It produces faster stimulation of uterine muscle than do the other ergot alkaloids, and probably exerts its effect by acting on α-adrenergic receptors, though it may also stimulate 5-HT receptors. Ergotamine (Figure 6.103) is a partial agonist of α-adrenoceptors and 5-HT receptors. It is not suitable for obstetric use because it also produces a pronounced peripheral vasoconstrictor action. This property is exploited in the treatment of acute attacks of migraine, where it reverses the dilatation of cranial blood vessels. Ergotamine is effective orally, or by inhalation in aerosol form, and may be combined with caffeine, which is believed to enhance its action. The semi-synthetic dihydroergotamine is produced by hydrogenation of the lysergic acid ∆9,10 double bond (giving C-10 stereochemistry as in ergoline) and is claimed to produce fewer side-effects, especially digestive upsets. The ‘ergotoxine’ alkaloid mixture also has oxytocic and vasoconstrictor activity but is only employed medicinally as the 9,10-dihydro derivatives dihydroergotoxine (co-dergocrine), a mixture in equal proportions of dihydroergocornine, dihydroergocristine,andthe dihydroergocryptines (α-andβ- in the ratio 2:1). In the case of these alkaloids, reduction of the double bond appears to reverse the vasoconstrictor effect, and dihydroergotoxine has a cerebral vasodilator activity. The increased blood flow is of benefit in some cases of senility and mild dementia, and helps to improve both mental function and physical performance. A number of semi-synthetic lysergic acid derivatives act by stimulation of dopamine recep- tors in the brain, and are of value in the treatment of neurological disorders such as Parkinson’s disease. Bromocriptine (2-bromo-α-ergocryptine), cabergoline, lisuride (lysuride), and per- golide (Figure 6.103) are all used in this way. Bromocriptine and cabergoline find wider use in that they also inhibit release of prolactin by the pituitary (see page 414), and can

(Continues) 376 ALKALOIDS

(Continued)

thus suppress lactation and be used in the treatment of breast tumours. Methysergide (Figure 6.103) is a semi-synthetic analogue of ergometrine, having a modified amino alcohol side-chain and an N-methyl group on the indole ring. It is a potent 5-HT antagonist and as such is employed in the prophylaxis of severe migraine headaches, though its administration has to be very closely supervised. Prolonged treatment with any of the ergot alkaloids is undesirable and it is vital that the clinical features associated with ergot poisoning are recognized. Treatment must be withdrawn immediately if any numbness or tingling develops in the fingers or toes. Side- effects will disappear on withdrawal of the drug, but there have been many cases where misdiagnosis has unfortunately led to foot or toe rot, and the necessity for amputation of the dead tissue. Undoubtedly the most notorious of the lysergic acid derivatives is lysergide (lysergic acid diethylamide or LSD) (Figure 6.103). This widely abused hallucinogen, known as ‘acid’, is probably the most active and specific psychotomimetic known, and is a mixed agonist–anatagonist at 5-HT receptors, interfering with the normal processes. An effective oral dose is from 30 to 50 µg. It was synthesized from lysergic acid, and even the trace amounts absorbed during its handling were sufficient to give its creator quite dramatic hallucinations. LSD intensifies perceptions and distorts them. How the mind is affected depends on how the user is feeling at the time, and no two ‘trips’ are alike. Experiences can vary from beautiful visions to living nightmares, sometimes lasting for days. Although the drug is not addictive, it can lead to schizophrenia and there is danger of serious physical accidents occurring whilst the user is under the influence of the drug.

Morning Glories

Lysergic acid derivatives have also been characterized in the seeds of morning glory (Ipomoea violacea), Rivea corymbosa, and other members of the Convolvulaceae. Such seeds formed the ancient hallucinogenic drug ololiuqui still used by the Mexican Indians in religious and other ceremonies. Extracts from the ground seeds are swallowed and the narcotic and hallucinogenic effects are said to provide contact with the gods. The active constituent has been identified to be principally ergine (lysergic acid amide) (Figure 6.99), and this has an activity about one-20th that of LSD, but is more narcotic than hallucinogenic. The alkaloid content of the seeds is usually low, at about 0.05%, but higher levels (0.5–1.3%) have been recorded. Minor ergot-related constituents include ergometrine (Figure 6.103), lysergic acid α-hydroxyethylamide (Figure 6.103), the inactive isolysergic acid amide (erginine), and some clavine alkaloids. Since morning glories are widely cultivated ornamentals and seeds are readily available, deliberate ingestion by thrill-seekers has been considerable. Although the biological activity is well below that of LSD, the practice is potentially dangerous.

ALKALOIDS DERIVED FROM of indole alkaloids. During this conversion, the ANTHRANILIC ACID anthranilic acid residue is decarboxylated, so that only the C6N skeleton is utilized. However, there are also many examples of where anthranilic acid Anthranilic acid (Figure 6.106) is a key inter- itself functions as an alkaloid precursor, using pro- mediate in the biosynthesis of L-tryptophan (see cesses which retain the full skeleton and exploit page 126) and so contributes to the elaboration the carboxyl (Figure 6.106). It should also be ALKALOIDS DERIVED FROM ANTHRANILIC ACID 377

5 4 5 4 8 9 1 CO2H 6 N 3 6 3 7 2 7 2 7 2 6 3 NH2 N NN4 8 1 8 1 5 10 anthranilic acidquinazoline quinoline acridine

Figure 6.106

L-Orn amide O O SCoA formation COSCoA HN HN N

NH2 N N H H anthraniloyl-CoA nucleophilic attack of amine on to iminium Peganum CO2H harmala

CO2H CO H 2 H2N N O H L-Asp N Justicia N H adhatoda OH N-acetyl- peganine anthranilic acid (vasicine)

Figure 6.107 appreciated that, in mammals, L-tryptophan can be pathway is not operative in Justicia adhatoda, degraded back to anthranilic acid (see page 127), and a much less predictable sequence from N- but this is not a route of importance in plants. acetylanthranilic acid and aspartic acid is observed (Figure 6.107).

Quinazoline Alkaloids Quinoline and Acridine Alkaloids Peganine (Figure 6.107) is a quinazoline alka- loid found in Peganum harmala (Zygophyllaceae), Alkaloids derived from anthranilic acid undoubt- where it co-occurs with the β-carboline alkaloid edly occur in greatest abundance in plants from harmine (see page 349). It is also responsible for the family the Rutaceae. Particularly well rep- the bronchodilator activity of Justicia adhatoda resented are alkaloids based on quinoline and (Adhatoda vasica) (Acanthaceae), a plant used acridine skeletons (Figure 6.106). Some quinoline in the treatment of respiratory ailments. As a alkaloids such as quinine and camptothecin have result, the alternative name vasicine is also some- been established to arise by fundamental rearrange- times used for peganine. Studies in Peganum har- ment of indole systems and have their origins in mala have clearly demonstrated peganine to be tryptophan (see page 359). A more direct route to derived from anthranilic acid, the remaining part the quinoline ring system is by the combination of the structure being a pyrrolidine ring supplied of anthranilic acid and acetate/malonate, and an by ornithine (compare Figure 6.1, page 292). The extension of this process also accounts for the ori- peganine skeleton is readily rationalized as a result gins of the acridine ring system (see Figure 3.47, of nucleophilic attack from the anthranilate nitro- page 81). Thus, anthraniloyl-CoA (Figure 6.108) gen on to the pyrrolinium cation, followed by can act as a starter unit for chain extension amide formation (Figure 6.107). Remarkably, this via one molecule of malonyl-CoA, and amide 378 ALKALOIDS

Claisen reaction; chain extension with malonate amide O formation O OH malonyl-CoA COSCoA di-enol tautomer

NH2 NH2 O N O N OH anthraniloyl-CoA SCoA H

favoured OMe OH OH OPP 4-hydroxy quinolone SAM tautomer

N O N O C-alkylation; position 3 N O H H is nucleophilic H cyclization on to dimethylallyl 4-hydroxy-2-quinolone side-chain; consider involvement of hydroxyimine tautomer

OMe oxidative cleavage of OMe OMe side-chain (Figure 4.35)

OH N O N O MeO N O platydesmine dictamnine OMe skimmianine

Figure 6.108

Claisen reaction; malonate chain extension of anthraniloyl starter OO Claisen O 3 x malonyl-CoA reaction O COSCoA

H O NHMe NMe O O N-methyl CoAS O NHMe anthraniloyl-CoA nucleophilic addition dehydration and enolization OOHto form aromatic ring OO

– H2O

N OH N O Me MeOH 1,3-dihydroxy-N-methylacridone

OOH O OMe O OMe OMe

N O N OMe N O Me Me Me OMe melicopicine acronycine rutacridone

Figure 6.109 formation generates the heterocyclic system, which these alkaloids. This allows formation of addi- will adopt the more stable 4-hydroxy-2-quinolone tional six- and five-membered oxygen heterocyclic form (Figure 6.108). Position 3 is highly nucle- rings, as seen with other systems, e.g. coumarins, ophilic and susceptible to alkylation, especially isoflavonoids, etc (see pages 145, 155). By an via dimethylallyl diphosphate in the case of analogous series of reactions, the dimethylallyl ALKALOIDS DERIVED FROM HISTIDINE 379 derivative can act as a precursor of furoquino- H H CO2H line alkaloids such as dictamnine and skimmi- N – CO2 N anine (Figure 6.108). These alkaloids are found NH NH N 2 histidine N 2 in both Dictamnus albus and Skimmia japonica L-His decarboxylase histamine (Rutaceae). To simplify the mechanistic interpre- tation of these reactions, it is more convenient to Figure 6.110 consider the di-enol form of the quinolone system. Should chain extension of anthranilyl-CoA (as presursor of alkaloids containing this ring system. the N-methyl derivative) incorporate three acetate/ There are relatively few examples, however, and malonate units, a polyketide would result definite evidence linking them to histidine is often (Figure 6.109). The acridine skeleton is then pro- lacking. duced by sequential Claisen reaction and C–N link- Histamine (Figure 6.110) is the decarboxyla- age by an addition reaction, dehydration, and eno- tion product from histidine and is often involved lization leading to the stable aromatic tautomer. in human allergic responses, e.g. to insect bites or Again, the acetate-derived ring, with its alternate pollens. Stress stimulates the action of the enzyme oxygenation, is susceptible to electrophilic attack, histidine decarboxylase and histamine is released and this can lead to alkylation (with dimethylal- from mast cells (Figure 6.110). Topical antihis- lyl diphosphate) or further hydroxylation. Alkaloids tamine creams are valuable for pain relief, and melicopicine from Melicope fareana, acronycine oral antihistamines are widely prescribed for nasal from Acronychia baueri, and rutacridone from allergies such as hay-fever. Major effects of his- Ruta graveolens (Rutaceae) typify some of the struc- tamine include dilation of blood vessels, inflam- tural variety that may then ensue (Figure 6.109). mation and swelling of tissues, and narrowing of airways. In serious cases, life-threatening anaphy- ALKALOIDS DERIVED FROM lactic shock may occur, caused by a dramatic fall in in blood pressure. HISTIDINE Histidine is a proven precursor of dolichothe- Imidazole Alkaloids line (Figure 6.111) in Dolichothele sphaerica (Cactaceae), the remaining carbon atoms orig- The amino acid L-histidine (Figure 6.110) con- inating from leucine via isovaleric acid (see tains an imidazole ring, and is thus the likely page 197). The imidazole alkaloids found in

HO2C

NH2 L-Leu

H H CO H H N 2 N N HO2C NH2 O N N L-Hisisovaleric acid dolichotheline

Figure 6.111

HO Me Me Me H N N N O O O N O N O N O pilocarpine isopilocarpine pilosine

Figure 6.112 380 ALKALOIDS

R R H Me N NH2 N H2NCO2H O CO2H N R = Me, L-Thr N O L-His R = Ph, L-Phe

Figure 6.113

Jaborandi leaves (Pilocarpus microphyllus and ophthalmic work as a miotic and as a treatment for P. jaborandi; Rutaceae)∗ are also probably derived glaucoma. Additional carbon atoms may originate from histidine, but experimental data are lacking. from acetate or perhaps the amino acid threonine in Jaborandi leaves contain primarily pilocarpine and the case of pilocarpine, whilst pilosine incorporates pilosine (Figure 6.112). Pilocarpine is valuable in a phenylpropane C6C3 unit (Figure 6.113).

Pilocarpus

Pilocarpus or jaborandi consists of the dried leaflets of Pilocarpus jaborandi, P. microphyllus, or P. pennatifolius (Rutaceae), small shrubs from Brazil and Paraguay. Pilocarpus microphyllus is currently the main source. The alkaloid content (0.5–1.0%) consists principally of the imidazole alkaloid pilocarpine (Figure 6.112), together with small amounts of pilosine (Figure 6.112) and related structures. Isomers such as isopilocarpine (Figure 6.112) and isopilosine are readily formed if base or heat is applied during extraction of the alkaloids. This is a result of enolization in the lactone ring, followed by adoption of the more favourable trans configuration rather than the natural cis. However, the iso alkaloids lack biological activity. The alkaloid content of the leaf rapidly deteriorates on storage. Pilocarpine salts are valuable in ophthalmic practice and are used in eyedrops as miotics and for the treatment of glaucoma. Pilocarpine is a cholinergic agent and stimulates the muscarinic receptors in the eye, causing constriction of the pupil and enhancement of outflow of aqueous humour. The structural resemblance to muscarine and acetylcholine is shown in Figure 6.114. Pilocarpine gives relief for both narrow angle and wide angle glaucoma. However, the ocular bioavailability of pilocarpine is low, and it is rapidly eliminated, thus resulting in a rather short duration of action. The effects are similar to those of physostigmine (see page 366) and the two agents are sometimes combined. Pilocarpine is antagonistic to atropine. It has been found that pilocarpine gives relief for dryness of the mouth that results in patients undergoing radiotherapy for mouth and throat cancers. As muscarinic agonists, pilocarpine and analogues are also being investigated for potential treatment of Alzheimer’s disease.

Me H OH H N Me H Me Me Me O N N N Me O Me OO H O muscarine acetylcholine pilocarpine (as conjugate acid)

Figure 6.114 ALKALOIDS DERIVED BY AMINATION REACTIONS 381

ALKALOIDS DERIVED BY AMINATION Acetate-derived Alkaloids REACTIONS The poison hemlock (Conium maculatum∗;Umbel- liferae/Apiaceae) accumulates a range of simple The majority of alkaloids are derived from amino piperidine alkaloids, e.g. coniine and γ-coniceine acid precursors by processes that incorporate into (Figure 6.115). These alkaloids would appear to be the final structure the nitrogen atom together with related to simple lysine-derived compounds such the amino acid carbon skeleton or a large propor- as pelletierine (see page 307), but, surprisingly, a tion of it. Many alkaloids do not conform with study of their biosynthetic origins excluded lysine this description, however, and are synthesized pri- as a precursor, and demonstrated the sequence marily from non-amino acid precursors, with the shown in Figure 6.115 to be operative. A fatty nitrogen atom being inserted into the structure at a acid precursor, capric (octanoic) acid, is uti- relatively late stage. Such structures are frequently lized, and this is transformed into the ketoalde- based on terpenoid or steroidal skeletons, though hyde by successive oxidation and reduction steps. some relatively simple alkaloids also appear to This ketoaldehyde is then the substrate for a be derived by similar late amination processes. In transamination reaction, the amino group origi- most of the examples studied, the nitrogen atom nating from L-alanine. Subsequent transformations is donated from an amino acid source through a are imine formation giving the heterocyclic ring of transamination reaction with a suitable aldehyde γ-coniceine, and then reduction to coniine. Pini- or ketone (see page 20). dine (Figure 6.116) from Pinus species is found

acetate O + HO2C HO2C OHC malonate O O octanoic acid L-Ala (capric acid) transamination

Schiff base pyruvic acid formation NADPH

N N H2N O H γ (+)-coniine -coniceine

Figure 6.115

O transamination, Schiff base formation O O O

CO2H O O N N H O or reduction and dehydration steps O OH

HO2C O O N N H H pinidine

Figure 6.116 382 ALKALOIDS

Conium maculatum Conium maculatum (Umbelliferae/Apiaceae) or poison hemlock is a large biennial herb indigenous to Europe and naturalized in North and South America. As a common poisonous plant, recognition is important, and this plant can be differentiated from most other members of the Umbelliferae/Apiaceae by its smooth purple-spotted stem. The dried unripe fruits were formerly used as a pain reliever and sedative, but have no medicinal use now. The ancient Greeks are said to have executed condemned prisoners, including Socrates, using poison hemlock. The poison causes gradual muscular paralysis followed by convulsions and death from respiratory paralysis. All parts of the plant are poisonous due to the alkaloid content, though the highest concentration of alkaloids is found in the green fruit (up to 1.6%). The major alkaloid (about 90%) is the volatile liquid coniine (Figure 6.115), with smaller amounts of structurally related piperidine alkaloids, including N-methylconiine and γ-coniceine (Figure 6.115). In North America, the name hemlock refers to species of Tsuga (Pinaceae), a group of coniferous trees, which should not be confused with the poison hemlock. to have a rather similar origin in acetate, and most of alkaloids (see page 315), L-phenylalanine is likely a poly-β-keto acid. During the sequence out- less frequently utilized, and usually it contributes lined in Figure 6.116, the carboxyl group is lost. only carbon atoms, e.g. C6C3,C6C2,orC6C1 Note that an alternative folding of the poly-β-keto units, without providing a nitrogen atom from its acid and loss of carboxyl might be formulated. amino group (see colchicine, page 342, lobeline, page 308, etc). Ephedrine (Figure 6.117), the main alkaloid in species of Ephedra∗ (Ephedraceae) and Phenylalanine-derived Alkaloids a valuable nasal decongestant and bronchial dila- tor, is a prime example. Whilst ephedrine con- Whilst the aromatic amino acid L-tyrosine is tains the same carbon and nitrogen skeleton as a common and extremely important precursor seen in phenylalanine, and L-phenylalanine is a

nucleophilic attack on to ester with side-chain degradation concomitant decarboxylation via cinnamic acid OOO

CO2H SCoA HO – CO2

NH2 O O pyruvic L-Phe acid transamination OH OH S R SAM O NHMe NH2 (–)-ephedrine (–)-norephedrine

NH2 OH OH (–)-cathinone S SAM S

NHMe NH2 (+)-pseudoephedrine (+)-norpseudoephedrine (cathine)

Figure 6.117 ALKALOIDS DERIVED BY AMINATION REACTIONS 383

nucleophilic attack of O reverse aldol-type enamine on to ester O O H reaction O H C OH Ph SCoA O 3 H3C H3C Ph CO2 C C pyruvic acid Ph R1 R1 R1 thiamine PP N S N S N S TPP anion (TPP) see Figure 2.15 regenerated R2 R2 R2 TPP/pyruvate-derived enamine

Figure 6.118

precursor, only seven carbons, a C6C1 fragment, from the more active cathinone, the corresponding are actually incorporated. It is found that pheny- carbonyl derivative. These natural compounds lalanine is metabolized, probably through cin- are structurally similar to the synthetic amfe- namic acid to benzoic acid (see page 141), and tamine/dexamfetamine; (amphetamine/dexamphe- this, perhaps as its coenzyme A ester, is acylated tamine) (Figure 6.119) and have similar properties. with pyruvate, decarboxylation occurring during the addition (Figure 6.117). The use of pyru- vate as a nucleophilic reagent in this way is OH O unusual in secondary metabolism, but occurs in primary metabolism during isoleucine and valine biosynthesis. A thiamine PP-mediated mechanism NH2 NH2 is suggested (Figure 6.118; compare decarboxy- (+)-norpseudoephedrine (–)-cathinone lation of pyruvate, page 21, and formation of (cathine) deoxyxylulose phosphate, page 170). This process yields the diketone, and a transamination reac- tion would then give cathinone (Figure 6.115). NH2 Reduction of the carbonyl group from either (+)-amfetamine (amphetamine) face provides the diastereomeric norephedrine or norpseudoephedrine (cathine). Finally, N- O O methylation would provide ephedrine or pseu- O NH2 NHMe doephedrine (Figure 6.117). Typically, all four of O the latter compounds can be found in Ephedra OMe species, the proportions varying according to MMDA MDMA (3-methoxy- (3,4-methylenedioxy- species. Norpseudoephedrine is also a major con- ∗ 4,5-methylenedioxy- methamfetamine; Ecstasy) stituent of the leaves of khat (Catha edulis; Celas- amfetamine) traceae), chewed in African and Arab countries as a stimulant. Most of the CNS stimulant action comes Figure 6.119

Ephedra

Ephedra or Ma Huang is one of the oldest known drugs, having being used by the Chinese for at least 5000 years. It consists of the entire plant or tops of various Ephedra species (Ephedraceae), including E. sinica and E. equisetina from China, and E. geriardiana, E. intermedia and E. major from India and Pakistan. The plants are small bushes with slender aerial stems and minute leaves, giving the appearance of being effectively leafless. The plants typically contain 0.5–2.0% of alkaloids, according to species, and from 30–90%

(Continues) 384 ALKALOIDS

(Continued)

of the total alkaloids is (−)-ephedrine (Figure 6.117). Related structures, including the diastereoisomeric (+)-pseudoephedrine and the demethyl analogues (−)-norephedrine and (+)-norpseudoephedrine (Figure 6.117) are also present. In E. intermedia, the proportion of pseudoephedrine exceeds that of ephedrine. Ephedrine is an indirectly acting sympathomimetic amine with effects similar to nor- adrenaline (see page 317). Lacking the phenolic groups of the catecholamines, it has only weak action on adrenoreceptors, but it is able to displace noradrenaline from storage vesicles in the nerve terminals, which can then act on receptors. It is orally active and has a longer duration of action than noradrenaline. It also has bronchodilator activity, giving relief in asthma, plus a vasoconstrictor action on mucous membranes, making it an effective nasal decongestant. Pseudoephedrine is also widely used in compound cough and cold preparations and as a decongestant. The ephedrine and pseudoephedrine used medicinally are usually synthetic. One commercial synthesis of ephedrine involves a fermentation reaction on benzaldehyde using brewer’s yeast (Saccharomyces sp.), giving initially an alcohol, then reductive condensation with methylamine yields (−)-ephedrine with very high enantioselectivity (Figure 6.120). The fermentation reaction is similar to that shown in Figure 6.118, in that an activated acetaldehyde bound to TPP is produced by the yeast by decarboxylation of pyruvate, and this unit is added stereospecifically to benzaldehyde in an aldol-like reaction. The herbal drug ephedra/Ma Huang is currently being traded as ‘herbal ecstasy’. Consumption gives CNS stimulation, but in high amounts can lead to hallucinations, paranoia, and psychosis.

OH OH CHO CH3COCO2H (i) MeNH2

yeast O (ii) H2/Pt NHMe (–)-ephedrine

Figure 6.120

Khat Khat, or Abyssinian tea, consists of the fresh leaves of Catha edulis (Celastraceae), a small tree cultivated in Ethiopia, East and South Africa, and the Yemen. The leaves are widely employed in African and Arabian countries, where they are chewed for a stimulant effect. This traditional use alleviates hunger and fatigue, but also gives a sensation of general well-being (compare coca, page 302). Users become cheerful and talkative, and khat has become a social drug. Prolonged usage can lead to hypertension, insomnia, or even mania. Khat consumption may lead to psychological dependence, but not normally physical dependence. There is presently little usage outside of Africa and Arabia, although this is increasing due to immigration from these areas. However, for maximum effects, the leaves must be fresh, and this somewhat restricts international trade. Dried leaves contain up to 1% cathine ((+)-norpseudoephedrine (Figure 6.119)), but young fresh leaves contain (−)-cathinone (Figure 6.119) as the principal CNS stimulant. Cathinone has similar pharmacological properties as the synthetic CNS stimulant (+)-amfetamine/dexamfetamine (amphetamine/dexamphetamine) (Figure 6.119), with a similar potency. Both compounds act by inducing release of catecholamines.

(Continues) ALKALOIDS DERIVED BY AMINATION REACTIONS 385

(Continued)

Medicinal use of amfetamine has declined markedly as drug dependence and the severe depression generated on withdrawal have been appreciated. Nevertheless, amfetamine abuse is significant. Amfetamines are taken orally, sniffed, or injected to give a long period of CNS stimulation (hours to days). Users often then take a depressant drug (alcohol, barbiturates, opioids) to terminate the effects. Users rapidly become dependent and develop tolerance. The consumption of khat is not yet restricted in the UK, even though both cathine and cathinone are now controlled drugs. It remains to be seen whether khat will be reclassified and its use restricted in any way. Other amfetamine-like derivatives of note are methoxymethylenedioxyamfetamine (MMDA) and methylenedioxymethamfetamine (MDMA) (Figure 6.119). MMDA is thought to be formed in the body after ingestion of nutmeg (Myristica fragrans; Myristicaceae), by an amination process on myristicin (see page 138), and it may be the agent responsible for the euphoric and hallucinogenic effects of nutmeg. MDMA is the illicit drug Ecstasy, a synthetic amfetamine-like stimulant popular among young people. The use of Ecstasy has resulted in a number of deaths, brought about by subsequent heatstroke and dehydration.

NH2 O

HO2C CoAS

L-Val isobutyryl-CoA L-Phe malonyl-CoA

transamination O

MeO CO2H MeO CHO MeO NH2 CoAS

HO HO HO ferulic acid vanillin vanillylamine

O MeO N H HO capsaicin

Figure 6.121

The amide capsaicin (Figure 6.121) constitutes transamination to give vanillylamine. The acid por- the powerfully pungent principle in chilli peppers tion of the amide structure is of polyketide origin, (Capsicum annuum; Solanaceae). Apart from its with a branched-chain fatty acyl-CoA being pro- culinary importance, it is also used medicinally duced by chain extension of isobutyryl-CoA. This in creams to counter neuralgia caused by her- starter unit is valine derived (see page 197). pes infections and in other topical pain-relieving preparations. The initial burning effect of cap- Terpenoid Alkaloids saicin is found to affect the pain receptors, mak- ing them less sensitive. The aromatic portion of A variety of alkaloids based on mono-, sesqui-, di-, capsaicin is derived from phenylalanine through and tri-terpenoid skeletons have been characterized, ferulic acid and vanillin (Figure 6.121, compare but information about their formation in nature is page 141), this aldehyde being the substrate for still somewhat sparse. Monoterpene alkaloids are in 386 ALKALOIDS the main structurally related to iridoid materials (see seen with coniine (see page 381). Reduction and page 187), the oxygen heterocycle being replaced methylation would yield β-skytanthine,whereas by a nitrogen-containing ring. β-Skytanthine from further oxidation could provide the pyridine ring Skytanthus acutus (Apocynaceae) and actinidine of actinidine. Valerian root (Valeriana officinalis; from Actinidia polygama (Actinidiaceae) serve as Valerianaceae) (see page 190) is known to contain examples (Figure 6.122). The iridoid loganin, so alkaloids such as that shown in Figure 6.122, as well important in the biosynthesis of terpenoid indole as iridoid structures. Whilst this alkaloid may be the alkaloids (see page 350) and the ipecac alkaloids result of N-alkylation on actinidine, an alternative (see page 343), is not a precursor of these structures, pathway in which tyramine is condensed with the and a modified series of reactions starting from dialdehyde could be proposed. In the latter case, this geraniol is proposed (Figure 6.122). The formation alkaloid could be regarded as an alkaloid derived of the dialdehyde follows closely elaboration of its from tyrosine, rather than in this group of terpenoid stereoisomer in loganin biosynthesis (see page 189). alkaloids. This could then act as a substrate for amination Gentianine (Figure 6.123) is probably the most via an amino acid, followed by ring formation as common of the monoterpene alkaloids, but it is

H compare Figure 5.22 transamination OH H H CHO CHO CHO H H NH CHO 2 geraniol O iridodial stereoisomer Schiff base formation

H N H H 2 reduction SAM H H OH ? N NMe tyramine O β-skytanthine

?

N N

actinidine Valerian alkaloid OH

Figure 6.122

hydrolysis - may be enzymic (β-glucosidase) nucleophilic addition or chemical (NH3); tautomerism to reactions with ammonia conjugated system

OGlc OH OGlc – H O OHC NH3 NH3 2 OO OOONH ON O MeO2C O O O O OH O secologanin gentiopicroside gentianine

Figure 6.123 ALKALOIDS DERIVED BY AMINATION REACTIONS 387 sometimes formed as an artefact when a suitable napellus has had some medicinal uses, plants plant extract is treated with ammonia, the base of both genera owe their highly toxic nature commonly used during isolation of alkaloids. Thus, to diterpenoid alkaloids. Aconite in particular is gentiopicroside (see page 190) from Gentiana regarded as extremely toxic, due to the presence lutea (Gentianaceae) is known to react with ammo- of aconitine (Figure 6.124) and related C19 nia to give gentianine (Figure 6.123). Many other norditerpenoid alkaloids. Species of Delphinium iridoid structures are known to react with ammo- accumulate diterpenoid alkaloids such as atisine nia to produce alkaloid artefacts. In some plants, (Figure 6.124), which tend to be less toxic than gentianine can be found when no ammonia treat- aconitine. An appreciation of their structural ment has been involved, and one may speculate relationship to diterpenes, e.g. ent-kaurene (see that loganin and secologanin may be precursors. page 208), is given in Figure 6.125, though Perhaps the most important examples of little experimental evidence is available. It terpenoid alkaloids from a pharmacological point appears feasible that a pre-ent-kaurene carbocation of view are those found in aconite∗ or wolfsbane undergoes Wagner–Meerwein rearrangements, and (Aconitum species; Ranunculaceae) and species that the atisine skeleton is then produced by of Delphinium (Ranunculaceae). Whilst Aconitum incorporating an N−CH2CH2−O fragment (e.g.

O H OH OH ≡ N H N H H O atisine OH OMe MeO H O HO OMe OCOPh H O ≡ MeO OH N OH O OAc OMe HO N H H OMe O HO H MeO MeO H aconitine

Figure 6.124

Aconite Aconites, commonly called wolfsbane or monkshood, are species of Aconitum (Ranuncu- laceae), valued ornamental herbaceous plants, grown for their showy blue or purple flowers, which are shaped like a monk’s cowl. Their alkaloid content, mainly in the roots, makes them some of the most toxic plants commonly encountered. The dried roots of Aconitum napellus were once used, mainly externally for relief of pain, e.g. in rheumatism. The toxic alkaloids (0.3–1.5%) are complex diterpene-derived esters. Aconitine (Figure 6.124) is the principal component (about 30%) and is a diester of aconine with acetic and benzoic acids. Hydrolysis products benzoylaconine and aconine are also present in dried plant material. These alkaloids appear to behave as neurotoxins by acting on sodium channels. All species of Aconitum and Delphinium are potentially toxic to man and animals and must be treated with caution. 388 ALKALOIDS

W–M 1,2-alkyl see Figure 5.47 shift H GGPP H H H

H H H

≡ ent-kaurene H W–M H W–M 1,3-hydride shift 1,2-alkyl shift OH

NH2 H H H atisine

H H H incorporation of additional 2-aminoethanol unit ♦ + ∇ ∇ ∗ ♦ N ∗ O N +

atisine-type aconitine-type

Figure 6.125 from 2-aminoethanol) to form the heterocyclic (Lycopersicon esculente) are typical examples of rings. The aconitine akeleton is probably formed such glycosides. from the atisine skeleton by further modifications As with the sapogenins, this group of steroidal as indicated. Note that a rearrangement process alkaloids is derived from cholesterol, with appro- converts two fused six-membered rings into a priate side-chain modifications during the sequence (7 + 5)-membered bicyclic system, and that one (Figure 6.127). Amination appears to employ carbon, that from the exocyclic double bond, L-arginine as the nitrogen source, probably via a is lost. substitution process on 26-hydroxycholesterol. A second substitution allows 26-amino-22-hydroxy- cholesterol to cyclize, generating a piperidine ring. Steroidal Alkaloids After 16β-hydroxylation, the secondary amine is Many plants in the Solanaceae accumulate steroidal oxidized to an imine, and the spiro-system can be envisaged as the result of a nucleophilic alkaloids based on a C27 cholestane skeleton, e.g. solasodine and tomatidine (Figure 6.126). These addition of the 16β-hydroxyl on to the imine are essentially nitrogen analogues of steroidal (or iminium via protonation). Whether the 22R saponins (see page 240) and have already been (as in solasodine)or22S (as in tomatidine) briefly considered along with these compounds. configuration is established may depend on this In contrast to the oxygen analogues, these com- reaction. pounds all have the same stereochemistry at A variant on the way the cholesterol side- C-25 (methyl always equatorial), but C-22 iso- chain is cyclized can be found in solanidine mers do exist, as solasodine and tomatidine exem- (Figure 6.126), which contains a condensed ring plify. They are usually present as glycosides which system with nitrogen at the bridgehead. Solanidine have surface activity and haemolytic properties is found in potatoes (Solanum tuberosum), typi- as do the saponins, but these compounds are cally as the glycoside α-solanine (Figure 6.126). also toxic if ingested. Solasonine from Solanum This condensed ring system appears to be produced species and tomatine (Figure 6.126) from tomato by a branch from the main pathway to solasodine/ ALKALOIDS DERIVED BY AMINATION REACTIONS 389

H 25 N H 22

D-Gal H O OH OH OH H β → HO O 1 3 O HH HO O O OH solasodine D-Glc O α1→2 O HO HO HO L-Rha

solasonine 25

22 D-Glc H NH D-Xyl OH O O HO HO β1→4 H O HO O O OH β1→3 H OH OH O HH O HO O HO H HO O OH tomatidine β → OH 1 2 D-Gal D-Glc

tomatine H H D-Gal H N OH OH OH H β → HO O 1 3 O HH HO O O OH O solanidine Glc Gal solanidine D-Glc α1→2 γ-solanine O HO β HO -solanine HO L-Rha α-solanine

Figure 6.126 tomatidine structures. Thus, a substitution process production. Similarly, other Solanum alkaloids∗ will allow generation of the new ring system such as solanidine with nitrogen in a condensed (Figure 6.128). ring system might also be exploited. Since the production of medicinal steroids from Several plants in the Liliaceae, notably the steroidal saponins (see page 266) requires prelimi- genus Veratrum (Liliaceae/Melanthiaceae), contain nary degradation to remove the ring systems con- a remarkable group of steroidal alkaloids in taining the original cholesterol side-chain, it is which a fundamental change to the basic steroid immaterial whether these rings contain oxygen or nucleus has taken place. This change expands nitrogen. Thus, plants rich in solasodine or tomati- ring D by one carbon at the expense of ring dine could also be employed for commercial steroid C, which consequently becomes five-membered. 390 ALKALOIDS

22 26 25

OH NH2 27 O L-Arg H

H H H H HO 26-hydroxycholesterol O cholesterol H H OH N N H H NH2 O OH nucleophilic substitution H H H O H nucleophilic addition on to H N imine; this reaction may N account for the different 22 H H stereochemistries at C-22 H O OH H H H H HO solasodine

Figure 6.127

H NH

H O nucleophilic attack of H hydroxyl on to imine HH HO H tomatidine H NH H OH H H N H nucleophilic displacement of hydroxyl by amine H H H HO solanidine

Figure 6.128

The resulting skeleton is termed a C-nor-D- (Figure 6.130) from Veratrum californicum, toxic homosteroid in keeping with these alterations in components in this plant that are responsible ring size. Cholesterol is a precursor of this group of for severe teratogenic effects. Animals grazing alkaloids, and a mechanism accounting for the ring on V. californicum and some other species of modifications is shown in Figure 6.129, where the Veratrum frequently give birth to young with changes are initiated by loss of a suitable leaving cyclopia, a malformation characterized by a single group from C-12. Typical representatives of C- eye in the centre of the forehead. The teratogenic nor-D-homosteroids are jervine and cyclopamine effects of jervine, cyclopamine, and cyclopamine ALKALOIDS DERIVED BY AMINATION REACTIONS 391

Solanum Alkaloids Solasodine (Figure 6.126) is a major component in many Solanum species, where it is present as glycosides in the leaves, and especially in the unripe fruits. Trial cultivations of a number of species, including Solanum laciniatum and S. aviculare (indigenous to New Zealand), S. khasianum (from India), and S. marginatum (from Ecuador), have been conducted in various countries. Alkaloid levels of 1–2% have been obtained, and these plants are especially suitable for long term cultivation if the fruits provide suitable quantities, being significantly easier to cultivate than disogenin-producing Dioscorea species. Solasodine may be converted into progesterone by means of the Marker degradation shown in Figure 5.119 (see page 266).

loss of leaving group produces OH2 R R carbocation; W–M 1,2-alkyl 12 migration follows D C D C C-nor-D-homosteroids

HO HO

Figure 6.129

H H H H N N H N H H OH O O H H O O H H H H OH O H R O 9 H H H HO O 4 HO HO O OAc H jervine cyclopamine OH OH OAc R = H, protoveratrine A R = OH, protoveratrine B

Figure 6.130 glucoside (cycloposine) on the developing fetus two more six-membered rings on to the C-nor- have now been well established. Other Veratrum D-homosteroid skeleton. This hexacyclic system alkaloids, especially those found in V. album is extensively oxygenated, and a novel hemiketal and V. viride, have been employed medicinally linkage bridges C-9 with C-4. Both the jervine as hypotensive agents, and used in the same and protoverine skeletons are readily rationalized way as Rauwolfia alkaloids (see page 352), through additional cyclization reactions involving often in combination with Rauwolfia.These a piperidine ring, probably formed by processes medicinal alkaloids, e.g. protoveratrine A and analogous to those seen with the Solanum alkaloids protoveratrine B (Figure 6.130), which are esters (Figure 6.127). These are outlined in Figure 6.131, of protoverine, are characterized by fusion of which suggests the participation of the piperidine 392 ALKALOIDS

F N HN N H E cholesterol C D D D C C HO e.g. protoverine piperidine intermediate H

(see Figure 6.127) ≡ N H N F E O D O D C C

e.g. jervine

Figure 6.131

20 O O

H H

H H H H

HO H2N pregnenolone holaphyllamine Me

H2N N 18 H H H H H H H Me N H N 2 2 conessine

Figure 6.132

OSO3H

H spermidine H H H2N N N OH H H H squalamine

Figure 6.133 intermediate from Figure 6.127. Typically, both pregnane derivatives are important animal hor- types of alkaloid are found co-occurring in mones (see page 273) or intermediates on the Veratrum species. way to other natural steroidal derivatives, e.g. Many steroidal derivatives are formed by cardioactive glycosides (see page 241). Alkaloids truncation of the original C8 side-chain, and C21 based on a pregnane skeleton are found in plants, ALKALOIDS DERIVED BY AMINATION REACTIONS 393 particularly in the Apocynaceae and Buxaceae, and The novel steroidal polyamine squalamine pregnenolone (Figure 6.132) is usually involved (Figure 6.133) has been isolated in very small in their production. Holaphyllamine from Holar- amounts (about 0.001%) from the liver of rhena floribunda (Apocynaceae) is obtained from the dogfish shark (Squalus acanthias), and is pregnenolone by replacement of the 3-hydroxyl with an amino group (Figure 6.132). Conessine CO2 (Figure 6.132) from Holarrhena antidysenterica is Gly also derived from pregnenolone, and requires two amination reactions, one at C-3 as for holaphyl- 6 7 5 L-Asp 1 N N lamine, plus a further one, originally at C-20, prob- 8 formate ably via the C-20 alcohol. The new ring system in 2 N 4 N 9 conessine is then the result of attack of the C-20 formate 3 amine on to the C-18 methyl, suitably activated, H. antidysenterica of course. The bark of has long L-Gln L-Gln been used, especially in India, as a treatment for amoebic dysentery. Figure 6.134

adenosine monophosphate guanosine monophosphate N-methylation of (AMP) (GMP) purine; positive Ad charge transferred to glycosylated nitrogen Me S O O O Me R N N HN N HN HN N N N N O N O N PO H purine PO H SAM PO biosynthetic O O O pathway HO OH HO OH HO OH inosine monophosphate xanthosine monophosphate 7-methyl XMP (IMP) (XMP) hydrolysis of phosphate ester

O O O O Me H H H Me N N N N N SAM HN HN SAM HN

O N N O N N O N N O N N H HO H HO H Me O O theophylline xanthine OH (1,3-dimethylxanthine) 2 HO OH HO OH xanthosine 7-methylxanthosine

SN2 displacement of purine leaving group D-ribose O Me O Me O Me Me N N SAM HN N SAM HN N

O N N O N N O N N H Me Me caffeine theobromine 7-methylxanthine (1,3,7-trimethylxanthine) (3,7-dimethylxanthine)

Figure 6.135 394 ALKALOIDS attracting attention because of its remarkable much more important as a drug compound because antimicrobial activity. This compound is a broad- of its muscle relaxant properties, utilized in the spectrum agent effective at very low concentrations relief of bronchial asthma. Theobromine is a against Gram-positive and Gram-negative bacteria, major constituent of cocoa∗, and related chocolate and also fungi, protozoa, and viruses including products. HIV. The sulphated side-chain helps to make The purine ring is gradually elaborated by squalamine water soluble. The polyamine portion piecing together small components from primary is spermidine, a compound widely distributed metabolism (Figure 6.134). The largest component in both animals and plants. Related aminosterol incorporated is glycine, which provides a C2N derivatives with similar high antimicrobial activity unit, whilst the remaining carbon atoms come from have also been isolated from the liver extracts. formate (by way of N 10-formyl-tetrahydrofolate (see page 126)) and bicarbonate. Two of the four PURINE ALKALOIDS nitrogen atoms are supplied by glutamine, and a third by aspartic acid. Synthesis of the nucleotides

The purine derivatives caffeine∗, theobromine∗, adenosine 5 -monophosphate (AMP) and guano- ∗ sine 5 -monophosphate (GMP) is by way of ino- and theophylline (Figure 6.135) are usually referred to as purine alkaloids. As alkaloids they sine 5 -monophosphate (IMP) and xanthosine 5 - have a limited distribution, but their origins are monophosphate (XMP) (Figure 6.135), and the very closely linked with those of the purine bases purine alkaloids then branch away through XMP. adenine and guanine, fundamental components of Methylation, then loss of phosphate, generates nucleosides, nucleotides, and the nucleic acids. the nucleoside 7-methylxanthosine, which is then Caffeine, in the form of beverages such as tea∗, released from the sugar. Successive methylations coffee∗,andcola∗ is one of the most widely on the nitrogens give caffeine by way of theo- consumed and socially accepted natural stimulants. bromine, whilst a different methylation sequence It is also used medicinally, but theophylline is can account for the formation of theophylline.

Caffeine, Theobromine, and Theophylline

The purine alkaloids caffeine, theobromine, and theophylline (Figure 6.135) are all methyl derivatives of xanthine and they commonly co-occur in a particular plant. The major sources of these compounds are the beverage materials such as tea, coffee, cocoa, and cola, which owe their stimulant properties to these water-soluble alkaloids. They competitively inhibit phosphodiesterase, resulting in an increase in cyclic AMP and subsequent release of adrenaline. This leads to a stimulation of the CNS, a relaxation of bronchial smooth muscle, and induction of diuresis, as major effects. These effects vary in the three compounds. Caffeine is the best CNS stimulant, and has weak diuretic action. Theobromine has little stimulant action, but has more diuretic activity and also muscle relaxant properties. Theophylline also has low stimulant action and is an effective diuretic, but it relaxes smooth muscle better than caffeine or theobromine. Caffeine is used medicinally as a CNS stimulant, usually combined with another therapeutic agent, as in compound analgesic preparations. Theobromine is of value as a diuretic and smooth muscle relaxant, but is not now routinely used. Theophylline is an important smooth muscle relaxant for relief of bronchospasm, and is frequently dispensed in slow- release formulations to reduce side-effects. It is also available as aminophylline (a more soluble preparation containing theophylline with ethylenediamine) and choline theophyllinate (theophylline and choline). The alkaloids may be isolated from natural sources, or obtained by total or partial synthesis.

(Continues) PURINE ALKALOIDS 395

(Continued)

It has been estimated that beverage consumption may provide the following amounts of caffeine per cup or average measure: coffee, 30–150 mg (average 60–80 mg); instant coffee, 20–100 mg (average 40–60 mg); decaffeinated coffee, 2–4 mg; tea, 10–100 mg (average 40 mg); cocoa, 2–50 mg (average 5 mg); cola drink, 25–60 mg. The maximal daily intake should not exceed about 1 g to avoid unpleasant side-effects, e.g. headaches, restlessness. An acute lethal dose is about 5–10 g. The biological effects produced from the caffeine ingested via the different drinks can vary, since its bioavailability is known to be modified by the other constituents present, especially the amount and nature of polyphenolic tannins.

Coffee Coffee consists of the dried ripe seed of Coffea arabica, C. canephora, C. liberica,orother Coffea species (Rubiaceae). The plants are small evergreen trees, widely cultivated in various parts of the world, e.g. Brazil and other South American countries, and Kenya. The fruit is deprived of its seed coat, then dried and roasted to develop its characteristic colour, odour, and taste. Coffee seeds contain 1–2% of caffeine and traces of theophylline and theobromine. These are mainly combined in the green seed with chlorogenic acid (see page 132) (5–7%), and roasting releases them and also causes some decomposition of chlorogenic acid to quinic acid and caffeic acid. The nicotinic acid derivative trigonelline is present in green seeds to the extent of about 0.25–1%; during roasting, this is extensively converted into nicotinic acid (vitamin B3, see page 313). Volatile oils and tannins provide odour and flavour. A proportion of the caffeine may sublime off during the roasting process, providing some commercial caffeine. Decaffeinated coffee, containing up to 0.08% caffeine, is obtained by removing caffeine, usually by aqueous percolation prior to roasting. This process provides another source of caffeine.

Tea Tea is the prepared leaves and leaf buds of Camellia sinensis (Thea sinensis) (Theaceae), an evergreen shrub cultivated in China, India, Japan, and Sri Lanka. For black tea, the leaves are allowed to ferment, allowing enzymic oxidation of the polyphenols, whilst green tea is produced by steaming and drying the leaves to prevent oxidation. During oxidation, colourless catechins (up to 40% in dried leaf) (see page 150) are converted into intensely coloured theaflavins and thearubigins. Oolong tea is semi-fermented. Tea contains 1–4% caffeine, and small amounts (up to 0.05%) of both theophylline and theobromine. Astringency and flavour come from tannins and volatile oils, the latter containing monoterpene alcohols (geraniol, linalool) and aromatic alcohols (benzyl alcohol, 2-phenylethanol). Theaflavins (see page 151) are believed to act as radical scavengers/antioxidants, and to provide beneficial effects against cardiovascular disease, cancers, and the ageing process generally. Tea leaf dust and waste is a major source of caffeine.

Cola Cola, or kola, is the dried cotyledon from seeds of various species of Cola (Sterculiaceae), e.g. C. nitida and C. acuminata, trees cultivated principally in West Africa and the West Indies. Seeds are prepared by splitting them open and drying. Cola seeds contain up to 3% caffeine

(Continues) 396 ALKALOIDS

(Continued)

and about 0.1% theobromine, partly bound to tannin materials. Drying allows some oxidation of polyphenols, formation of a red pigment, and liberation of free caffeine. Fresh cola seeds are chewed in tropical countries as a stimulant, and vast quantities of dried seeds are processed for the preparation of cola drinks, e.g. Coca-Cola and Pepsi-Cola.

Cocoa Although cocoa as a drink is now rather unfashionable, it provides the raw material for the manufacture of chocolate and is commercially very important. Cocoa (or cacao) is derived from the roasted seeds of Theobroma cacao (Sterculiaceae), a tree widely cultivated in South America and West Africa. The fruits develop on the trunk of the tree, and the seeds from them are separated, allowed to ferment, and are then roasted to develop the characteristic chocolate flavour. The kernels are then separated from the husks, ground up, and processed in various ways to give chocolate, cocoa, and cocoa butter. Cocoa seeds contain 35–50% of oil (cocoa butter or theobroma oil), 1–4% theobromine and 0.2–0.5% caffeine, plus tannins and volatile oils. During fermentation and roasting, most of the theobromine from the kernel passes into the husk, which thus provides a convenient source of the alkaloid. Theobroma oil or cocoa butter is obtained by hot expression from the ground seeds as a whitish solid with a mild chocolate taste. It is a valuable formulation aid in pharmacy where it is used as a suppository base. It contains glycerides of oleic (35%), stearic (35%), palmitic (26%), and linoleic (3%) acids (see page 44).

Mate´ Tea Mate´ tea is consumed in South America as a stimulant drink. Mate´ or Paraguay tea consists of the leaves of Ilex paraguensis (Aquifoliaceae), South American shrubs of the holly genus. The dried leaf contains 0.8–1.7% caffeine and smaller amounts of theobromine (0.3–0.9%) with little or no theophylline. Considerable amounts (10–16%) of chlorogenic acid (see page 132) are also present.

Guarana The seeds of the Brazilian plant Paullinia cupana (Sapindaceae) are used to make a stimulant drink. Crushed seeds are mixed with water to a paste, which is then sun dried. Portions of this are then boiled with hot water to provide a refreshing drink. The principal constituent, previously called guaranine, has been shown to be identical to caffeine, and the seeds may contain 3–5%. Small amounts of theophylline (0–0.25%) and theobromine (0.02–0.06%) are also present. Guarana is widely available as tablets and capsules, or as extracts, in health food shops where it is promoted to relieve mental and physical fatigue. Labels on such products frequently show the active constituent to be guaranine, but may not indicate that this is actually caffeine.

Saxitoxin and Tetrodotoxin described above. Not all features of its biosyn- thetic origin have been established, but the The structure of saxitoxin∗ (Figure 6.136) con- amino acid supplying most of the ring sys- tains a reduced purine ring system, but it is not tem is known to be L-arginine (Figure 6.136). biosynthetically related to the purine alkaloids Acetate and a C1 unit from methionine are PURINE ALKALOIDS 397

Met H N O OH 2 H H NH 2 O CO2H O N HN NH NH2 H N NH 2 2 NH H2N N OH L-Arg OH saxitoxin OH H2N CO2H HO H N O NH 2 H H N O 2 N NH2 OH N L-Arg H HO OH OPP IPP tetrodotoxin OH

Figure 6.136

also utilized. Saxitoxin contains two highly polar marine neurotoxin containing a polar guanidino guanidino functions, one of which is provided group. It has been established that the remain- by arginine, and is a fast acting neurotoxin der of the carbon skeleton in tetrodotoxin is a inhibiting nerve conduction by blocking sodium C5 isoprene unit, probably supplied as isopen- channels. It is one of a group of marine toxins tenyl diphosphate (Figure 6.136). Tetrodotoxin is referred to as paralytic shellfish poisons (PSP) well known as the toxic principle in the puffer found in a range of shellfish, but ultimately fish (Tetraodon species), regarded as delicacy derived from toxic strains of dinoflagellates con- in Japanese cuisine. As potent sodium chan- sumed by the shellfish. Arginine is also a pre- nel blockers, both saxitoxin and tetrodotoxin are cursor for tetrodotoxin∗ (Figure 6.136), another valuable pharmacological tools.

Saxitoxin Saxitoxin (Figure 6.136) was first isolated from the Alaskan butter clam (Saxidomus giganteus) but has since been found in many species of shellfish, especially bivalves such as mussels, scallops, and oysters. These filter feeders consume dinoflagellates (plankton) and can accumulate toxins synthesized by these organisms, particularly during outbreaks known as red tides, when conditions favour formation of huge blooms of the dinoflagellates (see also brevetoxin A, page 109). Species of the dinoflagellate Gonyaulax in marine locations, or the cyanobacterium Aphanizomenon in freshwater, have been identified among the causative organisms, and the problem is encountered widely in temperate and tropical areas (including Europe, North America, and Japan). Commercial production of shellfish is routinely monitored for toxicity, which will slowly diminish as conditions change and the causative organism disappears from the water. About a dozen natural saxitoxin-related structures have been characterized, and mixtures in various proportions are typically synthesized by a producer, with the possibility that the shellfish may also structurally modify the toxins further. Acute and often fatal poisonings caused by the consumption of contaminated shellfish are termed paralytic shellfish poisoning (PSP), which involves paralysis of the neuromuscular system, death resulting from respiratory failure. Saxitoxin is a cationic molecule, which binds to sodium channels, blocking the influx of sodium ions through excitable nerve membranes,

(Continues) 398 ALKALOIDS

(Continued)

and is a valuable pharmacological tool for the study of this process. Saxitoxin and tetrodotoxin (below) are some of the most potent non-protein neurotoxins known, and are active at very low concentrations (µgkg−1).

Tetrodotoxin Tetrodotoxin (Figure 6.136) is traditionally associated with the puffer fish, Tetraodon species, a fish known as fugu, a highly prized delicacy eaten in Japan. Preparation of fugu is a skilled operation in which organs containing the highest levels of toxin, e.g. liver, ovaries, and testes, are carefully separated from the flesh. Even so, deaths from fugu poisoning are not uncommon, and the element of risk presumably heightens culinary appreciation of the fish. As with saxitoxin, tetrodotoxin appears to be produced by microorganisms, and symbiotic marine bacteria, e.g. Vibrio species, have been implicated as the synthesizers. In addition to fugu, several other species of fish, newts, and frogs have been found to accumulate tetrodotoxin or related structures. The mode of action of tetrodotoxin is exactly the same as that of saxitoxin above, though there are some subtle differences in the mechanism of binding.

FURTHER READING Griffin WJ and Lin GD (2000) Chemotaxonomy and geographical distribution of tropane alkaloids. Phy- Biosynthesis, General tochemistry 53, 623–637. Gritsanapan W and Griffin WJ (1992) Alkaloids and Dalton DR (1991) Alkaloids. Kirk–Othmer Encyclope- metabolism in a Duboisia hybrid. Phytochemistry 31, dia of Chemical Technology, 4th edn, Vol 1. Wiley, 471–477. New York, pp 1039–1087. Lounasmaa M and Tamminen T (1993) The tropane Herbert RB (2001) The biosynthesis of plant alkaloids alkaloids. The Alkaloids, Chemistry and Pharmacol- and nitrogenous microbial metabolites. Nat Prod Rep ogy (ed Cordell GA) Vol 44. Academic, San Diego, 18,50–65. Earlier reviews: 1999, 16, 199–208; 1–114. 1997, 14, 359–372. Molyneux RJ, Nash RJ and Asano N (1996) Chemistry Kutchan TM (1995) Alkaloid biosynthesis – the basis and biological activity of the calystegines and related for metabolic engineering of medicinal plants. Plant nortropane alkaloids. Alkaloids, Chemical and Bio- Cell 7, 1059–1070. logical Perspectives (ed Pelletier SW) Vol 11. Else- Kutchan TM (1998) Molecular genetics of plant alkaloid vier, Amsterdam, pp 303–343. biosynthesis. The Alkaloids, Chemistry and Phar- O’Hagan D (2000) Pyrrole, pyrrolidine, pyridine, piperi- macology (ed Cordell GA) Vol 50. Academic, San dine and tropane alkaloids. Nat Prod Rep 17, Diego, pp 257–316. 435–446. Earlier review: 1997, 14, 637–651. Misra N, Luthra R, Singh KL and Kumar S (1999) O’Hagan D and Robins RJ (1998) Tropic acid ester Recent advances in biosynthesis of alkaloids. Com- biosynthesis in Datura stramonium and related prehensive Natural Products Chemistry, Vol 4. Else- species. Chem Soc Rev 27, 207–212. vier, Amsterdam, pp 25–59.

Cocaine Tropane Alkaloids Johnson EL and Emcho SD (1994) Variation in alkaloid Bernstein PR (1992) Antiasthmatic agents. Kirk–Othmer content in Erythroxylum coca leaves from leaf bud Encyclopedia of Chemical Technology, 4th edn, Vol 2. to leaf drop. Ann Bot 73, 645–650. Wiley, New York, pp 830–854. Lenz GR, Schoepke HG and Spaulding TC (1992) Anes- Evans WC (1990) Datura, a commercial source of thetics. Kirk–Othmer Encyclopedia of Chemical Tech- hyoscine. Pharm J 244, 651–653. nology, 4th edn, Vol 2. Wiley, New York, 778–800. FURTHER READING 399

Singh S (2000) Chemistry, design, and structure–activity Takahata H and Momose T (1993) Simple indolizidine relationship of cocaine antagonists. Chem Rev 100, alkaloids. The Alkaloids, Chemistry and Pharmacol- 925–1024. ogy (ed Cordell GA) Vol 44. Academic, San Diego, Woolverton WL and Johnson KM (1992) Neurobiology pp 189–256. of cocaine abuse. Trends in Pharmacological Sci- Ohmiya S, Saito K and Murakoshi I (1995) Lupine ences 13, 193–200. alkaloids. The Alkaloids, Chemistry and Pharmacol- ogy (ed Cordell GA) Vol 47. Academic, San Diego, pp 1–114. Epibatidine Robins DJ (1995) Biosynthesis of pyrrolizidine and quinolizidine alkaloids. The Alkaloids, Chemistry and Daly JW, Garaffo HM, Spande TF, Decker MW, Sulli- Pharmacology (ed Cordell GA) Vol 46. Academic, van JP and Williams M (2000) Alkaloids from frog San Diego, pp 1–61. skin: the discovery of epibatidine and the potential for developing novel non-opioid analgesics. Nat Prod Rep 17, 131–135. Castanospermine Szantay C, Kardos-Balogh Z and Szantay C Jr (1995) Epibatidine. The Alkaloids, Chemistry and Phar- Burgess K and Henderson I (1992) Synthetic approa- macology (ed Cordell GA) Vol 46. Academic, San ches to stereoisomers and analogues of castanosper- Diego, pp 95–125. mine. Tetrahedron 48, 4045–4066. Nash RJ, Watson AA and Asano N (1996) Polyhy- droxylated alkaloids that inhibit glycosidases. Alka- Pyrrolizidine Alkaloids loids, Chemical and Biological Perspectives (ed Pelletier SW) Vol 11. Elsevier, Amsterdam, Hartmann T (1999) Chemical ecology of pyrrolizidine pp 345–376. alkaloids. Planta 207, 483–495. Hartmann T and Witte L (1995) Chemistry, biology and chemoecology of the pyrrolizidine alkaloids. Alkaloids, Chemical and Biological Perspectives (ed Piperidine Alkaloids Pelletier SW) Vol 9. Wiley, New York, 155–233. Liddell JR (2000) Pyrrolizidine alkaloids. Nat Prod Rep O’Hagan D (2000) Pyrrole, pyrrolidine, pyridine, piperi- 17, 455–462. Earlier reviews: 1999, 16, 499–507; dine and tropane alkaloids. Nat Prod Rep 17, 1998, 15, 363–370. 435–446. Earlier reviews: 1997, 14, 637–651; Plun- Robins DJ (1995) Biosynthesis of pyrrolizidine and kett AO (1994) 11, 581–590. quinolizidine alkaloids. The Alkaloids, Chemistry and Schneider MJ (1996) Pyridine and piperidine alkaloids: Pharmacology (ed Cordell GA) Vol 46. Academic, an update. Alkaloids, Chemical and Biological Per- San Diego, pp 1–61. spectives (ed Pelletier SW) Vol 10. Elsevier, Ams- terdam, pp 155–299.

Piperidine Alkaloids Vitamin B3 O’Hagan D (2000) Pyrrole, pyrrolidine, pyridine, piperi- dine and tropane alkaloids. Nat Prod Rep 17, Van Arnum SD (1998) Vitamins (niacin, nicotinamide, 435–446. Earlier reviews: 1997, 14, 637–651; Plun- and nicotinic acid). Kirk–Othmer Encyclopedia of kett AO (1994) 11, 581–590. Chemical Technology, 4th edn, Vol 25. Wiley, New Schneider MJ (1996) Pyridine and piperidine alkaloids: York, pp 83 –99. an update. Alkaloids, Chemical and Biological Per- spectives (ed Pelletier SW) Vol 10. Elsevier, Ams- terdam, pp 155–299. Tobacco Baker R and Proctor C (2001) Where there’s smoke. Indolizidine and Quinolizidine Chem Brit 37(1), 38–41. Alkaloids Botting NP (1995) Chemistry and biochemistry of the kynurenine pathway of tryptophan metabolism. Michael JP (2000) Indolizidine and quinolizidine alka- Chem Soc Rev 24, 401–412. loids. Nat Prod Rep 17, 579–602. Earlier reviews: Hucko F, Tsetlin VI and Machold (1996) The emerg- 1999, 16, 675–696; 1998, 15, 571–594. ing three-dimensional structure of a receptor. The 400 ALKALOIDS

nicotinic acetylcholine receptor. Eur J Biochem 239, Herbert RB, Venter H and Pos S (2000) Do mam- 539–557. mals make their own morphine? Nat Prod Rep 17, Nathan A (1998) Smoking cessation products. Pharm J 317–322. 260, 340–343. Lenz GR, Schoepke HG and Spaulding TC (1992) Wills S (1994) Drugs and substance misuse: tobacco and Anesthetics. Kirk–Othmer Encyclopedia of Chem- alcohol. Pharm J 253, 158–160. ical Technology, 4th edn, Vol 2. Wiley, New York, pp 778–800. Nugent RA and Hall CM (1992) Analgesic, antipyretics, Catecholamines and antiinflammatory agents. Kirk–Othmer Encyclo- pedia of Chemical Technology, 4th edn, Vol 2. Wiley, Bernstein PR (1992) Antiasthmatic agents. Kirk–Othmer New York, pp 729–748. Encyclopedia of Chemical Technology, 4th edn, Schmidhammer H (1998) Opioid receptor antagonists. Vol 2. Wiley, New York, 830–854. Prog Med Chem 35,83–132. Cervoni P, Crandall DL and Chan PS (1993) Cardiovas- Szantay C, Dornyei G and Blasko G (1994) The mor- cular agents – antiarrhythmic agents. Kirk–Othmer phine alkaloids. The Alkaloids, Chemistry and Phar- Encyclopedia of Chemical Technology, 4th edn, macology (ed Cordell GA) Vol 45. Academic, San Vol 5. Wiley, New York, pp 207–238. Diego, pp 127–232. Cervoni P, Crandall DL and Chan PS (1993) Cardiovas- cular agents – antihypertensive agents. Kirk–Othmer Encyclopedia of Chemical Technology, 4th edn, Vol 5. Colchicine Wiley, New York, pp 262–289. Pugsley TA (1994) Epinephrine and norepinephrine. Boye O and Brossi A (1992) Tropolonic Colchicum Kirk–Othmer Encyclopedia of Chemical Technology, alkaloids and allo congeners. The Alkaloids, Chem- 4th edn, Vol 9. Wiley, New York, pp 715–730. istry and Pharmacology (ed Brossi A and Cordell GA) Vol 41. Academic, San Diego, pp 125–176.

Isoquinoline Alkaloids Ipecacuanha Bentley KW (2000) β-Phenylethylamines and the iso- quinoline alkaloids. Nat Prod Rep 17, 247–268. Ear- Fujii T and Ohba M (1998) The ipecac alkaloids and lier reviews: 1999, 16, 367–388; 1998, 15, 341–362. related bases. The Alkaloids, Chemistry and Biol- ogy (ed Cordell GA) Vol 51. Academic, San Diego, pp 271–321. Curare Klayman DL (1992) Antiparasitic agents (antipro- tozoals). Kirk–Othmer Encyclopedia of Chemical Bisset NG (1992) War and hunting poisons of the new Technology, 4th edn, Vol 3. Wiley, New York, World. Part 1. Notes on the early history of curare. pp 489–526. J Ethnopharmacol 36,1–26. Paul DB and Dobberstein RH (1994) Expectorants, anti- Bisset NG (1992) Curare. Alkaloids, Chemical and Bio- tussives, and related agents. Kirk–Othmer Encyclo- logical Perspectives (ed Pelletier SW) Vol 8. Wiley, pedia of Chemical Technology, 4th edn, Vol 9. Wiley, New York, pp 1–150. New York, pp 1061–1081. Menachery MD (1996) The alkaloids of South American Menispermaceae. Alkaloids, Chemical and Biological Perspectives (ed Pelletier SW) Vol 11. Amaryllidaceae Alkaloids Elsevier, Amsterdam, pp 269–302. Hoshino O (1998) The Amaryllidaceae alkaloids. The Alkaloids, Chemistry and Biology (ed Cordell GA) Opium Alkaloids Vol 51. Academic, San Diego, pp 324–424. Lewis JR (2001) Amaryllidaceae, Sceletium, imidazole, Brownstein MJ (1993) A brief history of opiates, opioid oxazole, thiazole, peptide and miscellaneous alka- peptides, and opiate receptors. Proc Natl Acad Sci loids. Nat Prod Rep 18,95–128. Earlier reviews: USA 90, 5391–5393. 2000, 17,57–84; 1999,16, 389–416. Chiara GD and North RA (1992) Neurobiology of opi- Scott LJ and Goa KL (2000) Galantamine: a review of ate abuse. Trends Pharmacol Sci 13, 185–193. its use in Alzheimer’s disease. Drugs 60, 1095–1122. FURTHER READING 401

Indole Alkaloids Stockigt¨ J, Obitz P, Falkenhagen H, Lutterbach R and Endreß S (1995) Natural products and enzymes from Ihara M and Fukumoto K (1996) Recent progress in plant cell cultures. Plant Cell Tissue Organ Culture the chemistry of non-monoterpenoid indole alkaloids. 43,97–109. Nat Prod Rep 13, 241–261. Earlier review: 1995, 12, 277–301. Catharanthus Alkaloids Psilocybin Kutney JP (1990) Biosynthesis and synthesis of indole and bisindole alkaloids in plant cell cultures: a Antkowiak R and Antkowiak WZ (1991) Alkaloids personal overview. Nat Prod Rep 7,85–103. from mushrooms. The Alkaloids, Chemistry and Kutney JP (1993) Plant cell culture combined with Pharmacology (ed Brossi A) Vol 40. Academic, San chemistry: a powerful route to complex natural Diego, pp 189–340. products. Account Chem Res 26, 559–566. Wills S (1993) Drugs and substance misuse: plants. Noble RL (1990) The discovery of the Vinca Pharm J 251, 227–229. alkaloids – chemotherapeutic agents against cancer. Biochem Cell Biol 68, 1344–1351. Verpoorte R, van der Heijden R and Moreno PRH Terpenoid Indole Alkaloids (1997) Biosynthesis of terpenoid indole alkaloids in Catharanthus roseus cells. The Alkaloids, Chemistry Hibino S and Choshi T (2001) Simple indole alkaloids and Pharmacology (ed Cordell GA) Vol 49. Aca- and those with a nonrearranged monoterpenoid unit. demic, San Diego, pp 221–299. Nat Prod Rep. 18,66–87. Earlier review: Lounas- maa M and Tolvanen A (2000) 17, 175–191. Kutchan TM (1993) Strictosidine: from alkaloid to Ibogaine enzyme to gene. Phytochemistry 32, 493–506. Popik P and Skolnick P (1999) Pharmacology of ibo- Leonard J (1999) Recent progress in the chemistry of gaine and ibogaine-related alkaloids. The Alkaloids, monoterpenoid alkaloids derived from secologanin. Chemistry and Biology (ed Cordell GA) Vol 52. Aca- Nat Prod Rep 16, 319–338. demic, San Diego, pp 197–231. Stockigt¨ J and Ruppert M (1999) Strictosidine – the biosynthetic key to monoterpenoid indole alkaloids. Comprehensive Natural Products Chemistry, Vol 4. Elsevier, Amsterdam, pp 109–138. Strychnine Toyota M and Ihara M (1998) Recent progress in the Bosch J, Bonjoch J and Amat M (1996) The Strychnos chemistry of non-monoterpenoid indole alkaloids. alkaloids. The Alkaloids, Chemistry and Pharmacol- Nat Prod Rep 15, 327–340. Earlier reviews: Ihara M ogy (ed Cordell GA) Vol 48. Academic, San Diego, 14 13 and Fukumoto K (1997) , 413–429; 1996, , pp 75–189. 241–261. Quetin-Leclercq J, Angenot L and Bisset NG (1990) Verpoorte R, van der Heiden R and Memelink J (1998) South American Strychnos species. Ethnobotany Plant biotechnology and the production of alka- (except curare) and alkaloid screening. J Ethnophar- loids: prospects of metabolic engineering. The Alka- macol 28,1–52. loids, Chemistry and Pharmacology (ed Cordell GA) Vol 50. Academic, San Diego, pp 453–508. Ellipticine

Rauwolfia Gribble GW (1990) Synthesis and antitumour activity of ellipticine alkaloids and related compounds. The Alka- Curzon G (1990) How reserpine and chlorpromazine loids, Chemistry and Pharmacology (ed Brossi A) act: the impact of key discoveries on the history Vol 39. Academic, San Diego, pp 239–352. of psychopharmacology. Trends Pharmacol Sci 11, 61–63. Stockigt¨ J (1995) Biosynthesis in Rauwolfia serpentina: Cinchona modern aspects of an old medicinal plant. The Alka- loids, Chemistry and Pharmacology (ed Cordell GA) Cervoni P, Crandall DL and Chan PS (1993) Cardiovas- Vol 47. Academic, San Diego, pp 115–172. cular agents – antiarrhythmic agents. Kirk–Othmer 402 ALKALOIDS

Encyclopedia of Chemical Technology, 4th edn, Quinoline and Acridine Alkaloids Vol 5. Wiley, New York, 207–238. Goodyer L (2000) Travel medicine (4): malaria. Pharm Michael JP (2000) Quinoline, quinazoline and acridone J 264, 405–410. alkaloids. Nat Prod Rep 17, 603–620. Earlier Klayman DL (1992) Antiparasitic agents (antipro- reviews: 1999, 16, 697–709; 1998, 15, 595–606. tozoals). Kirk–Othmer Encyclopedia of Chemical Tillequin F, Michel S and Skaltsounis A-L (1998) Technology, 4th edn, Vol 3. Wiley, New York, Acronycine-type alkaloids: chemistry and biology. pp 489–526. Alkaloids, Chemical and Biological Perspectives Lee M (1996) Malaria: in search of solutions. Chem Brit (ed Pelletier SW) Vol 12. Elsevier, Amsterdam, 32 (8), 28–30. pp 1–102.

Pilocarpine Camptothecin Maat L and Beyerman HC (1983) The imidazole alka- loids. The Alkaloids, Chemistry and Pharmacol- Cordell GA (1993) The discovery of plant anticancer ogy (ed Brossi A) Vol 22. Academic, San Diego, agents. Chem Ind 841–844. pp 282–331. Kawato Y and Terasawa H (1997) Recent advances in the medicinal chemistry and pharmacology of camptothecin. Prog Med Chem 34,69–109. Ephedra, Khat Wall ME and Wani MC (1998) History and future prospects of camptothecin and taxol. The Alka- Bernstein PR (1992) Antiasthmatic agents. Kirk–Othmer loids, Chemistry and Pharmacology (ed Cordell GA) Encyclopedia of Chemical Technology, 4th edn, Vol 50. Academic, San Diego, pp 509–536. Vol 2. Wiley, New York, pp 830–854. Crombie L, Crombie WML and Whiting DA (1990) Alkaloids of khat (Catha edulis). The Alkaloids, Chemistry and Pharmacology (ed Brossi A) Vol 39. Physostigmine Academic, San Diego, pp 139–164. Kalix P (1991) The pharmacology of psychoactive alka- Brossi A, Pei X-F and Greig NH (1996) Phenserine, loids from Ephedra and Catha. J Ethnopharmacol a novel anticholinesterase related to physostigmine: 32, 201–208. total synthesis and biological properties. Aust J Chem Wills S (1993) Drugs and substance misuse: plants. 49, 171–181. Pharm J 251, 227–229. Takano S and Ogasawara K (1989) Alkaloids of the calabar bean. The Alkaloids, Chemistry and Pharma- cology (ed Brossi A) Vol 36. Academic, San Diego, Capsaicin pp 225–251. Fusco BM and Giacovazzo M (1997) Peppers and pain: the promise of capsaicin. Drugs 53, 909–914.

Ergot Terpenoid and Steroidal Alkaloids

Groger D and Floss H (1998) Biochemistry of ergot Amiya T and Bando H (1988) Aconitum alkaloids. alkaloids: achievements and challenges. The Alka- The Alkaloids, Chemistry and Pharmacology (ed loids, Chemistry and Pharmacology (ed Cordell GA) Brossi A) Vol 34. Academic, San Diego, pp 95–179. Vol 50. Academic, San Diego, pp 171–218. Atta-ur-Rahman and Choudhary MI (1998) Chemistry Kren V (1991) Bioconversions of ergot alkaloids. Adv and biology of steroidal alkaloids. The Alkaloids, Biochem Eng Biotech 44, 123–144. Chemistry and Pharmacology (ed Cordell GA) Kren V, Harazim P and Malinka Z (1994) Claviceps Vol 50. Academic, San Diego, pp 61–108. purpurea (ergot): culture and bioproduction of ergot Atta-ur-Rahman and Choudhary MI (1999) Diterpenoid alkaloids. Biotechnology in Agriculture and Forestry, and steroidal alkaloids. Nat Prod Rep 16, 619–635. Vol 58 Medicinal and Aromatic Plants VII (ed Earlier reviews: 1997, 14, 191–203; 1995, 12, Bajaj YPS). Springer, Heidelberg, pp 139–156. 361–379. FURTHER READING 403

Cordell GA (1999) The monoterpene alkaloids. The (ed Brossi A) Vol 38. Academic, San Diego, Alkaloids, Chemistry and Biology (ed Cordell GA) pp 225–323. Vol 52. Academic, San Diego, pp 261–376. Baumann TW, Schulthess BH and Hanni¨ K (1995) Joshi BS and Pelletier SW (1999) Recent developments Guarana (Paullinia cupana) rewards seed dispersers in the chemistry of norditerpenoid and diterpenoid without intoxicating them by caffeine. Phytochem- alkaloids. Alkaloids, Chemical and Biological Per- istry 39, 1063–1070. spectives (ed Pelletier SW) Vol 13. Elsevier, Ams- Bernstein PR (1992) Antiasthmatic agents. Kirk–Othmer terdam, pp 289–370. Encyclopedia of Chemical Technology, 4th edn, Ripperger H (1998) Solanum steroid alkaloids: an Vol 2. Wiley, New York, pp 830–854. update. Alkaloids, Chemical and Biological Perspec- Houghton P (1995) Herbal products: guarana. Pharm J tives (ed Pelletier SW) Vol 12. Elsevier, Amsterdam, 254, 435–436. pp 103–185. Wills S (1994) Drugs and substance misuse: caffeine. Wang FP and Liang XT (1992) Chemistry of the diter- Pharm J 252, 822–824. penoid alkaloids. The Alkaloids, Chemistry and Phar- macology (ed Cordell GA) Vol 42. Academic, San Diego, 151–247. Saxitoxin, Tetrodotoxin

Purine Alkaloids Shimizu Y (1993) Microalgal metabolites. Chem Rev 93, 1685–1698. Atta-ur-Rahman and Choudhary MI (1990) Purine alka- Yasumoto T and Murata M (1993) Marine toxins. Chem loids. The Alkaloids, Chemistry and Pharmacology Rev 93, 1897–1909. Medicinal Natural Products. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 7 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

The fundamental structures of peptides and proteins are considered, and their biosynthesis by ribosomal and nonribosomal (multi-enzyme) processes are discussed. Ribosomal peptide biosynthesis leads to peptide hormones, and major groups of these are described, including thyroid, hypothalamic, anterior pituitary, posterior pituitary, and pancreatic hormones, as well as interferons, opioid peptides, and enzymes. Nonribosomal peptide biosynthesis is responsible for the formation of peptide antibiotics, peptide toxins, and modified peptides such as the penicillins, cephalosporins, and other β-lactams. Also covered in this chapter are some other amino acid derivatives, such as cyanogenic glycosides, glucosinolates, and the cysteine sulphoxides characteristic of garlic. Monograph topics giving more detailed information on medicinal agents include thyroxine, calcitonin, thyrotrophin-releasing hormone, luteinizing hormone-releasing hormone, growth hormone-releasing hormone, somatostatin, corticotropin, growth hormone, prolactin, gonadotrophins, oxytocin, vasopressin, insulin, glucagon, interferons, pharmaceutically important enzymes, cycloserine, polymyxins, bacitracins, tyrothricin and gramicidins, capreomycin, vancomycin and teicoplanin, bleomycin, cyclosporins, streptogramins, dactinomycin, death cap, ricin, botulinum toxin, microcystins, snake venoms, penicillins, cephalosporins, cephamycins, carbacephems, clavulanic acid, carbapenems, monobactams, and garlic.

Although the participation of amino acids in the biochemistry, the amide linkage is traditionally biosynthesis of some shikimate metabolites and referred to as a peptide bond. Whether the resultant particularly in the pathways leading to alkaloids has polymer is classified as a peptide or a protein is not already been explored in Chapters 4 and 6, amino clearly defined; generally a chain length of more acids are also the building blocks for other impor- than 40 residues confers protein status, whilst the tant classes of natural products. The elaboration of term polypeptide can be used to cover all chain shikimate metabolites and alkaloids utilized only a lengths. Although superficially similar, peptides limited range of amino acid precursors. Peptides, and proteins display a wide variety of biological proteins, and the other compounds considered in functions and many have marked physiological this chapter are synthesized from a very much wider properties. For example, they function as structural range of amino acids. Peptides and proteins repre- molecules in tissues, as enzymes, as antibodies, as sent another grey area between primary metabolism

and secondary metabolism, in that some materials 2 4 H O R H O R are widely distributed in nature and found, with sub- N N tle variations, in many different organisms, whilst N N 1 H 3 H others are of very restricted occurrence. O R OR peptide

PEPTIDES AND PROTEINS RCO2H RCO2H

HNH2 HNH2 Peptides and proteins are both polyamides com- L-amino acid D-amino acid posed of α-amino acids linked through their carboxyl and α-amino functions (Figure 7.1). In Figure 7.1 406 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

neurotransmitters, and acting as hormones can con- as are some plant toxins. These different activities trol many physiological processes, ranging from arise as a consequence of the sequence of amino gastric acid secretion and carbohydrate metabolism acids in the peptide or protein (the primary struc- to growth itself. The toxic components of snake ture), the three-dimensional structure which the and spider venoms are usually peptide in nature, molecule then adopts as a result of this sequence

Table 7.1 Amino acids: structures and standard abbreviations Amino acids encoded by DNA

CO2H CO2H Alanine Ala A Leucine Leu L NH2 NH2 NH H2N CO2H CO2H Arginine H2NN Arg R Lysine Lys K H NH2 NH2

H2NCO2H SCO2H Asparagine Asn N Methionine Met M O NH2 NH2

CO2H CO2H Aspartic acid HO2C Asp D Phenylalanine Phe F NH NH2 2

CO2H CO2H Cysteine HS Cys C Proline H Pro P NH2 NH

HO2CCO2H CO2H Glutamic acid Glu E Serine HO Ser S NH2 NH2 O OH

CO2H CO2H Glutamine H2N Gln Q Threonine Thr T

NH2 NH2 CO2H CO2H Glycine Gly G Tryptophan NH2 Trp W NH2 N H

CO2H CO2H Histidine N His H Tyrosine Tyr Y NH NH NH2 2 HO

Isoleucine CO2H Ile I Valine CO2H Val V

NH2 NH2

Some common amino acids not encoded by DNA

CO2H H CO2H H N Pyroglutamic NH Glp Ornithine 2 Orn acid oxoPro NH2 O (5-oxoproline)

CO2H CO2H H Hydroxyproline HO HPro Sarcosine (N- Sar NH methylglycine) NHMe RIBOSOMAL PEPTIDE BIOSYNTHESIS 407

amino- carboxyl- O O or N-terminus O H O or C-terminus H2N OH H2N H2N N OH H2N OH N OH O HO L-Phe OH OH L-Ala L-Ser Ala–Phe–Ser or H–Ala–Phe–Ser–OH or Ala→Phe→Ser or AFS N-terminus C-terminus O

N H

Figure 7.2

(the secondary and tertiary structures), and the appropriate abbreviations. Modified amino acids specific nature of individual side-chains in the may be represented by an appropriate variation molecule. Many structures have additional mod- of the normal abbreviation, e.g. N -methyltyrosine ifications to the basic polyamide system shown in as Tyr(Me). A frequently encountered modifica- Figure 7.1, and these features may also contribute tion is the conversion of the C -terminal carboxyl significantly to their biological activity. into an amide, and this is represented as Phe–NH2 The tripeptide formed from L-alanine, L-pheny- for example, which must not be interpreted as an lalanine, and L-serine in Figure 7.2 by two con- indication of the N -terminus. densation reactions is alanyl–phenylalanyl–serine, commonly represented as Ala–Phe–Ser, using the RIBOSOMAL PEPTIDE BIOSYNTHESIS standard three-letter abbreviations for amino acids as shown in Table 7.1, which gives the struc- Protein biosynthesis takes place on the ribosomes, tures of the 20 L-amino acids which are encoded and a simplified representation of the process by DNA. By convention, the left hand amino as characterized in Escherichia coli is shown in acid in this sequence is the one with a free Figure 7.3. The messenger RNA (mRNA) con- amino group, the N -terminus, whilst the right hand tains a transcription of one of the genes of DNA, amino acid has the free carboxyl, the C -terminus. and carries the information necessary to direct the Sometimes, the termini are emphasized by show- biosynthesis of a specific protein. The message is − − ing H and OH (Figure 7.2). In cyclic peptides, stored as a series of three-base sequences (codons) this convention can have no significance, so arrows in its nucleotides, and is read (translated) in the are incorporated into the sequence to indicate pep- 5 to 3 direction along the mRNA molecule. The tide bonds in the direction CO→NH. As sequences mRNA is bound to the smaller 30S subunit of the become longer, one letter abbreviations for amino bacterial ribosome. Initially, the amino acid is acti- acids are commonly used instead of the three- vated by an ATP-dependent process and it then letter abbreviations, thus Ala–Phe–Ser becomes binds via an ester linkage to an amino acid-specific AFS. The amino acid components of peptides and transfer RNA (tRNA) molecule through a termi- proteins predominantly have the L-configuration, nal adenosine group, giving an aminoacyl-tRNA but many peptides contain one or more D-amino (Figure 7.4). The aminoacyl-tRNA contains in its acids in their structures. Abbreviations thus assume nucleotide sequence a combination of three bases the L-configuration applies, and D-amino acids (the anticodon) which allows binding via hydro- must be specifically noted, e.g. Ala–D-Phe–Ser. gen bonding to the appropriate codon on mRNA. Some amino acids that are not encoded by DNA, In prokaryotes, the first amino acid encoded in but which are frequently encountered in peptides, the sequence is N -formylmethionine, and the cor- are shown in Table 7.1, and these also have their responding aminoacyl-tRNA is thus bound and 408 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

50S subunit

N-formylMet Ala

MeS H P site O A site (peptidyl) N H2N (aminoacyl) O H O O O A A C C C C ribosome

UAC CGC mRNA AUG GCG 5′ 3′

30S subunit

Figure 7.3

aminoacyl group transferred to ribose activation of amino acid by hydroxyl in 3′-terminal adenosine of formation of mixed anhydride tRNA; formation of ester linkage AMP O O ATP PP tRNA AMP RCO2H R R AMP tRNA

NH2 NH2 NH2

aminoacyl-AMP aminoacyl-tRNA

Figure 7.4 positioned at the P (for peptidyl) site on the a new aminoacyl-tRNA appropriate for the partic- ribosome (Figure 7.3). The next aminoacyl-tRNA ular codon, and a repeat of the elongation process (Figure 7.3 shows a tRNA specific for alanine) is occurs. The cycles of elongation and transloca- also bound via a codon–anticodon interaction and tion continue until a termination codon is reached, is positioned at an adjacent A (for aminoacyl) site and the peptide or protein is then hydrolysed and on the ribosome. This allows peptide bond for- released from the ribosome. The individual steps mation to occur, the amino group of the amino of protein biosynthesis all seem susceptible to dis- acid in the A site attacking the activated ester ruption by specific agents. Many of the antibiotics in the P site. The peptide chain is thus initiated used clinically are active by their ability to inhibit and has become attached to the tRNA located in protein biosynthesis in bacteria. They may inter- the A site. The tRNA at the P site is no longer fere with the binding of the aminoacyl-tRNA to required and is released from the ribosome. Then the A site (e.g. tetracyclines, see page 91), the for- the peptidyl-tRNA at the A site is translocated mation of the peptide bond (e.g. chloramphenicol, to the P site by the ribosome moving along the see page 130), or the translocation step (e.g. ery- mRNA a codon at a time, exposing the A site for thromycin, see page 99). RIBOSOMAL PEPTIDE BIOSYNTHESIS 409

amide (lactam) O O O O formation H H H N SH O H N S HS N H N H H H H S NH O O HO2C NH2 N-terminal O Cys residues disulphide bridge glutamic acid pyroglutamyl Glu– Glp– Cys Cys Cys Cys

Figure 7.5

Glp–Gly–Pro–Trp–Leu–Glu–Glu–Glu–Glu–Glu–Ala–Tyr–Gly–Trp–Gly–Trp–Met–Asp–Phe–NH2

human gastrin

Figure 7.6

Many of the peptides and proteins synthe- the γ-carboxylic acid and the α-amino of an N - sized on the ribosome then undergo enzymic terminal glutamic acid (Figure 7.5). The C -terminal post-translational modifications. In prokaryotes, the carboxylic acid may also frequently be converted N -formyl group on the N -terminal methionine is into an amide. Both modifications are exemplified removed, and in both prokaryotes and eukaryotes in the structure of gastrin (Figure 7.6), a peptide a number of amino acids from the N-terminus hormone that stimulates secretion of HCl in the may be hydrolysed off, thus shortening the origi- stomach. Such terminal modifications comprise a nal chain. This type of chain shortening is a means means of protecting a peptide from degradation by by which a protein or peptide can be stored in exopeptidases, which remove amino acids from the an inactive form, and then transformed into an ends of peptides, thus increasing its period of action. active form when required, e.g. the production of With the rapid advances made in genetic engi- insulin from proinsulin (see page 416). Glycopro- neering, it is now feasible to produce relatively teins are produced by adding sugar residues via large amounts of ribosome-constructed polypep- O-glycoside linkages to the hydroxyls of serine and tides by isolating or constructing DNA sequences encoding the particular product, and inserting these threonine residues or via N -glycoside linkages to into suitable organisms, commonly Escherichia the amino of asparagine. Phosphoproteins have coli. Of course, such procedures will not duplicate the hydroxyl groups of serine or threonine phos- any post-translational modifications, and these will phorylated. Importantly, post-translational modifi- have to be carried out on the initial polypeptide by cations allow those amino acids that are not encoded chemical means, or by suitable enzymes if these by DNA, yet are found in peptides and proteins, are available. Not all of the transformations can to be formed by the transformation of encoded be carried out both efficiently and selectively, thus ones. These include the hydroxylation of proline restricting access to important polypeptides by this to hydroxyproline and of lysine to hydroxylysine, means. Small peptides for drug use are generally N -methylation of histidine, and the oxidation of synthesized chemically, though larger peptides and the thiol groups of two cysteine residues to form proteins may be extracted from human and ani- a disulphide bridge, allowing cross-linking of mal tissues or bacterial cultures. In the design of polypeptide chains. This latter process (Figure 7.5) semi-synthetic or synthetic analogues for potential will form loops in a single polypeptide chain, or drug use, enzymic degradation can be reduced by may join separate chains together as in insulin the use of N -terminal pyroglutamic acid and C - (see page 416). Many peptides contain a pyrog- terminal amide residues, the inclusion of D-amino lutamic acid residue (Glp) at the N -terminus, a acids, and the removal of specific residues. These consequence of intramolecular cyclization between ploys to change recognition by specific degradative 410 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

I

HO I CO2H

NH2 R O I thyroxine R = I, thyroxine (levothyroxine; T4) R = H, tri-iodothyronine (T3)

I oxidative I I O coupling O O HN HN HN O O O I I I I O I I O O HN H O HN HN H O HO I I aromaticity restored by I elimination reaction

Figure 7.7 peptidases can increase the activity and lifetime of enzymes or proteins, by affecting the catalytic of the peptide. Very few peptide drugs may be activity of an enzyme, or by altering the permeabil- administered orally since they are rapidly inacti- ity of cell membranes. Hormones are not enzymes; vated or degraded by gastrointestinal enzymes, and they act by regulating existing processes. Fre- they must therefore be given by injection. quently, this action depends on the involvement of a second messenger, such as cyclic AMP (cAMP). PEPTIDE HORMONES Hormones are mammalian metabolites released Thyroid Hormones into the blood stream to elicit specific responses on a target tissue or organ. They are chemi- Thyroid hormones are necessary for the devel- cal messengers, and may be simple amino acid opment and function of cells throughout the derivatives, e.g. adrenaline (see page 317), or body. The thyroid hormones thyroxine∗ and polypeptides, e.g. insulin (see page 417), or they tri-iodothyronine∗ (Figure 7.7) are not peptides, may be steroidal in nature, e.g. progesterone (see but are actually simple derivatives of tyrosine. page 273). They may exert their effects in a variety However, they are believed to be derived by of ways, e.g. by influencing the rate of synthesis degradation of a larger protein molecule. One

Thyroxine

The thyroid hormones thyroxine (T4) and tri-iodothyronine (T3) (Figure 7.7) are derivatives of tyrosine and are necessary for development and function of cells throughout the body. They increase protein synthesis in almost all types of body tissue and + + increase oxygen consumption dependent upon Na /K ATPase (the Na pump). Excess thyroxine causes hyperthyroidism, with increased heart rate, blood pressure, over- activity, muscular weakness, and loss of weight. Too little thyroxine may lead to cretinism in children, with poor growth and mental deficiency, or myxoedema in adults, resulting in a slowing down of all body processes. Tri-iodothyronine is also

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produced from thyroxine by mono-deiodination in tissues outside the thyroid, and is actually three to five times more active than thyroxine. Levothyroxine (thyroxine)andliothyronine (tri- iodothyronine) are both used to supplement thyroid hormone levels, and may be administered orally. These materials are readily produced by chemical synthesis.

Cys–Gly–Asn–Leu–Ser–Thr–Cys–Met–Leu–Gly– Cys–Ser–Asn–Leu–Ser–Thr–Cys–Val–Leu–Gly– Thr–Tyr–Thr–Gln–Asp–Phe–Asn–Lys–Phe–His– Lys–Leu–Ser–Gln–Glu–Leu–His–Lys–Leu–Gln– Thr–Phe–Pro–Gln–Thr–Ala–Ile–Gly–Tyr–Gly– Thr–Tyr–Pro–Arg–Thr–Asn–Thr–Gly–Ser–Gly– Ala–Pro–NH2 Thr–Pro–NH2 human calcitonin calcitonin (salmon), salcatonin

Figure 7.8

Calcitonin Also produced in the thyroid gland, calcitonin is involved along with parathyroid hormone and 1α,25-dihydroxyvitamin D3 (see page 259) in the regulation of bone turnover and maintenance of calcium balance. Under the influence of high blood calcium levels, calcitonin is released to lower these levels by inhibiting uptake of calcium from the gastrointestinal tract, and by promoting its storage in bone. It also suppresses loss of calcium from bone when levels are low. Calcitonin is used to treat weakening of bone tissue and hypercalcaemia. Human calcitonin (Figure 7.8) is a 32-residue peptide with a disulphide bridge, and synthetic material may be used. However, synthetic salmon calcitonin (calcitonin (salmon); salcatonin) (Figure 7.8) is found to have greater potency and longer duration than human calcitonin. Calcitonins from different sources have quite significant differences in their amino acid sequences; the salmon peptide shows 16 changes from the human peptide. Porcine calcitonin is also available. hypothesis for their formation invokes tyrosine body, via the regulation of anterior pituitary residues in the protein thyroglobulin, which are hormone secretion. Thyrotrophin-releasing hor- iodinated to mono- and di-iodotyrosine, with mone (TRH)∗ is a tripeptide with an N -terminal suitably placed residues reacting together by a pyroglutamyl residue, and a C -terminal pro- phenolic oxidative coupling process (Figure 7.7). lineamide, whilst luteinizing hormone-releasing Re-aromatization results in cleavage of the side- hormone (LH-RH)∗ is a straight-chain decapep- chain of one residue, and thyroxine (or triiodothy- tide that also has both N -andC -termini ronine) is released from the protein by proteolytic blocked, through pyroglutamyl and glycineamide cleavage. Alternative mechanisms have been pro- ∗ ∗ respectively. Somatostatin (growth hormone- posed, however. Calcitonin (Figure 7.8) is also release inhibiting factor; GHRIH)merelydis- produced in the thyroid gland, and is involved in plays a disulphide bridge in its 14-amino acid the regulation of bone turnover and maintenance of chain. One of the largest of the hypothalamic calcium balance. This is a relatively simple peptide hormones is corticotropin-releasing hormone structure containing a disulphide bridge. (CRH) which contains 41 amino acids, with only the C -terminal blocked as an amide. This hor- Hypothalamic Hormones mone controls release from the anterior pitu- itary of corticotropin (ACTH) (see page 414), Hypothalamic hormones (Figure 7.9) can modulate which in turn is responsible for production of a wide variety of actions throughout the corticosteroids. 412 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

O H Glp–His–Trp–Ser–Tyr–Gly–Leu–Arg–Pro–Gly–NH2 N luteinizing hormone-releasing factor (LH-RH) O N N H (gonadorelin) H O H H Glp–His–Trp–Ser–Tyr–D-Trp–Leu–Arg–Pro–Gly–NH2 NNHCONH2 triptorelin

Glp–His–Pro–NH2 Glp–His–Trp–Ser–Tyr–D-Ser(tBu)–Leu–Arg–Pro–NHEt thyrotrophin-releasing hormone (TRH) buserelin (protirelin) t Glp–His–Trp–Ser–Tyr–D-Ser( Bu)–Leu–Arg–Pro–NH–NH–CONH2 goserelin

Tyr–Ala–Asp–Ala–Ile–Phe–Thr–Asn–Ser–Tyr– Glp–His–Trp–Ser–Tyr–D-Leu–Leu–Arg–Pro–NHEt Arg–Lys–Val–Leu–Gly–Gln–Leu–Ser–Ala–Arg– leuprorelin Lys–Leu–Leu–Gln–Asp–Ile–Met–Ser–Arg–NH2 sermorelin Glp–His–Trp–Ser–Tyr–3-(2-naphthyl)-D-Ala–Leu–Arg–Pro–Gly–NH2 nafarelin

Ala–Gly–Cys–Lys–Asn–Phe–Phe–Trp D-Phe–Cys–Phe–D-Trp 3-(2-naphthyl)-D-Ala– Cys–Tyr–D-Trp

Cys–Ser–Thr–Phe–Thr–Lys Thr(ol)–Cys–Thr–Lys H2N–Thr–Cys–Val–Lys growth hormone-release inhibiting factor (GHRIH) octreotide lanreotide (somatostatin)

Ser–Gln–Glu–Pro–Pro–Ile–Ser–Leu–Asp–Leu– Thr–Phe–His–Leu–Leu–Arg–Glu–Val–Leu–Glu– OH CO2H Met–Thr–Lys–Ala–Asp–Gln–Leu–Ala–Gln–Gln– CH2OH Ala–His–Ser–Asn–Arg–Lys–Leu–Leu–Asp–Ile– Ala–NH NH2 2 NH2 corticotropin-releasing hormone (CRH) 3-(2-naphthyl)-D-Ala Thr(ol)

Figure 7.9

Thyrotrophin-Releasing Hormone

Thyrotrophin-releasing hormone (TRH) (Figure 7.9) acts directly on the anterior pituitary to stimulate release of thyroid-stimulating hormone (TSH), prolactin, and growth hormone. Many other hormones may be released by direct or indirect effects. Synthetic material (known as protirelin) is used to assess thyroid function and TSH reserves.

Luteinizing Hormone-Releasing Hormone

Luteinizing hormone-releasing hormone (LH-RH) (also gonadotrophin-releasing hormone, GnRH) (Figure 7.9) is the mediator of gonadotrophin secretion from the anterior pituitary, stimulating both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release (see page 415). These are both involved in controlling male and female reproduction, inducing the production of oestrogens and progestogens in the female, and of androgens in the male. LH is essential for causing ovulation, and for the development and maintenance of the corpus luteum in the ovary, whilst FSH is required for maturation of both ovarian follicles in women, and of the testes in men.

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(Continued)

Gonadorelin is synthetic LH-RH and is used for the assessment of pituitary function, and also for the treatment of infertility, particularly in women. Analogues of LH-RH, e.g. triptorelin, buserelin, goserelin, leuprorelin,andnafarelin (Figure 7.9), have been developed and find use to inhibit ovarian steroid secretion, and to deprive cancers such as prostate and breast cancers of essential steroid hormones. All of these analogues include a D-amino acid residue at position 6, and modifications to the terminal residue 10, including omission of this residue in some cases. Such changes increase activity and half-life compared with gonadorelin, e.g. leuprorelin is 50 times more potent, and half-life is increased from 4 minutes to 3–4 hours.

Growth Hormone-Releasing Hormone/Factor Growth hormone-releasing hormone/factor (GHRH/GHRF) contains 40–44 amino acid residues and stimulates secretion of growth hormone (see page 414) from the anterior pituitary. Synthetic material containing the first 29-amino acid sequence has the full activity and potency of the natural material. This peptide (sermorelin) (Figure 7.9) is used as a diagnostic aid to test the secretion of growth hormone.

Somatostatin Somatostatin (growth hormone-release inhibiting factor; GHRIH) (Figure 7.9) is a 14- amino acid peptide containing a disulphide bridge, but is derived from a larger precursor protein. It is found in the pancreas and gastrointestinal tract as well as in the hypothalamus. It inhibits the release of growth hormone (see page 414) and thyrotrophin from the anterior pituitary, and also secretions of hormones from other endocrine glands, e.g. insulin and glucagon. Receptors for somatostatin are also found in most carcinoid tumours. Somatostatin has a relatively short duration of action (half-life 2–3 minutes), and the synthetic analogue octreotide (Figure 7.9), which is much longer acting (half-life 60–90 minutes), is currently in drug use for the treatment of various endocrine and malignant disorders, especially treatment of neuroendocrine tumours and the pituitary-related growth condition acromegaly. Octreotide contains only eight residues, but retains a crucial Phe–Trp–Lys–Thr sequence, even though the Trp now has the D-configuration. This prevents proteolysis between Trp and Lys, a major mechanism in somatostatin degradation. The C-terminus is no longer an amino acid, but an amino alcohol related to threonine. Radiolabelled octreotide has considerable potential for the visualization of neuroendocrine tumours. Lanreotide (Figure 7.9) is a recently introduced somatostatin analogue used in a similar way to octreotide. Lanreotide retains only a few of the molecular characteristics of octreotide, with the Phe–D-Trp–Lys–Thr sequence now modified to Tyr–D-Trp–Lys–Val and the N-terminal amino acid is the synthetic analogue 3-(2-napthyl)-D-alanine, also seen in the LH-RH analogue nafarelin.

Anterior Pituitary Hormones hormone, prolactin∗, which controls milk produc- tion in females, is structurally related to growth The main hormones of the anterior pituitary are hormone. corticotropin∗ and growth hormone∗, which each consist of a single long polypeptide chain, and the ∗ Posterior Pituitary Hormones gonadotrophins , which are glycoproteins con- taining two polypeptide chains. These hormones The two main hormones of the posterior regulate release of glucocorticoids, human growth, pituitary are oxytocin∗, which contracts the and sexual development respectively. A further smooth muscle of the uterus, and vasopressin∗, 414 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

Corticotropin

Corticotropin (corticotrophin; adrenocorticotrophin; ACTH) is a straight-chain polypeptide with39 amino acid residues, and its function is to control the activity of the adrenal cortex, particularly the production of corticosteroids. Secretion of the hormone is controlled by corticotropin-releasing hormone (CRH) from the hypothalamus. ACTH was formerly used as an alternative to corticosteroid therapy in rheumatoid arthritis, but its value was limited by variable therapeutic response. ACTH may be used to test adrenocortical function. It has mainly been replaced for this purpose by the synthetic analoguetetracosactide (tetracosactrin) (Figure 7.10), which contains the first 24 amino acid residues of ACTH, and is preferred because of its shorter duration of action and lower allergenicity.

Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–Pro–Val–Lys–Val–Tyr–Pro

tetracosactide (tetracosactrin)

Figure 7.10

Growth Hormone Growth Hormone (GH)(human growth hormone (HGH)orsomatotrophin) is necessary for normal growth characteristics, especially the lengthening of bones during development. A lack of HGH in children results in dwarfism, whilst continued release can lead to gigantism, or acromegaly, in which only the bones of the hands, feet, and face continue to grow. Growth hormones from animal sources are very species specific, so it has not been possible to use animal hormones for drug use. HGH contains 191 amino acids with two disulphide bridges, one of which creates a large loop (bridging residues 53 and 165) and the other a very small loop (bridging residues 182 and 189) near the C-terminus. It is synthesized via a prohormone containing 26 extra amino acids. Production of material with the natural amino acid sequence has become possible as a result of recombinant DNA technology; this is termed somatropin. This drug is used to improve linear growth in patients whose short stature is known to be caused by a lack of pituitary growth hormone. There is also some abuse of the drug by athletes wishing to enhance performance. Although HGH increases skeletal mass and strength, its use can result in some abnormal bone growth patterns.

Prolactin Prolactin has structural similarities to growth hormone, in that it is a single-chain polypeptide (198 amino acid residues) with three loops created by disulphide bonds. It is synthesized via a prohormone containing 29 extra amino acids. Growth hormone itself can bind to the prolactin receptor, but this is not significant under normal physiological conditions. Prolactin release is controlled by dopamine produced by the hypothalamus, and its main function is to control milk production. Prolactin has a synergistic action with oestrogen to promote mammary tissue proliferation during pregnancy, then at parturition, when oestrogen levels fall, prolactin levels rise and lactation is initiated. New nursing mothers have high levels of prolactin and this also inhibits gonadotrophin release and/or the response of the ovaries to these hormones. As a

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result, ovulation does not usually occur during breast feeding, preventing further conception. No prolactin derivatives are currently used in medicine, but the ergot alkaloid derivatives bromocriptine and cabergoline (see page 375) are dopamine agonists employed to inhibit prolactin release by pituitary tumours. They are not now recommended for routine suppression of lactation.

Gonadotrophins Follicle-stimulating hormone (FSH)andluteinizing hormone (LH) are involved in control- ling both male and female reproduction (see LH-RH, page 412). These are glycoproteins both composed of two polypeptide chains of 89 and 115 amino acid residues. The shorter chains are essentially identical, and differences in activity are caused by differences in the longer chain. Each chain has asparagine-linked oligosaccharide residues: the short chains each have one, the long chains two in the case of FSH or one for LH. FSH and LH together (human menopausal gonadotrophins; menotrophin) are purified from the urine of post-menopausal women and used in the treatment of female infertility. FSH alone is also available for this purpose, either natural material from urine termed urofollitropin (uro- follitrophin), or the recombinant proteins follitropin alfa and follitropin beta. Chorionic gonadotrophin (human chorionic gonadotrophin; HCG) is a gonad-stimulating glycopro- tein hormone which is obtained for drug use from the urine of pregnant women. It may be used to stimulate testosterone production in males with delayed puberty. the antidiuretic hormone. These nonapeptides classified as belonging to the vasopressin family containing a disulphide bridge are structurally very when the amino acid residue at position 8 is basic, similar, differing in only two amino acid residues e.g. Arg or Lys, or to the oxytocin family when (Figure 7.11). Structurally related peptides are this amino acid is neutral.

Cys–Tyr–Ile–Gln–Asn–Cys–Pro–Leu–Gly–NH2 (desamino)Cys–Tyr–Phe–Gln–Asn–Cys–Pro–D-Arg–Gly–NH2 oxytocin desmopressin (1-desamino-8-D-Arg-VP)

Cys–Tyr–Phe–Gln–Asn–Cys–Pro–Arg–Gly–NH2 Gly–Gly–Gly–Cys–Tyr–Phe–Gln–Asn–Cys–Pro–Lys–Gly–NH2 argipressin (arginine vasopressin) (human/bovine) terlipressin (Gly-Gly-Gly-8-L-Lys-VP)

Cys–Tyr–Phe–Gln–Asn–Cys–Pro–Lys–Gly–NH2 lypressin (lysine vasopressin) (porcine)

Figure 7.11

Oxytocin

Oxytocin (Figure 7.11) stimulates the pregnant uterus, causing contractions, and also brings about ejection of milk from the breasts. It thus plays a major role in the normal onset of labour at the end of pregnancy. Oxytocin for drug use is produced by synthesis, and is employed to induce or augment labour, as well as to minimize subsequent blood loss.

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(Continued) Vasopressin Vasopressin (antidiuretic hormone, ADH) is a hormone that has an antidiuretic action on the kidney, regulating the reabsorption of water. A deficiency in this hormone leads to diabetes insipidus, where the patient suffers increased urine output and intense thirst, typically consuming enormous quantities of fluid. Vasopressin is used to treat this condition. At high dosage, vasopressin promotes contraction of arterioles and capillaries, and brings about an increase in blood pressure. The structure of human and bovine vasopressin (arginine vasopressin; argipressin) (Figure 7.11) differs from that of oxytocin only in two amino acid residues. Lysine vasopressin (lypressin) (Figure 7.11) from pigs differs from arginine vasopressin in the second amino acid from the C-terminus. Both bovine and porcine peptides have been used medicinally, but these have been replaced by synthetic materials. The 1- desamino-8-D-Arg-vasopressin analogue desmopressin (Figure 7.11) has a longer duration of action than vasopressin and may also be administered orally. In contrast to the natural hormone, desmopressin has no vasoconstrictor effect. Terlipressin (Figure 7.11) is a lypressin pro-drug in which the polypeptide chain has been extended by three glycine residues. Enzymic hydrolysis liberates lypressin. It is mainly used for control of oesophageal bleeding.

Pancreatic Hormones polypeptide preproinsulin containing 100 amino ∗ acid residues. This loses a 16-residue portion of The hormone insulin (Figure 7.12) plays a key role in the regulation of carbohydrate, fat, its chain, and forms proinsulin with disulphide and protein metabolism. In particular, it has bridges connecting the terminal portions of the a hypoglycaemic effect, lowering the levels of chain in a loop (Figure 7.13). A central portion glucose in the blood. A deficiency in insulin of the loop (the C chain) is then cleaved out, synthesis leads to the condition diabetes, treatment leaving the A chain (21 residues) bonded to of which requires daily injections of insulin. the B chain (30 residues) by two disulphide Insulin is composed of two straight chain bridges. This is the resultant insulin. Mammalian polypeptides joined by disulphide bridges. This insulins (Figure 7.12) from different sources are structure is known to arise from a straight chain very similar, showing variations in the sequence

A chain Gly–Ile–Val–Glu–Gln–Cys–Cys–Thr–Ser–Ile–Cys–Ser–Leu–Tyr–Gln–Leu–Glu–Asn–Tyr–Cys–Asn 8 9 10 21

B chain Phe–Val–Asn–Gln–His–Leu–Cys–Gly–Ser–His–Leu–Val–Glu–Ala–Leu–Tyr–Leu–Val–Cys–Gly–Glu–Arg

Thr–Lys–Pro–Thr–Tyr–Phe–Phe–Gly 30 29 28 human insulin

AB 8 9 1028 29 30 insulin (human) Thr Ser IlePro Lys Thr His–Ser–Gln–Gly–Thr–Phe–Thr–Ser–Asp–Tyr– insulin (porcine) Thr Ser Ile Pro Lys Ala Ser–Lys–Tyr–Leu–Asp–Ser–Arg–Arg–Ala–Gln– insulin (bovine) Ala Ser ValPro Lys Ala Asp–Phe–Val–Gln–Trp–Leu–Met–Asn–Thr insulin lispro Thr Ser IleLys Pro Thr glucagon insulin aspart Thr Ser IleAsp Lys Thr

Figure 7.12 PEPTIDE HORMONES 417

Interferons The interferons∗ are a family of proteins secreted SS SS by animal cells in response to viral and parasitic B A B A B A infections, and are part of the host’s defence mech- C SS C SS anism. They display multiple activities, affecting the functioning of the immune system, cell prolif- eration, and cell differentiation, primarily by induc- ing the synthesis of other proteins. Accordingly, preproinsulin proinsulin insulin they have potential as antiviral, antiprotozoal, immunomodulatory, and cell growth regulatory Figure 7.13 agents. of amino acid residues 8–10 in chain A, and at Opioid Peptides ∗ amino acid 30 in chain B. Glucagon (Figure 7.12) Although the pain-killing properties of morphine is a straight-chain polypeptide hormone containing and related compounds have been known for a 29 amino acids that is secreted by the pancreas considerable time (see page 329), the existence when blood sugar levels are low, thus stimulating of endogenous peptide ligands for the receptors breakdown of glycogen in the liver. Unlike insulin, to which these compounds bind is a more recent its structure is identical in all animals. discovery. It is now appreciated that the body

Insulin Insulin is a hormone produced by the pancreas that plays a key role in the regulation of carbohydrate, fat, and protein metabolism. In particular, it has a hypoglycaemic effect, lowering the levels of glucose in the blood. If a malfunctioning pancreas results in a deficiency in insulin synthesis or secretion, the condition known as diabetes mellitus ensues. This results in increased amounts of glucose in the blood and urine, diuresis, depletion of carbohydrate stores, and subsequent breakdown of fat and protein. Incomplete breakdown of fat leads to the accumulation of ketones in the blood, severe acidosis, coma, and death. Where the pancreas is still functioning, albeit less efficiently, the condition is known as type 2 diabetes (non-insulin-dependent diabetes, NIDDM) and can be controlled by a controlled diet or oral antidiabetic drugs. In type 1 diabetes (insulin-dependent diabetes, IDDM) pancreatic cells no longer function, and injections of insulin are necessary, one to four times daily, depending on the severity of the condition. These need to be combined with a controlled diet and regular monitoring of glucose levels, but do not cure the disease, so treatment is lifelong. Mammalian insulins from different sources are very similar and may be used to treat diabetes. Porcine insulin and bovine insulin (Figure 7.12) are extracted from the pancreas of pigs and cattle respectively. Human insulin (Figure 7.12) is produced by the use of recombinant DNA technology in Escherichia coli to obtain the two polypeptide chains, and linking these chemically to form the disulphide bridges (such material is coded ‘crb’), or by modification of proinsulin produced in genetically modified E. coli (coded ‘prb’). Human insulin may also be obtained from porcine insulin by semi-synthesis, replacing the terminal alanine in chain B with threonine by enzymic methods (coded ‘emp’). Human insulin does not appear to be less immunogenic than animal insulin, but genetic engineering offers significant advantages over animal sources for obtaining highly purified material. Insulin may be provided in a rapid-acting soluble form, as suspensions of the zinc complex which have longer duration, or as suspensions with protamine

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(Continued)

(as isophane insulin). Protamine is a basic protein from the testes of fish of the salmon family, e.g. Salmo and Onchorhynchus species, which complexes with insulin, thereby reducing absorption and providing a longer period of action. Recently introduced recombinant human insulin analogues insulin lispro and insulin aspart have a faster onset and a shorter duration of action than soluble insulin. Insulin lispro has the reverse sequence for the 28 and 29 amino acids in the B chain, i.e. B28-Lys–B29-Pro, whilst insulin aspart has a single substitution of aspartic acid for proline at position 28 in the B chain (Figure 7.12). These changes in the primary structure affect the tendency of the molecule to associate into dimers and larger oligomers, thus increasing the availability of absorbable monomers. Insulin aspart is produced by expression in Saccharomyces cerevisiae (baker’s yeast). Insulin glargine is a new ultra- long-acting analogue that differs from human insulin by replacing the terminal asparagine at position 21 in chain A with glycine, and also adding two arginines to the end of the B chain, i.e. positions B31 and B32. These changes result in enhanced basicity, causing precipitation at neutral pH post-injection, and consequently a delayed, very gradual and prolonged activity profile (up to 24–48 hour duration of action) and allowing once-daily dosing.

Glucagon

Glucagon (Figure 7.12) is a straight-chain polypeptide hormone containing 29 amino acids that is secreted by the pancreas when blood sugar levels are low, thus stimulating breakdown of glycogen in the liver. It may be isolated from animal pancreas, or be produced by recombi- nant DNA processes using Saccharomyces cerevisiae. It may be administered for the emer- gency treatment of diabetes patients suffering from hypoglycaemia as a result of building up a dangerously high insulin level. Normally, a patient would counter this by eating some glucose or sucrose, but hypoglycaemia can rapidly cause unconsciousness, requiring very prompt action.

produces a family of endogenous opioid peptides, (‘endogenous morphine’) (Figure 7.14), which which bind to a series of receptors in different is several times more potent than morphine in locations. These peptides include enkephalins, relieving pain. Although β-endorphin contains the endorphins,anddynorphins, and are produced sequence for Met-enkephalin, the latter peptide primarily, but not exclusively, in the pituitary and Leu-enkephalin are derived from a larger gland. The pentapeptides Met-enkephalin and peptide proenkephalin A, whilst β-endorphin itself Leu-enkephalin (Figure 7.14) were the first to be is formed by cleavage of pro-opiomelanocortin. characterized. The largest peptide is β-endorphin The proenkephalin A structure contains four

Interferons Interferons were originally discovered as proteins that interfered with virus replication. When mice were injected with antibodies to interferons, they became markedly susceptible to virus-mediated disease, including virus-related tumour induction. Interferons can be detected at low levels in most human tissues, but amounts increase upon infection with viruses, bacteria, protozoa, and exposure to certain growth factors. Interferons were initially classified according to the cellular source, but recent nomenclature is based primarily on sequencing data. Thus leukocyte interferon (a mixture of proteins) is now known as interferon alfa, fibroblast interferon as interferon beta, and immune interferon as interferon gamma.

(Continues) PEPTIDE HORMONES 419

(Continued)

These typically range in size from 165 to 172 amino acid residues. The interferon system can often impair several steps in viral replication, and can modulate the immune system, and affect cell growth and differentiation. They have potential in treating many human diseases, including leukaemias and solid tumours, and viral conditions such as chronic infection with hepatitis B and C. Much of their current drug use is still research based. Interferon alfa is a protein containing 166 amino acid residues with two disulphide bridges and is produced by recombinant DNA techniques using Escherichia coli, or by stimulation of specific human cell lines. Variants with minor differences in sequence may be obtained according to the gene used, and are designated as alfa-2a, alfa-2b, etc. Interferon alfa is employed as an antitumour drug against certain lymphomas and solid tumours, and in the management of chronic hepatitis. Interferon beta is of value in the treatment of multiple sclerosis patients, though not all patients respond. Variants are designated beta-1a (a glycoprotein with 165 amino acid residues and one disulphide bridge) or beta-1b (a non-glycosylated protein with 164 amino acid residues and one disulphide bridge). Interferon gamma produced by recombinant DNA methods contains an unbridged polypeptide chain, and is designated gamma-1a (146 residues) or gamma-1b (140 residues) according to sequence. The immune interferon interferon gamma-1b is used to reduce the incidence of serious infection in patients with chronic granulomatous disease.

Met-enkephalin sequences and one of Leu- Enzymes enkephalin. The dynorphins, e.g. dynorphin A (Figure 7.14) are also produced by cleavage Enzymes are proteins that act as biological cata- of a larger precursor, namely proenkephalin B lysts. They facilitate chemical modification of sub- (prodynorphin), and all contain the Leu-enkephalin sequence. Some 20 opioid ligands have now been strate molecules by virtue of their specific binding characterized. When released, these endogenous properties, which arise from particular combina- opioids act upon specific receptors, inducing tions of functional groups in the constituent amino acids at the so-called active site. In many cases, analgesia, and depressing respiratory function and + several other processes. The individual peptides an essential cofactor, e.g. NAD ,PLP,orTPP, have relatively high specificity towards different may also be bound to participate in the transforma- receptors. It is known that morphine, β-endorphin, tion. The involvement of enzymes in biochemical and Met-enkephalin are agonists for the same reactions has been a major theme throughout this site. The opioid peptides are implicated in book. The ability of enzymes to carry out quite analgesia brought about by acupuncture, since complex chemical reactions, rapidly, at room tem- opiate antagonists can reverse the effects. The perature, and under essentially neutral conditions hope of exploiting similar peptides as ideal, is viewed with envy by synthetic chemists, who are non-addictive analgesics has yet to be attained; making rapid progress in harnessing this ability for repeated doses of endorphin or enkephalin produce their own uses. Several enzymes are currently of addiction and withdrawal symptoms. importance commercially, or for medical use, and

Tyr–Gly–Gly–Phe–Met–Thr–Ser–Glu–Lys–Ser– Tyr–Gly–Gly–Phe–Met Tyr–Gly–Gly–Phe–Leu Gln–Thr–Pro–Leu–Val–Thr–Leu–Phe–Lys–Asn– Met-enkephalin Leu-enkephalin Ala–Ile–Val–Lys–Asn–Ala–His–Lys–Lys–Gly– Gln β-endorphin Tyr–Gly–Gly–Phe–Leu–Arg–Arg–Ile–Arg–Pro–Lys–Leu–Lys–Trp–Asp–Asn–Gln dynorphin A

Figure 7.14 420 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

Table 7.2 Pharmaceutically important enzymes Enzyme Action Source Use Hydrolytic enzymes Pancreatin Hydrolysis of starch Porcine pancreas Digestive aid (amylase), fat (lipase), and protein (protease) Papain Hydrolysis of proteins Papaya fruit (Carica Meat tenderizer; papaya; Caricaceae) cleaning of contact lenses Chymotrypsin Hydrolysis of proteins Bovine pancreas Zonal lysis in cataract removal Hyaluronidase Hydrolysis of Mammalian testes Renders tissues more mucopolysaccharides permeable for subcutaneous or intramuscular injections Pepsin Hydrolysis of proteins Porcine stomach Digestive aid Trypsin Hydrolysis of proteins Bovine pancreas Wound and ulcer cleansing Fibrinolytic enzymes Streptokinase No enzymic activity, until it Streptococcus Treatment of venous complexes with and activates haemolyticus thrombosis and plasminogen in blood plasma pulmonary embolism to produce the proteolytic enzyme plasmin, which hydrolyses fibrin clots Urokinase A protease which activates Human urine, or Treatment of venous plasminogen in blood plasma human kidney tissue thrombosis and to form plasmin, which cultures pulmonary embolism; hydrolyses fibrin clots thrombolysis in the eye Alteplase (recombinant A protease which binds to Recombinant genetic Treatment of acute tissue-type plasminogen fibrin converting it to a potent engineering: human myocardial infarction activator; rt-PA) plasminogen activator; only gene expressed in active at the surface of the Chinese hamster blood clot ovary cells Anistreplase (acylated An inactive acylated form of Semi-synthesis from Treatment of acute plasminogen-streptokinase the plasminogen–strepto- urokinase myocardial infarction activator complex; APSAC) kinase activator complex; the acyl group (p-anisoyl) is slowly hydrolysed in the blood to give the active agent Reteplase A fibrinolytic protease; a Recombinant genetic Treatment of acute genetically-engineered human engineering myocardial infarction tissue-type plasminogen activator differing from alteplase at four amino acid residues (Continues) NONRIBOSOMAL PEPTIDE BIOSYNTHESIS 421

Table 7.2 (Continued) Enzyme Action Source Use Others Asparaginase Degradation of L-asparagine Erwinia chrysanthemi Treatment of acute (crisantaspase) lymphoblastic leukaemia; results in death of those tumour cells which require increased levels of exogenous L-asparagine; side-effects nausea and vomiting, allergic reactions and anaphylaxis Streptodornase Depolymerization of Streptococcus In combination with polymerized haemolyticus streptokinase as deoxyribonucleo-proteins desloughing agent to cleanse ulcers and promote healing these are described in Table 7.2. Enzymes are typi- gives a simplified representation), until the pep- cally larger than most of the polypeptides discussed tide is finally released from the enzyme. A typical above, and are thus extracted from natural sources. module consists of an adenylation (A) domain, a Recombinant DNA procedures are likely to make peptidyl carrier protein (PCP) domain, and a con- a very significant contribution in the future. densation (C) or elongation domain. The A domain activates a specific amino acid as an aminoa- cyl adenylate, which is then transferred to the NONRIBOSOMAL PEPTIDE PCP domain forming an aminoacyl thioester. Pan- BIOSYNTHESIS tothenic acid (vitamin B5, see page 31) bound to the enzyme as pantatheine is used to carry In marked contrast to the ribosomal biosynthesis of the growing peptide chain through its thiol group peptides and proteins, where a biological produc- (Figure 7.15). The significance of this is that the tion line interprets the genetic code, many natural long ‘pantatheinyl arm’ allows different active sites peptides are known to be synthesized by a more on the enzyme to be reached in the chain assem- individualistic sequence of enzyme-controlled pro- bly process (compare biosynthesis of fatty acids, cesses, in which each amino acid is added as a page 36, and polyketides, page 62). Nucleophilic result of the specificity of the enzyme involved. attack by the amino group of the neighbouring The many stages of the whole process appear to aminoacyl thioester is catalysed by the C domain be carried out by a multi-functional enzyme (nonri- and results in amide (peptide) bond formation. bosomal peptide synthase, NRPS) with a modular Enzyme-controlled biosynthesis in this manner is arrangement comparable to that seen with type I a feature of many microbial peptides, especially polyketide synthases (see page 114). The linear those containing unusual amino acids not encoded sequence of modules in the enzyme usually cor- by DNA and where post-translational modification responds to the generated amino acid sequence is unlikely, and also for the cyclic structures which in the peptide product. The amino acids are first are frequently encountered. As well as activating activated by conversion into AMP esters, which the amino acids and catalysing formation of the then bind to the enzyme through thioester linkages. peptide linkages, the enzyme may possess other The residues are held so as to allow a sequen- domains that are responsible for epimerizing L- tial series of peptide bond formations (Figure 7.15 amino acids to D-amino acids, probably through 422 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

activation of amino acid by formation of thioester formation of mixed anhydride linkage to enzyme AMP O O ATP PP Enz–SH AMP RCO2H R R AMP SEnz

NH2 NH2 NH2 aminoacyl–AMP

Module 1 Module 2 Module 3 Module 1 Module 2 Module 3

A PCP C A PCP C A PCP C A PCP C A PCP C A PCP C

S S SH SH S S O O O O R1 R2 growing peptide chain R2 R3 generated by sequentially H N H N HN H N 2 2 forming amide bond linkages 2 O A: adenylation domain R2 PCP: peptidyl carrier protein domain C: condensation domain NH2 'pantotheinyl arm' OO attached to growing bound to enzyme HS OH peptide chain N N through phosphate H H OH cysteamine pantothenic acid pantotheine

Figure 7.15

R H H R H terminating chain extension and releasing the pep- N N tide from the enzyme. Many of the medicinally N L N HO HOH useful peptides have cyclic structures. Cyclization epimerization through may result if the amino acids at the two termini of a intermediate enol-like linear peptide link up to form another peptide bond, tautomer but very often, ring formation can be the result of R H H N ester or amide linkages, which utilize side-chain N D functionalities in the constituent amino acids. As HO with polyketide synthases, genetic manipulation of Figure 7.16 nonribosomal peptide synthases allows production of peptide derivatives in which rational modifica- enol-like tautomers in the peptide (Figure 7.16). tions may be programmed according to the genes A terminal thioesterase domain is responsible for encoded.

PEPTIDE ANTIBIOTICS Cycloserine

D-Cycloserine (Figure 7.17) is produced by cultures of Streptomyces orchidaceus,ormay be prepared synthetically, and is probably the simplest substance with useful antibiotic

(Continues) PEPTIDE ANTIBODIES 423 activity. Cycloserine is water soluble and has a broad spectrum of antibacterial activity, but it is primarily employed for its activity against Mycobacterium tuberculosis. It behaves as a structural analogue of D-alanine, and inhibits the incorporation of D-alanine into bacterial cell walls. Since it can produce neurotoxicity in patients, it is reserved for infections resistant to first-line drugs.

NH2 NH3 NH3 NH2

O O O O O HO CO2H N N O H D-cycloserine D-Ala D-Ser

Figure 7.17

Polymyxins

The polymyxins are a group of cyclic polypeptide antibiotics produced by species of Bacillus. Polymyxins A–E were isolated from Bacillus polymyxa, though polymyxin B and polymyxin E were both subsequently shown to be mixtures of two components. A polypeptide mixture called colistin isolated from Bacillus colistinus was then found to be identical to polymyxin E. Polymyxin B and colistin (polymyxin E) are both used clinically. These antibiotic mixtures respectively contain principally polymyxin B1 with small amounts of polymyxin B2, or predominantly polymyxin E1 (≡ colistin A) with small amounts of polymyxin E2 (≡ colistin B) (Figure 7.18). These molecules contain ten amino acids, six of which are L-α,γ- diaminobutyric acid (L-Dab), with a fatty acid (6-methyloctanoic acid or 6-methylheptanoic acid) bonded to the N-terminus, and a cyclic peptide portion constructed via an amide bond between the carboxyl terminus and the γ-amino of one of the Dab residues. The γ-amino groups of the remaining Dab residues confer a strongly basic character to the antibiotics. This results in detergent-like properties and allows them to bind to and damage bacterial membranes. These peptides have been used for the treatment of infections with Gram- negative bacteria such as Pseudomonas aeruginosa, but are seldom used now because of

Dab Y Leu

H2NCO2H X Dab Thr Dab Dab Dab Dab =

Thr Dab NH2 L-α,γ-diaminobutyric acid XY

polymyxin B1 6-methyloctanoic acid D-Phe

polymyxin B2 6-methylheptanoic acid D-Phe

polymyxin E1 6-methyloctanoic acid D-Leu (colistin A)

polymyxin E2 6-methylheptanoic acid D-Leu (colistin B)

Figure 7.18

(Continues) 424 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

neurotoxic and nephrotoxic effects. However, they are included in some topical preparations such as ointments, eye drops, and ear drops, frequently in combination with other antibiotics.

Bacitracins Bacitracin is a mixture of at least nine peptides produced by cultures of Bacillus subtilis, with the principal component being bacitracin A (Figure 7.19). The structure contains a cyclic peptide portion, involving the carboxyl terminus and the ε-amino of lysine, and at the N-terminus an unusual thiazolinecarboxylic acid, which is a condensation product from isoleucine and cysteine residues (compare epothilones, page 105, and bleomycin, page 429). Bacitracin is active against a wide range of Gram-positive bacteria, and appears to affect biosynthesis of the bacterial cell wall by binding to and sequestering a polyprenyl diphosphate carrier of intermediates; this binding also requires a divalent metal ion, with zinc being especially active. It is rarely used systemically because some bacitracin components are nephrotoxic, but as zinc bacitracin, it is a component of ointment formulations for topical application. The vast majority of bacitracin manufactured is used at subtherapeutic doses as an animal feed additive, to increase feed efficiency, and at therapeutic dosage to control a variety of disorders in poultry and animals.

D-Orn Ile D-Phe CO2H

X Leu D-Glu Ile Lys N Cys Asn D-Asp His X = S

bacitracin A Ile NH2

Figure 7.19

Tyrothricin and Gramicidins

Tyrothricin is a mixture of polypeptide antibiotics produced by cultures of Bacillus brevis. The mixture contains about 20–30% linear polypeptides called gramicidins (Figure 7.20), and 70–80% of cyclic structures called tyrocidines (Figure 7.20). Tyrothricin is active against many Gram-positive bacteria, with the linear gramicidins being more active than the cyclic tyrocidines. The two groups are readily separated by solvent fractionation, and the gramicidin fraction, also termed gramicidin D, a mixture of at least eight closely related compounds, is used principally in ophthalmic preparations. The gramicidins are neutral polypeptides having the N-terminal amino group formylated, and the carboxy group linked to ethanolamine. Most of the gramicidin mixture is composed of valine-gramicidin A (about 80%) (Figure 7.20). Apart from the glycine residue, these compounds have a sequence of alternating D- and L-amino acids. Gramicidins act by producing ion channels in bacterial membranes. The tyrocidines are too toxic for therapeutic use on their own, but the tyrothricin mixture is incorporated into lozenges for relief of throat infections. Gramicidin S is a mixture of cyclic peptides obtained from another strain of Bacillus brevis. Its main component is gramicidin S1 (Figure 7.20), a symmetrical

(Continues) PEPTIDE ANTIBODIES 425

(Continued)

OHC–X–Gly–Ala–D-Leu–Ala–D-Val–Val–D-Val–Trp–D-Leu–Y–D-Leu–Trp–D-Leu–Trp–NH(CH2)2OH

XY gramicidin A Val-gramicidin A Val Trp D-Phe Pro X Y Asn D-Phe Pro Val Orn Leu Ile-gramicidin A Ile Trp gramicidin B Val-gramicidin B Val Phe Leu Orn Val Tyr Gln Leu Orn Val Pro D-Phe Ile-gramicidin B Ile Phe

XY gramicidin S1 gramicidin C Val-gramicidin C tyrocidine A Phe D-Phe Val Tyr Ile-gramicidin C Ile Tyr tyrocidine B Tyr D-Phe tyrocidine C Tyr D-Tyr

Figure 7.20

decapeptide known to be formed by the joining together of two separate chains. Gramicidin S is fairly toxic, and its use is restricted to topical preparations. Gramicidin S also acts on bacterial membranes, increasing permeability and loss of barrier function.

Capreomycin

Capreomycin is a mixture of cyclic polypeptides obtained from cultures of Streptomyces capreolus, and contains about 90% of capreomycins I, principally capreomycin IB (Figure 7.21). The capreomycin structures incorporate an L-capreomycidine moiety derived by cyclization of L-arginine, and three molecules of 2,3-diaminopropionic acid (Dap), which originate from serine via dehydroalanine. This antibiotic is given intramuscularly to treat tuberculosis patients who do not respond to first-line drugs, or where patients are sensitive to

Ser or Ala Dap β-lysine R O H ONH2 Dap H2N N NH2 N N H H O NH O H HN Dap = 2,3-diaminopropionic acid NNHNH2 O CO2H L-capreomycidine O Dap O H2N NH HO2CNH2 NH2 N NH H NH2 R = OH, capreomycin 1A CO2H CO2H = HO N NH R H, capreomycin 1B H NH2 NH2 L-Arg dehydroalanine L-Ser

Figure 7.21

(Continues) 426 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

streptomycin. It can cause irreversible hearing loss and impair kidney function. Capreomycin inhibits protein biosynthesis at the translocation step in sensitive bacteria.

Vancomycin and Teicoplanin

Vancomycin (Figure 7.22) is a glycopeptide antibiotic produced in cultures of Amycolatopsis orientalis (formerly Streptomyces orientalis), and has activity against Gram-positive bacteria, especially resistant strains of staphylococci, streptococci, and enterococci. It is an important agent in the control of methicillin-resistant Staphylococcus aureus (MRSA), with some strains now being sensitive only to vancomycin or teicoplanin (below). Vancomycin is not absorbed orally, and must be administered by intravenous injection. However, it can be given orally in the treatment of pseudomembranous colitis caused by Clostridium difficile,whichmay occur after administration of other antibiotics. Vancomycin acts by its ability to form a complex with terminal N-acyl–D-Ala–D-Ala residues of growing peptidoglycan chains (see Figure 7.36 and page 444), preventing their cross-linking to adjacent strands and thus inhibiting bacterial cell wall biosynthesis. The –D-Ala–D-Ala residues are accommodated in a ‘carboxylate-binding pocket’ in the vancomycin structure. By preventing peptidoglycan polymerization and cross-linking, it weakens the bacterial cell wall and ultimately causes cell lysis. The novel feature of vancomycin, and several other related antibiotics, is the tricyclic structure generated by three phenolic oxidative coupling reactions. The β- hydroxychlorotyrosine and 4-hydroxyphenylglycine residues in vancomycin originate from L-tyrosine, but the 3,5-dihydroxyphenylglycine ring is actually acetate derived. These modified aromatic rings are presumably present in the heptapeptide before coupling occurs (Figure 7.22). The teicoplanins (Figure 7.23) possess the same basic structure as vancomycin, but the N-terminal (4-hydroxyphenylglycine) and third (3,5-dihydroxyphenylglycine) amino acids are also aromatic, and this allows further phenolic oxidative coupling and generation of yet another ring system. Teicoplanin for drug use is a mixture of five teicoplanins produced by cultures of Actinoplanes teichomyceticus, which differ only in the nature and length of the fatty acid chain attached to the sugar residue. Teicoplanin has similar antibacterial activity to vancomycin, but has a longer duration of action, and may be administered by intramuscular as well as by intravenous injection. It is also used against Gram-positive pathogens resistant to established antibiotics. Vancomycin, teicoplanin, and structurally related glycopeptides are often referred to as dalbaheptides (from D-alanyl–D-alanine-binding heptapeptide), reflecting their mechanism of action and their chemical nature. Unfortunately, with increasing use of vancomycin and teicoplanin, there have even been reports of these agents becoming ineffective because resistant bacterial strains have emerged, particularly in enterococci. In resistant strains, the terminal –D-Ala–D-Ala residues, to which the antibiotic normally binds, have become replaced by –D-Ala–D-lactate. The incorporation of D-lactate into the peptide intermediates results in loss of crucial hydrogen-bonding interactions and a thousand-fold lowering in binding efficiency.

(Continues) PEPTIDE ANTIBODIES 427

(Continued)

NH2 L-vancosamine O HO O HO O D-glucose HO O HO Cl OO

HO OH Cl O O O H H O N N NHMe N N N H H H O O HN CO2H O

NH2

OH HO OH vancomycin

* oxidative coupling reactions Tyr OH Cl OH HO * * HO OH β-hydroxychloro- Cl O O tyrosine O H H O N N NHMe N N N H H H O O HN CO2H O 3,5-dihydroxyphenyl- * NH2 glycine Asn Leu OH HO OH β-hydroxychloro- acetate 4-hydroxyphenyl- tyrosine glycine

Tyr Tyr

Figure 7.22

Bleomycin

Bleomycin is a mixture of glycopeptide antibiotics isolated from cultures of Streptomyces verticillus, used for its anticancer activity. The major component (55–70%) of the mixture

(Continues) 428 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

teicoplanin T-A2-1, R =

O teicoplanin T-A2-2, R =

O D-glucosamine teicoplanin T-A2-3, R = NHR HO O O teicoplanin T-A2-4, R = HO O HO Cl O OOteicoplanin T-A2-4, R = NHAc HO O O HO Cl O O O O H H HO O N N NHMe N N N N-acetyl- H H H O O D-glucosamine HN CO2H

HO O OH OH HO O 4-hydroxyphenyl- OH O glycine OH 3,5-dihydroxyphenyl- OH glycine OH D-mannose

Figure 7.23

is bleomycin A2 (Figure 7.24), with bleomycin B2 constituting about 30%. The various bleomycins differ only in their terminal amine functions, the parent compound bleomycinic acid (Figure 7.24) being inactive. The molecules contain several unusual amino acids and sugars, an asparagine-derived pyrimidine ring, and a planar dithiazole ring system which has its origins in two cysteines (compare epothilones, page 105, and bacitracin, page 424). A C2 unit supplied by malonyl-CoA also forms part of the main chain as a component of the amino acid 4-amino-3-hydroxy-2-methylvaleric acid, which in addition features a methionine-derived methyl group. Bleomycin is a DNA-cleaving drug, causing single and double-strand breaks in DNA. The dithiazole system is involved in binding to DNA, probably by intercalation, whilst other parts of the molecule near the N-terminus are involved in chelating a metal ion, usually Fe2+, and oxygen, which are necessary for the DNA degradation reaction. More recently, bleomycin A2 has been shown to cleave RNA as well as DNA. Bleomycin is used alone, or in combination with other anticancer drugs, to treat squamous cell carcinomas of various organs, lymphomas, and some solid tumours. It is unusual amongst antitumour antibiotics in producing very little bone-marrow suppression, making it particularly useful in combination therapies with other drugs that do cause this response. However, there is some lung toxicity associated with bleomycin treatment. Various bleomycin analogues have been made by adding different precursor amines to the culture medium, or by semi-synthesis from bleomycinic acid. Peplomycin (Figure 7.24) is an example with some promise, in that it is more resistant to enzymes that cause in vivo hydrolysis of bleomycin at the N-terminal β-aminoalanine group.

(Continues) PEPTIDE ANTIBODIES 429

(Continued)

Ala + malonate + Me

Ser 4-amino-3-hydroxy- ONH2 NH 2-methylvaleric acid R H 2 O Asn N NH2 Thr Cys O N NN O O S Asn H H N N H2N N NH N H Cys Me O OH O O N HO S OH HO O N β-OH-His O H β-Ala L-gulose OH bleomycin A2 R = NH S OH O D-mannose OH H O NNH2 OH bleomycin B2 R = NH NH ONH2 bleomycinic acid R = OH

CO H β = 2 -Ala H2N peplomycin R = NH N H

Figure 7.24

Cyclosporins

The cyclosporins are a group of cyclic peptides produced by fungi such as Cylindrocarpon lucidum and Tolypocladium inflatum. These agents showed a narrow range of antifungal activity, but high levels of immunosuppressive and anti-inflammatory activities. The main component from the culture extracts is cyclosporin A (ciclosporin, cyclosporin) (Figure 7.25), but some 25 naturally occurring cyclosporins have been characterized. Cyclosporin A contains several N-methylated amino acid residues, together with the less common L-α-aminobutyric acid and an N-methylated butenylmethylthreonine. This latter amino acid has been shown to originate via the acetate pathway, and it effectively comprises a C8 polyketide chain, plus a methyl group from SAM. The sequence shown in Figure 7.26 is consistent with experimental data, and is analogous to other acetate-derived compounds (see Chapter 3). The assembly of the polypeptide chain is known to start from the D-alanine residue. Many of the other natural cyclosporin structures differ only with respect to a single amino acid (the α-aminobutyric acid residue) or the amount of N-methylation. Of all the natural analogues, and many synthetic ones produced, cyclosporin A is the most valuable for drug use. It is now widely exploited in organ and tissue transplant surgery, to prevent rejection following bone-marrow, kidney, liver, and heart transplants. It has revolutionized organ transplant surgery, substantially increasing survival rates in transplant patients. It may be administered orally or by intravenous injection, and the primary side-effect is nephrotoxicity, necessitating careful monitoring of kidney function. Cyclosporin A has the same mode of action as the macrolide FK-506 (tacrolimus) (see page 104) and is believed to inhibit T-cell activation in the immunosuppressive mechanism

(Continues) 430 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

(Me)Leu (Me)Val (Me)Bmt Abu

Me O Me O Me O N N N N N (Me)Leu H Sar (Me)Bmt = 4-(2-butenyl)-4,N-dimethyl-L-threonine Me O HO O O N Me Abu = L-α-aminobutyric acid N Me O O O H H Sar = sarcosine (N-methylglycine) N N N N D-Ala H OMe O (Me)Leu Ala (Me)Leu Val ciclosporin (cyclosporin A)

Figure 7.25

C-methylation before chain extension reduction of carbonyl then reduction OO O OH O acetate / malonate SEnz SEnz SEnz

hydroxylation oxidation

OH N-methylation OH O transamination OH O

CO2H SEnz SEnz NHMe NH2 O 4-(2-butenyl)-4,N-dimethyl-L-threonine

Figure 7.26

by first binding to a receptor protein, giving a complex that then inhibits a phosphatase enzyme called calcineurin. The resultant aberrant phosphorylation reactions prevent appro- priate gene transcription and subsequent T-cell activation. Cyclosporin A also finds use in the specialist treatment of severe resistant psoriasis.

Streptogramins

The names streptogramin and virginiamycin have been applied to antibiotic mixtures isolated from strains of Streptomyces virginiae, and individual components have thus acquired multiple synonyms; as a family these antibiotics have now been termed streptogramin antibiotics. These compounds fall into two distinct groups, group A, containing a 23-membered unsaturated ring with peptide and lactone bonds, and group B, which are depsipeptides (essentially peptides cyclized via a lactone). These structures contain many nonprotein amino acids (Figure 7.27). Until recently, most commercial production of these antibiotics was directed towards animal feed additives, but the growing emergence of antibiotic-resistant

(Continues) PEPTIDE ANTIBODIES 431

(Continued)

bacterial strains has led to the drug use of some streptogramin antibiotics. Thus, dalfopristin and quinupristin (Figure 7.27) are water-soluble drugs that may be used in combination for treating infections caused by Gram-positive bacteria that have failed to respond to other antibiotics, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin- resistant enterococci and staphylococci, and drug-resistant Streptococcus pneumoniae. They may need to be combined also with other agents where mixed infections involve Gram- negative organisms. Dalfopristin is a semi-synthetic sulphonyl derivative of streptogramin A (also termed virginiamycin M1, mikamycin A, pristinamycin IIA, and other names), and quinupristin is a modified form of streptogramin B (also mikamycin B, pristinamycin IA, and other names). Members of the A group tend to be less powerful antibiotics than those of the B group, but together they act synergistically, providing greater activity than the combined activity expected from the separate components. The dalfopristin and quinupristin combination is supplied in a 70:30 ratio, which provides maximum synergy (a 100-fold increase in activity compared to the single agents), and also corresponds to the natural proportion of group A to group B antibiotics in the producer organism. The streptogramins bind to the peptidyl transferase domain of the 50S ribosomal subunit; the remarkable synergism arises because initial binding of the group A derivative causes a conformational change to the ribosome, increasing affinity for the group B derivative and formation of an extremely stable

Gly 6 x acetate acetate O O OH OH N N H H Met Met O O O O O O N N O N O N Val O O O2S Pro Ser Et2N streptogramin A dalfopristin

NMe2 NMe2

Pro α N - -methyl- Me Me 4-dimethylamino-L-Phe N N NOD-amino- N NO S butyric acid O Et O Et N O O NH H N O O NH H Me H Me N N O O O 4-oxo- O O O Thr L-pipecolic acid O HN O O HN O

OH OH N N L-pipecolic acid phenylglycine 3-hydroxy- picolinic acid quinupristin

streptogramin B

Figure 7.27

(Continues) 432 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

ternary complex. This makes the streptogramin combination bactericidal, whereas the single agents provide only bacteriostatic activity. Streptogramin A is known to be biosynthesized from four amino acids, namely valine, glycine, serine, and proline, a polyketide-like chain containing six acetate units, a further isolated acetate unit, and two methionine-derived methyl groups. The oxazole ring is formed from serine by incorporating the carboxyl terminus of the polyketide chain (compare the thiazole rings in bleomycin, page 429). Streptogramin B is formed by a typical nonribosomal peptide synthase, utilizing several rare modified amino acid precursors. All except one are modified before assembly; oxidation of the pipecolic acid residue (see page 310) to 4-oxopipecolic acid is carried out post-cyclization. The starter unit is 3-hydroxypicolinic acid, which arises via picolinic acid in the kynurenine pathway (see page 312); p- aminophenylalanine has been met previously in chloramphenicol biosynthesis (page 129).

Dactinomycin

Dactinomycin (actinomycin D) (Figure 7.28) is an antibiotic produced by Streptomyces parvullus (formerly S. antibioticus), which has antibacterial and antifungal activity, but whose high toxicity limits its use to anticancer therapy. Several related natural actinomycins are known, but only dactinomycin is used medicinally. Dactinomycin has a planar phenoxazinone dicarboxylic acid system in its structure, to which are attached two identical cyclic pentapep- tides via amide bonds to threonine. The peptides are cyclized by lactone linkages utilizing the hydroxyl group of this threonine. The peptide portions of dactinomycin contain N-methylvaline and N-methylglycine (sarcosine) residues. In other actinomycins, the two peptides are not nec- essarily identical. The phenoxazinone ring system is known to be formed by fusing together two molecules of 3-hydroxy-4-methylanthranilic acid (Figure 7.28), which arises by C-methylation of 3-hydroxyanthranilic acid, a metabolite of tryptophan by the kynurenine pathway (see page 312). This planar phenoxazinone ring intercalates with double-stranded DNA inhibiting DNA-dependent RNA polymerases, but can also cause single-strand breaks in DNA. It is prin- cipally used to treat paediatric cancers, including Wilms’ tumour of the kidney, but produces several serious and painful side-effects. However, as a selective inhibitor of DNA-dependent RNA synthesis (transcription), it has become an important research tool in molecular biology.

Sar Sar L-Trp Pro (Me)Val Pro (Me)Val

D-Val O D-Val O Thr Thr O peptide O O CO2H CO2H COAMP NH2 SAM NH2 ATP NH2 NH2 x 2 N NH2

OH OH OH OH O O

3-hydroxyanthranilic 3-hydroxy-4-methyl- dactinomycin acid anthranilic acid (actinomycin D)

Figure 7.28

(Continues) PEPTIDE TOXINS 433

(Continued)

PEPTIDE TOXINS

Death Cap (Amanita phalloides)

The death cap, Amanita phalloides, is a highly poisonous European fungus with a mushroom-like fruiting body. The death cap has a whitish-green cap, and white gills. It has a superficial similarity to the common mushroom, Agaricus campestris,andmay sometimes be collected in error. Some 90% of human fatalities due to mushroom poisoning are attributed to the death cap. Identification of the death cap as a member of the genus Amanita, which includes other less poisonous species, is easily achieved by the presence of a volva at the base of the stem. This cuplike membranous structure is the remains of the universal veil in which the immature fruiting body was enclosed. Ingestion of the death cap produces vomiting and diarrhoea during the first 24 hours, followed after 3–5 days by coma and death. Some recoveries do occur, but the fatality rate is probably from 30 to 60%. There is no guaranteed treatment for death cap poisoning, though removal of material from the gastrointestinal tract, replacement of lost fluids, blood dialysis, and blood transfusions may be undertaken. The antihepatotoxic agent silybin (see page 153) has been used successfully. The toxic principles are cyclic polypeptides, which bring about major degeneration of the liver and kidneys. At least ten toxins have been identified, which may be subdivided into two groups, the phallotoxins and the amatoxins. The most extensively studied compounds are phalloin, a phallotoxin, and α-amanitin, an amatoxin (Figure 7.29). The phallotoxins are much less toxic than the amatoxins since after ingestion they are not well absorbed into the blood stream. When injected, they can cause severe damage to the membranes of liver cells, releasing potassium ions and enzymes. The amatoxins are extremely toxic when ingested, with a lethal dose of 5–7 mg for an adult human, and an average specimen of the death cap containing about 7 mg. The amatoxins cause lesions to the stomach and

O H N Ala Trp (γ-OH)Ile Pro Phe Phe Val Pro

S Ala HPro Pro Phe Phe Ala Pro N Cys Thr O S H antamanide phalloin Trp

HN Cys O H N [γ,γ-(OH)2]Ile (6-OH)Trp OH Gly O HPro S O O S N Ile H Asn Cys Gly (6-OH)Trp α -amanitin HN Cys

Figure 7.29

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(Continued)

intestine, and then irreversible loss of liver and kidney function. α-Amanitin has been shown to be a powerful inhibitor of RNA polymerase, blocking the elongation step. Structurally, the two groups of toxins appear similar. They both contain a γ-hydroxylated amino acid essential for toxic action, and have a sulphur bridge between a cysteine residue and the 2-position of a tryptophan or 6-hydroxytryptophan residue (Figure 7.29). In the case of the phallotoxins, this is a simple sulphide bridge, but in the amatoxins it is in the form of a sulphoxide. Interestingly, a rather more simple cyclic peptide antamanide (anti-amanita pep- tide) has also been isolated from Amanita phalloides. When pre-administered to laboratory animals, antamanide (Figure 7.29) provided prophylactic protection from the lethal affects of the phallotoxins.

Ricin The distinctive mottled-brown seeds of the castor oil plant Ricinus communis (Euphorbiaceae) are crushed to produce castor oil, which is predominantly composed of glycerides of ricinoleic acid (see page 47 and Table 3.2). The seed itself and the seed cake remaining after expression of the oil are highly toxic, due to the presence of polypeptide toxins, termed ricins. One or more forms of ricin are present according to the strain of plant. Seeds typically contain about 1mg g−1 ricin, representing about 5% of the protein content. The toxicity of ricins to mammals is so high that the ricin content of one seed (about 250 µg) is sufficient to kill an adult human, though, because of considerable variations in absorption and metabolism, consumption of a single seed might not be fatal, but would certainly lead to severe poisoning. The toxic symptoms include irritation and haemorrhage in the digestive tract, leading to vomiting and bloody diarrhoea, with subsequent convulsions and circulatory collapse. If death does not occur within 3–5 days, the patient may recover. Ricin is probably the best studied of a group of polypeptide toxins, known as ribosome- inactivating proteins (the acronym RIP seems most appropriate!). These toxins are potent inhibitors of eukaryotic protein biosynthesis by virtue of their cleavage of an N-glycosidic bond at a specific nucleotide residue in the 28S molecule of ribosomal RNA, which is part of the larger (60S) subunit of the eukaryotic ribosome. RIPs fall into two types. Type I proteins comprise a single polypeptide chain, sometimes glycosylated. Type II proteins have two chains linked by a disulphide bond: an A chain is essentially equivalent to type I proteins, whilst the B chain functions as a lectin, which means it has high affinity for specific sugar groups, in this case sugars (especially galactose) in glycolipids or glycoproteins on a cell membrane. Thus the B chain binds to the cell membrane, and, in so doing, facilitates entry of the A chain into the cytosol, where it inactivates the 60S ribosomal subunit and rapidly stops protein biosynthesis. The A chain is thus the toxic principle, but it is nontoxic to intact cells, and requires the B chain for its action. Ricin is a type II toxin. The A chain (ricin A) contains 267 amino acid residues, and the B chain (ricin B) 262 residues. Ricin A is exceptionally toxic, and it has been estimated that a single molecule is sufficient to kill an individual cell. This peptide can be prepared by genetic engineering using Escherichia coli. The potent action of this material on eukaryotic cells has been investigated in anticancer therapy. Ricin A has been coupled to monoclonal antibodies and successfully delivered specifically to the tumour cells. However, in vitro toxicity of ricin A-based immunotoxins is enhanced significantly if ricin B is also present.

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(Continued)

Similar toxic RIPs are found in other plants. Examples are trichosanthin, a type I toxin from the root tubers of Trichosanthes kirilowii (Cucurbitaceae), abrin, a type II toxin from the small brightly coloured red and black jequirity seeds (Abrus precatorius; Leguminosae/Fabaceae), and viscumin, a type II toxin from the leaf and stems of mistletoe (Viscum album, Loranthaceae).

Botulinum Toxin The Gram-positive bacterium Clostridium botulinum produces one of the most toxic materials known to man, botulinum toxin. Poisoning by the neurotoxins from this source, known as botulism, is not uncommon, and is a life-threatening form of food poisoning. It has been estimated that as many as 50 million people could be killed by one gram of the toxin. Clostridium botulinum is an anaerobic organism that is significantly heat resistant, though botulinum toxin is easily destroyed by heat. Food poisoning is almost always associated with foods such as canned meats and fish that have been incompletely sterilized, allowing growth of the bacterium, after which the food is then consumed without further cooking. Botulinum toxin is an extremely potent neurotoxin that acts by blocking calcium-dependent acetylcholine release at the peripheral neuromuscular junctions. Poisoning results in paralysis and death from respiratory failure. Damage to the nervous system is usually preceded by vomiting, diarrhoea, and severe abdominal pains. Seven different neurotoxins, types A–G, have been characterized, though only four of these, types A, B, E, and F, are clearly associated with human poisoning. A particular strain of the bacterium usually produces only one type of toxin. Each of these proteins is a single chain polypeptide (mass about 150 kDa) containing two subunits, a ‘light’ subunit with a mass of about 50 kDa, and a ‘heavy’ subunit with a mass of about 100 kDa. These subunits are linked by at least one disulphide bridge. The heavy subunit is responsible for toxin binding, whilst the light subunit possesses zinc metalloprotease activity, cleaving one of the proteins involved in the docking and release of synaptic vesicles. There is considerable structural similarity between botulinum toxins and tetanus toxin. Botulism antitoxin is available for the treatment of botulism food poisoning. This is a mixture of globulins raised against types A, B, andEtoxins. Botulinum toxin A complexed with haemagglutinin is currently employed medicinally to counter involuntary facial muscle spasms, e.g. around the eye. Very small (nanogram) amounts are injected locally and result in the destruction of the acetylcholine release mechanism at the neuromuscular junction. Since new nerve junctions will gradually be formed over two months or so, the result is not permanent, and the treatment will need to be repeated. It has also been found useful in easing muscle spasticity in children with cerebral palsy.

Microcystins

The microcystins are a group of some65 cyclic heptapeptides, produced by certain fresh water blue–green algae (cyanobacteria), including Microcystis aeruginosa, M. viridis, Anabaena flos- aquae, and others. These organisms form blooms on the surface of lakes and reservoirs during periods of calm hot weather, and can pollute drinking water for animals and humans. The microcystins cause acute hepatotoxicity and liver haemorrhage, and have been

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(Continued)

D-Glu Mhda

CO2H Me O N HN NH D-Ala Adda O OMe O O NH HN H H N N NH O Leu O O H2N N CO2H H CO2H Arg D-Masp O microcystin-LR Mhda = N-methyldehydroalanine NH 2 Masp = 3-methylaspartic acid Adda = 3-amino-9-methoxy-2,6,8-trimethyl L-Phe L-Met H3CCO2H 10-phenyldeca-4,6-dienoic acid

Figure 7.30

responsible for numerous animal deaths, though there is much less risk of human fatalities. They strongly inhibit protein phosphatases, and this activity may be associated with the hepatotoxicity and the promotion of liver tumours. The microcystins contain a mixture of D- and L-amino acids, and mainly differ in their combination of L-amino acids. The most frequently encountered compound, microcystin-LR (Figure 7.30) (LR = leucine–arginine) exemplifies the structures. Unusual amino acids are N-methyldehydroalanine (from N-methylserine), 3- methylaspartic acid (which originates from acetate and pyruvate), and the long chained aromatic-containing Adda. This amino acid is elaborated from phenylalanine, which supplies aC6C2 unit to act as a starter unit for a polyketide chain. Methyl substituents are provided by SAM (Figure 7.30).

Snake Venoms It is estimated that some 1300 of the 3200 species of snake are venomous. Snake venoms are used to immobilize prey, and to facilitate its digestion. Most of the material is polypeptide in nature, and can include enzymes and polypeptide toxins. A number of enzymes have been identified in all venoms, and these include hyaluronidase (see Table 7.2), which facilitates the distribution of the other venom components through the tissues. Peptidases, phosphodiesterases, phospholipases, ribonuclease, and deoxyribonuclease are all hydrolytic enzymes designed to digest the tissue of the prey. Some enzymes induce direct toxic effects, for example L-amino acid oxidase liberates hydrogen peroxide, a powerful oxidizing agent. In some venoms, the enzyme acetylcholinesterase disturbs the normal physiological response of the prey by hydrolysing acetylcholine. Major groups of polypeptide toxins found in snake venoms may be classified as neurotoxins, cytotoxins (or cardiotoxins), dendrotoxins, proteinase inhibitors, or acetylcholinesterase inhibitors. α- Neurotoxins (curaremimetic neurotoxins) found in many mambas (Dendroaspis) and cobras (Naja) are capable of interacting with nicotinic acetylcholine receptors in the postsynaptic membranes of skeletal muscles, leading to paralysis, an action similar to that of curare (see page 324). κ-Neurotoxins, on the other hand, are selective for neuronal nicotinic acetylcholine receptors. Typically, the neurotoxins contain 60–74 amino acid residues with four or five

(Continues) MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 437

(Continued)

disulphide bridges. Cytotoxins are present in cobra (Naja) and ringhal (Hemachatus) venoms and produce a rapid effect on the heart and circulation though the mode of action is not well established. Most have 60–62 amino acid residues. Dendrotoxins from mambas are characterized by their ability to facilitate the release of acetylcholine from nerve endings, and also act as highly potent and selective blockers of potassium channels. These contain about 60 amino acid residues, with three disulphide bridges. Anticholinesterase toxins have also been found in mamba venoms, typically with about 60 amino acids and four disulphide bridges.

MODIFIED PEPTIDES: PENICILLINS, valine residue is also inverted to the D-form. CEPHALOSPORINS, AND OTHER (Caution: ACV is an acronym, and does not β-LACTAMS refer to the systematic abbreviation described on page 407; ACV is actually δ-(L-α-aminoadipyl)- Penicillins L-cysteinyl-D-valine). ACV is then cyclized to isopenicillin N, with a single enzyme catalysing The penicillins∗ are the oldest of the clinical formation of the bicyclic ring system of the peni- antibiotics, and are still the most widely used. cillins. The reaction is oxidative and requires The first of the many penicillins to be employed molecular oxygen, and there is evidence that on a significant scale was penicillin G (ben- the four-membered β-lactam ring is formed first. zylpenicillin) (Figure 7.31), obtained from the The mechanism shown in Figure 7.31 is given fungus Penicillium chrysogenum by fermentation to simplify what is quite a complex reaction. in a medium containing corn-steep liquor. Peni- Penicillin G differs from isopenicillin N by the cillins contain a β-lactam-thiazolidine structure, nature of the side-chain attached to the 6-amino which has its biosynthetic origins in a tripep- group. The α-aminoadipyl side-chain of isopeni- tide, the components of which are L-aminoadipic cillin N is removed and replaced by another acid, formed in β-lactam-producing organisms according to its availability from the fermenta- from lysine via piperideine-6-carboxylic acid (see tion medium. Phenylethylamine in the corn-steep page 311), together with L-cysteine, and L-valine liquor medium was transformed by the fungus (Figure 7.31). Non-ribosomal peptide assembly into phenylacetic acid, which then reacted as its then leads to the tripeptide known as ACV, but, coenzyme A ester to produce the new amide during the condensation, the configuration of the penicillin G. Other penicillins are accessible by

Penicillins Commercial production of benzylpenicillin (penicillin G) (Figure 7.31) is by fermentation of selected high-yielding strains of Penicillium chrysogenum in the presence of phenylacetic acid. Although benzylpenicillin was the earliest commercially available member of the penicillin group of antibiotics, it still remains an important and useful drug for the treatment of many Gram-positive bacteria, including streptococcal, pneumococcal, gonococcal, and meningococcal infections. Benzylpenicillin is destroyed by gastric acid, and is thus not suitable for oral administration, and is best given as intramuscular or intravenous injection of the water-soluble sodium salt. Decomposition under acidic conditions leads to formation of penicillic acid and/or penicillenic acid, depending on pH (Figure 7.32). The β-lactam ring is opened by a mechanism in which the side-chain carbonyl participates, resulting in formation of an oxazolidine ring. Penicillic acid arises as the result of nucleophilic attack of the thiazolidine nitrogen on to the iminium function, followed by expulsion of the carboxylate leaving group.

(Continues) 438 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

+ NAD H2N CO2H CO H HO2CNHNH2 2 HO2C NH2 O HO2C N 2 L-Lys α-aminoadipic acid piperideine- L-α-aminoadipic acid δ-semialdehyde 6-carboxylic acid

A: adenylation domain Module 1 Module 2 Module 3 release PCP: peptidyl carrier protein domain C: condensation domain A PCP C A PCP C A PCP C E TE E: epimerization domain (Val module only) TE: thioesterase domain S S S H O O O H2N N SH i ii H N SH H N CO2HO NH 2 2 peptide bond formation; during O D L-Cys assembly, the L-valine residue is L-Val CO H H2N epimerized to the D-configuration ACV 2 CO2H O L-α-aminoadipic acid OH 2 Fe Enz O O H H H N S N SFeEnz N S Fe Enz

N N NH possible free radical O O O initiated cyclization CO H oxidation to 2 CO2H CO2H electrophilic C=S allows cyclization to β-lactam α H H -aminoadipic acid H H2N N S H2N S

CO2HO N hydrolysis of amide N O bond O CO2H CO2H PhCH CO H isopenicillin N 2 2 activation of acid 6-aminopenicillanic to CoA ester and acyl- acid (6-APA) transferase amide formation PhCH2COSCoA PhCH2COSCoA

α -aminoadipic acid H H 1 N 6 H 5 S N 2 RCO S O N 3 NH 7 O O 4 N CO H O HO C β-lactam 2 2 benzylpenicillin (penicillin G)

Figure 7.31

(Continued)

Alternatively, elimination of thiol accounts for formation of penicillenic acid. At higher pHs, benzylpenicillin suffers simple β-lactam ring opening and gives penicilloic acid (Figure 7.32). The strained β-lactam (cyclic amide) ring is more susceptible to hydrolysis than the unstrained side-chain amide function, since the normal stabilizing effect of the lone pair from the adjacent nitrogen is not possible due to the geometric restrictions (Figure 7.33).

(Continues) MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 439

(Continued)

H H H nucleophilic attack of amide oxygen on to N NH β S H S strained -lactam carbonyl with subsequent ring opening; the presence of O N pH < 5 O N electron-withdrawing heteroatoms in the O O side-chain will inhibit this reaction CO H H CO H benzylpenicillin 2 2

β HO or -lactamase elimination of pH > 8 (penicillinase) thiol leads to conjugation NH N H H H O H b O N S S HS a H b pH 4 O HN nucleophilic O N O HN HO2C attack of amine H CO2H on to iminium ion CO2H CO2H penicilloic acid penicillenic acid a pH 2 base-catalysed hydrolysis of strained β-lactam ring expulsion of H HO2C H S carboxylate as leaving S group HN OCO N N N CO H 2 CO2H

penicillic acid

Figure 7.32

H H H H R N S R N S

O N O N O O CO2H CO2H resonance involving the amide lone pair this resonance form is sterically normally decreases carbonyl character and impossible; the β-lactam carbonyl thus stabilizes the carbonyl against attack function is thus reactive towards by nucleophilic reagents nucleophilic reagents

Figure 7.33

Supplementation of the fermentation medium with acids other than phenylacetic acid was used to provide structurally modified penicillins, though the scope was limited to series of monosubstituted acetic acids by the specificity of the fungal enzymes involved in the activation of the acids to their coenzyme A esters. The most important new penicillin produced was phenoxymethylpenicillin (penicillin V), a result of adding phenoxyacetic acid to the culture (Figure 7.34). This new penicillin had the great advantage of being acid resistant, the introduction of an electron-withdrawing heteroatom into the side-chain inhibiting participation of the side-chain carbonyl in the reaction shown in Figure 7.32. Thus, penicillin V is suitable for oral administration, and still has particular value for respiratory tract infections and tonsillitis.

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(Continued)

OCO2H

H phenoxyacetic acid H H H2N S N O S N Penicillium O N O chrysogenum O CO2H culture CO2H 6-APA phenoxymethylpenicillin (penicillin V)

Figure 7.34

A much wider range of penicillins, many of which have become clinically useful, may be produced by semi-synthesis from 6-aminopenicillanic acid (6-APA). A multi-stage, but high-yielding, procedure has been developed to chemically hydrolyse a primary fermentation product like benzylpenicillin to 6-APA (Figure 7.35). This exploits the ability of the side-chain amide to adopt a resonance form, thus allowing conversion into an imidyl chloride, and then an imidyl ether, which is readily hydrolyzed. A new side-chain can then be added by simple esterification (Figure 7.35). Hydrolysis of penicillin G or penicillin V may also be accomplished enzymically in very high yield by using bacterial enzyme preparations from Escherichia coli, or species of Fusarium or Erwinia. Certain strains of Penicillium chrysogenum accumulate 6-aminopenicillanic acid, so that this compound may be produced by fermentation, though this is commercially less economic than the hydrolysis approach. Clinically useful penicillins produced by semi-synthesis or total synthesis are listed in Table 7.3. Penicillins with side- chains containing a basic amino group, e.g. ampicillin and amoxicillin (amoxycillin), are

silylation of carboxyl as chlorination of side-chain amide protecting group via imidyl alcohol tautomer H H H H H N N N S Me2SiCl2 S PCl5 S O N O N Cl N O O O CO2H CO2SiMe2Cl CO2SiMe2Cl benzylpenicillin imidyl chloride

BuOH

enzyme H N (penicillin acylase) H2O S OBu N O H RCOCl H H CO SiMe Cl H N 2 2 2 S R N S imidyl ether

N O N O O CO2H CO2H 6-APA

Figure 7.35

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(Continued)

Table 7.3 Semi-synthetic and synthetic penicillins

H H 1 R N S

O N O 2 CO2R R1 R2 Name Notes

Ph H benzylpenicillin acid sensitive (penicillin G) Ph O H phenoxymethyl- acid resistant penicillin (penicillin V)

NH2 H ampicillin acid resistant, broad spectrum Ph

NH2 −CH2OCOCMe3 pivampicillin pro-drug of ampicillin; better Ph absorbed, and then hydrolysed by esterases NH 2 RS bacampicillin pro-drug of ampicillin; better −CHMeOCO Et Ph 2 absorbed, and then hydrolysed by esterases

NH2 H amoxicillin acid resistant, broad (amoxycillin) spectrum; better absorption HO than ampicillin

CO2H RS H carbenicillin acid sensitive, broad Ph spectrum; active against Pseudomonas aeruginosa

CO2H RS H ticarcillin acid sensitive, broad spectrum; 2 × more active S than carbenicillin; used in combination with β-lactamase inhibitor clavulanic acid O O O HN N × NEt H piperacillin broad spectrum, 8–10 more active than carbenicillin against P. aeruginosa;used by injection for serious infections (continued overleaf )

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Table 7.3 (continued)

H H 1 R N S

O N O 2 CO2R R1 R2 Name Notes

O O HN N NH H azlocillin broad spectrum, more active than ticarcillin against P. aeruginosa;usedbyinjection for serious infections

H N N S

N pivmecillinam orally active pro-drug of O O mecillinam; β-lactamase OOO sensitive, active against Gram-negative organisms (except P. aeruginosa) OMe

H methicillin β-lactamase resistant, acid sensitive OMe O N H cloxacillin β-lactamase and acid resistant

Cl O F N H flucloxacillin β-lactamase and acid resistant

Cl

CO H 2 H OMe RS H N S temocillin β-lactamase resistant; active O N S O against Gram-negative (except CO2H P. aeruginosa) but not Gram-positive organisms

also acid resistant, since this nitrogen is protonated in preference to the lactam nitrogen. In addition, these agents were found to have a broader spectrum of activity than previous materials, in particular, activity against some Gram-negative bacteria which were not affected by penicillins G and V. The polar side-chain improves water-solubility and cell penetration into these microorganisms. Amoxicillin shows better oral absorption properties

(Continues) MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 443

(Continued) than ampicillin, though this can also be achieved by using the pro-drugs pivampicillin and bacampicillin. These are acyloxymethyl esters through the thiazolidine carboxyl, and are hydrolysed to ampicillin by esterases in the gut. Broad spectrum activity is also found with penicillins containing a carboxyl group in the side-chain, e.g. carbenicillin and ticar- cillin, and, although these compounds are still acid sensitive and are thus orally inactive, they demonstrate activity against pseudomonads, especially Pseudomonas aeruginosa. The activity of these agents is rather low, and it is necessary to inject very large doses. The acylureido penicillins azlocillin and piperacillin are much more active against Pseudomonas aeruginosa, and are also active against other Gram-negative bacteria such as Klebsiella pneumoniae and Haemophilus influenzae. Pivmecillinam is an acyloxymethyl ester pro-drug, and is hydrolysed to mecillinam after oral ingestion. It is unusual in being an amidino derivative instead of having an acyl side-chain; it has significant activity towards many Gram-negative bacteria. Despite the dramatic successes achieved with the early use of penicillin antibiotics, it was soon realised that many bacteria previously susceptible to these agents were subsequently able to develop resistance. The principal mechanism of resistance lies in the ability of organisms to produce β-lactamase (penicillinase) enzymes capable of hydrolysing the β- lactam ring in the same manner as shown for the base-catalysed hydrolysis in Figure 7.32. Several distinct classes of bacterial β-lactamases are recognized, the main division being into serine enzymes and zinc enzymes. The former have an active site serine residue, which attacks the β-lactam carbonyl, forming an acyl–enzyme intermediate. On the basis of characteristic amino acid sequences they are then subdivided into three classes, A, C, and D. The zinc metallo-enzymes form class B, and appear to involve only non- covalently bound intermediates. Class A β-lactamases are the most common amongst pathogenic bacteria. Most staphylococci are now resistant to benzylpenicillin. The discovery of new penicillins that were not hydrolysed by bacterial β-lactamases was thus a major breakthrough. Methicillin (Table 7.3), though no longer used, was the first commercial β- lactamase-resistant penicillin, and the steric bulk of the side-chain appears to contribute to this valuable property, hindering the approach of β-lactamase enzymes. Methicillin is acid sensitive, since it lacks an electron-withdrawing side-chain, but other penicillins were developed that combined bulk and electron-withdrawing properties, and could thus be used orally. These include a group of isoxazole derivatives termed the oxacillins, of which cloxacillin and flucloxacillin are first-choice agents against penicillin-resistant Staphylococcus aureus. Temocillin also has excellent resistance to β-lactamases as well as high activity towards Gram-negative organisms. It differs from all the other penicillins described in possessing a 6α-methoxyl group (compare the cephamycins, page 450). Another way of overcoming the penicillin-degrading effects of β-lactamase is to combine a β- lactamase-sensitive agent, e.g. amoxicillin or ticarcillin, with clavulanic acid (see page 452), which is a specific inhibitor of β-lactamase. Other mechanisms of resistance which have been encountered include modification of the binding sites on penicillin-binding proteins (see below), thus reducing their affinity for the penicillin, and decreased cell permeability, leading to reduced uptake of the antibiotic. Strains of Staphylococcus aureus resistant to both methicillin and isoxazolylpenicillins, e.g. cloxacillin and flucloxacillin, are known to have modified and insensitive penicillin-binding proteins; such strains are termed methicillin- resistant Staphylococcus aureus (MRSA). Penicillins and other β-lactam drugs exert their antibacterial effects by binding to proteins (penicillin-binding proteins) that are involved in the late stages of the biosynthesis of

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(Continued)

Cross-linking in peptidoglycan biosynthesis:

nucleophilic attack of active site serine formation of new amide bond at group on enzyme hydrolyses amide bond expense of ester hydrolysis H H peptide H peptide N Me peptide N Me H2N peptide N Me H O O N Me O O peptide O ON growing peptide chain: O O H H peptide–D-Ala–D-Ala H CO2H O Enzyme Ser cross-linked peptide chains: OH peptide–D-Ala–Gly–peptide

Enzyme Ser

Enzyme inhibition by β-lactams:

H common substructure H in β-lactam antibiotics H H R N R N

O N O HN O O CO H O hydrolysis of β-lactam H 2 CO2H penicillin / cephalosporin amide bond becomes irreversibly bound OH Enzyme Ser to enzyme Enzyme Ser

Figure 7.36

the bacterial cell wall. Cross-linking of the peptidoglycan chains which constitute the bacterial cell wall (see page 473) involves an acyl-D-Ala–D-Ala intermediate, which in its transition state conformation closely resembles the penicillin molecule (Figure 7.36). As a result, the penicillin occupies the active site of the enzyme, and becomes bound via an active site serine residue, this binding causing irreversible enzyme inhibition, and cessation of cell wall biosynthesis. Growing cells are then killed due to rupture of the cell membrane and loss of cellular contents. This binding reaction between penicillin-binding proteins and penicillins is chemically analogous to the action of β-lactamases, but in the latter case penicilloic acid is subsequently released from the β-lactamase, which can continue to function. The penicillins are very safe antibiotics for most individuals. The bacterial cell wall has no counterpart in mammalian cells, and the action is thus very specific. However, a significant proportion of patients can experience allergic responses ranging from a mild rash to fatal anaphylactic shock. Cleavage of the β-lactam ring through nucleophilic attack of an amino group in a protein is believed to lead to the formation of antigenic substances causing the allergic response. supplying different acids. The new amide link may Cephalosporins be achieved in two ways. Hydrolysis of isopeni- cillin N releases the amine 6-aminopenicillanic The cephalosporins∗,e.g.cephalosporin C acid (6-APA), which can then react with the coen- (Figure 7.37), are a penicillin-related group zyme A ester. Alternatively, an acyltransferase of antibiotics having a β-lactam-dihydrothiazine enzyme converts isopenicillin N into penicillin G ring system, and are produced by species of directly, without 6-aminopenicillanic acid actually Cephalosporium. The six-membered dihydroth- being released from the enzyme. iazine ring is produced from the five-membered MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 445

H H H H H S R H2N N S epimerase H2N N S S

CO2HO N CO2HO N N O O CO H CO H CO H isopenicillin N 2 penicillin N 2 2 ring expansion; hydroxylation of O2 a free radical methyl 2-oxoglutarate mechanism may occur O H H 2 H H H H2N N S 2-oxoglutarate H2N N S S

CO2HO N OH CO2HO N N O O CO H H2NCOP 2 CO2H CO2H desacetylcephalosporin C carbamoyl P

O2 / 2-oxoglutarate hydroxylation / methylation to acetyl-CoA introduce 7α-methoxy esterification SAM OMe H 1 H H H H2N N 7 6 S H2N N S 2 3 CO HO N 4 O CO HO N ONH 2 8 2 2 O 5 O CO2H O CO2H O cephalosporin C cephamycin C

Figure 7.37

thiazolidine ring of the penicillin system by an in Figure 7.37 to rationalize the transformation. oxidative process of ring expansion, incorporating Hydroxylation of the remaining methyl gives one of the methyl groups. The pathway desacetylcephalosporin C. Cephalosporin C is (Figure 7.37) diverges from that to penicillins the acetyl ester of this, whilst a further group at isopenicillin N,whichisfirst epimerized in of antibiotics termed the cephamycins∗ are char- the α-aminoadipyl side-chain to give penicillin acterized by a 7α-methoxy group, and are pro- N. Ring expansion then occurs, incorporating duced by hydroxylation/methylation, and, in the one of the methyls into the heterocyclic ring, case of cephamycin C, introduction of a carba- though the mechanism for this is not clearly mate group from carbamoyl phosphate on to the defined. A free radical mechanism is suggested hydroxymethyl function.

Cephalosporins

Cephalosporin C (Figure 7.37) is produced commercially by fermentation using cultures of a high-yielding strain of Acremonium chrysogenum (formerly Cephalosporium acremonium). Initial studies of the antibiotic compounds synthesized by C. acremonium identified penicillin N (originally called cephalosporin N) as the major component, with small amounts of cephalosporin C. In contrast to the penicillins, cephalosporin C was stable under acidic conditions and also was not attacked by penicillinase (β-lactamase). Antibacterial activity was rather low, however, and the antibiotic was poorly absorbed after oral administration. However, the structure offered considerable scope for side-chain modifications, more so than with the penicillins since it has two side-chains, and this has led to a wide variety of cephalosporin drugs, many of which are currently in clinical use. As with the penicillins, removal of the amide

(Continues) 446 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

H H H 1 H2N N S chemically H2N 7 6 S (see Figure 7.35) 2 8 3 CO2H O N O N 4 O O O 5 CO2H O CO2H O cephalosporin C 7-aminocephalosporanic acid (7-ACA)

Figure 7.38

side-chain by the hydrolysis of cephalosporin C to 7-aminocephalosporanic acid (7-ACA) (Figure 7.38) was the key to semi-synthetic modifications, and this may be achieved chemically by the procedure used for the penicillins (compare Figure 7.35). Removal of this side-chain by suitable microorganisms or enzymes has proved elusive. The ester side-chain at C-3 may be hydrolysed enzymically by fermentation with a yeast, or, alternatively, the acetoxy group is easily displaced by nucleophilic reagents. It is also possible to convert readily available benzylpenicillin into the deacetoxy derivative of 7-ACA through a chemical ring expansion process and enzymic removal of the side-chain. The semi-synthetic cephalosporins may be classified according to chemical structure, antibacterial spectrum, or β-lactamase resistance, but in practice, they tend to be classi- fied by a more abitrary system, dividing them into ‘generations’ (Table 7.4). Note that all the cephalosporin antibiotics begin with the prefix ceph-orcef-, the latter spelling now being preferred, though both spellings are still encountered for some drugs. The classifi- cation into generations is based primarily on the antibacterial spectrum displayed by the drugs but it is also more or less related to the year of introduction. However, drugs in the second generation may have been introduced after the third generation of drugs had been established. There is no intention to suggest that third generation drugs automatically supersede second and first generation drugs, and, indeed, agents from all generations are still currently used. First generation cephalosporins, e.g. cefalotin (cephalothin), cefalexin (cephalexin), cefradine (cephradine), cefadroxil,andcefazolin (cephazolin) have good activity against Gram-positive bacteria but low activity against Gram-negative organisms. They have comparable activity to ampicillin, and are effective against penicillinase-producing Staphylococcus. However, another β-lactamase (cephalosporinase) developed that inacti- vated these agents. Cefalotin, the first modified cephalosporin to be marketed, is poorly absorbed from the gut, and is thus not orally active. However, cefalexin, cefradine, and cefadroxil may be administered orally, a property that appears to be related to the 3-methyl side-chain. Second generation cephalosporins show a broader spectrum of activity, and are more active against aerobic Gram-negative bacteria like Haemophilus influenzae and Neisse- ria gonorrhoeae. This group of antibiotics includes cefaclor, cefuroxime,andcefamandole (cephamandole), and in general displays better resistance to β-lactamases that inacti- vated first generation cephalosporins. The third generation of cephalosporin antibiotics, e.g. cefotaxime, ceftazidime, ceftizoxime, cefodizime,andceftriaxone, have an extended Gram-negative spectrum, and are most active against enteric Gram-negative bacilli, but may be less active against some Gram-positive bacteria, especially Staphylococcus aureus.Many of the third generation cephalosporins are characterized by an aminothiazole ring on the amide side-chain, which appears to impart the high activity against Gram-negative bacteria. The O- substituted oxime group also improves potency, and confers resistance to β-lactamases. The oximes with syn stereochemistry as shown in Table 7.4 are considerably more active than the

(Continues) MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 447

(Continued)

Table 7.4 Cephalosporin antibiotics

H H R1 N S

O N 2 O R CO2H R1 R2 Name Notes First generation

2 CH2OCOMe cefalotin R group unstable to S (cephalothin) mammalian esterases; generally superseded

NH2

CH3 cefalexin orally active (cephalexin)

NH2

CH3 cefradine orally active; generally (cephradine) superseded

NH2

CH3 cefadroxil orally active

HO

S S N N cefazolin generally superseded N N NN (cephazolin) Second generation NH2

Cl cefaclor orally active

HO

NH2

CH CH CH3 cefprozil orally active

HO OMe N

CH2OCONH2 cefuroxime high resistance to β-lactamases; resistant to O mammalian esterases OH Me S N N cefamandole high resistance to (cephamandole) β-lactamases NN (continued overleaf )

(Continues) 448 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

Table 7.4 (continued)

H H R1 N S

O N 2 O R CO2H R1 R2 Name Notes Third generation OMe N

N CH2OCOMe cefotaxime unstable to mammalian

H2N esterases, but desacetyl S metabolite still has considerable antimicrobial activity OMe N N H ceftizoxime broad-spectrum Gram-negative

H2N activity; generally superseded S O N CO2H N N ceftazidime broad-spectrum Gram-negative H2N activity; good activity towards S Pseudomonas OMe N N cefpirome

H2N N S OMe N S S CO2H N cefodizime broad-spectrum Gram-negative N H2N activity S OMe N S NO N ceftriaxone broad-spectrum Gram-negative H N N activity; longer half-life than 2 MeN OH S other cephalosporins

O CO2H N

N CH=CH2 cefixime 2nd/3rd generation, orally H2N active; long duration of action S

CO2H N H ceftibuten 2nd/3rd generation, orally H2N active; generally superseded S (continued)

(Continues) MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 449

(Continued)

Table 7.4 (continued)

H H R1 N S

O N 2 O R CO2H R1 R2 Name Notes Pro-drugs OMe N H H N S

OONHO N 2 cefuroxime- 2nd generation, orally O axetil active; hydrolysed by O OOO esterases to liberate cefuroxime O

OMe N H H N N S H N 2 O N OMe S cefpodoxime 2nd/3rd generation, O -proxetil orally active; hydrolysed OOO by esterases to liberate cefpodoxime OO OMe H H R1 N S

O N 2 O R CO2H R1 R2 Name Notes Cephamycins H2N CH2OCONH2 cephamycin C CO2H

CH2OCONH2 cefoxitin stable to β-lactamases S and mammalian esterases Carbacephems NH2 H H N loracarbef improved chemical O N O Cl stability, longer half-life, CO2H better oral bioavailability compared to cefaclor

(Continues) 450 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

(Continued)

anti isomers. A disadvantage of many of the current cephalosporin drugs is that they are not efficiently absorbed when administered orally. This is to some extent governed by the nature of the side-chain on C-3. The orally active cefixime and ceftibuten have spectra of activity between the second and third generations. Orally active pro-drugs such as cefuroxime-axetil and cefpodoxime-proxetil have been developed with an additional ester function on the C-4 carboxyl. These compounds are hydrolysed to the active agents by esterases. Cephalosporin antibiotics are especially useful for treating infections in patients who are allergic to penicillins. Hypersensitivity to cephalosporins is much less common, and only about 5–10% of penicillin-sensitive patients will also be allergic to cephalosporins.

Cephamycins

The antibiotic cephamycin C (Figure 7.37) was isolated from Streptomyces clavuligerus and showntohavea7α-methoxy group on the basic cephalosporin ring system. Although cephamycin C and other natural cephamycins have only weak antibacterial activity, they are resistant to β-lactamase hydrolysis, a property conferred by the increased steric crowding due to the additional methoxy group. Semi-synthetic analogues have been obtained either by modification of the side-chains of natural cephamycins, or by chemical introduction of the 7α- methoxyl. Currently, the only cephamycin in general use is cefoxitin (Table 7.4), which is active against bowel flora including Bacterioides fragilis, and is used for treatment of peritonitis.

Carbacephems

Although cephalosporin analogues where the sulphur heteroatom has been replaced with carbon are not known naturally (contrast carbapenems, natural penicillin analogues, page 451), synthetically produced carbacephems have shown good antibacterial activity with considerably improved chemical stability over cephalosporins. The first of these to be produced for drug use is loracarbef (Table 7.4), which has similar antibacterial activity to cefaclor, but considerably greater stability, a longer half-life, and better oral bioavailability.

Other β-Lactams cephem respectively (Figure 7.39). Other variants containing the basic β-lactam ring system are also β The fused -lactam skeletons found in peni- found in nature. Of especial importance is the cillins and cephalosporins are termed penam and clavam (Figure 7.39) or oxapenam fused ring sys- tem typified by clavulanic acid∗ (Figure 7.40) S S S from Streptomyces clavuligerus. The weak antibac- N N N N O O O O terial activity of clavulanic acid is unimportant, β-lactam penam cephem penem for this compound is valuable as an efficient β H R2 inhibitor of -lactamases from both Gram-positive 1 O R N and Gram-negative bacteria. Despite the obvious N N O N structural similarity between clavulanic acid and O O OSO3H the penicillins, they are not derived from com- carbapenem clavam monobactam mon precursors, and there are some novel aspects Figure 7.39 associated with clavulanic acid biosynthesis. All MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 451

β-lactam ring formation via OP acyl–AMP HO NH NH NH ATP H2N HO2C NH N O N NH2 N NH2 O N NH2 glyceraldehyde H H H CO2H CO2H CO2H 3-P L-Arg side-chain O2 2-oxoglutarate hydroxylation H hydrolysis of O NH2 O2 guanidine function 2-oxoglutarate OH OH NH N N N O O NH2 O N NH2 CO H H 2 CO2H CO2H dihydroclavaminic acid proclavaminic acid

O2 2-oxoglutarate oxidative deamination to 2 epimerizations H aldehyde H H H O NH 5 O O O O O OH 2 NADPH N N 3 N N O O O O CO2H CO2H CO2H CO2H clavaminic acid clavulanic acid epimerization at C-3 via keto–enol ring opening tautomerism H H O O O O

N N ring reforms, but in opposite O O stereochemical sense CO2H CO2H

Figure 7.40 carbons are provided by two precursors, argi- of chirality at C-5 (which actually retains the C-5 nine and most likely glyceraldehyde 3-P. Glyc- hydrogen) must invoke opening of the oxazolidine eraldehyde 3-P supplies the β-lactam carbons ring (Figure 7.40). whilst the α-amino group of arginine provides the Another variant on the penicillin penam ring β-lactam nitrogen. In contrast to the penicillins, system is found in a group of compounds termed the β-lactam ring is formed via an acyl-AMP carbapenems∗ (Figure 7.39), where the sulphur activated intermediate. The sequence of reactions heteroatom has been replaced by carbon. This shown in Figure 7.40 leads to the monocyclic β- is exemplified by thienamycin (Figure 7.43), an lactam proclavaminic acid, which is the substrate antibiotic isolated from cultures of Streptomyces for oxidative cyclization to provide the oxazolidine cattleya. The sulphur-containing side-chain is a ring then dehydrogenation to clavaminic acid.The feature of many of the natural examples, and this three 2-oxoglutarate-dependent oxidations in this may feature sulphide or sulphone moieties, e.g. sequence are all catalysed by a single enzyme. The the olivanic acids (Figure 7.44) from S. olivaceus. final transformation into clavulanic acid requires The olivanic acids are potent β-lactamase inhibi- oxidative deamination of the terminal amine to an tors, especially towards the cephalosporinases, aldehyde and then reduction to an alcohol, but, which are poorly inhibited by clavulanic acid. Only more intriguingly, the two chiral centres C-3 and the broad outlines of the pathways to carbapenems C-5 have to be epimerized. These reactions have are established (Figure 7.43). The fundamental yet to be fully clarified. The unsaturated aldehyde ring system is derived from glutamic acid and is easily susceptible to inversion of configuration acetate. It is suggested that an activated form of at C-3 via keto–enol tautomerism, but a change glutamic acid, e.g. γ-glutamyl phosphate, is chain 452 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

Clavulanic Acid

Clavulanic acid (Figure 7.38) is produced by cultures of Streptomyces clavuligerus,the same actinomycete that produces cephamycin C. Although it has only weak antibacterial activity, it is capable of reacting with a wide variety of β-lactamase enzymes, opening the β-lactam ring, in a process initially analogous to that seen with penicillins (Figure 7.41). However, binding to the enzyme is irreversible, and the β-lactamase is inactivated. It seems likely that the side-chain with its double bond may contribute to this effect, causing further ring opening to give intermediates which react at the active site of the enzyme (Figure 7.41). Thus, dihydroclavulanic acid is no longer an enzyme inhibitor. Clavulanic acid is usually combined with a standard penicillin, e.g. with amoxicillin (as co-amoxyclav) or with ticarcillin, to act as a suicide substrate and provide these agents with protection against β-lactamases, thus extending their effectivity against a wider range of organisms. Similar success has been achieved using the semi-synthetic penicillanic acid sulphone, sulbactam (Figure 7.42), which is also a potent inhibitor of β-lactamase by a similar double ring opening mechanism (Figure 7.42). Sulbactam was used in combination with ampicillin, but has been superseded by the related sulphone tazobactam (Figure 7.42), which is a β-lactamase inhibitor used in combination with piperacillin.

nucleophilic attack of active-site serine on to β-lactam; subsequent Enz nucleophile on enzyme ring cleavage is facilitated by side-chain double bond attacks iminium ion and gives Nu irreversible binding H H OH O OH O O N N NH inactivation H OH O O H O H CO H O CO2H 2 O CO2H OH clavulanic acid Ser Ser Ser β-lactamase (class A)

Figure 7.41

H O O the sulphone group facilitates ring opening; S note that S=O is not quite analogous to C=O since d orbitals on sulphur are involved Enz N Nu O H O O H O O O O CO H 2 S S S sulbactam N N NH H O O N O O O N H CO H H CO H CO H S N 2 O 2 O 2 OH sulbactam N Ser Ser O Ser CO2H tazobactam β-lactamase inactivation

Figure 7.42 MODIFIED PEPTIDES: PENICILLINS, CEPHALOSPORINS, AND OTHER β-LACTAMS 453

Claisen-type reaction Schiff base formation PO acetyl-CoA O O L-Glu O O N CoAS CoAS H2N CO2H H2NCO2H hydroxyethyl side-chain constructed γ-glutamyl P CO2H via two SAM-dependent C-methylations at position 6 HO NH formation of H H 2 H H H O SAM SAM β-lactam ring 6 S 2 O N N N HN CoAS O L-Cys O O CO2H at position 2 CO2H CO2H CO2H thienamycin

Figure 7.43

olivanic acids: HH H H HN H H N H H N O O O HO3SO HO3SO HO3SO S S S N N N O O O O CO2H CO2H CO2H

O NMe2

NH2 HO N HO H H H H NH CO2H H S H S S N NH2 N N O O O CO2H CO2H CO2H imipenem meropenem cilastatin

Figure 7.44 extended with acetyl-CoA, then cyclized to the OH N imine (Figure 7.43). Reduction and β-lactam for- H N OH mation lead to the carbapenam, which yields the CO2H carbapenem by dehydrogenation. This material is O N H NO modified to produce thienamycin by insertion of 2 O CO H side-chains at C-2 and C-6; the detailed sequence nocardicin A 2 has yet to be established. Both carbons of the two- CO2H carbon side-chain at C-2 are known to be derived O from methionine, the result of a double methyla- N tion sequence reminiscent of the processes used for H OMe H N N N alkylating the side-chains of sterols (see page 254). H N ONN 2 O The C-2 cysteaminyl side-chain is supplied by the S OSO3H OSO3H amino acid cysteine. SQ 26,180 aztreonam The simple nonfused β-lactam ring is encoun- tered in a number of natural structures, such as Figure 7.45 the nocardicins, e.g. nocardicin A (Figure 7.45), from Nocardia uniformis, and the so-called isolated from strains of bacteria. The simplest of monobactams∗ (monocylic β-lactams) based on these monobactams is the compound referred to the structure shown in Figure 7.39, which was by its research coding SQ 26,180 (Figure 7.45) 454 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

Carbapenems

Thienamycin (Figure 7.44) is produced by cultures of Streptomyces cattleya, but in insufficient amounts for commercial use. This compound is thus obtained by total synthesis. In addition, thienamycin is relatively unstable, its side-chain primary amino group reacting as a nucleophile with other species, including the β-lactam group in other molecules. For drug use, thienamycin is converted into its more stable N-formimidoyl derivative imipenem (Figure 7.44). Imipenem has a broad spectrum of activity which includes activity towards many aerobic and anaerobic Gram-positive and Gram-negative bacteria, it is resistant to hydrolysis by most classes of β-lactamases, and it also possesses β-lactamase inhibitory activity. However, it is partially inactivated by dehydropeptidase in the kidney, and is thus administered in combination with cilastatin (Figure 7.45), a specific inhibitor of this enzyme. The newer carbapenem meropenem (Figure 7.45) is stable to dihydropeptidase and can be administered as a single agent. It has good activity against all clinically significant aerobes and anaerobes, except MRSA (methicillin-resistant S. aureus) and Enterococcus faecium, and stability to serine-based β-lactamases.

discovered in Chromobacterium violaceum. Whilst formed from L-serine and two molecules of many of the natural examples show a 3α- L-4-hydroxyphenylglycine, the latter having its methoxyl (corresponding to the 7α-methoxyl in origins in L-tyrosine (Figure 7.47). The inver- the cephamycins), the prominent feature in the sion of configurations probably occurs dur- monobactams is the N -sulphonic acid group- ing polypeptide formation (compare penicillins, ing. The fundamental precursor of the β-lactam page 438). The remaining portion in the carbon ring in the monobactams is serine (Figure 7.46). skeleton of nocardicin A is ether-linked to a In the simple structures, the β-lactam nitrogen 4-hydroxyphenylglycine unit, and actually derives presumably arises from ammonia, and cycliza- from L-methionine. This probably involves anSN2 tion occurs by displacement of the hydroxyl, in displacement on SAM as with simple methylation suitably activated form. The N -sulphonate func- reactions, though attack must be on the secondary tion comes from sulphate. For the more com- rather than primary centre (Figure 7.47). Again, plex structures such as the nocardicins, evidence the configuration of the L-amino acid is inverted points to a polypeptide origin with consider- at a late stage, as with the isopenicillin N to peni- able parallels with penicillin and cephalosporin cillin N conversion in cephalosporin biosynthesis biosynthesis. The postulated D,L,D-tripeptide is (see page 445).

Monobactams The naturally occurring monobactams show relatively poor antibacterial activity, but alteration of the side-chain as with penicillins and cephalosporins has produced many potent new compounds. Unlike those structures, the nonfused β-lactam ring is readily accessible by synthesis, so all analogues are produced synthetically. The first of these to be used clinically is aztreonam (Figure 7.45), which combines the side-chain of the cephalosporin ceftazidime (Table 7.4) with the monobactam nucleus. Aztreonam is very active against Gram-negative bacteria, including Pseudomonas aeruginosa, Haemophilus influenzae,andNeisseria meningitidis, but has little activity against Gram-positive organisms. It also displays a high degree of resistance to enzymatic hydrolysis by most of the common β-lactamases. Oral absorption is poor, and this drug is administered by injection. CYANOGENIC GLYCOSIDES 455

amide formation and transformation of hydroxyl into suitable leaving group OH OX H OMe H2N N H2N H2N NH O N CO2H CONH2 O OSO3H L-Ser SQ 26,180

Figure 7.46

NH2 OH OH

L-Tyr CO2H H2N H2N L-Tyr

HO CO2H CO2H L-4-hydroxyphenylglycineL-Ser L-4-hydroxyphenylglycine polypeptide assembly; configuration in 4-hydroxyphenylglycine residues is inverted during assembly oxidation of OH NH2 H N H D amine to oxime N L OH O N L OH O N O N HO HO O D O D CO H CO H CO H nocardicin G 2 2 nocardicin E 2 Me H2NS further modification includes SAM Ad epimerization at the chiral centre S 2 displacement; usually SAM N in the methionine- donates its S-methyl group, but here, derived side-chain the amino acid portion is donated OH N H N OH CO2H D O N H2NO O CO2H nocardicin A

Figure 7.47

CYANOGENIC GLYCOSIDES are brought into contact with the glycoside and begin to hydrolyse it. Thus, amygdalin is hydrol- Cyanogenic glycosides are a group of mainly ysed sequentially by β-glucosidase-type enzymes plant-derived materials, which liberate hydrocyanic to prunasin and then mandelonitrile, the lat- acid (HCN) on hydrolysis, and are thus of con- ter compound actually being the cyanohydrin cern as natural toxicants. The group is exempli- of benzaldehyde (Figure 7.48). Mandelonitrile is fied by amygdalin (Figure 7.48), a constituent in then hydrolysed to its component parts, benzalde- the kernels of bitter almonds (Prunus amygdalus hyde and toxic HCN, by the action of a further var. amara; Rosaceae) and other Prunus species enzyme. The kernels of bitter almonds, which such as apricots, peaches, cherries, and plums. contain amygdalin and the hydrolytic enzymes, When plant tissue containing a cyanogenic gly- are thus potentially toxic if ingested, whilst ker- coside is crushed, glycosidase enzymes also in nels of sweet almonds (Prunus amygdalus var. the plant but usually located in different cells dulcis) are not toxic, containing the enzymes 456 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

OH CHO O HO HCN HO O OH OH O O H2O HO β-glucosidase HO β-glucosidase HO O HO O H H HO H OH OH C Glc C Glc C N N N

amygdalin prunasin mandelonitrile (benzaldehyde cyanohydrin)

Figure 7.48

N-hydroxylationN-hydroxylation decarboxylation–elimination

O2 O CO2H CO2H 2 CO H NADPH NADPH 2

NH2 HN N N OH HO OH OH L-Phe N-hydroxy-L-Phe N,N-dihydroxy-L-Phe E-aldoxime trans–cis glucosylation dehydration of isomerization GlcO H HO H O2 oxime to nitrile UDPGlc NADPH C C C N N N N HO prunasin benzaldehyde phenylacetonitrile Z-aldoxime cyanohydrin (mandelonitrile)

Figure 7.49 but no cyanogenic glycoside. Amygdalin itself is N -hydroxylated and then converted into the alde- not especially toxic to animals; toxicity depends hyde oxime (aldoxime) by a sequence that involves on the co-ingestion of the hydrolytic enzymes. further N -hydroxylation and subsequent decar- Although formed by the hydrolysis of amygdalin, boxylation–elimination, with all of these reactions prunasin is also a natural cyanogenic glycoside and catalysed by a single cytochrome P-450-dependent may be found in seeds of black cherry (Prunus enzyme. The nitrile is formed by dehydration serotina), and in the seeds and leaves of cherry of the oxime, but this reaction actually proceeds laurel (Prunus laurocerasus). The food plant cas- on the Z -aldoxime produced by isomerization of sava (or tapioca) (Manihot esculenta; Euphorbia- the first formed E-aldoxime. (R)-Mandelonitrile cae) also produces the cyanogenic glycosides lina- is then the result of a stereoselective cytochrome marin and lotaustralin (see Figure 7.50), and P-450-dependent hydroxylation reaction. Finally, preparation of the starchy tuberous roots involves glycosylation occurs, the sugar unit usually being prolonged hydrolysis and boiling to release and glucose as in the case of prunasin and amygdalin. drive off the HCN before they are suitable for The stereoselectivity of the nitrile hydroxylation consumption. step varies depending on the plant system, so that Cyanogenic glycosides are produced from a epimeric cyanohydrins are found in nature, though range of amino acids by a common path- not in the same plant. Thus, the (S )-enantiomer way (Figure 7.49). The amino acid precur- of prunasin (called sambunigrin) is found in the sor of prunasin is phenylalanine, which is leaves of elder (Sambucus nigra; Caprifoliaceae). GLUCOSINOLATES 457

The main amino acids utilized in the biosyn- and then supplying linamarin, it has been possible thesis of cyanogenic glycosides are phenylala- to selectively generate toxic HCN in cancer cells. nine (e.g. prunasin, sambunigrin, and amygdalin), tyrosine (e.g. dhurrin from sorghum (Sorghum bicolor; Graminae/Poaceae)), valine (e.g. lina- GLUCOSINOLATES marin from flax (Linum usitatissimum; Linaceae)), isoleucine (e.g. lotaustralin, also from flax), and Glucosinolates have several features in common leucine (e.g. heterodendrin from Acacia species with the cyanogenic glycosides. They too are (Leguminosae/Fabaceae)) (Figure 7.50). Although glycosides which are enzymically hydrolysed in cyanogenic glycosides are widespread, they are damaged plant tissues giving rise to potentially particularly found in the families Rosaceae, Legu- toxic materials, and they share the early stages minosae/Fabaceae, Graminae/Poaceae, Araceae, of the cyanogenic glycoside biosynthetic pathway Compositae/Asteraceae, Euphorbiaceae, and Passi- for their formation in plants. A typical structure floraceae. It is highly likely that plants synthesize is sinalbin (Figure 7.51), found in seeds of white these compounds as protecting agents against her- mustard (Sinapis alba; Cruciferae/Brassicaceae). bivores. Some insects also accumulate cyanogenic Addition of water to the crushed or powdered glycosides in their bodies, again as a protective seeds results in hydrolysis of the glucoside bond device. Whilst many insects obtain these com- via the enzyme myrosinase (a thioglucosidase) to pounds by feeding on suitable plant sources, it give a thiohydroximate sulphonate (Figure 7.51). is remarkable that others are known to synthesize This compound usually yields the isothiocyanate cyanogenic glycosides themselves from amino acid acrinylisothiocyanate by a Lossen-type¨ rearrange- precursors. There is hope that cyanogenesis may ment as shown, prompted by the sulphate leaving provide a means of destroying cancer cells. By tar- group. Under certain conditions, dependent on pH, geting cancer cells with linamarase via a retrovirus or the presence of metal ions or other enzymes, related compounds such as thiocyanates (RSCN) or nitriles (RCN) may be formed from glucosi- GlcO H nolates. Acrinylisothiocyanate is a pungent-tasting material (mustard oil) typical of many plants in the L-Phe CN Cruciferae/Brassicaceae used as vegetables (e.g. cabbage, radish) and condiments (e.g. mustard, prunasin horseradish). Black mustard (Brassica nigra) con- tains sinigrin (Figure 7.51) in its seeds, which by GlcO H a similar sequence is hydrolysed to allylisothio- L-Tyr CN cyanate. Allylisothiocyanate is considerable more volatile than acrinylisothiocyanate, so that condi- HO dhurrin ment mustard prepared from black mustard has a pungent aroma as well as taste. OGlc L-Val The biosynthesis of sinalbin from tyro- CN sine is indicated in Figure 7.52. The aldoxime linamarin is produced from the amino acid by the early part of the cyanogenic glycoside path- OGlc way shown in Figure 7.49. This aldoxime incor- L-Ile CN porates sulphur from methionine (or cysteine) lotaustralin to give the thiohydroximic acid, perhaps by attack of a thiolate ion on to the imine sys- H OGlc tem, and this compound is then S -glucosylated L-Leu using UDPglucose. In nature, sulphate groups CN are provided by PAPS (3-phosphoadenosine-5- heterodendrin phosphosulphate), and sulphation features as the last step in the pathway. Similarly, phenylala- Figure 7.50 nine is the precursor of benzylglucosinolate 458 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

hydrolysis of S-glucoside Lössen-type rearrangement SGlc S H myrosinase N C N N S HO OSO3 K HO OSO3 K HO Glc sinalbin thiohydroximate sulphonate acrinylisothiocyanate

SGlc myrosinase N C N S OSO3 K sinigrin Glc allylisothiocyanate

Figure 7.51

L-Tyr probably via nucleophilic attack of suitable thiolate on to imine PAPS = 3′-phosphoadenosine-5′-phosphosulphate see Figure 7.49 Met? Cys? S-glucosylation

RS SH UDPGlc SGlc PAPS SGlc N N N N HO OH HO OH HO OH HO OSO3 K aldoxime thiohydroximic acid desulphoglucosinolate glucosinolate (sinalbin)

Figure 7.52

(Figure 7.53) in nasturtium (Tropaeolum majus; SGlc Tropaeolaceae), and tryptophan yields glucobras- L-Phe N sicin in horseradish (Armoracia rusticana;Cru- OSO3 K ciferae/Brassicaceae). Interestingly, chain exten- benzylglucosinolate sion of methionine to homomethionine and of phenylalanine to homophenylalanine is involved SGlc in the formation of sinigrin in Brassica nigra L-Trp N and gluconasturtiin in rapeseed (Brassica napus) N OSO K H 3 respectively (Figure 7.54). Two carbons derived glucobrassicin from acetate are incorporated into the side-chain in each case, together with loss of the orig- Figure 7.53 inal carboxyl, and this allows biosynthesis of the appropriate glucosinolates. For elaboration of similar crops. There is evidence that consumption the allyl side-chain in the biosynthesis of sini- grin, loss of methanethiol occurs as a late step of the hydrolysis products from glucosinolates in (Figure 7.54). food crops may induce goitre, an enlargement of Glucosinolates are found in many plants of the the thyroid gland. Thus, progoitrin in oil seed Cruciferae/Brassicaceae, Capparidaceae, Euphor- rape (Brassica napus; Cruciferae/Brassicaceae) on biacae, Phytolaccaceae, Resedaceae, and Tropae- hydrolysis yields the oxazolidine-2-thione goitrin olaceae, contributing to the pungent properties of (Figure 7.55), which is a potent goitrogen, inhibit- their crushed tissues. They are often at their high- ing iodine incorporation and thyroxine forma- est concentrations in seeds rather than leaf tissue. tion (see page 410). The goitrogenic effects of These compounds and their degradation products glucosinolates cannot be alleviated merely by presumably deter some predators, but may actually the administration of iodine. This severely limits attract others, e.g. caterpillars on cabbages and economic utilization for animal foodstuffs of GLUCOSINOLATES 459

E2 elimination of methanethiol MeS CO H CO H SGlc SGlc 2 MeS 2 MeS

NH2 NH2 H N N OSO K OSO K L-Met homomethionine 3 sinigrin 3 chain extension; carbons from acetate incorporated

CO2H CO2H SGlc NH2 NH N L-Phe 2 OSO K homophenylalanine gluconasturtiin 3

aldol isomerization; may reaction involve dehydration oxidative transamination then rehydration decarboxylation transamination CH2COSCoA CO2H HO CO H 2 O O CO2H H2N CO2H RCO2H RCO2H RCO2H – CO2 NH2 O OH R CO2H R R amino acid H homoamino acid C-α and carboxyl from acetate

Figure 7.54

H SGlc myrosinase N N C OH N OH S O S OSO K 3 goitrin progoitrin

O O myrosinase S SGlc S N C N S OSO3 K sulphoraphane glucoraphanin

Figure 7.55

H NH2 O H NH2 OH NH2 O alliinase S S S CO2H CO2H PLP CO2H S-allyl L-cysteine alliin allyl sulphenic acid dehydroalanine (S-allyl L-cysteine sulphoxide) x 2 – H2O H2O O O NH3 S S CO2H allicin (diallyl thiosulphinate)

Figure 7.56 460 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

the rapeseed meal remaining after oil expres- O NH2 O NH2 sion unless strains with very low levels of the S S CO H CO H glucosinolate are employed. On the other hand, 2 2 S-methyl S-propyl glucoraphanin, the glucosinolate precursor of L-cysteine sulphoxide L-cysteine sulphoxide sulphoraphane (Figure 7.55) from broccoli (Bras- sica oleracea italica; Cruciferae/Brassicaceae), has O NH2 S been shown to have beneficial medicinal prop- CO2H erties, in that it induces carcinogen-detoxifying S-1-propenyl enzyme systems and accelerates the removal of L-cysteine sulphoxide xenobiotics. Young sprouted seedlings contain Figure 7.57 some 10–100 times as much glucoraphanin as the mature plant, but, nevertheless, broccoli may be H regarded as a valuable dietary vegetable. O O S S CYSTEINE SULPHOXIDES 1-propenyl (Z)-propanethial S-oxide sulphenic acid (onion lachrymatory factor) The major flavour component of garlic∗ (Allium sativum; Liliaceae/Alliaceae) is a thiosulphinate Figure 7.58 called allicin (Figure 7.56). This compound is formed when garlic tissue is damaged as a risk of heart attacks. S -Alkyl cysteine sulphox- hydrolysis product of S -allyl cysteine sulphoxide ides are characteristic components of the onion (alliin) brought about by the pyridoxal phosphate- (Allium) genus. All Allium species contain S - dependent enzyme alliinase (Figure 7.56). Under methyl cysteine sulphoxide, though the S -propyl these conditions, alliin is cleaved by an elimina- analogue (Figure 7.57) predominates in chives tion reaction, and two molecules of the sulphenic (A. schoenoprasum), the S -1-propenyl derivative acid then form allicin. Pyruvic acid and ammo- in onions (A. cepa)andtheS -allyl compound nia are the other hydrolysis products. Allicin has alliin in garlic. Propenyl sulphenic acid derived considerable antibacterial and antifungal proper- by hydrolysis of S -1-propenyl cysteine sulphox- ties. There is widespread use of garlic, either ide provides a common kitchen hazard, rear- fresh, dried, or as garlic oil, as a beneficial ranging to the lachrymatory factor of onions agent to reduce cholesterol levels and reduce the (Figure 7.58).

Garlic Garlic (Allium sativum; Liliaceae/Alliaceae) has a long history of culinary and medicinal use. The compound bulb is composed of several smaller sections termed cloves. Allicin (Figure 7.56) is considered to be the most important of the biologically active components in the crushed bulb. It is not present in garlic, but is rapidly produced when the precursor alliin is cleaved by the action of the enzyme alliinase upon crushing the tissue. Both alliin and alliinase are stable when dry, and dried garlic still has the potential for releasing allicin when subsequently moistened. However, allicin itself is very unstable to heat or organic solvents degrading to many other compounds, including diallyl sulphides (mono-, di-, and oligo-sulphides), vinyldithiins, and ajoenes (Figure 7.59). Processed garlic preparations typically contain a range of different sulphur compounds. Garlic preparations used medicinally include steam-distilled oils, garlic macerated in vegetable oils (e.g. soybean oil), dried garlic powder, and gel-suspensions of garlic powder. Analyses indicate wide variations in the nature and amounts of constituents in the various preparations. Thus, freshly crushed garlic cloves typically contain allicin (about 0.4%) and other thiosulphinates (about 0.1%, chiefly allyl methyl thiosulphinate). The

(Continues) FURTHER READING 461

(Continued)

S

S x 2 S O S diallyl disulphide S 2-vinyl-4H-1,3-dithiin S S S S allicin thioacrolein S S x 2 S diallyl trisulphide

O O 3-vinyl-4H-1,2-dithiin SS S S S S (E)-ajoene (Z)-ajoene

Figure 7.59

garlic powder usually produces less of these materials, though high quality samples may be similar to the fresh samples. Oil-macerated powders appear to lose up to 80% of their sulphur compounds, and vinyldithiins and ajoenes predominate. Steam-distilled garlic oil has dialk(en)yl sulphides (i.e. diallyl sulphide, allyl methyl sulphide, etc) as the major sulphur components (0.1–0.5%). Bad breath and perspiration odours which often follow the ingestion of garlic, either medicinally or culinary, are due to allyl methyl sulphide and disulphide, diallyl sulphide and disulphide, and 2-propenethiol. Garlic is used for a variety of reasons, and some of the attributes associated with it, e.g. for cancer prevention, or to reduce heart attacks, may not be substantiated. Other properties such as antimicrobial activity, effects on lipid metabolism, and platelet aggregation inhibitory action have been demonstrated. Ajoene has been shown to be a potent antithrombotic agent through inhibition of platelet aggregation.

FURTHER READING Becker GW, MacKellar WC, Riggin RM and Wrob- lewski VW (1995) Hormones (human growth hor- General mone). Kirk–Othmer Encyclopedia of Chemical Technology, 4th edn, Vol 13. Wiley, New York, Lancini GC and Lorenzetti R (1993) Biotechnology pp 406–418. of Antibiotics and Other Microbial Metabolites. Engeland WC (1995) Hormones (survey). Kirk–Othmer Plenum, New York. Encyclopedia of Chemical Technology, 4th edn, Lancini G, Parenti F and Gallo GG (1995) Antibi- Vol 13. Wiley, New York, pp 357–370. otics – A Multidisciplinary Approach. Plenum, New Giannis A and Kolter T (1993) Peptidomimetics for York. receptor ligands – Discovery, development, and Niccolai D, Tarsi L and Thomas RJ (1997) The renewed medicinal perspectives. Angew Chem Int Edn Engl challenge of antibacterial chemotherapy. Chem Com- 32, 1244–1267. mun 2333–2342. Hirschmann R (1991) Medicinal chemistry in the golden Russell AD (1998) Mechanisms of bacterial resistance age of biology: lessons from steroid and peptide to antibiotics and biocides. Prog Med Chem 35, research. Angew Chem Int Edn Engl 30, 1278–1301. 133–197. Matteri RL (1995) Hormones (anterior pituitary hor- mones). Kirk–Othmer Encyclopedia of Chemical Technology, 4th edn, Vol 13. Wiley, New York, Peptide Hormones pp 370–380. Matteri RL (1995) Hormones (anterior pituitary-like Anon (1997) Thyroid and antithyroid preparations. hormones). Kirk–Othmer Encyclopedia of Chemical Kirk–Othmer Encyclopedia of Chemical Technology, Technology, 4th edn, Vol 13. Wiley, New York, 4th edn, Vol 24. Wiley, New York, pp 89–104. pp 381–391. 462 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

Spatola AF (1995) Hormones (posterior pituitary hor- Wiseman A (1994) Better by design: biocatalysts for the mones). Kirk–Othmer Encyclopedia of Chemical future. Chem Brit 571–573. Technology, 4th edn, Vol 13. Wiley, New York, pp 391–406. Nonribosomal Peptide Biosynthesis Insulin Cane DE, Walsh CT and Khosla C (1998) Harnessing the biosynthetic code: combinations, permutations, Brown A (1998) Diabetes mellitus (2): management of and mutations. Science 262,63–68. insulin-dependent diabetes mellitus. Pharm J 260, Kleinkauf H and von D ”ohren H (1996) A non- 753–756. ribosomal system of peptide biosynthesis. Eur J Shoelson SE (1995) Insulin and other diabetic agents. Biochem 236, 335–351. Kirk–Othmer Encyclopedia of Chemical Technology, Marahiel MA, Stachelhaus T and Mootz HD (1997) 4th edn, Vol 14. Wiley, New York, pp 662–676. Modular peptide synthetases involved in nonriboso- mal peptide synthesis. Chem Rev 97, 2651–2673. Interferons Rajendran N and Marahiel MA (1999) Multifunctional peptide synthetases required for nonribosomal bio- Sen GC and Lengyel P (1992) The interferon system: synthesis of peptide antibiotics. Comprehensive Nat- abird’s eye view of its biochemistry. JBiolChem ural Products Chemistry, Vol 4. Elsevier, Amster- 267, 5017–20. dam, pp 195–200. Wong S (1995) Immunotherapeutic agents. Kirk– von D ”ohrenH, Keller U, Vater J and Zocher R (1997) Othmer Encyclopedia of Chemical Technology,4th Multifunctional peptide synthetases. Chem Rev 97, edn, Vol 14. Wiley, New York, pp 64–86. 2675–2705.

Opioid Peptides Peptide Antibiotics

Brownstein MJ (1993) A brief history of opiates, opioid Nicolau KC, Boddy CNC, Br ”aseS and Winssinger N peptides, and opiate receptors. Proc Natl Acad Sci (1999) Chemistry, biology and medicine of the USA 90, 5391–5393. glycopeptide antibiotics. Angew Chem Int Edn 38, Childers SR (1995) Hormones (brain oligopeptides). 2096–2152. Kirk–Othmer Encyclopedia of Chemical Technology, Wise EM (1992) Antibiotics (peptides). Kirk–Othmer 4th edn, Vol 13. Wiley, New York, pp 418–433. Encyclopedia of Chemical Technology, 4th edn, Giannis A and Kolter T (1993) Peptidomimetics for Vol 3. Wiley, New York, pp 266–306. receptor ligands – discovery, development, and medicinal perspectives. Angew Chem Int Edn Engl 32, 1244–1267. Vancomycin, Teichoplanin Nugent RA and Hall CM (1992) Analgesic, antipyretics, and antiinflammatory agents. Kirk–Othmer Encyclo- Cavalleri B and Parenti F (1992) Antibiotics [glycopep- pedia of Chemical Technology, 4th edn, Vol 2. Wiley, tides (dalbaheptides)]. Kirk–Othmer Encyclopedia of New York, pp 729–748. Chemical Technology, 4th edn, Vol 2. Wiley, New Schiller PW (1991) Development of receptor-specific York, pp 995–1018. opioid peptide analogues. Prog Med Chem 28, Williams DH (1996) The glycopeptide story – how to 301–340. kill the deadly ‘superbugs’. Nat Prod Rep 13, Selley DE, Sim LJ and Childers SR (1995) Opioids, 469–477. endogenous. Kirk–Othmer Encyclopedia of Chemi- Williams DH and Bardsley B (1999) The vancomycin cal Technology, 4thedn, Vol 17. Wiley, New York, group of antibiotics and the fight against resistant pp 858–882. bacteria. Angew Chem Int Edn 38, 1172–1193.

Enzymes Cyclosporins

Cervoni P, Crandall DL and Chan PS (1993) Cardio- Clardy J (1995) The chemistry of signal transduction. vascular agents – thrombolytic agents. Kirk–Othmer Proc Natl Acad Sci USA 92,56–61. Encyclopedia of Chemical Technology, 4th edn, Lawen A (1996) Biosynthesis and mechanism of action Vol 5. Wiley, New York, pp 289–293. of cyclosporins. Prog Med Chem 33,53–97. FURTHER READING 463

Rosen MK and Schreiber SL (1992) Natural products as Bugg TDH (1999) Bacterial peptidoglycan biosynthesis probes of cellular function: studies of immunophilins. and its inhibition. Comprehensive Natural Products Angew Chem Int Edn Engl 31, 384–400. Chemistry, Vol 3. Elsevier, Amsterdam, pp 241–294. St ”ahelin HF (1996) The history of cyclosporin A Bugg TDH and Walsh CT (1992) Intracellular steps (Sandimmune) revisited: another point of view. of bacterial cell wall peptidoglycan biosynthesis: Experientia 52,5–13. enzymology, antibiotics, and antibiotic resistance. von D ”ohren H and Kleinkauf H (1999) Cyclosporin: Nat Prod Rep 9, 199–215. the biosynthetic path to a lipopeptide. Comprehensive Coulton S and Francois I (1994) ?-Lactamases: targets Natural Products Chemistry, Vol 1. Elsevier, Ams- for drug design. Prog Med Chem 31, 297–349. terdam, pp 533–555. Massova I and Mobashery S (1997) Molecular bases Wong S (1995) Immunotherapeutic agents. Kirk– for interactions between β-lactam antibiotics and β- Othmer Encyclopedia of Chemical Technology,4th lactamases. Account Chem Res 30, 162–168. edn, Vol 14. Wiley, New York, pp 64–86. Matagne A, Dubus A, Galleni M and Frere` J-M (1999) The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity. Nat Prod Rep 16,1–19. Bleomycin Niccolai D, Tarsi L and Thomas RJ (1997) The renewed challenge of antibacterial chemotherapy. Chem Com- Boger DL and Cai H (1999) Bleomycin: synthetic mun 2333–2342. and mechanistic studies. Angew Chem Int Edn 38, Page MI and Laws AP (1998) The mechanism of cataly- 448–476. Chem Commun Hecht SM (2000) Bleomycin: new perspectives on the sis and the inhibition of ?-lactamases. mechanism of action. J Nat Prod 63, 158–168. 1609–1617. Shen B, Du L, Sanchez C, Chen M and Edwards DJ (1999) Bleomycin biosynthesis in Streptomyces ver- ticillus ATCC15003: a model of hybrid peptide and Penicillins and Cephalosporins polyketide biosynthesis. Bioorg Chem 27, 155–171. Bryskier A (2000) Cephems: fifty years of continuous research. J Antibiot 53, 1028–1037. Streptogramins Luengo JM (1999) Enzymatic synthesis of penicillins. Comprehensive Natural Products Chemistry, Vol 4. Pechere` J-C (1996) Streptogramins: a unique class of Elsevier, Amsterdam, pp 239–274. antibiotics. Drugs 51(Suppl 1), 13–19. Nathwani D and Wood MJ (1993) Penicillins: a current Vannuffel P and Cocito C (1996) Mechanism of action review of their clinical pharmacology and therapeutic of streptogramins and macrolides. Drugs 51 (Suppl use. Drugs 45, 866–894. 1), 20–30. Ponsford RJ (1992) Antibiotics [β-lactams (penicillins and others)]. Kirk–Othmer Encyclopedia of Chemi- Peptide Toxins cal Technology, 4th edn, Vol 3. Wiley, New York, pp 129–158. Berressem P (1999) From bites and stings to medicines. Roberts J (1992) Antibiotics [β-lactams (cephalo- Chem Brit 35 (4), 40–42. sporins)]. Kirk–Othmer Encyclopedia of Chemical Carmichael WW (1992) Cyanobacteria secondary meta- Technology, 4th edn, Vol 3. Wiley, New York, bolites – the cyanotoxins. J Appl Bacteriol 72, pp 28–82. 445–459. Hucho F (1995) Toxins as tools in neurochemistry. Angew Chem Int Edn Engl 34,39–50. Clavulanic Acid, Carbapenems, Shimizu Y (1993) Microalgal metabolites. Chem Rev 93, 1685–1698. Monobactams Baggaley KH, Brown A and Schofield CJ (1997) Chem- β-Lactam Antibiotics istry and biosynthesis of clavulanic acid and other clavams. Nat Prod Rep 14, 309–333. Bentley R (1995) A secret arrangement. Chem Brit Coulton S and Hunt E (1996) Recent advances in the 793–796. (history of penicillin). chemistry and biology of carbapenem antibiotics. Brakhage A (1999) Biosynthesis of ?-lactam compounds Prog Med Chem 33,99–145. in microorganisms. Comprehensive Natural Products Lindner KR, Bonner DP and Koster WH (1992) Antibi- Chemistry, Vol 4. Elsevier, Amsterdam, pp 159–193. otics [β-lactams (monobactams)]. Kirk–Othmer 464 PEPTIDES, PROTEINS, AND OTHER AMINO ACID DERIVATIVES

Encyclopedia of Chemical Technology, 4th edn, Poulton JE (1990) Cyanogenesis in plants. Plant Physiol Vol 3, John Wiley, New York, pp 107–129. 94, 401–405. Southgate R and Osborne NF (1992) Antibiotics [?- lactams (carbapenems and penems)]. Kirk–Othmer Encyclopedia of Chemical Technology, 4th edn, Glucosinolates Vol 3. Wiley, New York, pp 1–27. Stam JG (1992) Antibiotics [?-lactams (?-lactamase Bones AM and Rossiter JT (1996) The myrosinase– inhibitors)]. Kirk–Othmer Encyclopedia of Chemi- glucosinolate system, its organisation and biochem- cal Technology, 4th edn, Vol 3. Wiley, New York, istry. Physiol Plant 97, 194–208. pp 83–107. Nestle M (1997) Broccoli sprouts as inducers of carci- nogen-detoxifying enzyme systems: Clinical, dietary, and policy implications. Proc Natl Acad Sci USA 94, Cyanogenic Glycosides 11 149–11 151.

Conn EE (1991) The metabolism of a natural product: lessons learned from cyanogenic glycosides. Planta Cysteine Sulphoxides, Garlic Med 57,S1–S9. Hickel A, Hasslacher M and Griengl H (1996) Hydrox- Block E (1992) The organosulfur chemistry of the genus ynitrile lyases: Functions and properties. Physiol Allium – Implications for the organic chemistry of Plant 98, 891–898. sulfur. Angew Chem Int Edn Engl 31, 1135–1178. Hughes MA (1999) Biosynthesis and degradation Lawson LD, Wang ZJ and Hughes BG (1991) Identi- of cyanogenic glycosides. Comprehensive Natural fication and HPLC quantitation of the sulfides and Products Chemistry, Vol 1. Elsevier, Amsterdam, dialk(en)yl thiosulfinates in commercial garlic prod- pp 881–895. ucts. Planta Med 57, 363–370. Jones DA (1998) Why are so many food plants cyano- McElnay JC and Li Wan Po A (1991) Dietary supple- genic? Phytochemistry 47, 155–162. ments: garlic. Pharm J 246, 324–326. Medicinal Natural Products. Paul M Dewick Copyright  2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 8 CARBOHYDRATES

Some fundamental modifications that occur in carbohydrate metabolism are outlined, then specific examples of monosaccharides, oligosaccharides, and polysaccharides are described. The formation of amino sugars follows, leading to a discussion of aminoglycoside antibiotics. Monograph topics giving more detailed information on medicinal agents include monosaccharides and disaccharides, vitamin C, polysaccharides, aminoglycoside antibiotics based on streptamine and 2-deoxystreptamine, acarbose, lincomycin, and clindamycin.

The main pathways of carbohydrate biosynthesis MONOSACCHARIDES and degradation comprise an important component of primary metabolism that is essential for all organ- Six-carbon sugars (hexoses) and five-carbon sugars isms. Carbohydrates are among the most abundant (pentoses) are the most frequently encountered constituents of plants, animals, and microorgan- carbohydrate units (monosaccharides) in nature. isms. Polymeric carbohydrates function as impor- Photosynthesis produces initially the three-carbon tant food reserves, and as structural components sugar 3-phosphoglyceraldehyde, two molecules of in cell walls. Animals and most microorganisms which are used to synthesize glucose 6-phosphate are dependent for their very existence on the car- by a sequence which effectively achieves the bohydrates produced by plants. Carbohydrates are reverse of the glycolytic reactions (Figure 8.1). the first products formed in photosynthesis, and Alternatively, by the complex reactions of the are the products from which plants then synthesize Calvin cycle, 3-phosphoglyceraldehyde may be their own food reserves as well as other chemi- used in the construction of the pentoses ribose cal constituents. These materials then become the 5-phosphate, ribulose 5-phosphate, and xylulose foodstuffs of other organisms. Secondary metabo- 5-phosphate. These sequences incorporate some lites are also ultimately derived from carbohydrate of the fundamental reactions which are used in metabolism, and the relationships of the acetate, the biochemical manipulation of monosaccharide shikimate, mevalonate, and deoxyxylulose phos- structures: phate pathways to primary metabolism have already • been indicated. Many of the medicinally important Mutation, where repositioning of a phos- secondary metabolites described in the earlier chap- phate group in the monosaccharide phosphate ters have been seen to contain clearly recognizable molecule, e.g. the isomerization of glu- carbohydrate portions in their structures, e.g. note cose 6-phosphate and glucose 1-phosphate the frequent occurrence of glycosides. In this chap- (Figure 8.2), is achieved via an intermediate ter, some of the important natural materials which diphosphate. can be grouped together because they are composed • Epimerization changes the stereochemistry at entirely or predominantly of basic carbohydrate one of the chiral centres, e.g. the interconver- units are discussed. Because of their widespread use sion of ribulose 5-phosphate and xylulose 5- in medicinal preparations, some materials with no phosphate (Figure 8.3). This reaction involves inherent biological activity, and which are clearly epimerization adjacent to a carbonyl group and of primary metabolic status, e.g. sucrose and starch, probably proceeds through a common enol are also included. tautomer, but some other epimerizations are 466 CARBOHYDRATES

CHO H OH CHO x 2 HO H PHOTOSYNTHESIS H OH HOH CH OP 2 HOH D-3-phosphoglyceraldehyde CH2OP D-glucose 6-P CALVIN CYCLE

CHO CH2OH CH2OH H OH O O HOH HOH HO H H OH H OH H OH

CH2OP CH2OP CH2OP D-ribose 5-P D-ribulose 5-P D-xylulose 5-P

Figure 8.1

6 OP OP OH HO O HO O HO O HO HO HO 1 HO HO HO OH OP OP D-glucose 6-P D-glucose 1,6-di P D-glucose 1-P

Figure 8.2

keto−enol enol−keto CH2OH tautomerism CH2OH tautomerism CH2OH O OH O achieves HOH OH HO H epimerization at position 3 H OH H OH H OH

CH2OP CH2OP CH2OP D-ribulose 5-P D-xylulose 5-P

Figure 8.3

known proceed through oxidation to an inter- Transketolase removes a two-carbon fragment mediate carbonyl, followed by reduction to from ketols such as fructose 6-phosphate (alter- give the opposite configuration. The substrate natively xylulose 5-phosphate or sedoheptu- for epimerization is often the UDPsugar rather lose 7-phosphate) through the participation of than the monosaccharide phosphate. thiamine diphosphate. Nucleophilic attack of • Aldose–ketose interconversions, e.g. glu- the thiamine diphosphate anion on to the car- cose 6-phosphate to fructose 6-phosphate bonyl results in an addition product which then (Figure 8.4), also proceed through a common fragments by a reverse aldol reaction, gener- enol intermediate. ating the chain-shortened aldose erythrose 4- • Transfer of C2 and C3 units in reactions catal- phosphate, and the two-carbon carbanion unit ysed by transketolase and transaldolase respec- attached to TPP (Figure 8.5) (compare the tively modify the chain length of the sugar. role of TPP in the decarboxylation of α-keto MONOSACCHARIDES 467

keto−enol enol−keto CHO tautomerism HOHtautomerism CH2OH achieves H OH OH O aldose−ketose HO H HO H HO H conversion HOH HOH HOH HOH HOH HOH

CH2OP CH2OP CH2OP D-glucose 6-P D-fructose 6-P

Figure 8.4

reverse aldol reaction C2 unit attached to CH OH CH OH TPP anion; this is CH2OH 2 2 nucleophilic TPP subsequently O H PPT COH PPTC OH attack of thiamine transferred to a transketolase diphosphate anion HO H HO H different aldose via on to carbonyl CHO HOH HOH the reverse reaction HOH HOH HOH HOH CH2OP CH2OP CH2OP D-fructose 6-P D-erythrose 4-P

Figure 8.5

ketose binds reverse aldol reaction to enzyme via CH2OH CH2OH CH2OH Schiff base CH2OH O NH Enz NH Enz NH Enz HO H HO H HO H HO H transaldolase HOH HOH C3 unit attached to enzyme as CHO HOH HOH enamine; this is subsequently HOH transferred to a different aldose via HOH HOH the reverse sequence HOH CH2OP CH2OP CH OP D-sedoheptulose 7-P 2 D-erythrose 4-P

Figure 8.6 acids, page 21). Then, in what is formally of this process, but transferring the C3 carban- the reverse of this reaction, this carbanion ion to another aldose such as glyceraldehyde 3- can attack another aldose such as ribose 5- phosphate (alternatively erythrose 4-phosphate phosphate (alternatively erythrose 4-phosphate or ribose 5-phosphate) and thus increasing its or glyceraldehyde 3-phosphate), thus extending length. its chain length by two carbons. Transaldolase • Oxidation and reduction reactions, typically removes a three-carbon fragment from a ketose employing the NAD/NADP nucleotides, alter such as sedoheptulose 7-phosphate (alterna- the oxidation state of the substrate. Oxidation tively fructose 6-phosphate) in a reverse aldol at C-1 converts an aldose into an aldonic acid, reaction, though this requires formation of a e.g. glucose 6-phosphate gives gluconolactone Schiff base between the carbonyl group and an 6-phosphate and then the open-chain gluconic active site lysine of the enzyme (Figure 8.6). acid 6-phosphate (Figure 8.7). Oxidation at Again, the reaction is completed by a reversal C-6 yields the corresponding uronic acids, 468 CARBOHYDRATES

hydrolysis oxidation at C-1 of lactone CO2H OP OP H OH + O HO O NADP HO H2O HO H HO HO 1 HOH HO HO O OH HOH D-gluconolactone 6-P D-glucose 6-P CH2OP D-gluconic acid 6-P

Figure 8.7

6 OH oxidation at C-6 other uncommon amino sugars are compo- CO2H HO O NAD+ HO O nents of the aminoglycoside antibiotics (see HO HO page 478). HO HO OPPU OPPU Monosaccharide structures may be depicted UDPglucose UDPglucuronic acid in open-chain forms showing their carbonyl Figure 8.8 character, or in cyclic hemiacetal or hemike- tal forms. The compounds exist predominantly in the cyclic forms, which result from nucle- but this takes place on UDPsugar derivatives, ophilic attack of an appropriate hydroxyl on to the e.g. UDPglucose to UDPglucuronic acid carbonyl (Figure 8.11). Both six-membered pyra- (Figure 8.8). Reduction is exemplified by the nose and five-membered furanose structures are conversion of both glucose and fructose into encountered, a particular ring size usually being the sugar alcohol sorbitol (glucitol), and of characteristic for any one sugar. Since the car- mannose into mannitol (Figure 8.9). bonyl group may be attacked from either side, two • Transamination reactions on keto sugars allow epimeric structures (anomers) are possible in each the introduction of amino groups as seen case, and in solution, the two forms are frequently in the amino sugars glucosamine and galac- in equilibrium. In natural product structures, sugar tosamine (Figure 8.10). These compounds, as units are most likely (but not always) to be encoun- their N -acetyl derivatives, are part of the struc- tered in just one of the epimeric forms. The two tures of several natural polysaccharides, and forms are designated α or β on the basis of the

reduction of reduction of CHO CH OH CH OH aldehyde 2 ketone 2 CHO CH2OH H OH H OH O HO H HO H

HO H NADH HO H NADH HO H HO H NADH HO H HOH HOH HOH HOH HOH HOH HOH HOH HOH HOH

CH2OH CH2OH CH2OH CH2OH CH2OH D-glucoseD-sorbitol D-fructose D-mannose D-mannitol (glucitol)

Figure 8.9

OH OH transamination OH OH OH O O HO O O HO O HO HO OH HO OH HO OH HO OH OH O NH2 NH2 D-glucose D-glucosamine D-galactosamine

Figure 8.10 OLIGOSACCHARIDES 469

OH hemiacetal 6 OH aldehyde6 OH hemiacetal 4 4 O 5 5 HO HO OH HO O HO H H 1 HO 2 O HO 2 OH HO 3 HO 1 3 HO OH H H α-D-glucose D-glucose β-D-glucose (α-D-glucopyranose) (open-chain) (β-D-glucopyranose)

hemiketalH ketone hemiketal 6 6 HO HO OH HO H HO O HO HO OH O O O 5 5 2 2 OH 4 3 OH 4 3 OH 1 1 HO HO HO α-D-fructose D-fructose β-D-fructose (α-D-fructofuranose) (open-chain) (β-D-fructofuranose)

Figure 8.11

OH OH OH OH OH HO S R R O R O R HO O HO R O HO OH HO R OHHO R OH HO HO HO HO OH β-D-glucose β-D-galactose β-D-mannose α-L-rhamnose (β-D-Glc) (β-D-Gal) (β-D-Mann) (α-L-Rha) [preferred conformation] OH R HO OH HO HO OH O S O R O R R O HO R HO R OH R HO OH OH HO HO HO OH HO β-D-xylose α-L-arabinose β-D-ribose β-D-fructose (β-D-Xyl) (α-L-Ara) (β-D-Rib) (β-D-Fru)

Figure 8.12 chiralities at the anomeric centre and at the high- CHO CHO R S est numbered chiral centre. If these are the same HOH HO H β α (RS convention) the anomer is termed ,or if CH2OH CH2OH they are different. The most commonly encoun- D-(+)-glyceraldehyde L-(–)-glyceraldehyde tered monosaccharides, and their usual anomers are CHO shown in Figure 8.12. Note that the D- and L- pre- H OH CHO fixes are assigned on the basis of the chirality (as HO H H OH depicted in Fischer projection) at the highest num- HOH HO H bered chiral centre and its relationship to D-(R)- H OH HO H (+)-glyceraldehyde or L-(S )-(−)-glyceraldehyde CH OH CH OH (Figure 8.13). 2 2 D-glucose L-arabinose

OLIGOSACCHARIDES Figure 8.13

The formation of oligosaccharides (typically two to ADP and GDP are sometimes involved. As five monomers) and polysaccharides is dependent outlined in Chapter 2 (see page 29), a UDPsugar on the generation of an activated sugar bound is formed by the reaction of a sugar 1-phosphate to a nucleoside diphosphate. The nucleoside with UTP, and then nucleophilic displacement of diphosphate most often employed is UDP, but the UDP leaving group by a suitable nucleophile 470 CARBOHYDRATES generates the new sugar derivative. This will be a maltose (Figure 8.15), a hydrolysis product from glycoside if the nucleophile is a suitable aglycone starch, contains two glucoses linked α1→4, whilst molecule, or an oligosaccharide if the nucleophile lactose, the main sugar component of cow’s is another sugar molecule (Figure 8.14). This milk, has galactose linked β1→4toglucose.In reaction, if mechanistically of SN2 type, should the systematic names, the ring size (pyranose give an inversion of configuration at C-1 in the or furanose) is also indicated. Sucrose (‘sugar’) electrophile, generating a product with the β- (Figure 8.15) is composed of glucose and fructose, configuration in the case of UDPglucose as shown. but these are both linked through their anomeric Many of the linkages formed between glucose centres, so the shorthand representation becomes monomers actually have the α-configuration, and α1→β2. This means that both the hemiacetal it is believed that a double SN2 mechanism structures are prevented from opening, and, in operates, which also involves a nucleophilic contrast to maltose and lactose, there can be no group on the enzyme (Figure 8.14). Linkages open-chain form in equilibrium with the cyclic are usually represented by a shorthand version, form. Therefore sucrose does not display any of which indicates the atoms bonded and the the properties usually associated with the masked configuration at the appropriate centre(s). Thus carbonyl group, e.g. it is not a reducing sugar.

OH ROH OH O HO O ROH HO HO H HO OR S 2 HO N HO OPPU H UDPglucose β-glucoside

OH Enz OH OH Enz O HO O B HO O ROH HO HO H HO B HO H SN2 SN2 HO HO HO OPPU H OR α-glucoside ROH

Figure 8.14

OH OH α → OH β → HO O 1 4 1 4 O OH HO 1 OH 4 4 HO 1 O O HO O O HO HO OH HO OH HO HO maltose lactose D-Glc(α1→4)D-Glc D-Gal(β1→4)D-Glc 4-O-(α-D-glucopyranosyl)-D-glucopyranose 4-O-(β-D-galactopyranosyl)-D-glucopyranose

OH HO O HO 1 HO HO OH HO OH O α1→β2 OH HO HO O O 4 O OH 2 HO 1 O β → 1 OH HO 1 4 HO sucrose lactulose D-Glc(α1→β2)D-Fru D-Gal(β1→4)D-Fru α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside β-D-galactopyranosyl-(1→4)-β-D-fructofuranoside

Figure 8.15 OLIGOSACCHARIDES 471

OH HO O OH OH HO O HO O HO HO OPPU HO HO UDPglucose HO HO PO HO O HO HO O O PO HO OH O O OH OH HO OH HO HO sucrose 6F-P sucrose fructose 6-P

Figure 8.16

Sucrose is known to be formed predominantly first formed product is sucrose 6F-phosphate by a slightly modified form of the sequence (F indicating the numbering refers to the fructose shown in Figure 8.16, in that UDPglucose is ring). Hydrolysis of the phosphate then generates attacked by fructose 6-phosphate, and that the sucrose (Figure 8.16).

Monosaccharides and Disaccharides D-Glucose (dextrose) (Figure 8.9) occurs naturally in grapes and other fruits. It is usually obtained by enzymic hydrolysis of starch, and is used as a nutrient, particularly in the form of an intravenous infusion. Chemical oxidation of glucose produces gluconic acid. The soluble calcium salt calcium gluconate is used as an intravenous calcium supplement. D-Fructose (Figure 8.13) is usually obtained from invert sugar (see below) separating it from glucose, and is of benefit as a food and sweetener for patients who cannot tolerate glucose, e.g. diabetics. Fructose has the sweetness of sucrose, and about twice that of glucose. High fructose corn syrup for use as a food sweetener is a mixture of fructose and glucose containing up to 90% fructose and is produced by enzymic hydrolysis/isomerization of starch. The sugar alcohol D-sorbitol (Figure 8.9) is found naturally in the ripe berries of the mountain ash (Sorbus aucuparia; Rosaceae) but is prepared semi-synthetically from glucose. It is half as sweet as sucrose, is not absorbed orally, and is not readily metabolized in the body. It finds particular use as a sweetener for diabetic products. D-Mannitol (Figure 8.9) is also produced from glucose, but occurs naturally in manna, the exudate of the manna ash Fraxinus ornus (Oleaceae). This material has similar characteristics to sorbitol, but is used principally as a diuretic. It is injected intravenously, is eliminated rapidly into the urine, and removes fluid by an osmotic effect. Sucrose (Figure 8.15) is obtained from a variety of sources, including sugar cane (Saccharum officinarum; Graminae/Poaceae), sugar beet (Beta vulgaris; Chenopodiaceae), and sugar maple (Acer saccharum; Aceraceae). It is a standard sweetening agent for foods, syrups, and drug preparations. Invert sugar is an equimolar mixture of glucose and fructose, obtained from sucrose by hydrolysis with acid or the enzyme invertase. During this process, the optical activity changes from + to −, hence the reference to inversion. The high sweetness of fructose combined with that of glucose means invert sugar provides a cheaper, less calorific food sweetener than sucrose. Honey is also mainly composed of invert sugar. Lactose (Figure 8.15) can comprise up to 8% of mammalian milk, and is extracted from cow’s milk, often as a by-product from cheese manufacture. It is only faintly sweet, and its principal use is as a diluent in tablet formulations. Lactulose (Figure 8.15)

(Continues) 472 CARBOHYDRATES

(Continued)

is a semi-synthetic disaccharide prepared from lactose, and composed of galactose linked β1 → 4 to fructose. It is not absorbed from the gastrointestinal tract, and is predominantly excreted unchanged. It helps to retain fluid in the bowel by osmosis, and is thus used as a laxative.

Vitamin C Vitamin C (ascorbic acid) (Figure 8.17) can be synthesized by most animals except humans, other primates, guinea pigs, bats and some birds, and for these it is obtained via the diet. Citrus fruits, peppers, guavas, rose hips, and blackcurrants are especially rich sources, but it is present in most fresh fruit and vegetables. Raw citrus fruits provide a good daily source. It is a water-soluble acidic compound (an enol; see Figure 8.18) and is rapidly degraded during cooking in the presence of air. Vitamin C deficiency leads to scurvy, characterized by muscular pain, skin lesions, fragile blood vessels, bleeding gums, and tooth loss. The vitamin is essential for the formation of collagen, the principal structural protein in skin, bone, tendons, and ligaments, being a cofactor in the hydroxylation of proline to 4-hydroxyproline, and of lysine to 5-hydroxylysine (see page 409), which account for up to 25% of the collagen structure. These reactions are catalysed by 2-oxoglutarate dioxygenases (see page 27), and the ascorbic acid requirement is to reduce an enzyme-bound iron–oxygen complex. Skin lesions characteristic of scurvy are a direct result of low levels of hydroxylation in the collagen structure synthesized in the absence of ascorbic acid. Ascorbic acid is also associated with the hydroxylation of tyrosine in the pathway to catecholamines (see page 316), and in the biosynthesis of homogentisic acid, the precursor of tocopherols and plastoquinones (see page 159). Ascorbic acid is usually prepared synthetically, and is used to treat or prevent deficiency. Natural ascorbic acid is extracted from rose hips, persimmons, and citrus fruits. Large doses have been given after surgery or burns to promote healing by increasing collagen synthesis. The benefits of consuming large doses of vitamin C to alleviate the common cold and other viral infections are not proven. Some sufferers believe it to be beneficial in the

reduction of open-chain oxidation of primary CH2OH alcohol to acid aldehyde form to primary alcohol 1 H OH OH HO C HO2C + 2 HO H NAD O OH 4 HO O HO NADH HO HO OH HO OH HO H OH OH 4 OH H OH OH OH D-glucose D-glucuronic acid L-gulonic acid 1 CO2H lactone formation between OH OH OH carboxyl and 4-hydroxyl enolization (note that gulonic acid is numbered differently from HO HO OH O HO OH OH O O O glucuronic acid) O O O

HO OH O L-ascorbic acid 2-oxogulonolactone L-gulonolactone (vitamin C)

Figure 8.17

(Continues) POLYSACCHARIDES 473

(Continued)

OH OH OH OH

HO HO HO HO O – H O O O O O O O

HO OH O OH O OH O OH L-ascorbic acid resonance forms of conjugate base (enolate anion)

Figure 8.18

prevention and therapy of cancer. Vitamin C does have valuable antioxidant properties, and these are exploited commercially in the food industries. In animals, ascorbic acid is synthesized in the liver from glucose, by a pathway which initially involves oxidation to glucuronic acid. This is followed by reduction of the carbonyl function, lactone formation, oxidation of the secondary alcohol to a carbonyl, with subsequent enolization (Figure 8.17). In plants, glucose or galactose can be converted into ascorbic acid by an analogous pathway, though other sequences from glucose have also been observed to operate. Man and other primates appear to be deficient in the enzyme oxidizing gulonolactone to the ketolactone, and are thus dependent on a dietary source of vitamin C.

POLYSACCHARIDES the Compositae/Asteraceae and Campanulaceae is inulin (Figure 8.19), which is a relatively small β → Polysaccharides fulfil two main functions in polymer of fructofuranose, linked through 2 1 living organisms, as food reserves, and as bonds. structural elements. Plants accumulate starch as Cellulose is reputedly the most abundant their main food reserve, a material that is organic material on earth, being the main con- composed entirely of glucopyranose units but in stituent in plant cell walls. It is composed of two types of molecule. Amylose (Figure 8.19) glucopyranose units linked β1→4inalinear is a linear polymer containing some 1000–2000 chain. Alternate residues are ‘rotated’ in the glucopyranose units linked α1→4. Amylopectin structure (Figure 8.19), allowing hydrogen bond- (Figure 8.19) is a much larger molecule than ing between adjacent molecules, and construction amylose (the number of glucose residues varies of the strong fibres characteristic of cellulose, widely but may be as high as 106), and it is as for example in cotton. The structure of a branched-chain molecule. In addition to α1→4 chitin (Figure 8.19) is rather similar to cellu- linkages, amylopectin has branches at about every lose, though it is composed of β1→4 linked N - 20 units through α1→6 linkages. These branches acetylglucosamine residues. Chitin is a major con- continue with α1→4 linkages, but then may have stituent in the shells of crustaceans, e.g. crabs subsidiary branching, giving a treelike structure. and lobsters, and insect skeletons, and its strength The mammalian carbohydrate storage molecule again depends on hydrogen bonding between adja- is glycogen, which is analogous to amylopectin cent molecules, producing rigid sheets. Chemical in structure, but is larger and contains more deacetylation of chitin provides chitosan, a valu- frequent branching, about every ten residues. The able industrial material used for water purifica- branching in amylopectin and glycogen is achieved tion because of its chelating properties, and in by the enzymic removal of a portion of the wound-healing preparations. Bacterial cell walls α1→4 linked straight chain consisting of several contain peptidoglycan structures in which car- glucose residues, then transferring this short chain bohydrate chains composed of alternating β1→4 to a suitable 6-hydroxyl group. A less common linked N -acetylglucosamine and N -acetylmuramic storage polysaccharide found in certain plants of acid (O-lactyl-N -acetylglucosamine) residues are 474 CARBOHYDRATES

OH D-Glc α → amylopectin O O 1 4 OH O (up to 106 residues; branching HO 1 OH O 4 HO HO 1 about every 20 residues) O O D-Glc HO α → HO 1 6 6 O HO O glycogen O O α1→4 (>106 residues; branching about D-Glc HO 1 OH amylose 4 HO O every 10 residues) (1000–2000 residues) D-Glc O HO OH HO β1→4 OH O O D-Glc O HO O HO 1 O O HO 4 OH HO HO O D-Glc O cellulose D-Glc D-Fru 2 (~8000 residues) 1 HO β → HO HO O 2 1 O OH β → 2 1 4 NHAc O D-Fru O HO O HO HO 1 O O O AcHN 4 OH GlcN = glucosamine D-GlcNAc inulin D-GlcNAc GlcNAc = N-acetylglucosamine (30–35 residues) chitin (500–5000 residues)

Figure 8.19

N-acetylmuramic acid D-GlcNAc OH β1→4 NHAc O O HO O O O O AcHN OH NHAc OH O O O HO O Ala O O O AcHN OH D-Glu O Ala Lys Gly Gly D-Ala D-Glu Gly Lys Gly Gly D-Ala peptidoglycan (20–130 monosaccharide residues)

Figure 8.20 cross-linked via peptide structures. The pepti- sequence, and the lysine from one chain is doglycan of Staphylococcus aureus is illustrated bonded to the penultimate D-alanine of another in Figure 8.20, showing the involvement of the chain through five glycine residues, at the lactyl group of the N -acetylmuramic acid to same time displacing the terminal D-alanine (see link the peptide with the carbohydrate via an Figure 7.36, page 444). The biological activi- amide/peptide bond. During the cross-linking pro- ties of the β-lactam antibiotics, e.g. penicillins cess, the peptide chains from the N -acetylmuramic and cephalosporins (see page 444) and of the acid residues have a terminal –Lys –D-Ala–D-Ala last-resort antibiotic vancomycin (see page 426) POLYSACCHARIDES 475

O MannA = mannuronic acid CO2H OH β → O HO2C 1 4 O HO 1 O HO O α1→4 HO 1 4 HO O O O HO2C OH D-GalA CO2H D-MannA 4 O D-MannA HO GalA = galacturonic acid HO alginic acid O − D-GalA (200 900 residues) pectin (400-1000 residues)

CO H α1→4 OSO H 2 β1→4 OSO H O O 3 O O 3 O O repeating units of HO O and HO O HO α1→4 HO α1→4 HO2C HO OSO3H HN HN O D-GalA O L-IdoA D-GlcN SO H D-GlcN SO3H note that sulphation is 3 IdoA = iduronic acid partial and variable

heparin (10−30 disaccharide residues)

Figure 8.21 stem from an inhibition of the cross-linking mech- in the cell walls of fruit, and the property of anism during the biosynthesis of the bacterial cell aqueous solutions under acid conditions forming wall, and relate to this terminal –D-Ala–D-Ala gels is the basis of jam making. Alginic acid sequence during biosynthesis. The subdivision (Figure 8.21) is formed by β1→4 linkage of man- of bacteria into Gram-positive or Gram-negative nuronic acid residues, and is the main cell wall reflects the ability of the peptidoglycan cell wall constituent of brown algae (seaweeds). Salts of to take up Gram’s dye stain. In Gram-negative alginic acid are valuable thickening agents in the organisms, an additional lipopolysaccharide cell food industry, and the insoluble calcium salt is membrane surrounding the peptidoglycan prevents the basis of absorbable alginate surgical dress- attack of the dye. ings. The mammalian blood anticoagulant heparin Polymers of uronic acids are encountered in (Figure 8.21) is also a carbohydrate polymer con- pectins, which are essentially chains of galac- taining uronic acid residues, but these alternate turonic acid residues linked α1→4 (Figure 8.21), with glucosamine derivatives. Polymers of this though some of the carboxyl groups are present kind are known as anionic mucopolysaccharides, as methyl esters. These materials are present or glycosaminoglycans. Heparin consists of two

Polysaccharides

Starch for medicinal and pharmaceutical use may be obtained from a variety of plant sources, including maize (Zea mays; Gramineae), wheat (Triticum aestivum; Gramineae), potato (Solanum tuberosum; Solanaceae), rice (Oryza sativa; Gramineae/Poaceae), and arrowroot (Maranta arudinacea; Marantaceae). Most contain about 25% amylose and 75% amylopectin (Figure 8.19), but these proportions can vary according to the plant tissue.

(Continues) 476 CARBOHYDRATES

(Continued)

Starch is widely used in the food industry, and finds considerable applications in medicine. Its absorbent properties make it ideal for dusting powders, and its ability to swell in water makes it a valuable formulation aid, being the basis for tablet disintegrants. Soluble starch is obtained by partial acid hydrolysis, and is completely soluble in hot water. Cellulose (Figure 8.19) may be extracted from wood pulp, and is usually partially hydrolysed with acid to give microcrystalline cellulose. These materials are used as tablet diluents. Semi-synthetic derivatives of cellulose, e.g. methylcellulose, hydroxymethylcellulose,and carboxymethylcellulose, are used as emulsifying and suspending agents. Cellulose acetate phthalate is cellulose with about half the hydroxyl groups acetylated, and the remainder esterified with phthalic acid. It is used as an acid-resistant enteric coating for tablets and capsules. Alginic acid (Figure 8.21) is obtained by alkaline (Na2CO3) extraction of a range of brown seaweeds, chiefly species of Laminaria (Laminariaceae) and Ascophyllum (Phaeophyceae) in Europe, and species of Macrocystis (Lessoniaceae) on the Pacific coast of the USA. The carbohydrate material constitutes 20–40% of the dry weight of the algae. The acid is usually converted into its soluble sodium salt or insoluble calcium salt. Sodium alginate finds many applications as a stabilizing and thickening agent in a variety of industries, particularly food manufacture, and the pharmaceutical industry, where it is of value in the formulation of creams, ointments, and tablets. Calcium alginate is the basis of many absorbable haemostatic surgical dressings. Alginic acid or alginates are incorporated into many aluminium- and magnesium-containing antacid preparations to protect against gastro-oesophageal reflux. Alginic acid released by the action of gastric acid helps to form a barrier over the gastric contents. Agar is a carbohydrate extracted using hot dilute acid from various species of red algae (seaweeds) including Gelidium (Gelidiaceae) and Gracilaria (Gracilariaceae) from Japan, Spain, Australasia, and the USA. Agar is a heterogeneous polymer, which may be fractionated into two main components, agarose and agaropectin. Agarose yields D- and L-galactose on hydrolysis, and contains alternating β1→3 linked D-galactose and α1→4 linked L-galactose, with the L-sugar in a 3,6-anhydro form. Agaropectin has a similar structure but some of the residues are methylated, sulphated, or in the form of a cyclic ketal with pyruvic acid. Agar’s main application is in bacterial culture media, where its gelling properties are exploited. It is also used to some extent as a suspending agent and a bulk laxative. Agarose is now important as a support in affinity chromatography. Tragacanth is a dried gummy exudate obtained from Astragalus gummifer (Legumi- nosae/Fabaceae) and other Astragalus species, small shrubs found in Iran, Syria, Greece, and Turkey. It is usually obtained by deliberate incision of the stems. This material swells in water to give a stiff mucilage with an extremely high viscosity, and provides a useful suspending and binding agent. It is chemically a complex material, and yields D-galacturonic acid, D-galactose, L-fucose, L-arabinose, and D-xylose on hydrolysis. Some of the uronic acid carboxyls are methylated. Acacia (gum arabic) is a dried gum from the stems and branches of the tree Acacia senegal (Leguminosae/Fabaceae), abundant in the Sudan and Central and West Africa. Trees are tapped by removing a portion of the bark. The gum is used as a suspending agent, and adhesive and binder for tablets. The carbohydrate is a complex branched-chain material, which yields L-arabinose, D-galactose, D-glucuronic acid, and L-rhamnose on hydrolysis. Occluded enzymes (oxidases, peroxidases, and pectinases) can cause problems in some formulations, unless inactivated by heat.

(Continues) AMINOSUGARS 477

(Continued)

Karaya or Sterculia Gum is a dried gum obtained from the trunks of the tree Sterculia urens (Sterculiaceae) or other Sterculia species found in India. It exudes naturally, or may be obtained by incising through the bark. It contains a branched polysaccharide comprising L-rhamnose, D-galactose, and a high proportion of D-galacturonic acid and D-glucuronic acid residues. The molecule is partially acetylated, and the gum typically has an odour of acetic acid. It is used as a bulk laxative, and as a suspending agent. It has proved particularly effective as an adhesive for stomal appliances, rings of the purified gum being used to provide a non-irritant seal between the stomal bag and the patient’s skin. Heparin is usually extracted from the mucosa of bovine lung or porcine intestines, where it is present in the mast cells. It is a blood anticoagulant and is used clinically to prevent or treat deep-vein thrombosis. It is administered by injection or intravenous infusion and provides rapid action. It is also active in vitro, and is used to prevent the clotting of blood in research preparations. Heparin acts by complexing with enzymes in the blood which are involved in the clotting process. Although not strictly an enzyme inhibitor, its presence enhances the natural inhibition process between thrombin and antithrombin III by forming a ternary complex. A specific pentasaccharide sequence containing a 3-O-sulphated D-glucosamine residue is essential for functional binding of antithrombin. Natural heparin is a mixture of glycosaminoglycans (Figure 8.21), with only a fraction of the molecules having the required binding sequence, and it has a relatively short duration of action. Partial hydrolysis of natural heparin by chemical or enzymic means has resulted in a range of low molecular weight heparins having similar activity but with a longer duration of action. Certoparin, dalteparin, enoxaparin,andtinzaparin are examples of these currently being used clinically. Protamine, a basic protein from the testes of fish of the salmon family, e.g. Salmo and Onchorhynchus species (see insulin, page 417), is a heparin antagonist, which may be used to counteract haemorrhage caused by overdosage of heparin. repeating disaccharide units, in which the amino modifications such as imine formation, the product functions and some of the hydroxyls are sulphated, will then be a polyhydroxy-piperidine or pyrroli- producing a heterogeneous polymer. The carboxyls dine. Any confusion with ornithine/lysine-derived and sulphates together make heparin a strongly alkaloids (see pages 292, 307) should be dispelled acidic water-soluble material. by the characteristic polyhydroxy substitution. The piperidine structures deoxynojirimycin and deoxymannojirimycin (Figure 8.22) from Strep- AMINOSUGARS tomyces subrutilis are good examples. The pathways to deoxynojirimycin and deoxy- Aminosugars are readily produced from ketosug- mannojirimycin (Figure 8.22) start from the ars by transamination processes. Whilst many of ketosugar fructose, which is aminated and then the natural examples, e.g. glucosamine and galac- oxidized to mannojirimycin. This can then form tosamine (Figure 8.10), demonstrate the results of a cyclic aminohemiacetal. Dehydration to the this transamination, there are some further struc- imine can follow, and reduction yields deoxy- tures where the newly introduced amino group mannojirimycin. Nojirimycin is an epimer of becomes part of a heterocyclic ring system. This mannojirimycin, and analogous modifications then arises by using the amino group as a nucleophile give deoxynojirimycin. Deoxynojirimycin is found to generate an aminohemiacetal linkage, rather in various strains of Streptomyces and Bacil- than a hydroxyl to produce a hemiacetal. This, lus, as well as some plants, e.g. Morus spp. of course, is the addition step in the formation of (Moraceae), and is attracting considerable atten- an imine (Schiff base) (see page 18). Should the tion in the search for anti-HIV agents. This and anomeric hydroxyl then be removed in subsequent related structures are inhibitors of glycosidase 478 CARBOHYDRATES

transamination CHO CH2OH CH2OH CH2OH H OH O H2N H H2N H HO H HO H HO H O HO H HOH HOH HOH HOH HOH HOH HOH HOH CH2OH CH2OH CH2OH CHO D-glucose D-fructose mannojirimycin nucleophilic addition of amino on to carbonyl epimerization HO HO OH HO NH HO NH HO OH HO OH nojirimycin HO

– H2O – H2O

OH HO HO N HO N HO HO HO HO reduction reduction OH OH HO OH HO OH HO HO OH NH NH ≡ HO HO ≡ OH HO HO OH N HO N H deoxynojirimycin deoxymannojirimycin H

Figure 8.22 enzymes (compare indolizidine alkaloids such as groups are bound as guanidino substituents making castanospermine, page 310). By altering the con- streptidine. Other medicinally useful aminogly- stitution of glycoproteins on the surface of the coside antibiotics are based on the aminocyclitol virus, such compounds interfere with the binding 2-deoxystreptamine (Figure 8.24), e.g. gentam- of the HIV particle to components of the immune icin C1 (see Figure 8.27) from Micromonospora system. purpurea. Streptamine and 2-deoxystreptamine are both derived from glucose 6-phosphate. The AMINOGLYCOSIDES route to the streptamine system can be for- mulated to involve oxidation (in the acyclic The aminoglycosides∗ form an important group form) of the 5-hydroxyl, allowing removal of of antibiotic agents and are immediately rec- a proton from C-6 and generation of an eno- ognizable as modified carbohydrate molecules. late anion (Figure 8.23). The cyclohexane ring Typically, they have two or three uncommon is then formed by attack of this enolate anion sugars attached through glycoside linkages to an on to the C-1 carbonyl. Reduction and hydrol- aminocyclitol, i.e. an amino-substituted cyclohex- ysis of the phosphate produces myo-inositol. ane system, which also has carbohydrate origins. The amino groups as in streptamine are then The first of these agents to be discovered was introduced by oxidation/transamination reactions. streptomycin (see Figure 8.25) from Streptomyces Streptidine incorporates amidino groups from griseus, whose structure contains the aminocyclitol arginine, by nucleophilic attack of the aminocy- streptamine (Figure 8.23), though both amino clitol amino group on to the imino function AMINOGLYCOSIDES 479

oxidation of formation of aldol-type OP open-chain form OP enolate anion reaction O O O NAD+ O HO HO HO OP HO OP HO OH HO O HO O HO OH HO HO HO HO D-glucose 6-P NH oxidations NADH transaminations HN NH2 ATP L-Arg HO OH HO OH HO OP HN OH HO OH H2N HO OH HO HO HO several steps: amino groups are HO HO NH streptidine introduced by transamination; myo-inositol myo-inositol 1-P the substrates for N-alkylation are aminocyclitol phosphates, hence requirement for ATP H N NH 2 H HO OH CO H H N OH 2 2 N-alkylation via nucleophilic H2NN HO H attack of amine on to imine; the NH L-Arg 2 streptamine other product is L-ornithine NH2

Figure 8.23

oxidation of E2 elimination favoured by reduction of carbonyl 4-hydroxyl formation of conjugated system restoring 4-hydroxyl OP OP 4 O + O HO O NAD O – HOP O NADH HO O HO OH HO OH HO OH HO OH HO HO HO HO D-glucose 6-P opening of hemiacetal ring generates enolate oxidation and aldol-type reaction transamination anion H2N O O 3 2 reactions O HO 4 HO 1 HO HO HO 6 NH2 HO OH HO O HO O 5 HO HO HO HO 2-deoxystreptamine 2-deoxy-scyllo-inosose

Figure 8.24 of arginine (Figure 8.23). However, streptamine cyclohexanone, and transamination reactions allow itself is not a precursor, and the first guani- formation of 2-deoxystreptamine. The pathway dino side-chain is built up before the second in Figure 8.24 is remarkably similar to that amino group is introduced. The N -alkylation operating in the biosynthesis of dehydroquinic steps also involve aminocyclitol phosphate sub- acid from the seven-carbon sugar DAHP in strates. The biosynthesis of 2-deoxystreptamine the early part of the shikimate pathway (see shares similar features, but the sequence involves page 122). loss of the oxygen function from C-6 of glu- The other component parts of streptomycin, cose 6-phosphate in an elimination reaction namely L-streptose and 2-deoxy-2-methylamino-L- (Figure 8.24). The elimination is facilitated by oxi- glucose (N -methyl-L-glucosamine) (Figure 8.25), dation of the 4-hydroxyl, which thus allows a con- are also derived from D-glucose 6-phosphate, jugated enone to develop in the elimination step, though the detailed features of these pathways but the original hydroxyl is reformed by reduc- will not be considered further. Undoubtedly, these tion after the elimination. The cyclohexane ring materials are linked to streptidine through stepwise is then formed by attack of an enolate anion on glycosylation reactions via their nucleoside sugars to the C-1 carbonyl giving a tetrahydroxy- (Figure 8.25). 480 CARBOHYDRATES

H2N H2N NH NH HN 2 HN 6 1 HO OH HO OH H2N HN OH H2N HN 4 OH streptidine 3 5 HO NH NH OHC O O OHC 1′ L-streptose O Me Me O UDP HO O HO OH HO 1″ O N-methyl-L-glucosamine HO NHMe 3″ (2-deoxy-2-methylamino-L-glucose) HO HO O O UDP HO NHMe streptomycin HO

Figure 8.25

Aminoglycoside Antibiotics

The aminoglycoside antibiotics have a wide spectrum of activity, including activity against some Gram-positive and many Gram-negative bacteria. They are not absorbed from the gut, and for systemic infections must be administered by injection. However, they can be administered orally to control intestinal flora. The widespread use of aminoglycoside antibiotics is limited by their nephrotoxicity, which results in impaired kidney function, and by their ototoxicity, which is a serious side-effect and can lead to irreversible loss of hearing. They are thus reserved for treatment of serious infections where less toxic antibiotics have proved ineffective. The aminoglycoside antibiotics interfere with protein biosynthesis by acting on the smaller 30S subunit of the bacterial ribosome. Streptomycin is known to interfere with the initiation complex, but most agents block the translocation step as the major mechanism of action. Some antibiotics can also induce a misreading of the genetic code to yield membrane proteins with an incorrect amino acid sequence leading to altered membrane permeability. This actually increases aminoglycoside uptake and leads to rapid cell death. Bacterial resistance to the aminoglycoside antibiotics has proved to be a problem, and this has also contributed to their decreasing use. Several mechanisms of resistance have been identified. These include changes in the bacterial ribosome so that the affinity for the antibiotic is significantly decreased, reduction in the rate at which the antibiotic passes into the bacterial cell, and plasmid transfer of extrachromosomal R-factors. This latter mechanism is the most common and causes major clinical problems. Bacteria are capable of acquiring genetic material from other bacteria, and in the case of the aminoglycosides this has led to the organisms becoming capable of producing enzymes that inactivate the antibiotic. The modifications encountered are acetylation, adenylylation, and phosphorylation. (Note adenylic acid = adenosine 5-phosphate). The enzymes are referred to as AAC (aminoglycoside acetyltransferase), ANT (aminoglycoside nucleotidyltransferase) (sometimes AAD (aminoglycoside adenylyltransferase)), and APH (aminoglycoside phosphotransferase). They differ with respect to the reaction catalysed, the position of derivatization (see numbering scheme in gentamicin, Figure 8.26), and the range of substrates attacked. Thus, some clinically significant inactivating enzymes are

(Continues) AMINOGLYCOSIDES 481

(Continued)

usual sites for derivatization of OH substituents in aminoglycoside 4″ 2′ 4′ 5″ O 3′ R1 antibiotics by inactivating enzymes Me 2″ H2N 1″ O MeHN 1′ 6′ 2 3″ HO 5′ NHR not applicable in case of gentamicin HO 6 5 4 O O H2N 2 NH2 1 3

2-deoxy- L-garosamine streptamine D-purpurosamine

1 2 R = Me, R = Me, gentamicin C1 1 2 R = Me, R = H, gentamicin C2 1 2 R = H, R = H, gentamicin C1a

Figure 8.26

• AAC(3) and AAC(6), which acetylate the 3- and 6-amino functions respectively in gentamicin, tobramycin, kanamycin, neomycin, amikacin, and netilmicin, • ANT(2), which adenylylates the 2-hydroxy group in gentamicin, tobramycin, and kanamycin, • APH(3), which phosphorylates the 3-hydroxyl in neomycin and kanamycin, and • APH(3), which phosphorylates the 3-hydroxyl of streptomycin.

Other changes which may be imparted include acetylation of groups at position 2, adenylylation of position 4 substituents, and phosphorylation of the position 2 substituent. Position 6 in the streptamine portion of streptomycin is also susceptible to adenylylation and phosphorylation. Aminoglycoside antibiotics are produced in culture by strains of Streptomyces and Micromonsopora. Compounds obtained from Streptomyces have been given names ending in -mycin, whilst those from Micromonospora have names ending in -micin.

Streptamine-containing Antibiotics

Streptomycin (Figure 8.25) is produced by cultures of a strain of Streptomyces griseus,and is mainly active against Gram-negative organisms. Because of its toxic properties it is rarely used in modern medicine except against resistant strains of Mycobacterium tuberculosis in the treatment of tuberculosis. Spectinomycin (Figure 8.27) is not strictly an aminoglycoside, but its structure does contain a modified streptamine portion linked by a glycoside bond to a deoxy sugar. It is sometimes written as a ketone at position 4, though this exists as a hydrate as shown in Figure 8.27. Spectinomycin is produced by cultures of Streptomyces spectabilis, and although it displays a broad antibacterial spectrum, it is only used against Neisseria gonorrhoea for the treatment of gonorrhoea where the organism has proved resistant to other antibiotics. It is known to inhibit protein biosynthesis on the 30S ribosomal subunit, but does not appear to cause any misreading of the genetic code.

(Continues) 482 CARBOHYDRATES

(Continued)

HO NHMe OH MeHN O HO O HO OH 4 O

spectinomycin Me

Figure 8.27

2-Deoxystreptamine-containing Antibiotics

Gentamicin is a mixture of antibiotics obtained from Micromonospora purpurea. Fermentation yields a mixture of gentamicins A, B, and C, from which gentamicin C is separated for medicinal use. This is also a mixture, the main component being gentamicin C1 (Figure 8.26) (50–60%), with smaller amounts of gentamicin C1a and gentamicin C2. These three components differ in respect to the side-chain in the purpurosamine sugar. Gentamicin is clinically the most important of the aminoglycoside antibiotics, and is widely used for the treatment of serious infections, often in combination with a penicillin when the infectious organism is unknown. It has a broad spectrum of activity, but is inactive against anaerobes. It is active against pathogenic enterobacteria such as Enterobacter, Escherichia,andKlebsiella, and also against Pseudomonas aeruginosa. Compared with other compounds in this group, its component structures contain fewer functionalities that may be attacked by inactivating enzymes, and this means gentamicin may be more effective than some other agents. Sisomicin (Figure 8.28) is a dehydro analogue of gentamicin C1a, and is produced by cultures of Micromonospora inyoensis. It is used medicinally in the form of the semi-synthetic N-ethyl derivative netilmicin (Figure 8.28), which has a similar activity to gentamicin, but causes less ototoxicity. The kanamycins (Figure 8.29) are a mixture of aminoglycosides produced by Streptomyces kanamyceticus, but have been superseded by other drugs. Amikacin (Figure 8.29) is a semi- synthetic acyl derivative of kanamycin A, the introduction of the 4-amino-2-hydroxybutyryl group helping to protect the antibiotic against enzymic deactivation at several positions, whilst

OH O Me H2N MeHN O HO HO NH O O 2 RHN NH2

2-deoxy- dehydro- L-garosamine streptamine purpurosamine C

R = H, sisomicin R = Et, netilmicin

Figure 8.28

(Continues) AMINOGLYCOSIDES 483

(Continued)

OH OH OH O O 3′ HO R OH HO NH2 OH H2N O H2N O HO HO NH2 HO HO NH2 O O O O H2N NH2 H2N NH2 6-amino-6-deoxy- 3-amino-3-deoxy- 2-deoxy- D-glucose 3-amino-3-deoxy- 2-deoxy- D-glucose streptamine (in kanamycin A) D-glucose streptamine D-nebrosamine R = OH, kanamycin A tobramycin R = NH2, kanamycin B OH O OH HO HO OH H2N O HO HO NH2 HO O O HN NH2 H2N O amikacin

Figure 8.29 still maintaining the activity of the parent molecule. It is stable to many of the aminoglycoside inactivating enzymes, and is valuable for the treatment of serious infections caused by Gram-negative bacteria which are resistant to gentamicin. Tobramycin (Figure 8.29) (also called nebramycin factor 6) is an analogue of kanamycin B isolated from Streptomyces tenebrarius, and is also less prone to deactivation in that it lacks the susceptible 3-hydroxyl group. It is slightly more active towards Pseudomonas aeruginosa than gentamicin, but shows less activity against other Gram-negative bacteria. Neomycin is a mixture of neomycin B (framycetin) (Figure 8.30) and its epimer neomycin C, the latter component accounting for some 5–15% of the mixture. It is produced by cultures of Streptomyces fradiae, and, in contrast to the other clinically useful aminoglycosides described, contains three sugar residues linked to 2-deoxystreptamine. One of these is the common sugar D-ribose. Neomycin has good activity against Gram-positive and Gram- negative bacteria, but is very ototoxic. Its use is thus restricted to oral treatment of intestinal infections (it is poorly absorbed from the digestive tract) and topical applications in eyedrops, eardrops, and ointments.

NH2 HO O D-neosamine HO NH2 H2N O 2-deoxystreptamine HO O NH2 O OH D-ribose OH O NH2

O * L-neosamine B H2N OH HO neomycin B (framycetin) (neomycin C is epimer at *)

Figure 8.30 484 CARBOHYDRATES

O dTDP-D-glucose H2N HO Schiff base formation; this HO CH2OH OPPdT facilitates epimerization at C-2 and 5,6-dehydration O dTDP-4-amino- HO CH2OH 4,6-dideoxy-D-glucose HO CH2OH HO H HO HO 5 H OH 2 O H OH HO O HO N HO H OH OH OH HO 2-epi-5-epi-valiolone OPPdT CH2OP OH sedo-heptulose 7-P HO maltose cyclization and other modifications analogous to those in the formation of O 3-dehydroquinic acid from DAHP HO N H HO (see Figure 4.1) OH OH HO O CO2H O O HO OH HO CO2H OH HO H H O O HO H HO acarbose HO OH HO H OH HO O HO NH H OH OH 2 OH CH2OP 3-dehydroquinic valienamine DAHP acid

Figure 8.31

The aminocyclitol found in acarbose∗ (Fig- epimerization and dehydration steps, and these ure 8.31) is based on valienamine, though this are readily seen to be facilitated by the imine is not a precursor, and the nitrogen is introduced function. Unusually, the two further glucose units via an imine with the aminosugar, 4-amino-4,6- are not added sequentially, but via the preformed dideoxyglucose in the form of its deoxyTDP dimer maltose. Acarbose is produced by strains of derivative. The cyclitol involved is 2-epi-5-epi- Actinoplanes sp., and is of clinical importance in valiolone, and this appears to be produced from the treatment of diabetes. the seven-carbon sugar derivative sedo-heptulose The antibiotic lincomycin∗ (Figure 8.32) from 7-phosphate. The reaction sequence is exactly Streptomyces lincolnensis bears a superficial analogous to that seen in the transformation similarity to the aminoglycosides, but has a of DAHP into 3-dehydroquinic acid at the rather more complex origin. The sugar fragment beginning of the shikimate pathway (page 122). is termed methyl α-thiolincosaminide, contains a The valienamine moiety requires subsequent thiomethyl group, and is known to be derived

Acarbose Acarbose is obtained commercially from fermentation cultures of selected strains of an undefined species of Actinoplanes. It is an inhibitor of α-glucosidase, the enzyme that hydrolyses starch and sucrose. It is employed in the treatment of diabetic patients, allowing better utilization of starch- or sucrose-containing diets, by delaying the digestion of such foods and thus slowing down the intestinal release of α-D-glucose. It has a small but significant effect in lowering blood glucose, and is used either on its own, or alongside oral hypoglycaemic agents, in cases where dietary control with or without drugs has proved inadequate. Flatulence is a common side-effect. AMINOGLYCOSIDES 485

NMe O OH NMe O Cl

HN HN HO HO O O 2 HO HO HO HO lincomycinSMe clindamycin SMe

Figure 8.32

oxidative cleavage Schiff base formation and loss of C-methylation of aromatic ring two-carbon fragment Ad R via SAM S HO CO2H OHC CO H 2 Me HO2C CO2H N H2 NH2 HO HO NH L-DOPA

• L-Met CO2H lincomycin N

4-propyl-N-methylproline

Figure 8.33 from two molecules of glucose, one of which ring (see page 27) provides all the carbons for the provides a five-carbon unit, the other a three-carbon pyrrolidine ring, the carboxyl, and two carbons of unit. The 4-propyl-N -methylproline fragment does the propyl side-chain. The terminal carbon of the not originate from proline, but is actually a propyl is supplied by L-methionine, as are the N - metabolite from the aromatic amino acid L-DOPA methyl, and the S -methyl in the sugar fragment. Two (Figure 8.33). Oxidative cleavage of the aromatic carbons from DOPA are lost during the biosynthesis.

Lincomycin and Clindamycin

Lincomycin (Figure 8.32) is obtained from cultures of Streptomyces lincolnensis. The semi- synthetic derivative clindamycin (Figure 8.32) obtained by chlorination of the lincomycin with resultant inversion of stereochemistry is more active and better absorbed from the gut, and has largely replaced lincomycin. Both antibiotics are active against most Gram-positive bacteria, including penicillin-resistant staphylococci. Their use is restricted by side-effects. These include diarrhoea and occasionally serious pseudomembraneous colitis, caused by overgrowth of resistant strains of Clostridium difficile, which can cause fatalities in elderly patients. However, this may be controlled by the additional administration of vancomycin (see page 426). Clindamycin finds particular application in the treatment of staphylococcal joint and bone infections such as osteomyelitis since it readily penetrates into bone. Clindamycin 2-phosphate is also of value, especially in the topical treatment of acne vulgaris and vaginal infections. Lincomycin and clindamycin inhibit protein biosynthesis by blocking the peptidyltransferase site on the 50S subunit of the bacterial ribosome. Microbial resistance may develop slowly, and in some cases has been traced to adenylylation of the antibiotic. 486 CARBOHYDRATES

FURTHER READING Franz G (1989) Polysaccharides in pharmacy: current applications and future concepts. Planta Med 55, General 493–497. BeMiller JN (1992) Carbohydrates. Kirk–Othmer Ency- clopedia of Chemical Technology, 4th edn, Vol 4. Heparin Wiley, New York, pp 911–948. Bell WR (1992) Blood, coagulants and anticoagulants. Weymouth-Wilson AC (1997) The role of carbohydrates Kirk–Othmer Encyclopedia of Chemical Technology, in biologically active natural products. Nat Prod Rep 4th edn, Vol 4. Wiley, New York, pp 333–360. 14,99–110. Petitou M and van Boeckel CAA (1992) Chemical synthesis of heparin fragments and analogues. Prog Chem Org Nat Prod 60, 143–210. Vitamin C Gunay NS and Linhardt RJ (1999) Heparinoids: struc- Emsley J (1995) A life on the high Cs. Chem Brit ture, biological activities and therapeutic applica- Planta Med 65 946–948. tions. , 301–306. Gordon MH (1996) Dietary antioxidants in disease van Boeckel CAA and Petitou M (1993) The unique prevention. Nat Prod Rep 13, 265–273. antithrombin III binding domain of heparin: a lead Angew Chem Int Kuellmer V (1998) Vitamins (ascorbic acid). Kirk– to new synthetic antithrombotics. Edn Engl 32 Othmer Encyclopedia of Chemical Technology, , 1671–1690. 4th edn, Vol 25. Wiley, New York, pp 17–47. Loewus FA (1999) Biosynthesis and metabolism of Aminosugars ascorbic acid in plants and of analogs of ascorbic acid in fungi. Phytochemistry 52, 193–210. Hughes Ab and Rudge AJ (1994) Deoxynojirimycin: Scott G (1995) Antioxidants – the modern elixir? Chem synthesis and biological activity. Nat Prod Rep 11, Brit 879–882. 135–162. Williams CM (1993) Diet and cancer prevention. Chem Ind 280–283. Aminoglycoside Antibiotics

Lancini GC and Lorenzetti R (1993) Biotechnology Polysaccharides, Gums of Antibiotics and Other Microbial Metabolites. Plenum, New York. Baird JK (1994) Gums. Kirk–Othmer Encyclopedia of Lancini G, Parenti F and Gallo GG (1995) Antibi- Chemical Technology, 4th edn, Vol 12. Wiley, New otics – a Multidisciplinary Approach. Plenum, New York, pp 842–862. York. Bugg TDH (1999) Bacterial peptidoglycan biosyn- McGregor D (1992) Antibiotics (aminoglycosides). thesis and its inhibition. Comprehensive Natural Kirk–Othmer Encyclopedia of Chemical Technology, Products Chemistry, Vol 3. Elsevier, Amsterdam, 4th edn, Vol 2. Wiley, New York, pp 904–926. pp 241–294. Bugg TDH and Walsh CT (1992) Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: Lincomycin, Clindamycin enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep 9, 199–215. Bannister B (1992) Antibiotics (lincosaminides). Kirk– Dea ICM (1989) Industrial polysaccharides. Pure Appl Othmer Encyclopedia of Chemical Technology, Chem 61, 1315–1322. 4th edn, Vol 3. Wiley, New York, pp 159–168.