The Synthesis and Biological Evaluation of Several Series Of

The Synthesis And Biological Evaluation Of Several Series Of Melatonin Agonists and Antagonists A Thesis Submitted by David J. Davies In partial fulfilment of the requirements for the degree of Doctor of Philosophy of the University of London Department of Chemistry University College London WC1E 6 BN September 1999 1 ProQuest Number: 10609324 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609324 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 iiSiv 7 “The changing of bodies into light, and light into bodies, is very comfortable to the course of nature, which seems delighted with transmutations. ” - Isaac Newton 2 Acknowledgements I wish to thank the following people for their support and contributions to this work:- Prof. Peter Garratt for his encouragement, understanding, patience and support, and for proof reading this thesis. Any remaining errors or omissions are mine alone. The Garratt group for their friendliness and encouragement. Dr. David Sugden and co-workers at Kings College London for carrying out all biological assays and for helpful discussions. Drs. Paul Doyle, C.J. Harris, Allen Miller, Rob West and Dave Madge of the Department of Medicinal Chemistry at the Wellcome Research Laboratories, for their support in starting this work, and Coffee producers of the world for enabling me to continue it. My partner Lucy, family, friends and colleagues for their support and encouragement. The Staff of the Physical Chemistry departments at the Wellcome Research Laboratories, and Glaxo-Wellcome, for providing NMR, Mass Spectrometry, Infra Red and Microanalytical services and for obtaining circular dichroism spectra. 3 Abstract The work contained in this thesis forms part of an ongoing programme to examine the structure of the melatonin receptor by synthesis of indole based melatonin analogues. The thesis is divided into five chapters. In Chapter One, an overview of the biosynthesis and physiological activity of the pineal hormone melatonin is presented. Receptor structure and distribution are also discussed, followed by a review of analogue studies reported in the literature up to the end of 1998. Chapter Two describes the results of studies aimed at the introduction of an element of conformational restriction to the C3 side chain of melatonin. A series of 1 -methylcycloalkan[b]indoles was synthesised which had high affinity in the2 - 1 J S [ I]-iodomelatonin radioligand binding assay and excellent biological potency in the Xenopus Laevis assay. Several racemic compounds from this series were resolved into enantiomers by chromatography and the melatonin receptor shown, for the first time, to be capable of chiral discrimination. The (R)-(-) enantiomers were demonstrated to be considerably better (up to 130 fold) at binding to the melatonin receptor and up to 400 times more potent than the (S)-(+) enantiomer. Several routes towards l-H-cycloalkan[b]indoles were investigated. Chapter Three reports the synthesis and results of an exploratory investigation into the potential of A^-acyltetrahydro-P-carbolines as ligands for the melatonin receptor. No significant new agonists were obtained and many of the compounds showed antagonist behaviour. Chapter Four includes the synthesis of several individual analogues or small sets of compounds aimed at fulfilling a specific requirement. Attempts were made to synthesise some novel analogues by direct substitution reactions on melatonin itself, and some attempts were made to alter postulated hydrogen bonding sites between ligand and receptor by replacing oxygen atoms in the pharmacophores with sulphur. Chapter Five contains the experimental details for Chapters Two to Four. 4 Abbreviations Ag Agonist Ant Antagonist Aq. Aqueous Bz Benzyl CDCI3 Deuterochloroform DCM Dichloromethane DMF N,N-dimethylformamide DMSO Dimethylsulphoxide EC50 Effective concentration required to achieve 50% of the maximal response. E.I.M.S. Electron impact mass spectrometry ETOAc Ethyl acetate Eq. Equivalent FAB Fast atom bombardment HOAt Hydroxyazabenzotriazole HPLC-MS High pressure liquid chromatography - Mass spectrometry h. Hour IC50 The concentration of test compound which blocks the maximal effect of melatonin by 50% IR Infra red Kd Dissociation constant Kj Inhibition constant MS Mass spectrometry NMR Nuclear magnetic resonance ppm Parts per million TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography 5 Contents Page Acknowledgements ........ 3 Abstract. ......... 4 Abbreviations ........ 5 Chapter 1: Review Physiological activity of the pineal hormone melatonin Aim. 11 1 .1 Introduction ....... 1 2 1 .2 Regulation of rhythmic melatonin production 17 1.3 Biosynthesis of melatonin ..... 19 1.4Regulation of melatonin biosynthesis 26 1.5 Melatonin metabolism ..... 27 1 .6 Receptor distribution ..... 28 1.7 Receptor classification ..... 32 1 .8 Circadian rhythms ...... 39 1.9Melatonin and reproduction .... 42 1 .1 0 Melatonin and sleep ..... 44 1 .1 1 Melatonin and thermoregulation .... 46 1 .1 2 Melatonin and the immune system. 47 1.13 Melatonin as an anti-oxidant and neuroprotective agent. 48 1.14 Melatonin as an oncostatic agent .... 49 1.15 Toxicity ....... 50 1.16 Structure activity relationship studies 51 1.17 C3 substituents. ...... 53 1.18 C2 substituents ...... 54 6 1.19 C5 substituents ...... 55 1.20 C6 substituents . 55 1.21 Indole bioisosteres ...... 57 1.22 Antagonists ....... 60 1.23 Review references ...... 63 Chapter 2: Conformationally Constrained Melatonin Analogues Aim ......... 73 Cy cloalkan [b] indoles 2.1 Introduction ....... 74 2.2 Synthetic approach. ...... 76 2.3 Mechanism of the Bischler reaction . 79 2.4 Synthesis . 81 2.5 Chiral chromatographic separation . 84 2.6 Synthesis of 9-H-tetrahydrocarbazoles . 89 T etrahy drocarbazolones: 2.7 Introduction ....... 93 2.8 Synthesis ....... 98 2.9 Solid phase attempts. ..... 105 Biology 2.10 Binding affinity assay . 107 2.11 Biological activity . 108 2.12 Data analysis. 109 7 Results and discussion 2.13 Binding affinity . Ill 2.14 Biological activity . 118 2.15 Summary ....... 128 Chapter 3: Tetrahydro-p-carbolines A i m .......................................................................................................... 129 3.1 Introduction ....... 130 3.2 Synthetic approach . 132 3.3 Mechanism of the Pictet-Spengler cyclisation . 134 3.4 Synthesis of tetrahydro-P-carbolines. 135 Acylation of tetrahydro-p-carbolines . 139 3.5 Coupling conditions. 139 3.6 Synthesis ....... 143 Biological Results 3.7 Results and discussion . 151 3.8 Summary . 156 Chapter 4: Miscellaneous Melatonin Analogues Aim . 158 8 2-Halomelatonin analogues 4.1 Introduction . 159 4.2 Synthesis . 162 4.3 Results and discussion . 163 Attempted synthesis of 2-fluoromelatonin 4.4 Introduction . 166 4.5 Synthesis . 169 4.6 Results and discussion . 172 O-Alkyl melatonin analogues 4.7 Introduction . 173 4.8 Synthesis . 174 4.9 Results and discussion . 179 Synthesis of N-acetyl-5-methoxy kynurenamine 4.10 Introduction ....... 181 4.11 Synthesis . 182 4.12 Results and discussion . 182 Sulphur containing analogues of melatonin 4.13 Introduction . 183 4.14 Synthesis . 188 4.15 Results and discussion ..... 202 4.16 Summary ....... 204 References for chapters 2,3 and 4. 205 9 Chapter 5: Experimental 5.1 General Information. 215 5.2 General Procedures . 216 5.3 Experimental for chapter 2 . .... 223 5.4 Experimental for chapter 3 . .... 272 5.5 Experimental for chapter 4 . 337 References for experimental procedures . 362 Appendices Selected NMR and CD spectra . 364 Ring Nomenclature . 378 X-ray crystallographic data . 379 Amino Acid Nomenclature . 380 10 Chapter 1 Physiological Activity of the Pineal Hormone Melatonin Aim This review constitutes a limited overview of aspects of melatonin research up to the end of 1998. It will attempt to focus mainly on data obtained in mammals, and in particular humans, which outline the mechanism of melatonin production and its effects on biological and endocrine function. The significance of melatonin in the establishment of circadian rhythms and sleep has been the main thrust of research to date, although in recent years a proliferation of reports have emerged suggesting effects in a wide variety of other areas. Publications can be arbitrarily divided into two types, those investigating the broad effects of melatonin on every living cell and those investigating the effects specifically mediated by high affinity cell membrane receptors. This chapter will concentrate largely on the latter. Some of the potential and actual clinical applications of melatonin will also be discussed. 11 1.1 Introduction tfi The 17 century mathematician and philosopher Rene Descartes sparked great interest in the pineal gland by identifying it as the seat of the human soul. The advent of empirical science, however, relegated the pineal gland to the role of a non-functioning vestigial organ, a view that was widely held as recently as the 1950’s. Since then the wheel has turned full circle and a

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