TRANSFORMATIVE REACTIONS ON CARBOCYCLIC COMPOUNDS

A Thesis submitted to the University of North Bengal

For the Award of

Doctor of Philosophy (Ph.D.) in Chemistry

By Jayanta Das

Research Guide Prof. Pranab Ghosh

DEPARTMENT OF CHEMISTRY UNIVERSITY OF NORTH BENGAL December, 2015

To my parents...

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DECLARATION

I hereby declare that the thesis entitled “ TRANSFORMATIVE REACTIONS ON CARBOCYCLIC COMPOUNDS ” has been prepared by myself under the guidance of Prof. Pranab Ghosh, Department of Chemistry, University of North Bengal, Drjeeling- 734013. No part of this thesis has formed the basis for the award of any degree or fellowship previously.

Jayanta Das Department of Chemistry University of North Bengal Darjeeling – 734 013, India

Date: 15.12.2015

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UNIVERSITY OF NORTH BENGAL

Prof. P. Ghosh , Ph: +91 3532776 381 (off)

DEPARTMENT OF CHEMISTRY +91 9474441468 (M) University of North Bengal, Fax: +91 353 2699 001 Darjeeling – 734 013, India. Email: [email protected]

Date: December 15 , 2015

CERTIFICATE

This is to certify that Sri Jayanta Das has prepared the thesis entitled

“TRANSFORMATIVE REACTIONS ON CARBOCYCLIC COMPOUNDS ”, for the

award of PhD degree of the University of North Bengal, under my guidance. He has carried

out the work at the Department of Chemistry, University of North Bengal, Darjeeling, West

Bengal-734013.

Professor Pranab Ghosh (Research Supervisor) Department of Chemistry University of North Bengal

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Abstract

The thesis entitled “Transformative reactions on carbocyclic compounds” comprises four chapters and a brief of their contents are as follows:

Chapter I: Carbocyclic compounds and their transformative reactions: A general perspective on natural products chemistry

Natural products include any substance produced by life. Now, due to the large extent of catenation capability of carbon, it can produce a large number of molecules made solely by them which in turn, implies that nature provides a huge number of carbocyclic compounds. And in virtue, nature itself is the richest source of a variety of carbocyclic compounds. Two or more carbocycles can be joined together in a number of different fashions to produce a number of different groups of carbocyclic compounds. And among the broad spectrum of all kinds of natural products, if we look for more abundant, easily available and highly useful carbocyclic natural products, we found mainly the steroids and terpenoids. Thus, having the opportunity of working in the field of natural products chemistry, the various known / new transformative reactions were carried out on some selective major steroids (cholesterol, b- sitosterol, ergosterol, diosgenin) and comparatively less-explored but highly potential pentacyclic triterpenoid- friedelin.

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As a consequence, to have a general and very brief overview on the transformative reactions on these substrates, the present chapter demonstrates the important findings revealed so far.

Chapter II: First report of solvent selective steroidal aromatization, efficient access to 4b,7 a-dihydroxy steroids, and syntheses of natural diaromatic ergosterols

This chapter elaborates the syntheses of natural diaromatic ergosterol derivatives and other steroidal analogues in an unprecedented simple, one-pot and convenient synthetic route. In the process, the key factor- the selectivity of the solvents (having 1,4-ethereal oxygens) towards aromatization has been established. Of note, only ethereal solvents with two oxygens such as 1,4-dioxane, 1,3-dioxalane, 1,2-dimethoxy ethane and 1,2-diethoxy ethane were found able to result selective aromatization. Thorough solvent-dependant study of the model reaction reveals valuable product composition which may be exploited, specially, for the synthesis of biologically important steroid molecules. Efficient access to 4 b,7 a-dihydroxy cholesterol is also described. By using the established solvent-selective steroidal methodology, the yield of the natural product, diaromatic ergosterol was optimized at 12%. Analogous chemistry of b-sitosterol and diosgenin is also reported. Furthermore, single crystal X-ray crystallography has resolved the molecular structures, for the first time in their class, of similar diaromatic cholesterol derivative and triacetylated 4 b,7 a-dihydroxy cholesterol derivative.

Chapter III: Polyhydroxy and epoxy-polyhydroxy steroids: design, synthesis and study of their preliminary gelation behaviour The present work associated with this chapter demonstrates basically two aspects of some new polyhydroxy steroids - designed synthesis and their preliminary gelation behavior.

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Altogether sixteen polyhydroxy steroids (PHS, 12 new) of cholesteryl and b-sitosteryl series were synthesized and characterized. Among them eight (all new) are, in precise, epoxy- polyhydroxy steroids (5,6-epoxy-3b,4 a-dihydroxy- and 5,6-epoxy-3b,4 a,7 a-trihydroxy-), of which the a-diastereomers were utilized further to synthesize novel new tetraols (3 b,4 b,5 a,6 b-tetrahydroxy-) and pentaols (3 b,4 b,5 a,6 b,7a -pentahydroxy steroids). Thus, epoxidation followed by alkaline-opening of the oxirane-ring of appropriate steroidal 5-ene- 3b,4 b-diols and -5-ene-3b,4 b,7 a-triols, to furnish novel steroidal tetrols and pentols respectively consequences, in practical, a three-step synthetic route for the transformation of 3b,4 b,5 a,6 b-tetrahydroxy- and 3 b,4 b,5 a,6 b,7a -pentahydroxy steroids starting directly from their corresponding basic steroids, cholesterol and b-sitosterol. As a new class of polyhydroxy steroids, preliminary gelation behavior of the molecules was evaluated. At 1% or below CGC (critical gelation concentration), five PHS derivatives were found to be gelators of some selective organic solvents. Some selective organogels were characterized through T gel (gel melting temperature) and related physical parameters ( DH etc.), rheological data, and by morphology analysis (electron microscopes).

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Chapter IV: Syntheses of new friedelane triterpenoids: A-ring modifications including 2-homo derivatives

Syntheses of a number of A-ring modified friedelane triterpenoids have been accomplished. These also include the 2-homo derivatives for which, as the key step, the transformation of friedelin with Vilsmeyer-Haack reagent was used. 3-Chloro-2-formylfriedel-2-ene, the main product isolated from the reaction was transformed suitably into various derivatives and hence, following two or three simple steps starting from friedelin, it rendered possible to produce a library of C2,C3-, C3,C4-, and C2,C3,C4- functionalized friedelane triterpenoids. Besides, some useful methodologies were thus established during the various transformative attempts. These include a two-step aromatization of friedelin by N-bromosuccinimide, a one- pot dechlorination with simultaneous C-23 activation, and selective 4 a-hydroxylation with simultaneous oxidation of allylic alcohol by selenium dioxide. Again, syntheses of some friedelane derivatives, viz. , 3b-amino-4a-hydroxy-, 2-carboxamide, 2,3-seco diol, 4a- hydroxy-3-chloro-2-formylfriedel-2-ene and 3-chloro-4a-hydroxy-2-hydroxymethylfriedel- 2-ene, in a few steps, were found very much effective to enrich the A-ring modifications of friedelane triterpenoids. On the other hand, heterocycle-linked (to C3 of friedelanes) 2- homofriedelane derivatives were achieved. We believe to use these friedelane triterpenoids for future biological applications as well as to explore more interesting and usefull multifunctionalized derivatives of the particular class of pentacyclic triterpenoids.

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Preface

The thesis is the compilation of the research work carried out by the author under the supervision of Professor Pranab Ghosh in the Department of Chemistry, University of North Bengal during the period 2008 to 2015. It comprises a number of transformative reactions on different carbocyclic compounds. And as the author had privilege to work in the natural products laboratory, the carbocyclic compounds chosen as the starting substrates are some selective steroids and pentacyclic triterpenoids having immense practical utilizations. In short, a chemical compound or substance produced by a living organism —thus found in nature, is a natural product. Within the periphery of organic chemistry, we consider natural products, typically, as pure organic compounds (thus pure molecules) isolated from natural sources related to lives, directly or indirectly. Due to the widespread and increasingly demandable applications of these natural molecules, supply at a required amount has lead their preparation in the laboratory by the process of chemical synthesis, which indeed has enriched organic chemistry enormously by following a huge number of challenging targets. And considering the prime importance of steroids and pentacyclic triterpenoids in medicinal chemistry as well as in materials science, we have taken an opportunity to enrich this particular area of the natural products chemistry by synthesizing a number of interestingly useful molecules through different known/ new transformative protocols. I feel it an imperative, in my first opportunity, to express my heartfelt and sincere gratitude to my supervisor, Professor Pranab Ghosh, Department of Chemistry, University of North Bengal, West Bengal for providing me the opportunity to carry out my research work with all the marvellous supervision and utmost important suggestions. His continuous encouragement and guidance has played a pivotal role in furnishing a good basis for the present thesis. I would like to thank all the faculty members of the Department of Chemistry, University of North Bengal, whose valuable suggestions helped me a lot during my entire research. Specially, the author expresses sincere gratitude to Professor S. K. Saha, Professor B. Basu, Professor A. Misra and Professor M. N. Roy for their continuous encouraging and utmost positive support. Special thanks are also to Dr. Asis Kumar Nanda and Dr. Sajal Das for their helpin NMR spectroscopy and Prof. P. S. Roy for his guidance in FTIR spectroscopy. I am also thankful to all the non-teaching staffs of the department, especially to Amit da, Yogen da, Sumit da and Amar da, for their valuable cooperation during my research work. The author is highly grateful to Professor Uday Maitra, IIsc., Bangalore for providing an opportunity to learn the gelation work under his fantastic supervision and utmost support by staying in his prestigious laboratory for one month. The active cooperation as well as pronounced support of Professor Maitra’s group (Sayantan, Mitasree, Raju, Bala, Tumpa, Shuva and Soumya) is really a wonderful experience for me and I thank them all a lot. Again, I am thankful to my dear friends

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Vadivel, Arun, Murali and Suhan over there at Bangalore who really made my stay outside very pleasant and enjoyable. I am immensely thankful to all of my labmates and my dear scholar friends for their moral support, energetic discussions and providing manifold strength continuously. In this regard, I must mention the name of few of them who sacrifices lot of their own schedules just for the sake of my need. My sincere thank thus goes to Antara, Bi ttu, Prosanjit, Gourab… I acknowledge the financial support of the Department of Chemistry, University of North Bengal and CSIR, New Delhi for providing me fellowships. I also acknowledge the support of SAIF-CDRI for providing me various spectral analyses. I am thankful to my friends Joyashish and Shibaji for their timely help and active support in my research. I feel fortunate enough to have friends like Raju (Biswakarma), Bhaskar, Prosenjit (Biswas), Sujit, Kinkar, Bipransh, Madhu, Pratik, Smitawho has made my life much easier during my reseaech, where I was busy in the lab making them awaiting elsewhere! I thank them all for their immense support personally as well as socially though I was able to pay time hardly for them. My heartfelt thanks goes again to my wife Antara for her valuable participation in providing suggestions, comments and for her precious help during my entire research period. Again I am thankful very much to my brother Dipu, for his valuable help, advice and potential support. I would like to express my sincere thanks to my parents for their unparalleled support and for their silent patience. I am again thankful to my elder sister, my sisters, and my in-laws for supporting me completing this work, although I was hardly capable to spent sufficient time with them. I hereby record my appreciation for them, last but not the least. Again it is my humble remark that all the shortcomings of the thesis are solely of the author’s.

Jayanta Das Department of Chemistry University of North Bengal Darjeeling-734013 15 th December, 2015

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Contents

Page No. Abstract……………………………………………………………………………………….iv Preface…………………………………………………………………………………….....viii List of Tables...... xvii List of Figures...... xviii List of Schemes...... xxxi List of Appendices Appendix A: Research papers published/ communicate d/ under preparation……...... xxxviii Appendix B: List of Research Papers in the Proceedings of National/ International Seminar/ Conference/Workshop...... xxxix Appendix C: Abbreviations...... xli

Chapter I Carbocyclic compounds and their transformative reactions: A general perspective on natural products chemistry

I.1 Carbocyclic compounds...... 2 I.2 Natural products and carbocyclic compounds...... 2 I.3 Steroids...... 5 I.3.1 Recent selectivee transformative reactions on cholesterol and b-sitosterol...... 6 I.3.1.1 Recent transformative reactions on cholesterol...... 7 I.3.1.2 Recent advances in the b-sitosterol chemistry...... 17 I.4 Pentacyclic Triterpenoids ...... 24 I.4.1 Different groups of pentacyclic triterpenoids...... 25 I. 4.1.1 Friedelane triterpenoids...... 25 I. 4.1.2 Lupane triterpenoids...... 26 I. 4.1.3 Ursane and oleanane triterpenoids...... 27 I. 4.1.4 Serratane and Ψ -taraxastane triterpenoids...... 28 I.4.2 Recent advances in the transformative reactions on pentacyclic triterpenoids...... 28 I.5 Some steroid- and PT-based marketed drugs ...... 58

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I.6 References...... 60

Chapter II First report of solvent selective steroidal aromatization, efficient access to 4 b,7 a- dihydroxy steroids, and syntheses of natural diaromatic ergosterols

II.A A short review on the action of selenium dioxide on steroids...... 63 II.A.1 Introduction of selenium dioxide as a reagent for organic syntheses...... 63 II.A.2 Application of the reagent on steroids...... 64 II.A.2.1 Biological consequences of 4 - and/ or 7-hydroxy steroids...... 71 II.B A brief review on steroidal aromatization reactions...... 72 II.B.1 From dieneones...... 73 II.B.2 From enone and diene...... 79 II.C Present work ...... 82 II.C.1 Background and abstract of the work...... 82 II.C.2 Results and discussion …………………………………………………….…… ...82

II.C.2.1 SeO 2-oxidation of cholesterol, cholesteryl acetate and cholesteryl benzoate...... 82 II.C.2.2 Study of the solvent dependent product ratio...... 84 II.C.2.3 Raising the yield of 4 b,7 a-dihydroxycholesterol...... 86 II.C.2.4 Application of the reaction protocol: syntheses of the diaromatic Natural ergosterols...... 87 II.C.2.5 Mechanistic concern towards aromatization...... 88 II.C.2.6 Further extention...... 90 II.C.2.7 Description of the molecular structures of 447 and 456...... 90

II.D Experimental ……………………………………………………………………………92 II.D.1 General ...... 92 II.D.2 X-ray crystallography...... 93 II.D.3 Representative procedure for the oxidation reactions...... 93 II.D.4 Product characterization...... 94 II.D.5 NMR Spectral Data Comparison of Natural and Synthetic Diaromatic Ergosterol

(457) in CDCl 3...... 99 II.E Conclusion...... 100

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II.F Supporting spectra ( 1H NMR, 13 C NMR, DEPT-135 NMR, mass)...... 100 II.G References...... 119

Chapter III Polyhydroxy and epoxy-polyhydroxy steroids: design, synthesis, and study of their preliminary gelation behaviour

III.A Introduction...... 122 III.A.1 A brief review on the polyhydroxy steroids ...... 122 III.A.1.1 Natural abundance...... 122 III.A. 1.1.1 Isolated from plants...... 122 III.A.1.1.2 Isolated from animals...... 124 III.A.1.1.2.a From coral...... 124 III.A.1.1.2.b From sponge...... 131 III.A.1.1.2.c. From fungus...... 139 III.A.1.1.2.d From starfish...... 139 III.A.1.1.2.e From mollusca...... 144 III.A.1.1.3 Miscellaneous...... 145 III.A.1.2 Synthetic polyhydroxy steroids...... 157 III.A.2 A brief review on the epoxy- and epoxy-polyhydroxy steroids...... 165 III.A.2.1 Natural abundance...... 165 III.A.2.1.1 Isolated from plants...... 165 III.A.2.1.2 Isolated from animals...... 166 III.A.2.1.2.a From coral...... 166 III.A.2.1.2.b From mushroom...... 169 III.A.2.1.2.c From algae...... 169 III.A.2.1.2.d From sponge...... 169 III.A.2.1.2.e From mollusc...... 172 III.A.2.1.2.f From starfish...... 172 III.A.2.2 Synthetic epoxy- and epoxy-polyhydroxy steroids...... 172 III.A.2.3 Biologically important epoxy- and epoxy-polyhydroxy steroids...... 193 III.A.2.4 Transformative reactions on epoxysteroids...... 194

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III.B Present work...... 197 III.B.1 Abstract of the work...... 197 III.B.2 Design and Synthesis...... 197 III. B.2.1Background and design of the work...... 197 III.B.2.2 Preparation of the starting materials...... 198 III.B.2.3 Synthesis of epoxy-polyhydroxy steroids...... 199 III.B.2.4 Synthesis of tetra- and pentahydroxy steroids...... 202 III.B.2.5 Structural feature: H bonding ...... 203 III.B.3 Application of the epoxy-polyhydroxy and polyhydroxy steroids: study of their preliminary gelation behaviour...... 204 III.B.3.1 Introduction: Gelation, low molecular-weight gelation (LMWG), and gelation of steroid molecules...... 204 III.B.3.2 Present work...... 206 III.B.3.2.1 Preparation of the gels: tests in different solvents...... 206 III.B.3.2.2 Phase selective gelation ability...... 207

III.B.3.2.3 Gel melting temperatures (T gel ) and related physical parameters...... 209 III.B.3.2.4 Rheological behaviour...... 210 III.B.3.2.5 Morphology through electron microscopes...... 212 III.B.3.2.6 Justification of the gelation abilities of the synthesized steroids through their presumable molecular interactions...... 217 III.C Experimental...... 219 III.C.1 General...... 219 III.C.2 Representative reaction for the syntheses of the epoxy-polyhydroxy steroids...219 III.C.3 Representative reaction for the syntheses of the tetra- and pentahydroxy steroids...... 220 III.C.4 Characterization of the products...... 220 III.D Conclusion...... 224 III.E Supporting spectra...... 225 III.F References...... 245

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Chapter-IV

Syntheses of new friedelane triterpenoids: A-ring modifications including 2- homo derivatives

IV.A Introduction: A brief review on the A-ring modified friedelane triterpenoids ……… .248 IV.A.1 Naturally occuring A-ring modified friedelane triterpenoids ……………….…248 IV.A.2 Synthetic A-ring modified friedelane triterpenoids ……………………………255 IV.A.2.1 Oxidative transformations...... 257 IV.A.2.2 Reductive transformations ...... 259 IV.A.2.3 Transformations based on both oxidation and reduction reactions...... 260 IV.A.2.4 Photochemical transformations ...... 261 IV.A.2.5 Rearrangement-based transformations...... 263 IV.A.2.6 Transformative reactions of bromo-friedelane triterpenoids...... 265 IV.A.2.7 Other transformations...... 266 IV.A.2.8 Additional bioactivities and concluding remarks...... 272 IV.B Present work ...... 274 IV.B.1 Background and abstract of the work...... 274 IV.B.2 Results and Discussion...... 274

IV.B.2.1 Extraction and isolation of friedelin and from Quercus suber bar k……. 274 IV.B.2.2 Action of Vilsmeyer-Haack reagent on friedelin: Syntheses of 3-chlorofriedel-2-ene ( 1327), 3-chloro-2-formylfriedel-2-ene ( 1328), 3-hydroxy-2-formylfriedel-2-ene ( 1329) and 4a-hydroxy-3-chloro- 2-formylfriedel-2-ene ( 1330 )...... 274 IV.B.2.2.1 A-ring modified friedelanes...... 276

IV.B.2.2.1.a Extraction and isolation of cerin ( 116) from Quercus suber bark. …………………………………… ……… ……….….…….276 IV.B.2.2.1.b Reaction of 3-chlorofriedel-3-ene ( 1327 ) with selenium dioxide...... 276 IV.B.2.2.1.c Reaction of 3-chlorofriedel-3-ene ( 1327 ) with m-CPBA...... 277 IV.B.2.2.1.d Reaction of 3-chlorofriedel-3-ene ( 1327 ) with

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N-bromosuccinimide...... 277 IV.B.2.2.1.e Synthesis of 3,4-secofriedelane-3,4-diol...... 278 IV.B.2.2.1.f Synthesis of 3-epi pachysandiol A...... 278 IV.B.2.2.1.g Synthesis of 3 b- amino -4a-hydroxyfriedelane from friedelin...... 279 IV.B.2.2.2 2-Homo friedelanes...... 280 IV.B.2.2.2.a Synthesis of 3-chloro-friedel-2-ene-2-carboxaldoxime...... 280 IV.B.2.2.2.b Reduction of 3-chloro-2-formylfriedel-2-eneinto its 2-hydroxymethyl derivative...... 280 IV.B.2.2.2.c Synthesis of 3-chlorofriedel-2ene-2-carboxamide...... 281 IV.B.2.2.2.d Allylic hydroxylation of 2-formyl derivative 1328 with

SeO 2 ...... 281 IV.B.2.2.2.e Transformation of 2-hydroxymethyl derivative 1344

with SeO 2...... 282 IV.B.2.2.2.f Preparation of the oxime derivative of 1330...... 282 IV.B.2.2.2.g Synthesis of 3-chloro-4a-hydroxy-2- hydroxymethylfriedel-2-ene...... 283 IV.B.2.2.3 Heterocycle-linked homofriedelanes...... 283 IV.C Experimental …………………………………………………………………………284 IV.C.1 General ……………………………………………………………………….. ..284 IV.C.2 General procedure for the Vilsmeier-Haack reaction of friedelin (d0)...... 285

IV.C.3 General procedure for the reduction with NaBH 4……………………………...285 IV.C.4 General procedure for the acetylation reactions ……………………………….285 IV.C.5 General procedure for the oximination reactions ………………………………286 IV.C.6 Procedure for the synthesis of nitrile 3-chlorofriedel-2-ene-2 -carboxamide 1346from oxime 1343...... 286 IV.C.7 Oxidation of 3-chlorofriedel-2-ene with mCPBA...... 286 IV.C.8 Allylic hydroxylation by selenium dioxide...... 286 IV.C.9 General procedure for the syntheses of heterocycle-linked 2-homofriedelanes ...... 287 IV.10 Characterization of the compounds...... 287 IV.D Conclusion...... 297 IV.E Supporting spectra...... 297

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IV.F References...... 337

Bibliogra phy……….....……………………………………………………………………..338 Index…………………….... .………………………………………………………………..380

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List of Tables

Chapter II

Entry Table No. Title of the table Page No. 1 Table 2.1 Solvent dependent product ratio. 84 2 Table 2.2 Optimization of the yield of 448 directly from cholesterol 87 3 Table 2.3 Optimization of the yield of the natural product 9 89 4 Table 2.4 Crystallographic data and refinement details for 447 and 456 92 5 Table 2.5 NMR Spectral Data Comparison of Natural and Synthetic 99 Diaromatic Ergosterol ( 457) in CDCl 3.

Chapter III

Entry Table No. Title of the table Page No. 1 Table 3.1 Diastereomeric distribution of various epoxides. 201 2 Table 3.2 Gelation-test results of the synthesized polyhydroxy steroids in 208 different solvents 3 Table 3.3 Dynamic rheology of the organogels. 211

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List of Figures

Chapter-I

Entry Figure No. Title of Figure Page No. 1 Figure 1.1 Some cyclic organic compounds ( 1-6: carbocyclic; 7: nitrogen- 2 based homocyclic; 8-11 : heterocyclic). 2 Figure 1.2 1,2-Cyclopentenophenanthrene ( 12 ), Cholesterol ( 13 ), b- 6 sitosterol ( 14 ) and stigmasterol ( 15 ). 3 Figure 1.3 Some natural phytosterols ( 75 , 81-83 ). 19 4 Figure 1.4 b-Sitosterol and cholesterol-based amphiphiles. 22 5 Figure 1.5 Major constituents of g-oryzanol. 23 6 Figure 1.6 Structural skeletons with carbon-numbering of various 25 pentacyclic triterpenoids. 7 Figure 1.7 Friedelin ( 115), cerin ( 116 ), celastrol ( 117 ) and correolide ( 118). 26 8 Figure 1.8 Betulinic acid ( 119), lupeol (120), ursolic acid ( 121) and 28 oleanolic acid ( 122). 9 Figure 1.9 Phlegmanol A ( 123) and arnidiol ( 124). 28 10 Figure 1.10 Betulin ( 125) and its amine dimer ( 126). 30 11 Figure 1.11 Synthesis of C3 neoglycosylation ( 128). 30 12 Figure 1.12 RPR103611 (129 ) and IC9564 ( 130 ). 31 13 Figure 1.13 Bevirimat ( 135) and C28-modified derivative 136. 32 14 Figure 1.14 A fluorescent cancer cell detector 137. 33 15 Figure 1.15 Compound 138 forthe co-delivery of anticancer drugs. 33 16 Figure 1.16 Piperazine derivatives of betulinic acid ( 142-143). 34 17 Figure 1.17 BA derivatives ( 145-148 ). 36 18 Figure 1.18 Natural betulinic acid saponins ( 149-150). 37 19 Figure 1.19 Lawesson’s reagent ( 151 ). 37 20 Figure 1.20 28-O-b-D-glucuronide betulinic acid and bevirimat ( 166 and 39 167). 21 Figure 1.21 C28-modified derivatives 171-173. 39 22 Figure 1.22 DMAP derivatives ( 174-180 ) of BA. 40 23 Figure 1.23 Triphenylphosphonium derivatives ( 181-189) of betulin and 41 betulinic acid. 24 Figure 1.24 Novel ester-triazole-linked triterpenoid –AZT conjugates ( 194- 43 205). 25 Figure 1.25 3,4-Seco betulinic acid (BA) derivatives ( 206-215). 44 26 Figure 1.26 Ionic derivatives ( 216-219 ) of betulinic acid. 44 27 Figure 1.27 Pentacyclic triterpenes ( 220-223) bearing O-[4-(1-piperazinyl)-4- 45

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oxo-butyryl moiety. 28 Figure 1.28 NO-releasing furoxan derivatives of betulinic acid (BA) ( 227- 45 229). 29 Figure 1.29 General skeleton of triazole-attached to BA at C3 through a linker 46 (230). 30 Figure 1.30 Polyamine derivatives ( 231-236) of betulinic acid. 46 31 Figure 1.31 Disubstituted lupeol derivatives ( 244-248) for antimalarial agents. 49 32 Figure 1.32 CDDU- (249-252) and CDDO-(253-257) esters . 50 33 Figure 1.33 C3 and C28-Modified nitric oxide-releasing derivatives ( 258-259 ) 51 of oleanolic acid. 34 Figure 1.34 Oleanolic acid dimmers ( 260-264). 51 35 Figure 1.35 Oleanolic acid saponins ( 265-273). 52 36 Figure 1.36 Hybrids ( 274-277) of O2-(2,4-dinitrophenyl)-diazeniumdiolate 53 and oleanolic acid (OA). 37 Figure 1.37 NO-donating oleanolic acid derivative ( 279-280 ). 54 38 Figure 1.38 3-O-acyl ursolic acid derivatives ( 286-294). 55 39 Figure 1.39 Glucoconjugates ( 297-300) of oleanolic acid. 56 40 Figure 1.40 Oleanolic acid saponins ( 301-306). 57 41 Figure 1.41 Bidesmosidic oleanolic acid saponins ( 307-309). 57 42 Figure 1.42 Some steroid-based marketed drugs. 59 43 Figure 1.43 Some of the PT-based marketed drugs ( 320-322 ). 59

Chapter-II

Entry Figure No. Title of Figure Page No.

1 Figure 2.1 SeO 2 oxidation products of 3 b-Benzoyloxy-5a-cholest-8(14)- 70 en -15-one ( 372). 2 Figure 2.2 Diketo aldehydes ( 382 ) and1,4-conjugated diene derivative of 71 diosgenin ( 383 ). 3 Figure 2.3 21-Acetoxy-9α,11 b-dichloro-17α-hydroxypregna-1,4-diene- 76 3,20-dione ( 405) and its derivatives ( 406-408), 21-acetoxy- 3,17α-dihydroxy-19-norpregna-l,3,5(10),6,8-pentaen-20-one (410) and its derivatives ( 411, 412 ) and 21-acetoxy-17α- hydroxypregna -l,4,8(14),9(1l)-tetraene-3,20-dione ( 409 ). 4 Figure 2.4 10 b-Hydroxy-19-nortestosterone ( 436), 17α -ethynyl-10b- 80 hydroxy -19-nortestosterone ( 437), 3-oxo-4-cholen-24-oic acid (438), 4-androstene-3,17-dione ( 370 ). 5 Figure 2.5 Natural diaromatic ergosterols. 87

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6 Figure 2.6 Molecular structure of 447 and atom labeling. 90 7 Figure 2.7 Molecular structure of 456 and atom labeling. 91 8 Figure 2.8 1H NMR Spectrum of 1-methyl-19-norcholesta- 101 1,3,5(10),6,8(9),14(15)-hexaene ( 447) in CDCl 3 9 Figure 2.9 1H NMR Spectrum of1-methyl-19-norcholesta- 101

1,3,5(10),6,8(9),14(15) -hexaene ( 447) in acetone-d6 10 Figure 2.10 13 C NMR Spectrum of 1-methyl-19-norcholesta- 102 1,3,5(10),6,8(9),14(15)-hexaene ( 447) in CDCl 3. 11 Figure 2.11 DEPT-135 Spectrum of 1-methyl-19-norcholesta- 102 1,3,5(10),6,8(9),14(15)-hexaene ( 447). 12 Figure 2.12 1H NMR Spectrum of3b, 4b-dihydroxycholest-5-ene ( 334 ). 103 13 Figure 2.13 13 C NMR Spectrum of 3b, 4b-dihydroxycholest-5-ene (334). 103 14 Figure 2.14 DEPT-135 NMR Spectrum of 3b, 4b-dihydroxycholest-5-ene 104 (334). 15 Figure 2.15 1H NMR Spectrum of 3b, 4b,7 a-trihydroxycholest-5-ene( 448). 104 16 Figure 2.16 13 C NMR Spectrum of 3b, 4b,7 a-trihydroxycholest-5- 105 ene( 448 ). 17 Figure 2.17 DEPT-135 NMR Spectrum of 3b, 4b,7 a-trihydroxycholest-5- 105 ene( 448 ). 18 Figure 2.18 1H NMR Spectrum of 3b ,4 b,7 a-triacetoxycholest-5-ene ( 456 ). 106 19 Figure 2.19 13 C NMR Spectrum of 3b ,4 b,7 a-triacetoxycholest-5-ene 106 (456). 20 Figure 2.20 DEPT-135 NMR Spectrum of 3b ,4 b,7 a-triacetoxycholest-5- 107 ene ( 456). 21 Figure 2.21 1H NMR Spectrum of 3b-benzoxy-4b-hydroxycholest-5-ene 107 (450). 22 Figure 2.22 13 C NMR Spectrum of 3b-benzoxy-4b-hydroxycholest-5-ene 108 (450). 23 Figure 2.23 1H NMR Spectrum of 3b-benzoxy-6a-hydroxycholest-4-ene 108 (452). 24 Figure 2.24 13 C NMR Spectrum of 3b-benzoxy-6a-hydroxycholest-4-ene 109 (452). 25 Figure 2.25 1H NMR Spectrum of 3b, 4b-dihydroxyspirost-5-ene( 335). 109 26 Figure 2.26 13 C NMR Spectrum of 3b, 4b-dihydroxyspirost-5-ene( 335). 110 27 Figure 2.27 DEPT-135 NMR Spectrum of 3b, 4b-dihydroxyspirost-5- 110 ene( 335 ). 28 Figure 2.28 1H NMR Spectrum of 3b, 4b,7a-trihydroxyspirost-5-ene ( 460 ). 111 29 Figure 2.29 13 C NMR Spectrum of 3b, 4b,7 a-trihydroxyspirost-5-ene ( 460 ). 111 30 Figure 2.30 DEPT-135 NMR Spectrum of 3b, 4b,7 a-trihydroxyspirost-5- 112 ene ( 460). 31 Figure 2.31 1H NMR Spectrum of 1-methyl-19-norb-sitosta- 112 1,3,5(10),6,8(9),14(15)-hexaene ( 453). 32 Figure 2.32 13 C NMR Spectrum of 1-methyl-19-nor-b-sitosta- 113 1,3,5(10),6,8(9),14(15)-hexaene ( 453). 33 Figure 2.33 1H NMR Spectrum of 3b, 4b-dihydroxy-b-sitost-5-ene( 454). 113

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34 Figure 2.34 13 C NMR Spectrum of 3b, 4b-dihydroxy-b-sitost-5-ene( 454). 114 35 Figure 2.35 1H NMR Spectrum of 3b, 4b,7 a-trihydroxy-b-sitost-5-ene 114 (455). 36 Figure 2.36 13 C NMR Spectrum of 3b, 4b,7 a-trihydroxy-b-sitost-5-ene 115 (455). 37 Figure 2.37 1H NMR spectrum of 19-norergosta-1,3,5,7,9,14,22-heptaene 115 (457). 38 Figure 2.38 13 C NMR spectrum of 19-norergosta-1,3,5,7,9,14,22-heptaene 116 (457). 39 Figure 2.39 1H NMR spectrum of the mixture of 19-norergosta- 116 1,3,5,7,9,14,22-heptaene ( 457 ) and 1-methylergosta- 1,3,5,7,9,14,22-heptaene ( 458). 40 Figure 2.40 13 C NMR spectrum of the mixture of 19-norergosta- 117 1,3,5,7,9,14,22-heptaene ( 457 ) and 1-methylergosta- 1,3,5,7,9,14,22-heptaene ( 458 ). 41 Figure 2.41 1H NMR spectrum of the petroleum fractions from column 118 chrom atography of the reaction of cholesterol with SeO 2 at 2h. Highlighted area shows negligible presence of cholesterol analogue of 19-norergosta-1,3,5,7,9,14,22-heptaene ( 457 ). 42 Figure 2.42 1H NMR spectrum of the petroleum fractions from column 118 chromat ography of the reaction of cholesterol with SeO 2 at 6h. Highlighted area shows negligible presence of cholesterol analogue of 19-norergosta-1,3,5,7,9,14,22-heptaene ( 457 ). 43 Figure 2.43 1H NMR spectrum of the petroleum fractions from column 119 chromatog raphy of the reaction of cholesterol with SeO 2 at 12h. Highlighted area shows negligible presence of cholesterol analogue of 19-norergosta-1,3,5,7,9,14,22-heptaene ( 457 ).

Chapter III

Entry Figure No. Title of the figure Page No. 1 Figure 3.1 Polyhydroxy steroids ( 461-463, 465-466) from plants. 123 2 Figure 3.2 Polyhydroxy steroids ( 464, 467-469) from plants. 123 3 Figure 3.3 Polyhydroxy steroids ( 78a, 79 and 470 ) from plants. 124 4 Figure 3.4 Polyhydroxy steroids ( 471-478) from plants. 124 5 Figure 3.5 25ξ-Cholestane-3b,5 a,6 b,26-tetrol-26-acetate ( 479), 24- 125 methylenecholestane -3b,5 a,6 b-triol ( 488 ), 1 a,3 b,5 a,6 b,11 a- pentahydroxy -24-methylene-5a-cholestane ( 489 ) and 4a,23,24(R)-trimethyl-5a-cholest-22E-ene-1a,3 b,6 b,11 a- tetrol ( 490 ). 6 Figure 3.6 Polyhydroxy sterols ( 480-487) from the soft coral 125 Sinulariadepressa . 7 Figure 3.7 Polyhydroxy sterols ( 491-500 ) from the soft coral Sinularia sp. 126 8 Figure 3.8 Polyhydroxy steroids ( 501-504) from soft coral. 127 9 Figure 3.9 Polyhydroxy steroids ( 505-508) from soft coral. 127 10 Figure 3.10 Polyhydroxy steroids 509-513 . 128

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11 Figure 3.11 Polyhydroxy steroids 488 , 518-532. 129 12 Figure 3.12 Polyhydroxy steroids 486 , 533-537. 129 13 Figure 3.13 Polyhydroxy steroids 538-551 from soft coral. 131 14 Figure 3.14 Polyhydroxy steroids 552-563 from soft coral. 132 15 Figure 3.15 Polyhydroxy steroids 564-572 . 132 16 Figure 3.16 Polyhydroxy steroids 573-583 . 133 17 Figure 3.17 Polyhydroxy steroids 584 and 594-596 from sponge. 133 18 Figure 3.18 Polyhydroxy steroids 585-593 from sponge. 134 19 Figure 3.19 Polyhydroxy steroids 597-606 from sponge. 134 20 Figure 3.20 Polyhydroxy steroids 607-614 . 135 21 Figure 3.21 Polyhydroxy steroids 615-622 . 136 22 Figure 3.22 Polyhydroxy steroids 623-635 . 136 23 Figure 3.23 Polyhydroxy steroids 636-638 from sponge. 137 24 Figure 3.24 Polyhydroxy steroids 639-641 from sponge. 137 25 Figure 3.25 Polyhydroxy steroids 642-644 . 138 26 Figure 3.26 Polyhydroxy steroids 645-648 from red sea marine sponge. 138 27 Figure 3.27 D-ring unsaturated steroids 649-653. 139 28 Figure 3.28 Polyhydroxy steroids 654-657 from fungus. 139 29 Figure 3.29 Polyhydroxy steroids 658-662 from starfish. 140 30 Figure 3.30 Polyhydroxy steroids 663-669 from starfish. 140 31 Figure 3.31 Polyhydroxysteroids 670-682 isolated from the starfish. 141 32 Figure 3.32 Polyhydroxy steroids 683-686 from starfish. 142 33 Figure 3.33 Polyhydroxysteroids 687-695 steroids fromstarfish. 142 34 Figure 3.34 Polyhydroxy steroids 696-703 from starfish. 143 35 Figure 3.35 Polyhydroxy steroids 704-713 from starfish. 143 36 Figure 3.36 Polyhydroxy steroids 714-716 . 144 37 Figure 3.37 Polyhydroxy steroids 717-722 . 145 38 Figure 3.38 Polyhydroxy steroids 723-725 from mollusca. 145 39 Figure 3.39 Polyhydroxy steroids 726-729 from marine sources. 146 40 Figure 3.40 Polyhydroxy steroids 730-739 from marine sources. 146 41 Figure 3.41 Polyhydroxy steroids 741-746 from marine sources. 147 42 Figure 3.42 Polyhydroxy steroids 747-749 . 147 43 Figure 3.43 Polyhydroxy steroids 750-756 . 148 44 Figure 3.44 Polyhydroxy steroids 757-774 from marine sources. 149 45 Figure 3.45 Polyhydroxy steroids 775-793 from marine sources. 150 46 Figure 3.46 Polyhydroxy steroids 794-803 from marine sources. 151 47 Figure 3.47 Polyhydroxy steroids 804-815 from marine sources. 152 48 Figure 3.48 Polyhydroxy steroids 816-828 from marine sources. 153 49 Figure 3.49 Polyhydroxy steroids 829-840 from marine sources. 154

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50 Figure 3.50 Polyhydroxy steroids 841-854 from marine sources. 155 51 Figure 3.51 Polyhydroxy steroids 855-859 from marine sources. 156 52 Figure 3.52 Polyhydroxy steroids 860-866 from marine sources. 156 53 Figure 3.53 Synthesis of compound 42 and 869. 157 54 Figure 3.54 Polyhydroxy steroids 907-913 . 163 55 Figure 3.55 Epoxy hydroxy steroids 922-924. 165 56 Figure 3.56 Epoxy steroids 925-931. 166 57 Figure 3.57 Epoxy steroids isolated from plants ( 932-933 ) and from coral 166 (934). 58 Figure 3.58 Epoxy polyhydroxy steroids 935-943 from soft coral. 167 59 Figure 3.59 Epoxy polyhydroxy sterols 944-946. 167 60 Figure 3.60 Epoxy polyhydroxy sterols 947-950 from soft coral. 168 61 Figure 3.61 Epoxy polyhydroxy sterols 951-954. 168 62 Figure 3.62 Polyhydroxyepoxy sterls from mushroom ( 956-959) and algae 169 (960). 63 Figure 3.63 Epoxy hydroxy sterols ( 961-962) from Dysidea sp. 170 64 Figure 3.64 Epoxy sterols ( 963-964) from sponge. 170 65 Figure 3.65 Epoxyhydroxy sterols 965-966. 170 66 Figure 3.66 Epoxy sterols 967-974. 171 67 Figure 3.67 Epoxy 9,11-seco sterols 975-982. 171 68 Figure 3.68 Epoxy sterols ( 983-988) isolated from Chinese sponge Ircinia 171 aruensis . 69 Figure 3.69 Epoxy sterols 989-990. 172 70 Figure 3.70 Epoxy steroids 1024-1026. 177 71 Figure 3.71 Epoxy derivatives( 328, 391, 1039-1044 )of steroids with 180 androstane skeleton. 72 Figure 3.72 Androsta-4,6-diene-3,17-dione ( 1134) and3a-hydroxy-6a,7 a- 190 epoxy-5a-androstan-17-one ( 1135). 73 Figure 3.73 D4-steroids and D5-steroids and their epoxides. 191 74 Figure 3.74 Compound 370 and 1145 and therir epoxides. 191 75 Figure 3.75 Cross conjugated and non-conjugated ketones and their 192 epoxides. 76 Figure 3.76 Non-Conjugated ketones and their different derivatives. 193 77 Figure 3.77 Biologically potent epoxy steroids. 194 78 Figure 3.78 Probable H-bonding arrangements in the tetrahydroxy steroids 204

(R= H, 1204; R= C 2H5, 1205). The bonds and atoms involved

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in H-bonding are shown in blue. 79 Figure 3.79 Probable H-bonding arrangements in the pentahydroxy steroids 204

(R= H, 1204; R= C 2H5, 1205). The bonds and atoms involved in H -bonding are shown in blue. 80 Figure 3.80 Images of different gels. 206 81 Figure 3.81 Solvent selective gelators 1206 and 1207 . 209 82 Figure 3.82 Phase selective gelation ability of 1200. 209

83 Figure 3.83 Tgel vs conc. (w/v%) and ln C vs 1/T gel of 1194/ DMSO gel 210 (above row) and 1198/ DMSO (bottom row) 84 Figure 3.84 Frequency sweep (left) and stress sweep (right) dynamic 211 rheological behavior of 1194 /DMSO gel (1%, w/v ). In 1Hz, G/= 21220 Pa, G // = 3720 Pa and σ*= 500 Pa. 85 Figure 3.85 Frequency sweep (left) and stress sweep (right) dynamic 212 rheological behavior of 1198 /DMSO gel (1%, w/v). In 1Hz, G/= 6960 Pa, G // = 1874 Pa, σ*= 40 Pa. 86 Figure 3.86 AFM images of 1194/ DMSO system at 0.05% (w/v, above) 213 and at 0.025% (w/v, below) 87 Figure 3.87a AFM images of 1198 / DMSO system at 0.2% (w/v). 213-214 88 Figure 3.87b AFM images of 1198 / DMSO system at 0.05% (w/v). 214 89 Figure 3.88 AFM images of 1200/ Dodecane system at 0.025% (w/v). 215 90 Figure 3.89 AFM images of 1201/ Dodecane system at 0.025%,w/v. 216 91 Figure 3.90 POM images of 1194/ DMSO (above row) and 1198 / DMSO 217 gels (bottom row) 92 Figure 3.91 Synthesized a and b-epoxy diols. 218 93 Figure 3.92 Synthesized a and b-epoxy triols. 218 94 Figure 3.93 1H NMR spectrum of 5 b-Cholestan-5a,6 a-epoxy-3b,4 b-diol 225 (1194). 95 Figure 3.94 Extended 1H NMR spectrum of 5 b-Cholestan-5a,6 a-epoxy- 226 3b,4 b-diol ( 1194 ). 96 Figure 3.95 13 C NMR spectrum of 5 b-Cholestan-5a,6 a-epoxy-3b,4 b- 226 diol( 1194). 97 Figure 3.96 FTIR spectrum of 5 b-Cholestan-5a,6 a-epoxy-3b,4 b- 227 diol( 1194). 98 Figure 3.97 1H NMR spectrum of5 b-Betasitostan-5a,6 a-epoxy-3b,4 b- 227 diol( 1195).

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99 Figure 3. 98 Extended 1H NMR spectrum of5 b-Betasitostan-5a,6 a-epoxy- 228 3b,4 b-diol ( 1195 ). 100 Figure 3.99 13 C NMR spectrum of5 b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol 228 (1195). 101 Figure 3.100 FTIR spectrum of5 b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol 229 (1195). 102 Figure 3.101 1H NMR spectrum of 5 b-Cholestan-5a, 6 a-epoxy-3b, 4 b, 7a - 229 triol ( 1196). 103 Figure 3.102 13 C NMR spectrum of5 b-Cholestan-5a, 6 a-epoxy-3b, 4 b, 7a - 230 triol ( 1196). 104 Figure 3.103 FTIR spectrum of 5 b-Cholestan-5a, 6a-epoxy-3b, 4b, 7a -triol 230 (1196). 105 Figure 3.104 1H NMR spectrum of 5 b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a - 231 triol ( 1197). 106 Figure 3.105 Extended 1H NMR spectrum of5 b-Betasitostan-5a,6 a-epoxy- 231 3b,4 b,7a -triol ( 1197). 107 Figure 3.106 13 C NMR spectrum of5 b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a - 232 triol ( 1197). 108 Figure 3.107 FTIR spectrum of5 b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a -triol 232 (1197). 109 Figure 3.108 1H NMR spectrum of5 a-Cholestan-5b,6 b-epoxy-3b,4 b-diol 233 (1198). 110 Figure 3.109 13 C NMR spectrum of5 a-Cholestan-5b,6 b-epoxy-3b,4 b-diol 233 (1198). 111 Figure 3.110 FTIR spectrum of5 a-Cholestan-5b,6 b-epoxy-3b,4 b-diol 234 (1198). 112 Figure 3.111 1H NMR spectrum of 5 a-Betasitostan-5b, 6b-epoxy-3b,4 b-diol 234 (1199). 113 Figure 3.112 Extended 1H NMR spectrum of5 a-Betasitostan-5b, 6b-epoxy- 235 3b,4 b-diol ( 1199 ). 114 Figure 3.113 13 C NMR spectrum of 5 a-Betasitostan-5b, 6b-epoxy-3b,4 b- 235 diol ( 1199). 115 Figure 3.114 FTIR spectrum of5 a-Betasitostan-5b, 6b-epoxy-3b,4 b-diol 236 (1199). 116 Figure 3.115 1H NMR spectrum of 5 a-Cholestan-5b,6 b-epoxy-3b,4 b,7a - 236 triol ( 1200).

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117 Figure 3.116 13 C NMR spectrum of 5 a-Cholestan-5b,6 b-epoxy-3b,4 b,7a - 237 triol ( 1200). 118 Figure 3.117 FTIR spectrum 238of 5 a-Cholestan-5b,6 b-epoxy-3b,4 b,7a - 237 triol ( 1200). 119 Figure 3.118 1H NMR spectrum of 5 a-Betasitostan-5b,6 b-epoxy-3b,4 b,7a - 238 triol ( 1201). 120 Figure 3.119 FTIR spectrum of 5 a-Betasitostan-5b,6 b-epoxy-3b,4 b,7a -triol 238 (1201). 121 Figure 3.120 1H NMR spectrum of 5 b-Cholestane-3b, 4b, 5a, 6b- tetrol 239 (1202). 122 Figure 3.121 Extended 1H NMR spectrum of 5 b-Cholestane-3b, 4 b, 5a, 6b- 239 tetrol ( 1202 ). 123 Figure 3.122 13 C NMR spectrum of 5 b-Cholestane-3b, 4b, 5a, 6b- tetrol 240 (1202). 124 Figure 3.123 FTIR spectrum of 5 b-Cholestane-3b, 4 b, 5a, 6b- tetrol ( 1202). 240 125 Figure 3.124 Extended 1H NMR spectrum of 5 b-betasitostane-3b, 241 4b, 5a, 6b-tetrol ( 1203). 126 Figure 3.125 FTIR spectrum of 5 b-betasitostane-3b, 4b, 5a, 6b-tetrol 241 (1203). 127 Figure 3.126 1H NMR spectrum of 5 b-Cholestane-3b, 4b, 5a, 6b, 7a - 242 pentol ( 1204). 128 Figure 3.127 Extended 1H NMR spectrum of 5 b-Cholestane-3b, 242 4b, 5a, 6b, 7a - pentol ( 1204). 129 Figure 3.128 13 C NMR spectrum of5 b-Cholestane-3b, 4b, 5a, 6b, 7a - 243 pentol ( 1204). 130 Figure 3.129 FTIR spectrum of5 b-Cholestane-3b, 4b, 5a, 6b, 7a - pentol 243 (1204). 131 Figure 3.130 1H NMR spectrum of5 b-Betasitostan-3b, 4b, 5a, 6b, 7a - 244 pentol ( 1205). 132 Figure 3.131 13 C NMR spectrum of5 b-Betasitostan-3b, 4 b, 5a, 6b, 7a - 244 pentol ( 1205). 133 Figure 3.132 FTIR spectrum of5 b-Betasitostan-3b, 4 b, 5a, 6b, 7a - pentol 245 (1205).

Chapter IV

Entry Figure No. Title of the figure Page No. 1 Figure 4.1 Friedelin ( 115), its chair form and cerin ( 116). 248 2 Figure 4.2 Diferent types of friedelanes ( Type A-E). 249 3 Figure 4.3 Friedelane-l,3-dione ( 1208)and friedelane ( 6). 250

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4 Figure 4.4 4-epi friedelin ( 1209). 250 5 Figure 4.5 3b-Hydroxy- and 3 a-hydroxy friedelane ( 1210 and 251 1211,respectively) and terminaline A ( 1212). 6 Figure 4.6 3,4-Secofriedelane-3,28-dioic acid ( 1213) and ( 1214-1216). 252 7 Figure 4.7 Lobatanhydride ( 1217 ), 3 a,25-dihydroxyfriedelan-2-one 253 (1218), 1 b,25-dihydroxyfriedelan-3-one ( 1219), 2 a-hydroxy- 3-oxofriedelan-30-oic acid ( 1220), 28-hydroxyfriedelane-1,3- dione ( 1221) and 29-hydroxyfriedelane-1,3-dione ( 1222 ). 8 Figure 4.8 1,2-Dehydro-2,3-secofriedelan-3-oic acid ( 1223), 1 b- 254 hydroxyfriedelin ( 1224), 3 b-hydroxyfriedelan-23-oic acid (1225), friedelin-3,4-lactone ( 1226). 9 Figure 4.9 Structures of norfriedelanes 1235-1237 . 254 10 Figure 4.10 29-Hydroxymethyl friedelin (1238), 3,4-Seco-friedelan-3-oic 255 acid ( 1239), 2-oxofriedoolean-3-en-29-oic acid ( 1240 ), its methyl ester derivative ( 1241 ) and salaspermic acid ( 1242 ). 11 Figure 4.11 2a,3 b-Dihydroxyfriedelan-28-oic acid ( 1243), 2 b-hydroxy-3- 256 oxofriedelan -30-oic acid ( 1244 ), dzununcanone ( 1245 ), trifloralactone 1246, triptocalline B 1247, triptocalline B 1248, milicifolines A –D ( 1249-1252) and euphorcinol (1253). 12 Figure 4.12 Compound 1254. 256 13 Figure 4.13 Norfriedelanone ( 1257), A(1)-norfriedel-4(23)-en-3-one 257 (1258) and A(4)-nor-23-norfriededel-1(10)-ene-2:3-dione (1259). 14 Figure 4.14 Friedel-3-ene ( 1262). 259 15 Figure 4.15 Tetrahydropyridine 1264 , iodo-ether 1265 and a- 259 acetoxytetrahydrofuran 1266. 16 Figure 4.16 2-Oxo-3-oxa-friedelane ( 1282 ), epoxyfriedelane ( 1283 ),and4- 263 epi shionone ( 1284). 17 Figure 4.17 b-Amyrine ( 1286, arrows show the consecutive 1,2 263 rearrangements toward friedelin). 18 Figure 4.18 Potent A-ring modified friedelane-based drugs: Celastrol ( 117 ) 272 and celasdin B ( 1324). 19 Figure 4.19 Bioactive netzahualcoyone ( 1325 ), compounds 1326 and 273 correolide ( 118). 20 Figure 4.20 Some biologically active aromatized friedelane triterpenoids. 278 21 Figure 4.21 1H NMR spectrum of friedelin ( 115). 298 22 Figure 4.22 13 C NMR spectrum of friedelin ( 115 ). 298 23 Figure 4.23 1H NMR spectrum of 3-chlorofriedel-3-ene ( 1327). 299 24 Figure 4.24 1H NMR spectrum (partially expanded) of 3-chlorofriedel-3- 299 ene ( 1327).

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25 Figure 4.25 13 C NMR spectrum of 3-chlorofriedel-3-ene( 1327). 300 26 Figure 4.26 FTIR spectrum of 3-chlorofriedel-3-ene( 1327). 300 27 Figure 4.27 1H NMR spectrum of 3-chloro-2-formylfriedel-2-ene ( 1328). 301 28 Figure 4.28 1H NMR spectrum (partially expanded) of 3-chloro-2- 301 formylfriedel-2-ene ( 1328). 29 Figure 4.29 13 C NMR spectrum of 3-chloro-2-formylfriedel-2-ene ( 1328). 302 30 Figure 4.30 FTIR spectrum of 3-chloro-2-formylfriedel-2-ene ( 1328). 302 31 Figure 4.31 1H NMR spectrum of 2-formyl-3-hydroxy-friedel-2-ene( 1329). 303 32 Figure 4.32 1H NMR spectrum (partially expanded) of 2-formyl-3- 303 hydroxy-friedel-2-ene ( 1329). 33 Figure 4.33 13 C NMR spectrum of 2-formyl-3-hydroxy-friedel-2-ene 304 (1329). 34 Figure 4.34 FTIR spectrum of 2-formyl-3-hydroxy-friedel-2-ene ( 1329). 304 35 Figure 4.35 1H NMR spectrum of 3-Chloro-2-formyl-4a-hydroxy-friedel- 305 2-ene ( 1330). 36 Figure 4.36 1H NMR spectrum (partially expanded) of 3-chloro-2-formyl- 305 4a-hydroxy-friedel-2-ene ( 1330). 37 Figure 4.37 13 C NMR spectrum of 3-chloro-2-formyl-4a-hydroxy-friedel- 306 2-ene ( 1330). 38 Figure 4.38 FTIRspectrum of 3-chloro-2-formyl-4a-hydroxy-friedel-2-ene 306 (1330). 39 Figure 4.39 1H NMR spectrum of friedel-3-ene-23-al ( 1331). 307 40 Figure 4.40 1H NMR spectrum (partially expanded) of friedel-3-ene-23-al 307 (1331). 41 Figure 4.41 13 C NMR spectrum of friedel-3-ene-23-al ( 1331). 308 42 Figure 4.42 FTIRspectrum offriedel-3-ene-23-al ( 1331). 308 43 Figure 4.43 1H NMR spectrum of 3 a,4 a-epoxy friedelane(1332). 309 44 Figure 4.44 Expanded 1H NMR spectrum of 3 a,4 a-epoxy 309 friedelane(1332 ). 45 Figure 4.45 13 C NMR spectrum of 3 a,4 a-epoxy friedelane(1332). 310 46 Figure 4.46 FTIRspectrum of 3a,4 a-epoxy friedelane(1332). 310 47 Figure 4.47 1H NMR spectrum of 24-norfriedel-1, 3, 5 (10), 6-tetraene 311 (1333). 48 Figure 4.48 1H NMR spectrum (partially expanded) of 24-norfriedel-1, 3, 5 311 (10), 6-tetraene ( 1333). 49 Figure 4.49 13 C NMR spectrum (partially expanded) of 24-norfriedel-1, 3, 312 5 (10), 6-tetraene ( 1333). 50 Figure 4.50 1H NMR spectrum of 3,4-seco-friedelane-3,4-diol ( 1337 ). 312 51 Figure 4.51 1H NMR spectrum (partially expanded) of 3,4-seco-friedelane- 313 3,4-diol ( 1337). 52 Figure 4.52 13 C NMR spectrum of 3,4-Seco-friedelane-3,4-diol ( 1337 ). 313 53 Figure 4.53 1H NMR spectrum of 3-epi pachysan diol-A ( 1338). 314 54 Figure 4.54 1H NMR spectrum (partially expanded) 3-epi pachysan diol-A 314 (1338). 55 Figure 4.55 13 C NMR spectrum of 3-epi pachysan diol-A ( 1338). 315 56 Figure 4.56 FTIRspectrum of 3-epi pachysan diol-A ( 1338). 315 57 Figure 4.57 1H NMR spectrum of friedel-3-enol acetate ( 1339 ). 316 58 Figure 4.58 13 C NMR spectrum of friedel-3-enol acetate ( 1339). 316 59 Figure 4.59 Mass spectrum of friedel-3-enol acetate ( 1339). 317 60 Figure 4.60 1H NMR spectrum of 4 a-acetate friedel-3-one ( 1340). 317 61 Figure 4.61 13 C NMR spectrum of 4 a-acetate friedel-3-one ( 1340). 318 62 Figure 4.62 1H NMR spectrum of 4 a-hydroxy friedelane-3-oxime ( 1341). 318

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63 Figure 4.63 13 C NMR spectrum of 4 a-hydroxy friedelane-3-oxime ( 1341). 319 64 Figure 4.64 1H NMR spectrum of 3b-amino-4a-hydroxyfriedelane (1342). 319 65 Figure 4.65 1H NMR spectrum (partially expanded) of 3b-amino-4a- 320 hydroxyfriedelane (1342). 66 Figure 4.66 13 C NMR spectrum of 3b-amino-4a-hydroxyfriedelane (1342). 320 67 Figure 4.67 1H NMR spectrum of 3-chlorofriedel-2-ene-2-carboxaldoxime 321 (1343). 68 Figure 4.68 1H NMR spectrum (partially expanded) of 3-chlorofriedel-2- 321 ene-2-carboxaldoxime ( 1343). 69 Figure 4.69 13 C NMR spectrum of 3-chlorofriedel-2-ene-2-carboxaldoxime 322 (1343). 70 Figure 4.70 FTIRspectrum of 3-chlorofriedel-2-ene-2-carboxaldoxime 322 (1343). 71 Figure 4.71 1H NMR spectrum of 3-chloro-2-hydroxymethyl-friedel-2-ene 323 (1344). 72 Figure 4.72 1H NMR spectrum (partially expanded) of 3-chloro-2- 323 hydroxymethyl-friedel-2-ene ( 1344). 73 Figure 4.73 13 C NMR spectrum of 3-chloro-2-hydroxymethyl-friedel-2-ene 324 (1344). 74 Figure 4.74 FTIR spectrum of 3-chloro-2-hydroxymethyl-friedel-2-ene 324 (1344). 75 Figure 4.75 1H NMR spectrum of 2-acetoxymethyl-3-chloro-friedel-2-ene 325 (1345). 76 Figure 4.76 1H NMR spectrum (with partial expansion) of 2- 325 acetoxymethyl-3-chloro-friedel-2-ene ( 1345). 77 Figure 4.77 13 C NMR spectrum of 2-acetoxymethyl-3-chloro-friedel-2-ene 326 (1345). 78 Figure 4.78 FTIR spectrum of 2-acetoxymethyl-3-chloro-friedel-2-ene 326 (1345). 79 Figure 4.79 1H NMR spectrum of 3-chlorofriedel-2-ene-2-carboxamide 327 (1346). 80 Figure 4.80 13 C NMR spectrum of 3-chlorofriedel-2-ene-2-carboxamide 327 (1346). 81 Figure 4.81 1H NMR spectrum of 3-chloro-4a-hydroxy-2-ene-2- 328 carboxaldoxime ( 1347). 82 Figure 4.82 13 C NMR spectrum of 3-chloro-4a-hydroxy-2-ene-2- 328 carboxaldoxime ( 1347). 83 Figure 4.83 FTIR spectrum of 3-chloro-4a-hydroxy-2-ene-2- 329 carboxaldoxime ( 1347). 84 Figure 4.84 1H NMR spectrum of 2-formyl-3-chloro-4a-hydroxy-2- 329 hydroxymethyl friedel-2-ene ( 1348). 85 Figure 4.85 13 C NMR spectrum of 2-formyl-3-chloro-4a-hydroxy-2- 330 hydroxymethyl friedel-2-ene ( 1348). 86 Figure 4.86 1H NMR spectrum of 2-formyl-3-(1 H-piperidin-1-yl)-friedel-2- 330 ene ( 1349). 87 Figure 4.87 13 C NMR spectrum of 2-formyl-3-(1 H-piperidin-1-yl)-friedel- 331 2-ene ( 1349). 88 Figure 4.88 1H NMR spectrum of 2-formyl-3-(1 H-morpholin-4-yl)-friedel- 331 2-ene ( 1350). 89 Figure 4.89 1H NMR spectrum (partially expanded) of 2-formyl-3-(1 H- 332 morpholin-4-yl)-friedel-2-ene ( 1350 ). 90 Figure 4.90 13 C NMR spectrum of 2-formyl-3-(1 H-morpholin-4-yl)-friedel- 332 2-ene ( 1350).

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91 Figure 4.91 1H NMR spectrum of 2-formyl-3-(1 H-piperazin-1-yl)-friedel- 333 2-ene (1351). 92 Figure 4.92 1H NMR spectrum of 2-formyl-3-(1 H-imidazol-1-yl)-friedel-2- 333 ene ( 1352). 93 Figure 4.93 13 C NMR spectrum of 2-formyl-3-(1 H-imidazol-1-yl)-friedel- 334 2-ene ( 1352). 94 Figure 4.94 1H NMR spectrum of 2-formyl-3-(1 H-benzimidazol-1-yl)- 334 friedel-2-ene ( 1353). 95 Figure 4.95 1H NMR spectrum (with partial expansion) of 2-formyl-3-(1 H- 335 benzimidazol-1-yl)-friedel-2-ene ( 1353). 96 Figure 4.96 13 C NMR spectrum of 2-formyl-3-(1 H-benzimidazol-1-yl)- 335 friedel-2-ene ( 1353). 97 Figure 4.97 1H NMR spectrum of 2-formyl-3-(1 H-1, 2, 3-benzotriazol-1- 336 yl)-friedel-2-ene ( 1354). 98 Figure 4.98 1H NMR spectrum (partially expanded) of 2-formyl-3-(1 H-1, 336 2, 3-benzotriazol-1-yl)-friedel-2-ene ( 1354). 99 Figure 4.99 13 C NMR spectrum of 2-formyl-3-(1 H-1, 2, 3-benzotriazol-1- 337 yl)-friedel-2-ene ( 1354).

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List of Schemes

Chapter-I

Entry Scheme No. Title of the scheme Page No. 1 Scheme 1.1 Synthesis of 3 a-fluorocholest-5-ene ( 16 ). 7 2 Scheme 1.2 A two-step synthesis of 3 a-epimer of cholesterol (17 ). 7 3 Scheme 1.3 Transformation of cholesterol into 3 b-nitrile derivative ( 18 ). 8 4 Scheme 1.4 Synthesis of 3 a- and 3 b-amino cholesterol ( 20 and 22 8 respectiv ely). 5 Scheme 1.5 Synthesis of N-3b-cholesterylbenzamide ( 23 ) by Ritter 9 reaction. 6 Scheme 1.6 Synthesis of N-3b-imidazolylcholesterol ( 24 ). 9 7 Scheme 1.7 Coupling of 6-chloropurine ( 25 ) with cholesterol by Mitsunobu 9 reaction. 8 Scheme 1.8 A one-pot deoxygenation of cholesterol. 10 9 Scheme 1.9 One-step synthesis of 5a-cholestanone ( 31 ) and cholest-5-ene- 10 3-one ( 32 ) respectively from 5a-cholestanol ( 30 ) and cholesterol ( 13 ). 10 Scheme 1.10 Catecholborane mediated reduction of cholesterol ( 13 ) and 11 cholesteryl benzoate ( 33 ). 11 Scheme 1.11 Synthesis of cholesterol-based monomer for radical 11 polymerization. 12 Scheme 1.12 Oxidative transformation of cholesterol ( 13 ) with 12 cetyltrimethylammonium dichromate. 13 Scheme 1.13 A copper-catalyzed allylic benzoyloxylation. 12 14 Scheme 1.14 Electrochemical allylic acetoxylation of cholesterol ( 13 ). 12 15 Scheme 1.15 Rhodium-catalyzed allylic oxidation of cholesterol and its 13 derivatives. 16 Scheme 1.16 Rhodium-catalyzed allylic oxidation of cholestenone ( 49 ) into 13 cholest - 4-ene-3,6-dione ( 50 ). 17 Scheme 1.17 Synthesis of squalamine-related polyaminocholesterol 14 derivatives ( 52-53 ). 18 Scheme 1.18 Iron-catalyzed intramolecular allylic amination in cholesteryl 14 sulfamate ( 54 ). 19 Scheme 1.19 Ruthenium-catalyzed alkylation of steroidal alkenes ( 30 and 15 56 ) with alcohols. 20 Scheme 1.20 Ruthenium-catalyzed dehydrative C-H alkylation of phenols 15 with cholesterol. 21 Scheme 1.21 Intramolecular allylic hydrogen abstraction by triplet 16 benzophenone. 22 Scheme 1.22 Rhodium-catalyzed decomposition of cholesteryl diazoacetate 17 (65 ). 23 Scheme 1.23 Regioselective oxygenation in cholesterol side-chain. 17

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24 Scheme 1.24 Synthetic routes to some oxygenated b-sitosterol ( 14 ) 19 derivatives. 25 Scheme 1.25 Proposed route for the conversion of b-sitosterol ( 14 ) to 20 cholesterol ( 13 ) in Silkworm. 26 Scheme 1.26 Synthesis of promising chemopreventive agent 1 a- 21 Hydroxyvitamin D5 ( 92 ) starting from b-sitosterol ( 14 ). 27 Scheme 1.27 Configurational assignment of amarasterone A ( 96 ) and 22 cyasterone ( 97 ) based on b-sitosterol ( 14 ). 28 Scheme 1.28 Synthesis of natural b-sitosteryl mono-oxime derivatives ( 100- 23 103). 29 Scheme 1.29 Biotransformation of b-sitosterol ( 14 ) by Mycobacterium sp. 24 30 Scheme 1.30 Synthesis of schottenol ( 109 ) starting from b-sitosterol ( 14 ). 24 31 Scheme 1.31 Key step for the synthesis of 24-nortriterpene derivatives from 32 betulin( 125). 32 Scheme 1.32 C3-Hydroxypropargylamine derivatives of betulinic acid 34 33 Scheme 1.33 Synthesis of betulin ( 125 ) and betulinal ( 144 ). 35 34 Scheme 1.34 Esters of L-amino acids 153-156. 38 35 Scheme 1.35 Synthesis of lupane derivatives (158-165). 38 36 Scheme 1.36 Semisynthesis of betulinic acid derivatives RS01 ( 168 ), RS02 39 (169) and RS03 ( 170). 37 Scheme 1.37 C, D and E-Ring-fused heterocyclic derivatives ( 190-193 ) of 42 betulin. 38 Scheme 1.38 NO-releasing nitrate derivatives of betulinic acid ( 225-226). 45 39 Scheme 1.39 Synthesis of lupeol esters ( 237). 47 40 Scheme 1.40 Synthesis of lupeol ( 120) from betulin ( 125). 48 41 Scheme 1.41 Synthesis of lupol based precursors for antimalarial agents 48 (241-243). 42 Scheme 1.42 Synthesis of NO-donating oleanolic acid derivative ( 278 ). 53 43 Scheme 1.43 Synthetic route of myriceric acid A ( 283) from oleanolic acid 54 (122). 44 Scheme 1.44 Synthesis of natural oleanolic acid saponins ( 284 ). 55 45 Scheme 1.45 Synthesis of prodrugs of oleanolic acid ( 285 ). 55 46 Scheme 1.46 Synthesis of oleanolic acid-based cyclic dimer ( 296 ). 56 47 Scheme 1.47 Seperation for ursolic acid ( 121) from oleanolic acid ( 122 ). 58 48 Scheme 1.48 Synthesis of oleanolic acid dimers ( 313 , 315). 58

Chapter II

Entry Scheme No. Title of the scheme Page No. 1 Scheme 2.1 Mechanism of allylic hydroxylation with SeO 2. 64 2 Scheme 2.2 Action of SeO 2 on ergosterol ( 83 ). 64

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3 Scheme 2.3 Action of SeO 2 on 5-dihydroergosterol ( 324b ) and its acetate 64 (324a). 4 Scheme 2.4 Action of SeO 2 on cholesterol ( 13 ) and other steroidal 5-ene- 65 3b-ols ( 328-330). 5 Scheme 2.5 Action of SeO 2 on cholestanone ( 31 ). 66 6 Scheme 2.6 Synthesis of 3b,7 a-dihydroxycholest-8 (14)-ene (339). 66 7 Scheme 2.7 Action of SeO 2 lanosteryl acetate ( 340). 67 8 Scheme 2.8 Action of SeO 2 on botogenin acetate ( 342 ), correlogenin ( 343) 67 and hecogenin acetate ( 344 ). 9 Scheme 2.9 Action of SeO 2/ MeOH on saturated steroidal 3-keto 68 derivatives ( 348-351 ). 3 10 Scheme 2.10 SeO 2 oxidation of methyl D -cholenate ( 357) and associated 68 transformations. 11 Scheme 2.11 Action of SeO 2 on 364. 69 12 Scheme 2.12 Synthesis of cholest-4-ene-7a,12a-diol-3-one ( 367). 69 13 Scheme 2.13 Action of SeO2 on 5 a-androstane-3,17-dione ( 368 ). 70 14 Scheme 2.14 Action of SeO 2 on steroidal 4-en-3-ones ( 49, 56, 370). 70 15 Scheme 2.15 Conversion of cholesterol ( 13 ) into 4-methyl-19-norcholesta- 73 1,3,5(10)-triene ( 384 ). 16 Scheme 2.16 Conversion of 3b-acetoxylanosta-5,8-diene-7-one ( 385) into 73 3b-acetoxy-19-norlanosta-5,7,9-trien-7-ol ( 386). 17 Scheme 2.17 Dienone-phenol rearrangement of cross-conjugated dienone or 74 trienone systems ( 387, 389 and 391 ). 18 Scheme 2.18 Dienone-phenole rearrangement. 74 19 Scheme 2.19 Transformtive reactions of bromohydrin 396, androsta-1,4,8- 75 triene -3,11,17-trione ( 400) and 17-acetoxyandrosta-1,4,9(11)- triene-3-one oxime ( 403) to result A-ring aromatized products. 20 Scheme 2.20 Transformation of dienone 413 into diaromatic steroid 414. 76 21 Scheme 2.21 A-ring aromation of ∆ 1,4,6 -22a-spirostatriene-3-one ( 415 ). 77 22 Scheme 2.22 A-ring aromatized cholesterol from 1,4,6-cholestatrien-3-one 77 (417). 23 Scheme 2.23 A-ring aromatization of 1,4,16-pregnatriene-3,20-dione. 78 24 Scheme 2.24 A-ring aromatized products from progesterone ( 56 ). 78 25 Scheme 2.25 Formation of estrone (59 ) from androsta-1,4-diene-3,17-dione 79 (371). + 26 Scheme 2.26 A-ring aromatization of some steroids by ‘(C 5Me 5)Ru ’ & 79 transformation of ergosterol ( 83 ) or 7-dehydrocholesterol ( 39 ) into B-ring aromatized products. 27 Scheme 2.27 Aromatization of ring-A of 17-b-acetoxy-1α,2α -epoxy-5α 80 androstan-3-one ( 434). 28 Scheme 2.28 Microbial transformation of some steroidal hormones. 81 29 Scheme 2.29 Steroidal aromatization by microorganisms. 81

30 Scheme 2.30 SeO 2 oxidation of cholesterol ( 13 ) and b-sitosterol ( 14 ). 83

31 Scheme 2.31 SeO 2 oxidation of cholesteryl acetate ( 40 ) and benzoate ( 33 ). 83 32 Scheme 2.32 Synthesis of natural diaromatic ergosterol derivatives 457 and 88 458. 33 Scheme 2.33 Action of SeO 2 on diosgenin ( 459). 90

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Chapter III

Entry Scheme No. Title of the scheme Page No. 1 Scheme 3.1 Synthesis of 24 (R)-and 24(S)-hydroxycholesterol ( 871 and 158 872) respectively. 2 Scheme 3.2 Cleavages of 874 and 876 by Dowex 50W X8. 158 3 Scheme 3.3 Treatment of epoxide 878 by SmI2 in THF. 159 4 Scheme 3.4 Synthesis of cytotoxic sterol 24 from stigmasterol ( 15 ). 159 5 Scheme 3.5 Synthesis of polyhydroxy steroids 887-888 by microorganism. 160

6 Scheme 3.6 Oxidation of 889 by BiO 3. 160 7 Scheme 3.7 Synthesis of polyhydroxy steroids 894-897. 161 8 Scheme 3.8 Synthesis of compounds 899-900. 161

9 Scheme 3.9 Synthesis ofnatural antitumor agent certonardalsterol D 3 (901). 161 10 Scheme 3.10 Synthesis of25(R)-spirostan-3b,5 a,6 b,19-tetrol ( 885 ) from 162 diosgenin ( 459 ). 11 Scheme 3.11 Synthesis of 914 . 163 12 Scheme 3.12 Synthesis of 7 a,12a-dihydroxy-4-cholesten-3-one ( 916). 164 13 Scheme 3.13 Synthesis of norcholyl-normalpropylketone ( 920) 164 andtrihydroxy-norsterocholane ( 921). 14 Scheme 3.14 Synthesis of a cytotoxic natural steroid 989 and its acetate 173 derivative 994. 15 Scheme 3.15 Synthesisofepoxysterols 878 , 998 and 1000. 173

16 Scheme 3.16 EpoxidationbyH 2O2/ MeOH . 174 17 Scheme 3.17 Synthesis of CDB-2914 1008. 174 18 Scheme 3.18 Synthesis of Melithasterol A 947 through microwave 175 irradiation. 19 Scheme 3.19 a-face epoxidation of 1012 with tert butyl hydroperoxide. 175 20 Scheme 3.20 Epoxidation of acetal 1014 . 176 21 Scheme 3.21 Synthesis of17 b -[[(1,1-Dimethylethyl)dimethylsilyloxy]- 176 6 b ,7 b -epoxy-3-methoxy-7-methylestra-1,3,5(10)-triene (1017). 22 Scheme 3.22 Epoxidation of7a-chloro-6b-ol 1018. 176 23 Scheme 3.23 Synthesis of3a,4 a-Epoxy-5a-androstan-17-one 1022and 177 3a,4a-epoxy-5a-androstan-17-one 1023. 24 Scheme 3.24 Synthesis of epoxy steroids 876, 1028 and 1030. 177 25 Scheme 3.25 Epoxidation of cholesterol derivatives. 178 26 Scheme 3.26 Epoxidation of3 b-acetoxy b-sitosterol. 179 27 Scheme 3.27 Epoxidation of methyl-3a,7 a-diacetylchol-11-enate 1049. 180 28 Scheme 3.28 Epoxidation of D4-3-ketosteroids. 180

5 29 Scheme 3.29 Epoxidation of D -3-ketosteroids by KMnO 4/Fe(ClO 4)3·nH2O. 181 30 Scheme 3.30 (22E)-3b-Acetoxy-14 a,15a-oxido-5a-ergosta-7,22-diene 181

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(1063) and 3b-acetoxy-14 a,15 a-epoxy-5a-cholest-8-en-7-one (1065). 31 Scheme 3.31 Epoxidation of 1068,acetate derivative of 1067 and 1070. 182 32 Scheme 3.32 Synthesis of 3b-Acetoxy-5,6b-epoxy stigmastane ( 1047 ) and 183 its acetate derivative 1048. 33 Scheme 3.33 m-CPBA oxidation of 3b-acetoxy-17-picolinylidene-androst-5- 184 ene ( 1072). 34 Scheme 3.34 m-CPBA oxidation of 3b-hydroxy-17-picolinylidene-androst- 184 5-ene ( 1077). 35 Scheme 3.35 m-CPBA oxidation of 3b,21-Diacetoxy-pregn-17(20)-ene 185 1084. 36 Scheme 3.36 Epoxidation of 5-ene-derivatives ( 1087) of cholesteyl skeleton. 185 37 Scheme 3.37 MW assisted epoxidation of 3 b,17 b-acetoxy androst-5-ene 186 (330) with m-CPBA. 38 Scheme 3.38 Synthesis of some epoxy steroids. 186 39 Scheme 3.39 Synthesis of 1095 from prednesolone ( 1094). 187

40 Scheme 3.40 Epoxidation of 1096 and 1099 in H2O2/ alkaline media. 187 41 Scheme 3.41 Synthesize of functionalised azasteroid derivatives. 188 42 Scheme 3.42 Synthesis of epoxy and 2-functionalised androstane 189 derivatives. 43 Scheme 3.43 Synthesis ofeplerenone( 1133). 190 44 Scheme 3.44 Selective5a,10a-epoxidation (inset: ulipristal, 1008.) 194 45 Scheme 3.45 Synthesis of novel steroidal molecules using epoxides. 195 46 Scheme 3.46 Ritter reaction on epoxy steroids. 196 47 Scheme 3.47 Transformative reactions on some epoxides. 196 48 Scheme 3.48 Synthesis of E-guggulsterone ( 1193 ). 197 49 Scheme 3.49 Project to synthesize novel steroidal terols and pentols. 198 50 Scheme 3.50 Route to tetraols and pentaols from diols and triols 199 respectively, via corresponding epoxides. 51 Scheme 3.51 Preparation of the starting diols and triols. 199 52 Scheme 3.52 Synthesis of epoxy-dihydroxy- and epoxy-trihydroxy steroids. 199 53 Scheme 3.53 Synthesis of tetrahydroxy- and pentahydroxy steroids. 203

Chapter IV

Entry Scheme No. Title of the scheme Page No. 1 Scheme 4.1 Derivatives of 1224 . 253 2 Scheme 4.2 Compound 1256. 257

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3 Scheme 4.3 Silylation of friedelin leading to different friedelane 258 derivatives. 4 Scheme 4.4 Treatment of friedelin ( 11 ) with t-BuOK and diazomethane. 259 5 Scheme 4.5 Conversion of friedelane-2,3-dione ( 1267) to friedelin ( 115). 260 6 Scheme 4.6 Conversion of friedelane-2,3-dione ( 1267) to 3-methoxy-2- 260 oxofriedelane ( 1269). 7 Scheme 4.7 Transformation of cerin ( 116) into different dihydroxy 261 derivatives. 8 Scheme 4.8 Transformation of friedelin into different dihydroxy 261 derivatives. 9 Scheme 4.9 Ultraviolet irradiation products of friedelin in ether and in 262 chloroform. 10 Scheme 4.10 Photochemical reactions on friedelin ( 115 ). 262 11 Scheme 4.11 Friedelin ( 115) to olean-l3(18)-ene ( 1287). 264 12 Scheme 4.12 Friedelene-oleanene rearrangement. 264 13 Scheme 4.13 Molecular rearrangement of 4α -bromofriedelin ( 1292) by 265 silver acetate. 14 Scheme 4.14 4-Aza-A-homofriedelan-3-one (1294) from both friedelin 265 oxime (1 255) and friedelin ( 115). 15 Scheme 4.15 Synthetic routes of different bromo-derivatives of friedelin. 267 16 Scheme 4.16 NBS on friedelin. 268 17 Scheme 4.17 Molecular rearrangements of bromo-derivatives of friedelin. 268 18 Scheme 4.18 A-ring modifications along with seco-friedelanes. 269 19 Scheme 4.19 Topoisomarase II a inhibitory seco-friedelanes. 270 20 Scheme 4.20 Biologically active derivatives of friedelin. 271 21 Scheme 4.21 Direct amidation of unactivated 24-methyl on friedelin. 272 22 Scheme 4.22 Action of Vilsmeyer-Haack reagent on friedelin. 275 23 Scheme 4.23 Synthesis of friedel-3-ene-23-al. 277 24 Scheme 4.24 Oxidation of 1327 with m-CPBA. 277 25 Scheme 4.25 Synthesis of A-ring aromatized friedelane triterpenoid by NBS. 278 26 Scheme 4.26 A two-step synthesis of 3,4-seco friedelane-3,4-diol from 278 friedelin. 27 Scheme 4.27 Synthesis of 3-epi pachysandiol A from cerin. 279 28 Scheme 4.28 A four-step synthesis of 3 b-amino-4a-hydroxyfriedelane from 279 friedelin. 29 Scheme 4.29 Oximination of 1330. 280 30 Scheme 4.30 Synthesis of 2-hydroxymethyl and its acetate derivative. 280 31 Scheme 4.31 Synthesis of 3-chlorofriedel-2-ene-2-carboxamide ( 1346 ). 281 32 Scheme 4.32 Synthesis of 4 a-hydroxy-3-chloro-2-formylfriedel-2-ene. 282 33 Scheme 4.33 Synthesis of 4 a-hydroxy-3-chloro-2-formylfriedel-2-ene. 282 34 Scheme 4.34 Oximination of 1328. 283 35 Scheme 4.35 Synthesis of 3-chloro-1a-hydroxy-2-hydroxymethylfriedel-2- 283 ene ( 1348).

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36 Scheme 4.36 Syntheses of aliphatic N-heterocycle-linked 2-homofriedelane 284 derivatives. 37 Scheme 4.37 Syntheses of aromatic N-heterocycle-linked 2-homofriedelane 284 derivatives.

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List of Appendices

Appendix A

Papers Published/ communicated/ under preparation:

1. Mercuric(II) acetate oxidation of steroidal exocyclic α,β -unsaturated ketone: transformation into a cyclic ether. Pranab Ghosh, Antara Sarkar,Jayanta Das , Molbank, 2009, 2009(3) , M616. 2. One-pot solid phase selective aromatization of cholesterol using N-bromosuccinimide: an optimized green methodology. Pranab Ghosh, Jayanta Das , Antara Sarkar, Green Chem. Lett. Rev., 2012, 2 (5), 173-177. 3. Oxidation with selenium dioxide: the first report of solvent selective steroidal aromatization, efficient access to 4 b,7 a-dihydroxy steroids, and syntheses of natural diaromatic ergosterols. Pranab Ghosh, Jayanta Das ,Antara Sarkar, Seik Weng Ng, Edward R.T. Tiekink, Tetrahedron , 2012, 68(32), 6485-6491. 4. Solid phase selective oxidation of b-sitosterol and 4 b-hydroxy cholesterol by N- bromosuccinimide-a green approach. Pranab Ghosh,Antara Sarkar, Jayanta Das , J. Ind. Chem. Soc. 2014, 91(12) , 2255-2257. 5. p-TsOH-Mediated green transformations of di- and trihydroxy steroids toward diverse A/B-ring oxo-functionalization. (Manuscript under preparation)

6. BF 3.OEt 2-Mediated oxidations toward phytotoxic A-ring modified friedelane triterpenoids. (Manuscript under preparation) 7. Polyhydroxy and epoxy-polyhydroxy steroids: design, synthesis, cytotoxicity and study of their preliminary gelation behaviour(Manuscript under preparation) 8. Syntheses of new bioactive friedelane triterpenoids: A-ring modifications including 2- homoderivatives(Manuscript under preparation)

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Appendix B

List of research papers presented in National/International Seminar/ Conference/Workshop.

1. Microwave-assisted one-pot solid phase aromatization of ring-A of cholesterol and its antimicrobial properties (Poster presentation). Jayanta Das , Antara Sarkar and Pranab Ghosh.12 th CRSI National Symposium in Chemistry. Feb. 4-7, 2010, IICT& NIPER, Hyderabad. 2. Concerted oxidation and reduction of 4β–hydroxy steroids (Poster presentation). Jayanta Das , Antara Sarkar and Pranab Ghosh. National Seminar on Frontiers in Chemistry 2011 & Celebration of the International Year of Chemistry 2011. Mar. 14- 16, 2011. Department of Chemistry, University of North Bengal. 3. 4β, 7α -Dihydroxy steroids: easy efficient synthesis, and p-toluenesulfonic acid induced ‘on -silica’ rearrangement (Poster presentation). Antara Sarkar, Jayanta Das and Pranab Ghosh. CRSI Eastern Zonal Meeting, 2011& Celebration of the International Year of Chemistry 2011. Jul. 22-24, 2011. Department of Chemistry, University of North Bengal. 4. Concerted oxidation and reduction of 4 b-hydroxy and 4 b,7 a-dihydroxy steroids (Oral presentation). JayantaDas and Pranab Ghosh. UGC Sponsored National Seminar on Frontier of Chemistry. Nov.15-16, 2011, Department of Chemistry, Gour Mahavidyalaya, Malda. 5. Nature’s green concept: Organicreactions “in water” and “on water” (Oral presentation). JayantaDas and Pranab Ghosh. UGC Sponsored Seminar on Green Chemistry for Environmentally Benign Chemical Synthesis. Feb. 14-15, 2012, Department of Chemistry, Raiganj Surendranath Mahavidyalaya & Department of Chemistry, University of North Bengal. 6. Diaromatic steroidal natural products: synthesis, antioxidant and oxidative DNA damage protective activities (Oral presentation). Pranab Ghosh, Jayanta Das , Antara Sarkar, Ramashish Kumar and Dipanwita Saha. National Seminar on Micro and Macro Resources in Biomolecular Technology. Feb. 25-26, 2013, Department of Biotechnology and Microbiology, University of North Bengal.

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7. Regioselective steroidal aromatization (Oral presentation). Jayanta Das and Pranab Ghosh. Workshop on Diversities and Frontiers in Chemistry under State University Network. Aug. 7-8, 2013, Department of Chemistry, University of North Bengal.

8. A-ring modified bioactive friedelane triterpenoids via BF 3.OEt 2-mediated oxidative transformation (Poster presentation). Antara Sarkar, Jayanta Das and Pranab Ghosh.16th CRSI National Symposium in Chemistry (NSC-16). Feb. 7-9, 2014, IIT Bombay, Powai, Mumbai.

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Appendix C

Abbreviations: ATP: adenosine triphosphate BA: betulinic acid

CDCl 3: deuteriated chloroform Calcd: calculated DART-MS: Direct Analysis in Real Time Mass spectroscopy DCM: dichloro methane DEPT: Distortionless enhancement of polarization transfer DMAP: N, N-dimethylamino pyridine DMF: dimethyl formamide DMSO: dimethyl sulphoxide FTIR: Fourier transformed infra-red h: hours LAH: lithium aluminium hydride mCPBA: m-chloro perbenzoic acid min: minute m.p: melting point NBS: N-bromosuccinimide NMR: nuclear magnetic resonance OA: oleanolic acid PT: pentacyclic triterpenoids Pet. ether: petroleum ether Pd-C: palladised charcoal p-TsOH: p-tolune sulphonic acid RT: room temperature. TBDMS: tertiarybutyldimethylsilyl TEMPO: 2,2,6,6-tetramethylpipyridinyloxy TLC: thin layer chromatography TMS: trimethyl silanne

SN 1: substitution nucleophilic unimolecular

SN 2: substitution nucleophilic bimolecular UA: ursolic acid

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Chapter I

Carbocyclic compounds and their transformative reactions: A general perspective on natural products chemistry

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I.1 Carbocyclic compounds Chemistry, in its widespread periphery, is basically full of unlimited molecules with their novel interactions which lets human know Nature closer. And organic chemistry is based typically on the innumerous organic compounds which, on their structural aspect, can broadly be divided in two ways- acyclic and cyclic. As the cyclic molecules represent those which are a combination of both literally ‘cyclic’ and ‘acyclic’ molecules (e.g., hexyl cyclohexane (1) - a cyclic compound, Figure 1.1), the numbers of cyclic entities are thus more in nature. In addition, cyclic molecules are classified into homocyclic and heterocyclic. The cyclic compounds where all the ring members are constituted by same element are termed as homocyclic compounds, e.g., cyclopropane ( 2), benzene ( 3), cholestane (5 a: 4, 5 b: 5), friedelane (6) pentazole ( 7) etc. And homocyclic compounds where all the ring members are carbon atoms, are termed as carbocyclic compounds (e.g., compounds 1-6). 1 On the other hand, cyclic compounds where the ring is made by one or more different elements, the system is termed as heterocyclic, e.g., aziridine ( 8), thiophene ( 9), morpholine ( 10 ), benzothiooxazoline ( 11 ), etc (Figure 1.1 and Chart 1.1 ).

H2C CH2 H2C 1 2 3 H 4; H= 5a 5; H= 5b

H N S N N O NH NH N N N S O H 8 9 10 11 6 7

Figure 1.1 Some cyclic organic compounds ( 1-6: carbocyclic; 7: nitrogen-based homocyclic; 8-11 : heterocyclic).

I.2 Natural products and carbocyclic compounds Humans, throughout the ages, have bind them together with Nature to cater for their basic needs —starting from foods for survival, to medicines for healthy survival. And Nature always

2 has provided the materials to fight against a wide spectrum of diseases. Particularly, the plant kingdom has formed the basis of traditional medicine systems.

Chart 1.1

A chemical compound or substance produced by a living organism —thus found in nature, is termed as a natural product. 2, 3 Natural products thus include any substance produced by life. 4 The commercially available materials used for the aid of daily-life, viz., cosmetics, dietary supplements, some kind of medicines, foods etc., which are produced from natural sources without any artificial additives are also named as natural products. 5 As a consequence, to supply at a required amount, many of the natural compounds have been prepared in the laboratory by the process of chemical synthesis, which indeed has enriched organic chemistry enormously by following a huge number of challenging targets.

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Within the periphery of organic chemistry, we consider natural products, typically, as pure organic compounds (thus pure molecules) isolated from natural sources related to lives, directly or indirectly. These natural compounds are produced mainly by the primary and secondary metabolism pathways. 6 Primary metabolites are defined as the components of basic metabolic pathways that are essentially required for life. Cellular functions such as nutrient assimilation, energy production, and growth/ development are associated with the primary metabolites. In short, the basic building blocks of life are the primary metabolite-based natural products which include carbohydrates, lipids, amino acids, and nucleic acids. 7-9 On the other hand, secondary metabolites, though have a broad range of functions, are not absolutely required for survival. Social signaling molecules ( pherom ones etc.), agents that solubilize and transport nutrients (siderophores etc.), competitive weapons which are used normally against competitors, prey, and predators ( repellants , venoms , toxins etc.) and immune system developing components are basically under the secondary metabolite-based natural products. 10, 11 Alkaloids, phenylpropanoids, polyketides, and terpenoids are the general structural classes of secondary metabolites. Natural products are prone to have pharmacological or biological activities and as a consequence, most traditional medicines as well as lots of modern medicines are based on them. The structural diversity of natural products exceeds that readily achievable by chemical synthesis, and on the other hand, synthetic analogs can be prepared with improved potency and safety. As a result, natural products are often inspirational and used as starting points for drug discovery. In fact, natural products are the inspiration for approximately one half of U.S. Food and Drug Administration-approved drugs. 12, 13 Now, due to the large extent of catenation capability of carbon, it can produce a large number of molecules made solely by them which in turn, implies that nature provides a huge number of carbocyclic compounds. And in virtue, nature itself is the richest source of a variety of carbocyclic compounds. Two or more carbocycles can be joined together in a number of different fashions to produce a number of different groups of carbocyclic compounds. And among the broad spectrum of all kinds of natural products, if we look for more common, easily available and highly useful carbocyclic natural products, we found mainly the steroids and terpenoids (Chart 1.1 ).

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Thus, having the opportunity of working in the field of natural products chemistry, the author intended to work on the steroids (which belong, at majority, to the primary metabolites class) and pentacyclic triterpenoids (as our laboratory is actively engaged enriching this particular terpenoid chemistry), specially on friedelane triterpenoids (which belong to the secondary metabolites class) as these two areas are drawing the major increasing attention among the carbocyclic natural products.

I.3 Steroids The important natural compounds which are constituted on the basis of 1,2- cyclopentenophenanthrene ( 12 ) skeleton with 17 carbon atoms are known as steroids ( Figure 1.2). Besides the widespread distribution of the steroids in nature, marine plants and animals are the richest source which display interesting biocidal activities. 14 Steroids include a large number of sterols, vitamin D, the bile acids, a number of sex hormones, the adrenal cortex hormones, some carcinogenic hydrocarbons, certain saponins, antibiotics, etc. Steroids can penetrate cells and bind to nuclear and membrane receptors. Nature has produced the steroid system to perform some selected fundamental biological functions and the fact directs to form the basis of new discoveries in this field which, in virtue, relates biology and chemistry with more practical applications. Rigidity, permeability, conformational order, and phase behavior of phospholipid membranes in cells are highly regulated by the sterols, and particularly by cholesterol ( 13 )14 which is a major component (present up to 50 mole%) of eukaryotic cell plasma membranes. 15,16 The alteration of the acquired concentrations of sterols are associated with diseases. 17 For example, excess cholesterol results human atherosclerosis. Patients afflicted with sitosterolemia also suffer from atherosclerosis, for an accumulation of excess plant sterols, mainly b-sitosterol (14 ) and stigmasterol ( 15 ). 18 (Figure 1.2 ) In healthy and hypercholesterolemic (but non- sitosterolemic) subjects, plant sterols are found valued for their ability to lower plasma cholesterol levels 19,20 as well as their potential as anticancer compounds. 21

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H H H

H H H H H H HO HO 12 HO 13 14 15

Figure 1.2 1,2-Cyclopentenophenanthrene ( 12 ), Cholesterol ( 13 ), b-sitosterol ( 14 ) and stigmasterol ( 15 ).

On the other hand, among the steroids, the compound considered as the most fundamental one is cholesterol ( 13 ) for the animals, and b-sitosterol ( 14 , structurally, 24 a-ethyl choleaterol) for the plants. As the author has the privilege to work with these molecules (and their derivatives), a brief general discussion about their recent advances in chemistry, giving emphasis especially on their transformative reactions, is provided below.

I.3.1 Recent selective transformative reactions on cholesterol and b-sitosterol Within the family of steroids, an important branch of polycyclic compounds, cholesterol ( 13 ) is the most abundant member. The essential structural component of animal cell membranes required in order to maintain proper membrane permeability as well as fluidity is the lipid molecule- cholesterol, more precisely cholest-5-en-3b-ol (13 ) which originates from Ancient Greek chole- (i.e., bile) and stereos (i.e., solid) followed by the chemical suffix -ol for alcohol. Cholesterol is also the precursor for the biosynthesis of a number of essentially important compounds including steroid hormones, bile acids, vitamin D etc. Cholesterol ( 13 ) is synthesized by animals and, in vertebrates it is formed predominantly in the liver. For more than a century, people have found it fascinating to work with the structure, biosynthetic pathway and metabolic regulation of cholesterol ( 13 ). Thirteen Nobel Prizes have 22 been awarded to scientists who devoted major parts of their careers to its study.  Cholesterol ( 13 ) was the first steroid ever isolated and was discovered by M.E. Chevreul in 1815. It was isolated from the non-saponifiable part of animal lipids. The structural elucidation of cholesterol was started in 1859 and its exact molecular formula (C 27 H46 O) was established in 1888 by F. Reinitzer. Proof for its structure was obtained chiefly through the novel work of A. Windaus and H.O. Wieland. The structure of cholesterol as was suggested by Windaus and Wieland in the 1920s was not exactly correct, but their contribution in the field was really

6 unparalleled. Depending on the X-ray diffraction data, in the 1930s, the exact structure of cholesterol was established. Woodward et al., in 1951, reported a total synthesis of cholesterol. 23 Thus, to start working with the transformative reactions of cholesterol and its derivatives, it was a bit essential to have a thorough study of the reports which are due to the various transformative reactions mainly. However, as the subject is an elaborative one, the major advances, excluding the simple derivatization reactions, since the last one and half decade is highlighted below.

1.3.1.1 Recent transformative reactions on cholesterol

Diethylaminosulfur trifluoride (Et 2NSF 3, commonly abbreviated as DAST) generally reacts with 24 alcohols to result the fluoro derivatives via an SN 2 or SN i mechanism. When cholesterol ( 13 ) was reacted with DAST, an SN 2 pathway furnished 3 a-fluorocholesterol ( 16 ) as the only product (Scheme 1.1). 25

Et2NSF3, Furan RT, 5 min., 56% HO F 13 16

Scheme 1.1 Synthesis of 3 a-fluorocholest-5-ene (16 ). Cholesterol ( 13 ) was converted to its 3 a-epimer 17 by following a two-step procedure via p- nitrobenzoate ( Scheme 1.2). 26

1. p-NO2PhCOOH, DEAD, PPh3 o HO 2. NaOH, H2O, 0 C HO 13 17

Scheme 1.2 A two-step synthesis of 3 a-epimer of cholesterol (17 ).  An established general method for the conversion of alcohols to the corresponding nitriles by using N-(p-toluenesulfonyl)imidazole (TsIm), was also applied to have 3 b-nitrile derivative

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18 of cholesterol at moderate yield. The method utilized NaCN, TsIm and triethylamine (TEA), and catalytic amounts of tetra-n-butylammonium iodide (TBAI) ( Scheme 1.3). 27

NaCN, TsIm, TBAI, TEA DMF, reflux, 6h HO NC 13 18

Scheme 1.3 Transformation of cholesterol into 3 b-nitrile derivative (18 ).

The 3 a-amino derivative of cholesterol and 3b-amino derivative of 5a-cholestane ( 20 and 22 respectively) were prepared respectively from cholesterol ( 13) and 5 a-cholestane ( 4) by following a two-step Mitsunobu procedure. A double inversion of configuration was utilized in the reaction sequences to afford ultimately the 3 b-amino derivative (22 ) via 3a-iodide (21 ) and 28 3b-azide (not shown in the Scheme 1.) followed by LiAlH 4 reduction ( Scheme 1.4).

DIAD, PPh3 LiAlH4 (PhO)2PON3, THF Et2O HO N3 H2N 13 19 20

o DIAD, PPh3 1. NaN3, DMSO, 90 C CH3I, THF 2. LiAlH4, Et2O, reflux HO I H2N H H H 4 21 22 Scheme 1.4 Synthesis of 3 a- and 3 b-amino cholesterol (20 and 22 respectively).

Mondal et al. found an interesting variation of the Ritter reaction where an inexpensive Ti(IV)/nitrile reagent was used for the transformation of cyclic secondary alcohols into the corresponding amides. 29 Retention of configuration was found to occur in the conversions e.g. cholesterol was converted into N-3b-cholesterylbenzamide ( 23) at a very good yield ( Scheme 1.5).

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PhCN, TiF4 O DCM, 0oC, 2h HO Ph N H 13 23

Scheme 1.5 Synthesis of N-3b-cholesterylbenzamide (23 ) by Ritter reaction.

Following a general reaction applicable to a variety of alcohols, cholesterol ( 13 ) was converted into the 3 b-imidazolyl derivative ( 24) in high yields by using N,N '- carbonyldiimidazole (CDI) as the heterocycle donor substrate ( Scheme 1.6). Similarly, N,N '- carbonylditriazole (CDT) was used to furnish the corresponding triazolyl derivative (Scheme not shown). 30 Some nucleo-cholesterols ( 26-28) was also prepared by the Mitsunobu coupling of 6- chloropurine ( 25) with cholesterol. The reaction resulted, besides the SN 2 major product 26, two isomeric compounds ( 27 and 28) formed via the mesomeric homoallylic carbocation ( Scheme 1.7).31

CDI, CH3CN , 3h HO N N 13 24

Scheme 1.6 Synthesis of N-3b-imidazolylcholesterol (24 ).

H N N DEAD, PPh3 + N + N N dioxane, RT, 48h N HO N N 13 Cl N 25 Cl N N N 26; 3a: 22.8% 27; 3b: 10.6% Cl 28 12.6% Scheme 1.7 Coupling of 6-chloropurine (25 ) with cholesterol by Mitsunobu reaction .

Primary and secondary alcohols were deoxygenated in one-pot via a combination of the Garegg –Samuelsson reaction, visible light-photoredox catalysis, and flow chemistry. The

9 reaction indeed attains importance due to the mild reaction condition, easy handling of the reagents, excellent functional group tolerance and good yield. Following the procedure, 5- cholestene ( 29) was obtained from cholesterol at 72% yield ( Scheme 1.8). 32

I2, PPh3, Imidazole, CH3CN, 2h fac-Ir(ppy) , i-Pr NEt, CH OH, flow LED HO 3 2 3 13 29

Scheme 1.8 A one-pot deoxygenation of cholesterol.

Rapid oxidation of alcohols into the corresponding carbonyl derivatives was achieved by using PhIO and catalytic amounts of TEMPO and Yb(OTf) 3. Following the oxidation protocol, 5a-cholestanol ( 30 ) furnished 5 a-cholestanone ( 31) whereas cholesterol ( 13 ) afforded cholest-5- en-3-one ( 32) by maintaining the double bond position unaltered ( Scheme 1.9). 33

PhIO, TEMPO, Yb(OTf)3

HO DCM, RT, 2.5h O H H 30 31

PhIO, TEMPO, Yb(OTf)3

HO DCM, RT, 2.5h O 13 32

Scheme 1.9 One-step synthesis of 5a-cholestanone ( 31 ) and cholest-5-ene-3-one ( 32 ) respectively from 5a-cholestanol ( 30 ) and cholesterol (13 ).

The organoborane derivative obtained from the reaction of cholesteryl benzoate ( 33) with two equivalents of catecholborane was treated with 4-tert -butylcatechol (TBC) in the presence of air to afford the reduced product 3 b-benzoyloxycholestane in excellent yield having a 5a/5 b = 9:1 mixture of diastereomers, 34 and 35, respectively. Following the same reaction protocol,

10 however, cholesterol afforded 5 a-cholestan-3b-ol ( 30) in 72% yield as a single diastereomer (Scheme 1.10 ). 34

O 1. HB O , DMA, 1,2-DCE BzO 2. TBC (2 equiv), air BzO 33 H 5a (34)/ 5b (35)= 9:1

O 1. HB O , DMA, 1,2-DCE HO 2. TBC (2 equiv), air HO H 13 30

Scheme 1.10 Catecholborane mediated reduction of cholesterol ( 13 ) and cholesteryl benzoate (33 ).

5a-Hydroxy-6b-acrylate derivative of cholesterol, 38 was prepared from the reaction of 3- alkoxy-5,6-epoxycholestane ( 36) with acrylic acid ( 37). (Scheme 1.11 ) Free radical polymerization was then carried out with the acrylate derivative which was studied thoroughly with the use of different methods. 35,36

O o + 60 C, 48h OH H2n+1CnO H C O O 37 2n+1 n HO 36 O O

38

Scheme 1.11 Synthesis of cholesterol-based monomer 38 for radical polymerization.

Cetyltrimethylammonium dichromate (CTADC), when was applied on cholesterol (13 ) in dichloromethane (DCM), 7-dehydrocholesterol ( 39) was the isolated product whereas addition of acetic acid in DCM produced 5-cholesten-3-one ( 32, Scheme 1.12 ). 37 The kinetics of the oxidation reaction suggests to follow the reversed micellar system and, involves the formation of an intermediate ester complex which ultimately was decomposed into the products.

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CTADC CTADC DCM DCM, CH COOH HO HO 3 O 39 13 32

Scheme 1.12 Oxidative transformation of cholesterol (13 ) with cetyltrimethylammonium dichromate.

A copper-catalyzed, stereoselective allylic benzoyloxylation of sterol derivatives was developed by Brunel et al.38 The complete stereoselectivity was justified through a rationale mechanism. Thus, following the reaction protocol, 7 a-hydroxycholesterol ( 42) was obtained in a two-step process from cholesteryl acetate ( 40 ) in 61% overall yield via the 7 a-benzoyloxy cholesteryl acetate 41 . ( Scheme 1.13).

t CuBr, BuOOCOPh LiAlH4 DCM, Reflux, 12h AcO AcO OBz HO OH 40 41 42

Scheme 1.13 A copper-catalyzed allylic benzoyloxylation.

Cholesterol ( 13 ), on electrochemical oxidation on a platinum electrode in glacial acetic acid furnished two major products: 7 a-acetoxycholesterol ( 43) and 7 b-acetoxycholesterol ( 44) in a ratio of 10:3. In the oxidation reaction, sodium perchlorate and sodium acetate was also used as the supporting electrolytes ( Scheme 1.14). 39

Anodic oxidation + CH COOH, RT HO 3 HO OAc HO OAc 13 43 44

Scheme 1.14 Electrochemical allylic acetoxylation of cholesterol (13 ). 

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Dirhodium caprolactamate with t-butyl hydroperoxide (TBHP) was used to oxidize cholesterol (13 ), its OTBDMS ( 45 ), 3b-acetoxycholesterol ( 40) into their respective 7-oxo derivatives 46-48 (Scheme 1.15).40,41 It was observed that oxygen-protected steroids provide yields better. This oxidizing system was also used for the allylic oxidations of steroidal enones into the corresponding enediones in moderate to high yields, e.g. cholest-4-en-3-one ( 49) was oxidized into cholest- 4-ene-3,6-dione ( 50) at 50% yield. 42 (Scheme 1.16).

TBHP, Rh2(cap)4 DCE, Reflux, 20h R R O 13; R= OH 46; R= OH 45; R= OTBDMS 47; R= OTBDMS 40; R= OAc 48; R= OAc

Scheme 1.15 Rhodium-catalyzed allylic oxidation of cholesterol and its derivatives.

TBHP, Rh2(cap)4 DCE, Reflux, 20h O O 49 50 O

Scheme 1.16 Rhodium-catalyzed allylic oxidation of cholestenone (49 ) into cholest- 4-ene-3,6-dione (50 ). A number of squalamine-related polyaminosterols were synthesized from cholesterol. 43 In the key-step of the transformations, the azido group was introduced on the allylic C-7 position of cholesteryl acetate ( 40 ) with the treatment of trimethylsilyl azide in the presence of Pb(IV) acetate ( Scheme 1.17). The yield of the 7-azido cholesteryl acetate ( 51) was obtained at 68% with the epimeric ratio of 7 a/7 b = 77:23. The epimers were separated and transformed into the corresponding squalamine analogs ( 52 , 53 ) which were evaluated with their biological activities.

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Pb(OAc)4, (CH3)3SiN3 DCM, RT, 2h HO NH AcO AcO N3 H N 2 N 40 51; 7a/ 7b= 77: 23 H 52; 7a 53; 7b Scheme 1.17 Synthesis of squalamine-related polyaminocholesterol derivatives ( 52 and 53 ).

An intramolecular allylic C-H amination catalyzed by iron(III) has recently been reported. 44 The inexpensive commercial compound, [Fe(III)pc]Cl (pc = phthalocyaninato), typically used as an industrial additive in ink and rubber manufacturing, was employed as the iron-catalyst. In the reaction protocol, allylic C-H amination was found to be favored to the aziridination and amination at other C-H bond types. Employing the reaction, cholesteryl sulfamate ( 54 ) furnished a single diastereomer of the allylic intramolecular amination product 55 at 58% yield. (Scheme 1.18)

1. ClSO2NCO HCOOH, CH3CN [FePc]Cl, AgSbF6

2. NaH, DMF, CHCl PhI(OPiv)2, toluene, CH3CN HO 3 O O 13 O S NH2 O S NH O 54 O 55 Scheme 1.18 Iron-catalyzed intramolecular allylic amination in cholesteryl sulfamate (54 ).

Catalytic selective alkylation of alkenes with the alcohols to form a C-C bond between vinyl 45 C–H and C-OH centers with the concomitant loss of H 2O was reported. The catalyst used for the transformation was a cationic ruthenium complex and a broad range of substrate functionality including amines and carbonyls were found to be tolerated. With p-methoxybenzyl alcohol, both cholesterol ( 13 ) and progesterone ( 56) under the optimum reaction conditions, afforded the corresponding 6- and 4-p-methoxybenzylated products ( 57 and 58 respectively), without affecting either the alcohol or carbonyl functional groups. (Scheme 1.19)

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+ - HO [(C6H6)(PCy3) (CO)RuH] BF4 o p-MeO-C4H4CH2OH, PhCl, 90 C, 6h HO 57 13 OMe

COCH3

+ - [(C6H6)(PCy3) (CO)RuH] BF4 O p-MeO-C H CH OH, PhCl, 90oC, 6h O 4 4 2 56 58 OMe

Scheme 1.19 Ruthenium-catalyzed alkylation of steroidal alkenes (30 and 56 ) with alcohols.

O OH

+ Condition A HO HO 60 13 59

O

H N OH OH Condition B + HO HN 13 61 62

Scheme 1.20 Ruthenium-catalyzed dehydrative C-H alkylation of phenols with cholesterol: Condition A: + - o [(C 6H6)(PCy 3)(CO)RuH] BF 4 , cyclopentene (0.15 equiv), toluene/ DMSO 9:1, 100 C, 10h; Condition B: + - o [(C 6H6)(PCy 3)(CO)RuH] BF 4 , cyclopentene (1.5 equiv), toluene/ C 6H5Cl= 1:1, 100 C, 8h.

Another interesting dehydrative C –H alkylation reaction of phenols with alcohols to produce ortho -substituted phenol derivatives was also reported by using the same cationic ruthenium

15 complex. 46 And utilizing this transformation, C-H alkylation of estrone ( 59) with cholesterol (13 ) was carried out to form a 1:1 diastereomeric mixture of the coupling product 60 . On the other hand, the analogous C-H alkenylation of 2-hydroxycarbazole ( 61 ) with cholesterol (13 ) resulted the desired oxidative coupling product 62 only. (Scheme 1.20 ) Employing the triplet excited benzophenone, abstraction of the allylic hydrogen from C-7 of cholesterol was studied. 47 When the reaction was considered for intramolecular transformation (e.g., from 63 to 64), very high diastereoselectivity (7 b-H was abstracted only) was found to occur ( Scheme 1.21 ). 48,49 Cholest-5-en-3b-yl diazoacetate ( 65 ) was designed and prepared at a good yield (68%) from cholesterol ( 13 ) by diketene condensation following by diazo transfer and deacetylation. The diazoacetate derivative of cholesterol, 65 was then subjected to standard conditions for diazo decomposition with a series of chiral dirhodium(II) carboxamidate catalysts ( Scheme 1.22 ). 50 The products ( 66 and 67 ) from C –H insertion were found in high yields and selectivities. Intereatingly, insertion into the 3-position to form b-lactone derivatives was preferred by the S- configured catalysts whereas insertion to the equatorial C 2-H bond took place preferentially by the R-configured catalysts. However, present of substituents or functional groups at the 5/6- position were found to prevent C –H insertion at the 4-position.

hn O O O OH R , R = H, Me O 1 2 O R1 R1 R2 R2 63 64

Scheme 1.21 Intramolecular allylic hydrogen abstraction by triplet benzophenone.

Cholest-5-en-3b-yl diazoacetate ( 65) was designed and prepared at a good yield (68%) from cholesterol ( 13 ) by diketene condensation following by diazo transfer and deacetylation. The diazoacetate derivative of cholesterol, 65 was then subjected to standard conditions for diazo decomposition with a series of chiral dirhodium(II) carboxamidate catalysts (Scheme 1.22). 50

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Rh L O 2 4 DCM, reflux, 5h O + O O N 66 67 2 65 COOMe O 5R H N O Rh2(5R-MEPY)4: 81% (94:6) Rh Rh (5S-MEPY) : 74% (33:67) Rh 2 4

Scheme 1.22 Rhodium-catalyzed decomposition of cholesteryl diazoacetate (65 ).

A regioselective hydroxylation at the tertiary C 25 –H bond in cholesterol derivatives such as in 5 a,6 b-dibromocholestan-3b-yl acetate ( 68) was found to occur by ethyl(trifluoromethyl) dioxirane (ETDO, 71 ), generated in situ from the treatment of 1,1,1-trifluorobutan-2-one ( 69) with potassium peroxymonosulfate ( 70 ) ( Scheme 1.23). 51 Thus following the key-step of selective hydroxylation (by forming 72 ), concise syntheses of naturally occurring alkyl chain- based oxysterols, viz. , 25-hydroxycholesterol ( 73) and 24-oxocholesterol ( 74 ) were achieved starting from cholesterol (13 ).

OH ETDO AcO Br AcO HO Br Br Br 13 68 72

O

70 69 71 OH

HO HO 74 73

Scheme 1.23 Regioselective oxygenation in cholesterol side-chain.

1.3.1.2 Recent advances in the b-sitosterol chemistry The sterols synthesized in plants are termed as phytosterols, of which the most prevalent are b- sitosterol (14 ) and campesterol ( 75 ) (Figure 1.3) (comprising 95% of total plant sterols).

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Recently, in the direction to lower cholesterol levels, incorporation of phytosterol esters into foodstuffs has increased which in turn has increased the interest in phytosterols (and b- sitosterol!). 52,53 Dietary intake of phytosterols is projected to increase in Western countries as consumers respond to health messages to increase vegetable oil consumption at the expense of animal fats. 54 b-Sitosterol is structurally 24 a-ethyl cholesterol, and hence it may undergo similar oxidation processes and products to cholesterol. The adverse effects of cholesterol oxidation products is well documented which also include the harmful role in the development of atherosclerosis. 55-57 The consumption of dietary phytosterols in increased quantities has lead to the possibility of increased levels of phytosterol oxides in the blood. It is assumed, generally, that the absorption of the phytosterols as well as the phytosterol oxidation products (POPs) occurs poorly from the diet, however, the oxidation products have been isolated recently from the plasma of healthy human subjects. 58 Discussion continues as to whether these POPs are absorbed as such or transformed into, in vivo, from the parent phytosterols. 59 The mutation of the ATP-binding cassette transporter G5 (ABCG5) or ABCG8 gene causes an autosomal recessive disease termed as phytosterolemia (or, b-sitosterolemia). 60 ABCG5/ ABCG8 proteins are important to regulate the intestinal absorption as well as the biliary excretion of phytosterols. And, a defect in the protein leads to increased absorption and decreased excretion of the phytosterols, derived from the diet. As a result, an accumulation of the phytosterols occurs in serum and tissues except for the brain, thereby inducing tendon and tuberous xanthomas and premature coronary atherosclerosis. 61 Hence, for the diagnosis of phytosterolemia it is required to determine the serum phytosterols, such as b-sitosterol and 62 campesterol. Recently, British Journal of Nutrition has found it important to conclude that, “the development of accurate and sensitive methods for qualitative and quantitative analyses of oxysterols and oxyphytosterols in food, dietary products and biological samples has become a new challenge.” 63 Synthesis of multi-gram quantities of pure b-sitosterol ( 14 ) along with its oxygenated derivatives (76-80 ) are reported in the literature ( Scheme 1.24).64,65

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a c d

HO AcO HO O HO OH 76 77 14 78a, 78b; 7a: 7b= 3: 97

f e b

Conditions: (a) Ac2O/ py (b) i. CuSO4/ KMnO4, tBuOH, H2O; ii. K2CO3, MeOH o (c) i. CrO3, dimethylpyrazole, DCM, -20 C; ii. K2CO3, MeOH (d) CeCl .7H O, NaBH HO HO 3 2 4 HO O (e) m-CPBA, DCM, 0oC OH o 80 (f) i. HCOOH, 80 C, 10 min.; ii.H2O2; iii.MeOH, NaOH 79 for b: 5a,6a: 5b,6 b= 7: 93 for e: 5a,6a: 5b,6 b= 6: 1 Scheme 1.24 Synthetic routes to some oxygenated b-sitosterol (14 ) derivatives.

High-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been reported, by Hakamata et al., to use successfully in the determination of the phytosterols such as b-sitosterol ( 14 ), stigmasterol ( 15 ), campesterol ( 75), and brassicasterol ( 81 ) ( Figure 1.3). 66

H H

H H H H HO HO 75 81

H H HO HO H 83 82

Figure 1.3 Some natural phytosterols (75 , 81-83 ). People have used NMR spectroscopy also as an useful tool to measure the solubility limit of a number of biologically relevant sterols in electroformed giant unilamellar vesicle membranes containing phosphatidylcholine (PC) lipids in different sterol ratios. The findings conclude the solubility limits of cholesterol ( 13 ), lanosterol ( 82), ergosterol ( 83), (Figure 1.3) stigmasterol

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(15 ), and b-sitosterol ( 14 ) to be 65 –70 mol%, ~35 mol%, 30 –35 mol%, 20 –25 mol%, and ~40 mol%, respectively. Clearly, cholesterol possessed, in the experimental model, highest solubility in comparison to the similar sterols viz. , stigmasterol and b-sitosterol, which differ from cholesterol only in their alkyl tails. Thus, subtle differences in alkyl chain structure could strongly affect sterol solubility. 67 In accordance with the experimental results, Mancera et al. showed, with a series of molecular dynamics simulations, the effect of b-sitosterol ( 14 ) on some important bio- membranes containing DMSO. As DMSO is one of the most widely used and important cryoprotective agents, the group considered it valuable to determine the nature of the interactions of DMSO with cell membranes at the molecular level and they found that b-sitosterol ( 14 ) was more effective, at ordering the bilayer, than stigmasterol ( 15 ). 68 In the conversion of b-sitosterol ( 14 ) to cholesterol ( 13) in Silkworm, Ikekawa et al. proposed fucosterol-24,28-epoxide ( 85) to be a probable intermediate. Through some valuable experiments and with the comparison of corresponding in vivo findings, the group proposed that b-sitosterol (14 ) was first transformed into fucosterol ( 84) followed by epoxidation to form 85. This Fucosterol-24,28-epoxide ( 85) was then dealkylated to furnish desmosterol ( 86) which produced finally cholesterol ( 13 ) (Scheme 1.25). 69

O

H H H

H H H H H H HO HO HO 14 84 85

H H

H H H H HO HO 13 86

Scheme 1.25 Proposed route for the conversion of b-sitosterol (14 ) to cholesterol (13 ) in Silkworm.

Moriarty and Albinescu synthesized (overall yield 1.2%) 1 a-Hydroxyvitamin D5 ( 92, Scheme 1.26), a promising chemopreventive agent for breast cancer which is under development as a drug, by utilizing 7-dehydrositosteryl acetate ( 90 ) as the precursor. They started with

20 stigmasterol ( 15 ) to result 89 which furnished finally the potential compound 92 by following the photochemical step (from 89 to 90, condition f, Scheme 1.26) as the key step for the B-ring opening. 70

H H H b c H H H H H H RO OMe OMe 15 R= H 87 88 R= Ts d

H

f H H e H H AcO AcO 89 76

RO 90

OH HO OH 91 92 Scheme 1.26 Synthesis of promising chemopreventive agent 1 a-Hydroxyvitamin D5 (92 ) starting from b-sitosterol (14 ). Conditions: (a) TsCl, DMAP, py, CH 2Cl 2, RT; (b) CH 3COOK, MeOH, reflux; (c) H 2,

10%, Pd/C, CH 3COOEt, RT; (d) (OAc) 2Zn, CH 3COOH, reflux; (e) i. dibromantin, hexane, NaHCO 3, o o reflux; ii. LiBr, acetone, toluene, 0 C; iii. PhSH, Et 3N, RT; iv. m-CPBA, CH 3COOH, 0 C; v. PhMe, o t o o Et 3N, 70 C;(f) i. hn, BuOMe, ethyl 4-(dimethylamino)benzoate, -20 C to 0 C, ii. uranium glass filter, 9- o o acetylanthracene, -20 C to 0 C; iii. CH 3COOH, reflux.

β-Sitosterol (14 ) has recently been utilized to synthesize a novel third-generation designer amphiphile/surfactant, named as “Nok” (i.e., SPGS -550-M; structurally, β -sitosterol methoxypolyethyleneglycol succinate, 93, Figure 1.4) by Lipshutz and Klumphu. In water, it

21 readily forms nanomicelles which serve as nanoreactors. This comparatively far less costly material has been evaluated as highly potential catalyst for a number of transition-metal- catalyzed reactions run under micellar conditions. Two other amphiphiles (named as CPGS-750- M, 94 and PSS, 95; Figure 1.4) were also prepared and studied. The novel surfactant “Nok” has also been comertialized recently (Aldrich catalog number 776033). 71

O O O O O O O O O O O O H H H n H H n H H 4 n O O O H H H H H H

(n= ca. 16) (n= ca. 13) 94 95 (n= ca. 14) 93 Figure 1.4 b-Sitosterol and cholesterol-based amphiphiles.

Recently, Fujimoto et al. proposed b-sitosterol ( 14 ) as the biosynthetic precursor for other phytosteroids amarasterone A ( 96) and cyasterone ( 97). The C-24 configuration of the recently isolated phytosteroids was also found identical with b-sitosterol (14 ) (Scheme 1.27). 72

OH OH OH OH H H OH O 24 R s H O H HO HO H H H OH H OH HO HO HO H H 14 O 96 O 97 Scheme 1.27 Configurational assignment of amarasterone A (96 ) and cyasterone (97 ) based on b- sitosterol (14 ).

b-Sitosterol (14 ) together with g-oryzanol component (actually a fraction containing ferulate esters of triterpene alcohols and plant sterols 73 , and ca 80% of this is constituted by cycloartenyl ferulate ( 98a), 24-methylenecycloartanyl ferulate ( 98b) and campesteryl ferulate ( 98c)74 ; Figure 1.5) were found to be self-assembled to produce organogel systems. The influence of the type of oil-phase on the gelation was also studied. A decreasing polarity of the oil was found to promote the self-assembly leading to formation of nano-tubules at higher temperatures and at lower structurant concentrations. 75

22

O O O O O O HO HO 98a 98b HO 98c OMe OMe OMe

Figure 1.5 Major constituents of g-oryzanol.

(6 E)-Hydroximino-24-ethylcholest-4-en-3-one ( 101 ), a natural steroidal oxime isolated from Cinachyrella alloclada and C. apion , was synthesized from b-sitosterol. 76,77 Synthesis of a number of oxyphytosterols (99-103) was also reported from stigmasterol (15 ) (Scheme 1.28). 78

H PCC NH2OH.HCl H H + CH2Cl2 CH3COONa HO O HON O 14 O O NOH 99 100 101

NaBH4/CH3OH Jones’ CoCl2·6H2O reagent, acetone H NH2OH·HCl CH COONa. 3H O H H 3 2 HO HO EtOH HO 15 O 102 NOH 103 Scheme 1.28 Synthesis of natural b-sitosteryl mono-oxime derivatives (100-103 ).

Biotransformation of b-sitosterol by Mycobacterium sp. was found to result 4-stigmasten-3- one ( 104), 26-hydroxy-4-stigmasten-3-one ( 105) and 3-oxo-4-stigmasten-26-oic acid ( 106) (Scheme 1.29). 79

23

R H H Mycobacterium sp. H H H H HO O 14 104: R= CH3 105: R= CH2OH 106: R= COOH

Scheme 1.29 Biotransformation of b-sitosterol (14 ) by Mycobacterium sp.

Very recently, Schottenol ( 109), a phytosterol present in argan oil and in cactus pear seed oil, and which revealed reduced mitochondrial activity on 158N murine oligodendrocytes (50%) and C6 rat glioma cells (10 –20%), was synthesized from b-sitosterol (14 ) (Scheme 1.30 ). 80

H H H a b H H H H H H HO HO O HO O H 14 77 107

Conditions: (a) CuCl(OH)-TMEDA (5 mol%), tBuOOH (5M c in decane), CH2Cl2-MeOH (4: 1), RT, 40h; (b) 10% Pd/C, ammonium formate, CH3COOEt- o H MeOH (1:1), 70 C, 1h; (c) TsNHNH2, MeOH, o o 75 C, 3h; (d) LiH, toluene-THF (1:1), 110 C, 5h. d H H H H HO HO NNHTs H H 109 108 Scheme 1.30 Synthesis of schottenol (109) starting from b-sitosterol (14 ).

I.4 Pentacyclic Triterpenoids When five carbocyclic rings are fused one-by-one to produce some 6-6-6-6-6, 6-6-6-6-5 or 6-6- 7-6-6 type of structure along with some other functional groups (not must) and few –Me groups, to have altogether 30 carbon atoms obeying isoprene rule is known as pentacyclic triterpenoid (PT, Figure 1.6). 81 These are the secondary metabolites which are widely distributed in plants and are traditionally used as medicines. 82 Natural PTs are found to possess unique biological activities. A number of important pharmacological and related mechanistic studies including

24 antitumor, antiviral, anti-inflammatory, antimicrobial, antidiabetic, antiparasitic, cardio-hepato- and gastro-protective, analgesic and wound-healing effects have been carried out. Several PTs are now being marketed as therapeutic agents, and some synthetic PT derivatives are now under clinical trials. 83 The large number of continuous research works in the field, with the evaluation of their practical utilization, implies the increasing interest of the chemical as well as pharmacological research of the pentacyclic triterpenoids.

I.4.1 Different groups of pentacyclic triterpenoids Pentacyclic triterpenoids are an widespread group of compounds which altogether, depending on their basic structural skeletons, can be divided into six broad classes- 1) friedelane ( 6), 2) lupane (110), 3) ursane ( 111), 4) oleanane ( 112), 5) serratane ( 113 ) and 6) Ψ-taraxastane ( 114 ) ( Figure 1.6). Very brief discussion on these classes of compounds are provided below.

29 30 29 30 20 30 29 19 20 21 21 20 21 27 19 19 12 18 12 22 12 22 13 13 22 11 11 18 11 18 25 13 17 25 26 17 25 26 28 17 1 9 14 28 1 14 1 14 28 2 16 9 16 9 16 2 10 8 15 2 10 8 15 10 8 15 5 5 3 26 4 27 5 27 4 7 3 7 3 4 7 6 6 6 24 23 24 24 23 6 110 23 111 30 20 21 30 29 22 30 19 18 29 29 17 19 21 20 21 20 12 19 12 16 12 11 13 28 11 13 22 11 13 22 25 18 25 26 18 26 17 14 15 25 26 17 1 1 1 28 14 28 14 9 16 9 16 2 2 8 27 9 10 8 15 10 2 10 8 15 5 27 5 3 3 4 4 5 27 4 7 7 3 7 6 6 6 23 24 23 24 23 24 112 113 114

Figure 1.6 Structural skeletons with carbon-numbering of various pentacyclic triterpenoids.

I.4.1.1 Friedelane triterpenoids Friedelane triterpenoids possess [6-6-6-6-6]-fused pentacyclic basic skeleton with eight -Me groups situated at C(4), C(5), C(9), C(13), C(14), C(17), and C(20) (a geminal-dimethyl), respectively. The most fundamental compounds of the class are friedelin (115 ) and cerin ( 116 )

25

(Figure 1.6) which are obtained, at major amount, from the cork smoker washed solids. A large number of friedelanes with different functional/ active groups 84 and their potential biological activities have been reported. 85 Recently, Zhan et al. have reviewed the family of friedelane triterpenoids beautifully by giving emphasis on the fascinating structure and their interesting bioactivities. This review essentially concludes friedelane triterpenoids as ‘prime candidates for developing new drugs.’86 Some very active compounds of the group, such as celastrol ( 117 ) and correolide ( 118 ), are being used in pharmacy or in further drug development ( Figure 1.7) .86 As the author worked with friedelane triterpenoids giving emphasis on their transformative reactions, a brief review on their bio-availability as well as their transformative reactions are produced separately in Chapter IV which describes the syntheses of new friedelane triterpenoids focusing on A-ring modifications including 2-homo derivatives.

H H H H H H HO

O O

115 116 COOH O COOMe H HO O O OAc OAc O OAc HO AcO OAc 117 118

Figure 1.7 Friedelin ( 115), cerin ( 116 ), celastrol ( 117 ) and correolide ( 118 ).

I.4.1.2 Lupane triterpenoids The pentacyclic triterpenoids having [6-6-6-6-5]-fused carbocyclic rings with six -Me groups situated as C(4) (geminal-dimethyl), C(8), C(10), C(14), C(17), and an isopropyl group at C(20) are known as lupane triterpenoids.

26

Betulinic acid (BA, 119 ), a naturally occurring plant-derived pentacyclic triterpenoid found in many fruits and vegetables 87-88 is the most important member of this group ( Figure 1.8). It exhibits a wide spectrum of pharmacological and biochemical activities including anti- inflammatory and anticancer, 87-89 anti-HIV 90-93 activities, with low toxicity to normal cells. 94 The natural compound BA shows potential anticancer effects through the activation of mitochondrial pathway of apoptosis in cancer cells. To enhance its antitumor effects through combination protocols, in chemo- or radiotherapy, or in the death receptor ligand TRAIL, BA was found to result as a potential additive. 95 BA is a promisingly new experimental anticancer agent for the treatment of human cancer due to its relative selectivity towards the cytotoxicity against malignant cells compared to normal cells. 96 Currently, BA is in second phase of clinical trials for dysplastic nervus treatment. 97 Again, a number of betulinic acid derivatives were prepared and evaluated for their bio-activities. 98-100 Among the other important lupane triterpenoids, lupeol (120 ) (Figure 1.8) and its derivatves were found to have a broad spectrum of biological activities including but not limited to, antiurolithic, 101 antioxidative, 102 anti-inflammatory, 103 hepatoprotective, 104 and antilipidemic activities. 105,106

I.4.1.3 Ursane and oleanane triterpenoids Both the ursane and oleanane group of pentacyclic triterpenoids contain a [6-6-6-6-6]-fused carbocyclic skeleton along with eight methyl groups. Thus, these are structurally isomeric. In ursane PTs, the methyl groups are situated at C(4) (geminal-dimethyl), C(8), C(10), C(14), C(17), C(20) and C(21) whereas in the oleanane PTs, no methyl group is there at C(21) and geminal dimethyls are present at C(20). The most fundamental compounds of the group are ursolic acid (UA, 121 ) and oleanolic acid (OA, 122 ) ( Figure 1.8). Oleanolic acid, ursolic acid, and their derivatives along with some other PTs of the group are also found to possess useful bioactivities such as anti-inflammatory, 107-110 diuretic and anti-tumor, 111-112 hepatoprotective 113- 114 and anti-HIV 115-116 activities. Ursolic acid itself is cytotoxic against A-549, L-1210 and KB tumor cells. 117 There are useful reviews highlighting also the broad spectrum of biological activities including phytotoxic 118 and photosynthesis inhibition activities 89 and low toxicities of oleanane and ursane triterpenoids. 119-123

27

COOH COOH COOH

HO HO HO HO 122 119 120 121 Figure 1.8 Betulinic acid ( 119 ), lupeol ( 120 ), ursolic acid ( 121) and oleanolic acid ( 122).

I.4.1.4 Serratane and Ψ -taraxastane triterpenoids A [6-6-7-6-6]-fused carbocyclic skeleton having seven -Me groups at C(4) (geminal-dimethyl), C(8), C(10), C(18), and C(22) (geminal-dimethyl) to result a pentacyclic triterpenoid is known as serratane triterpenoids. On the other hand, the ursane skeletal-based groups of PTs where the stereochemistry of the methyl groups at C-20 and C-21 are just the reverse to that of ursane itself, is known as Ψ -taraxastane triterpenoids. However, these PTs are somehow limited in their reports so far. Few of the members of these groups are phlegmanol A ( 123 ), 124 faradiol, heliantriol B o, heliantriol C, arnidiol ( 124 ) and many of them are found to be cytotoxic ( Figure 1.9). 125

OH

O OH O HO HO 123 124 OH

Figure 1.9 Phlegmanol A ( 123 ) and arnidiol ( 124 ).

1.4.2 Recent advances in the transformative reactions on pentacyclic triterpenoids Though this is a very broad and elaborate subject, the present title is an effort to summarize, in very brief, the different transformative scopes on some major and selective pentacyclic triterpenoids. Again, as all of these are associated with some useful/ probable biological importance, the bioactivities are also mentioned. Of note, most of the derivatization processes are consist of a number of steps as well as a number of different transformative protocols. Thus, in

28 most of the instances the major derivatization step(s) or the skeletal presentation of the resultant compound(s) is provided below to have a very brief overview. Betulinic acid (BA), a natural pentacyclic triterpene exhibits a variety of biological activities including antitumor properties. 126 Betulinic acid was also found to show selective cytotoxicity on tumor cell lines, but not on normal cells. 127 BA (3 b-hydroxy-lup-20(29)-en-28-oic acid) is widely distributed in the plant kingdom throughout the world. 124 And these two factors, easy availability and promising bioactivity render the compound very much suitable for a number of chemical transformative reactions. The cooperative effect of betulinic acid for the sensitization of anticancer drug-induced apoptosis was also studied by Fulda et al. 128 Aiken et al. reviewed BA derivatives as HIV-1 antivirals 129 and Fulda reviewed the anticancer potential of betulinic acid. 130 A number of modified betulinic acid derivatives were achived which include cytotoxic A- seco derivatives, 131 C28-modified HIV-2 inhibitors, 132 C28-modified alphavirus replication inhibitors, 133 C3-modified BA derivatives for anti-AIDS agents. 100 A-ring modifications including the synthesis of betulin amine dimer (126) (Figure 1.10), 134 C3-neoglycosylation (128)135 are reported (Figure 1.11). Synthetic routes for C28,C30-disubstituted derivatives, C3,C28-disubstituted 3 b-amino derivatives and C3,C28-disubstituted C28-piperidine derivatives were found useful to result ultimately a number of potent anti-HIV betulinic acid triterpenoids. 136 After having a library of these derivatives, SAR were revealed to be used efficiently for further designed transformations. 100 Simple modifications at C3, C20, C28 and C30 in betulinic acid lead the derivatives towards potent Topoisomerase inhibitors. 137 Some natural C3 modified BA derivatives were also found to show DNA topoisimerase II inhibitory activity.138 C3 and C28- modified BA derivatives showed anticancer activities 139 as well as activation and inhibition of the proteasome. 140C3, C20 and C29- Modified derivatives of BA were synthesized and were found to be anti-diabetec. 141 Bidesmosidic betulin and betulinic acid saponins were also synthesized and were evaluated for their cytotoxicity. 142 C28 and/ or C3-Modified derivatives were found active as antitumor agents. 143, 144 A number of heterocyclic ring-fused betulinic acid derivatives were also synthesized and evaluated for their biological activities. 145 

29

O H H H NH OH H

H

HO H H 125 AcO H

2 126

Figure 1.10 Betulin ( 125) and its amine dimer ( 126).

H H H H COOH COOH O O H O H (HO) N X n X H H 119; X= OH 128; X= O, NH 127; X= NH2 Figure 1.11 C3-Neoglycosylation of betulinic acid and its 3 b-amino derivative.

Mayaux et al. reported a betulinic acid derivative RPR103611 ( 129 , Figure 1.11) which was found to be a fusion inhibitor active at a submicromolar level. 146 Results also showed that epimeric IC9564 (130, Figure 1.12) inhibits HIV-1 at the membrane fusion step 145 and further analysis showed that HIV-1 gp120 was the molecular target for IC9564. In analogy, Lee and his group discovered more potent anti-fusion derivatives of betulinic acid where the modifications were carried out in the isopropylene and C-28 side chain. 99

30

O H H H COOH N N H OH O H X 129; RPR103611 H

O H H H COOH N N H OH O H 130; IC9564 X H

Figure 1.12 RPR103611 ( 129 ) and IC9564 ( 130).

Betulin (125) was utilized to furnish a number of 24-nortriterpene derivatives. The key steps in the transformation were a Sua´rez cleavage of the A-ring followed by SmI 2-mediated pinacol- type coupling to reclose the A-ring. The derivatives were then screened for their anticancer activities. 147 (Scheme 1.31 ) Bevirimat ( 135, BVM, also known as PA-457, DSB, and MPC- 4326, Figure 1.13) is an anti-HIV agent that blocks HIV-1 replication by interfering with HIV-1 Gag-SP1 processing at a late stage of viral maturation. However, clinical trials of 135 have revealed a high baseline drug resistance that is attributed to naturally occurring polymorphisms in HIV-1 Gag. 129,148-155 Modification of the structure of 135 was thus essential to overcome the resistance. Improved activity of the compound was found by attaching a side chain at the C28 position of 135.156 In analogy, C28-modified derivative, compound 136 was found to be at least 20-fold more potent than 135 against the replication of NL4-3/V370A. Later on though it was found to be inactive against BVM-R viruses, its improved anti-HIV-1 activity against NL4-3 strain suggested that C28 modifications could impact drug-target interaction. Thus, a library of compounds of 135 analogues were synthesized systematically focusing C28 modifications. 157

31

H H H H

CH2OH CH2OP Saurez OHC OHC cleavage H H [O] HO H H H O 125 131 132 Pinacol coupling

H H H H

CH2OH CH2OH HO H H O HO H HO 134 133 Scheme 1.31 Key step for the synthesis of 24-nortriterpene derivatives from betulin (125 ).

H H H H R R

O O O O H H HOOC O HOOC O H H H 135, Bevirimat; R= OH N / (CH2)8 CONH2 R= -H2N-(CH2)n-COR , n= 6-9 / 136; R= R= -H2N-(CH2)n-NHR , n= 7-9 O N CO2Me H H

Figure 1.13 Bevirimat ( 135 ) and C28-modified derivative 136.

Very recently, a fluorescent cancer cell detector as well as potent anticancer agent has been synthesized based on betulinic acid (119). The compound 137 (Figure 1.14) is actually an example and use of isatins as betulinic acid conjugate for selective detection of cancer and subsequent killing of cancer cells via apoptosis. 158

32

OH H H NH O

O H O HO H N MeO

137

Figure 1.14 A fluorescent cancer cell detector 137.

Recently, self-assembled targeted folate-conjugated eight-arm-polyethylene glycol –betulinic acid nanoparticles 138 were synthesized for the co-delivery of anticancer drugs. 159 (Figure 1.15)

H H OH

O COOH O O O H H N O O O N PEG O H H 6-7 N O 1-2 N N H 138 H2N N N H

Figure 1.15 Compound 138 for the co-delivery of anticancer drugs.

Biotransformation of betulinic acid (119) and its derivatives were also achieved. Microbial transformations of betulinic and betulonic acids are reported. 160, 161 Liu et al. optimized the biotransformation process of betulin (125) into betulinic acid (119) catalysed by fungus Armillaria luteo-virens Sacc ZJUQH100-6. 162 Following copper-catalyzed Mannich reactions, Csuk et al. synthesized a library of C3- hydroxypropargylamine derivatives of betulinic acid (141, as the general skeleton), some of which showed significant cytotoxicity. 163 (Scheme 1.32)

33

H H H H OR OR

O HC CMgBr O H H THF, 25oC HO O 138; R= H H H 139; R= CH3 140

H H H H OR OR

O Secondary amine (R2NH) O H H HO formalin, CuI (cat.) o H DMSO, 40 c H 140 R1= H/ CH3 141 R 2 Scheme 1.32 C3- Hydroxypropargylamine derivatives of betulinic acid.

The semisynthesis of piperazine derivatives 142-143 of betulinic acid are described recently and these were evaluated biologically for their antimalarial activity, cytotoxicity and the mechanism of their action. 164 (Figure 1.16)

NH N 2 H H H N N

O H O RO H 142; X; R= O 143; Y; R=

Figure 1.16 Piperazine derivatives of betulinic acid 142-143.

Synthetic route for the synthesis of betulinic acid ( 119) from betulin ( 125 ) was developed by utilizing the selective oxidation of the primary alcohol function of betulin ( 125 ) without affecting

34 the secondary hydroxyl group. The corresponding aldehyde, betulinal ( 144 ) was also achieved exclusively by applying shorter reaction times and lower temperatures. 165 (Scheme 1.33)

H H H H OH R

O TEMPO, NaCl2O, NaClO H H 86- 92% HO HO H H 119 125; R= COOH 144; R= CHO

Scheme 1.33 Synthesis of betulin ( 125) and betulinal ( 144).

C-20 Modified betulinic acid derivatives were prepared and were evaluated for structure – activity relationship study which revealed that the C-20 position was found to be sensitive to the size and the electron density of the substituents in retaining the cytotoxicity of betulinic acid and was found to be undesirable position for derivatization. 166 Betulinic acid and its derivatives (modified at C3 and C20), as anticipated earlier, were found to possess anti-angiogenic effects and cytotoxic activity of 3-O-acyl/3-hydrazine/2- bromo/20,29-dibromo betulinic acid derivatives were also studied by Mukherjee et al. 167, 168 Some other A-ring modified betulinic acid derivatives were synthesized and evaluated for their cytotoxic activities. 169 3b-O-Phthalic esters of betulinic acid and its esters were also synthesized. The evaluation of cytotoxicity of the prepared compounds revealed that hemiphthalic esters possess better cytotoxicity in comparison to the starting materials. 170 Betulinic acid derivatives with a side chain at C-3 were found to inhibit HIV-1 maturation. On the other hand, BA derivatives with a side chain at C-28 were active to block HIV-1 entry. In order to combine the anti-maturation and anti-entry activities in a single molecule, bi-functional BA derivatives (147 ) containing side chains at C-3 and C-28 were synthesized by Lee and his coworkers. Screening resulted compound ([[N-[3b-O-(30,30-dimethylsuccinyl)-lup-20(29)-en- 28-oyl]-7-aminoheptyl]-carbamoyl]methane, 148) to be the most potent which inhibited HIV-1 at an EC 50 of 0.0026 mM and was at least 20 times more potent than either the anti-maturation

35 lead compound DSB (145 ) or the anti-entry lead compound IC9564 (146). This bi-functional BA derivative was active against both HIV entry and maturation. 171 (Figure 1.17)

H H H H OH O O O O HN COOH H H (CH2)7 N H O HO OH HOOC H H IC9564; 146 DSB; 145

anti-HIV maturation anti-HIV fusion

H H O O O HN COOH H (CH2)7 N H HOOC O OH H

Bifunctional derivative, 147

H H O H O HN N H (CH2)7 HOOC O O H 148 Figure 1.17 BA derivatives 145-148 .

Natural betulinic acid saponins (149-150) which were isolated from Pulsatilla koreana and Schefflera rotundifolia , were synthesized using a stepwise glycosidation approach involving eight linear steps. 172 (Figure 1.18) Sulfur derivatives of lupane and oleanane triterpenoids were also achieved 173 by using the Lawesson’s reagent ( 151), 174 (Figure 1.19) a widely used thionation reagent. 175

36

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

O OH O HO O OH HO H H OH O O O O HO H HO H O O HO O HO O HO 149 HO 150 HO HO OH OH Figure 1.18 Natural betulinic acid saponins ( 149-150).

S MeO P S SP OMe S 151

Figure 1.19 Lawesson’s reagent (151 ).

Nitrogen-containing derivatives of betulin and betulinic acid, such as amine derivatives, 169,176,177 oxime derivatives, 169,170,176 amino acid conjugates, 178 amide derivatives, 179- 180 hydrazine 181 and hydrazone derivatives, 170-171,177,181 imidazolide derivatives, 182 and other N- heterocyclic derivatives 183-188 have been reported to possess antiproliferative effect against tumour cell lines. Imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid were also synthesized and were evaluated for their cytotoxic activities. 189 To achieve better water solubility without the loss of the observed earlier anticancer properties, betulin and betulinic acid were undergone simple transformation to mono- and disubstituted esters of L-amino acids (153-156).190 (Scheme 1.34) Alphitolic acid ( 158), a naturally occurring lupane type pentacyclic triterpene with various pharmacological properties, was synthesized in 10 steps with an overall yield of 19% starting from the readily available diketone 157 . Seven other isomeric 2,3-dihydroxy lupanes 159-165 were also synthesized. The synthesized triterpenes 158-165 were also evaluated for their bio- activities. 191 (Scheme 1.35 )

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R O R H H 2 H H 2 O + O N O NH3 C H l- O HCl O H dioxane H R O R1O 1 H H 153 154 O H O N CDI, THF Betulin OH Reflux O R 24-48h Betulinic acid H H H H 152 OH O H O HCl O O H O H H - + O N dioxane Cl H3N O H O H O R1 R1 155 156

R and R2 = side chain of amino acid R1 = H or amino acid for 1 and 2; and side chain of amino acid for 3 and 4. Scheme 1.34 Esters of L-amino acids 153-156.

158; R1= a-OH, R2= b-OH, X= O 159; R1= a-OH, R2= b-OH, X= H2 H H H H 160; R1= a-OH, R2= a-OH, X= O 161; R = a-OH, R = a-OH, X= H OTr OH 1 2 2 162; R1= b-OH, R2= a-OH, X= O HO R 163; R = b-OH, R = a-OH, X= H 1 X 1 2 2 H H 164; R1= b-OH, R2= b-OH, X= O 165; R1= b-OH, R2= b-OH, X= H2 O R2 H 157 H Scheme 1.35 Synthesis of lupane derivatives (158-165).

Stereoselective synthesis of 28-O-b-D-glucuronide betulinic acid ( 166) was carried out under phase-transfer conditions. The methodology was also applicable for the preparation of the major acyl glucuronide metabolite of bevirimat ( 167) or other carboxylic acid-containing drug metabolites.192 (Figure 1.20) C3- and C28-Modified carbamate derivatives of betulinic acid and betulin possessing selective cytotoxic activity were also achieved by Kommera et al. 193 Semisynthesis of betulinic acid derivatives RS01 ( 168), RS02 ( 169) and RS03 ( 170) were carried out, which showed 18-45 times improved cytotoxic activity against HepG2 cells. 194 (Scheme 1.36 )



38

H H

2 CO2R

H R1O H OH 1 2 HO 166; R = H, R = O OH CO2H O OH 167; R1= R2= HO O OH CO2H, CO2H

Figure 1.20 28-O-b-D-glucuronide betulinic acid and bevirimat ( 166 and 167).

H H H H H H O O OH

N N O BA H and H and H 119 O N O N N O H H N H 168; RS01 169; RS02 170; RS03 Scheme 1.36 Semisynthesis of betulinic acid derivatives RS01 ( 168), RS02 ( 169) and RS03 ( 170).

Bevirimat, a C3-modified BA derivative exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative drug binding site. Towards the improvement of the drug, a library of C28-modified derivatives were synthesized and among them, comp 171, 172 and 173 were found to improve markedly the microsomal stability compared to A43D, a C28-modified HIV-1 entry inhibitors. 195 (Figure 1.21)

H H H H H H O O O O H HN N HN R R HN (CH ) (CH ) 1 2 (CH ) H 2 7 H 2 n H 2 8 R 1 HO O HO HO H H H 171 172; n= 6-9; R1-R2 = -C(=O)-AA esters 173; R1= -Gln-OMe Figure 1.21 C28-modified derivatives 171-173.

39

Chemical transformation of betulinic acid, through concise 1,2,3-triazole synthesis via click chemistry approach at C-3 position in ring A, was achieved to result 3-O-propargylated betulinic acid and its 1,2,3-triazoles which were found to be potential apoptotic agents. 196 A number of DMAP derivatives of BA (174-180) were synthesized and the compounds were active for promoting cell death through directly targeting mitochondria of breast cancer cells. 197 (Figure 1.22)

H H H H

N+ O O N+ O O N+ H H N+ N+ N+ O O O O Cl- Cl- H - H - 174 2Cl 175 2Cl

H H H H O N+ O N+ + N N+ O O O O H Cl- H N+ N+ 2Cl- O O H H Cl- 176 2Cl- 177

N+

H H + N - O 2Cl H H O N+ O O H N+ N+ O O N+ O H H N+ 2Br- O N+ 178 H - 2Cl 2Br- 179

H H O N+ N+ + O O N H N+ 2Br- O H - 2Br 180 Figure 1.22 DMAP derivatives ( 174-180) of BA.

40

Synthesis of triphenylphosphonium derivatives (181-189) of betulin and betulinic acid, characterized by a covalently linkage of the hydrophobic fragment of the triterpenoid at C2- or C30-position with the triphenylphosphonium moiety via a hydrocarbon bridge, were achieved and found potent against Schistosoma mansoni , in vitro and in vivo .198 (Figure 1.23)



H H H H H H

CO2Me CO2Me CO2Me Ph +P Ph +P Ph +P 3 H 3 H 3 H Br- Br- I- AcO AcO HO H H H 181 182 183

H H H H H H

CO2Me CO2H CO2Bn Ph +P Ph +P Ph +P 3 H 3 H 3 H - Br- I- I HO AcO AcO H H H 184 185 186

+ + Ph3 P Ph3 P I Br- Br- H H H H H H OAc CO2Me CO2M e Ph +P H H 3 H I- AcO AcO AcO H H H 187 188 189

Figure 1.23 Triphenylphosphonium derivatives ( 181-189 ) of betulin and betulinic acid.

Simultaneous C, D and E-ring-fused heterocyclic derivatives (190-193) of betulin (125) were synthesized and found to impair Leishmania braziliensis viability and host –parasite interaction. 199 (Scheme 1.37) Novel ester-triazole-linked triterpenoid –AZT conjugates ( 194-205) were synthesized by transforming the triterpenoid first into the corresponding propargyl esters and subsequently deployed as substrates for a click chemistry-mediated coupling with azidothymidine (AZT) en route and the derivatives were evaluated for their useful cytotoxicity. 200 (Figure 1.24) 3,4-Seco betulinic acid (BA) derivatives ( 206-215 ) were synthesized and some derivatives exhibited enhanced chemopreventive ability. 201 (Figure 1.25)

41

O O N N O N O N N N H OH OH O HCOOH (excess) O reflux, 2.5h O H H H HO HO H O H H H 125 190 191

10% TPAP, O2 PCC, CH2Cl2 4A MA, RT, 6d RT, 3.5h O N O N O N N O N N CHO CHO H H O H HO 193 H 192 Scheme 1.37 C, D and E-Ring-fused heterocyclic derivatives ( 190-193) of betulin .

A series of novel 3-oxo-23-hydroxybetulinic acid derivatives were synthesized and evaluated for their SAR-based antitumor activities. 202 Lupane-type 3 b-O-monodesmosidic saponins with an extended C-28 side chain, were synthesized and structure-activity relationship study of their cytotoxic activities were evaluated. 203 Ionic derivatives ( 216-219 ) of betulinic acid were synthesized and were found to exhibit antiviral activity against herpes simplex virus type-2 (HSV-2), but not HIV-1 reverse transcriptase. 204 (Figure 1.26) Pentacyclic triterpenes ( 220-223) bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety were synthesized and biofunctional evaluation was carried out as their antiproliferative activities. Compounds OA-4 ( 222) and AA-5 ( 221) showed potentiality for further optimization as antitumor drugs. 205 (Figure 1.27) NO-releasing derivatives of betulinic acid (BA) bearing two types of NO-donors [nitrates (225-226) ( Scheme 1.38 ) and furoxans ( 227-229) ( Figure 1.28)] were synthesized and evaluated for their antitumor activity. Both C3 and C28-positions were modified beautifully to result the bioactive derivatives. 206

42

O O

NH NH HO HO N O N O O O

R2 N R1 N N H N O N O N

O O H H RO HO H H 194; R= H 198; R1= Me, R2= H 195; R= CO(CH2)2COOH 199; R1= H, R2= Me 196; R= CO(CH2)3COOH 197; R= COCH2CH(CH3)CH2COOH O

O NH HO NH N O O HO N O O N H N N O N R2 H N H R1 O N O H H O RO H H O 202; R= CO(CH2)2COOH H 203; R= CO(CH2)3COOH 200; R1= R2= H 204; R= H 201; R1, R2= O O

NH HO N O O

N H N HO O N H O H O H 205

Figure 1.24 Novel ester-triazole-linked triterpenoid –AZT conjugates ( 194-205).

43

H H O R1 O R1 H H R O R O 2 H 2 H R H 3 H

206; R1= OH, R2= OMe 210; R = OH, R = OMe, R = OH O 207; R1= OH, R2= OH 1 2 3 208; R1= NH(CH2)7NHCOCH3, R2= OMe HO 211; R = OH, R = OMe, R = 209; R1= NH(CH2)7NHCOCH3, R2= OH 1 2 3 O O

H H O R1 O R H H HO O MeO O H H R AcO 2 H H 214; R = NH(CH ) NHCOCH OH, R = OH 212; R= OH 1 2 7 3 2 O 213; R= NH(CH2)7NHCOCH3 215; R = NH(CH ) NHCOCH , R = HO 1 2 7 3 2 O O

Figure 1.25 3,4-Seco betulinic acid (BA) derivatives ( 206-215).

O O H H H H CH (CH ) CH N - N - 2 2 n 3 O O + H N+ H N O HO O H H HO HO H H 216; Cholinium salt of BA-glycine 217; Benzalkonium salt of BA-glycine

CH2(CH2)nCH3 H OH H N+ O- N+ O- H H O O H H HO HO H H 218; Cholinium betulinate 219; Benzalkonium betulinate Figure 1.26 Ionic derivatives ( 216-219 ) of betulinic acid.

44

COOCH3

O O O H H O HN O N OCH3 H H N HN O O HO H H AA-5 O 220 221 HO

O O H H HN O OCH3 HN O OCH3 H H N N O O H H O 222 O 223 OA-4 UA-4

Figure 1.27 Pentacyclic triterpenes ( 220-223) bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety.

H H H O O O(CH2)nBr

o OH Jones, 0 C OH Br(CH2)nBr O H H H HO O O H H H 224 119 138

AgNO3

H H O(CH2)nONO2 O(CH2)nONO2

O NH2OH.HCl. O H H HON O 226 H H 225

Scheme 1.38 NO-releasing nitrate derivatives of betulinic acid ( 225-226).

N O O O N O H H N R OH N O O SO2P O R h PhO2S O O O O O N O 229 O N O 227, R= H 228; R=CH3

Figure 1.28 NO-releasing furoxan derivatives of betulinic acid (BA) ( 227-229).

45

A family of betulinic acid analogues, carrying a triazole unit at C-3 attached through a linker (230, Figure 1.29) was synthesized by the application of azide-alkyne “click reaction.” The derivatives (follow structural skeleton, Figure 1.29) were evaluated biologically as inducers of apoptosis in human colon carcinoma cells (HT-29).207

OH

O N N N Linker O R 230

Figure 1.29 General skeleton of triazole-attached to BA at C3 through a linker ( 230).

Pyrazine-fused 23-hydroxybetulinic acid derivatives were synthesized by introducing a pyrazine ring between C-2 and C-3 position and further modifications were carried out by substitution of C-28 carboxyl group by ester and amide linkage to enhance the antitumor activity. 208 Cytotoxic 2,2-difluoroderivatives of dihydrobetulinic acid and allobetulin were also synthesized. 209 Polyamine derivatives (231-236 ) of betulinic acid were synthesized and evaluated for their cytotoxic and antimicrobial activities. 210 (Figure 1.30)

O O H N H 231; R= O N 232; R= O N H R1 N H O O O H H N N 233; R= N N HO H H O

H H H N HN N N 234; R= H N 235; R= 236; R= H2N N 2 N H Figure 1.30 Polyamine derivatives 231-236 of betulinic acid.

46

Synthesis of 28 a-homolupane triterpenes and the corresponding saponins containing D- mannose, D-idose, D-arabinose, and L-rhamnose moieties was elaborated and the results indeed were found to correlate the synthesis of various lupane-type triterpene and saponin derivatives as potential anticancer compounds. 211 Lupeol, a lupane series of natural pentacyclic triterpenoid with potential pharmaceutical activities, has been reviewed. 212-213 A concise enantioselective total synthesis of lupeol was accomplished recently by Corey et al. 214 Long-chain fatty acid esters of the pentacyclic triterpenoid were found to exhibit antimalarial activities. 215 A-ring modifications as well as A- ring-fused heterocyclic derivatives of lupeol were synthesized and found to be nitric oxide and pro-inflammatory cytokine inhibitors. 216 Structure modifications of lupeol at the isopropylene moiety was described via allylic oxidation using selenium dioxide and the derivatives were evaluated for their glucose uptake stimulatory effect. 217 A variety of lupeol esters (237) were synthesized by using the appropriate acids and the derivatives were evaluated for their in vivo antihyperglycemic and antidyslipidemic activity. 218 (Scheme 1.39)

H H

H H Appropriate acid O H H DCC, DMAP HO CH Cl , RT, 4h R O H 2 2 H 120 237

Scheme 1.39 Synthesis of lupeol esters ( 237 ).

A convenient synthesis of lupeol (120) from betulin (125) isolated from white birch species, was also established with an overall yield of 50% in 4 steps, which indeed enriched the natural availability of lupeol. 219 (Scheme 1.40 )

 

47

H H H OH OAc OH H H H Ac O, DMAP NaOH, CH3OH H 2 H H quant. 86% HO AcO AcO H H H 125 238 239

PCC, CH2Cl2 quant.

H H

H H CHO N2H2/ H2O, KOH H H diglyme, reflux HO AcO H 58% H 120 240

Scheme 1.40 Synthesis of lupeol ( 120) from betulin ( 125 ).

Lupeol-based libraries of antimalarial agents were synthesized under solid-phase synthesis methodology. Lupeol (120 ) was first anchored to a solid support (Rink amide/ Sieber Amide) through aliphatic dicarboxylic acid moieties. The resulting polymer linked 3 b-O (resin- alkanoyl)-lup-20(29)-ene 241 was then used to generate the key intermediates 3 b-O (resin- alkanoyl)-30-bromo-lup-20(29)-ene 242 and 3 b-O (resin-alkanoyl)-30-amino-lup-20(29)-ene 243 for the generation of a large library based on disubstituted lupeol derivatives (244-248).220 (Scheme 1.41 and Figure 1.31)

Br

O O

N O O O H 242 N n OH H O O O H2N DIC, DMAP HO DMF/ THF (1:1) N n O 120 H 241 n= 2, 3, 4, 6

O O

N n O H 243 Scheme 1.41 Synthesis of lupol-based precursors for antimalarial agents ( 241-243).

48

R' R H N R O R'' N

O O O O O O

H N 2 O H2N O H2N n O n n 246 244 245

H R H R N H N N

O O

O O O O

H N O H N 248 2 n 247 2 n O

Figure 1.31 Disubstituted lupeol derivatives ( 244-248) for antimalarial agents.

Oleanolic acid (OA) and ursolic acid (UA) are ubiquitous pentacyclic triterpene compounds in plants with great interest as anti-inflammatory therapeutics. 115,221-226 Recently Shanmugam et al. have reviewed oleanolic acid and its synthetic derivatives for the prevention and therapy of cancer. 227 A number of oleanolic acid derivatives are synthesized and evaluated for their biological activities. The transformative modifications include glycosyl derivatives, 228 glycosylated diazeniumdiolate-based derivatives, 229 synthesis of heterocyclic derivatives, 230-232 oleanolic acid derivative –chalcone conjugates, 233 furoxan-based nitric oxide (NO) releasing derivatives etc. 234 Besides, a large number of derivatives were synthesized depending on the transformative access to C3, C28 and C12-C13 positions. 235-248 The semi-syntheses of taraxerane triterpenoids by diverse bromination processes and other reactions, from oleanolic acid and some closely related derivatives, was also performed. 249 Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei is also reported. 250 Ursolic acid has also been modified suitably to produce a number of pharmaceutically active derivatives. 247,250-252 Methyl 2-cyano-3,12-dioxoursol-1,9-dien-28-oate (CDDU-methyl ester, 249) was synthesized from commercially available ursolic acid (121) which was based on an oxidative ozonolysis-mediated C-ring enone formation, and the route provided the access to ursolic acid-

49 derived cyano enone analogues (250-252) with C-ring activation. The biological activities were found approximately five-fold less than the corresponding oleanolic acid (122) derivatives (253- 257). 253 (Figure 1.32)

O O O H H H H H OMe N N CF H H H 3 NC NC NC O O O

O O O H H H

CDDU-Methyl Ester (249) CDDU-Methyl Amide (250) CDDU-Trifluoroethyl Amide (251)

O O O H H H N H N OR N H H H NC NC NC O O O

O O O H H H CDDO-Me (253); R= Me CDDU-Imidazolide (252) CDDO (254); R= H CDDO-Ethyl Amide (255)

O O H H H N CF N N H 3 H NC NC O O

O O H H

CDDO-Trifluroethyl Amide (256) CDDO-Imidazolide(257)

Figure 1.32 CDDU- (249-252) and CDDO-(253-257) esters .

C3- and C28-Modified nitric oxide-releasing derivatives (258-259) of oleanolic acid were synthesized and were found to be potential anti-colon cancer agents. 254 (Figure 1.33) Synthesis of oleanolic acid dimmers ( 260-264) were also accomplished and were biologically evaluated as glycogen phosphorylase inhibitors. 255 (Figure 1.34)

50

R' N O- R'' N+ N O OH O N HO HO 2 O H O OH N O O H NO N 2 O H 258

R' N O- R'' N+ N OH O HO HO O O O2N H OH O H R''' O NO2 H 259

Figure 1.33 C3 and C28-Modified nitric oxide-releasing derivatives ( 258-259) of oleanolic acid.

H N O O H H H O O O O O NH O AcO HO HO O NH O H O H H O O O O HO AcO HO 262 260 261

O H H CO2R O N HO N N O N NN

N N N H CO2R N O N H N O O 264; (a, b: R= H/ Bn) 263 HO

Figure 1.34 Oleanolic acid dimers ( 260-264).

51

A series of oleanolic acid saponins (265-273) were synthesized and found to be a- glucosidase and a-amylase inhibitors. 256 (Figure 1.35)

O O HO O HO OH O HO O O OH O O O OH HO HO O O O O O OH OH HO HO OH OH HO OH 265 OH HO OH OH 266 HO OH

O O HO O HO HO O O OH OH O O O O OH HO HO O O O O O OH OH HO HO OH O HO OH OH HO O OH O 267 HO OH OH 268 O OH OH HO OH OH HO OH

O O

O O O O HO O O OH HO OH HO OH OH OH 269 HO OH 270 HO OH

O O O

O O OH O O OH HO OH HO RO O O HO HO OH OH 273; R= Different monosaccharides 271 O 272 O and polysaccharides OH OH OH OH O HO OH OH O HO HOHO Figure 1.35 Oleanolic acid saponins ( 265-273).

52

A series of hybrids (274-277) from O2-(2,4-dinitrophenyl)-diazeniumdiolate and oleanolic acid (OA) were synthesized, and biologically evaluated as novel nitric oxide (NO)-releasing prodrugs. 257 (Figure 1.36)

N O HO N N O H O N HO OH 2 O HO O N O O H OH NO N 2 O H 274

OH N H H HO OH O2N HO HO OH O HO O O O O N O O O H OH H H NO2 N HO O H H 275 276

O2N H HO HO OH R O N O NO N 2 O OH O OH N linker H COO H 277 Figure 1.36 Hybrids ( 274-277) of O2-(2,4-dinitrophenyl)-diazeniumdiolate with oleanolic acid.

H H OH O X O Y .HCl O O O H H O NO HO 2 O H H 278 122 X= L-Val, L-Ile, or L-Ala Y= H, L-Val, or L-Ile Scheme 1.42 Synthesis of NO-donating oleanolic acid derivative ( 278 ).

53

A series of amino acid/ dipeptide diester prodrugs of NO-donating oleanolic acid derivatives (278-280) were synthesized and evaluated biologically as PepT1 targeting antitumor prodrugs. 116 (Scheme 1.42 and Figure 1.37) A partial large-scale synthesis (14 steps, 31% yield) of myriceric acid A ( 283 ), an endothelin receptor antagonist, from oleanolic acid ( 122) was accomplished. 258 (Scheme 1.43 )

H O X O Y O O H O NO 2 O H

279; X= L-Val, Y= L-Val-Boc; 7b, X= L-Val, Y= L-Ala-Boc 280; X= L-Val, Y= L-Val-HCl; 8b, X= L-Val, Y= L-Ala.HCl

Figure 1.37 NO-donating oleanolic acid derivative ( 279-280).

OH OH

O O OH HO O 122 281

OH OH

O O O O O O O OH P(OEt) O O 2

283 OH 282

Scheme 1.43 Synthetic route of myriceric acid A ( 283 ) from oleanolic acid ( 122).

A number of natural oleanolic acid saponins (284) were also synthesized. 259 (Scheme 1.44)

54

OH HO OH O 284a; R = H, R = HO O O OH 1 2 HO OH HO O OH OH OH 284b; R1= O R = O O O HO 2 R O HO HO 2 OH OH OR1 Scheme 1.44 Synthesis of natural oleanolic acid saponins ( 284).

Propylene glycol-linked amino acid/ dipeptide diester prodrugs of oleanolic acid (285) for PepT1-mediated transport were also synthesized. 260 (Scheme 1.45)

H H OH O X O Y .HCl O O H H HO HO H 122 H 285 X= L-Val, L-Phe, L-Ile, D-Val, L-Ala Y= H, L-Val, L-Ile

Scheme 1.45 Synthesis of prodrugs of oleanolic acid ( 285).

A number of 3-O-acyl ursolic acid derivatives (286-294) were prepared and were then evaluated as anti-AIDS agents. 116 (Figure 1.38)

O O

286; R= H 287; R= COOH 288; R= COOH OH H O O O H 291; R= COOH 289; R= COOH RO H O O O O COOH 290; R= O COOH 292; R= 293; R= 294; R=

Figure 1.38 3-O-acyl ursolic acid derivatives ( 286-294 ). One oleanolic acid-based cyclic dimer (296) was synthesized using click chemistry approach and it showed remarkable selectivity and affinity to bind fluoride ion. 261 (Scheme 1.46)

55

O

O O O N OH O N N N N O a O N O O O HO HO 122 295 O

296

Scheme 1.46 Synthesis of oleanolic acid-based cyclic dimer ( 296).

Synthesis of glucoconjugates (297-300) of oleanolic acid, linked by either a triazole moiety or an ester function, as inhibitors of glycogen phosphorylase were accomplished by Cheng et al. 262 (Figure 1.39)

H O O N H O RO OR O N N RO RO OMe HO O O 297; R= Bn O 298; R= H O O O RO O 299; R= Bn RO 300; R= H RO OMe

Figure 1.39 Glucoconjugates ( 297-300 ) of oleanolic acid.

Synthesis of oleanolic acid saponins (301-306) were also achieved by Huang et al. 263 (Figure 1.40) Concise synthesis of bidesmosidic oleanolic acid saponins ( 307-309) with strong inhibitory activity on pancreatic lipase were also accomplished. 264 (Figure 1.41) A convenient transformative reaction-based separation technique for ursolic acid ( 121) from oleanolic acid ( 122) were also achieved by Csuk et al. 265 (Scheme 1.47 ) A series of oleanolic acid dimers ( 313, 315) linked at C-28 by 1,6-hexanediamine, or built around the carbon chains of varying lengths between two carboxyl groups were synthesized and biologically evaluated as their anti-tumor activities. 266 (Scheme 1.48 )

56

O O 2 O O O O RR3 1 R O OH HO O O OH HO O HO O O O HO O HO OH HO O OH HO O O OH HO O OH HO OH HO HO O O OH HO HO HO HO OH OH 304

1 2 3 301; R =OH, R =H, R =CH2OH 302; R1 =H, R2 =OH, R3 =H 303; R1 =OH, R2 =H, R3 =H

O O O O O O HO O OH O HO OH O OH 306 HO HO O HO O 305 HO HO OH OH Figure 1.40 Oleanolic acid saponins ( 301-306).

H H O

O H O O HO O n-Bu HO H O O OH O HO HO O RO OH OH HO HO OH

307; R= Xyl 308; R= Glc 309; R= Glc (1 4) Glc

Figure 1.41 Bidesmosidic oleanolic acid saponins ( 307-309).

57

OH OH O OH O O OH OH m-CPBA 121 122 121 O O + + + (60: 40) (57%) + HO HO HO 310 311 312 Scheme 1.47 Seperation for ursolic acid ( 121) from oleanolic acid ( 122 ).



H H H O O OH (CH3)n

O Br(CH2)nBr O O K2CO3, DMF HO HO OH 313 (a-d; n= 2, 4, 6, 8) 122

H H H H H N N OH (CH3)6 O (COCl)2, Et2N O O 1,6-hexanediamine AcO AcO 315 OAc 314 Scheme 1.48 Synthesis of oleanolic acid dimers ( 313, 315).

I.5 Some steroid- and PT-based marketed drugs There are many steroid- and PT-based drugs known globally for their diversified therapeutic applications. For example, to treat the acute leukemia in children a steroidal drug named prednisone ( 316) is used with combination of the other antineoplastic agents. 267-270 To treat postmenopausal breast cancer, a steroidal drug exemestane ( 317) is used as aromatase inhibitors. 271 Steroids dutasteride ( 318), is used in clinic for the treatment of androgen-dependent prostate cancer and fluoxymesterone ( 319) is used to treat advance- and metastatic neoplasm of the breast ( Figure 1.42). 272-275

58

COCH2OH OH O OH HO

F O O 316 317 O H CF N 3 O

F3C

O O N H 318 319

Figure 1.42 Some steroid-based marketed drugs.

H H COOH HO HO O H H O HO HO HO H H 321 OH HO O CH2 O O O OH O H OH OH O O O H OH OH HO OH O H OH OH O 320 322

Figure 1.43 Some of the PT-based marketed drugs (320-322).

A number of PT-based drugs are also available in the market e.g., oleanolic acid ( 122) for lever disease, asiaticoside ( 320 ) for wound healing and Perkinson’s disease, corosolic acid ( 321) for diabetes and obesity, carbenoxolone ( 322) for oesophageal ulceration and inflammation, etc. (Figure 1.43)

59

I.6 References The references associated with this chapter are provided in the Bibliography section of the thesis. Please follow page 338 onwards for theses references.

60

Chapter II

First report of solvent selective steroidal aromatization, efficient access to 4 b,7 a-dihydroxy steroids, and syntheses of natural diaromatic ergosterols

61

Abstract

Firstly, the transformative scope of selenium dioxide on steroids was reviewed, and as the present work has a significant contribution on the one-pot synthesis of diaromatic steroids including two natural products, the subject ‘steroidal aromatization’ wa s also reviewed. And, the present work demonstrates selenium dioxide oxidation of cholesterol which reveals a solvent-dependent product selectivity and facile one-pot synthesis of three derivatives, including aromatic analogues of naturally occurring ergosterol. Of note, only ethereal solvents with two oxygens such as 1,4-dioxane, 1,3-dioxalane, 1,2-dimethoxy ethane and 1,2-diethoxy ethane were found able to result selective aromatization. Efficient access to 4 b,7 a-dihydroxy cholesterol is also described. Analogous chemistry of b-sitosterol and diosgenin is also reported. The protocol is found effective to synthesize two diaromatic ergosterol natural products. A brief description of the molecular structures of the representative diaromatic cholesterol derivative and the triacetylated 4 b,7 a-dihydroxy cholesterol derivative were proven by X-ray crystallography.

62

II.A A short review on the action of selenium dioxide on steroids II.A.1 Introduction of selenium dioxide as a reagent for organic syntheses Selenium dioxide was introduced as a new oxidising agent in the year 1933 by H. L. Riley, J. F. Morley and N. A. C. Friend. They isolated 1,2-ketoaldehydes and 1,2-diketones from aldehydes and ketones respectively.1-2 The reagent was then found to convert D-ethyl tartrate into ethyl D- ketolhydroxysuccinate. 3 Astin, Newman and Riley reported that when ethanol was heated with selenium dioxide at 230 oC, glyoxal was the product at 5% yield, while n-propyl and n-butyl alcohols resulted complex reaction products including some alkyl selenites. 4 The three functional groups, most often which are subjected to oxidation by this reagent are olefins, ketones and aldehydes. Methyl or methylene groups adjacent to benzene nucleus was converted easily to 5-6 aldehydes, ketones or carboxylic acids by SeO 2. Acetylenic compounds such as 1-heptyne or 7 6 1-octyne reacted with SeO 2 to form the corresponding 3-hydroxy derivatives. Guillemonat showed that on reaction with selenium dioxide, 1-ethylcyclohexene yielded the acetate derivative of l-ethylcyclohexen-6-ol; 1,6-dimethylcyclohexene yielded o-xylene and 2,3-dimethyl-1,3- cyclohexadiene; while 1,2-dimethylcyclohexene yielded o-xylene and 2,3-dimethyl-1,3- cyclohexadiene; and cyclohexene yielded the acetate derivative of l-cyclohexen-3-ol; 3- methylcyclohexene gave 6-methylcyclohexanol and small amounts of toluene, 4- methylcyclohexene, and 4-methylcyclohexen-3-ol; and 4-methylcyclohexene forms a mixture of the acetates of 4-, 5-, and 6-methylcyclohexen-3-ol. Campbell and Haris showed the variation of 9,10 the reaction products of SeO 2 and D -octalin in acetic anhydride with temperature; the isolated products were D9,10 -octalol-1-acetate, D9,10 -octalindiol-1,5-diacetate and hexahydronaphthalin diol-1,5-diacetate respectively at 5 o, 30o and 120 o C. 8-9 Besides the other transformative utilizations, selenium dioxide- mediated allylic hydroxylation comes out to be the most reliable and predictable method. 10-16 Useful regio- and stereoselectivity to furnish the (E)-allylic alcohols predominantly was found to occur using the reagent. 17 Sharpless proposed a well established mechanism for the allylic hydroxylation which involves two consecutive pericyclic reactions- an electrophilic ene reaction followed by the 2,3- sigmatropic rearrangement ( Scheme 2.1). 18, 19

63

OH R 1 R1 Se R1 R Ene reaction O [2, 3] - Sigmatropic 1 + SeO2 OSeOH R R OH 2 R2 2 R2

Scheme 2.1 Mechanism of allylic hydroxylation with SeO 2.

II.A.2 Application of the reagent on steroids Having the usefulness of selenium dioxide as a versatile reagent for the transformation of general organic molecules, the application on steroids and triterpenoids were readily envisioned. The reaction of ergosterol (83 ) with selenium dioxide in benzene/ ethanol for 19 hours was a complex one, whereas the main product isolated at room temperature was dehydroergosterol (323) (Scheme 2.2) which was previously obtained by the dehydrogenating action of mercuric(II) acetate on ergosterol in boiling alcohol. 20

SeO2 H H H C6H6/ EtOH HO HO 83 RT, 19h 323

Scheme 2.2 Action of SeO 2 on ergosterol (83 ).

Similarly, when 5-dihydroergosteryl acetate (324a) was treated with SeO 2 in acetic acid 8(14), 22 D - ergostadiene-3b, 7 a-diol diacetate (325 ) was formed, but when SeO 2 was used in benzene/ ethanol along with acetic anhydride at 0 oC, 3 b-acetoxy-7a-ethoxy-D8(14),22 -ergostadiene (326) was formed. Corresponding 3 b-hydroxy derivative (327) was obtained by using 5- dihydroxyergosterol (324b) as the starting material ( Scheme 2.3). 21, 22

SeO2/ AcOH SeO2/ EtOH AcO OAc RO Ac2O RO OEt 325 324a; R= Ac 326; R= Ac 324b; R=H 327; R= H

Scheme 2.3 Action of SeO 2 on 5-dihydroergosterol (324b ) and its acetate (324a ).

64

Cholesterol, cholesteryl acetate, cholesteryl chloride, cholesteryl bromide, allocholesterol, y-cholesterol, a-cholestantriol, cholesteryl ether, a-cholesterol oxide, y-cholestane, cholestene, y-cholestene, cholesterilene, cholestan-6-one, cholestenone, cholestan-6-on-3-ol acetate, cholestan-3,6-dione-5-ol, oxycholesterylene, oxycholestenone, and coprosterol were found to be unreactive with SeO 2 in boiling 90% EtOH. But the reactions took place in acetic acid and at 100oC with all these substances with the exception of a-cholestantriol, y-cholestane, cholestan- 6-one-3-ol acetate, and oxycholesterylene. Cholestan-3,6-dione and coprostanone reacted both in 90% EtOH and in AcOH. 21 With selenium dioxide, cholesterol (13 ) was found to form cis - D5,6 cholestene-3,4-diol (or, 3 b,4 b-dihydroxycholest-5-ene) (334) either in benzene, nitrobenzene or in glacial acetic acid ( Scheme 2.4). cis -Cholestene-3,4-diol-3-benzoate was also prepared from choleseryl benzoate using the same reaction protocol. The author also assumed the formation of trans -diol from the corresponding diacetate via the treatment of selenium dioxide on cholesteryl acetate, 23 although later on Butenandt and Hausmann showed that it was not the trans -diol but 6-hydroxy cholesterol. 24 Recently also, selenium dioxide was found to be effective to produce 4 b-hydroxy steroids (331-335) from the corresponding steroidal 5-ene-3b-ols (13 , 328-330) when refluxed with the reagent in dioxane at 80 o C for 18 hours ( Scheme 2.4). 25

O R3 R3 R2 R2 O SeO2 Dioxane 80oC, 18h R1O R1O HO OH OH 328; R1= H, R2=R3= O 331; R1= H, R2=R3= O 329; R1= H, R2=H,R3= COCH3 335; 4b-Hydroxy diosgenin 332; R1= H, R2=H,R3= COCH3 330; R1= COCH3, R2= H, R3= COCH3 333; R1= COCH3, R2= H, R3= COCH3 13; R1= H, R2= H, R3= C8H17 334; R1= H, R2= H, R3= C8H17

Scheme 2.4 Action of SeO 2 on cholesterol (13 ) and other steroidal 5-ene-3b-ols (328-330).

Cholestanone (31 ) was found to form cholestan-2,3-dione (336) by the action of SeO 2 in 3,5 boiling alcohol ( Scheme 2.5). With SeO 2, 3b-acetoxycholestan-7-one, D -cholestadien-7-one and 12-ketocholanic acid were found to react neither in alcohol nor in glacial acetic acid. 26

65

O SeO2 O Alcohol/ H2O O Reflux 31 336

Scheme 2.5 Action of SeO 2 on cholestanone ( 31 ).

When D7-cholestenyl acetate (337) was treated with selenium dioxide in acetic acid/ benzene at 0 oC, D8(14) -cholestene-3b,7 a-diol diacetate (338 ) was obtained and its diol derivative (339) 22 was prepared from it by LiAlH 4 reduction ( Scheme 2.6).

SeO2 LiAlH4 HOAc, C6H6 AcO AcO OAc HO OH 337 338 339 Scheme 2.6 Synthesis of 3b,7 a-dihydroxycholest-8 (14)-ene (339).

Sitosterol and stigmasterol did not react in 90% EtOH, but reacted in acetic acid at 100 oC.

Stigmasterol formed 6-hydroxy and 4-hydroxy derivatives when was treated with SeO 2 in acetic acid/ benzene at 90 o C. Sigmasteryl acetate formed 3-acetoxy-4-hydroxy stigmasterol by the same process. 27 The corresponding cholesterol derivative was reported by the same group in the year 1939 where they used benzene as the solvent. 28 Lanosteryl acetate (340) in ethanol when refluxed with selenium dioxide in water for 1 hour, a diol monoacetate (341) was found to be formed ( Scheme 2.7). Similarly a- dihydrolanosteryl acetate formed g- lanosteryl acetate. 29 The reagent was employed on a number of ergosterol derivatives also. 21 The transformative capability of the reagent in different reaction conditions was tested on a number of steroids as well as triterpenoids. 21 Again, preparation of pregnenolone and androsterone derivatives using selenium dioxide was reported by Marker, Crooks and Wittbecker, 30 Ruzicka and Plattner 31 and others. 32

66

H HO H

O SeO2 O Ethanol/ H O O 2 O H Reflux, 1h H 340 341

Scheme 2.7 Action of SeO 2 lanosteryl acetate (340).

Selenium dioxide was used as dehydrogenating agent in the bile acid series where D9,(11) -12 ketones were prepared from 12-ketones in presence of acetic acid. 33 Botogenin acetate ( 342) and correlogenin ( 343, 25 b-epimer of botogenin) were converted to the corresponding 9,11-dehydro derivatives ( 345 and 346, respectively) by selenium dioxide in t-butanol. 34 Similarly hecogenin acetate ( 344, reduced 5 a-H) formed 9(1l)-dehydrohecogenin acetate ( 347 ) by the same in presence of little acetic acid ( Scheme 2.8). 35

O O O O O O t SeO2/ BuOH

RO OR 342; R= Ac (25a) 345; R= Ac (25a) 343; R= H (25b) 346; R= H (25b) 344; R= Ac, Reduced 5a-H 347; R= Ac, Reduced 5a-H

Scheme 2.8 Action of SeO 2 on botogenin acetate (342 ), correlogenin (343) and hecogenin acetate (344 ).

In 1954, Oliveto, Gerold and Hershberg showed that saturated steroidal 3-keto derivatives, such as pregnane-17 a,21-diol-3,11,20-trione-21-acetate ( 348), pregnan-17 a-ol-3,11,20-trione (349), pregnane-3,11,20-trione ( 350) and pregnane-l1 b,17a-diol-3,20-dione (351) reacted with

SeO 2 in methanol to form the corresponding 3,3-dimethoxy ketals (352-355) while compounds having ketones at C-11, C-17, C-20 or at C-3 with conjugated double bond at C-4 e.g.; cortisone acetate ( 356), D4-androstene-3,17-dione and pregnan-3a-ol-11,20-dione did not react ( Scheme 2.9). 36

67

R R OAc O O R2 R2 O R1 R1 O OH SeO2/ MeOH MeO O MeO H H O 352; R= OAc, R = OH, R =O 348; R= OAc, R1= OH, R2= O 1 2 356 349; R= H, R1= OH, R2= O 353; R= H, R1=O H, R2= O 350; R= H, R1= H, R2= O 354; R= H, R1= H, R2=O 351; R= H, R1= OH, R2= OH 355; R= H, R1= OH, R2= OH

Scheme 2.9 Action of SeO 2/ MeOH on saturated steroidal 3-keto derivatives (348-351).

Ring-A contracted acids were found to be formed when steroidal 3-ketones of 5 a- and 5b- series were treated with selenium dioxide and the bond on either side of carbonyl groups were broken. And the stereochemistry of ring A/B juncture was found to be unaffected in the course of the reaction. 37 Terpene hydrocarbons having methyl or methylene group adjacent to the double bond when were treated with selenium dioxide in glacial acetic acid or in acetic anhydride, resulted acetate at ordinary temperature with intact double bond, while the double bond got saturated at high temperature. 38

O

O

SeO2 + + HO HO H 357 358 359 360

SeO2

TsO HO H H H 361 362 363

3 Scheme 2.10 SeO 2 oxidation of methyl D -cholenate ( 357 ) and associated transformations.

68

3 4 SeO 2 oxidation of methyl D -cholenate (357), in acetic acid, resulted 3b-and 3 a-hydroxy-D - cholenates (358 and 359 respectively) along with D3,5 -diene 360. 3b-Hydroxy-D1-cholenate 363 was also found using the same process from methyl D2-cholenate (362, Scheme 2.10). 39 When 3 b-acetoxy-D7,9(11) -diene of diosgenin (364 ) was treated with selenium dioxide in acetic acid/ benzene at 0 oC, a 14-hydroxy derivative (365) was isolated ( Scheme 2.11). 22

O O O O SeO2/ CH3COOH H OH AcO AcO H H 364 365

Scheme 2.11 Action of SeO 2 on compound 364.

5b-Cholestane-7a,12a-diol-3-one (366) yielded cholest-4-ene-7a,12a-diol-3-one (367) 40 when treated with SeO 2 in ethanol ( Scheme 2.12).

OH OH

SeO2 EtOH O OH O OH H 366 367

Scheme 2.12 Synthesis of cholest-4-ene-7a,12a-diol-3-one (367).

When 5a-androstane-3,17-dione (368) was refluxed with selenium dioxide in t-amyl alcohol, acetic acid and water, the isolated products were found to be 5a-androst-l-ene-3,17- dione (369), androst-4ene-3,17-dione (370), and androsta-l,4dien-3,l7dione (371) (Scheme 2.13). 41

Vitamin D 3 acetate on treatment with selenium dioxide and t-butyl hydroperoxide yielded a 42 mixture from which a Diels-Alder dimer of 1-oxotransvitamin D 3 acetate was isolated.

69

O O O O

H H H H SeO2 H H H H + H H + H H tAmyl alcohol O O O O H CH COOH/ H O H 368 3 2 Reflux 369 370 371 Scheme 2.13 Action of SeO2 on 5 a-androstane-3,17-dione (368).

3b-Benzoyloxy-5a-cholest-8(14)-en-15-one (372) on treatment with selenium dioxide in 2- metyl-2-propanol resulted 3 b-benzoyloxy-5a-cholest-8(14),16-dien-15-one (373), 3 b- benzoyloxy-5a-cholest-8(14),16-dien-15-one 16-selenenic acid (374) and 3 b-benzoyloxy-9a- hydroxy-5a-cholest-6,8(14),16-trien-15-one (375, Figure 2.1). 43

SeOH O OH O RO O O H BzO BzO BzO H H H 372; R=COC6H5 373 374 375 376; R=H

Figure 2.1 SeO 2 oxidation products of 3b-Benzoyloxy-5a-cholest-8(14)-en-15-one (372).

44 SeO 2 was found to furnish 6-hydroxycorticosteroids, 6β -hydroxy derivatives of progesterone and testosterone via allylic oxidation. 45 Besides, 3β -benzoyloxy-5α -cholest-8(14)- en-15-one ( 372) was reacted with SeO 2 to form 3β -hydroxy-5α -cholest-8(14),16-dien-15-one (376) as conjugated products (Figure 2.1).43

R2 R2 R2 R1 R1 R1

SeO2 HO Dioxane + o O 80 C, 18h O O 370; R1=R2= O 371; R =R = O 1 2 379; R1=R2= O 56; R1= H, R2= COCH3 377; R = H, R = COCH 1 2 3 380; R1= H, R2= COCH3 49; R1= H, R2= C8H17 378; R = H, R = C H 1 2 8 17 381; R1= H, R2= C8H17

Scheme 2.14 Action of SeO 2 on steroidal 4-en-3-ones (49, 56, 370).

70

When selenium dioxide in dioxane was employed to the steroidal 4-en-3-ones ( 49 , 56 , 370), the corresponding 1,4-conjugated-dienes ( 371, 377-378, 383) along with 2-hydroxy-1,4-diene derivatives ( 379-381) were isolated. In case of progesterone ( 56 ), along with the two products, a small amount of diketo aldehyde ( 382) was also isolated as is expected ( a-oxidation of carbonyl) (Scheme 2.14 and Figure 2.2 ). 25

O O O H O

O O  

Figure 2.2 Diketo aldehydes ( 382) and 1,4-conjugated diene derivative of diosgenin ( 383).

II.A.2.1 Biological consequences of 4 - and/ or 7-hydroxy steroids Among the oxysterols in human circulation, the major one, 4 b-hydroxy cholesterol, is, along with other oxysterols, degraded generally to bile acids via 7a-hydroxylation as the rate-limiting step. 46, 47 In addition, other hydroxy derivatives of cholesterol are drawing attention due to their profound importance in human metabolism. This motivates the investigation of their structure activity relationship in order to correlate disease with drug treatment. 40, 47-53 Clearly, there exists an enormous demand to synthesize 4 b,7 a-dihydroxy steroids, preferably in more direct and easier routes than existing synthetic methodologies. 54 55 In vitro cholesterol oxidation products were found to be good markers of LDL oxidation. In addition, the oxysterol 7 b-hydroxycholesterol, together with an increased oxidation susceptibility of VLDL+LDL, were the strongest predictors of progression of carotid atherosclerosis in Finnish men 56 and it was also reported that this could be the possible predictor for lung cancer risk. 51 7a-, 24- and 27-Hydroxy cholesterol formed via some enzymatic reactions, are the important circulating oxysterols in human. 4 a- and 4 b-hydroxy cholesterols are also found in human and it was supposed that these are formed during the in vitro LDL oxidation but in lower concentration compared to 7-oxocholesterol. But later it was reported that antiepileptic drugs

71 increase the concentration of 4 b-hydroxycholesterol in human circulation by influencing cytochrome P450 enzyme and thus converting cholesterol into 4 b-hydroxycholeterol. 57 7a- Hydroxycholesterol was reported to convert into some natural C-24 bile acids with special reference to Chenodeoxycholic Acid Biogenesis by fortified rat mitochondria. 46 The exceptionally slow elimination of 4 b-hydroxycholesterol results indeed the high plasma concentration of itself, probably in part because of the low rate of 7 a-hydroxylation of the steroid. The fact was established by the observation that 4 b-hydroxycholesterol was converted into acidic products at a much slower rate than 7 a-hydroxycholesterol in primary human hepatocytes, and 4 b-hydroxycholesterol was 7 a-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 b- hydroxycholesterol. Again, the findings were justified on the basis of the potential role of 4 b- hydroxycholesterol as a ligand for the nuclear receptor LXR, discussed. 47 Diczfalusy et al. reported 4 b-hydroxycholesterol as an in vivo marker of CYP3A4 inhibition. 58 Again, results suggest that 7 b-hydroxy cholesterol promotes HUVECs survival and proliferation following a mechanism independent of ROS production and involving calcium-dependent activation of ERK. 59

II.B A brief review on steroidal aromatization reactions 1 Though Marker and co-workers first reported the conversion of steroidal nucleus into its aromatic analogue 60 but Inhoffen and Zuhhdarff established the fact in the year 1938 by carrying out the transformation of 1,4-cholestadien-3-one to a phenol moeity. This was actually an acid- catalysed dienone-phenol rearrangement and later the structure was confirmed by the others as the phenol entity. 61-68 Romo and his group reported the same rearrangement thermally with the aromatic derivative of 1,4-cholestadien-3-one.69, 70 A-ring aromatized cholesterol was formed from cholesterol itself. 65 Hanson and Organ showed that treatment of 1,3-dibromo-5,5- dimethylhydantoin on cholesterol (13 ) produced 4-methyl-19-norcholesta-1,3,5(10)-triene (384).71-73 They also reported the formation of 4-methyloestra-l,3.5(10)-trien-l7-one and 4- methyl-19-norpregna-l,3,5(10)-trien-20-one respectively from dehydroisoandrosterone and pregnenolone, while 3 b-acetoxyandrosta-4,6-diene-17-one and 3 b-acetoxypregna-5,7-diene-20-

1 This review was prepared only after the serendipitous isolation of the diaromatic steroids via the single-step SeO 2 oxidation on steroids. Please follow the present work, Section xyz, for details of the work. 72 one respectively from dehydroisoandrosterone acetate and pregnenolone acetate by the same process. They found NBS as a poorer alternative for the same transformation ( Scheme 2.15).

Hydantoin deriv.

HO 13 384

Scheme 2.15 Conversion of cholesterol ( 13 ) into 4-methyl-19-norcholesta-1,3,5(10)-triene ( 384).

II.B.1 From dieneones Barton and Thomas treated 3 b-acetoxylanosta-5,8-diene-7-one ( 385) with zinc in acetic acid to form a phenolic compound which was later characterized as 3 b-acetoxy-19-norlanosta-5,7,9- trien-7-ol ( 386) by K.Tsuda, E. Ohki, S. Nozoe ( Scheme 2.16). 74, 75

Zn/ AcOH Reflux AcO O AcO OH 385 386

Scheme 2.16 Conversion of 3b-acetoxylanosta-5,8-diene-7-one ( 385) into 3 b-acetoxy-19-norlanosta- 5,7,9-trien-7-ol ( 386).

On the other hand, when androsta-1,4,9(11)-trien-3,17-dione ( 387) was refluxed with zinc in pyridine or ethylene glycol, ∆ 9-estrone (388) was resulted. ( Scheme 2.17). 76 As was expected, similar results were shown by compounds 389 and 391 to form 390 and 392 respectively (Scheme 2.17). 77-79 p-Cresol type rearrangement product 393 and estrone ( 59 ) were isolated from dieneone 371; and phenolic 395 from dieneone 39466, 68 were formed by using zinc in pyridine. These transformations indeed described a new type of dienone-phenol rearrangement of cross- conjugated dienone or trienone systems ( Scheme 2.18 ). 77

73

O O

Zn/ Py Reflux O OH 387 388 O O OAc OAc OH OH

Zn/ Py Reflux O HO 389 390 O O

Zn/ Py Reflux O HO 391 392

Scheme 2.17 Dienone-phenol rearrangement of cross-conjugated dienone or trienone systems (387, 389 and 391 ).

O O O OH

Zn/ Py + Reflux O HO 59 CH 371 3 393

O O HO HO HO

Zn/ Py Reflux O 394 395

Scheme 2.18 Dienone-phenol rearrangement.

74

Designed bromohydrin 396 and epoxide 39780, 81 separately produced derivative 388 on reaction with Zn in pyridine. Again 90 , when was reacted with collidine, yielded derivative 398, which formed readily 3,11 b-dihydroxyestra-1,3,5(10),8-tetraen-17-one ( 399). Again, when androsta-1,4,8-triene-3,11,17-trione ( 400) was treated with Zn in DMF, 401 and 402 were formed. ( Scheme 2.19). 81 Similarly zinc in DMF in presence of a little amount of water was found to be effective to aromatize oxime 403 into 404 (Scheme 2.19). 82

O O O HO O

KOAc Zn, py Br MeOH O O HO 396 397 388 Collidine or, AgNO3, Dioxane RT O O HO HO

Zn/ DMF

O HO 398 399

O O O O O O

Zn/ DMF + O HO HO 400 401 402

OAc OAc

Zn/ DMF/ H2O

HON H2N 403 404

Scheme 2.19 Transformtive reactions of bromohydrin 396, androsta-1,4,8-triene-3,11,17-trione ( 400) and 17-acetoxyandrosta-1,4,9(11)-triene-3-one oxime ( 403) to result A-ring aromatized products.

75

OH OAc OH O O O Y OH OH OH X

O O RO Z 409 Y 405, X=Y=Cl; Z=H 410, R=Y=H 406, X=Y=Cl; Z=F 411, R=Ac; Y=F 407, X=Y=Cl; Z=CH3 412, R=Ac; Y=CH3 408, X=Br;Y=OH; Z=H

Figure 2.3 21-Acetoxy-9α,11 b-dichloro-17α-hydroxypregna-1,4-diene-3,20-dione ( 405 ) and its derivatives (406-408), 21-acetoxy-3,17α-dihydroxy-19-norpregna-l,3,5(10),6,8-pentaen-20-one ( 410) and its derivatives (411, 412) and 21-acetoxy-17α-hydroxypregna-l,4,8(14),9(1l)-tetraene-3,20-dione ( 409).

Pd-C catalysis or acid elimination of an allylic hydroxyl group and subsequent rearrangement or pyrolysis by selenium dioxide etc. were the different protocols for carrying out the aromatization of either ring-A or -B or both ring-A and -B of the steroids. When 21-acetoxy- 9α,11 b-dichloro-17α-hydroxypregna-1,4-diene-3,20-dione ( 405) was refluxed with DMF or pyridine, 21-acetoxy-17α -hydroxypregna-l,4,8(14),9(1l)-tetraene-3,20-dione ( 409) and 21- acetoxy-3,17α-dihydroxy-19-norpregna-l,3,5(10),6,8-pentaen-20-one ( 410 ) were formed. The same protocol was applied to 406, 407 and 408, to form 411, 412 and 410, respectively, all consisting of aromatized AB ring were obtained, though compound 412 from 407 was obtained after acetylation and compound 410 was also obtained from compound 408 on refluxing with DMF ( Figure 2.3). Similar treatment yielded the diaromatic steroid derivative 414 from dienone 413 (Scheme 2.20 ). 83

O O Cl Cl Zn, DMF Reflux O HO 413 414

Scheme 2.20 Transformation of dienone 413 into diaromatic steroid 414 .

76

The authors in their next communication, reported that pyridine alone was also effective for aromatization though it took a longer period of time. 84 F. Sondheimer and his group showed that ∆ 1,4,6 -22a-spirostatriene-3-one ( 415) on vapour phase aromatization at 600 0C in mineral oil or in tetralene afforded the corresponding 3- hydroxy-∆1,3,5(10),6 -tetraene ( 416) with the elimination of C-19 methyl group ( Scheme 2.21). 85

O O O O

600oC Mineral oil O HO 415 416

Scheme 2.21 A-ring aromatizati on of ∆ 1,4,6 -22a-spirostatriene-3-one ( 415).

Djerassi and his group investigated the aromatization experiments in the cholesterol series. 69,70,86 They synthesized the aromatic cholesterol compounds following dienone-phenol rearrangement. 1,4,6-cholestatrien-3-one ( 417) on heating with p-TsOH in acetic anhydride for 4 hours resulted a phenolic acid which on saponification gave 6-dehydro-1-methyl phenol (418) and on methylation gave the methyl ether 419 . When the 6-dehydro acetate 419 was dehydrogenated with selenium dioxide in acetic acid solution, the naphthalenic analog, l-methyl- 3-hydroxy-19-nor-1,3,5,6,8-cholestapentaene (420 ) was formed. 420 rapidly decomposed on exposure to light and air (Scheme 2.22 ).

C8H17 C8H17 C8H17

p-TsOH SeO2 Ac2O, 4 h CH COOH O RO 3 HO 417 418; R=H, 420 419; R=CH3CO Scheme 2.22 A-ring aromatized cholesterol from 1,4,6-cholestatrien-3-one ( 417).

77

Using mineral oil vapor phase aromatization , 1,4,16-pregnatriene-3,20-dione ( 421) was also converted to 3-hydroxy-l7-acetyl-1,3,5,16-estratetraene (422).87 Compound 422 was transformed into the aromatic analogs 423 and 425 of the corpus luteum hormone, progesterone ( 56 ), and the adrenal hormone, 17 b-hydroxyprogesterone respectively. ( Scheme 2.23 ). 86

O O O

Mineral oil H2/ Pd-C 600oC O HO HO 421 422 423

p-TsOH, Ac2O Slow distillation

O OAc OH

PhCO3H RO AcO 425 a R=H 424 b R= CH3CO Scheme 2.23 A-ring aromatization of 1,4,16-pregnatriene-3,20-dione.

Progesterone ( 56 ) was transformed into the cross congjugated trienone 426, structurally, 1,4,6,16-pregnatetraene-3,20-dione which after dienone-phenol rearrangement or on 4 hours distillation with p-tolunesulphonic acid in acetic anhydride, produced an aromatic analog 427. (Scheme 2.24 ). 86

O O O

p-TsOH, Ac2O , 4 h O O AcO 56 426 427

Scheme 2.24 A-ring aromatized products from progesterone (56 ).

78

Dryden, Webber and Wieczorek reported that when androsta-1,4-diene-3,17-dione ( 371) was pyrolysed in mineral oil-tetralene solution, estrone ( 59 ) was found to be formed. ( Scheme 2.25 ). 88

O O

H H H H , Mineral oil/ Tetralene solution H H

O HO 371 59

Scheme 2.25 Formation of estrone (59 ) from androsta-1,4-diene-3,17-dione ( 371).

II.B.2 From enone and diene

[Cp*Ru(OMe)] 2 on treatment with trifluorosulfonic acid (CF 3SO 3H), was found to produce + ‘(C 5Me 5)Ru ’ which was found effective to aromatize the A-ring of the steroid molecules having enone functionality. 89, 90 Some of the reactions are shown in the following Scheme (Scheme 2.26). Again, using the same ruthenium species, B-ring aromatized steroids (432-433) were also prepared ( Scheme 2.26 ). 91

R R H H + H (C5Me5)Ru [Ru]+ H THF, Reflux H H H H 40 h O HO 428; R= OH, 429; R= OH, 49; R=C8H17 430; R=C8H17 56; R=COCH3 431; R=COCH3 R R

+ (C5Me5)Ru THF, Reflux H H H 40 h HO HO Ru+Cp+ 83; R=C9H17 432; R=C9H17 39; R=C8H17 433; R=C8H17

+ Scheme 2.26 A-ring aromatization of some steroids by ‘(C 5Me 5)Ru ’ and transformation of ergosterol (83 ) or 7-dehydrocholesterol ( 39 ) into B-ring aromatized products.

79

Epoxy steroids were also found to be aromatized with acids e.g. 17-b-acetoxy-1α,2α -epoxy- 5α -androstan-3-one ( 434) formed the diacetate of 1-methylestradiol ( 435, Scheme 2.27 ). 92

OAc OAc

O p-TsOH Ac O O 2 AcO H 434 435

Scheme 2.27 Aromatization of ring-A of 17-b-acetoxy-1α,2α -epoxy-5α androstan -3-one ( 434).

Preparation of the aromatized steroids of stomach was also reported. The rate of aromatization of 10 b-hydroxy-19-nortestosterone ( 436) and 17α -ethynyl-10 b-hydroxy-19- nortestosterone ( 437) in HCl at 37 oC was studied by R. Y. Kirdani and D. S. Layne. 93 Selective A-ring aromatization of some steroids viz. , 3-oxo-4-cholen-24-oic acid ( 438 ), 4-androstene-3,17- dione ( 370) and cholesterol ( 13 ) were also found to occur by human gut bacteria ( Figure 2.4). 94,95 Apart from chemical transformations, steroidal hormones were also found to be degraded by microorgsnisms. 96-98 A species of Pseudomonas isolated from cotton 100 was able to ferment 4- androstene-3-17-dione ( 370) and yielded 9,10-seco-3-hydroxy-1,3,5(10)-androstatriene-9,17- dione ( 439). 99 19-Hydroxyandrost-3, 17-dione ( 440) was transformed into estrone ( 59 ) by Pseudomonas sp. 99 and by Nocardia restrictus . Nocardia restrictus was also able to degrade androst-4-ene-3,17-dione ( 370) into 9,10-seco-3-hydroxy-1,3,5(10)-androstatriene-9,17-dione (439, Scheme 2.28 ). 101

O O H OH R H OH H H H H H H H H O O O 436; R= OH 437 438 370; R= H

Figure 2.4 10 b-Hydroxy-19-nortestosterone ( 436), 17α -ethynyl-10 b-hydroxy-19-nortestosterone ( 437), 3-oxo-4-cholen-24-oic acid ( 438) and 4-androstene-3,17-dione ( 370).

80

O O

AcO Pseudomonas sp. O or, Nocardia restrictus O HO 370 439

O O HO H Pseudomonas sp. H H or, Nocardia restrictus O HO 59 440

Scheme 2.28 Microbial transformation of some steroidal hormones.

It was reported that estrogen ( 59 ) was formed, separately, from 441, when incubated with N. restrictus and from 442 when treated with CSD-10, an organism isolated from soil utilizing cholesterol as a sole carbon source. 102 19-Hydroxyprogesterone ( 443), 3b,19-dihydroxy-pregn- 5-en-20-one 3-acetate ( 444 ) and pregn-5-ene-3b,19,20 b-triol 3-acetate ( 445 ) were transformed into 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one ( 446) when treated with Nocardia sp. (Scheme 2.29 ). 103 Thus, a number of steroid molecules having several different functionalities have been utilized to produce the corresponding aromatized steroids by following both of chemical as well as by bio-transformative processes.

O HO H HO H H H H H CSD-10 N. restrictus H H O HO O 442 59 441

O O R

HO HO H H H Nocardia sp. Nocardia sp. H H H H H H O HO AcO 446 443 444; R= CH(OH)CH3 445; R= COCH3

Scheme 2.29 Steroidal aromatization by microorganisms.

81

II.C Present work II.C.1 Background and abstract of the work Selenium dioxide-mediated oxidation is regarded as one of the most reliable and predictable methods for allylic hydroxylation, especially in the steroid field. 21-23,25-28,40,45,104-105 Despite the proven biological importance of oxygen bearing functionalities in steroidal systems 40,46-53,57,106- 110 no systematic approach has been adopted to explore the oxidizing ability of selenium dioxide

(SeO 2) in steroidal systems. The present work demonstrates there is ample opportunity to elaborate the chemistry of steroidal systems based on the oxidizing ability of SeO 2. Under these circumstances, we were encouraged to synthesize oxysterols and to explore the oxidation behaviour of SeO 2 on steroids. For the present study, cholesterol was chosen as the representative molecule. The results thus obtained were subsequently extended to ergosterol, b- sitosterol and diosgenin. In brief, selenium dioxide oxidation of cholesterol reveals a solvent-dependent product selectivity and facile one-pot synthesis of three derivatives, including aromatic analogues of naturally occurring ergosterol. Efficient access to 4 b,7 a-dihydroxy cholesterol is described. Analogous chemistry of b-sitosterol and diosgenin is also reported. The protocol is found effective to synthesize two diaromatic ergosterol natural products. A brief description of the molecular structures of the representative diaromatic cholesterol derivative and the triacetylated 4b,7 a-dihydroxy cholesterol derivative are proven by X-ray crystallography.

II.C.2 Results and discussion

II.C.2.1 SeO 2-oxidation of cholesterol, cholesteryl acetate and cholesteryl benzoate

The reaction of SeO 2 with cholesterol was reported to produce 4 b-hydroxy cholesterol as the only product 23,25 whereas with the same reagent, cholesteryl acetate and benzoate yielded 4 b- hydroxylated and 6 a-hydroxylated products, respectively. 23 The observation was supported by 105 Marker et al. who studied the action of SeO 2 on stigmasterol, stigmasteryl acetate and sitosteryl acetate.

Herein, we wish to report the results of a solvent-dependent investigation of SeO 2 oxidation on cholesterol (13 ) which resulted in the formation of naphthalene analogue (447) via selective aromatization of rings A and B of cholesterol, together with 4 b-hydroxy- (334) and 4 b, 7 a- dihydroxy ( 448) derivatives ( Scheme 2.30 ). Cholesteryl acetate (40 ) and benzoate (33 ) also

82 yielded the naphthalene analogues along with the other two products as reported 23 (Scheme 2.31 ).

R R R R H H H H

H H SeO2, Solvent + H H + H H HO Temp, Time HO R'O OR/ 13, 14 OH OR/ 13; R= 447; 3-6% 334; 10-75% 448; R= a, R/ =H; 5-27% 453; 7% 454; 51% Ac2O, 455; R= b, R/= H; 22% Py / 14; R = 456; R=a, R = COCH3; (94% from 448)

Scheme 2.30 SeO 2 oxidation of cholesterol ( 13) and b-sitosterol ( 14).

R R R H H H

SeO2, Dioxane H H 447 + H H + H H 100oC, 24 h R1O R O 3% from 40 R1O 1 R= 4% from 33 OH OH

449; R1=COCH3, 35% 451; R1=COCH3, 37% 40; R1=COCH3 450; R1=COPh, 32% 452; R1=COPh, 34% 33; R1=COPh

Scheme 2.31 SeO 2 oxidation of cholesteryl acetate ( 40 ) and benzoate ( 33 ).

Steroidal dehydrogenation of a maximum two hydrogen atoms using selenium dioxide 25,41,43,111-112 as well as steroidal aromatization (using other reagents) and their biological 113 implications have been reported. But, achieving dehydrogenation using SeO 2 by removing as many as nine hydrogen atoms from a cholesterol molecule to induce aromatization ( e.g . formation of naphthalene analogue, 447) in a solvent-specific reaction is unprecedented. Of note, only ethereal solvents with two oxygens such as 1,4-dioxane, 1,3-dioxalane, 1,2-dimethoxy ethane and 1,2-diethoxy ethane are able to yield 447. To the best of our knowledge, this is the first report of the solvent-selective aromatization using selenium dioxide. The products have been fully characterized (see Experimental Section and supporting spectra) by elemental analysis and spectroscopy (IR, 1H, 13 C, DEPT-135 NMR, mass), and in the cases of 447 (to confirm the structure) and 456 (to confirm the a-configuration of the 7-OH

83 group in 448 and, by analogy, that in 453 and 460), by single crystal X-ray crystallography (Figure 2.6 and Figure 2.7). The molecular structures of 447 and 456 are also described.114

II.C.2.2 Study of the solvent dependent product ratio The results of the systematic study have revealed that the composition of the products is largely solvent dependent ( Table 1 ). Four different classes of solvents viz. (i) ethers (entries 1-8), ii) alcohols (entries 9-13), iii) basic (entries 14-16) and iv) polar aprotic solvents (entries 17-19) were used. To obtain the diol, 334 , the use of ether as solvent, particularly THF (entry 8), was found to be best, whereas for triol, 448, 1,4-dioxane (moist, entry 2) gave maximum yield. Lower alcohols appeared to give better yield than their long chain analogues (entries 9-12). Dihydroxy alcohol, e.g . ethylene glycol, were also examined but were found to be ineffective (entry 13). Basic solvent, e.g . pyridine (entry 14) was found effective in converting cholesterol into the diol and triol whereas triethylamine and morpholine (entries 15 and 16) were found to be ineffective. This is presumably due to the higher basicity of morpholine ( pKb = 8.33) and triethylamine ( pKb = 11.01) compared to pyridine ( pKb = 5.21). The reaction was also attempted in strong basic medium with the use of K 2CO 3 and NaOH in aqueous ethanol, 1,4-dioxane and acetonitrile. However, the extent of transformation was negligible in each case. Among the polar aprotic solvents used, DMSO resulted in good overall transformation (entry 19), whereas acetonitrile (entry 17) and DMF (entry 18) gave poor yields.

Table 2.1 Solvent dependent product ratio.

Entry Solvent system Reaction conditions Yield (%) b, c

447 334 448

1 1,4-Dioxane 100ºC, 24 h 3 35 6

2 1,4-Dioxane: water (50:1) 100ºC, 24 h 5 50 26

Reflux, 1 h 6 34 27

Reflux, 3 h trace 46 8

3 1,4-Dioxane: water (5:1) 100ºC, 24 h trace 64 5

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Table 2.1 (contd.)

Entry Solvent system Reaction conditions Yield (%) b, c

447 334 448

4 1,3-Dioxalane Reflux, 24 h 6 32 5

Reflux, 6 h trace 42 8

5 2-Methoxy ethanol 100ºC, 24 h NF 48 trace

6 1,2-Dimethoxy ethane Reflux, 24 h 6 54 6

7 1,2-Diethoxy ethane Reflux, 24 h 5 51 11

8 Tetrahydrofuran Reflux, 24 h NF 75 17

9 Ethanol Reflux, 24 h NF 35 11

10 tButanol Reflux, 24 h NF 57 15

11 Octanol Reflux, 40 h NR NR NR

12 Decanol Reflux, 40 h NR NR NR

13 Ethylene glycol 100ºC, 24 h NR NR NR

Reflux, 5 h NF 10 trace

14 Pyridine Reflux, 24 h NF 62 13

15 Triethylamine Reflux, 40 h NR NR NR

16 Morpholine 100ºC, 40 h NR NR NR

17 Acetonitrile Reflux, 24 h NF 21 5

18 DMF 100ºC, 24 h NF 28 11

19 DMSO 100ºC, 24 h NF 60 5

20 On solid support d MW e, 2 min NF 30 trace aAll the reactions were performed on 580 mg, 1.5 mmol of cholesterol. b Yield refers to isolated pure compounds. c NF= not found, NR= no reaction. dOn preactivated silica gel 60-120, after making dust. eDomestic microwave, at 600W.

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It was observed that the aromatized product 447 was formed when the oxidation was carried out in 1,4-dioxane (entries 1-3), 1,3-dioxalane (entry 4), 1,2-dimethoxy ethane (entry 6) and in 1, 2-diethoxy ethane (entry 7). All other solvents used (Table 1) failed to furnish 447 indicating the likely participation of both ethereal oxygens situated at 1,4-positions in the aromatization process. In a separate experiment, carried out under solvent-free microwave induced conditions, the reaction produced diol ( 334) but not 447 (entry 20).

II.C.2.3. Raising the yield of 4 b,7 a-dihydroxycholesterol By contrast, the synthesis of 44854 (Scheme 2.30 ) is a simple and convenient one-step reaction. From the solvent dependant study ( Table 1 ), a maximum of 27% yield of the triol 448 was obtained using 1,4-dioxane (moist, entry 2) as the solvent. It was clear that SeO 2 induced allylic hydroxylation in 334 to form 448 and hence, we tried reacting 334 with SeO 2 in the identical reaction conditions which produced 448 in 72% yield. As the formation of 448 is one of the important findings, we changed the reaction conditions little bit to raise the yield of 448 by carrying out the reaction directly with cholesterol ( Table 2 ). Firstly, we conducted the reactions with longer period, viz., 48 h, 72 h and 96 h. Among these conditions, maximum of 46% yield was obtained at 72 h (entry 2). The composition of the reactants along with reaction time was then also varied taking different mole ratios of cholesterol to selenium dioxide. Interestingly, it was seen that 1:7 mole ratio of the reactants furnished better result (Table 2, entry 5) than the 1:3.5 mole ratio ( Table 1 , entry 2) toward the formation of 448 . However, increased reaction time or, the other mole compositions taking even excess selenium dioxide could not result satisfactory yield of the product 448 (entries 6-8). Besides, we could not isolate diaromatic cholesterol 447 from the reactions in these conditions and the diol 334 was isolated at poor yields. Thus it may be concluded that, though these reaction conditions furnished better results toward the yield of 448 , the conditions rendered unsuitable for the formation of both the aromatized product as well as the diol. Hence it seemed to be advantageous to have 448 via 334.

The reactions of 4 b-hydroxy steroids (i.e., the diols) with SeO 2 were conducted for 48 h. After usual work up a deep red gummy residue was obtained which seemed apperantly, not to contain any solid product. The corresponding 4 b,7 a-dihydroxy steroids (i.e., the triols) were finally purified from it after repeated column chromatography.

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Table 2.2 Optimization of the yield of 448 directly from cholesterol

Entry Reactant composition a Reaction condition b Yield (%) c

1 1: 3.5 100oC, 48 h 32 d

2 1: 3.5 100oC, 72 h 46

3 1: 3.5 100oC, 96 h 43

4 1: 3.5 Reflux, 1h 20 d

5 1: 7 100oC, 24 h 44

6 1: 7 100oC, 48 h 37

7 1: 10.5 100oC, 24h 38

8 1: 14 100oC, 48 h 28

aMole ratio of cholesterol to selenium dioxide; on 580 mg, 1.5 mmol of cholesterol bAll the reactions were carried out in 1,4-dioxane: water = 50:1. cAromatized product 447 were not found, diol 334 were isolated at <6% yield except entries 1 and 4. d334 was isolated at 10% and 28% yield respectively from entry 1 and 4.

II.C.2.4 Application of the reaction protocol: syntheses of the diaromatic natural ergosterols In 2004, Qin and Liu, isolated two aromatized ergosterol derivatives ( 457 and 458) from the ascomycete Daldinia concentrica , and described the long-sought after biological precursor steroids for organic matter in Earth’s subsurface. Of the two compounds isolated, the latter, 458, bears an unusual methyl group at position 1. According to the authors, the aromatized products arise due to microbial action on the precursor molecule ergosterol. 115 Prior to its isolation from nature, diaromatic ergosterol 457 was synthesized earlier in 1966 (Figure 2.5) .116

R

457; R= H 458; R= CH3

Figure 2.5 Natural diaromatic ergosterols.

87

Having the natural diaromatic ergosterol analogue of cholesterol, we obviously were interested to apply the reaction protocol on ergosterol ( 83). As anticipated, the reaction furnished both the natural compounds ( Scheme 2.32; 457 and 458) in relatively better yields compared to cholesterol. It is important to mention here that when we formed 457 from ergosterol, attention was directed to the reaction of cholesterol. The reaction was conducted (with cholesterol) again at 2h, 6h and 12h. And it was found that although 1H NMR indicated the formation of the corresponding analogue, the amount was negligible (see supporting spectra, Figure 2.39 – 2.43). Of note, the fact that the major product was 457 (12%) rather than 458 (1.3%) provided plausible mechanistic hints of the involvement of the 7-dehydro skeleton in the formation of 1-hydro derivative ( 457) rather than 1-methyl product ( 458 ). However, we could not isolate 458 as a single compound rather it was in a mixture with 9 (9:1, by NMR). 18 In order to optimize the yield, different reaction conditions were explored using selective solvents ( Table 2 ). Ethanol and 2-methoxy ethanol failed to furnish 457 and/ or 458. However, the synthesis of these naturally occurring compounds using our methodology ruled out the possibility of incidental formation of the aromatized cholesterol analogue in our previous experiments.

R1 R1 R

SeO H H 2 HO Dioxane 83 100oC, 6h 457; R = H, 12% R1= 458; R = CH3, 1.3%

Scheme 2.32 Synthesis of natural diaromatic ergosterol derivatives 457 and 458.

II.C.2.5 Mechanistic concern towards aromatization Again , it is noted that the yield of 447 is rather poor (maximum reproducible yield 6%) compared to those of the other two products ( 334 and 448). As has been pointed out earlier, the formation of 447 is very significant because the reaction (of SeO 2 and cholesterol) involves a selective aromatization of rings A and B of the tetracyclic skeleton with simultaneous regioselective formation of a double bond between C-14 and C-15.

88

Table 2.3 Optimization a of the yield of the natural product 9

Entry Solvent System Reaction condition Yield (%) 457 458 1 1,4-Dioxane: water (50:1) a) 100ºC, 2 h 5.5 < 1

b) 100ºC, 6 h 12 1.3

c) 100ºC, 10 h 7 < 1

2 Ethanol Reflux, 24 h NF b NF b

3 2-Methoxy ethanol 100ºC, 24 h NF b NF b a Thorough solvent study of the reaction of cholesterol with SeO 2 (Table 1 above) has prompted us to choose selective solvents and reaction conditions. bNF = Not found.

On the basis of the previous literature,68-70 it may be anticipated that the aromatization is accompanied with the methyl migration (from 13, 14 and 83) as well as demethylation (from 83) of the methyl situated at the ring juncture (C-19) in the starting materials. The solvent selectivity suggests a possible explanation towards the regioselective dehydrogenation. It was assumed that a transient dioxonium intermediate of the ethers may be formed with selenium moiety offering the furnished regioselectivity. 4b-Hydroxy cholesterol ( 334) did not furnish the corresponding diaromatic derivative 447 whereas ergosterol ( 83, possessing C5=C6 as well as C7=C8 double bond in ring-B) yielded the diaromatic ergosterol derivatives ( 457 and 458). So there exists a possibility of the involvement of A-ring and more specifically the 4 b-hydrogen towards the formation of the aromatized products. Under identical reaction conditions, neither the diol 334 nor the triol 448 could be converted to the aromatized product 447. Rather 334 produced, as mentioned previously, 448 in a very good yield whereas 448 did not react at all. The mechanism for the conversion of 13 to 447 is unclear at this stage, although it may be concluded that hydroxylation (as in 334 and 448) is, apparently, not a precursor to form 447. Moreover, since the yield of 447 does not vary substantially when prepared from 13 or its acetate/benzoate derivatives under identical conditions ( Scheme 2.31 ), the groups at the 3 b position are not likely acting as leaving groups in these reactions. However, a detail experimental investigation with a number of differently substituted starting materials may lead to a clear mechanistic approach for the aromatization process.

89

II.C.2.6 Further extention To extend the scope of the protocol, we carried out analogous reactions on b-sitosterol ( 14, Scheme 2.30 ) and diosgenin ( 459, Scheme 2.33 ) under identical conditions. On reaction, b- sitosterol, like cholesterol, produced (in 1,4-dioxane at 100ºC) the aromatic analogue ( 453, 7%), 4b-hydroxy b-sitosterol ( 454, 51%) and 4 b,7 a-dihydroxy b-sitosterol ( 455, 22%). By contrast, diosgenin (both at elevated temperature and microwave induced solvent-free conditions, Scheme 2. 4) yielded 4 b-hydroxy diosgenin ( 335, 30-58%) and 4 b,7 a-dihydroxy diosgenin ( 460, trace- 17%) but no aromatic analogue. Conversions of the corresponding diols ( 454 and 335) to the triols were also achieved in high yields (455, 72%; 460, 69%).

O

O H SeO2 H H a or b + H H HO HO OH HO OH OH

459 335 460 a= 1,4-dioxane, 100oC, 24 h 58% 17% b= on silica, MW, 600W 30% Trace

Scheme 2.33 Action of SeO 2 on diosgenin ( 459 ).

II.C.2.7 Description of the molecular structures of 447 and 456. (The numbering of the carbon atoms in this description follows the X-ray structure atom labeling as depicted on Figure 2.6 and Figure 2.7)

Figure 2.6 Molecular structure of 447 and atom labeling.

90

The molecular structure of 447, Figure 2.6, has been established unambiguously by X-ray crystallography on crystals grown from the slow evaporation of its chloroform/methanol solution held at room temperature. The crystallographic analysis confirms the spectroscopic results and determines the C16=C17 double bond distance as 1.333(3) Å (of note, according to the usual numbering of the carbon atoms of the steroid skeleton, this is actually C14=C15 double bond). The five-membered ring is not planar, rather it is an envelope with the flap atom being C19. The d1 puckering parameters for this ring are q 2 = 0.274(3) Å and f2 = 143.5(5)º. The only non-planar six-membered ring (C8,C9,C12-C14,C16) is in the form of a half-chair with puckering parameters: Q = 0.472(2) Å, q = 50.9(2)º and f = 210.3(4)º. The dihedral angle formed between the two unsaturated rings is 3.44(11)º. In the side arm, the methylene groups are in the open or extended form as seen in the values of the C20 –C22 –C23 –C24 and C22 –C23 –C24 –C25 torsion angles of -174.2(2) and 175.4(2)º, respectively.

Figure 2.7 Molecular structure of 456 and atom labeling.

The molecular structure determination of 456, Figure 2.7, establishes the molecular connectivity for the crystal grown from the slow evaporation of its chloroform/methanol solution held at room temperature. The five-membered ring is twisted about the C18 –C19 bond and has d1 puckering parameters q2 = 0.437(2) Å and f2 = 204.0(3)º. Each of the six-membered rings (C1-C6) and C(12,C13, C18-C21) adopt very nearly chair conformations while that for the (C3,C4,C12-C15) is in the form of a half-chair with puckering parameters: Q = 0.494(2) Å, q = 133.2(2)º and f = 323.7(3)º. In the (C1-C6) ring the O1- and O3-acetyl groups occupy equatorial and axial positions, respectively, whereas with reference to the (C3,C4,C12-C15) ring, O5-acetyl group is axial. As with the side chain in 447, in 456 , it adopts an extended

91 conformation with C25 –C26 –C28 –C29 and C28 –C29 –C30 –C31 torsion angles of 177.73(18) and 173.8(2)º, respectively. Crystallographic data and final refinement details are given in Table 3. Table 2.4 Crystallographic data and refinement details for 447 and 456 Compound 447 456

Formula C27 H36 C33 H52 O6 Formula weight 360.56 544.75 Crystal system orthorhombic monoclinic

Space group P212121 P21 a/Å 5.9494(7) 12.0015(11) b/Å 11.5746(12) 10.1282(9) c/Å 31.351(3) 12.7563(11) b,/° 90 101.335(1) V/Å 3 2158.9(4) 1520.3(2) Z 4 2 -3 Dc/g cm 1.109 1.190 F(000) 792 596 m(Mo Ka)/mm -1 0.062 0.080 Measured data 20266 14690 q range/° 2.2 – 27.5 1.6 – 27.5 Unique data 2864 3699 Observed data ( I ³ 2.0s(I)) 2202 3225 R, obs. data; all data 0.047; 0.073 0.037; 0.047 a; b in weighting Scheme 2. 0.078; 0 0.055; 0.087

Rw, obs. data; all data 0.111; 0.125 0.089; 0.094

II.D Experimental II.D.1 General Melting points were measured in open capillary methods and were uncorrected. The FAB Mass spectra were recorded on a Jeol SX 102/Da-600 mass spectrometer/ Data System using Argon/ Xenon as the FAB gas. The DART-MS was recorded on a JEOL-AccuTOF JMS-T100LC mass

92 spectrometer having a DART (Direct Analysis in Real Time) source. 1H NMR and 13 C NMR spectra were recorded on Brucker Avance 300MHz FT-NMR spectrometer using 5 mm BBO probe. Either CDCl 3 or DMSO-d6 or CD 3COCD 3 was used as solvent and TMS as reference material. Data are presented as follows: Chemical shift -in ppm on the scale relative to δ TMS = 0; coupling constant- J/Hz. Elemental analysis was performed using a Vario EL-III elementary analyser. Infrared spectra were recorded on Shimudzu FT-IR 8300 Spectrometer as neat or thin films (KBr or Nujol) as indicated in the experimental procedures, and at room temperature. Frequencies are given in wave numbers (cm -1). For column chromatography silica gel G, 60-120 mesh was used with petroleum ether- ethyl acetate mixture as the eluent. For thin layer chromatography (TLC), freshly made silica gel plates (using silica gel for TLC + petroleum ether) were used and visualization was achieved by staining with iodine.

II.D.2 X-ray crystallography 117-124 Intensity data for 447 and 456 were measured at 100 K on a Bruker SMART APEX diffractometer with Mo K a radiation. Data processing (APEX2 and SAINT) 1x and absorption correction for 447 (SADABS) 2x were accomplished by standard methods. The structures were solved by direct-methods with SHELXS-97 3x and refinement (anisotropic displacement parameters, hydrogen atoms in the riding model approximation and a weighting Scheme 2. of the 2 2 2 2 2 2 3x form w = 1/[ s (Fo ) + ( aP ) + bP ] for P = ( Fo + 2 Fc )/3) was on F by means of SHELXL-97. The absolute structures of both 447 and 456 could not be determined experimentally and so for each, it was assigned based on that of cholesterol. As such, 2082 ( 447) and 3934 ( 456 ) Friedel pairs were averaged in the final refinements. For the refinement of 447, high thermal motion was displayed by the C27 atom. However, multiple sites were not resolved for this atom. For the refinement of 456 , the O4 atom displayed elongated anisotropic displacement parameters and hence, this atom was refined with the ISOR command in SHELXL-97.3x Fig. 1 and 2 were drawn with ORTEP 4x at the 50% probability level. Data manipulation and interpretation were with WinGX 5x and PLATON 6x .

II.D.3 Representative procedure for the oxidation reactions To a solution of 13 (770 mg, 2 mmol) in dioxane (15 mL) was added selenium dioxide (777 mg, 7 mmol), the mixture was heated at 100 0C for 24h. The reaction mixture was then cooled and the

93 black selenium deposited was filtered off. To the filtrate ether (50 mL) was poured and was washed successively with water and then with saturated brine solution, dried over Na 2SO 4 and concentrated in vacuo to give a reddish gummy residue. The compounds presented therein, were then separated by column chromatography eluted successively by petroleum ether, petroleum ether/ ethyl acetate= 17:3 and petroleum ether/ ethyl acetate= 3:2 to afford 447 (44 mg, 6%), 334 (403 mg, 50%) and 448 (217 mg, 26%) respectively. The proportion of reactants to selenium dioxide, for the other oxidation reactions, was kept constant throughout the various experiments.

II.D.4. Product characterization II.D.4.1 1-Methyl-19-norcholesta-1,3,5(10),6,8(9),14(15)-hexaene (447). Needle shaped white 0 1 crystals (CHCl 3 -MeOH), mp 96-97 C; 3-6% yield; R f (petroleum ether) 0.95; H NMR (300

MHz, CD 3COCD 3): 0.89 (d, J=6.6 Hz, 6H), 1.05 (s, 3H), 1.08 (d, J= 6.0 Hz, 3H), 2.50-2.59 ( m, 1H), 2.95 (s, 3H), 3.55-3.59 (m, 2H), 6.23 (s, 1H), 7.25 (m, 2H), 7.59-7.64 (m, 2H), 7.72 (d, 13 J=9.0 Hz, 1H); C NMR (75 MHz, CDCl 3): 14.8 (CH 3), 19.0 (CH3), 22.6 (CH 3), 22.8 (CH3),

23.8 (CH2), 27.3 (CH3), 28.0 (CH), 28.7 (CH 2), 34.1(CH), 35.9 (CH 2), 36.1 (CH 2), 37.2 (CH2),

39.6 (CH2), 44.4 (C), 57.5 (CH), 120.4 (CH), 123.2 (CH), 124.8 (CH), 127.5 (CH), 127.8 (CH), 129.2 (C), 130.7 (CH), 132.5 (C), 133.7 (C), 134.6 (C), 135.5 (C), 148.8 (C); IR (Nujol, cm -1): 3057, 1380, 1365, 1278, 1257, 1103, 1060, 987, 935, 894, 821, 788, 754. DART-MS (ESI+), m/z: 362 ([M+2H] +, 27%), 361([M+ H] +, 100), 360 ([M] +, 30), 359 (23), 347 (5). Elemental analyses: Found: C, 89.88; H, 10.15. C 27 H36 requires C, 89.93; H, 10.07%.

II.D.4.2 4b-Hydroxycholesterol (or, 3 b,4b-dihydroxycholest-5-ene, 334). Needle shaped 0 white crystals (CHCl 3-MeOH), mp 168-170 C; 10-75% Yield; R f (30% ethyl acetate/petroleum 1 ether) 0.45; H NMR (300 MHz, CDCl 3): 0.68 (s, 3H), 0.86 (d, J=6.0 Hz, 6H), 0.90 (d, J=6.3 Hz), 1.18 (s, 3H), 1.25-1.66 (m, 13H), 1.81-2.10 (m, 8H), 3.54-3.56 (m, 1H), 4.13 (s, 1H), 5.67 13 (s, 1H); C NMR (75 MHz, CDCl 3): 11.9 (CH 3), 18.7 (CH3), 20.5 (CH 2), 21.1 (CH3), 22.6

(CH3), 22.8 (CH3), 23.8 (CH2), 24.3 (CH2), 25.4 (CH2), 28.0 (CH), 28.2 (CH 2), 31.9 (CH), 32.1

(CH2), 35.8 (CH), 36.0 (C), 36.2 (CH 2), 36.9 (CH2), 39.5 (CH2), 39.7 (CH2), 42.3 (C), 50.2 (CH), 56.1 (CH), 56.9 (CH), 72.5 (CH), 77.3 (CH), 128.8 (CH), 142.8 (C); IR (KBr, cm -1): 3382 (br.), 1168, 978; FAB-MS (ESI+), m/z: 402 (12%), 401 (11), 399 (13), 386 (29), 385 (100), 384 (50),

94

383 (48), 368 (28), 367 (62). Elemental analyses: Found: C, 80.44; H, 11.59. C 27 H46 O2 requires C, 80.53; H, 11.52%.

II.D.4.3 4b,7 a-Dihydroxycholesterol (or, 3 b,4b,7 a-trihydroxycholest-5-ene, 448). White 0 1 amorphous solid, mp 193-194 C; 5-78% Yield; R f (70% ethyl acetate/petroleum ether) 0.55; H

NMR (300 MHz, CDCl 3): 0.69 (s, 3H), 0.86 (d, J= 6.6 Hz, 6H), 0.92 (d, J= 6.6 Hz, 3H), 1.18 (s, 3H), 3.57-3.65 (m, 1H), 3.94 (t, J=3.0 Hz, 1H), 4.18 (d, J= 3.0 Hz, 1Hz), 5.86 (d, J=3.0 Hz, 1H); 13 C NMR (75 MHz, CDCl 3): 11.6 (CH 3), 18.7 (CH 3), 19.4 (CH3), 20.1 (CH 2), 22.6 (CH 3), 22.8

(CH3), 23.8 (CH2), 24.4 (CH2), 25.1 (CH2), 28.0 (CH), 28.3 (CH 2), 35.8 (CH), 36.2 (CH 2), 36.7

(CH2), 37.0 (C), 37.6 (CH), 39.1 (CH 2), 39.5 (CH 2), 42.1 (C), 42.6 (CH), 49.4 (CH), 55.9 (CH), 65.3 (CH), 72.1 (CH), 77.0 (CH), 129.7(CH), 147.0 (C); IR (KBr, cm -1): 3349, 1153, 1066, 965. DART-MS (ESI+), m/z: 402 (6%), 401 (26), 385 (5), 384 (28), 383 (100), 366(4), 365(10).

Elemental analyses: Found: C, 77.37; H, 11.17. C 27 H46 O3 requires C, 77.45; H, 11.08%.

II.D.4.4 1-Methyl-19-nor b-sitosta-1,3,5(10),6,8(9),14(15)-hexaene (453). Needle shaped 0 1 white crystals (CH 2Cl 2-MeOH), mp 87-88 C, 7% yield; R f (petroleum ether) 0.95; H NMR

(300 MHz, CDCl 3): 0.87 (d, J=7.2 Hz, 6H), 0.94 (d, J=7.2 Hz, 3H), 1.07 (s, 3H), 1.10 (s, 3H), 1.15-1.35 (m, 6H), 1.39-1.60 (m, 4H), 1.68-1.74 (m, 4H), 2.19-2.35 (m, 2H), 2.94 (s, 3H), 3.51- 3.55 (m, 2H), 6.15 (s, 1H), 7.20-7.25 (m, 2H), 7.55-7.60 (m, 2H), 7.66 (d, J=8.7 Hz, 2H); 13 C

NMR (75 MHz, CDCl 3): 12.0, 14.8, 19.0, 19.9, 23.1, 26.0, 27.3, 28.7, 29.2, 29.4, 33.9, 34.5, 36.0, 37.2, 44.5, 45.9, 57.4, 120., 123.2, 124.8, 127.6, 127.8, 129.2, 130.7, 132.5, 133.7, 134.6, 135.6, 148.9. IR (Nujol, cm -1): 3049, 1367, 1278, 1112, 987, 896, 821, 844, 789, 755. Elemental analyses: Found: C, 89.69; H, 10.31. C 29 H40 requires C, 89.62; H, 10.38%.

II.D.4.5 4b-Hydroxy-b–sitosterol (or, 3 b,4b-dihydroxy-b-sitost-5-ene, 454). Needle 0 shaped white crystals (CHCl 3-MeOH), mp 162-164 C, 51% yield; Rf (30% ethyl 1 acetate/petroleum ether) 0.45; H NMR (300 MHz, CDCl 3): 0.68 (s, 1H), 0.81 (d, J= 6.9 Hz, 3H), 0.86 (d, J= 6.9 Hz, 6H), 0.92 (d, J= 6.6 Hz, 3 H), 1.18 (s, 3H), 3.51-3.61 (m, 1H), 4.15 (s, 13 1H), 5.68 (s, 1H); C NMR (75 MHz, CDCl 3): 11.9, 12.0, 18.8, 19.0, 19.8, 20.5, 21.1, 23.1, 24.3, 25.4, 26.0, 28.2, 29.1, 31.8, 32.1, 33.9, 36.0, 36.1, 36.9, 39.7, 42.3, 45.8, 50.2, 56.0, 56.9,

95

72.5, 77.3, 128.8, 142.8; IR (Nujol, cm -1): 3392, 1172, 1069, 977; Elemental analyses: Found: C,

80.89; H,11.66. C 29 H50 O2 requires C, 80.86; H, 11.71%.

II.D.4.6 4b,7 a-Dihydroxy b-sitosterol (or, 3 b,4b,7 a-trihydroxy-b-sitost-5-ene, 455). 0 1 White solid. mp 192-193 C; 22-72% yield; Rf (70% ethyl acetate/petroleum ether) 0.55; H

NMR (300 MHz, DMSO-d6):0.63 (s, 3H), 0.79 (d, J=6.3 Hz, 3H), 0.83 (d, J=6.0 Hz, 6H), 0.91 (d, J=6.3 Hz, 3H), 1.07 (s, 3H), 3.26-3.38 (m, 1H), 3.64 (t, J=3.0 Hz, 1H), 3.89 (d, J=3 Hz, 1H), 13 5.58 (d, J=4.8Hz, 1H); C NMR (75 MHz, DMSO-d6): 11.9, 12.2, 12.3, 19.1, 19.4, 19.6, 20.2, 23.0, 24.2, 25.4, 25.8, 28.4, 29.1, 33.8, 36.0, 36.9, 37.4, 37.9, 41.9, 42.3, 45.6, 49.4, 55.8, 63.8, 72.1, 77.1, 129.2, 145.7; IR (Nujol, cm -1): 3392, 1165, 1078, 967. Elemental analyses: Found: C,

77.88; H, 11.32. C 29 H50 O3 requires C, 77.96; H, 11.29 %.

II.D.4.7 3b,4 b,7 a-Triacetoxycholest-5-ene (456). Colourless cubic crystals (CHCl 3- 0 1 MeOH), mp 170-171 C; 94% yield from 4a ; R f (5% ethyl acetate/petroleum ether) 0.75; H

NMR (300 MHz, CDCl 3): 0.66 (s, 3H), 0.86 (d, J=6.3 Hz, 6H), 0.92 (d, J=6.6 Hz, 3H), 1.14 (s, 3H), 2.01 (s, 3H), 2.04 (s, 3H), 2.06 (s, 3H), 4.77-4.82 (m, 1H), 5.06 (t, J=4.5 Hz, 1H), 5.52 (s, 13 1H), 5.93 (d, J=6.0 Hz, 1H); C NMR (75 MHz, CDCl 3): 11.4 (CH 3), 18.8 (CH 3), 20.2 (CH2),

21.2 (CH3), 21.3 (CH3), 21.6 (CH3), 22.3 (CH 2), 22.6 (CH3), 22.8 (CH3), 23.9 (CH2), 24.1 (CH2),

28.0 (CH), 28.1 (CH 2), 35.7 (CH), 35.8 (CH), 36.2 (CH 2), 36.3 (CH2), 37.0 (C), 38.9 (CH 2), 39.5

(CH2), 42.2 (CH), 43.5 (CH), 49.0 (CH), 55.9 (CH), 67.6 (CH), 72.2 (CH), 75.0 (CH), 128.4 (CH), 143.9 (C), 169.7 (C), 170.2 (C), 170.4 (C); IR (Nujol, cm -1): 1734, 1365, 1246, 1044, 1012, 976, 941, 889. DART-MS (ESI+), m/z: 485 (6%), 426 (29), 425(100), 384 (5), 383 (17),

366 (5), 365 (16). Elemental analyses: Found: C, 79.69; H, 10.62. C 33 H52 O3 requires C, 79.77; H, 10.56%.

II.D.4.8 3b-Acetoxy-4b-hydroxycholest-5-ene (449). White solid, mp 175-1760C, 35% 1 yield; Rf (15% ethyl acetate/petroleum ether) 0.45; H NMR (300 MHz, CDCl 3): 0.68 (s, 3H), 0.86 (d, J=6.6 Hz, 6H), 0.91 (d, J=6.6 Hz, 3H), 1.22 (s, 3H), 2.11 (s, 3H), 4.25 (s, 1H), 4.69-4.76 13 (m, 1H), 5.71 (s, 1H); C NMR (75 MHz, CDCl 3): 11.9 (CH 3), 18.7 (CH 3), 20.5 (CH 2), 21.1

(CH3), 21.4 (CH3), 21.7 (CH2), 22.6 (CH3), 22.8 (CH 3), 23.8 (CH2), 24.2 (CH2), 28.0 (CH), 28.2

(CH2), 31.7 (CH), 32.1 (CH 2), 35.8 (CH), 36.2 (CH 2), 36.6 (C), 36.9 (CH 2), 39.5 (CH2), 39.6

96

(CH2), 42.3 (C), 50.2 (CH), 56.1 (CH), 56.8 (CH), 75.5 (CH), 75.6 (CH), 129.5 (CH), 141.5 (C), 170.2 (C); IR (Nujol, cm -1): 3412, 1737, 1279, 1046. DART-MS (ESI+), m/z: 429 (11%), 428 (61). 427 (100%), 385(16), 368 (18), 367 (58). Elemental analyses: Found: C, 78.39; H, 10.79.

C29 H48 O3 requires C, 78.31; H, 10.89%.

II.D.4.9 3b-Acetoxy-6a-hydroxycholest-4-ene (451). White solid, mp 139-1400C, 32% 1 yield; Rf (20% ethyl acetate/petroleum ether) 0.45; H NMR (300 MHz, CDCl 3): 0.68 (s, 3H), 0.87 (d, J=6.6 Hz, 6H), 0.91 (d, J=6.6 Hz, 3H), 1.18 (s, 3H), 2.08 (s, 1H), 3.60-3.69 (m, 1H), 13 5.38 (d, J= 2.7 Hz, 1H), 5.85 (d, J=3.0 Hz, 1H); C NMR (75 MHz, CDCl 3): 11.8 (CH 3), 18.7

(CH3), 20.4 (CH3), 20.6 (CH2), 21.6 (CH3), 22.6 (CH3), 22.8 (CH 3), 23.8 (CH2), 24.2 (CH2), 25.8

(CH2), 28.0 (CH), 28.2 (CH 2), 31.6 (CH), 32.1 (CH 2), 35.8 (CH), 36.0 (C), 36.2 (CH2), 36.8

(CH2), 39.5 (CH2), 39.6 (CH 2), 42.3 (C), 50.2 (CH), 56.1 (CH), 56.8 (CH), 71.7 (CH), 79.3 (CH), 128.8(CH), 138.8 (C), 171.2 (C); IR (Nujol, cm -1): 3398, 1738, 1260, 1237, 1074; DART- MS (ESI+), m/z: 428 (14%), 427 (43), 385 (77), 368 (29). Elemental analyses: Found: C, 78.38;

H, 10.96. C 29 H48 O3 requires C, 78.31; H, 10.89%.

II.D.4.10 3b-Benzoxy-4b-hydroxycholest-5-ene (450). White feather like crystals (CHCl 3- 0 1 MeOH), mp 202-204 C, 37% yield; Rf (10% ethyl acetate/petroleum ether) 0.55; H NMR (300

MHz, CDCl 3): 0.74 (s, 3H), 0.92 (d, J=6.3 Hz, 6H), 0.97 (d= 6.3 Hz, 3H), 1.31 (s, 3H), 2.21 (s, 1H), 4.45 (s, 1H), 5.01-5.06 (m, 1H), 5.79 (s, 1H), 7.49 (t, J=7.5 Hz, 2H), 7.61 (t, J=6.6 Hz, 1H), 13 8.11 (d, J=7.5 Hz, 2H); C NMR (75 MHz, CDCl 3): 11.3, 18.1, 20.0, 20.5, 21.3, 22.0, 22.3, 23.3, 23.7, 27.5, 27.7, 31.2, 31.6, 35.2, 35.6, 35.7, 36.5, 39.0, 39.1, 41.8, 49.7, 55.5, 56.3, 75.1, 75.5, 127.8 (2), 129.1 (3), 129.7, 132.5, 140.9, 165.1; IR (Nujol, cm -1): 3533(sharp), 1694, 1682, 1282, 1127, 1068, 1026, 967, 920, 845, 709; DART-MS (ESI+), m/z: 491 (7%), 490 (37), 489

(100), 368 (3), 367 (10). Elemental analyses: Found: C, 80.65; H, 9.84. C 34 H50 O3 requires C, 80.57; H, 9.95%.

II.D.4.11 3b-Benzoxy-6a-hydroxycholest-4-ene (452). Needle shaped white crystals 0 1 (CHCl 3-MeOH), mp 142-144 C, 34% yield; Rf (12% ethyl acetate/petroleum ether) 0.55; H

NMR (300 MHz, CDCl 3): 0.66 (s, 3H), 0.86 (d, J= 6.6 Hz, 6H), 0.91 (d, J=6.3 Hz, 3H), 1.20 (s, 3H), 3.75 (t, J= 6 Hz, 1H), 5.67 (s, 1H), 5.93 (d, J= 3 Hz, 1H), 7.44 (t, J= 7.5, 2H), 7.55 (t, J= 7.2

97

13 Hz, 1H), 8.04 (d, J= 8.1 Hz, 2H); C NMR (75 MHz, CDCl 3): `11.8, 18.7, 20.7, 22.6, 22.8, 23.8, 24.2, 26.0, 28.0, 28.2, 31.7, 32.1, 35.8, 36.0, 36.2, 36.8, 39.5, 39.6, 42.3, 50.2, 56.1, 56.8, 72.0, 80.0, 128.4, 128.4, 129.6, 129.6, 130.5, 132.0, 133.0, 138.9, 166.8; IR (Nujol, cm -1): 3442, 3392, 1686, 1674, 1273, 1170, 1110, 977, 899, 759, 711. DART-MS (ESI+), m/z: 491 (4%), 490 (36), 489 (100), 386 (26), 385 (89), 368 (24), 367 (83). Elemental analyses: Found: C, 80.48; H,

9.86. C34 H50 O3 requires C, 80.57; H, 9.95%.

II.D.4.12 4b-Hydroxy diosgenin (or, 3 b,4b-dihydroxyspirost-5-ene, 335). White solid, 0 1 mp 171-173 C, 30-58% yield; R f (30% ethyl acetate/petroleum ether) 0.45; H NMR (300 MHz,

CDCl 3): 0.79 (d, J=3.0 Hz, 3H), 0.80 (s, 3H), 0.97 (d, J=6.8 Hz, 3H), 1.21 (s, 3H), 2.32 (s, 1H), 2.43 (d, J= 6Hz, 1H), 3.37 (t, J=10.8 Hz, 1H), 3.46 (d, J=3.0 Hz, 1H), 3.48-3.55 (m, 1H), 4.13 (d, J=3Hz, 1H), 4.40 (dd, J=15.0 Hz and 7.5 Hz, 1H), 5.66 (d, J=2.7 Hz,1H); 13 C NMR (75 MHz,

CDCl 3): 14.5 (CH 3), 16.3 (CH 3), 17.1 (CH3), 20.3 (CH 2), 21.0(CH 3), 25.3 (CH 2), 28.8 (CH2),

30.3 (CH), 31.4 (CH 2), 31.8 (CH 2), 32.2 (CH), 36.2 (C), 36.9 (CH 2), 39.7 (CH2), 40.3 (C), 41.6

(CH), 50.1 (CH), 56.6 (CH 2), 62.0 (CH), 66.9 (CH 2), 72.4 (CH), 77.2 (CH), 80.8 (CH), 109.3 (C), 128.3 (CH), 142.9 (C); IR (Nujol, cm -1): 3392, 1169, 1047, 976. FAB-MS, m/z: 432 (37%), 431(90), 430(39), 429(77), 414(49), 413(100), 412(27), 411(28), 395(25). Elemental analyses:

Found: C, 75.21; H, 9.79. C 27 H42 O4 requires C, 75.29; H, 9.84 %.

II.D.4.13 4b,7 a-Dihydroxy diosgenin (or, 3 b,4b,7 a-trihydroxyspirost-5-ene, 460). 0 1 White solid, mp 201-202 C, 17-69% yield; Rf (70% ethyl acetate/petroleum ether) 0.50; H NMR

(300 MHz, CDCl 3): 0.79 (d, J=3.0 Hz, 3H), 0.80 (s, 3H), 0.98 (d, J=6.9 Hz, 3H), 1.20 (s, 3H), 3.37 (t, J=10.8 Hz, 1H), 3.45-3.49 (m, 1H), 3.52-3.64 (m, 1H), 3.94 (t, J=4.5 Hz, 1H), 4.09-4.19 (m, 1H), 4.48 (dd, J=15.0 Hz and 7.5 Hz, 1H), 5.86 (d, J= 4.8 Hz, 1H); 13 C NMR (75 MHz,

CDCl 3): 14.6 (CH 3), 16.1 (CH 3), 17.2 (CH 3), 19.4 (CH 3), 20.0 (CH 2), 25.1 (CH 2), 28.8 (CH2),

30.3 (CH), 31.4 (CH 2), 31.9 (CH2), 36.6 (CH 2), 37.2 (CH), 39.1 (CH 2), 40.4 (C), 41.7 (CH), 42.5

(CH), 49.0 (CH), 62.0 (CH), 65.2 (CH), 66.9 (CH 2), 72.1 (CH), 76.9 (CH), 80.8 (CH), 109.3 (C), 129.6 (CH), 146.9 (C); IR (Nujol, cm -1): 3395, 1172, 1054, 978. Elemental analyses: Found: C,

72.49; H, 9.41. C 27 H42 O5 requires C, 72.59; H, 9.48%.

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II.D.4.14. 19-Norergosta-1,3,5,7,9,14,22-heptaene (457). Pale yellow needles, mp 125- o 1 126 C (Chloroform- methanol), 5.5-12% yield; R f (petroleum ether) 0.95; H NMR (300 MHz,

CDCl 3): 0.84 (d, J= 4.2 Hz, 3H), 0.88 (d, J= 4.2, 3H), 0.95 (s, 3H), 0.98 (d, J= 5.1, 3H), 1.15 (d, J= 6.6 Hz, 3H), 1.52 (m, 1H), 1.76 (m, 1H), 1.90 (m, 1H), 1.61 and 2.33 (m, 2H), 2.33 (m, 1H), 2.21 and 2.36 (m, 2H), 3.14 and 3.29 (m, 2H), 5.24 (m, 1H), 5.30 (m, 1H), 6.12 (t, J=3 Hz, 1H), 7.42 (m, 1H), 7.45 (m, 1H), 7.50 (m, 1H), 7.64 (m, 1H), 7.77 (d, J= 7.8 Hz, 1H), 8.01 (d, J= 9.0 13 Hz, 1H); C NMR (75 MHz, CDCl 3): 15.4 (CH 3), 17.7 (CH3)19.7 (CH 3), 20.0 (CH 3), 21.2(CH 3),

23.7 (CH 2), 33.1 (CH), 36.5 (CH 2), 36.9 (CH2), 39.0 (CH), 42.9 (CH), 45.2 (C), 57.2 (CH), 120.7 (CH), 123.6 (CH), 123.7(CH), 125.2 (CH), 126.1 (CH), 126.3 (CH), 128.5 (CH),128.5 (C), 130.2 (C), 132.5 (CH), 132.8 (2C), 135.3 (CH) , 148.1 (C). These spectral data match that originally reported by Qin and Liu 115 , a summary of which is found below in Table 1

II.D.5 Comparison of NMR Spectral Data of Natural and Synthetic Diaromatic Ergosterol Table 2.5 NMR Spectral Data Comparison of Natural and Synthetic Diaromatic Ergosterol (457) in

CDCl 3; Coupling Constants ( J) in Hz.

Position 1H NMR 13 C NMR Synthetic diaromatic Natural diaromatic Synthetic diaromatic Natural diaromatic ergosterol ergosterol ergosterol ergosterol (300MHz) (500 MHz) (75 MHz) (125 MHz) 1 8.01 d, 9.0 7.97 d, 8.4 123.7 CH 123.7 CH 2 7.45 m 7.45 m 126.1 CH 126.0 CH 3 7.42 m 7.39 m 125.2 CH 125.2 CH 4 7.76 d, 7.8 7.73 d, 8.0 128.5 CH 128.4 CH 5 132.8 C 132.7 C 6 7.50 m 7.58 m 126.1 CH 126.3 CH 7 7.64 m 7.63 m 123.6 CH 123.6 CH 8 128.5 C 128.4 C 9 130.2 C 130.1 C 10 132.8 C 132.7 C

11 3.29 m; 3.14 m 3.27 m; 3.10 m 23.7 CH2 23.7 CH2

12 2.33 m; 1.61 m 2.32 m; 1.65 m 36.5 CH2 36.5 CH2 13 45.2 C 45.2 C 14 148.1 C 148.1 C 15 6.12 t, 3.0 6.09 bs 120.7 CH 120.7 CH

16 2.36 m; 2.21 m 2.36 m; 2.15 m 36.9 CH 2 36.9 CH2

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17 1.76 m 1.73 m 57.2 CH 57.1 CH

18 0.95 s 0.95 s 15.4 CH3 15.4 CH3 20 2.33 m 2.32 m 39.0 CH 39.0 CH

21 1.15 d, 6.6 1.11 d, 6.5 21.2 CH3 21.7 CH3 22 5.24 m 5.22 m 135.3 CH 135.3 CH 23 5.33 m 5.29 m 132.5 CH 132.4 CH 24 1.90 m 1.85 m 42.9 CH 42.9 CH 25 1.52 m 1.46 m 33.1 CH 33.1 CH

26 0.84 d, 4.2 0.79 d, 6.9 20.0 CH3 20.0 CH3

27 0.88 d, 4.2 0.83 d, 6.9 19.7 CH3 19.7 CH3

28 0.98 d, 5.1 0.92 d, 6.8 17.7 CH3 17.7 CH3

II.E Conclusion In summary, we have accomplished the syntheses of natural diaromatic ergosterol derivatives and other steroidal analogues in an unprecedented simple, one-pot and convenient synthetic route. In the process, we have established the key factor as the selectivity of the solvents (having 1,4-ethereal oxygens) towards the formation of the aromatized products. Thorough solvent- dependant study of the model reaction reveals valuable product composition which may be exploited, specially, for the synthesis of biologically important steroid molecules. By using the established solvent-selective steroidal methodology, the yield of the natural product, diaromatic ergosterol ( 457) was optimized at 12%. The same reaction is also found to be an easy and efficient access to the human metabolism research demanded 4 b,7 a-dihydroxy steroids. Furthermore, single crystal X-ray crystallography has resolved the molecular structures, for the first time in their class, of similar diaromatic cholesterol derivative ( 447 ) and triacetylated 4b,7 a-dihydroxy cholesterol derivative ( 456).

II.F Supporting spectra ( 1H NMR, 13 C NMR, DEPT-135 NMR)

100

Figure 2.8 1H NMR Spectrum of 1-methyl-19-norcholesta-1,3,5(10),6,8(9),14(15)-hexaene (447) in

CDCl 3.

Figure 2.9 1H NMR Spectrum of 1-methyl-19-norcholesta-1,3,5(10),6,8(9),14(15)-hexaene (447) in acetone-d6.

101

Figure 2.10 13 C NMR Spectrum of 1-methyl-19-norcholesta-1,3,5(10),6,8(9),14(15)-hexaene (447) in

CDCl 3.

Figure 2.11 DEPT-135 Spectrum of 1-methyl-19-norcholesta-1,3,5(10),6,8(9),14(15)-hexaene (447).

102

Figure 2.12 1H NMR Spectrum of 3b, 4b-dihydroxycholest-5-ene (334).

Figure 2.13 13 C NMR Spectrum of 3b, 4b-dihydroxycholest-5-ene (334 ).

103

Figure 2.14 DEPT-135 NMR Spectrum of 3b, 4b-dihydroxycholest-5-ene (334 ).

Figure 2.15 1H NMR Spectrum of 3b, 4b,7 a-trihydroxycholest-5-ene (448 ).

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Figure 2.16 13 C NMR Spectrum of 3b, 4b,7 a-trihydroxycholest-5-ene (448 ).

Figure 2.17 DEPT-135 NMR Spectrum of 3b, 4b,7 a-trihydroxycholest-5-ene (448 ).

105

Figure 2.18 1H NMR Spectrum of 3b ,4 b,7 a-triacetoxycholest-5-ene (456 ).

Figure 2.19 13 C NMR Spectrum of 3b ,4 b,7 a-triacetoxycholest-5-ene (456 ).

106

Figure 2.20 DEPT-135 NMR Spectrum of 3b ,4 b,7 a-triacetoxycholest-5-ene (456 ).

Figure 2.21 1H NMR Spectrum of 3b-benzoxy-4b-hydroxycholest-5-ene (450).

107

Figure 2.22 13 C NMR Spectrum of 3b-benzoxy-4b-hydroxycholest-5-ene (450).

Figure 2.23 1H NMR Spectrum of 3b-benzoxy-6a-hydroxycholest-4-ene (452).

108

Figure 2.24 13 C NMR Spectrum of 3b-benzoxy-6a-hydroxycholest-4-ene (452).

Figure 2.25 1H NMR Spectrum of 3b, 4b-dihydroxyspirost-5-ene (335).

109

Figure 2.26 13 C NMR Spectrum of 3b, 4b-dihydroxyspirost-5-ene (335).

Figure 2.27 DEPT-135 NMR Spectrum of 3b, 4b-dihydroxyspirost-5-ene (335).

110

Figure 2.28 1H NMR Spectrum of 3b, 4b,7 a-trihydroxyspirost-5-ene (460).

Figure 2.29 13 C NMR Spectrum of3b, 4b,7 a-trihydroxyspirost-5-ene (460).

111

Figure 2.30 DEPT-135 NMR Spectrum of 3b, 4b,7 a-trihydroxyspirost-5-ene (460).

Figure 2.31 1H NMR Spectrum of 1-methyl-19-norb-sitosta-1,3,5(10),6,8(9),14(15)-hexaene (453 ).

112

Figure 2.32 13 C NMR Spectrum of 1-methyl-19-norb-sitosta-1,3,5(10),6,8(9),14(15)-hexaene (453).

Figure 2.33 1H NMR Spectrum of 3b, 4b-dihydroxy-b-sitost-5-ene (454).

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Figure 2.34 13 C NMR Spectrum of 3b, 4b-dihydroxy-b-sitost-5-ene (454).

Figure 2.35 1H NMR Spectrum of 3b, 4b,7 a-trihydroxy-b-sitost-5-ene (455).

114

Figure 2.36 13 C NMR Spectrum of 3b, 4b,7 a-trihydroxy-b-sitost-5-ene (455).

Figure 2.37 1H NMR spectrum of 19-norergosta-1,3,5,7,9,14,22-heptaene (457).

115

Figure 2.38 13 C NMR spectrum of 19-norergosta-1,3,5,7,9,14,22-heptaene (457).

Figure 2.39 1H NMR spectrum of the mixture of 19-norergosta-1,3,5,7,9,14,22-heptaene (457 ) and 1- methylergosta-1,3,5,7,9,14,22-heptaene ( 458).

116

Calculation of the amount (in %) of 458 in the mixture:

If 457:458 were in the ratio of 1:1, then the singlet at d2.936 ppm (for 1-methyl in 458) would correspond to three protons i.e., if d2.936 ppm were for 3 protons then 458 would be 50% in the mixture (other 50% would be 457) So, 0.577 ppm protons give rise to ( 50 ´ 0.577 ) % of 458 in the mixture = 9.62% 3

In the same way if we consider the multiplet at d3.65 ppm (for two protons at C11 in 458): if d3.65 were for 2 protons then 458 would be 50% in the mixture So, 0.429 ppm protons give rise to ( 50 ´ 0.429 ) % of 458 in the mixture = 10.73% 2

From the above calculation, we have considered the amount of 458, on an average, as 10% in the mixture, or in other words, the ratio of 457 to 458 is 9:1.

Figure 2.40 13 C NMR spectrum of the mixture of 19-norergosta-1,3,5,7,9,14,22-heptaene ( 457) and 1- methylergosta-1,3,5,7,9,14,22-heptaene ( 458).

117

Figure 2.41 1H NMR spectrum of the petroleum fractions from column chromatography of the reaction of cholesterol with SeO 2 at 2h. Highlighted area shows negligible presence of cholesterol analogue of 19- norergosta-1,3,5,7,9,14,22-heptaene ( 457).

Figure 2.42 1H NMR spectrum of the petroleum fractions from column chromatography of the reaction of cholesterol with SeO 2 at 6h. Highlighted area shows negligible presence of cholesterol analogue of 19- norergosta-1,3,5,7,9,14,22-heptaene ( 457).

118

Figure 2.43 1H NMR spectrum of the petroleum fractions from column chromatography of the reaction of cholesterol with SeO 2 at 12h. Highlighted area shows negligible presence of cholesterol analogue of 19-norergosta-1,3,5,7,9,14,22-heptaene ( 457).

II.G References The references associated with this chapter are provided in the Bibliography section. Please follow page 353 onwards for these references.

119

Chapter III

Polyhydroxy and epoxy-polyhydroxy steroids: design, synthesis and study of their preliminary gelation behaviour

120

Abstract

First, polyhydroxy- and epoxypolyhydroxy steroids are reviewed thoroughly giving emphasis on their different structural variations. The major contribution of these classes of compounds was found to be due to the nature, in comparison to the synthetic results. And, the present work associated with this chapter demonstrates basically two aspects of some new polyhydroxy steroids - designed synthesis and their preliminary gelation behavior. Altogether sixteen polyhydroxy steroids (PHS, 12 new) of cholesteryl and b-sitosteryl series were synthesized and characterized. Among them eight (all new) are, in precise, epoxy- polyhydroxy steroids (5,6-epoxy-3b,4 a-dihydroxy- and 5,6-epoxy-3b,4 a,7 a-trihydroxy-), of which the a-diastereomers were utilized further to synthesize novel new tetraols (3 b,4 b,5 a,6 b- tetrahydroxy-) and pentaols (3 b,4 b,5 a,6 b,7a -pentahydroxy steroids). As a new class of polyhydroxy steroids, preliminary gelation behavior of the molecules was evaluated. At 1% or below CGC (critical gelation concentration), five PHS derivatives were found to be gelators of some selective organic solvents. Some selective organogels were characterized through T gel (gel melting temperature) and related physical parameters ( DH etc.), rheological data, and by morphology analysis (electron microscopes).

121

III.A Introduction Among the oxysteroids of natural abundance as well as synthetic targets, hydroxylated derivatives are the major contributing steroids. Along with the various hydroxyl groups attached directly to the steroidal skeleton, these are simultaneously frequently associated with other functionalities. And, both due to biogenetic as well as synthetic ease of transformation of epoxides into the corresponding hydroxy derivatives, epoxy and more specially epoxy-hydroxy steroids draw immense attention. Thus, herein we have focused on the background of two types of steroidal derivatives, viz., polyhydroxy steroids and epoxypolyhydroxy steroids.

III.A.1 A brief review on the polyhydroxy steroids III.A.1.1 Natural abundance In nature, both the plant and animal kingdom are the major and richest source of polyhydroxy steroids or polyhydroxysterols. Polyhydroxysteroids have been found in a number of algae, and virtually in every marine invertebrate phyla, i.e. Porifera, Coelenterata, Bryozoa, Mollusca, Echinodermata, Arthropoda, and Tunicata as well as in fish. III.A.1.1.1 Isolated from plants In the plant kingdom, algae, mushroom, phylum and other different parts of various plants were found to contain a good number of polyhydroxy steroids. Red algae Asparagopsis armata 1, Rissoella verruculosa 2 and Rhodymenia palmate 3 were found to possess dihydroxy steroids 461 and 462. Again, Liagora distenta 4 and Scinaia furcellata 4 were found to contain 462. Of note, the authors suggested that there was a possibility of formation of these side chain oxygenated sterols as artifacts by autoxidation during the isolation process as these were never found in fresh red alga A. armata . Steroids 461 and 462 were also formed from desmosterol 1 (structurally cholesta-5,24-diene-3b-ol) by autoxidation process. ( Figure 3.1) Ikekawa et al .5 first reported the isolation of a dihydroxy sterol saringosterol 463 from two brown algae. ( Figure 3.1) Later it was again found in different brown algae 6-8, such as in Hazikia fusiformis 7 and Ascophyllum nodosum 6, although its origin in some brown algae was doubtful and in case of the last alga 463, it was considered as an artifact during the air-drying process. Desmarestia aculeate 9 was reported to contain a dihydroxy steroid 464 having a novel side chain. ( Figure 3.2)

122

From the edible mushroom Grifola gargal, polyhydroxysterol 465 was isolated in 2011. 10 (Figure 3.1)

R

HO HO HO 461; R= OH O 465 OH HO

462; R= HO HO HO HO 463; R= H O 466

Figure 3.1 Polyhydroxy steroids (461-463 , 465-466) from plants.

The leaves of Cantella asiantica, a medicinal herb commonly used as memory enhancer and immunomodulator was found to contain a tetrahydroxysterol castasterone (466). 11 (Figure 3.1) In 2011, the roots of Breneyia fructicosa were reported to possess comparatively less common 4a-methyl sterols 467 and 468.12 (Figure 3.2)

HO R1 O HO OH OH HO OH HO HO HO 464 469 467; R1= CH2OH 468; R1= COOH

Figure 3.2 Polyhydroxy steroids (464 , 467-469) from plants.

Deacetyl metaplexigenin ( 469), a pentahydroxy C-21 steroid was isolated from the roots of Cynanchum otophyllum .13 (Figure 3.2) 7α-Hydroxysitosterol ( 78a ), 3β,5 α,6 β-trihydroxy-(24R)-24-ethyl-5α-cholestane ( 79 ) and 7 α- hydroxystigmasterol ( 470 ) were isolated from Helianthus tuberosus , grown in China. 14 (Figure

123

3.3)

HO OH HO HO OH HO 78a OH 470 79

Figure 3.3 Polyhydroxy steroids (78a , 79 and 470) from plants.

Pinnasterol 471 and acetylpinnasterol 472 were isolated from Laurencia pinnatan . ( Figure 3.4) 471 and 472 were ecdysone-like sterols having 22(S) configuration. 15 From the flower of the woody climber Dregea volubilis , found in hotter part of India, three new polyhydroxy pregnanes dregealol ( 473), volubilogenone ( 474) and volubilol ( 475), and three known pregnanes drevogenin D ( 476), iso-drevogenin P ( 477) and 17a-marsdenin ( 478) were isolated.16 (Figure 3.4)

OH HO H Me OH OR2 OH O HO HO O R1 R1 / R2 / 3 RO tigloyl = 2 1/ OH OH 4/ HO HO HO O 476; R = OH, R = H 473; R1= H, R2= tigloyl 1 2 471, R=H 474; R = OH, R = H 477; R1= R2= H 472, R=Ac 1 2 478; R = H, R = OH 475; R1= R2= H 1 2 Figure 3.4 Polyhydroxy steroids (471-478 ) from plants.

III.A.1.1.2 Isolated from animals: Marine species such as molluscs, sponges, starfishes, echinoderms are the rich sources of polyhydroxy steroids. III.A.1.1.2.a From coral: 25ξ-Cholestane-3b,5 a,6 b,26-tetrol-26-acetate ( 479) was collected from the octocoral Carijoa (Telesto ) riiseii .17 (Figure 3.5)

124

R2 HO R1 HO OAc H H

H H H H HO HO HO HO HO OH OH OH 488; R = R = H 479 1 2 490 489; R1=R2=OH Figure 3.5 25ξ-Cholestane-3b,5 a,6 b,26-tetrol-26-acetate ( 479), 24-methylenecholestane- 3b,5 a,6 b-triol ( 488), 1a,3 b,5 a,6 b,11a-pentahydroxy-24-methylene-5a-cholestane ( 489) and

4a,23,24(R)-trimethyl-5a-cholest-22E-ene-1a,3 b,6 b,11a-tetrol ( 490 ).

Eight polyhydroxysteroids ( 480-487), compound 480 being new, were isolated from the Hainan soft coral Sinularia depressa Tixier-Durivault. 18 (Figure 3.6)

R4 H H H HO R1 R3 H H R1 H H H H H H

R2 HO OAc HO H R2 484 R3 480; R1= R2=R3= OH, R4= H 485; R1= H, R2=a-OH, R3= b-OH 481; R1= R4= H, R2= R3= OH 486; R1= OH, R2=a-OH, R3= b-OH 482; R1= H, R2= R3= R4= OH 487; R1= OAc, R2=a-OH, R3= b-OH 483; R1=R2= R3= R4= OH Figure 3.6 Polyhydroxy sterols (480-487) from the soft coral Sinularia depressa .

Recently in 2013, from the Red sea soft coral Sinularia polydactyla, 24- methylenecholestane-3b,5 a,6 b-triol ( 488), and 1 a,3 b,5 a,6 b,11a-pentahydroxy-24-methylene- 5a-cholestane ( 489) were isolated by Shaaban et. al. 19 (Figure 3.5) Acerosterol, structurally 4 a,23,24(R)-trimethyl-5a-cholest-22E-ene-1a,3 b,6 b,11a-tetrol (490) was found from the gorgonian octocoral Pesudopterogorgia acerosa .20 (Figure 3.5) Another Hainan soft coral of Sinularia sp. was found to possess 24-methylcholesta-5, 24(25)- diene-3b,7 b,19-triol-19-monoacetate ( 491) along with a known steroid 24-methylcholesta-5, 24(25)-diene-3b,7 b,19-triol-7b-monoacetate ( 484). 21 (Figure 3.7). Again, from the Sinularia sp. of soft coral, Kobayashi et al . isolated 24-methylenecholestane-1a,3 b,5 a,6 b,16b-pentol ( 492) in

125 the year 1992. 22 (Figure 3.7). Similar skeletal polyhydroxy sterols 493-497 were also isolated from the Indunasian soft coral Sinularia sp. 23 (Figure 3.7).

AcO OH OH OH H R H H Sinularia sp. HO OH HO HO HO H OH 493, R= H 491 492 494, R= OH H

AcO HO H H OH H

H H H H H H HO HO HO HO HO HO OH O O 496 497 495

OH H OH H H H

H H H H HO HO R HO O O 498, R= b-OH 500 499, R= a-H Figure 3.7 Polyhydroxy sterols (491-500) from the soft coral Sinularia sp.

The ethanol extract of Sinularia gibberosa were found to possess two new steroids, gibberoketosterols B ( 498) and -C ( 499) and one known steroid, gibberoketosteroid ( 500).24 (Figure 3.7). Gegardia savaglia was reported to contain ecysterone 50125 , ecdysone 502, ajugasterone 50326 in large amounts and gegardiasterone 50427 in small amount. ( Figure 3.8)

126

HO OH OH

OH OH HO HO OH OH OH OH H H O O 501 502 HO OH HO OH

HO HO OH HO HO OH OH OH OH H H O O 503 504

Figure 3.8 Polyhydroxy steroids (501-504 ) from soft coral.

Soft coral genus Naphthea was reported to be a good source of 19-hydroxylated polyhydroxy steroids.28-29 Among these, Naphthea chabroli , collected from the Mandapam coast of India in the Gulf of Mannar, was found to possess 24-methylene cholest-5-en-1a, 3b,19-triol (505) and 24-methylene cholest-5-en-3b,7 b,9 a,19-tetrol ( 506), and three known steroids, ergost- 5,24(28)-dien-3b,19-diol (litosterol), cholest-5-en-3b,7 b,19-triol, and ergost-5,24 (28)-dien- 3b,7 b,19-triol. 28 (Figure 3.9).

O

HO HO HO HO O R OH HO HO OH HO 505 507, R=OAc 506 508, R=H

Figure 3.9 Polyhydroxy steroids (505-508 ) from soft coral.

Another steroid named hippurin-1 507 was isolated from Isis hippuris .30 Its desacetoxy derivative ( 508) was also described.30 (Figure 3.9). A novel glycoside A 509 (Figure 3.10) was isolated from the gorgonian Anthoplexaura dimorpha 31 , found in the southern coast of Japan. Three new 9,10-secosterols 510-512 with

127 conventional 24-methylene, 24-methyl and cholestanol side chains were isolated from Sinularia sp. 32-33 The C-8 epimer 513 of 509 was also found from the same species but in minor amounts. (Figure 3.10)

HO R HO O HO O H O H OH O OH HO O HO HO OH 510, R= O 509 513 OH

O OOH 511 R= HO 512, R=

Figure 3.10 Polyhydroxy steroids 509-513.

Nine new (518, 521, 525-528, 530-532) and eleven known (488, 514-517, 519-520, 522- 524, 529 ) polyhydroxysterols were isolated from the south china sea gorgonian Menella kanisa by a chinese research group in 2013. 34 (Figure 3.11). Nebrosteroids N ( 533) and nebrosteroids P ( 534) were isolated from the soft coral Nephthea chabrolii in 2012. 35 (Figure 3.12 ).

128

R6 H R1 R2 H H H HO R3 R5 R4

514; R1= R2= R5= H, R3= R4= OH, R6= a a = 515; R1= R2= R5= H, R3= R4= OH, R6= b 516; R1= R2= R5= H, R3= R4= OH, R6= c 517; R = R = R = H, R = R = OH, R = d b = 1 2 5 3 4 6 488; R1= R2= R5= H, R3= R4= OH, R6= g 518; R1= R2= R5= H, R3= R4= OH, R6= h c = 519; R1= R3= R4= OH, R2= R5= H, R6= a 520; R1= R3= R4= OH, R2= R5= H, R6= b 521; R1= R3= R4= OH, R2= R5= H, R6= c d = 522; R1= R3= R4= OH, R2= R5= H, R6= d 523; R1= R3= R4= OH, R2= R5= H, R6= g e = 524; R1= R3= OH, R2= H, R4,R5= O, R6= a 525; R1= R3= OH, R2= H, R4,R5= O, R6= b f = 526; R1= R3= OH, R2= H, R4,R5= O, R6= c 527; R1= R3= OH, R2= H, R4,R5= O, R6= g 528; R1= R3= OH, R2= H, R4,R5= O, R6= d g = 529; R1, R2= O, R3= R4= OH, R5= H, R6= a 530; R1, R2= O, R3= R4= OH, R5= H, R6= b 531; R1, R2= O, R3= R4= OH, R5= H, R6= c h = OOH 532; R1, R2= O, R3= R4= OH, R5= H, R6= g

Figure 3.11 Polyhydroxy steroids 488, 518-532 .

H H HO AcO HO H H OH

H H H H H H HO OH HO HO MeO H 533 OH 535 534 OH HO

HO H H H

H H H H H H HO HO HO HO HO 536 OH OH 486 537

Figure 3.12 Polyhydroxy steroids 486, 533-537 .

129

From the same coral a 4 a-methylated steoid named nebrosteroid 535 was isolated by Duh et al. in 2009. 36 A novel sterol sarcophytosterol 536 was found from the soft coral Lobophytum sarcophytoids. 37 In the year 1997 Venkateswarlu et al. isolated one polyhydroxysterol 537 from the soft coral Lobophytum crassum .38 24-Methylene-cholesta-3b,5 a,6 b,19-tetrol ( 486) was reported from the soft corals Nephthea albida and N. tiexieral verseveldt 39 in 1992. (Figure 3.12). Polyhydroxysterols having 3 b,5 a,6 b-trihydroxy groups 538, was first isolated from the soft coral Sarcophyton elegance 40 and its 25-hydroxy analog 539, was found from Sclerophytum sp 41 which also contained the rare (24S)-24-methylcholestane-3b,5 b,6 a,25-tetrol 540. 541 was also found from the former coral.42 A series of 1 b,3 b,5 a,6 b-tetrahydroxysteroids 542-546 were found from Sarcophyton glaucum 43-45 , Lobophytum pauciflorum 46 and Sclerophytum sp. 41 Among these compounds 546 was actually the first isolated polyoxygenated androstane derivative from marine invertebrates.44 Kobayashi and Mitsuhashi isolated (24S)-24-methyl-5a-cholestane- 3b,5 b,6 b,25ξ,26 -pentol 547 from S. glaucum .45 Another soft coral Sarcophyton subviride 47 was found to contain polyhydroxysterols with 1 b,3 b,5 a,6 b-tetrol such as 548 and 549. From the soft coral Asterospicularia randalli 48 , 3 b,5 a,6 b-trihydroxysterols with C-22 and C-24 hydroxy groups 550 were isolated and from the Red Sea soft coral Lobophytum depressum 49 lobophytosterol 551 and depresosterol 552 were found. The same trihydroxy moiety with the fourth hydroxy group at 7 b-position 553 and the 7 b-acetate analogs 554-556 were found respectively in Anthelia glaucia 50 and Xenia sp. 51 19-Hydroxysterols 484, 55752-53 were found in Liptophyton viridis . Among these compounds, 484 containing 7 b-acetoxy group, was the first reported naturally occurring 19-hydroxy steroid. From the same species, Bortolotto et al. 54 isolated 4a-methyl-3b,8 b-dihydroxy-5a-ergost-24(28)-en-23-one 558. The first reported marine sterols having A/B-cis ring functionality was compound 559 found in Lobophytum pauciflorum 55 and Sclerophytum .56-57 Anandamansterol 560 and nicobarsterol 561 were found from another species of Sclerophytum .57 The 7 b- and 7 a-hydroxy derivatives 562 and 563 of 24- methylenecholest-5-ene-3b,16b-diol-3-O-a-L-fucoside were found in Alcyonium sp. 57 (Figure 3.13 and Figure 3.14 )

130

R R

OH 1 R1 OH R

542; R=Me, R1=OAc HO HO HO 543; R=Me, R =OH OH OH OH 1 OH OH OH 544; R=Me, R1= H 538; R=H, R1=OAc 541 CH 545; R= 2 ,R1=H 539; R=OH, R1= OH C 540; R=OH, R1= OAc H O HO OH OHOH OH OAc

HO HO OH HO HO OH OH OH 548 547 OH 546 OH OH O OH OH OH OH

HO HO HO OH OH OH OAc 551 549 550

Figure 3.13 Polyhydroxy steroids 538-551 from soft coral.

Anthiopathes subpinnata 59-60 , commonly called black coral was reported to possess highly oxygenated sterols 564-570. (Figure 3.15 ). From Sinularia numerosa 61 numersterol A and B, structurally, 24-methylenecholestane- la,3 b,5 a,6 b-tetrol 571 and 25-methylene-22-homocholestane1b ,3 b,5 a-triol 572 respectively were isolated. ( Figure 3.15 ). Polyhydroxylated nuclei 573-576, with different side chains, as well as compounds 577-580 all containing 11 a-hydroxy were isolated from Sinularia dissecta .62-63 From the same source four polyhydroxysterols ( 541, 581-583) were also found by Ramesh and Venkateswarlu in 1999 among which 581-583 were new. 64 (Figure 3.16 )

III.A.1.1.2.b From sponge: In the year 1981 Carl Djerassi isolated a steroidal heptol 584 from the sponge Dysedea etheria .65 (Figure 3.17 ).

131

553; R1=H, R= OH OH R 554; R1=Ac, R= OH HO OR1 555; R1=Ac, R= HO HO 552 OH

556; R1=Ac, R=

HO O OAc OH HO R HO 558 HO 484; R=OAc OH 557; R=H HO O 559 OH O HO OH

HO O H O R R1 OH O OH 562; R=OH, R1=H 560 HO 1 HO 561 OH 563; R=H, R =OH OH HO

Figure 3.14 Polyhydroxy steroids 552-563 from soft coral.

OH OH R R OH HO HO R

H R HO H 1 H H H H H R H O O HO OH 564; R= H 566; R= H, R1= OH 22 565; R= Me 567; R= H, , R1= OH 570; R= H/ OH 22 568; R= Me, , R1= OH 569; R= H, R1= O

OH OH H H

H H H H HO HO HO HO OH 572 571

Figure 3.15 Polyhydroxy steroids 564-572.

132

R side chain R HO HO OH OH HO OH

HO HO 573, R= CH 577, R=CH HO 3 3 HO 574, R=CH2OAc 578, R=CH2OAc OH 575, R=CHO 579, R=CHO 576, R=COOH 580, R=COOH 581

HO HO HO HO

HO HO HO HO OH OH 582 583 Figure 3.16 Polyhydroxy steroids 573-583 .

H HO HO R1 HO R 2 H OH HO H O HO HO HO OH OH OH

584 594; R1= R2= H 595, R1= R2= OH 596, R1= H, R2= OH

Figure 3.17 Polyhydroxy steroids 584 and 594-596 from sponge.

Marine sponge Theonella swinhoei was found to contain 4-methylenesterols-theonellasterols D, E, F, G, H ( 585-589) and conicasterols C, D ( 590-591), conicasterols G ( 592), conicasterols J (593). 66 (Figure 3.18 ) Xestobergsterol A 594 and B 595 were isolated from the Okinawan marine sponge Xestospongia bergquistia , as unique polyhydroxysteroid having a cis -C/D ring junction and and additional carbocyclic E ring 67 . Xestobergsterol C 596 was found from the Okinawan marine sponge Ircinia sp. 68 (Figure 3.17 ).

133

R/ HO

R OH OH OH OH HO HO OH HO H H H 589 590 585; R= OMe, R/= 586; R= OH, R/= 587; R= OMe, R/= 588; R= OH, R/= HO

OH OH O OH HO HO O HO OH H H H 592 593 591

Figure 3.18 Polyhydroxy steroids 585-593 from sponge.

Seven new agosterol congeners, agosterols E ( 597), -E3 (598), -C2 (599), -C7 (600), -F ( 601),

-G ( 602) and -A3 (603) along with three known sterols, agosterols A ( 604 ), -C ( 605) and -D2 (606) were isolated from the sponge Acanthodendrilla sp. by Tsukamoto et al. 69 in 2003 although compound 604-606 were previously reported from Spongia sp. 70,71 (Figure 3.19 ).

OH R4 R3 H A =

R2 H OH R1O H B = AcO OAc

597; R1= Ac, R2=OH, R3= H, R4= A OH 598; R1= Ac, R2=OH, R3= H, R4= B HO 599; R1= R2= R3= H, R4= C 600; R1= R2= R3= H, R4= D C = 601; R1= R2= H, R3= OH, R4= A 602; R = R = H, R = OAc, R = A 1 2 3 4 OH 603; R1= Ac, R2= H, R3= OH, R4=B HO 604; R = Ac, R = H, R = OH, R =A 1 2 3 4 D = 605; R1= R2= R3= H, R4= A 606; R1= Ac, R2= R3= H, R4= C

Figure 3.19 Polyhydroxy steroids 597-606 from sponge.

134

Piccialli and Sica isolated eight 3 b,5 a,6 b-trihydroxysterols from the sponge Spongionella gracilis. Among these 607, 608, 613 and 614 were new and 609-612 were previously found from bryozoans Myriapora truncate as 3,6-diacetyl derivatives. 72 (Figure 3.20)

R

609; R= 610; R= HO HO OH 611; R= 612; R= 607; R=

613; R= 614; R= 608; R=

Figure 3.20 Polyhydroxy steroids 607-614.

Sponges of the genus Disedia was found to be a good source of polyhydroxysteroids. From the methanol extract of Disedia herbacea 73, a polyhydroxylated-9,11-secosterol, named herbasterol 615 possessing antimicrobial and ichthyotoxic activities was isolated. From the same sponge, collected from Ethiopea, compound 616-617 were isolated.74 Other sterols with the nucleus of 615 were also isolated from Spongionella gracilis 75 , Hippospongia communis 75, Spongia officinalis 75, Ircinia variabilis 75, Myriapora truncate 76 and from the scallop Patinopecten yessoensis 77. From the sponge Spirastrella incostans 24-ethylcholestane-3b,5 a,6 b- triol 78 and its 6-keto derivative 79 were isolated. (Figure 3.21 ) Dysidea etheria 80 , collected in Bermuda was reported to have eight new polyhydroxysterols possessing a framework of 5 a-cholest-7-ene-2a,3 b,5,6b,9 a,l1 a,19-heptol 584 and various conventional side chains. Three minor sterols 618-620 were also isolated from the same sponge. Two other sterols 621 and 622, structurally similar to 617 but lacking the 19-hydoxy group, were isolated from Dpidea fragilis 81, collected in Black sea. ( Figure 3.21 )

135

HO

HO O H HO HO HO H HO OH H OH OH HO HO HO 615 OAc HO 616 OAc 617 R HO RO HO HO HO HO HO HO 618; R= H HO HO HO 621; R= H OH 619; R= Me OH 620; R= Et 622; R= Ac

Figure 3.21 Polyhydroxy steroids 615-622.

The first report of isolation of pregnane derivative 623 and 624 from marine sources was from the sponge Heliclona rubens .82 D7-3b,6 a-Dihydroxysterol 625, D7-3b,5 a,6 b,9 a-tetrahydroxysterol 626 and 5,6-secosterols 627-634 with various side chains were isolated respectively from Spongionella gracilis 72 , Spongia officinalis 83 and Hippospongia communis .84-85 Spongia officinalis 86 was reported to contain 9,11-secosterol 635, previously found in gorgonian,87 a soft coral of the genus Sinularia 32,33 and in the sponge Dysidea herbacea 73. Another seven di/ tri-hydroxy 9,11- secosterols were also isolated from the gorgonian Tripalea clavaria .88 (Figure 3.22 )

R R1 OHC

O

HO H HO HO HO HO HO H 1 OH 623; R=H, R =OH 625 OH OH 1 635 624; R=OH,R =H 626

R

633; R= 627; R= , 629; R= , 631; R= ,

HO HO OH 628; R= , 630; R= , 632; R= , 634; R=

Figure 3.22 Polyhydroxy steroids 623-635.

136

The sponge Petrosia contignata 89, collected from New Guana was reported to contain a highly oxygenated steroid contignasterol 636, with a cyclic hemiacetal side chain. This compound is the first naturally occurring steroid with 14 b-H configuration. Xesobergosterol A 637 and -B 638, with 14 b-H configurations were isolated from Xestospongia bergquistia 64 by Shoji et al. (Figure 3.23 ).

OH O

OH R1 R2 H H O H O HO OH HO OH OH OH OH 636 637; R1=R2= H 638; R1= R2= OH

Figure 3.23 Polyhydroxy steroids 636-638 from sponge.

Petrosia weinbergi 90-91 was reported to have compound 639 and 640, having an unprecedented cyclopropane-containing side chain. (Figure 3.24 ).

R1 R2 H

+ - H OH Na O3SO HO H H Na+ -O SO HO O-(4-OAc)-Xyl 3 H O-Gal 639; R = OH, R = H 1 2 641 640; R1= H, R2= OH

Figure 3.24 Polyhydroxy steroids 639-641 from sponge.

A novel steroidal saponin, pachastrelloside A 641 was found in the sponge Pachastrella sp .This glycoside is capable of inhibiting cell division of fertilized starfish eggs.92 (Figure 3.24 )

137

Callyspongia fibrosa , collected from the western part of Bay of Bengal (India) was reported to contain one known steroid 24S-24 methyl-cholestane-3b,5 a,6 b,25-tetraol-25-mono acetate (538) and four new sterols 642-644, 540. Except 644 all possessed 3 b,6 b-dihydoxy system and 25-O-acetate as common feature. 93 (Figure 3.25 )

R 3 R4

A = R2 OAc

B = HO R 1 OH

642; R1= R2= R3= H, R4= A 643; R1= R3= H, R2= OH, R4= A 644; R1= R3= OH, R2= H, R4= B

Figure 3.25 Polyhydroxy steroids 642-644.

The dichloromethane extract of the red sea marine sponge Lamellodysidea herbacea contained four polyhydroxysteroids cholesta-8-en-3b,5 a,6 a,25-tetrol ( 645 ), cholesta-8(14)-en- 3b,5 a,6 a,25-tetrol ( 646), cholesta-8,24-dien-3b,5 a,6 a-triol ( 647) and cholesta-8(14)-24-dien- 3b,5 a,6 a-triol ( 648). 94 (Figure 3.26)

H H 14 OH 14 9 9 8 H 8 H HO HO HO HO OH OH 645; 8,9-ene 647; 8,9-ene 646; 8,14-ene 648; 8,14-ene

Figure 3.26 Polyhydroxy steroids 645-648 from red sea marine sponge.

D8(14) -3b,7 a-Dihydroxysterols 339, 649-650 from Pellina semitubulosa 95 and D-ring unsaturated steroid 3 b,6 a-diols 651-653 from Topsentia aurantiaca were isolated. 96 (Figure 3.27)

138

651; R= OH 649, 652; R= HO OH HO H 650, 653; R= 649-650 651-653

Figure 3.27 D-ring unsaturated steroids 649-653 .

III.A.1.1.2.c From fungus: Two ergosterol derivatives, D7-3b,5 a,6 b-triol ( 654) and D7- 3b,5 a,6 b,9 a-tetrol ( 655) were found in the terrestial fungus Polyporus versicolor .97 (Figure 3.28)

OH OH OH

H H H H H R HO HO HO O OH OH OH OH 656, R= b-OH 654 655 657, R= a-OH . Figure 3.28 Polyhydroxy steroids 654-657 from fungus.

From the marine fungus Rhizopus sp. two new ergosterols, 3 b,15b-dihydroxyl-(22E, 24R)- ergosta-5,8(14),22-trien-7-one ( 656) and 3 b,15 a-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22- trien-7-one ( 657) were isolated by Gu and Zhu et. al in 2008. 98 (Figure 3.28 ) III.A.1.1.2.d From starfish: The main metabolites of starfishes are polyhydroxy steroids and oligosides. Compound 658 was isolated from the far eastern starfish Hippasteria kurilensis by Stonik et. al in 2008. 99 (Figure 3.29 ) Astropecten polyacanthus , a species of starfish was found to contain steroidal compounds 339, 658-662.100 (Figure 3.29 ) A novel polyhydroxy sterol 663 and 664 were isolated from the starfish Asterina pectenifira .101 (Figure 3.30 )

139

OH

OH OH HO OH OH OH HO CHO 659 658

R2 OH HO O R HO O 1 H 661, R1= H, R2= OH 660 662, R1= OH, R2= H Figure 3.29 Polyhydroxy steroids 658-662 from starfish.

OH OH

OH OH O OH H O H OH HO OH HO OH H H HO OH OH 663 664 OH OH

OH OH OH OH H OH H OH HO R HO H 1 H R OH R OH 665; R= R1= H 666; R= R1= OH 668; R= H 667; R= OH, R1= H 669; R= OH, 22 -trans

Figure 3.30 Polyhydroxy steroids 663-669 from starfish.

From the starfish Protoreaster nodosus , one research group, in 1982 isolated the polyhydroxysterols, 664, 5α -cholestane-3β,6α,8,15α,16β,26 -hexol 665, and 5α -cholestane- 3β,4β,6α,7α,8,15α,16β,26 -octol 666102 , while the other group, in 1984 103 found 5a-cholestane-

140

3b,4 b,8,15a,16b, 26-heptol 667 24 ξ-methyl-5a-cholestane-3b,6 a,8,15a,16b,26-hexol 668 and (22E) 24 ξ-methyl-5a-cholest-22-en-3b,4 b,8,15a,16 b,26-heptol 669 . (Figure 3.30 ) Polyhydroxysteroids isolated from starfish though were generally found to contain 8 b- hydroxyl group, but compounds 670-678 were exceptional. Hacelia attenuate 104 was the source of 670. Luidia maculate 105 was found to contain compound 671, 673 and 674 although Solaster borealis 106 and Myxoderma platyacanthum 107 were other sources of compound 671. Compound 672 was isolated from Myxoderma platyacanthum 104 and Roaster sp. 107 Tremaster novaecaledoniae 108 was the source of compound 675, 676 and 678 and Sphaerodiscus placenta 109 was for 677. (Figure 3.31 )

R'

OH OH OH OR2 HO R1 OH H HO OH H OH R1 2 OH Side chain R R 678

OH 670; R'= OH H H H OH HO OH OH H H 671; R'= H OH 679 - + OH H SO3 Na 672; R'= OH

OH H OH OH OH OH 673; R'= H OH OH OH OH HO 674; R'= H OH OH OH OH H 680 675; R'= H OH OH OH OH OH H SO -Na+ 676; R'= 3 OH HO R OH H H H 677; R'= OH 681; R=H 682; R=OH

Figure 3.31 Polyhydroxysteroids 670-682 isolated from the starfish.

141

Polyhydroxysteroids 658, 679-682 were isolated from the starfish Culcita Novaeguineae in 1991.110 (Figure 3.31 ) Polyhydroxysterols containing amide linkage in their side chains 683-684 and side chain methyl oxidized to carboxyl group 685-686 were encountered in starfish Myxoderma platyacanthum .107 (Figure 3.32 )

O - + SO Na CO2H N 3 H 683 685 CO2H O OH - + HO SO3 Na HO N OH H 684 686

Figure 3.32 Polyhydroxy steroids 683-686 from starfish.

Another starfish Archaster typicas 111,112 was reported to contain highly hydroxylated steroids 687-695, few of them being moderately cytotoxic also. (Figure 3.33 )

OH OH OH OH OH OH OH OH OH OSO -Na+ HO OH HO 3 OH OH HO OH OH OH OR R OH OH OH O 694; R=H OH 695; R=OH 687; R=H 690 691 OH - + 689; R=SO -Na+ 688; R=SO3 Na 3

OH OH OH

692 693 Figure 3.33 Polyhydroxy steroids 687-695 steroids fromstarfish.

Styracaster caroli , a deep-water starfish was reported to contain a good number of polyhydroxysterols 696-706113 with unusual side chains. Another series of polyhydroxysterols

142

679 , 707-713 were encountered from different starfishes as shown below. Some of these possessed sulphate groups in their side chains. (Figure 3.34 and Figure 3.35 )

R2

R R1 OH HO HO HO HO OH OH

side chain R R1 R2 side chain 696 - + OH OSO3 Na OH H OH H

701

697 - + OSO3 Na OH H H OH - + OSO3 Na 702 OH 698 - + OSO3 Na OH H OH OH OH + - Na O3SO 703 699 - + OSO3 Na OH H H OH

700 - + OSO3 Na OH H H OH OH

Figure 3.34 Polyhydroxy steroids 696-703 from starfish.

OH O OH

N - + H SO3 Na OH OH OH OH 15

OH OH OH HO HO HO R 1 R OH OH OH R side chain OH R R1 712 OH 704 OH OH 707; 15a; R= H; 679; 15b; R= H 705 OH O OSO -Na+ OH 706 H OH 3 708; 15a; R= H; 709; 15b; R= OH, 710; 15b; R= H; OH HO OH OH 711; 15b; R= H 713

Figure 3.35 Polyhydroxy steroids 704-713 from starfish.

143

From China sea starfish Asterina pectinifera five new polyhydroxysterols 664, 666, 714-716 were isolated by Liu et al. in 2010. 114 (Figure 3.36)

OR

OH OH OH OH

H OH H OH HO OH HO H H OH OH OH 715; R= H O 714 OMe 716; R= HOH2C OH

Figure 3.36 Polyhydroxy steroids 714-716.

Starfish Patiria miniata Brandt (Asterinidae) was found to cotain patiriosides A and -B and pectinioside G115 and acanthaglycoside C.116 The former two are chemically hexaglycoside sulphates and the latter two are asterosaponins. Two known steroid monoglycoside sulfates 717 and 718, were isolated from Patiria pectinifera 117 and Oreaster reticulates 118 respectively . Starfish Patiria miniata Brandt (Asterinidae) was found to contain steroid oligoglycoside- sulfates, miniatosides A 719 and -B 720, which co-occurance with 717 and 718 and seven polyhydroxysteroids, among which two (721 and 722) compounds were reported first time.119 (Figure 3.37)

III.A.1.1.2.e From mollusca: Mollusca, in comparison to the other marine animals, are found to contain a lesser number of polyhydroxysterols. The skin extract of droid nudibranch Diaulula sandiegensis was found to contain diaulusterols A 723 and -B 724.120 The 2 a,3 a-diol array of diaulusterols are not commonly found in natural steroids. These were also found in Eudendrium glomeratum. 121-123 Trihydroxysteroids with the rare 9 a-hydroxylation 725 was found in the scallop Patinopecten yessonensis .124 (Figure 3.38)

144

OR' OMe RO HO O O HO O HO HO O O HO OSO -Na+ O +- O 3 Na O3SO OH O

OH OH OH OH OH HO OH OH OH HO HO - + OH OH 717; R=Me, R'=SO3 Na - + 719 720 718; R=SO3 Na , R'=Me

OH OH OH HO OH OH OH

OH OH HO OH HO OH - + OH OSO3 Na OH OH 721 722 Figure 3.37 Polyhydroxy steroids 717-722.

HO OR HO HO HO HO O OH

723; R= COCH2CHOHCH3 725 724; R= H

Figure 3.38 Polyhydroxy steroids 723-725 from mollusca.

III.A.1.1.3 Miscellaneous: Eight compounds 665-667, 669, and 726-729 (Figure 3.39 ) with 3b,6 a,8,15a,16b-hydroxy groups and different side chains were found from various marine sources as shown in Table 1 .125 Another group of sterols 680, 714, 722 and 730-739 (Figure 3.40) with basic hydroxylation patterns as 3 b,6 a,8,15 b,16b with different side chains were also reported. Compound 740 was found from Darmasterias imbricate.126 Sphaerodiscus placenta 109 was a good source of compound 665 and 641 though the former was also available in Crossaster papposus 127 and Culcita novaeguineae.110 Rosaster sp. 128 was found to contain compound 664

145 and 666. Hacelia attenuate 129 possessed compounds 742-744. All these compounds 664-666 and 741-745 possessed common hydroxy groups at 3 b,6 b,8,15a,16b in basic skeleton with difference in their side chain. Compound 745 and 746 were isolated from Solaster borealis.106 (Figure 3.41 )

side chain

a= OH OH OH

OH b= OH HO R1

R OH - + OSO3 Na

c=

726; R= H; R1= OH; side chain= a 727; 6-O-sulphate, R= OH; R1= OH; side chain= a 728; R= R1= H; side chain=b 729; R= R1= H; side chain= c

Figure 3.39 Polyhydroxy steroids 726-729 from marine sources.

OH OH

2 side chain R OH HO R1 OH a= R OH

730; R= R1= R2= H; side chain= a b= OH 731; R= OH; R1= R2= H; side chain= a 732; 3-O-Sulfate; R= OH, R1= R2= H; side chain= a 733; R= H; R = OH; R = H; side chain= a 1 2 c= OH 734; R= R1= H; R2= OH; side chain= a 735; 3-O-Sulfate;R= R1= OH; R2= H; side chain= a 736; R= R1= R2= H; side chain= b 737; R= R1= R2= H; side chain= c d= OH 738; R= R1= H; R2= OH; side chain= c OH 739; 6-O-Sulfate; R= R1= OH; R2= H; side chain= d

Figure 3.40 Polyhydroxy steroids 730-739 from marine sources.

146

R a= OH OH OH OH OH

OH b= OH OH HO R3 HO R2 R1 OH OH 740 741; R = R = R = H; R= a c= OH 1 2 3 OH 742; R1= R2= R3= H; R= b OH OH 743; R1= OH; R2= R3= H; R= b 744; R1= OH; R2= R3= H; R= c OH HO OH R OH 745; R= H 746; R= OH

Figure 3.41 Polyhydroxy steroids 741-746 from marine sources.

3-Oxocholest-4-ene-4,16b,18,22 (R)-tetrol-16,18-diacetate 747 was isolated by Cimino et al. in Mediterranean hydroids of the genus Eudendrium sp .130 (Figure 3.42 )

OH HO AcO

O OAc H

OH O HO HO HO OH OH 749 747 748 Figure 3.42 Polyhydroxy steroids 747-749.

One of first polyhydroxysteroids isolated from marine species Pseudopterogorgia elisabethae .131 was 5 a-choletane-3b,5,6b,9-tetrol 748. Pseudopterogorgia americana 87 was reported to possess 3 b,11-dihydroxy-9,11-secogorgost-5-en-9-one 749. (Figure 3.42 ) Amurensoside A 750, -B 751 and -C 752, sharing the same hydroxylation pattern 3b,6 a,15a- were found from Asterias amurensis 132 whereas granulatoside B 753 and laeviuscoloside F 754 with 3 b,6 b,15a- hydroxylation pattern were isolated from Choriaster granulates 133 and Henricia laeviuscola 134 respectively. Amurensoside D 755 was also found

147 from the same species of Asterias 132 but with hydroxylation pattern as 3 b,6 a,15b. Compound 756 with 3 b,6 b,15b-pattern was isolated from Fromia monilis.135 (Figure 3.43 )

2 side chain R R1 R OR2 HO O - + 750 H SO3 Na HO OH OR2 HO O 1 - + OR 751 OH SO3 Na HO HO R OH OR2 OH +Na-O SO O H H 3 752 HO OH

side chain R R1 R2 OR2 O HO O OH H MeO 753 OH HO OH 2 OH OR O RO MeO O OH 1 OH R OH 754 HO HO OH OH

HO O OH OH O OH

OH HO O OH HO OH O HO OH OMe OH OH 755 756

Figure 3.43 Polyhydroxy steroids 750-756.

Compounds 757-769, collected from different marine sources shared common hydroxylation pattern 3 b,6 a,8,15a- and differed in their side chains only, whereas compound 770-774 shared 3b,6 a,8,15a,16b- pattern. A large group of sterols 775-807 having common hydroxylation pattern as 3 b,6 b,8,15b- were encountered from different marine sources. In 1990 Minale et al.

148 isolated pisasteroid E 808 from Pisaster giganteus. 136 A group of polyhydroxysteroids with glycoside linkage were found from different starfishes. These were grouped according to their basic hydroxylation pattern, e.g., compound 809-822 contained 3 b,6 a,8,15b,16b- pattern in the aglycon part whereas 3 b,6 a,8,16a- was present in compounds 823-836 . Another group of polyhydroxy steroids were the compounds 837 -851. The smallest group of compounds was with compound 852-854. (Figure 3.44 -Figure 3.50 )

OR OR

OH OH OH OH OH OH OH OH HO OH HO O O R HO H H HO H H OH H OH OCH3 H OH R R 770; R= H 771; R= OH OR O HO 757 + - H CO Na O3SO 3 O H CO OH 765 H3CO O 3 HO O OH OH OH O 758 HO HO OH H3CO HO H + - Na O3SO OH O H OH 766 HO O R HO OH O OH O + - Na O3SO 772 HO 759 HO O O OH HO O HO OH OR O 773 HO HO H3CO OH HO OSO -Na+ O 3 760 H3CO O HO O OH OH HO OH H OCH3 H OH HO OH - + R OSO3 Na HO O O H CO O O 761 3 O HO O HO OH 767 HO OH OH OH - + OSO3 Na HO HO O OH 768 H CO HO O 3 HO H 762 O OH HO O OH OH OCH3 774 OH H CO - + 3 OH OSO3 Na O HO O 763 HO O O O HO O O OH OH OH OH

OH HO O HO H 764 H3CO OH H OH 769 OH Figure 3.44 Polyhydroxy steroids 757-774 from marine sources.

149

OR HO O OH O OH OH OH OH HO H OH HO H - + H OH OSO3 Na +- 786 R Na O3S OH O OH 775 HO

HO OH HO O OH HO O H O OH H OH 787 HO 776 + - Na O3SO OH O O OH HO OH

O OH + - 777 Na O3SO HO O OH HO OH HO O H HO O OH H OH 788 OH 778 + - O Na O3SO O OH OH H3CO OH OH HO O 779 HO HO O OH OH HO O H - + OSO3 Na OH H OH 789 HO O 780 HO OH R OR + - Na O3SO O O 781 HO HO OH OH 790 HO OH +Na-O SO O 782 3 OH H3CO HO H OH OH OH OH OH HO HO O HO 791 O HO O 783 HO O O OCH3 HO O OCH 3 OH OH 792 HO HO H CO O 3 O 784 H CO O HO O 3 O HO O OH OH OH OH HO HO 793 H3CO O O 785 H CO O HO O 3 O HO O OCH3 OCH3

Figure 3.45 Polyhydroxy steroids 775-793 from marine sources.

150

OH H CO 3 OMe HO O OH O Me OH O O O O OH OH H - + OH OSO3 Na OH + - Na O3SO H OH OH OH HO OH OH H OH 794 H CO OR 3 OMe 795 O O Me OH O O OH OH H - + HO H OH OSO3 Na H OH R OH HO OH +Na-O SO O 797 3 OH OH 796 HO OH HO OH OH O O OH HO H CO O 798 3 O HO O OH OCH3

HO O OH HO O H OH OH H OH OH H3CO OH 799 HO O OCH3 O O O O O OH OH OH OH HO HO H OH OH OH 801 HO H OH OH OH OH OCH OCH3 800 3 HO HO O O O O OH OH O OH O OH O O OH OH OH OH HO H HO H OH OH OH OH 802 803 Figure 3.46 Polyhydroxy steroids 794-803 from marine sources.

151

H3CO OH OH O OCH3 O O O O O OH OH OH HO OMe OH HO OH

OH OH HO H HO OH OH OH H OH 804 806 805; 6-sulphate OH H3CO OCH3 O O - + O OSO3 Na O OH OH HO OH OH HO O OH HO O OH OH OH HO OH OH OH 808 + - 807 Na O3SO HO OH O OH OH O O OH OCH3 O OH OH OH OH HO H OH OH OH HO H 810 H OH 809

- + OSO3 Na - + HO OH OH OSO3 Na HO HO O OH OH O OH HO OH O OH O OH OH - + OSO3 Na H OH HO H OH H3CO OCH H OH 812 3 811 HO HO O O O OH MeO O O O OH OH OH O OH OH OH O OH OH OH HO H HO H R OH H OH 814; R=H 813 815; R=OH Figure 3.47 Polyhydroxy steroids 804-815 from marine sources.

152

OR

OR R

HO OH - + OH OH OH OH 816 OSO3 Na O OH OH OH HO OH HO H HO H 817 OH H OH H OH O - + OSO3 Na R HO OR OH 818 OH O - + OSO3 Na

O H3CO OH OH 819 OH O OH OH O HO OH HO H - + HO O OSO3 Na HO OH OH R O OH O HO O O 820 OH MeO OH OH OH HO O 821 MeO O O O O OH OH HOO HO H OHHO OH H OH 822 - + OSO3 Na HO OH O OH O OH

OH OH O HO O H H - + HO OSO3 Na OH H OH HO H HO H 824 H OH OH HO OH 823 O OH O O O OH

OH OH

OH OH +Na-O SO H O O H 3 H HO H H OH HO H OH 826 HO OCH3 OH 825 OH O O OH

OH OH

OH OH O O H O O H H H OH OH H3CO OCH HO OCH OH OH HO 3 HO 3 828 827

Figure 3.48 Polyhydroxy steroids 816-828 from marine sources.

153

HO HO OH OH O O O OH O OH OH OH OH OH O O H O O H OH H HO H OH OCH3 H3CO OH OH HO OCH3 HO 830 829 OH HO H3CO OH OH O OH O O O OH OH O O OH - + OSO3 Na HO H OH H OH H OH OR3 834 RO H OH R2 R1 OH R R1 R2 R3 OH O O H 831 HH OH H H H3CO OCH OH OH 832 H OH H H HO 3 835 833 H OH H COCH3

OH OH

HO - + O OSO3 Na OH OH MeO O HO O OMe H OH 837 OH OH O OH O O H H HO OCH OH OH HO 3 836 OH OH

OSO -Na+ O 3 O OH OH OH HO HO OMe H OH OH 839 O HO O HO HO OMe R OH OH O OH 838 O

OH OH

- + OSO3 Na HO H OH 840

Figure 3.49 Polyhydroxy steroids 829-840 from marine sources.

154

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

OH OSO -Na+ O 3 OH OH HO O HO OMe H OH OH HO OH 843 R OH OH O OH 841; R= H O 842; R= OH OH OH OH O OH OH OH OH - + O OSO3 Na - + HO OSO3 Na 845 HO H OH H OH 844 - + OSO3 Na OH

OH H OH OH

OH OSO -Na+ O 3 O O O HO OH OH HO OH OH HO HO 846 847 OH

OH OH OH OH OH OSO -Na+ 3 - + O O OSO3 Na O O HO OH OCH3 HO OH HO OH OCH3 848 HO 849

OH OH OH OH OH OSO -Na+ O 3 O OH O HO OCH OH 850 O HO 3 HO OCH OH 851 OH HO 3 O MeO O HO OCH 3 O HO O OH

OH OH

OH OH O HO O R OH 852; R= H HO OCH OH OH 854 853; R= OH HO 3

Figure 3.50 Polyhydroxy steroids 841-854 from marine sources.

Moniloside A 855 and -B 856 were isolated from Fromia monilia .135 (Figure 3.51 )

155

Polyhydroxysteroid hormones, also called ecdysones 501-502, 857-859 were found in arthropods. Karlson and Skinner isolated compound 502 in crabs in 1960.137 In the year 1966 Horn et al. first reported the isolation of crustecdysone 501 138 and 2-deoxycrustecdyson 857139 from crayfish Jesus lalandei . From Calinectes sapidus 140 , two 20-hydroxyecdysteroids, inokosterone 858 and makisterone A 859 were isolated. 20-Hydroxyecdysones were also found in Ajuga turkestanica (Labiatae), Vitex glabrata (Verbenaceae), Tapinella panuoides (Fungi) etc. 141 (Figure 3.51 )

OH OHOH

OH

OH OH HO O O HO HO R H O 855; R= H O 856; R= OH 857

OHOH OHOH

CH2OH OH HO HO OH OH HO HO H H O O 858 859

Figure 3.51 Polyhydroxy steroids 855-859 from marine sources.

OH OH OH OAc R OH OH HO HO HO O OH HO OH OH H O HO OH 4 H 863; , R= Ac RO 860 861; R= OH 864; R= Ac 862; R= H 865; R= H 866; 4, R= H Figure 3.52 Polyhydroxy steroids 860-866 from marine sources.

156

The first bile alcohol isolated from shark bile was scymnol 860.142-145 Another two bile alcohols myxinol 861 and deoxymyxinol 862 were found from hagfish.145-147 Tachibana, Nakanishi and their co-workers isolated steroid glycoside, mosesins 863-866 from P. marmoratus .148 These compounds are released by some fishes while they are about to be bitten by sharks. (Figure 3.52 )

OH OH OH OAc R OH OH HO HO HO O OH HO OH OH H O HO OH 4 H 863; , R= Ac RO 860 861; R= OH 864; R= Ac 862; R= H 865; R= H 866; 4, R= H Figure 3.52 Polyhydroxy steroids 860-866 from marine sources.

III.A.1.2 Synthetic polyhydroxy steroids Syntheses of some dihydroxy- as well as trihydroxy steroids were discussed also in the review part in connection with the work described in Chapter II. Again, some more of the syntheses of polyhydroxy steroids, in general, are summarized herein in brief. Cholest-5-en-3b,7 a-diol ( 42) was prepared from 3 b-O-sulphated-cholest-5-ene-7a-ol ( 867) by treating with 1:1 dioxane-pyridine. 26,27-Dinor-24-methyl-5a-cholest-22-ene-3b,6 a,15a,25- tetrol ( 869) was obtained by refluxing 868 with acetic acid at 100 oC for 2 hours. 149 (Figure 3.53 )

OR2 H H H H OR1 H H HO H RO OH OH 868; R = H, R = b-D-glucose 867; R= Na2SO3 1 2 42; R= H 869; R1= R2= H

Figure 3.53 Synthesis of compound 42 and 869.

157

D5-3b,7 a- and D5-3b,7 b-Hydroxysteroid were the autooxidation products of D5-sterols.150 Anion of 870 (derived from C-22 phenyl sulphone, 870) was used separately with (2S)-1,2- epoxy-3-methylbutane I and (2R)-1,2-epoxy-3-methylbutane II , to yield 24 (R)-and 24(S)- hydroxycholesterol ( 871 and 872) respectively. 151 (Scheme 3.1 )

OH OH SO2Ph H a I= a H HO HO II= 871 872 OCH3 870

a) i) n-BuLi,-780C, 2h; ii) Li, NH (l), -780C, 30 mint; iii) TsOH, dioxane-H O, 800C, 1h 3 2 Scheme 3.1 Synthesis of 24 (R)-and 24(S)-hydroxycholesterol ( 871 and 872 ) respectively.

Hydrolytic cleavage of (25R)-5,6-epoxyspirostan-22 a-O-3b-ol ( 873) by Dowex 50W X8 in aqueous methanol resulted (25R)-6b-Methoxyspirostan-22 a-O-3b,5 a-diol 874 and (25R)- spirostan-22a-O-3b,5 a,6 b-triol 875. Similarly, (25R)-spirost-5-en-22 a-O-3b,4 b-diol, 876 on cleavage with Dowex 50W X8 yielded (25R)-3b,6 b-dihydroxy-5a-spirostan-22 a-O-4-one 877.152 (Scheme 3.2 )

O O O

O O O Dowex 50W X8 Dowex 50W X8 MeOH/ H O MeOH/ H2O HO 2 HO HO HO O HO OMe OH 875 874 873 O O

O O Dowex 50W X8

HO HO O H OH O OH 876 877

Scheme 3.2 Cleavages of 874 and 876 by Dowex 50W X8.

158

When the epoxide 878 was treated with SmI 2 in THF, followed by carbonyl reduction and desilylation 153 , a trihydroxy steroid (23)R,25(S)-dihydroxycholesterol 879 was formed.154 (Scheme 3.3 )

OH O O O OH 1. SmI2, THF, -78 C O BH, THF, -10oC 2. O

TBSO 3.Bu4NF HO 878 879

Scheme 3.3 Treatment of epoxide 878 by SmI 2 in THF.

A cytotoxic, polyhydroxylated sterol 24-methylene-cholesta-3b,5 a,6 b,19-tetrol ( 24 ) was synthesized from stigmasterol (15 ) by 10 steps with overall 9% yield. 155a (Scheme 3.4 )

O O

I II

HO AcO AcO 15 Br 880 OH 881

O O III O

HO HO

IV V O HO AcO AcO HO Br OH 883 882 884 + O

HO HO

VI HO HO OH OH OH OH 885 24

Scheme 3.4 Synthesis of cytotoxic sterol 24 from stigmasterol ( 15 ). I) Ref. 155b; II) NB/HClO 4/ H 2O;

III) Pb(OAc) 4, I 2/ light; IV) Zn/ 95% EtOH; V) a. HCOOH/H 2O2, b.CH 3OH/ KOH; VI) Ph 3P=CH 2

159

Trichoderma viride , a microorganism converted 5 a,6 a-epoxy-3b-hydroxy-16-pregnen-20- one ( 886) into 3 b,5 a,6 b-trihydroxy-16-pregnen-20-one ( 887) and 3 b,5 a,6 b, 15 b-tetrahydroxy- 16-pregnen-20-one ( 889) in aerobic condition. 156 (Scheme 3.5 )

O O O

Trichoderma viride + OH HO HO HO O HO HO OH 886 OH 887 888 Scheme 3.5 Synthesis of polyhydroxy steroids 887-888 by microorganism.

The 20, 21-ketol group of a 21-chloromethylpregnane derivative (889) was oxidized by o 157 BiO 3/ AcOH at (50-60) C while refluxed for 3h with the reagent. (Scheme 3.6 )

OH O O O Cl HO OH HO OH

Bi2O3 (4.7 eq.) AcOH, 50-60oC, 3h O O 889 52% 890

Scheme 3.6 Oxidation of 889 by BiO 3.

12,23-Dihydroxy-22-oxocholaic acid 894-897 were synthesized as shown beow. 158 (Scheme 3.7) The metabolite of ORG OD14 (generic name: tibolone, structurally: (7 a,17 a)-17-hydroxy- 7-methyl- 19-norpregn-5(10)-en-20-yn-3-one, 898 ) which is of great interest in hormone replacement therapy in menopausal women at risk of osteoporosis 159, 160 was found to be a 3a,7 b,17a-triol ( 899). The 7-epimer of the triol, 900, was also synthesized ( Scheme 3.8 ). 161

160

O O O OH 22 23 22 23

12 12 O OAc HO OAc X X OAc f OAc Y Y H H 891; X= H, Y= OAc (64%) 894; X= H, Y= b-OAc 892; X= H, Y= OTs (65%) 895; X= H, Y= b-OTs e 2 896; X, Y= 2 893; X, Y= (82%) g 897; X= Y= a-OH (67%)

e) LiBr, Li2CO3, DMF; f) NaBH4, MeOH; g) OsO4, NMO

Scheme 3.7 Synthesis of polyhydroxy steroids 894-897 .

OH OH OH

H H H

H H H H H H HO OH O OH HO OH 900 898 899 Scheme 3.8 Synthesis of compounds 899-900 .

Certonardalsterol D 3 (901 ), a highly potent natural antitumor agent was synthesized stereoselectively from (+)-dehydroepiandrosterone ( 328) via 22-aldehyde.162 (Scheme 3.9 )

OH

O

OH HO HO 328 OH 901

Scheme 3.9 Synthesis of natural antitumor agent certonardalsterol D 3 (901).

161

25(R)-spirostan-3b,5 a,6 b,19-tetrol (885 ) which is capable of suppressing proliferation and migration of C6 malignant glioma cells, was synthesized from diosgenin (459) at 8.55% overall yield as shown in Scheme 3.10 below. 163

O O O O O O

Ac2O NBS Pyridine HClO HO AcO 4 AcO Br 459 902 903 OH

O O light Pb(OAc)4/I2 O AcO HO O

Ac2O Zn O Pyridine C H OH AcO AcO 2 5 AcO 905 883 904 mCPBA O O

AcO HO

1.H2SO4/ THF

AcO 2.Na2CO3/MeOH HO O OH 906 OH 885

Scheme 3.10 Synthesis of 25(R)-spirostan-3b,5 a,6 b,19-tetrol ( 885) from diosgenin ( 459 ).

D5- Steroids were directly converted into their corresponding 5 a,6 b-dihydroxysterols by using magnesium bis(monoperoxophthalate) hexahydrate with bismuth triflate(III) in acetone. The process was of two steps although there was no need to work up the intermediate step and was carried out in same solvent. This protocol was applied on cholesterol, cholesteryl acetate, dehydroepiandosterone, sitosterol, stigmasterol, pregnenolone, 3 b-hydroxyetiocholenic acid, 16- dehydropregnenolone, 16 a-hydroxypregnenolone and 16 a,17a-epoxy-21-acetoxypregnenolone to form their corresponding 5 a,6 b-diols (514, 907, 79 , 908-912, 887, 913). 164 (Figure 3.54 )

162

Step I Step II MMPA Bi(OTf) HO HO 3 HO O HO OH

O 22 R2 R O OAc

R1

HO R1 HO HO HO HO OH OH OH 913 907; R=OAc; R2= H 909; R= O (ketonic), R1= H 980; R1= OH; R2= C2H5; 22-ene 1 910; R= COCH3; R = H 1 911; R= COCH3; R = OH 912; R= COOH; R1= H

Figure 3.54 Polyhydroxy steroids 907-913.

Sodium borohydride reduction of 391 resulted 4,6-androstadiene-3b,17b-diol 914 as shown below. 165 (Scheme 3.11 )

O OH

NaBH4 O HO 391 914

Scheme 3.11 Synthesis of 914.

7a-Position of cholesterol was hydroxylated by cholesterol-7a-hydroxylase Cyp7A1 which was then oxidised followed by concomitant double bond migration by HSD3B7 to form 7 a- hyroxy-4-cholesten-3-one ( 915). 915 was then transformed to 7 a,12a-dihydroxy-4-cholesten-3- one ( 916) by CYP8B1 .166 (Scheme 3.12 )

163

H CYP7A1 H H HO HO OH 13 42

OH HSD3B7

CYP7B1

O OH O OH

916 915

Scheme 3.12 Synthesis of 7 a,12a-dihydroxy-4-cholesten-3-one ( 916).

Triacetyl cholylamide ( 917) was treated with Grignard reagent isopropyl magnesium bromide to form norcholyl-isopropylketone ( 918) which after Wolf-Kishner’s reduction yielded trihydroxy coprostane ( 919 ). Compound 917 on treatment with normal propyl bromide resulted norcholyl-normalpropylketone ( 920 ) and this on Wolf-Kishner’sreduction again yielded trihydroxy-norsterocholane ( 921). 167 (Scheme 3.13 )

O O CH3COO CH3COO

NH2 Br

H3COCO OCOCH3 H3COCO OCOCH3 917 918

CH CH CH Br Wolf-Kishner's 3 2 2 Reduction

O CH3COO OH OH

Wolf-Kishner's Reduction

H3COCO OCOCH3 HO OH HO OH 920 921 919

Scheme 3.13 Synthesis of norcholyl-normalpropylketone ( 920 ) and trihydroxy-norsterocholane ( 921).

164

Huppi et al. undertook the challenging synthesis of a number of polyhydroxy steroids which also include crustecdysone, a moulting hormone of insects (501). 168 (Figure 3.18 )

III.A.2 A brief review on the epoxy- and epoxy-polyhydroxy steroids In nature, many compounds with different functional groups are found. One of such frequently occurring functional group is epoxide. Their importance lie on the fact that these can be easily prepared from various starting materials as well as these are very susceptible to reaction with a large number of reagents due to their inherent polarity and the strain of the three-membered ring. 169-170 Plants and marine animals are the major sources of this group of compounds.

III.A.2.1 Natural abundance All marine organisms have been proven to provide unusual steroid metabolites, but people believe that marine sponges may provide the most diverse and biogenetically unprecedented array of unconventional steroids in the entire animal kingdom.

III.A.2.1.1 Isolated from plants Though the number is not so high, steroidal epoxides are found in different parts of various plants. Serratula wolffi is one of the richest sources of ecdysteroids. A novel natural epoxysteroid, 14 a,15a-epoxy-14,15-dihydrostachysterone B ( 922 ) ( Figure 3.55 ) was isolated from the roots of S. wolffi .171

OH H H OH O O H O H HO OH O O OH O HO O H HO H O 922 OH O 924 OH 923

Figure 3.55 Epoxy hydroxy steroids 922-924 .

Withania somnifera leaves were reported to contain withaferin A ( 923 )172 possessing antitumour, COX-2 enzyme inhibitory, chemopreventive, antioxidant and the inhibition of

165 human lung, colon, CNS and breast cancer cell proliferation, etc activities. 173-177 Along with compound 923 withanone ( 924 ) was also isolated from the plant. 172 (Figure 3.55 ) Physalis philadelphica Lam. was found to contain epoxy steroids 925-931.178 (Figure 3.56 )

R' R OH OH OH OHOH

O O O H O H OH O O O O O O O H OR H OH H H OH

H H H H H H H H

O O O O OR OH HO 925; R=H; R'=OH 928; R=OH 930 931 926; R=Ac; R'=OH 929; R=H 927; R=H; R'=H

Figure 3.56 Epoxy steroids 925-931 .

Two new epoxy steroids, 5 α,8 α-epidioxy-22 β,23β-epoxyergosta-6-en-3β-ol ( 932 ) and 5α,8 α-epidioxy-22 α,23α-epoxyergosta-6-en-3β-ol ( 933 ) were isolated from the plant Helianthus tuberosus .179 (Figure 3.57 )

O O H

H H HO HO O HO OH O O O O 932 933 934

Figure 3.57 Epoxy steroids isolated from plants ( 932-933 ) and from coral ( 934).

III.A.2.1.2 Isolated from animals Different marine organisms, like coral, mollusk, sponge, starfish, mushroom, algae are the major sources of epoxy steroids. And marine sponges are the richest source of these metabolites.

III.A.2.1.2.a From coral A number of oxysteroids including epoxysteroiods are known to be produced from a number of different soft corals. A new cytotoxic sterol 934 was isolated from the soft coral Gersemia fruticosa in the year 1998. 179 (Figure 3.57 )

166

From the soft coral Sinularia disecta a polyhydroxyepoxysteroid 935 was isolated in 1999.64 (Figure 3.58 )

R R O HO H HO OH O O H H

H H H H HO HO O OH O 935 936; R= 940; R= H OH H 937; R= 941; R= H H HO O 938; R= 943 942; R=

939; R=

Figure 3.58 Epoxy polyhydroxy steroids 935-943 from soft coral.

Four new epoxy sterols 936-939 were isolated from the soft coral Clavularia viridis .180 Stoloneferone L-N ( 940-942 ) was isolated from the bioassay-guided fraction of dichloromethane extract of Clavularia viridis .181 (Figure 3.58 ) The ethanol extract of Sinularia gibberosa possessed gibberoepoxysteroid 943 .24 (Figure 3.58)

O OH OH OH OH

OH OH OH HO HO HO O O O 944 945 946

Figure 3.59 Epoxy polyhydroxy sterols 944-946.

The Red Sea soft coral Lobophytum depressum 49 was found to contain three polyhydroxysterols 944-946 with 5 b,6 b-epoxy groups. ( Figure 3.59 ) Melithasterol A ( 947), a 5 a,6 a-epoxy natural steroid was isolated from a gorgonian coral Melithaea ocracea .182 (Figure 3.60 )

167

OR AcO O HO

H HO OH RO HO O 948; R=Ac O 947 949 R=H 950

Figure 3.60 Epoxy polyhydroxy sterols 947-950 from soft coral.

More than 30 of the genus Sarcophyton occurring in different areas were examined. One of the soft coral, Sarcophyton crassocaule isolated from the Indian Ocean was reported to contain 17 different compounds of which 948 and 949 were novel 17 b, 20 b-epoxy steroids. 183 (Figure 3.60) From the MeOH extract of the gorgonian soft coral Isis hippuris polyhydroxy-epoxy natural steroid 950 (structually 11 a-trihydroxygorgosta-5a,6 a-epoxy-12 b-monoacetate) was isolated. 184 (Figure 3.60) Among the most prolific sources of sterols in marine environment, octocorals were well recognized. One of such genus Plexaurella grisea was reported to contain the epoxy steroid 5b,6 b-epoxyergost-24(28)-ene-3b,7 b-diol ( 951 ) along with other sterols 952 and 934 , and compound 946 was isolated infact first time from natural sources. Compound 951 showed 185 activity against the HT 29 cell line (ED 50 = 0.1 mg/mL). (Figure 3.61 )

R 951; R= H OR OH H H H

952; R= H H H H HO OH RO OR HO OH O O O 953; R=H 955; R=Ac 954

Figure 3.61 Epoxy polyhydroxy sterols 951-954.

Sinugrandisterol C ( 953 ) and -D ( 954 ), identified respectively as 5 b,6 b-epoxy-24- methylenecholesta-1a,3 b,7 b-triol and 5 b,6 b-epoxy-24-methylenecholesta-22E-en-1a,3 b,7 b-

168 triol were isolated from the ethanol extract of soft coral Sinularia grandilobata . The acetyl derivative ( 955 ) of compound 953 was also prepared. 186 (Figure 3.61 )

III.A.2.1.2.b From mushroom: From the edible mushroom Grifola gargal, polyhydroxyepoxysterols 956-959 were isolated in 2011. 10 (Figure 3.62 ) change 957

R

OH HO O HO O HO OH O O 957 956 960; R=

O 85; R= O O HO O O HO O 958 959

Figure 3.62 Polyhydroxyepoxy sterls from mushroom ( 956-959) and algae ( 960).

III.A.2.1.2.c From algae 24,25-Epoxycholesterol 960was isolated from red algae Asparagopsis armata 1 and Rissoella verruculosa .2 Hizikia fusiformis , a brown alga was found to possess 24,28-epoxyfucosterol 85 . (Figure 3.62)

III.A.2.1.2.d From sponge 9a,ll a-Epoxycholest-7-ene-3b,5 a,6 b,19-tetrol-6-acetate 961 was isolated from an unidentified species of Dysidea , collected in Guam.187 Dysidea herbacea , collected from Ethiopea was found to contain one epoxy-trihydroxy sterol 962.74 (Figure 3.63 ) Compounds 963-964 were found in the Pacific sponge Jereicopsis graphidiophora and 964 was the first reported D9(11) -8,14 naturally occurring epoxide.188 (Figure 3.64 )

The 5 b,6 b-epoxide 965, having antileukemic activity (IC 50 = 0.5 mg/mL) against P 388 leukemia cells in vitro was found in Liptophyton viridis. 52-53 Marine gastropod Planaxsis sulkatus

169 possessed a cytotoxic epoxy-trihydroxy sterol 966 which was active against L 1210 cell line and metabolite 966 was suspected to be the precursor of 960.189 (Figure 3.65 )

HO HO O

HO HO OH HO O OAc 961 962

Figure 3.63 Epoxy hydroxy sterols ( 961-962) from Dysidea sp.

O

O MeO MeO 963 964

Figure 3.64 Epoxy sterols ( 963-964) from sponge.

HO O

HO H HO O HO OH 965 966

Figure 3.65 Epoxyhydroxy sterols 965-966.

Epoxy steroid 967 was from Microscleroderma spirophora 190; epoxy steroids 968-973 were from Spongia officianalis 191 and 974 was isolated from Theonella swinhoei .192 (Figure 3.66 ). Glaciasterols A ( 975) and -B ( 976), were found in Aplysilla glacialis 193 and two of their analogues compounds 977 and 978 were collected from Dysidea fragilis .194 These were actually 9,11-secosterols. Another group of secosterols, compounds 979-981 were isolated from Lufariella sp. 195 and compound 982 from Spongia matamata .196 (Figure 3.67 ) and many more can be found from the paper of Aiello A. 197

170

R R

O AcO OAc AcO OAc O O HO 967 969; R= 972; R=

970; R= 973; R= O OMe HO 971; R= 974; R= 968

Figure 3.66 Epoxy sterols 967-974.

975; R= 979; R'=Ac, R= HO CHO R R 976; R= O O 980; R'=Ac, R=

977; R= H H 981; R'=Ac, R= AcO AcO O O 978; R= 982; R'=H, R=

Figure 3.67 Epoxy 9,11-seco sterols 975-982 .

5a,6 a-Epoxy-26,27-dinorergosta-7,22-en-3b-ol ( 983), 5a,6 a-epoxycholesta-7,22-en-3b-ol (984), 5a,6 a-epoxyergosta-7,24(28)-en-3b-ol ( 985), 5a,6 a-epoxyergosta-7-en-3b-ol ( 986), 5a,6 a-epoxystigmasta-7,22-en-3b-ol ( 987) and 5 a,6 a-epoxystigmasta-7-en-3b-ol ( 988 ) were isolated from Chinese sponge Ircinia aruensis .198 (Figure 3.68 )

R

983; R= 985; R= 987; R= H H HO 984; R= 986; R= 988; R= O

Figure 3.68 Epoxy sterols ( 983-988) isolated from Chinese sponge Ircinia aruensis .

171

III.A.2.1.2.e From mollusc 9a,11a-Epoxy-5a-cholest-7-en-3b,5,6b-triol ( 989 ), a cytotoxic epoxy-trihydroxy sterol was isolated from the marine mollusk Planaxis sulcatus .189 (Figure 3.69 )

III.A.2.1.2.f From starfish The first case of marine polar steroids containing 4,5-epoxy functionality was reported to be the aglycon moiety of kurilensoside H ( 990) which was found from the alcoholic extract of the Far Eastern starfish Hippasteria kurilensis collected near Kuril Islands. 199 (Figure 3.69 )

C8H17

O OH OH OH OH HO O HO O HO O OH HO OH 989 OH 990

Figure 3.69 Epoxy sterols 989-990.

III.A.2.2 Synthetic epoxy- and epoxy-polyhydroxy steroids A cytotoxic natural steroid, 9a,11a-epoxy-5a-cholest-7-en-3b,5,6b-triol ( 989 ) was synthesized from cholesta- 5,7-dien-3-yl acetate, 991 via a number of intermediate compounds as shown in the scheme. 200 5,6a-Epoxy-5a-cholest-7-en-3b-yl acetate ( 994) was also synthesized from compound 991 by treating with MTO, UHP and pyridine in diethyl ether solution. 200 (Scheme 3.14) 3b-tButyldimethylsilyloxy-bisnor-5-cholenaldehyde 995 on treatment with arsonium ylide 201 996 in THF, followed by epoxidation with H 2O2-NaOH and EtOH-THF yieled compound 878.154 Compound 998 and 1000 were obtained respectively from 997 and 999 by the t o diasteroselective epoxidation process where V(O)(acac) 2 and BuOOH were used in DCM at 0 C. 202 In case of synthesis of compound 1000, desilylation was followed after epoxidation. 154 (Scheme 3.15 )

172

MTO/ UHP

o AcO Et2O, 25 C AcO 991 HO OH 992 MTO/ UHP Py, Et2O, RT

1. Ac2O, Py, RT 2. Hg(OAc)2, RT AcOH/CHCl3 AcO O 994

1. m-CPBA o CHCl3, 0 C 989 2. K2CO3 AcO HO MeOH/H2O OAc 993

Scheme 3.14 Synthesis of a cytotoxic natural steroid 989 and its acetate derivative 994.

O

1. Ph3As O O H 998 THF, 23oC,16h 2. H O , NaOH, TBSO 2 2 TBSO 995 2:1 EtOH-THF 878 35oC, 3h

OH OH

22 O o V(O)(acac)2, 0 C t-BuOOH, CH2Cl2

TBSO HO 997; 22b-OH 998; 22a-OH; 24a,25a-epoxy 999; 22a-OH 1000; 22b-OH; 24b,25b-epoxy (desilylation o was needeed: Bu4NF, THF, 23 C, 3h)

Scheme 3.15 Synthesis of epoxy sterols 878, 998 and 1000 .

173

Yin et al. produced 1 a,2 a-epoxy-17,17-ethylenedioxyandrost-4,6-dien-3-one ( 1002) from 203 17,17-ethylenedioxyandrost-1,4,6-trien-3-one ( 1001) by H 2O2-MeOH/NaOH. (Scheme 3.16 )

O O O O O H2O2/ MeOH NaOH O O 1001 1002

Scheme 3.16 Epoxidation by H2O2/ MeOH .

CDB-2914 (structurally 17 a-acetoxy-11 b-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9- diene-3,20-dione; 1008 ), an orally usable antiprogestin, was found to be eight times more potent than mifepristone (in rat),204 though was synthesized earlier, 205 a large-scale synthesis was required for clinical trials of CDB-2914 (1008 ). And this was accomplished by following a selective epoxidation and then ring opening of the epoxidation in the intermediate steps. 206 (Scheme 3.17 )

O O O O O OH OH OH Ethlylene glycol HC(OEt)3 (CF3)2CO, H2O2 O Na HPO , CH Cl O O p-TsOH, CH Cl 2 4 2 2 O 2 2 O O 1003 1004 1005

Mg, cat, CuCl Br NMe2 THF

N O N O N O

OAc OH OH

H2SO4, EtOH p-TsOH, CH2Cl2 Heat O (CF3CO)2O, AcOH O O OH 1008 (CDB-2914) 1007 1006 Scheme 3.17 Synthesis of CDB-2914 1008.

174

Melithasterol A ( 947), a 5 a,6 a-epoxy natural steroid was synthesized through microwave irradiation and the epoxidation was achieved from the isomerization of 5 a,8 a-epidioxy steroid (1009 ). 182 (Scheme 3.18 )

R R R R

Ac2O/Py MW 10% KOH O O HO AcO AcO OH HO OH O O O O 1011 947 1009 1010 (R= C8H17, Cholesteryl)

Scheme 3.18 Synthesis of Melithasterol A 947 through microwave irradiation.

When 17(20)-pregnene linked to an ester carrying an a,a-dimethylbenzyl alcohol group (1012) was treated with tert butyl hydroperoxide it led to epoxidation from the a-face in 60% yield (1 013). 207-208 (Scheme 3.19 )

H O

tBuOOH

cat. Mo(CO)6/ C6H6 O O H H O n 1012 O n 1013

HO HO

Scheme 3.19 a-face epoxidation of 1012 with tert butyl hydroperoxide.

3-Methoxyestra-1,3,5(10),6-tetraen-17-one cyclic 1,2-ethanediyl acetal (1014) when was treated with 3-chloroperbenzoic acid in ethyl acetate solution for 6 hours at room temperature epoxy steroid 1015, structurally, 6a,7 a-epoxy-3-methoxyestra-1,3,5(10) –trien-17-one cyclic 1,2- ethanediyl acetal was produced. 209 (Scheme 3.20 )

175

O O O O

m-CPBA/ EtOAc O O O 1014 1015

Scheme 3.20 Epoxidation of acetal 1014 .

7 a -Chloro-17 b -[[(1,1-dimethylethyl)dimethylsilyl]oxy]-3-methoxy-7-methylestra- 1,3,5(10)-trien-6 b -ol 1016, obtained from (17b)-17-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-3- methoxy-7-methylestra-1,3,5(10),6-tetraene was dissolved in THF and was reacted with potassium tert -butoxide to result ultimely 17 b -[[(1,1-Dimethylethyl)dimethylsilyloxy]-6 b ,7 b - epoxy-3-methoxy-7-methylestra-1,3,5(10)-triene (1017).209 (Scheme 3.21 )

OTBDMS OTBDMS

oxalic acid, acetone Cl O water (50%) O O OH 1016 1017

Scheme 3.21 Synthesis of 17 b -[[(1,1-Dimethylethyl)dimethylsilyloxy]-6 b ,7 b -epoxy-3-methoxy-7- methylestra-1,3,5(10)-triene (1017).

Steroid 7 a-chloro-6b-ol 1018 was treated with tBuOK in THF result 6 b,7 b-epoxysteroid 1019 as shown below. 209 (Scheme 3.22 )

O O O O

tBuOk Cl THF O O O OH 1018 1019

Scheme 3.22 Epoxidation of 7a-chloro-6b-ol 1018 .

176

H2O2/ formic acid mixture in DCM transformed 1020 and 1021 respectively into 3a,4 a- Epoxy-5a-androstan-17-one 1022 and 3 a,4a-epoxy-5a-androstan-17-one 1023.210 (Scheme 3.23)

O

H2O2/ HCOOH +

H O O 1019; 5a 1022 1023 1021; 5b

Scheme 3.23 Synthesis of 3a,4 a-Epoxy-5a-androstan-17-one 1022 and 3 a,4a-epoxy-5a-androstan-17- one 1023.

Previously, compound 1024-1026 were prepared from their corresponding 4-ene-3-one derivatives by reacting with H 2O2 in methanol in presence of NaOH (in case of 1024 and 1025) 211-213 or NaHCO 3 (in case of 1026). (Figure 3.70 )

O

R

O O

1024; R=CH3 1025; R=CH2OH 1026; R=CHO

Figure 3.70 Epoxy steroids 1024-1026 .

When (25R)-spirost-5-en-22 a-O-3b,4 b-diol, 335 was treated with m-CPBA in DCM, (25R)- spirost-5-en-22 a-O-3b,4 b-diol, 876 was formed as enantiomeric mixture. 152 (Scheme 3.24 ) 2,3a-Epoxy-5a-cholestane 1028 was prepared from 5 a-cholest-2-ene 1027 by m-CPBA in water-DCM mixture. 214 (Scheme 3.24 ) 17β-Hydroxy-3-methoxy-14β,15β-epoxy-7α -methylestra-1,3,5(10)-triene ( 1030) was prepared from the homoallylic alcohol 1029 by treating with mCPBA in chloroform. 215 (Scheme 3.24)

177

O O

O O mCPBA DCM HO HO O OH OH 335 876

mCPBA O H2O/ DCM H H 1027 1028

OH OH

mCPBA O MeO MeO 1029 1030

Scheme 3.24 Synthesis of epoxy steroids 876, 1028 and 1030.

A number of epoxy derivatives of cholesterol were synthesized either by using H 2O2 in

MeOH or m-CPBA in CHCl 3. Such as 5α,6α -epoxy-cholestan-3β -ol ( 1031) from cholest-5-en- 3b-ol ( 13 ) and 4β,5β -epoxy-6-cholesten-3β -ol ( 1033) and 4β,5β -epoxy-6α,7α -epoxycholestan-

3β -ol ( 1034) from 4,6-cholestadien-3β -ol ( 1032) were prepared by using m-CPBA in CHCl 3. 6α,7α -Epoxy-1,4-cholestadien-3-one ( 1036) was also fomed from 1,4,6-cholestatrien-3-one

(1035) by similar treatment. But when the same (comp 1035) was treated with H2O2, 5%NaOH/MeOH in MeOH at rt, it yielded 1α,2α -Epoxy-4,6-choestadien-3-one ( 1037) which on reduction with NaBH 4 in absolute ethanol resulted 1α,2α -Epoxy-4,6-cholestadien-3β -ol (1038 ). 216 (Scheme 3.25 )

178

H2O2 mCPBA MeOH CHCl No Reaction 3 HO HO O 13 1031

H2O2 mCPBA MeOH CHCl3 No Reaction + HO HO HO O O O 1032 1033 1034

mCPBA O O H2O2 CHCl3 NaBH MeOH 4 O O HO O HO 1037 1036 1035 1038

Scheme 3.25 Epoxidation of cholesterol derivatives.

Similarly, 3b-hydroxy-5a,6 a-epoxyandrostan-17-one (1039 ) from dehydroepiandrostone (328) and 4β,5β -epoxy-androst-6ene-3β, 17b -diol ( 1041) from 4,6-androstadien-3β,17b -diol

(1040) were prepared by using m-CPBA in CHCl 3. 6α,7α -Epoxy-1,4-androstadien-3,17-dione (1042) was also formed from 1,4,6-androstatrien-3,17-dione (391) by similar treatment. But when the same (comp 391) was treated with H2O2, 5%NaOH/MeOH in MeOH at room temperature, it yielded 1α,2α -epoxy-4,6-androstadien-3,17-dione ( 1043) which on reduction with 217 NaBH 4 in absolute ethanol resulted 1α,2α -epoxy-4,6-androstadien-3β ,17 b-diol ( 1044). (Figure 3.71 )

179

O O OH OH

HO HO HO HO O O 328 1039 1040 1041

O O O OH

O O

O O O HO O 391 1042 1043 1044 Figure 3.71 Epoxy derivatives (328, 391, 1039-1044) of steroids with androstane skeleton.

5,6a- and 5,6 b-epoxysitosterols ( 1045-1046 and 1047-1048 respectively) were prepared from b-sitosterol ( via the acetate derivative 76) by m-CPBA followed by deacetylation. 218 (Scheme 3.26)

H H H

mCPBA, NaHCO3 H H H H + H H CH Cl AcO 2 2 RO RO O O 76 1045; R= Ac 1047; R= Ac 1046; R= H 1048; R= H

Scheme 3.26 Epoxidation of 3b-acetoxy b-sitosterol.

O O O OMe OMe

mCPBA/ DCM AcO OAc AcO OAc H H 1049 1050

Scheme 3.27 Epoxidation of methyl-3a,7 a-diacetylchol-11-enate 1049.

180

mCPBA in DCM converted methyl-3a,7 a-diacetylchol-11-enate 1049 into methyl 3 a,7 a- diacetyl-11 a,12a-epoxy-5b-cholan-24-oate ( 1050). 219 (Scheme 3.27) mChloroperbezoic acid was also used to convert D4-3-ketosteroids ( 428, 56, 1051-1052) into their corresponding seven-membered A-ring epoxy lactones ( 1053-1056 & 1057-1060) as shown below. 220 (Scheme 3.28)

R1 R1 R1 R3 R2 R3 R2 R3 R2

m-CPBA/ CH2Cl2 o 0 C, 24h O + O O O O O O 1 2 3 428; R = OH, R =R =H 1053; R1= OH, R2=R3=H 1057; R1= OH, R2=R3=H 56; R1= COCH , R2=R3=H 1 2 3 1 2 3 3 1054; R = COCH3, R =R =H 1058; R = COCH3, R =R =H 1051; R1= COCH , R2= OH, R3=H 1 2 3 1 2 3 3 1055; R = COCH3, R = OH, R =H 1059; R = COCH3, R = OH, R =H 1052; R1= OH, R2= COCH OH, R3=OH 1 2 3 1 2 3 2 1056; R = OH, R = COCH2OH, R =OH 1060; R = OH, R = COCH2OH, R =OH Scheme 3.28 Epoxidation of D4-3-ketosteroids.

Another new reagent KMnO 4/Fe(ClO 4)3·nH2O, in the presence of NaH 2PO 4·3H 2O was applied on D5-steroids to produce the corresponding 5 b,6 b-epoxy steroids. 20-Oxo-pregn-5-ene- 3b-yl acetate 330 in DCM was transformed to 5,6-epoxy-20-oxopregnane-3b-yl acetate 1061.

This methodology, excluding the use of NaH 2PO 4·3H 2O, was also used to prepare the corresponding a-ketols both from D5-steroids and the epoxides seperately. 221 (Scheme 3.29)

O O

KMnO4/ Fe(ClO4)3.nH2O/ NaH2PO4. 2H2O) CH Cl / tBuOH/ H O, RT AcO 2 2 2 AcO O 330 1061

5 Scheme 3.29 Epoxidation of D -3-ketosteroids by KMnO 4/Fe(ClO 4)3·nH2O.

(22E)-3b-Acetoxy-5a-ergosta-7,14,22-triene ( 1062) was converted into (22E)-3b-Acetoxy- 14 a,15a-oxido-5a-ergosta-7,22-diene ( 1063) by m-CPBA in DCM. 222 Applying the same

181 procedure 3b-acetoxy-5a-cholesta-8,14-dien-7-one ( 1064) was transformed into 3b-acetoxy- 14 a,15 a-epoxy-5a-cholest-8-en-7-one (1065). 223 (Scheme 3.30 )

mCPBA/ DCM O AcO AcO H H 1062 1063

mCPBA/ DCM O AcO O AcO O 1064 1065

Scheme 3.30 (22E)-3b-Acetoxy-14 a,15a-oxido-5a-ergosta-7,22-diene ( 1063 ) and 3b-acetoxy-14 a,15 a- epoxy-5a-cholest-8-en-7-one (1065 ).

3a,4 a-Epoxy-5a-androstan-17 b-ol ( 1068) was synthesized from 5 a-androst-3-en-17 b-ol (1066) when the latter in DCM was treated overnight with performic acid and hydrogen peroxide mixture at RT. Using the same methodology acetate derivative of 1067 (1069 ) from the acetate derivate of 1066 (1068) were prepared. The oxime derivative ( 1070) of compound 1022 was also synthesized by the standard oximination process. The aromatase inhibitory activity was tested with the synthesized oxysteroids and it was found that at least one carbonyl group was found to be essential for having effective inhibitory activity. 224 (Scheme 3.31) 3b-Acetoxy-5a-bromo-6b-hydroxystigmastane ( 1071) was refluxed with sodium acetate with ethanol to result 3 b-Acetoxy-5,6b-epoxy stigmastane ( 1047). Its 3 b-hydroxy derivative (1048) was also prepared. 225 (scheme 3.32 )

182

OH OH

H2O2, HCO2H DCM, RT H O H 1066 1067

OCOCH3 OCOCH3

H2O2, HCO2H DCM, RT H O H 1068 1069

O NOH

NH2OH

O H O H 1022 1070

Scheme 3.31 Epoxidation of 1068, acetate derivative of 1067 and 1070.

H H CH COONa H H 3 H H EtOH AcO RO Br O OH 1071 1047; R= Ac 1048; R= H Na2CO3/ MeOH

Scheme 3.32 Synthesis of 3b-Acetoxy-5,6b-epoxy stigmastane ( 1047) and its acetate derivative 1048.

mCPBA oxidation of 3b-acetoxy-17-picolinylidene-androst-5-ene ( 1072) resulted 5 a,6 a- epoxy-N-oxide ( 1073), 5b,6 b-epoxy-N-oxide ( 1074), 5a,6 a:17 a,20a-diepoxy derivative 1075 and 5 b,6 b:17 a,20a-diepoxy derivative 1076. (Scheme 3.33) Again the same treatment on 3b- hydroxy-17-picolinylidene-androst-5-ene ( 1077) at 1:1 or 1:2 mole ratio yielded a mixture of epoxides 1078 and 1079 , whereas at 1:6 ratio, a mixture of 5 a,6 a-epoxy-N-oxide 1080 and 5b,6 b-epoxy-N-oxide 1081, 5a,6 a:17 a,20a-diepoxy-N-oxide 1082 and 5 b,6 b:17 a,20a- diepoxy-N-oxide 1083 were isolated.226 (Scheme 3.34)

183

H H N N

O mCPBA/ CH2Cl2 165: mCPBA=1:2 AcO AcO 1072 O 1073

mCPBA/ CH2Cl2 165: mCPBA=1:6

H H

N O N

O O + AcO AcO O O 1074 1075; 5a, 6a-epoxy 1076; 5b,6b-epoxy

Scheme 3.33 m-CPBA oxidation of 3b-acetoxy-17-picolinylidene-androst-5-ene ( 1072).

H H H H

N N N O N

O O mCPBA/ CH2Cl2 170: mCPBA=1:6 + + HO HO HO HO O O O 1077 1078; 5a,6a-epoxy 1080; 5a,6a-epoxy 1082; 5a,6a-epoxy 1079; 5b,6b-epoxy 1081; 5b,6b-epoxy 1083; 5b,6b-epoxy

Scheme 3.34 m-CPBA oxidation of 3b-hydroxy-17-picolinylidene-androst-5-ene ( 1077).

3b,21-Diacetoxy-pregn-17(20)-ene 1084 was epoxidised with m-CPBA to furnish 3 b,21- diacetoxy-17 a,20a-epoxy-5a-pregnane 1085 and 3 b,21-diacetoxy-17 b,20b-epoxy-5a-pregnane 1086.227 (Scheme 3.35)

184

OAc OAc OAc H H H O O

mCPBA + AcO AcO AcO H H H 1084 1085 1086

Scheme 3.35 m-CPBA oxidation of 3b,21-Diacetoxy-pregn-17(20)-ene 1084.

A number of 5,6-epoxy cholesterol derivatives having different 3 b-ether linkages were prepared from the corresponding 5-ene-derivatives (1087) using m-CPBA. Thus, cholesteryl 3 b- methoxy-5a,6 a-epoxy, cholesteryl 3 b-ethoxy-5a,6 a-epoxy, cholesteryl 3 b-butoxy-5a,6 a- epoxy, cholesteryl 3 b-hexyloxy-5a,6 a-epoxy, cholesteryl 3 b-octyloxy-5a,6 a-epoxy, cholesteryl 3b-decyloxy-5a,6 a-epoxy, cholesteryl 3 b-dodecyloxy-5a,6 a-epoxy were prepared having nuclei of 36 .228 (Scheme 3.36)

H H mCPBA H H H H CH2Cl2, RT H CnO H CnO 2n+1 2n+1 O 1087 36 R=CnH2n+1, n=1, 2, 4, 6, 8, 10, 12

Scheme 3.36 Epoxidation of 5-ene-derivatives ( 1087) of cholesteyl skeleton.

In 2011 Borah and Chowdhury reported the MW assisted epoxidation of 3b,17b-acetoxy androst-5-ene ( 330) with m-CPBA to yield 3 b,17b-acetoxy-5a,6 a-epoxy-5a-androstane ( 1061). The reaction took two minutes to complete while the classical method needed 9 days. The yield of the MW process was also better (71%) in comparison to the traditional way (43%). 229 (Scheme 3.37)

185

O OAc

mCPBA

AcO AcO O 330 1061

Scheme 3.37 MW assisted epoxidation of 3 b,17 b-acetoxy androst-5-ene ( 330) with mCPBA.

mCPBA in DCM/ NaHCO 3 converted 3b-Acetoxy-12 b-benzyloxy-5a-furostan-14, 16-diene (1088) into 3 b-Acetoxy-12 b-benzyloxy-5a-furostan-14-epoxy-17-ene ( 1089 ) and 3 b-Acetoxy- 12 b-benzyloxy-5a-furostan-14-hydroxy-17-ene ( 1090) into 3 b-Acetoxy-12 b-benzyloxy-5a- furostan-14-hydroxy-16-epoxide ( 1091). 230 (Scheme 3.38)

H BzO BzO O O H H mCPBA / CH2Cl2 H H O NaHCO AcO 3 AcO H H 1088 1089

BzO BzO O O O H H mCPBA / CH Cl H OH 2 2 H OH NaHCO AcO 3 AcO H H 1090 1091

mCPBA O

H H OH OH 1092 1093

Scheme 3.38 Synthesis of some epoxy steroids.

186

Again, the same reagent converted 5a-cholest-2-en-6a-ol ( 1092) into the corresponding epoxide 1093.231 (Scheme 3.38) Prednisolone, a synthetic glucocorticoid used to treat a variety of inflammatory and auto- immune conditions, (1094) was converted to 17 a,21-dihydroxy-9b,11b-epoxy-16 a- methylpregna-1,4-diene-3,20-dione 21-acetate ( 1095), a key synthetic intermediate in the synthesis of a range of highly useful medicinal glucocorticoids, by modified Mattox rearrangement in six steps and at 13% overall yield. 232 (Scheme 3.39)

O O HO OH OAc OH OH H O

H H H H O O 1094 1095

Scheme 3.39 Synthesis of 1095 from prednesolone ( 1094 ).

4a,5 a-Epoxy-17-picolinylidene-androstane-3-one ( 1097) and 4b,5 b-epoxy-17- picolinylidene-androstane-3-one ( 1098) were obtained by the oxidation of compound 1096 with hydrogen peroxide in alkaline medium. Similarly, 17 a-picolyl derivative 1099 gave a mixture of epoxides 1100 and 1101 as below. 226 (Scheme 3.40 )

H N N

H2O2/ NaOH

O O O 1096 1097, 4a,5a- 1098, 4b, 5b-

OH N OH N

H2O2/ NaOH O O O 1099 1100, 4a,5a- 1101, 4b, 5b-

Scheme 3.40 Epoxidation of 1096 and 1099 in H 2O2/ alkaline media.

187

Epoxy azasteroids ( 1102-1103) were very useful to synthesize a number of functionalised azasteroid derivatives ( 1108, 1111-1119). 233 (Scheme 3.41)

H H H H OCOC6H5 H OCOC6H5 H OCOC6H5

H H H a b H H H H H H RO O RO O RO N 1102; R=H 1104; R=H HO 1103; R=Ac 1105; R=Ac 1106; R=H 1107; R=Ac b H H H 2 H OCOC6H5 H OCOC6H5 H OR

H H H c c H H H H H H 1 MsO N O RO N OH R O N X H H 1109; R=H 1111; X=O,R1=Ac, R2= COC H 1108 6 5 1110; R=Ac 1112; X=O, R1=R2=H g 1 2 1113; X=H2, R =R =H d H H OCOC6H5 H H COCH3 COCH3 H H H h H H e or f H H H H RO N O RO N X O N X H H H 1118; X=H 1114; R=H 1116; X=H2 2 1115; R=MEM 1117; X=O 1119; X=O

o a) BF3.Et2O, 64% or 94%; b) NH2OH. HCl, KHCO3, MeOH, 52%; c) MsCl, py, 0 C, o 4h, 97%; d) KNO2, DMSO, 1150 C, 26%; e) KOH, then PCC, then HCl, 72%;f) LAH, then CrO3, then HCl, 20%; g) CrO3, 57%; h) H2IrCl6, H3PO3, (Me)2CHOH, 57% or 83%

Scheme 3.41 Synthesize of functionalised azasteroid derivatives.

1a,2 a-Epoxy-4,6-androstadiene-3,17-dione ( 1043) was prepared from 1,4,6-androstatriene- 3,17-dione ( 391) by treating it with with hydrogen peroxide in 5% NaOH/ MeOH. This compound 1043 was important for preparing 2-functionalised derivatives ( 1120-1124). When compound 391 was treated with m-CPBA in CHCl 3 epoxy steroid 1042 was formed. Under

188 reflux condition, with sodium cyanide and ammonium chloride in ethanol compound 1042 resulted 2 a-cyano-6a,7 a-epoxy-4-androstene-3,17-dione ( 1125). Cytotoxicity of compound 391 and 1125 was tested. Compound 1043 showed a selective dose dependent and high cytotoxicity on T47D cells (IC 50 7.1 μM) , better than that of tamoxifen (IC 50 13.0 μM) and cisplatin (IC 50 9.63 μM) 234, but was non-cytotoxic on MDA-MB231 cells. Thus, the cytotoxicity on estrogen- dependent breast cancer cells seemed to be dependent on 1 a,2 a-epoxy ring. However, cyanoepoxysteroid 1125 showed no appreciable inhibitory activity. 235 (Scheme 3.42)

O O O

O X H2O2 HX O NaOH/ MeOH Reflux, 3h 391 RT, 12h O O 1043 1120; X=Cl 1121; X=Br NaCN, NH4Cl EtOH, H2O O Reflux, 24h O O OH NC NC N3 + O O O 1122 1124 1123

O O O

NC mCPBA NaCN/ NH4Cl CHCl3 O O EtOH, Reflux O O 391 O 1042 1125

Scheme 3.42 Synthesis of epoxy and 2-functionalised androstane derivatives.

Eplerenone ( 1133), a cardiovascular epoxysteroidal drug was synthesized in seven steps from commercially available 11 a-hydroxyl canrenone ( 1126) with overall 48% yield. 236 (Scheme 3.43)

189

O O O HO MsO O O O

O O O 1126 1127 1128

O O O O O O

O O O O O O 1131 O OH 1130 1129

O O O O O

O COOMe O COOMe 1132 1133 Scheme 3.43 Synthesis of eplerenone (1133 ).

This drug was also synthesized from 17 b-Hydroxy-7a-carbomethoxy-3-oxo-pregna-4,9(11)- diene-21-carboxylic acid, g-lactone ( 1132) by treating it with Cl 3CCONH 2/ H 2O2 followed by o 237-238 CH 2Cl 2/ K 2HPO 4 at (10-20) C. (Scheme 3.43) Biotransformation of androsta-4,6-diene-3,17-dione ( 1134) into 3a-hydroxy-6a,7 a-epoxy- 5a-androstan-17-one ( 1135 ) was found to occur during the elution of ethyl acetate/petrol from the broth extract. 239 (Figure 3.72 )

O O H H H H

H H H H H HO O H O 1134 1135

Figure 3.72 Androsta-4,6-diene-3,17-dione ( 1134) and 3a-hydroxy-6a,7 a-epoxy-5a-androstan-17-one (1135).

190

Epoxydation with [Mn(tdcpp)Cl] and [Fe(tpfpp)Cl] was reported by Rebelo et al. in 2005. They showed that the Mn-complex resulted preferentially the b-epoxide ( 1139, 1141, and 1143) of D4- and D5-steroids respectively from 17b-acetoxy-4-androstene 1136, 4-cholestene 1137 and 3b -acetoxy-5-cholestene 40 whereas the Fe-complex catalyzes preferentially the formation of a- epoxids (1138 and 1140) of D4-steroids (1136, 1137), and increases the a-stereoselectivity in the 5 240 epoxidation (1142) of D from 40 in the presence of an oxygen donor viz., H2O2. (Figure 3.73 )

R C8H17 R O

H

H H AcO O O O 1136; R=OAc 40 1138; R=OAc, 4a,5a- 1142; 5,6-a-epoxy 1137; R=C8H17 1139; R=OAc, 4b,5b- 1143; 5,6-b-epoxy 1140; R=C8H17, 4a,5a- 1141; R=C8H17, 4b,5b-

Figure 3.73 D4-steroids and D5-steroids and their epoxides.

Oxidation of 4-androstane-3,17-dione ( 370) with hydrogen peroxide and Fe(bpmen)(OTf)2 was found to result the epoxides 1144 and 1024.241 (Figure 3.74)

O O O

H H H

H H H H H H O O O O O 370 1144 1024 O O O

H H H

H H H H H H O O O O O O 1145 1146 1147

Figure 3.74 Compound 370 and 1145 and therir epoxides.

191

The same reaction with 6-methylen-4-androsten-3,17-dione 1145 yielded the external epoxide 1146 quantitatively, but by increasing the charge of the catalyst, a mixture of 1146 and 1147 were isolated. 241 (Figure 3.74) Similarly cross-conjugated ketones, e.g., 1,4-androstadien-3,17-dione 371 and 17a-acetoxy- 21-hydroxy-16a-methyl-3,11,20-trioxo-1,4-dien-21-propionate 1148 furnished 4,5-a-poxides 1149 and 1150 respectively whereas non-conjugated ketones 4,9(11)-androstadien-3,17-dione 1151 afforded compounds 1152 and 1153 with 5% of catalyst charge and products 1152, 1153, 1154 and 1155 with 15% of catalyst charge. 241 (Figure 3.75 )

OC(O)Et

O O O O OAc H H H

H H H H H H O O O O 371 1148 1149 OC(O)Et

O O O O OAc H H H

H H H H O O O O O 1150 1151 1152

O O O O O O O H H H

H H H O O O O 1153 1154 1155

Figure 3.75 Cross conjugated and non-conjugated ketones and their epoxides.

Another non-conjugated ketone, 1156 quantitatively produced epoxide 1157 with 5% of catalyst charge but 1158, 1159 and 1160 at increased catalyst charge. The oxidation of 3 b-

192 acetoxy-5-cholestene 40 yielded the a-epoxide 1161 in low catalytic charge and both a - and b- epoxide ( 1161 and 1162) in high catalytic charge condition. 241 (Figure 3.76 )

O O O OAc OAc OAc H H H H H H O H H H H H H O O O O O 1156 1157 1158

O O OAc OAc H H H H H H O O H H H H H H O O AcO O O 1159 1160 40

H H H H

H H H H AcO AcO O O 1161 1162

Figure 3.76 Non-Conjugated ketones and their different derivatives.

Ulipristal, an FDA approved emergency contraceptive pill was recently synthesized totally at approximately 27% yield and the process utilized selective epoxidation, at the first step, to achieve the 5 a,10a-epoxide ( 1164) (a:b=80:20) at 60% diastereomeric excess. This selective epoxidation indeed increased the yield of the target molecule ( 1008). 242 (Scheme 3.44)

III.A. 2.3 Biologically importance of epoxy- and epoxy-polyhydroxy steroids Epoxysteroids 1024 and 1026 were found to make aromatase inhibitors inactive by suicide and affinity-lebeling mechanism respectively. 243 (Figure 3.77)

193

N O O O O OAc

H O ,(CF ) CO 2 2 3 2 O + O Na2HPO4/ CH2Cl2 O O O O O O O 1165 1163 1164 1008

Scheme 3.44 Selective 5a,10a-epoxidation (inset: ulipristal, 1008 .)

O OAC OH O NH R 2 O S O O O O HO O O O O O 1168 1024; R=CH3 1166 1167 1026; R=CHO Figure 3.77 Biologically potent epoxy steroids.

Steroidal a-epoxides 1166 and 1167 were found to show potent HIV-1 Tat inhibition. Tat is a HIV-1 nuclear regulatory protein functioning as an essential activator of transcription. Tat having no known cellular counterpart loses its ability of replication of HIV-1 when its genes were deleted. 244 (Figure 3.77 ) Performing SAR studies it was found that whereas 4,5-unsaturation was not active against Tat, 4,5-epoxidation induced the inhibitory potential and a-epoxides were much more active in comparison to the b-epoxides. 245 Again, 6 b,19-epoxy steroid 1168 was found to show the 246 inhibitory activity against the human placental aromatase (Ki value: 2.2 mM). (Figure 3.77 )

III.A.2.4 Transformative reactions on epoxysteroids Epoxides are also utilized in the syntheses of a number of novel steroidal molecules. Using aromatic amies ( 1170-1174 ) in ionic liquids 2 a,3 a-epoxy-5a-androstan-17-one ( 1169) was converted respectively to 3a-hydroxy-2b-((4'-methyl-phenyl)-amino)-5a-androst-17-one ( 1175), 3a-hydroxy-2b-((4'-hydroxy-phenyl)-amino)-5a-androst-17-one ( 1176), 3 a-hydroxy-2b-(phenyl amino)-5a-androst-17-one ( 1177), 3 a-hydroxy-2b-((4'-acetyl-phenyl)-amino)-5a-androst-17- one ( 1178), 3 a-hydroxy-2b-((4'-methyl-phenyl)-amino)-5a-androst-17-one ( 1179), together with

194

2b,3 a-dihydroxy-5a-androst-17-one ( 1180) as a side product. Reaction of 1169 in dioxane with 4-methyl aniline ( 1170) afforded an imine derivative 1181.247 (Scheme 3.45)

R

O R HN HO O + + + - [bmin] [BF4] HO HO H H H NH2 1169 1175; R=Me 1180 1170; R=Me 1176; R=OH 1171; R=OH 1177; R= H 1172; R= H 1178; R=COMe 283, 1173; R=COMe 1179; R=NO2 Dioxane 1174; R=NO2

N Me

O + 1175 H 1181

Scheme 3.45 Synthesis of novel steroidal molecules using epoxides.

BiBr 3-Mediated Ritter reaction on 5 b,6 b-epoxycholestan-3b-yl acetate 1162 with MeCN resulted 5a-Acetamido-6b -hydroxycholestan-3b-yl-acetate ( 1182). The same reactant 1162 was found to be rearranged into 6 b-hydroxy-5b-methyl-19-norcholest-9(10)-en-3b-yl acetate ( 1183) and 6 b-Hydroxy-5b,14b-dimethyl-18,19-dinorcholest-13(17)-en-3b-l acetate ( 1184) when was o treated with Bi(OTf) 3·xH2O in MeNO 2 at 80 C, and only into 1183 in dioxane. (Scheme 3.46) Similar type of rearrangement was seen in case of 5 b,6 b-epoxy-17-oxoandrostan-3b-yl acetate 1185 in 1,4-dioxane at 50 o C affording 6 b-hydroxy-5b-methyl-17-oxo-19-norandrost-9(10)-en- o 3b-yl acetate ( 1186). But changing the solvent from dioxane to MeNO 2 at 0 C yielded compound 1186 along with 6 b-hydroxy-5b-methyl-17-oxo-19-norandrost-8(14)-en-3b-yl acetate ( 1187). o 248 Similarly, compound 1188 produced 1189 in dioxane and 1190 in MeNO 2 at 50 C. (Scheme 3.47)

195

C8H17 C8H17

H H BiBr3 (20 mol%) H H H H MeCN, RT AcO AcO O AcHN OH 1162 1182

C8H17 C8H17 C8H17

H H H H Bi (III) Salt H H H + H MeNO AcO 2 AcO AcO O OH OH 1162 1183 1184 Scheme 3.46 Ritter reaction on epoxy steroids.

O O O

H H H a c H H H 1186 + H AcO AcO AcO O OH OH 1186 1185 1187

O O O

H H H H a b H H H H AcO AcO AcO O OH OH 1189 1188 1190

Scheme 3.47 Transformative reactions on some epoxides. Reaction condition: a= Bi(OTf)3.xH2O (5 o o mol%), 1,4-dioxane, Molecular Sieves 4Å (0.25g/ml), 80 C; b= Bi(OTf) 3.xH2O (5 mol%),MeNO 2, 50 C; o c= Bi(OTf) 3.xH2O (5 mol%),MeNO 2, 0 C.

E-guggulsterone ( 1193), an important plant steroid used in many nutritional supplements was synthesized from 16,17-epoxypregnenolone ( 1191) through a hydrazine reduction 249-250 and Oppenauer oxidation.251-253 (Scheme 3.48

196

O O OH Al(O-i-propyl)3, O KOH, NH2NH2.H2O cyclohexanone Di(ethylene glycol) benzene HO HO o O o 80 C,2h 1191 160 C, 2h 1192 (cis- only) 1193 Scheme 3.48 Synthesis of E-guggulsterone ( 1193).

III.B Present work The present work demonstrates basically two aspects of some new polyhydroxy steroids - i) designed synthesis (section III.B.1) and their gelation behavior (section III.B.2).

III.B.1. Abstract of the work Altogether eighteen polyhydroxy steroids (PHS, 12 new) of cholesteryl and b-sitosteryl series were synthesized and characterized. Among them eight (all new) are, in precise, epoxy- polyhydroxy steroids (5,6-epoxy-3b,4 a-dihydroxy- and 5,6-epoxy-3b,4 a,7 a-trihydroxy-), of which the a-diastereomers were utilized further to synthesize novel new tetraols (3 b,4 b,5 a,6 b- tetrahydroxy-) and pentaols (3 b,4 b,5 a,6 b,7a -pentahydroxy steroids). As a new class of polyhydroxy steroids, gelation behavior of the molecules was evaluated. At 1% or below CGC (critical gelation concentration), five PHS derivatives were found to be gelators of some selective organic solvents. The organogels were characterized through FTIR analysis, T gel (gel melting temperature) and related physical parameters ( DH etc.), rheological data, and by morphology analysis (electron microscopes).

III.B.2 Design and Synthesis III.B.2.1 Background and design of the work Starting from the basic natural steroids (e.g., cholesterol, b-sitosterol etc.) we successfully demonstrated, in the previous chapter ( Chapter II ), the easy and efficient access to the 3 b,4 b- dihydroxy- (the diols) and 3 b,4 b,7 a-trihydroxy (the triols) steroids. To further the valuable utilization of the diols and triols, we envisioned the synthesis of novel 3 b,4 b,5 a,6 b- tetrahydroxy- and 3 b,4 b,5 a,6 b,7a -pentahydroxy steroids by utilizing somehow the C5-C6

197 double bond present in the steroid molecules, for that would be the minimum effort to achieve, from the natural steroids (in three steps! Scheme 3.49). So, dihydroxylation of the C5-C6 double bond was the assigned tip-target which was indeed not possible directly because most of the single-step easy processes utilize acidic conditions which obviously would be sensitive due to the cis -3,4-diol structure ( Scheme 3.50 ). In this juncture, we planned first to have the epoxides which can then be opened easily, in basic medium, to the corresponding polyhydroxylated products. Again, it is interesting to mention that the present route was indeed a valuable one, as the so-formed epoxy-polyhydroxy steroids would also be new class of steroid molecules.

R R1 3 Steps! OH OH 1 HO HO

OH R Cholesterol (13); R1= H Tetrols; R= H, R1= H or, -C2H5 b-Sitosterol (14); R1= C2H5 Pentols; R= OH, R1= H or, -C2H5

Scheme 3.49 Project to synthesize novel steroidal terols and pentols.

R1 OH OH OH Epoxidation Oxirane opening HO HO AB

2 (alkaline) 2 2 HO R OH R R O 1 R = H/ C2H5 HO 2 2 R2= H/ OH R = H/ OH R = H/ OH

Scheme 3.50 Route to tetraols and pentaols from diols and triols respectively, via corresponding epoxides.

III.B.2.2 Preparation of the starting materials The steroidal biomolecules which were used as the starting compounds for the present work are diols and triols (4 b-hydroxy- and 4 b,7 a-dihydroxy steroids respectively). These were prepared directly from the basic natural steroids (cholesterol and b-sitosterol), by employing selenium dioxide as the oxidizing agent ( Scheme 3.51). The complete discussion can be found in the Chapter II of the present thesis.

198

R1 OH OH SeO , Dioxane 2 HO + HO Other HO + 100oC, 24h product 1 OH 334; R = H 448; R1 = H 1 1 Cholesterol (13); R = H 454; R = C2H5 1 455; R = C2H5 b-Sitosterol (14); R1 = C H 2 5 SeO2, Dioxane 100oC, 48h

Scheme 3.51 Preparation of the starting diols and triols.

III.B.2.3 Synthesis of epoxy-polyhydroxy steroids Epoxidation of 4 b-hydroxy cholesterol (334) with m-chloroperbenzoic acid yielded the diastereomeric mixture of the 5,6-epoxy-4b-hydroxy derivatives (1194 and 1198). Separation and purification of the diastereomeric steroidal epoxides seems to be quite difficult and in many occasions, people have desired to isolate those as mixtures. After some time-consuming efforts the a- and b-epoxy-diastereomers were separated by repeated and careful column chromatography and were then further purified by repeated recrystallization (please follow the experimental section). The reactions were also applied to the b-sitosterol analogue. Thus, to the point, on epoxidation with m-CPBA, 4 b-hydroxy cholesterol furnished 5,6 a-epoxy-3b,4 b- dihydroxy-5a-cholestane (1194) and 5,6 b-epoxy-3b,4 b-dihydroxy-5b-cholestane (1198), and 4b-hydroxy-b-sitosterol, furnished 5,6 a-epoxy-3b,4 b-dihydroxy-5a-stigmastane (1195) and 5,6b-epoxy-3b,4 b-dihydroxy-5b-stigmastane (1199). (Scheme 3.52)

R R mCPBA R CHCl3 O OH Reflux, 6h OH HO HO + HO R O 1 R1 R1 HO

1194; R= a, R1= H 1198; R= a, R1= H R= a: b: 1195; R= b, R1= H 1199; R= b, R1= H 1196; R= a, R1= C2H5 1200; R= a, R1= C2H5 1197; R= b, R1= C2H5 1201; R= b, R1= C2H5 Scheme 3.52 Synthesis of epoxy-dihydroxy- and epoxy-trihydroxy steroids.

Due to the trans-fused-A/B-ring functionality of the steroid molecule, in case of 5,6 a-epoxy- 4b-hydroxy cholesterol (1194), the epoxide oxygen, in no way can undergo H-bonding with the 3b,4 b-diol functionality. This implies the greater chance to attribute intermolecular H-bonding.

199

On the other hand, in case of 5,6 b-epoxy-4b-hydroxy cholesterol (1198), the ring-A/B of the steroid molecule is cis -fused which brings account the possibility of extended H-bond formation involving the epoxide oxygen and the 3 b,4 b-diol functionality. This possibility of intramolecular H-bonding reduces somewhat the polarity of the b-epoxy-diol in comparison to the a- diastereomer which was observed practically in the column chromatographic separation. While increasing the polarity in the column, b-epoxy-diol 1198 comes always first followed by the a- epoxy-diol 1194. Thus, the b-epoxy-diastereomer was somehow more stable than the a-epoxy isomer. Now, if we consider the mechanism of epoxidation, it looks easier to attack the C5-C6 double bond from the a-face, resulting the 5,6 a-epoxide as the major product. But in practice, the diastereomric ratio, a- to b- was found to be approximately 1:3, i.e., 5,6 b-epoxide was the major product. Hence, presumably it is more likely that, after the nucleophillic attack by m- CPBA, rapid ring flipping involving the C5-C10 bond occurs to achieve the more stable b- isomeric conformation. One more interesting point here is that, in case of the 5,6 b-epoxy-4b- hydroxy steroids, like the natural bile acids, the lipophobic polar groups (three –OH) are surrounded only to the b-face of the molecule which we can consider as the synthetic partial mimic to the natural bile acids. Epoxidation of 4 b,7a -dihydroxy cholesterol (448) with m-chloroperbenzoic acid yielded the diastereomeric mixture of the 5,6-epoxy-4b,7a -dihydroxy derivatives (1196 and 1200), which are basically the epoxy-triols. The a- and b-epoxy-diastereomers were separated by repeated and careful column chromatography. The reactions were also applied to the b-sitosterol analogue. Thus, to the point, on epoxidation with m-CPBA, 4 b,7a -dihydroxy cholesterol (448) furnished 5,6 a-epoxy-3b,4 b,7a -trihydroxy-5a-cholestane (1196) and 5,6 b-epoxy-3b,4 b,7a - trihydroxy-5b-cholestane (1200), and 4 b,7 a-dihydroxy-b-sitosterol (455) furnished 5,6 a-epoxy- 3b,4 b,7a -dihydroxy-5a-stigmastane (1197) and 5,6 b-epoxy-3b,4 b,7a -dihydroxy-5b- stigmastane (1201). The diastereomeric ratio of the a- to b-epoxytriol was found approximately just the reverse one in comparison to the epoxydiols. In this case the isolated yield ratio of a- to b- diastereomers was 1:3. In 5,6 a-epoxy-3b,4 b,7a -trihydroxy-5a-cholestane (1196), their remains a great possibility of two sets of (five-membered) strong H-bond formation- one between 3 b- and 4 b-OH and another one by involving the epoxide oxygen and 7 a-OH. On the

200 other hand, in 5,6b-epoxy-3b,4 b,7a -trihydroxy-5b-cholestane (1200), the epoxide oxygen can form H-bond with the 4b-OH which is already H-bonded with the 3 b-OH. So, though the epoxide oxygen extends the H-bonding of 3 b,4 b-diol functionality, the 7 a-OH remains completely free. So, in short, whereas the H-bond functionalities in a-epoxide will remain more as intramolecularly bonded, in case of the b-epoxide there are major chances to form intermolecular H-bonding involving 7 a-OH. As a consequence, the a-epoxytriol is somewhat less polar than the b-isomer and the fact is well evident from the column chromatographic separation. In column, the a-diastereomer always comes first followed by the b-isomer, when the polarity of the eluent was increased gradually. However, the isolation of the a-isomer as the major product demonstrates that the initial attack of the nucleophile (the m-CPBA molecule) occurs from the a-face of the substrate molecule. The formation of the b-isomer is attributed only due to the b-face attack, which is possible at minor account. But the C5-C10 bond flipping here is presumably undesirable, as the b-face attack which might result bond flipping, produces less stable diastereomer.

Table 3.1 Diastereomeric distribution of various epoxides.

b a

O mCPBA R2 R1 Conditions R2 HO HO R3 + 3 R O R3 HO 2 R Entry Substrate Conditions Diastereomeric ratio ( a: b)

1 2 3 1 13 : R = R = R = H CHCl 3, reflux, 1h ~ 3:1 1 2 3 2 14 : R = C 2H5; R = R = H CHCl 3, reflux, 1h ~ 3:1 1 2 3 3 334: R = H; R = OH; R = H CHCl 3, reflux, 6h ~ 1:3 1 2 3 4 454: R = C 2H5; R = OH; R = H CHCl 3, reflux, 6h ~ 1:3 1 2 3 5 448: R = H; R = R = OH CHCl 3, reflux, 6h ~ 3:1 1 2 3 6 455: R = C 2H5; R = R = OH CHCl 3, reflux, 6h ~ 3:1

If we follow these molecules structurally, the arrangement of the polar groups looks quite fascinating. In the a-isomer two lipophobic polar groups are there on the a-face and two others

201 are on the b-face which makes the two faces of the steroid molecule (considering ring-A and -B) approximately equally available for the micelization and related issues. On the other hand, in the b-isomer, the b-face of the molecule possesses three lipophobic polar groups while there is only one on the a-face. As a result, the b-face is more polar and the fact can be utilized in the study of face-selective micelization and related issues of the surface-science. ( Table 3.1 )

III.B.2.4 Synthesis of tetra- and pentahydroxy steroids According to our plan, the next step was to convert the steroidal epoxydiols and epoxytriols into the corresponding tetrahydroxy- and pentahydroxy steroids. As is mentioned earlier, it was not possible to open the oxiran-ring by acid-mediated reactions, the epoxy-polyhydroxy steroids were treated with methanolic KOH which after usual work-up and column chromatographic separation yielded the corresponding polyhydroxy derivatives. In 2012, Poirot and his group reported the valuable results on the surprising unreactivity of 5,6b-epoxy cholesterol with nucleophiles whereas the a-diastereomer could undergo the nucleophilic ring opening. They established the fact quite beautifully by analyzing some very relevant parameters as well as by some computational energy calculations. 254 This led us to try working only with the a-epoxides toward the oxirane ring opening by hydroxide ion as the nucleophile. Though no catalyst was used to achieve the transformations, the reaction time was little bit longer (48h) to have appreciable amount of the corresponding products. Thus, on treatment with aqueous methanolic KOH, 5,6 a-epoxy-3b,4 b-dihydroxy-5a- cholestane (1194) furnished 3 b,4 b,5 a,6 b-tetrahydroxy-5a-cholestane (1202 ) and 5 a,6 a-epoxy- 3b,4 b-dihydroxy-5a-stigmastane (1195) yielded 3 b,4 b,5 a,6 b-tetrahydroxy-5a-stigmastane (1203). Again, by employing the similar reaction conditions, 5,6 a-epoxy-3b,4 b,7a -trihydroxy- 5a-cholestane (1196) yielded 3 b,4 b,5 a,6 b,7a -pentahydroxy-5a-cholestane (1204) and 5,6 a- epoxy-3b,4 b,7a -trihydroxy-5a-stigmastane (1197) furnished 3 b,4 b,5 a,6 b,7a -pentahydroxy-5a- stigmastane (1205). (Scheme 3.53)

202

R R2 OH 2 KOH, CH OH OH OH HO 3 HO Reflux, 48h O R1 OH R1

1194; R1= R2= H 1202; R1= R2= H 1195; R1= H, R2= C2H5 1203; R1= H, R2= C2H5 1196; R1= OH, R2= H 1204; R1= OH, R2= H 1197; R1= OH, R2= C2H5 1205; R1= OH, R2= C2H5

Scheme 3.53 Synthesis of tetrahydroxy- and pentahydroxy steroids.

III.B.2.5 Structural feature: H bonding The tetrols (1202 and 1203) are structurally 3 b,4 b,5 a,6 b-tetrahydroxy-5a-steroids which demonstrate the high possibility of two strong intramolecular H-bond formation involving the 3b-, 4 b- and 6 b-hydroxy functionalities- one of them being able to furnish six-membered cyclic arrangement and another as five-membered cyclic ( Figure 3.78). And moreover, these two arrangements may be treated as the extended H-bonding capability in respect to one another. In these structures, clearly, the 5 a-hydroxy group can undergo intermolecular H-bond formation. On the other hand, the pentahydroxy steroids (1204 and 1205), where the –OH group distributions are- 3b- , 4 b- , 5 a- , 6 b- and 7a-, can undergo structurally three strong intramolecular H-bonding involving all the hydroxy groups. Among these, two can produce six- membered cyclic structure and another one as five-membered cyclic. Notably, the five- membered arrangement and one of the six-membered arrangements can be treated as the extended H-bonding capability in respect to one another ( Figure 3.79). The large extent of intramolecular H-bonding, due to the partially zig-zag distribution of the hydroxy groups in both the tetraols and pentaols, is reflected, presumably, in the physical state of the compounds- all being sticky gum (which on standing for few days in air is transformed into sticky solid (completely amorphous), due to the expected hygroscopic nature of the polyhydroxy compounds). Again, of note, the polarity of these tetra- and pentahydroxy steroids were found a bit low (during the column chromatographic separation) in comparison to the starting a-epoxides which possess lesser number of appropriate groups. This may also be attributed due to the intramolecular H-bonds coming in function.

203

H H R H R O H O O H O H O O 1202; R= H OH 1203; R= C2H5 OH

Figure 3.78 Probable H-bonding arrangements in the tetrahydroxy steroids (R= H, 1204; R= C 2H5, 1205). The bonds and atoms involved in H-bonding are shown in blue.

H H H R H R O O O H O H O O

O O H H O H O H

H H R H R O H O O H O H O O

O H O O H O H H

Figure 3.79 Probable H-bonding arrangements in the pentahydroxy steroids (R= H, 1204 ; R= C 2H5, 1205). The bonds and atoms involved in H-bonding are shown in blue.

III.B.3 Application of the epoxy-polyhydroxy and polyhydroxy steroids: study of their preliminary gelation behavior

III.B.3.1 Introduction: gelation, low molecular-weight gelation (LMWG), and gelation of steroid molecules Gelation is a physical phenomenon where compound(s) (pure or mixture) produce(s), with a liquid system (pure or mixture), gelly-like material, termed as gel. The compounds are termed as gelators and the liquid system as solvent of gelation. In contrast to the polymeric gels which are indeed rigid (i.e. thermally irreversible and cannot be re-dissolved) due to their covalent (and thus permanent) cross-linking networks, supramolecular gels are basically soft (i.e. thermally

204 reversible and can be re-dissolved), viscoelastic solid-like materials which owe special interest because of their unique novel properties and potential practical applications in the fields of drug delivery, separations, cosmetics, sensors, biomimetics, templates for metal oxide nanotubes, immobilizing protein arrays, optical and semiconducting nanostructured materials, etc.255-262 Low molecular mass organo gelators (LMOGs) are small organic molecules which make strong, entangled three-dimensional networks through their self-aggregation resulting higher surface area, which thus can efficiently entrap different solvent molecules. 1 The LMOGs get self-assembled through non-covalent interactions including hydrogen bonding, p–p stacking, van der Waals, or hydrophobic interactions. When the solvent component, in a gel material is an organic solvent, the gel is named as an organogel and where the solvent component is water, the gel is a hydrogel. There have been a number of important contributions, over the last few decades, describing the gels derived from LMOGs. 255-267 These fascinating materials have been studied extensively enriching the knowledge of the self-organization of gelator molecules into a 3-D network structure, the gelation phenomenon, the dependence of their stability and property on the various physical as well as chemical factors. 268-277 People are making them engaged, increasingly, in the study of different aspects of gelation including the preparation of new gels which lead to a better understanding of the properties of gels formed by LMOGs and hence to find out practical applications. Target-based-designing of new gelators has, till date, been remained much complicated and challenging due to the specifications of a number of physical as well as chemical parameters. Specially, the structural contribution of both the gelator and the solvent of gelation is found to be highly sensitive and subtle structural changes can practically alter the results enormously. Nevertheless, it may be assumed, presumably, that aliphatic or aromatic molecules having hetero atoms (may be as polar groups) resulting altogether a large surface area can provide a probability of gelation. 278 But still hardly surprisingly, in such situations, it is again unable to predict the solvent of gelation. Due to the ‘in -built’ larger hydrophobic structure along with the diffe rent hydrophilic polar groups in different suitable positions, steroids and steroid-derived molecules show significant possibility in the formation of supramolecular gels. Among the large number of steroidal molecules/ analogues, cholesterol and different cholic acids have possessed special attention due to the wide scope of nice modifications/ derivatizations as well as for their relatively higher

205 scope of direct applications. Thus, by tailoring the hydrophobic-hydrophilic interactions, the overall polarity profile of these steroid-based compounds can be adjusted suitably to result self- assembling characteristics, which may direct them towards the potential components of supramolecular gels. Recently, Kolehmainen and his group, in their beautiful review, have accounted the recent advances in the field of steroidal supramolecular gels. 279 Along with their potential applications, the broad areas of the subject, viz. , steroid-based hydro- and organogels, steroidal metallogels, two-component steroidal gels, and stimuli-responsive steroidal gels, are covered in the article. 279

III.B.3.2 Present work Thus, as a new class of polyhydroxy steroids, the synthesized molecules associated with the present chapter, preliminary gelation behavior was evaluated. Besides, starting substrates which are still hydroxy steroids ( 334, 454 , 448 and 455 ) and two known steroids ( 514 and 79 ) were considered for the study.

III.B.3.2.1 Preparation of the gels: tests in different solvents The gelation ability of the synthesized oxysteroids was tested in a number of different common aromatic as well as aliphatic solvents. The rapid advancement of the subject supramolecular gelation of small molecules has led the preparation of the gels to much lower values of critical gelation concentration (CGC) as to 0.1% w/v or even lower. Considering the fact, the gelation ability of the compounds was tested primarily, only at 1% w/v and conclusion was drawn by following the ‘inverse test tube method’ all owed for 24h. A summary of the gelation tests in different solvents are given below in Table 3.2.

Figure 3.80 Images of different gels.

206

Among the epoxydiols, it was found that, in DMSO, the a-epoxy diols both of cholesterol and b-sitosterol (1194 and 1195 respectively), produced transparent gels whereas b-epoxy diol of cholesterol (1198) resulted translucent gel. Of note, the cholesteryl compounds acted as gelators at much lower CGC values in comparison to the b-sitosteryl analogues ( Table 3.2). On the other hand, among the epoxytriols, in long chain alkanes viz. , decane, dodecane etc., the b-epoxy triols both of cholesterol and b-sitosterol (1201 and 1202 respectively), produced transparent gels at quite lower CGC values ( Table 3.2) whereas a-epoxytriols (1196 and 1197 ) remained only as clear solutions. Except DMSO and long chain alkanes, all the other solvents tested for gelation resulted either of the clear solutions, precipitates, partial gels or weak gels. Whereas the epoxy steroids were found able to gel some selective organic solvents, the tetra- and pentahydroxy derivatives were found inactive, within the organic solvents tested, in organogelation at the concentration of 1% w/v. The variance of the gelation ability of the oxysteroids studied can be justified by the analysis of the stereo-arrangements of the different oxyfunctionalizations present in the molecules.

III.B.3.2.2 Phase selective gelation ability Phase selective gelation (PSG) is nothing but the selective gelation of one component of a binary immiscible solvent system which typically results two phases- oil and water. Such gelation ability is remarkably useful for practical applications, one major interest being the oil spill recovery where the gelators can selectively gel organic solvents (oil) from oil – water mixtures. The first phase selective gelation of organic solvents in presence of water was reported by Bhattacharya and co-workers. 280 They synthesized an alanine-based amphiphile ( 1206) to reveal the novel selective gelation property. Banerjee et al. reported an aromatic amino acid based organogelator ( 1207) which can potentially be used for oil spill recovery due to the very rapid and selective gelation of the oil phase from the oil-water mixture at room temperature. 281 Again Sureshan and his group discovered a mannitol-based excellent super organogelator potential for practical applications in oil-spills recovery, through the phase-selective gelation ability. 282 Besides, there are a number of other reports of phase selective gelator molecules. 283-292 (Figure 3.81)

207

Table 3.2 Gelation-test results of the synthesized polyhydroxy steroids in different solvents .

Solvents

DCB Dioxane - - -Xylene -Xylene Compounds Compounds 1,2 Toluene p DMSO 1,4 Acetonitrile Methanol Isopropanol Butanol Octanol Decane Dodecane 334 S S S I S I S S S S C C 454 S S S P S C C S S S C C 448 P P P P S I S S S S P P 455 P P P P S I S S S S P P 1194 C S S G S P S S S S P P (0.2) 1197 C S S G S I I S S S P P (1.0) 1196 C I C C S I P C C S I C 1197 C P C C S I P C S S P C 1198 S S S G S C P S S S C PG (0.5) 1199 S S S P S P P S S S P P 1200 P P S P S P S S S S G G (0.25) (0.2) 1201 P P S P S P S S S S G G (0.3) (0.2) 1202 S S S P S I S S S S S S 1203 S S S P S I S S S S S S 1204 S S S P S I S S S S P PG 1205 S S S P S I S S S S P PG

208

O COOH O O N N H H O 1206 1207

Figure 3.81 Solvent selective gelators 1206 and 1207.

Interestingly, among the synthesized compounds, gelators 1200 and 1201 were found to gel selectively the organic solvent of an organic –water mixture keeping the water part intact (Figure 3.82). Though the gelators 1200 and 1201 produced transparent gels with the long chain hydrocarbons, presence of water molecules in the organogel medium made the gels translucent. In addition, the gels formed in these cases were found to be less stable (qualitatively). These facts imply that the H-bond formation ability of the gelators is affected when they are in contact with water, although the effects cannot completely result the compounds to become non-gelators. Compounds 1200 and 1201 possess polar groups both on the a- as well as b-face of the respective steroids which makes the molecules critically amphoteric. And the phase-selective gelation ability of these PHSs may thus be explained on the basis of such structural contribution.

Figure 3.82. Phase selective gelation ability of 1200.

III.B.3.2.3 Gel melting temperatures (T gel ) and related physical parameters

The temperature at which a gel melts to a solution is known as its gel-melting temperature ( Tgel ).

This provides information regarding the stability of a gel. Higher Tgel value corresponds to the higher thermal stability of a gel. The Tgel values of these gels were measured by inverted tube experiments. The experiments were performed by heating the organogels in a water bath (thermostat controlled) maintaining the heating rate at 2 o C/1 min until these were melted. The

Tgel values of the gels at different concentrations were measured and error range in gel melting

209

o temperature ( Tgel ) determination was found to be ±1 C. Then applying the Schröder-van Laar equation, the enthalpy changes ( ΔH) for the gel-sol transitions of two organogels were determined. 258

According to the Schroder-van Laar equation, ln C= - (ΔH/ R). 1/ T gel + K, Slope = - ΔH/ R So, for 1194/ DMSO gel: ΔH= 32 KJmole -1 And for 1198/ DMSO gel: ΔH= 54 KJmole -1

T gel (oC) ln C Linear Fit of Book1_B 62

-3.7 60 Equation y = a +

Adj. R-Sq 0.998 58 -3.8 Value Standard

56 Book1_B Interce 7.81493 0.22024 -3.9 Book1_B Slope -3845.7 71.65937

54

-4.0

52 lnC T gel(oC) T 50 -4.1

48

-4.2 46

44 -4.3 0.6 0.7 0.8 0.9 1.0 0.00300 0.00302 0.00304 0.00306 0.00308 0.00310 0.00312 0.00314

Conc. (w/v %) 1/ Tgel (K-1)

T gel (oC) ln C Linear Fit of ln C

-3.7 324

-3.8 Equation y = a + b

322 Adj. R-Squa 0.99137

Value Standard Err

-3.9 ln C Intercept 16.33638 1.09408

ln C Slope -6499.547 349.55891

320 -4.0 lnC T gel(oC) T

318 -4.1

-4.2

316

-4.3 0.6 0.7 0.8 0.9 1.0 0.00308 0.00310 0.00312 0.00314 0.00316 0.00318

conc. (w/v %) 1/ T gel (K-1)

Figure 3.83 Tgel vs conc. (w/v%) and ln C vs 1/T gel of 1194/ DMSO gel (above row) and 1198/ DMSO gel (bottom row).

III.B.3.2.4 Rheological behaviour Gels are viscoelastic soft materials which in principle enable them to store as well as to dissipate energy. 293-294 These mechanical properties of the organogels were studied by dynamic rheology

210 of two selective organogels. The rheology experiments on the gels were performed by following two usual mode: (i) frequency sweep at a fixed oscillatory stress of 1.0 Pa and (ii) stress sweep at a fixed frequency of 1.0 Hz. The dynamic storage modulus (G ') and the loss modulus (G '' ) are measured by the frequency sweep experiments. For soft-solids, like gels, dynamic storage modulus (G ') remains greater than the loss modulus (G '' ); i. e., G ' > G '' and the ratio of these two characterizes the stiffness of the gels. On the other hand, the stress sweep experiments provide the yield stress which is indeed the limit beyond which the gel starts to flow. 295 (Table 3.3 , Figure 3.84-Figure 3.85 )

Table 3.3 Dynamic rheology of the organogels. [The G ' and (G '/G '' ) values shown in the table were obtained from frequency sweep experiments performed at a fixed stress of 1.0 Pa. The values are at frequency of 1.0 Hz. The s* values shown were obtained from stress sweep experiments at a fixed frequency of 1.0 Hz (error ± 10 –15%)].

Entry Gel system G'/ Pa G'' / Pa G'/ G'' s*/ Pa 1 1194/DMSO 21220 3720 5.7 500 2 1198/DMSO 6960 1874 3.7 40

5 dmso 1 wv-0001o 5 dmso 1 wv-0001o 5 dmso 1 w v-0001o, Frequency sw eep step 5 dmso 1 w v-0001o, Stress sw eep step 1.000E6 1.000E6 1.000E5 1.000E5

1.000E5 1.000E5 10000 10000

10000 10000 1000 1000 '(Pa) a P ( '' G '(Pa) a P ( '' G 1000 1000 G' (Pa) G' (Pa)

100.0 100.0 100.0 100.0

10.00 10.00 10.00 10.00

1.000 1.000 1.000 1.000 0.1000 1.000 10.00 100.0 1.000 10.00 100.0 1000 frequency (Hz) osc. stress (Pa) Figure 3.84 Frequency sweep (left) and stress sweep (right) dynamic rheological behavior of 1194/DMSO gel (1%, w/v). In 1Hz, G /= 21220 Pa, G // = 3720 Pa and σ*= 500 Pa.

211

9 dmso 1 wv 231214-0005o 9 dmso 1 w v 231214-0005o, Frequency sw eep step 1.000E5 1.000E5 1.000E5 9 dmso 1 w v 231214-0005o, Stress sw eep step 1.000E5

10000 10000

1000 1000 10000 10000

100.0 100.0 '(Pa) a P ( '' G '(Pa) a P ( '' G G' (Pa) G' (Pa) 10.00 10.00

1000 1000 1.000 1.000

0.1000 0.1000

0.01000 0.01000 100.0 100.0 1.000 10.00 100.0 1000 10000 1.000E5 0.1100 frequency (Hz) 100.0 osc. stress (Pa) Figure 3.85 Frequency sweep (left) and stress sweep (right) dynamic rheological behavior of 1198/DMSO gel (1%, w/v). In 1Hz, G /= 6960 Pa, G // = 1874 Pa, σ*= 40 Pa.

III.B.3.2.5 Morphology through electron microscopes A. Atomic force microscopy (AFM). The self-assembly of the epoxy-polyhydroxy steroids was also studied by atomic force microscope (AFM). Investigation by AFM revealed the formation of fibrillar networks for the 1194/DMSO and 1198/DMSO systems. The 1194 /DMSO fibres were found to have diameter ca 130 nm at 0.025% (w/v) whereas at higher concentration (0.05% (w/v), the fibres were found to make highly entangled bundles of diameter ranging from ca 350- 400 nm. At higher concentration (0.2% w/v), the 1198/DMSO fibers were found only in the form of bundles of diameter ca 1.5 mm and close inspection of the bundles showed to be formed by two smaller bundles of approximately equal diameter. Again, 1198/DMSO, at lower concentration (0.05% w/v), showed quite a bit interesting surface topography where it was found that a number of microspheres were arranged in an interconnected array showing like a necklace formation, prior to form fibers. The microspheres here were of diameter ca 400- 500 nm. For 1200/dodecane and 1201 /dodecane systems (both at 0.025%, w/v), microsphere-like morphology was revealed by AFM analysis, and more concentrated system produced lumps only. 11/ Dodecane showed spheres of diameter ca 300- 450 nm whereas 1201/dodecane possessed spheres of diameter ca 400- 600 nm with the morphology like interconnected/ aggregated- spheres. (Figure 3.86-Figure 3.89 )

212

Figure 3.86 AFM images of 1194/ DMSO system at 0.05% (w/v, above) and at 0.025% (w/v, below).

Figure 3.87a AFM images of 1198/DMSO system at 0.2% (w/v).

213

Figure 3.87a (contd.) AFM images of 1198/DMSO system at 0.2% (w/v).

Figure 3.87b AFM images of 1198/DMSO system at 0.05% (w/v).

214

Figure 3.88 AFM images of 1200/Dodecane system at 0.025%, w/v.

215

Figure 3.89 AFM images of 1201/dodecane at at 0.025%, w/v.

B. Polarizing optical microscopy (POM). Through the polarizing optical microscope, 1194/DMSO, 1198/DMSO, and 1195/DMSO gels were found to be optically anisotropic whereas 1200/dodecane and 1201 /dodecane gels did not show any birefringence (the 1195/DMSO gel showed very weak birefringence). Thus, as per the AFM images, the birefractive behaviour of the DMSO gels was possibly arose due to the immobilized solvent inside the self-assembled nanofibres, whereas the microsphere-network in cases of the dodecane gels resulted a net isotropic phases and hence rendered non-refringerent. (Figure 3.90 )

216

Figure 3.90 POM images of 1194/DMSO (above row) and 1198/ DMSO gels (bottom row).

IV.B.3.2.6 Justification of the gelation abilities of the synthesized steroids through their presumable molecular interactions If we follow the structural difference between the a -epoxy diols and b-epoxy diols, it is readily clear that in the a-diastereomers (1194 and 1195), the epoxy group by no way can undergo intramolecular H-bonding (and thus remains available completely for intermolecular H-bonding) which implies to facilitate self-assembly. On the other hand, in case of the b-epoxy diols (1198 and 1199) the epoxy oxygen can undergo weak H-bondig with the 3 b-OH due to the inappropriate distance between the two active groups. Hence, the epoxy oxygen remains partially available for intermolecular H-bonding providing a chance of self-assembly. So, if we compare the two diastereomeric epoxy diols, the a-epoxy diols clearly provide better positive interactions toward overall intermolecular H- bonding capability which is consequences by the observed CGC values. Thus, whereas the a - epoxy diol of cholesterol gels DMSO at a concentration of 0.2% w/v, the CGC for the b- diastereomer is 0.5% w/v. Comparing the cholesterol and b-sitosterol (structurally 24- ethylcholesterol) analogues, it may presumably be assumed that the compound of the latter series undergo intermolecular weak fit due to the branched lypophilic alkyl chain (note, that the solvent is DMSO). (Figure 3.91 )

217

R R O OH HO

O HO HO 1194; R= H 1198; R= H 1195; R= C2H5 1199; R= C2H5

Figure 3.91 Synthesized a and b-epoxy diols.

Considering the diastereomeric epoxide functionality, the ability of gelation of the epoxy triols were found just to be reversed in comparison to the epoxy diols. If a 7 a- OH is added to the epoxy diols, the resultants are the corresponding epoxy triols. Considering the functional/active group distribution of the a-epoxy triols (1196 and 1197), the H-bond acceptor functionalities as well as the H-bond donor functionalities are engaged very suitably through the intramolecular H-bonding, leaving poor opportunity towards self-assembly. As a consequence, these are found to be non-gelators of the organic solvents tested. (Figure 3.92 )

R R O OH HO OH O OH HO HO 1196; R= H 1200; R= H 1197; R= C2H5 1201; R= C2H5

Figure 3.92 Synthesized a and b-epoxy triols. But the b- epoxy triols (1200 and 1201) possess 7 a-OH group which is very much available for intermolecular H-bonding and like the b-epoxy diols, the epoxy oxygen can provide further space for the same raising the huge opportunity for self-assembly. As a result, the b-epoxy triols are found to be good gelators of long chain aliphatic hydrocarbons. It is interesting to note again as the epoxy triols gel long chain alkanes which are completely hydrophobic, the difference in side chain of the cholesteryl and b-sitosteryl derivatives cannot make significant difference in their CGC values.

218

III.C Experimental III.C.1 General Melting points were measured in open capillary methods and were uncorrected. 1H NMR and 13 C NMR spectra were recorded on Brucker Avance 300MHz FT-NMR spectrometer using 5 mm

BBO probe. Either CDCl 3 or DMSO-d6 was used as solvent and TMS as reference material. Data are presented as follows: chemical shift -in ppm on the scale relative to δ TMS = 0; coupling constant- J/Hz. Elemental analysis was performed using Thermo Finnigan FLASH EA 1112 CHNS Analyser. Fourier transform infrared (FT-IR) spectra were taken on a Bruker Optics ALPHA-E spectrometer with a universal Zn-Se ATR (attenuated total reflection) accessory in the 600-4000 cm –1 region or using a Diamond ATR (Golden Gate). The Mass spectra were recorded on Thermo Scientific Q-exactive, accela 1250 Pump. For column chromatography silica gel G, 60-120 mesh was used with petroleum ether- ethyl acetate mixture as the eluent. For thin layer chromatography (TLC), freshly made silica gel plates (using silica gel for TLC + petroleum ether) were used and visualization was achieved by staining with iodine. AFM images were recorded on JPK Nanowizard II in tapping mode. The solution of the corresponding derivative in appropriate gelating solvent was aged for 12 h and drop casted on a cleaved mica- sheet and allowed to dry in air for 12h followed by vacuum drying at 25°C for 3-4 h. Olympus BX 51 polarizing optical microscope was used for recording POM images of the gels. In preparing POM samples, the gels were carefully scooped up and placed over a clean microscope slide covering the sample with a thin cover slip.

III.C.2 Representative reaction for the syntheses of the epoxy-polyhydroxy steroids. To a solution of the appropriate diol (or triol) (1 mmol) in chloroform (20 mL) was added mCPBA (206 mg, 1.2 mmol) and the mixture was allowed to reflux for 6h. The reaction mixture was then cooled, 10 mL more chloroform was added, and washed successively with saturated solution of NaHCO 3 (2x 25 mL), with saturated brine solution (2x 25 mL) and finally with water

(1x 25 mL), and then concentrated (vaccuum) and dried (Na 2SO 4) to give a yellowish gummy residue. The compounds presented therein, were then separated by slow column chromatography eluted by petroleum ether and ethyl acetate, with successive increase in polarity. Recrystallization from appropriate sovents produced the corresponding pure products (please

219 follow the characterization section for exact eluent polarity and the solvent of recrystallisation for each isolated products).

III.C.3 Representative reaction for the syntheses of the tetra- and pentahydroxy steroids.

To a 2% (w/v) KOH solution in aqueous methanol (MeOH: H 2O= 9: 1; 40 mL) the appropriate 5a-epoxy steroid (epoxy diol or epoxy triol, 0.5- 0.7 mmol) was added and the mixture was allowed to reflux for 48h. The reaction mixture was then cooled and methanol was evaporated out (rota-vap). Chloroform (30 mL) was then added into the reaction mixture, and washed successively with water (3x 25 mL), saturated brine solution (2x 25 mL) and again with water

(1x 25 mL), and then concentrated (vaccuum) and dried (Na 2SO 4) to give a deep yellowish gummy residue. The compounds presented therein, were then separated by column chromatography eluted by petroleum ether and ethyl acetate (please follow the characterization section for exact eluent polarity).

III.C.4 Characterization of the products III.C.4.1 5b-Cholestan-5a,6 a-epoxy-3b,4 b-diol (or, 5 a,6 a-epoxy-4b-hydroxycholesterol, 1194): Eluent in column chromatography: 25% ethyl acetate in petroleum ether (initial o 1 fractions). Yield: 16-20%. m.f. C 27 H46 O3, white powedered solid. m.p. 160-161 C. H NMR (300

MHz, CDCl 3): δ 0.61 (s, 3H, Me -19), 0.85 (d, J=3 Hz, 3H, Me-27), 0.86 (d, J= 3 Hz, 3H, Me- 26), 0.89 (s, 3H, Me-21), 1.21 (s, 3H, Me-18), 1.93- 1.96 (m, 4H, H-2 and H-7), 3.05 (d, J=3.6 13 Hz, 1H, H-4) 3.19 (s, 1H, H-3) 3.82-3.86 (m, 1H, H-6). C NMR: (75 MHz, CDCl 3): δ 11.9

(CH3-18), 15.3 (CH3-19), 18.7 (CH3-21), 19.9, 22.6 (CH3-26), 22.8 (CH3- 27), 23.9, 24.0, 25.5, 28.0, 28.1, 28.3, 30.0, 32.7, 34.3, 35.8, 36.2, 39.4, 39.5, 42.4,, 43.4, 55.9, 56.9, 59.4 (C-6), 64.9 (C-5), 70.1 (CH-3), 77.3 (CH-4). FTIR (neat, cm -1): ν 3344, 3211, 2924, 2847, 1454, 1362, 1070, 960, 754. Analysis calcd: C, 77.46; H, 11.07. Found: C, 73.72; H, 10.53.

III.C.4.2 5b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol (or, 5a,6 a-epoxy-4b-hydroxy-b-sitosterol, 1195): Eluent in column chromatography: 25% ethyl acetate in petroleum ether (initial o 1 fractions). Yield: 13-15%. m.f. C 29 H50 O3, white powedered solid. m.p. 172-174 C. H NMR (300

MHz, CDCl 3): δ 0.62 (s, 3H, Me -19), 0.80 (s, 3H, Me-27), 0.82 (s, 3H, Me-26), 0.84 (s, 3H, Me- 29) 0.90 (s, 3H, Me-21), 1.22 (s, 3H, Me-18), 1.93- 1.97 (m, 4H, H-2 and H-7) 3.04 (d, J=3.6

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Hz, 1H, H-4) 3.20 (d, J= 2.4 Hz, 1H, H-3), 3.84-3.88 (m, 1H, H-6). 13 C NMR: (75 MHz,

CDCl 3): δ 11.9 (CH 3-18), 12.0 (CH 3-29), 15.3(CH 3-26), 18.7 (CH3-21), 19.0 (CH 3-19), 19.8

(CH3-27), 19.9, 23.1, 24.0, 25.5, 26.1, 28.1, 28.3, 29.1, 29.9, 32.7, 33.9, 34.2, 36.1, 39.4, 42.4, 43.3, 45.8, 55.8, 56.9, 59.3 (C-6), 64.8 (C-5), 70.1 (C-3), 77.31 (C-4). FTIR (neat, cm -1): ν 3404, 2932, 2865, 1459, 1379, 957. Analysis calcd: C, 77.97; H, 11.28. Found: C, 75.74; H, 10.61.

III.C.4.3 5b-Cholestan-5a, 6 a-epoxy-3b, 4 b, 7a-triol (or, 5 a, 6 a-epoxy-4b, 7a-dihydroxy cholesterol, 1196): Eluent in column chromatography: 35% ethyl acetate in petroleum ether o (final fractions). Yield: 52-55%. m.f. C 27 H46 O4, white crystalline solid. m.p. 168-170 C 1 (chloroform-ethanol). H NMR (300 MHz, DMSO-d6): 0.56 (s, 3H, Me-19), 0.83 (s, 3H, Me-27), 0.85 (s, 3H, Me-26), 0.89 (s, 3H, Me-21), 1.13 (s, 3H, Me-18), 3.73 (m, 1H, H-3), 4.05 (d, 1H, 13 H-4), 4.44 (dd, J=6 Hz, 18Hz, 1H, H-6), 4.81 (m, 1H, H-7). C NMR: (75 MHz, DMSO-d6): δ

12.0 (CH3-18), 15.3 (CH3-19), 19.0 (CH3-21), 19.6, 22.8 (CH3-26), 23.1 (CH3- 27), 23.6, 24.7, 26.0, 27.8, 27.8, 28.2, 33.6, 35.1, 35.6, 36.1, 37.2, 38.1, 38.8, 42.0, 48.9, 55.6, 62.8 (C-6), 64.2 (C-7), 67.0 (C-5), 69.8 (C-3), 76.7 (C-4). FTIR (neat, cm -1): ν 3311, 2926, 2862, 1605, 1479, 1409, 1051, 896. Analysis calcd: C, 74.61; H, 10.67. Found: C, 74.33; H, 10.38.

III.C.4.4 5b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a-triol (or, 5 a,6 a-epoxy-4b,7a-dihydroxy-b- sitosterol, 1197): Eluent in column chromatography: 35% ethyl acetate in petroleum ether (final o fractions). Yield: 50-55%. m.f. C 29 H50 O4, white crystalline solid. m.p. 238-240 C (chloroform- 1 methanol). H NMR (300 MHz, DMSO-d6): δ 0.56 (s, 3H, Me -18), 0.78 (s, 3H, Me-27), 0.80 (d, J= 2.1 Hz, 3H, Me-26), 0.82 (d, J= 9.0 Hz, 3H, Me-29), 0.84 (d, J= 2.1 Hz, 3H, Me-21), 1.12 (d, J= 4.2 Hz, 3H, Me-19), 3.73 (d, J=3.0 Hz, 1H, H-3), 4.05 (d, J= 2.4 Hz, 1H, H-4), 4.43 (dd, J=6 13 Hz, 18 Hz, 3H, H-6), 4.82 (m, 1H, H-7). C NMR: (75 MHz, CDCl 3): δ 12.0 (CH 3-18), 12.3

(CH3-29), 15.3(CH 3-26), 19.1 (CH3-21), 19.4 (CH 3-19), 19.7, 20.1 (CH3-27), 23.1, 24.7, 25.9, 26.1, 28.3, 29.1, 33.7, 33.9, 35.1, 36.0, 37.2, 38.1, 38.9, 42.0, 45.7, 48.9, 55.4, 62.8 (C-6), 64.3 (C-7), 67.1 (C-5), 69.8 (C-3), 76.8 (C-4). FTIR (neat, cm -1): ν 3443, 2928, 2864, 1464, 1375, 1067, 921, 750. Analysis calcd: C, 75.28; H, 10.89. Found: C, 72.64; H, 10.66.

III.C.4.5 5a-Cholestan-5b,6 b-epoxy-3b,4 b-diol (or, 5 b,6 b-epoxy-4b-hydroxycholesterol, 1198): Eluent in column chromatography: 25% ethyl acetate in petroleum ether (final fractions).

221

o 1 Yield: 55-62%. m.f. C 27 H46 O3, white crystalline solid. m.p. 164-165 C (CHCl 3/ C 2H5OH). H

NMR (300 MHz, CDCl 3): δ 0.63 (s, 3H, Me -19), 0.85 (s, 3H, Me-27), 0.87 (s, 3H, Me-26), 0.90

(s, 3H, Me-21), 1.13 (s, 3H, Me-18), 1.92- 2.02 (m, 3H, H-2 and H b-7), 2.13 (dt, J=3 Hz and 15

Hz, 1H, H a-7 ), 2.54 (br s, -OH groups), 3.19 (s, 1H, H-4), 3.36 (d, J=3 Hz, 1H, H-3), 3.59-3.62 13 (m, 1H, H-6). C NMR: (75 MHz, CDCl 3): δ 11.7 (CH 3-18), 17.8 (CH3-19), 18.7 (CH3-21),

21.3, 22.6 (CH3-26), 22.8 (CH3- 27), 23.8, 24.3, 24.8, 28.0, 28.1, 30.0, 32.6, 34.8, 35.7, 36.2, 36.9, 39.5, 39.7, 42.5, 51.6, 56.2, 64.0 (C-6), 65.5 (C-5), 70.3 (C-3), 77.2 (C-4). FTIR (neat, cm - 1): ν 3484, 3412, 3222, 2941, 2859, 1463, 1373, 1237, 1092, 911. Analysis calcd: C, 77.46; H, 11.07. Found: C, 77.54; H, 10.78.

III.C.4.6 5a-Betasitostan-5b,6b-epoxy-3b,4 b-diol (or, 5 b,6 b-Epoxy-4b-hydroxy-b-sitosterol, 1199): Eluent in column chromatography: 25% ethyl acetate in petroleum ether (final fractions). o 1 Yield: 50-60%. m.f. C 29 H50 O3, white powered solid. m.p. 192 C. H NMR (300 MHz, CDCl 3): δ 0.56 (s, 3H, Me-18), 0.75 (s, 3H, Me-27), 0.77 (s, 3H, Me-26), 0.82 (s, 3H, Me-29) 0.84 (s, 3H,

Me-21), 1.06 (s, 3H, Me-19), 1.84- 1.95 (m, 3H, H-2 and H b-7), 2.06 (dd, J= 2.4 Hz and 14.4Hz,

1H, H a-7), 2.12- 2.70 (br hump, -OH groups), 3.12 (s, 1H, H-4), 3.29 (s, 1H, H-3) 3.54 (dt, J=3 13 Hz and 7.2 Hz, 1H, H-6). C NMR: (75 MHz, CDCl 3): δ 10.7 (CH 3-18), 11.0 (CH 3-29), 16.8

(CH3-26), 17.7 (CH3-21), 18.0 (CH3-19), 18.8 (CH 3-27), 20.2, 22.0, 23.2, 23.8, 25.0, 27.1, 28.1, 28.6, 31.5, 32.9, 33.7, 35.1, 35.9, 38.6, 41.2, 44.8, 50.6, 55.0, 55.2, 62.9 (C-6), 64.5 (C-5), 70.2 (C-3), 76.1 (C-4). FTIR (neat, cm -1): ν 3365, 2926, 2855, 1454, 1383, 1084, 915. Analysis calcd: C, 77.97; H, 11.28. Found: C, 76.32; H, 10.87.

III.C.4.7 5a-Cholestan-5b,6 b-epoxy-3b,4 b,7a-triol (or, 5 b,6b-epoxy-4b,7a-dihydroxy cholesterol, 1200): Eluent in column chromatography: 35% ethyl acetate in petroleum ether o 1 (initial fractions). Yield: 12-15%. m.f. C 27 H46 O4, white powdered solid. m.p. 158-160 C. H

NMR (300 MHz, CDCl 3): 0.65 (s, 3H, Me-19), 0.85 (s, 3H, Me-27), 0.87 (s, 3H, Me-26), 0.91 (s, 3H, Me-21), 1.14 (s, 3H, Me-18), 1.86- 2.00 (m, 5H, H-1, H-2, H-8), 2.31 (br s, -OH groups), 3.21 (s, 1H, H-6), 3.38 (s, 1H, H-7), 3.68 (d, J=11.1 Hz, 1H, H-3) 4.08 (s, 1H, H-4). 13 C NMR:

(75 MHz, DMSO-d6): δ 11.6 (CH 3-18), 18.0 (CH 3-19), 18.7 (CH3-21), 21.1, 22.6 (CH3-26), 22.8

(CH3- 27), 23.8, 24.6, 28.0, 28.2, 34.4, 35.7, 36.1, 36.6, 39.2, 39.5, 39.5, 42.0, 42.1, 49.2, 56.0,

222

65.01 (C-6), 66.1 (C-5), 66.9 (C-7), 71.0 (C-3), 77.2 (C-4). FTIR (neat, cm -1): ν 3376, 2921, 2853, 1454, 1364, 1057, 924. Analysis calcd: C, 74.61; H, 10.67. Found: C, 73.05; H, 10.20.

III.C.4.8 5a-Betasitostan-5b,6 b-epoxy-3b,4 b,7a-triol (or, 5 b,6 b-epoxy-4b,7a-dihydroxy-b- sitosterol, 1201): Eluent in column chromatography: 35% ethyl acetate in petroleum ether o 1 (initial fractions). Yield: 12-15%. m.f. C 29 H50 O4, white powdered solid. m.p. 192 C. H NMR

(300 MHz, CDCl 3): δ 0.65 (s, 3H, Me -18), 0.80 (d, J= 1.2 Hz, 3H, Me-27), 0.82 (s, 3H, Me-26), 0.84 (d, J= 9.0 Hz, 3H, Me-29), 0.92 (s, 3H, Me-21), 1.14 (s, 3H, Me-19), 1.82- 2.01 (m, 5H, H- 1, H-2 and H-8), 2.15 (br s, -OH groups), 3.21 (s, 1H, H-4), 3.38 (s, 1H, H-3) 4.08 (s, 1H, H-3) 13 4.19 (s, 3H, H-6). 4.82 (s, 1H,H-7) C NMR: (75 MHz, CDCl 3): δ 11.5 (CH 3-18), 12.0 (CH3-

29), 18.0 (CH3-26), 18.7 (CH3-21), 19.0 (CH3-19), 19.8 (CH 3-27), 21.1, 23.1, 23.1,23.7, 24.7, 26.0, 28.2, 29.2, 33.9, 34.4, 34.4, 36.1, 36.6, 39.2, 42.1, 42.2, 45.9, 49.3, 56.0, 65.0 (C-6), 66.1 (C-5), 66.9 (C-7), 71.0 (C-3), 77.2 (C-4). FTIR (neat, cm -1): ν 3364, 2935, 2861, 1454, 1 373, 1057, 941, 621. Analysis calcd: C, 75.28; H, 10.89. Found: C, 72.86; H, 10.57.

III.C.4.9 5b-Cholestane-3b, 4 b, 5a, 6b- tetrol (1202): Eluent in column chromatography: 35% 1 ethyl acetate in petroleum ether. Yield: 78-85%. m.f. C 27 H48 O4, pale yellow sticky gum. H

NMR (300 MHz, DMSO-d6) 0.67 (s, 3H, Me-19), 0.89 (s, 6H, Me-27 & 26), 0.91 (s, 3H, Me- 21), 1.27 (s, 3H, Me-18), 3.72 (s, 1H, H-6), 3.85 (m, 1H, H-3), 4.20 (d, J= 12 Hz, 1H, H-4). 13 C

NMR: (75 MHz, DMSO-d6): δ 12.3 (CH 3-18), 15.2 (CH 3-19), 19.0 (CH3-21), 20.2, 22.9 (CH3-

26), 23.1 (CH3- 27), 23.8, 24.3, 26.6, 27.9, 27.9, 28.3, 28.8, 30.7, 32.2, 35.7, 36.1, 36.6, 38.2, 42.7, 45.6, 56.1, 56.2, 57.1, 67.8 (C-7), 72.7 (C-3), 79.3 (C-4), 87.6 (C-5). FTIR (neat, cm -1): ν 3423, 2926, 2855, 1463, 1359, 1066, 942. Analysis calcd: C, 74.26; H, 11.08. Found: C, 71.9; H, 10.54.

III.C.4.10 5b-betasitostane-3b, 4 b, 5a, 6b-tetrol (1203): Eluent in column chromatography: 1 35% ethyl acetate in petroleum ether. Yield: 75-80%. m.f. C29 H52 O4, pale yellow sticky gum. H

NMR (300 MHz, DMSO-d6): δ 0.63 (d, J= 7.2 Hz, 3H, Me-18), 0.80 (s, 3H, Me- 27), 082 (s, 3H, Me- 26), 0.84 (s, 3H, Me- 29), 0.91 (d, J= 5.7 Hz, 3H, Me- 21), 1.22 (d, J= 5.7 Hz, Me- 19), 3.67 13 (s, 1H, H-6), 3.80 (m, 1H, H-3), 4.12-4.15 (m, J= 1H, H-4). C NMR: (75 MHz, DMSO-d6): δ

12.2 (CH3-18), 12.3 (CH3-29), 15.2 (CH3-26), 19.0(CH 3-21), 19.4(CH 3-19), 20.2 (CH 3-27), 23.1,

223

24.3, 26.1, 26.6, 28.3, 28.9, 29.2, 29.2, 30.7, 32.2, 33.9, 36.0, 38.2, 42.7, 45.7, 45.7, 56.1, 56.1, 57.1, 67.8 (C-7), 72.7 (C-3), 79.3 (C-4), 87.6 (C-5). FTIR (neat, cm -1): ν 3442, 2912, 2865, 1445, 1374, 1028, 976. Analysis calcd: C, 74.95; H, 11.28. Found: C, 73.12; H, 10.73.

III.C.4.11 5b-Cholestane-3b, 4 b, 5a, 6b, 7a- pentol (1204): Eluent in column chromatography: 1 50% ethyl acetate in petroleum ether. Yield: 70-78%. m.f. C 27 H48 O5, pale yellow sticky gum. H

NMR (300 MHz, CDCl 3): 0.70 (d, J= 10.8 Hz, 3H, Me-19), 0.86 (s, 3H, Me-27), 0.88 (s, 3H, Me-26), 0.89 (s, 3H, Me-21), 1.28 (d, J= 10.8 Hz, 3H, Me-18), 1.89-2.05 (m, 2H, H-2), 3.83- 13 4.13 (m, 4H, H-3, H-4, H-6, H-7). C NMR: (75 MHz, CDCl 3): δ 11.8 (CH 3-18), 14.7 (CH3-19),

18.7 (CH3-21), 19.9, 22.6 (CH 3-26), 22.8 (CH 3- 27), 23.6, 23.8, 26.1, 28.0, 28.2, 31.5, 35.6, 35.8, 36.2, 38.7, 38.9, 39.4, 39.5, 42.6, 49.8, 56.0, 58.2, 68.2 (C-7), 68.3 (C-6), 74.8 (C-3), 78.4 (C-4), 88.3 (C-5). FTIR (neat, cm -1): ν 3338, 2950, 1463, 1355, 1051, 942. Analysis calcd: C, 71.64; H, 10.69. Found: C, 68.88; H, 10.08.

III.C.4.12 5b-Betasitostan-3b, 4 b, 5a, 6b, 7a- pentol (1205): Eluent in column chromatography: 50% ethyl acetate in petroleum ether. Yield: 67-72%. m.f. C 29 H52 O5, pale yellow sticky gum. 1H NMR (300 MHz, DMSO-d6): δ 0.62 (s, 3H, Me -18), 0.78 (s, 3H, Me-27), 0.80 (s, 3H, Me-26), 0.82 (s, 3H, Me-29), 0.90 (s, 3H, Me-21), 1.18 (s, 3H, Me-19), 1.61- 1.93 13 (m, 2H, H-2), 3.67-3.96 (m, 4H, H-3, H-4, H-6, H-7). C NMR: (75 MHz, CDCl 3): δ 12.1 (CH 3-

18), 12.2 (CH3-29), 15.0 (CH 3-26), 19.0 (CH3-21), 19.4 (CH 3-19), 20.0, 20.1 (CH 3-27), 23.1, 23.5, 25.9, 26.3, 28.3, 29.1, 29.1, 32.4, 33.8, 35.3, 36.1, 38.8, 42.4, 45.6, 49.8, 55.9, 58.8, 67.1 (C-7), 68.1 (C-6),75.0 (C-3), 78.9 (C-4), 88.2 (C-5). FTIR (neat, cm -1): ν 3400, 2936, 2856, 1454, 1364, 1057, 962. Analysis calcd: C, 72.46; H, 10.90. Found: C, 69.85; H, 10.52.

III.D Conclusion Epoxidation followed by alkaline-opening of the oxirane-ring of appropriate steroidal 5-ene- 3b,4 b-diols and -5-ene-3b,4 b,7 a-triols led to furnish novel steroidal tetrols and pentols respectively. As the effect, syntheses of a new series of polyhydroxy and epoxy-polyhydroxy steroids were accomplished. The overall reaction sequences thus let out, in practical, a three-step synthetic route for the transformation of 3b,4 b,5 a,6 b-tetrahydroxy- and 3 b,4 b,5 a,6 b,7a - pentahydroxy steroids starting directly from their corresponding basic steroids, cholesterol and

224 b-sitosterol. As a new series of polyhydroxy steroids, gelation behavior of the synthesized molecules was evaluated. At 1% or below CGC (critical gelation concentration), five PHS derivatives (epoxy-diols and -triols) were found to be gelators of some selective organic solvents viz. , DMSO and long chain hydrocarbons. The organogels were characterized by T gel (gel melting temperature) and related physical parameters ( DH etc.), rheological data, and by morphology analysis (electron microscopes). The thermal stability of the 11 (or, 12 )/ dodecane gels were found higher in comparison to the 5 (or, 9)/ DMSO gels. The viscoelastic soft behavior of the organogels was proved by rheology experiments whereas atomic force microscopy reveals fibrillar (or, bundles of fibres) networks in the DMSO gels and microspheres in case of dodecane gels. Polarizing optical microscopy showed the DMSO gels to be birefringent. Biological evaluation of the synthesized PHS derivatives is in progress and the results are found to be promisingly cytotoxic towards cancer cells.

III.E Supporting spectra

Figure 3.93 1H NMR spectrum of 5 b-Cholestan-5a,6 a-epoxy-3b,4 b-diol ( 1194 ).

225

Figure 3.94 Extended 1H NMR spectrum of 5b-Cholestan-5a,6 a-epoxy-3b,4 b-diol ( 1194 ).

Figure 3.95 13 C NMR spectrum of 5 b-Cholestan-5a,6 a-epoxy-3b,4 b-diol (1194 ).

226

Figure 3.96 IR spectrum of 5 b-Cholestan-5a,6 a-epoxy-3b,4 b-diol (1194).

Figure 3.97 1H NMR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol (1195).

227

Figure 3.98 Extended 1H NMR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol ( 1195).

Figure 3.99 13 C NMR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol ( 1195).

228

Figure 3.100 IR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b-diol ( 1195 ).

Figure 3.101 1H NMR spectrum of 5b-Cholestan-5a, 6a-epoxy-3b, 4b, 7a -triol ( 1196).

229

Figure 3.102 13 C NMR spectrum of 5b-Cholestan-5a, 6a-epoxy-3b, 4b, 7a -triol ( 1196).

Figure 3.103 IR spectrum of 5 b-Cholestan-5a, 6a-epoxy-3b, 4b, 7a -triol ( 1196).

230

Figure 3.104 1H NMR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a -triol ( 1197).

Figure 3.105 Extended 1H NMR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a -triol ( 1197).

231

Figure 3.106 13 C NMR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a -triol ( 1197).

Figure 3.107 IR spectrum of 5b-Betasitostan-5a,6 a-epoxy-3b,4 b,7a -triol ( 1197).

232

Figure 3.108 1H NMR spectrum of 5a-Cholestan-5b,6 b-epoxy-3b,4 b-diol ( 1198).

Figure 3.109 13 C NMR spectrum of 5a-Cholestan-5b,6 b-epoxy-3b,4 b-diol ( 1198).

233

Figure 3.110 IR spectrum of 5a-Cholestan-5b,6 b-epoxy-3b,4 b-diol ( 1198).

Figure 3.111 1H NMR spectrum of 5a-Betasitostan-5b, 6b-epoxy-3b,4 b-diol ( 1199 ).

234

Figure 3.112 Extended 1H NMR spectrum of 5a-Betasitostan-5b, 6b-epoxy-3b,4 b-diol ( 1199 ).

Figure 3.113 13 C NMR spectrum of 5a-Betasitostan-5b, 6b-epoxy-3b,4 b-diol ( 1199 ).

235

Figure 3.114 IR spectrum of 5a-Betasitostan-5b, 6b-epoxy-3b,4 b-diol ( 1199 ).

Figure 3.115 1H NMR spectrum of 5a-Cholestan-5b,6 b-epoxy-3b,4 b,7a -triol ( 1200 ).

236

Figure 3.116 13 C NMR spectrum of 5 a-Cholestan-5b,6 b-epoxy-3b,4 b,7a -triol ( 1200 ).

Figure 3.117 IR spectrum of 5 a-Cholestan-5b,6 b-epoxy-3b,4 b,7a -triol ( 1200 ).

237

Figure 3.118 1H NMR spectrum of 5a-Betasitostan-5b,6 b-epoxy-3b,4 b,7a -triol ( 1201).

Figure 3.119 IR spectrum of 5 a-Betasitostan-5b,6 b-epoxy-3b,4 b,7a -triol ( 1201).

238

Figure 3.120 1H NMR spectrum of 5b-Cholestane-3b, 4 b, 5a, 6b- tetrol ( 1202 ).

Figure 3.121 Extended 1H NMR spectrum of 5 b-Cholestane-3b, 4 b, 5a, 6b- tetrol ( 1202 ).

239

Figure 3.122 13 C NMR spectrum of 5 b-Cholestane-3b, 4 b, 5a, 6b- tetrol ( 1202 ).

Figure 3.123 IR spectrum of 5 b-Cholestane-3b, 4b, 5a, 6b- tetrol ( 1202).

240

Figure 3.124 Extended 1H NMR spectrum of 5b-betasitostane-3b, 4 b, 5a, 6b-tetrol ( 1203 ).

Figure 3.125 IR spectrum of 5 b-betasitostane-3b, 4b, 5a, 6b-tetrol ( 1203).

241

Figure 3.126 1H NMR spectrum of 5b-Cholestane-3b, 4 b, 5a, 6b, 7a - pentol ( 1204 ).

Figure 3.127 Extended 1H NMR spectrum of 5b-Cholestane-3b, 4 b, 5a, 6b, 7a - pentol ( 1204).

242

Figure 3.128 13 C NMR spectrum of 5b-Cholestane-3b, 4 b, 5a, 6b, 7a - pentol ( 1204 ).

Figure 3.129 IR spectrum of 5b-Cholestane-3b, 4b, 5a, 6b, 7a - pentol ( 1204).

243

Figure 3.130 1H NMR spectrum of 5b-Betasitostan-3b, 4 b, 5a, 6b, 7a - pentol ( 1205 ).

Figure 3.131 13 C NMR spectrum of 5b-Betasitostan-3b, 4 b, 5a, 6b, 7a - pentol ( 1205 ).

244

Figure 3.132 IR spectrum of 5b-Betasitostan-3b, 4 b, 5a, 6b, 7a - pentol ( 1205).

III.F References The references associated with this chapter are provided in the Bibliography section of the thesis. Please follow page-359 onwards for these references.

245

Chapter-IV

Syntheses of new friedelane triterpenoids: A-ring modifications including 2-homo derivatives

246

Abstract

First, the A-ring modified friedelane triterpenoids are reviewed in brief, which forms indeed the present day basis of the different type of A-ring modifications of this particular PT either of natural occurrence or due to various transformative reactions. And, the practical work associated with the chapter constitutes the syntheses of a library of A-ring modified friedelane triterpenoids. The modifications also include the all new 2- homoderivatives. The syntheses of the novel 2-homofriedelanes are based on the transformative reactions of the designed triterpenoid 3-chloro-2-formylfriedel-2-ene ( 402) which was isolated as the major product from the reaction of friedelin ( 000) with the novel Vilsmeyer-Haack reagent. Some new derivatives of the friedelane series were also prepared from cerin ( 0000, a naturally occurring PT; structurally 2 a-hydroxy friedelin) as well as using one of the new derivative 401, structurally 3-chlorofriedel-2-ene, isolated as a side product from the key reaction. Moreover, considering the beauty of 3-chloro-2-en-al moiety, associated with the A-ring of the triterpenoid, a number of heterocycle-linked- (bonded to C3) 2-homofriedelane triterpenoids were synthesized.

247

IV.A Introduction: A brief review on the A-ring modified friedelane triterpenoids A pentacyclic triterpenoid was extracted from the bark of Querques Suber as far back as 1807 by Chevreul 1,2 and was named friedelin ( 115) in honour of Friedel who was probably the first one to disclose the presence of a ketone group in the triterpenoid. 3 That one of the compound extracted contained a ketone functional group was again confirmed by Istrati and Ostrogovich. 4 Later, Drake et al. established the molecular formula of friedelin and prepared the oxime, enol esters and carbonyl derivatives, and it was converted to the saturated parent hydrocarbon friedelane. The oxime was transformed into the oxime acetate (by refluxing with acetic anhydride),

Beckmann rearrangement product (with PCl 5) and was reconverted to friedelin on hydrolysis 5-9 with H 3PO 4 in n-amyl alcohol. But it was the success of Corey and Ursprung, and Dutler, Jeger and Ruzicka, separately, in 1956 to provide the complete structures of friedelin (115) and cerin ( 116) and then Brownnlie and his collaborators produce the stereochemistry of these triterpenoids ( Figure 4.1 ). 10-12 Thus, friedelin and associated triterpenoids which are generally named as friedelane triterpenoids are a group of triterpenoids bearing a [6-6-6-6-6]-fused pentacyclic skeleton with eight methyl groups at C(4), C(5), C(9), C(13), C(14), C(17), and C(20) (geminal-dimethyl), respectively ( Figure 4.1 ). The final structural knowledge indeed made available a broad scope of study of the transformative reactions on friedelin and associated friedelane triterpenoids.

O Me Me Me H H H Me H H Me HO H Me H Me O O Me 115 115 in chair structure 116 Figure 4. 1 Friedelin ( 115), its chair form and cerin ( 116 ).

IV.A.1 Naturally occuring A-ring modified friedelane triterpenoids More than 400 natural friedelane triterpenoids are isolated till date. The friedelane triterpenoids are found to be distributed more commonly in the families such as Celastraceae , Hippocrateaceae , Euphorbiaceae , Flacourtiaceae , and Guttiferae and among them the first two

248 are the richest sources of it. Based on the structural aspects, the FTs can be classified into five major classess such as i) normal friedelanes ( Type A ); ii) secofriedelanes ( Type B ); iii) norfriedelanes ( Type C ); iv) dimeric friedelanes ( Type D ) and v) rearranged friedelanes ( Type E) ( Figure 4.2 ). Leaf and twig parts provide mainly the first two classess of FTs whereas the classess (iii) and (iv) are isolated mainly from the root parts. Interestingly, friedelanes are also found to be present in some fungi. 13-14 Surprisingly, though there are reports of other triterpenoids which are found in nature as saponins, friedelanes are not being found as glycosides in nature. 15-17 Zhan et al. have summarized the natural sources of friedelane triterpenoids recently in their beautiful review. 18 Again, some selective interesting naturally occuring FTs where the A- ring got modified are described herein in short.

COOH MeOOC

OH

O O O OH O HO HO Type B Type C Type A MeOOC

O OBz O O HO MeO O AcO OAc O OAc O Type D

Type E

COOMe Figure 4.2 Diferent types of friedelanes ( Type A-E).

Friedelin ( 115) and friedelane-l,3-dione ( 1208) (Figure 4.3 ), 19 along with some other friedelane triterpenoids were isolated from the stem and bark extract of Peritassa compta .

249

O H H

O 1208 6

Figure 4.3 Friedelane-l,3-dione ( 1208) and friedelane ( 6). Friedelin was also found to be a major constituent of Grapefruit epicuticular Wax, 20 A. indica 21 and also the stem bark exudates of Maytenus macrocarpa, which produced friedelane (6) also (Figure 4.3 ). The biocidal activities of 6 were studied and found to be inactive to the antitumor activity against P-388 lymphoid neoplasm, A-549 human lung carcinoma, HT-29 human colon carcinoma, or MEL-28 human melanoma cell lines, but showed weak activity against aldose reductase. 22 4-Epi friedelin ( 1209) along with twelve other known terpenoids (Figure 4.4 ) were found in the leaves of Syzygium formosanum. 23

H H H

O 1209

Figure 4 .4 4-epi friedelin ( 1209 ).

Friedelin ( 115) and 3 b-hydroxy friedelane ( 1210) ( Figure 4.5 ) were isolated from Artocarpus altilis 24 and friedelin ( 115) along with 4-epi friedelin ( 1209) were isolated from the leaves of Salacia chinensis. 25 Friedelin ( 115) and 3 b-hydroxy friedelane ( 1210 ) were again isolated from Celastrus vulcanicola and the photosynthetic inhibitory activity of these compounds revealed 1210 to be an “energy transfer inhibitor, interacting and enhancing the light activated Mg 2+ -ATPase” 26 .

250

The foliar epicuticular waxes of the leaves of A. Esperanzae were found to contain 4- epi friedelin ( 1209). 27,28 Baskar et al. isolated friedelin ( 115) from the leaves of Azima tetracantha and found friedelin to result 75.28% antifeedant, 66% larvicidal and 66.66% pupicidal activities against H. armigera and S. Litura respectively. 29 During the phytochemical investigation of Maytenus truncata Reiss, both the epimers of 3- hydroxy friedelane ( b: 1210 and a : 1211) were isolated (Figure 4.5 ).30

H H H H H H

HO O 1210; 3b-hydroxy friedelane 1211; 3a-hydroxy friedelane OH 1212 Figure 4.5 3b-Hydroxy- and 3 a-hydroxy friedelane ( 1210 and 1211, respectively) and terminaline A (1212).

The plant extracts of Maytenus aquifolium and M. ilicifolium show anti-ulcer activity and these are indeed used medicinally in Brazil under the name “espinheirasanta”. Lancas et al., showed that friedelin ( 115) and 3b-hydroxy friedelane (1210) are the markers of these plants. 31-32 The leaves and root barks of M. aquifolium and S. campretris were found to accumulate friedelin and quinonemethide respectively. From the enzymatic extracts of the leaves it was found that cyclase converted the substrate oxidosqualene to the triterpenes, 3 b-hydroxy friedelane ( 1210) and friedelin ( 115). Moreover, when mevalonolactone was administrated to the leaves of M. aquifolium seedlings, radio labelled friedelin was found to be produced in the leaves, twigs and stems, while labelled maytenin and pristimerin were found in the root bark. From these important observations it was concluded that triterpenes once biosynthesized in the leaves were translocated to the root bark and further transformed into the antitumoral quinonemethide triterpenoids. 32-33 Investigation of the leaves and barks of the Indian L. racemosa collected from Sundarban and Kakinada 33 and the stems of the same collected from the Indian Bhiravapalem Island 34

251 revealed the presence of friedelin. Terminalia glaucescens was also found to contain an A-ring modified secotriterpene terminaline A ( 1212, Figure 4.5 ) and friedelin. 35 Faure et al. isolated 3,4-secofriedelane-3,28-dioic acid ( 1213) and two other new friedelane- type triterpenoids from the leaves of Calophyllum inophyllum (Clusiaceae) found in French Polynesia ( Figure 4.6 ). 36

O H H H COOH H H H R1 HO R2 1214; R1= R2= O, R3= R5= H, R4= OH R4 1215; R1= R2= H, R3= R4= O, R5= OH 1216; R = R = R = H, R = R = OH R3 1 4 5 2 3 1213 R5 Figure 4.6 3,4-Secofriedelane-3,28-dioic acid ( 1213) and ( 1214-1216 ).

The stem and bark of the Mangrove plant Hibiscus tiliaceus was found to contain a number of A-ring modified friedelane triterpenoids ( 115, 1210 , 1214-1216) (Figure 4.6 ). 37 Lobatanhydride ( 1217) is the first example of a triterpenoid anhydride which contains a seven-membered A-ring. It was isolated, along with friedelin ( 115), 3 a,25-dihydroxyfriedelan-2- one ( 1218) and 1 b,25-dihydroxyfriedelan-3-one ( 1219), 2a-hydroxy-3-oxofriedelan-30-oic acid (1220 ), 28-hydroxyfriedelane-1,3-dione ( 1221), and 29-hydroxyfriedelane-1,3-dione ( 1222) (Figure 4.7 ) from Crossopetalum lobatum Lundell. 38 1,2-Dehydro-2,3-secofriedelan-3-oic acid ( 1223), 1b-hydroxyfriedelin ( 1224), and 3 b- hydroxyfriedelan-23-oic acid ( 1225) and friedelin-3,4-lactone ( 1226) were isolated from the leaves of Garcia pariflora (Figure 8 ).39 A number of derivatives ( 1208, 1227-1234) of 1224 were then prepared using various oxidation, reduction and esterification strategy ( Scheme 4.1 ). Cytotoxicity of the synthetic as well as the natural compounds was also tested against human cancer cell lines U251, PC-3, K562, HCT-15, MCF-7 and SKLU-1.

252

OH OH OH OH O H H H H H H H O O HO O O 1217 1218 1219

HOOC R2

O H H H H H H R HO 1

O O O 1221; R1= CH2OH, R2= Me 1222; R1= Me, R2= CH2OH 1220 Figure 4.7 Lobatanhydride ( 1217), 3 a,25-dihydroxyfriedelan-2-one ( 1218), 1 b,25-dihydroxyfriedelan-3- one ( 1219), 2 a-hydroxy-3-oxofriedelan-30-oic acid ( 1220), 28-hydroxyfriedelane-1,3-dione ( 1221 ) and 29-hydroxyfriedelane-1,3-dione ( 1222).

OH OH OH H H H H H

O + + HON O O O O 1227 1232 1233 HO 1234 1224

OH O OH OH OAc H H H H H

O + HN O HO AcO AcO 1228 1230 1231 1208 1229

Scheme 4.1 Derivatives of 1224 1

1 Details of scheme, ref. 39. 253

OH H H H H H H H H H H H H

CHO O HOOC O HO O COOH 1223 1224 1225 1226

Figure 4.8 1,2-Dehydro-2,3-secofriedelan-3-oic acid ( 1223), 1 b-hydroxyfriedelin ( 1224), 3 b- hydroxyfriedelan-23-oic acid ( 1225 ), friedelin-3,4-lactone ( 1226 ).

Very recently, Liu and his group isolated three novel norfriedelanes ( 1235-1237) (Figure 4.9) from the branches and roots of Malpighia emarginata . Among them norfriedelanes 1235 and 1237 were found to show acetylcholine esterase inhibitory effects having IC 50 values of 10.3 and 28.7 μM, respectively. 40

O H H OH H H H H O O O O O

O O 1235 1236 1237 Figure 4.9 Structures of norfriedelanes 1235-1237.

Friedelin ( 115), 29-hydroxymethyl friedelin ( 1238) and 3,4-seco-friedelan-3-oic acid ( 1239) (Figure 4.10 ) were isolated from the bark extract of Chinese Heritiera littoralis .41 Luis and his group isolated a friedo-olean triterpenoid, from the root extract of Schaefferia cuneifolia, which was proved structurally to be 2-oxofriedoolean-3-en-29-oic acid ( 1240). 42 The corresponding methyl ester derivative ( 1241) was also prepared ( Figure 4.10 ). The choloroform extract of Tripterygium wilfordii was found to contain salaspermic acid (1242, Figure 4.10 ), a friedelane triterpene acid. The compound resulted the inhibition of HIV

254 replication in H9 lymphocytes with an IC 50 value of 5 mg/mL (10 mM), and the inhibition of 43 uninfected H9 cell growth had an IC 50 value of 53 mM.

COOR CH2OH COOH

H H H H H O H H H

HOOC O HO 1238 1240; R= H O 1239 1242 1241; R= CH3 Figure 4.10 29-Hydroxymethyl friedelin (1238), 3,4-Seco-friedelan-3-oic acid ( 1239), 2-oxofriedoolean- 3-en-29-oic acid ( 1240 ), its methyl ester derivative ( 1241 ) and salaspermic acid ( 1242 ).

In addition, among the large number of natural A-ring modified friedelane triterpenoids, 2a,3 b-dihydroxyfriedelan-28-oic acid ( 1243) from the leaves of Marila pluricostata ,44 2b- hydroxy-3-oxofriedelan-30-oic acid ( 1244) from Dichapetalum barteri ,45 Dzununcanone ( 1245), a 3,24-dinor-2,3-seco-friedelane derivative from Hippocrate excels, 46 1246 from Passiflora wilsonii 47 were isolated. Other norfriedelane derivatives such as trifloralactone 1247 and triptocalline B 1248 were isolated from Microtropis triflora, 48 and milicifolines A –D ( 1249- 1252) from Maytenus ilicifolia. 49 Milicifolines B ( 1250) and -C ( 1251) related to the cheiloclines were also reported by the same group in 2005. (Figure 4.11) Besides the wide distribution of friedelane triterpenoid in the plant origin, the fermented broth of the fungus Leptosphaeria maculanns was found to contain euphorcinol ( 1253) and this was actually the first report of the occurrence of a friedelane triterpenoid in fungi ( Figure 4.11).13 Compound 1254 (Figure 4.12 ) was also found in marine endophytic fungus. 14

IV.A.2 Synthetic A-ring modified friedelane triterpenoids Chemical transformation is a very important useful tool in organic chemistry towards a number of diverse attempts including structural elucidation, structural modification, target-based structure-synthesis (semisynthesis and total synthesis), evaluation of the scope and limitations of a reaction/ reagent, etc. And it is very much obvious that these all imply enriching chemistrty to lead humans and their sourroundings toward a better existence from all the directions starting from food to technology and health to healthy imagination!

255

Besides the natural friedelane triterpenoids, a number of synthestic derivatives have so far been reported which include numerous transformative protocols. Though many transformations include different strategies (viz., oxidation, reduction etc.) simultaneously, herein an attempt is made to arrange the main transformative reactions under some different suitable titles.

HOOC COOMe OH H H H COOH HO H H H H H HO H H MeOOC HOOC HO O O 1243 O 1244 1245 1246

O O OH O O

OH H H H H HO H H H HO H HO O O O MeO O HO O O O O HO 1247 1248 1249 1250

O OH O O

O O H H H H O H H O O HO Me 1251 COOMe 1252 1253 Figure 4.11 2a,3 b-Dihydroxyfriedelan-28-oic acid ( 1243 ), 2 b-hydroxy-3-oxofriedelan-30-oic acid (1244), dzununcanone ( 1245 ), trifloralactone 1246 , triptocalline B 1247, triptocalline B 1248, milicifolines A –D ( 1249-1252) and euphorcinol (1253).

COOH

HO 1254

Figure 4.12 Compound 1254 .

256

IV.A.2.1 Oxidative transformations Stevenson, in 1961, described a convenient isolation method for friedelin from cork smoker wash solids. He prepared its oxime ( 1255) which on treatment with nitrous acid readily yielded compound 1256 (Scheme 4.2 ) which was converted again to friedelin by heating in aqueous dioxane. 50

H H H H H H O 1256 HNO2

HON (O2N)N 1256 1255

Scheme 4.2 Compound 1256.

Chromic acid oxidation of friedelin to produce friedelonic acid 8,9 and dehydrogenation of one of the epimeric secondary alcohols (derived from reduction of friedelin) by selenium at 315oC to produce 1,2,7-trimethylnaphthalene, 1,2,8-trimethylphenanthrene and l,8- dimethylpicene were studied earlier. 7 Oxidation of friedelin ( 115) yielded a dicarboxylic acid and anhydride of the acid on pyrolysis produced norfriedelanone ( 1257). This on reflux in glacial acetic acid with selenium dioxide gave norfriedelenone, established as A(1)-norfriedel-4(23)-en- 3-one ( 1258) and more drastic oxidation with selenium dioxide in dioxane at 200 oC furnished norfriedelendione 51-53 which was later identified as A(4)-nor-23-norfriededel-1(10)-ene-2,3-dione (1259) (Figure 4.13 ). 54

H H H H H H

O O O O 1257 1258 1259

Figure 4.13 Norfriedelanone ( 1257), A(1)-norfriedel-4(23)-en-3-one ( 1258) and A(4)-nor-23- norfriededel-1(10)-ene-2:3-dione ( 1259).

257

In another set of reactions, controlled silylation first transformed friedelin ( 115) into 3- trimethylsiloxyfriedel-2-ene ( 1260) in high yields. The silyl ether 1260 was then oxidized with

OsO 4/NMMO to produce 2 a-hydroxyfriedelan-3-one (another natural friedelane triterpenoid known as cerin, 116) which by periodic acid oxidation resulted 2,3-secofriedelan-2-al-3-oic acid (1261). On the other hand, oxidation of 1260 with DDQ afforded friedel-1-en-3-one ( 1233). Compound 1260 on reductive ozonolysis furnished 2 a,3 b-dihydroxyfriedelane, also known as pachysandiol A ( 1216). The seco-acid 1261 was found to be a potent inhibitor of human lymphocyte proliferation (IC 50 10.7 mM) and of the growth of a human cancer cell line (GI 50 5.4- 17.2 mM) (Scheme 4.3 ).55

HO

O O Me3SiO 1260 116 115

HO OHC HOOC O HO

1233 1216 1261 Scheme 4.3 Silylation of friedelin leading to different friedelane derivatives (Ref. 78).

Brownlie, Spring, Stevenson and Strachan in their thorugh study with friedelin revealed that dehydration of friedelanol produces an unsaturated hydrocarbon, friedel-3-ene ( 1262) ( Figure 4.14). 12 Again, an unsaturated ester 1263 was isolated by the oxidation of friedelin ( 115) with potassium tert -butoxide followed by treatment with diazomethane (Scheme 4.4 ).56

LTA/I 2 (Lead tetraacetate/iodine) oxidized 3b -Hydroxy friedelane (1210) in dry benzene to furnish a tetrahydropyridine 1264, an iodo-ether 1265 and an a-acetoxytetrahydrofuran 1266 derivatives (Figure 4.15 ).57

258

H H H

1262

Figure 4.14 Friedel-3-ene ( 1262).

H H H H H H i) O2, t-BuOK H3CO2C ii) CH2N2 O O 115 1263

Scheme 4.4 Treatment of friedelin ( 11 ) with t-BuOK and diazomethane.

H H

H R' H O 1264; R=R'=H R 1265; R=H, R'=I 1266; R=OAc, R'=H Figure 4.15 Tetrahydropyridine 1264 , iodo-ether 1265 and a-acetoxytetrahydrofuran 1266.

IV.A.2.2 Reductive transformations Kane and Stevenson, in 1960, isolated friedelane-2,3-dione ( 1267) from cork smoker wash solids and they characterized it as monoacetate, monobenzoate, monomethyl and quinoxaline derivatives. Friedelane was isolated from 2,3-dione 1267 by following the Huang-Minlon reduction condition whereas selective reduction was found able to produce friedelin ( Scheme

259

4.5). The monomethyl ether 1269 was obtained from friedelane-2,3-dione ( 1267) by refluxing it with boron trifluride etherate in methanol solution ( Scheme 4.6 ). 58

H H H H H H H H H O O i) LAH/ ether Zn dust

O ii) Ac2O, Py AcO CH3COOH O

1267 1268 115 Scheme 4.5 Conversion of friedelane-2,3-dione ( 1267) to friedelin ( 115).

H H H H H H O O BF3OEt2/ CH3OH O H3CO 1267 1269

Scheme 4.6 Conversion of friedelane-2,3-dione ( 1267) to 3-methoxy-2-oxofriedelane ( 1269).

IV.A.2.3 Transformations based on both oxidation and reduction reactions Cerin ( 116) was primarily assumed to be 2 b-hydroxyfriedelin, which was later on proved, by Stevenson and his group, actually to be the 2 a-hydroxy isomer. 59 They reduced cerin ( 116) with sodium borohydride and thus isolated 2 a,3 b-dihydroxy friedelane ( 1216) which is indeed a natural friedelane triterpenoid named as pachysandiol-A. When cerin, after acetylation, was allowed for prolonged heating with potassium acetate, 2 b-acetoxyfriedelin ( 1271) was isolated which on reduction with lithium aluminium hydride produced 2 b,3 b-dihydroxy friedelane (1272). Friedelin ( 115) was also converted to friedelane-3b-yl benzoate ( 1273) by reduction followed by benzoylation. On pyrolysis, this benzoate produced friedel-2-ene ( 1274) which upon treatment with mCPBA, followed by perchloric acid treatment and acetylation resulted the diacetate of pachysandiol-A ( 1275). And then diol 1216 was again formed by hydrolyzing the diacetate. On the other hand, 2 a,3 a-dihydrxyfriedelane ( 1276) was prepared from friedel-2-ene

260

(1274) by the action of osmium tetraoxide, followed by cleavage of the ester by lithium aluminium hydride ( Scheme 4.7 and Scheme 4.8 ). Thus analyzing and comparing all the results, cerin was established as 2 a-hydroxyfriedelin. 59

H H H H H HO HO AcO AcO HO NaBH Ac O 4 A 2 KOAc LiAlH4 Py heat HO O O O HO 116 1216 1270 1271 1272

Scheme 4.7 Transformation of cerin ( 116 ) into different dihydroxy derivatives.

H H H H H H H H H HO i) Reduction i) OsO4 115 ii) Benzoylation BzO ii) LAH HO 1274 1273 1276 i) Perchloric acid ii) Acetylation

H H H H H H HO AcO Hydrol.

HO AcO 1216 1275 Scheme 4.8 Transformation of friedelin into different dihydroxy derivatives.

IV.A.2.4 Photochemical transformations Stevention and his group showed that the ultraviolet irradiation of friedelin ( 115) was able to result different products depending on the solvents. In ether solution, they isolated 5-ethyl-10β- vinyl-des-A-friedelane ( 1277) and norfriedelane ( 1279) whereas in chloroform solution, ethyl 3,4-secofriedelane-3-oate ( 1278) was found to be produced. Corresponding acid and the alcohol were also produced from the ethyl ester of the seco acid 1278 by following hydrolysis and reduction, separately (Scheme 4.9 ).60

261

UV UV 115 + EtO CHCl Ether O 3 1277 1278 1279 Scheme 4.9 Ultraviolet irradiation products of friedelin in ether and in chloroform.

Phototransformative reactions on friedelin were also carried out by many other research groups. 61 Shirasaki, Tsuyuki, Takahashi and Stevenson irradiated friedelin ( 115) with a high pressure mercury lamp under nitrogen atmosphere in a quertz vessel in ether containing acetone. The isolated product was A-seco keto-ol 1280 which was successively treated with LAH and

Pb(OAC) 4 to result seco-aldehyde 1281 which was further oxidised with Ag 2O to yield the seco- acid 1239 and the successive transformations, in fact, confirmed compound 1280 structurally as 5α -ethyl-10β-(4-hydroxy-4-methyl-3-oxopentyl)-des-A-friedelane. 62 The other major a-ring modified friedelane triterpenoids which were produced photochemically are 2-oxo-3-oxa- friedelane ( 1282), 63 epoxyfriedelane ( 1283), 64,65 3β -hydroxy friedelane ( 1210) and 3α -hydroxy friedelane ( 1211), 66,67 and 4-epi friedelin ( 6) and 4-epi shionone ( 1284), 68,69 and some nitrogen containing friedelanes. 70 A one-step synthesis of putranjivic acid ( 1285) was also accomplished by following a photochemical oxidation of friedelin, in benzene under oxygen ( Scheme 4.10 and Figure 4.16). 71

ether and acetone O C6H6, O2 hv Irradiation with HO HOOC O high pressure Hg-lamp; N atm 2 1280 1285 25 i) LAH ii) Pb(OAc)4

Ag2O

HOOC OHC

1239 1281 Scheme 4.10 Photochemical reactions on friedelin ( 115 ).

262

O

O

O O

1282 1283 1284 Figure 4.16 2-Oxo-3-oxa-friedelane ( 1282), epoxyfriedelane (1283), and 4-epi shionone ( 1284).

IV.A.2.5 Rearrangement-based transformations The transformation of a-amyrine or b-amyrine ( 1286) into friedelin ( 115) was explained by a series of consecutive 1,2-rearrangements of methyl groups and hydrogens (Figure 4.17). Reduction of friedelin ( 115) with lithium aluminum hydride produces 3 b-hydroxy friedelane (1210). Treatment of 1210 with hydrogen chloride in phenol at 110 oC caused a remarkable multi-group rearrangement which afforded olean-l3(18)-ene ( 1287). Oleanene 1287 was again prepared from a- or b-amyrine by a set of consecutive reactions viz., oxidation, Wolff-Kishner reduction and acid catalysed isomerization (Scheme 4.11 )72

H+

H H H H

H H HO O H 1286 115

Figure 4.17 b-Amyrine ( 1286, arrows show the consecutive 1,2-rearrangements toward friedelin).

When friedel-3-ene ( 1262) in boiling acetic acid was treated with hydrogen chloride, glutin- 5(10)-ene ( 1289) was obtained. This on vigorous acid treatment yielded a mixture of olean- l3(18)-ene ( 1287) and 18a-olean-12-ene ( 1291). Glutin-5(10)-en-3-one ( 1290) on several hours of treatment with HCl in boiling glacial acetic acid produced a mixture of glutin-5(10)-ene (1289) and olean-12-ene, i.e.; b-amyrine ( 1286). Glutin-5(10)-ene ( 1289 ) after 145 hrs of

263 treatment with HCl in chloroform at room temperature yielded olean-12-ene ( 1286), though 1286 itself remained unaffected by the same condition. ( Scheme 4.12 ). 12,73,74

H H H H H H HCl, Phenol LiAlH4 H o O HO 110 C H 115 1210 1287

mCPBA

O

H 1288 H

Scheme 4.11 Friedelin ( 115 ) to olean-l3(18)-ene ( 1287 ).

H H H H H H H HCl, boil HCl, boil glacial glacia AcOH lAcO O H HCl HC glacial 1262 l 1290 boil 14 /C AcOH 1289 5 HC h, l RT 3 ,

H H H+ H H H

H H H 1291 1287 1286

Scheme 4.12 Friedelene-oleanene rearrangement.

264

The action of silver acetate on 4α -bromofriedelin ( 1292) was reported to yield a a mixture of rearranged friedel-en-ones 1293 and 1290 (Scheme 4.13 ).75

H H H H H H AgOAc + O O O Br 1292 1293 1290 Scheme 4.13 Molecular rearrangement of 4α -bromofriedelin ( 1292) by silver acetate.

Drake and Shrader reported the Beckmann rearrangement of friedelin oxime ( 1255)6 and the structure was established actually as 4-aza-A-homofriedelan-3-one ( 1294) by Stevenson in the year 1963. 76 He also isolated the same product by the treatment of p-tolunesulfonyl chloride on the oxime in pyridine solution. The same product was also isolated by applying the Schmidt reaction condition on friedelin ( 115) (Scheme 4.14 ). 76

H H H H Beckmann H H Schmidt Reaction Rearrangement O or O HN p-TsCl/ py HON 115 1294 1255 Scheme 4.14 4-Aza-A-homofriedelan-3-one ( 1294) from both friedelin oxime (1 255 ) and friedelin ( 115).

IV.A.2.6 Transformative reactions of bromo-friedelane triterpenoids Friedelin ( 115) yielded 4α -bromofriedelin ( 1292) on treatment with N-bromosuccinimide in 77 CCl 4, but produced 2α -bromofriedelin ( 1295) by either monobromination with bromine in 10 CHCl 3 in presernce of HBr or, base catalysed monobromination in acetic acid or by treatment with pyridinium bromide-dibromide in acetic acid. 78 Again, friedelin ( 115 ) when treated with 10 HBr in CHCl 3, 2α,4α -dibromo friedelin ( 1296) was produced which was transformed readily into the more stable 2 a,4 b-dibromo isomer ( 1297) in HBr-AcOH at 20 oC. 75,79 The 2 a,4 a-

265 dibromo derivative 1296 was also obtained from the 4 a-bromo compound 1292 by either the treatment of Br 2 in acetic acid in presence of HBr or with excess potassium acetate, and again from the enol benzoate derivative of friedelin ( 1298) by dibromination in pyridine- dichloromethane solution. 78 2a-Bromo derivative 1295 was also the other product obtained in 78 the last process. 4α -Bromofriedelin ( 1292) when treated with Br 2 in acetic acid yielded 2α - bromofriedelin ( 1295)78,79 which when treated again with the same reagent formed either 2α,4β -dibromo friedelin ( 1297) or 2,2-dibromo friedelin ( 1299), as was assumed by Djerassi et al., 80 though Shoppee and Johnston obtained the latter compound by the same process and also by dibromination of friedelin ( 115). 78 This 2,2-dibromo compound when refluxed with KOH in ethanol yielded the 2,3-diketo derivative ( 1267), 77 identical with that obtained from cerin ( 116) by chromium trioxide oxidation in acetic acid. 51 Again, this 2,2-dibromo compound upon treatment with 2,4,6-collidine at 180 oC resulted 2 a-bromo friedel-1-ene-3-one ( 1300). The same reagent under nitrogen atmosphere converted 2 a-bromo derivative into friedel-1-ene-3-one

(1233) and bromination of it followed by Al 2O3 treatment resulted 2-bromofriedel-1-ene-3-one (1300) via 1a,2 b-dibromo friedelin ( 1301). 78 All these transformations are illustrated in Scheme 4.15. Pradhan et al. also reported a group of bromo- derivatives ( 1299, 1302-1306 ) of friedelin, using NBS, and their associated rearrangements as shown below 81-83 (Scheme 4.16 ). Kane and Stevenson also reported that friedel-18-ene-3-one ( 1307) was obtained by debromination of 1308 and 1309, produced respectively from 2α -bromofriedelin ( 1295) and 4α - bromofriedelin ( 1292) by the action of NBS. And compound 1307 regenerated 1309 on treatment with NBS ( Scheme 4.17 ). 77

IV.A.2.7 Other transformations Amyrin ( 1286), 84 or a readily available tetracyclic triterpene 85 was the starting material for chemical synthesis of friedelin. A total synthesis of friedelin was accomplished successfully in 31 steps and at 0.3% yield by Ireland and Walba in 1976. 86

266

Br2/AcOH

Br H H H Route a or b or c NBS O O CCl4 Br O 1295 115 1292

2,4,6-Collidine monobromination o HBr Br2/AcOH, HBr 180 C, N2 Atm. Py-DCM Chloroform or excess KOAc or Route c Py-DCM Br O solution Br HBr-AcOH dibromination BzO O 1233 Br 20oC O 1298 1296 Br 1297 O

O 1267 KOH, EtOH Br Br Br Br Br /AcOH 2 + O O O Br 1299 1295 1297 Dibromination Br Bromination Br Br Al2O3

O O O

1301 1300 115 lidine 6-col 2,4, o 180 C 1299 Scheme 4.15 Synthetic routes of different bromo-derivatives of friedelin. [ Route a : monobromination with Br 2 in chloroform in presence of HBr, Route b : base catalysed monobromination in AcOH and Route c : pyridinium bromide-dibromide in acetic acid.]

267

H H H H H H H H H R2 Br O NBS Friedelin R1 + + 115 DMSO O O HO

R3 1305 1306 1302; R1=R3=Br, R2=H 1303; R2=R3=Br, R1=H 1304; R1=R3=H, R2=Br 1299; R1=R2=Br, R3=H Scheme 4.16 NBS on friedelin.

H H H H H H H Debromination NBS NBS O O O Br Br 1292 1309 1307

Debromination

H H H H H Br Br NBS

O O

1295 1308 Scheme 4.17 Molecular rearrangements of bromo-derivatives of friedelin.

Moiteiro et al. synthesized secofriedelane triterpenoids stereoselectively in high yields. The ability of the compounds to inhibit the growth in vitro of three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (nonsmallcell lung cancer), and SF-268 (CNS cancer) were evaluated and only compounds 11246, 1312 and 1313 were found to possess significant growth inhibitory effects, exhibiting GI 50 values that range from 24.6 to 32.8 mM and 10.9 to17.6 mM, respectively ( Scheme 4.18 ). 87

268

H H H H H A A TMIS, HMDS A + Me3SiO O 25oC, 48h Me3SiO 115 1310 1260 mCPBA, 2h

H H

H H H H IO A A ROOC 5 6 + O O HO HO 1215 1311 O 1312; R=H 1313; R=Me

H H H H H H HO KMnO4 HOOC A ((C H ) (C H ) N+)Cl- O 6 5 2 5 3 O 116 1246 Scheme 4.18 A-ring modifications along with seco-friedelanes.

Our laboratory also has reported the synthesis of five oxygenated friedelane triterpenoids, four of them being seco derivatives. Lactone 1282 was synthesized both from cerin ( 116) and friedelin ( 115); seco dioic acid 1314 and its dimethyl ester (1315) were synthesised using friedelin ( 115); and seco keto acid 1246 and its methyl ester were prepared using cerin ( 116). The 3D molecular docking of the derivatives in the central catalytic domain of topoisomerase II a (1bgw PDB for topoisomerase II a) was also performed which described the binding nature and type of interactions between the enzyme and the synthesized friedelane triterpenoids and the topoisomerase II a inhibitory activity was confirmed by in vitro experiments ( Scheme 4.19 ). 88

269

Dry CrO3, O glacial AcOH Pb(OAc)4 116 HOOC ice cold water O O ice cold water CHCl3, anh.Al2(SO4)3 CHCl3, anh.Al2(SO4)3 1246 1282

LTA, glacial AcOH, CHCl3, anh.Na2SO4

i) glacial AcOH, V2O5 CH2N2, dry ether, HNO3,cold H2O, glacial AcOH MeOOC CHCl3, anh.Na2SO4 HOOC anh.Na2SO4 115 HOOC MeOOC ii)H O 2 2 1315 1314 Scheme 4.19 Topoisomarase II a inhibitory seco-friedelanes.

It was observed that in most of the studies, A-ring of friedelin was modified, though there are reports of changes in ring-B or even in ring C, D or E. A number of A-ring modified friedelin derivatives were found to possess potent biocidal activities as were studied by a large number of research groups. Synthesis, bioactivity screening and structure-activity relationships of various natural and synthetic triterpenoids such as friedelin ( 115), 2α -trimethylsiloxyfriedelan-3-one (1318), 3-hydroxyfriedel-3-en-2-one ( 1306), friedelin-2,3-lactone ( 1226), friedelin-3-oxime (1255) and friedelin-3,4-lactam ( 1294) were studied. Insecticidal and phytotoxic potential of these compounds, their selective cytotoxic effects on insect and mammalian cells, and their antiparasitic effects were also described. Structurally modified A-ring derivatives such as 116, 2,3-secofriedelan-2-al-3-oic acid ( 1319), its acetylated derivative 1320, 3 β- and 3α - hydroxyfriedelane ( 1210 and 1211), 3α -hydroxyfriedel-2-one ( 1214), 4β-hydroxyfriedel-3-one (1311), 3,4-secofriedelan-4-oxo-3-oic-acid ( 1312), lactone 1226, and the oxime 1255 were found to be stronger insecticides than the parent compound. Methyl-3-nor-2,4-secofriedelan-4-oxo-2- oic acid ( 1246) and its acetylated derivative 1321 also showed insecticidal activity in contrast to their inactive parent compound 1306. The post ingestive effects and cytotoxicity of these compounds suggested a multifaceted insecticidal mode of action. These structural modifications did not result in better phytotoxic agents than the parent compounds except for lactam 1294 and yielded several moderately active antiparasite derivatives (seco acids 1319, 1312, 1321 and 4β - hydroxyfriedel-3-one 1311 ) with cytotoxic effects on mammalian cells (Scheme 4.20 ).89

270

H H

HON H O N H 1255 1294 HO H

1210 O O H 1226 O H 115 RO

1211; R=H H O H 1316; R=Ac 1233

Me3SiO Me SiO 1260 3 H 1310 RO

H H H RO RO 1216; R=H + 1317; R=Me O O O H HO HO 116; R=H 1215 OHC 1318;R=SiMe3 1311 ROOC H H HOOC 1319; R=H O 1320; R=CH3 O HO 1214 1312

H H H H O ROOC O HO 1246; R=H 1306 1321; R=Me

Scheme 4.20 Biologically active derivatives of friedelin.2

2 Details of the scheme, ref. 89.

271

Very recently, in 2014, Kang et al. revealed a novel process of direct amidation of sp 3 C-H bonds and they applied the technique also on friedelin ( 115) (Scheme 4.21 ).90

TsN3 (2 equiv) (IrCp*Cl2)2 (5 mol%) H H H H H H AgNTf (20 mol%) H H H MeONH2.HCl 2 NaOAc AgOAc (10 mol%) A MeO MeOH/ H2O 1,2-DCE MeO O N O N 75oC, 3h 60 C, 24h 115 80% H 1322 75% HN 1323 Ts Scheme 4.21 Direct amidation of unactivated 24-methyl on friedelin.

IV.A.2.8 Additional bioactivities and concluding remarks Apart from these triterpenoids many more biologically potent derivatives of friedelane triterpenoids are also reported. Some of them are reproduced here in short. A series of synthesized seco-friedelin triterpenoids were found to have moderate cytotoxicity and insecticidal activities. 91-93 Celastrol 117 (Figure 4.18 ) was found to have potent anti-inflammatory and neuroprotective effect and investigation was carried out for the treatment of Parkinson’s and Alzheimer’s diseases. Anticancer activity of this compound was also tested for different tumor cell lines, as well as in preclinical animal model. 94-102

COOH HOH2C

H H H H O OH

HO O

117 1324

Figure 4.18 Potent A-ring modified friedelane-based drugs: Celastrol ( 117) and celasdin B ( 1324).

Another biologically important friedelane triterpenoids is celasdin B ( 1324) (Figure 18 ). From the biological evaluation, compound 1324 was found to exhibit anti-HIV replication 103 activity in H9 lymphocyte cells with an EC 50 value of 0.8 mg/ml.

272

Friedelin ( 115) was found to be capable of binding the human receptors for endothelin A (ETA) and angiotensin 1 (AT1) 104 and it also showed significant protective activities under different conditions, against AA, CCl 4, CdCl 2 induced hepatotoxicity or in the case of cyclophosphamide, cardiotoxicity, in mouse or rat models. 105-110 Against Staphylococcus aureus , Netzahualcoyone ( 1325, Figure 4.19 ) exhibited stronger inhibitory activity (with a MIC value of 1.5 – 1.6 mg/ml) in comparison to some of the antibiotics used in clinical practice. 111

COOMe OH H O H O COOMe COOMe H H OH O H H H H OAc O OAc O O OAc OAc O OAc O OAc HO AcO OAc OAc AcO 1325 1326 118 Figure 4.19 Bioactive netzahualcoyone ( 1325 ), compounds 1326 and correolide ( 118).

Compound 1326 was found to possess potential analgesic and potassium channel-blocking activity. 112 Correolide ( 118) was found to block KV1.3 voltagegated potassium channel with an 113 IC 50 value of 86 nM, and the compound also inhibited human T-cell proliferation with an EC 50 value of 307 nM (Figure 4.19 ).113,114 Many more natural friedelane triterpenoids showed potent biocidal activities which also include a-glucosidase inhibitory, 115 anti-inflammatory, 116 anti-HIV, 117 anti-tumor-promoting 118 activities etc. In summary, studies have shown that the natural compound friedelin ( 115) and particularly, its derivatives have anti-cancer activity, analgesic and anti-inflammatory capability, anti- bacterial activity and can act as vascularizing agent. Some derivatives can potentially be used in phermaceuticals and functional foods for the treatment or prevention of cardiovascular and cerebrovascular diseases and tumors. Their use in cosmetics and as agro chemicals are also well pronounced. 119 And it is noteworthy to mention that the synthetic bioactive friedelane triterpenoids are mainly due to the various modifications on the A-ring of the PT, although nature has provided a number of bioactive analogues which have different functionalities distributed in all the rings.

273

IV.B Present work IV.B.1 Background and abstract of the work After the thorough review on the A-ring modified friedelane triterpenoids (as depicted above) and analyzing the highly valued potential scope of the chemical transformations associated with biological evaluation towards the practical utilizations, the author became interested in this particular field of the natural products chemistry, and hence undertook the present work as discussed below. This chapter constitutes the syntheses of a library of A-ring modified friedelane triterpenoids. The modifications also include the all new 2-homoderivatives. The syntheses of the novel 2-homofriedelanes are based on the transformative reactions of the designed triterpenoid 3- chloro-2-formylfriedel-2-ene ( 1328) which was isolated as the major product from the reaction of friedelin ( 115) with the novel Vilsmeyer-Haack reagent. Some new derivatives of the friedelane series were also prepared from cerin ( 116, a naturally occurring PT; structurally 2 a- hydroxy friedelin) as well as using one of the new derivative 1327, structurally 3-chlorofriedel-2- ene, isolated as a side product from the key reaction. Moreover, considering the beauty of 3- chloro-2-en-al moiety, associated with the A-ring of the triterpenoid, a number of heterocycle- linked- (bonded to C3) 2-homofriedelane triterpenoids were synthesized.

IV.B.2 Results and Discussion IV.B.2.1 Extraction and isolation of friedelin from Quercus suber bark 3 Kg of finely powdered cork ( Quercus suber ) was extracted with petroleum ether in a soxhlet apparatus for 72h. The crude yellowish solid obtained, after removal of the solvent by distillation, was dissolved in minimum volume of chloroform and chromatographed over silica gel column (200 g). Eluent of the column with 2% ethyl acetate in petroleum ether yielded pure white solid characterized as friedelin ( 115).

IV.B.2.2 Action of Vilsmeyer-Haack reagent on friedelin: Syntheses of 3-chlorofriedel-2- ene (1327), 3-chloro-2-formylfriedel-2-ene (1328), 3-hydroxy-2-formylfriedel-2-ene (1329) and 4a-hydroxy-3-chloro-2-formylfriedel-2-ene (1330). Vilsmeyer-Haack reaction is a well-known transformative protocol for 2-formylation of carbonyl compounds and it uses phosphorus oxychloride along with N,N -dimethylformamide mixture as

274 the reagent. Considering the wide scope of application of the novel reagent, we targetted friedelin as the substrate, to prepare the 2-formylated and thus 2-homo derivative. And this derivative was indeed envisioned to be used to achieve a number of all new 2-homofriedelane triterpenoids by employing simple transformative reactions. Thus first, when friedelin was treated with phosphorus oxychloride and N, N- dimethylformamide (please follow Section xyz for detailed reaction procedure), the four products isolated were characterized (please follow section fgh for detailed characterization) as 3-chlorofriedel-3-ene ( 1327 ), 3-chloro-2-formylfriedel-2-ene ( 1328), 3-hydroxy-2-formylfriedel- 2-ene ( 1329) and 1a-hydroxy-3-chloro-2-formylfriedel-2-ene ( 1330). (Scheme 4.22 )

H H H H H H H H H H DMF/ POCl O O O 3 + + + O CHCl3 Cl Cl HO Cl HO 115 1327 1328 1329 1330 Scheme 4.22 Action of Vilsmeyer-Haack reagent on friedelin.

Each of the products isolated (please follow section IV.C.10 for the detailed characterizations) from the above key reaction were then utilized to achieve a library of friedelane triterpenoids having different functional/ active group distributions. Product 1327, on further simple transformative protocols was found to result new A-ring modified friedelane triterpenoids. To add some more derivatives to this particular group of A-ring modified compounds, friedelin and cerin were again utilized. On the other hand, the products 1328, 1329 and 1330 which belong to the all new 2-homofriedelane series were also used for further transformative reactions. Moreover, considering the beauty of 3-chloro-2-en-al moiety, associated with the A-ring of the triterpenoid, towards the easy access to the nucleophilic substitution at C3 as well as to the formation of heterocycles fused with the A-ring, a number of interesting heterocycle-linked- (bonded to C3) as well as heterocycle-fused (using C2-C3) homofriedelane triterpenoids were synthesized. Thus, the synthesized compounds can be divided into four broad sections viz., A. A-ring modified friedelanes

275

B. 2-Homo friedelanes C. Heterocycle-linked homo friedelanes.

IV.B.2.2.1 A-ring modified friedelanes According to the previous reports of the action of Vilsmeyer-Haack reagent on the ketosteroids, besides the a-formylated major product, a chloro-ene derivative was also isolated. In our case, the substrate friedelin likewise could furnish two isomeric chloro-enes viz., 3-chlorofriedel-2-ene (1327a) and 3-chlorofriedel-3-ene ( 1327); where in practice only the latter was isolated which can be attributed to the stability of the more substituted alkene. Next, this new derivative was used to undergo some transformative reactions to result the unprecedented derivatives. Moreover, to enrich the library of A-ring modified compounds, friedelin and cerin were transformed into some new derivatives.

IV.B.2.2.1.a Extraction and isolation of cerin (116) from Quercus suber bark Cerin, a friedelane triterpenoid, structurally 2 a-hydroxy friedelin ( 116, Figure 1)1-2 is available from the same source where from friedelin was isolated. Ethyl acetate as eluent in the column chromatography of the cork extracts (please follow section IV.B.2.1 for detailed extraction and isolation method) produced pure white crystalline solid characterized as cerin (m.p. 252-256oC, after repeated recrystallization from CHCl 3).

IV.B.2.2.1.b Reaction of 3-chlorofriedel-3-ene (1327) with selenium dioxide: Besides other transformative scopes, selenium dioxide is a well known reagent for allylic hydroxylation (please follow section lkj, (Chapter II) for the brief review on the action of the reagent, especially on steroids). Very recently, Mugesh et al. have reported a selenium-mediated dehalogenation of halogenated nucleosides which implies to help understanding the metabolism of halogenated nucleosides in DNA and RNA. 120 When the reagent was employed on 3-chlorofriedel-3-ene (1327), the 23-methyl of the friedelane skeleton, an allylic one, was found to get oxidized into an aldehyde along with simultaneous dechlorination to furnish the product friedel-3-ene-23-al (1331). (Scheme 4.23 )

276

H H H H

SeO2, dioxane 100oC, 36h Cl H

1327 O 1331

Scheme 4.23 Synthesis of friedel-3-ene-23-al.

IV.B.2.2.1.c Reaction of 3-chlorofriedel-3-ene (1327) with m-CPBA: Oxidation of 3- chlorofriedel-3-ene ( 1327) with m-CPBA was found to result the a- epoxidation of the 3-ene functionality. Thus the compound produced was structurally 3-chloro-3a,4 a-epoxyfriedelane (1332, Scheme 4.24 ).

H H H H

mCPBA/ CHCl3 Cl Reflux, 4h Cl O 1327 1332

Scheme 4.24 Oxidation of 1327 with m-CPBA.

IV.B.2.2.1.d Reaction of 3-chlorofriedel-3-ene (1327) with N-bromosuccinimide: synthesis of 24-Nor friedel-1, 3, 5 (10), 6-tetraene (1333): NBS is a well reagent occasionally used for allylic bromination although we were able to result the A-ring aromatization of steroids by using the same. 121, 122 Here, we aimed to achieve the corresponding 2-bromo derivative by employing NBS on 3-chlorofriedel-3-ene ( 1327), but indeed, it was our surprise to isolate the interesting A- ring aromatized titled friedelane 1333 as the only product ( Scheme 25 ). There are actually a number of important biologically active aromatized friedelanes, or having their quininoid structure, available in nature. 123-128 Pristimerin (1334), celastrol ( 117), 6-oxopristimerol ( 1335) and demethylzeylasteral (1336) are some of the important compounds of this class (Figure 4.20 ).

277

CO2Me COOH CO2Me COOH

H H H H O O HO HO

HO HO HO HO O CHO O 117 1334 1335 1336 Figure 4.20 Some biologically active aromatized friedelane triterpenoids.

H H H H H

NBS/ CHCl3 Cl RT, 24h

1327 1333

Scheme 4.25 Synthesis of A-ring aromatized friedelane triterpenoid by NBS.

IV.B.2.2.1.e Synthesis of 3,4-seco friedelane-3,4-diol (1337): Friedelin was oxidized into the lactone 1226 with m-CPBA. The lactone was then treated with NaBH 4 to produce the 3,4- seco friedelane-3,4-diol (1337). (Scheme 4.26 )

H H H mCPBA H H NaBH4 CHCl3 THF HO O Reflux, 2h Reflux, 4h HO O O

115 1226 1337

Scheme 4.26 A two-step synthesis of 3,4-seco friedelane-3,4-diol from friedelin.

IV.B.2.2.1.f Synthesis of 3-epi pachysandiol A (1338): Pachysandiol A, structurally 2 a,3 b- dihydroxy friedelane, is a natural compound available in many plants. 129 The compound can easily be achieved by the NaBH 4 reduction of cerin ( 116). Here, we have employed the reduction

278 of cerin with sodium in alcohol to furnish actually a cis -diol, the 3-epimer of pachysandiol A, structurally 2 a,3 a-dihydroxy friedelane (1338, Scheme 4.27 ).

H H H H HO HO Na/ Isopropanol Reflux, 2h O HO 116 1338

Scheme 4.27 Synthesis of 3-epi pachysandiol A from cerin.

IV.B.2.2.1.g Synthesis of 3 b-amino -4a-hydroxyfriedelane from friedelin: The titled compound was synthesized in four steps starting from friedelin. Selective activation of the C-4 was actually the key reaction steps (step 1 and 2 here) which were achieved by following BF3- mediated oxidation followed by mCPBA oxidation. Friedelin was thus transformed first into the enol acetate 1339 which, on oxidation with mCPBA showed the migration of the acetoxy group to C4-a to result 1340. Oximination of the 4 a-acetoxyfriedel-3-one (to furnish 1341) followed by reduction with LiAlH 4 finally yielded the desired new compound 3 b- amino -4a- hydroxyfriedelane (1342, Scheme 4.28 ).

H H H H H

mCPBA BF3.OEt2/ OAc2 CH Cl , aq. NaHCO CHCl , RT, 24 h 2 2 3 O 3 AcO RT, 2h O AcO 115 1339 1340

NH2OH.HCl/ Py 100oC, 4h H H

LiAlH4 Dry THF, Reflux H2N HON HO HO 1342 1341

Scheme 4.28 A four-step synthesis of 3 b-amino-4a-hydroxyfriedelane from friedelin.

279

IV.B.2.2.2 2-Homo friedelanes A series of ‘all new’ 2 -homofriedelane triterpenoids were synthesized following two or more sequential steps starting from friedelin. The key reaction, as is mentioned earlier, was the 2- formylation of the triterpene by applying the novel Vilsmeyer-Haack reagent. As the scheme demonstrates, the reaction produced four homo- derivatives, 1328 being the major product (Scheme 22 ). Next, 402 was transformed into some more new derivatives to enrich the friedelane triterpenoid series.

IV.B.2.2.2.a Synthesis of 3-chlorofriedel-2-ene-2-carboxaldoxime (1343): The 2-formyl group of the 2-homo-derivative 1328 was transformed, by usual common procedure, with hydroxylamine hydrochloride into the corresponding oxime to furnish the titled compound (1343, Scheme 4.29).

H H H H OHC NH2OH.HCl, Py HON o Cl 100 C, 2h Cl

1328 1343

Scheme 4.29 Oximination of 1330. IV.B.2.2.2.b Reduction of 3-chloro-2-formylfriedel-2-ene (1328) into its 2-hydroxymethyl derivative 1344: The 2-formyl functionality of compound 1328 was reduced with NaBH 4 to result the allylic alcohol, 3-chloro-2-hydroxymethylfriedel-2-ene ( 1344, 92%). The 2-methanol friedelane derivative was again acetylated (with acetic anhydride) to yield 3-chloro-2- acetoxymethylfriedel-2-ene ( 1345) quantitatively ( Scheme 4.30).

O H H H H H OHC NaBH4 HO Ac O, Py O CHCl3/ MeOH 2 o Cl RT, 24h Cl 100 C, 2h Cl

1328 1344 1345

Scheme 4.30 Synthesis of 2-hydroxymethyl and its acetate derivative.

280

IV.B.2.2.2.c Synthesis of 3-chlorofriedel-2-ene-2-carboxamide (1346): This was synthesized from the 2-formyl derivative 1328 following two steps. First, the oxime of 1328 (1343, 98%) was obtained by usual procedure (with hydroxylamine hydrochloride), which after purification through recrystallisation, was allowed to reflux with anhydrous FeCl 3 in dry DMF ( Scheme 30 ). The titled compound 1346 was isolated at 58% yield from the oxime. Though we could expect the corresponding 2-nitrile derivative in the reaction condition, formation of the 2-carboxamide derivative which is actually the Beckmann rearranged product was probably formed via the 2- nitrile derivative in course of the work-up procedure. Thus, from friedelin, the titled compound was synthesized in three steps. (Scheme 4.31 )

O H H H H H OHC NH2OH.HCl, Py HON FeCl3, DMF H2N o Reflux, 5h Cl 100 C, 2h Cl Cl

1328 1343 1346 Scheme 4.31 Synthesis of 3-chlorofriedel-2-ene-2-carboxamide (1346 ).

IV.B.2.2.2.d Allylic hydroxylation of 2-formyl derivative 1328 with SeO 2: Compound 1328 possesses two allylic positions available for further functionalization (at C1 and C4), in what context selenium dioxide was employed to it for allylic hydroxylation. Among the two allylic positions available, only the C1 was found to be hydroxylated, leaving C4 completely. Thus the reaction of SeO 2 on compound 1328 furnished 4a-hydroxy-3-chloro-2-formylfriedel-2-ene (1330, Scheme 4.32), which was again a minor reaction product obtained from the key reaction of friedelin with the novel Vilsmeyer-Haack reagent. In this context, it may be concluded that the allylic C1 is much more reluctant towards hydroxylation, in comparison to the allylic C4.

281

H H H H OHC SeO2 OHC 1,4-Dioxane o Cl 100 C, 24h Cl HO 1328 1330

Scheme 4.32 Synthesis of 4a-hydroxy-3-chloro-2-formylfriedel-2-ene.

IV.B.2.2.2.e Transformation of 2-hydroxymethyl derivative 1344 with SeO 2: Like compound 402, 3-chloro-2-hydroxymethylfriedel-2-ene ( 1344 ) also possesses two allylic positions for further functionalization (at C1 and C4). The effort to transform 1344 into the allylic hydroxylated products by using selenium dioxide resulted only the C4-a-hydroxylated 1330 where the primary allylic alcohol functionality of the reactant got simultaneously oxidized into the aldehyde group ( Scheme 4.33).

H H H H SeO2 OHC HO 1,4-Dioxane o Cl 100 C, 24h Cl HO 1344 1330

Scheme 4.33 Synthesis of 4a-hydroxy-3-chloro-2-formylfriedel-2-ene.

IV.B.2.2.2.f Preparation of the oxime derivative of 1330: The 2-formyl group of compound 1330 was transformed into the corresponding oxime ( 1347) by usual common procedure, with hydroxylamine hydrochloride. Thus the synthesized compound was 3-chloro-4a-hydroxy-2-ene- 2-carboxaldoxime ( 1347, Scheme 4.34).

282

H H H H OHC NH2OH.HCl, Py HON o Cl 100 C, 2h Cl HO HO 1330 1347

Scheme 4.34 Oximination of 1328.

IV.B.2.2.2.g Synthesis of 3-chloro-4a-hydroxy-2-hydroxymethylfriedel-2-ene (1348): The aldehyde functionality of compound 1330 was reduced into the corresponding primary alcoholic group by using sodium borohydride. Thus, the titled derivative of the 2-homofriedelane series was obtained from 1330 at 72% yield ( Scheme 4.35).

H H H H OHC NaBH4 HO CHCl3/ MeOH Cl RT, 24h Cl HO HO 404 420

Scheme 4.35 Synthesis of 3-chloro-1a-hydroxy-2-hydroxymethylfriedel-2-ene (1348).

IV.B.2.2.3 Heterocycle-linked homo friedelanes The C3(sp 2)-Cl of the homofriedelane 1328 is susceptible to nucleophilic substitution reactions thanks due to the associative conjugated ene-formyl functionality. Thus, some suitable nitrogen heterocycles of biological relevance were used as the nucleophiles to achieve the resultants which are, indeed, the new group of heterocycle-linked 2-homo friedelane derivatives. The heterocycles used for the preparation of such interesting molecules are rather simple and common, where we have used imidazole, benzimidazole and 1,2,3-benzotriazole as the aromatic N-heterocycles, and morpholine, piperidine and piperazine as the aliphatic N-heterocycles. Of note, the attempted reactions under air produced poor yields (<10%) of the desired products

283 leaving compound 1329 as the major yields (>75%) whereas the yield distribution was found to be reversed (approx) when the reactions were carried out under nitrogen. The reaction schemes and the products are shown below (Scheme 4.36 and Scheme 4.37 ).

O H H H N-Heterocycle, K2CO3 H H H O dry DMF o Cl N2, 80 C, 3h N X 1328 1349; X= CH2 1350; X= O 1351; X= NH

Scheme 4.36 Syntheses of aliphatic N-heterocycle-linked 2-homofriedelane derivatives.

O H H H N-Heterocycle, K2CO3 H H H O dry DMF

o Y Cl N2, 80 C, 3h N X 1328 N 1352; X= CH2, Y= H2 1353; X= CH2, Y= Ph 1354; X= NH, Y= Ph

Scheme 4.37 Syntheses of aromatic N-heterocycle-linked 2-homofriedelane derivatives.

IV.C Experimental IV.C.1 General: Melting points were measured in open capillary methods and were uncorrected. 1H NMR and 13C NMR spectra were recorded on BruckerAvance 300MHz FT-NMR spectrometer using 5 mm BBO probe. CDCl 3 or DMSO-d6 were used as solvent and TMS as reference material. Data are presented as follows: Chemical shift -in ppm on the scale relative to δ TMS = 0; coupling constant- J/Hz. Infrared spectra were recorded either in Shimudzu FT-IR 8300 Spectrometer or in Perkin Elmer FT-IR Spectrum RX 1 Spectrometer as neat or thin films (KBr or Nujol) as indicated in the experimental procedures, and at room temperature. Frequencies are given in

284 wave numbers (cm-1). Mass spectra were recorded on a Qtof Micro YA263 high-resolution mass spectrometer. For column chromatography, silica gel G, 60-120 mesh was used with petroleum ether- ethyl acetate mixture as the eluent. For thin layer chromatography (TLC), freshly made silica gel plates (using silica gel for TLC and petroleum ether) were used and visualization was achieved by staining with iodine.

IV.C.2 General procedure for the Vilsmeier -Haack reaction of friedelin (115): A solution of friedelin ( 115, 1 g, 2.34 mmol) in dry chloroform (25 mL) was added dropwise to a cold and stirred solution of phosphorus oxychloride (5 mL) and dry dimethylformamide (5 mL). The mixture was allowed to attain room temperature and then refluxed under nitrogen for 6 h. It was then concentrated under reduced pressure and poured onto ice followed by extraction with chloroform (3 × 20 mL). The combined extracts were washed with brine (3 × 25 mL) and dried

(Na 2SO 4), and solvent was removed to give a yellowish solid. Purification by column chromatography yielded the four products- 3-chlorofriedel-3-ene ( 1327), 3-chloro-2- formylfriedel-2-ene ( 1328), 3-hydroxy-2-formylfriedel-2-ene ( 1329 ) and 1 a-hydroxy-3-chloro- 2-formylfriedel-2-ene ( 1330 ).

IV.C.3 General procedure for the reduction with NaBH 4:

Compound (10 mg) was dissolved in CH 2Cl 2-MeOH (1:1, 10 mL) and NaBH 4 (1.2 equivalent) was added. The solution was stirred for 4 hours at room temperature. Sodium hydroxide (1 M, 10 mL) was added and the reaction mixture was extracted with CHCl 3 and after usual work-up (washed, dried, solvent evaporated), silica gel column chromatography furnished the expected pure products.

IV.C.4 General procedure for the acetylation reactions 10 mg of the compound was dissolved in pyridine (1 mL) and acetic anhydride (0.5 mL) was added to it and allowed to heat at 100 oC for specific time (please follow the respective Schemes for the reaction times). The reaction mixture was cooled, poured into ice cold water (50 mL), filtered and washed with cold water repeatedly and the solid was vacuum dried. The residue was column chromatographed and further recrystalised from chloroform-methanol to obtain the corresponding pure acetyl derivative.

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IV.C.5 General procedure for the oximination reactions: 10 mg of the compound was dissolved in pyridine (1 mL) and hydroxylamine hydrochloride (1.5 eqv.) was added to it and allowed to heat at 100 oC for 4h. The reaction mixture was cooled, poured into ice cold water (50 mL), filtered and washed with water repeatedly and the solid was vacuum dried. The residue was recrystallised from chloroform-ethanol or ethanol to obtain the corresponding pure oxime derivative.

IV.C.6 Procedure for the synthesis of 3-chlorofriedel-2-ene-2-carboxamide 1346 from oxime 1343:

In a solution of oxime 1343 (20 mg, 0.04 mmol) in dry DMF (5 mL) was added anhydrous FeCl 3 (5 eqv) and the mixture was allowed to reflux for 5h. The reaction was cooled and water (20 mL) was added. It was then extracted with diethyl ether (3 × 15 mL) and the combined organic solvent was washed successively with water (2 × 25 mL) and brine solution (2 × 25 mL), dried

(Na 2SO 4), and solvent was removed at reduced pressure to give a yellowish solid. Finally, column chromatography furnished the 2-carboxamide friedelane derivative 1346.

IV.C.7 Oxidation of 3-chlorofriedel-2-ene (1327) with mCPBA: 3-Chlorofriedel-2-ene (40.0 mg, 0.09 mmol) was dissolved in chloroform (10 mL), and then m- CPBA (20 mg, 0.12 mmol) was added to the solution. The mixture was then allowed to reflux for 2h. The reaction was cooled, little more chloroform (10 mL) and water (15 mL) was poured and the organic layer was separated, washed with saturated solution of NaHCO 3 (2 x 15 mL), and with water (2 x 15 mL), and then concentrated (vaccuum) and dried (Na 2SO 4). Column chromatography followed by recrystallization yielded the desired pure compound 1332.

IV.C.8 Allylic hydroxylation by selenium dioxide: To a solution of 1328 (or, 1344, 0.1 mmol) in dioxane (10 mL) was added selenium dioxide (0.15 mmol), the mixture was heated at 100 0C for 24h. The reaction mixture was then cooled and the black selenium deposited was filtered off through Whatman 41. To the filtrate chloroform (50 mL) was poured and was washed successively with water and then with saturated brine solution, dried over Na 2SO 4 and concentrated in vacuo to give a reddish gummy residue. The compounds presented therein, were then separated by column chromatography.

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IV.C.9 General procedure for the syntheses of heterocycle-linked 2-homo friedelanes:

A mixture of compound 1328 (0.1 mmol), suitable heterocycle (0.25 mmol), and K 2CO 3 (0.5 mmol) in dry DMF (2 mL) was heated at 80 °C under N 2 for 3 h. After cooling to room temperature, the reaction mixture was poured onto ice-cold water (30 mL), and was extracted with chlorororm (3 × 15 mL) and the combined organic solvent was washed successively with water (2 × 25 mL) and brine solution (2 × 25 mL), dried (Na 2SO 4), and solvent was removed at reduced pressure to give a yellowish solid. Finally, column chromatography furnished the heterocycle-linked 2-homo friedelane derivatives.

IV.C.10 Characterization of the compounds IV.C.10.1 Friedelin (115): Eluent in column chromatography: 2% ethyl acetate in petroleum ether. White crystals, m. p. 262-263oC (Pet. ether- ethyl acetate), (lit. 130 262-263oC). 1H NMR

(300 MHz, CDCl 3): δ 0.73 (s, 3H, Me-24), 0.87 (s, 3H, Me-25), 0.89 (s, 3H, Me-23), 0.95(s, 3H, Me-30), 1.00 (s, 6H, Me-26 and Me-29), 1.05 (s, 3H, Me-27), 1.18 (s, 3H, Me-28), 1.91- 2.02 13 (m, 1H, H-1), 2.20-2.47 (m, 3H, H-2 and H-4). C NMR (75 MHz, CDCl 3): δ 6.80 (C -23), 14.61 (C-24), 17.91 (C-25), 18.18 (C-7), 18.64 (C-27), 20.22 (C-26), 22.24 (C-1), 28.12 (C-20), 29.93 (C-17), 30.45 (C-12), 31.74 (C-29), 32.04 (C-28), 32.35 (C-21), 32.69 (C-15), 34.99 (C-30), 35.28 (C-19), 35.55 (C-11), 35.94 (C-16), 37.37 (C-9), 38.23 (C-14), 39.20 (C-22), 39.63 (C-13), 41.21 (C-6), 41.49 (C-2), 42.11 (C-5), 42.70 (C-18), 53.03 (C-8), 58.15 (C-4), 59.38 (C-10), 213.37 (C-3). FTIR (nujol, cm -1): ν 1714, 1380, 1302, 1257, 1103, 1071, 1046, 1004, 795, 719.

IV.C.10.2 3-Chlorofriedel-3-ene (1327): Eluent in column chromatography: petroleum ether. o 1 Yield: 12%. White cube-like hard crystals, m.p. 260-262 C. m.f. C 30 H49 Cl . H NMR (300 MHz,

CDCl 3): δ 0.77 (s, 3H, Me-24), 0.87 (m, 3H, Me-25), 0.93 (m, 12H, Me-26, Me-27, Me-29, Me- 30), 1.10 (s, 3H, Me-28) 1.63 (d, 3H, J=2.1 Hz, Me-23), 1.80 (dd, 1H, J= 3 Hz, 12 Hz H-1), 13 2.23-2.36 (br m, 2H, H-2). C NMR (75 MHz, CDCl 3): δ 13.03 (C-24), 17.27 (C-23), 17.27, 17.54 (C-25), 17.80 (C-27), 19.04, 19.51(C-26), 27.18, 29.06, 29.55, 30.82, 31.13, 31.32 (C-29), 31.88 (C-28), 33.99, 34.26, 34.38, 34.49 (C-30), 35.07, 36.02, 37.34, 38.06, 38.27, 38.78, 39.66, 41.94, 51.72, 55.21, 125.83 (C-3), 138.20 (C-4). FTIR (neat, cm -1): ν 3409, 2926, 2847, 1444, 1380, 1072, 973.

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IV.C.10.3 3-Chloro-2-formylfriedel-2-ene (1328) : Eluent in column chromatography: 5% ethyl o 1 acetate in petroleum ether. Yield: 52%. m.f. C 31 H49 ClO, crystalline solid, m.p. 218 C. H NMR

(300 MHz, CDCl 3): δ 0.77 (s, 3H, Me-24), 0.93 (s, 3H, Me-25 ) 0.94 (s, 3H, Me-30), 0.99 (s, 6H, Me-26 and Me-29), 1.01 (s, 3H, Me-27), 1.14 (s, 3H, Me-28), 1.16 (d, 3H, J=2.4 Hz Me-23), 13 1.91-2.05 (m, 2H, H ax -1 and H-10) 2.39-2.45 (m, 2H, Heq and H-4). C NMR (75 MHz, CDCl 3): δ 11.77 (C-23), 14.43 (C-24), 17.39 (C-25), 18.31, 18.61 (C-27), 20.41 (C-26), 21.53, 28.18, 30.03, 30.27, 31.79 (C-29), 32.12 (C-28), 32.43, 32.80, 35.03 (C-30), 35.17, 35.31, 35.99, 36.79, 38.17, 38.17, 39.27, 39.66, 41.84, 42.75, 52.88, 53.21, 54.23, 133.08 (C-2), 155.10 (C-3), 192.18 (-CHO). FTIR (neat, cm-1): ν 2932, 2855, 1675, 1611, 1 394, 1232, 953.

IV.C.10.4 2-Formyl-3-hydroxy-friedel-2-ene (1329): Eluent in column chromatography: 7% o ethyl acetate in petroleum ether. Yield: 10%. m.f. C 31 H50 O2, powdered solid, m.p. 258-260 C. 1 H NMR (300 MHz, CDCl 3): δ 0.93 (s, 3H, Me-24), 0.94 (s, 3H, Me-25), 0.986 (s, 3H, Me-26), 0.994 (s, 3H, Me-29), 1.01 (s, 3H, Me-30), 1.14 (s, 3H, Me-27), 1.18 (s, 3H, Me-28), 1.79-1.85

(m, 1H, Hax -1), 1.99- 2.07 (m, 3H, Me-23), 2.37- 2.52 (m, 1H, H eq 1), 2.64 (dd, 1H, J= 3 Hz and 13 18 Hz, H-4), 9.97 (d, 1H, J= 4.8 Hz, -CHO). C NMR (75 MHz, CDCl 3): δ 16.52 (CH 3-23),

17.63 (CH3-24), 18.10 (CH 3-25), 18.51, 18.78 (CH 3-27), 20.10 (CH3-26), 28.14, 28.14, 30.01,

30.20, 31.79 (CH3-29), 32.11, 32.16, 32.74 (CH 3-28), 34.60, 34.64, 34.99 (CH 3-30), 35.31, 35.88, 36.85, 38.23, 38.42, 39.21, 39.70, 42.76, 42.76, 52.44, 54.59, 129.02 (C-2), 166.02 (C-3), 192.02 (-CHO). FTIR (KBr, cm -1): ν 3443, 2933, 2863, 1687, 1587, 1462, 1383, 1282, 1177, 972, 675, 628, 573. Analysis calcd: C, 81.88; H, 11.08. Found: C, 81.40, H, 10.95.

IV.C.10.5 3-Chloro-2-formyl-4a-hydroxy-friedel-2-ene (1330): Eluent in column chromatography: 10% ethyl acetate in petroleum ether. Yield: 7%. m.f. C 31 H49 ClO 2, powdered o 1 solid, m.p. 195 C. H NMR (300 MHz, CDCl 3): δ 0.90 (s, 3H, Me-25), 0.94 (s, 3H, Me-30), 0.97 (s, 3H, Me-24), 1.00 (s, 6H, Me-26 and Me-29), 1.01 (s, 3H, Me-27), 1.18 (s, 3H, Me-28), 1.40 (s, 3H, Me-23), 1.94- 2.14 (m, 3H, H-6 and H-10), 2.41 (dd, J= 4.2 Hz & 18.3 Hz, 2H, H-1), 13 10.24 (s, 1H, -CHO). C NMR (75 MHz, CDCl 3): δ 17.42 (CH 3-24), 17.89 (CH3-25), 17.98

(CH3-23), 18.70 (CH3-27), 20.43 (CH3-26), 20.66, 22.03, 28.16, 30.02, 30.26, 31.74 (CH 3-29),

32.09 (CH3-28), 32.39, 32.76, 33.59, 35.03 (CH 3-30), 35.32, 35.40, 35.96, 36.61, 38.13, 39.26,

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39.66, 42.60, 42.71, 46.43, 52.04, 78.4 (C-4), 133.7 (C-2), 153.2 (C-3) 192.8 (-CHO). FTIR (neat, cm -1): ν 3480, 2926, 2855, 1675, 1373, 1113, 945.

IV.C.10.6 Friedel-3-ene-23-al (1331): Eluent in column chromatography: petroleum ether. o Yield: 56%. m.f. C 30 H48 O, White small-needle-shaped crystals. m.p. 232 C. (petroleum ether- 1 ethyl acetate). H NMR (300 MHz, CDCl 3): δ 0.88 (s, 3H, Me -25), 0.95 (s, 3H, Me-30), 1.00 (s, 6H, Me-26 and Me-29), 1.01 (s, 3H, Me-27), 1.15 (s, 3H, Me-24), 1.18 (s, 3H, Me-28), 2.26-

2.52 (m, 3H, H-1 and H-10), 2.72 (td, J= 3Hz and 15 Hz, 1H, H eq -2), 6.55 (d, J=2.7 Hz, 1H, H- 13 3), 9.30 (s, 1H, -CHO). C NMR (75 MHz, CDCl 3): δ 16.73 (C -24), 17.96 (C-25), 18.33 (C-27), 18.62, 20.05 (C-26), 21.12, 28.19, 28.87, 30.07, 30.57, 31.83 (C-29), 32.14 (C-28), 32.27, 32.86, 35.03 (C-30), 35.39, 35.51, 36.07, 37.28, 37.54, 37.93, 38.37, 39.29, 39.77, 42.93, 53.09, 56.95, 151.84 (C-3), 151.98 (C-4), 194.23 (-CHO). FTIR (KBr, cm -1): ν 3449, 2929, 2862, 2713, 1680, + 1628, 1455, 1384, 1170, 1042, 991, 825, 690. ESI-MS: [C 30 H48 O + Na] requires 447.35; found 447.29. Analysis calcd: C, 84.84; H, 11.39. Found: C, 84.38, H, 11.07.

IV.C.10.7 3a,4 a-Epoxy friedelane (1332): Eluent in column chromatography: petroleum ether. 1 Isolated yield: 56%. White powdered solid. m.f. C 30 H50 O. H NMR (300 MHz, CDCl 3): δ 0.82

(s, 3H, CH 3-25), 0.934 (s, 3H, CH 3-24), 0.944 (s, 3H, CH 3-30), 0.99 (d, J= 1.8 Hz, 3H, CH 3-26),

1.00 (s, 3H, CH 3-29), 1.02 (s, 3H, CH 3-27), 1.17 (s, 3H, CH 3-28), 1.26 (s, 3H, CH 3-23), 2.19 (q, 13 J= 6.6 Hz, 1H, H ax -2), 2.89 (br s, 1H, H eq -2), 4.221 (t, J= 3 Hz, 1H, H-3), C NMR (75 MHz,

CDCl 3): d 9.52 (CH3-23), 15.23 (CH 3-24), 17.68 (CH3-25), 18.20 (CH 3-7), 18.64 (CH 3-27),

20.00 (CH3-26), 23.81 (C-1), 28.16 (C-20), 30.01 (C-17), 30.51 (C-12), 31.79 (CH 3-29), 32.13

CH 3-28), 32.29 (C-21), 32.83 (C-15), 34.99 (CH 3-30), 35.24 (C-19), 35.31 (C-11), 36.03 (C-16), 36.41 (C-9), 38.36 (C-14), 39.25 (C-22), 39.72 (C-13), 40.73 (C-6), 41.78 (C-2), 42.87 (C-18), 50.42 (C-5), 52.83 (C-10), 53.36 (C-8), 76.88 (C-3), 102.35 (C-4). FTIR (KBr, cm -1): ν 2919, 2850, 1466, 1376, 1309, 723, 655. Analysis calcd: C, 84.44; H, 11.81. Found: C, 84.66, H, 11.95.

IV.C.10.8 24-Nor friedel-1, 3, 5 (10), 6-tetraene (1333): Eluent in column chromatography: 1 petroleum ether. Isolated yield: 42%. White sticky gum. m.f. C 29 H42 . H NMR (300 MHz,

CDCl 3): δ 0.97 (s, 3H, CH 3-30), 1.02 (s, 6H, Me-26 and CH 3-29), 1.03 (s, 3H, CH 3-27), 1.21 (d,

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J= 2.4 Hz, CH 3-28), 1.25 (d, J= 4.2 Hz, CH 3-25), 2.25 (d, J= 3Hz, CH 3-23), 6.06 (dd, 1H, J= 2.7Hz and 9.9 Hz, H-7), 6.83 (dd, 1H, J= 3.0 Hz and 9.9 Hz, H-6), 6.92-7.17 (m, 3H, H-1, H-2 13 and H-3). C NMR (75 MHz, CDCl 3): d 15.01 (C-25), 18.81 (C-27), 20.45 (C-26), 21.80, 21.85, 21.22 (C-23), 30.29, 30.96, 31.49, 31.61 (C-29), 32.09 (C-28), 32.22, 33.02, 34.77, 35.37 (C-30), 35.73, 37.69, 38.00, 38.94, 39.36, 42.83, 47.93, 119.14, 124.95, 128.98, 130.95, 131.13, 131.84, 133.87, 147.12. Analysis calcd: C, 89.16; H, 10.84. Found: C, 88.97, H, 11.01.

IV.C.10.9 Friedelin-2,3-lactone (1226): Eluent in column chromatography: 5% ethyl acetate in o 89 o 1 petroleum ether. White crystalline solid, mp: 287-289 C (lit. 288-290 C), m.f. C 30 H50 O2. H

NMR (300 MHz, CDCl 3): d 0.83 (s, 3H, Me-25), 0.89 (s, 3H, Me-24), 0.95 (s, 3H, Me-26), 0.99 (s, 6H, Me-27 and Me-30), 1.00 (s, 3H, Me- 29), 1.17 (s, 3H, Me-28), 1.20 (d, 3H, J = 6.3 Hz,

Me-23), 1.94 (m, 1H, H-1ax ), 2.52 (td, 1H, J= 1.5, 13.0 and 13.0 Hz, H-2ax ), 2.63 (ddd, 1H, J = 13 1.5, 7.0 and 13.0 Hz, H-2eq ), 4.22 (q, 1H, J = 6.3 Hz, H-4). C NMR (75 MHz, CDCl 3): d 13.45 (C-23), 16.22 (C-24), 17.90 (C-25), 18.03 (C-7), 18.55 (C-1), 18.59 (C-26), 20.20 (C-27), 28.16 (C-20), 29.98 (C-17), 30.59 (C-12), 31.75 (C-29), 32.05 (C-28), 32.35 (C-15), 32.73 (C-21), 34.35 (C-2), 35.03 (C-30), 35.29 (C-11), 35.40 (C-16), 35.95 (C-19), 38.18 (C-9), 38.37 (C-14), 38.44 (C-6), 39.22 (C-22), 39.33 (C-13), 40.76 (C-5), 42.71 (C-18), 52.72 (C-8), 63.94 (C- 10), -1 84.91(C-4), 175.64 (C3). FTIR: nmax (KBr, cm ): 2945, 1734 (C=O), 1072 (CO), 752.

IV.C.10.10 3,4-Seco-friedelane-3,4-diol: (1337): Eluent in column chromatography: 35% ethyl o 1 acetate in petroleum ether. White powdered solid, m.p. 249 C m.f. C 30 H54 O2. H NMR (300

MHz, CDCl 3): δ 0.88 (s, 3H, Me -24), 0.93 (s, 3H, Me-25), 0.95 (s, 3H, Me-30), 0.98 (s, 3H, Me- 23), 1.00 (s, 6H, Me-26 and Me-29), 1.01 (s, 3H, Me-27), 1.18 (s, 3H, Me-28), 3.51-3.65 (m, 3H, 13 H-3 and H-4). C NMR (75 MHz, CDCl 3): δ 16.37 (C -23), 17.81 (C-24), 18.02, 18.65 (C-27), 18.79(C-25), 20.16 (C-26), 21.98, 28.16, 30.00, 30.23, 31.85 (C-29), 32.10 (C-28), 32.26, 32.82, 34.94 (C-30), 35.07, 35.29, 36.06, 38.30, 39.28, 39.56, 41.91, 42.81, 52.84, 58.53, 63.25 (C-3), 75.87 (C-4). FTIR (neat, cm -1): ν 3392, 2937, 2868, 1652, 1458, 1388, 1068. Analysis calcd: C, 80.65; H, 12.18. Found: C, 80.20, H, 12.01.

IV.C.10.11 3-Epi pachysandiol A (1338): Eluent in column chromatography: 50% ethyl acetate in petroleum ether. Yield: 62%. White needle-shaped crystals, m.p. 282- 283oC (ethyl acetate,

290

59 o 1 lit. 281.5- 283 C), m.f. C 30 H52 O2. H NMR (300 MHz, CDCl 3): δ 0.80 (d, J= 6.3 Hz, 3H,

CH 3-24), 0.90 (d, J= 6.6 Hz , 3H, CH 3-25), 0.95 (s, 1H, CH 3-30), 0.99 (s, 6H, CH 3-26 and CH 3-

29 ), 1.00 (s, 1H, CH 3- 2), 1.17 (s, 1H, CH 3- 28), 1.26 (s, 1H, CH 3-23), 3.18 (dd, J= 9 Hz and 10.5 Hz, C-3), 3.38- 3.47 (m, 1H, C-2). 13 C NMR (75 MHz, CDCl3): δ 9.71, 14.76, 17.77, 18.15, 18.60, 20.13, 27.91, 28.15, 29.34, 30.00, 30.55, 31.76, 32.09, 32.37, 32.81, 35.00, 35.35, 35.53, 36.04, 36.96, 38.26, 38.32, 39.26, 39.70, 41.17, 42.83, 49.78, 53.12, 57.41, 77.19, 77.50. -1 FTIR (KBr, cm ): ν 3416, 2934, 2866, 1702, 1635, 1461, 1386, 1034, 592. ESI-MS: [C 30 H48 O + Na + + H +] requires 468.39; found 468.55. Analysis calcd: C, 81.02; H, 11.79. Found: C, 81.40, H, 11.92.

IV.C.10.12 Friedel-3-enol-acetate (1339): Eluent in column chromatography: 2% ethyl acetate o 1 in petroleum ether. Yield: 8-60%. m.f. C 32 H52 O2 white crystals, m.p. 262 C (CHCl 3-MeOH) . H

NMR (300 MHz, CDCl 3): δ 0.85 (s, 3H, Me-25), 0.95 (s, 3H, Me-30), 1.00 (s, 6H, Me-26 and Me-29), 1.01 (s, 3H, Me-27), 1.02 (s, 3H, Me-24), 1.18 (s, 3H, 28-Me), 1.59 (s, 3H, Me-23), 13 2.12 (s, 3H, -CH 3 of -OAc). C NMR (75 MHz, CDCl 3): δ 9.55, 17.46, 18.16, 18.26, 18.67, 20.10, 20.69, 20.88, 28.20, 28.39, 30.07, 30.61, 31.83, 32.16, 32.39, 32.90, 35.04, 35.25, 35.42, 36.11, 37.08, 38.22, 38.39, 38.66, 39.31, 39.84, 42.95, 52.75, 56.10, 130.57 (C-4), 141.31 (C-3), 168.98 (>C=O of -OAc). FTIR (nujol, cm -1): ν 667, 759, 1070, 1222, 1382, 1456, 1749 (>C=O of -OAc), 2337. Mass: 491.32 (M ++ Na+), (33%), 489.31 (100%), 413.22 (13%), 301.13 (14%), 149.01 (14%), 100.10 (12%). Analysis calcd: C, 81.99; H, 11. 18. Found: C, 81.56; H, 10.89.

IV.C.10.13 4 a-Acetoxyfriedel-3-one (1340) : Eluent in column chromatography: 2% ethyl o acetate in petroleum ether. Yield: 12-41%. m.f. C 32 H52 O3 white crystals, m.p. 174-176 C 1 (CHCl 3-MeOH). H NMR (300 MHz, CDCl 3): δ 0.80 (s, 3H, Me-25), 0.86 (s, 3H, Me-24), 0.96 (s, 3H, Me-30), 1.00 (s, 6H, Me-26 and Me-29), 1.07 (s, 3H, Me-27), 1.18 (s, 3H, Me-28), 1.30 13 (s, 3H, Me-23), 1.93- 2.02 (m, 1H, H-1), 2.26-2.52 (m, 2H, H-2), 2.14 (-CH 3 of -OAc). C NMR

(75 MHz, CDCl 3): δ 12.64, 15.68, 17.95, 18.13, 18.73, 20.19, 21.50, 22.98, 28.20, 30.03, 30.55, 31.84, 32.12, 32.38, 32.85, 34.29, 35.00, 35.34, 35.95, 36.05, 37.29, 38.03, 38.32, 39.28, 39.70, - 42.88, 46.05, 50.34, 52.47, 89.09 (C-4), 170.11 (-CH 3 of -OAc), 208.30 (C-3). FTIR (nujol, cm 1 + + ): ν 722, 1119, 1245, 1377, 1508, 1736, 2345. ESI-MS: [C 30 H48 O + Na + H ] requires 508.38; found 508.54. Analysis calcd: C, 79.29; H, 10.81. Found: C, 79.52; H, 10.63.

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IV.C.10.14 4 a-Hydroxy friedelane-3-oxime (1341) : Yield: 88%. m.f. C 30 H51 NO 2, pale yellow solid. 1H NMR (300 MHz, DMSO-d6): δ 0.73 (s, 3H, Me-24), 0.81 (s, 3H, Me-25), 0.96 (s, 3H, Me-30), 0.99 (s, 6H, Me-26 and Me-29), 1.12 (s, 3H, Me-27), 1.17 (s, 3H, Me-28), 1.26 (s, 3H, Me-23), 1.70-2.01 (m, 3H, H-2 and H-10), 4.39 (br s, 1H, -OH), 10.38 (br s, 1H, -NOH). 13 C NMR (75 MHz, DMSO-d6): δ 17.16, 18.12, 18.67, 18.93, 19.85, 20.36, 28.32, 30.06, 30.59, 32.16, 32.35, 32.99, 33.75, 35.24, 35.80, 36.11, 36.97, 38.25, 39.50, 39.77, 40.05, 40.33, 40.61, 40.89, 42.90, 43.47, 49.44, 52.37, 76.62 (C-4), 161.14 (C-3). FTIR (nujol, cm -1): ν 34 42, 3301, 2723, 1378, 1302, 1178, 1045, 992, 944, 919, 769, 725, 572.

IV.C.10.15 3b- Amino -4a-hydroxyfriedelane (1342): Recrystallized from chloroform-ethanol 1 to result pale yellow, small-flower-like crystals. m.f. C 30 H53 NO. H NMR (300 MHz, CDCl 3): δ 0.84 (s, 3H, Me-24), 0.88 (s, 3H, Me-25), 0.93 (s, 3H, Me-30), 0.95 (s, 6H, Me-29), 1.10 (s, 3H, Me-27), 1.14 (s, 3H, Me-26), 1.18 (s, 3H, Me-28), 2.08 (m, 3H, Me-23), 2.17- 2.28 (m, 1H, H- 13 10), 2.47- 2.56 (m, 1H, H-2), 3.61- 3.83 (br hump, -OH), 5.01- 5.29 (br hump, -NH 2). C NMR

(75 MHz, CDCl 3): δ 16.44 (CH 3-24), 16.81 (CH 3-25), 16.94 (CH3-23), 17.66 (CH 3-27), 19.15

(CH3-26), 23.78, 24.78, 27.14, 28.68, 28.96, 29.02, 30.80, 31.11, 31.32, 31.79 (CH3-29), 33.94

(CH3-28), 34.29, 34.36, 34.93 (CH3-30), 37.27, 37.33, 38.23, 38.62, 41.81, 51.36, 52.26, 52.94, 54.8 (C-3), 76.20 (C-4). Analysis calcd: C, 81.20; H, 12.04, N, 3.16 Found: C, 81.40, H, 11.95, N, 3.19.

IV.C.10.16 3-Chlorofriedel-2-ene-2-carboxaldoxime (1343): Purified by recrystallization with chloroform- methanol mixture to obtain off-white needle-shaped crystals, m.p. 164 oC, m.f. 1 C31 H50 ClNO. H NMR (300 MHz, CDCl 3): δ 0.78 (s, 3H, Me-24), 0.93 (s, 3H, Me-25), 0.94 (s, 3H, Me-30), 0.99 (s, 6H, Me-26 and Me-29), 1.01 (s, 3H, Me-27), 1.09 (d, J= 7.2 Hz, 3H, Me- 23), 1.17 (s, 3H, Me-28), 1.92 (d, J= 13.2 Hz, 1H, H-1), 2.13 (dt, J= 3.3 Hz & 12.9 Hz, 1H, H-4), 13 2.31-2.45 (m, 2H, H-10), 8.40 (s, 1H, -CH=NOH). C NMR (75 MHz, CDCl 3): δ 12.47 (CH 3-

23), 14.28 (CH3-24), 17.39 (CH3-25), 18.33, 18.63 (CH3-27), 20.43 (CH 3-26), 23.09, 28.17,

30.02, 30.35, 31.77 (CH 3-29), 32.10, 32.41, 32.77 (CH 3-28), 35.06 (CH 3-30), 35.15, 35.35, 35.97, 36.73, 38.04, 38.18, 39.26, 39.65, 41.81, 42.74, 52.32, 52.87, 54.32, 126.29 (C-2), 141.69 (C-3), 150.28 (-C=NOH). FTIR (KBr, cm -1): ν 3353, 2931, 2862, 1623, 1458, 1386, 1294, 1182,

292

+ 1135, 985, 958, 749, 549. ESI-MS: [C 30 H48 O + Na ] requires 510.34; found 510.56. Analysis calcd: C, 76.27; H, 10.32, N, 2.87. Found: C, 76.67, H, 10.67, N, 2.77.

IV.C.10.17 3-Chloro-2-hydroxymethyl-friedel-2-ene (1344): Eluent in column o chromatography: 15% ethyl acetate in petroleum ether. White crystals, m.p. 244-246 C (CHCl 3- 1 methanol), m.f. C 31 H51 ClO. H NMR (300 MHz, CDCl 3): δ 0.77 (s, 3H, Me-24), 0.79 (s, 3H, Me- 25), 0.93 (s, 3H, Me-23), 0.94 (s, 3H, Me-30), 0.99 (s, 3H, Me-27), 1.01 (d, J=2.1 Hz, 6H, Me- 26 & 29), 1.17 (d, J= 3 Hz, 3H, Me-28), 1.90 (dd, J= 3 Hz and 10.5 Hz, 1H, H-1), 2.07-2.27 (m, 13 3H, H-1, H-4 and H-10), 4.25 (dd, J= 12 Hz and 26.7 Hz, 2H, -CH2OH). C NMR (75 MHz,

CDCl 3): δ 12.43 (CH 3-23), 14.09 (CH3-24), 17.49 (CH3-25), 18.37, 18.64 (CH 3-27), 20.39 (CH3-

26), 26.05, 28.19, 30.04, 30.35, 31.80 (CH 3-29), 32.12 (CH3-28), 32.42, 32.82, 35.04 (CH 3-30), 35.33, 36.01, 36.69, 37.97, 38.20, 39.28, 39.68, 41.87, 42.77, 51.31, 52.87, 54.96, 64.07 (- -1 CH 2OH), 132.14 (C-2), 133.10 (C-3). FTIR (KBr, cm ): ν 3384, 2938, 2864, 1662, 1461, 1385, + + 1183, 1133, 1045, 1007, 948, 755, 696. ESIHRMS: [C 30 H48 O + Na + 2H ] requires 499.36; found 499.56. Analysis calcd: C, 78.35; H, 10.82. Found: C, 78.87, H, 10.96.

IV.C.10.18 2-Acetoxymethyl-3-chloro-friedel-2-ene (1345): Eluent in column chromatography: 2% ethyl acetate in petroleum ether. White crystals, m.p. 180-182oC (Pet. 1 ether- ethyl acetate), m.f. C 33 H53 ClO 2. H NMR (300 MHz, CDCl 3): δ 0.77 (s, 3H, Me-24), 0.92 (s, 3H, Me-25), 0.94 (s, 3H, Me-23), 1.00 (s, 3H, Me-30), 1.01 (s, 6H, Me-26 and Me-29), 1.06

(s, 3H, Me-27), 1.17 (s, 3H, Me-28), 1.90 (d, J= 13.2 Hz, 1H, H-1), 2.09 (s, 3H, -CH 3 of -OAc), 13 4.75 (s, 2H, -CH2OH ). C NMR (75 MHz, CDCl 3): δ 12.38 (CH 3-23), 14.09 (CH3-24), 17.45

(CH3-25), 18.33, 18.58 (CH3-27), 20.33 (CH 3-26), 20.87, 25.67, 28.15, 30.03, 30.26, 31.82

(CH3-29), 32.10 (CH3-28), 32.38, 32.83, 34.96 (CH 3-30), 35.20, 35.32, 35.99, 36.66, 37.89,

38.20, 39.23, 39.67, 41.83, 42.82, 51.39, 52.83, 54.75, 65.30 (-CH2OAc), 127.69 (C-2), 134.98 -1 (C-3), 171.02 (CH 3 of -OAc). FTIR (KBr, cm ): ν 3444, 2933, 2866, 1745, 1459, 1383, 1225, + + 1026, 954, 910, 759. ESIHRMS: [C 30 H48 O + Na + 2H ] requires 541.37; found 541.58. Analysis calcd: C, 76.63; H, 10.33, O, 6.19. Found: C, 76.13; H, 10.75, O, 6.05.

IV.C.10.19 3-Chlorofriedel-2-ene-2-carboxamide (1346): Eluent in column chromatography: 10% ethyl acetate in petroleum ether. Pale yellow crystals, m.p. 233-236 oC (Pet. ether- ethyl

293

1 acetate), m. f. C 31 H50 ClNO. H NMR (300 MHz, CDCl 3): δ 0.94 (s, 3H, Me-24), 0.96 (s, 3H, Me-25), 0.97 (s, 3H, Me-23), 0.99 (s, 3H, Me-30), 1.005 (s, 3H, Me-27), 1.014 (s, 6H, Me-26 and Me-29), 1.17 (s, 3H, Me-28), 1.99 (d, J= 12.3 Hz, 1H, H-1), 5.08 (s, 1H, -CONH 2), 5.48 (s, 13 1H, -CONH 2). C NMR (75 MHz, CDCl 3): δ 16.7 (CH 3-24), 17.49 (CH3-25), 17.53 (CH3-23),

19.35 (CH3-27), 21.57 (CH3-26), 26.58, 27.16, 28.69, 29.03, 29.25, 30.84, 31.12, 31.37, 31.87

(CH3-29), 33.95 (CH3-28), 34.04, 34.29, 35.02 (CH 3-30), 36.16, 37.24, 38.24, 38.65, 38.65,

39.38, 41.83, 44.05, 51.82, 52.20, 132.02 (C-2), 153.58 (C-3), 161.70 (-CONH 2). Analysis calcd: C, 76.27; H, 10.32; N, 2.87; O, 3.28. Found: C, 75.87; H, 10.76; N, 2.90, O, 3.35.

IV.C.10.20 3-Chloro-4a-hydroxy-2-ene-2-carboxaldoxime (1347): Purified by recrystallization with chloroform- methanol mixture to obtain pale yellow crystals, m.p. 240oC. 1 m.f. C 31 H50 ClNO 2. H NMR (300 MHz, CDCl 3): δ 0.92 (s, 3H, Me-25), 0.94 (s, 3H, Me-30), 0.98 (s, 3H, Me-24), 1.00 (s, 3H, Me-27), 1.01 (s, 6H, Me-26 and Me-29), 1.18 (s, 3H, Me-28), 1.35 (s, 3H, Me-23), 1.95- 2.19 (m, 4H, H-6 and H-10), 2.46 (dd, J= 4.5 Hz and 17.7 Hz, 2H, H- 13 1), 8.37 (s, 1H, -CH=NOH). C NMR (75 MHz, CDCl 3): δ 17.43 (CH 3-24), 17.89 (CH3-25),

18.04 (CH3-23), 18.72 (CH 3-27), 20.43 (CH3-26), 21.30, 23.50, 28.17, 30.03, 30.30, 31.75 (CH 3-

29), 32.09 (CH3-28), 32.37, 32.75, 33.59, 35.03 (CH 3-30), 35.32, 35.47, 35.96, 36.59, 38.15, 39.25, 39.67, 42.56, 42.72, 46.56, 52.05, 77.97 (C-4), 128.70 (C-2), 141.20 (C-3), 150.15 (C=NOH). FTIR (KBr, cm -1): ν 3422, 2939, 2868, 1627, 1459, 1385, 1295, 1181, 1073, 992, + 972, 751, 696. ESI-MS: [C 30 H48 O + Na] requires 526.34; found 526.55. Analysis calcd: C, 73.85; H, 10.00; N, 2.78; O, 6.35. Found: C, 73.87; H, 10.76; N, 2.90, O, 6.00.

IV.C.10.21 3-Chloro-4a-hydroxy-2-hydroxymethylfriedel-2-ene (1348): Eluent in column chromatography: 25% ethyl acetate in petroleum ether. Pale yellow crystals, m.p. 160oC, m.f. 1 C31 H51 ClO 2. H NMR (300 MHz, CDCl 3): δ 0.87 (s, 3H, Me -25), 0.91 (s, 3H, Me-24), 0.95 (s, 3H, Me-30), 1.00 (s, 9H, Me-26, Me-27 and Me-29), 1.15 (s, 3H, Me-28), 1.26 (s, 3H, Me-23), 13 1.75 (s, 3H, Me-23), 1.87- 1.91 (m, 3H, H-1 and H-10), 4.19 (s, 1H, -CH2OH). C NMR (75

MHz, CDCl 3): δ 14.1 5 (CH 3-24), 18.14 (CH 3-25), 18.62 (CH 3-27), 19.04 (C-23), 20.05 (CH 3-

26), 27.13, 28.16, 29.71, 30.02, 30.45, 31.81 (CH 3-29), 32.13 (CH 3-28), 32.26, 32.78, 35.01

(CH3-30), 35.16, 35.30, 35.98, 36.48, 38.30, 38.47, 39.24, 39.77, 41.55, 42.83, 50.43, 52.59,

70.27 (-CH 2OH), 77.21 (C-4), 128.95 (C-2), 144.11 (C-3).

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IV.C.10.22 2-Formyl-3-(1 H-piperidin-1-yl)-friedel-2-ene (1349): Eluent in column 1 chromatography: 10% ethyl acetate in petroleum ether. Pale yellow solid. m.f. C 36 H59 NO. H

NMR (300 MHz, CDCl 3): δ 0.95 (s, 6H, Me-24 and CH 3-30), 1.01 (s, 6H, CH 3-26 and CH 3-29),

1.03 (s, 3H, Me-25), 1.16 (s, 3H, Me-27), 1.18 (s, 3H, Me-28), 1.27 (d, J= 3.3 Hz, CH 3-23), 1.50- 1.55 (m, 6H, H-3', H-4' and H-6'), 3.25- 3.80 (m, 4H, H-2' and H-6'). 13C NMR (75 MHz,

CDCl 3): δ 14.06 (CH3-24), 17.64 (CH3-25), 18.26 (CH3-27), 18.51, 18.72 (CH 3-23), 20.12 (CH3-

26), 22.68, 24.50, 26.45, 28.17, 29.71, 30.04, 30.25 31.79 (CH 3-29), 32.14 (CH 3-28), 32.32,

32.81, 34.66, 35.00 (CH3-30), 35.36, 35.96, 35.96, 38.12, 38.30, 38.44, 39.25, 39.74, 42.84, 46.51, 46.86, 52.53, 53.18, 54.66, 142.95, 165.84, 199.80. Analysis calcd: C, 82.85; H, 11.40; N, 2.68. Found: C, 82.10; H, 10.99; N, 2.26.

IV.C.10.23 2-Formyl-3-(1 H-morpholin-4-yl)-friedel-2-ene (1350): Eluent in column chromatography: 10% ethyl acetate in petroleum ether. Pale yellow solid. m.p. 170oC, m.f. 1 C35 H57 NO 2. H NMR (300 MHz, CDCl 3): δ 0.91 (s, 3H, CH 3-24), 0.94 (s, 3H, CH 3-30), 1.00 (s,

6H, CH 3-26 and CH 3-29), 1.01 (s, 6H, CH 3-25 and CH 3-27), 1.07 (s, 3H, CH 3-23), 1.18 (s, 3H,

CH 3-28), 1.91-2.06 (m, 4H, H-1, H-2), 2.33- 2.37 (t, J= 6 Hz, 1H, H-4), 2.96 (br s, 4H, H-3' and 13 H-5'), 3.71 (br s, 4H, H-2' and H-6'). C NMR (75 MHz, CDCl 3): δ 13.13 (CH 3-24), 17.60

(CH3-25), 18.14, 18.54 (CH 3-27), 19.04 (CH 3-23), 20.13 (CH 3-26), 28.14, 29.68, 30.02, 30.24,

31.76 (CH3-29), 32.11 (CH3-28), 32.23, 32.78, 34.60, 35.00 (CH 3-30), 35.34, 35.64, 35.95, 36.90, 38.22, 38.63, 39.24, 39.80, 40.50, 42.81, 50.34 (2), 52.54, 54.80, 66.78 (2), 141.22 (C-2), 166.66 (C-3), 198.38 (-CHO). Analysis calcd: C, 80.25; H, 10.97; N, 2.67. Found: C, 80.46; H, 11.22; N, 2.22.

IV.C.10.24 2-Formyl-3-(1 H-piperazin-1-yl)-friedel-2-ene (1351): Eluent in column chromatography: 20% ethyl acetate in petroleum ether. White powdered solid. m.f. C 35 H58 N2O. 1 H NMR (300 MHz, CDCl 3): δ 0.87 (s, 3H, CH 3-24), 0.92 (s, 6H, CH 3-26 and CH 3-29), 0.93 (s,

3H, CH 3-30), 1.01 (s, 3H, Me-25), 1.11 (s, 3H, CH 3-27), 1.19 (s, 6H, CH 3-23, CH 3-28), 3.25- 3.70 (m, 8H, H-2', H-3', H-5' and H-6').

IV.C.10.25 2-Formyl-3-(1 H-imidazol-1-yl)-friedel-2-ene (1352): Eluent in column chromatography: 50% ethyl acetate in petroleum ether. Pale yellow crystal. m.p. 210oC

295

1 (decomp.), m.f. C 34 H52 N2O. H NMR (300 MHz, CDCl 3): δ 0.95 (s, 3H, CH 3-30), 0.99 (s, 3H,

CH 3-26), 1.00 (s, 3H, CH3-29), 1.03 (s, 3H, CH3-27), 1.04 (s, 3H, CH 3-25), 1.19 (s, 3H, CH 3-28),

1.26 (s, 3H, CH 3-24), 1.74 (s, 3H, CH 3-23), 1.91- 2.03 (m, 1H, H-10), 2.48- 2.85 (m, 3H, H-2 and H-4 ), 6.83 (br s, 1H, H-4' ), 7.27 (m, associated with the CHCl 3 peak (from CDCl 3), 1H, H- 13 5'), 7.72 (br s, 1H, H-2'). C NMR (75 MHz, CDCl 3): δ 14.16 (CH 3-24), 17.69 (C-25), 18.00,

18.56 (C-27), 19.05 (C-23), 20.15 (CH3-26), 28.15, 29.68, 30.03, 30.19, 31.75 (CH3-29), 32.11

(CH3-28), 32.23, 32.75, 34.04, 34.65, 34.98 (CH 3-30), 35.36, 35.89, 37.08, 38.03, 38.29, 39.23, 39.76, 41.42, 42.81, 52.49, 54.66, 121.20, 130.49, 130.53, 130.57, 168.52, 193.35. Analysis calcd: C, 80.90; H, 10.38; N, 5.55. Found: C, 81.16; H, 10.29; N, 5.71.

IV.C.10.26 2-Formyl-3-(1 H-benzimidazol-1-yl)-friedel-2-ene (1353): Eluent in column chromatography: 25% ethyl acetate in petroleum ether. Pale yellow crystals. m.p. 220oC, m.f. 1 C38 H54 N2O. H NMR (300 MHz, CDCl 3): δ 0.96 (s, 3H, CH 3-30), 1.01 (s, 3H, CH 3-29), 1.03 (s,

3H, CH 3-26), 1.06 (s, 3H, CH 3-27), 1.20 (s, 3H, CH 3-28), 1.26 (d, J= 3.3 Hz, 3H, CH 3-25), 1.38

(s, 3H, CH 3-24), 1.65 (s, 3H, CH 3-23). 7.20- 7.91 (m, 4H, H-4', H-5', H-6' and H-7'), 8.13 (d, J= 13 5.1 Hz, 1H, H-2'). C NMR (75 MHz, CDCl 3): δ 14.39 (CH 3- 24), 17.78 (CH 3- 25), 18.09, 18.59

(CH3- 27), 19.47 (CH3- 23), 20.19 (CH 3- 26), 28.20, 29.70, 30.09, 30.25, 31.81 (CH 3- 29), 32.16

(CH3- 28), 32.30, 32.83, 34.35, 34.74, 35.01 (CH3- 30), 35.40, 35.95, 37.23, 37.98, 38.36, 39.27, 39.81, 41.77, 42.89, 52.55, 54.86, 109.86, 120.08 (2), 124.14, 127.96 (2), 134.50 (C-2), 145.24, 171.95 (C-3), 192.97 (-CHO). Analysis calcd: C, 82.26; H, 9.81; N, 5.05. Found: C, 82.11; H, 9.72; N, 5.13.

IV.C.10.27 2-Formyl-3-(1 H-1, 2, 3-Benzotriazol-1-yl)-friedel-2-ene (1354): Eluent in column chromatography: 50% ethyl acetate in petroleum ether. Pale yellow crystalline solid. m.p. 237oC 1 (decomp.), m.f. C 37 H53 N3O. H NMR (300 MHz, CDCl 3): δ 0.76 (d, J= 4.5 Hz, 3H, CH 3-25),

0.93 (s, 3H, CH 3-24), 0.95 (s, 3H, CH 3-30), 1.00 (s, 3H, Me-26), 1.01 (d, J= 1.5 Hz, CH 3- 29),

1.03 (s, 3H, CH 3-27), 1.18 (d, 3H, J= 2.7 Hz CH 3-28), 1.95 (s, 3H, CH 3-23), 6.84 (t, J= 7.5 Hz, 1H), 7.02 (d, J= 7.8 Hz, 1H), 7.22- 7.28 (m, 1H), 7.98 (s, 1H), 10.22 (-CHO). 13C NMR (75

MHz, CDCl 3): δ 14.39 (CH 3-24), 17.56 (CH3-25), 18.24, 18.35 (CH 3-23), 18.46 (CH3-27), 20.39

(CH3-26), 28.19, 30.07, 30.29, 31.81 (CH 3-29), 31.90, 32.15 (CH3-28), 32.48, 32.88, 34.99

(CH3-30), 35.37, 35.96, 36.05, 36.81, 38.30, 39.29, 39.76, 42.87, 42.94, 52.39, 52.98, 54.34,

296

112.70, 120.02, 129.17, 129.26, 137.66 (C-2), 144.77, 155.01 (C-3), 192.12 (-CHO). Analysis calcd: C, 79.95; H, 9.61; N, 7.56. Found: C, 80.06; H, 9.48; N, 7.62.

IV.D Conclusion Syntheses of a number of A-ring modified friedelane triterpenoids have been accomplished. These also include the 2-homo derivatives for which, as the key step, the transformation of friedelin with Vilsmeyer-Haack reagent was used. 3-Chloro-2-formylfriedel-2-ene the main product isolated from the reaction was transformed suitably into various derivatives and hence, following two or three simple steps starting from friedelin, it rendered possible to produce a library of C2,C3-, C3,C4-, and C2,C3,C4- functionalized friedelane triterpenoids. Besides, some useful methodologies were thus established during the various transformative attempts. These include a two-step aromatization of friedelin by N-bromosuccinimide, a one-pot dechlorination with simultaneous C-23 activation, and selective 4 a-hydroxylation with simultaneous oxidation of allylic alcohol by selenium dioxide. Again, syntheses of some friedelane derivatives, viz. , 3b- amino-4a-hydroxy-, 2-carboxamide, 2,3-seco diol, 4a-hydroxy-3-chloro-2-formylfriedel-2-ene and 3-chloro-4a-hydroxy-2-hydroxymethylfriedel-2-ene, in a few steps, were found very much effective to enrich the A-ring modifications of friedelane triterpenoids. On the other hand, heterocycle-linked (to C3 of friedelanes) 2-homo friedelane derivatives were achieved. We believe to use these friedelane triterpenoids for future biological applications as well as to explore more interesting and usefull multifunctionalized derivatives of the particular class of pentacyclic triterpenoids.

IV.E Supporting spectra

297

Figure 4 .21 1H NMR spectrum of friedelin ( 115 ).

Figure 4 .22 13 C NMR spectrum of friedelin ( 115).

298

Figure 4.23 1H NMR spectrum of 3-chlorofriedel-3-ene ( 1327).

Figure 4.24 1H NMR spectrum (partially expanded) of 3-chlorofriedel-3-ene ( 1327).

299

Figure 4.25 13 C NMR spectrum of 3-chlorofriedel-3-ene (1327 ).

Figure 4.26 FTIR spectrum of 3-chlorofriedel-3-ene (1327).

300

Figure 4.27 1H NMR spectrum of 3-chloro-2-formylfriedel-2-ene ( 1328).

Figure 4.28 1H NMR spectrum (partially expanded) of 3-chloro-2-formylfriedel-2-ene ( 1328).

301

Figure 4.29 13 C NMR spectrum of 3-chloro-2-formylfriedel-2-ene ( 1328).

Figure 4.30 FTIR spectrum of 3-chloro-2-formylfriedel-2-ene ( 1328).

302

Figure 4.31 1H NMR spectrum of 2-formyl-3-hydroxy-friedel-2-ene (1329).

Figure 4.32 1H NMR spectrum (partially expanded) of 2-formyl-3-hydroxy-friedel-2-ene ( 1329).

303

Figure 4.33 13 C NMR spectrum of 2-formyl-3-hydroxy-friedel-2-ene ( 1329).

Figure 4.34 FTIR spectrum of 2-formyl-3-hydroxy-friedel-2-ene ( 1329).

304

Figure 4.35 1H NMR spectrum of 3-Chloro-2-formyl-4a-hydroxy-friedel-2-ene ( 1330).

Figure 4.36 1H NMR spectrum (partially expanded) of 3-chloro-2-formyl-4a-hydroxy-friedel-2-ene (1330).

305

Figure 4.37 13 C NMR spectrum of 3-chloro-2-formyl-4a-hydroxy-friedel-2-ene ( 1330).

Figure 4.38 FTIR spectrum of 3-chloro-2-formyl-4a-hydroxy-friedel-2-ene ( 1330 ).

306

Figure 4.39 1H NMR spectrum of friedel-3-ene-23-al ( 1331).

Figure 4.40 1H NMR spectrum (partially expanded) of friedel-3-ene-23-al ( 1331).

307

Figure 4.41 13 C NMR spectrum of friedel-3-ene-23-al ( 1331).

Figure 4.42 FTIR spectrum of friedel-3-ene-23-al ( 1331).

308

Figure 4.43 1H NMR spectrum of 3a,4 a-epoxy friedelane(1332 ).

Figure 4.44 Expanded 1H NMR spectrum of 3 a,4 a-epoxy friedelane(1332).

309

Figure 4.45 13 C NMR spectrum of 3 a,4 a-epoxy friedelane(1332 ).

Figure 4.46 FTIR spectrum of 3 a,4 a-epoxy friedelane(1332).

310

Figure 4.47 1H NMR spectrum of 24-norfriedel-1, 3, 5 (10), 6-tetraene ( 1333).

Figure 4.48 1H NMR spectrum (partially expanded) of 24-norfriedel-1, 3, 5 (10), 6-tetraene ( 1333 ).

311

Figure 4.49 13 C NMR spectrum (partially expanded) of 24-norfriedel-1, 3, 5 (10), 6-tetraene ( 1333 ).

Figure 4.50 1H NMR spectrum of 3,4-seco-friedelane-3,4-diol ( 1337).

312

Figure 4.51 1H NMR spectrum (partially expanded) of 3,4-seco-friedelane-3,4-diol ( 1337 ).

Figure 4.52 13 C NMR spectrum of 3,4-Seco-friedelane-3,4-diol ( 1337).

313

Figure 4.53 1H NMR spectrum of 3-epi pachysan diol-A ( 1338).

Figure 4.54 1H NMR spectrum (partially expanded) 3-epi pachysan diol-A ( 1338).

314

Figure 4.55 13 C NMR spectrum of 3-epi pachysan diol-A ( 1338).

Figure 4.56 FTIR spectrum of 3-epi pachysan diol-A ( 1338).

315

Figure 4.57 1H NMR spectrum of friedel-3-enol acetate ( 1339).

Figure 4.58 13 C NMR spectrum of friedel-3-enol acetate ( 1339).

316

Figure 4.59 Mass spectrum of friedel-3-enol acetate ( 1339 ).

Figure 4.60 1H NMR spectrum of 4 a-acetate friedel-3-one ( 1340).

317

Figure 4.61 13 C NMR spectrum of 4 a-acetate friedel-3-one ( 1340).

Figure 4.62 1H NMR spectrum of 4 a-hydroxy friedelane-3-oxime ( 1341).

318

Figure 4.63 13 C NMR spectrum of 4 a-hydroxy friedelane-3-oxime ( 1341).

Figure 4.64 1H NMR spectrum of 3b-amino-4a-hydroxyfriedelane (1342).

319

Figure 4.65 1H NMR spectrum (partially expanded) of 3b-amino-4a-hydroxyfriedelane (1342 ).

Figure 4.66 13 C NMR spectrum of 3b-amino-4a-hydroxyfriedelane (1342).

320

Figure 4.67 1H NMR spectrum of 3-chlorofriedel-2-ene-2-carboxaldoxime ( 1343).

Figure 4.68 1H NMR spectrum (partially expanded) of 3-chlorofriedel-2-ene-2-carboxaldoxime ( 1343).

321

Figure 4.69 13 C NMR spectrum of 3-chlorofriedel-2-ene-2-carboxaldoxime ( 1343).

Figure 4.70 FTIR spectrum of 3-chlorofriedel-2-ene-2-carboxaldoxime ( 1343).

322

Figure 4.71 1H NMR spectrum of 3-chloro-2-hydroxymethyl-friedel-2-ene ( 1344).

Figure 4.72 1H NMR spectrum (partially expanded) of 3-chloro-2-hydroxymethyl-friedel-2-ene (1344).

323

Figure 4.73 13 C NMR spectrum of 3-chloro-2-hydroxymethyl-friedel-2-ene ( 1344).

Figure 4.74 FTIR spectrum of 3-chloro-2-hydroxymethyl-friedel-2-ene ( 1344).

324

Figure 4.75 1H NMR spectrum of 2-acetoxymethyl-3-chloro-friedel-2-ene ( 1345).

Figure 4.76 1H NMR spectrum (with partial expansion) of 2-acetoxymethyl-3-chloro-friedel-2-ene (1345).

325

Figure 4.77 13 C NMR spectrum of 2-acetoxymethyl-3-chloro-friedel-2-ene ( 1345).

Figure 4.78 FTIR spectrum of 2-acetoxymethyl-3-chloro-friedel-2-ene ( 1345).

326

Figure 4.79 1H NMR spectrum of 3-chlorofriedel-2-ene-2-carboxamide ( 1346).

Figure 4.80 13 C NMR spectrum of 3-chlorofriedel-2-ene-2-carboxamide ( 1346).

327

Figure 4.81 1H NMR spectrum of 3-chloro-4a-hydroxy-2-ene-2-carboxaldoxime ( 1347).

Figure 4.82 13 C NMR spectrum of 3-chloro-4a-hydroxy-2-ene-2-carboxaldoxime ( 1347).

328

Figure 4.83 FTIR spectrum of 3-chloro-4a-hydroxy-2-ene-2-carboxaldoxime ( 1347 ).

Figure 4.84 1H NMR spectrum of 2-formyl-3-chloro-4a-hydroxy-2-hydroxymethyl friedel-2-ene ( 1348).

329

Figure 4.85 13C NMR spectrum of 2-formyl-3-chloro-4a-hydroxy-2-hydroxymethyl friedel-2-ene ( 1348).

Figure 4.86 1H NMR spectrum of 2-formyl-3-(1 H-piperidin-1-yl)-friedel-2-ene ( 1349).

330

Figure 4.87 13 C NMR spectrum of 2-formyl-3-(1 H-piperidin-1-yl)-friedel-2-ene ( 1349).

Figure 4.88 1H NMR spectrum of 2-formyl-3-(1 H-morpholin-4-yl)-friedel-2-ene ( 1350).

331

Figure 4.89 1H NMR spectrum (partially expanded) of 2-formyl-3-(1 H-morpholin-4-yl)-friedel-2-ene (1350).

Figure 4.90 13 C NMR spectrum of 2-formyl-3-(1 H-morpholin-4-yl)-friedel-2-ene ( 1350).

332

Figure 4.91 1H NMR spectrum of 2-formyl-3-(1 H-piperazin-1-yl)-friedel-2-ene ( 1351).

Figure 4.92 1H NMR spectrum of 2-formyl-3-(1 H-imidazol-1-yl)-friedel-2-ene ( 1352 ).

333

Figure 4 .93 13 C NMR spectrum of 2-formyl-3-(1 H-imidazol-1-yl)-friedel-2-ene ( 1352 ).

Figure 4.94 1H NMR spectrum of 2-formyl-3-(1 H-benzimidazol-1-yl)-friedel-2-ene ( 1353 ).

334

Figure 4.95 1H NMR spectrum (with partial expansion) of 2-formyl-3-(1 H-benzimidazol-1-yl)-friedel-2- ene ( 1353).

Figure 4.96 13 C NMR spectrum of 2-formyl-3-(1 H-benzimidazol-1-yl)-friedel-2-ene ( 1353).

335

Figure 4.97 1H NMR spectrum of 2-formyl-3-(1 H-1, 2, 3-benzotriazol-1-yl)-friedel-2-ene ( 1354).

Figure 4.98 1H NMR spectrum (partially expanded) of 2-formyl-3-(1 H-1, 2, 3-benzotriazol-1-yl)-friedel- 2-ene ( 1354).

336

Figure 4.99 13 C NMR spectrum of 2-formyl-3-(1 H-1, 2, 3-benzotriazol-1-yl)-friedel-2-ene ( 1354).

IV.F References: The references of this chapter are provided in the Bibliography section of the thesis. Please follow page-372 onwards for these references.

337

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Index

A B acyclic, 2 Bevirimat, 31-32, 38-39 7a-acetoxycholesterol, 12 Bidesmosidic, 29, 56-57 bile acids, 5-6, 71-72, 200 3b-acetoxycholestan-7-one, 65 bromohydrin, 75 3b-acetoxycholesterol, 13 2α -bromofriedelin, 267-268 7b-acetoxycholesterol, 12 Bryozoa, 122, 135 4a-acetoxyfriedel-3-one, 280, 292

Algae, 122, 166, 169 amarasterone A, 22 C 3b- Amino -4a-hydroxyfriedelane, 280, Carbenoxolone, 59 293 Campesterol, 17-19 4-androstene-3-17-dione, 80 carbocyclic, 1-2, 4-5, 24, 26-28, 130 androsterone, 66 celastrol, 26, 273, 279 anti-AIDS, 29, 55 chalcone, 49 anticancer, 5, 27, 29, 31-33, 37, 47, 273 D2-cholenate,69 anti-colon cancer, 50 5a-cholestane, 8, 125 antidiabetic, 25 D3-cholenate, 68-69 anti-inflammatory, 25, 27, 49, 273 3b-hydroxy-D1-cholenate, 69 antimalarial, 34, 47-49 cholestan-2,3-dione, 65 antiparasitic, 25, 271 cholest-4-en-3-one, 13 antiproliferative, 37, 42 cholest-5-en-3-one, 10 antitumor, 25, 27, 29, 42, 46, 54 CDDO, 50 antiviral, 25, 29, 42 CDDU, 50 apoptosis, 27, 29, 32, 46 cis -cholestene-3,4-diol-3-benzoate, arnidiol, 28 65cholesteryl acetate, 12-13,65, 82-83, 162 aromatase inhibitors, 98, 193 3-chlorofriedel-2-ene, 248, 275-277, 287 aromatization, 61-62, 72, 76-84, 86, 88-89, 3-chloro-2-formylfriedel-2-ene 278, 298 collidine, 75 asiaticoside, 59 coral, 124-128, 130-132, 166-169 azasteroids, 188 corosolic acid, 59

380 correlogenin, 67 fucosterol, 20 cortisone acetate, 67 fungus, 33, 139, 256 crystallographic, 91-92 cyasterone, 22 G cyclic, 2, 8, 55-56, 137, 175, 203 Garegg –Samuelsson, 9 cytochrome P450, 72 Gelation, 22, 120-121, 197, 204, 207-209, cyclopentenophenanthrene, 5-6 217-218, 225 cytotoxicity, 27, 29, 33-35, 41, 253, 271, Glucocorticoids, 187 273 H D Hepatoprotective, 27 Dehydrogenation, 83, 89, 258 19-hydroxyandrost-3, 17-dione, 80 dehydroisoandrosterone, 72-73 7b-hydroxycholesterol, 71 dehydroergosterol, 64 25-hydroxycholesterol, 17 desmosterol, 20, 122 17b-hydroxyprogesterone, 78 9(1l)-dehydrohecogenin acetate, 67 3b-hydroxyfriedelane, 251-252, 259, 263- diaromatic, 61-62, 72, 76, 82, 87-89, 99- 264 100 K 3,3-dimethoxy ketals, 67 12-ketocholanic acid, 65 dutasteride, 58 L E lanosterol, 19 4-epi friedelin, 251-252, 263 lanosteryl acetate, 66-67 ergosterol, 19, 61-62, 64, 66, 69, 82, 88- Lawesson’s reagent , 56 90, 99-100 Lipophobic, 200-202 estrone, 16, 73, 79-80 M ∆9-estrone, 73 4-methyl-19-norcholesta-1,3,5(10)-triene, eplerenone, 189-190 72, 73 exemestane, 59 micelization, 202 F Mitsunobu coupling, 9 faradiol, 28 mollusca, 122, 144, 145

3a-fluorocholesterol, 7 morphology, 121, 197, 212, 213, 225 friedel-3-ene-23-al, 278, 290, 308-309 mushroom, 122, 166, 169 friedelinoxime, 266-267 friedelonic acid, 258

381

N serratane, 25, 28 naphthalene analogues, 83 siderophores, 4 naturalproduct, 1-5, 62, 82, 89, 100, 275 sigmatropic, 63, 64 norfriedelanes, 250, 255 sponge, 124, 131, 133-138, 165, 166, 169- nucleo-cholesterols, 9 171 O starfish, 124, 137, 139-144, 149, 166, 172 7-oxocholesterol, 71 stereoselectivity, 12, 16, 63, 191 24-oxocholesterol, 17 submicromolar, 30 organogels, 121, 197, 206, 209-211, 225 supramolecular, 204-206 g-oryzanol, 22, 23 T Ψ-taraxastane, 25, 28 P Taraxerane, 49 Pentahydroxy, 123, 125, 197, 202- 204, 207, 220, 225 tetracyclic, 88, 269 testosterone, 70, 80 pheromones, 4 tetrahydroxy, 121, 123, 130, 136, 160, pregnenolone, 66, 72, 73, 162, 196 197, 202-204, 225 prodrugs, 53-55 topoisomerase, 29, 271 progesterone, 14, 70, 71, 78, 81 U putranjivic acid, 263 ulipristal, 193, 194 Q ursolic acid, 27, 28, 49, 55, 56, 58 quertz, 263 V R VilsmeyerHaack, 275-277, 281-282, 286, regioselective, 17, 88, 89 296 repellants, 4 rheological, 121, 197, 211, 212, 225

Ritter reaction, 8, 9, 195, 196

S Saponins, 5, 29, 36, 37, 42, 47, 52, 54, 55-

57, 144, 250 3,4-secofriedelane-3,28-dioic acid, 253 selenium dioxide, 47, 62-71, 76, 77, 82,

83, 86, 93, 94, 258, 277, 282, 283, 287, 298

382

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RESEARCH LETTER One-pot solid phase selective aromatization of cholesterol using N-bromosuccinimide: an optimized green methodology Pranab Ghosh*, Jayanta Das and Antara Sarkar

Natural Product and Polymer Chemistry Laboratory, Department of Chemistry, North Bengal University, Darjeeling -734013, West Bengal, India (Received 28 October 2010; final version received 11 May 2011)

One-pot silica promoted selective aromatization of ring-A of cholesterol using N-bromosuccinimide (NBS) has been performed. The structure of the compound has been characterized by spectral analysis (UV, FT-IR, NMR, and Mass). Keywords: cholesterol; aromatization; N-bromosuccinimide

Introduction starting material. Developmental studies in this area 15,21 23 Á26 27 Á32 Selective aromatization of steroidal skeleton has been are still in progress ( , ). Microbial ( ) as well as enzymatic ( 33,34) steroidal aromatization regarded as an efficient and useful chemical transfor- has also been attempted, and the compounds are mation both in terms of synthetic and biological found to have potent biological implications. As a consideration. Many of the steroidal hormones have consequence of the importance given toward the been aromatized by using various transformation selective aromatization of a steroidal skeleton, we protocols. Although Marker and co-workers (1) first started working in this area and finally could able to reported the transformation of steroidal nucleus into report herein a novel synthesis of A-ring aromatized its aromatic analog, it was the work of Inhoffen and cholesterol via a very simple one-pot solid phase Zuhhdarff which firmly established the involvement reaction. of such type of chemical transformation ( 2Á5). They reported an acid-catalyzed dienone-phenol rearrange- ment of 1, 4-cholestadien-3-one to furnish a phenol Results and discussion entity, the structure of which was later on confirmed A mixture of cholesterol ( 1a , 0.38 g, 1.0 mmol) and Á by others ( 2 8). Again, Romo and his group showed NBS (0.42 g, 5.0 mmol) in activated silica furnished a a thermal transformation of 1, 4-cholestadien-3-one selective aromatic product 1-methyl-19-norcholesta- into aromatic derivatives and the dieneone-phenol 1,3,5(10)-triene, 2 (Scheme 1). To the best of our rearrangement in the cholesterol series ( 9,10). Hanson knowledge the isolated compound is being reported

Downloaded by Bengal University ] at 23:05 09 March 2012 Downloaded [North and Organ (11,12) have reported a steroidal aroma- for the first time and it is actually isomeric to the tization reaction which includes a transformation of compound reported by Hanson and Organ ( 11 ). cholesterol into A-ring aromatized product 4-methyl- Compound 2 appeared as pale greenish yellow 19-norcholesta-1, 3, 5(10)-triene on treatment with 1, gum (46%, 0.17 g, 0.46 mmol) with characteristic 3-dibromo-5, 5-dimethylhydantoin. But they were odor which on standing (about 30 days at room unable to isolate the compound in its pure form temperature) slowly solidifies. The solidified mass (11 ). They reported NBS as a poorer alternative to was analyzed (TLC, IR, and NMR) to be a mixture hydantoin. Urbanos and co-workers (13,14) showed of compounds which could not be isolated and an efficient selective transformation of steroids into moreover, it did not contain the aromatized product aromatized derivatives by an electrophilic ruthenium 2 (by comparison with the TLC, IR, and NMR complex. However, in order to aromatize steroidal spectra of the gummy sample ( 2) and its solidified compounds selectively, people have used several mass). steroidal enones (15 ), hydroxy ketones ( 16 ), epoxy The isolated product 2 (as a gum) was characterized steroids (10,17,18), dieneones ( 19 Á22 ), etc. as the on the basis of UV, IR, 1H, 13 C NMR spectroscopy,

*Corresponding author. Email: [email protected]

ISSN 1751-8253 print/ISSN 1751-7192 online # 2012 Taylor & Francis http://dx.doi.org/10.1080/17518253.2011.607472 http://www.tandfonline.com 174 P. Ghosh et al.

NBS, Solid Support Room temp or heat R or MW, time R= 1a : OH 2, upto 60% 1b : OCOCH 3 1c : OCOPh 1d: Cl

Scheme 1. Selective synthesis of A-ring aromatized cholesterol.

mass spectrometry, and elemental analysis. The UV aromatization reaction was also performed, and it spectrum of 2 showed a peak, the pattern of which is was established that 1 g mmol 1 of silica is best similar to the benzenoid derivatives ( lmax 277 nm, suitable for the transformation (Table 3). t 2360). The IR spectrum showed absorptions at Variation of solid supports (Table 4) indicated that Á1 nmax 2951, 1462, 738, and 815 cm characteristic for an silica gel 60 Á120 mesh (used normally for column aromatic ring. 1H NMR spectrum showed peaks at chromatography) was found to be the most effective d2.19 (3 H, s, CH3), 6.97(1 H, d, J 7.2 Hz, CH), support for the transformation. Silica gel F254 gave 7.05(1H, t, J7.5 Hz, CH), 7.17(1H, d, J8.1 Hz, poor yield, alumina and KF-alumina did not yield CH) ppm which correspond to the aromatic A-ring of the product (2) at all, and bentonite and 4 A ˚ molecular 1 13 the product. The H decoupled C NMR spectrum of sieves showed very little transformation. After each 2 showed 27 distinct resonances of which six belong to and every experiment the unreacted starting material, the aromatic carbons. The mass spectrum displayed a cholesterol, has been recovered quantitatively. Cata-  molecular ion peak (M ) and base peak at m/z 366 lytic role of silica for the above transformation has also  (38%) and 365 (M -H), respectively. been tested (Table 5); other solid supports used in the The reaction was attempted both on solid support study gave very poor yield of the product. as well as in solution phase. However, no aromatiza- Recyclability of the catalyst was tested by the tion was observed in solution phase. following way Á after the reaction is over, chloroform For a preliminary study of optimization we was added to the reaction mixture and the silica gel performed the reaction using three different proto- was filtered off followed by successive washings with cols, viz., (1) reaction at room temperature; (2) by the methanol (2), acetone (1), and was activated Á use of microwave irradiation (taking silica gel 60 120 (1508C/1 mm Hg, 1 h). The recovered activated silica mesh and F254, both); and (3) at elevated tempera- was then used in a fresh transformation as the Á tures (taking only silica gel 60 120 mesh). Although supporting medium for the reaction and it was found the conversion is found to be rapid both in microwave that upto six consecutive runs, there was no appreci- irradiation and at higher temperature the yield was able change in the percentage yield of the desired not satisfactory following either of the protocols. It Downloaded by Bengal University ] at 23:05 09 March 2012 Downloaded [North product. Thus silica may be considered as a very was further established that the optimized time (in effective reusable solid support for this specific terms of percentage yield) at room temperature is 72 transformation (Table 5). h, at thermal condition 5 min, and in microwave it Á 8 was 5 7 min (150 W, 100 C) (Tables 1 and 2). The Table 1. Optimization of the reaction condition taking slow reaction rate of the transformation is quite cholesterol and NBSa Á at room temperature and MW obvious from the fairly longer optimized reaction irradiation. time at room temperature. Investigation indicated Room temperature MW induced that beyond 72 h there is a gradual decrease of c c percentage yield of the product (Table 1). This may be yield (%) yield (%) explained on the basis of the observation that beyond Silica usedb 12 h 24 h 48 h 72 h 120 h 5 Á7 min 72 h it slowly started solidifying and consequently analysis of the solid mass indicated that it is different 60 Á120 mesh 11 22 25 46 18 30 from aromatized product 2 and finally identified as a F254 5 13 13 11 4 27 mixture of compounds as stated above. aReactions were performed on 0.25 g, 0.65 mmol substrate. Optimization study for the amount of silica bOne gram *per millimole of activated silica gel was used. support (silica gel 60 Á120 mesh) for the above cYield refers to isolated pure product. Green Chemistry Letters and Reviews 175

Table 2. Optimization of the reaction condition a taking Table 4. Optimization for the solid support a Á taking cholesterol and NBSb Á at elevated temperatures. cholesterol and NBSb.

Yield (%)c in various time intervals Yield Recovered cholesterol Solid support (%)c (%)c Temperature 1 min 5 min 15 min 30 min 60 min Silica gel (60Á120 46 35 50 8C 18 36 33 25 21 mesh) 1008C 15 35 27 24 19 Silica gel F254 11 87

a 1 Alumina neutral NR 96 Reactions were performed on 1 g mmol of activated silica gel KF-Alumina NR 97 (60Á120 mesh). 4 A ˚ molecular sieve 9 87 bOn 0.25 g, 0.65 mmol substrate. cYield refers to isolated pure product. Bentonite 12 85 Neat NR 98

In order to generalize the findings, we extended Abbreviation: NR, no reaction observed. the above reaction on another steroidal system, aOne gram *per millimole of activated solid support was used. diosgenin, but surprisingly we could not isolate bReactions were performed on 0.25 g, 0.65 mmol substrate. c similar aromatic analog of diosgenin using NBS, Yield refers to isolated pure compounds. although Sondheimer and his group (35 ) has reported its aromatization via another route. silica gel plates using silica gel G for thin layer Replacement of C-3 ÁOH by ÁCl ( 1d ) showed chromatography (Merck). similar result but introduction of OAc ( 1b ) gave better yield compared to cholesterol itself. On the Procedure for the preparation of 2 from cholesterol other hand the benzoate derivative (1c ) gave poor Silica gel (60Á120 mesh, 1.0 g) was activated (150 8C/1 yield of the product (Table 6).When we carried out mm Hg, 1 h) and mixed with cholesterol (0.38 g, 1.0 the same reaction on a 4-substituted cholesterol mmol) and NBS (recrystalized from hot water to have derivative, 4- b-hydroxycholesterol, we could not white crystals, 0.42 g, 5.0 mmol) taking in a dry isolate the aromatic product. Thus the transforma- mortar. The mixture was then pasted well to dust by a tion cannot be correlated with nature as well as on pestle, transferred to a round bottom flask, and was the position of the substituent in ring-A of choles- kept in stirring for 72 h. Next, Chloroform (50 mL) terol. was poured, filtered, and the filtrate was washed with water (3 30 mL) and dried over Na 2SO 4. The solvent was evaporated and the residue was purified Experimental by column chromatography using petroleum ether as UV spectrum was measured on JASCO V-530 UV/VIS the eluent. Compound 2 appeared as pale greenish Spectrophotometer. Infrared spectrum was measured yellow gum (46%, 0.17 g, 0.46 mmol) with character- in neat with a Shimadzu FT-IR 8300 Spectrometer. istic odor. The NMR spectra were recorded on a 300 MHz Bruker Avance FT-NMR spectrometer with CDCl 3 Compound 2

Downloaded by Bengal University ] at 23:05 09 March 2012 Downloaded [North as solvent and TMS as internal reference, chemical t shifts are expressed as d ppm. Mass spectrum was UV (Petroleum ether): lmax 277 nm, 2360. IR 1 measured on a JEOL-AccuTOF JMS-T100LC Mass (neat) (nmax /cm ): 2951 (Ar ÁH str.), 2867 and 2929 Spectrometer. Analytical-TLC was performed with (CH3 str.), 1462 (Arom. ring vib.), 1455 and 1380 1 (CH3 def.), 815 and 738 (Arom. H). H NMR (300 Table 3. Optimization for the silica support a Á taking cholesterol and NBSb. Table 5. Recycling experiment using cholesterol and NBS catalyzed by silica a. Entry Silica in g mmol Á1 Isolated yield of 2 (%) Run Isolated yield of 2 (%) 1 0.25 22 2 0.50 37 1 43 3 0.75 40 2 45 4 1.00 46 3 43 5 1.50 46 4 42 6 2.00 44 5 40 6 42 aReactions were performed on activated silica gel (60 Á120 mesh). bOn 0.25 g, 0.65 mmol substrate and at room temperature (72 h). aOne gram * per millimole of silica gel (60 Á120 mesh) was used. 176 P. Ghosh et al.

Table 6. Effect of the 3-substituent on yield of 2. References Reaction Yield (1) Marker, R.E.; Kamm, O.; Oakwood, T.S.; Laucius, Entry Substratea conditionsb (%)c J.F. J. Am. Chem. Soc . 1936, 58 , 1503 Á1504. (2) Inhoffen, H.H.; Minlon, H. Naturwissenschaften 1938, 26 , 756. (3) Inhoffen, H.H.; Zuehlsdorff, G. Ber. 1941, 74 , 604 Á 1 RT, 72 h 46 616. HO (4) Clemo, G.R.; Haworth, R.D.; Walton, E. J. Chem Soc. 1930, 1110 Á1115. (5) Inhoffen, H.H. Angew. Chem. 1940, 53 , 471 Á475. 2 RT, 72 h 60 (6) Wilds, A.L.; Djerassi, C. J. Am. Chem. Soc . 1946, 68 , 1712Á1715. AcO (7) Dreiding, A.S.; Voltman, A. J. Am. Chem. Soc . 1954, 20 , 537 Á539. 3 RT, 72 h 32 (8) Morand, P.; Lyall, J. Chem. Rev. 1968, 68 , 85 Á124. (9) Romo, J.; Djerassi, C.; Rosenkranz, G. J. Org. Chem . PhOCO 1950, 15 , 896 Á900. (10) Romo, J.; Rosenkranz, G.; Djerassi, C. J. Org. Chem . 3 RT, 72 h 32 1950, 15 , 1289 Á1292. (11) Hanson, J.R.; Organ, T.D. J. Chem. Soc. C 1970, 513 Á Cl 515 Hanson, J.R.; Organ, T.D. J. Chem. Soc. C 1970, aReactions were performed on 1 mmol scale. 1313Á1314. bOne gram of activated silica gel (60 Á120 mesh) was used. (12) Hanson, J.R. J. Chem. Soc. D 1971, 1119. cYield refers to isolated pure product. (13) Halcrow, M.A.; Urbanos, F.; Chaudret, B. Organ ometallics 1993, 12 , 955 Á957. (14) Urbanos, F.; Halcrow, M.A.; Fernandez-Baeza, J.; Dahan, F.; Labroue, D.; Chaudret, B. J. Am. Chem. MHz, CDCl3): d 0.68 (3 H, s, CH 3), 0.87 (3 H, s, CH 3), Soc. 1993, 115, 3484 Á3493. 0.85 (3 H, s, CH 3), 1.25 (3 H, s, CH 3), 2.19 (3 H, s, (15) Alvarez, F.S.; Ruiz, A.B. J.Org. Chem. 1965, 30 , CH3), 6.97 (1 H, d, J 7.2 Hz, CH), 7.05 (1 H, t, 2047Á2049. J7.5 Hz, CH), 7.17 (1 H, d, J8.1 Hz, CH). 13 C (16) Kirdan, R.Y.; Layne, D.S. J. Med. Chem . 1964, 7, NMR (300 MHz, CDCl ): d 11.9, 18.6, 19.8, 22.5, and 592Á595. 3 Á 22.8 (5CH ), 23.8, 23.9, 26.8, and 27.2 (4CH ), 28.0 (17) Kaufmann, S. J. Org. Chem . 1966, 31 , 2395 2397. 3 2 (18) Anastasia, M.; Ciuffreda, P.; Puppo, M.D.; Fiecchi, (CH), 28.3 and 29.7 (2CH ), 35.8 (CH), 36.2 (CH ), 2 2 A. J. Chem. Soc. Perkin Trans . 1983, 1, 587 Á590. 37.8 (CH), 39.5 and 40.0 (2CH2), 42.6 (C), 44.4, 55.6, (19) Heller, M.; Lenhard, R.H.; Bernstein, S. J. Am. Chem. 56.3, 123.0, 125.2 and 127.1 (6CH), 135.1, 136.1 and Soc. 1964, 86 , 2309 Á2310; Heller, M.; Lenhard, R.H.; 140.5 (3C). Anal. Calcd for C27 H42 (366.34): C, 88.44; Bernstein, S. J. Am. Chem. Soc . 1967, 89, 1911Á1918. H 11.56%. Found: C, 88.51; H 11.47%. DART-MS, (20) Libman, J.Q.; Mazur, Y. J.Chem. Soc. D 1971, 1146 Á m/z (%): 366 (M , 38), 365 (100), 364 (17), 363 (32), 1147. 361 (9), 353 (4), 351 (4). (21) Dryden, H.L.; Webber, G.M.; Wieczorek, J.J. J. Am. Downloaded by Bengal University ] at 23:05 09 March 2012 Downloaded [North Chem. Soc. 1963, 86 , 742 Á743. (22) Djerassi, C.; Rosenkranz, G.; Iriarte, J.; Berlin, J.; Romo, J. J. Am. Chem. Soc . 1951, 73 , 1523 Á1527. Conclusion (23) Tsuda, K.; Ohki, E.; Nozoe, S. J.Org. Chem. 1963, 28 , A one-pot green approach has been developed to 786Á789. aromatize A-ring of cholesterol selectively using NBS. (24) Tsuda, D.; Nozoe, S.; Okada, Y. J. Org. Chem . 1963, The simplicity of the reaction condition and a cost- 28 , 789 Á792. effective reaction protocol is highly encouraging for (25) Tsuda, D.; Nozoe, S.; Tatezawa, T.; Sharif, S.M. J. Org. Chem . 1963, 28 , 795 Á798. the environmentally benign chemical transforma- (26) Suginome, H.; Senboku, H.; Yamada, S. Tetrahedon tions. Lett. 1988, 29 , 79 Á80. (27) Dodson, R.M.; Muir, R.D. J. Am. Chem. Soc . 1961, Á Acknowledgements 83 , 4627 4631; Dodson, R.M.; Muir, R.D. J. Am. Chem. Soc. 1958, 80, 5004Á5005. The authors are thankful to CDRI, Lucknow, India, for the (28) Thomson, E.A.; Jr.; Siiteri, P.K. J. Biol. Chem . 1974, mass spectrometry and elemental analysis. A.S. and J.D. 249, 5373 Á5378. thank UGC, New Delhi, and CSIR, New Delhi, respec- (29) Singh, K.; Marshal, D.J.; Vezina, C. Appl. Environ. tively, for awarding Junior Research Fellowship. Microbial. 1970, 20 , 23 Á25. Green Chemistry Letters and Reviews 177

(30) Sih, C.J.; Wang, K.C. J. Am. Chem. Soc . 1965, 87 , (33) Payne, A.H.; Hales, D.B. Endocr. Rev. 2004, 25 , 947 Á 1387Á1388. 970. (31) Kautsky, M.P.; Hagerman, D.D. Steroids 1976, 28 , (34) Caspi, E.; Arunachalam, T.; Nelson, P.A. J. Am. 247Á259. Chem. Soc. 1986, 108, 1847 Á1852. (32) Moortgy, K.B.; Meigs, R.A. Biochim. Biophys. Acta (35) Sondheimer, F.; Neumann, F.; Ringold, H.J.; Ro- 1978, 528, 222 Á229. senkranz, G. J. Am. Chem. Soc . 1954, 76 , 2230 Á2233. Downloaded by Bengal University ] at 23:05 09 March 2012 Downloaded [North This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy

Tetrahedron 68 (2012) 6485 e6491

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Tetrahedron

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Oxidation with selenium dioxide: the first report of solvent-selective steroidal aromatization, ef ficient access to 4 b,7 a-dihydroxy steroids, and syntheses of natural diaromatic ergosterols

Pranab Ghosh a,*, Jayanta Das a, Antara Sarkar a, Seik Weng Ng b, Edward R.T. Tiekink b a Natural Product and Polymer Chemistry Laboratory, Department of Chemistry, North Bengal University, Darjeeling 734013, India b Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia article info abstract

Article history: Selenium dioxide oxidation of cholesterol reveals a solvent-dependent product selectivity and facile one- Received 28 February 2012 pot synthesis of three derivatives, including aromatic analogues of naturally occurring ergosterol. Ef fi- Received in revised form 20 May 2012 cient access to 4 b,7 a-dihydroxy cholesterol is described. Analogous chemistry of b-sitosterol and dio- Accepted 27 May 2012 sgenin is also reported. The protocol is found effective to synthesize two diaromatic ergosterol natural Available online 1 June 2012 products. A brief description of the molecular structures of the representative diaromatic cholesterol derivative and the triacetylated 4 b,7 a-dihydroxy cholesterol derivative are proven by X-ray Keywords: crystallography. Selenium dioxide Ó Aromatization 2012 Elsevier Ltd. All rights reserved. 4b,7 a-Dihydroxy steroids Dehydrogenation Natural products

1. Introduction metabolism. This motivates the investigation of their structur- eeactivity relationship in order to correlate disease with drug Selenium dioxide-mediated oxidation is regarded as one of the treatment.4,8,9 Clearly, there exists an enormous demand to syn- most reliable and predictable methods for allylic hydroxylation, thesize 4b,7 a-dihydroxy steroids, preferably in more direct and e especially in the steroid field.1 5 Despite the proven biological easier routes than existing synthetic methodologies. 10 Motivated by importance of oxygen bearing functionalities in steroidal sys- the above, we were encouraged to synthesize oxysterols and to 4,6e9 tems no systematic approach has been adopted to explore the explore the oxidation behaviour of SeO 2 on steroids. For the present oxidizing ability of selenium dioxide (SeO 2) in steroidal systems. study, cholesterol was chosen as the representative molecule. The The present report demonstrates there is ample opportunity to results thus obtained were subsequently extended to ergosterol, b- elaborate the chemistry of steroidal systems based on the oxidizing sitosterol and diosgenin. ability of SeO 2. The reaction of SeO 2 with cholesterol was reported to produce 4b-hydroxy cholesterol as the only product 2,3 whereas with the 2. Results and discussion same reagent, cholesteryl acetate and benzoate yielded 4 b-hy- droxylated and 6 a-hydroxylated products, respectively. 2 The ob- In particular, we wish to report the results of a solvent- servation was supported by Marker et al. 5 who studied the action of dependent investigation of SeO 2 oxidation on cholesterol, which resulted in the formation of naphthalene analogue ( 2a ) via selective SeO2 on stigmasterol, stigmasteryl acetate and sitosteryl acetate. Among the oxysterols in human circulation, the major one, 4 b- aromatization of rings A and B of cholesterol, together with 4 b- hydroxy cholesterol, is, along with other oxysterols, degraded hydroxy ( 3a ) and 4 b,7 a-dihydroxy ( 4a ) derivatives ( Scheme 1 ). generally to bile acids via 7 a-hydroxylation as the rate-limiting Cholesteryl acetate and benzoate also yielded the naphthalene 2 step.7,8 In addition, other hydroxy derivatives of cholesterol are analogues along with the other two products as reported (Scheme drawing attention due to their profound importance in human 2). Steroidal dehydrogenation of a maximum two hydrogen atoms using selenium dioxide3,11 as well as steroidal aromatization (using * Corresponding author. Tel.: þ91 353 2776 38; fax: þ91 353 2699 001; e-mail other reagents) and their biological implications have been repor- 12 address: [email protected] (P. Ghosh). ted. But, achieving dehydrogenation using SeO2 by removing as

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R R R R H H H H

H H SeO 2, Solvent + H H + H H HO Temp, Time HO R'O OR / 1a, 1b OH OR / R: a = 2a : 3-6% 3a : 10-75% 4a: R= a, R / =H; 5-27% 2b : 7% 3b : 51% Ac 2O, 4b : R= b, R /= H; 22% Py / b = 4aa: R=a, R = COCH 3; 94% from 4a

Scheme 1. SeO2 oxidation of cholesterol ( 1a ) and b-sitosterol (1b ).

R R R H H H

H H SeO 2, Dioxane o 2a + H H + H H 1 100 C, 24 h R O 1 R1O 3% from 5a, R O R= 4% from 5b OH OH 6a : R 1=COCH , 35% 7a : R 1=COCH , 37% 5a : R 1=COCH 3 3 3 6b : R 1=COPh, 32% 7b : R 1=COPh, 34% 5b : R 1=COPh

Scheme 2. SeO2 oxidation of cholesteryl acetate ( 5a ) and benzoate ( 5b ). many as nine hydrogen atoms from a cholesterol molecule to in- aprotic solvents (entries 17 e19) were used. To obtain the diol, 3a , duce aromatization (e.g., formation of naphthalene analogue, 2a ) in the use of ether as solvent, particularly THF (entry 8), was found to a solvent-speci fic reaction is unprecedented. Of note, only ethereal be best, whereas for triol, 4a , 1,4-dioxane (moist, entry 2) gave solvents with two oxygens such as 1,4-dioxane, 1,3-dioxalane, 1,2- maximum yield. Lower alcohols appeared to give better yield than dimethoxy ethane and 1,2-diethoxy ethane are able to yield 2a . their long chain analogues (entries 9e12). Dihydroxy alcohol, e.g., To the best of our knowledge, this is the first report of the solvent- ethylene glycol, was also examined but was found to be ineffective selective aromatization using selenium dioxide. (entry 13). Basic solvent, e.g., pyridine (entry 14) was found ef- The products have been fully characterized (see Experimental fective in converting cholesterol into the diol and triol whereas section and Supplementary data) by elemental analysis and spec- triethylamine and morpholine (entries 15 and 16) were found to troscopy (IR, 1H, 13 C, DEPT-135 NMR, mass), and in the cases of 2a be ineffective. This is presumably due to the higher basicity (to confirm the structure) and 4aa (to confirm the a-configuration of morpholine (p Kb¼8.33) and triethylamine (p Kb¼11.01) com- of the 7-OH group in 4a and, by analogy, that in 4b and 13 ), by pared to pyridine (p Kb¼5.21). The reaction was also attempted in single crystal X-ray crystallography ( Figs. 1 and 2 ). The molecular strong basic medium with the use of K 2CO 3 and NaOH in aqueous structures of 2a and 4aa are described in Supplementary data.13 ethanol, 1,4-dioxane and acetonitrile. However, the extent of

Fig. 1. Molecular structure of 2a and atom labelling.

The results of the systematic study have revealed that the transformation was negligible in each case. Among the polar composition of the products is largely solvent dependent ( Table 1 ). aprotic solvents used, DMSO resulted in good overall trans- Four different classes of solvents viz. (i) ethers (entries 1 e8), (ii) formation (entry 19), whereas acetonitrile (entry 17) and DMF alcohols (entries 9e13), (iii) basic (entries 14 e16) and (iv) polar (entry 18) gave poor yields. Author's personal copy

P. Ghosh et al. / Tetrahedron 68 (2012) 6485 e6491 6487

Fig. 2. Molecular structure of 4aa and atom labelling.

Table 1 composition of the reactants along with reaction time was then also a Product composition for the reaction of cholesterol with selenium dioxide in dif- varied taking different mole ratios of cholesterol to selenium di- ferent solvents oxide. Interestingly, it was seen that 1:7 mole ratio of the reactants b,c Entry Solvent system Reaction condition Yield (%) furnished better result ( Table 2 , entry 5) than the 1:3.5 mole ratio 2a 3a 4a (Table 1 , entry 2) towards the formation of 4a . However, increased 1 1,4-Dioxane 100 C, 24 h 3 35 6 reaction time or, the other mole compositions taking even excess 2 1,4-Dioxane/water (50:1) 100 C, 24 h 5 50 26 selenium dioxide could not result satisfactory yield of the product Re flux, 1 h 6 34 27 4a (entries 6e8). Besides, we could not isolate diaromatic choles- fl Re ux, 3 h Trace 46 8 terol 2a from the reactions in these conditions and the diol 3a was 3 1,4-Dioxane/water (5:1) 100 C, 24 h Trace 64 5 4 1,3-Dioxalane Re flux, 24 h 6 32 5 isolated at poor yields. Thus it may be concluded that, though these Re flux, 6 h Trace 42 8 reaction conditions furnished better results towards the yield of 4a , 5 2-Methoxy ethanol 100 C, 24 h NF 48 Trace the conditions rendered unsuitable for the formation of both the fl 6 1,2-Dimethoxy ethane Re ux, 24 h 6 54 6 aromatized product as well as the diol. Hence it seemed to be ad- 7 1,2-Diethoxy ethane Re flux, 24 h 5 51 11 8 Tetrahydrofuran Re flux, 24 h NF 75 17 vantageous to have 4a via 3a . 9 Ethanol Re flux, 24 h NF 35 11 10 tert-Butanol Re flux, 24 h NR 57 15 11 Octanol Re flux, 40 h NR NR NR Table 2 12 Decanol Re flux, 40 h NR NR NR Optimization of the yield of 4a directly from cholesterol 13 Ethylene glycol 100 C, 24 h NR NR NR a b c Re flux, 5 h NF 10 Trace Entry Reactant composition Reaction condition Yield (%) 14 Pyridine Re flux, 24 h NF 62 13 1 1:3.5 100 C, 48 h 32 d 15 Triethylamine Re flux, 40 h NR NR NR 2 1:3.5 100 C, 72 h 46 16 Morpholine 100 C, 40 h NR NR NR 3 1:3.5 100 C, 96 h 43 17 Acetonitrile Re flux, 24 h NF 21 5 4 1:3.5 Re flux, 1 h 20 d 18 DMF 100 C, 24 h NF 28 11 5 1:7 100 C, 24 h 44 19 DMSO 100 C, 24 h NF 60 5 6 1:7 100 C, 48 h 37 20 On solid support d MW, e min NF 30 Trace 7 1:10.5 100 C, 24 h 38 8 1:14 100 C, 48 h 28 a All the reactions were performed on 580 mg, 1.5 mmol of cholesterol. b Yield refers to isolated pure compounds. a Mole ratio of cholesterol to selenium dioxide; on 580 mg, 1.5 mmol of c NF ¼not found, NR ¼no reaction. cholesterol. d On preactivated silica gel 60 e120, after making dust. b All the reactions were carried out in 1,4-dioxane/water ¼50:1. e Domestic microwave, at 600 W. c Aromatized product 2a was not found, diol 3a was isolated at 6% yield except entries 1 and 4. d Compound 3a was isolated at 10% and 28% yields, respectively, from entries 1 It was observed that the aromatized product 2a was formed and 4. when the oxidation was carried out in 1,4-dioxane (entries 1 e3), 1,3-dioxalane (entry 4), 1,2-dimethoxy ethane (entry 6) and in 1,2- In 2004, Qin and Liu, 16 isolated two aromatized ergosterol de- diethoxy ethane (entry 7). All other solvents used ( Table 1 ) failed to rivatives ( 9 and 10 ) from the ascomycete Daldinia concentrica, and furnish 2a indicating the likely participation of both ethereal oxy- described the long-sought after biological precursor steroids for gens situated at 1,4-positions in the aromatization process. In organic matter in Earth ’s subsurface. Of the two compounds iso- a separate experiment, carried out under solvent-free microwave lated, the latter, 10 , bears an unusual methyl group at position 1. induced conditions, the reaction produced diol (3a ) but not 2a According to the authors, the aromatized products arise due to (entry 20). microbial action on the precursor molecule ergosterol. By contrast, the synthesis of 4a 10 (Scheme 1) is a simple and Having the natural diaromatic ergosterol analogue of choles- convenient one-step reaction. From the solvent-dependant study terol, we obviously were interested to apply the reaction protocol (Table 1 ), a maximum of 27% yield of the triol 4a was obtained using on ergosterol ( 8). As anticipated, the reaction furnished both the 1,4-dioxane (moist, entry 2) as the solvent. It was clear that SeO 2 natural compounds ( Scheme 3; 9 and 10 ) in relatively better yields induced allylic hydroxylation in 3a to form 4a and hence, we tried compared to cholesterol. 17 Surprisingly, the fact that the major reacting 3a with SeO2 in the identical reaction conditions, which product was 9 (12%) rather than 10 (1.3%) gives plausible mecha- produced 4a in 72% yield. 14 As the formation of 4a is one of the nistic hints of the involvement of the 7-dehydro skeleton in the important findings, we changed the reaction conditions little bit to formation of 1-hydro derivative ( 9) rather than 1-methyl product raise the yield of 4a by carrying out the reaction directly with (10 ). However, we could not isolate 10 as a single compound rather cholesterol (Table 2 ). 15 Firstly, we conducted the reactions with it was in a mixture with 9 (9:1, by NMR). 18 In order to optimize the longer period, viz., 48 h, 72 h and 96 h. Among these conditions, yield, different reaction conditions were explored using selective maximum of 46% yield was obtained at 72 h (entry 2). The solvents (Table 3 ). Ethanol and 2-methoxy ethanol failed to furnish Author's personal copy

6488 P. Ghosh et al. / Tetrahedron 68 (2012) 6485 e6491

9 and/or 10 . However, the synthesis of these naturally occurring 4b-Hydroxy cholesterol ( 2a ) did not furnish the corre- compounds using our methodology ruled out the possibility of sponding diaromatic derivative whereas ergosterol ( 8, possess- incidental formation of the aromatized cholesterol analogue in our ing C5 ]C6 as well as C7 ]C8 double bond in ring-B) yielded the previous experiments. diaromatic ergosterol derivative. So there exists a possibility of the involvement of A-ring and more speci fically the 4 b-hydro- gen towards the formation of the aromatized products. Under 1 1 R R identical reaction conditions, neither the diol 3a nor the triol 4a R could be converted to the aromatized product 2a . Rather 3a produced, as mentioned previously, 4a in a very good yield whereas 4a did not react at all. The mechanism for the conver- SeO H H 2 sion of 1a to 2a is unclear at this stage, although it may be HO Dioxane concluded that hydroxylation (as in 3a and 4a ) is, apparently, o 8 100 C, 6h not a precursor to form 2a . Moreover, since the yield of 2a does 9; R = H, 12% not vary substantially when prepared from 1a or its acetate/ benzoate derivatives under identical conditions ( Scheme 2 ), the R1= 10 ; R = CH 3, 1.3% groups at the 3 b position are not likely acting as leaving groups in these reactions. However, a detail experimental investigation Scheme 3. Synthesis of natural diaromatic ergosterol derivatives 9 and 10 . with a number of differently substituted starting materials may lead to a clear mechanistic approach for the aromatization process. Table 3 To extend the scope of the protocol, we carried out analogous Optimization a of the yield of the natural product 9 reactions on b-sitosterol ( 1b , Scheme 1 ) and diosgenin ( 11 , Entry Solvent system Reaction condition Yield (%) Scheme 4 ) under identical conditions. On reaction, b-sitosterol, like cholesterol, produced (in 1,4-dioxane at 100 C) the aro- 9 10 matic analogue ( 2b , 7%), 4 b-hydroxy b-sitosterol ( 3b , 51%) and 1 1,4-Dioxane/water (50:1) (a) 100 C, 2 h 5.5 <1 (b) 100 C, 6 h 12 1.3 4b,7 a-dihydroxy b-sitosterol ( 4b , 22%). By contrast, diosgenin (c) 100 C, 10 h 7 <1 (both at elevated temperature and microwave induced solvent- 2 Ethanol Re flux, 24 h NF b NF b free conditions, Scheme 4 ) yielded 4 b-hydroxy diosgenin ( 12 ,  b b 3 2-Methoxy ethanol 100 C, 24 h NF NF 30 e58%) and 4 b,7 a-dihydroxy diosgenin ( 13 , trace-17%) but no a Thorough solvent study of the reaction of cholesterol with SeO 2 (Table 1 above) aromatic analogue. Conversions of the corresponding diols ( 3b has prompted us to choose selective solvents and reaction conditions. and 12 ) to the triols were also achieved in high yields ( 4b , 72%; b NF ¼not found. 13 , 69%). 14

O

O H SeO 2 H H a or b + H H HO HO OH HO OH OH

11 12 13 a= 1,4-dioxane, 100 oC, 24 h 58% 17% b= on silica, MW, 600W 30% Trace

Scheme 4. Action of SeO 2 on diosgenin ( 11 ).

Again, it is noted that the yield of 2a is rather poor (maximum 3. Conclusion reproducible yield 6%) compared to those of the other two products (3a and 4a ). As has been pointed out earlier, the formation of 2a is In summary, we have accomplished the syntheses of natural very signi ficant because the reaction (of SeO2 and cholesterol) in- diaromatic ergosterol derivatives and other steroidal analogues in volves a selective aromatization of rings A and B of the tetracyclic an unprecedented simple, one-pot and convenient synthetic route. skeleton with simultaneous regioselective formation of a double In the process, we have established the key factor as the selectivity bond between C-14 and C-15. of the solvents (having 1,4-ethereal oxygens) towards the forma- On the basis of the previous literature, 19 it may be antici- tion of the aromatized products. Thorough solvent-dependant pated that the aromatization is accompanied with the methyl study of the model reaction reveals valuable product composi- migration (from 1a , 1b and 8) as well as demethylation (from 8) tion, which may be exploited, specially, for the synthesis of bi- of the methyl situated at the ring juncture (C-19) in the starting ologically important steroid molecules. By using the established materials. The solvent selectivity suggests a possible explana- solvent-selective steroidal methodology, the yield of the natural tion towards the regioselective dehydrogenation. It was as- product, diaromatic ergosterol ( 9) was optimized at 12%. The same sumed that a transient dioxonium intermediate of the ethers reaction is also found to be an easy and ef ficient access to the hu- may be formed with selenium moiety offering the furnished man metabolism research demanded 4 b,7 a-dihydroxy steroids. regioselectivity. Furthermore, single crystal X-ray crystallography has resolved the Author's personal copy

P. Ghosh et al. / Tetrahedron 68 (2012) 6485 e6491 6489 molecular structures, for the first time in their class, of similar The proportion of reactants to selenium dioxide, for the other diaromatic cholesterol derivative ( 2a ) and triacetylated 4 b,7 a- oxidation reactions, was kept constant throughout the various dihydroxy cholesterol derivative ( 4aa). experiments.

4.4. Product characterization 4. Experimental section 4.4.1. 1-Methyl-19-norcholesta-1,3,5(10),6,8(9),14(15)-hexaene  4.1. General (2a ). Needle shaped white crystals (CHCl 3/MeOH), mp 96 e97 C; 1 3e6% yield; Rf (petroleum ether) 0.95; H NMR (300 MHz, Melting points were measured in open capillary methods and CD 3COCD 3): 0.89 (d, J¼6.6 Hz, 6H), 1.05 (s, 3H), 1.08 (d, J¼6.0 Hz, were uncorrected. The FAB mass spectra were recorded on a Jeol SX 3H), 2.50e2.59 (m, 1H), 2.95 (s, 3H), 3.55e3.59 (m, 2H), 6.23 (s, 1H), 102/Da-600 mass spectrometer/Data System using Argon/Xenon as 7.25 (m, 2H), 7.59 e7.64 (m, 2H), 7.72 (d, J¼9.0 Hz, 1H); 13 C NMR the FAB gas. The DART-MS was recorded on a JEOL-AccuTOF JMS- (75 MHz, CDCl3): 14.8 (CH 3), 19.0 (CH 3), 22.6 (CH 3), 22.8 (CH 3), 23.8 T100LC mass spectrometer having a DART (Direct Analysis in Real (CH2), 27.3 (CH 3), 28.0 (CH), 28.7 (CH 2), 34.1 (CH), 35.9 (CH 2), 36.1 1 13 Time) source. H NMR and C NMR spectra were recorded on (CH2), 37.2 (CH 2), 39.6 (CH 2), 44.4 (C), 57.5 (CH), 120.4 (CH), 123.2 Brucker Avance 300 MHz FT-NMR spectrometer using 5 mm BBO (CH), 124.8 (CH), 127.5 (CH), 127.8 (CH), 129.2 (C), 130.7 (CH), 132.5 À1 probe. Either CDCl 3 or DMSO- d6 or CD 3COCD 3 was used as solvent (C), 133.7 (C), 134.6 (C), 135.5 (C), 148.8 (C); IR (Nujol, cm ): 3057, and TMS as reference material. Data are presented as follows: 1380, 1365, 1278, 1257, 1103, 1060, 987, 935, 894, 821, 788, 754. þ þ þ Chemical shiftdin parts per million on the scale relative to dTMS¼0; DART-MS (ESI ), m/z: 362 ([M þ2H] , 27%), 361 ([M þH] , 100), 360 coupling constantdJ/Hz. Elemental analysis was performed using ([M]þ, 30), 359 (23), 347 (5). Elemental analyses: found: C, 89.88; H, a Vario EL-III elementary analyser. Infrared spectra were recorded 10.15. C 27 H36 requires C, 89.93; H, 10.07%. on Shimadzu FT-IR 8300 Spectrometer as neat or thin films (KBr or Nujol) as indicated in the experimental procedures, and at room 4.4.2. 4b-Hydroxy cholesterol (3 b,4 b-dihydroxy-5-cholestene, À1  temperature. Frequencies are given in wave numbers (cm ). For 3a ). Needle shaped white crystals (CHCl3/MeOH), mp 168 e170 C; e 1 column chromatography silica gel G, 60 120 mesh was used with 10 e75% yield; Rf (30% ethyl acetate/petroleum ether) 0.45; H NMR petroleum ether/ethyl acetate mixture as the eluent. For thin layer (300 MHz, CDCl 3): 0.68 (s, 3H), 0.86 (d, J¼6.0 Hz, 6H), 0.90 (d, chromatography (TLC), freshly made silica gel plates (using silica J¼6.3 Hz), 1.18 (s, 3H), 1.25 e1.66 (m, 13H), 1.81 e2.10 (m, 8H), gel for TLCþpetroleum ether) were used and visualization was 3.54e3.56 (m, 1H), 4.13 (s, 1H), 5.67 (s, 1H); 13 C NMR (75 MHz, achieved by staining with iodine. CDCl3): 11.9 (CH 3), 18.7 (CH 3), 20.5 (CH 2), 21.1 (CH 3), 22.6 (CH 3), 22.8 (CH3), 23.8 (CH 2), 24.3 (CH 2), 25.4 (CH 2), 28.0 (CH), 28.2 (CH 2), 31.9 (CH), 32.1 (CH ), 35.8 (CH), 36.0 (C), 36.2 (CH ), 36.9 (CH ), 39.5 4.2. X-ray crystallography 20 2 2 2 (CH2), 39.7 (CH 2), 42.3 (C), 50.2 (CH), 56.1 (CH), 56.9 (CH), 72.5 (CH), 77.3 (CH), 128.8 (CH), 142.8 (C); IR (KBr, cm À1): 3382 (br), 1168, 978; Intensity data for 2a and 4aa were measured at 100 K on FABMS (ESI þ), m/z: 402 (12%), 401 (11), 399 (13), 386 (29), 385 a Bruker SMART APEX diffractometer with Mo K a radiation. Data (100), 384 (50), 383 (48), 368 (28), 367 (62). Elemental analyses: processing (APEX2 and SAINT) 1x and absorption correction for 2a found: C, 80.44; H, 11.59. C H O requires C, 80.53; H, 11.52%. (SADABS)2x were accomplished by standard methods. The struc- 27 46 2 tures were solved by direct-methods with SHELXS-97 3x and re- 4.4.3. 4 b,7 a-Dihydroxy cholesterol (3 b,4 b,7 a-trihydroxy-5- finement (anisotropic displacement parameters, hydrogen atoms in cholestene, 4a ). White amorphous solid, mp 193 e194 C; 5 e78% the riding model approximation and a weighting scheme of the yield; R (70% ethyl acetate/petroleum ether) 0.55; 1H NMR (300 MHz, form w ¼ 1=½s2ðF2Þ þ ð aP Þ2 þ bP Š for P ¼ ð F2 þ 2F2Þ=3) was on F2 f o o c CDCl ): 0.69(s,3H), 0.86(d, J¼6.6 Hz, 6H), 0.92(d, J¼6.6 Hz, 3H),1.18(s, by means of SHELXL-97. 3x The absolute structures of both 2a and 3 3H), 3.57 e3.65 (m, 1H), 3.94 (t, J¼3.0 Hz, 1H), 4.18 (d, J¼3.0 Hz, 1 Hz), 4aa could not be determined experimentally and so for each, it was 5.86 (d, J¼3.0 Hz,1H); 13 C NMR (75 MHz, CDCl ): 11.6 (CH ),18.7 (CH ), assigned based on that of cholesterol. As such, 2082 ( 2a ) and 3934 3 3 3 19.4 (CH ), 20.1 (CH ), 22.6 (CH ), 22.8 (CH ), 23.8 (CH ), 24.4 (CH ), (4aa) Friedel pairs were averaged in the final re finements. For the 3 2 3 3 2 2 25.1 (CH ), 28.0 (CH), 28.3 (CH ), 35.8 (CH), 36.2 (CH ), 36.7 (CH ), 37.0 re finement of 2a , high thermal motion was displayed by the C27 2 2 2 2 (C), 37.6(CH), 39.1 (CH ), 39.5 (CH ), 42.1 (C), 42.6 (CH), 49.4 (CH), 55.9 atom. However, multiple sites were not resolved for this atom. For 2 2 (CH), 65.3 (CH), 72.1 (CH), 77.0 (CH), 129.7 (CH), 147.0 (C); IR (KBr, the re finement of 4aa, the O4 atom displayed elongated anisotropic cm À1): 3349,1153,1066, 965. DART-MS (ESI þ), m/z: 402 (6%), 401 (26), displacement parameters and hence, this atom was re fined with 385 (5), 384 (28), 383 (100), 366 (4), 365 (10). Elemental analyses: the ISOR command in SHELXL-97. 3x Figs. 1 and 2 were drawn with found: C, 77.37; H, 11.17. C H O requires C, 77.45; H, 11.08%. ORTEP4x at the 50% probability level. Data manipulation and in- 27 46 3 terpretation were with WinGX 5x and PLATON.6x 4.4.4. 1-Methyl-19-nor b-sitosta-1,3,5(10),6,8(9),14(15)-hexaene  (2b ). Needle shaped white crystals (CH 2Cl 2/MeOH), mp 87 e88 C, 1 4.3. Representative procedure for the oxidation reactions 7% yield; Rf (petroleum ether) 0.95; H NMR (300 MHz, CDCl 3): 0.87 (d, J¼7.2 Hz, 6H), 0.94 (d, J¼7.2 Hz, 3H), 1.07 (s, 3H), 1.10 (s, 3H), To a solution of 1a (770 mg, 2 mmol) in dioxane (15 mL) was 1.15 e1.35 (m, 6H), 1.39 e1.60 (m, 4H), 1.68 e1.74 (m, 4H), 2.19 e2.35 added selenium dioxide (777 mg, 7 mmol), the mixture was heated (m, 2H), 2.94 (s, 3H), 3.51e3.55 (m, 2H), 6.15 (s, 1H), 7.20 e7.25 (m, at 100 C for 24 h. The reaction mixture was then cooled and the 2H), 7.55 e7.60 (m, 2H), 7.66 (d, J¼8.7 Hz, 2H); 13 C NMR (75 MHz, black selenium deposited was filtered off. To the filtrate, ether CDCl3): 12.0, 14.8, 19.0, 19.9, 23.1, 26.0, 27.3, 28.7, 29.2, 29.4, 33.9, (50 mL) was poured and was washed successively with water and 34.5, 36.0, 37.2, 44.5, 45.9, 57.4, 120.4, 123.2, 124.8, 127.6, 127.8, À1 then with saturated brine solution, dried over Na 2SO 4 and concen- 129.2, 130.7, 132.5, 133.7, 134.6, 135.6, 148.9; IR (Nujol, cm ): 3049, trated in vacuo to give a reddish gummy residue. The compounds 1367, 1278, 1112, 987, 896, 821, 844, 789, 755. Elemental analyses: presented therein, were then separated by column chromatography found: C, 89.69; H, 10.31. C 29 H40 requires C, 89.62; H, 10.38%. eluted successively by petroleum ether, petroleum ether/ethyl acetate ¼17:3 and petroleum ether/ethyl acetate ¼3:2 to afford 2a 4.4.5. 4b-Hydroxy b-sitosterol (3 b,4 b-dihydroxy-5-b-sitostene,  (44 mg, 6%), 3a (403 mg, 50%) and 4a (217 mg, 26%), respectively. 3b ). Needle shaped white crystals (CHCl 3/MeOH), mp 162 e164 C, Author's personal copy

6490 P. Ghosh et al. / Tetrahedron 68 (2012) 6485 e6491

1 1 51% yield; Rf (30% ethyl acetate/petroleum ether) 0.45; H NMR ethyl acetate/petroleum ether) 0.55; H NMR (300 MHz, CDCl 3): (300 MHz, CDCl 3): 0.68 (s, 1H), 0.81 (d, J¼6.9 Hz, 3H), 0.86 (d, 0.74 (s, 3H), 0.92 (d, J¼6.3 Hz, 6H), 0.97 (d, J¼6.3 Hz, 3H), 1.31 (s, J¼6.9 Hz, 6H), 0.92 (d, J¼6.6 Hz, 3H), 1.18 (s, 3H), 3.51 e3.61 (m, 1H), 3H), 2.21 (s, 1H), 4.45 (s, 1H), 5.01 e5.06 (m, 1H), 5.79 (s, 1H), 7.49 (t, 13 13 4.15 (s, 1H), 5.68 (s, 1H); C NMR (75 MHz, CDCl 3): 11.9, 12.0, 18.8, J¼7.5 Hz, 2H), 7.61 (t, J¼6.6 Hz, 1H), 8.11 (d, J¼7.5 Hz, 2H); C NMR 19.0, 19.8, 20.5, 21.1, 23.1, 24.3, 25.4, 26.0, 28.2, 29.1, 31.8, 32.1, 33.9, (75 MHz, CDCl3): 11.3, 18.1, 20.0, 20.5, 21.3, 22.0, 22.3, 23.3, 23.7, 36.0, 36.1, 36.9, 39.7, 42.3, 45.8, 50.2, 56.0, 56.9, 72.5, 77.3, 128.8, 27.5, 27.7, 31.2, 31.6, 35.2, 35.6, 35.7, 36.5, 39.0, 39.1, 41.8, 49.7, 55.5, 142.8; IR (Nujol, cm À1): 3392, 1172, 1069, 977. Elemental analyses: 56.3, 75.1, 75.5, 127.8 (2), 129.1 (3), 129.7, 132.5, 140.9, 165.1; IR À1 found: C, 80.89; H,11.66. C 29 H50 O2 requires C, 80.86; H, 11.71%. (Nujol, cm ): 3533 (sharp), 1694, 1682, 1282, 1127, 1068, 1026, 967, 920, 845, 709. DART-MS (ESI þ), m/z: 491 (7%), 490 (37), 489 (100), 4.4.6. 4b,7 a-Dihydroxy b-sitosterol (3 b,4 b,7 a-trihydroxy-5-b-sitos- 368 (3), 367 (10). Elemental analyses: found: C, 80.65; H, 9.84.  tene, 4b ). White solid, mp 192 e193 C; 22 e72% yield; Rf (70% ethyl C34 H50 O3 requires C, 80.57; H, 9.95%. 1 acetate/petroleum ether) 0.55; H NMR (300 MHz, DMSO- d6): 0.63 (s, 3H), 0.79 (d, J¼6.3 Hz, 3H), 0.83 (d, J¼6.0 Hz, 6H), 0.91 (d, 4.4.11. 3 b-Benzoxy-6 a-hydroxy-4-cholestene ( 7b ). Needle shaped  J¼6.3 Hz, 3H), 1.07 (s, 3H), 3.26 e3.38 (m, 1H), 3.64 (t, J¼3.0 Hz, 1H), white crystals (CHCl 3/MeOH), mp 142 e144 C, 34% yield; Rf (12% 13 1 3.89 (d, J¼3 Hz, 1H), 5.58 (d, J¼4.8 Hz, 1H); C NMR (75 MHz, ethyl acetate/petroleum ether) 0.55; H NMR (300 MHz, CDCl 3): DMSO-d6): 11.9,12.2, 12.3, 19.1, 19.4, 19.6, 20.2, 23.0, 24.2, 25.4, 25.8, 0.66 (s, 3H), 0.86 (d, J¼6.6 Hz, 6H), 0.91 (d, J¼6.3 Hz, 3H), 1.20 (s, 28.4, 29.1, 33.8, 36.0, 36.9, 37.4, 37.9, 41.9, 42.3, 45.6, 49.4, 55.8, 63.8, 3H), 3.75 (t, J¼6 Hz, 1H), 5.67 (s, 1H), 5.93 (d, J¼3 Hz, 1H), 7.44 (t, 72.1, 77.1, 129.2, 145.7; IR (Nujol, cm À1): 3392, 1165, 1078, 967. El- J¼7.5, 2H), 7.55 (t, J¼7.2 Hz, 1H), 8.04 (d, J¼8.1 Hz, 2H); 13 C NMR emental analyses: found: C, 77.88; H, 11.32. C 29 H50 O3 requires C, (75 MHz, CDCl3): 11.8, 18.7, 20.7, 22.6, 22.8, 23.8, 24.2, 26.0, 28.0, 77.96; H, 11.29%. 28.2, 31.7, 32.1, 35.8, 36.0, 36.2, 36.8, 39.5, 39.6, 42.3, 50.2, 56.1, 56.8, 72.0, 80.0, 128.4, 128.4, 129.6, 129.6, 130.5, 132.0, 133.0, 138.9, 166.8; 4.4.7. 3b,4 b,7 a-Triacetoxy-5-cholestene ( 4aa). Colourless cubic IR (Nujol, cm À1): 3442, 3392, 1686, 1674, 1273, 1170, 1110, 977, 899,  þ crystals (CHCl3/MeOH), mp 170 e171 C; 94% yield from 4a ; Rf (5% 759, 711. DART-MS (ESI ), m/z: 491 (4%), 490 (36), 489 (100), 386 1 ethyl acetate/petroleum ether) 0.75; H NMR (300 MHz, CDCl 3): (26), 385 (89), 368 (24), 367 (83). Elemental analyses: found: C, 0.66 (s, 3H), 0.86 (d, J¼6.3 Hz, 6H), 0.92 (d, J¼6.6 Hz, 3H), 1.14 (s, 80.48; H, 9.86. C 34 H50 O3 requires C, 80.57; H, 9.95%. 3H), 2.01 (s, 3H), 2.04 (s, 3H), 2.06 (s, 3H), 4.77 e4.82 (m, 1H), 5.06 (t, J¼4.5 Hz, 1H), 5.52 (s, 1H), 5.93 (d, J¼6.0 Hz, 1H); 13 C NMR (75 MHz, 4.4.12. 4 b-Hydroxy diosgenin (3 b,4 b-dihydroxy-5-spirostene,  CDCl3): 11.4 (CH 3), 18.8 (CH 3), 20.2 (CH 2), 21.2 (CH 3), 21.3 (CH 3), 21.6 12 ). White solid, mp 171 e173 C, 30 e58% yield; Rf (30% ethyl ace- 1 (CH3), 22.3 (CH 2), 22.6 (CH 3), 22.8 (CH 3), 23.9 (CH 2), 24.1 (CH 2), 28.0 tate/petroleum ether) 0.45; H NMR (300 MHz, CDCl 3): 0.79 (d, (CH), 28.1 (CH 2), 35.7 (CH), 35.8 (CH), 36.2 (CH 2), 36.3 (CH 2), 37.0 J¼3.0 Hz, 3H), 0.80 (s, 3H), 0.97 (d, J¼6.8 Hz, 3H), 1.21 (s, 3H), 2.32 (C), 38.9 (CH2), 39.5 (CH 2), 42.2 (CH), 43.5 (CH), 49.0 (CH), 55.9 (CH), (s, 1H), 2.43 (d, J¼6 Hz, 1H), 3.37 (t, J¼10.8 Hz, 1H), 3.46 (d, J¼3.0 Hz, 67.6 (CH), 72.2 (CH), 75.0 (CH), 128.4 (CH), 143.9 (C), 169.7 (C), 170.2 1H), 3.48e3.55 (m, 1H), 4.13 (d, J¼3 Hz, 1H), 4.40 (dd, J¼15.0 and À1 13 (C), 170.4 (C); IR (Nujol, cm ): 1734, 1365, 1246, 1044, 1012, 976, 7.5 Hz, 1H), 5.66 (d, J¼2.7 Hz, 1H); C NMR (75 MHz, CDCl 3): 14.5 þ 941, 889. DART-MS (ESI ), m/z: 485 (6%), 426 (29), 425 (100), 384 (CH3), 16.3 (CH 3), 17.1 (CH 3), 20.3 (CH 2), 21.0 (CH 3), 25.3 (CH 2), 28.8 (5), 383 (17), 366 (5), 365 (16). Elemental analyses: found: C, 79.69; (CH2), 30.3 (CH), 31.4 (CH 2), 31.8 (CH 2), 32.2 (CH), 36.2 (C), 36.9 H, 10.62. C 33 H52 O3 requires C, 79.77; H, 10.56%. (CH2), 39.7 (CH 2), 40.3 (C), 41.6 (CH), 50.1 (CH), 56.6 (CH 2), 62.0 (CH), 66.9 (CH 2), 72.4 (CH), 77.2 (CH), 80.8 (CH), 109.3 (C), 128.3 4.4.8. 3b-Acetoxy-4b-hydroxy-5-cholestene (6a ). White solid, mp (CH), 142.9 (C); IR (Nujol, cm À1): 3392, 1169, 1047, 976. FABMS, m/z:  1 175 e176 C, 35% yield; Rf (15% ethyl acetate/petroleum ether) 0.45; H 432 (37%), 431 (90), 430 (39), 429 (77), 414 (49), 413 (100), 412 (27), NMR (300 MHz, CDCl 3): 0.68 (s, 3H), 0.86 (d, J¼6.6 Hz, 6H), 0.91 (d, 411 (28), 395 (25). Elemental analyses: found: C, 75.21; H, 9.79. J¼6.6 Hz, 3H), 1.22 (s, 3H), 2.11 (s, 3H), 4.25 (s, 1H), 4.69 e4.76 (m, 1H), C27 H42 O4 requires C, 75.29; H, 9.84%. 13 5.71 (s,1H); C NMR (75 MHz, CDCl 3): 11.9 (CH 3),18.7 (CH 3), 20.5 (CH 2), 21.1(CH 3), 21.4 (CH 3), 21.7 (CH 2), 22.6 (CH 3), 22.8 (CH 3), 23.8 (CH 2), 24.2 4.4.13. 4 b,7 a-Dihydroxy diosgenin (3 b,4 b,7 a-trihydroxy-5-  (CH 2), 28.0 (CH), 28.2 (CH 2), 31.7 (CH), 32.1 (CH 2), 35.8 (CH), 36.2 (CH 2), spirostene, 13 ). White solid, mp 201 e202 C, 17 e69% yield; Rf 1 36.6 (C), 36.9 (CH 2), 39.5 (CH 2), 39.6 (CH 2), 42.3 (C), 50.2 (CH), 56.1(CH), (70% ethyl acetate/petroleum ether) 0.50; H NMR (300 MHz, 56.8 (CH), 75.5 (CH), 75.6 (CH),129.5 (CH),141.5 (C),170.2 (C); IR (Nujol, CDCl3): 0.79 (d, J¼3.0 Hz, 3H), 0.80 (s, 3H), 0.98 (d, J¼6.9 Hz, 3H), þ cm À1): 3412,1737,1279,1046. DART-MS (ESI ), m/z: 429 (11%), 428 (61). 1.20 (s, 3H), 3.37 (t, J¼10.8 Hz, 1H), 3.45 e3.49 (m, 1H), 3.52e3.64 427 (100%), 385 (16), 368 (18), 367 (58). Elemental analyses: found: C, (m, 1H), 3.94 (t, J¼4.5 Hz, 1H), 4.09e4.19 (m, 1H), 4.48 (dd, J¼15.0 13 78.39; H, 10.79. C 29 H48 O3 requires C, 78.31; H, 10.89%. and 7.5 Hz, 1H), 5.86 (d, J¼4.8 Hz, 1H); C NMR (75 MHz, CDCl 3): 14.6 (CH 3), 16.1 (CH 3), 17.2 (CH 3), 19.4 (CH 3), 20.0 (CH 2), 25.1 (CH 2), 4.4.9. 3b-Acetoxy-6a-hydroxy-4-cholestene (7a ). White solid, mp 28.8 (CH2), 30.3 (CH), 31.4 (CH 2), 31.9 (CH 2), 36.6 (CH 2), 37.2 (CH),  139 e140 C, 32% yield; Rf (20% ethyl acetate/petroleum ether) 0.45; 39.1 (CH 2), 40.4 (C), 41.7 (CH), 42.5 (CH), 49.0 (CH), 62.0 (CH), 65.2 1 H NMR (300 MHz, CDCl 3): 0.68 (s, 3H), 0.87 (d, J¼6.6 Hz, 6H), 0.91 (CH), 66.9 (CH 2), 72.1 (CH), 76.9 (CH), 80.8 (CH), 109.3 (C), 129.6 (d, J¼6.6 Hz, 3H), 1.18 (s, 3H), 2.08 (s, 1H), 3.60 e3.69 (m, 1H), 5.38 (CH), 146.9 (C); IR (Nujol, cm À1): 3395, 1172, 1054, 978. Elemental 13 (d, J¼2.7 Hz, 1H), 5.85 (d, J¼3.0 Hz, 1H); C NMR (75 MHz, CDCl 3): analyses: found: C, 72.49; H, 9.41. C 27 H42 O5 requires C, 72.59; H, 11.8 (CH 3), 18.7 (CH 3), 20.4 (CH 3), 20.6 (CH 2), 21.6 (CH 3), 22.6 (CH 3), 9.48%. 22.8 (CH3), 23.8 (CH 2), 24.2 (CH 2), 25.8 (CH 2), 28.0 (CH), 28.2 (CH 2), 31.6 (CH), 32.1 (CH 2), 35.8 (CH), 36.0 (C), 36.2 (CH 2), 36.8 (CH 2), 39.5 4.4.14. 19-Norergosta-1,3,5,7,9,14,22-heptaene ( 9). Pale yellow  (CH2), 39.6 (CH 2), 42.3 (C), 50.2 (CH), 56.1 (CH), 56.8 (CH), 71.7 (CH), needles, mp 125 e126 C (Chloroform/methanol), 5.5 e12% yield; Rf À1 1 79.3 (CH), 128.8 (CH), 138.8 (C), 171.2 (C); IR (Nujol, cm ): 3398, (petroleum ether) 0.95; H NMR (300 MHz, CDCl 3): 0.84 (d, þ 1738, 1260, 1237, 1074. DART-MS (ESI ), m/z: 428 (14%), 427 (43), J¼4.2 Hz, 3H), 0.88 (d, J¼4.2 Hz, 3H), 0.95 (s, 3H), 0.98 (d, J¼5.1 Hz, 385 (77), 368 (29). Elemental analyses: found: C, 78.38; H, 10.96. 3H), 1.15 (d, J¼6.6 Hz, 3H), 1.52 (m, 1H), 1.76 (m, 1H), 1.90 (m, 1H), C29 H48 O3 requires C, 78.31; H, 10.89%. 1.61 and 2.33 (m, 2H), 2.33 (m, 1H), 2.21 and 2.36 (m, 2H), 3.14 and 3.29 (m, 2H), 5.24 (m, 1H), 5.30 (m, 1H), 6.12 (t, J¼3 Hz, 1H), 7.42 (m, 4.4.10. 3 b-Benzoxy-4b-hydroxy-5-cholestene (6b ). White feather 1H), 7.45 (m, 1H), 7.50 (m, 1H), 7.64 (m, 1H), 7.77 (d, J¼7.8 Hz, 1H),  13 like crystals (CHCl3/MeOH), mp 202 e204 C, 37% yield; Rf (10% 8.01 (d, J¼9.0 Hz, 1H); C NMR (75 MHz, CDCl 3): 15.4 (CH 3), 17.7 Author's personal copy

P. Ghosh et al. / Tetrahedron 68 (2012) 6485 e6491 6491

(CH3)19.7 (CH 3), 20.0 (CH 3), 21.2 (CH 3), 23.7 (CH 2), 33.1 (CH), 36.5 Bertilsson, L.; Broome, U.; Einarsson, C.; Diczfalusy, U. J. Biol. Chem. 2001 , 276 , e e € (CH ), 36.9 (CH ), 39.0 (CH), 42.9 (CH), 45.2 (C), 57.2 (CH), 120.7 38685 38689; (e) Breuerr, O. J. Lipid Res. 1995 , 36 , 2275 2281; (f) Lutjohann, 2 2 D.; Marinova, M.; Schneider, B.; Oldenburg, J.; Bergmann, K. v.; Bieber, T.; (CH),123.6 (CH),123.7 (CH),125.2 (CH), 126.1 (CH), 126.3 (CH),128.5 Bj orkhem,€ I.; Diczfalusy, U. Int. J. Clin. Pharmacol. Ther. 2009 , 47 , 709 e715. (CH),128.5 (C), 130.2 (C),132.5 (CH),132.8 (2C),135.3 (CH) , 148.1 (C). 7. Ayaki, Y.; Yamasaki, K. J. Biochem. 1970 , 68 , 341 e346. These spectral data match that originally reported by Qin and Liu, 16 8. Bodin, K.; Andersson, U.; Rysledt, E.; Ellis, E.; Norlin, M.; Pikulova, I.; Eggersten, G.; Bj orkhem,€ I.; Diczfalusy, U. J. Biol. Chem. 2002 , 277 , 31534 e31540. a summary of which is found in Table S1 in Supplementary data . 9. (a) Ares, M. P. S.; Pom-Ares, M. I.; Moses, S.; Thyberg, J.; Juntti-Berggren, L.; Berggren, P.-O.; Hultgardh-Nilsson, A.; Kallin, B.; Nilsson, J. Atherosclerosis 2000 , 153 , 23 e35; (b) Bertolotti, M.; Carulli, N.; Menozzi, D.; Zironi, F.; Digrisolo, A.;

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