31295013018931.Pdf (6.180Mb)

SYNTHESIS OF STEROL BIOSYNTHESIS INHIBITORS

AND METABOLISM OF ISOTOPICALLY LABELED

STEROLS BY Saccharomyces cerevisiae

WEN ZHOU, B.S., M.S.

A DISSERTATION

CHEMISTRY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY

Approved

May, 1998 ACKNOWLEDGMENTS

I would like to express my deepest thanks to my advisor, Dr. W. David Nes, for his expert support and helpful advice during my graduate education at Texas Tech University. I would also like to thank Dr. Edward J. Parish and Dr. George I. Makhatadze for serving on my doctoral committee. I am grateful to former and current postdoctoral associates and graduate students in Dr. Nes' laboratory for their kind help, including: Dr. Dean Guo, Dr. Yusen Tong, Dr. Zhonghua Jia, Monica Lopez, Brian S. McCourt, Wenxu Zhou, Anil T. Mangla, Derek S. Nichols, Ling He. I would also like to thank Dr. John N. Marx for his technical advice and help for this research. I am also in debt to Mr. David Purkiss for his assistance in performing NMR spectral work for this research. Finally, I would like to thank my parents, Lijun Wang and Bingxian Zhou for their constant support and encouragement. The support of the Welch Foundation is greatly appreciated.

11 CONTENTS

ACKNOWLEDGMENTS 11 ABSTRACT ix LIST OF TABLES xi LIST OF FIGURES xii LIST OF SCHEMES xiv CHAPTER 1. INTRODUCTION 1 1.1. Structure and nomenclature 1 1.2. Background of sterol biosynthesis 2 1.3. Isopentenoid biosynthesis 4 1.4. §qualene biosynthesis 5 1.5. Lanosterol and cycloartenol biosynthesis 6 1.6. Pathways of sterol biosynthesis in fungi, animals, and plants 7 1.7. Sterol methylation mechanism 10 1.8. X*-group mechanism 12 1.9. Structure-fiinction relationships 13 1.10. Sterol methylation inhibitors 15 2. STUDIES ON STEROL METHYLATION MECHANISM 24 2.1. Mechanism studies of the sterol methylation in yeast 24 2.2. Mechanism studies of phytosterol methylation 28 2.3. Mechanismstudiesof the second sterol methylation 29 2.4. Molecular modeling and analysis of C-20 configuration 32 3. STUDIES ON STEROL METHYLATION INHIBITORS 37 3.1. Design, preparation and inhibition studies of C-20, C-21 and C-22 related sterol inhibitor 37 3.2. Design, preparation and inhibition studies of C-23 related inhibitors 42

111 3.3. Design, preparation and inhibition studies of C-24 related inhibitor 43 3.4. Design, preparation and inhibition studies of C-25 related inhibitors 44 3.5. Design, preparation and inhibition studies of C-26 and C-27 related inhibitors 45 3.6. Design, preparation and inhibition studies of C-28 related inhibitors 50 3.7. Summary 54 MATERLALS AND METHODS 60 4.1. Chemical reagents 60 4.2. Methods 60 NATURAL PRODUCTS ISOLATION AND BIOORGANIC SYNTHESIS 62 5.1. Isolation of cycloartenol from y-oryzanol (Figure 5.1) 62 5.2. Protection of 3 P hydroxyl group of sterols (Figure 5.2) 64 5.2.1 Acetate ester 66 5.2.1.1. Formation of acetate ester 66 5.2.1.2. Cleavage of acetate ester 66 5.2.2. (3a, 5)-Cyclo-6p-methoxy ether 66 5.2.2.1. Formation of (3a, 5)-cyclo-6p-methoxy ether 66 5.2.2.2. Cleavage of (3a, 5)-cyclo-6p-methoxy ether 68 5.2.3. Tetrahydropyranyl ether 69 5.2.3.1. Formation of tetrahydropyranyl ether 69 5.2.3.2. Cleavage of tetrahydropyranyl ether 69 5.2.4. Summary 70

13 5.3. Preparation of [27- C]lanosterol (Scheme 2.1) and [27-^^C]zymosterol 70 5.3.1 2-[^^C]Methyl-1 -acetoxy-methyl triphenylphosphonium iodide 44 70

IV 5.3.2. 24-al Lanosterol 46 71 5.3.3. 3p-Acetoxy-26-ester-[27-^^C]lanosterol47 71 5.3.4. 3p-Acetoxy-26-ol-[27-^^C]lanosterol48 72 5.3.5. [27-'^C]Lanosterol 49 72 5.3.6. [27-^^C]Zymosterol 73 5.4. Biosynthesis of [27-^^C]ergosterol 50 in S. cerevisiae strain GL7 (Scheme 2.2.1) 74 5.5. Preparation of [24-^H]lanosterol 53 and biosynthesis of [25-^H]ergosterol 54 in S. cerevisiae strain GL7 (Scheme 2.2) 75 5.5.1. 3p-Acetoxy-24-keto lanosterol 51 75

5.5.2. [245-^H]-245-ol-3p-Acetoxy lanosterol 52 75 5.5.3. [24-^H]Lanosterol 53 76 5.5.4. Biosynthesis of [25-^H]ergosterol 54 76 5.6. Biosynthesis of [28-^H2]ergosterol 56 in S. cerevisiae strain GL7 (Scheme 2.3) 76 5.7. Preparation of 20i?-22-aza-cholest-5-en-3P-ol 58a and 205'-22-aza-cholest-5-en-3P-ol 58b (Scheme 3.1) 76 5.8. Preparation of 3P-ol-cholesta-5(6), 20(22)£, 24(25)- trien-20-one 61, 3p-ol-cholesta-5(6), 20(21), 24(25)- trien-20-one 62 and 3p-ol-cholesta-5(6), 17(20)Z, 24(25) -trien-20-one 63 (Scheme 3.2) 78 5.8.1. 3p-Tetrahydropyranylooxy-21 -nor-cholesta- 5, 24-dien-20-one 59 78 5.8.2. 3 p-Tetrahydropyranylooxy-cholesta- 5(6), 20(21), 24(25)-trien-20-one 60 79 5.8.3. 3p-ol-Cholesta-5(6), 20(22)^, 24(25)-trien-20-one 61, 3p-ol-cholesta-5(6), 20(21), 24(6)-trien-20-one 62 and 3p-ol-cholesta-5(6), 17 (20)Z, 24(25)-trien-20-one 63 79 5.9. Preparation of 20-epidesmosterol 72b (Scheme 3.3) 81 5.9.1. i-Pregnenolone 64 81 5.9.2. 20-Keto-24(25)-en-i-sterol 65 81 5.9.3. 20(21), 24(25)-dien-i-Sterol 66 82 5.9.4. 20(21)-en-24(25)-Epoxide-i-sterol67 82 5.9.5. 20(R, 5)-24(25)-Epoxide-i-sterol 68 83 5.9.6. 20(i?, 5)-25-ol-i-Sterol 69 83 5.9.7. 20iR, S)-24(25)-en- i-Sterol 70 and 20(R, S)- 25(27)-en- i-sterol 71 83 5.9.8. Desmosterol 72a and 20-epidesmosterol 72b 84 5.10. Preparation of 23-aza-cholest-5-en-3p-ol 76 and 25-aza-cholesta-5, 22-dien-3p-ol 77 (Scheme 3.4) 86 5.10.1. (3a, 5)-Cyclo-6p-methoxy-cholest-22-al 75 S6 5.10.2. 23-Aza-cholest-5-en-3-ol 76 86 5.10.3. 25-Aza-cholesta-5, 22-dien-3-ol 77 87 5.11. Preparation of 24-aza-cholest-8-en-3p-ol 82 (Scheme 3.5) 88 5.11.1. 3p-Acetoxy-cholest-8-en-24-al 79 88 5.11.2. 3p-Acetoxy-24-[(tert-butyldimethylsilyl)oxy] -cholesta-8(9), 23(24)-dien 80 88 5.11.3.3p-Acetoxy-cholest-8-en-23-al81 89 5.11.4. 24-Aza-cholest-8-en-3p-ol 82 89 5.12. Preparation of 25-aza sterols. (Scheme 3.6) 90 5.12.1. 25-Aza sterols 90 5.12.2. 26-Nor-25-aza-cholest-8-en-3P-ol 85 90 5.13. Preparation of 26, 27-dinor-cholesta-8(9), 24(25) -dien-3p-ol 86 (Scheme 3.6) 92 5.14. Preparation of 26, 27-cyclopropylidene sterols (Scheme 3.7) 92 3 5.14.1. Preparation of [3a- H]-26, 27-cyclopropylidene -cholesta-8, 24-dien-3P-ol 92a 92 5.14.1.1. 26, 27-Cyclopropylidene-cholesta-8, 24- dien-3P-ol90 92 5.14.1.2. 3-Keto-26, 27-cyclopropylidene- cholesta-8(9), 24(25)-dien-3p-ol 91 93

VI 5.14.1.3. [3a-^H]-26, 27-Cyclopropylidene- cholesta-8, 24-dien-3p-ol 92 94 5.14.2. Preparation of 26, 27-cyclopropylidene-9p, 19- cyclopropyl-cholesta-24-en-3p-ol 93 94 5.14.3. Preparation of 26, 27-cyclopropylidene-4,4-dimethyl- 14a-methyl-cholesta-8(9), 24(25)-dien-3p-ol 94 95 5.14.4. Preparation of 26, 27-cyclopropylidene-cholesta- 5(6), 24(25)-dien-3p-ol 95 95 5.15. Preparation of 24(25), 26(26')-diene sterols (Scheme 3.8) 95 5.15.1.3 p-Acetoxy-26-al zymosterol 96 96 5.15.2. 24(25)-26(26')-Diene zymosterol 97 96 5.15.3. 24(25)-26(26')-Diene cycloartenol 98 97 5.15.4. 24(25)-26(26')-Diene lanosterol 99 97 5.15.5. 24(25)-26(26')-Diene desmosterol 100 97 5.16. Preparation of 8(9), 14(15), 24(25)-trien-25-ethylnyl- cholesta-3P-ol 103 (Scheme 3.9) 97 5.17. Preparation of 24(R, S), 25-methano sterols (Scheme 3.10) 100 5.17.1. 3p-Acetoxy-24, 25-dichloromethano zymosterol 104 100 5.17.2. 24 (R, S), 25-Methano-zymosterol 105 100 5.17.3. 24 (R, S), 25-Methano-cycloartenol 106 101 5.17.4. 24 (R, S), 25-Methano-lanosterol 107 101 5.17.5. 24 {R. 5), 25-Methano-desmosterol 108 102 5.18. Preparation of 24(.^, 5), 25-epimino lanosterol 110, 25-amino lanosterol 111 and 28-A^-methyl-24(i^, S), 25-epimino lanosterol 112 (Scheme 3.11) 102 5.18.1. Preparation of 24 (R, S), 25-epimino lanosterol 110 102 5.18.2. Preparation of 25-amino lanosterol 111 103 5.18.3. 28-A^-Methyl-24(i?, S), 25-epimino lanosterol 112 103 5.18.4. 24(R, S), 25-Epimino zymosterol 113 104 5.19. 24, 28-Methano cholesterol US (Scheme 3.12) 104

Vll 5.20. 24(R, S), 28(i?, 5)-Methano fucosterol 117 (Scheme 3.12) 104 5.21. 24(i?, 5), 28(i?, 5)-Epimino fucosterol 118 (Scheme 3.12) 105 5.22. Preparation of 24a-amino lanosterol 120a and 25P-amino lanosterol 120b (Scheme 3.13) 106 5.22.1. 3p-Acetoxy-24-oximo lanosterol 119 106 5.22.2. 24a-Amino lanosterol 120a and 24p-amino lanosterol 120b 106 5.23. Preparation of 25a-amino-26-nor cholesterol 122a and 25p-amino-26-nor cholesterol 122b (Scheme 3.13) 107 5.24. Preparation of 24-vinyl lanosterol 125, 29', 29"-cyclopropylidene lanosterol 126 and 24-methylene zymosterol 127 (Scheme 3.14) 108 5.24.1. 3P-Acetoxy-24-methylene lanosterol 123 108 5.24.2. 3P-Acetoxy-24(i?, 5)-28-al-lanosterol 124 109 5.24.3. 24(R, 5)-Vinyl-lanosterol 125 110 5.24.4. 24(R, S)-29\ 29"-CyclopropyHdene lanosterol 126 110 5.24.5. 24-Methylene zymosterol 127 111 5.25. Preparation of [26-^H]lanosterol 131 (Scheme 5.1) 111 5.25.1. 3p-Acetoxy-26-al-lanosterol 129 111 5.25.2. [26-^H]-3p-Acetoxy-26-ol lanosterol 130 112 5.25.3. [26-^H]Lanosterol 131 112 REFERENCES 114 APPENDDC A. ABBREVIATIONS 121 APPENDIX B. INDEX OF CHEMICALS 124

Vlll ABSTRACT

The dissertation focuses on the chemistry and mechanism of sterol biomethylation catalyzed by (S)-adenosyl-L-methionine: A^'*^^^^ -sterol methyl transferase (SMT) enzyme. The research is composed of two parts: synthesis of sterol methylation inhibitors and isotopically labeled substrates and mechanism studies of the SMT enzyme. Part 1 describes preparation of ^H and ^^C-isotopically labeled sterols, and the design, synthesis and enzymatic evaluation of three types of sterol methylation inhibitors: (1) substrate analogs which act as product inhibitors of the reaction; (2) substrate analogs which act as mechanism-based inactivators; and (3) transition state analogs. The synthetic work focused on modification of the side chains of cholesterol, zymosterol, lanosterol, and cycloartenol which are natural substrates in C-methylation reactions. The sterol side chain was modified at C-20, C-21, and C-22 positions to study the effect of configuration and conformation of the sterol side chain on biomethylation. A series of novel sterol methylation inhibitors containing aza, aziridine, and ammonium groups at positions C-22 to C-25 were synthesized to serve as transition state analogs. Substrate analogs with the 26, 27-cyclopropylidene functional group were also synthesised and discovered to be potent irreversible mechanism-based inhibitors of the SMT enzyme. Several new 24, 25- methano and 24, 28-methano sterols, 24-vinyl and 29', 29"-cyclopropylidene lanosterols, and 24(25), 26(26')-diene and 24(25)-en-25-ethylnyl sterols have been synthesized. All the inhibitors were characterized by gas chromatography-mass spectrometry, high pressure liquid chromatography, and 'H and ^^C nuclear magnetic resonance spectroscopy. Part 2 describes research on the coupled methylenation-deprotonation of C-24 of the sterol side chain in plant and fungal sterol C-methylation reactions. These studies involved determination of the stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation and of C-28 deprotonation. To accomplish our aims, [27-'^C], [24- ^H] and [28-^H2] labeled sterols were prepared and assayed with SMT enzymes from a fungus {Saccharomyces cerevisiae) and a plant (Arabidopsis thaliana). As a result.

IX migration of the hydrogen from C-24 to C-25 was found to be introduced from Re-facQ of the 24, 25-double bond of the sterol side chain to generate the similar 25R stereochemistry in S. cerevisiae and A. thaliana, suggesting a similar topography of the SMT active site. LIST OF TABLES

1.1. Sterol substrate specificity for plant and fungal A^"^^^^^ -SMT 16 3.1. Inhibition of the SMT enzyme from S. cerevisiae by substrate and inhibitors 55 5.1 NMR spectra of cycloartenol 65 5.2 ^^C-NMR (75 MHz) of 20iJ-desmosterol and 205'-desmosterol 85 5.3 Characterization of 25-aza sterols 91 5.4 ^^C-NMR data of 103 (8(9), 14(15), 24(25)-trien-cholesta-3P-ol) and 128 (8(9), 14(15)-dien-cholesta-3p-ol) 99

XI LIST OF FIGURES

1.1. Stereochemical features and numbering system of sterols 1 1.2. Conformational perspective of a generic sterol 2 1.3. Isopentenoid biosynthetic pathway 3 1.4. Squalene biosynthetic pathway 5 1.5. Lanosterol biosynthetic pathway 6 1.6. Hypothetical mechanism for the cyclization of squalene oxide to cycloartenol g 1.7. Sterol biosynthetic pathways in fungi, animals, and plants 9 1.8. Mechanism of Re-face and 5'/-face "methyl cation attack" 11 1.9. Hypothetical X"-group mechanism for sterol methylation 13 1.10. Two possible conformation (bent and flat) of cycloartenol compared with the conformation of lanosterol 14 1.11. Synthesis of sterol methylation inhibitors by Pascal 18 1.12. Synthesis of 23-thia sterol by Pascal 18 1.13. Synthesis of sulfur substituted sterols by Ator 19 1.14. Synthesis of sterol methylation inhibitors by Benveniste 21 1.15. Synthesis of sterol methylation inhibitors by Ator 23 1.16. Synthesis of 25-thia sterol by Benveniste 23 2.1. ^H NMR (300 MHz) spectra of: (A) ergosterol; (B) [28-^H2] ergosterol; ( C) [25-^H]ergosterol; (D) [27-*^C]ergosterol 27 2.2. Partial ^^C-NMR (75 MHz) spectra of ['^C-27] sterols 27 2.3. Methylation catalyzed by microsome-bound SMT enzyme from com seedlings 29 2.4. Proposed mechanism for the second biomethylation by the SMT enzyme 30 2.5. First and second methylation steps catalyzed by the recombinant SMT enzyme from A. Thaliana 31 2.6. Newman projection (C-20 to C-17) of desmosterol and its epimers 33

xu 2.7. Optimization geometry of desmosterol calculated by CS MOP AC Pro vision 3.5 34 2.8. Optimization geometry of left-handed 20-epidesmosterol calculated by CS MOP AC Pro vision 3.5 34 2.9. Optimization geometry of right-handed 20-epidesmosterol calculated by CS MOP AC Pro vision 3.5 35 3.1. C-23 and C-25 carbon cations during the process of sterol methylation 42 3.2. Hypothetical mechanism of inactivating SMT enzyme with suicide substrate inhibitor 26, 27-cyclopropylidene sterol 48 3.3. Hypothetical mechanism of inactivating SMT enzyme with suicide substrate inhibitor 24(25), 26(26')-dien sterol 49 3.4. Hypothetical two possible inhibition pattern (Reversible/Irreversible) determined by two different bases at the active site of the SMT enzyme 56 3.5. Similarity of amino acid sequence in the SMT enzymes from different sources 57 3.6. Hypothetical model illustrating the sterol-enzyme-AdoMet tertiary complex at the beginning of the sterol methylation reaction 59 5.1. Isolation of cycloartenol from y-Oryzanol 63 5.2. Protection of 3p-OH of sterols 67

XIU LIST OF SCHEMES

2.1 Preparation of [27-^^C]lanosterol and biosynthesis of 25i?-[27-'^C] ergosterol in S. cerevisiae strain GL7 25 2.2 Preparation of [24-^H]lanosterol and biosynthesis of [25-^H]ergosterol in S. cerevisiae strain GL7 26 2.3 Biosynthesis of [28-^H2]ergosterol in S. cerevisiae strain GL7 26 3.1 Preparation of 22-aza sterols 3 8 3.2 Preparation of 20(17/21/22), 24(25)-dien sterols 39 3.3 Preparation of 20-epidesmosterol 40 3.4 Preparation of 23-aza sterols 42 3.5 Preparation of 24-aza sterol 43 3.6 Preparation of 25-aza sterols 44 3.7 Preparation of 26, 27-cyclopropylidene sterols 46 3.8 Preparation of 24(25), 26(26')-dien sterols 47 3.9. Preparation of 8(9), 14(15), 24(25)-trien-25-ethylnyl-cholesta-3P-ol 47 3.10 Preparationof24(25)-methano sterols 50 3.11 Preparation of 24(25)-epimino sterol and its derivatives 51 3.12 Preparationof24(28)-methano and 24(28)-epimino sterols 52 3.13 Preparation of 24-amino and 25-amino sterols 53 3.14 Preparationof 24-methylene and 24-vinyl sterols 54 5.1 Preparation of [26-^H]lanosterol 112

XIV CHAPTER 1. INTRODUCTION

1.1. Structure and nomenclature In this dissertation, the sterol nomenclature follows the system proposed by Fieser and Fieser^^^ and subsequently modified by Nes and McKean^^^ and Popjak et al,^^^ see Figures 1.1 and 1.2. hi 1989, the lUPAC commission recommended another nomenclature system. However, there are several confusion areas with the lUPAC system and therefore is not used consistently by many natural products chemists.^"^^

20R ("right-handed") 29 fromC-2ofMVA o asymmetric center

P and axial, lies to C-25 prochiral center front of molecule

fromC-6(3')ofMVA

24a-alkyl is R, but P and equatorial, S when A2 2 present lies in plane of molecule. \ a and axial, lies a, lies to front HO to back of of side chain molecule in this conformation a and equatorial, \. i 4 P ^^ axial, lies lies in plane of ^~^ \ y out of plane of 24P-alkyl is S, but/? p, lies to back molecule molecule 122 of side chain when A present

Figure 1.1. Stereochemical features and numbering system of sterols (adapted from Ref 5)

According to the system described by Fieser and Fieser and modified by Nes and McKean, the two geminal methyl groups, attached to C-4 with the a-configuration and P- configuration, and the methyl group attached to C-14, are numbered 30, 31 and 32, 19.1 A -^- p face (top or front side)

5.8A

a face (rear or back side) 7.7A

Figure 1.2. Conformational perspective of a generic sterol respectively. The nucleus a/p system refers to equatorial (lies in plane of molecule) and axial (lies out of plane of molecule). Alternatively, in the side chain, the a/p system is different. Groups oriented in front of the plane of the side chain juxtaposed in the staggered conformation are regarded as a-oriented; whereas those in back are P-oriented. The systematic name of cholesterol is cholest-5-en-3p-ol, and lanosterol is named as 4, 4, 14a-trimethyl-5a-cholesta-8, 24-dien-3p-ol, or 5a, 13p, 14a, 17p, 20R-lanosta-8, 24- dien-3p-ol. For select sterols prepared synthetically, the lUPAC system is used to locate the position of the additional carbon atoms on the side chain here, extra carbon atoms attached to C-26, C-27, and C-28 are referred to 26', 27' and 28', respectively.^"^^

1.2. Background of sterol biosynthesis Sterols play vital hormonal, regulatory, and architectural roles in all living organisms.^^^ Cholesterol has been the subject of an enormous amount of research for its physiological importance. Over the last six decades 13 Nobel Prizes have been awarded to scientists who made discoveries concerning the structure, biosynthesis, and metabolic regulation of cholesterol.^^^^^^ The first milestone in the elucidation of cholesterol biosynthesis was the discovery that all the carbon atoms in cholesterol are derived from acetyl CoA, a fact based on radioisotope labeling experiments.^^^ It is generally accepted that cholesterol biosynthetic pathway is composed of three stages: (1) acetyl CoA to Mevalonate Pathway Non-Mevalonate Pathway

O OHO S CH3 H- OH CoA HOK CH2OP Acetyl-CoA Pyruvate ^^^ / D-1-deoxy- Glyceraldehyde ^"2 POH2G Xylulose-5P 3-Phosphale Acetoacetyl-CoA Thiolase CoA

,x> PO - 0H^„ S CH3 CoA OH O Acetoacetyl-CoA Hypothetical Branched Chain Q hiteoTiediate Acetyl-CoA HMG-CoA Synthase v^ CoA

O CK-: GOGH PPG S OH CoA Isopentenyl Diphosphate Hydroxymethylglutaryl-CoA (HMG-CoA) NADPH HMG-CoA Mevalonate Pyrophosphate^ ^^^^ ADP Reductase Decarboxylase K NAD? , CoA

CH3 1. Mevalonate Kinase GOGH 2. Phosphomevalonate Kinase PPOH2G' ^^x^CGGH

2 ATP Mevalonate Pyrophosphate ADP Figure 1.3. Isopentenoid biosynthesis pathway isopentenyl diphosphate (C5); (2) isopentenyl diphosphate to squalene (C30); and (3) squalene to lanosterol (C30). The first and second stages universally exist in plants, animals, and fiangi. Based on the progress in this field, a few drugs, like Merck's Zocor and Warner-Lambert's Lipitor, are currently marketed to help people to lower serum cholesterol levels. These drugs are designed to inhibit some enzymes which play critical roles in cholesterol biosynthesis, such as squalene synthase and squalene epoxide cyclase (lanosterol synthase). We will discuss sterol biosynthesis from acetyl CoA to sterol in the following sections.

1.3. Isopentenoid biosynthesis The fundamental building block of isopentenoids (syn= isoprenoids, terpenoids) is isopentenyl diphosphate (syn= pyrophosphate), hi biological systems this metabolite is synthesized through the mevalonate pathway, also called the Bloch-Comforth pathway (Figure 1.3), in which production of isopentenoids involves irreversible carbon flux fi-om acetyl CoA to hydroxymethylglutaryl CoA (HMG-CoA) to mevalonate to isopentenyl diphosphate, see reviews in.^^^**^^ Although this mechanism has been generally accepted for several decades, there are still various hypotheses which have been proposed to explain the role of mevalonate in the terpene biosynthetic pathway. The uniqueness of the mevalonate pathway has been challenged by several research groups. From studies on [^'^C-]mevalonate incorporation into fatty alcohols, Nes' group found that cycloartenol production can be influenced either by carbon flux via the "Popjak shunf. This novel shunt can explain the reason of introduction of amino acids and sugars into isopentyl diphosphate by a route that bypasses mevalonic acid as an intermediate.^^^^ Recent studies by Rohmer and his co-workers demonstrated that the classical mevalonate pathway for the formation of isoprenoids does not exist in a number of bacteria and in thylakoids of a cyanobacterium. They proposed a novel pathway (or non-mevalonate pathway) involving the condensation of a triose phosphate with activated acetaldehyde and a subsequent skeletal rearrangement of the condensation product,^^^^^*^^ see Figure 1.3. They found that the chloroplast-bound isoprenoids (p-carotene, lutein, prenyl chains of chlorophylls and plastoquinone-9) were synthesized via a novel IPP biosynthesis pathway which involves glyceraldehyde 3-phosphate and pyruvate as precursors rather than mevalonate.^'^^ In recent work fi-omArigon i and coworkers, 1- deoxy-D-xylulose, not mevalonate, was found to be the predominant isoprenoid precursor of phytol, p-carotene, and lutein. Arigoni suggested that the mevalonate pathway may be operational in the cytosol of plant cells, and the deoxyxylulose pathway may be segregated in plastids with some flux of metabolites between the different isoprenoid pools.^^^1

IPP H^ PPj PFO IPP Isomerase Isopentenyl PPO ^^ A, GPP Synthase PP^ Diphosphate (IPP) Dimethylallyimeth l Diphosphate Geranyl Diphosphate (DMAPP) (GPP) -IPP FPP synthase W. PPi

H CHSR NADP

Famesyl Diphosphate (FPP) FPP Presqualene Diphosphate (PSPP) " ^

Figure 1.4. Squalene biosynthetic pathway

1.4. Squalene biosynthesis Squalene biosynthesis is well established. As shown in Figure 1.4, isopentenyl diphosphate is converted to its isomer, dimethylallyl diphosphate (DMAPP), by an isomerase. DMAPP then condenses in head-to tail pattern with IPP to form the Cio molecule geranyl diphosphate (GPP). This basic reaction is then repeated: GPP condenses in head-to-tail pattern with IPP to form the C15 molecule famesyl diphosphate (FPP). Finally, squalene synthase catalyzes the head-to-tail condensation of two molecules of FPP to form the C30 molecule squalene via the unusual rearrangement of presqualene diphosphate (PSPP).^^^^ The intermediate of PSPP is supported by recent findings by Poulter and his co-workers.^^^^ They trapped a tertiary cyclopropylcarbinyl cationic intermediate which is identified as major intermediate in the rearrangement between presqualene diphosphate and the straight-chain squalene by squalene synthase.

1.5. Lanosterol and cycloartenol biosynthesis Squalene epoxidase catalyzes the epoxidation of squalene to (S)-2, 3- oxidosqualene. This is the first step during sterol biosynthesis that requires oxygen. The conversion of (S)-2, 3-oxidosqualene to lanosterol by lanosterol synthase is remarkable as one of the most complex of all enzyme catalyzed reactions. It is also one of the most • n 71 complicated one-step transformations known in biochemistry or synthetic chemistry.^ Proton induced cyclization of (S)-2, 3-oxidosqualene in the preformed chair-boat-chair- boat conformation gives the intermediate protosterol cation which has the structure with 17P-side chain ensuring stereochemical control at C-20 of lanosterol.^^^^""^^ Recent work in Corey's group has shown that cyclization reaction occurs in discrete stages: (1) C-ring of the steroid nucleus is formed by ring closure to a five-membered structure; (2) the six- membered C-ring is formed by ring expansion of this five-membered cyclopentylcarbinyl cation precursor.^^^^ ^^"^"^^ Finally, the intrinsic low activation energies and a little assistance from the enzyme drives the rearrangements of hydrogen and methyl groups on the pathway from the protosterol cation to lanosterol till the deprotonation step which fixes the 8, 9-locafion of the nucleus double bond,^^^''^ see Figure 1.5. In higher plants, cycloartenol is generated from squalene oxide by a cycloartenol cyclase (synthase). Cycloartenol passes through a protosterol cation intermediate followed by 1, 2-hydrogen, methyl groups shift and 19-H loss,^^^^^'^ see Figure 1.6. The formation

Q of cyclopropane ring on cycloartenol is different from that of A -double bond on Squalene epoxidase

(S)-2, 3-Oxidosqualene

Lanosterol synthase

Lanosterol Protosterol cation Figure 1.5. Lanosterol biosynthetic pathway

lanosterol. A bridged cation intermediate between C-9 and C-19 was proposed to be critical for the migration of C-9 hydrogen and formation of the cis cyclopropane ring since it could meet the requirement of the biogenetic isoprene rule.^^^^

1.6. Pathways of sterol biosynthesis in fungi, animals, and plants The end products of sterol biosynthesis in vertebrates, plants, and fungi are A^- sterols accumulate in cell membranes, see Figure 1.7. Cholesterol is synthesized predominantly in mammalian cells, whereas, the major sterols synthesised by plants are campesterol, dihydrobrassicasterol, sitosterol and stigmasterol. The plant sterols are commonly distinguished from those occurring in animals, by the presence of a methyl or Hv"iw ^

1. proton initiated attack ENZ—B —H 0 on the 2,3-epoxy group 2. ring annulation to 1 form the protosteroid (S)-2,3-Epoxysqualene 17(20)-Protosteryl Bridged Carbenium Ion (the 5-coiIed substrate binds with P-face toward active site) Wagner-Meerwein shifts 1. 17a H to 20P H = 20/? H 2. 13a H to 17a H 3. 14p CHsto 13pCH3 4. 8a CH3 to 14a CH3 5. 9P H to 8p H. with 9p hydrogen abstraction to form 8,9-double bond and stable intermediate

20/?-"Left-handed" BBB

ibstraction of H. /H- (delivered from P-face) HO HO • Enz 4 C-19 Protosterol Lanosterol

20i?-"Right-handed"

Cycloartenol

Figure 1.6. Hypothetical mechanism for the cyclization of squalene oxide to cycloartenol (adapted from Ref 20) (S)-2, 3-Oxidosqualene Fungi and Animals ^ \ Plants

HO Campesterol (24a-Me) HO' Dihydrobrassicasterol (24P-Me) Sitosterol Stigmasterol Figure 1.7. Sterol biosynthesis pathways in fimgi, animals, and plants

ethyl group at C-24 in the side chain. Fimgal sterol biosynthesis follows the vertebrate pathway to the point of zymosterol, which is then methylated at C-24 in fimgi, and affords the major fungal sterol ergosterol as the end product. Differences in the sterol structure from animals, plants and fungi are used to design sterol biosynthesis inhibitors for medicinal and agricultural purposes.^^^^

NH,

HOOC

OH OH S-adenosylmethionine (AdoMet, SAM)

1.7. Sterol Methylation Mechanisms Methylation of the sterol side chain is the characteristic feature that distinguishes sterol biosynthesis in fungi and plants from animals.^^'^^"''^ The (S)-adenosyl-L- methionine: A^"^^^^^-sterol methyl transferase (SMT: EC: 2.1.1.41) catalyzes the transfer of the methyl group from AdoMet (SAM) to the 24, 25-double bond of the sterol side chain. ^^^ ^^^^"^^ The formation of 24-methylene sterols involves a I, 2-hydrogen shift from the C-24 to C-25 position and loss of one hydrogen atom from the methyl group originating from AdoMet. Stereochemically, C-24 and C-25 positions become the stereogenic centers during the methylation step controlled by SMT.^^^^ The direction of the transfer of the methyl group, either Re-face or 5'/-face attack on C-24 of A^"^^^^^ precursors, determines the stereochemistry of the C-24 position, see Figure 1.8. 5'/-face attack at C-24 (24p-orientation of the methyl group) has been proposed in higher plants by Nes.^^^^ This mechanism was further confirmed by Fujimoto ^^^^ in plants and by Arigoni^^^^^ and Nes ^^^^ in yeast. The opposite methylation mechanism was reported in the formation of isofucosterol in Pinus pineaP^^ On the other hand, the steric course of the hydrogen migration from the C-24 to C-25 position leads to the metabolic fate of the C-26 and C-27 methyl groups of A^"^^^^^ sterol and the C-25 prochilarity of the final product 24-methylene sterol. Seo and coworkers investigated the formation of 24- methylene cholesterol in Physalisperviana^^^^ and 24-methylene cycloartenol in

10 Trichosanthes kirilowiiP^^ They proposed Re-face hydrogen migration in higher plants. The same steric course of hydrogen migration was suggested for the biosynthesis of poriferasterol in Ochromonas malhamensisP^^

pro-E C-26

N^/N/'N/V pro-Z C-27

Re-fdiCQ "methyl cation attack" .S/-face "methyl cation attack" H

\/^\/sysys^

s/W^/V' Rotate Around C-25 pro-S C-27

H 25-R v^/^v'N/^s/' Figtire 1.8. Mechanism of i?^-face and iSz-face "methyl cation attack"

During the previous studies of ergosterol biosynthesis, Arigoni relied on the NMR spectra of an authentic specimen [^^C-27]ergosterol to identify the isotopically labeled

11 carbon, C-26 or C-27, formed biosynthetically from incubation with [2-^^C]mevalonate fed to ClavicepspaspaliP^^^ The '^C labeled ergosterol was biosynthesized from lanosterol, which should possess the a ^^C-labeled C-26 group. Based on the NMR analysis, Arigoni suggested that the pro-.£: methyl group on lanosterol become the pro-5 methyl group on ergosterol, i.e. C-255'-configuration in ergosterol. Seo and co-workers investigated the same steric course of hydrogen migration from C-24 to C-25 in Saccharomyces cerevisiae by using ['^C^HsJacetate as substrate to produce ergosterol.^^^^ ^^"^^ They reported the ^^C-NMR signals for C-26 and C-27 of isotopically labeled ergosterol at 6 19.89 and 19.61 ppm, respectively. Their results agreed with the other groups' work, which assigned the NMR chemical shift of C-26 downfield from that of C- 27 in 24-alkyl sterols.^^^^"^^ Although the '^C-enhanced signals in their spectrum of ergosterol were not obvious, Seo et al., seemingly confirmed the Arigoni's proposal on the mechanism of fungal sterol methylation.

1.8. X'-group mechanism Alternatively, in comparison with the concerted mechanism proposed in Figure 1.8, Comforth and Wokciechowski once suggested that the sterol methylation reaction might follow an so called X' -group mechanism, in which a basic group from the active site of SMT enzyme covalently binds with a C-25 carbocation intermediate,^^^^"''^ see Figure 1.9. The X'-group mechanism is based on a model for a normal chemical reaction, i.e., an electrophilic addition from a "methyl cation" to an alkene (24, 25-double bond) which proceeds by a stepwise modification in the bond-making-bond-breaking steps. Therefore, the resulting tertiary carbocation generated at C-25 of the sterol side chain must combine with a nucleophile on the face of the double bond opposite to the face attacked by the electrophile. As a result of their proposal, the hydrogen migration from C- 24 to C-25 must proceed from the Si-fa.ce of the original double bond, showing the 1, 2- hydrogen shift of H-24 to C-25 is stereoselective. However, recent studies by Nes and co workers confirmed that the SMT enzyme-catalyzed reaction proceeds with inversion of configuration at the methyl center of AdoMet and they demonstrate a random bi bi kinetic

12 mechanism. Obviously, the X'-group mechanism, which operates stepwise during the methylation, conflicts with this observations. ^^^^

Figure 1.9. Hypothetical X'-group mechanism for sterol methylation

1.9. Structure-function relationships The structural features for sterol binding to the SMT enzymes from the different sources have been studied in several laboratories.^'^^^'^^''^ [38a-f][39a-b] gj^Q,^^^ ^^ Table 1.1, cycloartenol appears to be the preferred substrate for the plant SMT, while zymosterol appears to be the preferred structure for the fungal SMT. In the study on the structural requirement for transformation of substrates by the SMT enzyme from S. cerevisiae, the SMT enzyme was found to be regio-selective and stereo-selective for sterol.^^^^ Three

13 Cycloartenol Bent Conformation

Cycloartenol Flat Conformation

H H H

Lanosterol Conformation Figure 1.10. Two possible conformation (bent and flat) of cycloartenol compared with the conformation of lanosterol structural parameters were found to be obligatory for sterol binding to the enzyme: an unhindered 24, 25-bond, an equatorially-oriented polar group at C-3 attached to a planar nucleus and a freely rotating side chain. ^"^^^'^^ Methyl groups at C-4 were used to prevent productive sterol binding to the SMT from the yeast. It was also found that the introduction of an 8(9)-bond, 14a-methyl or 9p, 19-cyclopropyl group had no essential requirement or significantly harmfiil effects for the SMT enzyme catalyzed reaction,

14 whereas a free 3P-OH and 24, 25-double bond were obligatory for the substrate binding and methylation.^'^^^'''^ Comparative studies with 3P-OH and 3-keto cyclopropyl sterols indicated the oxygen atom at C-3 most likely acted as a proton acceptor in hydrogen bonding with the enzyme. From recent studies on the SMT enzyme from Arabidopsis thaliana using 24(28)-methylene lophenol as substrate indicated C-4 methyl sterols may be preferred substrates for the second methylation step.^"^^^ To explain the different sterol specificity for the SMT enzyme from plant and fimgi, some investigators assumed that it is because the plant sterols have bent structures which is unfavorable for binding with the enzyme.^'^^^^'*^^^'^'*^ In Figures 1.5 and 1.6, both cycloartenol and lanosterol are shown to arise from a common intermediate squalene epoxide. The only difference between the mechanism of cyclization is a 19-H loss and cyclopropane ring formation for cycloartenol and a 9-H loss and a double bond formation for lanosterol. Thus, it is reasonable to consider that the conformation of cycloartenol may be similar to that of lanosterol, which is flat, see Figure 1.10.^^^^^^^^ ^"^^^ This hypothesis has been confirmed by two experimental results: I) the X-ray crystallography study on the solid state conformation of lanosterol and cycloartenol which showed they had a similar conformation^^^^; and 2) the high field ID- and 2D-NMR (500 MHz) analysis of the cycloartenol in solution indicated the conformation of cycloartenol was flat and that no significant population of molecules interconvert from the flat to the bent shapeP^^

I.IO. Sterol methylation inhibitors Sterol methylation inhibitors are valuable tools to study the topology of the SMT enzyme active site. Sterol methylation inhibitors targeted to critical slow steps in the pathway are also used to develop potential antifungal drugs.^^^ Disruption of ergosterol production is a major source of antifungal therapy. It was discovered that the methylation of lanosterol is a rate-limiting step of sterol biosynthesis pathway in some fungL^"*^^ Hence, sterol methylation inhibitors have been designed to serve as antifungal compounds. Synthetic strategies developed by Nes, Ator, Pascal, Benveniste and

15 *

o o ^ * o o m CO o

* 00 ^ r-^ vo c3

* ••—1 CO en ^ un

* 00 o O O :«: O ON B d • f—( * 00 o O o td a o m o a d

+-> •t—> * -' un o •d ,(D IJ O O ON T3 o d (D <^ ^ ^ d 0) .3 o CO P. O o O > a o (N o ^^ ^^ • I—( O O o o .B-B 13 B o a I c3 ^ • 1—1 o o 'a 00 -t-> OO 00 d p d) ^ o (D o 00 o cd O 1—1 00 to •*—> o cd CO CO 'o I o 1 o o 00 'T3 Xi o I o oo o 'o 1 Y b (u ^ S en 13 d 'T:' ?5 -h; o cj ^^ w o oo d ^^^ (Nr i p^i^ I d t^ o >. so "^ • <—> 00 (U o 1 u^ r^ oM ti •<-' r u (U cd d CO •«—a> ••-> +-> -B B 00 ^ cd OO O 00 CO 00 ro ro •*-; O O O OO

1—4 9 5I o o q a >^ es m hol e hol e 1 CO OH * Q ^ Q u Nl U m o-i "^ U C J •X- -x- H I ^

16 Oehlschlager have lead to novel sterol side chains with heteroatoms that demonstrably interfere with C-methylation.f^^^f^^'"^ Pascal^"^^^^ and a coworker synthesized (20, 21), (24, 25)-bis(methylene)-5a- cholestan-3p-ol 2 from a diene precursor 1_ by Simmons-Smith reaction followed with deprotection of THP ether. They also synthesized 22-aza-5a-20§-cholestan-3p-ol 4 from 3p-THP-5a-pregnan-20-one 3, see Figure 1.11. When 2 and 4 were tested with the culture Crithidia fasciculata, the ergosterol synthesis was suppressed. 22-aza-cholstanol 4 was suggested to be the specific inhibitor of the A^'^^^^^ side chain reductase. They also hydrogenated 20-hydroxymethyl-pregna-l, 4-diene-3-one 5 to give 6. The tosylate sterol 7 was heated with isobutylamine ethanol solution to produce an azasterol followed by reduction to produce diastereoisomers 23-aza-sterol 8, see Figure 1.12.. 7 was also used to react with sodium isobutylthiolate and then reduced with sodium borohydride to afford diastereoisomers 23-thiasterol 10. When C. fasciculata was cultured in the presence of 23-aza-cholestanol 8, the levels of ergosterol and ergost-7-en-3p-ol were suppressed, but the amounts of the precursors of these sterols, ergosta-7, 24(28)-dien-3p-ol and ergosta-5, 7, 22, 24(28)-tetraen-3P-ol, were relatively increased. Thus, 8 was suggested more likely to inhibit reduction of 24, 28-double bond rather than the methylation of 24, 25-double bond by SMT enzyme. However, 23-thia-cholestaol was found to have no significant effect on sterol biosynthesis in C. fasciculata. These resuUs indicate that nitrogen substituted sterols are preferred inhibitors for the SMT enzyme and A^'*^^^^ reductase because of positively charged nitrogen at physiological pH. Ator^'^^'*^ and coworkers synthesized several sulfur substituted sterols to study with the SMT enzyme from Candida albicans, see Figure 1.13. 3p-Hydroxyl group of bisnorcholenic acid methyl ester was esterified to give 11 by rer/-butyldimethylsilyl trifluromethanesulfonate in the presence of 2, 6-lutidine. Lithium aluminum hydride reduction and subsequent Swem oxidation of methyl ester U afforded an aldehyde 12. 23-Aldehyde sterol 13 was prepared by Wittig condensation with (Ph)3P=CH0Me and hydrolysis of the intermediate enol ether in Hg(0Ac)2 THF-H2O solution. The

17 THPO

38% THPO

(a) I2, Zn-Cu, ether, CH2I2, reflux, 5 days; (b) HCl, reflux, MeOH; (c) isoamyl amine, NaBCNH3, then HCl

Figure 1.11. Synthesis of sterol methylation inhibitors by Pascal

(a) H2, Pd/C; (b) TsCl, pyr., -l(fC; (c) isobutylamine, EtOH, l^Q 50 h, then NaBHj; (d) Sodium isobutylthiolate, EtOH, reflux 3h; (e) NaBI^, EtOH Figure 1.12. Synthesis of 23-thia sterol by Pascal

18 %^COOMe

R^TBDMS

(a) LAH, THF; (b) DMSO, (C0C1)2, Et3N; (c) Ph3P^CH20CH3Br-, potassium amylate, toluene; (d) Hg(OAc)2; (e) t-BuNH2.BH3, CH2CI2; (f) TsCl, pyr; (g) KSCHMe2; (h) THF-H2O-ACOH 1:1:1.5; (i) Mel, CH2CI2; 0) EtI, CH2CI2; (k) mCPBA, CH2CI2 Figure 1.13. Synthesis of sulfur substituted sterols by Ator

homologated aldehyde 13 was reduced and then converted to tosylate 14. 24-Thia cholesterol 15 was accomplished via displacement of the tosylate with the potassium salt of (CH3)2CHSH in DMF and acid hydrolysis of the tert-butyldimethylsilyl protecting group. Alkylation of the sulfide 15 yielded sulfonium salts 16 and 17. Further oxidation of 15 with mCPBA gave sulfoxide 18. As a result, 24-thia cholesterol 38 was only a modest competitive inhibitor of the enzyme with Kt values of 1.5 i^M. Methylsulfonium sterol 16 also exhibited strong binding affinity to the SMT enzyme with Ki values of 16 nM, indicating its resemblance to the transition state intermediate proposed in Figure 1.8. Ethylsulfonium sterol 17 had 30-fold decrease in affinity, suggesting its side chain less fit for the portion of the enzyme active site because of its bulkier size. However, sulfoxide sterol 17 was not an effective inhibitor with lower binding affinity {Ki, 20 |LIM).^'*^''^ In the inhibition studies on C. fasciculata, Pascal and coworker prepared 24-thia-5a-cholestanol as the similar

19 procedure as Figure 1.13. They found 24-thia-5a-cholestanol was a specific inhibitor for the SMT enzyme, because when incubating it with the culture C. fasciculata, ergosterol biosynthesis was suppressed, and the principal sterol observed was cholesta-5, 7, 24(25)- trien-3p-ol.^'*^^^ In comparison with 24-thiacolestanol, 23-thiacholestanol had no inhibition effect on the SMT enzyme. Therefore, it was suggested that 24-thia sterol could be charged by methylation of the sulfur from the AdoMet since 24-sulfur is isoelectronic and at the center of the SMT enzyme catalyzed reaction. Benveniste^"^^^ and coworkers used 3-acetoxy cycloaudenol 19 as the starting agent to prepare 24iJ-24 methyl-25, 26, 27-trisnor-24-trimethyl-ammonium-cycloartenol iodide 27, see Figure 1.14. Ozonolysis of 19 gave a ketone 20. 21 was generated by the oxidation of the ketone 20 with mCPBA. Base hydrolysis of di-acetate compound 21 afforded 3-acetoxy-24-hydroxyl sterol 22. Tosylate compound 23 reacted with sodium azide and followed by hydrogenation gave 24-amino sterol 24, of which 24 methyl configuration was reported to reverse from the 24S to the 24R. 25-Aza-247?-methyl cycloartenol 26a was accomplished by dimethylation of compound 24 and then reduction with lithium aluminum hydride. 24i?-Methyl-25, 26, 27-trisnor-24-trimethyl-ammonium- cycloartenol iodide 27a was prepared from 26a by the reaction with methyl iodide. 24S- Methyl-25-aza cycloatemol 26b and 245'-24 methyl-25, 26, 27-trisnor-24-trimethylammonium-cycloartenol iodide 27b were synthesized by the same method. In their synthesis of diastereoisomer 24-ethyl-25-azacycloartemol 31,, 3p-acetoxy cycloartenol 24-aldehyde 28 was used as starting material. 29 was prepared by reaction with dimethyl amine in the presence of catalyst titanium tetrachloride. Rearrangement of double bond was achieved by addition of trifiuoroacid to give 30. Reaction of Grignard reagent with 30 yielded diastereoisomer 24-ethyl-25-azacycloartemol 31. In the inhibition studies of SMT enzyme from maize seedling microsome, all 25- aza sterols showed very strong inhibition effect. The Kt values are 20 nM for 24i?-methyl- 25-aza cycloartenol 26a, 30 nM for 245'-methyl-25-aza-cycloartenol 26b, 35 nM for 24R- methyl-25, 26, 27-trisnor-24-trimethylammonium cycloartenol iodide 27a, 45 nM for 245'-methyl-25, 26, 27-trisnor-24-trimethylammonium cycloartenol iodide 27b, 85 nM for

20 Me 0

AcO 11 c 40%

22 Me

V^/^/^ I

27a

Me Me \ N S/S^/x I

26b 27b

(a) O3, CHCI3, Zn-AcOH; (b) mCPBA, diClj; (c) K2CO3, MeOH, CH2CI2; (d) TsCl, pyr.; (e) NaN3, MeOH-THF-l^O; {mi, Pt02; (g) Mel, ether; (h) LAH, ether

^ N^0C0CF3-

AcO

(a) Me2NH, TiCl,; (b) CF3COOH; (c) EtMgl, then HCl Figure 1.14. Synthesis of sterol methylation inhibitors by Benveniste

21 24(ie,5)-ethyl-25-aza cycloartenol 31 and 50 nM for 24(R, S:)-methyl-25, 26, 27-trisnor- 24-trimethylsulfonium cycloartenol iodide 42. These results demonstrates: (1) positively charged C-25 heteroatoms (N and S) are analogs of a proposed carbocationic high-energy intermediate in the SMT enzyme catalyzed reaction; (2) 24i?-methyl group is a preferred unit for the enzyme, which corresponds the Re-face mechanism proposed in Figure 1.6; (3) the bulkier group at C-24 is not preferred by the enzyme; and (4) add bulk group at C-25 interferes with productive binding to the enzyme.^"*^^ Ator and coworkers synthesized several novel C-25 related sterol methylation inhibitors in their work on C. albicans,^'^'^'^^ see Figure 1.15. The 24, 25-double bond of 3p-Trifluoroacetate lanosterol 33 was cleaved by ozonolysis to afford aldehyde 33. Amide 34 was achieved by Jones oxidation of 33 produced an intermediate carboxylic acid, which was converted to its acid chloride and then treated with ammonia solution in THF. The trifluroacetyl group was restored by treatment of the crude amide mixture with excess trifluoroacetic anhydride since it was partially removed during amide formation. Methyl imidate 35 was derived from amide 34 by addition of excess methyl triflate. Amidines 36 and 37 were generated from imidate 35 by reaction with ammonia and dimethylamine in ethanol. The addition of 35 to the amine also led to the loss of the trifluoroacetyl group. Imidazole 38 was accomplished by the reaction of 35 with aminoacetaldehyde dimethyl acetal and subsequent treatment with hydrochloric acid. Methylation of the imidazole moiety in the presence of the unprotected 3-hydroxyl yielded 39, using sodium hydride and excess methyl iodide in DMF. As shown in Figure 1.16, diastereoisomer 24-methyl-25, 26, 27-trinor-24-dimethylsulfonium-cycloartenol iodide 42 was achieved via the reaction of 24-methyl-24-tosylate-3-acetoxy cycloartenol 40 with CHsSLi and subsequent methylation of compound 41 with methyl iodide. In the inhibition studies of SMT enzyme from C. albicans, amidine analogs 36 and 37 as well as the imidazole analogs 38 and 39 were found to be extremely effective inhibitors. The Ki values were 11 nm for 36, 6 nM for 37, 11 nM for 38 and 5 nM for 39. Though all these compounds have a nitrogen atom at C-25, the mechanism of their strong binding affinities toward the SMT enzyme is consistent with the HEI model or bridged

22 carbocation model. It can be hypothesized that amino acids in the active site of the SMT enzyme may have several residues involved with hydrogen bonding with the amino and imidazole groups of these inhibitors, which increases their binding efficacy. ^'^^'^^

CONHo

F3COCd^ >C

NH2^TfO-

35% from • 34

NH2^TfO'

(a) O3, CH2CI2, then Me2S; (b) CrO,; (c) (C0C1)2, CH2CI2, then NH3; (d) (CF3CO)20, pyr; (e) MeOTf, CH2CI2; (f) NH3, EtOH; (g) Me2NH, EtOH; (h) NH2CH2CH(OMe)2, EtOH; (i) HCl, THF; G) NaH, DMF, Mel. Figure 1.15. Synthesis of sterol methylation inhibitors by Ator

o.\ s^-

AcO '"// 40 c^ 41 42

(a) CHsSLi (5.7 equivalent), HMPA, 60°C, 4 h; (b) CH3I, MeOH, 25°C, 72 h Figure 1.16. Synthesis of 25-thia sterol by Benveniste

23 CHAPTER 2. STUDIES ON STEROL METHYLATION MECHANISM

2.1. Mechanism studies of the sterol methylation reaction in yeast In our studies on the mechanism of sterol methylation in yeast, we re-examined the hydrogen migration from C-24 to C-25 in ergosterol biosynthesis in S. cerevisiaeP'^^ Considering the possibility that C. paspali and S. cerevisiae might operate different methylation pathways, we prepared [27-^^C]lanosterol 49^^*^^^^°^ and [24-^H]lanosterol 53 which were incubated with the sterol auxotroph S. cerevisiae strain GL7 to produce sterospecifically '^C-labeled ergosterol, see Schemes 2.1 and 2.2. We also incubated zymosterol and [ Hs-me/Zzy/]methionine (which is converted to [ Hs-wer/zyflAdoMet by GL7) with GL7 to produce [28-^H2]ergosterol 56,^^^^ see Scheme 2.3. To prepare [27-*^C]lanosterol, the ^^C labeled Wittig reagent 44 was freshly prepared from C-methyl iodide (99% C) and phophorane 43 in 63% yield. Condensation of the Wittig reagent 44 with aldehyde 46, prepared by ozonolysis of lanosterol acetate 45, gave in 80% yield, the pure ester 47. Reduction of the ester group of 47 with lithium aluminium hydride and aluminium chloride (1; 1) 48 in 80% yield. Aluminium chloride was added in order to prevent 1, 4-addition to the conjugated system. 49 was prepared by acetylation of 48, followed by reduction with lithium in ethylamine at 0°C. To prepare [24-^H]lanosterol, 51 was converted from lanosterol acetate 45 by hydroboration and subsequent oxidation of PCC. Reduction of the 24-keto group of 51 with sodium borodeuteride afforded 52. Dehydrogenation of 24-OH group of 52 followed by reduction of the acetate gave [24-^H]lanosterol 53. Total yield is about 45%. Figure 2.1 shows four different ergosterol samples recovered from the incubation cells. Spectrum A represents native ergosterol (control spectrum) and its spectrum is an identical match with that of other spectra we obtained on ergosterol isolated from related fimgi and plants. ^^^^'''^ The intensity of the doublet for C-28 at 6 0.912 originating from the methyl on [^H3-w^r/2>'/]AdoMet (derived from [^H3-we%/]methionine) is reduced by addition of the two deuterium atom (B, Figure 2.1), and the doublets for C-26 and C-27

24 H Ph3P = c< l+/^TT/13, COOCH. Ph3P"CH('XH3)COOCH3r 43 44

AcO AcO

Synthetically Metabolically Prepared Formed 49 50

a) 'XH3I13, ; b) O3, then Zn, HOAc; c) II, «-BuLi, THF; d) LAH, AlC^; e) AC2O, pyridine, then Li, EtNH2; f) S. cerevisiae strain GL7

Scheme 2.1. Preparation of [27- 13C]lanostero/ l and biosynthesis of 25i?-[27- 13CJergostero l in S. cerevisiae strain GL7 resonating at 5 0.82 and 0.84 collapsed into siglets after the hydrogen at C-25 is replaced with a deuterium (C, Figure 2.1). Additionally, the ^^C-NMR of the [25-^H]ergosterol was similar to the control spectrum, except the resonance for C-25 was absent. The latter

25 AcO

a) BH3-THF, 0°C, then PCC, CH2CI2; b) NaB^H4, ethanol; c) POCI3, pyr.; d) S. cerevisiae strain GL7

Scheme 2.2. Preparation of [24-^H]lanosterol and biosynthesis of [25- H]ergosterol in S. cerevisiae strain GL7

C^H2H

7 [ H3-/wer/3'>'/]Methionine S. cerevisiae strain GL7

55 56 Scheme 2.3. Biosynthesis of [28- H2jergosterol in S. cerevisiae strain GL7 results confirm that AdoMet attacks C-24 and the hydrogen originally at C-24 migrates to C-25. Figure 2.1 D shows H-NMR spectrum of C-labeled ergosterol. The proton signal of C-27 was characterized by the pair of doublets with a coupling constant JH-C~ 250 Hz. Compared with the natural ergosterol, the doublet at 0.837 ppm was assigned for C-27, while the upfield doublet at 0.823 ppm was assigned for C-26. Meanwhile, ^^C-NMR spectrum of C-labeled ergosterol showed that there appeared an enhanced signal at 19.925, which was shifted downfield from its position at 17.629 ppm of the [27- '^C]lanosterol spectrum. Thus, '^C chemical shift (Figure 2.2) of ergosterol C-26 and C-

26 2 r- o o CO o a CO "3 H^ i "

-«^ c^ -^ ^O .2 "^

1 en . o (N O

d &£)

(N I CO

CO rl^ O O tJD ^ S3 o^ Li I 1) r-

o ^ 2 2

ex o CO GO

"^ IT) Oo CN m U P< Z 2 LC •<-• I—I CO "" O ^ CD . »-i O to

XjISUSJUJ

27 27 should be assigned as 19.616 and 19.925 ppm rather than the previous assignment, respectively. These results demonstrated that pro-Z methyl (C-27) on lanosterol becomes the pro-R methyl (C-27) on ergosterol, i.e., the C-26 and C-27 positions of ergosterol are reversed from their original positions on lanosterol by rotation around C-25. Therefore, the 1, 2-hydrogen shift in ergosterol biosynthesis should involve Re-face mechanism which is the same mechanism of C-methylation reported in C. paspali.

2.2. Mechanism studies of phytosterol methylation The stereochemistry of phytosterol methylation was considered as Re-face mechanism of methyl cation attack from AdoMet instead of 5'/-face mechanism in fungal sterols by some investigators.^^^^^^^"'^^^^'^^''"^^^"*'^ But shown in Figure 1.8, we proposed that phytosterol might have the similar 5'/-face mechanism like ergosterol in fimgi. To confirm our hypothesis, 27-methyl ^^C-labeled lanosterol and AdoMet were incubated with a microsome-bound SMT enzyme prepared from 4-day old Zea mays seedlings,^^"^^^ see Figure 2.3. Although cycloartenol is the natural substrate for the SMT enzyme from com, lanosterol may be served as the universal substrate for the SMT enzyme from com and fungi if the methylation has the same .Sz-face mechanism. As we expected, the major product which was isolated and identified by chromatographic and spectra methods was [27-^^C]24(28)-methylene-24,25-dihydro-lanosterol. Using the coupling effect between ^H and ^^C atoms in proton NMR, the chemical shifts of C-26 and C-27 were assigned as 1.024 (d'd, J= 5.2 Hz, 6.8 Hz) and 1.017 (d'd, J= 125.5 Hz, 6.8 Hz) in the first time, respectively. In comparison with ^^C-NMR spectmm of 27-^^C-labeled ergosterol in which C-27 resonates downfield from the C-26, we found the similar signal pattem of C- 26 and C-27 in the ^^C-NMR spectrum of [27-^^C]-24(28)-methylene-24,25-dihydro- lanosterol (1:10 mixture with nonlabeled 24(28)-methylene-24,25-dihydrolanosterol), i.e., C-27 (21.97 ppm) downfield from C-26 (21.84 ppm). This results demonstrated that the 25R stereochemistry of ergosterol is retained after it is generated from 24(28)-methylene-24(25)-dihydro-lanosterol during normal C- methylation of sterols, see Figure 1.8. Thus, we confirmed that the pro-Z methyl unit (C-

28 27) on cycloartenol should be transformed into the pro-R methyl unit at C-25 on 24(28)- methylene cycloartenol during hydrogen migration from C-24 to C-25 under physiological conditions of phytosterol synthesis. It also shows that SMT enzyme from com operates the same Re-face methylation mechanism as in fimgi and marine [27] organisms.

Microsome-hound SMT Rnzym^ from Com Seedlings, AdoMet HO"

13 [27- C]Lanosterol 257?-[27-'X]-24(28)-Methylene-24(25)13 - Dihydro-Lanosterol Figure 2.3. Methylation catalyzed by microsome-bound SMT enzyme from com seedlings

2.3. Mechanism studies of the second sterol methylation The mechanism of the second C-methylation may be significant in the evolution of sterol biosynthesis of vascular (advanced) plants and non-vascular (primitive) plants. As shown in mechanism proposed in Figure 2.4, some research groups hypothesize that the C-29 phytosterol (E2) was converted from 24(28)-methylene sterol ( C) via an intermediate (D) through path b.^^^^^^"^^ Nonetheless, there remain unanswered questions in this area. The first question is whether the first and second methylation mechanism operate mechanistically in a similar way. Shown in path a of Figure 2.4, an SN2 mechanism may proceed during the successive transmethylations, such that in the second C-methylation step a 24-vinyl sterol (Ei) may be formed. However, only three types of side chains, 24(28)Z-ethylidene (E2), 24(28)£-ethylidene (E3), and 24p-ethyl-25(27)-en (E4), occur in nature. Thus, only path b and path c are possible mechanisms to produce the E2, E3, and E4. The second question is whether there is single enzyme or two different enzymes involved in the two-step methylation. We designed several experiments to get at these questions. First, the recombinant SMT enzyme of ^. thaliana was successfiilly

29 X 'o,

Figure 2.4. Proposed mechanism for the second methylation by the SMT enzyme overexpressed in E. coli cells under the control of Ty promoter, and [27- CJzymosterol was prepared from the natural zymosterol, using the same method as described in Scheme 2.1.^"^'^ Second, the sterol methylation reaction was found to be catalyzed using the recombinant enzyme with [27-'^CJzymosterol and AdoMet, see Figure 2.5. The products were found to be a mixture of [27-'^C]fecosterol and [27-'^C]24(28)Z-ethylidene

30 CH3

Recombinant SMT Enzvm^ of A. thaliana, AdoMet HO

[27- C]Zymosterol 25/?-[27-'X]Fecostero13 l

Recombinant SMT Enzyme of A. thaliana, AdoMet

25/?-[27-'X]-24(28)Z-Ethyliden13, e 24(25)-Dihydro-Zymosterol Figure 2.5. First and second methylation steps catalyzed by the recombinant SMT enzyme from A. thaliana zymosterol. [27- C]Fecosterol served as the substrate to perform the same methylation reaction catalyzed by the recombinant SMT enzyme. [27-'^C]24(28)Z-Ethylidene zymosterol was isolated as the major product of the reaction. Clearly, these results indicate that there is only single SMT enzyme which catalyzes the methylation reaction to afford mono and double alkylation products. On the other hand, the chemical shifts of C- 26 and C-27 of [27-'^C]fecosterol were assigned as 1.028 (d'd, J= 6.8 Hz each, H-26) and

1.019 (d'd, J= 125.5 Hz, 6.9 Hz, H-27) in 'H-NMR spectrum, 21.84 (C-26) and 21.98 (C- 27 enhanced peak) in '^C-NMR spectrum. Meanwhile, the chemical shifts of C-26 and C- 27 of [27-'^C]24(28)Z-ethylidene-24(25)-dihydrozymosterol were also assigned as 0.973

(d'd, J= 6.7 Hz each, H-26) and 0.972 (d'd, J= 125.2 Hz, 6.8 Hz, H-27) in 'H-NMR spectrum, 21.00 (C-26) and 21.071 (C-27 enhanced peak) in '^C-NMR spectmm. hi both compounds, C-27 of proton NMR resonates downfield from the C-26, while C-27 of '^C-

31 NMR resonates upfield from the C-26. The results show that 25R configuration is retained during the second C-methylation.

2.4. Molecular modeling and analysis of C-20 configuration C-20 is the only chiral center on the side chain of sterols, of which the stereochemistry is considered as 20^ for most of the natural sterols. Shown in Figure 1.5, the cyclization product of squalene epoxide is 20R lanosterol instead of 205* lanosterol. Thus, the 20R configuration appears to be more significant functionally than that of the 20iS' orientation as show below.

HO Left-handed 20-epidesmosterol Right-handed 20-epidesmosterol

During our synthesis of 20-epidesmosterol, it was found not to crystallize easily. Therefore, we attempted to use the molecular modeling method to study the configuration of C-20. The molecular modeling and analysis program we used here is CS MOP AC Pro vision 3.5 from Cambridgesoft Corporation. The computational calculations were based on the potential function AMI program, which is a semiempirical computational method developed in the late 1980's by Dewar et al.^^^^ AMI is a proper choice when dealing with large molecules like sterols. Our major focus of molecular modeling is the side chain orientation of 20-epidesmosterol. Although the stmcture of right-handed 20-episterol is not theoretically forbidden, and no X-ray stmcture was obtained to against it, many investigators always considered tiie left-handed side chain as a reasonable orientation for 20-episterol. To elucidate its absolute configuration by molecular modeling, we inputted

32 H H G(22)H2Ri RiH2(22)G.^^-K^^G(21)H3

R5R4R3(13)G"V]-y^G(16)H2R2 R5R4R3(13)S G(16)H2R2 H C(21)H3' ' Left-handed 20-epidesmosterol Right-handed 20-epidesmosterol Heat of formation: -91.184 kcal/mol Heat of formation: -91.415 kcal/mol Total steric energy: 79.989 Kcal/mol Total steric energy: 81.128 kcal/mol

H H3(21)G.^^^;^'^G(22)H2Ri

R5R4R3(13)G'V. ^G(16)H2R2 H Right-handed desmosterol Heat of formation: -99.167 kcal/mole Total steic energy: 72.202 kcal/mol Figure 2.6. Newman projection (C-20 to C-17) of desmosterol and its epimers the 2D chemdraw stmctures of left-handed 20-epidesmosterol, right handed 20- epidesmosterol, and desmosterol (right-handed), to the CS MOP AC Program to calculate their minimal energies and optimisation geometry. As a result. Figure 2.7, 2.8, and 2.9, show the optimizaiton geometry of desmosterol, the right-handed 20-epidesmosterol, and left-handed 20-epidesmosterol, with minimal energies (heat of formation) of-99.167, - 91.184, and -91.415 kcal/mole, respectively. Therefore, in term of thermodynamics, desmosterol is clearly more favotirable than its 20 epimer, demonstrating why it is the only product of squalene epoxide cyclization. On the other hand, the total steric energy of left-handed 20-epidesmosterol is 1.139 kcal/mol less than that of its right-handed although the heat of formation of left-handed 20-epidesmosterol is 0.231 kcal/mol higher than that of the right-handed, showing the left-handed stmcture is more favorable than the right-handed stmcture. Based on the optimization geometry of Figures 2.7, 2.8, and 2.9, Figure 2.6 shows the Newman projection of desmosterol and its epimers in the top view from C-20 to C-

33 Figure 2.7. Optimization geometry of desmosterol calculated by CS MOPAC Pro vision 3.5. Note: Big circle: Carbon atom. Small circle: Oxygen atom. Hydrogen atoms are hidden. Heat of formation: -99.167 kcal/mol. Total steric energy: 72.202 kcal/mol.

Figure 2.8. Optimization geometry of left-handed 20-epidesmosterol calculated by CS MOPAC Pro vision 3.5. Note: Big circle: Carbon atom. Small circle: Oxygen atom. Hydrogen atoms are hidden. Heat of formation: -91.184 kcal/mol. Total steric energy: 79.989 kcal/mol

34 Figure 2.9. Optimization geometry of right-handed 20-epidesmosterol calculated by CS MOPAC Pro vision 3.5. Note: Big circle: Carbon atom. Small circle: Oxygen atom. Hydrogen atoms are hidden. Heat of formation: -9l.415kcal/mol. Total steric energy: 81.128 kcal/mol

17. In comparison with the Newman projection of desmosterol, C-21 and C-22 of the right- handed 20-epi desmosterol overlap with C-16 and H(C-17), respectively. Thus, the steric interaction between C-21 and C-16 may lead to the significant change of the NMR chemical shift of C-20. On the other hand, C-21 and C-22 of the left-handed 20-epi desmosterol stagger among the C-16, H(C-17), and C-13. In comparison with Newman projection of desmosterol, C-22 of the left-handed 20-epi desmosterol has more steric hindrance from C-13 group than from C-16 group, vice versa, C-21 has the less steric hindrance. Table 5.2 gives rise to the assignment of '^C-NMR for the desmosterol and its 20 epimer, showing C-22, C-20 and C-17 have the largest different chemical shift between desmosterol and 20-epi desmosterol, while the difference of C-21 is slightly higher than the background. Obviously, the model of the right-handed side chain does not match the NMR assignment, but the model of the left-handed side chain fully supports the current NMR assignment of 20-epidesmosteroL Therefore, combining the resuhs of NMR spectmm and Newman projection of these molecules, it is believed that the model of the left-handed side chain is more likely the geometry of the side chain where physiological conditions. Our enzyme study confirms the model of the left-handed side

35 chain of 20-epidesmosterol since 20-epidesmosterol was neither a substrate nor an inhibitor of the SMT enzyme from Prototheca wickerhamii (Nes and Mangla, unpublished result), whereas desmosterol was a suitable sterol substrate for the algal SMT enzyme.

36 CHAPTER 3. STUDIES ON STEROL METHYLATION INHIBITORS

As shown in Figure 1.8, the positive charge on the sulfiir atom on AdoMet is the key point to complete sterol side chain alkylation since it generates an electrophilic methyl group through the cleavage of C-CH3 bond and renders an nucleophilic reaction possible between alkene (24, 25-double bond of sterols) and AdoMet. The design of inhibitors is based on the putative transition state of a C-24, C-25 bridged carbocation and a complementary base in the active site of SMT enzyme. Based on the mechanism of methylation, we designed, synthesized and tested three types of SMT enzyme inhibitors: (1) substrate analogs which act as product inhibitors of the reaction; (2) substrate analogs which act as mechanism-based inhibitors; and (3) transition state analogs. In this chapter, we will discuss the design, preparation and inhibition studies of the inhibitors as follows: (I) C-20, C-21 and C-22 related inhibitors, (2) C-23 related inhibitors, (3) C-24 related inhibitors, (4) C-25 related inhibitors, (5) C-26 and C-27 related inhibitors, and (6) C-28 related inhibitors.

3.1 Design, preparation and inhibition studies of C-20, C-21 3.2 and C-22 related sterol inhibitors

20R Carbon is the only chiral center on the side chain of desmosterol, zymosterol, lanosterol and cycloartenol. As discussed in Chapter 2, the stereochemistry of carbon 20 is fimctionally significant since it determines the orientation of the side chain to be right- handed. To probe further effect of tetrahedral character and stereochemistry of C-20 on SMT enzyrne activity, sterols were prepared by: (1) adding new double bonds between C- 20, C-21 and C-22; (2) preparing novel 20-epidesmosterol; and (3) preparing 20R and 205'-22-aza sterols. The preparation of C-20, C-21 and C-22 related inhibitors is illustrated in Schemes 3.1 and 3.2. Pregnenolone 57 was initially converted to 3p-acetate by reaction with acetic anhydride and pyridine. Schiff base was formed by reaction between the keto group in the

37 side chain of pregnenolone and isoamyl amine. Reduction of Schiff base by sodium cyanoborohydride generated 20 epimers. Deprotection of 3P-acetate afforded 22-aza sterols 58a and 58b which could be separated by reverse phase HPLC, see Scheme 3.1 3P-Hydroxy group pregnenolone was converted to the tetrahydropyranyl ether (THP). Enolate alkylation of 3p-THP pregnenolone with 4-bromo-2-butene extended the side chain to give ketone 59. Diene 60 was achieved by a Wittig reaction. Deprotection of THP ether with concentrated hydrochloric acid led to the isomerization of C-20, 21 double bond to give 61, 62 and 63, see Scheme 3.2.

a,b,c ^ -T ^^^ + 60%

HO HO 57 58a 58b Yield Ratio: 1.65 : 1 Ki Ratio: 1 : 4.5

ibition Pattem: Competitive Non-Competitive

(a) AC2O, pyr., 70°C; (c) (CH3)2CH(CH2)2NH2.HCI, NaBH3CN, THF/MeOH, r.t. (c) LAH, ether r.t. Scheme 3.1. Preparation of 22-aza sterols

Desmosterol was originally synthesized by dehydration of 25-hydroxy cholesterol.^^^^ Dehydration usually gave 5, 24- and 5, 25-diene cholesterol, which could be separated by AgNOs-Silica gel chromatography, although they could not be separated by GLC. Since 25-hydroxyl cholesterol is not easily available, several research groups reported the preparation of desmosterol from different starting materials. Morisaki and coworkers reported a simple method to synthesize desmosterol from commercial cholenic acid.^^^^ Takano and coworkers synthesized desmosterol from dehydro-epiandrosterone,

38 using a stereocontrolled protonation method. ^^^^ Rosenstein also reported the preparation of desmosterol from 3p-acetoxy-bisnorchola-5-en-22-al.f^^^

65%

THPO

HO 25% HO HO 61 Competitive Inhibitor

(a)l) DHP, p-TsOH. 2) LDA, 4-bromo-2-butene, THF, -78''C; (b) Ph3P^CH3Br", n-BuLi, THF; (c) HCl, reflux, MeOH Scheme 3.2. Preparation of 20(17/21/22), 24(25)-dien sterols

We developed a different synthetic route to synthesize 20-epidesmosterol from pregnenolone. Compared with the starting materials used by others, pregnenolone is easily available and very cheap (about $2 /g from Aldrich). In our preparation of 20- epidesmosterol, the 20S chiral center was generated by reduction of 20, 21 double bond instead of reduction of 20, 22 double bond that was a common approach used by many

39 a, b 55%

OCH, 57 64

90% yield OCH, 20R:20S=3:1

+ 65% yield OCH3 24(25)en:25(27)en-4:l ^^^y^

69 70

90%

a) TsCl, Pyridine, r.t. overnight; b) NaOAc, MeOH, reflux 3h; c) LDA, THF, (Cli)2C=CHCH2Br, -78''C; d)n-BuLi, THF, P1^P(I)CH3 reflux; e)m-CPBA, CH^CIj, 0°C; f) H2/Pt02, 50 psi, r.t. 40 h; g) LAH, THF, reflux 5h; h) POC|,'Pyridine, r.t. 2h; i) p-TsOH, dioxane/l|o 85/15, 80°C, 2h; J) epimers separated by rp HPLC. Scheme 3.3. Preparation of 20-epidesmosterol

40 researchers.f^°^'^ The i-sterol 64 was prepared by formation of 3p-tosylate group and base catalyzed rearrangement. Diene 65 and 66 were synthesized by the same approach as described above. Regio-selective epoxidation of 66 gave epoxide 67 with wCPBA. Hydrogenation of 20, 21-double bond with platinum oxide led to the 20-epimer 68. Lithium aluminium hydride reduced the epoxide 68 to 25-hydroxyl i-sterol 69 through delivery of hydride to the less substituted carbon. Then phosphoryl chloride (POCI3) was used to eliminate 25-hydroxyl group to give 24, 25-double bond i-sterol 70 with isomer 25, 27-double bond i-sterol 71. Finally, i-sterol was treated by p-toluenesulfonic acid in dioxane aqueous solution to generate desmosterol 72a and epidesmosterol 72b which could only be separated by HPLC. In the mean time, 20i?-25(27)-en-cholesterol 73a and 20S-25(27)-en-cholesterol 73b were also prepared as the by-products. In the inhibition studies of SMT enzyme from S. cerevisiae, 20i?-22-azacholesterol 58a has a four to fivefold lower Kt value than 205'-22-aza-cholesterol 58b. This result indicates that inversion of the configuration at C-20 from the 20R to the 205' should change the conformation of side chain from its natural position to the unnatural that binds the enzyme less productively. Furthermore, 58b performs non-competitive-type kinetics, while 58a shows competitive-type kinetics, demonstrating different binding pattems of the 22-azasterol diastereoisomers with the SMT enzyme. As described in our modeling study in Figures 2.8 and 2.9, the right -handed and left -handed side chains may not interact with the same residues in the SMT active site because of their different orientation in the binding pocket. We also found that triene sterol 61 (cholesta-5, 20(22), 24(25)-trienol) binds the enzyme with an apparent K^ of 100 |j,m and apparent Vmax of 5 pmol/min/mg protein. It is a competitive inhibitor with a Kj of 120 )^M. In comparison with 61,, triene sterols 62 and 63 were not considered as substrates or inhibitors after testing with the SMT enzyme. ^"^^^^ Obviously, the orientation of their side chains are critical in determining the different binding modes of three types of sterols. Based on these results, we believe that the side chain of sterols productively binds the SMT enzyme in the right-handed conformation. This result is also supported by our recent test that 20- epidesmosterol 72b is neither a substrate nor an inhibitor for the SMT enzyme.

41 CH, CH, K X SML ^^o. K \dc AdoMet + + s/\/\/\/^ Figure 3.1. C-23 and C-25 Carbon cations during the process of sterol methylation

(a) O3, CH2CI2, -78°C, then Me2S; (b) (CH3)2CHCH2NH2 HCl, NaBH3CN; (c) n-BuLi, THF, Ph3P"'CH2CH2N(CH3)2Br; (d) Zn(0Ac)2, AcOH, reflux, then LAH/ether, r.t. Scheme 3.4. Preparation of 23-aza sterol

3.2. Design, synthesis and inhibition studies of C-23 related inhibitors C-23 in the sterol side chain is close to the methylation center C-24 in the SMT enzyme catalyzed reaction. When the methyl group form AdoMet attacks the sterol 24, 25- double bond, C-23 and C-25 carbon cations may be formed in the mean time, as shown above. Because it is more stable than C-23 carbon cation, C-25 carbon cation is always presented as the major intermediate rather than that of C-23. Hence, to elucidate the role of C-23 in the reaction, heteroatoms of nitrogen and sulfur were usually used to substitute the C-23. Scheme 3.4 show the synthesis of 23-azasterols and 23-thiasterol. i-

42 Sterol 74 were prepared from stigmasterol by the same method as described above. Ozonolysis of sterol 74 gave an aldehyde 75. 23-Aza-cholesterol 76 was achieved by formation and reduction of a Schiff base and then deprotection of i-sterol. In the inhibition studies of SMT enzyme from S cerevisiae, 23-azacholesterol 76 had moderate inhibition effect with Kf value of 75 nm.f'*^^^

CHO

79 AcO ^ OTBDMS

v'V^/V^ 80

(a) O3, CH2CI2, -78°C, then Zn, AcOH or Me2S; (b) TBDMSiOTf, Et3N, CH2CI. (c) (CH3)2CHNH2 HCl, NaBH3CN, THF/MeOH, r.t.; (d) LAH, ether, r.t. Scheme 3.5. Preparation of 24-aza sterol

3.3. Design, preparation and inhibition studies of C-24 related inhibitors C-24 of the sterol side chain is the position on which the methyl group from the AdoMet attacks. To elucidate the role of this key position in the SMT enzyme catalyzed reaction, C-24 can not only be substituted by heteroatoms, but also designed bulkier by extending the side chains around C-24. A series of C-24 related inhibitors were prepared as illustrated in Scheme 3.5. A^"^^^^^ double bond of 3p-acetoxy zymosterol 78 was cleaved by ozonolysis to give an aldehyde 79. Further degradation of the side chain from 79 to 81 was accomplished by conversion of aldehyde 79 to its corresponding tert- butyldimethylsilyl enol ether 80 and subsequent ozonolysis of 23, 24-double bond. 24-

43 aza-zymosterol 82 was finally prepared by a procedure similarly used for 22-aza and 23- aza sterols. During the inhibition studies of SMT enzyme from S cerevisiae, 24-aza zymosterol performed strong binding affinity with Kt values of 20 nM.f'*^^^

AcO

(a) O3, CH2CI2, then Zn/AcOH; (b) Me2NH-HCl, MeOH/THF, NaBl^CN, then LAH, ether; (c) MeNH2, MeOH-THF, NaBH3CN, then LAH, ether; (d) Ph3P^CH3r, «-BuLi, THF Scheme 3.6. Preparation of 25-aza sterols

3.4. Design, preparation and inhibition studies of C-25 related inhibitors As shown in Figure 1.8, the transition state of sterol methylation with a bridged carbocation between C-24 and C-25 is the basis for the design of the SMT enzyme inhibitors, although a high-energy intermediate (HEI) model which includes a methyl at C-24 and a carbonium ion at C-25 was suggested as a putative mechanism for the SMT enzyme catalyzed reaction.^ ' In our approach to prepare 25-azasterols, ozonolysis of

44 3p-acetoxy zymosterol 78 gave 83, see Scheme 3.6. 25-Aza sterols 84 and 85 were generated from the aldehyde 43 by the reaction with dimethyl amine and methyl amine followed by reduction with sodium cyanoborohydride. Diene sterol 86 was prepared by a Wittig reaction. 25-aza cycloartenol 87 and 25-aza lanosterol 88 were prepared as the same method. 25-Aza desmosterol 89 was prepared from the i-sterol. During the inhibition studies of SMT enzyme from S. cerevisiae, 25-aza sterols was found to exhibit strong binding affinities with Ki values of 15 nM for 25-aza zymosterol 84, 20 nM for 25-aza cholesterol 89, 45 nM for 25-aza lanosterol 88 and 50 nM for 25-aza cycloartenol 87. The order of the Ki values indicates the specificity of the sterol nucleus for the SMT enzyme from S. cerevisiae. Zymosterol nucleus is clearly the preferred stmcture for molecular recognition of the SMT enzyme from S. cerevisiae. 25- Aza-27-nor-zymosterol 85 showed less binding affinity and 26, 27-binor zymosterol 86 was not observed any inhibition effect, indicating that 26, 27 dimethyl groups are functionally significant for the enzyme.^"^^^^

3.5. Design, preparation and inhibition studies of C-26 and C-27 related inhibitors The isopropyl C-26 and C-27 methyl groups are two carbons away from the reaction center C-24 of the SMT enzyme catalyzed reaction. If the terminal dimethyl groups C-26/C-27 can be eliminated or otherwise modified, the hydrophobic features of sterol side chain will be different. As a result, methyl group attack from the AdoMet to the C-24 may not proceed during the SMT enzyme catalyzed reaction. Based on this assumption, we prepared three types of C-26/C-27 related sterol methylation inhibitors. As shown in Scheme 3.7, 26, 27-cyclopropylidene zymosterol 90 was accomplished by a Wittig reaction with an aldehyde 83 generated by ozonolysis. PCC oxidation of 90 gave 3-keto sterol 91. Reduction of 91 with sodium borotritide afforded [3a-^H]26, 27-cyclopropylidene zymosterol 92 for SMT enzyme active site mapping.^"^^^^ 26, 27-Cyclopropylidene cycloartenol 93, 26, 27-cyclopropylidene lanosterol 94 were

45 prepared by the same method. 26, 27-cyclopropylidene desmosterol 95 was prepared from the i-sterol followed by the same Wittig reaction.^^'^ Shown in Scheme 3.8, C-26 trans methyl group of 3p-acetoxy zymosterol was selectively oxidized by selenium dioxide to afford an aldehyde 96. 24(25), 26(26')-dien zymosterol 97 was generated from a Wittig reaction with 74. Deprotection with lithium aluminum hydride generated the free alcohol.f^^^t^^"^^! 24(25), 26(26')-dien Cycloartenol 98, 24(25), 26(26)-dien lanosterol 99 and 24(25), 26(26)-desmosterol 100 were prepared by the same method.

Ratio:

(a) O3, CH2CI2, then Zn-AcOH; (b) Cyclopropyltriphenylphophonium bromide, /7-BuLi, THF, reflux 2h, then LAH, ether; (c) PCC, Cl^Clj; (d) NaB^li,, then separated by HPLC Scheme 3.7. Preparation of 26, 27-cyclopropylidene sterols

46 AcO

(a) Se02, ethanol reflux overnignt; (b) Ph3P^CH3r, n-BuLi, THF, reflux 3h, then LAH, ether Scheme 3.8. Preparation of 24(25), 26(26')-dien sterols

AcO

HO'

a) Se02, ethanol; b) CBr4, PPh3, Zn; c) n-BuLi, THF, -7^C; d) LAH, ether. Scheme 3.9. Preparation of 8(9), 14(15), 24(25)-trien- 25-ethylnyl-cholesta-3 p-ol

In Scheme 3.9, the Corey-Fuchs procedure was used for the transformation of the vinyl aldehyde 101 to the corresponding enyne 103.^^^^ The dibromide 102 was prepared

47 in excellent yield upon treatment of 101 with CBr4-PPh3/Zn in dichloromethane. The reaction of the dibromide with «-BuLi led to the enyne 103 also in excellent yield. The 26, 27-cyclopropylidene sterols are considered mechanism-based inactivators or suicide substrate inhibitors as indicated in the mechanism illustrated in Figure 3.2.^"*^^^ Shown in Figure 3.2, the arrangement of the bridged carbocation generated after the methyl group attack leads to open the cyclopropyl ring and form a new carbon cation at C-27 or C-26. This results in formation of a new bond between a base of the active site and the C-27 or C-26. Thus, the SMT enzyme will be inactivated. Direct evidences for the inactivation of the SMT enzyme by 26, 27-cyclopropylidene sterols was obtained by the active site mapping studies.^'^'^^^ The inhibition study of 8(9), 14(15), 24(25)-trien-25- ethylnyl-cholesta-3p-ol is in progress in our laboratory.

Figure 3.2. Hypothetical mechanism of inactivating SMT enzyme with suicide substrate inhibitor 26, 27-cyclopropylidene sterol

48 During the inhibition studies of SMT enzyme from the alga P. wickerhamii, 24(25), 26(26')-dien sterols was foimd to exhibit strong binding affinity to the enzyme (Nes and Mangla, unpublished results). Figure 3.3 shows a hypothetical mechanism of inactivating SMT enzyme with the suicide substrate inhibitor, 24(25), 26(26')-dien sterol. As shown in Figure 1.8, Si-face "methyl cation" attack from AdoMet to the 24, 25-double bond of sterol will lead to a C-25 carbocation. Consequently, a C-26' vinyl cation may be formed because of resonance. As a result, a covalent bonding should occur between C-26' and a base in the active site of the SMT enzyme. Therefore, 24(25), 26(26')-dien sterol can be used as a suicide substrate in SMT active site mapping.

Figure 3.3. Hypothetical mechanism of inactivating SMT enzyme with suicide substrate inhibitor 24(25), 26(26')-dien sterol

49 3.6. Design, preparation and inhibition studies of C-28 related inhibitors As shown in Figure 2.4, C-28 is a reaction center during the second Ci-transfer. Although we have confirmed that only one SMT enzyme catalyzes the two-step methylation, the mechanism of first and second methylation is different. The hypothetical transition state of second sterol methylation can be used as a model for preparing second methylation inhibitors. On the other hand, marine sterol biosynthesis always has more than one-step methylation and their side chain alkylation pattems are also different from plant sterols. ^^^^ '^^^ '^^^ ^^^^ ^^^^ For example, the side chain stmctures of marine sterols have cyclopro group and vinyl groups. Therefore, these marine sterol side chain stmctures may function in nature as substrate analogues for the inhibition of second C-methylation step.

AcO

•''/// 106 {^V^-'V^ 107 HO

(a) Benzyltriethylammonium chloride, CHCI3, KOH, 0°C, overnight; (b) Li, liquid NH3 Scheme 3.10. Preparation of 24(25)-methano sterols

As shown in Scheme 3.10, the dichlorocyclopropane 104 was prepared by the addition of dichlorocarbene to the 24, 25-double bond of 3p-acetoxy lanosterol.^^^^ Dissolving metal reduction of 104 yielded 24{R, S)-24, 25-methano-lanosterol 105 in excellent yield. 24{R,

50 S)-24, 25-methano-cycloartenol 106, 24{R, S)-24, 25-methano-zymosterol 107, and 24{R, S)-24, 25-methano-desmosterol 108 were prepared by the same method. To prepare aziridine sterols,^^^^ 109 was prepared by the addition of iodo isocyanate to the 24, 25-double bond of 3p-acetoxy lanosterol 45. Reduction of 109 yielded 24{R, 5)-25-epimino-lanosterol 110. 24{R, 5)-25-epimino-zymosterol 113 and 24{R, S), 2S{R, AS)-epimino-fucosterol 118 was prepared by the similar method as 110. Hydrogenation of 110 afforded 25-amino lanosterol m. 24{R, 5)-25-epimino-28-A^- methyl-lanosterol 112 was accomplished by the reaction of 110 with formaldehyde and sodium cynoboro hydride. 24, 28-Methano-cholesterol 115, 24{R, S), 2S{R, 5)-methano- fucosterol 117 were prepared from 3p-acetoxy-24-methylene cholesterol 114 and 3p- acetoxy-fucosterol 116, using the same method as 105, see Schemes 3.11 and 3.12.

NCO

NH X, 90%

CH3 N NHo >/v^/» wv^^ 112 111

(a) ICl, NaN3, CH3CN, r.t., 48 h; (b) LAH, ether, 12 h; (c) Hj, Ni, benzene, 12h; (d) HCHO, NaBH3CN, CH3CN/MeOH, AcOH

Scheme 3.11. Preparation of 24(25)-epimino sterol and hs derivatives

As shown in Scheme 3.13, 3p-acetoxy 24-keto lanosterol 51 was generated by the hydroboration of 3p-acetoxy lanosterol 45 and subsequent oxidation by PCC.^^^^ 3p- Acetoxy-24-oximolanosterol 119 was prepared by the reaction between 51. and

51 hydroxylamine hydrochloride. Reduction of 119 afforded a mixture of 24a- aminolanosterol 120a and 24p-aminolanosterol 120b which were resolved by TLC. 25a- Amino cholesterol 122a and 25p-amino cholesterol 122b were prepared from 25-keto cholesterol 121 using the same method, shown in Scheme 3.13. 24-Methylene sterol 123 was prepared by a Wittig reaction with 51. Hydroboration and oxidation of 123 gave 24^-28-aldehyde sterol 124. The racemic mixture was reacted with Wittig reagent and deprotected to yield 24^-vinyl-lanosterol 125 and 24^-28', 28"-cyclopropylidene lanosterol 126. Fecosterol 127 was synthesized by the same method. Unfortunately, 28, 28'-cyclopropylidene sterol 132 was not accomplished. The steric hindrance of C-26 and C-27 dimethyl groups prevent 24-keto unit from Wittig cyclopropylidene formation.

AcO

(a) Benzyltriethylammonium chloride, CHCI3, KOH, 0°C, overnight; (b) Li, liquid NH3; (c)AgOCN, I2, ether; (d) LAH, ether, 12 h Scheme 3.12. Preparation of 24(28)-methano and 24(28)-epimino sterols

52 In the inhibition studies of SMT enzyme from S. cerevisiae, inhibitors 110,113 and 118, which have the aziridine groups at their side chains, were found to generate Ki value of 10 nm, 5 nm and 3 |j,m, respectively. The results show that 24(25) epimino sterol is a highly potent inhibitor and added bulk at C-24 decreases the sterol specificity. 25- Amino-26-nor cholesterol 122a and 122b were used to explain the importance of branching at C-26. Comparing 25-aza cholesterol with 25-amino-26-nor cholesterol shows the former is about 10 times less efficient in binding to the SMT enzyme. These results indicate that the stmcture of the distal portion of the side chain, favors sterols that possess a gem dimethyl group at C-25 and a hydrogen atom at C-24. The remaining inhibitors will be studied shortly with plant and fungal SMT enzyme.

NOH

AcO >v

120a 120b

e,f + NH^ NH-, 29.3% 122a 122b

(a) BH3-THF, O^C; (b) PCC, CH2CI2; (c) Ph3P^CH3r, n-BuLi, THF; (e) NH2OH-HCI, NaAc, ethanol; (f) LAH, ether, reflux 4h. Scheme 3.13. Preparation of 24-amino and 25-amino sterols

53 HO

(a) BH3-THF, 0°C; (b) PCC, Cl^Clj; (c) Ph3P^CH3r, n-BuLi, THF; (d) Cyclopropyltriphenylphophonium bromide, n-BuLi, THF Scheme 3.14. Preparation of 24-methylene and 24-vinyl sterols

3.7 Summary The goals of this dissertation were to prepare and characterize sterol methylation inhibitors and to elucidate the mechanism of sterol methylation reaction catalyzed by the SMT enzyme from S. cerevisiae. These sterol methylation inhibitors are expected to mimic the stmctural and electronic properties of the carbocation intermediates formed during the normal sterol methylation reaction, see Figure 1.8. Table 3.1 is a summary of inhibition effects of the inhibitors toward the SMT enzyme from S. cerevisiae. We discovered that the modes of interactions of the inhibitors with the enzyme may be different according to the orientation of the side chain and nature of the charged species introduced into the sterol side chain.^^^ As shown in Table 6, 24{R, S), 25-epimino lanosterol 110 and 24{R, S), 25-epimino zymosterol 113 are the most potent inhibitors tested.

54 Table 3.1. Inhibition of the SMT enzyme from S. cerevisiae by substrate and inhibitors

Inhibitor # Ki Kinetic Pattem Lanosterol NA NA Cycloartenol NA NA Zymosterol 55 200 ^im C/R 20i?-22-aza-cholest-5-en-3p-ol 58a 220 nm C/R 205'-22-aza-cholest-5-en-3 P-ol 58b 1 fim NC/R Cholesta-5(6), 20(22)£, 24(25)-trien-20-one-3p-ol 61 120 \xm C/R Cholesta-5(6), 20(21), 24(6)-trien-20-one-3p-ol 62 NA NA Cholesta-5(6), 17(20)Z, 24(25)-trien-20-one-3p-ol 63 NA NA 23-Aza-cholest-5-en-3p-ol 76 75 nm NC/R 25-Aza-cholesta-5, 22-dien-3p-ol 77 250 nm NC/R 24-Aza-cholest-8-en-3 P-ol 82 20 nm NC/R 25-Aza zymosterol 84 15 nm NC/R 25-Aza-26-nor zymosterol 85 NA NA 26, 27-Dinor zymosterol M NA NA 25-Aza cycloartenol 87 50 nm NC/R 25-Aza lanosterol 88 45 nm NC/R 25-Aza desmosterol 89 25 nm NC/R 26, 27-Cyclopropylidene zymosterol 90 48 um C/I 26, 27-Cyclopropylidene cycloartenol 93 NA NA 26, 27-Cyclopropylidene lanosterol 94 NA NA 26, 27-Cyclopropylidene desmosterol 25 120 ^m C/I 24{R, S), 25-epimino lanosterol 110 lOnm NC/R 25-Amino lanosterol 111 50 jam ND 24(7?, S), 25-epimino zymosterol 113 5 nm NC/R 24{R, S), 2S{R, 5)-epimino fucosterol 118 3 [im NC/R 24-Methylene zymosterol 127 54 fxm C/R

Note. NA= not active; C= competitive inhibition; NC= non-competitive inhibition; ND= not determined; R= reversible type of inhibition; 1= irreversible type of inhibition. Enzyme kinetics were determined from Lineweaver-Burk plots. Kj values were determined in inhibition tested (range I nM to 100 |Lim) with a soluble SMT enzyme preparation ofS. cerevisiae: tested relative zymosterol as the sterol acceptor molecule.

Aziridine-containing sterols may act as either transition state analogues or as mechanism-based inhibitors,^'^^^^^'^^^^ depending on the amino acid composition in the

55 active site and on possible differences in protein stmcture that give the sterol access to different enzyme conformations during turnover, see Figure 3.4. Two possible bases are hypothesized to attach to SMT that participate in catalysis during the progress of the reaction: Base Bi (top) can fimction as a deprotonating agent to generate a A^"^^^^^- methylene stmcture; while Base B2 (bottom) may function as a nucleophile agent to form a covalent bond between a positive charged ion on the sterol side chain and B2. This hypothesis can be confirmed by studies on the active site of the SMT enzyme by genetic engineering.

E + S ES-complex EP I I B, I 1 r tn S tn CH3

~~Ternary complex: Transition State Intermediate enzyme-coenzyme- sterol acceptor molecule!~~

~~El-complex ^^ methvl from azridine grounp T H"\ ^' ^^ ^^ 7 I "*^ : M Tight Nu'^'T'^ , CH3 ^ : ) binding _ + N'nteract.on~~

-•EI Bi or B2 Neutral form Salt form Salt form (mechanism based inactivator) (transition state analog) (mechanism-based inactivator) EI Reversible inhibition Reversible inhibition Irreversible inhibition (slow-release) Figure 3.4. Hypothetical two possible inhibition pattem (ReversibleArreversible) determined by two different bases at the active site of the SMT enzyme. E= enzyme; S= substrate; P= product; 1= inhibitor, N= nucleus.(adapted from Ref 5)

~~56 SMTi enzyme from: Regions Deduced ( 1 ) and expected (2 ) reaction products II III IV Sacchromyces , cerevisiae H3N COO" 79-92 123-133 181-202 209-222 79 DF \YEYGWG JSFHFS 92~~

~~Nicotiana H.N tabacum COO" ^83-96^428-138 186-207 y^ ir^^-^ 210-223 83 DI YEWGWG QSFHFS96~~

~~Arabidopsis + thaliana H3N COO" 83-96^27-137 185-206 213-226 83 DI ^EWGWG bsFHFS 96~~

~~Glycine ^, + COO" max^ H3N (1) .81-94 125-135 183-204 X -....___;;-_^^ 211-224~~

~~""^ I *--l^ ^ (2) 8i SF ^EFGWG ^SFHFA 94~~

~~Triticum + aestivum H3N COO" (2) 79-9^123-133 183-204~~

~~79 SFIYEYGWG (2)~~

~~Zea mays j^ N COO' (1) 60-73 104-114 162-183 190-203~~

~~60 SFIYEYGWG iSFHFA 73 (2)~~

~~Figure 3.5. Similarity of amino acid sequence in the SMT enzymes from different sources (adapted from Ref 74)~~

57 According to the resuhs from sterol methylation mechanism studies, we found the similar mechanism occurred in plants and yeast,^^^^^^^^^^^ i.e.. Si-face methyl cation attack from AdoMet to 24, 25 double bond of sterol side chain, and Re-face hydrogen migration from C-24 to C-25. On the other hand, based on our recent progress on overexpression, purification and stereochemical studies of recombinant SMT enzyme from S. cerevisiae, we also found the similar amino acid sequence in several SMT enzymes from different sources,^^^^ see Figure 3.5. There are four regions in the SMT protein of conspicuous similarity, which we operationally refer to Regions I, II, III, and TV. Regions II, III, and IV have been recognized and are present generally in AdoMet-dependent enzymes and the SMT enzymes possess these three regions.^^^^ However, we discovered by examination of the gene sequences that all SMT enzymes possess an additional Region I, which proceeds from residues 79 to 92 in the yeast SMT enzyme, see Figure 3.5. Such conservation of amino acid residues in the primary stmcture of SMT enzymes suggests this domain may be unique to enzyme that act on sterols. Region I appears to contain an aromatic-rich YEXGWG motif that we have speculated may exist in the sterol binding site of the SMT enzyme for mechanistic reasons.^^^^^^^^^ Among this motif, tryptophan (W) has been considered to play two functions in the sterol binding site: as a counter ion to interact with the positive charge on the sulfur atom on the AdoMet thereby stabilizing specific cationic transition states along the methylation pathway to A^'^^^^^-sterol formation and as a deprotonating agent to generate a A^'^^^^^-methylene stmcture following methylation of the sterol side chain; however, other amino acids in Region I, such as Glu^^, could also conceivably fimction as the terminal deprotonating agent. This result is consistent with hypothetical mechanism in Figure 3.4. Tyrosine (Y) in the N-terminal of Region I may form a hydrogen bonding between its terminal hydroxyl group and sterol 3 p-hydroxyl group^^^^ to stabilize the enzyme-substrate complex. Histidine (H) which is three amino acid residues away from the C-terminal of Region I may play a role as another deprotonating agent or a nucleophile agent, as described in Figure 3.4. Combining all the results we achieved. Figure 3.6 is concluded to demonstrate the sterol-enzyme-AdoMet complex at the beginning of the sterol methylation reaction.

~~5i Figure 3.6. Hypothetical model illustrating the sterol-enzyme-AdoMet tertiary complex at the beginning of the sterol methylation reaction (adapted from W. D. Nes, with permission)~~

~~59 CHAPTER 4. MATERL\LS AND METHODS~~

4.1. Chemical reagents Lanosterol and stigmasterol were purchased from Sigma Chemical. Desmosterol was purchased from Steraloids. Cycloartenol was isolated from y-Oryzanol. Zymosterol was from the Nes steroid collection. AdoMet iodide salt was purchased from Sigma. [^H3-w^%/]AdoMet (10-15 Ci/mmol) was purchased from NEN dupont. Unless otherwise indicated, all the chemical reagents were purchased from Aldrich Chemical Co. Fresh THF and diethyl ether were prepared by heating the liquid at reflux, under nitrogen, in a recirculation still over sodium. Triethylamine, dichloromethane and DMSO were distilled from calcitmi hydride. Pyridine was purified by distillation under reduced pressure and stored over molecular sieve 4A or KOH pellets. Diisopropylamine was purified by distillation from calcium hydride and stored over KOH pellets. Benzene and hexanes was distilled from sodium. Dry methanol was obtained by distillation from magnesium tuming. Dry acetone was obtained by distillation from potassium carbonate. Yields refer to chromatographically and spectroscopically ('H-NMR (300 MHz)) homogeneous materials, unless otherwise stated. Reagents were purchased at highest commercial quality and used without further purification unless otherwise stated. The waste sodium was treated with methanol or ethanol. Cooling conditions were obtained as follows: 0-5°C, using cmshed ice; -5 to - 18°C, using a mixture of one part of sodium chloride and three parts of ice; -20 to -30°C, adding dry ice to CCU; -40 to -50°C, using a mixture of five parts of CaCl2-6H20 and 3.5-4 parts of cmshed ice; -50 to -78°C, adding dry ice to ethanol or acetone.

~~4.2. Methods NMR spectra was recorded in deutereochloroform at 300 MHz for ^ H-NMR and 75 MHz for '^C-NMR, using a Bmker AF300 spectrometer. Chemical shifts (5, ppm) are~~

60 referenced to TMS (6, 0).The following abbreviations were used to explain the multiplicities: s= singlet, d= doublet, t= triplet, q= quartet, m= multiplet, b= broad. IR spectra were obtained from thin films of the material between NaCl plates using a Perkin Elmer 600 series FT-IR spectrometer. Mass spectra were recorded on a Hewlett-Packard 6890 GC interfaced to a 5973 mass spectrometer. Capillary GLC was performed using a DB-5 column (J & W, Folsom, CA), 30 m x 250 jxm, film thickness 0.25 |xm; flow rate helium 1.2 ml/min; injector temperature 250°C; initial column temperature 170°C, hold for 1 min, ramp at 20°C/min to 280°C. Gas-liquid chromatography (GLC) was performed on a Hewlett-Packard 5890 series II instmment equipped with an HP3395 integrator, using a 3% SE-30 packed column operated isothermally with flame ionization detection. Oven temperature 245°C, injector A temperature 275°C, detector A temperature 300°C, column head pressure 60 psi, helium (carrier gas) pressure 60 psi, compressed air pressure 60 psi, and hydrogen pressure 45 psi, injection voltime 1 |xl. Reversed-phase high performance liquid chromatography (HPLC) was operated on 25 cm analytical and semipreparative CI8 columns at 1 ml/min and 4 ml/min, respectively. Sterols eluting in HPLC were detected by UV monitor set at 205 nm. Thin-layer chromatography (TLC) was performed on commercially prepared analytical plates (0.25 and 0.5 mm, Analtech) with visualization by spraying with 50% (v/v) aqueous H2SO4 and heating the plates slowly. Flash chromatography was performed on silica gel 60-200 mesh using eluants as indicated. The column was prepared by wet or dry packing. The eluting was performed under a little pressure to keep the rate at about 100 drops per minute. Melting points were determined in open capillary tubes on a Electrothermal apparatus and are uncorrected.

~~61 CHAPTER 5. NATURAL PRODUCTS ISOLATION AND BIOORGANIC SYNTHESIS~~

5.1. Isolation of cycloartenol from y-oryzanol (Figure 5.1) y-Oryzanol (100 g) was heated to reflux with 10% methanolic potassium hydroxide aqueous solution (1000 ml, KOH/H20/MeOH, 1:1:8, w/v/v/) for 30 min. The mixture was filtered by cheese clothes, extracted with diethyl ether (3 x 700ml). The combined extracts were concentrated by distilling ether and then diluted with toluene (150 ml) The solution was concentrated again by removal of toluene-water azeotropic mixture. The solid was dissolved in acetone and the precipitate was removed by centrifuge. The acetone solution was concentrated to give non-saponifiable fraction (NSF) (3 g). The NSF material was stored at 0°C. NSF (3 g) dissolved in acetone (30 ml) and silica gel (5 g) was added to the solution, Removal of solvent gave dry NSF-Silica gel mixture. The mixture was loaded on the top of a 5 cm x 100 cm silica gel column packed with hexanes. The column was eluted with hexanes (500 ml), 1:1 hexanes: benzene (500 ml), benzene (500 ml), 1:1 benzene: ether (1000 ml), ether (500 ml), 1:1 ether: methanol (500 ml) and methanol (500 ml). The eluants were collected by Erlenmeyer flasks 200 ml/100 min. 4,4 -Dimethyl sterol and desmethyl sterol fractions was determined by TLC, using cholesterol and lanosterol as references. These fractions were concentrated by removal of solvent. At this stage, the sterols should be separated from the other lipids. The sterols were dissolved in benzene (10 ml) and then loaded on the top of a 5 cm x 100 cm silica gel column packed with benzene. The column was eluted with benzene, 95:5 benzene: ether (500 ml), 90:10 benzene: ether (500 ml), 85:5 benzene: ether (500 ml), 80:20 benzene: ether (500 ml), 75:25 benzene: ether (500 ml), 70:30 benzene: ether (500 ml). The fractions were collected by an automatic collector with 30 ml/30 min per tube. The fractions were checked by TLC and then concentrated. At this step, 4,4-dimethyl sterol can be separated from the 4-desmethyl sterol. 4,4-Dimethyl sterols consist of 24-methylene cycloartanol and cycloartenol, which are separated from one another by chromatography on silver nitrate-impregnated silica gel columns.^ ^

~~62 100 g y-Oryzanol~~

~~1000ml KOH:Il20:MeOH 1 1:1:8, w/v/v. Reflux, 30 mini 1' NSF Material |~~

~~100% Hexane, 50% Hexane/Benzene, 100% Benzene, 50% Benzene/Ether, 100% Ether, 50°/ Ether/MeOH, 100% MeOH, Silica Gel Column~~

~~Triterpenoid Fractions~~

~~Benzene/Ether from 95:5 to 70:30 Silica Gel Column~~

~~Mixture of 24-Methylene Cycloartanol & Cycloartenol~~

~~Acetylation of 4,4-Dimethyl Sterol Mixture I~~

~~Silver Nitrate Impregnated Silica Gel Column I~~

~~3p-Acetoxy Cycloartenol \~~

~~Cycloartenoll~~

~~Figure 5.1. Isolation of cycloartenol from y-Oryzanol~~

63 The silver nitrate-silica gel column was pi-epared as follows: silver nitrate (27 g) was dissolved in water (30 ml) and then silica gel (100 g) was added in the solution. The mixture was concentrated by removal of water. The wet solid was dried in an oven at 90°C overnight. The dry silver nitrate-siliCa gel mixture was poured into a 4cm x 80cm column. The column was washed by 7:3 cyclohexane: toluene till the column became clear. 4,4-Dimethyl sterols was acetylated by the reaction with acetic anhydride in the presence of pyridine. 3P-Aetoxy-4,4-dimethyl sterols was then dissolved in 7:3 cyclohexane: toluene (5 ml) and then loaded on the top of the silver nitrate-silica gel column. The column was eluted with 7:3 cyclohexane:toluene (400 ml), 6:4 cyclohexane:toluene (400 ml), 5:5 cyclohexane: toluene (400 ml), 4:6 cyclohexane: toluene (400 ml), 3:7 cyclohexane: toluene (400 ml) and 2:8 cyclohexane: toluene (400 ml). The fractions were collected by an automatic collector with 25 ml/ 25 min per tube. The fractions were first checked by TLC. The fractions containing sterols were then examined by GC to confirm 3p-acetoxy-24-methlene cycloartanol and 3p-acetoxy cycloartenol with different retention time. The final products were reduced using LAH.

~~Table 5.1 is the Chemical Shifts of '^C- NMR (125 MHz) and 'H-NMR (500 MHz) for Cycloartenol (in CDCI3 at 303k)f^'^~~

5.2. Protection of 3p hydroxyl group of sterols (Figure 5.2) 3P Hydroxyl group of sterols is functionally significant in the temary complex, since it acts as a proton acceptor in hydrogen bonding interactions with residues from the active site.^^^^ The hydroxyl group is nucleophilic, acidic (pKa 10-18), and can be easily oxidized by a wide range of reagents. Because it can participate in many reactions under mild conditions, 3p hydroxyl group was protected by three types of protecting groups during our preparation of sterol biosynthesis inhibitors. ^^^^^^^^

~~64 Table 5.1. NMR spectra of cycloartenol~~

#Carbon ^^C-NMR ^H-NMR 1 32.00 1.25, 1.55 2 30.42 1.57,1.75 m 3 78.87 3.28 m 4 40.51 — 5 47.15 — 6 21.14 0.78 dd, 1.62 7 26.03 1.08, 1.32 8 47.99 1.51 dd (12.2,4.9)* 9 20.05 — 10 26.12 — 11 26.53 1.10, 1.98 dd 12 32.94 1.62 13 45.32 — 14 48.83 — 15 35.60 — 16 28.16 1.27,1.99 17 52.32 1.58 18 18.03 0.97 19 29.91 0.33 d (4.0) 0.56 d (4.3) 20 35.91 — 21 18.26 0.89 d (7.0) 22 36.38 1.05,1.44 23 24.97 1.86,2.40 24 125.29 5.10m 25 130.89 — 26 17.64 1.68 d (1.0) 27 25.72 1.61s 30 25.46 0.97 s 31 14.02 0.81 s 32 19.33 0.89 s (): coupling constant, Hz

65 5.2.1 Acetate ester 5.2.1.1. Formation of acetate ester 3p Hydroxyl sterol (1 g) was dissolved in anhydrous pyridine (8 ml) and then acetic anhydride (8 ml) was added to the solution. The mixture was heated at 70°C for 30 min. While still hot, the solution was decanted to ice-cold water (50 ml) and filtered. The solid was dried at 60°C under vacuum condition to yield the acetate in quantitative yield.

~~NMR data for the acetate protecting group: 'H-NMR (300 MHz), 2.02(s, 3H, 3- OCOCH3); ^^C-NMR(75 MHz), 170.65 (3-OCOMe)~~

5.2.1.2. Cleavage of acetate ester Method 1: 3p-Acetoxy sterol (500 mg) was dissolved in anhydrous diethyl ether (50 ml) and then LAH (25 mg) was added to the solution. The mixture was stirred at r.t. for 30 min. Water was added dropwise to decompose LAH till no bubbles in the reaction flask. The mixture was filtered. The filtrate was evaporated to give 3p hydroxyl sterol in 80% yield. Method 2: 3p-Acetoxy sterol (500 mg) was dissolved in 1:2 THF-MeOH solution (50 ml). Saturated aqueous K2CO3 (5 ml) was added to the solution. The reaction mixture was refluxed for I h under a nitrogen atmosphere and poured into diethyl ether (150 ml). The mixture was washed by water and brine and concentrated by removal of the solvents. The cmde product was purified by chromatography with 9:1 hexane-ethyl acetate.

5.2.2. (3a, 5)-Cvclo-6(3-methoxv ether 5.2.2.1. Formation of (3a, 5)-cyclo-6p-methoxy ether To protect the 5, 6-double bond from ozonalysis and hydrogenation, the 3p hydroxyl group in A^ sterols is usually converted to a (3a, 5)-cyclo-6p-methoxy ether form, so called "i-sterol". The following method was used to form and deprotect i- stigmasterol.

~~66 Ac20, Pyr, 70°C~~

~~LAH, ether, r.t. AcO~~

~~Dihydropyran, p-TsOH, Dioxane, 25°C~~

~~p-TsOH, Dioxan/H20 85:15, reflux, 2h THPO~~

~~1) TsCl or MeS02Cl, Pyr; 2) KHCO3, MeOH~~

~~Zn(0Ac)2, AcOH, reflux then LAH, ether; or p-TsOH, dioxane/H20 85/15, reflux OCH.~~

~~i - Sterol Nucleus Figure 5.2. Protection of 3P-0H of sterols~~

Step one: To a solution of stigmasterol (7.4 g, 18 mmol) in dry pyridine (100 ml) was added/7-toluene sulfonyl chloride (8.7 g, 45.6 mmol, recrystalized in hexane). The mixture was stirred in dark at r.t. overnight and then was added to ice-cold 5% sodium bicarbonate solution (500 ml). The solid was collected by filtration, washed with water and dried by vacuum oven, affording 3p-tosyl stigmasterol (9.5 g, 93% yield). GC RRTc: 0.64 (21.8%) & 0.85 (76.7%). HPLCac: 1.37.

^H-NMR (300 MHz): 7.775 (d, 2H, J= 8.34 Hz, 2, 6 H- benzene), 7.308 (d, 2H, J= 8.46 Hz, 3, 5 H-benzene), 5.27 (d, IH, 6-H), 5.04 (m, 2H, 22-H & 23-H), 2.425 (s, 3H, 3-methyl-benzene), 0.984 (d, J= 6.6 Hz, 21-H), 0.943 (s, 3H, 19-H), 0.651 (s, 3H, 18-H).

67 Step two: A solution of sodium acetate (6.7 g) in dry methanol (400 ml) was heated to reflux and then was added finely pulverised 3-tosyl stigmasterol (8.0 g, 14.1 mmol). The solution continued to reflux for 3 h and then was concentrated. Ether (350 ml) was added to the product and the solution was washed sequentially with water (300 ml), 5% sodium bicarbonate (200 ml), and water (200 ml), and then dried by magnesium sulfate. The solvent was evaporated and the cmde product purified by chromatography (hexane : ethyl acetate 9:1) to yield i-stigmasterol (4.9 g, 82% yield). GC RRTc: 0.82. HPLCac: 1.62.

~~^H-NMR (300 MHz): 5.125 & 4.96 (m'm, 2H, 22-H & 23-H), 3.290 (s, 3H, 6p- OCH3), 0.992 (s, 3H, 19-H), .981 (d, 3H, J= 6.6 Hz, 21-H), 0.701 (s, 3H, 18-H), 0.615 & 0.396 (t&q,lH&lH,4-H).~~

^^C-NMR (75 MHz): 82.37 (6P-OCH3), 21.41 (C-3), 19.25 (C-19), 13.05 (C-4), 12.23(0-18), 40.52 (C-20), 21.07 (C-21), 138.36 (C-22), 129.16 (C-23), 51.22 (C-24), 31.85 (C-25), 21.19 (C-26), 18.97 (C-27), 25.38 (C-28), 12.40 (C-29).

5.2.2.2. Cleavage of (3a, 5)-cyclo-6p-methoxy ether Method 1: i-Sterol (100 mg) was dissolved in 85:15 dioxane: water (8 ml) and then/7-TsOH (5 mg)was added to the solution. The reaction mixture was stirred and heated to reflux for 2 h. 5%> Sodium bicarbonate aqueous solution (20 ml) was added to the mixture and thoroughly extracted with diethyl ether. The combined ether extracts were dried with magnesium sulfate and evaporated to dryness to yield 70 % of 3 p hydroxyl sterol. Method 2: i-Sterol (100 mg) was added to the mixture of zinc acetate (500 mg) and glacial acetic acid (2 ml). The mixture was stirred at reflux for 2 h and then cooled, diluted with water (5 ml) and extracted thoroughly with hexane:benzene 50:50 (50 ml). The combined organic extracts were then washed with water, 5%) sodium bicarbonate, and sodium chloride. After the solution was dried over anhydrous magnesium sulfate, the

68 solvent was removed. The cmde product was dissolved in anhydrous diethyl ether and LAH (20 mg) was added to the solution. The mixture was stirred at r.t. for 30 min. Water was added dropwise to decompose the excess LAH. The mixture was filtered and the filtrate was evaporate to yield 3p hydroxyl sterol. The cmde sterol was then purified by chromatography.

5.2.3. Tetrahydropyranyl ether 5.2.3.1. Formation of tetrahydropyranyl ether To a slurry of pregnenolone (l.Og, 3.16 mmol) in dioxane (9 ml, distilled from sodium) was added dihydropyran (1.5 ml) and p-toluenesulfonic acid monohydrate (10 mg). The solution was stirred at ambient temperature for 3.5 h and then diluted with 100 ml of aqueous saturated sodium bicarbonate. The aqueous layer was extracted with ether (3 X 50 ml) and combined extracts were washed with water (2 x 50 ml) and concentrated under vacuum. The cmde product was purified by silica gel column with hexanes: ethyl acetate 95:5 to yield 3p-THP pregnan-5-en-20-one (1.1 g, 93%) GCRRTc: 1.384.

~~m.p.: 116-117°C.~~

~~'H-NMR (300 MHz): 0.628 (s, 3H, 18-H), 1.009 (s, 3H, 19-H), 2.128 (s, 3H, 21- H), 3.49 & 3.92 (m'm, 2H, THP OCH2), 3.54 (m, IH, 3-H), 4.71 (m, IH, THP methine),~~

~~5.348 (m, IH, 6-H).~~

5.2.3.2. Cleavage of tetrahydropyranyl ether 3P-THP pregnenolone (100 mg) was dissolved in 4:2:1 ACOH-THF-H2O solution (10 ml). The solution was stirred at 45°C for 2 h and then poured to 5% sodium bicarbonate aqueous solution (30 ml). The deprotected product was extracted with diethyl ether. The combined extracts were concentrated by removal of the solvent.

69 5.2.4. Summarv These three types of reaction are often used for protection of the 3p-hydroxyl group. Acetates are easily formed and cleaved. Acetates are stable to pH from 1-2 up to 8, organometallics, catalytic hydrogenation, borohydrides, Lewis acids, and oxidizing agents. They are not stable under mildly basic conditions and can be partially cleaved during the Wittig reaction. i-Sterol is used widely for protecting 3p hydroxyl group and 5, 6-double bond for A^ sterols, such as desmosterol, stigmasterol, and pregnenolone. i-Sterol is stable to base (pH 6- 12), organometallics, ylids, hydrogenation, and hydride reducing agents. Formation of the i- sterol is not a high yield generating reaction and three isomers are formed during the reaction of the second step which complicates purification of the desired product. Tetrahydropyranyl ether is stable to base (pH 6-12), hydrogenation, hydrides, and oxidizing agents, but unstable to aqueous acid and to Lewis acid. It is relatively stable to nucleophiles and organometallics.^

13 5.3. Preparation of [27- C]lanosterol (Scheme 2.1) and [27-'^C]zymosterol 5.3.1. 2-r CI methvl-1 -acetoxv-methvl triphenvbhosphonium iodide 44 A solution of methyl (triphenylphosphoranylidene) acetate 43 (Ig, 3 mmol) in 15

13 ml of ethyl acetate was heated at reflux and stirred, as a solution of [ C] - methyl iodide 13 (0.43g, 3mmol, 99%> C) was added at once. The reaction mixture was stirred and heated at reflux for 2 h. The solution was finally cooled and the resulting solid was filtered and purified by crystallization (chloroform - hexane) to give pure product 44 (0.9g, 63% yield). m.p.: 134-1350C

~~'H-NMR (200MHZ): 1.71 (3H, m, 'jp.c.H= 160 Hz, ^^CHj), 3.59 (3H, s, -OCH3), 6.67 (IH, m, J= 40 Hz, P-CH), 7.73 (lOH, m, J= 28 Hz, Cfl,), 7.97 (5H, m, J= 22 Hz, C6H5).~~

70 5.3.2. 24-al Lanosterol 46 A solution of lanosterol acetate 45 (500mg, 1.07 mmol) and pyridine (0,1 ml) in 25 ml of dichloromethane was cooled to -78 and treated with ozone at the rate of 1,500 mg per h for 40 min. After the solution was warmed to r.t., 1.2 ml of acetic acid and 600 mg of zinc dust was added. The mixture was stirred for 1 h. The remaining dust was filtered. Filtrate was washed with water (4 x 30 ml) and dried magnesium sulfate. The solvent was evaporated under reduced pressure and the residue purified by flash chromatography (20 mm column, 50 g of silica gel, packed by hexane) eluting with benzene. The appropriate fractions were combined and concentrated to yield pure aldehyde 46 (250 mg, 50% yield). GCRRTc: 1.79. m.p.: 74°C. ^H-NMR (300 MHz): 0.69 (s, 3H, I8-H3), 0.88 (each s, 9H, 19-H, 31-H, 32-H), 0.904 (d, 3H, J= 4 Hz, 21-H), 100 (s, 3H, 30-H), 2.05 (s, 3H, 3-OCOCH3), 4.50 (q, IH, J= 10.6 Hz, 3-H), 9.77 (t, IH, J= 2.5 Hz, 24-H). ^^C-NMR(75 MHz): 203.12 (C-24), 170.91 (3-OCOCH3), 134.26 (C-8), 134.20 (C-9).

13 5.3.3. 3p-Acetoxv-26-ester-r27- Cl-lanosterol 47 44 (286mg, 0.6mmol) suspended in 25ml dry THF was reacted with «-BuLi solution in heptane (1.6 M, 0.6mmol) at r.t. for 1 h with dry nitrogen with a serological septum. After 1 h, lano aldehyde (88mg, 0.2mmol) was added. The mixture was then stirred at r.t. for 12 h. Excess reagent was decomposed with moist ether and the reaction worked up in the usual manner. The solid was filtered and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (20 mm column, 40 g of silica gel, hexane-ethyl acetate 10:1). It gave labeled ester 47 (82mg, 80% yield). GC RRTc: 5.78.

~~71 1 H-NMR (300 MHz): 0.69 (s, 3H, 18-H), 0.88 (each s, 9H, 19-H, 31-H, 32-H),~~

0.93 (d, 3H, J= 5.7 Hz, 21-H), 1.00 (s, 3H, 30-H), 1.71 (d, 3H, 'JC-H= 181 Hz, 27-H), 2.05 (s, 3H, 3-OCOCH3), 3.73 (s, 3H, C-27OOCH3), 4.53 (q, IH, J= 18 Hz, 3-H), 6.78 (q, IH, J= 24 Hz, 24-H). 13 C-NMR (75 MHz) showed two signals: 12.30 (C-27), 20.69 (C-26) with 10.2:1 intensity ratio.

5.3.4. 3B-Acetoxv-26-ol-r27- Cl-lanosterol 48 A solution of aluminum chloride (3.5mg, 0.026mmol) in 5 ml dry ether was stirred at 0 C, when LAH (4mg, 0.1 mmol) was added immediately. The mixture was stirred for 30 min, when a solution of 47 (50 mg) in dry ether (10 ml) was added. The mixture was stirred at O^C for 1 h before successive addition of water (10 |LII), 10% aqueous sodium hydroxide (20 |al), water (15 ml) and 10% aqueous sodium hydroxide (10 |a,l). The resulting mixture was stirred an additional hour, the white precipitate was filtered and the filtrate was washed with aqueous saturated sodium chloride (2 ml). Evaporation of the solvent under reduced pressure and gave 48 (40 mg, 94% yield). GC RRTc: 3.44. ^H-NMR (300 MHz): 0.691 (s, 3H, 18-H), 0.812 (s, 3H, 31-H), 0.878 (s, 3H, 32- H), 0.924 (d, 3H, J= 5.95, 21-H), 0.983 (s, 3H, 19-H), 1.000 (s, 3H, 30-H), 1.62 (d, 3H, ^Jc-H= 146 Hz, 27-H), 3.25 (d'd, IH, J= 20 Hz, 3-H), 3.71 (m, IH, J= 22 Hz, C-26H2), 5.42(q, lH,J=22Hz,24-H).

~~C-NMR (75 MHz) showed two signals: 13.863 (C-27) & 21.271 (C-26) with 10.2:1 intensity ratio.~~

~~13 5.3.5. [27- ClLanosterol 49 Pyridine (4 ml) and acetic anhydride (4 ml) were added to the residue 48 (30mg,~~

~~0.090mmol). The mixture was heated at 70°C for 30 min. The solution was then poured~~

72 into 30 ml ice-cold water and extracted by hexane 3 x 30 ml. The combined hexane layers was evaporated under reduced pressure to give diacetate. Then a solution of the labeled diacetate in 5 ml ethyl amine was stirred at O^C, as small pieces of freshly cut lithium (10 mg) were added. When the initial effervescence had ceased, the mixture was stirred vigorously for 40 min. An aqueous solution of ammonium chloride (10 ml) was decanted and the mixture extracted with ether (3x15 ml). The organic extracts were combined, washed with saturated sodium chloride solution (15 ml). Evaporation of the solvent under 13 reduced pressure and purification of the residue by HPLC to give [27- C]lanosterol 49 (15 mg). GCRRTc: 1.63. HPLC ac: 0.957 MS: 427 (M""), 412, 394, 356, 341, 329, 311, 273, 259, 241, 215, 187,161, 145, 119,95,81,70,41. 'H-NMR (300 MHz): 0.688 (s, 3H, 18-H), 0.811 (s, 3H, 31-H), 0.874 (s, 3H, 32- H), 0.912 (d, 3H, J= 5.95 Hz, 21-H), 0.9.81 (s, 3H, 19-H), 1.00 (s, 3H, 30-H), 1.686 (d, 3H, ^Jc.H=3.98, 26-H), 1.603 (d, 3H, ^JC.H= 125 Hz, 27-H), 3.24 (d'd, IH, J= 22 Hz, 3-H), 5.10(q, lH,J=24Hz,24-H). ^^C-NMR (75 MHz) showed two signals: 17.629 (C-27) & 25.719 (C-26) with 10.2:1 intensity ratio.

5.3.6. r27-^^C1Zvmosterol [27-^^C]Zymosterol was prepared by the same method as described above for [27- '^C] lanosterol, but containing 5 % nucleus isomer. 'H-NMR (200 MHz): 0.608 (s, 3H, 18-H), 0.950 (s, 3H, 19-H), 0.944 (d, J= 6.3

Hz, 21-H), 1.600 (d, 'Jc-H= 125.0 Hz, 3H, 27-H), 1.683 (d, 'JC-H= 3.0 Hz, 3H, 26-H), 3.492 (m, IH, 3-H), 5.094 (m, IH, 24-H). '^C-NMR (75 MHz): two enhanced signals 17.628 (C-13) and 25.717 (C-26) with 5.7:1 intensity ratio.

73 13 5.4. Biosynthesis of [27- CJergosterol 50 in S. cerevisiae strain GL7 (Scheme 2.1) S. cerevisiae strain GL7 was grown on a medium of 2%) peptone, 1% yeast extract and 2% glucose. Experiment was carried out with 6 x 300 ml of medium in 6 x 1 liter 13 Erlenmeyer flasks for studies to isolate minor metabolites of [27- C]lanosterol 49, 100 ml of medium in a 250 flask for ergosterol (0.5 mg/L) and 100 ml of medium in a 250 ml 13 flask for blank controlling. [27- C]Lanosterol (6x1.5 mg) in absolute ethanol and Tween 80 (6 x 4.5 ml) was added to medium (6 x 300 ml medium). Culture was inoculated with ca. 0.5 x 10^ cells/ml and shaken continuously for 72 h at 180 rpm on a o GIO gyrator shaker at 27 C. Growth was monitored by counting the cells with a hemocytometer. Cultures were centrifuged at 8,000 rpm for 15 min and gave cells (19 g). The cells were saponified at reflux temperature for 30 min in aqueous methanolic KOH (10% KOH, 10%) H2O and 80%) MeOH, w/v/v) to produce a nonsaponifiable lipid fraction (NSF). The NSF obtained by dilution with water and extraction with diethyl ether was first injected into a GC column packed 3% SE-30 to determine the lipid profile and then purified by reversed-phase HPLC (semipreparative Zorbax column), eluting with methanol (4ml/min). Finally, [27-^^CJergosterol 50 (1.9 mg, 21% yield) was achieved. HPLC ac: 0.82 (C-18-Whatman, 96:4 methanol-water). EI-MS: 397 (M^), 364, 338, 271.

~~'H-NMR (300 MHz): 0.631 (s, 3H, 18-H), 0.823 (q, 3H, ^JC-H= 6.7 Hz, JH-H= 5.3~~

Hz, 26-H), 0.837 (q, 3H, 'JC-H= 124.3 Hz, JH-H= 6.7 Hz, 27-H), 0.918 (d, 3H, J= 6.8 Hz, 28-H), 0.947 (s, 3H, 19-H), 1.037 (d, 3H, J= 6.6Hz, 21-H), 3.627 (m, IH, 3-H), 5.223 (m, 2H, 22-H, 23-H), 5.385 (m, IH, 7-H), 5.574 (m, IH, 6-H). ^^C-NMR (75 MHz) shows two signals: 19.616 (C-26) & 19.925 (C-27) with 1:11.4 intensity ratio.

74 5.5. Preparation of [24- H]lanosterol 53 and biosynthesis of [25- H]ergosterol 54 in S. cerevisiae strain GL7 (Scheme 2.2) 5.5.1. 3P-Acetoxv-24-keto lanosterol 51 3p-Acetoxy lanosterol 45 (500 mg), purified from commercial lanosteryl acetate by silver nitrate silica gel, was dissolved in dry THF (15 ml) at 0°C under a nitrogen atmosphere. Borane-tetrahydrofiiran complex solution (1.0M,1.5 ml) was added dropwise. The reaction was stirred for 1 h at 0°C under nitrogen. The mixture was poured into 60 ml water and extracted thoroughly with diethyl ether. The combined extracts were dried over anhydrous magnesium sulfate, evaporated at reduced pressure. The residue was dried in a vacuum dessicator over phosphoms pentoxide at 50°C for 3 h and ovemight at r.t.. The residue was then dissolved in dry dichloromethane (15 ml), PCC (500 mg) and molecular sieves (10 mg, 4A) were added. The reaction mixture was stirred and heated to reflux for 2 h. The mixture was concentrated by removal of the solvent and dissolved in diethyl ether (150 ml). The ether solution was washed by 5% sodium bicarbonate aqueous solution (50 ml), water (50 ml) and brine (50 ml). The cmde product was concentrated and purified by chromatography with 9:1 hexane-ethyl acetate to afford 3p-acetoxy-24- keto lanosterol 51 (310 mg, 60% yield). GCRRTc: 3.257. 'H-NMR (300 MHz): 0.650 (s, 3H, 18-H), 0.874-0.881 (m, 12H, 30-H, 31-H, 32- H, 21-H), 1.001 (s, 3H, 19-H), 1.082 (s, 3H, 26-H), 1.104 (s, 3H, 27-H), 2.054 (s, 3H, - OCOCH3), 4.506 (m, IH, 3-H). '^C-NMR (75 MHz): 24.25 (C-27), 22.90 (C-26), 30.18 (C-25), 215.66 (C-24), 40.92 (C-23), 37.58 (C-22), 18.58 (C-21), 36.18 (C-20), 18.19 (C-19), 15.85 (C-18), 50.56 (C-17), 134.52 & 134.30 (C-8 & C-9), 171.14 (-OCOCH3), 81.01 (C-3).

5.5.2. r24a-^H1-24a-ol-3P-Acetoxv lanosterol 52 24-Keto sterol 51 (100 mg) was dissolved in absolute ethanol (15 ml). Sodium borodeuteride (10 mg) was added and the mixture stirred at 0 C. After 3 h at r.t., the

~~75 mixture was poured into saturated aqueous NH4CI solution and extracted thoroughly with ether. The cmde product 52 was dried by MgS04 and used for next step.~~

5.5.3. r24-^HlLanosterol 53 To a solution of 52 (50 mg) in dry pyridine (3 ml) was added POCI3 (0.1 ml) and the mixture was stirred for 3 h at r.t.. The mixture was poured to water (20 ml) and extracted thoroughly by ether. The combined extracts was dried by MgS04 and then reduced by LAH to give [24-^H]lanosterol 53 (25 mg). 'H-NMR of 53 shows absence of peak at 5.2 ppm.

5.5.4. Biosynthesis of [25-^H]ergosterol 54 Using the same method to biosynthesize [25-^H]ergosterol 54 in S. cerevisiae strain GL7 as section 5.4.. [24-^H]lanosterol 53 (5 mg/L) instead of [27-'^C]lanosterol 49 (5 mg/L) was added to in one liter medium. ' H-NMR of 54 shows two siglet peaks for H-26 and H-27, see Figure 17 spectrum C.

5.6. Biosynthesis of [28-^H2]ergosterol 56 in S. cerevisiae strain GL7 (Scheme 2.3) Using similar method as section 5.4.. The only change is to replace [27- C]lanosterol 49 (5 mg/L) with zymosterol 55 (5 mg/L) and [ H3-mg^/i>^/]methionine

~~(625 mg/L) in one liter of medium. 'H-NMR of [28-^H2]ergosterol shows significantly reduced doublet peaks for H-28, see Figure 17 spectrum B.~~

5.7. Preparation of 20i?-22-aza-cholest-5-en-3p-ol 58a and 205'-22-aza-cholest-5-en-3p-ol 58b (Scheme 3.1) HCl solution in THF (1.0 M, 5.2 ml) (Aldrich) was added to isoamyl amine (0.6 ml). Evaporation of the solvent in a nitrogen stream gave isoamyl amine hydrochloride solid (500 mg, 4.07 mmol). Pregnenolone 57 (250 mg) was acetylated to its acetate (270 mg, 0.75 mmol) was then added to isoamyl amine hydrochloride. The mixture was dissolved in methanol (8 ml) and THF (4 ml) and then treated with sodium

76 cyanoborohydride THF solution (1.0 M, 2 ml). The mixture was stirred imder nitrogen at r.t. for 72 h. The mixture was concentrated and put in ether (100 ml) and then washed by 5% sodiimi bicarbonate aqueous solution (2 x 50 ml). The ether solution was dried by magnesium sulfate and then treated with LAH (100 mg) for 30 min at r.t.. Water was added to stop the reaction and residue was filtered. GC indicated that there were two isomers in cmde product with 1.62 : 1.00 ratio and 55.8%), 34.4%) yields. The two isomers were separated by semipreparative RP-HPLC column, eluting with methanol plus 0.1% NH4OH. 20i?-22-Aza-cholest-5-en-3p-ol 58a: GCRRTc: 0.895. HPLC ac: 0.42. MS: 387 (M^), 386, 372, 356, 330, 314, 283, 284, 256, 243, 213, 199, 173, 159, 133,114,105,91,58,53. ^H-NMR (300 MHz): 0.721 (s, 3H, 18H), 0.895 & 0.905 (d'd, 3H & 3H, J= 6.55 Hz & J= 6.56 Hz, 26-H & 27-H), 0.980 (d, 3H, J= 6.13 Hz, 21-H), I.OIO (s, 3H, 19-H), 2.36 - 2.78 (m, 3H, 20-H & 23-H), 3.50 (m, IH, 3-H), 5.35 (d, IH, 6-H). ^^C-NMR (75 MHz): 56.2 (C-20), 19.3 (C-21), 45.1 (C-23), 40.3 (C-24), 26.2 (C- 25), 22.4 (C-26), 22.9 (C-27), 71.7 (C-3), 140.7 (C-5), 121.6 (C-6).

~~205-22-Aza-cholest-5-en-3 P-ol 58b: GCRRTc: 1.032. HPLC ac: 0.47. MS: 386 (M^ 372, 330, 283, 243, 215, 199, 173, 145, 133, 114, 91, 58, 55.~~

^H-NMR (300 MHz): 0.693 (s, 3H, 18-H), 0.891 (d,.6H, J= 6.57 Hz, 26-H & 27- H), 1.005 (s, 3H, 19-H), 1.083 (d, 3H, J= 6.15 Hz, 21-H), 2.44 (m, 2H, 23-H), 2.69 (m, IH, 20-H), 3.50 (m, IH, 3-H), 5.35 (m, IH, 6-H). "c-NMR (75 MHz): 56.4 (C-20), 19.4 (C-21), 45.0 (C-23), 39.3 (C-24), 26.2 (C- 25), 22.6 (C-26), 22.8 (C-27), 71.7 (C-3), 140.8 (C-5), 121.6 (C-6).

77 5.8. Preparation of 3p-ol-cholesta-5(6), 20(22)£, 24(25)- trien-20-one 61, 3p-ol-cholesta-5(6), 20(21), 24(25)- trien-20-one 62 and 3p-ol-cholesta-5(6), 17(20)Z, 24(25)- trien-20-one 63 (Scheme 3.2) 5.8.1. 3p-Tetrahvdropvranvlooxv-21-nor-cholesta-5. 24-dien-20-one 59 Pregnenolone 57 was converted to 3p-tetrahydropyranylooxy pregnenolone, using the same method as section 5.2.3.1.. n-BuU in heptane(1.6 M, 0.94 ml, 1.5 mmol) was added to a solution of diisopropylamine (0.21 ml, 1.5 mmol) in dry THF (8 ml) under 99.999% nitrogen at

-78^0. After 15 min, 3p-Tetrahydropyranylooxy-pregnolone (0.6 g, 1.5 mmol) in THF (3ml) was added, and after 30 min this was followed by 4-bromo-2-methyl-2-butene (0.195 ml, 1.5 mmol). The solution was allowed to warm to room temp, ovemight. Then methanol (15 ml) was added and the mixture was poured into ether (100 ml). The ethereal solution was washed with water and 5%) sodium bicarbonate, and it was dried over magnesium sulfate. Evaporation of the solvent left a yellow oil which was chromatographed on silica gel (10:1 hexane / ethyl acetate). Concentration of the fractions exhibiting Rf= 0.69 on silica gel TLC with 10:1 hexane/ethyl acetate gave 3p-tetrahydropyranylooxy-21-nor-cholesta-5, 24-dien-20-one 59 (0.35 g, 50% yield).

~~m.p.: 104- 105°C. GCRRTc: 4.63. HPLC ac: 0.63 (semipreparative Zorbax column with methanol).~~

^H-NMR (300 MHz): 0.608 (s, 3H, 18-H), 1.005 (s, 3H, 19-H), 1.611 and 1.669 (s's, 6H, 26-H & 27-H), 3.499 & 3.907 (m'm, 2H, THP OCH2), 3.534 (m, IH, 3-H), 4.715 (m, IH, THP methine), 5.054 (m, IH, 24-H), 5.349 (m, IH, 6-H). '^C-NMR(75 MHz): 211.32 (C-20), 140.84 (C-5), 132.41 (C-25), 123.14 (C-24), 121.19 (C-6), 13.34 (C-18), 17.63 (C-26), 19.34 (C-19), 25.65 (C-27), 25.45 (C-23), 31.25 (C- 22).

78 5.8.2. 3p-Tetrahvdropvranvlooxv-cholesta-5(6). 20(211 24(25Vtrien-20-nne 60 «-BuLi (0.634 ml of 1.6M solution in heptane, 1.02 mmol) was added to a solution of methyltriphenylphosphonium bromide (412 mg, 1.02 mmol) in dry THF (10 ml), and the mixture was refluxed for 1 h. 3 P-Tetrahydropyranylooxy-2 l-nor-cholesta-5, 24-dien-20-one 59 (150 mg, 0.34 mmol) was added, and reflux was continued ovemight. The mixture was then filtered though the short silica gel column. The product was recrystalized from methanol to give 60 (130 mg, 86% yield).

~~m.p.: 92-930C. GCRRTc: 3.71. HPLC ac: 1.61 (semipreparative Zorbax column with methanol).~~

^H-NMR (300 MHz): 0.575 (s, 3H, 18-H), 1.010 (s, 3H, 19-H), 1.685 & 1.609 (s's, 6H, 26-H & 27-H), 3.52 (m, IH, 3-H), 3.50 & 3.894 (m'm, 2H, THP OCH2), 4.713 (m, IH, THP methine), 4.787 & 4.874 (s's, 2H, 21-H), 5.12 (m, IH, 24-H), 5.353 (m,lH, 6-H).

~~^^C-NMR(75 MHz): 149.346(C-20), 140.939 (C-5), 131.431 (C-25), 124.373 (C-24), 121.478 (C-6), 109.394 (C-21), 12.754 (C-18), 19.415 (C-19).~~

5.8.3. 3p-ol-Cholesta-5(6). 20(22)£. 24(25)-trien-20-one 61. 3P-ol-cholesta-5(6). 20(21). 24(6)-trien-20-one 62 and 3B-ol- cholesta-5(6). 17 (20)Z 24r25)-trien-20-one 63 3p-Tetrahydropyranylooxy-cholesta-5, 20 (21), 24-trien-20-one (20 mg) was refluxed in methanol (5 ml) containing two drops of 37.5% hydrochloric acid for 3 h. The product was poured into dry ether (80 ml) and washed with 5% sodium bicarbonate. The residue was recrystalized from methanol to give isomers 61, 62, 63 (total 12 mg, 75% yield). The three isomers were further separated by waterman RP-HPLC column with acetonitrile : isopropanol 9:1. 3P-ol-Cholesta-5, 20{22)E, 24-trien-20-one 61: GCRRTc: 1.08.

~~m.p.: 98-99°C (melts), 108°C.~~

~~79 HPLC ac: 0.438. MS: 382 (IVT), 367, 349, 339, 313, 300, 271, 255, 229, 213, 199, 185, 159, 136, 121, 109, 93, 67, 55.~~

~~'H-NMR (300 MHz): 0.549 (s, 3H, 18-H), 1.01 l(s, 3H, 19-H), 1.594 (s, 3H, 21-H), 1.638 (d, 3H, 27-H), 1.695 (d, 3H, 26-H), 3.533 (m, IH, 3-H), 5.13 (m, 2H, 22-H & 24-H), 5.36 (m, IH, 6-H).~~

~~^^C-NMR(75 MHz): 140.80(C-5), 134.35 (C-20), 131.23 (C-25), 124.13 & 123.62 (C- 22 & C-24), 121.67 (C-6), 12.86 (C-18), 19.42 (C-19).~~

~~3p-ol-Cholesta-5, 20(21), 24-trien-20-one 62: GCRRTc: l.OU. HPLC ac: 0.419.~~

~~m.p.: 98-99°C (melts), 108°C. MS: 382 (M""), 367, 349, 313, 295, 272, 271, 239, 213, 211, 185, 159, 145, 119, 105, 93, 69, 53.~~

'H-NMR(300 MHz): 0.581 (s, 3H, 18-H), 1.012 (s, 3H, 19-H), 1.612 (s, 3H, 27-H), 1.690 (s, 3H, 26-H), 3.531 (m, IH, 3-H), 4.790 & 4.878 (s's. 2H, 21-H), 5.120(m, IH, 24-H), 5.36(d, IH, 6-H). '^C-NMR (75 MHz): 149.31 (C-20), 140.8l(C-5), 131.43(0-25), 124.38(0-24), 121.63(0-6), 109.43(0-21), 12.757(0-18), 19.422(0-19).

~~3p-ol-Cholesta-5, 17 (20)Z, 24-trien-20-one 63: GC RRTc: 0.96. HPLC ac: 0.404. m.p.: 98-99°C (melts), 108°C. MS: 382 (M^ 368, 349, 339, 313, 295, 271, 253, 239, 211, 199, 187, 159, 145, 133,~~

~~105,95.~~

~~80 ^H-NMR (300 MHz): 0.867 (s, 3H, 18-H), 1.016 (s, 3H, 19-H), 1.564 (s, 3H, 21-H), 1.617 (s, 3H, 27-H), 1.692 (s, 3H, 26-H), 3.517 (m, IH, 3-H), 5.133 (m, IH, 24-H), 5.370 (m, IH, 6-H).~~

~~'^C-NMR (75 MHz): 143.6 (C-17), 140.8 (C-5), 133.6 (C-25), 125.0 (C-20), 124.9 (C- 24), 121.6 (C-6), 17,71 (C-18), 19.35 (C-19).~~

5.9. Preparation of 20-epidesmosterol 72b (Scheme 3.3) 5.9.1. i-Pregnenolone 64 Pregnenolone 57 (2.0 g, 6.32 mmol) was dissolved in 20 ml dry pyridine and thenp- Toluenesulfonyl chloride (2.1 g, 11.02 nmiol) was added to the solution. The reaction mixture was stirred at r.t. under darkness ovemight. The mixture was poured into 50 ml ice-cold water and filtered. The solid was dried under vacuo to give 3-tosyl pregnenolone. GC RRTc: 0.252, TLC (10:1 hexane-ethyl acetate) Rf: 0.25. The cmde product (3.0 g) was dissolved in anhydrous methanol (150 ml) and then sodium acetate (3.2 g) was added to the solution. The reaction mixture was stirred and heated to reflux for 2 h. The mixture was concentrated by evaporating the solvents and then poured into a solution of diethyl ether (150 ml). The solution was washed by water (2 x 50 ml) and brine (50 ml). The cmde product was concentrated by removal of the solvents and purified by chromatography with 95:5 hexane-ethyl acetate to afford i-pregnenolone 64 (1.2 g, 55% yield). TLC (10:1 hexane-ethyl acetate) Rf: 0.64. GCRRTc: 0.186. MS: 330 (M^), 315, 298, 275, 255, 228, 213, 187.

5.9.2. 20-Keto-24r25)-en-i-sterol 65 Diisopropylamine (0.5 ml) was added to dry THF (15 ml) at -78°C under a 99.999% nitrogen atmosphere and then n-BuU solution in heptane (1.6 M, 2.4 ml) was added dropwise to the solution. The mixture was stirred at -78°C for 30 min. i-Pregnenolone 64 (1.0 g) in dry THF (3 ml) was added dropwise to the LDA solution. The reaction mixture continued to stir at -78°C for 30 min. 4-Bromo-2-methyl-2-butene (0.5 ml) was added dropwise and the mixture

81 was stirred at -78°C for 2 h then at r.t. for 12 h. The reaction mixture was poured to ether (120 ml) and washed by 5% sodium bicarbonate aqueous solution (50 ml), water (50 ml) and brine (50 ml). The cmde product was concentrated by removal of the solvents and purified by chromatography though gradient solvent system with 97:3, 94:6, and 90:10 hexane-ethyl acetate to give 20-keto-24(25)-en-i-sterol 65 (600 mg, 50% yield). TLC (9:1 hexane-ethyl acetate) Rf: 0.57. GC RRTc: 0.722, MS: 398 (M^), 383, 366, 343, 315, 298, 281, 255, 229, 213, 187.

5.9.3. 20an, 24(25)-dien-i-Sterol 66 A2-BuLi solution in heptane (1.6 M, 0.95 ml) was added to a solution of methyltriphenyl phosphonium iodide (300 mg) in dry THF (10 ml) under a nitrogen atmosphere. The mixture was stirred and heated to reflux for 45 min. 20-Keto-24(25)-en-i-sterol 65 (100 mg) in dry THF (3 ml) was added to the suspended solution. The reaction mixture continued to stir and reflux for 3 h. The mixture was poured to ether (100 ml) and washed by 5% sodium bicarbonate aqueous solution (40 ml), water (40 ml) and brine (40 ml). The cmde product was concentrated by removal of the solvents and purified by chromatography with 4:1 hexane-ethyl acetate to afford 20(21), 24(25)-diene-i-sterol 66 (90 mg, 90% yield). GC: 0,562. MS: 396 (M""), 381, 364, 341, 321, 307, 285, 267.

5.9.4, 20r2n-en-24a5)-Epoxide-i-sterol 67 3-Chloroperoxybenzoic acid (wCPBA, 57-86% grade, 60 mg) was added to a ice-cold solution of 20(21), 24(25)-diene-i-sterol 66 (90 mg) in CH2CI2 (5 ml), and the mixture was allowed to warm slowly to r.t,. After 12 h, the mixture was diluted with ether (20 ml) and washed with IM NaOH (2x5 ml), 10% sodium bicarbonate (5 ml), and brine (5 ml) and then dried with magnesium sulfate and concentrated. The cmde product was purified by chromatography with 8:1 hexane-ethyl acetate to give 20(21)-en-24(25)-epoxide-i-sterol 67 (80 mg, 85% yield) as a colourless oil.

82 GC RRTc: 0,776. HPLC ac: 0,49. MS: 412 (M^), 397, 380, 365, 357, 339, 308, 281, 253. 'H-NMR (300 MHz): 0,442 (t, J= 13.1 Hz, IH), 0.626 (s, 3H, 18-H), 0,649 (t, J= 14 Hz, IH), 1,027 (s, IH, 19-H), 1.274 (d, J= 2.8 Hz, 3H), 1.311 (d, J= 1.2 Hz, 3H), 3.333 (s, 3H, 6-OCH3), 4.866 (d, J= 21.0 Hz, 21-H). '^C-NMR (75 MHz): 18.78 (C-27), 24.91 (C-26), 58,49 (C-25), 64.21 (C-24), 25.86 & 25,93 (C-23 R, S), 34,19 & 34,29 (C-22 R, S), 148,52 (C-20), 109,91 (C-21), 13.07 (C-18), 13.13 (C-4), 19,30 (C-19), 82,31 (-OCH3),

5.9.5, 20(R. 5)-24r25VEpoxide-i-sterol 68 A solution of 20(21 )-en-24(25)-epoxide-i-sterol 67 (50 mg) in dry dioxane (10 ml) was hydrogenated (50 psi) with Pt02 (5 mg) in a Parr apparatus for 24 h at r.t.. The reaction mixture was filtered and concentrated to dryness to give the oily residue 68 (45 mg, 90%) yield). GC RRTc: 0,777 for 205-24(25)-epoxide-i-sterol, 0.853 for 20i?-24(25)-epoxide-i- sterol. MS: 414 (M^), 399, 382, 359, 341, 325, 301, 285, 253, 229, 213.

5.9.6, 20(R. 5)-25-ol-i-Sterol 69 LAH (10 mg) was added to a solution of 20(i?, 5)-24(25)-epoxide-i-sterol 68 (30 mg) in dry THF (10 ml). The reaction mixture was stirred and heated to reflux for 5 h. Water was added dropwdse to the mixture till no bubbles appeared in the solution. The mixture was filtered and filtrate was concentrated to the oily residue 69 (27 mg, 90%) yield). GC RRTc: 0.820 for 20S-25-hydroxyl- i-sterol, 0.924 for 20R-25-hydroxyl- i-sterol. MS: 416 (M^), 401, 384, 361, 341, 311, 281, 253, 227, 207.

5.9.7, 20(R. 5^-24(25)-en-i-Sterol 70 and 20(R, 5)-25(27Ven-i-sterol 71 To a solution of 20(i?, 5)-25-hydroxyl-i-sterol 69 (20 mg) in dry pyridine (3 ml) was added POCI3 (0,1 ml) and the mixture was stirred for 3 h at r.t,. The mixture was poured to

83 water (20 ml) and extracted thoroughly by ether. The combined extracts was concentrated to give the desired olefins 20{R, 5)-24(25)-en-i-sterol 70 and their isomers 20{R, S)-25{27)-en- i- sterol 71. GC RRTc: 0,616 for 20R-24(25)-en-i-sterol, 0.569 for 20S-24(25)-en~i-sterol. MS: 398 (M^^), 383, 366, 351, 343, 314, 285, 253, 227, 199,

5.9.8, Desmosterol 72a and 20-epidesmosterol 72b 20{R, S)-24{25)-en- i-sterol 70 (10 mg) was dissolved in 85:15 dioxane-water (5 ml) and thenp-Toluenesulfonic acid monohydrate (1 mg) was added to the solution. The reaction mixttire was stirred and heated to 80°C for 3 h. The mixture was poured to ether (30 ml) and washed by 5% sodium bicarbonate aqueous solution (10 ml) and water (10 ml). The solution was concentrated and the cmde product was dissolved in 5 ml HPLC methanol, Desmosterol 72a (5 mg) and 20-epidesmosterol 72b (1.3 mg) was separated by semi-preparative Zorbax HPLC column with methanol as eluting solvent and analytical Waterman HPLC column with 95:5 methanol-water, Desmosterol 72a: GCRRTc: 1,070, HPLC ac: 0,787. MS: 384 (M^), 369, 351, 327, 300, 281, 271, 253, 229, 207. 'H-NMR (300 MHz): 0,679 (s, 3H, 18-H), 0,935 (d, J= 6.55 Hz, 3H, 21-H), 1,009 (s, 3H, 19-H), 1,603 (s, 3H, 27-H), 1.684 (s, 3H, 26-H), 5,088 (m, IH, 24-H), 5.353 (d, J= 5.16 Hz, IH, 6-H),

~~20-Epidesmosterol 72b: GC RRTc: 0,989. HPLC ac: 0,748. MS: 384 (M^ 369, 351, 327, 300, 281, 271, 253, 229, 207.~~

84 'H-NMR (300 MHz): 0,675 (s, 3H, 18-H), 0,838 (d, J= 6.55 Hz, 3H, 21-H), 1.008 (s, 3H, 19-H), 1.605 (s, 3H, 27-H), 1.688 (s, 3H, 26-H), 3.528 (m, IH, 3-H), 5,093 (m, IH, 24-H), 5,354 (d,J= 5,16 Hz, IH, 6-H).

Table 5,2, C-NMR (75 MHz) of desmosterol and 20-epidesmosterol #0 ODesmosterol S20-Epidesmosterol (ppm) 5epi - 5des (ppm) (ppm) 1 37.20 37,33 0,13 2 31.62 31,74 0,12 3 71.78 71.90 0.12 4 42.26 42.38 0.12 5 140.71 140.84 0.13 6 121.70 121.83 0.13 7 31.86 31.99 0.13 8 31.86 31.99 0.13 9 50.06 50,20 0.14 10 36.47 36.59 0.12 11 21.05 21.19 0.14 12 39.72 39.85 0.13 13 42.26 42.43 0.17 14 56.71 56.87 0.16 15 24.27 24.29 0.02 16 28.19 28,23 0.04 17 56.01 55,76 -0.25* 18 11.83 12.16 0.33 19 19,38 19.50 0.12 20 36.06 35.56 -0.50* 21 18.60 18.76 0.16 22 35.58 35,16 -0.42* 23 24.67 24.91 0.24 24 125.19 125.26 0.07 25 130.94 131,20 0.26 26 17.63 17.79 0.16 27 25.72 25.86 0.14 *Chemical shift of C-17, 20, 22 has the significant difference between desmosterol and 20-epidesmosterol. Range of background difference: 0.12 to 0.14.

85 5.10. Preparation of 23-aza-cholest-5-en-3p-ol 76 and 25-aza-cholesta-5, 22-dien-3p-ol 77 (Scheme 3.4) 5.10.1. (3a, 5)-Cvclo-6B-methoxv-cholest-22-al 75 i-Stigmasterol ((3a, 5)-cyclo-6p-methoxy-stigma-22-en) 74 was prepared by the same method as section 5.2.2.1.. A solution of 74 (1 g, 2.36 mmol) in dry CH2CI2 (30 ml) and pyridine (0.1 ml) was cooled to -78^0 and ozone was passed though the solution. Same workup as section gave 75 (480 mg, 52%) yield). GCRRTc: 0.31.

~~'H-NMR (300 MHz): 9.556 (d, IH, J= 3,2 Hz, 22-H), 3.303 (s, 3H, 6P-OCH3), 1,099 (d, 3H, J= 6.8 Hz, 21-H), 1.005 (s, 3H, 19-H), .744 {s, 3H, 18-H), 0.633 & 0.418 (t &q, 1H&1H,4-H).~~

~~'^C-NMR (75 MHz): 205,32 (C-22), 82,37 (6P-OCH3), 19,37 (C-19), 12,70 (C- 18),~~

5.10.2, 23-Aza-cholest-5-en-3-ol 76 A solution of (3a, 5)-cyclo-6P-Methoxy-cholest-22-al (70 mg) in 5 ml dry MeOH:THF 2:1 was treated with isobutylamine hydrochloride (200 mg) and sodium cyanoborohydride (0.48 ml I .OM THF solution) at r.t. for 40 h. The mixture was concentrated and added to ether (50 ml) and the solution washed with 5% NaHC03 (30 ml) and water (30 ml). The solvent was removed and the cmde product (3a, 5)-cyclo-23- aza-cholest-5-en-3-ol was added to the mixture of zinc acetate (200 mg) and glacial acetic acid (2 ml). The mixture was stirred at reflux for 2 h and then cooled, diluted with water (5 ml) and extracted thoroughly with hexane:benzene 50:50 (50 ml). The combined organic extracts were then washed with water, 5% sodium bicarbonate, and brine. After the solution was dried over anhydrous magnesium sulfate, the solvent was removed. The residue was purified by preparative TLC (20 x 20 cm, 1000 microns, eluting twice with hexane:ether:diethylamine 10:10:1, Rf= 0.45) to give 76 (30 mg, 40% yield).

~~GC RRTc: 0,97 (50%) & 1.07 (50%).~~

~~86 MS: 387 (M^), 372, 344, 300,271, 267, 239, 215, 199, 173, 156, 145, 119, 105,~~

~~^H-NMR (300 MHz): 5.323 (d, IH, J= 5 Hz, 6-H), 3.49 (m, IH, 3-H), .981 (d, 3H, J= 6,4 Hz, 21-H), .978 (s, 3H, 19-H), ,889 (d, 6H, J= 6,6 Hz, 26-H & 27-H), .676 & .671 (s's, 3H, 18-H),~~

~~^^C-NMR (75 MHz): 140,73 (C-5), 121,58 (C-6), 39,6 (C-20), 17,6 (C-21), 54.3 (C-22), 54,1 (C-24), 28,0 (C-25), 24,1 (C-26), 24,1 (C-27).~~

5,10.3, 25-Aza-cholesta-5, 22-dien-3-ol 77 (2-Dimethylaminoethyl) triphenylphosphonium bromide (300 mg, 0.72 mmol), suspend in dry THF (15 ml), was reacted with «-BuLi solution in heptane (1.6M, 0.45 ml) at r.t. for 1 h under dry nitrogen. (3a, 5)-Cyclo-6p-Methoxy-cholest-22-al (100 mg) was then added to the solution. The mixture was heated to reflux and stirred ovemight. Excess reagent was decomposed with moist ether and the mixture was eluted though a short silica gel column with ether. The cmde product (3a, 5)-cyclo-25-aza-cholest-22-en was added to the mixture of freshly fused zinc acetate (500 mg) and glacial acetic acid (2 ml). The mixture was stirred at reflux for 2 h and then cooled, diluted with water (5 ml) and extracted thoroughly with hexane:benzene 50:50 (50 ml). The combined organic extracts were then washed with water (30 ml), 5% sodium bicarbonate aqueous solution (30 ml), and brine (30 ml). After the solution was dried over anhydrous magnesium sulfate, the solvent was removed. The residue was purified by preparative TLC (20 x 20 cm, 1000 microns, eluting twice with hexane:ether:diethylamine 10:9:1, Rf= 0.48), yield 25-aza- cholesta-5, 22-dien-3-ol 75 (35 mg, 30% yield). GC RRTc: 0.99. MS: 385 (M^), 370, 341, 340, 307, 272, 239, 229, 199, 171, 145, 133, 105, 94, 58.

~~^H-NMR (300 MHz): 5.64 (t, IH, 22-H), 5.395 (m, IH, 23-H), 5.31 (d, IH, 6-H), 2.684 (s, 6H, 26-H & 27-H), 0,986 (s, 3H, 19-H), 0.983 (d, 3H, J= 6.47 Hz, 21-H), 0,697 (s,3H, 18-H).~~

~~87 ^^C-NMR (75 MHz): 140.74 (C-5), 121.50 (C-6), 71.66 (C-3), 35.09 (C-20), 21.14 (C-21), 145.43 (C-22), 115.38 (C-23), 56.62 (C-24), 42.86 (C-27), 42.55 (C-26).~~

5.11, Preparation of 24-aza-cholest-8-en-3p-ol 82 (Scheme 3,5) 5.11.1.3 P-Acetoxv-cholest-8-en-24-al 79 To a solution of zymosterol (1 g, 2,60 mmol) in pyridine (10 ml) was added acetic anhydride (10 ml). The reaction mixture was in 70 C for 30 min, and then it was decanted to ice cold water. The solid was filtered and dried in 60 C oven. Ozone was passed into a solution of the acetate zymosterol (0.55 g, 1.29 mmol) in CH2CI2 (35 ml) and pyridine

(0,1 ml) at -78 ^0. Using GC to monitor the reaction till the end. The solution was allowed to r,t, and then was added zinc (500 mg) and acetic acid (1,2 ml). The mixture was stirred for a h and then filtered. Filtrate was washed by saturated aqueous sodium bicarbonate (50 ml) and water (2 x 50 ml). The solvent was evaporated. The product was purified by chromatography (hexane:ethyl acetate 5:1), affording 79 (350 mg, 70% yield). GCRRTc: 1,26, ^H-NMR (300 MHz): 0,610 (3H, s, 18-H), 0.936 (3H, d, J= 4.2 Hz, 21-H), 0.963 (3H, s, 19-H), 2,029 (3H, s, 3-OCOCH3), 4,688 (IH, dd, J= 30 Hz, 3-H), 9,772 (IH, s,

~~24-H), ^^C-NMR (75 MHz): 128,20 (C-8), 134,73 (C-9), 170,65 (3-OC-Ac), 203.16 (C- 24)~~

5.11,2. 3B-Acetoxv-24-r(tert-butvldimethvlsilvl)oxv1- cholesta-8(9V 23r24Vdien 80 To an ice-cooled solution of 79 (200 mg, 0.45 mmol) in CH2CI2 (8 ml) containing dry triethylamine (1 ml) was added tert-butyldimethylsilyl trifluoromethanesulfate (0.15 ml, 0.66 mmol). The reaction mixture was stirred at r.t. for 1 h and then heated at 35 ^C for I h. The solvent was evaporated in vacuuo, and the residue purified by chromatography (3:10 CH2CI2: hexane) to yield 80 (105 mg, 0,19 mmol, 42% yield). A

~~88 cis/trans mixture of silyl enol ethers was determined by GC (RRTc 3.49), and by HPLC (RP column with methanol, ac: 1.2 & 2,02),~~

~~'H-NMR (300 MHz): 6,225 (d, IH, J= 6,0 Hz, 24-H), 4,687 (m, IH, 3-H), 4.445 (m, IH, 23-H), 0,958 (s, 3H, 19-H), 0,605 (s, 3H, 18-H), 0.117 & 0,073 (s, 3H each, SiMe),~~

~~^^C-NMR (75 MHz): 139.02 (C-24), 134.72 (C-9), 128.38 (C-8), 108.62 (C-23), 72,99 (C-3).~~

5.11.3. 3p-Acetoxv-cholest-8-en-23-al 81 A solution of 80 (105 mg, 0,19 mmol) in CH2CI2 containing pyridine (0.1 ml) was cooled to -78 ^C and ozone was added. Same workup as section 5.11.1. to yield 81 (56 mg, 0,14 mmol, 71% yield). GCRRTc: 1.01.

5.11.4. 24-Aza-cholest-8-en-3B-ol 82 A solution of 81 (56 mg, 0,14 mmol) in dry MeOH/THF 2:1 (6 ml) was treated with 200 mg of isopropylamine hydrochloride and 0,4 ml 1.0 M sodium cyanoborohydride THF solution at r.t. for ovemight. A product was determined by GC (RRTc 1,65), The mixture was concentrated and put in ether (50 ml) and then washed by 5% aqueous sodium bicarbonate (30 ml) and water (30 ml). The ether solution was dried by magnesium sulfate and then treated with LAH (10 mg) for 30 min at r.t.. Water was added to stop the reaction and the residue filtered. The solvent was evaporated and the cmde product purified by semi-preparative RP-HPLC column eluted with methanol treated with 0.1% NH4OH, affording 82 (30 mg, 0,08 mmol, 50% yield). GCRRTc: 1,14. HPLC ac: 0,60. MS: 387 (M""), 372, 344, 328, 313, 295, 271, 246, 213, 201, 170, 154, 131, 105, 91,72,55,

~~89 'H-NMR (300 MHz): 0.612 (s, 3H, 18-H), 0.948 (s, 3H, 19-H), 0.957 (d, 3H, 21- H), 1.064 (d, 6H, 26-H & 27-H), 2.795, 2.658 & 2.514 (m, IH each, 25-H & 23-H), 3.618 (m, 1H,3-H).~~

~~^ WMR (75 MHz): 134.99 (C-9), 128,21 (C-8), 71,18 (C-3), 45,0 (C-23), 48.9 (C-25), 22,9 (C-26), 23,0 (C-27).~~

~~Isomer 24-aza-chol-7(8)-en-3p-ol:~~

~~'H-NMR (300 MHz): 0.537 (s, 3H, 18-H)~~

~~'^C-NMR(75 MHz): 139,45 (C-8), 117,52 (7-C), 13.03 (C-19), 11.82 (C-18).~~

5.12, Preparation of 25-aza sterols, (Scheme 3.6) 5.12.1, 25-Aza sterols General method: Firstly, 3p-acetoxy-24-al sterols were prepared by the same method as section 5.11.1..Then a solution of 3p-acetoxy-24-al sterols (50 mg) in dry MeOH: THF (2:1 v/v) (5 ml) was treated with (CH3)2NH-HCl (200 mg) and sodium cyanoborohydride THF solution (1,0 M, 0,4 ml) under N.2 at r,t, for ovemight. The mixture was then poured into ether (40 ml). The ether layer was washed twice with 40 ml 5%) aqueous sodium bicarbonate and dried with magnesium sulfate. Removal of ether in vacuo gave 95%) yield, 3p-acetoxy groups was reduced to 3 p-OH by LAH. i-Sterol was deprotected to 3P-0H by zinc acetate and acetic acid.

~~Table 5.3 shows GC RRTc, MS, 'H-NMR and '^C-NMR data of 25-aza sterols.~~

5.12.2, 26-Nor-25-aza-cholest-8-en-3B-ol 85 To a solution of 3p-acetoxy 24-al zymosterol 79 (50 mg) in dry MeOH:THF (2:1 v/v) (3.5 ml) was added CH3NH2-HCI (200 mg) and sodium cyanoborohydride THF solution (1,0 M, 0.4 ml) and stirred under a nitrogen atmosphere ovemight at r.t.. The reaction mixture was poured into diethyl ether (50 ml). The ether was washed with 5% sodium bicarbonate aqueous solution (25 ml) and brine (25 ml) and then dried over

90 Table 5,3. Characterization of 25-aza sterols Inhibitors GC RRTc MS(M^) 'H-NMR (300 MHz) 25-Aza-cholesterol 1.00 387 0.946 (d, 3H, 21-H), 2.220 (s, 6H, 26-H and 27-H) 89 25-Aza-zymosterol 1.00 387 0.948 (d, 3H, 21-H), 2.222 (s, 6H, 26-H and 27-H) M 25 - Aza-cycloartenol 1.87 429 0.882 (d, 3H, 21-H), 2.208 (s, 6H, 26-H and 27-H) 87 25-Aza-lanosterol 1.07 429 0.907 (d, 3H, 21-H), 2.218 (s, 6H, 26H and 27-H) 88

Inhibitors '^C-NMR(75 C-21 C-22 C-23 C-24 C-25 C-26 O^ MHz) C-20 25-Aza-cholesterol 36.1 18.7 33.7 24.3 60.4 NP 45.5 45.5 89 25-Aza-zymosterol 36.2 18.7 33.6 24.3 60.5 NP 45.5 45.5 M 25-Aza-cycloartenol 36.0 18.3 33.9 24.4 60.4 NP 45.4 45.4 87 25-Aza-lanosterol 36.4 18.7 33.9 24.5 60.5 NP 45.5 45.5 88 magnesium sulfate. The mixture was filtered and concentrated. LAH (10 mg) was added to a solution of cmde product in dry ether. The deprotected product was purified by HPLC to give 85 (30 mg, 55% yield). GCRRTc: 1.17. HPLC ac: 0.98. MS: 373 (M^), 358, 340, 313, 277, 213, 201, 'H-NMR (300 MHz): 0.609 (s, 3H, H-18), 0.907 (d, 3H, H-21), 0,950 (s, 3H, H- 19), 2.429 (s, 3H, H-27) (H-26, missing). '^C-NMR (75 MHz): 36.52 (C-20), 18.72 (C-21), 36.18 (C-22), 26,47 (C-23), 52.47 (C-24), C-25 (absent), C-26 (absent), 33.47 (C-27).

91 5.13, Preparation of 26, 27-dinor-cholesta-8(9), 24(25)- dien-3p-ol 86 (Scheme 3,6) 3p-Acetoxy-24-al zymosterol 79 was prepared as section 5,11.1.. To a solution of methyltriphenylphosphonium bromide (412 mg, 1.02 mmol) in dry THF (10 ml) was added «-BuLi (0.634 ml, 1.6 M solution in heptane, 1.02 mmol) under nitrogen. The reaction mixture was stirred and heated to reflux for 45 min. 79 (130 mg) was then added to the mixture. The mixture continued to stir and reflux for 2 h. The mixture was poured to ether (150 ml) and washed by 5% sodium bicarbonate aqueous solution (50 ml), water (50 ml) and brine (50 ml). The solution was concentrated and the cmde product was purified by chromatography with 9:1 hexane-ethyl acetate, followed by deprotection with LAH to afford 26, 27-dinor-cholesta-8, 24-dien-3P-ol 86 (101 mg, 90% yield). GC RRTc: 0.67. HPLC ac: 0.64 (semipreparative Zorbax column eluted with methanol). MS: 356 (M""), 341, 323, 299, 273, 229, 213. 'H-NMR (300 MHz): 0.609 (s, 3H, H-18), 0.950 (s, 3H, H-19), 0.94 (d, 3H, H- 21). '^C-NMR (75 MHz): 35.854 (C-20), 18.538 (C-21), 35.180 (C-22), 30.588 (C- 23), 139.654 (C-24), 113.873 (C-25) (C-26 and C-27 are missing).

~~5,14, Preparation of 26, 27-cyclopropylidene sterols (Scheme 3,7)~~

5.14.1, Preparation of [3a- H1-26. 27-cvclopropvlidene-cholesta-8, 24-dien-3B-ol 92a 5,14,1.1. 26, 27-cyclopropylidene-cholesta-8, 24-dien-3P-ol 90 3p-Acetoxy-24-al zymosterol 79 was prepared as section 5.11,1..To a suspension of cyclopropyltriphenylphosphonium bromide (200 mg, 0.52 mmol) in dry THF (30 ml) rt-BuLi in heptane (1.6 M, 0.35 ml) was added. The mixture was heated and stirred for 1 h. 79 (70mg, 0.16 mmol) was added to the reaction. The mixture was stirred at r.t, for 2 h and then moist ether (30 ml) was decanted to decompose the reagent. The solid was filtered and the filtrate was dried by magnesium sulfate. The solvent was evaporated

92 under reduced pressure. The residue was purified by silica gel column chromatography, eluting with benzene. The fractions were collected and combined and the solvent was evaporated under reduced pressure. LAH (10 mg, 0.26 mmol) was added to the product in dry ether (40 ml). The mixture was stirred at r.t, for 15 min and then add water to it until no bubbles in the solution. The solvent was evaporated. According to GC, there are two components in the residue (RRTc: 1,48 & 1.57), Based on the 'H and ^^C NMR, the component eluting at RRTc 1.57 should be the product. The two components were separated by HPLC (semi-preparative column). 10 mg of 90 was generated, 30 % yield. m,p,: 100-101°C, MS: 382 {M"), 349, 349, 339, 297, 271, 257, 229, 213, 173, 145, 119, 107, 81, 55.

~~^H-NMR (300 MHz): 0,609 (s, 3H, 18-H), 0.953 (d, 3H, J= 6,42 Hz, 21-H), 0.950 (s, 3H,19-H), 1.012 (each s, 4H, 26-H2, 27-H), 3.619 (d'd, IH, J= 30 Hz, 3-H), 5.734 (m, lH,J=25Hz,24-H),~~

~~'^C-NMR (75 MHz): 1,777 (C-27), 2,165 (C-26), 118.80 (C-25), 120.59 (C-24), 28,63 (C-23), 35,69 (C-22), 18, 55 (C-21), 35.93 (C-20), 128,254 (C-8), 134.981 (C-9).~~

5.14,1,2, 3-Keto-26, 27-cyclopropylidene-cholesta-8(9), 24(25)-dien-3p-ol91 A suspension of 90 (10 mg), PCC (10 mg), powdered NaOAc (0,5 mg) in 1,0 ml dry CH2CI2 (purged O2 by N2 and stored with molecular sieves), was stirred at r,t, for 30 min. Following dilution with dry ether, the solution was stirred for another 10 min, and solution and the solid precipitate were placed on a short silica gel column and the column was eluted with diethyl ether. The solvents were removed from the filtrate. The residue 91 was dried by MgS04 and used directly for next step.

93 5.14.1.3. [3a- H]-26, 27-Cyclopropylidene-cholesta-8, 24- dien-3 P-ol 92a 21 (10 mg) was dissolved in absolute ethanol (2 ml). The contents of one vial of sodium borotritide (5 mCi) were added and the mixture stirred at 0°C. After 3 h at r.t,, sodium borohydride (2 mg) was added to the mixture and the reaction was allowed to proceed for an additional 2 h. The mixture was poured into saturated aqueous NH4CI solution and extracted thoroughly with ether. The cmde product was purified by analytical RP-HPLC column with methanol. HPLC ac: 0,7 ([3a-^H]-3p-ol-26, 27-cyclopropylidene Zymosterol, 92a), 0.59 ([3p- H]-3a-ol-26, 27-cyclopropylidene zymosterol, 92b),

~~5.14,2, Preparation of 26, 27-cvclopropvlidene-9B, 19-cvclopropvl-cholesta-24-en-3 P-ol 93 Using the same method described in section 5,14,1,,Yield was 68%.~~

~~m.p. 78.0-79.0 ^C (from MeOH). GC RRTc: 2.26. IRnmax: 3436.3,2931.0,2860.5, 1648.7, 1625.2, 1460.7, 1372.0, 1096.4, 1049,4,~~

~~685,497, EI-MS (m/z): 424 {M\ 5%), 409 (M" -CH3, 15%), 391 (M^ -CH3-H2O, 32%), 363 (12%), 337 (8%), 315 (7%), 284 (24%), 259 (10%).~~

'H-NMR (300 MHz): 0,965 (s, 3H, 18-H), 0.551 & 0.333 (d'd, 2H, 19-H), 0.905 (d, 3H, 21-H), 5,742 (m, IH, 24-H), 1,010 (bs, 4H, 26-H & 27-H), 0,984 (s, 3H, 28-H), 0,891 (s, 3H, 29-H), 0,891 (s, 3H, 30-H), ''c-NMR (75 MHz): 35.76 (C-20), 18,12 (C-21), 35.86 (C-22), 28.79 (C-23), 118,86 (C-24), 120.59 (C-25), 2.19 (C-26), 1,80 (C-27).

~~5.14.3. Preparation of 26, 27-cvclopropylidene-4.4-dimethvl-~~

~~94 14a-methvl-cholesta-8r9\ 24(25)-dien-3B-ol 94 Using the same method described in section 5.14.1., HPLC purification afforded the desired product 70%) yield,~~

~~m,p,: 125-127°C (from MeOH), GC RRTc: 2.0, HPLC ac: 0.88.~~

~~IRnmax: 3424, 2942,7, 1572,3, 1454.8, 1372,0, 1196, 1020,0, 720,3, 608,7 cm'^~~

~~EI-MS (m/z, relative intensity) 424 (M"", 12%), 409 (M"'-CH3, 39%), 391 (M^- CH3-H2O, 18%), 259 (14%), 241 (16%),~~

~~^H-NMR (300 MHz): 0.686 (s, 3H, 18-H), 0.980 (s, 3H, 19-H), 5.740 (m, IH, 24- H), l.lOl (bs, 4H, 26-H & 27-H), 0.999 (s, 3H, 28-H), 0.809 (s, 3H, 29-H), 0.876 (s, 3H, 30-H),~~

~~^^C-NMR (75 MHz): 36.14 (0-20), 18,54 (C-21), 35,86 (C-22), 28.77 (C-23), 118.84 (C-24), 120,59 (C-25), 2.19 (C-26), 1.80 (C-27).~~

5,14,4. Preparation of 26, 27-cvclopropvlidene-cholesta-5(6), 24(25)-dien-3B-ol95 24-Al-i-desmosterol was prepared by conversion of desmosterol to i-sterol and followed by ozonolysis. 26, 27-Cyclopropyhdene-cholest-5(6), 24(25)-dien-3p-ol 95 was accomplished by a Wittig reaction with 24-aldehyde i-desmosterol and ylid. From 50 mg starting material desmosterol, 5 mg of 95 was generated. GCRRTc: 1.33.

~~'H-NMR (300 MHz): 0,679 (s, 3H, 18-H), 0,944 (d, 3H, J= 6,5 Hz, 21-H), 1.010 (bs, 7H, 19-H, 26-H, 27-H), 5.731 (m, IH, 24-H), 5.353 (d, IH, 6-H).~~

~~'^C-NMR(75 MHZ): 140.75 (C-5), 121.71 (C-6), 71,80 (C-3), 35.61 (C-20), 18.53 (C-21), 35,48 (C-22), 28,57 (C-23), 118.82 (C-24), 120.60 (C-25), 2.18 (C-26), 1.79(0-27).~~

95 5,15, Preparation of 24(25), 26(26')-diene sterols (Scheme 3,8) 5,15,1,3 p-Acetoxv-26-al zymosterol 96 To a solution of 3p-acetoxy zymosterol 78 (400 mg) in absolute etiianol (50 ml) was added Se02 (180 mg). The mixture was stirred and heated to reflux for 15 h. After the mixture was concentrated by removal of the solvent in vacuo, it was poured into diethyl ether (150 ml). The ether was washed by water (70 ml) and 70 ml brine (70 ml). Evaporation of the organic layer gave the cmde product, which was purified by chromatography with 9:1 hexane-ethyl acetate to afford 96 (390 mg, 73% yield), GC RRTc: 3.29. MS: 438 (IVT), 423, 410, 395, 378, 363, 339, 312, 297, 279, 251, 238, 223, 199.

5.15,2, 24r25)-26r26'VDiene zvmosterol 97 To a suspended solution of methyltriphenyl phosphonium bromide (300 mg) in dry THF (15 ml) was added dropwise «-BuLi solution in heptane (1.6M, 0.45 ml). The reaction mixture was stirred and heated to reflux for 45 min under nitrogen. 96 (150 mg) was added to the reaction. The mixture continued to stir and reflux for 2 h. The reaction mixture was then poured to diethyl ether (150 ml). The ether was washed by 50 ml 5% sodium bicarbonate aqueous solution (50 ml), water (50 ml) and brine (50 ml). The organic layer was concentrated by removal of the solvents. The cmde product was purified by chromatography with 9:1 hexane-ethyl acetate and then dried over magnesium sulfate. The solid was then dissolved in anhydrous ether (20 ml) and LAH (15 mg) was added into the solution. The reaction mixture was stirred at r,t. for 30 min. Water was added dropwise to decompose excess LAH till no bubbles appeared. The mixture was filtered and the filtrate was dried by evaporating solvents. Recrystallization afforded 97 (120 mg, 90% yield). GCRRTc: 1,65. MS: 394 (M^), 380, 361, 297, 285, 270, 255, 237, 218.

~~96 5.15.3. 24r25)-26r26'VDiene cvcloartenol 98 24(25)-26(26')-Diene cycloartenol 98 was prepared by the same method as described above.~~

5.15.4. 24(25)-26r26'VDiene lanosterol 99 24(25)-26(26')-Diene lanosterol 99 was prepared by the same method as described above. GC RRTc: 2.49. MS: 438 (M^), 423, 405, 377, 355, 341, 313, 281, 246, 229, 207, 187. 'H-NMR (300 MHz): 0.689 (s, 3H, 18-H), 0,810 (s, 3H, 31-H), 0,876 (s, 3H, 32- H), 0,927 (d, J= 6.14 Hz, 3H, 21-H), 0.981 (s, 3H, 19-H), 1.001 (s, 3H, 30-H), 1.754 (s, 3H, 27-H), 3.236 (m, IH, 3-H), 4,913 (d, J= 10,62 Hz, IH, 26'-H), 5.068 (d, J= 17.32 Hz, IH, 26'-H), 5.481 (t, IH, 24-H), 6.371 (q, IH, 26-H). '^C-NMR (75 MHz): 29.69 (C-27), 110.19 (C-26'), 141.67 (C-26), 133.59 (C-25), 133.94 (C-24), 25.12 (C-23), 35.91 (C-22), 18.58 (C-21), 36.29 (C-20), 18.23 (C-19), 15.74 (C-18), 50.37 (C-17), 134.35 & 134.38 (C-8 & C-9), 78.96 (C-3), 24.24 (C-32), 15.41(0-31), 27.95(0-30).

5.15.5. 24(25V26a6'VDiene desmosterol 100 24(25)-26(26')-Diene desmosterol 100 was prepared by the same method as described above except that desmosterol was converted to i-sterol before the Se02 oxidation step and deprotection step. GCRRTc: 1.626.

5.16. Preparation of 8(9), 14(15), 24(25)-trien-25-ethylnyl- cholesta-3p-ol 103 (Scheme 3.9) 26-al Sterol 101 was prepared as the same method as section 5.15.1. To a suspended solution of zinc dust (163 mg, 2.5 mmol) and triphenylphosphine (650 mg, 2.5 mmol) in methylene chloride (20 ml) was added CBr4 (880 mg, 2.5 mmol) in methylene

97 chloride (3 ml) at r.t.. The resulting suspension was stirred for 24 h under a nitrogen atmosphere. 101 (150 mg)in CH2CI2 (5 ml) was then added to the reaction mixture, and the stirring was continued for an additional 5 h. Workup was accomplished by dilution of the mixture with pentane (50 ml) followed by filtration of the resulting mixture though Celite to remove the insoluble material and evaporation of the solvent. The insoluble material was subjected to additional CH2CI2 (2 x 30 ml) extraction. The methylene chloride-pentane mixture after an additional Celite filtration was concentrated, and the residue was dissolved in 9:1 hexane-ethyl acetate and filtered though a short silica gel column to remove any remaining insoluble material. After solvent evaporation the dibromide was obtained in essentially quantitative amounts as a colorless oil. The vacuum dried, unstable material was used directiy for the next step. TLC, 20:1 hexane- ethyl acetate, dibromide 102 sterol Rf= 0.45 (101 Rf= 0.18). To a solution of dibromide 102 (310mg, 0.52 mmol) in dry ether (15 ml) cooled to -78°C was added «-BuLi (1.60 M in hexanes, 0.9 ml, 1.44 mmol), and the resulting mixture was stirred for 10 min. After removing the cold bath, the mixture was stirred for one h, and then the reaction was quenched with ammonium chloride aqueous solution (20 ml). The mixture was extracted with ether (3 x 30 ml), and then the combined organic layers were washed with brine, dried with Na2S04, and concentrated, TLC (10:1 ethyl acetate), Rf= 0,10, The product was then reduced by LAH in ether. Flash chromatography (silica gel, 10% EtOAc/hexane) of the reduced residue afforded 180 mg (90% yield) of the enyne 103. The sample for spectral characterization was purified by RP-HPLC (Cig Zorbax, methanol), (Nucleus stmcture determination of 8(9), 14(15), 24(25)-trien-25- ethylnyl-cholesta-3p-ol 103 by '^C-NMR data, see Table 5.4) m.p,: 92-94°C GCRRTc: 1,604 HPLC ac: 0.447 MS: 392 (M^), 377, 359, 297, 270, 255, 237, 207

~~98 'H-NMR (300 MHz): 0,818 (s, 3H, 18-H), 0,963 (d, J= 6,38 Hz, 3H, 21-H), 0.992 (s, 3H, 19-H), 1,800 (s, 3H, 27-H), 2,760 (s, IH, 26'-H), 3,634 (m, IH, 3-H), 5.357 (s, IH,15-H), 5,953 (m,lH, 24-H)~~

~~Table 5,4. '^C-NMR data of 103 and 128~~

#0 5l28 Sl03 6103- 6128 I 35.2 35.3 0,1 2 31,3 31.7 0,4 3 70,7 71.0 0,3 4 37,9 38.3 0,4 5 40,8 40.9 0,1 6 25.1 25.1 0 7 26.4 26.6 0.2 8 122.7 123 0.3 9 140.4 140.9 0.5 10 36.3 36.5 0,2 11 21.7 21.8 0,1 12 36.8 36.9 0.1 13 44.8 45.1 0.3 14 150.7 151.0 0.3 15 117.1 117.3 0.2 16 35.7 35.8 0.1 17 57.0 57.0 0 18 15.6 15.7 0.1 19 18.2 18.4 0.2 20 33.8 33.8 0 21 18.7 18.7 0 22 35.9 34,9 1.0 23 23.6 25.2 1.6 24 39.3 140,4 25 27.8 116.4 26 22.4 87,0 26' 73,3 27 22.6 16,9 Note: From C-1 to C-21, 13'X-NM/ R chemical shift 0=<6io3- 6i28<=0.5, indicating that nucleus structures of 103 and 128 are same. Stmcture of 128; ' C-NMR data is compared with data on a similar sterol reported in Ref 79,

~~99 128~~

5,17, Preparation of 24(i?, S), 25-methano sterols (Scheme 3.10) 5.17.1. 3p-Acetoxv-24, 25-dichloromethano zvmosterol 104 To a solution of 3p-acetoxy zymosterol 78 (400 mg) in chloroform (10 ml) was added benzyltriethylammonium chloride (200 mg) at O'^C, After 10 min, 50% aqueous sodium hydroxide solution (6 ml) was added dropwise to the solution. The reaction mixture was stirred ovemight at 0°C under nitrogen. The mixture was poured into a separatory fimnel containing water (20 ml). The organic layer was diluted with diethyl ether (50 ml) and washed by 5% sodium bicarbonate aqueous solution (15 ml) and water (15 ml), dried over magnesium sulfate, and filtered. The solvents were removed using a rotary evaporator. The cmde product was purified by flash chromatography with 9:1 hexane-ethyl acetate to afford clear solid 104 (280 mg, 59% yield). GC RRTc: 3,914,

5.17.2. 24 (R, S), 25-Methano-zvmosterol 105 To a predried flask under a dry ice condenser was charged ammonia gas at -70°C. When enough liquid ammonia (50 ml) was in the flask, freshly cut lithium metal was added in small pieces. The blue color developed soon. After 30 min, 104 (100 mg) in dry THF (8 ml) was added dropwise to the dissolving metal solution. The mixture was stirred I h at the temperature maintained between -70 and -40°C, and then quenched with 30 ml of ethanol/ether 1:1 until disappearance of the blue color. The mixture was warmed to r.t. ovemight in a open flask for evaporation of the ammonia and solvents. The residues was dissolved in diethyl ether (100 ml) and then washed with water (50 ml) and brine (50 ml). After removal of the solvents, the cmde product was purified by chromatography with

100 95:5 hexane-ethyl acetate to afford 75 mg of 24 {R, S), 25-methano-zymosterol 105 (75 mg, 95% yield), GCRRTc: 1,28, HPLC: ac: 0.87. MS: 398 (M^), 383 (M^-CH3), 365 (M^-CH3-H20), 299, 285, 271, 255, 229, 213, 199. 'H-NMR (300 MHz): -0.158 , 0.344, 0.383 (m, 3H, H-28 & H-24), 0,609 (s, 3H, H-18), 0,920 (d, 3H, H-21), 0,950 (s, 3H, H-19), l.Ol I & 1,023 (d, 6H, H-26 & H-27), 3.618 (m,H-6), '^C-NMR (75 MHz): 36.32 & 36.42 (C-20), 18.85 & 18.77 (C-21), 36.89 (C-22), 26.27 & 26.54 (C-23), 25.14 & 24.98 (C-24), 19,89 & 19,98 (C-25), 19,75 & 19,49 (C- 28), 27,69 (C-27), 36.15 (C-26),

~~5.17.3. 24 (R, S). 25-Methano-cvcloartenol 106 24 {R, S), 25-Methano-cycloartenol 106 was prepared by the same procedure as described above.~~

5.17.4, 24 (R. S), 25-Methano-lanosterol 107 24 {R, S), 25-Methano-lanosterol 107 was prepared by the same procedure as described above. GC:RRTc: 1,80, HPLC ac: 0.87. 'H-NMR (300 MHz): 0.67 (s, 3H, H-18), 1,01 (s, 3H, H-19), 0,88 (d, 3H, H-21), 1,02 (d, 3H, H-26), 1.00 (d, 3H, H-27), -0.16 & 0.35 (m, 2H, H-28). '^C-NMR (75 MHz): 36:3 (C-20), 18.80 & 18.74 (C-21), 36.66 & 36,72 (C-22), 26,46 & 26,68 (C-23), 25,02 & 25,20 (C-24), 20.00 (C-25), 19.81 & 19.95 (C-26), 27.42 & 27.61 (C-27), 19.74 & 19.50 (C-28).

~~101 5,17,5. 24 (R. S), 25-Methano-desmosterol 108 24 {R, S), 25-Methano-desmosterol 108 was prepared by the same procedure as described above.~~

5,18. Preparation of 24(i?, S), 25-epimino lanosterol 110, 25-amino lanosterol m and 28-A^-methyl-24(7?, S), 25-epimino lanosterol 112 (Scheme 3.11) 5.18.1. Preparation of 24 (R. S). 25-epimino lanosterol 110 A suspension of iodine £izide was prepared by slowly adding iodine monochloride (0.4 g) to a stirred mixture of sodium azide (l.lg) in acetonitrile (6 ml). The reaction temperature was maintained at r,t, and stirring was continued for 1 h. A solution of 3p- acetoxy-lanosterol in THF (25 ml) was added over a 10 min period and the resulting reaction mixture stirred for 48 h. The mixture was evaporated to dryness and the residue was dissolved in ethyl ether (60 ml) and LAH (0,6 g) added. The reaction was allowed to stand for 12 h and ice cautiously added to decompose the remaining hydride. Water was added and extracted with ether. The residue obtained upon evaporation of ether was subjected to silica gel column chromatography. Elution with a solvent system of 5% triethylamine in ethyl acetate : toluene 1:1 resulted in the recovery of a product which was recrystalized from acetone-water to yield 24 {R, S), 25-epiminolanosterol 110 (370 mg, 45% yield) GC RRTc: 2,62, MS: 441 {M\ 62%), 426 (M^-CH3,100%), 424 (M^-NH3, 7%), 409 (6%), 166 (27%), 126 (14%), 109 (7%), 99(61%), 98 (57%), 'H-NMR (300 MHz): 0.692 (s, 3H, 18-H), 0.995 (s, 3H, 19-H), 0.906 (d, 3H, J= 6,3 Hz, 21-H), 1,249 (s, 3H, 26-H), 1.163 (s, 3H, 27-H), 0.98 (s, 3H, 30-H), 0,81 (s, 3H, 31-H), 0.874 (s,3H, 32-H). '^C-NMR (75 MHz): 36.1 (C-20), 18.6 (C-21), 34.2 (C-22), 26.4 (C-23), 43.8 (C- 24), 50,4(0-25), 27,5 (C-26), 19,4 (C-27).

102 5.18.2, Preparation of 25-amino lanosterol 111 24 {R, S), 25-epimino lanosterol 110 (200 mg) in benzene (20 ml) was hydrogenated for 12 h at 55 p,s,i. over a Raney Nickel catalyst (approx. 40 mg). The catalyst was removed by filtration though celite and the resulting filtrate was evaporated to dryness to yield a residue which was subjected to column chromatography using 5% triethylamine in ethyl acetate-toluene (1:1) as the eluting solvent. Evaporation of the solvent and crystallization from acetone-water yielded 25-aminolanosterol IH (182 mg, 90%). GC RRTc: 2.37. MS: 443 (M"", 10%), 428 (]Vr-CH3, 12%), 411 (M^-CH3-NH3, 8%), 393 (7%), 109 (4%). 'H-NMR (300 MHz): 0,682 (s, 3H, 18-H), 1.003 (s, 3H, 19-H), 0.914 (d, 3H, 21- H), 1,300 (s, 6H, 26-H & 27-H). '^C-NMR (75 MHz): 36,4 (C-20), 18.7 (C-21), 36,8 (C-22), 21,2 (C-23), 45,5 (C- 24), 49,4 (C-25), 30.3 (C-26 & C-27).

5.18.3. 28-A^-Methvl-24(ig. S), 25-epimino lanosterol 112 To a solution of nO (50 mg) in 5:1 CH3CN-MeOH (6 ml) was added aqueous HCHO (0.2 ml, 37%), NaBCNH3 (41 mg) and I drop of 99.9% acetic acid. The reaction mixture was stirred and heated to reflux ovemight and then 48 h at r.t.. The mixture was poured into 5% sodium bicarbonate aqueous solution (20 ml) and extracted thoroughly with diethyl ether. The combined extracts were concentrated and the cmde product was purified by RP-HPLC (Cig Zorbax, methanol containing 0.1% NH4OH) to afford 28-iV- methyl-24(i?, S), 25-epimino lanosterol 112 (1 mg, 2 % yield), GCRRTc: 2,10. HPLC ac: 0,60. MS: 455 (M^), 440 (M^-CH3), 422 (M^-CH3-H20), 207, 180, 159, 140, 112.

103 'H-NMR (300 MHz): 0,693 (s, 3H, 18-H), 1,001 (s, 3H, 19-H), 0,906 (d, 3H, J= 6,3 Hz, 21-H), 1,254 (s, 3H, 26-H), 1,174 (s, 3H, 27-H), 0.980 (s, 3H, 30-H), 0.811 (s, 3H, 31-H), 0.873 (s, 3H, 32-H), 2.202 & 2.371 (s's, 3H, N-CH3).

~~5.18,4, 24(R, S). 25-Epimino zvmosterol 113 24{R, S), 25-Epimino zymosterol 113 was prepared by the same procedure as described as above.~~

5,19. 24, 28-Methano cholesterol 115 (Scheme 3,12) Using the same method as section 5,17 to prepare 28-methano cholesterol 115 (78 mg, 75% yield) from 3P-acetoxy 24-methylene cholesterol 114 (100 mg). GCRRTc: 1.761. HPLC ac: 0.972. MS: 412 (M"), 397 (M^-CH3), 394 (M^-H20), 384, 379 (M^-H20-CH3), 369, 351, 327,300,271,255,231,213,187. 'H-NMR (300 MHz): 0.157 & 0.229 (eacht, H-28'), 0.6662 (s, H-18), 0.856 & 0,834 (d'd, J= 6.9 Hz, H-26 & H-27), 0.888 (d, J= 6.5 Hz, H-21), 1,006 (s, H-19), 3.493 (m, H-3), 5,352 (d, H-6), '^C-NMR (75 MHz): 9,912 & 10,002 (C-28, C-28'), 19.470 (C-27), 19.585 (C- 26), 30,111 (C-25), 32.556 (C-24), 24.146 (C-23), 32.556 (C-24), 18.811 (C-21), 36.123 (C-20),

5,20, 24{R, S), 2S{R, 5)-Methano fiicosterol 117 (Scheme 3.12) Using the same method as section 5.17 to prepare 24{R, S), 2S{R, S)-methano fiicosterol 117 (85 mg, 75% yield) from 3p-acetoxy fiicosterol 116 (120 mg), GCRRTc: 1.986. HPLCac: 1.033. MS: 426 (M^), 411 (M^-CH3), 408 (M^-H20), 393 (M^-H20-CH3), 365, 341, 314, 271,255,229,213,185,161.

104 ^H-NMR (300 MHz): -0,236, 0,374 & 0,598 (each m, H-28', H-28' & H-28), 0.6742 (s, H-18), 0.834 (d, J= 6.95 Hz, H-26 & H-27), 0.920 & 0.916 (d'd, J= 6.5 Hz, H- 21), 1.008 (s, H-19), 1,044 & 1,035 (d'd, J= 6,3 Hz, H-29), 3,53 (m, H-3), 5,354 (d, H-6). '^C-NMR (75 MHz): 19,612 & 19,531 (C-27), 19.792 (C-26), 28.118 & 27.941 (C-25), 34,054 & 33,925 (C-24), 25,679 & 25.537 (C-23), 34.434 & 34.291 (C-22), 18,866 & 18,793 (C-21), 36,705 & 36,609 (C-20), 14,465 & 14,394 (C-28'), 17.902 & 17.752 (C-28), 16,175 & 15,828 (C-29).

~~5.21. 24(i^, S), 2S{R, 5)-Epimino fiicosterol 118 (Scheme 3.12) Silver cyanate (108 mg) was added to the solution of 3p-acetoxy fucosterol 116~~

(100 mg) in anhydrous ether (6 ml) at 0 0, To the stirred suspension, solid iodine (135 mg) was added and the mixture continued to stir for 2 h and then 6 h at r,t,. The ether solution was filtered though Celite to remove the inorganic salts, then evaporated to give a light tan solid. The cmde iodo isocyanate was dissolved again in dry ether and the solution was treated with LAH (80 mg). The reaction mixture was allowed to warm at r.t. and stirring was continued for 10 h. A solution of sodium hydroxide (200 mg) in dry methanol (1 ml) was then added dropwise over a period of 5 min. The white precipitate was filtered and the salt was washed with ether and the solvent was washed with water and dried with magnesium sulfate. Solvent evaporation gave cmde product 118 (50 mg). The product was purified by semi-preparative C^ Zorbax HPLC column with methanol: isopropanol 9:1 plus 0,05%)NH4OH, GC RRTc: 2.65. HPLCac: 1,73, MS: 428 (M^), 412 (M^-CH3), 410 (M^-H20), 395 (M^-H20-CH3), 384, 368, 343, 317,300,285,271,257,213,211. ^H-NMR (300 MHz): 0.645 (s, 3H, 18-H), 0.854 & 0.875 (d'd, 6H, 26-H & 27- H), 0.902 (d, 3H, J= 6.5 Hz, 21-H), 0.976 (s, 3H, 19-H), 1.142 & 1.161 (d'd, 3H, 29-H), 3.482 (m, IH, 3-H), 5.328 (d, IH, 6-H).

105 ^^C-NMR(75 MHz): 140,78 (C-5), 121,62 (C-6), 71.65 (C-3),19,37 (C-19), 11.80 (C-18), 36.4 (C-20), 18.7 (C-21), 34.1 (33,6) (C-22), 26.4 (26.1) (C-23), 55.8 (C-24), 30.9 (C-25), 19.1 (C-26), 19.4 (C-27), 55.5 (C-28), 14.8 (C-29),

5,22. Preparation of 24a-amino lanosterol 120a and 24p-amino lanosterol 120b (Scheme 3.13) 5.22.1. 3p-Acetoxv-24-oximo lanosterol 119 3p-Acetoxy-24-keto lanosterol 51 was prepared in section 5,5,1,. 51 (100 mg) in EtOH (15 ml) was stirred with NH2OH-HCI (120 mg) and anhydrous NaOAc (140 mg) for 3 h to yield the corresponding oxime which was crystallized from MeOH to afford 92 mg of 3p-Acetoxy-24-oximolanosterol 119 (92 mg, 90%)). MS: 457(M^ 1%), 442 (]Vr-CH3, 9%), 426 (M^'-N-OH, 14%), 425 (2%), 424 (M"'-H20-CH3, 6%), 398 (6%), 364(10%), 215 (4%), 187 (6%), 175 (4%), 171 (5%), 161 (7%), 159 (13%), 157 (5%), 99 (9%), 98 (100%), 43 (49%). 'H-NMR (300 MHz): 0,688 (s, 3H, 18-H), 0.992 (s, 3H, 19-H), 0,964 (d, 3H, 21- H), 1,117 (s, 3H, 26H), 1,090 (s, 3H, 27-H). '^C-NMR (75 MHz): 37.1 (C-20), 18,4 (C-21), 32.1 (C-22), 23.6 (C-23), 167.1 (C-24), 33.5 (C-25), 20.1 (C-26 & C-27).

5.22.2. 24a-Amino lanosterol 120a and 24B-amino lanosterol 120b 3P-Acetoxy-24-oximo lanosterol 119 (60 mg) in anhydrous diethyl ether (25 ml) was refluxed with LAH for 4 h. Usual work up gave 54 mg of a residue which was purified by preparative TLC method (2 silica gel plates, 0.5 mm thickness, 20 x 20 cm. Chloroform : Methanol: Ammonia (85:15:0.25)) to give 24a-amino lanosterol 120a (3.7 mg, 6.5% yield) and 24p-amino lanosterol 120b (48.1 mg, 80% yield). 24a-Amino lanosterol 120a: GC RRTc: 2.23. MS: 443 (M^ 24%), 429 (M^-14, 5%), 428 (M^-CH3, 12%), 401 (6%), 400 (20%), 355 (3%), 168 (5%), 44 (100%).

106 'H-NMR (300 MHz): 0.691 (s, 3H, 18-H), 1.000 (s, 3H, 19-H), 0.904 (d, 3H, 21- H), 0.882 (s, 3H, 26-H), 0.925 (s, 3H, 27-H). '^C-NMR (75 MHz): 36.6(0-20), 19.0 (C-21), 34.9 (C-22), 21.0 (C-23), 58.2 (C- 24), 28.3 (C-25), 20.7 (C-26 & C-27).

24p-Amino-lanosterol 120b: GC RRTc: 2.62. MS: 443 (M^ 24%), 429 (M^-14, 5%), 428 (M^-CH3, 16%), 402 (5%), 401 (30%), 400 (100%), 184 (6%), 168 (1%), 86 (9%), 72 (18%). 'H-NMR (300 MHz): 0.691 (s, 3H, 18-H), 1.001 (s, 3H, 19H), 0.909 (d, 3H, 21- H), 0.887 (s, 3H, 26H), 0.925 (s, 3H, 27-H). '^C-NMR (75 MHz): 36.6 (C-20), 18.7 (C-21), 33.0 (C-22), 31.3 (C-23), 57.0 (C- 24), 19.2(0-26), 17.4(0-27).

5.23. Preparation of 25a-amino-26-nor cholesterol 122a and 25p-amino-26-nor cholesterol 122b (Scheme 3.13) 25-Keto-26-nor cholesterol was treated with NH2OH-HCI in dry EtOH and then with LAH in dry ether as the Preparation of 24-a-aminolanosterol and 25-p-amino lanosterol to afford 25a-amino-26-nor cholesterol 122a (29.3% yield) and 25p-amino-26- nor cholesterol 122b {52.1% yield). 25a-Amino-26-nor cholesterol 122a: GCRRTc: 1.45. MS: 387 (M^ 64%), 372(M''-CH3, 32%), 271(39%), 252 (13%), 173 (21%), 145 (39%), 105(100%). 'H-NMR (300 MHz) (in CDCI3 and CD3OD): 0.569 (s, 3H, 18-H), 0,893 (s, 3H, 19-H), 0,818 (d, 3H, 21-H), 1,127 (s, 3H, 27-H), '^C-NMR (75 MHz) (in CDCI3 and CD3OD): 35,5 (C-20), 18.3 (C-21), 35.5 (C- 22), 22.1 (C-23), 36.5 (C-24), 47.4 (C-25), 19.5 (C-27).

107 25p-Amino-26-nor cholesterol 122b: GCRRTc: 1.45, MS: 387 {W, 64%), 372 (M^-CH3, 32%), 271(39%), 252 (13%), 173 (21%), 145 (39%), 105 (100%), 'H-NMR (300 MHz) (in CDCI3 and CD3OD): 0,592 (s, 3H, 18-H), 0.918 (s, 3H, 19-H), 0.835 (d, 3H, 21-H), 0.918 (s, 3H, 27-H), '^C-NMR (75 MHz) (in CDCI3 and CD3OD): 35,6 (C-20), 18.5 (C-21), 35.7 (C- 22), 22,7 (C-23), 39,6 (0-24), 46.7 (C-25), 22.6 (C-27).

5.24. Preparation of 24-vinyl lanosterol 125, 29', 29" -cyclopropylidene lanosterol 126 and 24-methylene zymosterol 127 (Scheme 3.14) 5.24,1. 3p-Acetoxv-24-methvlene lanosterol 123 3p-Acetoxy-24-keto lanosterol 51. was prepared in section 5.5.1..To a suspended solution of methyltriphenyl phosphonium bromide (l.O g) in 25 ml dry THF was added dropwise «-BuLi solution in heptane (1,6 M, 1,6 ml). The reaction mixture was stirred and heated to reflux for 45 min under nitrogen, 51 (300 mg) was added to the reaction. The mixture continued to stir and reflux for 2 h. The reaction mixture was then poured to diethyl ether (150 ml). The ether was washed by 5%) sodium bicarbonate aqueous solution (50 ml), water (50 ml) and brine (50 ml). The organic layer was concentrated by removal of the solvents. The cmde product was purified by chromatography with 9:1 hexane-ethyl acetate and then dried over magnesium sulfate, Recrystallization afforded 270 mg 3p- acetoxy-24-methylene lanosterol 123 (270 mg, 90% yield). GCRRTc: 2.616, MS: 483 (M^"), 467, 439, 423, 407, 383, 341, 323, 301, 283, 255, 'H-NMR (300 MHz): 0,691 (s, 3H, 18-H), 0,881 (bs, 9H, 30-H, 31-H, 32-H), 0,923 (d, J= 6.25 Hz, 21-H), 1,004 (s, 3H, 19-H), 1.016 (d, J= 1.73 Hz, 3H, 26-H), 1.039 (d, J= 1,73 Hz, 3H, 27-H), 2.054 (s, 3H, -OCOCH3), 4.502 (m, IH, 3-H), 4.691 (d, J= 15.17 Hz, 2H, 28-H).

108 '^C-NMR (75 MHz): 24,24 (0-32), 16.52 (C-31), 24.15 (C-30), 105.89 (C-28), 21.98 (C-27), 21.84 (C-26), 33.78 (C-25), 156.91 (C-24), 30.94 (C-23), 34.94 (C-22), 18.69 (C-21), 36.47 (C-20), 18.10 (C-19), 15.74 (C-18), 50.34 (C-17), 134.22 & 134.45 (C-8 & C-9), 80.92 (C-3), 171.04 (-OCOCH3), 21.34 (-OCOCH3).

5,24,2, 3B-Acetoxv-24(i?. ^-28-al-lanosterol 124 123 (250 mg) was dissolved in dry THF (15 ml)at 0°C under a nitrogen atmosphere, Borane-tetrahydrofiiran complex solution (1,0 M, 0,8 ml) (Aldrich) was added dropwise. The reaction was stirred for 1 h at 0°C under nitrogen. The mixture was poured into water (60 ml) and extracted thoroughly with diethyl ether. The combined extracts were dried over anhydrous magnesium sulfate, evaporated at reduced pressure. The residue was dried in a vacuum dessicator over phosphoms pentoxide at 50°C for 3 h and ovemight at r.t,. The residue was then dissolved in dry dichloromethane (15 ml), PCC (250 mg) and molecular sieves (10 mg, 4A type) were added. The reaction mixture was stirred and heated to reflux for 2 h. The mixture was concentrated by removal of the solvent and dissolved in diethyl ether (150 ml). The ether solution was washed by 5% sodium bicarbonate aqueous solution (50 ml), water (50 ml) and brine (50 ml). The cmde product was concentrated and purified by chromatography with 9:1 hexane-ethyl acetate to afford 3p-acetoxy-24(i?,5)-28-al-lanosterol 124 (165 mg, 65% yield). GCRRTc: 4.135. 'H-NMR (300 MHz): 0.680 (s. 3H, 18-H), 0.861 & 0,880 (s & s, 3H & 6H, 30-H, 31-H, 32-H), 0,913 (d'd, J= 6.22 Hz, 21-H), 0,957 (d, J= 2,60 Hz, 3H, 26-H), 0.978 (d, J= 3,17 Hz, 3H, 27-H), 1,000 (s, 3H, 19-H), 2,054 (s, 3H, -OCOCH3), 4,498 (m, IH, 3-H), 9,597 (d'd, J= 12,70 Hz, IH, 28-H). '^C-NMR (75 MHz): 24.32 (C-32), 16,62 (C-31), 24.25 (C-30), 206,36 (C-28), 19.94 & 20,03 (C-27), 20,20 & 20,53 (C-26), 28.25 & 28.33 (C-25), 58.72 & 52.90 (C- 24), 22,98 & 23.08 (C-23), 34.03 & 34.17 (C-22), 18,61 & 18.78 (C-21), 36.88 & 36.97 (C-20), 18.19 (C-19), 15.83 (C-18), 50.23 & 50.36 (C-17), 134.51 & 134.32 (C-8 & C-9), 81.02 (C-3), 21.44 & 171.14 (-OCOCH3).

109 5.24.3. 24(R. 5^-Vinvl-lanosterol 125 To a suspended solution of methyltriphenyl phosphonium bromide (100 mg) in 8 ml dry THF (8 ml) was added dropwise. «-BuLi solution in heptane (1.6 M, 150 |il). The reaction mixture was stirred and heated to reflux for 45 min imder nitrogen. 124 (50 mg) was added to the reaction. The mixture continued to stir and reflux for 2 h. The reaction mixture was then poured to diethyl ether (50 ml). The ether was washed by 5% sodium bicarbonate aqueous solution (15 ml), water (15 ml) and brine (15 ml). The organic layer was concentrated by removal of the solvents. The cmde product was purified by chromatography with 9:1 hexane-ethyl acetate and then dried over magnesium sulfate. The solid was then dissolved in anhydrous ether (20 ml) and LAH (5 mg) was added into the solution. The reaction mixture was stirred at r,t. for 30 min. Water was added dropwise to decompose excess LAH till no bubbles appeared. The mixture was filtered and the filtrate was dried by evaporating solvents, HPLC purification afforded 24{R,S)- vinyl-lanosterol 125 (39 mg, 85% yield), GCRRTc: 2,135, HPLCac: 1,077, MS: 454 (M^^), 439, 421, 405, 381, 355, 327, 297, 281, 259. 'H-NMR (300 MHz): 0.6785 & 0,6835 (s's, 3H, H-18), 0,813 & 0,826 (d'd, J= 6.7 Hz, 3H, H-27(26)), 0,872 & 0,870 (d'd, J= 6.7 Hz, 3H, H-26(27)), 0,8094 (s, 3H, H- 31), 0,8691 (s, 3H, H-32), 0.893 (d, J= 6,7 Hz, 3H, H-21), 0.9787 (s, 3H, H-30), 0.9994 (s, 3H, H-19), 3,235 (m, IH, H-3), 5,532 (m, IH, H-28), 4,945 (m, 2H, H-29), '3C-NMR(75 MHz): 141,380 & 141.286 (C-28), 115.177 & 115.071 (C-29), 134.504 & 134,456 (C-8, C-9), 20,912 & 20,759 (C- 26), 18,725 & 18.603 (C-27), 28,833 & 28.579 (C-25), 51.167 & 50,950 (C-24), 26,584 (C-23), 34.190 & 34.088 (C- 22), 18,340 (C-21), 35.669 (C-20)

5.24.4. 24(R. S)-29\ 29"-Cvclopropvlidene lanosterol 126 To a suspension of cyclopropyltriphenylphosphonium bromide (150 mg, 0.39 mmol) in dry THF (10 ml) «-BuLi solution in heptane (1.6 M, 0.25 ml) was added The

110 reaction mixture was stirred and heated to reflux for 45 min under nitrogen. 124 (50 mg) was added to the reaction. The mixture continued to stir and reflux for 2 h and then ovemight at r.t.. The reaction mixture was then poured to diethyl ether (50 ml). The ether was washed by 15 ml 5% sodium bicarbonate aqueous solution (15 ml), water (15 ml) and brine (15 ml). The organic layer was concentrated by removal of the solvents. The cmde product was purified by chromatography with 9:1 hexane-ethyl acetate and then dried over magnesium sulfate. The solid was then dissolved in anhydrous ether (20 ml) and LAH (5 mg) was added into the solution. The reaction mixture was stirred at r.t. for 30 min. Water was added dropwise to decompose excess LAH till no bubbles appeared. The mixture was filtered and the filtrate was dried by evaporating solvents, RP-HPLC purification afforded 24{R, S)-29\ 29"-cyclopropylidene lanosterol 126 (8 mg, 20% yield), GC RRTc: 5.426. HPLCac: 1.267. MS: 480 (M^), 465, 447, 429, 405, 355, 341, 327, 281, 253, 241, 207. 'H-NMR (300 MHz): 0,687 & 0.674 (s's, H-18), 0.809 (s, H-31), 0.8667 (s, 3H, H-32), 0.842 (d'd, 3H, H-27), 0.863 (d, 3H, H-26), 0.886 (d, 3H, H-21), 0.977 (s, 3H, H- 30), 0.998 (s, 3H, H-19), 1.253 (s, H-29', 29"), 3.242 (m, IH, H-3), 5.525 (m, IH, H-28). '^C-NMR (75 MHz): 0.089 & 2.446 (C-29' & C-29"), 121,493 (C-28), 120.819 (C-29),

~~5,24.5. 24-Methvlene zvmosterol 127 24-Methylene zymosterol ^27 was prepared by the same method as described above, GCRRTc: 1,317.~~

~~5,25. Preparation of [26-^H]lanosterol 131 (Scheme 5.1) 5.25.1. 3B-Acetoxv-26-al-lanosterol 129 3p-Acetoxy-26-al-lanosterol 129 was prepared by the same method as described in 5.15.1.~~

~~Ill GC RRTc: 4.380 HPLC ac: 0.687~~

5.25.2. [26-^H1-3p-Acetoxv-26-Ql lanosterol 130 129 (100 mg) in 95% ethanol (20 ml) were stirred with ^aB^U4 (10 mg) for 4 h at r.t.. Standard work-up afforded [26-2H]-3p-acetoxy-26-ol lanosterol 130 (90 mg, 90% yield), GC RRTc: 9.383 HPLC ac: 0,580

~~AcO 129~~

~~CH2^H~~

~~s/\^\/^~~

~~(a) NaB^H4, 95% ethanol; (b) Pyridine-SO^ complex, then LAH Scheme 5.1. Preparation of [26- H]lanosterol~~

5,25,3, r26-^H1Lanosterol 131 Pyridine-S03 complex (100 mg) were added to a solution of 130 (50 mg) in 25 ml of dry THF, and the mixture was stirred ovemight at r.t.. After analysis of TLC indicated that the formation of the sulfate ester was completed, LAH (15 mg) was added to the

112 stirred mixture at 0°C, After 3 h, 2-3 drops of water was added to destroy the excess LAH, The solvent was removed in vacuo and the product extracted with ethyl ether. The organic layer was washed with water (20 ml). The residues were purified by HPLC with methanol to give [26-^H]lanosterol 131 (30 mg, 33% yield). GCRRTc: 1,628 HPLC ac: 0,957 MS: 427 (M""), 412, 394, 359, 341, 311, 281, 'H-NMR (300 MHz): 0.688 (s, 3H, 18-H), 0,810 (s, 3H, 31-H), 0,874 (s, 3H, 32- H), 0,911 (d, 3H, J= 6,0 Hz, 21-H), 0,981 (s, 3H, 19-H), 1.001 (s, 3H, 30-H), 1,604 (s, 3H, 27-H), 3,235 (m, IH, 3-H), 5.101 (m, IH, 24-H). '^C-NMR (75 MHz): 17.609 (C-27, 1/3 of the normal intensity), 25. 693 (C-26, 1/5 of the normal intensity)

~~113 REFERENCES~~

~~1. Fieser, L. F., and Fieser, M, Steroids, Reinhold, New York, 1959,~~

~~2. Nes, W. R,, and McKean, M. L. Biochemistry of Steroids and Other Isopentenoids, University Park Press, Baltimore, 1977, 1-36.~~

~~3. Popjak, G., Edmond, J., Anet, F. A. L., and Easton, N. A. J. Amer. Chem. Soc. 1977, 99,931-936.~~

~~4. Parker, S. R., and Nes, W. D. in Isopentenoid Metabolism (Nes, W. D., Parish, E. J., and Trzaskos, J. M,, eds), Amer. Chem. Soc. Symp. Ser. 1992, 497: 110-145.~~

5. Guo, D., Mangla, A, T,, Zhou, W., Lopez, M., Jia, Z., Nichols, D., and Nes, W.D. in Cholesterol: its Metabolism and Functions in Biology and Medicine (Brittman, R. ed.) Plenum Press, New York 1997, 91-114,

~~6. Nes, W. D, and Venkatramesh, M. Amer. Chem. Soc. Symp. Ser. 1994, 562, 55-89,~~

~~7. Brown, M. S., Goldstein, J. L. Angew. Chem. Int. Ed Engl., 1986, 25, 583-602.~~

~~8. Gibbons, G. F., Mitropoulos, K. A., Myant, N. B. in Biochemistry of Cholesterol, Elsevier Biomedical, Amsterdam, 1982.~~

9. (a) Spurgeon, S. L,, and Porter, J, W. in Biosynthesis of Isoprenoid Compounds (Porter, J. W. and Spurgeon, S, L,, eds), vol. I, 1-46, 1981, John Wiley and Sons, New York; (b) Qureshi, N, and Porter, J, W. in Biosynthesis of Isoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., eds), vol. 1, 47-93, 1981, John Wiley and Sons, New York; ( c) Bloch, K. Steroids, 1992, 57, 378-383.

~~10. Nes, W. D., and Bach, T. J. Proc. Royal Soc. Lond B. 1985, 225, 425-444.~~

~~11. Rohmer, M., Seemann, M., Horbach, S., Bringer-Meyer, S,, Sahm, H, J. Amer Chem. Soc. 1996,118,2564-2566.~~

~~12. Rohmer, M,, Knani, M,, Simonin, P., Sutter, B. & Sahm, H. Biochem. J. 1993, 295, 517-524.~~

~~13. Lichtenthaler, H.K., Schwender, J., Disch, A. and Rohmer, M. FEBSletts 1997, 400, 271-274.~~

~~114 14, Arigoni, D., Sagner, S., Latzel, C, Eisenreich, W., Bacher, A., and Zenk, M. H. Proc. Natl. Acad Sci. USA, 1997, 94, 10600-10605.~~

~~15, Scrimgeour, K. G., Moran, L. A., Horton, H. R., Ochs, R. S., and Rawn, J. D. Biochemistry, 2"'' Edition, Neil Patterson Publishers, Prentice Hall, 1994, pp20.42- 20.47~~

~~16, Jarstfer, M.B.; Blagg, B.S.J.; Rogers, D.H.; Poulter, CD. J. Amer. Chem. Soc, 1996, 118, 13089-13090.~~

~~17, Abe, I.; Rohmer, M., Prestwich, G. D. Chem. Rev. 1993, 93, 2189.~~

18, (a) Corey, E. J., Virgil, S. 0. J. Amer. Chem. Soc, 1991, 113, 4025; (b) Corey, E. J., Virgil, S. C, Sarshar, S. J. J. Amer Chem. Soc, 1991, 113, 8171; ( c) Corey, E. J., Virgil, S. C, Liu, D. R., Sarshar, S. J. J. Amer. Chem. Soc.,, 1992, 114, 1524; (d) Corey, E. J., Virgil, S. C. J. Amer. Chem. Soc,, 1990, 112, 6429,

~~19, Corey, E, J,, Cheng, H, Tetrahedron Lett., 1996, 37 (16), 2709-2712,~~

20, (a) Corey, E, J,, Daley, D, C, Cheng, H, Tetrahedron Lett., 1996, 37 (19), 3287-3290; (b) Corey, E, J,, Cheng, H., Baker, C. H., Matsuda, S. P. T., Li, D., Song, X. J. Amer. Chem. Soc, 1997, 119, 1277-1288.

~~21, Nes, W. D., Koike, K,, Jia, Z,, Sakamoto, Y., Satou, T,, Nikaido, T., and Griffin, J. F. J. Amer. Chem. Soc, 1998, in press,~~

~~22, Eschenmoser, A,, Ruzicka, L,, Jeger, O,, and Arigoni, D, Helv. Chim. Acta, 1955, 38, 1890,~~

~~23, Oehlschlager, A, C, and Czyzewaska, E. in Emerging Targets and Antifungal Therapy (J, Sutcliffe and N. H, Georgopapadakou, eds.), Routiedge, Chapman & Hall Press, New York, 1992, 437-475.~~

24, (a) Nes, W, R, in Ecology and Metabolism of Plant Lipids (Fuller, G. and Nes, W. D., eds,), Amer. Chem. Soc Symp. Ser., 1987, 325, 252-267; (b) Nes, W. R., Sekula, B. C, Nes, W, D,, and Adler, J, H. J. Biol. Chem., 1978, 253, 6218-6225.

25, (a) Barton, D, H, R,, Harrison, D, M,, Moss, G, P, Chem. Commun., 1966, 595-596; (b) Lederer, E. Quart. Rev. Chem. Soc, 1969, 23, 453-481; (c) Akhtar, M., hunt, P. F„ Parvez, M, A, Biochem. J., 1967, 103, 616-622; (d) Moore, J. T,, Gaylor, J. L. J. Biol. Chem., 1969, 244, 6334-6340; (e) Arigoni, D. Ciba Found Symp., 1978, 243- 261,

~~115 26. Nes, W. D., Janssen, G. G., Bergenstrahle, A. J. Biol. Chem., 1991, 266, 15202- 15212.~~

~~27. Guo, Jia, Z., Nes, W. D, J. Amer. Chem. Soc, 1996, 118, 8507-8508,~~

~~28. Yagi, T,, Morisaki, M,, Kushiro, T,, Yoshida, H., Fujimoto, Y, Phytochemistry, 1996, 41, 1057-1064,~~

~~29. Zhou, W., Guo, D., Nes, W, D. Tetrahedron Lett., 1996, 37, 1339-1342.~~

~~30. Nicotra, F., Ronchetti, F., Russo, G., Lugaro, G., Casellato, M. J. Chem. Soc, Perkin Trans. I, 1981,498-502.~~

~~31. Seo, S., Uomori, A., Yoshimura, Y., Takeda, K. J. Chem. Soc, Chem. Commun., 1984,1174-1176.~~

~~32. Seo, S., Uomori, A,, Yoshimura, Y,, Sankawa, U., Ebizuka, Y., Seto, H., Takeda, K. J. Chem. Soc, Perkin Trans. I, 1987, 1876-1878,~~

~~33. Nicotra, F., Ronchetti, F., Russo, G., Toma, L. Magn. Reson. Chem., 1985, 23, 134- 136,~~

~~34. Seo, S., Uomori, A,, Yoshimura, Y., Takeda, K., Seto, H., Ebizuka, Y., Noguchi, H., Sankawa, U, J. Chem. Soc, Perkin Trans. I, 1988, 2407-2414.~~

35. (a) Wright, J. L. C, Mclnnes, A. G., Shimizu, S., Smith, D. G., Walter, J. A., Idler, D., Khalil, W. Can. J. Chem., 1978, 56, 1898-1903; (b) Rubinstein, I., Goad, J. L., Mulhein, L. J. Phytochemistry, 1976, 15, 195-200.

~~36. (a) Comforth, J, W, Agnew. Chem. Int. Ed Engl, 1968, 7, 903-911; (b) Wokciechowski, Z. A., Goad, L. J,, and Goodwin, T, W. Biochem. J., 1973, 136, 405- 412.~~

~~37. Venkatramesh, M,, Guo, D,, Jia, Z., and Nes, W. D. Biochim. Biophys. Acta, 1996, 1299,313-324,~~

116 38. (a) Goad, L. J., Lenton, J, R„ Knapp, F, F., and Goodwin, T. W. Lipids, 1974, 9, 582- 595; (b) Goodwin, T. W. in Biochemistry of Isoprenoids Compounds, Vol. I, 1981, 443-480, John Wiley and Sons, New York; ( c) Nishino, T., Hata, S., Taketani, S., Yabusaki, Y,, and Katsuki, W, J. Biochem., 1981, 89, 1391-1396; (d) Bansal, S, K„ and knoche, H. W, Phytochemistry, 1981, 20, 1269-1277; (e) Moore, J, R„ and Gaylor, J. L, J. Biol. Chem., 1970, 245, 4684-4688; (f) Rahier, A„ Taton, M„ Bouvier-Nave, P„ Schmitt, P„ Benveniste, P„ Schuber, F., Namla, A, S., Cattel, L., Anding, C, and Place, P, Lipids, 1986, 21, 51-62.

39. (a) Fonteneau, P., Hartmann-Bouillon, M. A., and Benveniste, P, Plant Sci. Letts., 1977, 10, 147-155; (b) Malhotra, H, C„ and Nes, W. R. J. Biol. Chem., 1971, 246, 4934-4837; ( c) Janssen, G, G., Kalinowska, M., Norton, R. A., and Nes, W. D. in Physiology and Biochemistry of Sterols (Patterson, G. W. and Nes, W. D., eds), Amer. Oil Chem. Soc, 1991, 83-117.

~~40. (a) Janssen, G. G., and Nes, W. D. J. Biol. Chem., 1992, 267, 25856-25863; (b) Nes, W. D,, Norton, R, A,, and Benson, M. Phytochemistry, 1992, 31, 805-811.~~

~~41. Tong, Y,, McCourt, B. S., Guo, D., Mangla, A.T., Zhou, W.-Z., Jenkins, M. D., Zhou, W., Lopez, M. and Nes, W. D. Tetrahedron Lett., 1997, 38(35): 6115-6118.~~

~~42. Bloch, K. E. CRC Crit. Rev. Biochem., 1983, 14, 47-92.~~

~~43. Rahier, A., Genot, J-C, Schubler, F., Benveniste, P., and Namla, A. S. J. Biol. Chem., 1984,259, 15215-15223.~~

~~44. Ourisson, G. J. Plant Physiol, 1994, 243, 434-439.~~

~~45. Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. Proc Natl. Acad. Sci. USA, 1988,85,5759-5763.~~

~~46. Griffin, J. F., Nes, W. D., and Allinger, N. L. INFORM, 1994, 5, 509 (A).~~

47. (a) Nes, W. D., Guo, D., and Zhou, W. Arch. Biochem. Biophys., 1997, 342, 68-81; (b) Namla, A. S., Rahier, A., Benveniste, P., and Schuber, F. J. Amer. Chem. Soc, 1981, 103, 2408-2409; ( c) Ator, M. A., Schmidt, S. J., Adams, J. L., and Dolle, R. E. Biochemistry, 1989, 28, 9633-9640; (d) Ator, M. A., Schmidt, S. J., Adams, J. L., and Dolle, R. E., Kmse, L. L., Frey, C. L., and Barone, J. M. J. Med. Chem., 1992, 35, 100-106; (e) Rahman, M. D,, and Pascal, R. A, J. Biol. Chem., 1990, 265, 4986-4996.

~~48. Nes, W. D., and Le, P, H, Pest. Biochem. Physiol, 1988, 30, 87-94,~~

~~117 49. Mihailovic, M, M., Ph.D. Dissertation, Eidgenossische Technische Hochschule, Zurich, 1984, Nr. 7535.~~

~~50. Yagi, T.; Kobayashi, N.; Morisaki, M.; Hara, N.; Fujimoto, Y. Chem. Phar. Bull. (Japan) 1994, 42, 680-682.~~

~~51. Raab, K. H.; DeSouza, N. J.; Nes, W. R. Biochim. Biophys. Acta 1968, 152, 742-748.~~

~~52. (a) Nes, W, D.; Le, P. H. Biochim. Biophys. Acta 1990, 1042, 119-1125; (b) Nes, W. D.; Norton, R. A.; Cmmley, F. G.; Madigan, S. J.; Katz, E. R. Proc Natl Acad Sci. (USA) 1990, 87, 7665-7569.~~

53. (a) Goodwin, T. W.. In The Biochemistry of Plants: A Comprehensive Treatise (Stumpf, P. K., ed.); Academic Press: New York, 1980, Vol. 4,485-507; (b) Seo, S.; Uomori, A.; Yashimura, Y.; Takeda, K.J. J. Amer. Chem. Soc, 1983, 105, 6343; ( c) Nicotra, F.; Ronchetti, F.; Russo, G.; Toma, L. J. Chem. Soc, Perkin Trans. I, 1985, 521.

~~54. Seo, S., Uomori, A., Yoshimura, Y., Seto, H., Ebizuka, Y., Noguchi, H., Sankawa, U., Takeda, K. J. Chem. Soc Perkin Trans 1, 1990, 105-109~~

~~55. Dewar, M. J. S., Zoebisch, E. G., Healy, E. F., Stewart, J. P. J. Amer. Chem. Soc, 1985,107,3902.~~

~~56. Ryer, A., L., Gebert, W. H., Murrill, N. M. J. Amer. Chem. Soc, 1950, 72, 4247.~~

~~57. Morisaki, M., Shibata, M,, Duque, C, Imamura, N,, and Ikekawa, N. Chem. Pharm. Bull Japan, 1980, 28(2), 606-611.~~

~~58. Takano, S., Yamada, S,, Numata, H., and Ogasawara K. J. Chem. Soc, Chem. Commun., 1983,760-761.~~

~~59. Kircher, H, W„ and Rosenstein, F, U. J. Org Chem., 1987, 2586-2588.~~

60. (a) Ikan, R., Markus, A,, and Bergmann, E, D. J. Org Chem., 1971, 36, 3944; (b) Schmit, J. P., Piraux, M., and Pilette, J. F. J. Org Chem., 1975, 40, 1586; ( c) McMorris, T. C, and Schow, S. R. J. Org Chem., 1976, 41, 3759; (d) Naewid, T. A., Cooney, K. E., and Uskokovic, M. R. Helv. Chim. Acta, 1974, 57, 771; (e) Tschesche, R., Goo'ssens, B., Piesteri, G., and Topfer, A. Tetrahedron, 1977, 33, 735; (f) Nes, W. R.,' Varkey, T. E., and Krevitz, K. J. Amer. Chem. Soc, 1977, 99, 250.

~~61. Jia Z., Zhou, W., Guo, D., and Nes, W. D. Syn. Commun., 1996, 26, 3841-3846.~~

~~118 62. Plattner, J. J.; Bhalerao, U. T.; Rapoport, H. J. Am. Chem. Soc 1969, 91, 4933-4934.~~

~~63. Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis; Wiley: New York, 1974; Vol. 4, p 424.~~

~~64. Nicotra, F., Ronchetti, F. and Russo, G. Journal of Labelled Compounds and Radiopharmaceuticals 1978, XIV (4), 541-548.~~

~~65. Corey, E. J., and Fuchs, P. L, Tetrahedron Lett., 1972, 3769.~~

~~66. Tarchini, C, Rohmer, M,, and Djerassi, C, Helv. Chim. Acta, 1979, 62, 1210-1214.~~

~~67. Catalan, C. A. N., Kokke, W. C. M. C, Duque, C, and Djerassi, 0, J. Org. Chem., 1983,48,5207-5214,~~

~~68. Ha, T, B„ and Djerassi, C, Steroids, 1989, 53, 487-499,~~

~~69. Giner, J. L., Silva, 0, J., and Djerassi, C. J Amer. Chem. Soc, 1990, 112, 9626-9627.~~

~~70. Zielinski, J„ Li, H-T,, and Djerassi, 0. J Org Chem., 1982, 47, 620-625.~~

~~71. Parish, E, J,, Honda, H,, Chitrakom, S,, and Taylor, F, R, Chem. Phys. Lipids, 1988, 48, 255-259,~~

~~72. Giner, J., Zimmerman, M. P., and Djerassi, 0. J Org Chem., 1988, 53, 5895-5902~~

~~73. a) Fujimoto, Y., Morisaki, M., and Ikekawa, N. Steroids, 1974, 24, 367-375; b) Giner, J., and Djerassi, 0. J. ^mer. Chem. Soc, 1991, 113, 1386-1393.~~

~~74. Nes, W. D., McCourt, B. S„ Zhou, W.-X„ Ma, J„ Marshall, J, A,, Peek, L, A„ and Brennan, M., Arch. Biochem. Biophys., 1998, in press.~~

~~75. Kagan, R. M., and Clarke, S. Arch. Biochem. Biophys., 1994, 310, 417-427,~~

~~76. Xu, S„ Norton, R, A., Crumley, F. G., Nes, W. D. J Chromatogr. 1988, 452, 377- 398.~~

~~77. Kocienski, P. J. Protecting Groups, Georg Thieme Veriag Stuttgart, New York, 1994, 21-94.~~

~~78. Smith, M. B. Organic Synthesis, McGraw-Hill, Inc., 1994, 629-672.~~

~~119 79. Akihisa, T. in Analysis of Sterols and other Biologically Significant Steroids (Nes, W. D, and Parish, E, J,, eds.) Academic Press, Inc., New York, 1989, 251-266.~~

120 APPENDIX A ABBREVIATIONS ac Retention time relative to cholesterol in HPLC Ac Acetyl (CH3CO) ADP Adenosine 5'-diphosphate AdoMet iS-adenosylmethionine ATP Adenosine 5'-triphosphate Bu «-Butyl (-CH2CH2CH2CH3) °C Degree Celsius CoA Coenzyme A DHP 3, 4-Dihydro-2//-pyran

DMAPP Dimethylallyl diphosphate DMSO Dimethyl sulfoxide ((CH3)2SO) DMF A^, iV-Dimethylformamide (HCON(CH3)2) Et Ethyl (-CH2CH3) FPP Famesyl diphosphate GC Gas liquid chromatography GPP Geranyl diphosphate h Hour, hours HMG Hydroxymethylglutaryl-CoA HMPA Hexamethylphosphoramide((Me3N)3P=0) IPP Isopentyl diphosphate iPr Isopropyl (-CH(CH3)2) IR Infrared LAH Lithium aluminium hydride (LiAlIL^)

~~121 LDA Lithium diisopropylamide (LiN(iPr)2) mCPBA me^a-Chloroperoxybenzoic acid COOOH~~

CI Me Methyl (-CH3) Min Minutes mp Melting point MS Mass spectroscopy NADP^ Nicotinamide adenine dinucleotide phosphate NADPH Nicotinamide adenine dinucleotide phosphate NMR Nuclear magnetic resonance NSF Nonsaponifiable lipid fraction P Phosphate

~~O )i~~

~~O' PCC Pyridinium chlorochromate~~

~~ClCrOj"~~

~~Ph Phenyl~~

~~PSPP Presqualene diphosphate pyr Pyridine~~

~~Nf' RP-HPLC Reverse phase high performance liquid chromatography RRTc Retention time relative to cholesterol in GC~~

~~122 rt Room temperature SAM S'-adenosylmethionine SO Side Chain SMT Sterol methyl transferase TBDMS /-Butyldimethylsilyl (/-BuMe2Si) Tf Triflate (-SO2CF3) THF Tetrahydrofiaran~~

~~THP Tetrahydropyranyl~~

~~TLC Thin layer chromatography TMS Tetramethylsilane ((CH3)4Si) Ts Tosyl (p-Toluenesulfonyl)~~

~~"'^-\ /r%7>2 UV Ultraviolet~~

~~123 APPENDIX B INDEX OF CHEMICALS~~

CH3COOH Acetic acid (CH3CO)20 Acetic anhydride CH3CN Acetonitrile (CH3)2C=CHCH2Br 4-Bromo-2-methyl-2-butene BH3-THF Borane-tetrahydrofiiran complex CaCl2 Calcium chloride CBr4 Carbon tetrabromide CHCI3 Chloroform CH2CI2 Dichloromethane (CH3CH2)2NH Diethylamine CH3CH2NH2 Ethylamine EtOH Ethanol NH2OH-HCI Hydroxylamine hydrochloride ICl Iodine monochloride CH3I lodomethane (CH3)2CHCH2CH2NH2 Isoamylamine (CH3)2CHCH2NH2 Isobutylamine (CH3)2CHOH Isopropanol (CH3)2CHNH2 Isopropylamine MgS04 Magnesium sulfate MeOH Methanol (CH3)2S Methyl sulfide POCI3 Phosphoms oxychloride P2O5 Phosphoms pentoxide Pt02 Platinum(IV) oxide (Adam's Catalyst)

124 KOH Potassium hydroxide NaOAc Sodium acetate NaN3 Sodium azide NaHC03 Sodium bicarbonate NaBH4 Sodium borohydride NaB^H4 Sodium borodeuteride NaB^H4 Sodium borotritide NaCl Sodium Chloride NaBCNHs Sodium cyanoborohydride NaOH Sodium hydroxide (CH3CH2)3N Triethylamine (C6H5)3P Triphenylphosphine Zn(0Ac)2 Zinc acetate

~~125~~