Steroids Reactions and Partial Synthesis A. B. Turner Chemistry Department University of Aberdeen Aberdeen AB9 2UE Scotland Reviewing the literature published during the period November 1987 to October 1988 (Continuing the coverage of literature in Natural Product Reports 1989 Vol. 6 p. 539) 1 Reactions acetoxymethyl- 17p-tosylate (5) in acetic acid or dimethyl-1.1 Alcohols and Carboxylic Acids and their Derivatives sulphoxide gives the 17a-acetoxy-derivative (6) via participation Halides and Epoxides of the neighbouring acetoxymethyl group as demonstrated by 1.1.1 Oxidation Substitution and Reduction studies of the solvolysis of the tosylate (7).8 1.1.2 Ethers and Esters 1.1.3 Opening of Epoxide Rings 1.2 Unsaturated Compounds 1.2.1 Electrophilic Addition 1.2.2 Other Reactions of Olefinic and Aromatic Steroids 1.3 Carbonyl Compounds 1.3.1 Reduction and Dehydrogenation 1.3.2 Other Reactions 1.3.3 Reactions of a$-Unsaturated Carbonyl Compounds and Enols or Enolic Derivatives 1.4 Compounds of Nitrogen Phosphorus Sulphur and other Hetero-elements 1.5 Molecular Rearrangements 1.6 Remote Functionalization Reactions 1.7 Photochemical Reactions 2 Partial Synthesis 2.1 Derivatives and Analogues of Cholestane 2.2 Vitamins D their Derivatives and their Metabolites 2.3 Cholanes Norcholanes and Dinorcholanes 2.4 Pregnanes 2.5 Androstanes and Oestranes 2.6 Cardenolides and Bufadienolides 2.7 Heterocyclic Compoounds 2.8 Cyclopropano-steroids 2.9 Microbiological Transformations 3 References During the year the following topics have been reviewed backbone rearrangements,l aromatase inhibitors,2 synthesis of naturally occurring polyhydroxy~teroids,~ synthesis of vitamin D metabolite^,^ and stereo-controlled side-chain con~truction.~ Other reviews are mentioned in the relevant sections.Me 1 Reactions 1.1 Alcohols and Carboxylic Acids and their Derivatives Halides and Epoxides 1.1.1 Oxidation Substitution and Reduction The iodoxybenzene method for the direct oxidation of 5p-cholan-3a-01s to 1,4-dien-3-ones has been applied to a wider (5) R = CH20COMe range of cholanoic esters.6 The oxidation involves the use of benzene seleninic anhydride as catalyst in refluxing toluene (7) R = H and good yields are obtained when hydroxyl groups in rings B and c are protected.Both 6- and 7-hydroxy esters give complex mixtures of products whereas methyl deoxycholate (1) gives the OCOMe 1,4-diene-3,12-dione (2) cleanly. N,N'-Sulphinyldiimidazole prepared from imidazole and thionyl chloride can be used to dehydrate steroidal alcohol^.^ Thus the 1lp-alcohol(3) is converted into the dienedione (4) in 45% yield by brief reaction in tetrahydrofuran at room temperature. Tertiary alcohols are also dehydrated without rearrangement or ester formation. Solvolysis of the 16a-17 2-2 NATURAL PRODUCT REPORTS 1991 I CF3 R’O (8) R’ = COMe R2 = F 19) R’ = H R2 = OMe R’ & k2 (10) R’ =BOH R2= H (11) R’=aCI R2= H (12) R’ =PCl R2= H (14) R’ = POH R2 = BOCOMe (75) R’ = aCI R2 = BOCOMe (16) R’ = POCOMe R2 = aCI (17) R’ = BOCOMe R2 = POH (13) 0 (18) (20) (22) Aerial oxidative defluorination of 6-fluoroandrost-5-en-3-one occurs with phenylhydrazine to give the 3-phenylhydrazone of the 4-ene-3,6-dione in 70 YOyield.gA related oxidation to the 4-ene-3,6-dione itself occurs in toluene containing sodium methoxide and also in chloroform.One fluorine atom of the hexafluoride (8) is mysteriously replaced by a methoxyl group during hydrolysis of the ester groups with methanolic potassium hydroxide.lo The methoxy- pentafluoride (9) can be isolated in 20 % overall yield from the original cholesterol derivative via bromination dehydro-bromination and saponification. Reactions of steroidal alcohols with triphenylphosphine- carbon tetrachloride can involve neighbouring group par- ticipation of acetoxyl groups and allylic and homoallylic double bonds. Thus 3P-hydroxy-androst-Sen- 17-one (10) gives both 301- and 3~-chloro-derivatives (1 1) and (12) and 3cc,5-cycloandrost-6-en-17-one (1 3) a product mixture similar to that originally obtained from cholesterol. Only minor amounts of 17-dichloromethylene derivatives are formed. l14p-Acetoxydehydroepiandrosterone (14) by contrast gives the (19) R = H,OH,CI,OCOMe C8H17 MeOCH20& H (21) gH20CHzR Bu‘Me2SiO (23)R = H,Ph 3a- and 4a-chloro-derivatives (1 5)and (1 6) and its isomer (1 7) gives the 4,6-diene (18) as the major product probably via a 6P-chloro intermediate.Cholestene epoxides (19) are readily reconverted to 5-enes in ethereal solution by treatment with alumina impregnated with silver nitrate. l2 I .I.2Ethers and Esters Treatment of the stannylmethyl ether (20) with an excess of n- butyllithium in tetrahydrofuran causes [2,3] sigmatropic re-arrangement to the 14a-hydroxymethyl compound (21) in 79 % yield.13 1,2-Glycol monoethers of type (23) are readily obtained from the 17-ketone (22) and a-alkoxyacid chlorides by samarium diiodide mediated decarbonylation. l4 Reaction occurs within a few minutes at room temperature and isolated yields are 52% for the 17a-methoxymethyl-l7~-ol and 60% for the 17a-benzyloxymethyl- 17p-01.Reaction of unsaturated esters of type (24) with lithium NATURAL PRODUCT REPORTS 1991-A. B. TURNER (24) (25) Reagents i PriNLi THF; ii MeI THF HMPA Scheme 1 (26) R = H (27) R = CI Reagents i CH,=CHCH,NCS AlCl, DMF R R + + 4? 0 -37% R=OCOMe 50% -38% R =CI 46% -36% R=H 47% 51% R=OH 36% Scheme 2 for alkylation of the enolate anions. The absolute configuration of the C(20)methyl groups in these A16-steroids can be determined by 'H NMR analysis. In the 20-Ha-isomers the C(20)methyl groups consistently resonate at higher field (0.05-0.1 ppm) than those in the 20-H~-compounds.Fur- thermore the C(20) stereochemistry of ethyl 20-alkylpregn- 16- en-21-oates can be assigned from CD data. 1.1.3 Opening of Epoxide Rings The 5P,6P-epoxide (26) is oxidized to the ketol(28) by chromium trioxide whereas the 3a-chloroepoxide (27) is unreactive. l7 The latter epoxide is obtained by peracid oxidation of 3a-chloroandrost-5-en- 17-one and the chlorine atom can be removed by reduction with tributyltin hydride. Trans-diaxial opening of 7a 8a-epoxy- 5a-choles tan- 3/3-01 acetate with lithium in ethylamine gives the 7a-alcohol whereas the 8P-01 is formed from the epimeric 7P,8P-epoxide with lithium aluminium hydride. No trans-diaxial products were isolated on treatment of the 7a,8a-epoxide with mineral acids or Lewis acids.Instead the C-8 carbocation gives rise to a mixture of the 7-ketone allylic 7a-o1s and dienes. Cleavage reactions of 5a,6a-epoxycholestanes with lead tetraacetate,l9 and ally1 isothiocyanate in the presence of aluminium chloride,20 lead to products such as isothiocyanates (29) and oxazolidine thiones (30) (Scheme 2). Copper catalysed 1,4-addition of methyl magnesium iodide to the unsaturated epoxide (31) gives the 3/3,15a-diol (32).,l Similar stereospecific addition occurs with benzyl alcohol and benzyl chloride to give related 15-oxygenated sterols. diisopropylamide followed by treatment with alkyl halides gives predominantly the A16-(20S)alkylation products (25) which can be isolated in 82-92 % ~ie1d.l~. l6 This means that this type of alkylation can now be used to construct both 20s and 20R side chains since the analogous alkylation of the saturated esters is well known to give the opposite configuration at C-20 (Scheme 1).The remarkable selectivity difference between the saturated and unsaturated esters is explained by the differing degrees of steric congestion in the transition states NATURAL PRODUCT REPORTS 1991 (33) f N\ OR (40) R = Me,Et 1.2 Unsaturated Compounds I .2.1 Electrophilic Addition Epoxidation of the allylic alcohol (33) with 3-chloroperbenzoic acid or with (+)-or (-)-diethy1 tartrate-t-butyl peroxide-titanium isopropoxide leads stereospecifically to the spiro- epoxide (34).22 Thus the usual directive effect of the hydroxyl group in this type of oxidation is absent the oxygen being delivered to the methylene group exclusively from the a-face.This is probably due to steric hindrance to formation of a p-face peracid complex by the angular methyl group and the axial 2p-and 6p-hydrogens. Unlike normal steroids the A-norandrost-5-enes (35) and (36) react with 3-chloroperbenzoic acid to give the 5p,6P-epoxide~.~~ A further difference is that in the normal steroid series the directive effect of a neighbouring hydroxyl group is lost upon acetylation and epoxidation then occurs from the a-face whereas in the A-nor series epoxidation still occurs from the p-face. Reaction of cholesteryl acetate with the pyridine-trifluoro- acetic anhydride-molecular oxygen system gives a mixture of the a-and p-epoxides and the 5-en-7-one together with various trifluor~acetates.~~ The mechanism involves a hydroperoxide intermediate.A ruthenium porphyrin is an efficient catalyst for the stereospecific epoxidation of cholest-5-enes by oxygen. 25 Cholesteryl acetate gives nearly pure (> 99 YO)5P,6P-epoxide although cholesterol itself does not react. Photo-oxygenation of cholesterol in the presence of titanium tetraisopropoxide provides a convenient route to the epoxyalcohols (37) and (38).26Oxygen transfer in the intermediate cholesterol hydro- peroxide takes place much faster than radical promoted ( M eCOO12 HC (35) (36) *\ OR isomerization. In dichloromethane the product ratio is approx. 1 :1 whereas the alcohol (37) predominates after oxidation in chloroform or pyridine.Neighbouring group participation of allylic and homoallylic ester groups occurs in the addition of hypobromous acid to the A5-bond of chole~tenes.~’ All the stereoisomers of the 3,7- diacetates as well as some 3,7,19-triacetates have been used in studies of the relative importance of steric electronic and stereoelectronic effects in controlling the product distribution. The results have predictive value and establish that in-troduction of a neighbouring group is a useful method for directing the course of the electrophilic addition. The stereochemistry of cis-dihydroxylation of 3a,5-cyclo- androst-6-en- 17-one (13) via permanganate or osmate cyclic ester intermediates is controlled by the cyclopropane ring leading to predominant p-face attack upon the double bond.28 The conformation (39) of the cyclo-steroid allows much greater access to the p-face of the A6-bond than is possible in the corresponding 5a-cholest-6-enes (which undergo a-face attack).The presence of the cyclopropane ring forces the angular methyl group away from the p-face of the double bond and at the same time increases steric hindrance on the a-face of the molecule. Hydroboration/oxidation gives the 6a,7a- and 7p- alcohols in yields of 7 and 55 % (1 1 mixture) respectively. This also indicates some degree of control by the three- membered ring. Hydroxylation of stigmastadienone oximes (40) by silver acetate and iodine in aqueous acetic acid followed by acetylation with acetic anhydride in pyridine gives 4548 % of the cis-diacetates (41) and l&l 1 YOof the trans-iodoacetates (42).29 NATURAL PRODUCT REPORTS 1991-A.B. TURNER HO (48) (52) (54) X = H2 (55) x = 0 Cycloaddition of dichloroketene (generated from trichloro- acetyl chloride and zinc) to 3- 7- 17- and 20-methylene steroids affords the corresponding cyclobutanones.30 Yields reflect the steric crowding around the double bond. Thus 3-methylene-5a-cholestane (43) gives the epimeric cyclo-butanones (44)and (49 in yields of 17 YOand 64 YOrespectively. 1.2.2 Other Reactions of OleJinic and Aromatic Steroids The suprafacial rearrangements of the allylic peroxide (46) to its isomer (47) does not involve exchange of oxygen with the atmosphere suggesting a concerted mechanism whereas the subsequent rearrangement of the 7a-hydroperoxide (47) to the 7P-hydroperoxide is susceptible to exchange indicating a dissociative me~hanism.~~ Regioselective hydroboration of the sterol 8,14-diene system followed by deoxygenation via thiocarbonate formation and subsequent treatment with tributyltin hydride in the presence of a radical initiator is a key sequence in the introduction of the A8-bond in the synthesis of zymosterol (48).32 Reproducible conditions for the isomerization of ergosterol or its benzoate to (49) R = No2 0 NO2 (56) (57) the 8,14-dienes (49) with hydrogen chloride in chloroform have been e~tablished.~~ In the case of the benzoate the SP-isomer is formed.7-Dehydrocholesterol likewise gives the 8,14-diene (50) in 65 YOyield on a lOOg scale. A revised mechanism is proposed for the low temperature isomerization of 7-dehydrocholesteryl- benzoate to the 5a-7,14-diene (51).34 Two by-products the 5P-isomer and the 5,8-8,14-diene were isolated from the reaction together with a new intermediate the 3~-benzoyloxy-6a-chloro-5a-cholest-7-ene. An improved procedure for the synthesis of the diene (51) minimizes the levels of these and other contaminants. Under the optimum conditions this sterol can be obtained with only minor amounts of the 5a-8,14-diene and 5-15% of the SP-8,14-diene which can be removed by recrystallization. Activated tritium generated by microwave discharge in tritium gas has been used to label the steroid nucleus.35 Addition to isolated double bonds occurs in the order A' > A4 > A5.The oestrapentaene (52) has been converted into the ring D-tritiated norgestrel (53) in four The action of chromic acid upon 6-nitrocholesta-3,5-diene (54) gives the oxidation products (55)-(57).37 NATURAL PRODUCT REPORTS 1991 OAc 0 Ac OAc (58) (59) OAc Reagents i HBr AcOH (1 :10) (60) Scheme 3 OSiMe2Bu' Me0@ (c0)3& Me0 (62) (63) C8H17 \(\/C02Me HO& 0& @FCozMe 0 0 0 (65) (66) 7p 17~-Diacetoxy-4-methyleneandrost-5-ene (58) reacts with hydrogen bromide in acetic acid to give the 174-dimethyl-oestratriene (59) in 95% yield in spite of the presence of an apparent blocking methylene group at C-4.38 Incorporation of deuterium from deuterium bromide/deuterioacetic acid at the C-4 methyl group is consistent with methyl migration from C- 10 to C-1 (Scheme 3).The 3,4-dimethyl isomer (60) is also isolated in 3% yield but no anthrasteroids are obtained. The reaction between the lithium anion of 173-dithione and a mixture of a and p complexes (61) leads to insertion of the heterocycle predominantly at the C-1 .39 The resulting dithiane (62) can be converted into several 1-substituted derivatives. The isoindole (63) has been developed as a precolumn derivatization reagent for oestr~gens.~~ It reacts with the phenolic hydroxyl group under alkaline conditions to give fluorescent products which are readily separated by HPLC on a reversed-phase column using aqueous methanol as eluent.1.3 Carbonyl Compounds 1.3.1 Reduction and Dehydrogenation The proportion of the 6a-alcohol in the product mixture from aqueous potassium borohydride reduction of the ketone (64) absorbed into polymer supports (e.g.polystyrene and Amberlite XAD2) varies from zero to 90% according to the type of polymer Phase transfer agents also affect the product ratio and the greatest variations arise when substrates are adsorbed onto the inner surfaces of the polymers. Similar selectivities are found in the reduction of other ketones using this system. The 3,6-diketocholanate (65) gives the A2-6-ketone (66) in 67 '/O yield with trimethylsilyl chloride and zinc amalgam.42 Reduction of unconjugated carbonyl groups occurs selectively in alkoxide reductions catalysed by zirconocene and hafnocene complexes.43 Thus 4-androstene-3,17-dione and progesterone give 17-hydroxyandrost-4-en-3-oneand 20-hydroxypregn-4- en-3-one7 respectively in 80 YOand 67 YOyields.1.3.2 Other Reactions Ketones having an adjacent tertiary carbon are oxidized by superoxide anion radicals in an enol dependent process to give tertiary a-hydroperoxide which are readily reduced to a-ketokg4 Thus pregnan-20-ones gives 17a-hydroperoxy-20- ones with potassium superoxide and 18-crown-6 in benzene at 67 "C. Reaction is faster in the presence of oxygen under NATURAL PRODUCT REPORTS 1991-A. B. TURNER OR @+ 0' (80) R =COMe (67) R = COCH20H (81) R = SiMe3 (82) (68) R=C02H (69) R = H iio N iii I # bMe iv [aOMe pressure and the yields of hydroperoxide are somewhat enhanced.The hydroperoxides can be efficiently reduced by triphenylphosphine to the tertiary a-ketols (64-72 YOoverall yield). 3P-Hydroxy-5a-cholestan-6-oneis similarly converted into 3P,5a-dihydroxycholestan-6-onein an overall yield of 66 YO.The isolation of the hydroperoxides supports mechanisms previously proposed for the reaction of steroidal 3-lactones with superoxide ion. Oxidative degradation of (67) gives the acid (68) in 88% yield together with the androstane (69) in 7.5 YOyield.45 Baeyer-Villiger oxidation of the ketone (70) with perbenzoic acid in chloroform in the presence of toluene-p-sulphonic acid gives the lactones (71) and (72).46 The 4-en-3-one (73) behaves in a similar way.Oxidation of the ketones (74) and (75) with perbenzoic acid gives a range of lactone-derived products including the acid chloride (76).47 Details of the rearrangement of 16P-hydroxy- 17-ketones to 17P-hydroxy- 16-ketones have appeared.48 Rate studies on the acid- and base-catalysed rearrangement of the ketols (77) and (78) to the isomer (79) reveal a marked kinetic isotope effect (K,/K = 4.5or 3.0). The ketol (79) derived from the labelled precursor (78) retains deuterium to the extent of 16-65 YO.The stereospecific intramolecular 1,2-hydride shift is the principal (73) R' = H R2 = CI (74) R' = CI R2 = H (75) R' = Br R2 = H (76) mechanism involved in the rearrangement and the 16-0x0 group enolizes preferentially towards the 17-position under these conditions.The enol acetate (80) and enol silyl ether (81) of oestrone have been fluorinated with fluorine gas xenon difluoride fluoroxytrifluoromethane and caesium fluoroxysulphate. 49 The reaction between the silyl ether (81) and xenon difluoride exhibits high selectivity for the a-isomer (82). Bromination of 5a-cholestan-6-one oxime with N-bromosuccinimide in carbon tetrachloride in the presence of dibenzoyl peroxide gives 6P- nitro-7a-bromocholest-4-ene 7-bromocholesta-4,6-dien-6-one 5a-cholestan-6-one and 6-nitro~holest-5-ene.~~ Similar product mixtures are formed from 3P-acetoxy-and 3P-chloro-5a-cholestan-6-one oximes. Bromination of 5a-cyanocholestan-6- one (83) and treatment of the resulting 7a-bromo de'rivative H (77) R = H H (79) (84) with potassium cyanide gives the Sa,7P-dicyano-6-one (85).51 An alternative route to this product involves the epoxide (85) (Scheme 4).Similar reactions can be carried out with the (78) R = D corresponding 3P-chloro- and -hydroxy compounds.NATURAL PRODUCT REPORTS 1991 CH2 I SH (88) (89) (91) x=s (92) X = 0 OR’ 0 R’ (94) R’ = R2 = H (95)R’ = R2 = H (93) (96)R’ = Ac R2 = Tos (97)R’ = Ac R2 = Tos OAC (98) 0 R’ (100) R’ = H R2=Ph R’ = R2 = Me R’ = Me R2 = Et (991 Reaction of the cyclocholestenone (87) with 1,2-ethanethiol in acetic acid containing boron trifluoride etherate gives the dithiolanes (88) and (89) together with the thi~ether.~~ The 12,12-ethane-dithioacetal(91) can be selectively formed from the triketone (90).The 12-monoacetal(92) can also be prepared by exchange dioxolanation. 53 1.3.3 Reactions of a#-Unsaturated Carbonyl Compounds and EnoIs or EnoIic Derivatives Borohydride reduction of the acetoxymethylene ketone (93) gives epimeric diols (94) and (99 which can be converted to the tosylates (96) and (97).54 Solvolysis of (96) in dry acetic acid gives diacetate (98) by inversion at C-17 via a cyclic cation which can be trapped as the orthoester (99). This neighbouring group participation is absent in the solvolysis of the tosylate (97). Peracid oxidation of 16-alkylidene- and arylidene- 17-ones (100) leads to products of direct oxidation of the olefinic double No bond mainly epoxide~.~~ unsaturated &lactones are obtained.NATURAL PRODUCT REPORTS 1991-A. B. TURNER Yo AcO& (101) (107) R' = R2 = H 0& (108) R' =OMe R2 = H (109) R' = H R2 = OMe (110) Reaction of the enone (101) with iodosobenzene diacetate in methanolic potassium hydroxide gives the epoxides (102) and (103) together with the ether (104).56 The 16-methyl derivative (105) under similar conditions undergoes Favorskii rearrange- ment to the carboxylic acid (106) and its methyl ester.57 The reaction of the enedione (107) with o-iodosylbenzoic acid in methanolic potassium hydroxide gives methoxy products (1 08) and (109) together with the dienone (110).5s The 17P-alcohol (1 1 1) gives similar products. Oxidation of enones by tetrazolium {fi=OMe (103) (104) vo OH 0dP 0d?' (1 12) OH salts and related oxidants leads to y-oxygenated Thus enone (1 11) gives enedione (1 12) in 8 1 YOyield with Blue Tetrazolium.Kinetic studies lead to a postulated mechanism involving addition of the 3,5-dienolate anion to the tetrazolium salt to give the adduct (1 13) which is cleaved by an ionic process allowing attack of hydroxide ion at C-6. Oxidation of S-cis and S-trans a$-unsaturated ketones with 3-chloroperbenzoic acid show the former to be more reactive producing the corresponding a,P-epoxy ketones in higher NATURAL PRODUCT REPORTS 1991 (1 16) Me0@C8H17 (120) R’ =OH R2 = H (121) R’ = H R2 =OH OAc MeO*’ @ 0& (125) ( 126) OR ‘I 0 OR (131) yield.60 Baeyer-Villiger products are formed only in low yield.The dienone (1 14) gives predominantly the epoxyketone (1 15) in violation of the rule that linear conjugated dienones are oxidized at the double bond more distant from the carbonyl group. This is in accord with the behaviour of S-cis a,P-unsaturated ketones. vie-Diols of P-seco-cholestanes arise from Baeyer-Villiger oxidation of the 7-ketone (1 16) to the lactone (1 17) followed by lithium aluminium hydride reduction to the diol (1 1Q61 Dehydration of the derived monoacetate (1 19) with phosphoryl chloride and osmium tetroxide oxidation of the resulting mixture of olefins gives the vic-diols (1 20)-( 122). The rearranged cholestane (123) has been degraded to the (117) (118) R= H (119) R=Ac C8H17 Me0&N\ OAc CH2 (1 23) (1 24) OAc OH (127) R’ 7 H R2 = CH2CH=CH2 (129) (128) R1 = CH2CH=CH2 R2 = H (130) I I\ OmC H 2C H =C H bicyclic ketone (124) by an improved route involving acid catalysed fragmentation of the 9,lO-seco derivative (125).62 7a-Allyloestradiol(l29) and its 7p-epimer (1 30) are obtained from oestra-4,6-dien-3-one (1 26) by allylation with allyl-trimethylsilane in the presence of tetra-n-butylammonium fluoride the intermediates (127) and (128) being aromatized with cupric bromide-lithium Attempted allylation of dienones (126) using titanium tetrachloride as catalyst leads to 6P,6P’-dimers (1 3 1).63+64 The related dienone (1 32) affords the 7a-ally1 derivative (133) in 73 YO yield under similar condition^.^' The angular methyl group at C-10 inhibits dimerization on the p-face at C-6.Allylation at C-7 of 19-nor- dienone (126) is also possible using allyltributyltin in the NATURAL PRODUCT REPORTS 1991-A. B. TURNER COCH20Ac X N-N 0 Ph/ (136) X=H2 (138) X=H2 (137) X=O (139) X=O GPN-N Ph' (142) 0& (144) (146) R' =OH R2 = H (147) R' = H R2 = OH (149) presence of aluminium chloride as catalyst with the 7a-ally1 derivative (127) being isolated in 35% yield.64 Addition of 3- mercaptopropanoic acid to the dienone (134) gives the 7a- adduct (135) which is suitable for conjugation with bovine serum albumin.65 Addition of phenylhydrazine to stigmast-4-en-6-ones (1 36) and (137) gives the pyrazoles (138) and (139).66 Cyclization of the dienone (140) with phenylhydrazine gives the pyrazole (141).Reduction of testosterone with potassium tri-(R,S)-sec-butylborohydride gives mainly the allylic 3/3-alcoho17 whereas 2a-fluoro-testosterone and -androstenedione give only the allylic 3a-al~ohols.~~ Tritium-labelled canrenone (142) is ob- tained by catalytic reduction of a 1,4-dien-3-0ne precursor followed by exchange of the enolizable label at C-2.6s Canrenone labelled with 14C at the lactone carbonyl group is formed by reaction of a 17-ethynyl precursor with 14C0,. The stable trienol (143) ketonizes in aqueous solution to give the 4,7- and 5,7-dien-3-ones (144) and (145).69 At pH 2-8 the unconjugated ketone (145) is the major product while at pH -1 the conjugated ketone (144) predominates.1.4 Compounds of Nitrogen Phosphorus Sulphur and Other Hetero-elements 17a-Amino-5a-androstan-3a-01 (147) has been prepared from the 3/3-01 (146) by a sequence involving N,O-diformylation selective 0-deformylation Mitsunobu reaction and 0-de-f~rmylation.~~ The -corresponding 17P-amine (148) has been prepared from 5a-androstan-3/3-01- I7-one by tosylation epi- merization formamidation and hydr~lysis.~' Further bis-quaternary androstanes (149) active as neuromuscular blockers have been prepared by standard method^.'^ Some new amino- and nitro-cholestanes have been evaluated as inhibitors of 4-methylsterol ~xidase.~~ These are obtained from NATURAL PRODUCT REPORTS 1991 CSH17 R R (150) R = H (152) R=N02 (151) ( 153) (154) R = NO;! (157) R = NO2 (155) R=NH2 (158) R=NH2 (156) R = CH2NH2 R Br G Rd? N @;HO R’ J$ R2 I No2 NO2 ‘0 H No2 (159) R = OAc ( 160) (161) R=OAc (162) R = H (164) R’ =OH R2=N02 (166) R = CI (167) R =CI (163) R = Br (165) R’ =OH RZ= NH2 (168) R’ =CI R2 = NO2 0 02N 0Ac No2 NO2 (1 69) (171) (1 72) \/ NHCONHPh Ph R&N’ 5a-cholest- 1-en-3-one (150) by interaction of the derived dienol acetate (1 51) with ammonium nitrate-trifluoroacetic anhydride.The resulting 4-nitro compound (1 52) gives the nitroketone (1 53) upon hydrogenation over palladium and this is reduced to the nitro-alcohol (1 54) with borohydride. Reduction of the nitro group itself to give the amino-alcohol (1 55) requires protection of the 3-hydroxyl group in the form of its tetrahydropyranyl ether.The amine (1 56) has also been prepared. Catalytic transfer hydrogenation of the axial nitro group of the cholestane (157) using ammonium formate and palladium-charcoal gives the 6P-amine (1 58) in 82 YOyield.74 This and other examples show this method of reduction to be stereospecific and there is no inversion at C-6 via an oximino intermediate leading to the more stable 6a-amine. Lithium aluminium hydride reduction of the nitrocholestene (1 59) in ether gives the products (160)-(165).75 The stability of the enamine (165) which is the major product (37% yield) is surprizing. The other products are formed in 8-20% yield.The corresponding 3P-chloro compound (1 66) is similarly reduced to the oxime (167) and the debrominated product (168) in yields of 41 YOand 29 YO,respectively. Oxidation of the dinitrocholestadiene (1 69) with lead tetraacetate gives the lactones (170) and (171) together with the triene (172).76 Reaction of cholestenes with N-amino-phthalimide in dichloromethane in the presence of lead tetraacetate leads to phthalimidoaziridines of type (1 73). 77 Cyclization of the phenylsemicarbazones (1 74) with chloro- acetic acid in the presence of anhydrous sodium acetate gives the N-phenyloxazolidones (1 75). 78 The related thiazolidones (1 76) and (177) are obtained by cyclizing thiosemicarbazones NATURAL PRODUCT REPORTS 1991-A. B. TURNER N& (177) (178) (179) &c8H17 (jp H H ( 180) (181) H13 with chloroacetic acid.7s Isomeric structures are excluded by the spectral data.Rearrangement of the dithioacetals (178) with phenylselenenyl chloride in dichloromethane gives the dihydrodithiin (1 79) in 95 YOyield.*O In the androstan- 17-one series the yield is reduced to 80 %. 1.5 Molecular Rearrangements Acid-catalysed rearrangement of 5a-cholest-7-ene leads initially to 5a-cholest-8(14)- and -14-ene~.~' In the presence of boron trifluoride etherate or anhydrous toluene-p-sulphonic acid further transformation to the ring c/D-rearranged cholestene (180) and its 20-isomer occurs. The (20S)-isomers (181) and (182) are also formed as well as the 14p-methyl-18-nor-cholestenes (1 83).Minor products in the backbone rearrange- ment of cholest-Sene with boron trifluoride etherate include H (182) 78H17 (187) R = H (189) R =Me Additional products formed with anhydrous toluene-p-sul- phonic acid include some which are isomeric at C-20 as well as C-10 and have a spiro C/D ring junction e.g. (184) and (185). Backbone rearrangement of des-A-cholestene (1 86) and con- geners yields products (187) isomeric at C-20 with the methyl group at C-10 in the more stable equatorial The 5-methylene compound (1 88) yields similar isomeric products (189) with both C-5 and C-10 methyl groups equatorial. Allylic bromination of the alkene (186) with three equivalents of N-bromosuccinimide followed by dehydrobromination with collidine in boiling p-xylene gives a mixture of products containing the des-~-cholesta-5,7,9-triene (190).Treatment of this mixture with lithium aluminium hydride gives a product containing 90 YOof the aromatic hydrocarbon (1 90). These compounds have been used as standards to confirm their widespread occurrence in a variety of marine shales having a the lop-isomers of the well known diacholest-13( 17)-ene~.~~ mild thermal history and with ages ranging up to the Jurassic NATURAL PRODUCT REPORTS 1991 Me P a' HO OCHMe2 COMe OCHMe2 (191) R = H (192) ( 194) (193)R = COMe Me COMe (195) OCOCMe3 0 CI 0& I COMe (200) R = Et (201) R=COMe period (1-2 x lo6 years). Their origin may lie in micro- biological degradation of the sterols of organisms present at the time of sediment deposition.Solvolysis of the tosylate (191) in isopropanol gives the B-homocholestenol (192) as the sole product but a complex mixture is formed from the corresponding 3P-acetate (193).s4 If pyridine is present the alcohol (191) gives the mixture of products but in the case of the acetate (193) only the cyclosteroid (194) is Acetolysis of the cyclo-OH propane tosylate (195) gives the rearranged products (196)-( 198).86 (207) Oxidation of the 19-iodoandrostane (199) with 3-chloro- perbenzoic acid in ether gives the rearranged acetates (200)-(202) via a transient hypervalent iodine i~~termediate.~~ The thermal rearrangement of 4-chlor0-4~5-epoxides (203) and (204) leads to diosphenols (205) and (206),88 after removal of chlorine with zincsopper couple.Treatment of the cyclosteroid (207) with hydrogen bromide in acetic acid gives the anthrasteroid (208) as a minor product whereas the l3a-methyl epimer of (207) gives the enantiomeric 13a 14a-anthrasteroid without isomerization at C-14?' The stereochemistry of the anthrasteroids is established by NOE measurements. Use of deuterium bromide leads to extensive deuteriation. The major aromatic steroid formed in each case is (2091 the oestratriene (209). Heating a solution of the pregnadienone NATURAL PRODUCT REPORTS 1991-A. B. TURNER vo (210) HO** (2lo) and 2-acetylcyclopent-4-ene-I73-dione in benzene in a sealed tube yields c-nor-D-homosteroid (21 I).’’ Carbanion formation at C-12 could lead to bond formation between C-12 and C-14 but the precise mechanism is not clear.Dienone- phenol rearrangement of androsta- I ,4-diene-3,12,17-trione (90) with toluene-p-sulphonic acid in boiling benzene gives the phenol (212) in 13 YOyield.53 The kinetics of dehydrogenation of both rearranged (21 3) and unrearranged (214) C-aromatic hydrocarbons to the phenanthrenes (215) have been studied in connection with maturity and thermal history assessment of sedimentary organic material.’l In sealed tubes in the presence of sulphur-treated cretaceous carbonate sediment at moderate temperatures the aromatization rate for the non-rearranged isomers is greater than that for the rearranged series. 1.6 Remote Functionalization Reactions The (3-benzoylphenyl)acetoxy group at the 6P-position is an excellent template for selective functionalization at C- 15.92 It allows ready conversion of the cyclocholestane (216) into 15-HO&Po OH A A (222) 0J3Pe & H (221) oxocholesterol (2 17).The androstenolone (2 19) prepared in 95% yield from the relay chlorinated ester (218) has been converted into the fluorohydrin (220) in four Oxidation of the non-activated 5a714a-androstane (221) and its 3p-acetoxy derivative with chromium trioxide in a mixture of dichloromethane acetic acid and acetic anhydride gives the androstenones (222) and the corresponding acetate in yields of 47 YOand 68 YO,re~pectively.’~ The initial site of oxidation presumably is the tertiary 14a-CH bond.1.7 Photochemical Reactions Transformations leading to the four-membered carbocyclic ring have been reviewed.’j Details of the extensive work formation of an additional fused to the steroid nucleus on the photolysis of steroid ketols have ap~eared.’~-’~ 5-Hydroxy-Scr-and -i&cholestan-6- ones rearrange stereospecifically with retention of configuration at C-5 to give the corresponding lactones 6-oxa-~-homo- cholestan-7-0nes.~~ The corresponding 7a-deutero compounds NPR X NATURAL PRODUCT REPORTS 1991 H (223) (224) H also undergo stereospecific rearrangement to the 5-deuterio- lactones and irradiation of the 5-deuterioxy-6-ketones give 1 :3 and 7 1 mixtures respectively of the 7aa- and 7aP-deuterio- lactones.Photolysis of the 5a-methoxy-6-ketone in ethanol leads to stereoselective formation of ethyl 5-methoxy-5,6-seco- 5a-cholestan-6-oate while that of 3P-acetoxy-5a-cholestan-6-one gives mainly products of photoreduction. Irradiation of acetone or dioxan solutions of 3P-acetoxy-(E)- and (2)-19-nor-5,1 O-seco-cholest- 1 (1 O)-en-5-ones prepared from 19-norcholesterol gives 9-29 % E/Z isomerization product and 1 1-28 YOla-hydroxy- 19-norcholesteryl acetate." Irradiation through pyrex of 2P-nitro-5a-cholestan-3-one in ethanol gives 5a-cholestane-2,3-dione and its 3-oxime. loo A nitro-nitrite pathway is proposed for the formation of the oxime. The 4,4-dimethyl derivative of the 2P-nitro-3-one similarly gives the a-hydroxyimino ketone and 3-nitro-501- cholestan-2-one gives the a-diketone.The 4P-nitro-SP-3-0ne and the 6a-nitro-5a-7-one give only the a-hydroxy-iminoketones. These arise through hydrogen abstraction by the n,n* excited nitro group followed by elimination of water. The a-diketones are formed by nitro-nitrite rearrangement of the excitged nitro group of the enol forms. Photolysis of nitroamines in the presence of iodine and various oxidants affords neutral nitroaminyl radicals which undergo intramolecular hydrogen abstraction to give N-nitroimines.lO1 Thus the 6P-nitroamine (223) gives the bridged N-nitro compound (224). The most efficient combination is that of iodine with iodosobenzene diace ta te. Photodeconjugation of the oxime (225) involves an in-tramolecular stereospecific transfer of the hydroxyimino hy- drogen atom.lo2 The product (226) is formed in 69-75 yield O/O in aprotic as well as protic solvents.Thus the presence of the methyl group at C-1 prevents the ring opening to the nitrile oxide and the subsequent isoxazole formation observed earlier A (229) (230) with the C-1 unsubstituted analogue. The mechanism is again based upon deuterium labelling studies. Photolysis of three isomeric cholestenone oximes (E)-and (Z)-cholest-4-en-3-one oximes (E)-2,2-dimethylcholest-4-en-3-oneoxime and (E)-cholest-5-en-7-one oxime in protic solvents takes place regio- selectively to give in each case < 33% enamino-lactam e.g. (227) from the first oxime pair as the sole product.The alternative enone-lactams are not formed from the photo- excited a$-unsaturated ketone oximes although they are the sole products from Beckmann rearrangement of these oximes. lo3 The photochemical behaviour of various conjugated ketones in concentrated sulphuric acid has been examined.lo4 Some (e.g. 4-en-3-ones and 5-en-7-ones) are recovered unchanged but the 1-en-3-one (228) gives the rearranged products (229) rather than the [2 +2lphotodimer usually formed in neutral solution. Organoselenium reagents are useful for the generation of alkoxyl radicals from alcohols prior to intramolecular hydrogen abstraction reactions.105 Thus photolysis of 2P-hydroxy-cholestane in the presence of diphenylselenium hydroxyacetate and iodine gives the cyclic ether (230) in 89% yield.Similar functionalization of the C-10 angular methyl group occurs with the 4P-alcohol (74%) and the 6P-alcohol (97%). In the case of 20-hydroxypregnanes functionalization of the C-13 angular methyl group gives the cyclic ethers in yields of 54% and 68 Oh for the 20R and 20s epimers respectively. The p-scission of alkoxyl radicals has been used as a method for the aromatization of ring A of cholesterol and androst-5-en- 3P-01-17-one (Scheme 5). lo6 The cholesterol-derived cyclic hemiacetal (23 1) undergoes regioselective p-scission through photolysis of the corresponding hypoiodite to give a 5 1 mixture of the formates (232) and (233) in good yield. Reductive elimination with zinc in acetic acid affords the dienol acetate (234) which after hydrolysis and Swern oxidation gives the 33 NATURAL PRODUCT REPORTS 1991-A.B. TURNER 5steps + 2\ HO AcO Br AcO Br AcO Br OCHO OCHO ,CSH17 R = H or =O (2311 (232) (233) 'H Reagents i HgO I, PhH; ii hv;iii Zn AcOH 115 "C; iv KOH MeOH EtOH; v TFAA DMSO Et,N CH,Cl, -78 "C Scheme 5 {:'I {so '0. yp OH OH OH h'l 0tJ (240) 26% OH - 7 huorheat' (239) f0L0 CP-OOH & OH Scheme 6 (241) 20% phenol (235) in an overall yield of 16%. In the 17-0x0-slowly converted into its 6a-epimer with hydrochloric acid in androstane series the overall yield of oestrone is 27%. The acetic acid.lo7 trifluoroacetate is a by-product in the final step of this Irradiation of the cardenolide epoxide (238) prepared from sequence.gitoxigenin gives 16-ketone (239) as the primary photo-Photolysis of the triflate (236) in pyridine in the presence or product.lo8 This undergoes oxa-di-m-methane rearrangement absence of trifluoroiodomethane gives the enone (237) which is (Scheme 6) to give the bicycloheptanes (240) and (241). 3-2 NATURAL PRODUCT REPORTS 1991 0& 0GP (242) X=OH (243) (244) (245) X = H OAc OAc OAc 0Ac (249) OCOCMe3 I (2511 On treatment with titanium(1v)tetraisopropoxide the oes-trone-derived hydroperoxide (242) gives the epoxyquinols (243) and (244) together with the quinol (245).lo9 The epoxides (243) and (244) are readily obtained by Sharpless oxidation of the quinol (245) with t-butylhydroperoxide in the presence of titanium(1v) or vanadium(v) catalysts.Use of 3-chloro-perbenzoic acid leads mainly to Baeyer-Villiger oxidation in ring D. The 5a-alcohol (246) is confirmed as the major photo- oxidation product of norethindrone (247) following its synthesis from the ketone.ll" Lead tetraacetate oxidation of the 9a-alcohol (248) in benzene solution by irradiation in the presence of calcium carbonate gives the secosteroid (249) as the major product (-61 9'" yield) whereas photochemically in-duced oxidation with mercuric oxide and iodine involves mainly a-epoxidation of the double bond to give the epoxide (250) in 58 % yield."l The hydroxyl group at the 9a-position appears to control the course of this oxidation perhaps by (252) coordination with the reagent prior to attack upon the olefinic bond.Only one of five different crystalline forms of the prednisolone ester (251) suffers aerial oxidation catalysed by ultraviolet light.'12 The reactive one is a solvated and loosely packed crystal form (space group P6,) in which the molecules are arranged in a helix along the six-fold axis. The reactivity towards oxygen may be due to the presence of a large tunnel parallel to the hexagonal axis which allows air to penetrate the crystal. In studies on the origin of the wavelength dependent photochemical behaviour of the homoannular diene (252) the fluorescence spectrum and quantum yield of the ring-opened triene have been determined at 302 and 307 nm and both the intensity distribution and fluorescence quantum yields are found to be independent of excitation energy.'13 The large Stokes shift which is observed is consistent with an excited state torsional relaxation about the central double bond of the NATURAL PRODUCT REPORTS 1991-A.B. TURNER OH @'0 (255) (256) 0 (258) (259) H Me I CHCH20Si-CH 2Br I Me @ -I OMe HvH X e VlI --X=OH -01 HOS' 0 @ AcO AcO VIII IX X=H \ H Reagents i Bu,SnH AIBN PhH 80 "C; ii KOBuL DMSO 20 "C; iii Ac,O pyridine; iv H,O, KF DMF; v BuLi THF toluene-p-sulphonyl chloride; vi BuLi THF ;vii LiC-CCMe,OTHP BF .Et,O; viii toluene-p-sulphonyl chloride pyridine; ix LiAIH, THF; x H, Pd-CaCO, EtOH ;xi toluene-p-sulphonic acid aqueous dioxane Scheme 7 product triene.Photolysis of the oestradiene (253) gives an equilibrium mixture of 5,7-dienes and 9 IO-seco-5( 10),6,8- trienes together with the thermally unstable bicyclohexene (254).l14 Divergence from the photochemistry of the 10-methyl series can be ascribed to conformational differences. The epoxyketones (255) photolyse in the same manner as the ketone (256) in methan01.l'~ The major products are the methyl 5- isopropyl-4-nor-3,5-secoesters. In ether the 5P76P-epoxyketone (255) photodecarbonylates via a souble hydrogen shift (C- 2 -,C-4 and C-1 -,C-2) suggesting that the epoxide cleavage takes place under stereoelectronic control. Similar photo- reactions occur in the cholestane series. 2 Partial Synthesis 2.1 Derivatives and Analogues of Cholestane The deuteriated cholesterol (257) has been prepared for use as an internal monitor of cholesterol oxidation products in biological fluids and tissues.116 Palladium-catalysed reaction of alkanes with the acid chloride (258) in the presence of N-ethylmorpholine gives 3-alkenylcholest-2-enes (259) in yields of 15-20 Reductive radical cyclization of bromomethyl silyl ethers involves the 5-ex0 mode allowing the stereoselective synthesis of 20(S),25-hydroxycholesterol (Scheme 7).11* NATURAL PRODUCT REPORTS 1991 OH YCHO I OMe (261) (263) I ii liii n iv -v,vi {P BrMkId a= 00 U (265) Reagents i NaOCl; ii; ButMe,SiC1 4-dimethylaminopyridine Et,N CH,Cl,; 111 LDA (Ph,P+Et)Br- THF; iv CuCN reagent a THF; v H,.pt-c EtOAc ;vi toluene-p-sulphonic acid aq. acetone; vii ;Ac,O pyridine ;viii NaH (EtO),P(O)CH,CN THF ;ix (Me,CHCH,),AlH hexam PhMe; x Ac,O pyridine; xi MeCOOCHO pyridine; xii CrO, CH,Cl, 3,5-dimethylpyrazole; xiii aq. K,CO Scheme 8 can be prepared from the 14a-The four epimers of 7,22-dihydroxycholesterol (260) have 14P-Cholesta-5,7-dien-3P-ol been prepared from the aldehyde (261).11' A high degree of epimer in six stages.'" Inversion at C-14 is best achieved by asymmetric induction is observed in its condensation with an selective hydration of the A5-bond prior to selenium dioxide arsenic ylide to give the 22(S)-alcohol. Deprotection of the iso- oxidation and reduction of the resulting 8( 14)-en-7a-o1 with ether acetylation of the 22-hydroxyl group and allylic oxidation zinc and sulphuric acid.A four-step synthesis of 3P-hydroxy- are achieved in one pot. The allylic oxidation at C-7 is best 5a-cholest-8( 14)-en- 15-one (262) from 7-dehydrocholesterol achieved with sodium chromate or dichromate in acetic acid giving an overall yield of 39% can be carried out on the with final hydride reduction in the presence of cerium trichloride kilogram scale.lZ1 A major by-product of the final stage in methanol for the 7P-alcohol or L-selectride for the 7a- hydrolysis of the epoxide (31) to the enone (262) is the C-alcohol. aromatic sterol (263). NATURAL PRODUCT REPORTS 1991-A. B. TURNER (270) X = N,S (271) (273) 3P-Acetoxyandrost-5-en- 17-one has been converted via 15p-hydroxy-24-oxocholesterol(264)into 1SP,29-dihydroxy-7-0~0-fucosterol (265) (Scheme 8).lZ2Dehydromarthasterone (266) is prepared from asterone diacetate (267) in eight steps via coupling of the ally1 chloride (268) with the anion of the dithiane (269).lZ3 The configuration of the A17(20)-bond in the starfish sterol (266) was established by NOE studies.The 24- heteroatom-substituted cholestanols (270) have been prepared from 501-pregan-20-one (27 1) via treatment of the tosylate (272) with the sodium salt of 2-propane-thiol or with 2-(methyl- amino)propane. 12* (274) (275) 22R,23R Key intermediates (e.g. 273) for brassinosteroid synthesis have been prepared stereoselectively and in high yield by reaction of arsonium salts Ph,AsCH,COR Br-with C-22 aldehydes (274) under solid-liquid phase transfer conditions.125 The effect of temperature variation upon the sterochemistry of the products of the aldol reaction of this aldehyde (274) with 3- methylbut-2-enolide anion have been studied. 126 The optimum temperature for production of the brassinolide intermediate (275) is -78 "C. A number of new brassinolides bearing hydroxyl groups at C-20 and C-28 have been prepared from pregnen01one.l~' A new route for the synthesis of the 2a,3a- 38 NATURAL PRODUCT REPORTS 1991 OH I OSiMe3 (276) (277) HO& (279) (280) R = H,Me,Et,Pr,CH2CHMe2,CH2CMe20H C8H17 AcOl$P0 OH (281) (282) (283) R' = R2 = H (284) R' =OH R2 = OCO(CH2),Me HO&A HO R A R (285) t I 0 0 Reagents i (CF,CO),O 2,6-di-t-butyl-4-methylpyridine CH,Cl ; ii Bu,N HCOOH Pd(OAc),(Ph,P), DMF ;iii NaOMe MeOH PhMe Scheme 9 NATURAL PRODUCT REPORTS 1991-A.B. TURNER OH \ CHO ‘4 dihydroxy-7-oxa-6-oxo-~-homo structural unit of brassinolide involves ozonolysis of enol silyl ethers e.g. (276).12* The naturally occurring 25-methyl dolichosterone (277) and the tetrol-lactone (278) a more potent plant growth regulator than brassinolide itself have been synthesized from stigmasterol. lZ9 Acetylenic cholesterol derivatives (279) and (280) have been prepared from pregnenolone and stigmasterol. 130 These C-22 alkynes were devised as suicide inhibitors of the cytochrome P- 450 dependent monooxygenase responsible for the C-22 hydroxyfation involved in ecdysone biosynthesis.The trio1 (279) inhibits ecdysone synthesis in follicular cells under in vitro conditions the inhibition being selective for the C-22 hydroxyl- ase system. Two routes have been developed for the conversion of 5a-cholest-7-en-3p-01 into the ecdysteroid precursor (28 l).I3l By one of these 5,6-dihydroergosterol can be converted into the AZ2-24-methyl analogue (282) which has the complete range of functionalities required for the synthesis of naturally occurring ecdysteroids. 2,22-Dideoxyecdysone (283) another putative precursor in the biosynthesis of ecdysone has been prepared from ergoster01.l~~ Several fatty acid esters of type (284) are available from ecdysone via acylation of its 2,3- acetonide at C-22 with the appropriate anhydride.133 The protecting acetonide group can be removed using O.1M hydrogen chloride in dioxan with overall yields up to 70%.The long-standing problem of the regio-controlled synthesis of the ergosterol B-isomers (285)-(287) has been solved in the regiospecific genera tion of trienol triflate intermediates (Scheme 9).13“Previously mixtures of these isomers have always been obtained under acidic conditions and the realization that the relative order of stability of these isomeric trienes is (285) > (286) > (287) suggested their possible synthesis from suitable enones via enol triflate formation. Reaction of the appropriate enones under thermodynamic control gives the corresponding trienol triflates in excellent yield and these intermediates are readily reduced to the pure trienes in 75-80 YO overall yield.Routes from the cholenediol derivative (288) to zymosterol and related compounds have been developed. 135 Oxidation with pyridinium chlorochromate gives the 24-aldehyde which is protected as its acetal during modifications in ring B prior to the final Wittig reaction with isopropylidenetriphenyl phos- phorane. 2.2 Vitamins D their Derivatives and their Metabolites The C-22 aldehyde (289) and its la-hydroxy derivatives have been prepared from bisnorcholenic They are useful for the synthesis of side-chain modified analogues. The dienyne (290) which is readily labelled in the side-chain is prepared via enol-triflate coupling catalysed by bis(tripheny1phosphine)-palladium (11) ~hloride.’~’ An efficient synthesis of 10,19-dihydroercalciol and analogues (291) and (292) involves regioselective hydrometalation with titanocene dichloride in combination with lithium aluminium hydride or Red-Al.138 Under optimal conditions for the hydrotitanation-protonation process the former system affords good stereoselectivity and allows efficient labelling at the C-19 position.la-Hydroxy- ercalciol(293) is prepared from the diacetate (294) by irradiation with a high-pressure mercury lamp followed by thermal AcO isomerization and saponification. 13’ The diacetate (294) is prepared from ergosta-4,6,22-trien-3-one (295) in eleven steps. iii I Bu ‘Me$30°0~OSiMe2Bu (290) H (291) R’ = Me R2 = H (292) R‘=H R2=Me H0’.AH (293) (294) 40 A multistep conversion of ergosterol into a 24-hydroxycalcidiol precursor makes use of 1,4-dihydrophthalazine- 1,4-dione as the diene protecting agent.lgO It is compared with an established route which uses 4-phenyl- 1,2,4-triazoline-3,5-dionefor pro- tection of the Giene system in ring B. The new sequence allows the overall yield to be doubled. The 1,4-dioxo- 1,2,3,4-tetra- hydrophthalazin-2,3-ylenegroup proved to be the better diene protecting group in the ozonolytic degradation of the ergosterol side-chain and maintaining the protecting group in the next steps in the sequence is advantageous in diminishing the sensitivity of the intermediates towards air and light. Among the intermediates prepared this year are 3-deoxy- A9(11)-calcitriol,141 radiolabelled photoaffinity analogue of a side-chain modified calciol~,~~~~ calcitri01,~~~ lg4 di ethyl ana-logues,lg5 2-0xa-analogues,~~~ side-chain fluorinated ercal-cidio1,1g7 and 24(R)-fluorocalcitriol.lg8 The vitamin precursors 1a,3P-diacetoxy- lp-methylcholesta- 5,7-diene,lg9 and la 1~,25-triacetoxy-21-norcholesta-5,7-diene,150 have been prepared from cholesterol and 3P-hydroxy- androst-5-en-1T-one respectively. Conversion of (62)-tacalciol into calciol at 20 "C is acceler- ated at high pressure (15 K bar) in various s01vents.l~~ Rate constants are 3-5 times larger than the corresponding rate constants at 1 bar. A concerted [1,7] sigmatropic hydrogen shift mechanism is indicated by AVt of -5.14 cm3 mol-' or lower by analogy with the values obtained for concerted [1,5] sigmatropic shifts.The significant rate increase together with the quantitative recovery is of potential use in the production of calciol analogues when the [1,7] hydrogen shift is slow or when the compounds are thermally labile. Kinetic studies of this type of shift in the transformation of 3-deoxy- 1 -hydroxy- (6Z)-tacalciol epimers to 3-deoxy- 1 -hydroxycalciol show a primary deuterium kinetic isotope effect of approximately 6.152 This is very much lower than that (-45) reported previously for the isomerization of the naturally occurring (62)-tacalciol to calciol. Complete assignment of the 'H and 13C NMR chemical shifts of (6Z)-tacalciol are reported. 153 Two-dimen-sional NOE experiments provide the first direct experimental evidence for the existence of two types of conformation in solution.2.3 Cholanes Norcholanes and Dinorcholanes The four C-24,25-diastereomers of varanic acid (296) have been separated by a combination of flash chromatography and preparative HPLC on a reversed phase column.154 These diastereomers were all converted into cholic acid by incubation with rat liver micro some^,'^^ showing that the configuration at the 24- and 25-positions has no effect upon the efficiency of the side-chain degradation. New routes to the three stereoisomers of 3a,6a-dihydroxy-5P-cholanic acid (297) involve inversion at C-3 of 3a-hydroxy-6-ones with diethylazodicarboxylate-triphenylphosphine-formic acid and with dimethylformamide without epimerization at C-5.156 Samples of the acid (297) and its methyl ester previously isolated are shown to be mixtures epimeric at C-3.Among conjugates prepared by standard methods are disulphates of unconjugated and glycine- and taurine-con- jugated cholic and glycine conjugates of the 3-0x0- derivatives of bile The condensation is effected by the peptide coupling reagent N-ethoxycarbonyl-2-ethoxy-1,2-di hydroquinoline. An efficient one-carbon degradation of the bile acid side- chain which involves treatment of formylated bile acids with sodium nitrite in a mixture of trifluoro-acetic acid and its anhydride gives 24-nor-23-nitriles via a second order Beckmann rearrangement. 159 Hydrolysis with alkali gives the nor-bile acids in high yield.The nor- and bisnor-lactones (298) have been prepared from the acid (297) and its homologue for evaluation of their growth-promoting activity.160 Treatment of the chiral acetal (299) (S-R,R-isomer) with allyltrimethylsilane 9-allyl-9-borabicyclononane,or allyltributylstannane in the NATURAL PRODUCT REPORTS 1991 HO (296) (2971 (3 (298) R = C02H,CH2C02H OH I SiMe2Bu' (299) presence of titanium (IV) chloride gives the (S,S)-isomer of the alcohol (3O0).l6l The corresponding reaction of the acetal (301) (S-S,Sisomer) leads to the same homoallylalcohol with the first two reagents but the isomeric alcohol (302) [(S,R)isomer] is obtained preferentially with allyltributylstannane. Similar results are obtained with a stannylacetylene.Thus the extent of NATURAL PRODUCT REPORTS 1991-A. B. TURNER 41 OH I OMe (304) (303) AcO.ci.’i (306) (307)R = CI (308) R = H II 1:-1-1 C02Me (309) vii viii @ C02Me (310)1 :1 mixture of ! 17aand 170 epimers Ratio 1-7:1 Reagents:i dichlorodicyanobenzoquinone,PhH ;ii MeOH thallium (111) nitrate trihydrate ;iii KH THF BEt, BrCH,CO,Me; iv (CH,SH),, BF;Et,O CH,Cl,; v NaOH EtOH; vi H,PO, P,O,; vii thallium (111) nitrate trihydrate MeOH THF; viii H, Pd-C AcOH Scheme 10 asymmetric induction depends upon the nucleophilicity of the organometallic reagents and the results show that bond formation and cleavage must be concerted in the reactions in which a high degree of asymmetric induction is achieved.The ene reaction of (Z)-A17(20)-01efin (303) with acetylenic aldehydes in the presence of dimethylaluminium chloride gives (22R)-22-hydroxy-23-ynes (304) thereby allowing control over both chiral centres (C-20 and C-22).162 The ability of the a-face ene reaction to control the stereochemistry at C-20 is known and similar diastereofacial selectivity has been reported in 1986 for titanium (IV) mediated alkynylation with stannylacetylenes. Glyoxylate ene reactions promoted by certain Lewis acids also proceed with high erythro or threo diastereoselection.163 The utility of this ene reaction is demonstrated by the stereo-controlled synthesis of the 22R-hydroxy-23-carboxylate side-chain e.g. in the conversion of alkene (303) into the ester (305) which was isolated as a single stereoisomer in 67 YOyield using dimethylaluminium chloride as catalyst.Reaction of dichloroketene with the 20-methylene derivative of 164 17a-epoxy-3P-acetoxy- 5-pregnene (306) gives the lactone (307) in 85 YOyield.164 Zinc dust dechlorinates the lactone (307) completely but tributyltin hydride gives the monochloro derivative (308) in 80% yield. The structure and C-20 configuration of the lactone (308) was established by X-ray analysis. 2.4 Pregnanes 2,2,3,4,4-Pentadeuteriopregnanediol 3-gl~curonide’~~ and 1,1,19,19,19-pentadeuteriocortisol 166 have been synthesized from progesterone and prednisone respectively. Phospholipid- linked cortisol and dexamethasone with widely varying length and degree of unsaturation of the lipid side-chains have been prepared.16’ In the synthesis of the aldosterone biosynthesis inhibitors 18- vinyl- and 18-ethynyl-progesterone the protective group at C- 3 influences the efficiency of diisobutylaluminium hydride reduction of the 18-cyanomethyl group.16s The 18-carbox-aldehyde intermediates are formed in good yield in the presence of the 3P-t-butyldimethylsilyl ether and 3,3-ethylenedithio groups but not with the 3P-acetoxy or 3,3-ethylenedioxo groups.These appear to be the first examples of conformational transmission outside the steroid nucleus at an angular methyl group. Methyldehydroabietate (309) has been converted into the epimeric C-aromatic- 18-norpregnanes (3 10) via initial func- NATURAL PRODUCT REPORTS 1991 (311) OH OH (31 3) A (316) (317) R = H (319) (318) R = Br tionalization of the isopropyl group by oxidation with dichlorodicyanobenzoquinone (Scheme lo).169 This oxidation is selective enough to be used as a preparative method although unreacted starting material has to be separated by chromatography. Oxidation of 3a75a-cyc10cho1estanes using the Gif system gives the corresponding pregnan-20-ones as the major products in -7% yield.170 2.5 Androstanes and Oestranes SP-Andro~t-6-ene-3~17-dione (3 1 1) is obtained in 84 YOyield by cyclization of the all-trans cyclotetradecatriene (3 12) in xylene at 180 "C,using a catalytic amount of methylene blue.171 Sa-Androst-2-ene-5a 17P-diol (3 13) is the common inter-mediate in the synthesis of the 2-deuteriated androstenes (314) and (3 1 5).172The diol(3 13) prepared by an improved route is converted via epoxidation reductive epoxide ring-opening HO& HO oxidation and dehydration to the 2P-labelled-4-en-3-ones.Use of hexadeuteriodiborane in a similar sequence of reactions leads to both 2a- and 2P-labelled 4-en-3-ones. 174-Additionof methyl magnesium iodide to the D-homo- androstenone (3 16) gives the C/D-C~S 13a-androstane (3 17). 173 Favorskii rearrangement of the bromo derivative (3 18) gives 13a-androstane- 16a-carboxylic acid (3 19) and its 16P-epimer. The ketone (317) is oxidized to the acid (319) by thallium (III) salts. These acids can be converted into methyl ketones alcohols and 13a-androstan- 16-one.The sandaracopimaric acid derivative (320) can be converted into the 1 Sketoandrostadiene (321) by acid-catalysed cycliza- ti~n.'~~ 2,3,17- and 19-Oxygenated androstanes e.g. the tetrol (322) and deuterium or deuterium-tritium labelled analogues have been prepared from 19-hydroxyandrostenedione. Jones oxidation of androsta-5,16-dien-3/3-01(323) gives androsta-4,16-diene-3,6-dione (324) which has a penetrating urinous odour similar to that of andr0~tenone.l~~ The 1 la- NATURAL PRODUCT REPORTS 1991-A. B. TURNER OA & 0& (325) (326) OH (327) (328) OH MeOcs;”’ OH OH HO HO k2 (331) (332) (3331 OH Reagents i MeMgI Et,O; ii Br, H,O; iii LiAlH Scheme 11 hydroxyandrostenone (325) and its 11 a-ylhemisuccinate have been prepared from adrenosterone (326).177 New spirolactones (327) and (328) have been synthe-179 An efficient ring D expansion of 17-0x0-androstanes and -0estranes involves condensation of ketones with ethyl- diazo(1ithio)-acetate to give a-diazo-P-hydroxy esters which are smoothly converted into p-oxoesters by reaction with dirhodium (11) tetra-acetate.lsO This sequence allows the preparation of D-homo- 17a-ketones. The 16,8-alcohols (329) and (330) have been prepared from the respective 15-hydroxymethylene- 16-ketones. 181 Nucleophilic attack by the cyanomethylene carbanion on the diastereomeric chromium tricarbonyl complexes of 3-methoxy- oestra- 1,3,5-trienes gives 3-cyanomethyl complexes which are readily converted into 3-alkylated oestratrienes.182 Birch re- duction (lithium-liquid ammonia-ethanol) of the ether (33 l) followed by hydrolysis with acetic acid bromination-dehydro- bromination with pyridinium perbromide in pyridine and dehydration catalysed by hydrochloric acid in chloroform gives trenbolone (332).183 2-Isopropyloestradiol (333) is ob- tained from 2-acetyloestradiol by standard reactions. la42- and 4-Methoxyequilin (334) and (335) respectively have been prepared from 2-iodo- and 4- bromoequilin derivatives by nucleophilic displacement of the halide ion by methoxide ion in the presence of copper(r1) chloride and 15-cr0wn-5.’~~ 2- and 4- Methoxyequilenin have been obtained in similar fashion from 2- and 4-iodo-7,8-epoxyoestrone.Selenium dioxide oxidation of the equilins can also be employed. Chlorination of oestradiol with N-chloro imides such as N-chlorosuccinimide and trichloroisocyanuric acid in acetonitrile gives 1 OP-chloro- 17P-hydroxyoestra- 1,4-dien-3-0ne which is readily reduced to the starting phenol. lE6 Also produced are the 2 lop- and 4,l OP-dichloro- and 2,4,1 OF-trichloro-oestra- 1,4- dien-3-ones. Thus only substitution of the aromatic ring occurs in contrast to the results of bromination with N-bromoimide reagents. Preferential removal of the 1OP-chlorine atom of the di-and tri-chloro-compounds using sodium borohydride allows efficient syntheses of 2-or 4-chloro-and 2,4-dichloro-oestradiol. 18’ 16,16-Dimethyl- 17P-hydroxy- androstanes and -0estranes have been prepared by treatment of 16-methylene- 17-ketones with methylmagnesium iodide.la8 In-itial 1,2-addition of the Grignard reagent gives the 17a-methyl derivatives which add hypobromous acid and rearrange (Scheme 11). Ring B functionalized oestrane and oestradiol NATURAL PRODUCT REPORTS 1991 0 OH (336) (337) OAc OAc AcO (339) (340) MeO (345) R’= H R2=OAc (347) (346) R’R2 = 0 analogues (336) and (337) suitable for coupling to proteins have been prepared from adrenosterone by introduction of the carboxymethyl side-chain at C-7 followed by aromatization of ring In further studies on placental aromatase support for the proposed a-hydroxy-iron (1x1) peroxide mechanism is provided by model studies of the action of hydrogen peroxide on the intermediate (338).lgo Aromatization of this compound is accompanied by formation of one equivalent of formic acid per mole of oestrogen obtained.In the absence of peroxide little conversion occurs. The (2)-and (E)-l9-nor-seco ketones (339) and (340) both undergo acid catalysed transannular cyclization with con-comitant aromatization of ring A to give the oestratriene (341) in yields of 76 % and 83 YO,respectively. In the 19-methyl series the (2)-form is rather inert towards acid whereas the (E)-isomer gives the A-nor-B-homo derivative (342) in 68 YOyield.lgl Transannular cyclization of the (E)-seco ketone having the 19- methyl group also occurs thermally in the absence of protonation.0&0/ca I SiMe2But (338) AcO’ (3411 (342) OH Me0 (349) (+)-Dehydroabietic acid can be converted into the hexa- hydrophenanthrene (343) by introduction of oxygen functions at C-5 and C-l7a and migration of the methyl group to C-13.Ig2 Ring contraction gives the des-A steroid (344). 2.6 Cardenolides and Bufadienolides Substituted 14,2 1-epoxy-SP,14P-card-20(22)-enolides(345) and (346) are formed from furylandrostanediols by chromic acid 0xidati0n.l~~ The former is dehydrogenated to the 9(11)-en- 12- one by selenium dioxide. Elimination of methane sulphonic acid from 12a-or 12/3-methanesulphonyloxy-14P-hydroxy steroids proceeds with rearrangement of the steroid nucleus. lg4 Thus the 12P-mesylate of (347) gives the bridged abeo-cardadienolide (348) whereas its 12a-epimer gives the abeo derivative (349).Elimination of the 12~-methylsulphonyloxy group to give the All-alkene only occurs when there is steric fixation of the migrating carbon atoms at positions 14 and 17 as in the case of the 20,14P-lactone. Reaction of gomphoside (350) and its 3’-epimer with NATURAL PRODUCT REPORTS 1991-A B. TURNER Q HO HO HO & )nyjkd40H Ac AcO HO H H (353) (354) I Reagents i. HCl gas CHCl,; ii 0, CH,Cl, then Zn/AcOH; iii BuiN-BH, CH,C1 then aq. HCl followed by Ac,O 4-N,N-dimethylaminopyridine; iv 0, CH,Cl, then Me$; v Pr'COMe LDA THF; vi H, Pd-CaCO,/Pb; vii KzCO3 MeOH; viii (PhO),P(O)N3 EtO,CN=NCO,Et Ph,P THF ; ix Ph,P; x Ph,P+MeBr- KOCMe,Et PhMe Scheme 12 diazomethane gives two products in each case.lS5 These are 2'-epimeric spiro-oxiranes formed by reaction of diazomethane with the ring-opened 2'-keto forms.Digitoxigenin (351)can be efficiently converted into synthetically useful derivatives of 2,6- dideoxy-D-ri bohexose (the terminal sugar residue) by a sequence involving exhaustive benzoylation. lS6 The synthesis of y-isobufalin from testosterone involves the oxidation of the furan ring of the intermediate (352) to a carboxyl group.lS7 Synthesis of the unnatural 1401-and 16a- epimers of bufotalin acetate (353) and 16-deacetylcinobufagin (354) involves reduction of the corresponding 16-ketones. lS8 2.7 Heterocyclic Compounds The preparation of steroidal tetrazoles by the Schmidt reaction of ketones has been reviewed,lS9 and reports have appeared on related syntheses of tetrazoles and Beckmann rearrangements leading to lactams have been reviewed205 and further ,07 The antifungal antibiotic (355) has been NATURAL PRODUCT REPORTS 1991 CO R (356) (357) R = OH,NEt*,OMe,NHBu' CH(Me) Et,0SiMe3 HO M eO HA (358) (359) SO2QMe-0' (362) R =CHO v vi \ OSOzCF3 x xi vii-ix 0RP c-- I IA I Me Me Me (360) Reagents i NaBH, EtOH ;ii toluene-p-sulphonyl chloride pyridine ;iii toluene-p-sulphonic acid Me,CO; iv LDA THF hexane ;v KMnO, NaIO, aq.ButOH; vi MeNH, (CH,OH),; vii H, Pt AcOH; viii CrO, H,SO ;ix (CF,SOJ,O 2,6-di-t-butyl-4-methylpyridine CH,Cl ; x Pd(PPh,),(OAc), CO PriNH CH,Cl,; xi H, Pt EtOAc Scheme 13 prepared from ergosterol acetate using an intramolecular aza- Wittig reaction in the penultimate cyclization step (Scheme 12)? 4-Aza-3-oxosteroids (356) give the corresponding Al-lactams (357) with dichlorodicyano benzoquinone and N,N-bis(tri-methylsily1)trifluoroacetamide in 85-90 % yields.209 These silylation-mediated oxidations are shown to involve quinone- substrate adducts (358) which have been isolated as two diastereomers one of which undergoes thermolysis to the A1-lactam at four times the rate of the other.Under similar conditions the methyl ether (359) is unreactive showing that formation of the 0-trimethylsilyl imidate is a prerequisite for C-C bond formation. This could involve either a single electron transfer mechanism between the quinone and the 0-silylated imidate or nucleophilic attack by its enamine tautomer upon the quinone ring.Collapse of the adducts (358) is likely to be an ionic process involving loss of a proton from C-1 rather than the long-accepted hydride ion transfer mechanism. The bridged lactam (360) has been prepared for evaluation as a 5a-reductase inhibitor by a route (Scheme 13) starting from 19-hydroxyandrostenedione, 210 via intermediates (36 1)-(365). The structure of the bridged enone (361) was confirmed by X-ray analysis. It differs little from androstenedione as far as the conformations of rings B c,and D are concerned but there are significant differences between the A-rings particularly with regard to the position of C-1.NATURAL PRODUCT REPORTS 1991-A. B. TURNER (366)R' = R2 = H R3 = bond (367)R' =OH R2 = H R3 = bond (368)R' = OH R2 = Me R3 = bond (369)R' = OH R2= H R3 = 0 R AcO I Me AcO 0 (373) (374) (375) Cephalostatin (366) a powerful cell growth inhibitor isolated from a marine worm apparently results from condensation of two 2-amin0-3-ketocholestanes.~~~~ Three related dimers (367)-(369) have been isolated from the same source.212 The bis-quaternary salts (370) and (371) exhibit much less neuromuscular blocking activity than chandonium iodide.213 The structures of isoxazolidines (372) produced from the secosteroid (373) by reaction with N-methyl hydroxylamine (376) (377) have been established by X-ray analysis.214Thallium (111) nitrate reacts with y,b-unsaturated alcohols to give tetrahydrofuran derivatives and y-hydroxyketones.Oxidation of the latter leads via 1,4-dicarbonyl compounds to pyrrole or furan deriv-ative~.'~~ Thus the alcohol (374) gives the pyrrole (375). Reaction of 3,6-dioxocholest-4-ene and 6-oxocholest-4-ene with ethyl acetoacetate in the presence of fused zinc chloride gives pyrans (376) and (377).?-16 Many other compounds having a NPR 8 NATURAL PRODUCT REPORTS 1991 (378)X = O (379)x = s 00 (379) // Reagents i NaH THF DMSO; ii Me,SOI THF DMSO; iii Al(OPr'), PhMe cyclohexanone; iv Ph,PS PhH picric acid; v CF,CO,H Scheme 14 (380) variety of nitrogen oxygen and sulphur-containing hetero- ~~ ~ cyclic systems attached to rings A,~ A/B,~" -B ~ ~ ~ ~ and~ ~224-229 ha ve been prepared.1OP-Oxiranyl steroids (378) are prepared from 19-0x0-androstanes by ylide reactions (Scheme 14).230 The oxiranes are converted into thiiranes (379) with inversion at C-19 using triphenylphosphine and picric acid. The R isomers have aromatase inhibiting activity. 2.8 Cyclopropano-steroids Four stereoisomers of the cholanoic acid (380) a side-chain cyclopropyl analogue of ursodeoxycholic acid prepared by rhodium-catalysed decomposition of ethyl diazoacetate in the presence of a A22-24-norcholene have been separated by medium-pressure chromatography. Their configurations have been determined by 13C NMR spectroscopy and each was The synthesized independentl~.~~~ four isomers differ sig-nificantly in physical properties and in their interaction with intestinal bacterial enzymes.Only one is conjugated with glycine or taurine in the liver and secreted into bile in the form of these conjugates. ~~ -~The 1,24-dihydroxycalciol analogue (38 1) is prepared from ercalciol in twelve steps. 232 The side-chain is constructed by addition of cyclopropylcarbonylmethylenetriphenyl-phosphorane to a 22-aldehyde intermediate. The compound shows comparable activity to calcitriol with regard to inhibition of proliferation and induction of differentiation of cells but is at least two orders of magnitude less potent in its effects on NATURAL PRODUCT REPORTS 1991-A. B. TURNER OH OAc OH I H0" & (382) calcium metabolism.Hence it is a promising drug for the treatment of psoriasis. A stereoselective synthesis of glaucasterol (382) involves complete chirality transfer from C-22 of the benzoate (383) to C-24 of the cyclopropane (382) by means of a zerovalent palladium 2.9 Microbiological Transformations Two hydrolytic enzymes Chromobacterium viscosum lipase and Bacillus subtilis protease can be used to esterify 5a-androstane- 3p 17P-diol in acetone.234 Whereas the lipase exclusively acylates the 3P-hydroxyl group the protease acts principally upon the 17P-hydroxyl group Gram quantities of 3P- and 17P- monobutyryl derivatives can be obtained. In the case of the 3p- ester of 5a-pregnane-3/3,20P-diol, the remaining free hydroxyl group was oxidized with pyridinium chlorochromate in dichloromethane prior to alkaline hydrolysis to give 3P- hydroxy-5a-pregnan-2O-onein 63 YOisolated yield.This com- bined microbiological and chemical route provides a useful alternative to enzymic oxidation catalysed by hydroxysteroid dehydrogenases. Incubation of ring A-aza-androstanes with Cunninghamella elegans leads to hydroxylation at positions la lp 6/3 7P 9a and lSP in transformed yields ranging from 615Y0.~~~ The 6P-hydroxylation of the 4-aza-androstane (384) resembles that previously observed with ring-D-aza analogues but lp-hy-droxylation was not noted with the latter compound. 9a-(385)R = H (386)R =OH OAc Hydroxylation occurs both with 3-aza- and 4-aza-~-homo- steroids this being the major site of hydroxylation of 17a-aza- D-homoandrostanes.In the case of the 5a-hydroxy compound (385) hydroxylation is directed to ring D and the 15P-alcohol (386) is obtained in 15% yield whereas with the 5P-acetoxy compound (387) the unusual 7P-hydroxylation is observed. In both cases the more usual 7a-hydroxylation is subject to steric hindrance. Inversion of the C-3 hydroxyl group of a range of bile acids can be achieved in two steps through sequential use of 301- and 3P-hydroxy steroid dehydrogenases in free or immobilized forms. Transformations are almost quantitative and products > 98 YOpure. Low concentrations of organic solvents such as ethanol and ethyl acetate produce favourable effects on reaction rates.236 The preparative use of cholesterol oxidase has been extended to polyphoric The enzyme is active in micro- emulsions with an organic phase consisting of mixtures of cyclohexane and chloroform.Kinetic data for the oxidation of 7a- and 7P-hydroxycholestero1 in microemulsions with the enzymes from Streptomyces are similar to those obtained for cholesterol itself. The oxidase is active in the two-phase system of butyl acetate and aqueous buffer and preparative enzymic conversion of 7P-hydroxycholestero1 to 7P-hydroxycholest-4- en-3-one can be carried out in this medium. Conversion of cholesterol to cholestenone can also be performed in liquid/ solid systems in buffer with cholesterol adsorbed on silica gel in an organic medium using cholesterol oxidase and catalase entrapped in silica.3 References 1 A. V. 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