NZ231604A - Pharmaceutical compositions useful as hypocholesterolemic agents - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £31 604 23 1 6 0 4 WO DRAWINGS Priority Date(s): Complete Specification Filed; Class: J~)l fo.l.K-sw-.l ...

[ Publication Date: !?yP.

P.O. Journal. No: . W&?, Under the provisions of Regulation 23 (1) the ..«&>f.bj& — Specification has been ante-dated to 19 Patents Form No. 5 Initiate^ This is a divisional out of application No. 223344 da'ted-—_ 28 January 1988. '] NEW ZEALAND _ i _ i\ , / PATENTS ACT 1953 COMPLETE SPECIFICATION PHARMACEUTICAL COMPOSITIONS USEFUL AS HYPOCHOLESTEROLEMIC AGENTS J %/We, E.I. DCJ PONT DE NEMOURS AND COMPANY a corporation organized and existing under the laws o£ the State of Delaware, United States of America, located at 10th & Market Streets, Wilmington, Delaware, U.S.A., hereby declare the invention, for which ^"we pray that a patent may be granted to jjt^/us, and the method by which it is to be performed, to be particularly described in and by the following statement: (fdTlowed by page la) 23 16 04 1 a CR-8 489-A The present invention relates to pharmaceutical compositions containing substituted lanosterols and to the use of these compositions to inhibit the activity of lanosta-8,24-dien-3$-ol 14c<-methyl-demethylase and suppress the activity 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), two enzymes which are important in cholesterol biosynthesis. The overall effect of the substituted lanosterols is to decrease cholesterol formation/ thereby resulting in lower serum cholesterol levels in mammals.

Elevated concentrations of serum cholesterol have been demonstrated by a number of clinical studies to be a major contributing factor in the development and progression of atherosclerosis, a disease characterized by the formation of cholesterol-containing plaques in the aorta and lesser arteries. The plaques tend to clog the arterial passage ways, making it difficult, if not impossible, for blood to flow from the heart to various tissues in the body. This pathobiological condition can ultimately lead to Coronary Heart Disease (CDH). See, e.g., Kannel et al. Ann. Intern. Med., 9_0: 8 5—91 (1979); Final Report of the Pooling Project, J. Chron. Dis., _3_1: 201-306 ( 1978 ). By maintaining low cholesterol levels in the blood, arterial plaque formation and CHD can potentially be avoided. See, e.g., Brensike et al., Circulation, £9:313-324 (1984) and Levy et al. Circulation, 69:325-337 (1984). (followed by page 2) - '■'W- ,.i^ 23 16 04 In mammals, serum cholesterol is derived from exogenous dietary sources as well as through endogenous de novo synthesis. Endogenous synthesis of cholesterol involves a complex set of enzyme-catalyzed reactions and regulatory mechanisms which to date are only partially understood. As Rodwell et al., Adv. Lipid Res., 14:1-74 (1976) indicate, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is generally accepted as the rate-limiting enzyme which controls cholesterol biosynthesis from acetyl-CoA in all organisms. Brown et al., J.

Lipid Res., 21:505-517 (1980) have shown that regulation of HMGR is a complex process which is under a feedback control mechanism involving both steroidal as well as nonsteroidal isoprenoid metabolites. The authors point out that under normal conditions, the ability of cholesterol to regulate its own biosynthesis when associated with lipoprotein particles is one aspect of this feedback control mechanism. In addition, it has been demonstrated that various oxygenated sterols, when used in a highly purified state, are even more effective than cholesterol in attenuating the amount of HMGR activity, see Breslow et al., Biochem. Biophys. Acta, 398:10-17 (1975), Kandutsch et al., J. Biol. Chem., 252:409-415 (1977), and Chen et al., J. Biol. Chem., 254:715-720 (1979), leading tothe hypothesis that oxysterols may also be endogenous mediators which regulate HMGR activity and cholesterol synthesis in situ. See Kandutsch et al., Science, 201:498-501 (1978).

This proposition stimulated considerable research activity. See, e.g., Chen et al., J. Biol. Chem., 254: 715-720 (1979); Havel et al., J. Biol. Chem., 254:9573-9582 (1979); Chang et al., J. Biol. Chem., 255:7787-7795 (1980); Chorvat, U.S. Patent 4,230,626 (1980); Gibbons et al., J. Biol. Chem., 255: 395-400, (1980); Kandutsch et al., J. Biol. Chem., 255:10814- 10821 (1980); Cavenee 23 16 04 3 et al., J. Biol. Chem., 256:2675- 2681 (1981); Tanaka et al., J. Biol. Chem., 258: 13331-13339 (1983); and Trzaskos et al.. Fed. Proc., 4_41656, (1985). As a result, a number of inhibitors of HMGR activity have been found.

Gibbons et al., J. Biol. Chem., 255:395-400 (1980), for example, have shown that certain synthetic oxygenated lanosterol derivatives are active inhibitors of HMGR activity. Trzaskos et al., Ted. Proc., 44:656 (1985) have established that in situ generation of the Gibbons compounds leads to attenuated HMGR activity and decreased cholesterol biosynthesis.

In addition, Schroepfer et al., U. S. Patent 4,202,891 and Schroepfer et al., Proc. Natl. Acad. Sci. USA, 8JL: 6861-6865 (1984) have revealed that other oxygenated lanosterol derivatives may be successfully employed to lower serum cholesterol levels in animals.

Additional compounds which affect HMGR and/or other enzymes critical to serum cholesterol biosynthesis are needed. The present invention is directed to this end.

Thus, the present invention provides tnerapeutic pharmaceutical compositions for inhibiting lanosta-8,24-dien-3(J-ol 14a-methyl-demethylase activity, suppressing hmgr activity, decreasing cholesterol formation and lowering serum cholesterol levels in mammals. The pharmaceutical compositions comprise (i) an effective amount of a compound of the formula: II] (followed by page 3a) 231im 3a in which the substituent at carbon position 14 is alpha in configuration; the ring structure may be unsaturated between carbon positions 6 and 7, between carbon positions 7 and 8, between carbon positions 8 and 9 or between both carbon positions 6 and 7 and carbon positions 8 and 9; Rx is -0, 0WX, or OCOW1; R2 is H, C^-Cj alkyl, C2-C6 alkenyl, or aryl-Cj-C6-alkyl; Rj is H, cx-c6 alkyl, C,-Ct alkenyl, Cj-Cj alkynyl, aryl-C^-Cs -alkyl, CHO, CH2OR4, CHj CH, OR, , CHNOR, , CH, SR4 , CH, CH, SR4 , CHOHW, , CHOR4W3 , CHORjWJ, CN, CHZ2 , CHjZ, CHS, CHR4N(R4)2, CH,CHjN(R4 ), , CHJCHJZ, N(R4)2, SR4 , 0R4, CH-NNHR4, poly-(OR4, 0RS , epoxy) Cj-C, alkyl, N(R5)2, NR4Rs, SRj, 0R5, CH-NNHRj , CH2OR5, CH2CH2ORs, CHNOR, , CH, SRs , ch2ch2sr5, CHR4N(R5)2, chr4nr4r5 , CH2CHaN(R5)2 , CH2CH2NR4R5, CH 2 CH2 NR4 0R( , CHR4NR4OR5, cow4 , csw4, C(NR4 )W4, C(NR4)SR4, C(S)SR4, CHR4NR4N(R4 }2, CHR4NR5N(R4)2, (followed by page 4) r) 10 4- 231604 chr4nr4nr4r5 , chr4nr5nr4r5 , chr4nr4n(r5 )j , CHR4NR5N(Rj )2 , chj ch2nr4or5 , chr4nr4or4, ^ chr4 nrj ORj , chr4 nr5 or4 , chj cha nrs or, , ch2ch2nr5or4 , cr4-cr4r5, c=cr5 , CR4-CR4C(R4 )2z, CsC-C1R4)jZ, CR4-CR4C(R4 )aOR5 , c=c-c(r4 )2or5 , cr4-cr4c<r4 )2or4 , C5C-C(R4 )jOR4 , C(0)NR40R4, C(0)NR40R, , C(S)NR4OR4, C(S)NR4ORj, C(R4)2OR4, C(R4)JOR5, chr4nr4 so2w4 , ch2chr4nr4so2w4 , c(r4 )2cr4nor4 , c(r4 )2cr4nor5 , c(r4 )2cr4nor4 , c(R4 )2CR4NORs , C(R4)2W5, CR4WsOR4, or CR4WsSR4; R4 is H, Cl-C4 alkyl, C2-Cs alkenyl, aryl, aryl-Cx-Cs-alkyl or C2-Cs alkynyl? Rs is COW, , CSWj , or C(NR4)W,; X and Y, independently, are H, Cj-C^ alkyl, Z, OR4 , OR5 , SR4 , SR5 , N(R4)2, N(R, )2 nr4rs, nr4or4, nr4ors, nr4n(r4)2, nr4nr4r3, NR4N(R5)j, NRsN(R4)2, NR5NR4Rs, or NR5N(R5)2; or X and Y, taken together, are NR4, NR5, nor4 , nor5, s, c(r4)2, c(r5)2, cr5r4, nn(r4)2, NNR4Rs , NN(R5 )2 , or 0; Z is halogen; Wj is H, Ca —C2 0 alkyl, C2-C20 alkenyl, O 25 aryl, aryl-Cj-Cj0-alkyl, or C2-C20 alkynyl, W2 is H, Cx-C4 alkyl, C2~Cf alkenyl, aryl, aryl-Cj-Cs-alkyl, or C2-C6 alkynyl; W3 is H, Cx-C4 alkyl, C2-C6 alkenyl, aryl, aryl-C1-Cfi-alkyl, C2-C6 alkynyl, OR4, or N<R4 )2 ; W4 is C1-Cs alkyl, C2-C6 alkenyl, aryl, aryl-Cj-Cs-alkyl, C2-C6 alkynyl, OR4, or N(R4)3 ; and W5 is a 5- or 6-membered aromatic 35 heterocyclic ring containing 1 or 2 nitrogen atoms as part of the ring, said ring optionally substituted with substituents 231604 223344- © 5 v. selected from the group consisting of Cx-Ct alkyl and C3-Ct alkenyl; and their physiologically acceptable salts; ^ provided that a) vhen Rs is CHO, and X and Y are both H, and carbons 7-8 and 8-9 are unsaturated, then Rx is other than OH or OCOCH3, and Kj is other than CH5 ; b) when Ra is CHj and carbons 7-8 or 8-9 are unsaturated, then Ra is other than OH or OCOCHj, R3 is other than CHS or H, and X and Y are other than OH, OCOCHj or H; c) when Rj is -0, or is OWa where Wx is H or Cj-C4 alkyl, or is OCOWx when Wx is C1-CJ0 alkyl or phenyl, and X is OR4 where R4 is H and OR5 is OCOWj where W3 is Cj-Cjg alkyl or phenyl, and Y is H or OH, then r, is other then b or a ci~ct alkyl; d) when R3 is CHaOH or CH2 OCOCHj, and Ra is H or CHj, and carbons 6-7, 7-8 or 8-9 are unsaturated, then Ra is other than -0 or OH or OCOCHj, and X is other than H or OH; e) when X and Y are both H, then R3 is other than H or CHj; f) when X and Y are both H, then R5 is other than OH, and Ra is other than H; g) when Ra is H, and Rs is o OH, and X is p OH, and Y is a H, and carbons 6-7, 7-8, and 8-9 are saturated, then Rx is other than OH; h) when X is OH, 0R5 , N(R4)2, N(R5)2, NR4R5, 35 SR., NR,OR., or NR.OR., then Y is other 4 4 4 5 than 2, OH, ORj , SR& , NR4OR4|, NR4 ^ NU,),, or N (Rs ) j; and (f *\\ [si oj] " 9 MAR 1990*7/ V, 23160 D iO. •i) when R3 is H, OH, or Cx-Ct alkyl, then X and Y, taken together, are other than 0; and (ii) a pharraaceutically acceptable carrier or diluent.

In addition, the present invention encompasses methods for inhibiting lanosta-8,24-dien-30-ol l<Jcc-methyl-deraethylase activity, suppressing HMGR activity, decreasing cholesterol formation and lowering serum cholesterol levels comprising administering to a non-human mammal an effective amount of a compound of the formula: C.H.

[I] wherein ~7 23 16 04 chr4nr4or5, cow4, csw4 , c(nr4)w4, c(nr4)sr4, c(s)sr4/ chr4nr4n(r4 )2 , chr4nr5n(r4 )2 , chr4nr4nr4R5 , CHR4NR5NR4Rj , chr4nr4N(R5 ) 2, chr4nr5n(r5 )2, ch2ch2nr4or5 , chr4nr4or4, CHR4NR5ORs, CHR4NRsOR4, CHjCHJNRJORJ , ch2 ch2 nr5 or4, cr4-cr4 rs, chcr5, cr4-cr4c<r4 )2z, csc-c(r4)2z, cr4-cr4c(r4 )2or5 , c5c-c(r4 )2or5 , cr4-cr4c(r4 )2or4 , C=C-C(R4 )jOR4 , C(0)NR40R4, C(0)NR40Rs, C(S)NR4OR4, C(S)NR4ORs, C(R4)2OR4, C(R4)2OR5, chr4nr4 so2w4 , ch2chr4 nr4so2w4 , c(r4 )2cr4nor4 , c(r4 )2cr4nor5 , c(r4)2W5, cr4w5or4, or CR4W5SR4 ; R4 is B, alkyl, C2-Cs alkenyl, aryl, aryl-Cx-C,-alkyl, or C2-Cs alkynyl; Rs is COW3, CSW3, or C(NR4)W3; X and Y, independently, are H, Cl-Cf alkyl, Z, or4 , or5 , sr4, sr5 , n(r4)2, n(r3 )2 , nr4rs, nr4or4, nr4ors, nr4n(r4)2, nr4nr4r5, NR4N(R5)2, NR5N(R4)2, NR5NR4R5, orNR5N(Rs)2; or X and Y, taken together, are NR4, NR,, NORt , NOR,, S, C(R4)2, C(Rs)2, CR,R4, NN{R4)2, :NNR4Rs/ NN(R5)2, or 0; Z is halogen; is H, C1-C20 alkyl, C2-C20 alkenyl, aryl, aryl-Cj-Cjg-alkyl, or C2-C20 alkynyl, W2 is H, Cj-Cj alkyl, C2-C6 alkenyl, aryl, aryl-Cj-C6-alkyl, or C2-Cs alkynyl; w3 is H, cx-cs alkyl, C2-Cfi alkenyl, aryl, aryl-Cj-C6-alkyl, C2~CS alkynyl, OR4 , or N(R4)2 ; W4 is C1-C6 alkyl, C2-Cs alkenyl, aryl, aryl-Cj-C6-alkyl, C2-C6 alkynyl, 0r4 , or n{r4)2; and W, is a 5- or 6-membered aromatic heterocyclic ring containing 1 or 2 nitrogen -3 J* 231 atoms as part of the ring, said ring optionally substituted with substituents selected from the group consisting of C1-C< alkyl and C2-C6 alkenyl; and their physiologically acceptable salts; provided that a) when R3 is CHO, when X and Y are both H, and carbons 7-8 and 8-9 are unsaturated/ then Rx is other than OH or OCOCHj, and Rj is other than CH3; b) when R3 is CH3 and carbons 7-8 and 8-9 are unsaturated, then Rx is other than OH or OCOCHj, Rj is other than CHj or H, and X and Y are other than OH# OCOCHj or H; c) when Rx is -0, or is 0WX where Wx is H or Cj-Cj alkyl, or is OCOMj^ when Wr is Cj-Cjg alkyl or phenyl, X is 0R4 where R4 is H or OR. is OCOW. where W. is C, -C, „ 3 3 x 2 0 alkyl or phenyl, and Y is H or OH, then Rj is other then H or *Cl-C6 alkyl; d) when R, is CH2 OH or CHa OCOCHj , R2 is H or CH,, and carbons 6-7, 7-8 or 8-9 are ; unsaturated, then Rx is other than -0 or OH or OCOCHj, and X is other than H or OH; e) when X and Y are both H, then Rj is other than H or CH3; f) when X and Y are both H, then R3 is other than OH an R2 is other than H; g) when R2 is H, R3 is oOH, X is 0OH and Y is a H, and carbons 6-7, 7-8, and 8-9 are saturated, then R^ is other than OH; and h) when X is OH, 0R5 , N(R4 )2 , N(R5 )2 , NR4Rs , SRs , NR4 0R4 , NR4 ORj then Y is other than 2, or OH, 0R5 , SRs , NR4 0R4 , NR4OR5, N(R4)2, N(RS)2; and 231604 i) when R3 is H, oh, or Cl-Ci alkyl, then X and Y, taken together, are other than O. In the above formulas, the ring structure may be fully saturated, or may be unsaturated between one of carbon positions 6-7, 7-8 or 8-9 or between both carbon positions 6-7 and 8-9. For convenience, the compounds are designated herein as the "d" compound when fully saturated, the "a" compound when unsaturated in the 8-9 position, the "b" compound when unsaturated in the 7-8 postion, the "c" compound when unsaturated in the 6-7 position, and the "a/c" compound when unsaturated in both positions 6-7 and 8-9.

As used herein, the substituent designated as "poly-(OR4, OR, , expoxy) Cx-Ct alkyl" shall be taken to mean a Cj^ to C$ alkyl chain substituted with one or more of any combination of OR4, OR, and epoxy.' As used herein, the term "alkyl", employed either alone or in combination with other terms such as "poly-(OR4, OR,, expoxy) C1-C< alkyl" or "arylalkyl", denotes straight chain or branched alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, and the different butyl, pentyl or hexyl isomers.

As used herein, the term "alkenyl", employed either alone or in combination with other terms, denotes straight chain or branched mono- or poly-unsaturated alkyl, e.g., vinyl, propenyl (allyl), crotyl, isopentenyl, and different butenyl, pentenyl, hexadienyl and hexenyl isomers.

As used herein, the term "alkynyl", employed either alone or in combination with other terms, denotes straight chain or branched mono- or poly-unsaturated alkyl, e.g., ethynyl, propynyl (propargyl), 2-butynyl and other butynyl isomers, and the different pentynyl, hexadiynyl and hexynyl isomers.

As used herein, the term "acyl", employed either alone or in combination with other terms, denotes a 4 V 23 16 04 carbonyl group attached to an alkyl, alkenyl, alkynyl, arylalkyl or aryl group e.g. acetate, butyrate, benzoate, and different alkyl, alkenyl, alkynyl, or aryl isomers.

As used herein, the term "halogen" denotes fluorine, chlorine, bromine and iodine.

With respect to the above ■ •* composition, and method of use formulas, preferred categories of compounds are: 1. Compounds wherein is H, Cj-Cj alkyl, or aryl-CA-C(-alkyl? R3 is H, Cj-Cj alkyl, C2-Cs alkenyl, Cj-Cj alkynyl, aryl-^-Cfi-alkyl, CHO, CH2OR4 , ch2ch2or4, chnor4, ch2sr4, ch2ch2sr4, chor4w2, chor5w2, cn, chz2 , ch2z, chs, chr4n(r4)2, ch2CHjn(r4 )2 , ch2ch2z, n(r4)2, sr4 , or4 , ch-nnhr4 , poly-(or4 , or, , epoxy) ^-c, alkyl, n(r5)2, nr4r5, srj, ors , ch-nnhrj , ch2 ors , ch2 ch2 or5 , chnor5 , ch2 sr3 , ch2 ch2 sr5 , chr4n(r5)2, chr4nr4r5, ch2ch2n(r5 )2, ' ch2ch2nr4r5, ch2ch2nr4or4 , or chr4nr4or5; and X and Y, independently, are H, C1-C( alkyl, Z, OR4 , ORs , SR4 , SR, , N(R4)jr N(R5)2 NR4R}, NR4OR4, or NR4 OR, ; or X and Y, taken together, are NR4 , NRS, NOR4 , NOR, , S, C(R4)2, C(R5)2, or CRj R4 . 2. Compounds wherein R2 is C2-Cs alkenyl, or C2-C6 alkynl. 3. Compounds wherein Rj is COW4 r CSW4 , C(NR4 )W4 , C(NR4)SR4, c(s)sr4, chr4nr4n(r4 )2, chr4nr5n{r4 )2 , CHR4NR4NR4 R5 , CHR4NR5NR4 R5 , CHR4NR4N(RS )2, chr4nr5n(r5 )2 , ch2ch2nr4 or5 , chr4nr4or4, chr4 nr5 or5, chr4 nr5 or4, ch2ch2nr5or5, ch2ch2nr5or4 , cr4-cr4r5, c=cr5 , v ^\\ 23 1 6 0 4 CR4-CR4C(R4 )2Z, C=C-C(R4)2Z, CR4-CR4C(R4 )2ORs , C3>C(R4 )2OR5 , CR4-CR4C(R4 )jOR4 , C3£-C(R4 )2OR4 , C(0)NR40R4, C{0)NR40Rs, 5 C(S)NR4OR4, C(S)NR4OR5, C(R4),OR4, C(R4)20Rs, chr4nr4 so2w4, ch2chr4nr4so2w4 , c(r4 )2cr4nor4 , C(R4 )2CR4NORs , CR4W5OR4> or CR4W3SR4. 4. Compounds wherein Rj is COW4 , CSW4, C(NR4)W4, c(nr4)sr4, 10 c(s)sr4, CHR4NR4N(R4 )2 , chr4nr5n(r4 )2, chr4nr4nr4r5 , chr4nr5nr4r5 , chr4nr4n(r5 )2 , chr4nr1n(r5 )2, ch2ch2nr4or5 , chr4nr4or4, chr4nr5or5, chr4nr5or4, ch2 ch2 nr5 or5 , ch2ch2nr5or4 , cr4-cr4r5, CSCRj , cr4-CR4C(R4 )2Z, C=C-C(R4 )2Z, CR4-CR4C(R4 )2ORs , CSC-C(R4 )2or5 , cr4-cr4c(r4 )2or4 , C^C-C(R4 )2or4 , C(S)NR4OR4, C(S)NR4OR5, C(R4)2OR4, c(r4)2or5, chr4nr4so2w4 , ch2chr4nr4so2w4 , c(r4 )2cr4nor4 , c(r4 )2cr4nor5 , ,20 CR4W5OR4, or cr4w5sr4 .- Compounds wherein X and Y, independently, are NR4N(R4)2, NR4NR4R5, NR4N(Rs)2, nr5n(r4)2, nr5nr4r5, or JJR, N {R5 )2 ; or X and Y, taken together, are NN(R4)2, NNR4R5, NN(Rs)2, or 0. 6. Compounds wherein R3 is H, Cx-C6 alkyl, C2-C6 alkenyl, aryl-C1-Cs-alkyl, C2-Cs alkynyl, CHO, CH2OR4 , ch2ch2or4, chnor4 , ch2sr4, ch2ch2sr4, chor4w2 CH0R5W2 , CN, CHZ2 , CH2Z, CHS, CHR4N(R4)2, CH2CH2N(R4 )2 , CHjCHjZ, N{R4)2, SR4 , CH-NNHR4, poly-(OR4 , OR,, epoxy) C1-Cfi alkyl, N(R,)2, NR4R5, SRs , OR, , CH-NNHR, , CH20R5 , CH2 CH2 OR, , CHNOR, , CH2SR5, CH2CH2SR5, CHR4N(R, )2, CHR4NR4Rs, CH2CH2N(R5 )j , CH2CH2NR4R5, CH2 CH2NR4OR4 , or CHR4NR4OR, . 1-2 J* 23 7. Compounds wherein Rj is H, Cj-Cj alkyl, C2-Cfi alkenyl, aryl-Cj -Cs -alkyl, CHOR4W2, CHOR,W2, Ct-C4 alkynyl, CHO, CH2OR4, CH2CH2OR4, CHN0R4, CH2SR4, CH2CH2SR4, CHOHOWj , CHZ2 , CHjZ, CHS, CHR4N(R4)2, CH2CHjN(R4 )s , CHaCHaZ, N(R4)jf SR4 , CH«NNHR4 , poly-(OR4, OR, , epoxy) Cx -C, alkyl, N(R5)2, NR4Rs, SRs , OR,, CH-NNHR,, CH20R5, CHjCHjOR,, CHNOR, , CH2 SR, , CH2CH2SR,, chr4n(r, )2, chr4nr4r,, ch2ch2n(r, )2 ? ch2ch2nr4r,, ch2ch2nr4or4 , or ch2r4nr4or5. 8. Compounds wherein X and Y, independently, are H, Cj-Cj alkyl, or4 , or,, sr, , SR4 , n(r4 )2 , n(r,)2, nr4r5, nr4or4, or nr4or, ; or X and y, taken together, are NR4 , NR,, NOR4 , NOR, , S, C(R4)2, C(R,)j, or CR4R,. 9. Compounds wherein X and Y, independently, are H, Cj-Cj alkyl, Z, OR4 , OR, , SR,, SR4 , N(R4 )2 , NR40R4, NR4ORj, N(R5)2 or NR4R,; and or X and y, taken together, are nr4 , nr,, :nor5, S, C(R4)2, C(Rs)2, or CR4R, .

. Compounds wherein ^ R, is OH; Rj is H, or CH, ; R3 is CHF2 , CHOHCHCHj , CHNOH, CN, lCH3 , CH2CH-CH2, or OH; and X and y, independently, are H, F, or OH; or X'and y, taken together, are NOH. 11. Compounds wherein Rx is OH; R2 is H, or CHj; 35 R3 is CHF2, CHOHCHCHj, CHNOH, or dtfBfeh. © Jr 231604 CHa CH-CHj; and X and Y, independently, are H or OH. 12. Compounds wherein ^ Rj is OH, or OCOWj where is phenyl or CH3 ; R3 is H, or CH3 ; Rj is H, CHO, CH2OR4 where R4 is H or Cl-C( alkyl, CHNOH, CH3 , CN, OH, or CHR4N(R4)2 where R4 is H or CH3; and X and Y independently are H, F or OH; or X and Y, taken together, is NOH. 13. Compounds wherein R1 is OH; rj is ch3 ; R3 is CH3 , CN, or OH; and X and Y, independently, are H, F, or OH; or X and Y, taken together, is NOH. 14. Compounds wherein X and Y, independently, are CA-C4 alkyl, ors , sr4, srs , n(R4>2, n(r5 )3 , nr4r5, nr4or4, nr4or5, nr4n(r4)j, nr4nr4r5, NR4N(R5)j, NR5N(R4)j, nr5nr4r5, NR5N(R5)j, or :0R4 , where R4 is Cx-C6 alkyl, C2-Cs alkenyl, aryl, aryl-^-Cg-alkyl, or C2-Cs alkynyl, or X and Y, taken together, are nr4, nr5, nor4 , norj , s, c(r4)2, c(R5)j, crsr4, nn(r4)2, nnr4r5 , or nn(r5 )j .

Many of the above compounds are preferable for reasons of increased ease of synthesis and/or greater efficacy.

Specifically preferred for reasons of greatest ease of synthesis and/or greatest efficacy are: • 32, 32-di fluoro-lanost-8-en-30-ol; • 32,32-difluoro-lanost-7-en-30-ol; • 4,4-dimethyl-14a-(1'-hydroxy-2'- '•V'kv 231 propenyl)-5a-cholest-8-en-30-ol; 14a-allyl-4,4-dimethyl-5a-cholest-8-en-30-ol-15-oxime; lanost-8-en-32-aldoxime-30-ol; lanost-7-en-32-aldoxime-30~ol; 14a-cyano-4,4-dimethyl-5a-cholest-8-en-30-ol; 15a-fluoro-lanost-7-en-30-ol; 15a-fluoro-14a-methyl-5a-cholest- 7-en-30-ol; -hydroxy-lanost-8-en-15-oxime; 3 0-hydroxy-lanost-7-en-15-oxime; 4,4-dimethyl-5a-cholest-8-en-30,14a,15a-triol; 5a-cholest-8-en-30,14 a,15a-triol; 3 0-hydroxy-lanost-8-en-32-ohydroxamic acid; 3 0,15a-dihydroxy-lanost-8-en-32-al; 3 0-hydroxy-lanost-8-en-32-aldoxime-15-oxime; 3 0-acetoxy-lanost-8-en-32-aldoxime; 3 0-acetoxy-lanost-7-en-15-oxime; lanost-6-en-32-aldoxime-30-ol; 15a-anino-lanost-8-en-30-ol 14a-amino-4,4-dimethy1-5 a-cholest- 8-en-30-ol; 4,4-dimethyl-14a-(N-formyl-amino)-5a-cholest-8-en-30-ol; 4,4-dimethyl-14a-(N-ethoxycarbonyl-amino)-5a-cholest-8-en-3 0-ol; and 32-ethynyl-lanost-8-ene-3 0,32-diol.

The compounds of the above formulas can be employed to inhibit lanosta-8,24-dien-30-ol ,5 2 3 1 6 0 4 14a-methyl-demethylase activity, suppress HMGR activity, decrease cholesterol formation and lower serum cholesterol levels in mammals. These compounds can be administered alone, or in combination with pharmaceutically acceptable carriers or diluents appropriate to the indicated route of administration. Administration can be oral, sublingual, buccal, topical and parenteral such as intravenous, subcutaneous or intramuscular. Acceptable carriers and diluents are well-known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Gennaro, A. R., ed., Mack Publishing Co., Easton, PA (1985). The useful dosage to be administered and the mode of administration will vary depending upon the age, weight and species of mammal treated.

Briefly, the mechanism by which the active 14,15-substituted lanosterol compounds of the present invention are believed to function is as follows.

First, the observed decrease in HMGR activity is thought to occur as a result of a decreased synthesis of HMGR protein and/or an enhanced rate of HMGR degradation (collectively termed herein as "suppression"). The observed decrease in lanosta-8,24-dien-3p-ol 14a-methyl demethylase activity is thought to occur as a result of a direct action of the compounds on the demethylase enzyme (termed herein as "inhibition"). The inhibition of lanosta-8 ,24-dien-3g-ol 14a-methyl demethylasie activity is also thought to result in the production of molecules which in turn act as suppressors of HMGR activity as described above. These actions in turn are thought to collectively result in a decrease in cholesterol synthesis and a reduction in serum cholesterol levels. lb 231 General Procedure for the Preparation of Unsaturated 14,15-Substituted Lanosterols The compounds of the present invention accomodate the necessary requirements for lanosta-8,24-dien-3{3-ol 14a-methyl demethylase inhibition and suppression of HMGR activity. To prepare these compounds a three-prong synthetic approach was employed, i.e., monosubstitution at the 14- or 15-position and disubstitution at the 14-and 15-positions. 14-Monosubsti tution (SCHEMES I-V) Introduction of the appropriate substituent at the 14a-position required the elaboration of lanosta-8,24-dien-30-ol {SCHEME I, Compound 1) into the protected 3 0-hydroxy-14a-hydroxymethyl-dihydrolanosterol (Compound 6).

I~~7 J* 23 1 6 SCHEME I CHj CHj PhC S''l7 PhC PhCO CH CH 3 3 6 n & jar 23 16 04 Transformation of the commercially available lanosta-8,24-dien-30-ol (obtained from Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178) to the diol (Compound 2) was achieved using the procedure described in Parish et al., J. Lipid Res., 22: 859-868 (1981). To utilize this intermediate in our studies, a novel synthetic route that allows selective protection of the 30-hydroxy group while the 14a-hydroxymethyl group is free for elaboration was employed. Taking advantage of the sterically encumbered secondary alcohol at the 7a position, the diol (Compound 2) was treated with freshly distilled benzoyl chloride in dry pyridine (obtained from Aldrich Chemical Co., Inc., 940 West St. Paul Ave., 15 Milwaukee, WI 53233) at 40® for 25 min. The reaction resulted, after silica gel chromatography, in the recovery of 30-benzoate (Compound 3) in a 48% yield. Using the directing effects of the 7a-hydroxyl group, the resulting Compound 3 was oxidized in refluxing benzene under a nitrogen atmosphere by means of recrystallized (from acetic acid) lead tetraacetate (obtained from Aldrich) treatment. After 17 hr of refluxing followed by silica gel chromatography, the desired furan, Compound 4, was obtained in a yield of 70%. Furan ring cleavage of Compound 4 was achieved by exposing 4 to excess pyridine hydrochloride (obtained from Fluka A.G., Buchs. Switzerland) in refluxing acetic anhydride for 18 hr under an atmosphere of nitrogen. Three olefin isomers of the newly generated acetate Compound 5 (Compounds 5a, 5b and 5c) were obtained in an overall 60% yield.

Separation of the isomers by silica gel chromatography (mplc) provided 5c (16% yield), and separation by high pressure liquid chromatography (hplc) afforded 5a (19% yield) and 5b (27% yield). With all three olefinic isomers separated, the final selective hydrolysis was achieved by treatment of 5a with 23 16 04 ethanolic potassium hydroxide for 2 hr at 10° to generate Compound 6a in 68% yield. In the same manner, both Compounds 6b (73% yield) and 6c (62% yield) were also obtained, providing entry into the double bond isomers of the 14a-substituted series.

Following preparation of the critical monoprotected diol, Compound 6, the next goal is the elaboration of that compound into the desired 14a-substituted dihydrolanosterols. This is shown in SCHEME II.

SCHEME II C H PhO chj chj 8 R1 - PhCOj 9 rx - oh Oxidation of the 14a-hydroxymethyl group to the 14a-carboxaldehyde Compound 7a was achieved in a 93% e oo 23 1 yield by treating Compound 6a with Jones reagent (prepared as described in Meinwald et al., Org. Syn., £5:77-79 (1965)) at -10" for 15 min. Compound 7a was ^ then recovered using medium pressure silica gel chromatography (MPLC). in the same manner Compound 7b was prepared in 85% yield. The 14a-carboxyaldehyde Compound 7c was prepared in a 60% yield by treatment at room temperature of a dichloromethane solution of Compound 6c with pyridinium dichromate (Aldrich) and ^ powdered 4A molecular sieves (Aldrich) followed by silica gel purification on MPLC.

Exposing the azeotropically (benzene) dried aldehyde Compound 7a to neat DAST (diethylaaino sulfur trifluoride, Aldrich) under an argon atmosphere for 4.5 hr at 80° afforded the 14a-difluoromethyl dihydro-lanosterol, Compound 8a, in a 76% yield. In the same manner, Compound 8b was prepared from Compound 7b in a 48% yield. An anhydrous (distilled over benzophenone and sodium) diethyl ether solution of Compound 8a was exposed to lithium aluminum hydride (obtained from Alfa Prod., Danver, MA) at room temperature for 20 min, resulting in the desired 3f5-hydroxy-14a-difluoromethyl dihydrolanosterol, Compound 9a, in an 86% yield. The difluoride, Compound 9a, along with another olefin isomer Compound 9b prepared in a similiar fashion in an 86% yield constitute two examples of 14a-substituted lanosterols within the scope of the present invention.

Further elaboration of the 14a position (SCHEME III) was achieved by treating the hydroxy aldehyde Compound 10 (prepared as described by Shafiee et al., J. Lipid Res., 27:1-10 (1986)) with a variety of alkyl and alkenyl anions (Grignards, lithium reagents).

G a\ 23160 11 12 13 w - ch-ch. w - ch-ch! w » &lkyl, ^2~^6 alkeny1» ary^» C2~Cg alkynyl, aryl-C1-C6-alkyl Exposure of an anhydrous tetrahydrofuran solution of Compound 10a to an excess of a vinyl magnesium bromide tetrahydrofuran solution (Aldrich) at room temperature resulted in formation of the diastereomeric diols, Compound 11a and Compound 12a, in 87% combined yield. The diastereomers were separated by MPLC to provide Compound 11a in a 66% yield and Compound 12a in * a 21% yield. Compounds 11a and 12a are two additional examples of 14a-substituted lanosterols of the present invention. In the same manner, Compounds lib, 11c, 12b, and 12c may be prepared.

Compounds 13, compounds also within the scope of the invention, may be prepared in a similar fashion by adding CH3 or any alkyl, alkenyl, aryl, arylalkyl, or alkynyl anion to Compound 10.

Nitrogen was introduced into the steroid compound (SCHEME IV) by treating aldehyde Compound 7b with hydroxylamine hydrochloride (Aldrich) in pyridine for 16 hr. oa 231 scheme iv .t> I CH-NOH 14 R2 - PhC02 R2 - OH CH, CHj 16 R1 - PhCOj 17 R: - OH The resulting oxime, Compound 14b, was obtained in a yield of >95*. In a similar fashion, Compounds 14a (96% yield) and 14c (70% yield at 80°) were prepared from Compounds 7a and 7c, respectively.

Benzoate was removed from Compound 14b with ethanolic potassium hydroxide resulting in the hydroxy oxime 15b in a 94% yield. In the same manner oximes 15a and 15c were prepared from Compounds 14a and 14c, respectively. Compounds 15a, 15b and 15c are all within the scope of the present invention.

O 10 a3 23 1 6 0 4 & Preparation of the 14a-carbonitrile was achieved (SCHEME IV) by dehydrating the oxime Compound 14a directly with phenylisocyanate (Aldrich) and freshly distilled triethylamine to afford, after silica gel chromatography, the carbonitrile, Compound 16a, in 85% yield.

Similarily, Compounds 16b and 16c were prepared from Compounds 14b and 14c, respectively. Exposure of Compound 16a to ethanolic potassium hydroxide resulted in the removal of the benzoate and generated, after silica gel chromatography, the desired nitrile, Compound 17a (a compound of the present invention), in a 90% yield. In the same manner, nitrile compounds 17b and ^ 17c, also within the scope of the present invention, may be prepared.

To prepare a variety of 14a substituted lanosterols, the enones, Compounds 18 and 19 (SCHEME V), were prepared using the procedures previously reported in Woodward et al., J. Biol. Chem., 241:1502-1510 (1966). vJ nnx+fl-i /—•x SCHEME V 2316 04 18 both R2 - CH; 19 both R- - H " 20b both R2 - CH3 r3 - ch2ch-ch2 C»2CH-CH2 Compound 18 was alkylated with freshly distilled allyl bromide (Aldrich) in the presence of potassium tertiary butoxide in tertiary butanol to afford, after silica gel chromatography, the 14a-allyl-30-benzoyloxy- 4,4-dimethyl-5a-cholest-7-ene (Compound 20b), in 20% yield (SCHEME V). The 15-ketone was reduced by treating Compound 20b with excess hydrazine in hot (180-220®) diethylene glycol with excess sodium to generate the deprotected 14a-substituted dihydrolanosterol Compound 21b, a Formula I compound, as a mixture of 14a- and 7a-substituted lanosterols. The 14a and 7a compounds 23 1 can then be separated by chemical chromatographic methods such as argenic chromatography readily known to those skilled in the art. In the same fashion, Compound 21a can also be prepared.

Using the above method, and modifications thereof which would be obvious to those skilled in the art, other examples of the lanosterols series within the scope of the present invention can be prepared. 15-Substitution (Schemes VI-VIIA) Introduction of substituents at the 15-position required preparation of Compound 22b, 30-benzoyloxy-15a-hydroxy-lanost-7-ene, and Compound 23b, 3(3-benzoyloxy-15a-hydroxy-14a-methyl-5a-cholest-7-ene, using the methods reported in Woodward et al., J. Chem. Soc., 1131-1143 (1957) and Knight et al., J.' Biol.

Chem., 241:1502-1510 (1966)). Conversion (SCHEME VI) of the 15a-hydroxy substituent of Compound 22b to the 15a-fluoro steroid, Compound 24b, was achieved with the addition of DAST (diethylamino sulfur trifluoride, obtained from Aldrich) at -78°. This retention of stereochemistry was confirmed by a series of NMR studies (Nuclear Overhauser Effect).

G © a<=> J* 231 6 SCHEME VI ?«HW 22b both R2 - CH3 23b both R2 ■ H CH, -."n CH, CH i \ Rj R, 24b both R2 - CH3 25b both R2 - H o 26b both R2 - CH3 27b both R2 - H Reductive removal of the 15a-fluoro benzoate, 30 Compound 24b, to the alcohol, Compound 26b, was performed by the addition of lithium aluminum hydride (Aldrich) to a cold (0°) ethereal solution (diethylether:tetrahydrofuran at a ratio of 4:1) of Compound 24b. Following the above procedure in the 35 4,4-desmethyl series, the fluoride, Compound 27b was prepared from Compound 23b. Both Compounds 26b and 27b an J* 231 are compounds of the present invention.

Starting from Compounds 22a and 23a, which were prepared in the same manner as Compounds 22b and 23b, respectively, the corresponding 15a fluorides, Compounds f"*} 5 26a and 27a can be similarily prepared.

The ketones, Compounds 28b, 29b and 30b, previously reported by Woodward et al., J. Chem. Soc., 1131-1143, (1957); Knight, et al., J. Biol. Chem., 241; 1502-1510, (1966), can be elaborated to oximes and other heteroatom r~\ 10 compounds substituted at the 15 position (Formula lb) within the scope of the present invention (SCHEME VII).

SCHEME VII y 7 6 R2 2 28b R1 - oh, both Rj or r3 ch3 29b R1 - PhC02, both or r3 - ch3 30b R1 - PhCOj/ both or r3 « ch3 "^OH ^2 ^2 lib) J* For example, exposure of 30-hydroxy-lanost-7~en-15-one (Compound 28b) to hydroxylamine hydrochloride (Aldrich) in hot pyridine (80°) for 18 hr provided, after silica ^ gel chromatography, the 15-oxime, Compound 31b in a yield of 85%, In the same manner, compound 31a was prepared in an 85% yield. Oxime Compound 31b provides another example of a compound of the present invention and in addition, along with Compounds 29b and 30b, (Oi ^ provides a synthetic entry using conventional techniques to other compounds of within the scope of the present claims.

The ketones (SCHEME VIIA), Compounds 29a, 30a and 42a, are prepared by the elaboration of Compounds 38a or 231604 .20 PhCO HO SCHEME VIIA C."i7 38a B2 - CH3 39a S2 - H 40a R2 " ch3 CHj CH, 31a 30a 42a R, » CH3' R2 - H; R3 - ch3 r2 - H, or CH3 r3 - crcealky1, c _C6 alkenyl, c2-C6 alkynyl' CH2OCB2^h» • • • (see legend I) t - 23 16 04 M 3' Preparation of epoxide 38a was performed using a process analogous to that described for the preparation of 39a in Anastasia et al., J. Org. Chen. j!6: 3265-3267 (1981). Exposure of the epoxide 38a to excess boron trifluoride etherate (Aldrich Chemical Co.) at 0* for 1 hour provided in 75% yield the new ketone 40a with the 14(3 hydrogen substitution. Alkylation of ketone 40a with methyl iodide in potassium tertiary butoxide in tertiary butanol supplied Compound 29a in 75% yield after recrystallization. Hydrolysis of the benzoate in Compound 29a using 5% potassium hydroxide in ethanol at 80"C for 18 hours provided the hydroxy ketone, Compound 28a. Exposure of Compound 28a to hydroxylamine hydrochloride (Aldrich) in hot pyridine (85*) for 18 hours afforded 15 oxime Compound 31a in 85% yield after recyrstallization. In this manner Compounds 38a, and 39a can be converted into Compounds 30a, and 42a which are included in the scope of the invention.

Elaboration of Compounds 29a, 30a, and 42a in the similar fashion as Compounds 24b, 21b, provides entry into other compounds of the present invention. In addition, this process allows 14-15 difunctional modification of Compound 42a to generate additional compounds within the present scope. 14,15-Disubstitution (SCHEMES VIII AND VII B) Introduction of 14,15-heteroatoms was achieved by exposing the diene, Compound 32a, prepared as reported in Woodward et al., J. Chem. Soc., 1131-1143 (1957) and Knight et al., J. Biol. Chem., 241; 1502-1510 (1966), with osmium tetroxide in pyridine and benzene. -5' 231 32 both R2 . CH, 34 both *2 * cu3 33 both R2 • B 35 both F2 " a This reaction resulted, after silica gel chromatography, in the formation of the vicinal diol, Compound 34a, in an 81% yield. This 14,15-heteroatom substitution has been incorporated into the desmethyl series, Compound 33a, to make the triol, Compound 35a. Compounds 33a and 35a are two additional examples of compounds of the present invention. In this manner, starting with the 7,14 dienes (Compounds 32b and 33b), the corresponding compounds. Compounds 34b and 35b, may be prepared.

An example of 14,15 difunctional elaboration is illustrated in Scheme VII B.

-J* ••32. 2 3 16 0 4 SCHEME VII B 9s^i: PhC02 CH2OCH2Ph 47a R2 = CH, 9B^I: HO ** OH = ch3 CeHn- ** OH CH2OCH2Ph C8H1: HO CH5OH * OH 49a R2 = CH3 Compound 47a, prepared as described in Scheme VIIA, was treated with lithium aluminum hydride (Aldrich) to generate the diol, Compound 48a in 64% yield. This diol was further treated with 10% palladium on activated carbon (Aldrich) in tetrahydrofuran, ethanol and acetic 35 acid (25:25:1) to provide the triol Compound 49a in 61% yield. At this point, oxidizable functionality has been introduced at both the 15 and 32 position of lanostenol.

• Jfn'- o ^3 0 J V7 S~ W .>7 Selective oxidation of the 32 alcohol vas achieved by the treatment with pyridium dichromate (Aldrich) in dichloromethane to give after chromatography only f**) ^ Compound 50a in 63% yield. In this fashion this route offers a selective method for the preparations of oxidized functionality at the 32 position of lanostenol while Keeping the 15 position in a functionalized form. Thus affording a method for the preparation of a variety of 14,15 disubstituted lanosterols. Ketone removal in compound 47a as indicated previously offers an alternative approach to prepare 14 substituted lanosterols.

General Procedure for the Preparation of Saturated 14,15-Substituted Lanosterols To prepare the saturated steroid ring system (Scheme IX), the 3p-benzoyloxy-lanostan-7-one (Compound 36) in Parish et al., 3. Lipid Res., 2_2: 859-868 (1981) was exposed to Wolf-Kishner conditions (see Knight et al. J. Am. Chem. Soc. 88(4): 790-798). *2Pc A* 2316 04 SCHEME IX n VU«" CH, CH, 36 37 [ I] PhO CHj CH, CHj CH, 5c 5d To a solution of sodium disolved in diethylene glycol was added Compound 36 followed by excess anhydrous hydrazine. After heating (180®) for 48 hours, the excess hydrazine was distilled off over 48 hours at 220® 35 resulting in lanostan-3£-ol (Compound 37) in approximately 50% yield.

-A*- o s* 23 1 6 04 ?* Compound 5c is also a convenient starting material for the saturated lanostane ring system. Treatment of Compound 5c with 10% palladium on carbon in ethanol at 80° for 24 hours under 200 atmospheres of hydrogen afforded, after HPLC purification, 32-acetoxy-33-benzoyloxy-lanostane (compound 5d) in 76% yield.

Compound 5d may be elaborated in the same fashion as Compound 5a-5c to generate the corresponding alcohol, Compound 6d. Compounds 6 and 37 afford synthetic entry to the saturated compounds of the present invention using the General Procedures described for the preparation of the corresponding unsaturated compounds. Preparation of Salts • .

Physiologically acceptable salts of the compounds are also within the scope of the present invention and can be prepared in a number of ways apparent to those skilled in the art.

For example, metal salts can be made by contacting compounds of the invention with a solution of an alkali or alkaline earth metal salt having a sufficiently basic anion (e.g., hydroxide, alkoxide or carbonate). Quaternary amine salts can be made by similar techniques.

Salts can also be prepared by exchange of one cation for another. Cationic exchange can be effected by direct contact of an aqueous solution of a salt of a compound of the invention (e.g. alkali or quaternary amine salt) with a solution containing the cation to be exchanged. This method is most effective when the desired salt containing the exchange cation is insoluble in water and can be separated by filtration.

Exchange may also be effected by passing an aqueous solution of a salt of a compound (e.g., an alkali metal ^ or quaternary amine salt) through a column packed with a cation exchange resin containing the cation to be exchanged for that of the original salt and the desired ,-t-v 23 1 6 0 4 product is eluted from the column. This method is particularly useful when the desired salt is water-soluble, e.g., a potassium, sodium or calcium salt.

Acid addition salts, useful in this invention, can be obtained by reacting a compound of the present invention with a suitable acid, e.g., p-toluenesulfonic acid, acetic acid or the like.

The preparation of compounds within the scope of the invention is further illustrated by the following specific examples. in 23 16 04 Synthetic Examples The following Examples describe synthetic procedures employed in production of specific compounds within the scope of the present invention. Unless otherwise indicated, all parts and percentages in the following Examples and.in the General Procedures set forth above are by weight and all temperatures are reported in degrees Celsius (*C). All chromatography solvents percentages were determined by volume. All proton NMR spectra are referenced to tetramethylsilane (TMS) at 0.00 ppm while all fluorine NMR spectra are referenced to freon-11 (F-ll) at 0.00. ppm.

The following abbreviations are employed in the Examples: NMR: nuclear magnetic resonance spectroscopy IR: infrared spectroscopy MS: mass spectrometry HRMS: high resolution mass spectrometry EI: electron impact CI: chemical ionization EA: elemental analysis [a]25.: optical rotation at 25°C at the sodium D line m.p.: melting point MPLC: medium pressure liquid chromatography HPLC: high pressure liquid chromatography * Rf: retention factor on silica gel thin layer chromatography GC: ' gas chromatography Particular intermediates or products are identified by reference to the numbered compounds in the general synthetic procedures summarized above. Physical data for various compounds produced by procedures substantially corresponding to the description contained in each Example are provided following the individual 3^ 231 Examples.

Example 1 (A-V): Preparation of 14,15-Substituted Lanosterols A. Preparation of 30-benzoyloxy-lanost-7a-ol (Compound 3) Lanostane-30,7a-diol (Compound 2) (4.6 g, 10.3 mmol) was dissolved iri'anhydrous pyridine (100 mL) at 40°. Benzoyl chloride (6.0 mL, 51.7 mmol) was added and the mixture was stirred at 40® for 25 min. The cooled reaction mixture (0®) was diluted with ice cold ether (200 mL) and acidified with IN HCl to pH 6.5. The organic fraction was washed with 10% aqueous cupric sulfate (2 x 100 mL), water (1 x 50 mL), dried over anhydrous magnesium sulfate and evaporated under reduced pressure to afford the crude residue. The residue was subjected to medium pressure liquid chromatography (100 psi, 100 cm X 2.7 cm) using 0.5% ethyl acetate in toluene (4 L) and then 2% ethyl acetate in toluene (fractions: 27 mL). The contents of fractions 113 through 202 were pooled and evaporated under reduced pressure, providing 2.72 g (48%, corrected 64%) of pure Compound 3.

Physical Data (Compound 3): [a]25 - +25.8® +/- 3.1° (C - 0.64, CHCl,); m.p. « 190-190.5® (white flakes, acetone); Rf - 0.55 (10% ethyl acetate in toluene); NMR (300 MHz CDC13): 8.06 (d, J - 7.5 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 4.79 (dd, J - 11.4 Hz, 4.5 Hz, 1H, 3-CHOR), 4.11 (s, 1H, 7-CHOH), 2.0-0.85 (m, 27H), 1.11 (s, 3H, CHj ) , 1.06 (s, 3H, CHJ, 1.01 (s, 3H, CHj), 0.93 (S, 3H, CH3), 0.88 (d, J - 6.6Hz, 6H, 26-CHj and 27-CH3), 0.88 (d, J - 6.5 Hz, 3H, 21-CH3), 0.76 (s, 3h, 18-chj); IR (CHClj solution, cm"1): 3520 (bw, OH), 2950 (s, CH -39 >3 sat), 2870 (s, CH sat), 1710 (s, C-O), 1600(w), 1580(w), 1465(m), 1450(m), 1275(vs), 1115(b); MS (EI): 550 (3%, M+), 517 (21%, M -HjO, -CH3), 395 (96%, M -H2 0, -CHjf -C6H5COOH); HHMS for Cj,H510j (M+): calculated 550.4386, found 550.4354.

B. Preparation of 30-benzoyloxy-7a,32-epoxylanostane (Compound 4) -benzoyloxy-lanost-7a-ol (Compound 3) (2.72 g, 4.95 mmol) was dissolved in benzene (1.1 L). About 150 mL of the solvent was distilled off to remove any traces of water'. Lead tetraacetate (12.8 g, 28.9 mmol) (recrystallised from acetic acid) was added and the resulting mixture was refluxed under a nitrogen atmosphere for 17 hours. After cooling to room temperature, the reaction mixture was treated with a 20% aqueous potassium iodide solution (200 mL) then a saturated solution of sodium thiosulfate was added (until the yellow precipitate had dissolved), and the resultant mixture was extracted with diethyl ether (4 x 150 mL). The combined organic extracts were dried over anhydrous.magnesium sulfate and evaporated under reduced pressure. The resulting residue was subjected to MPLC (35 psi; 50 cm x 2.5 cm) using 5% diethyl ether in toluene as the eluting solvent (fraction size: 28 mL). The content of fractions 17 through 29 were pooled and the solvent was evaporated under reduced pressure, giving 1.90 g of Compound 4 (70% yield).

Physical Data (Compound 4): [a]25 - +41.0e +/- 0.8° (c - 1.01, CHClj); m.p. - 225-227® (fine needles, acetone); Rf - 0.60 (10% ethyl acetate in toluene); nmr (300 MHz, CDClj ): 8.05 (d, J - 7.2 Hz, 2h, phenyl), 7.64-7.39 (m, 3H, phenyl), 4.75 (dd, J - 11.5 Hz, 4.6 V;'" 0 23 Hz, 1H, 3-CHOR), 4.22 (n, 1H, 7-CHOR), 4.02 (d, J - 7.5 Hz, 1H, 32~CH2OR), 3.38 (d, J - 7.5 Hz, 1H, 32-CHjOR), ^ 2.1-0.8 (in, 26H), 1.03 (s, 3H, 31-CH,), 0.92-0.87 (m, - 5 18H, CHj s); IR (CHClj solution, cm"1): 2955 (s, CH sat), 2870 (5, CH sat) 1710 (s, C-O), 1278(vs) , 1116(e), 1025(m), 962(m). HRMS for CjtHj302 (M -CHjOH): calculated 517.4045, found 517.3994; MS (EI): 517 (38*, M -CHjOH), 403 (641, M -CHa OH-Ct Hx, ) , 395 (100%, M -CH2OH-CtH5COOH).

CN 10 C C. Preparation of 32-acetoxy-30-benzoyloxy-lanost-8-ene (Compound 5a), 32-acetoxy-3g-benzoyloxy-lano6t-7-ene -c (Compound 5b) and 32-acetoxy-3fJ-benzoyloxy-lanost- 6-ene (Compound 5c) 3fS-benzoyloxy-7a-32-epoxy-lanostane (Compound 4) (1.90 g, 3.47 mmol) was refluxed for 18 hr in acetic anhydride (380 mL) with pyridine hydrochloride (3.8 g, 2q 32.9 mmol) under nitrogen. After cooling (25°), the mixture was poured into ice-water (400 mL) apd stirred for 2 hr. This aqueous mixture was then extracted with diethyl ether (4 x 150 mL) and the combined ether extracts were washed successively with cold (0#) aqueous 25 5* hydrochloric acid (300 mL), saturated aqueous sodium bicarbonate (8 x 200 mL), water (2 x 100 mL) and brine (100 mL). The extract was dried over anhydrous magnesium sulfate and evaporation was carried out under reduced pressure. The crude product was subjected to 2q MPLC (100 psi, 100 cm x 2.5 cm) using toluene (4 L) and O then 0.5% ethyl acetate in toluene as the eluting solvent (fraction size: 27 mL). The contents of the following fractions were pooled and evaporated to dryness: (1) fractions 274-311 (Compound 5c), 329 mg (16%); (2) fractions 312-327 (Compound 5a), 249 mg; (3) fractions 366-402 (Compound 5b), 245 mg and (4) fractions 328-365 (Compounds 5a and 5b in a 32:68 t 231604 mixture), 461 mg. The mixture of Compounds 5a and 5b was resolved by repeating HPLC (400 psi, 50 cm x 2.5 cm) using 0.25% ethyl acetate in toluene as the eluting ** solvent. Total yields for Compound 5a - 19%, Compound 5b - 27% and Compound 5c - 16%.

Physical Data (Compound 5a): [a]35 - +61 ® +/- 2° (C- 1.00, CHClj); 10 m.p. ■ 109.5-110° (ethanol - 5% water, needles); V'. / Rf - 0.64 (10% ethyl acetate in toluene) NMR (300 MHz, CDCl,): 8.06 (d, J - 7.2 Hz, 2H, phenyl), 7.60 - 7.40 (a, 3H, phenyl), 4.75 (dd, J - 11.4 Hz, 4.2 Hz, 1H, 3-CHOR), 4.08 (d, J 10.5 Hz, 1H, 32-CHaOR), 15 3.97 (d, J - 10.5 Hz, 1H, 32-CHjOR), 2.2-0.85 (m, 26H),. 2.06 (s, 3H, acetate), 1.08 (s, 3H, CH3 ), 1.06 (s, 3H, CH3), 0.97 (s, 3H, 30-CHj), 0.90 (d, J - 6.3 Hz, 3H, 21-CHj), 0.88 (d, J - 6.6 Hz, 6H, 26-CH, and 27-CH,), 0.73 (s, 3H, 18-CHj); ~20 IR (CHClj solution, cm"1): 2950 (s, CH sat), 2860 (s, CH sat), 1710 (s, C-O), 1600 (m), 1465(s), 1450'(s), 1275 (vs), 1115(s) , 1025(s)f 980(s), 970(s); MS (EI): 530 (2%, M -CH3C02H), 517 (22%, M -CH2 OCOCHj), 395 (100%, M -CH2 OCOCH3, -CsHsC02H); HRMS for C37H5402 (M -CHjCOjH): calculated 530.4142, found 530.4116.

Physical Data (Compound 5b): [a]23 - +50° +/- 2° (c - 1.03, CHClj); nup. - 154-155° (ethanol - 5% water, needles); Rf - 0.63' (10% ethyl acetate in toluene); NMR (300 MHz, CDClj): 8.06 (d, J - 7.2 Hz, 2H, phenyl), 7.60 - 7.40 (m, 3H, phenyl), 5.25 (d, J - 4.8 Hz, 1H, 7-CH), 4.79 (dd, J - 10.8 Hz, 4.2 Hz, 1H, 3-CHOR)/ 4.59 35 (d, J - 10.8 Hz, 1H, 32-CH2OR), 3.73 (d, J - 10.8 Hz, 1H, 32-CH2 OR), 2.15 - 0.85 (m, 25H), 1.99 (s, 3H, acetate), 1.13 (s, 3H, 31-CH3), 0.95 (s, 3H, CH3), 0.94 4^ 23 1 6 04 (T) (s, 3H, CHj), 0.90 (d, J - 6.0 Hz, 3H, 21-CHa), 0.88 (d, J - 7.2 Hz, 6H, 26-CHj and 27-CH3), 0.73 (s, 3H, 18-CH3 ) ; IR (CHC13 solution, cm"1): 2950 (s, CH sat), 2860 (s, CH sat), 1710 (s, C-O), 1600 (m), 1465 (s), 1450(8), -1380 ( s ), 1365 (s ) e 1275 (vs), 1115(b)-, 1025(5), 965(s). MS (EI): 517 (25%, M -CH2C02CH3 ), 395 (100%, m -CH2 C02 CH, ) ; HRMS for CJ6HJ0O2 (M -CH2C02CHj ) : calculated 517.4045, found 517.3999.

Physical Data (Compound 5c): [a]25 - -36.6# +/- 2° (c - 1.01, CHClj); m.p. - 141-141.5° (ethanol - 10% water, very fine ^ needles); Rf - 0.66 (101 ethyl acetate in toluene); NMR (300 MHz, CDCl,): 8.06 (d, J - 7.2 Hz, 2H, phenyl), 7.60 - 7.40 {m, 3H, phenyl), 5.61 (d, J - 10.2 Hz, 1H, olefinic H), 5.52 (d, J - 10.2 Hz, 1H, olefinic H), 4.77 (dd, J - 11.4 Hz, 4.8 Hz, 1H, 3-CHOR), 4.56 (d, J - 11.5 Hz, 1H, 32—CHj OR), 3.96 (d, J - 11.5 Hz, 1H,'32-CHaOR), 2.42 - 0.85 (m, 24H), 1.99 (s, 3H, acetate), 1.03 (c, 3H, 31-CHj), 0.97 (s, 3H, CH,), 0.91-0.87 (m, 15H, CHj s ); ; ^ 3^R (CHClj solution, cm"1): 2950 (s, CH sat), 2860 (s, CH sat), 1720 (s, C-O), 1600(m), 1470(s), 1450(b), 1385(s), 1370(B), 1315(s), 1275(vs), 1115(b), 1025(s), 970(s). MS (EI): 517 (9%, M ~CH2 OCOCHj ) , 453 (32%, M-CH3C02"H, -C6HS), 408 (30%, M -CHjCOjH, -CSH5C02H), 395 (100%, M -CH2 OCOCHj , -CsH5C02H); HRMS for CjjHjjOj (M -CHjOCOCHj): calculated 517.4045, found 517.4042.

D. Preparation of 33-benzoyloxy-lanost-8-en-32-ol 35 (Compound 6a) 32-acetoxy-30-benzoyloxy-lanost-8-ene (Compound 5a) ( 330 mg, 559 j/mol) was dissolved in ethanol (100 mL) and r . *a. '•*> ' f. o 4-3 ^4T- 23 16 04 treated with potassium hydroxide (87%, 8.3 g) in ethanol (23 mL) and water (7 raL) at 5°. The mixture was stirred at 10° for 2 hr and quenched with ice water (40 mL). "* The mixture was then extracted with dichloromethane (3 x 100 mL) with the combined organic fractions dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The residue .was subjected to MPLC (55 psi, 50 cm x 1.8 cm) using 2% ethyl acetate in toluene as the eluting solvent (fractions 18 mL). The contents of fractions 13 through 35 were pooled, and after evaporation under reduced pressure provided 209 mg (68%) of (Compound 6a).

^ Physical Data (Compound 6a): [ct]2J - + 79 9 +/- 2° (C - 1.02, CHClj); m.p.- 167-168.5® (ethanol -5% water); Rf - 0.50 (10% ethyl acetate in toluene); NMR (300 MHz, CDClj): 8.06 (d, J - 7.2 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 4.75 (dd, J - 11.7 Hz, 4.2 Hz, 1H, 3-CHOR), 3.66(dd, J - 10.8 Hz, 9.3 Hz, 1H, 32-CHjOH), 3.25 (dd, J - 10.8 Hz, 3.8 Hz, 1H, C-32-H), 2.15-0.85 (m, 26H), 1.13 (s, 3H, CH3), 1.08 (s, 3H, CH3), 0.98 (s, 3H, 30—CHj), 0.90 (d, J- 6.5 Hz, 3H, 21-CHj), 0.88 (d, J - 6.6 Hz, 6H, 26-CHj and 27-CH3), 0.72 (s, 3H, 18-CHj).

IR (CHClj solution, cm"1): 3480 (bw, OH), 2950 (s, CH sat), 2860 (s, CH sat), 1710 (s, C-O), 1460(s), 1445(s), 1305(s)f 1275(vs), 1115(B).

MS (EI): 530 (8%, M -H,0), 518 (42%, M -CHa0), 395 (68%, X -CHjOH,' -Ct Hs C02H) , 105 (100%, CsH,.C0+).

HRMS for Cj7H5402 (M -H20): calculated 530.4124 , found 530.4162.

Physical Data (Compound 6b; 731 yield from Compound 5b): la]25 - +51.5° +/- 2.0° (c - 1.01, CHClj); m.p.- 209.5-211° (acetone, needles); 4"/— <44- 231 6 Rf - 0.50 (10% ethyl acetate in toluene); NMR (300 MHz, CDCl,): 8.06 (d, J - 7.5 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 5.39 (m, 1H, 7-CH), 4.75 (dd, 5 J - 4.7 Hz, 11.3 Hz, 1H, 3-CHOR), 3.66 (d, J - 10.2 Hz, 1H, 32-CHjOH), 3.26 (t, J - 10.2 Hz, 1H, 32-CHjOH), 2.15-0.85 (m, 26H), 0.96 (s,3H, CH,}, 0.89 (d, J - 6.5 Hz, 3H, 21-CHj), 0.87 (d, J - 6.8 Hz, 6H, 26-CH, and 27-CHj), 0.74 (s, 3H, 18-CH,); _IR (CHClj solution, cm"1): 3500 (bw, OH), 2940 (s, CH sat), 2860 (s, CH sat), 1705 (s, C-O), 1600(m), 1465(b), 1315(b), 1275(vs), 1115(b), 1020(b), 970(b); MS (EI): 518 (22%, M -CHaO), 395 (37%, K -CH,OH, -C6H5COaH), 381 (19%, M -CH2-0, -CjH5C02H, -CH, ) , 105 15 (100%, C6H5CO+); HRMS for C3SHS402 (M -CH20): calculated 518.4124, found 518.4161.

Physical Data (Compound 6c; 62% yield from Compound 5c): 20 [a]25 - -0.9° +/- 2.0° (c - 1.02, CHClj); m.p. ■ 222-223° (ethanol, needles); Rf - 0.55 (10% ethyl acetate in toluene); NMR (300 MHz, CDCl3 ) : 8.05 (d, J - 7.5 Hz, 2H, phenyl), 7.60-7.40.(m, 3H, phenyl), 5.85 (d, J - 10,2 Hz, 1H, 2^ olefinic H), 5.69 (d, J - 10.2 Hz, 1H, olefinic H), 4.78 (dd, J - 11.6 Hz, 4.7 Hz, 1H, 3-CHOR), 4.20 (d, J - 11.5 Hz, 1H, 32-CH2OH), 3.46 (t, J - 11.5 Hz, 1H, 32-CH2OH), 2.40 (m, 1H), 2.12-0.85 (m, 24H), 1.04 (s, 3H, CH, ), 0.98 (s, 3H, CH,), 0.93 (s, 3H, CH,), 0.90-0.84 (m, 12H, 30 CH,s); IR (CHCl, solution, cm"1): 3690 (w, OH), 3540 (bw, OH) 2960 (s, CH sat), 2870 (s, CH sat), 1710 (s, C-O), 1600(w), 158Q (w), 1470 (m), 1450 (m), 1315 (m), 1280 (vs), 1120 (s); MS (EI): 530 (5%, M -HjO), 517 (17%, M -CH2OH), 408 (15%, M -H20, -C6H5COOH), 403 (8%, M -CH2 OH,-C, Hx , ) , 395 (100%, M -CH2OH, -C5H6COOH); t ^ 23 1 6 04' HRMS for Cj,H5 4 02 (M -H2 0): calculated 530.4124, found 530.4093.

El. Preparation of 3g-benzoyloxy-lanost-8-en-32-al (7 (Compound 7a) 3|3-benzoyloxy-lanost-8-en-32-ol (Compound 6a) (200 mg, 365 fjmol) was dissolved in acetone (100 mL) and treated with Jones reagent (2.3 mL) at -10°. The mixture was stirred for 15 min at -10°. The reaction mixture was diluted with water (100 mL) and quickly extracted with toluene (3 x 50 mL). The combined toluene fractions were washed with water (2 x 50 mL), dried over anhydrous magnesium sulfate, and removed by ^ evaporation under reduced pressure. The resulting residue was subjected to MPLC (50 psi, 50 cm x 1.8 cm) being eluted with toluene. The procedure resulted in 185 mg (93%) of Compound 7a.

Physical Data (Compound 7a): [a)15 - -243° +/- 4° (c - 1.00, CHCl,); m.p.- 206-207° (acetone, fine needles); Rf - 0.60 (toluene); NMR (300 MHz, CDClj): 9.47 (s, IE, 32-CHO), 8.05 (d, J -7.5 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 4.73 (dd, J - 11.6 Hz, 5.1 Hz, 1H, 3-CHOR), 2.4-0.85 (m, 26H), 1.14 (s, 3H, CH3), 1.07 (s, 3H, CH, ) , 0.94 (s, 3H, CH, ) , 0.90 (d, J - 6.3 Hz, 3H, 21-CH, ) , 0.87 (d,*J - 6.6 Hz, 6H, 26-CH, and 27-CH, ), 0.77 (s, 3H, 18-CH,). IR (CHC1, solution, cm-1): 2940 (s, CH sat), 2860 (s, CH sat), 1710 (s, C-O), 1690 (s, C-O), 1465(s), 1450(e), 1275(vs), 1115(s); MS (EI): 517 (37%, M -CHO), 395 (100%, M -CHO, -C6H5C02H); HRMS for C,sH5,02 (M -CHO): calculated 517.4046, found 517.4076. 4-g jtrt Physical Data (Compound 7b, 85% yield from Compound 6b; reaction time - 1.5 hours at 0°): [a]2 5 - +46.5° +/- 2 • (c - 0.99, CHCl,); m.p.- 193.5-195.5* (acetone, fine needles); Rf 0.6 (toluene ); NMR (300 MHz, CDC1,): 9.66 (s, 1H, 32-CHO), 8.05 (d, J -7.5 Hz, 2H, phenyl), 7,60-7.40 (a, 3H, phenyl), 5.45 (a, 1H, 7-CH), 4.76 (dd, J - 11.1 Hz, 4,2 Hz, 1H, 3-CHOR), 2.2-0.85 (m, 25H), 1.14 (s, 3H, CH,), 0.98 (s, 3H, CH, ), 0.95 (s, 3H, CH,), 0.93 (d, J - 6.5 Hz, 3H, 21-CH,), 0.87 (d, J - 6.6 Hz, 6H, 26-CH, and 27-CH,), 0.75 (s, 3H, 18-CH, ) ; IR (CHCl, solution, cm"*): 2950 (s, CH sat), 2860 (s, CH sat), 1705 (s, C-O), 1600(a), 1275(vs), 1115(b), 970(b); MS (EI): 518 (51, M -CO), 517 (13*, M -CHO), 395 (68%, M -CHO, -CtH5C02H), 105 (100\, CjHjCO-f); HRMS for C,,H5402 (M -CO): calculated 518.4124, found 518.4115.

E2. Preparation of 3{3-benzoyloxy-lanost-6-en-32-al (Compound 7c) 3{3-benzoyloxy-lanost-6-en-32-ol (Compound 6c) (73 mg, 133 //mole) was dissolved in dichloromethane (distilled from phosphorous pentoxide) (5 mL) and treated with pyridinium dichromate (981, Aldrich) (74 mg, 193 //mole) and 4 powdered molecular sieves (Aldrich) (72 mg) at room temperature under dry nitrogen atmosphere. After stirring 2 hr, diethyl ether was added (40 mL) and the mixture was filtered through florisil and Celite (obtained from Manville Products Corp., Denver, CO). Evaporation of solvents under reduced pressure gave a residue that was subjected to MPLC (70 psi, 50 cm x 1.2 cm) using toluene as the elutant, to afford 42.9 mg (60%) of Compound 7c. 'iw.&r.v ",.-v \ ' ' 23 /xd Physical Data (Compound 7c): la]'* - -26.8° +/- 2.0° (c - 1.01, CHCl,); m.p. - 175-177° (acetone, very fine needles}? Rf - 0.8 (5% ethyl acetate in toluene); NMR (300 MHz, CDClj): 9.97 (s, 1H, 32-CHO), 8.05 (d, J -7.5 Hz, 2H, phenyl), 7.60-7.40 (m, 3fl, phenyl), 5.69 (d, J - 10.7 Hz, 1H, olefinic H), 5.64 (d, J - 10.7 Hz, 1H, olefinic H), 4.74 (dd, J - 11.7 Hz, 4.8 Hz, 1H, 3-CHOR), 2.46 (m, 1H), 2.23-0.85 (m, 23H), 1.03 (s, 3H, CH,), 0.97 (s, 3H, CH,), 0.95-0.90 (m, 12H, CH,s), 0.87 (d, J - 6.6 Hz, 6H, 26-CH, and 27-CH,); IR (KBr vafer, cm-1): 2950 (s, CH sat), 2870 (s, CH sat), 1718 (s, C-O), 1710 (s, C-O), 1600(v), 1580(v), 1465(m), 1450(a), 1275(vs), 1115(s); MS (EI) : 546 (1%, M+), 518 (29%, M -CO), 396 (44%, M -CO, -CtH5COOH), 381 (100%, M -CO, -C6H5C00H, -CH3); HRMS for C,,HS40, (M+): calculated 546.4073, found 546.4099.

F. Preparation of 3(J-benzoyloxy-32 , 32-difluoro-lanost-8-ene (Compound 8a) Azeotropically (benzene) dried 3j3-benzoyloxy-lanost-8-en-32-al (180 mg, 330 //mol) (Compound 7a) was stirred at 80° in neat DAST (diethylaaino sulfur trifluoride) (5 mL) under an argon atmosphere for 4.5 hr. After cooling (0°), the reaction mixture vas cautiously added, dropwise, to cold (0°) saturated aqueous sodium bicarbonate (100 mL) and dichloromethane (100 mL). After separation, the aqueous phase was reextract'ed with dichloromethane (2 x 100 mL) and the combined dichloromethane layers were washed with water (1 x 100 mL), dried over anhydrous magnesium sulfate, and filtered. Then the dichloromethane was removed by evaporation under reduced pressure. The residue was dissolved in dichloromethane, filtered through silica gel and the solvent once again removed by evaporation, t- _ f?*N * 2 3 16 0 4 this time in vacuum. This residue was subjected to MPLC (35 psi, 50 cm x 1.8 cm) using 25% toluene in hexane as the eluting solvent (fractions: 9 mL) giving 141 mg (75%) of Compound 8a of 91% purity determined by capiliary GC (DB-1, 320°, H2).

Physical Data (Compound 8a; repurified by HPLC to 98.8% purity): [a]25 - +74.3° +/- 2.0° (c - 0.99, CHC13)? m.p. - 188-190° (needles, acetone); Rf - 0.80 (toluene); NMR (300 MHz, CDC1,): 8.05 (d, J -7.2 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 5.72 (dd, J - 57.5 Hz, 56.0 Hz, 1H, 32-CHF2), 4.76 (dd, J - 11.1 Hz, 7.5 Hz, 1H, 3-CHOR), 2.2-0.85 (m, 26H), 1.09 (s, 3H, CH,), 1.06 (s, 3H, CH,), 0.97 (s, 3H, 30-CH3 ) , 0.90 (d, J - 6.3 Hz, 3H, 21—CH,) , 0.87 (d, J - 6.8 Hz, 5h, 26-CH, and 27-CH,), 0.74 (s, 3H, 18-CH,); 19F-NMR (188.2 MHz, CDCl,): -115.97 (dd, J - 277.1 Hz, 57.5 Hz, IF 32-CHFj), -122.35 (dd, J - 277.1 Hz, 56.0 Hz, IF, 32-CHF2); IR (KBr wafer, cm"1): 2950 (s, CH sat), 2860 (s, CH sat), 1710 (s, C-O), 1600(w), 1280(vs), 1115(s), 1020(s) ; MS (EI): 568 (1%, M+), 528 (4%, M -2HF), 517 (20%, M —CHFj ) , 395 (47*, M -CHF2 , -CsH5C02H), 105 (100%, C4 HjCO+); HRMS for C37H5402F2 (M+): calculated 568.4092, found 568.4079.

Physical Data (Compound 8b; 48% yield from Compound 7b; repurified by HPLC, 97% pure): [a]25 . +47.5" +/" 2.0° (c - 1.02, CHCl,); m.p. - 207.5-209.5° (acetone); Rf - 0.80 (toluene); NMR (300 MHz, CDCl,): 8.05 (d, J - 7.8 Hz, 2H, 1% 23 1 6 04 phenyl), 7.60-7.40 (m, 3H, phenyl), 5.98 (t, J - 56.3 Hz, 1H, 32-CHFj), 5.39(m, 1H, 7-CH), 4.77 (dd, J - 11.0 Hz, 3.8 Hz, 1H, 3-CHOR), 2.15-0.85 (m, 25H), 1.14 (s, 3H, 31-CH3), 0.97(s, 3H, CH3), 0.96(s, 3H, CH,), 0.93 (d, J - 6.3 Hz, 3H, 21-CHj), 0.88 (d, J - 6.6 Hz, 6H, 26-CH, and 27-CH.,), 0.75 (s, 3H, 18-CH,); 19F-NMR (188.2 MHz, CDCl, ): -118.15 (dd, J - 277 Hz, 56 Hz, IF, 32-CHF2), -123.26 (dd, J - 277 Hz, 56 Hz, IF, 3 2-CHF2 ); IR (KBr wafer, cm'1 ): 2940 (s, CH sat), 2880 (s), 2860 (s, CH sat), 1708 (vs, C-O), 1600(w), 1280(vs), 1115 (s), 1045(s); MS (EI): 517 (5%, M -CHF2 ) , "395 (20%, M -CHF2 , -cf Hs COj H); HRMS for C,6H5,02 (M -CHFj): calculated 517.4045 found 517.4024.

G. Preparation of 32,32-difluoro-lanost-8-cn-30-ol 20 (Compound 9a) Lithium aluminum hydride (28 mg, 738 //mol, obtained from Alfa Products) was added, at room temperature, to 30-benzoyloxy-32,32-difluoro-lanost-8-ene (Compound 8a) (141 mg, 248 pmol) which had been dissolved in anhydrous (distilled from sodium and benzophenone) diethyl ether (20 mL). The resulting mixture was stirred for 20 min. The cooled (0°) reaction was quenched with the careful addition of ice water then a saturated solution of ammonium chloride. After separation, the aqueous layer was extracted with dichloromethane (2 x 100 mL) and the combined 'organic extracts were pooled, dried over anhydrous magnesium sulfate and the solvent removed by evaporation under reduced pressure. The resulting mixture of residues was subjected to MPLC (45 psi, 50 cm x 1.8 cm), being eluted with 4% ethyl acetate in toluene, and collected to provide a sample for final purification by HPLC (60 psi, 60 cm x 2.5 cm). For r- 23 1 6 04' HPLC, the eluting solvent was hexane:toluene:ethyl acetate (74:21:5). HPLC afforded 99 mg of Compound 9a in 86% yield.

Physical Data (Compound 9a): laj2 5 - +68° +/- 29 (c - 1.00, CHCl,)? m.p.- 119.5-120.5° (powder); Rf - 0.3 (10% ethyl acetate in toluene); NMR (300 MHz, CDCl,): 5.70 (dd, J - 55.8 Hz, 57.5 Hz, 1H, 32-CHF3), 3.26 (m, 1H, 3-CHOH), 2.15-0.85 (m, 26H), 1.02 (s, 3H, CH,), 1.01 (s, 3H, CH, ), 0.90 (d, J - 6.6 Hz, 3H, 21-CH,), 0.87 (d, J - 7.2 Hz, 6H, 26-CH, and 27-CH, ), 0.83 (s, 3H, 30-CH,-), 0.73 (s, 3H, 18-CH,). 19F-NMR (188.2 MHz, CDCl,): -116.06 (dd, J - 276.7 Hz, 57.5 Hz, IF, 32-CHFj), -122.26 (dd, J - 276.7 Hz, 55.8 Hz, IF, 32-CHFj); IR (CHCl, solution, cm"1): 3600 (m, OH), 3460 (wb, OH), 2950 (s, CH sat), 2860 (s, CH sat), 1470(s), 1375(s), 1105(s), 1090(s), 1015(s) , 910(s ); MS (EI): 464 (10%, M+), 447 (4%, M -OH), 431' (12%, M -CH,, -H20), 413 (1001, M -CHFj), 395 (46%, M -H20, ' -CHF2 ) ; HRMS for CJ0HJ0OF2 (M+): calculated 464.3830, found 25 464.3794; EA for C,0H50OF2: calculated, C 77.54%, H 10.85%, F 8.18%; found, C 77.38%, H 10.86%,, F 7.87%.

Physical Data (Compound 9b; 86% yield from Compound 8b): 30 [o]JS - +17.7* +/- 3.2° (c - 0.62, CHCl,); m.p.- 138-139° (powder); Rf - 0.30 (10% ethyl acetate in toluene); NMR (300 MHz, CDCl,): 5.96 (t, J - 56.4 Hz, 1H, 32-CHF2), 5.38 (m, 1H, 7-CH), 3.26 (dd, J - 10.2 Hz, 5.1 35 Hz, 1H, 3-CHOH), 2.2-0.85 (m, 26H), 1.00 (s, 3H, 31-CH,), 0.92 (d, J - 6.5 Hz, 3H, 21-CH,), 0.90 (s, 3H, CH,), 0.89 (d, J - 6.5 Hz, 6H, 26-CH, and 27-CH,), 0.86 a* 23160 (s, 3H, CHj ), 0.73 (s, 3H, 18-CHj); 19F-NMR (188.2 MHz, CDClj ) : -118.02 (dd, J - 277 Hz, 56 Hz, IF, 32-CHF3), -123.23 (dd, J - 277 Hz, 56 Hz, IF, 32-CHF2 ); IR (CHClj solution, cm"1): 3620 (m, OH), 3450 (bw, OH), 2960 (s, CH sat), 2940 (s, CH sat), 2870 (s, CH sat), 1470(m), 1380(a), 1365(a), 1095(a), 1050U), 665(a); HRMS for Cj 0 H5 0 OFj (M+): calculated 464 .3830, found 464.3792; MS (EI): 464 (5%, M+), 431 (10%, M -H20, -CHj), 413 (100%, M —CHFj), 395 (60%, M -H20, -CHF2 ) ; EA for C30H50OF2: calculated, C 77.54%, H 10.85%, F 8.18%; found, C 77.19%, H 10.70%, F 8.13%.

H. Preparation of both diastereoaers of 4,4-dimethyl-14a-( 1 '-hydroxy-2 '-propenyl )-5a-cholest-8-en-3j3-ol (Compounds 11a and 12a) A 1.6 M solution of vinyl aagnesium bromide in tetrahydrofuran (1.0 aL, 1.60 mmol) was added to a solution of lanost-8-en-32-al-30-ol (prepared as described by Shafiee et al., J. Lipid Res., 27:1-10 (1986)) (Compound 10a) (74.5 ag, 168 //mol) in dry (distilled from sodium and benzophenone) tetrahydrofuran (5 mL) at room temperature. After stirring for 120 ain at room temperature, saturated ammonium chloride solution (10 mL) was added and the resulting reaction mixture was extracted with diethyl ether (2 x 50 mL). The combined organic fractions were washed with water (1 x 50 aL) dried over anhydrous magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was subjected to MPLC (80 psi, 50 cm x 1.2 cm) being eluted with 5% ethyl acetate in toluene to afford 52.1 mg of Compound 11a (66% yield) and 16.4 mg of Compound 12a (21% yield). .... v m 2 3 16 0 4 Physical Data (Compound 11a): la]25 - +54.7* +/- 2.0° (c - 1.05, CHCl,); m.p. « 155-157® (acetone, prisms); Rf - 0.6 (50% ethyl acetate in toluene); NMR (300 MHz, CDCl,): 6.04-5.92 (m, 1H, olefinic CH), .22-5.05 (m, 2H, olefinic CH2 ), 4.11 (m, 1H, 32-CHOH), 3.22 (m, 1H, 3-CHOH), 2.2-0.85 (m, 28H), 1.01 (s, 3H, CH3), 1.00 (s, 3H, CHj), 0.90 (d, J - 6.5 Hz, 3H, 21-CHj ), 0.87 (d, J - 6.8 Hz, 6H, 26-CHj and 27-CH3 ), 0.82 (s, 3H, 30-CHj), 0.71 (s, 3H, 18-CH3); IR (CHClj solution, cm"1 ): 3600 (m, OH), 3470 (bw, OH), 2950 (s, CH sat), 2870 (s, CH sat), 1465 (m), 1375 (a), 1115(B), 990(a), 920(m); MS (EI): 452 (1%, M -HaO), 437 (1%, M -HjO, -CHj), 413 (100%, M -CjH^OH), 395 (45%, M -CjH^OH, -C4H5COOH); HRMS for CJ2HS20 (M -Hj 0): calculated 452.4018, found 452.3999; EA for Cj2H5402: calculated, C 81.64%, H 11.56%; found, C 81.63%, H 11.46%, Physical Data (Compound 12a): [a]25 - +49.2° +/- 4.8° (c - 0.42, CHCl,); m.p. - 133-135° (powder); Rf - 0.5 (50% ethyl acetate in toluene); NMR (300 MHz, CDClj): 5.93-5.78 (m, 1H, olefinic CH), 5.16-5.01 (m, 2H, olefinic CH2), 4.10 (m, 1H, 32-CHOH), 3.25 (dd, J - 11.3 Hz, 4.7 Hz, 1H, 3-CHOH), 2.32 .(a, 1H), 2.18-0.85 (m, 27H), 1.07 (s, 3H, CH3), 1.02 (s, 3H, CH,), 0.92 (d, J - 6.3 Hz, 3H, 21-CH3), 0.88 (d, J -6.6 Hz, 6-H, 26-CH3 and 27-CH3 ) , 0.84 (s, 3H, 31-CH3), 0.74 (s, 3H, 18-CHj); IR (KBr wafer, cm-1): 3470 (broad, OH), 2950 (s, CH sat), 2930 (s, CH sat), 2865 (s, CH sat), 2855 (s, CH 35 sat), 1470(a), 1375(a), 920(m); MS (EI): 408 ( 30%, M -2HzO, -CHj ) , 395 ( 100%, M -C3H4OH, -H O) ; S3 23 16 04 HRMS for CJ0H47 (M -2h20, -CHj): calculated 400.3756, found 408.3715; EA for Cj 2H5 4 Oj: calculated, C 81.64%, H 11.56%; found, C 81.73%, H 11.60%.

I. Preparation of 30-benzoyloxy-lanost-7-en-32-aldoxiae (Compound 14b) Hydroxylamine hydrochloride (250 mg, 3.60 mmol) was added to 30-benzoyloxy-lanost-7-en-32-al (Compound 7b) (98 mg, 179 //mol) in anhydrous pyridine (10 mL). The mixture was stirred for 16 hr, diluted with diethyl ether (30 mL), and washed with aqueous hydrochloric acid IN (2 x 50 mL). The aqueous layer was reextracted with diethyl ether (30 mL) and the combined organic layers were washed with 10% cupric sulfate (3 x 30 mL), water (30 mL) and brine (30 mL), dried over anhydrous magnesium sulfate and evaporated under reduced pressure, giving 105 mg of Compound 14b.

Physical Data (Compound 14b): [a]2 5 - +30 . 4 8 +/- 2° (c - 1.02, CHClj); m.p.- 200-202° (acetone, needles); Rf - 0.5 (toluene); NMR (300 MHz, CDClj): 8.05 (d, J - 7.8 Hz, 2H, phenyl), 7.78 (s, 1H, 32-CH-NOH), 7.60-7.40 (m, 3H, phenyl), 7.18 (bs, 1H, 32-CH-NOH), 5.42 (m, 1H, 7-CH), 4.77 (dd, J - II.0 Hz, 4.1 Hz, IB, 3-chor), 2.2-0.85 (m, 25H),V1.14 (s, 3H, 31-CHj), 0.97 (s, 3H, CH,), 0.95 (s, 3H, CH3), 0^90 (d, J - 6.6 Hz, 3H, 21-CH3), 0.88 (d, J - 6.6 Hz, 6H, 26-CHj and 27-CH,), 0.74 (s, 3H, 18-CH3); IR (CHCl, solution, cm-1): 3570 (m, OH), 3280 (bm, OH), 2950 (s, CH sat), 2860 (s, CH sat), 1710 (s, C-O), 1600 (m), 1580 (m), 1470 (s), 1450 (s), 1315(s), 1280(vs), 1115(s), 970(s ) ; HRMS for C,, H, ,O, (M -H,0, —HCN): calculated 516.3967, ■ i " 4 O 3 I Z « found 516.3932; ^Si^f?^ri;.i: s4, 2 3 1 j*? MS (EI): 516 (10%, » -H30, -HCN), 403 (14%, M -H20, -HCN, -C,H17 ), 379 (20%, M -H20, -HCN, -C6HsCOOH, -CH,), 105 (100%, C6HsC0+).

Physical Data (Compound 14a; 96% yield from Compound 7a) : [a]25 - -56.6° +/- 2.0°. (c - 1.06, CHCl,); m.p. - 193-1966 (ethanol, flakes); Rf - 0.5 (toluene); NMR (300 MHz, CDCl}): 8.05 (d, J - 7.8 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 7.33 (s, 1H, 32-CHNOH), 7.05 (bs, 1H, 32-CHNOH), 4.75 (dd, J - 11.6 Hz, 4.2 Hz, 1H, 3-CHOR), 2.22-0.85 (m, 26H), 1.10 (s, 3H, CH,), 1.06 (s, 3H, CH, ) , 0.95 (s, 3H, CHa), 0.89 (d, J - 6.6 Hz, 3H, 21-CH3), 0.87 (d, J - 6.6 Hz, 6H, 26-CHj and 27-CH, ), 0.77 (s, 3H, 18-CHj); IR (KBr vafer, cm"1 ): 3280 (bm, OH), 2950 (s, CH sat), 2870 (s, CH sat), 1718 (s, C-O), 1600 (w), 1580(w), 1465(m) , 1450(m), 1310(m), 1270(vs), 1110(b) ; HRMS for Cj7H54N02 (M -OH): calculated, 544.4155; found 544.4252; MS (EI): 544 (100%, M -OH), 406 (70%, M -HjO, -CHj, — cf h} COOH) , 380 (25%, M -H20, -CN, -CH, , -C£H5COOH), 379 25 (75%, M -H20, -HCN, -CH,, -CsH5COOH) 105 (71%, -CfcH5CO+).

Physical Data (Compound 14c; 70% yield from Compound 7c; reaction time - 16 hr at 80°): Ice)2 5 - -77. 9° +/-2.0° (c - 1.00, CHCl,); m.p. - 204-205° (acetone, needles); Rf - 0.5 (toluene); NMR (300 MHz, CDCl,): 8.05 (d, J - 7.5 Hz, 2H, phenyl), 7.78 (s, 1H, 32-CHNOH), 7.60-7.40 (m, 3H, phenyl), 5.68 35 (d, J - 10.2 Hz, 1H, olefinic H), 5.57 (d, J - 10.2 Hz, 1H, olefinic H), 4.75 (dd, j - 11.5 Hz, 4.8 Hz, 1H, 3-CHOR), 2.43 (m, 1H), 2.18 (m, 1H), 1.95-0.85 (m, 22H), -5a- *2* 231 (C, 10 1.03 (s, 3H, CHS),0.97 (s, 3H, CHj ), 0.92-0.88 (m, 12H, CHjS), 0.86 (s, 3H, 18-CHj); IR (KBr wafer, cm"1 ): 3280 (bra, OH), 2950' (c, CH sat), 5 2930 (s, CH sat), 2860 (s, CH sat), 1718 (b, C-O), 1600(w), 1580(w), 1465(m), 1450(m), 1310(m),'1275(ve), 1110(e); HRMS for C37H55N0j (M+): calculated 561.4182, found 561.4135; MS (EI): 561 (6%, M+) , 544 (15%, M-OH), 421 (201, H -H20, -C6H5COOH), 406 (33%, M-H30, -CsHsCOOH, -CH, ) .

J. Preparation of lanost-7-en-32-aldoxime-33-ol (Compound 15b) Potassium hydroxide (6.5 g) in ethanol (48 mL) and water (3 mL) was added to a solution of 3f5-benzoyloxy-lanost-7-en-32-aldoxime (Compound 14b) (218 mg, 389 j/mol) in ethanol (50 mL) which had been warmed to 50*. The mixture was stirred at 50° for 2 hr, then cooled and quenched with water (200 mL). The reaction mixture was extracted with dichloromethane (3 x 100 mL) and the combined organic extracts were dried over anhydrous magnesium sulfate, filtered and subjected to evaporation under reduced pressure. The resulting residue was subjected to MPLC (50 psi, 50 cm x 1.8 cm), being eluted with 10% ethyl acetate in toluene, giving 168 mg (94%) of Compound 15b.

Physical Data (Compound 15b): ( 30 [a]25 - -2.0° +/- 2.0° (c - 1.00, CHClj); m.p.- 212.5-214.5° (ethanol-water); Rf - 0.55 (50% ethyl acetate in toluene); NMR ( 300 MHz, CDClj ) : 7.77 (s, 1H, 32-CH-NOH), 7.40 (bs, 1H, 32-CH-NOH), 5.41 (m, 1H, 7-CH), 3.26 (dd, J - 10.7 35 Hz, 5.0 Hz, 1H, 3-CHOH), 2.10-0.85 (m, 26H), 0.99 (s, 3H, CHj), 0.90-0.86 (m, 15H, rest of CHj), 0.72 (s, 3H, 18-CHj ) ; * 15 56 23 1 IR (CHClj solution, csf1): 3560 (m, OH), 3300 (bm, OH), 2950 (s, CH sat), 2860 (s, CH sat), 1470(e), 1380(s); HRMS for Cj 0 Hs 0 NO (M -OH): calculated 440.3892, found 440.3877; MS (EI): 440 (100%, M -OH), 412 (52%, M -H20, -HCN), 406 (33%, M —2H2O, -CHj), 397 ( 49%, M -HjO, -HCN, -CH,), 379 (34%, M-2HaO, -HCN, -CHj ); EA for CjoH51N02: calculated C 78.72%, H 11.23%, N 3.06%; found, C 78.50%, H 11.29%, N 2.95%.

Physical Data (Compound 15a; yield >95% from Compound 14a): [a]25 - —87.2* +/- 2.0° (c - 1.09,.CHCl,); m.p. - 226-229® (acetone, needles); Rf - 0.55 (50% ethyl acetate in toluene); NMR (300 MHz, CDClj): 7.30 (s, 1H, 32-CH-NOH), 7.18 (bs, 1H, 32-CH-NOH), 3.25 (dd, J - 11.1 Hz, 4.3 Hz, 1H, 3-CHOH), 2.20-0.85 (m, 27H), 1.02 (s, 3H, CH,), 0.99 (s, 3H, CHj), 0.88 (d, J - 6.6 Hz, 3H, 21-CHj), 0.87 (d, J - 6.6 Hz, 6H, 26-CHj and 27-CH,), 0.82 (s, 3H,'CHS), 0.76 (s, 3H, 18-CHj ); IR (CHClj solution, cm"1): 3580 (m, OH,), 3340 (bw, OH), 2950 (s, CH sat), 2930 (s, CH sat), 2870 (s, CH sat), 1465(m) , 1455(a), 1375(b); HRMS for CJ0H5eN0 (M -OH): calculated, 440.3892; found 440.3885; MS (EI): 440 (65%, M -OH), 412 (28%, M -H20, —HCN)., 397 (22%, M -H20, -HCN, -CHj), 394 (31%, M -2HaO, -HCN), 380 (29% M -2H2 O, -CN, -CHj), 379 ( 94%, M -2H20, -HCN, —CHj ) ; EA for CJOH51N02: calculated, C 78.72%, H 11.23%, N 3.06%; found, C 78.90%, H 11.32%, N 2.87%.

Physical Data (Compound 15c; yield 89% from Compound 14c): [a]25 - -109.1® +/- 2.0° (c - 0.92, CHClj); ;(/• ■A © ^ 231 m m.p. - 198-200° (powder); Rf - 0.55 (50% ethyl acetate in toluene); NMR (300 MHz, CDClj): 7.76 (s, 1H, 32-CH-NOH), 5.67 (d, j - 10.1 Hz, 1H, olefinic H), 5.54 (d, J - 10.1 Hz, lH, olefinic H), 3.25 (dd, J - 11.3 Hz, 4.8 Hz, 1H, 3-CHOH), 2.39 (m, 1H), 2.17 (m, 1H), 1.96-0.85 (m, 23H), 1.02 (s, 3H, CH, ) , 0.91 (s, 3H,.CH3 ), 0. 89-0.85 (m/,12H, CHjS), 0.79 (s, 3H, 18-CHj); O 10 2B (CHC13 solution, cm"1): 3580 (m, OH), 3360 (bw, OH), 2950 (s, CH sat), 2930 (s, CH sat), 2865 (b, CH sat), 1465(m), 1380(m), 1365(m); HRMS for Cj0HjeNO (M -OH): calculated 440.3892, found 440.3895; MS (ei): 440 (6%, M -oh), 406 (7%, M -2hao, -chj), 379 (6%, M -2h20, -hcn, -ch, ) ; EA for C,eH5lN0a: calculated, C 78.72%, H 11.23%, N 3.06%; found, C 78.69%, H 10.88%, N 2.82%.

K. Preparation of 33-benzoyloxy-4,4-dimethyl-5a,143-cholest-8-en-15-one (Compound 40a) To a solution of 30-benzoyloxy-4,4-dimethyl-5a-cholest-8,14-diene (5.0 g, 10.2 mmol) in diethyl ether solution (300mL) and 0.5 M aqueous sodium bicarbonate (300 mL) was added meta-chloro-perbenzoic acid (2.44 g, 11.3 mmol) (Aldrich). After 30 min., the ether fraction was separated from the sodium bicarbonate layer and the aqueous fraction was repeatedly extracted with diethyl ether (5 x 500 mL). The organic fractions were combined, washed with brine (1 x 100 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to about 150 ml. This ether solution of 38a was promptly cooled (0°) and treated dropwise with boron trifluoride etherate (26 mL, 22.6 mmol) (Aldrich).

After 30 min, the reaction was quenched with ice water (200 mL) and the aqueous phase was reextracted with ether (3 x lOOmL). The combined either fractions were ~5. /f ^ 231 extracted with saturated sodium bicarbonate solution (1 x 200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was ^ crystallized from isopropanol (75 mL) to afford 4.10 g of 3 0-benzoyloxy-4 , 4-dime thy 1-5a, 14|3-cholest-8-en-15-one (Compound 40a) in 75% yield.

Physical Data (Compound 40a) [a]25 - -21.9° +/- 5.0° (C - 0.40, CHClj) m.p. - 154-155° (isopropanol) Rf - 0.75 (5% ethyl acetate in hexanes) NMR (300 MHz, CDCl,): 8.05 (d, J -7.2 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 4.80 (dd, J - 11.1 Hz, 4.5 Hz, 1H, 3-CHOR), 2.60-0.85 (m,25H), 1.08 (6, 3H, 31—CHj), 1.07 (s, 3H, 19-CHS), 1.02 (d, J - 6.3 Hz, 3H, 21-CHj), 1.00 (s, 3H, 30-CHj), 0.97 (s, 3H, 18-CHj), 0.89 (d, J - 6.6Hz, 6H, 26-CHj and 27-CH3).

HRMS for Cj s H$2Oj (M+): calculated 532.3917, found 532.3923. 2 5 L. Preparation of 30-benzoyloxy-lanost-8-en-15-one (Compound 29a) To a 0.5 M solution of potassium tertiary butoxide in tertiary butanol (72 mL, 37.6 mmol) was added 3 0-benzoyloxy-4,4-dimethyl-5a,143-cholest-8-en-l5-one (Compound 40a) (2.0 g, 3.76 mmol) in anhydrous tetahydrofuran (5.0 mL) rapidly followed by the addition of methyl iodide (2.24mL, 35.8 mmol). After 15 min. the reactions mixture was poured into ice cold water (50 mL), extracted with toluene: ethyl acetate (1:1) (4 X 200 mL), &nd the combined organics were washed with saturated sodium chloride (100 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The resultant residue was crystallized from isopropanol (75 mL) to provide 1.40 g of pure 3 |3-benzoyloxy-lanost-8-en-15-one (Compound 29a) in 68% yield. % 2 316 0 4 Physical Data (Compound 29a): m.p. - 199-200® (ethyl acetate-methanol) Rf - 0.77 (5* ethyl acetate in hexane); NMR (300 MHz, CDCl,): B.05 (m, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 4.75 (dd, J - 11.1 Hz, 4.5 Hz, 1H 3-CHOR), 2.80-0.85 (m,24H), 1.12 (s, 3H, 32-CH, ), 1.06 (s, 6H, 19-CH, and 31-CH,), 0.98 (d, J - 6.3 Hz, 3H, 21-CHj), 0.97 (s, 3H, 30-CHj), 0.87 (d, J - 6.6Hz, 6H, 26-CH, and 27-CH,), 0.78 (s, 3H, 18-CH,); HRMS for C,7H54O, (M+): calculated 546. 4074, found 546.4137.

LI. Preparation of 33-benxoyloxy-32-benxyloxy-lanost-8-15 en-15 one (Compound 47a).

The same procedure as used for the preparation of Compound 29a was employed with the following exceptions: Compound 40a was added in benzene rather than tetrahydrofuran; benzyl chloromethyl ether prepared by the method of Connor (D. S. Connor, G. W. Klein, G. N. Taylor, Org. Syn. ^2: 16-19 (1972)) was utilized instead of methyl iodide; and resultant reaction mixture was chromatographed (hexane:ethylacetale, 98:2) instead of crystallized directly. Compound 47a, in a 64* yield, resulted.

Physical Data (Compound 47a): [a]25- +84.9+2.0°(c-1.02, CHCl,); m.p.- 49-50®(methanol); Rf- 0.37 (5% ethyl acetate in hexane); NMR (300 MHz, CDCl,): 8.05 (d, J-7.2HZ, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 7.40-7.20 (m, 5H, phenyl), 4.76 (dd, J-11.3HZ, 4.4Hz, 1H, 3-CHOR), 4.48 (d, J-12.3HZ, 1H, OCHjPh), 4.40 (d, J-12.3HZ, 1H, OCHjPh), 35 3.69 (d, J-9.3HZ, 1H, 32-CH2OR), 3.40 (d, J-9.3HZ, 1H, 32-CH2 OR), 2.70-2.45 (m, 2H, 16-CHa), 2.15-0.85 (M, 22H), 1.05 (S, 6H, 19-CH, AND 31-CH,), 0.98 (S, 3H, •-Oj* /-N I 23 1 6 04 ^nc -CHj), 0.96 (D, J-6.3HZ, 3H, 21-CHj) , 0.84 (d, J-6.6Hz, 6H, 26-CHj and 27-CHj), 0.77 (s, 3H, 18-CHj); IR (CHClj solution, cm-1): 2950 (s, CH sat), 2883 (s, CH sat), 1739 (s, C-O), 1716, (s, C-O), 1452(s), 1274(b), 1113(s); MS (El): 652 (9%, M+) , 409 (79%, M-PhCOOH, -CHjOCHjPh), 105 (100%, CsH,CO+).

HRMS for C44H<0O4 (M+): calculated 652.4493, found 652.4549.

M. Preparation of 30-benzoyloxy-14a-cyano-4,4-dimethyl-5a-cholest-8-ene (Compound 16a) Phenyl isocyanate (35 pL, 322 //mol) vas added to 30-benzoyloxy-lanost-8-en-32-aldoxime (Compound 14a) (72.5 mg, 129 /mol) and triethylamine (5 drops) in azeotropically dried benzene (8 mL) at room temperature. The mixture was stirred at reflux for 1.5 hr. The cooled reaction mixture (room temperature) was quenched with water (40 mL) and extracted with dichloromethane (3 x 100 mL). The combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and the solvent evaporated under reduced pressure. The residue was subjected to MPLC (70 psi, 50 cm x 1.8 cm), using toluene as the eluting solvent, to afford 59.7 mg (85%) of Compound 16a.

Physical Data (Compound 16a): [a]25 - +15.0° +/- 2.0® (c - 1.04, CHClj); m.p. - 211.5-213.5° (acetone, very fine needles); Rf - 0.6 (toluene); NMR (300 MHz, CDClj): 8.06 (d, J - 7.2 Hz, 2H, phenyl), 7.60-7.40 (a, 3H, phenyl), 4.76 (dd, J - 11.1 Hz, 4.5 35 Hz, 1H, 3-CHOR), 2.35-0.85 (a, 26H), 1.08 (s, 3H, CHj), 1.07 (S, 3H, CHj ) , 0.99 (s, 3H, CHj ), 0.96 (d, J - 6.3 Hz, 3H, 21-CHj ), 0.88 (d, J - 6.6 Hz, 6H, 26-CHj and r-:.

L- 23 £i 27-CH,), 0.74 (s/ 3H, 18-CHj); IR (KBr wafer, cm"1): 2950 (s, CH sat), 2865 (s, CH sat), 2220 (w, CN), 1715 (s, C-O), 1465(a), 1450(a), 1275(vs), 1115(m); HRMS (isobutane CI) for CJ7H5JN02 (M-t-): calculated 543.4076, found 543.4055; MS (CI): 543 (3%, M+), .517 (20%, M -CN), 395 (33%, M -CN, -CjHJCOOH).

N. Preparation of 14a-cyano-4,4-dimethyl-5a-cholest-8-en-33-ol (Compound 17a) 3p-benzoyloxy-14 a-cyano-4,4-dimethyl-5a-cholest-8-ene (Compound 16a) (45.3.mg, 83.4 fjmol) was dissolved in 1 N ethanolic potassium hydroxide (30 mL) containing 5% water at room temperature. The mixture was stirred for 30 min. at 50®, poured into ice water (200 mL) and extracted with dichloromethane (3 x 200 mL). The combined extracts were dried over anhydrous magnesium sulfate and solvent removed by evaporation under reduced pressure. The residue was subjected to MPLC (80 psi, 50 cm x 1.2 cm), using 8% ethyl acetate in toluene as the eluting solvent, to afford 33.0 mg (90%) of Compound 17a.

Physical Data (Compound 17a): [a]25 - -9.5® +/" 2.0° (c - 1.08, CHCl,); m.p. - 167.5-169° (powder); Rf - 0.6 (50% ethyl acetate in toluene); NMR (300 MHz, CDClj): 3.27 (dd, J - 11.3 Hz, 4.4 Hz, 1H, 3-CHOH), 2.3-0.85 (m, 26H), 1.02 (s, 3H, CH,), 1.01 (s, 3H, CH,), 0.95 (d, J - 6.3 Hz, 3H, 21-CH,), 0.88 (d, J -6.6 Hz, 6H, 26-CH, and 27-CH,), 0.83 (s, 3H, 30-CHj), 0.73 (s, 3H, 18-CHj); IR (CHCl, solution, cm"1): 3610 (m, OH), 3840 (bw, OH), 2960 (s, CH sat), 2940 (s, CH sat), 2870 (s, CH sat), 2220 (w, CN), 1465(m), 1380(m); .-65- * 2 3 1 MS (EI): 439 (14*, M+), 424 (14%, M -CH, ) , 406 (100%, M -CHj, -HaO), 397 (44%, M -HCN, -CHj ) ; HRMS (EI) for CJ0H4JNO (M+): calculated 439.3814, found 5 439.3852; EA for Cj0H4>NO: calculated, C 81.94%, H 11.23%, N 3.19%; found, C 82.15%, H 11.28%, N 3.15%.

Nl. Preparation of 30-acetoxy-lanost-8-en-32-al 10 (Compound 4 3a) Acetic anhydride (2 mL) was added to a solution of lanost 8-en-32-al-3fJ-ol (prepared as described by Shafiee et al., J. Lipid Res., 2J7: 1-10 (1986)) (Compound 10a) (208 mg, 471 -fjmol) in anhydrous pyridine 1 ^ (4 mL) at room temperature. After stirring for 17 hours at room temperature the mixture was poured into water (25 mL) and extracted with diethyl ether (3 X 40 mL). The combined organic fractions were washed with IN hydrochloric acid (50 mL), 10% aqueous cupric sulfate (50 mL), water (50 mL) and brine (50 mL), dried over ~T anhydrous magnesium sulfate and evaporated under reduced pressure giving 226 mg (<95%) of Compound 43a.

Physical Data (Compound 43a): [a]25—258.7+2.0° (c-0.95, CHClj); f"''= m.p.-144-146° (methanol/acetone 1:1); r Rf-0.75 (25% ethyl acetate in toluene); NMR ( 300 MHz, CDCl,): 9..44 (s, 1H, 32-CHO), 4.47 >(dd, J-11.6, Hz, 4.4 Hz, 1H, 3-CHOR), 2.35-0.85 (m, 26H), 2^06 (s, 3H, acetate), 1.08 (s, 3H, CH,), 0.90 (s, 3H, CH,), 0.89 (d, J-6.5HZ, 3H, 21-CHj), 0.86 (s, 3H, CHj ) , 0.86 (d, H- 6.5Hz, 6H, 26-CHj and 27-CHj), 0.75 (s, 3H, 18-CHj); IR (CHCl, solution, cm-1): 2955 (s, CH sat), 2865 (s, CH sat), 1725 (s, C-O), 1700 (s, C-O), 1465 (m), 1365 (m), 1255 (s), 1025 9m); MS (El): 455 (42%, M-CHO), 395 (100%, M-CHO, -CHjCOOH) '-S 6** « <3 23 1 HRMS for C3lH5102 (M-CHO): calculated 455.3890, found 455.3900.

^ N2. Preparation of 33-acetoxy-lanost-8-en-32-aldoxime (Compound 4 4a) Hydroxylamine hydrochloride (300 mg, 4.32 mmol) was added to 3p-acetoxy-lanost-8-en-32-al (Compound 43a) (205 mg, 424 pmol) in anhydrous pyridine (5 mL). The mixture was stirred at 40" for 16 hours, poured into water (50 mL) and extracted with diethyl ether (3 X 40 mL). The combined organic layers were washed with IN hydrochloric acid, 10% aqueous cupric sulfate, water and brine (50 mL each), dried over anhydrous magnesium ^ sulfate and evaporated under reduced pressure giving 210 mg (95%) of Compound 2s.

Physical Data (Compound 44a): [a]25- -105.5+2.0° (c-1.05, CHCl,); m.p.- 185-186® (powder from evaporation of acetone); Rf- 0.6 (25% ethyl acetate in toluene ); NMR (300 MHz, CDClj): 7.30 (s, 1H, 32-CH-NOH), 4.49 (dd, J-11.6 Hz, 4.4Hz, 1H, 3-CHOR), 2.20-0.85 (m, 27H), 2.06 (s, 3H, acetate), 1.04 (s, 3H, CH3), 0.89-0.85 (m, 15H, CH,s), 0.75 (s, 3H, 18-CHj); IR (CHClj solution, cm-1): 3580 (v), 3300 (bw), 2950 (s, CH sat), 2865 (s, CH sat), 1720 (s, C-O), 1465 (m), 1375 (m), 1260 (s), 1030 (m). c 2 3 1 >s" 0. Preparation of 3g-benzoyloxy-15a-fluoro-lanost-7-ene (Compound 24b) Diethylamino sulfur trifluoride (DAST, 5.75 ^ mL, 46 mmol) was added to a solution of 30-benzoyloxy-lanost-7-en-15a-ol (Compound 22b, 1.6 g, 2.92 mmol) in dry (distilled from phosphorus pentoxide) dichloromethane (100 »L) under .argon at -78°. The reaction was stirred at -78° for 0.5 hr, then diluted with dichloromethane 1100 mL). Aqueous sodium bicarbonate was added until the aqueous phase was neutral and the mixture was extracted with toluene:ethyl acetate 1:1 (3 X 100 mL). The combined organic solutions were dried over anhydrous magnesium sulfate, filtered, and solvents removed in vacuo to give 1.7 g of a complex mixture.

Purification by chromatography on silica gel (eluant 5% ethyl acetate in hexane), followed by HPLC on silica gel (eluant 25% toluene in hexane) yielded 294 mg (18% yield) of 30-benzoyloxy-15a-fluoro-lanost-7-ene (Compound 24b).

Physical Data (Compound 24b): [al25 - +45.9° +/- 2-0° (c - 1.06, CHCl,); m.p. - 226-227° (powder); Rf - 0.50 (5% ethyl acetate in hexane); 0.15 (25% toluene in hexane); NMR (300 MHz, CDC13): 8.06 (d, J - 7.4 Hz, 2H phenyl), 7.55 (m, 1H, phenyl), 7.45 (m, 2H, phenyl), 5.51 Jm, 1H, 7-CH), 5.05 (ddd, J - 56.6, 9.2, 5.6 Hz, 1H, 15-CHF), 30 4^78 (dd, J - 9.4, 5.6 Hz, 1H, 3-CH), 2.18-1.27 (m, 23H), 1.15 (s, 3H, 31-CHj), 1.10 (d, J - 3.6 Hz, 3H, 32-CHj), 0.95 (s, 3H, CHj), 0.94 (s, 3H, CH,), 0.87 (unresolved d, 9H, 21, 26, 27-CH3), 0.70 (s, 3H, 18-CHj)? IR (KBr, cm"1): 2960 (m), 2930 (m, CH sat), 1705 (s, C-O), 1460 (m), 1450 (m), 1390 (m), 1380 (m), 1365 (m), 1280 (s, C-O), 1120 (m, C-F), 1035 (m), 1025 (m); ■h-'. >'t e £ 23 16 MS (EI): 550 (20%, M+), 413 (100%, M -C,H#02); HRMS for C31H5j02F (M+): calculated 550.4186, found 550.4163.

Physical Data (Compound 25b; 32% yield from Compound 23b) s [a]25 - +19.0° +/-2.0# .(c - 1.02, CHCl,); m.p. » 202-203° (powder); Rf - 0.42 (1:1, toluene:hexane) NMR (300 MHz, CDCl,): 8.05 (d, J - 7.4 Hz, 2H phenyl), C 7.55 (m, 1H, phenyl), 7.45 (m ,2H, phenyl), 5.45 (m, 1H, 7-CH), 5.05 (ddd, J - 56.8, 9.3, 5.5 Hz, 1H, 15-CHF), 4.95 (m, 1H), 2.1-1.0 (m, 25H), 1.15 (d, J - 3.5 Hz, 3H, 32-CH,), 0.90 (m, 12H, CH,), 0.75 (s, 3H, 18-CH,); 19F NMR (188.2 MHz, CDCl,): -193.2 (dd, 56.6, 27.8 Hz); MS (EI): 522 (10%, M+), 385 (95%, M -C,H,02); HRMS for C,5H5102F (M+): calculated 522.3873, found 522.3873; IR (K3r, cm"1): 2960 (m), 2930 (m, CH sat), 1710 (s, C-O), 1460 (m), 1450 (m), 1390 (m), 1380 (m), 1365 (m) , 1275 (s, C-O), 1120 (m, C-F), 1035 (n), 1025 (m) .

P. Preparation of 15a-fluoro-lanost-7-en-30-ol 25 (Compound 26b) Lithium aluminum hydride (290 mg, 7.6 mmol) was added slowly to a solution of 30-benzoyloxy-15a-fluoro-lanost-7-ene (Compound 24b, 294 mg, 0.534 mmol) in diethyl ether (40 mL) and tetrahydrofuran (10 aL) at 0° under nitrogen. The reaction was stirred for 10 min., then quenched with sodium sulfate decahydrate (1 g) addition. The mixture was diluted by the addition of diethyl ether (50 mL). Then, ethyl acetate (20 mL) was added, the solution filtered though a sintered glass funnel and the solvents removed in vacuo. The resulting solid was crystallized from isopropanol to give 230 mg (96% yield) of 15o-fluoro-lanost-7-en-3fJ-ol (Compound foLv-.J'W " .« *&* ^ 23 1 >o 26b). The crystals had a m.p.- 159-160°, and the NMR showed co-crystallization with isopropanol {2:1, 26b:isopropanol). The crystals were then dissolved in benzene and the solvent removed to give an amorphous solid.

Physical Data (Compound 26b): [a]2 5 - +24.6° +/- 4.0° (c - 0.5, CHClj); m.p. - 172.5-173° (powder); fr H Rf - 0.18 (1:2:7, ethyl acetate:toluene:hexane); NMR (300 MHz, CDCl,): 5.50 (m, 1H, 7-CH), 5.05 (ddd, J -56.6, 9.2, 5.6 Hz, 1H, 150-CHF), 3.27 (dd, J - 10.6, 4.9 Hz, 1H, 3—CH), 2.18-1.27 (m/ 23H), 1.10 (d, J - 3.6 Hz, 32-CHj), 1.01 (s, 3H, CHj), 0.92-0.87 (m, 12H, CH3s), 0.71 Is, 3H, 18-CHj); 19f NMR (188.2 MHz, CDClj): -192.9 (ddq, J - 56.6, 27.5, 3.6 15-CHF); IR (CHClj, eta"1 ): 3620 (w, OH), 2960 (s), 2935 (s), 2870 (m, CH sat), 1467 (m), 1448 (m), 1384 (m), 1367 (m), 1090 (w, CF), 1028 (m), 995 (m), 670 (m); MS (EI): 446 (65%, M+), 431 (40%, M -CH,), 413 (50%, M -CH, , -H;0), 306 (100%, M-C,H1S0); HRMS for CJ0H51OF (M+): calculated 446.3924, found 446.3917; C: EA for CJ0H51OF: calculated, C 80.66%, H 11.51%, F W 4.25%; found, C 80.83%, H 11.29%, F 4.11%.

Physical Data (Compound 27b from Compound 25b; 93% yield) : [a]25 - +15.3° +/- 2.4° (c - 0.85 CHClj); m.p. - 68-69° Rf - 0.11 (1:2:7; ethyl acetate:toluene:hexane); NMR (300 MHz, CDClj): 5.45 (m, 1H, 7-CH), 5.05 (ddd, J ■ 56.8, 9.3, 5.5 Hz, 1H, 15-CHF), 3.62 (m, 1H, 3-CH), 2.1-1.0 (m, 25H), 1.15 (d, J-3.5 Hz, 3H, 32-CHj), 0.88 (m, 9H, CHjS), 0.83 (s, 3H, CH,), 0.73 (s, 3H, 18-CHj); ■10~ <T7 2 3 1 6 0 4 19r NMR (188.2 MHz, CHCl,): -193.2 (dd, 56.7, 27.8 Hz); MS (EI): 418 (100%, M), 403 (90%, M -CH,); HRMS for CJgH41F: calculated 418.3611, found 418.3592? IR (CHCl,, cm"1 ): 2960 (s), 2935 (s, CH sat), 1465 (m) , 1445 (m), 1385 (m), 1365 (m), 1115 (w), 1035 (m), 1025 (m), 1000 (w).

Q. Preparation of 30-hydroxy-lanost-7-en-15-oxime (Compound 31b) -hydroxy-lanost-7-en-15-one (120 mg, 0.27 mmol) was dissolved in dry (distilled from potassium hydroxide) pyridine (3 mL) under argon. Hydroxylamine hydrochloride (Aldrich) (200 mg, 2.92 mmol) was added. The solution was heated at 80° for 18 hr. After the solution had cooled to room temperature, it was diluted with ethyl acetate arid washed, in order, with water (1 X 50 mL), 10% hydrochloric acid (1 X 50 mL), water (1 X 50 mL), aqueous sodium bicarbonate (1 X 50 mL), and aqueous sodium chloride (1 X 50 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered. The solvents were removed in vacuo. Purification of the residues by means of silica gel chromatography (eluant, 15% ethyliacetate in hexanes) gave 106 mg (85% yield) of 33-hydroxy-lanost-7-en-15-oxime (Compound 31b).

Physical Data (Compound 31b): la]25 - +46.0° +/- 2.3° (c - 0.86, CHCl,); m.p. - 185-186° (powder); Rf - 0.26 (20% ethyl acetate in toluene); NMR ( 300 'MHz, CDCl,): 6.85 (m, 1H( 7-CH), 6.44 (s, 1H), 3.26 (dd, J - 12, 5 Hz, 1H, 3-CH), 2.76 (dd, J - 18, 9 Hz, 1H), 2.28 (dd, J - 20, 9 Hz, 1H) 2.1-1.1 (m, 22H), 1.21 (s, 3H, 32-CHj), 0.99 (s, 3H, 31-CH,), 0.94 (d, J - 6 Hz, 3H, 21-CH,), 0.88 (bs, 9H, CH,s), 0.86 (s, 3H, CH,), 0.70 (s, 3H, 18-CH,); IR (KBr, cm"1): 3350 (bs, OH), 2990(s), 2840(s, CH sat), *5 c L is- 23 1 6 0 4 1670 (w, C-N), 1470 (in) , 1382(s), 920(m, NO); MS (EI): 457 (5%, M+), 442 (25%, M+ -CH,), 440 (100%, M -OH) ; HRMS for CjjHj^OjN (M+): calculated 457.3920, found 57.3917; EA for C30H51OjN: calculated, C 78.72%, H 11.23%, N 3.06%; found, C 78.63%, H 10.98%, N 2.76% R. Preparation of'14a-allyl-3|3-benzoyloxy-4, 4-dimethyl-\ 5o-cholest-7-en-15-one (Compound 20b) '•l ^ , -benzoyloxy-4,4-dimethyl-5a-cholest-7-en-15-one (Compound 18) (1.13 g, 2.13 mmol) was added to a 0.5 M solution of potassium • in tert-butanol (45 mL) ^ under argon at 25°. Freshly distilled allyl bromide (200 iiL, 2.30 mmol) was added and the solution stirred for 1 hr. The reaction mixture was quenched with aqueous ammonium chloride (100 mL) and extracted with toluene (3 X 100 mL). The combined organic fractions were washed with water (1 X 100 mL) then saturated aqueous sodium chloride (1 X 100 mL), dried over anhydrous magnesium sulfate, filtered and solvents removed in vacuo. The residues were purified by means of silica.gel chromatography (eluant, 3:20:77, ethyl ^ acetate:toluenerhexane) followed by HPLC (eluant, 1.5:20:78.5, ethyl acetate:toluene:hexane). Two fractions resulted: 14«-allyl-3|3-benzoyloxy-4 ,4-dimethyl-5a-cholest-7-en-15-one (Compound 20b) 240 mg (yield 19.7%), Rf » 0.39, and 7o-allyl-3(3-benzoyloxy-4,4-dimethyl-5a-cholest-8(14)-en-l5-one, 600mg (yield 49.2%), Rf - 0.37 (1:4:15; ethyl acetate:toluene:hexane).

Physical Data (Compound 20b): NMR (300 MHz, CDCl,): 8.06 (d, 7.4 Hz, 2H, phenyl), 7.55 (m, 1H, phenyl), 7.45 (m, 2H, phenyl), 6.51 (m, 1H, 7-CH), 5.61 (m, 1H, allyl, CH), 4.98 (m, 2H, allyl, ri 2316 0 4 eq CHj), 4.77 (dd, J - 9.4, 4.6 Hz, 1H, 3-CHOR), 2.7-1.1 (m, 23H), 1.15 (s, 3H, CH,), 0.99 (m, 9H, CH,s), 0.91 (S, 3H, CH,), 0.88 (s, 3H, CH, ) , 0.82 (s, 3H, 18-CH,). 3 HRMS for C,sH560, (M+): calculated 572.4229, found 572.4214.

R1. Preparation of 14a-allyl-30-benzoyloxy-4,4-dimethyl-5a-cholest-8-en-5-one (Compound 20a) (7) The same preparative procedure as employed for v"'' 10 Compound 20b was used, with the following exceptions: Compound 40a was utilized as the starting material, instead of Compound 18; and crude product was crystallized from ethylacetate to give pure 14a-allyl-33-benzoyloxy-4,4-dimethyl-5a-cholest-8-en-5-one.

Physical Data (Compound 20a): m.p.— 172-174'C (ethyl acetate) Rf- 0.42 (5% ethyl acetate/hexanes) NMR (300 MHz, CDCl,): 8.06 (d, J-7.2 Hz, 2H, phenyl), 7.60-7.40 (m, 3H, phenyl), 7.75 (m, 1H, olefinic CH), 5.01 (m, 2H, olefinic CH2)4.75 (dd, J-12, 5Hz, 1H, 3-CHOH), 2.61 (dd, J- 20, 9, 1H), 2.5-1.1 (ax, 26H), l.J (s, 3H, CH, ) 1.05 (s, 3H, CH,), 1.00 (m, 6H), 0.9 (d, J-7Hz, 6H, 26-CH, and 27-CH,), 0.80 (s, 3H, 18-CH,).

S. Preparation of 14a-allyl-4,4-dimethyl-5a-cholest-7-en-3f3-ol (Compound 21b) Sodium (20 mg, 0.87 mmol) was added to dry (distilled from calcium hydride) diethylene glycol (3 mL) under' nitrogen. Compound 20b, 14a-allyl-30-benzoyloxy-4,4-dimethyl-5a-cholest-7-en-15-one (54 mg, 0.094 mmol) was then added followed by anhydrous hydrazine (1 mL). The solution was heated under a reflux condenser to 180® for 48 hr. The condenser was removed and the excess hydrazine was distilled off at 220°. The solution was allowed to cool to room SK.-'rtv » ^ v„ -7® 2 3 1 6 0 4 temperature then diluted with dichloromethane and water. The aqueous phase was neutralized by addition of 10% hydrochloric acid. The mixture was extracted with dichloromethane and the combined organic fractions were dried over anhydrous magnesium sulfate, filtered and the solvents removed in vacuo to give 25 mg of a complex mixture of 14a-allyl-4 , 4-dimethyl-5a-cholest-7-en-3{3-ol (Compound 21b), and 7o-allyl-4,4-dimethyl-5a-cholest-8(14)-3p-ol.

T. Preparation of 4 ,4-dimethyl-5a-cholest-8-ene-3(5,14a, 15a-triol (Compound 34a) Pyridine (6.4 mL) was added to a solution of 4,4-dimethyl-5a-cholest-8,14-dien-3p-ol (Compound 32a) (3.25 g, 7.87 mmol) in 150 mL of benzene and the mixture was cooled to 5* in an ice bath. A solution of osmium tetroxide (2.1 g, 8.26 mmol) in 42 mL of dichloromethane was added dropwise to the mixture over a period of 0.5 hr and the resulting dark brown solution stirred for 1 hr at room temperature. Hydrogen sulfide gas was then bubbled through the solution for about 15 min and the black precipitates were removed by filtration through Celite (Manville Products Corp., Denver CO). The filtercake was washed with diethyl ether three times and the solvents were evaporated from the filtrate to give a dark brown oil. The residue was dissolved in ether and dichloromethane (9:1) and passed through a bed of silica gel with elution by diethyl ether. Evaporation of the solvents afforded a white crystalline solid and recrystal'lization from benzene and hexane provided 2.85 g (81% yield) of 4,4-dimethyl-5a-cholest-8-ene-30,14a, 15a-triol (Compound 34a).

Physical Data (Compound 34a): m.p. - 133-134° (dec); Rf « 0.23 (2:3, ethyl acetate :hexane); r- -~7< 3* 23 16 04 NMR (300 MHz, CDClj-DjO (5%)): 4.11 (1H, dd, j - 5 Hz, 9 Hz), 3.22 (1H, dd, j - 5 Hz, 11 Hz), 2.33 (1H, m), 2.26 (1H, bm), 2.09 (2H, bra), 2.00-1.05 (20H, m), 1.01 (6H, d, j - 3 Hz), 0.87 (3H, s), 0.86 (3H, d, j - 8 Hz), 0.85 (3H, s), 0.82 (3H, b), 0.69 (3H, s); IR (Neat, cm"1): 3422 (bs, OH), 2950(s), 1652(g), 1465(a), 1036(a); EA for C2>HS0Oj: calculated, C 77.97%, H 11.28%; found C 78.05%, H 11.19%.

Physical Data (Compound 35a from Compound 33a): NMR (300 MHz, CDCl,): 4.11 (m, 1H), 3.61 (m, 1H), 2.50 (d, J - 9.0 Hz, 1H), 2.45-0.8 (a, 26H), 1.63 (d, J « 6.0 Hz, 1H), 0.98 (s, 3H), 0.87 (s, 6H), 0.85 (s, 3H), 0.71 (s, 3H); EA for CJ7H4S03: calculated, C 77.46%, H 11.08%; found 76.27%, H 11.15%.

U. Preparation of 30-hydroxy-lanost-8-en-15~one (Compound 28a) To a solution of 3|3-benzoyloxy-lanost-8-en-15-one (Compound 29a) (0.25 g, 0.46 mmol) in toluene (2.5 aL) and methanol (10 mL) was added anhydrous potassium carbonate (0.7 g). After stirring for 4 hours, toluene (2.5 mL) was added followed 2 hours later by the addition of 5% potassium hydroxide in ethanol. The reaction mixture was heated to 80° for 18 hours, then diluted with water (50 mL) and repeatedly extracted with toluene:ether (1:1) (4 X 100 mL). The combined organic extracts were washed with saturated sodium chloride (1 X 100 mL), dired over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The resultant residue was purified by silica gel chromatography (10% ethyl acetate in hexanes) to afford (180 mg) 30-hydroxy-lanost-8-en-15-one (Compound 28a) in 89% yield. ~tx a* 231 Ul. Preparation of 14a-allyl-4,4«dimethyl-5a-cholest-8-en-30-ol-15-one. (Compound 45a) The same procedure as used for Compound 28a was 0 employed with the following exceptions: Compound 20a was used as the starting material? and the solution was stirred at 40°C for 4 hours instead of 80°C for 18 hours. The procedure afforded Compound 45a in a 92% yield.

Physical Data (Compound 45a): m.p.- 139-1409C (ethyl acetate); Rf- 0.26 (10* ethyl acetate, 20%.toluene, 70% hexanes); NMR (300 MHz, CDClj): 5.71 (m, 1H, olefinic CH), 4.96 (m, 2H, olefinic CH2), 3.24 (dd, J«12.5 Hz, 1H, 3-CHOH), 2.57 (dd, J-20, 9Hz, 1H), 2.4-1.1 (m, 26H), 1.01 <s, 6H, CHjS), 0.97 (d, J-6HZ, 3H, 21-CH,), 0.89 (d, J-7Hz, 6H, 26-CHj and 27-CHj), 0.82 (s, 3H, 4<x-CHj), 0.76 (s, 3H, 18-CHj); MS (EI): 468 (10%, m) , 427 (100%, m-CjHs); HRMS for CjjHjjOj (H* ): calculated 468.3967, found 468.4087.

V. Preparation of 3p-hydroxy-lanost-8-en-15-oxime (Compound 31a) -hydroxy-lanost-8-en-15-one (Compound 28a) (180 mg, 0.41 mmol) was dissolved in anhydrous pyridine (3 mL) then treated with hydroxylamine hydrochloride (300 mg) (Aldrich). The reaction mixture was heated (85°) for 18 hours at which time an additional hydroxylamine hydrochloride (400 mg) sample was introduced. After quenching the reaction with water (50 mL), and extracting with toluene.-ether (1:1) (2 X 100 mL), the conbined organics were washed with IN hydrochloric acid (2 X 50 mL), water 50 mL), 10% copper sulfate solution (50 mL), water (50 mL), saturated sodium bicarbonate (50 ' •* ' ' '****■ • „ . . ... . . 13 2 3 1 6 0 4' mL), and saturated sodium chloride (50 mL), then dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The residue was crystallized from ethyl acetate in methanol (1:1) to provide 160 mg 5 of pure 33-hydroxy-lanost-8-en-15-oxime (Compound 31a) in 85% yield.

Physical Data (Compound 31a) O [a]35 - + 88.7° +/- 2.0° (c - 1.02, CHCl.) v.; J m.p. - 187-188° (ethyl acetate: methanol, 1:1) Rf - 0.25 (10% ethyl acetate in hexane) NMR (300 MHz, CDCl,):6.90 (bs, 1H, NOB), 3.27 (dd, J -11.1 Hz, 4.5 Hz, 1H, 3-CHOR), 2.77. (m, 2H), 2.30 (m, 2H), 2.10 (m, 2H), 1.80-0.80 (m, 24H), 1.13 (s, 3H, 15 14-CHj), 1.03 (S, 3H, 19-CH,), 1.02 (s, 3H, 31-CH,), 0.96 (d, J - 6.3 Hz, 3H, 21-CH3), 0.87 (d, J - 6.6 Hz, 6H, 26-CHj and 27-CH3) , 0.84 (s, 3H, 30-CH3), 0.73 (s, 3H, 18-CH3 ).

IR (CHClj, cm"1 ):3600 (bw, OH), 3300 (vbm, OH), 2960 20 (vs, CH sat), 1470 (m), 1375 (m), 1035 (m), 940 (m).

VI. Preparation of 14a-allyl-4,4-dimethyl-5a-cholest-8-en-3p-ol-15-oxime (Compound 46a) The same procedure as used for Compound 31a was 25 employed, with the following exceptions: the reaction mixture was heated at 60*C for 96 hours instead of 85°C for 18 hours; and Compound 45a was utilized as the starting material instead of Compound 28a. Compound 46a was provided in 40% yield.

Physical Data (Compound 45a): m.p.- 116-117°C (ethyl acetate); Rf- 0.20 (10% ethyl acetate, 20% toluene, 70% hexanes); NMR (300 MHz, CDCl,): 8.2 (s, 1H, NOH) 5.82 (m, 1H, olefinic CH), 4.96 (m, 2H, olefinic CH2), 3.24 (dd, J-12, 5Hz, 1H, 3-CHOH), 2.82 (dd, J-20, 9Hz, 1H), n 4- -T8 2.5-1.1 (to, 26H) , 1.02 (s, 6H, CH, S) , 0.95 (d, J-6Hz, 3H, 21-CHj) 0.89 (d, J-7Hz, 6H, 26-CH, and 27-CH,), 0.83 (s, 3H, CH3), 0.72 (s, 3H, 18-CH,); ms (ei): 465 (301, m-h,0), 442 (90%, m-hjo -c3h5) HRMS for C3aHslNO (M-HjO): calculated 465.3971, found 465.3932.

V?. Preparation of 32-benzyloxy-lanost-8-ene-3f$, 15a-diol (Compound 48a) To a cool (0°C), stirred tetrahydrofuran solution (10 mL) of 3f5, benzoyloxy-32-benzyloxy-lanost-8-ene-15-one (compound 47a) (1.0 g, 1.52 mmol) was added lithium aluminum hydride (Aldrich) (230 mg, 6.0 mmol) portionwise. The reaction was stirred at 0"C for 1 hour then diluted with 50 hL of diethyl ether and the reaction was quenched with dropwise addition of water (ca. 1 mL). With aluminum ester hydrolysis complete, the reaction mixture was dried over anhydrous magnesium sulfate, filtered and concentrated. The resultant residue was subjected to silica gel chromatography (15% ethyl acetate in hexanes) to give 537 mg (64%) of pure Compound 48a.

Physical Data (Compound 48a): NMR (300 MHz, CDCl,): 7.35 (m, 5H, phenyl), 4.62 (d, J - 13.8 Hz, 1H, CHjPh), 4.43 (d, J - 13.8 Hz, 1H, CHjPh), 4.38 (m, 1H, 15-CHOH), 3.90 (d, J - 8.0Hz, 1H, 32-CH2 OR), 3.85 (d, J - 10.0 Hz, 1H, OH), 3.60 (d, J -8.0Hz, 1H, 32-CH2OR), 3.25 (dd, J - 11.0 Hz, 4.5 Hz, 1H, 3-CHOH), '2.40 (m, 2H), 2.00 (m, 3H), 1.80 - 0.85 (m, 24H), 1.08 (s, 3H, CH3), 1.03 (S, 3H, CH, ), 0.92 (d, J -7.0Hz, 6H, 26-CH3 and 27-CH,), 0.90 (d, J - 7.0Hz, 3H, 21-CH,), 0.88 (s, 3H, CH,), 0.75 (s, 3H, 18-CH,).

X. Preparation of lanost-8-ene-3(J-15a,32-triol (Compound 49a). wfeamwsfcvtfw// - ,, - • j, , ■ x~s.

IS' j* 231604 A cold (08C) solution (tetrahydrofuranrethanol: acetic acid; 25 mL:25mL:lmL) of 32-benzyloxy-lanost-8-ene-30,15a-diol (Compound 48a) (330 sag, 0.6 mmol ) was degassed with nitrogen followed by the addition of 10* 5 palladium on activated carbon (Aldrich) (75 »g). At this point, the nitrogen line was replaced with a hydrogen gas source which permitted the bubbling of hydrogen gas through the reaction mixture for 1 hour. Examination of the reaction mixture by thin layer 10 chromatography showed complete conversion to desired triol Compound 49a. After removal of hydrogen source, the reaction was diluted with tetrahydrofuran 100 mL and filtered through celite to remove palladium. The filtered solution was concentrated and then diluted with 15 dichloromethane (300 mL) and washed with water (3x100 mL); saturated sodium bicarbonate solution (2x50 mL), saturated sodium chloride (100 mL) and dried over magnesium sulfate then filtered and concentrated. The residue was purified by silica gel chromatography (20% 20 ethyl acetate in hexanes) to afford 170 mg (61%) of pure Compound 49a.

Physical Data (Compound 4 9a): NMR (300 MHz, CDClj): 4.45 (dd, J - 7.0Hz, 11.0Hz, 1H, 25 15-CHOH), 4.10 (d, J - 12.0Hz, 1H, 32-CHjOH), 3.60 (d, J - 12.0 Hz, 1H, 32-CHaOH), 3.23 (dd, J - 11.0 Hz, 4.5 Hz, 1H, 3-CHOH), 3.00 (bm, 3H, OH), 2.70 (m, 1H), 2.30 (m, 6H), 2.10-0.85 (m, 23H), 1.03 (s, 6H, CH3), 0.97 (s, 3H, CHj), 0.95 (d, J - 7.0Hz, 3H, 21-CH3), 0.93 (d, J -30 7.0Hz, 6H, 26-CHj and 27-CH3), 0.73 (s, 3H, 18-CH,).

Y. Preparation of 30,15a-dihydroxy-lanost-8-en-32-al (Compound 50a) To a cold (-50®C) dichloromethane solution (1.5 mL) 35 of lanost-8-ene-30,15a,32-triol (Compound 49a) (30 mg, 70 pmol) was added recrystalized (water then dried under 23 16 04 IS vacuum and phosphorous pentoxide) pyridiniura dichromate (25 mg, 70 //mol). The reaction mixture was permitted to reach —30®C and followed by thin layer chromatography until complete (ca. 1 hour) at which time the reaction was quenched with isopropanol (1 mL) then diluted with diethyl ether (50 aL). The solution was filtered through a bed of fluorsil (Fischer) then concentrated to give a residue that was purified by silica gel chromatography (15% ethylacetate in hexanes). In this fashion pure Compound 50a was obtained in 63% yield (18 mg).

Physical Data (Compound 50a): NMR (300 MHz, CDCl,): 9.58 (s, 1H, CHO), 4.20 (m, 1H, -CHOH), 4.00 (d, J - 10.0Hz, 1H, OH, exchangable with Dj 0), 3.28 (m, 1H, 3-CHOH), 2.40-0.85 (a, 24H), 1.10 (s, 3H, CH3), 1.03 (s, 3H, CH3 ), 0.89 (d, J - 7.0Hz, 3H, CHj ), 0.86 (d, J - 7.0Hz, 6Hz, 26-CHj and 27-CH}), 0.85 (s, 3H, CHj), 0.78 (s, 3H, 18-CHj).

Examples 2-13: Inhibition an3 Suppression of Cholesterogenic Enzymes In Mammalian Cells A. 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) Suppression Assay The ability of the compounds of Formula I to" suppress the activity of HMGR, the rate limiting enzyme of cholesterol biosynthesis, was tested'as follows. Chinese Hamster Ovary (CHO) cells were divided twice weekly and were maintained in McCoy's 5A medium supplemented with 1% carbosil delipidated Fetal Bovine Serum (FBS) (obtained from Gibco Laboratories, Chagrin Falls, OH). Cells were harvested during the logarithmic phase of growth and cell cultures were prepared by adding 0.5 x 10s cells to each well in a 24 well cluster r\.

S —i~) 23 1 6 0 4 dish (obtained from Costar, Data Packaging Corp., Cambridge, MA) employing 1 mL of the above medium per each well. The cell cultures were incubated for 48 hr at 37® in a 5% COa , 95% air environment. The test 5 compounds in a 2.5% suspension of bovine serum albumin (BSA) (fatty acid free) in ethanol were then added to the cultures such that,the final ethanol and BSA concentrations in the incubation medium were 0.5% and 0.25% respectively. Treated cells were incubated with 10 the indicated compounds for 6 hr at 37® in a 5% COa , 95% air environment. Control cells were treated in an identical fashion to those which received test compound, except they were incubated with the BSA and ethanol suspension only.

HMGR activity was then measured in digitonin- permeabilized cells by the method developed by Leonard et al., J. Biol. Chem., 262: 7914-7919 (1987).

Specifically, the medium in each well was aspirated and the cells rinsed with a 50 mM solution of phosphate 20 buffered saline (PBS). One mL of 30 //g/mL of digitonin in CSK buffer (prepared using 10 mM Pipes [piperazine-N,N'-bis(2- ethansulfonic) acid], 100 mM KCl, 2.5 mM MgCla , 300 mM sucrose, 1 mM EGTA, pH 6.8) was added to each well and incubated for 10 min at 22° 25 to permeabilize the cells. The buffer was carefully aspirated and the wells were rinsed twice each time with 1 mL of PBS. HMGR activity was measured directly by adding 75 p~L of PIB buffer (50 mM potassium phosphate, 1 mM NaaEDTA, 10 mM dithiothreitol, pH 7.4) to each well 30 and incubating the cells for 30 minutes at 37* as described above. The enzyme assay was initiated by the addition of 83 //L of substrate/cof actor mixture such that the final assay contained the following: 0.1 M potassium phosphate, 5 mM dithiothreitol, 20 mM 35 glucose-6-phosphate, 2.5 mM NADP, 0.175 units of glucose-6-phosphate dehydrogenase, 150 pM lJ4C] 23 16 04 -fzr KHG-Coenzyrae A (15 DPM/pmol), pH 7.4. The assay mixture was incubated for 30 min at 37° and terminated by the addition of 70 pL of [3H]-mevalonic acid (35,000 DPM/assay), 0.15 mg/mL in 3 N HCl. The reaction was '<• i''' left to lactonize for an additional 30 min at 37" or overnight at room temperature.

Reaction products were separated by thin layer chromatography on silica gel G (obtained from Analtech, (—• Newark, DE) developed in an unsaturated environment with s ' 10 acetone:benzene (3:2, v:v). The band corresponding to mevalonolactone was identified by exposure to iodine vapor and was scraped into counting vials. The extent of conversion of starting substrate, HMG-CoA, to mevalonic acid was determined by liquid scintillation 15 counting in Biofluor (obtained from New England Nuclear, Boston, MA). Corrections for recovery and blank values were made for each sample. Protein determinations were made by the Bio-rad (Bio-Rad, Richmond, CA) dye binding assay according to the manufacturer's instruction using 20 bovine serum albumin as standard. Cellular protein was Bolubilized from culture dishes by the addition of 20 pL of 16 N KOH and assayed directly for protein amount. Suppression values are expressed as the amount of compound required to supress HMGR activity by 50% relative to that of the controls.

B. Lanosta-8,24-dien-30-ol 14a-Methyl Demethylase Inhibition Assay Lanosta-8,24-dien-30-ol 14a-methyl demethylase activity was determined in rat hepatic microsomal preparations as described in Trzaskos et al., J. Biol Chem., 261, 16937-16942 (1986). Specifically, test compounds were added to assay mixtures as detergent 35 suspensions with substrate employing 5 mg Triton WR-1339, 100 pK lanost-8-en-3g-ol substrate, and test -a 231 compounds ranging from 0 to 100 //M. The inhibitor/substrate/detergent suspension was incubated with 2 mg microsomal protein for 5 min at 37" followed by a 10 min incubation initiated by the addition of cofactors required for the reaction. Inhibition values were calculated by comparing the enzymic activity in inhibitor-containing assays with those of control assays which did not receive added compounds. Inhibition values are expressed as the amount of compound required to inhibit lanosta-8,24-dien-3f3-ol 14o-methyl demethylase activity by 50* relative to that of the controls.

The results of Assays A and B are reported in Table I below. 23 16 04 "80 yju--> Table I Effect of Various Test Compounds Upon Measured HMGR and Lanosta-8,24-dien-30-ol 14a-methyl Demethylase Activities Example Lanosterol HMGR Demethylase NO.

Compound IC50 (//M) 2 26b 1.0 >100 3 34a 0.5 3.0 4 17a 1.4 11.0 9a 1.3 16.0 6 35a 0.4 >100 7 15b 1.4 3.0 8 27b 0.5 >100 9 31b 0.2 55.0 9b 1.4 37.0 11 11a 0.06 0.8 12 12a 0.13 3.2 13 15a 0.04 1.1 The ability of compounds of this invention to effectively suppress HMGR activity and inhibit lanosta-8,24-dien-30-ol 14a-methyl demethylase activity is demon- strated by the data in Table I. As a comparison, it should be noted that cholesterol, lanosta-8,24-dien-30-ol (Compound 1), and lanost-8-en-30-ol, when tested under these same conditions, were without effect on measured HMGR activity. In addition, with respect to ™ 23160 lanosta-8,24-dien-30-ol 14a-methyl demethylase activity, cholesterol was also found to be without effect, although lanosta-8,24-dien-33-ol and lanost-8-en-30-ol clearly served as substrates for the demethylase enzyme.

The dual mechanism of inhibition at the point of lanosta-8,24-dien-30-ol 14o-raethyl demethylation coupled with HMGR suppression makes these compounds attractive as hypocholesteroleraic agents.

Examples 14-21: Decreasing Cholesterol Synthesis In Mammalian Cells The ability of the compounds of Formula I to decrease c[e novo cholesterol synthesis was tested by measuring the incorporation of radiolabeled acetate into cholesterol synthesized in CHO cells. To conduct this experiment, CHO cells were plated at 0.3 x 10s cells per 15 x 60 mm tissue culture dish in 5 mL of McCoy's modified medium (Gibco Laboratories) containing 1% Cab-O-Sil delipidated FBS and incubated for 48 hours at 37° in a 5* co}, 95% air environment. Test compounds in a 2.5% suspension of BSA (fatty acid free) in ethanol were then.added to the cultures such that the final ethanol and BSA concentrations were 0.5% and 2.5%, respectively. Control cultures received ethanol and BSA alone. The cultures containing test compounds were then incubated at 37° in a 5% C02, 95% air environment for 17 hr before the addition of [1t2-1*C]-acetic acid (57 //Ci/mmol; obtained from New England Nuclear, Boston, MA) at a concentration of 10//Ci/mL for 2 additional hr.

The cells were harvested by washing the cells twice with 5 mL of cold PBS and scraping the cells into 1 mL PBS. The cell containing solution was then transferred to 15 mL extraction tubes and treated with 1 mL of 90% methanol (containing 15% KOH, 100|/g/mL butylated hydroxytoluene). All samples were saponified for 1 hr «&2- 23 1 6 at SO0 and the non-saponifiable lipids then extracted with petroleum ether. The petroleum ether fractions were dried under nitrogen, resuspended in ethanol, counted and analyzed by reverse phase HPLC at 45" using an Ultrasphere Octyl column (from Altex Scientific Inc., Berkeley, CA.} with a mobile phase of acetonitrile:methanol:water (44:44:12). The results are reported in Table II. The amount of cholesterol synthesis is measured by the amount of radiolabeled acetate incorporated into the cholesterol peak. The percent of cholesterol synthesis in the treated culture is calculated based upon the control culture.

Table II Effect of Various Test Compounds Upon Measured Cholesterol Synthesis Example Compound and Cholesterol Cholesterol No. Concentration* Synthesis Synthesis (pH) (DPMs) (%) 14 Control 38855 100 26b (10) 12279 32 16 9a (10) 3615 9 . 17 9b (10) 10125 26 18 11a (1) 360 1 19 ' 12a (1) 636 2 34a (1) 16521 43 21 31b (0.5) 24064 62 22 15b (0.5) 1534 4 "Concentration in brackets. ^3 23 1 The ability of compounds of the present invention to decrease de novo cholesterol synthesis is demonstrated by the data in Table II.

Examples 23-24: Lowering Blood Cholesterol Levels in Hamsters The ability of the compounds of Formula I to lower blood cholesterol levels has been demonstrated in hamsters utilizing the following protocol. Male Golden Syrian hamsters (50-60 grams) were obtained from Charles River, Inc. (Wilmington, MA). Animals were housed in individual suspension cages and were maintained on a light cycle consisting of 12 hours of light followed by 12 hours of dark. Animals were allowed free access to water and feed (Agway ground chow, RMH 3200, Agway, Syracuse, N.Y.) containing 1% (w/w) corn oil) for a minimum of 4 weeks. Following this stabilization period a sample of blood was collected by orbital sinus bleeding under light ether anesthesia into heparinized capillary tubes. Plasma was separated by centrifugation (600 x g for 10 minutes) and plasma cholesterols were determined by an autoanalyzer (Centrifichem 600, Baker Instruments, Allentown, PA). Based upon measured plasma cholesterol values, the animals were randomized into two groups such that the mean plasma cholesterol values were identical for both groups.

Animals in the two groups were then placed on one of two diets: (1) Diet A, consisting of ground chow plus 1% (w/w) corn oil, as described above; or (2) Diet B, consisting of Diet A plus 0.2% (w/w) of a test compound. Animals on Diet B, the treated animals, were allowed free access to feed and water, while animals on Diet A were pair-matched with Diet B animals and served as pair-fed controls. The animals were kept on their respective diets for 7-days at which time they were bled ^ 23 1 6 04 by cardiac puncture under C02 anesthesia. Total plasma cholesterol levels were determined as described above.

The results are presented in Table III. The data is reported as means + SEM (standard error of the mean), r 5 in units of mg/dl. The value "N" represents the number of animals in each group.

Table III Effect Of Various Test Compounds Upon Plasma Cholesterol Levels In Hamsters Example Compound Plasma Cholesterol No . Control Treated mg/dl 23 34a 136.0 + 3.5 119.3 + 2.3 (N-16) (N-16) r"*\ c 24 31b 136.0 + 4.8 69.6 + 2.6 (N-16) (N-16) As the data in Table III indicates, blood cholesterol levels can be significantly lowered by administration of compounds of Formula I. 231604 223344

Claims (4)

WHAT WE CLAIM IS:

1. A composition suitable for inhibiting lanosterol-8,24-dien-3y$-ol 14e<-methyl-demethy2ase activity comprising (i) an effective amount of a compound of N the formula Ra R2 r V^«*' in which the substituent at carbon position 14 is alpha in configuration; the ring structure may be unsaturated between carbon positions 6 and 7, between carbon positions 7 and 8/ between carbon positions 8 and 9 or between both carbon positions 6 and 7 and carbon positions 8 and 9; is -0, OW. or OCOW, Rj is H, cl-c6 alkyl, c2-c6 alkenyl, c2-c6 alkynyl or aryl-C2-C6-alkyl; r3 is h, cj— c4 alkyl, ^2-<"6 a^kenyl, c2-c6 alkynyl, aryl-cx-c6-alkyl, cho, ch20r4, ch2ch2or4, chnor4, ch2sr4 , ch2ch2sr4, CHOR4W2, CH0R5W2 , CN, CHZ2 , CH2Z, CHS, CHR4N(R4)2, CH2CH2N(R4 )2 , CH2CH2Z, N(R4)2# SR4, 0R4, CH-NNHR,, poly-(0R4, 0R5, epoxy) 'V* c^/ { 23UoCh - 86 - Cj^-Cg alkyl, N(R5)2, NR^, SRg, ORj, CH=NNHR,., CH2OR5, ch2ch2or5, chnor5, ch2sr5, ch2ch2sr5, chr4n(r5)2, chr4nr4r5, ch2ch2n(r5)2, ch2ch2nr4r5, ch2ch2nr4or4, ckr4nr4or5, cow4, csw4, c(nr4)w4, c(nr4)sr4, c(s)sr4, chr4nr4n(r4)2, chr4nr5n(r4)2/ chr4nr4nr4r5, chr4nr5nr4r5, chr4nr4n(r5)2, chr4nr5n(r5)2, ch2ch2nr4or5, chr4nr4or4, chr4nr5or5, chr4nr5or4, ch2ch2nr5or5, ch2ch2nr5or4, cr4=cr4r5, CECRj, cr4=cr4c(r4)2z, c=c-c(r4)2z, cr4=cr4c(r4)2or5, cec-c<r4)2or5, cr4=cr4c(r4)2or4, c=c-c(r4)20r4, c(0)nr40r4/ c(0)nr40r5, c(s)nr4or4, c(s)nr4or5, c(r4)2or4, c(r4)2or5/ chr4nr4so2w4, ch2chr4nr4so2w4, c(r4)2cr4nor4/ c(r4)2cr4nor5/ c(r4)2cr4nor4, c(r4)2cr4n0r5/ c(r4)2w5, cr4w5or4, or CR4W5SR4; R4 is H, C^-Cg alkyl, Cj-Cg alkenyl, aryl, aryl-C^-Cg-alkyl or C2~Cg alkynyl; R5 is COW3/ CSW3, or C(NR4)W3; X and y, independently, are H, C^-Cg alkyl, Z, OR4, SR4, or5, sr5, n(r4)2, n(r5)2, nr4r5, nr4or4, nr4or5, nr4n(r4)2/ NR4NR4R5, NR4N(R5)2, NR5N(R4)2, NR5NR4R5, or NR5N(R5)2; or x and y, taken together, are nr4, nr,., nor4, nor^, s, c(r4)2, c(r5)2, cr5r4, nn(r4)2, nnr4r5, nn(r5)2, or 0; Z is halogen; W1 is H, C^-Cjq alkyl, C2~C20 alJcenyl, arY1/ aryl-C^,-C2Q-al3cyl, or C2-C20 a^cynylf W2 is H, C1-Cg alkyl, C2~Cg alkenyl, aryl, aryl-C^-Cg-alkyl, or C2~Cg alkynyl; is H, alkyl, C2-Cg alkenyl, aryl, aryl-C^-Cg- alkyl, C2~Cg alkynyl, 0R4, or N(R4)2; W4 is C1~Cg alkyl, C2~Cg alkenyl, aryl, aryl-C^-Cg-alkyl, C2~Cg alkynyl, 0R4, or N(R4)2; and Wj. is a 5- or 6-membered aromatic heterocyzriic--ring„,., containing 1 or 2 nitrogen aroms as part of the -ri ngfr.i^iri ring optionally substituted with substituents ^ n \ 16 JUL 1990S ■,9j ■*i\ «<» // -81- 231 604?iHB4*! X / selected from the group consisting of Cj-Cj alkyl and C3-Cf alkenyl; and their physiologically acceptable salts; provided that a) when Rj is CHO, and X and Y are both H, and carbons 7-8 and 8-9 are unsaturated, ^ then Rx is other than OH or OCOCHj, and Rj is other than CHj; b) when Rj is CHj and carbons 7-8 or 8-9 are unsaturated, then R2 is other than OH or OCOCHj, Rj is other than CHj or H, and X and Y are other than OH, OCOCHj or B; c) when Rj is -O, or is OWj where is H or Cj-Cj alkyl, or is OCOWx when Wx is Cj-Cjg alkyl or phenyl, and X is 0R4 where R4 is H and 0R5 is 0C0W3 where W3 is Cj-Cj0 alkyl or phenyl, and Y is H or OH, then Rj is other then H or a Cj-C4 alkyl; d) When Rj is CH2OH or CHjOCOCHj, and R2 is H or CHj, and carbons 6-7, 7-8 or 8-9 are unsaturated, then Rx is other than -0 or OH or OCOCHj, and X is other than H or OH; e) when X and Y are both H, then Rj is other than H or CHj ; f) when X and Y are both H, then R3 is other than OH, and R2 is other than H; g) when R2 is H, and Rj is a OH, and X is & OH, and Y is a H, and carbons 6-7, 7-8, and 8-9 are saturated, then Rj^ is other than OH; -&z- fjO 1 O ~ < J x t > U • i h) i) j) wh sr. en X is OH, 0RS , N ( R4 ) 3 , N(R5)2, NR4R} , nr4or4 or NR4 ORj, then Y is other than Z, OH, ORj , SRs , NR4OR4, NR4 ORs N(R4 )2 , or N(RS )2 ;

2 . when Rj is H, OH, or Cx-Cs alkyl, then X and Y, taken together, are other than 0; and when Rj is Ca-c6 alkyl, then X and Y are other than OH or halogen; and (ii) a pharmaceutically acceptable carrier or diluent. A composition suitable for inhibiting

3-hydroxy-3-methylglutaryl coenzyme A reductase activity comprising (i) an effective amount of a compound of the formula 2 "2 as defined in claim 1 . 231004 -si- 3, a composition suitable for decreasing cholesterol formation comprising (i) an effective amount of a compound of the formula as defined in claim 1 m 3 i(>04 -c\o- 4. A composition suitable for lowering serum cholesterol levels comprising (i) an effective amount of a compound of the formula C.H Q as defined in claim 1 231C04 -ii- n 5. A method for inhibiting lanosterol-8,2

4-dien-30-ol 14a-methyl-deraethylase activity in non-humans comprising administering an effective amount of a compound of the formula as defined in clain. 1. O , .. . -. ...._ .y. ,^,. •''- r . ' 23.1CCM f\ -11- 6. a method for suppressing 3-hydroxy-3- methylglutaryl coenzyme A reductase activity in non-humans cxrnprising adrninistering an effective amount of a compound of the formula -k~ A method for decreasing cholesterol formation in non-humans comprising administering an effective amount of a compound of the formula as defined in claim 1. c 231004 t j -m- 8. A method for lowering serum cholesterol levels in non-humans aarorising administering an effective amount of a compound of the formula as defined in claim 1-. H nvw 9. A composition according to claim 1 and substantially as described in this specification with reference to the examples. 0 *"■ T„, o E.fr. DU PONT DE NEMOURS AND COMPANY