Intermediates for the Synthesis of Bile Acid Derivatives, in Particular of Obeticholic Acid
20210179661 · 2021-06-17
Assignee
Inventors
- Alexander Weymouth-Wilson (Reading, GB)
- Zofia Komsta (Reading, GB)
- Laura Wallis (Reading, GB)
- Timothy Evans (Reading, GB)
- Ieuan Davies (Reading, GB)
- Carl Otter (Reading, GB)
- Rhys Batchelor (Reading, GB)
Cpc classification
C07J41/0094
CHEMISTRY; METALLURGY
C07J41/0055
CHEMISTRY; METALLURGY
C07J17/00
CHEMISTRY; METALLURGY
C07J41/0061
CHEMISTRY; METALLURGY
C07J9/005
CHEMISTRY; METALLURGY
C07J9/00
CHEMISTRY; METALLURGY
International classification
C07J43/00
CHEMISTRY; METALLURGY
C07J17/00
CHEMISTRY; METALLURGY
C07J21/00
CHEMISTRY; METALLURGY
C07J31/00
CHEMISTRY; METALLURGY
C07J33/00
CHEMISTRY; METALLURGY
C07J41/00
CHEMISTRY; METALLURGY
C07J51/00
CHEMISTRY; METALLURGY
C07J71/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to compounds which are intermediates in the synthesis of bile acid derivatives with pharmacological activity. The invention relates to compounds of general formula (I):
##STR00001##
wherein: , R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and Y are as defined herein.
The compounds are intermediates in the synthesis of synthetic bile acids which are useful in the treatment of conditions such as liver disease. In addition, the invention relates to a method of synthesizing these intermediates and a method of preparing obeticholic acid and obeticholic acid analogues from the compounds of the invention.
Claims
1-15. (canceled)
16. A compound of general formula (I): ##STR00126## wherein: is a carbon-carbon single or double bond; R.sup.1 is C.sub.1-4 alkyl optionally substituted with one or more substituents selected from halo, OR.sup.7a and NR.sup.7aR.sup.7b; where each of R.sup.7a and R.sup.7b is independently selected from H and C.sub.1-4 alkyl; R.sup.2 is ═O or OH or a protected OH; R.sup.3 is H, halo or OH or a protected OH; when
is a carbon-carbon double bond, Y is a bond or an alkylene, alkenylene or alkynylene linker group having from 1 to 20 carbon atoms and optionally substituted with one or more groups R.sup.13; when
is a carbon-carbon single bond, Y is a bond or an alkylene linker group having from 1 to 20 carbon atoms and optionally substituted with one or more groups R.sup.13; each R.sup.13 is independently halo, OR.sup.8 or NR.sup.8R.sup.9; where each of R.sup.8 and R.sup.9 is independently selected from H and C.sub.1-4 alkyl; R.sup.4 is halo, CN, C(O)R.sup.10, CH(OR.sup.10)(OR.sup.11), CH(R.sup.10)(OR.sup.11), CH(SR.sup.10)(SR.sup.11), NR.sup.10R.sup.11, BR.sup.10R.sup.11, C(O)CH.sub.2N.sub.2, —CH═CH.sub.2, —C≡CH, CH[C(O)OR.sup.10].sub.2, CH(BR.sup.10R.sup.11).sub.2, azide or a carboxylic acid mimetic group; where each R.sup.10 and R.sup.11 is independently: a. hydrogen or b. C.sub.1-20 alkyl, C.sub.2-20 alkenyl or C.sub.2-20 alkynyl, any of which is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, C(O)OR.sup.19, C(O)N(R.sup.19).sub.2, SO.sub.2R.sup.19, SO.sub.3R.sup.19, OSO.sub.3R.sup.19, N(R.sup.19).sub.2 and a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group, either of which is optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, C(O)OR.sup.19, C(O)N(R.sup.19).sub.2, SO.sub.2R.sup.19, SO.sub.3R.sup.19, OSO.sub.3R.sup.19 and N(R.sup.19).sub.2; or c. a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, C(O)OR.sup.19, C(O)N(R.sup.19).sub.2, SO.sub.2R.sup.19, SO.sub.3R.sup.19, OSO.sub.3R.sup.19 and N(R.sup.19).sub.2; or d. a polyethylene glycol residue; or e. when R.sup.4 is CH(OR.sup.10)(OR.sup.11), CH(R.sup.10)(OR.sup.11), CH(SR.sup.10)(SR.sup.11), NR.sup.10R.sup.11, BR.sup.10R.sup.11, CH[C(O)OR.sup.10].sub.2 or CH(BR.sup.10R.sup.11).sub.2 and R.sup.10 and an R.sup.11 group, together with the atom or atoms to which they are attached, may combine to form a 3 to 10-membered heterocyclic ring; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl and a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or when Y is a double bond, R.sup.4 is CH.sub.2; R.sup.5 is H or OH or a protected OH group; R.sup.6 is ═O; or a salt or an isotopic variant thereof.
17. The compound according to claim 16, selected from the group consisting of: a compound of general formula (IB): ##STR00127## wherein R.sup.1, R.sup.3, Y, R.sup.4 and R.sup.5 are as defined in claim 16; or a compound of general formula (IC): ##STR00128## wherein R.sup.1, R.sup.3, Y, R.sup.4 and R.sup.5 are as defined in claim 16; or a compound of general formula (ID): ##STR00129## wherein R.sup.1, R.sup.3, Y, R.sup.4 and R.sup.5 are as defined in claim 16; or a compound of general formula (IE): ##STR00130## wherein R.sup.1, R.sup.3, Y, R.sup.4 and R.sup.5 are as defined in claim 16; or a salt or an isotopic variant of any of these.
18. The compound according to claim 16, wherein R.sup.1 is ethyl.
19. The compound according to claim 17, wherein the compound is a compound of general formula (IB) and Y is selected from the group consisting of a bond, an unsubstituted C.sub.1-3 alkylene group, a C.sub.1-3 alkylene group substituted with OH, and a C.sub.1-3 alkenylene group.
20. The compound according to claim 17, wherein the compound is a compound of general formula (IC), a compound of general formula (ID) or a compound of general formula (IE) and Y is selected from a bond and an alkylene group having 1 to 3 carbon atoms and is optionally substituted with one or two OH groups.
21. The compound according to claim 16, wherein each R.sup.10 and R.sup.11 is independently: a. hydrogen or b. C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl, any of which is optionally substituted with one or more substituents as defined in claim 16; or c. a 6- to 10-membered aryl or 5 to 10-membered heteroaryl group either of which is optionally substituted with one or more substituents as defined in claim 16; or d. a polyethylene glycol residue; or e. when R.sup.4 is CH(OR.sup.10)(OR.sup.11), CH(R.sup.10)(OR.sup.11), CH(SR.sup.10)(SR.sup.11), NR.sup.10R.sup.11, BR.sup.10R.sup.11, CH[C(O)OR.sup.10].sub.2 or CH(BR.sup.10R.sup.11).sub.2 an R.sup.10 and an R.sup.11 group, together with the atom or atoms to which they are attached, may combine to form a 3- to 10-membered heterocyclic ring; or when R.sup.4 is NR.sup.10R.sup.11, R.sup.10 may be H or C.sub.1-4 alkyl and R.sup.11 may be a 5-10 membered heteroaryl group.
22. The compound according to claim 16, wherein: when one or more of R.sup.2, R.sup.3 and R.sup.5 is a protected OH group, the protected OH group comprises i. OC(O)R.sup.14, where R.sup.14 is a group R.sup.10; or ii. OSI(R.sup.16).sub.3, and wherein: one or more of R.sup.10, R.sup.11 and R.sup.16 is: a. C.sub.1-20 alkyl, C.sub.2-20 alkenyl or C.sub.2-20 alkynyl, any of which is optionally substituted with halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, C(O)OR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19, OSO.sub.3R.sup.19, N(R.sup.19).sub.2 or a 6- to 10-membered aryl or 5 to 14-membered heteroaryl group, either of which is optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, halo, NO.sub.2, CN, OR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; where R.sup.19 is as defined in claim 16; or b. a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group wherein said aryl or heteroaryl group is optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, halo, NO.sub.2, CN, OR.sup.19, SR.sup.19 or N(R.sup.19).sub.2; where R.sup.19 is as defined in claim 16.
23. The compound according to claim 16, wherein R.sup.19 is selected from H, methyl, ethyl, trifluoromethyl and phenyl optionally substituted with one or more substituents selected from fluoro, chloro, methyl, ethyl and trifluoromethyl.
24. The compound according to claim 16, wherein R.sup.4 is selected from the group consisting of halo, CN, C(O)R.sup.10, CH(OR.sup.10)(OR.sup.11), NR.sup.10R.sup.11, BR.sup.10R.sup.11, —CH═CH.sub.2, —C≡CH, CH[C(O)OR.sup.10].sub.2, azide, and CH(BR.sup.10R.sup.11).sub.2; or wherein Y is a double bond and R.sup.4 is CH.sub.2, and wherein R.sup.10 and R.sup.11 are as defined in claim 16.
25. The compound according to claim 24, wherein R.sup.4 is selected from the group consisting of halo, CN, C(O)R.sup.10, CH(OR.sup.10)(OR.sup.11), NR.sup.10R.sup.11, CH[C(O)OR.sup.10].sub.2, and azide; wherein R.sup.10 is selected from H and C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl, any of which is optionally substituted with halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, C(O)OR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19, OSO.sub.3R.sup.19, N(R.sup.19).sub.2, and a 6- to 10-membered aryl or 5 to 14-membered heteroaryl group, either of which is optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, halo, NO.sub.2, CN, OR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; where R.sup.19 is as defined in claim 16; or when R.sup.4 is CH(OR.sup.10)(OR.sup.11), the OR.sup.10 and OR.sup.11 groups together with the carbon atom to which they are attached may form a cyclic acetal group; or when R.sup.4 is NR.sup.10R.sup.11, R.sup.10 is selected from H and C.sub.1-4 alkyl and R.sup.11 is a 5-10 membered heteroaryl group.
26. The compound according to claim 25, wherein R.sup.4 is selected from the group consisting of: Cl, Br, CN, C(O)H, CH(OR.sup.10).sub.2, 1,3-dioxane, 1,3-dioxolane and CH[C(O)OR.sup.10].sub.2; where R.sup.10 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, iso-butyl and t-butyl.
27. The compound according to claim 25, wherein R.sup.4 is azide.
28. The compound according to claim 16, wherein the compound is selected from: (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-cholen-23-carboxy-24-oic acid dimethyl ester; (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic acid dimethyl ester; (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester; (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester; (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid; (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile; (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile; (5β, 6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile; (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile; (6β, 7α, 20R)-20-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one; (5β, 6β, 7α, 20R)-20-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one; (5β, 6β, 20R)-20-cyanomethyl-6-ethyl-7-oxo-pregna-3-one; (6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one; (5β, 6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one; (5β, 6β, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione; (5β, 6α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione; or a salt thereof.
29. The compound according to claim 16, wherein R.sup.4 is a carboxylic acid mimetic group selected from tetrazole, —C(O)NHSO.sub.2R.sup.30 and —NHC(O)NHSO.sub.2R.sup.30; wherein R.sup.30 is H, C.sub.1-6 alkyl or aryl.
30. The compound according to claim 29, wherein R.sup.4 is tetrazole.
Description
ABBREVIATIONS USED IN EXAMPLES
[0270]
TABLE-US-00001 AcOH Acetic acid Aq. Aqueous nBuLi n-Butyl lithium DCM Dichloromethane DMF N,N-dimethylformamide DMAP 4-Dimethylaminopyridine DMP Dess-Martin periodinane EtOAc Ethyl acetate EtOH Ethanol EtMgBr Ethyl magnesium bromide h Hour HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol HMPO (20S)-20-hydroxymethyl-pregna-4-en-3-one also known as 20-hydroxymethylpregn-4-en-3-one and 3-keto-bis- norcholenol IPA Isopropanol mCPBA meta-chloroperoxybenzoic acid MeCN Acetonitrile MeOH Methanol Mesyl Methane sulfonyl MsCl Methane sulfonylchloride MTO Methyltrioxorhenium(VI) NaOMe Sodium methoxide PhMe Toluene PTFE Polytetrafluoroethylene Py Pyridine TBDMSCl tert-Butyldimethylsilyl chloride TBME tert-butyl methyl ether TEPA Triethyl phosphonoacetate THF Tetrahydrofuran TLC Thin layer chromatography TMSOTf Trimethyl silyl trifluoromethanesulfonate Tosyl Toluene sulfonyl pTSA•H.sub.2O para toluene sulfonic acid monohydrate UHP Urea hydrogen peroxide
Example 1—Preparation of Compounds of General Formula (II)
[0271] Scheme 3 below shows the conversion of an analogue of a compound of general formula (III) in which the side chain is —CH.sub.2OH to analogues of a compound of general formula (II) in which the side chain is —CH.sub.2OC(O)CH.sub.3 and —CH.sub.2OH and the subsequent conversion of this compound into other compounds of general formula (II) with different side chains.
[0272] As shown in Scheme 3, the general formula (II) analogue with the —CH.sub.2OH side chain can be converted to compounds of general formula (II) with side chains including —CH.sub.2-9-borabicyclo(3.3.1) nonyl, —CH.sub.2CH.sub.2CH[B(alkyl).sub.2].sub.2, —CH.sub.2CN, —CH.sub.2CH.sub.2CN, —CH.sub.2Br, —CH.sub.2CH[C(O)OEt].sub.2, —CH.sub.2—C≡CH, —CH.sub.2—CH═CH.sub.2, ═CH.sub.2, —C(O)H, —CH.sub.2NH.sub.2,
##STR00032##
where X is O or S
alkyl may be C.sub.1-6 alkyl and Et is ethyl; and also carboxylic acid mimetic groups including —C(O)NHSO.sub.2R.sup.30 and —NHC(O)NH—SO.sub.2R.sup.30.
[0273] Synthesis of compounds of general formula (II) shown in Scheme 3 is described below.
A. Synthesis of (20S)-20-hydroxymethyl-pregna-4-en-3-one
[0274] ##STR00033##
[0275] (20S)-20-Hydroxymethyl-pregna-4-en-3-one (HMPO) can be prepared by chemoselective reduction of dinorcholenaldehyde ((20S)-20-formyl-pregn-4-en-3-one) with NaBH.sub.4 in primary alcohol (Barry M. Trost, Alvin C. Lavoie J. Am. Chem. Soc., 1983, 105 (15), 5075-5090).
B. Synthesis of (20S)-20-acetoxymethyl-pregna-4,6-dien-3-one
[0276] ##STR00034##
[0277] HMPO (300 g, 0.913 mol) was charged to a reaction vessel, followed by AcOH (0.9 L) and toluene (0.3 L) with stirring. p-Chloranil (245 g, 1.00 mol) was then charged and the reaction mixture heated to 110° C. and maintained at this temperature for 6 h. The mixture was then cooled to 5° C. and held at that temperature for 2 h. The resulting solid was filtered and the filter-cake washed with cold, premixed 3:1 AcOH:Toluene (4×150 mL) and the filtrate was concentrated in-vacuo. The residue was dissolved in acetone (900 mL), then 3.5% w/w aqueous NaOH (3.0 L) was charged dropwise with stirring, maintaining the temperature below 30° C. The resulting solids were collected by filtration and the filter cake was washed with premixed 1:1 acetone:water (1.5 L). The filter cake was then slurried in 1:1 acetone:water (600 mL) at 20° C., filtered and washed with premixed 1:1 acetone:water (1.0 L). The solid was dried under vacuum at 65-70° C. to give the desired product (224 g, 67%) as a tan solid. δH (400 MHz, CDCl.sub.3); 6.17-6.12 (1H, m, C6-CH), 6.10 (1H, dd, J 9.9, 2.0, C7-CH), 5.68 (1H, s, C4-CH), 4.10 (1H, dd, J 10.7, 3.5, C22-CH.sub.aH.sub.b), 3.79 (1H, dd, J 10.7, 7.4, C22-CH.sub.aH.sub.b), 2.58 (1H, ddd, J 17.9, 14.4, 5.4, C2-CH.sub.aH.sub.b), 2.49-2.39 (1H, m, C2-CH.sub.aH.sub.b), 2.20 (1H, brt, J 10.2, C8-CH), 2.10-1.97 (1H, m), 2.06 (3H, s, OC(O)CH.sub.3), 1.96-1.66 (4H, m), 1.62-1.53 (1H, m), 1.52-1.16 (8H, m), 1.12 (3H, s, C19-CH.sub.3), 1.04 (3H, d, J 6.6, C21-CH.sub.3), 0.79 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.6, 171.3, 163.8, 141.2, 127.9, 123.6, 69.4, 53.2, 52.6, 50.7, 43.6, 39.4, 37.7, 36.1, 35.8, 33.9, 33.9, 27.6, 23.8, 21.0, 20.7, 17.1, 16.3, 11.9.
C. Synthesis of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one
[0278] ##STR00035##
[0279] (20S)-20-Acetoxymethyl-pregna-4,6-dien-3-one (25 g, 67.5 mmol) was suspended in MeOH (250 mL) and sodium methoxide (25% w/v solution in MeOH) was added until pH 12 was achieved. The resulting mixture was stirred at room temperature for 4 h. The pH was adjusted to pH 4 by addition of Finex CS08GH.sup.+ resin. The mixture was filtered and the filtrate was concentrated under reduced pressure, co-evaporating with PhMe (2×250 mL). The residue was dried in a vacuum oven at 30° C. for 48 h to give the desired product (22.15 g, 99%) as a light brown solid. δH (400 MHz, CDCl.sub.3); 6.16-6.11 (1H, m, C7-CH), 6.09 (1H, dd, J 9.9, 2.3, C6-CH), 5.67 (1H, s, C4-CH), 3.65 (1H, dd, J 10.5, 3.3, C22-CH.sub.aH.sub.b), 3.59 (1H, dd, J 10.5, 6.7, C22-CH.sub.aH.sub.b), 2.57 (1H, ddd, J 18.0, 14.4, 5.5, C2-CH.sub.aH.sub.b), 2.45-2.38 (1H, m, C2-CH.sub.aH.sub.b), 2.19 (1H, brt, J 10.4, C8-CH), 2.11-1.76 (5H, m), 1.71 (1H, td, J 13.9, 5.3, C1-CH.sub.aH.sub.b), 1.65-1.16 (9H, m), 1.11 (3H, s, C19-CH.sub.3), 1.06 (3H, d, J 6.6, C21-CH.sub.3), 0.78 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.7, 164.0, 141.4, 127.9, 123.5, 67.8, 53.2, 52.3, 50.7, 43.5, 39.4, 38.7, 37.8, 36.1, 33.9, 33.9, 27.6, 23.8, 20.7, 16.7, 16.3, 12.0;
D. Synthesis of (20S)-20-tertbutyldimethylsilyloxymethyl-pregna-4,6-dien-3-one
[0280] ##STR00036##
[0281] (20S)-20-Hydroxymethyl-pregna-4,6-dien-3-one (1.00 g, 3.04 mmol) was dissolved in anhydrous CH.sub.2Cl.sub.2 (10 mL) and the solution was cooled to 0° C. Imidazole (414 mg, 6.09 mmol) and TBDMSCI (551 mg, 3.65 mmol) were added and the reaction was stirred at 0° C. for 4 h. The reaction was warmed to room temperature and CH.sub.2Cl.sub.2 (10 mL) and water (20 mL) were added. The layers were separated and the organic phase was washed with water (20 mL), saturated aqueous sodium chloride (20 mL), dried over sodium sulfate and was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-25% EtOAc in heptane) to give the desired product (890 mg, 66%) as a light yellow solid. δH (400 MHz, CDCl.sub.3); 6.14 (1H, dd, J 9.9, 1.3, C7-CH), 6.09 (1H, dd, J 9.8, 2.4, C6-CH), 5.66 (1H, s, C4-CH), 3.58 (1H, dd, J 9.7, 3.4, C22-CH.sub.aH.sub.b), 3.28 (1H, dd, J 9.7, 7.2, C22-CH.sub.aH.sub.b), 2.57 (1H, ddd, J 17.9, 14.4, 5.4, C2-CH.sub.aH.sub.b), 2.47-2.37 (1H, m, C2-CH.sub.aH.sub.b), 2.19 (1H, brt, J 10.3, C8-CH), 2.07 (1H, dt, J 12.9, 3.3), 2.00 (1H, dd, J 8.5, 2.1), 1.94-1.63 (3H, m), 1.60-1.15 (9H, m), 1.11 (3H, s, C19-CH.sub.3), 1.00 (3H, d, J 6.7, C21-CH.sub.3), 0.89 (9H, s, SiC(CH.sub.3).sub.3), 0.77 (3H, s, C18-CH.sub.3), 0.03 (6H, s, Si(CH.sub.3).sub.2); δC (100 MHz, CDCl.sub.3); 199.6, 163.9, 141.5, 127.8, 123.5, 67.7, 53.2, 52.5, 50.7, 43.5, 39.4, 39.0, 37.8, 36.1, 34.0, 33.9, 27.6, 25.9, 25.9, 25.9, 23.9, 20.7, 18.4, 16.9, 16.3, 12.0, −5.3, −5.4; (IR) v.sub.max(cm.sup.−1): 3027, 2956, 2930, 2891, 2857, 1677, 1077, 753; HRMS (ESI-TOF) m/z: (M+H).sup.+ calculated for C.sub.28H.sub.46O.sub.2Si 442.3267, found 443.3338.
E. Synthesis of (20S)-20-formyl-pregna-4,6-dien-3-one
[0282] ##STR00037##
[0283] (20S)-20-Hydroxymethyl-pregna-4,6-dien-3-one (3.01 g, 9.16 mmol) was dissolved in anhydrous CH.sub.2Cl.sub.2 (60 ml) and the solution was cooled to 0° C. Dess-Martin periodinane (5.83 g, 13.7 mmol) was added portion-wise over 10 minutes and the reaction was allowed to slowly warm to room temperature and was stirred for 22 h. The mixture was cooled to 0° C. and a 1:1 mixture of 10% aq. Na.sub.2S.sub.2O.sub.3 and 2% aq. NaHCO.sub.3 (75 ml) was added portionwise. CH.sub.2Cl.sub.2 (50 mL) was added and the layers were separated. The aqueous phase was extracted with CH.sub.2Cl.sub.2 (2×50 mL) and the combined organics were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-25% EtOAc in heptane) to give the desired product (1.23 g, 41%) as a pale yellow solid. δH (400 MHz, CDCl.sub.3); 9.59 (1H, d, J 3.2, CHO), 6.12 (2H, s, C6-CH and C7-CH), 5.68 (1H, s, C4-CH), 2.58 (1H, ddd, J 17.9, 14.4, 5.4), 2.49-2.36 (2H, m), 2.22 (1H, t, J 10.6, C8-CH), 2.08-1.81 (4H, m), 1.73 (1H, td, J 13.8, 5.1, C1-CH.sub.aH.sub.b), 1.65-1.20 (8H, m), 1.15 (3H, d, J 6.9, C21-CH.sub.3), 1.13 (3H, s, C19-CH.sub.3), 0.82 (3H, d, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 204.6, 199.5, 163.6, 140.8, 128.1, 123.7, 52.8, 50.8, 50.7, 49.4, 44.0, 39.2, 37.6, 36.0, 33.9, 33.9, 27.0, 24.1, 20.6, 16.3, 13.5, 12.3; (IR) v.sub.max(cm.sup.−1): 3030, 2934, 2706, 1717, 1655, 1615, 15811; HRMS (ESI-TOF) m/z: (M+H).sup.+ calculated for C.sub.22H.sub.30O.sub.2 326.2246; found 327.2318.
F. Synthesis of (20S)-20-(ethylenedioxymethyl)-pregna-4,6-dien-3-one
[0284] ##STR00038##
[0285] To a solution of (20S)-20-formyl-pregna-4,6-dien-3-one (3.89 g, 12 mmol) in CH.sub.2Cl.sub.2 (5 vol, 20 mL) under an argon atmosphere was added 1,2-bis(trimethylsilyloxy) ethane (2.94 mL, 12 mmol). The reaction mixture was cooled to −78° C. and TMSOTf (108 μL, 0.6 mmol) was added. After 2 h the reaction mixture was diluted with CH.sub.2Cl.sub.2 (100 mL) and washed with water (2×100 mL) and 5% aq. NaCl (100 mL). The organic phase was dried over Na.sub.2SO.sub.4 and was concentrated under reduced pressure. Purification by column chromatography on silica gel gave the desired product (2.42 g, 55%) as a colourless crystalline solid. δH (700 MHz, CDCl.sub.3); 6.12 (2H, m), 5.67 (1H, m), 4.86 (1H, d, J 2.0), 3.94 (2H, m), 3.86 (2H, m), 2.56 (1H, m), 2.43 (1H, m), 2.19 (1H, t, J 10.6), 2.05-1.95 (3H, m), 1.85 to 1.20 (12H, m), 1.11 (3H, s), 0.95 (3H, d, J=6.7), 0.77 (3H, s). δC (176 MHz, CDCl.sub.3); 199.7, 163.9, 141.4, 127.9, 123.6, 105.6, 65.3, 65.1, 52.9, 52.2, 50.6, 43.7, 39.3, 39.3, 37.8, 36.1, 34.0, 33.9, 27.3, 23.9, 20.67, 16.3, 11.7, 11.6.
G. Synthesis of (20S)-20-(1-aminomethyl)-pregna-4,6-dien-3-one
[0286] ##STR00039##
Synthesis of (20S)-tosyloxymethyl-pregna-4,6-dien-3-one
[0287] ##STR00040##
[0288] To a solution of (20S)-hydroxymethyl-pregna-4,6-dien-3-one (1.50 g, 4.58 mmol) in pyridine (50 mL) at 0° C. was added p-toluenesulfonyl chloride (1.79 g, 9.39 mmol). The reaction was stirred at 0° C. for 1 h and ambient for 17 h. The reaction was quenched with 1 M aq. HCl (75 mL) and was diluted with ethyl acetate (150 mL). The organic phase was separated and washed with water (50 mL), 5% aq. sodium bicarbonate (75 mL), 5% aq. NaCl (50 mL) and was concentrated in vacuo. The residue was purified by column chromatography on silica gel (heptane-EtOAc) to give the desired product (1.59 g, 72%) as a yellow powder. R.sub.f: 0.36 (3:2, heptane:ethyl acetate); .sup.1H NMR (700 MHz, CDCl.sub.3): δ=7.78 (2H, d, J 8.2, Ar—H), 7.35 (2H, d, J 8.2, Ar—H), 6.10 (2H, br. s, C6H and C7H), 5.67 (1H, s, C4H), 3.97 (1H, dd, J 9.3, 3.2, C22H), 3.80 (1H, dd, J 9.3, 6.4, C22H), 2.56 (1H, ddd, J 17.6, 14.6, 5.6, C2H), 2.45-2.41 (4H, m, C2H and Ts-CH.sub.3), 2.17 (1H, t, J 10.5), 2.01-1.96 (2H, m), 1.80-1.67 (4H, m), 1.54 (1H, dq, J 13.5, 3.1), 1.41 (1H, qd, J 13.1, 3.9), 1.30-1.23 (3H, m), 1.23-1.17 (3H, m), 1.10 (3H, s, C19H), 1.00 (3H, d, J 6.7, C21H), 0.73 (3H, s, C18H). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=197.9, 162.0, 142.9, 139.2, 131.3, 128.0, 126.2, 126.1, 121.9, 73.6, 51.3, 49.9, 48.8, 41.7, 37.4, 35.9, 34.4, 34.3, 32.2, 32.1, 25.6, 21.9, 20.0, 18.8, 15.1, 14.5, 10.1.
(ii) Synthesis of (20S)-azidomethyl-pregna-4,6-dien-3-one
[0289] ##STR00041##
[0290] To a suspension of (20S)-tosyloxymethyl-pregna-4,6-dien-3-one (1.58 g, 3.27 mmol) in DMF (24 mL) and water (59 μL) was added sodium azide (273 mg, 4.20 mmol). The reaction was heated to 70° C. and stirred for 1 h. The reaction was quenched with 2% aq. sodium bicarbonate solution (50 mL) at 40° C., and was diluted with ethyl acetate (100 mL). The layers were separated and the organic layer was washed with 2% aq. sodium bicarbonate (50 mL), 5% aq. NaCl (50 mL) and was concentrated in vacuo. The residue was purified by column chromatography on silica gel (heptane-EtOAc) to give the desired product (1.01 g, 91% yield) as a colourless crystalline solid. R.sub.f: 0.54 (3:2, heptane:ethyl acetate); .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.12 (1H, d, J 9.9, C6H), 6.10 (1H, dd, J 9.9, 2.1, C7H), 5.67 (1H, s, C4H), 3.38 (1H, dd, J 11.9, 3.3, C22H), 3.07 (1H, dd, J 11.9, 7.3, C22H), 2.57 (1H, ddd, J 17.8, 14.7, 5.4, C2H), 2.46-2.41 (1H, m, C2H), 2.17 (1H, t, J 10.6), 2.04 (1H, dt, J 12.8, 3.3), 2.00 (1H, ddd, J 13.2, 5.4, 2.1), 1.93-1.86 (1H, m), 1.86-1.81 (1H, m), 1.75-1.65 (2H, m), 1.56 (1H, dq, J 13.4, 3.7), 1.44 (1H, qd, J 13.0, 4.0), 1.40-1.28 (6H, m), 1.11 (3H, s, C19H), 1.06 (3H, d, J 6.7, C21H), 0.77 (3H, s, C18H). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.9, 163.8, 141.1, 128.0, 123.6, 57.9, 53.2, 53.0, 50.6, 43.6, 39.3, 37.7, 36.9, 36.0, 34.0, 33.9, 27.8, 23.8, 20.6, 17.8, 16.3, 12.0.
(iii) Synthesis of (20S)-aminomethyl-pregna-4,6-dien-3-one
[0291] ##STR00042##
[0292] To a solution of (20S)-azidomethyl-pregna-4,6-dien-3-one (99 mg, 0.29 mmol) and triphenylphosphine (106 mg, 0.40 mmol) in THF (1.1 mL) under an argon atmosphere, acetone (300 μL) was added. The reaction mixture was stirred at 18° C. for 64 h. The reaction mixture was diluted with EtOAc (10 mL) and aq. hydrochloric acid solution (10 mL, 2M). The aq. phase was basified with aq. sodium hydroxide solution (6.5 mL, 2M) to pH 11, and extracted with EtOAc (10 mL). The organic phase was separated and concentrated in vacuo. The residue was purified by flash chromatography on silica gel to afford (20S)-aminomethyl-pregna-4,6-dien-3-one as an off-white powder (28 mg, 30% yield), R.sub.f 0.23 (4:1, CH.sub.2Cl.sub.2:MeOH); .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.12-6.07 (2H, m, C6H and C7H), 5.67 (1H, s, C4H), 3.05 (1H, dd, J 12.7, 3.1, C22H.sub.aH.sub.b), 2.74 (1H, dd, J 12.7, 8.3, C22H.sub.aH.sub.b), 2.58 (1H, ddd, J 17.9, 14.5, 5.4, C2H.sub.aH.sub.b), 2.46-2.41 (1H, m, C2H.sub.aH.sub.b), 2.18 (1H, t, J 10.5), 2.05-1.94 (3H, m), 1.90-1.81 (2H, m), 1.68 (1H, td, J 13.9, 5.6), 1.55 (1H, dq, J 13.4, 3.4), 1.45-1.17 (9H, m), 1.20 (3H, obscured d, J 6.7, C21H), 1.11 (3H, s, C18H), 0.78 (3H, s, C19H). .sup.13C NMR (140 MHz, CDCl.sub.3): δ=199.5, 163.6, 140.8, 128.0, 123.7, 53.2, 52.8, 50.6, 45.3, 43.6, 39.3, 37.6, 36.0, 36.0, 35.1, 34.0, 33.9, 27.8, 23.7, 20.7, 17.3, 16.3.
H. Synthesis of (20S)-20-(1-mesyloxymethyl)-pregna-4,6-dien-3-one
[0293] ##STR00043##
[0294] To a solution of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (1.00 g, 3.05 mmol) in pyridine (10 mL) was added DMAP (19 mg, 0.15 mmol). MsCl (1.18 mL, 15.2 mmol) was added dropwise and the reaction was stirred at room temperature for 18 h. The reaction was cooled in an ice bath and water (10 mL) was added dropwise. EtOAc (20 mL) was added and the layers were separated. The aqueous layer was extracted with EtOAc (3×20 mL). The combined organic phases were washed with 2 M aq. HCl (20 mL), dried over sodium sulfate and were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-50% EtOAc in heptane) to give the desired product (1.01 g, 82%) as an orange solid. δH (400 MHz, CDCl.sub.3); 6.12 (2H, brs, C6-CH and C7-CH), 5.68 (1H, s, C4-CH), 4.21 (1H, dd, J 9.4, 3.2, C22-CH.sub.aH.sub.b), 4.01 (1H, dd, J 9.4, 6.6, C22-CH.sub.aH.sub.b), 3.01 (3H, s, OS(O.sub.2)CH.sub.3), 2.58 (1H, ddd, J 18.0, 14.4, 5.5, C2-CH.sub.aH.sub.b), 2.49-2.39 (1H, m, C2-CH.sub.aH.sub.b), 2.21 (1H, brt, J 10.5, C8-CH), 2.09-1.80 (5H, m), 1.73 (1H, td, J 13.8, 5.2, C1-CH.sub.aH.sub.b), 1.63-1.53 (1H, m), 1.52-1.18 (7H, m), 1.13 (3H, s, C19-CH.sub.3), 1.12 (3H, d, J 6.1, C21-CH.sub.3), 0.80 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.5, 163.6, 140.9, 128.0, 123.7, 74.8, 53.1, 51.8, 50.6, 43.6, 39.3, 37.7, 37.2, 36.3, 36.0, 33.9, 33.9, 27.5, 23.8, 20.6, 16.9, 16.3, 12.0.
I. Synthesis of (20R)-20-(1-cyanomethyl)-pregna-4,6-dien-3-one
[0295] ##STR00044##
(i) Synthesis of (20S)-20-bromomethyl-4-pregnen-3-one
[0296] ##STR00045##
[0297] To a solution of (20S)-hydroxymethyl-4-pregnen-3-one (50 g, 0.15 mol) in CH.sub.2Cl.sub.2 (350 mL) at 0° C. was added triphenylphosphine (43.6 g, 0.17 mol). N-bromosuccinimide (29.6 g, 0.17 mol) was added portionwise and the reaction mixture was stirred at 18° C. After 18 h, the reaction mixture was cooled to 0° C. and triphenylphosphine (19.8 g, 0.08 mol) was added, followed by N-bromosuccinimide (13.5 g, 0.08 mol) portionwise. The mixture was warmed to 18° C. After 2 h the reaction mixture was washed with water (350 mL) and the aqueous phase extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic phases were washed with 5% aq. sodium bicarbonate (350 mL), and the aqueous phase extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were washed with 5% aq. sodium chloride (150 mL), dried over sodium sulfate and were concentrated in vacuo. The residue was purified by column chromatography on silica gel (heptane-EtOAc) to give the desired product (47.1 g, 79%) as a yellow solid. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.72 (1H, s), 3.50 (1H, dd, J=9.8, 2.7, C22-CH.sub.aH.sub.b), 3.35 (1H, dd, J=9.8, 5.9, C22-CH.sub.aH.sub.b), 2.45-2.32 (3H, m), 2.27 (1H, ddd, J=14.6, 4.1, 2.5), 2.04-1.98 (2H, m), 1.91-1.82 (2H, m), 1.72-1.64 (3H, m), 1.56-1.50 (2H, m), 1.43 (1H, qd, J=13.1, 4.1), 1.33-1.27 (2H, m), 1.22 (1H, dd, J=13.0, 4.2), 1.20-1.13 (1H, m), 1.18 (3H, s), 1.09 (3H, d, J=6.4), 1.09-1.00 (2H, m), 0.94 (1H, ddd, J=12.3, 10.9, 4.1), 0.74 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=197.5, 169.3, 121.8, 53.5, 51.6, 51.6, 41.4, 40.4, 37.3, 36.5, 35.7, 33.6, 33.6, 31.9, 30.8, 29.9, 25.5, 22.0, 18.9, 16.6, 15.3, 10.3.
(ii) Synthesis of (20R)-cyanomethyl-4-pregnen-3-one
[0298] ##STR00046##
[0299] To a suspension of (20S)-20-bromomethyl-4-pregnen-3-one (15 g, 38.1 mmol) in DMF (225 mL) was added potassium cyanide (7.5 g, 114 mmol). The suspension was stirred at 80° C. for 41 h before cooling to room temperature. EtOAc (250 mL) and water (500 mL) were added and the layers were separated. The aqueous layer was extracted with EtOAc (2×250 mL) and the combined organic phases were washed with 5% aq. NaCl (250 mL) and were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (heptane/EtOAc) to afford the desired product (9.7 g, 75%) as a white solid. δH (700 MHz, CDCl.sub.3); 5.73 (1H, s, C4-CH), 2.45-2.32 (4H, m), 2.27 (1H, ddd, J=14.6, 4.2, 2.7), 2.24 (1H, dd, J=16.8, 7.1), 2.04-1.99 (2H, m), 1.89-1.78 (3H, m), 1.72-1.65 (2H, m), 1.57-1.51 (2H, m), 1.43 (1H, qd, J=13.2, 4.0), 1.31-1.16 (4H, m), 1.18 (3H, s), 1.17 (3H, d, J=6.7), 1.11-1.01 (2H, m), 0.94 (1H, ddd, J=12.3, 10.7, 4.1), 0.74 (3H, s); δC (176 MHz, CDCl.sub.3); 199.5, 171.2, 123.9, 118.9, 55.7, 54.7, 53.6, 42.5, 39.2, 38.5, 35.7, 35.6, 34.0, 33.6, 32.8, 31.9, 28.0, 24.8, 24.1, 20.9, 19.3, 17.4, 12.1.
(iii) Synthesis of (20R)-cyanomethyl-4,6-pregnadien-3-one
[0300] ##STR00047##
[0301] To a suspenstion of (20R)-cyanomethyl-4-pregnen-3-one (9.1 g, 26.8 mmol) in toluene (36 mL) and acetic acid (0.15 mL) was added p-chloranil (7.2 g, 39.5 mmol). The mixture was heated at reflux for 90 minutes before allowing to cool to room temperature. The suspension was filtered, washing with toluene (25 mL). The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (heptane/EtOAc). The material was then dissolved in acetone (35 mL) and methanol (23 mL) and 0.5 M aq. NaOH (200 mL) was added dropwise. Water (100 mL) was added and the resulting solid was filtered, washing with water (2×50 mL) and 2:1 acetone:water (2×20 mL). The solid was dried in vacuo to afford the desired product (5.4 g, 60%) as a pale brown solid. δH (700 MHz, CDCl.sub.3); 6.11 (2H, s), 5.67 (1H, s), 2.57 (1H, ddd, J=18.0, 14.4, 5.4), 2.45-2.42 (1H, m), 2.37 (1H, dd, J=16.7, 3.7), 2.25 (1H, dd, J=16.7, 7.2), 2.01 (1H, t, J=10.4), 2.03 (1H, dt, J=12.8, 3.3), 2.00 (1H, ddd, J=13.2, 5.4, 2.1), 1.96-1.91 (1H, m), 1.88-1.81 (1H, m), 1.74-1.70 (1H, m), 1.58 (1H, dq, J=13.4, 3.6), 1.44 (1H, qd, J=4.4, 3.9), 1.36-1.20 (7H, m), 1.18 (3H, d, J=6.7), 1.11 (3H, s), 0.79 (3H, s); δC (176 MHz, CDCl.sub.3); 199.6, 163.67, 140.8, 128.1, 123.7, 118.8, 54.6, 53.2, 50.5, 43.5, 39.1, 37.6, 36.0, 33.9, 33.9, 33.5, 28.0, 24.8, 23.6, 20.6, 19.3, 16.3, 12.0.
J. Synthesis of (20S)-20-(1-bromomethyl)-pregna-4,6-dien-3-one
[0302] ##STR00048##
[0303] To a solution of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (1.00 g, 3.05 mmol) in anhydrous CH.sub.2Cl.sub.2 (10 mL) was added carbon tetrabromide (1.52 g, 4.57 mmol). Triphenylphosphine (1.20 g, 4.57 mmol) was added and the mixture was heated at reflux for 2 h. The reaction was allowed to cool to room temperature and water (20 mL) was added. The layers were separated and the organic layer was washed with 5% aq. NaHCO.sub.3 (20 mL), 10% aq NaCl (20 mL) and was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-25% acetone in heptane) to give the desired product (980 mg, 82%) as a light yellow crystalline solid. δH (400 MHz, CDCl.sub.3); 6.09-6.00 (2H, m, C6-CH and C7 CH), 5.59 (1H, s, C4-CH), 3.43 (1H, dd, J 9.8, 2.7, C22-CH.sub.aH.sub.b), 3.29 (1H, dd, J 9.8, 5.8, C22-CH.sub.aH.sub.b), 2.50 (1H, ddd, J 17.9, 14.4, 5.4, C2-CH.sub.aH.sub.b), 2.40-2.30 (1H, m, C2-CH.sub.aH.sub.b), 2.13 (1H, brt, J 9.8, C8-CH), 2.01-1.57 (5H, m), 1.55-1.45 (1H, m), 1.44-1.10 (8H, m), 1.05 (3H, s, C19-CH.sub.3), 1.03 (3H, d, J 6.5, C21-CH.sub.3), 0.72 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.2, 163.6, 141.0, 127.9, 123.6, 53.5, 53.1, 50.6, 43.4, 43.3, 39.2, 37.7, 37.6, 36.0, 33.9, 33.9, 27.4, 23.6, 20.6, 18.6, 16.3, 12.3.
K. Synthesis of 23-ethoxyformyl-3-oxo-4,6-choladien-24-oic Acid Ethyl Ester
[0304] ##STR00049##
[0305] Sodium hydride (60% dispersion in mineral oil, 226 mg, 5.64 mmol) was suspended in anhydrous THF (10 mL) and the mixture was cooled to 0° C. Diethyl malonate (1.17 mL, 7.68 mmol) was added drop-wise and the mixture was stirred at 0° C. for 15 minutes. A solution of (20S)-20-(bromomethyl)-pregna-4,6-dien-3-one (1.00 g, 2.56 mmol) in anhydrous THF (10 mL) was added drop-wise and the reaction was heated at reflux for 18 h. The reaction was allowed to cool to room temperature and water (10 mL) was added. EtOAc (25 mL) was added and the layers were separated. The aqueous layer was extracted with EtOAc (3×50 mL) and the combined organics were washed with 10% aq. NaCl (50 mL), dried over sodium sulfate and were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-25% acetone in heptane) to give the desired product (1.00 g, 83%) as a clear oil. δH (400 MHz, CDCl.sub.3); 6.17-6.07 (2H, m, C6-CH and C7-CH), 5.67 (1H, s, C4-CH), 4.29-4.14 (4H, m, 2×C(O)OCH.sub.2), 3.44 (1H, dd, J 10.9, 3.7, EtO.sub.2CCH), 2.57 (1H, ddd, J 17.9, 14.4, 5.4, C2-CH.sub.aH.sub.b), 2.43 (1H, dddd, J 17.8, 5.1, 2.0, 0.8, C2-CH.sub.aH.sub.b), 2.24-2.12 (2H, m), 2.10-1.93 (3H, m), 1.87-1.77 (1H, m), 1.71 (1H, td, J 16.2, 5.2, C1-CH.sub.aH.sub.b), 1.59-1.35 (4H, m), 1.34-1.14 (12H, m), 1.11 (3H, s, C18-CH.sub.3), 0.96 (3H, d, J 6.2, C21-CH.sub.3), 0.75 (3H, s, C19-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.5, 170.0, 169.6, 163.8, 141.3, 127.9, 123.6, 61.4, 61.2, 56.2, 53.4, 50.6, 49.8, 43.5, 39.5, 37.7, 36.1, 35.0, 34.3, 34.0, 33.9, 28.0, 23.7, 20.7, 18.2, 16.3, 14.2, 14.1, 11.9.
L. Synthesis of (20S)-20-(5-Tosyltetrazol-1-yl)methyl-pregna-4,6-dien-3-one
[0306] ##STR00050##
[0307] To a solution of (20S)-azidomethyl-pregna-4,6-dien-3-one (500 mg, 1.41 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added p-toluenesulfonyl cyanide (282 mg, 1.55 mmol). Copper(I) trifluoromethanesulfonate benzene complex (71 mg, 0.141 mmol) was added and the mixture was stirred at room temperature for 18 h. Toluene (5 mL), added p-toluenesulfonyl cyanide (128 mg, 0.708 mmol) and copper(I) trifluoromethanesulfonate benzene complex (71 mg, 0.141 mmol) were added and the mixture was heated to 60° C. for 24 h. Water (10 mL) and CH.sub.2Cl.sub.2 (30 mL) were added and the layers were separated. The organic layer was washed with 10% aq. Na.sub.2S.sub.2O.sub.3/2% aq. NaHCO.sub.3 (2×20 mL), 10% aq. NaCl (20 mL), was dried over sodium sulfate and was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-50% EtOAc in heptane) to give the desired product (381 mg, 50%) as a light yellow solid. δH (400 MHz, CDCl.sub.3); 8.03-7.97 (2H, m, ArH), 7.46 (2H, m, ArH), 6.14 (2H, brs, C6-CH and C7-CH), 5.69 (1H, s, C4-CH), 4.80 (1H, dd, J 13.4, 3.9, C22-CH.sub.aH.sub.b), 4.45 (1H, dd, J 13.4, 10.5, C22-CH.sub.aH.sub.b), 2.26-2.53 (1H, m), 2.51 (3H, s, ArCH.sub.3), 2.49-2.28 (2H, m), 2.24 (1H, appt, J, 10.5), 2.13-1.97 (2H, m), 1.96-1.87 (1H, m), 1.79-1.63 (2H, m), 1.53-1.18 (8H, m), 1.13 (3H, s, C19-CH.sub.3), 0.89 (3H, d, J 6.6, C21-CH.sub.3), 0.86 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.5, 163.6, 147.5, 140.8, 134.3, 130.4, 129.3, 128.1, 123.7, 55.1, 53.9, 53.2, 50.7, 44.0, 39.4, 37.8, 37.6, 36.0, 33.9, 33.9, 31.9, 27.5, 23.8, 22.7, 21.9, 20.6, 16.5, 16.3, 12.0.
M. Synthesis of N-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)cyclopropyl Sulfonamide
[0308] ##STR00051##
(i) Synthesis of (22E)-3-Oxo-4,6,22-cholatrien-24-oic Acid
[0309] ##STR00052##
[0310] (22E)-3-Oxo-4,6,22-cholatrien-24-oic acid ethyl ester (10 g, 25.2 mmol) was suspended in IPA (100 mL) and the mixture was heated to 60° C. 0.5 M aq. NaOH (60 mL, 30 mmol) was added and the mixture was stirred at 60° C. for 3 h. The volatiles were removed under reduced pressure and EtOAc (250 mL) was added. The mixture was acidified to pH 1 using 2 M aq. HCl, and further EtOAc (100 mL) was added. The layers were separated and the organic layer was washed with water (3×100 mL) and concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) with heating and was then cooled to −20° C. for 18 h. The solid formed was filtered, washing with EtOAc (20 mL). The solid was then dried under reduced pressure to give the desired product (4.55 g, 49%) as a tan solid. δH (400 MHz, CDCl.sub.3); 6.94 (1H, dd, J 15.6, 9.0, C23-CH), 6.11 (2H, brs, C6-CH and C7-CH), 5.77 (1H, dd, J 15.6, 0.6, C22-CH), 5.68 (1H, s, C4-CH), 2.58 (1H, ddd, J 18.0, 14.4, 5.4, C2-CH.sub.aH.sub.b), 2.51-2.40 (1H, m, C2-CH.sub.aH.sub.b), 2.40-2.28 (1H, m), 2.21 (1H, appt, J 10.1), 2.10-1.95 (2H, m), 1.89-1.65 (3H, m), 1.64-1.53 (1H, m), 1.53-1.39 (1H, m), 1.38-1.18 (7H, m), 1.12 (3H, s, C19-CH.sub.3), 1.12 (3H, d, J 6.6, C21-CH.sub.3), 0.81 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 199.7, 171.8, 163.9, 156.9, 141.1, 128.0, 123.6, 118.6, 54.7, 53.2, 50.7, 43.7, 39.7, 39.3, 37.7, 36.1, 33.9, 33.9, 27.8, 23.7, 20.6, 19.1, 16.3, 12.1.
(ii) Synthesis of N-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)cyclopropylsulfonamide
[0311] ##STR00053##
[0312] To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid (2.00 g, 5.43 mmol) in CH.sub.2Cl.sub.2 (40 mL) was added EDCI (1.69 g, 10.9 mmol) and DMAP (1.33 g, 10.9 mmol). Cyclopropane sulfonamide (1.97 g, 16.3 mmol) was added and the reaction was stirred at room temperature for 22 h. Water (25 mL) was added and the layers were separated. The aqueous layer was extracted with CH.sub.2Cl.sub.2 (2×25 mL) and the combined organics were washed with 2 M aq HCl (20 mL), 10% aq. NaCl (10 mL), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (0-10% acetone in toluene) to give the desired product (1.68 g, 66%) as an off-white solid. δH (400 MHz, CDCl.sub.3); 8.90 (1H, s, NH), 6.95 (1H, dd, J 15.5, 9.0, C23-CH), 6.11 (2H, brs, C6-CH and C7-CH), 5.86 (1H, dd, J 15.5, 0.5, C22-CH), 5.68 (1H, s, C4-CH), 3.00 (1H, dddd, J 12.8, 9.5, 8.1, 4.8, SO.sub.2CH), 2.64 (1H, ddd, J 18.1, 14.4, 5.4, C2-CH.sub.aH.sub.b), 2.51-2.41 (1H, m, C2-CH.sub.aH.sub.b), 2.40-2.28 (1H, m), 2.25-2.15 (1H, m), 2.09-1.96 (2H, m), 1.85-1.64 (3H, m), 1.63-1.52 (1H, m), 1.51-1.17 (9H, m), 1.17-1.07 (5H, m), 1.12 (3H, s, C19-CH.sub.3), 0.80 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 200.0, 164.2, 164.1, 155.5, 141.3, 127.9, 123.6, 119.4, 54.7, 53.2, 50.6, 43.8, 39.8, 39.3, 37.8, 36.1, 33.9, 33.9, 31.5, 28.1, 23.7, 20.6, 19.1, 16.3, 12.2, 6.3, 6.3.
N. Synthesis of N-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)-4-(trifluoromethoxy)benzenesulfonamide
[0313] ##STR00054##
[0314] To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid (2.00 g, 5.43 mmol) in CH.sub.2Cl.sub.2 (40 mL) was added EDCI (1.69 g, 10.9 mmol) and DMAP (1.33 g, 10.9 mmol). 4-(Trifluoromethoxy)benzene sulfonamide (3.93 g, 16.3 mmol) was added and the reaction was stirred at room temperature for 22 h. Water (25 mL) was added and the layers were separated. The aqueous layer was extracted with CH.sub.2Cl.sub.2 (2×25 mL) and the combined organics were washed with 2 M aq HCl (20 mL), 10% aq. NaCl (10 mL), dried over sodium sulfate and concentrated under reduced pressure. The residue was used in the next step without purification. A portion was purified by column chromatography on silica gel (0-50% EtOAc in heptane) to give the desired product as an off-white solid. δH (400 MHz, MeOD); 8.16-8.11 (2H, m, ArH), 7.52-7.46 (2H, m, ArH), 6.82 (1H, dd, J 15.4, 9.0, C23-CH), 6.20 (1H, brdd, J 9.8, 1.4, C6-CH), 6.15 (1H, dd, J 9.9, 1.4, C7-CH), 5.82 (1H, dd, J 15.4, 0.7, C22-CH), 5.64 (1H, s, C4-CH), 2.62 (1H, ddd, J 18.2, 14.5, 5.4, C2-CH.sub.aH.sub.b), 2.42-2.20 (3H, m), 2.12-1.98 (2H, m), 1.88-1.63 (3H, m), 1.63-1.55 (1H, m), 1.49 (1H, dd, J 12.6, 3.8), 1.40-1.18 (7H, m), 1.14 (3H, s, C19-CH.sub.3), 1.08 (3H, d, J 6.6, C21-CH.sub.3), 0.81 (3H, s, C18-CH.sub.3); δC (100 MHz, MeOD); 202.3, 167.2, 165.9, 156.7, 154.0, 143.3, 139.7, 131.8, 128.8, 123.9, 123.0 (q, J 254), 121.9, 120.6, 56.0, 54.6, 52.2, 44.9, 40.9, 40.6, 39.1, 37.4, 35.0, 34.7, 30.2, 29.0, 24.7, 21.7, 19.5, 16.6, 12.5.
O. Synthesis of (20S)—(N-benzyl)aminomethyl-pregna-4,6-dien-3-one
[0315] ##STR00055##
[0316] (20S)-Formyl-pregna-4,6-dien-3-one (98 mg, 0.30 mmol) and benzylamine (21 μL, 0.30 mmol) were dissolved in 1,2-dichloroethane (1.0 mL) under an argon atmosphere. Sodium triacetoxyborohydride (96 mg, 0.45 mmol) was added. The reaction mixture was stirred at 20° C. for 2 h, then quenched with aq. sodium bicarbonate solution (5%, 2 mL). The mixture was diluted with EtOAc (10 mL) and water (5 mL). The aq. phase was separated and extracted with EtOAc (2×5 mL). The organic phases were combined and concentrated in vacuo. The residue was purified by silica column chromatography (heptane-EtOAc) to yield (20S)—(N-benzyl)aminomethyl-pregna-4,6-dien-3-one as a beige powder (51 mg, 41% yield). R.sub.f 0.15 (EtOAc); .sup.1H NMR (500 MHz, CDCl.sub.3): δ=7.34 (4H, d, J 4.5, Bn-CH), 7.29-7.23 (1H, m, Bn-CH), 6.15 (1H, d, J 10.2, C6), 6.11 (1H, dd, J 9.6, 2.0, C7H), 5.68 (1H, s, C4H), 3.84 (1H, d, J 13.1, Bn-CH.sub.aH.sub.b), 3.75 (1H, d, J 13.1, Bn-CH.sub.aH.sub.b), 2.69 (1H, dd, J 11.6, 3.0, C22H.sub.aH.sub.b), 2.58 (1H, ddd, J 17.2, 14.5, 5.3, C2H.sub.aH.sub.b), 2.44 (1H, dd, J 17.4, 4.4, C2H.sub.aH.sub.b), 2.35 (1H, dd, J 11.5, 8.3, C22H.sub.aH.sub.b), 2.20 (1H, t, J 10.7, H8), 2.07 (1H, dt, J 12.6, 3.0), 2.04-1.97 (1H, m, C1H.sub.aH.sub.b), 1.92-1.68 (3H, m), 1.68-1.60 (1H, m, C20H), 1.60-1.52 (1H, m), 1.44 (1H, qd, J 12.8, 3.9), 1.40-1.18 (7H, m), 1.13 (3H, s, C18H), 1.04 (3H, d, J 6.6, C21H), 0.78 (3H, s, C19H). .sup.13C NMR (126 MHz, CDCl.sub.3): δ=199.7, 164.0, 141.4, 140.5, 128.4, 128.1, 127.8, 126.9, 123.5, 54.9, 54.2, 54.0, 53.3, 50.7, 43.5, 39.5, 37.7, 36.5, 36.0, 34.0, 33.9, 27.9, 23.8, 20.7, 17.8, 16.3, 12.0.
Example 2—Preparation of Compounds of General Formula (IA)
A. Epoxidation of (22E)-3-oxo-4,6,22-cholatrien-24-oic Acid Ethyl Ester Using Methyltrioxorhenium to Form (6α, 7α, 22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic Acid Ethyl Ester
[0317] ##STR00056##
[0318] To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid ethyl ester (5.00 g, 12.6 mmol) in HFIP (20 mL, 4 volumes) and EtOAc (10 mL, 2 volumes) was added MTO (37 mg, 0.126 mmol) and 3-methylpyrazole (122 μl, 1.51 mmol) and the mixture was cooled to 5° C. UHP (1.30 g, 13.9 mmol) was added portion-wise and the mixture was stirred at 5° C. for 24 h. After 24 h, a second addition of MTO (37 mg, 0.126 mmol) and UHP (1.30 g, 13.9 mmol) was conducted and the reaction was stirred at 5° C. for 18 h. The reaction was then quenched by the portion-wise addition of 12% aq. NaHSO.sub.3 (15 mL) maintaining the temperature <25° C. The mixture stirred for 0.5 h whilst warming to ambient temperature, to ensure all peroxide was quenched. Water (12.5 mL) and EtOAc (5 mL) were added and the layers separated. The organic phase was washed with 5% aq. NaHCO.sub.3 (20 mL), water (20 mL) and then concentrated under reduced pressure. The crude material (5.72 g) was crystallised from EtOAc (15 mL).
Further Epoxidation Reactions of Compounds of Formula (II)
General Procedure A: MTO Catalyzed Epoxidation
[0319] To a solution of a compound of general formula (II) (1 eq.) and MTO (1 mol %) in EtOAc (2 vol) and HFIP (4 vol) was added 3-methylpyrazole (0.12 eq.) and the mixture was cooled to 5° C. UHP (1.1 eq) was added and the mixture was stirred for 18-50 h until deemed complete by TLC analysis. The reaction mixture was then quenched with the addition of 12% aq. NaHSO.sub.3 (3 vol) then partitioned between water (2.5 vol) and EtOAc (1 vol). The phases were separated and the organic phase washed with 5% aq. NaHCO.sub.3 (4 vol) and water (4 vol). After concentration under reduced pressure the crude residue was purified by column chromatography (SiO.sub.2, eluting with heptane: EtOAc gradient).
B. Epoxidation of (20S)-20-Hydroxymethyl-Pregna-4,6-Dien-3-One to Form (6α, 7α, 20S)-6,7-epoxy-20-hydroxymethyl-pregn-4-en-3-one
[0320] ##STR00057##
[0321] (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (500 mg, 1.52 mmol) was epoxidized using MTO according to the General Procedure A to yield the title compound (210 mg, 40%) as a light yellow solid.
[0322] δH (400 MHz, CDCl.sub.3); 6.11 (1H, s, C4-CH), 3.66 (1H, dd, J 10.4, 3.3, C22-CH.sub.aH.sub.b), 3.45 (1H, d, J 3.7, C6-CH), 3.42-3.32 (2H, m, C7-CH and C22-CH.sub.aH.sub.b), 2.56 (1H, ddd, J 18.2, 14.1, 5.5, C2-CH.sub.aH.sub.b), 2.45 (1H, dddd, J 18.0, 5.3, 2.0, 0.8, C2-CH.sub.aH.sub.b), 2.02 (1H, dt, J 12.8, 2.7, C12-CH.sub.aH.sub.b), 1.98-1.83 (4H, m), 1.71 (1H, td, J 13.6, 5.5, C1-CH.sub.aH.sub.b), 1.65-1.16 (10H, m), 1.10 (3H, s, C19-CH.sub.3), 1.06 (3H, d, J 6.6, C21-CH.sub.3), 0.77 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 198.3, 162.7, 131.1, 67.8, 54.6, 52.5, 52.5, 51.1, 43.2, 40.6, 39.2, 38.8, 35.6, 34.7, 34.1, 33.9, 27.8, 23.8, 19.9, 17.2, 16.7, 11.9.
C. Epoxidation of (20S)-20-(1-bromomethyl)-pregna-4,6-dien-3-one to form (6α, 7α, 20S)-20-(1-bromomethyl)-6,7-epoxy-pregn-4-en-3-one
[0323] ##STR00058##
[0324] (20S)-20-(1-bromomethyl)-pregna-4,6-dien-3-one (500 mg, 1.28 mmol) was epoxidized using MTO according to General Procedure A to yield the title compound (290 mg, 56%) as a light brown solid.
[0325] δH (400 MHz, CDCl.sub.3); 6.12 (1H, s, C4-CH), 3.52 (1H, dd, J 9.8, 2.6, C22-CH.sub.aH.sub.b), 3.46 (1H, d, J 3.7, C6-CH), 3.39-3.17 (2H, m, C7-CH and C22-CH.sub.aH.sub.b), 2.56 (1H, ddd, J 18.1, 14.0, 5.4, C2-CH.sub.aH.sub.b), 2.47 (1H, dddd, J 18.0, 5.5, 2.2, 0.9, C2-CH.sub.aH.sub.b), 2.05-1.84 (5H, m), 1.79-1.66 (2H, m), 1.58-1.46 (1H, m), 1.44-1.19 (7H, m), 1.11 (3H, d, J 6.3, C21-CH.sub.3), 1.10 (3H, s, C19-CH.sub.3), 0.78 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 198.2, 162.6, 131.2, 54.5, 53.5, 52.5, 51.2, 43.1, 43.0, 40.6, 39.0, 37.8, 35.6, 34.7, 34.1, 33.9, 27.6, 34.6, 19.9, 18.6, 17.2, 12.2.
D. Epoxidation of (20S)-20-(1-mesyloxymethyl)-pregna-4,6-dien-3-one to form (6α, 7α, 20S)-20-(1-mesyloxymethyl)-6,7-epoxy-pregn-4-en-3-one
[0326] ##STR00059##
[0327] (20S)-20-(1-mesyloxymethyl)-pregna-4,6-dien-3-one (500 mg, 1.24 mmol) was epoxidized using MTO according to General Procedure A to yield the title compound (460 mg, 88%) as a light yellow solid.
[0328] δH (400 MHz, CDCl.sub.3); 6.12 (1H, s, C4-CH), 4.22 (1H, dd, J 9.4, 3.2, C22-CH.sub.aH.sub.b), 3.99 (1H, dd, J 9.4, 6.9, C22-CH.sub.aH.sub.b), 3.46 (1H, brd, J 3.7, C6-CH), 3.34 (1H, brd, J 3.6, C7-CH), 3.01 (3H, S, OS(O.sub.2)CH.sub.3), 2.56 (1H, ddd, J 18.2, 14.1, 5.5, C2-CH.sub.aH.sub.b), 2.50-2.41 (1H, m), 2.05-1.80 (6H, m), 1.72 (1H, td, J 13.6, 5.6, C1-CH.sub.aH.sub.b), 1.65-1.17 (8H, m), 1.11 (3H, d, J 6.5, C21-CH.sub.3), 1.10 (3H, s, C19-CH.sub.3), 0.76 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 198.2, 162.5, 131.2, 74.7, 54.5, 52.5, 51.8, 51.1, 43.3, 40.6, 39.1, 37.3, 36.4, 35.6, 34.7, 34.1, 33.9, 27.7, 23.7, 19.9, 17.2, 16.8, 11.9.
E. Epoxidation of (20S)-20-(1-tertbutyldimethylsilyloxymethyl)-pregna-4,6-dien-3-one to form (6α, 7α, 20S)-20-(1-tert-butyldimethylsilyloxymethyl)-6,7-epoxy-pregn-4-en-3-one
[0329] ##STR00060##
[0330] (20S)-20-(1-tertbutyldimethylsilyloxymethyl)-pregna-4,6-dien-3-one (500 mg, 1.13 mmol) was epoxidized using MTO according to General Procedure A to yield the title compound (100 mg, 19%) as a light brown solid.
[0331] δH (400 MHz, CDCl.sub.3); 6.11 (1H, s, C4-CH), 3.58 (1H, dd, J 9.6, 3.3, C22-CH.sub.aH.sub.b), 3.45 (1H, d, J 3.7, C6-CH), 3.42 (1H, brd, J 3.5, C7-CH), 3.28 (1H, dd, J 9.6, 7.2, C22-CH.sub.aH.sub.b), 2.55 (1H, ddd, J 18.2, 14.1, 5.5, C2-CH.sub.aH.sub.b), 2.49-2.40 (1H, m, C2-CH.sub.aH.sub.b), 2.02 (1H, td, J 12.8, 3.0, C12-CH.sub.aH.sub.b), 1.98-1.82 (4H, m), 1.71 (1H, td, J 13.6, 5.5, C1-CH.sub.aH.sub.b), 1.61-1.14 (9H, m), 1.10 (3H, s, C19-CH.sub.3), 1.00 (3H, d, J 6.6, C21-CH.sub.3), 0.89 (9H, s, SiC(CH.sub.3).sub.3), 0.75 (3H, s, C18-CH.sub.3), 0.06 (6H, d, J 0.6, 2×SiCH.sub.3); δC (100 MHz, CDCl.sub.3); 198.3, 162.8, 131.1, 67.7, 54.7, 52.6, 52.3, 51.1, 43.1, 40.7, 39.2, 39.0, 35.6, 34.7, 34.1, 33.9, 27.8, 26.0, 26.0, 26.0, 23.8, 19.9, 18.4, 17.2, 16.9, 11.9, −5.3, −5.4.
F. Epoxidation of (20S)-20-acetoxymethyl-pregna-4,6-dien-3-one to form (6α, 7α, 20S)-20-acetoxymethyl-6,7-epoxy-pregn-4-en-3-one
[0332] ##STR00061##
[0333] The product was prepared according to the general procedure for MTO catalysed epoxidation on 200 g scale, isolated in 50% yield (105 g) as a tan solid.
[0334] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.11 (1H, s), 4.09 (1H, dd, J 10.7, 3.4), 3.79 (1H, dd, J 10.7, 7.4), 3.45 (1H, d, J 3.7), 3.34 (1H, d, J 3.5), 2.55 (1H, m), 2.46 (1H, m), 2.05 (3H, s), 2.02-1.85 (5H, m), 1.78-1.68 (2H, m), 1.55-1.20 (8H, m), 1.10 (3H, s), 1.02 (3H, d, J 6.6), 0.76 (3H, s); .sup.13C NMR (175 MHz, CDCl.sub.3): δ=198.3, 171.3, 162.7, 131.1, 69.3, 54.6, 52.5, 52.4, 51.1, 43.2, 40.6, 39.1, 35.8, 35.6, 34.6, 34.1, 33.9, 27.7, 23.7, 21.0, 19.9, 17.2, 17.1, 11.8.
G. Epoxidation of (20S)-20-(ethylenedioxymethyl)-pregna-4,6-dien-3-one (Example 1F) to form (6α, 7α, 20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregn-4-en-3-one
[0335] ##STR00062##
[0336] (20S)-20-(ethylenedioxymethyl)-pregna-4,6-dien-3-one (3.15 g, 8.5 mmol) and BHT (57 mg, 0.26 mmol) were charged to a flask under argon, followed by EtOAc (8 vol, 25 mL) and water (2.5 vol, 7.9 mL) and the mixture heated to 80° C. mCPBA 70% (3.69 g, 15 mmol) in EtOAc (5 vol, 16 mL) was added dropwise over 10 minutes and the reaction mixture then stirred at 70° C. for 1 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain). The reaction mixture was allowed to cool to room temperature and washed with 1M aq. NaOH (3×50 mL) and 10% aq. Na.sub.2S.sub.2O.sub.3 (3×50 mL). After a negative test for peroxides the organic phase was dried over Na.sub.2SO.sub.4 and concentrated in-vacuo at 40° C. Purification by column chromatography and concentration in-vacuo at 40° C. gave (6α, 7α, 20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregna-4-en-3-one as a white crystalline solid (1.15 g). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.31 (1H, s), 4.85 (1H, d, J 2.0), 4.0-3.8 (2H, m), 3.45 (1H, d, J 3.7), 3.35 (1H, d, J 3.6), 2.59-2.43 (2H, m), 2.05-1.68 (8H, m), 1.55-1.20 (10H, m), 1.10 (3H, s), 0.93 (3H, d, J 6.6), 0.75 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=198.6, 163.0, 131.0, 105.9, 65.2, 65.0, 54.7, 52.5, 51.9, 50.8, 43.4, 40.6, 39.3, 39.0, 35.6, 34.6, 34.1, 33.8, 27.4, 23.8, 19.9, 17.2, 11.6, 11.6.
H. Epoxidation of (20S)-azidomethyl-pregna-4,6-dien-3-one to form (6α, 7α, 20S)-6,7-epoxy-20-azidomethyl-pregna-4-en-3-one
[0337] ##STR00063##
[0338] To a solution of (20S)-azidomethyl-pregna-4,6-dien-3-one (203 mg, 0.598 mmol) and 3-methylpyrazole (3 μL, 0.04 mmol) in HFIP (0.8 mL) under argon atmosphere at 10° C., MTO (3.2 mg, 0.013 mmol) and UHP (64 mg, 0.68 mmol) were added. The reaction was stirred at 10° C. for 2 h, and quenched with 5% aq. sodium bisulfite solution (1.0 mL). The reaction was diluted with ethyl acetate (10 mL) and washed with water (10 mL) and 10% aq. sodium bicarbonate solution (10 mL). The organic phase was separated and concentrated in vacuo. The residue was purified by column chromatography on silica gel (heptane-EtOAc, R.sub.f in 3:2 heptane:EtOAc=0.42) to the desired product (99 mg, 47%) as a white powder. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.11 (1H, s, C4-CH), 3.46 (1H, d, J=3.7, C6-CH), 3.39 (1H, dd, J=11.9, 3.3, C22-CH.sub.aH.sub.b), 3.34 (1H, d, J=3.7, C7-CH), 3.06 (1H, dd, J=11.9, 7.5, C22-CH.sub.aH.sub.b), 2.55 (1H, ddd, J=18.0, 14.3, 5.5, C2-CH.sub.aH.sub.b), 2.48-2.44 (1H, m, C2-CH.sub.aH.sub.b), 2.00 (1H, dt, J=11.9, 3.3), 1.97-1.90 (3H, m), 1.87 (1H, td, J=10.8, 1.4, C8-CH), 1.74-1.63 (2H, m), 1.53 (1H, dq, J=12.7, 3.5), 1.49-1.45 (1H, m), 1.41-1.23 (5H, m), 1.22 (1H, td, J=12.7, 3.5), 1.10 (3H, s, C18-CH.sub.3), 1.06 (3H, d, J=6.6, C21-CH.sub.3), 0.78 (3H, s, C19-CH.sub.3). .sup.13C NMR (140 MHz, CDCl.sub.3): δ=198.3, 162.6, 131.1, 57.9, 54.6, 52.9, 52.5, 51.2, 43.2, 40.6, 39.1, 36.9, 35.6, 34.6, 34.1, 33.9, 28.0, 23.7, 19.9, 17.7, 17.2 11.9.
I. Epoxidation of N-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)cyclopropyl Sulfonamide to Form N-((6α, 7α, 22E)-3,24-dioxo-6,7-epoxy-4,22-choladien-24-yl)cyclopropylsulfonamide
[0339] ##STR00064##
[0340] The product was prepared according to the general procedure for MTO catalysed epoxidation on 1 g scale, isolated in 68% yield (697 mg) as an off white solid. δH (400 MHz, CDCl.sub.3); 8.69 (1H, brs, NH), 6.93 (1H, dd, J 15.4, 9.6, C23-CH), 6.12 (1H, s, 04-CH), 5.83 (1H, m, 022-CH), 3.47 (1H, d, J 14.7, C6-CH), 3.36-3.32 (1H, m, C7-CH), 3.00 (1H, dddd, J 12.8, 9.5, 8.1, 4.8, SO.sub.2CH), 2.67-2.40 (2H, m), 2.39-2.27 (1H, m), 2.09-1.64 (7H, m), 1.62-1.18 (11H, m), 1.11 (3H, d, J 6.1, C21-CH.sub.3), 1.10 (3H, s, C19-CH.sub.3), 0.78 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 198.6, 164.0, 162.8, 156.6, 131.1, 119.3, 54.6, 54.5, 52.6, 51.2, 43.4, 40.6, 39.8, 39.1, 35.6, 34.6, 34.1, 33.9, 31.5, 28.2, 23.7, 19.9, 19.1, 17.2, 12.1, 6.3, 6.3.
J. Epoxidation of N-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)-4-(trifluoromethoxy)benzenesulfonamide to form N-((6α, 7α, 22E)-3,24-dioxo-6,7-epoxy-4,22-choladien-24-yl)-4-(trifluoromethoxy)benzenesulfonamide
[0341] ##STR00065##
[0342] The product was prepared according to the general procedure for MTO catalysed epoxidation on 1 g scale, isolated in 5% yield (50 mg) as a colourless solid.
[0343] δH (400 MHz, MeOD); 8.17-8.09 (2H, m, ArH), 7.52-7.46 (2H, m, ArH), 6.82 (1H, dd, J 15.4, 8.9, 3.7, C23-CH), 6.07 (1H, s, C4-CH), 5.84 (1H, dd, J 15.4, 0.7, C22-CH), 3.49 (1H, d, J 3.8, C6-CH), 3.37-3.33 (1H, m, C7-CH), 2.62 (1H, ddd, J 18.2, 14.6, 5.6, C2-CH.sub.aH.sub.b), 2.44-2.27 (2H, m), 2.08-1.88 (3H, m), 1.85-1.60 (2H, m), 1.60-1.49 (1H, m), 1.48-1.17 (9H, m), 1.12 (3H, s, C19-CH.sub.3), 1.07 (3H, d, J 6.6, C21-CH.sub.3), 0.80 (3H, s, C18-CH.sub.3); δC (100 MHz, MeOD); 201.0, 166.2, 166.1, 156.5, 153.9, 139.8, 131.8, 131.4, 122.0, 121.7 (q, J 256), 120.8, 55.9, 55.7, 53.6, 52.8, 44.6, 42.3, 41.0, 40.5, 36.9, 35.9, 35.2, 35.0, 29.2, 24.6, 21.0, 19.5, 17.3, 12.4.
K. Epoxidation of (20S)-20-(5-Tosyltetrazol-1-yl)methyl-pregna-4,6-dien-3-one to Form (6α, 7α, 20S)-20-(5-Tosyltetrazol-1-yl)methyl-6,7-epoxy-pregna-4-en-3-one
[0344] ##STR00066##
[0345] The product was prepared according to the general procedure for MTO catalysed epoxidation on 300 mg scale, isolated in 33% yield (103 mg) as a colourless solid. δH (400 MHz, CDCl.sub.3); 8.00-7.94 (2H, m, ArH), 7.47-7.41 (2H, m, ArH), 6.10 (1H, s, C4-CH), 4.77 (1H, dd, J 13.4, 3.9, C22-CH.sub.aH.sub.b), 4.42 (1H, dd, J 13.4, 3.9, C22-CH.sub.aH.sub.b), 3.46 (1H, d, J 3.7, C6-CH), 3.37-3.33 (1H, m, C7-CH), 2.61-2.37 (3H, m), 2.48 (3H, s, ArCH.sub.3), 2.37-2.24 (1H, m), 2.11-1.80 (3H, m), 1.76-1.61 (2H, m), 1.58-1.17 (8H, m), 1.09 (3H, s, C19-CH.sub.3), 0.85 (3H, d, J 7.0, C21-CH.sub.3), 0.81 (3H, s, C18-CH.sub.3); δC (100 MHz, CDCl.sub.3); 198.2, 162.5, 153.3, 147.5, 134.4, 131.1, 130.4, 129.3, 55.1, 54.5, 53.8, 52.5, 51.2, 43.6, 40.6, 39.1, 37.7, 35.5, 34.6, 34.1, 33.9, 27.6, 23.8, 21.9, 19.9, 17.2, 16.4, 11.9.
Example 3—Preparation of Compounds of General Formula (XXI) Via Compounds of General Formula (I) with Malonate Side Chain
[0346] The compounds of general formula (II) may be converted to compounds of general formula (IA) as described above and these compounds may then be converted to compounds of general formula (IB), (IC), (ID), (IE) and (IF) by the methods described below. A compound of general formula (IF) may be converted to a compound of general (XXI).
[0347] The following illustrates the conversion of a compound of general formula (II) in which —YR.sup.4 is CH.sub.2OH via compounds of formulae (IA), (IB), (IC), (ID) and (IE) in which —YR.sup.4 is —CH.sub.2CH[C(O)OMe]2 to a compound of general formula (XXI) in which R.sup.4a is C(O)OH is shown in Scheme 4 below.
##STR00067##
A. Synthesis of 23-carboxy-3-oxo-4-cholen-24-oic Acid Dimethyl Ester
[0348] ##STR00068##
[0349] To a suspension of (20S)-20-bromomethyl-4-pregnen-3-one (15 g, 38.1 mmol), tetrabutylammonium bromide (1.2 g, 3.8 mmol) and potassium carbonate (26.3 g, 191 mmol) in toluene (150 mL) was added dimethylmalonate (13.1 mL, 114 mmol) and the reaction mixture was stirred at 80° C. for 91 h. The reaction mixture was then cooled to room temperature and was poured onto water (150 mL). The layers were separated and the aqueous phase was extracted with EtOAc (2×100 mL). The combined organic phases were washed with 5% aq. sodium chloride (100 mL) and were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (heptane-EtOAc) to give the desired product (14.8 g, 87%) as a yellow solid. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.72 (1H, s), 3.75 (3H, s), 3.72 (3H, s), 3.48 (1H, dd, J=11.0, 4.0), 2.44-2.36 (2H, m), 2.33 (1H, dt, J=17.0, 3.6), 2.27 (1H, ddd, J=14.6, 4.1, 2.4), 2.18 (1H, ddd, J=13.7, 11.1, 2.5), 2.03-2.00 (2H, m), 1.95-1.89 (1H, m), 1.85-1.82 (1H, m), 1.71-1.67 (1H, m), 1.64-1.60 (1H, m), 1.54-1.39 (4H, m), 1.37-1.30 (2H, m), 1.19-1.09 (3H, m), 1.18 (3H, s), 1.05-0.99 (2H, m), 0.94-0.90 (1H, m), 0.93 (3H, d, J=6.5), 0.70 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.6, 171.5, 170.4, 170.0, 123.8, 56.3, 55.8, 53.7, 52.6, 52.4, 49.4, 42.5, 39.6, 38.6, 35.7, 35.6, 35.1, 34.3, 34.0, 32.9, 32.0, 28.0, 24.1, 21.0, 18.1, 17.4, 11.9.
B. Synthesis of 23-carboxy-3-oxo-4,6-choladien-24-oic Acid Dimethyl Ester
[0350] ##STR00069##
[0351] 23-Carboxy-3-oxo-4-cholen-24-oic acid dimethyl ester (14.5 g, 32.7 mmol) was suspended in toluene (60 mL) and acetic acid (0.19 mL, 3.3 mmol). p-Chloranil (8.8 g, 35.9 mmol) was added and the mixture stirred at reflux for 65 min. The reaction mixture was cooled to room temperature and filtered. The filter cake was washed with toluene (45 mL) and the filtrate concentrated under reduced pressure. The residue (21.6 g) was used without further purification. A small portion was purified by column chromatography on silica gel (heptane-EtOAc) to give the product. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.12 (1H, d, J=10.8), 6.08 (1H, dd, J=9.8, 2.2), 5.65 (1H, s), 3.74 (3H, s), 3.71 (3H, s), 3.47 (1H, dd, J=11.0, 3.9), 2.58 (1H, dd, J=14.3, 5.3), 2.53 (1H, dd, J=14.3, 5.3), 2.44-2.38 (1H, m), 2.21-2.15 (2H, m), 2.05-1.92 (3H, m), 1.83-1.77 (1H, m), 1.69 (1H, td, J=13.9, 5.2), 1.55-1.34 (5H, m), 1.31-1.11 (5H, m), 1.10 (3H, s), 0.93 (3H, d, J=6.3), 0.73 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.6, 170.4, 170.0, 163.9, 141.4, 127.8, 123.5, 56.1, 53.4, 52.6, 52.4, 50.6, 49.4, 43.5, 39.5, 37.7, 36.0, 35.1, 34.3, 33.9, 33.9, 28.0, 23.7, 20.6, 18.1, 16.3, 11.9.
C. Synthesis of (6α, 7α)-6,7-epoxy-3-oxo-4-cholen-23-carboxy-24-oic Acid Dimethyl Ester
[0352] ##STR00070##
[0353] 23-Carboxy-3-oxo-4,6-choladien-24-oic acid dimethyl ester (8.94 g, 19.5 mmol) was dissolved in HFIP (35.8 mL) and EtOAc (17.9 mL) and the solution was cooled to 10° C. MTO (51 mg, 0.195 mmol) and 3-methylpyrazole (97 μL, 1.17 mmol) were charged to the solution followed by UHP (2.08 g, 21.4 mmol) in 2 portions over 5 minutes. After 2 h further MTO (51 mg, 0.195 mmol) and 3-methylpyrazole (97 μL, 1.17 mmol) were charged and the solution stirred for 16 h. Further MTO (51 mg, 0.195 mmol), 3 methylpyrazole (97 μL, 1.17 mmol) and UHP (0.38 g, 3.90 mmol) were charged and the solution stirred for 2 h. The reaction was quenched by addition of 5% aq. NaHSO.sub.3 (36 mL) over 5 minutes. The phases were separated and the organic phase washed with 5% aq. NaHSO.sub.3 until a negative test for peroxides was observed. The organic phase was washed with 5% aq. NaHCO.sub.3 (40 mL) and water (40 mL), then dried over sodium sulfate and was concentrated in vacuo. The residue was purified by column chromatography on silica gel to give the desired product (7.07 g, 76%) as a white crystalline solid. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.10 (1H, s), 5.31 (2H, s), 3.75 (3H, s), 3.73 (3H, s), 3.48 (1H, dd, J=11.1, 4.0), 3.45 (1H, d, J=4.0 Hz), 3.34 (1H, d, J=3.6 Hz), 2.55 (1H, ddd, J=18.1, 14.4, 5.6), 2.45 (1H, m), 2.19 (1H, ddd, J=13.6, 11.1, 2.4), 2.05-1.85 (5H, m), 1.70 (1H, td, J=13.9, 5.2), 1.53-1.25 (6H, m), 1.22-1.17 (2H, m), 1.09 (3H, s), 0.49 (3H, d, J=6.5), 0.72 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=198.4, 170.3, 170.0, 162.8, 131.1, 56.0, 54.6, 53.4, 52.6, 52.5, 52.4, 51.3, 49.3, 43.1, 40.6, 39.2, 35.5, 35.1, 34.5, 34.3, 34.1, 33.8, 28.1, 23.6, 19.9, 18.1, 17.2, 11.8.
D. Synthesis of (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-cholen-23-carboxy-24-oic Acid Dimethyl Ester
[0354] ##STR00071##
[0355] To a solution of 0.5 M ZnCl in THF (15.7 mL, 0.6 eq) in THF (24 mL, 4 vol) under argon at −15° C. was added 1 M EtMgBr in TBME (23.6 mL, 1.8 eq) dropwise over 20 mins. CuCl (65 mg, 0.05 eq) was added in a single portion and the suspension stirred for 10 mins. (6α, 7α)-6,7-epoxy-3-oxo-4-cholanen-23-carboxy-24-oic acid dimethyl ester (6 g) dissolved in THF (24 mL, 4 vol) was added dropwise over 30 mins and the mixture stirred for 90 mins. Sat. aq. NH.sub.4Cl (15 mL, 2.5 vol) was added dropwise and the mixture warmed to ambient temperature. The solids were removed by filtration and the filter cake washed with EtOAc (2×25 mL). The filtrate was washed with sat. aq. NH.sub.4Cl (2×100 mL) and water (2×100 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. Purification by column chromatography afforded (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-cholanen-23-carboxy-24-oic acid dimethyl ester as a white crystalline solid (55%).
[0356] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.77 (1H, s), 3.75 (3H, s), 3.74 (1H, s), 3.73 (3H, s), 3.48 (1H, dd, J=11.1, 4.0), 2.47 (1H, ddd, J=17.5, 15.0, 5.0), 2.37 (1H, m), 2.31 (1H, m), 2.19 (1H, m), 2.05-1.94 (4H, m), 1.81-1.41 (11H, m), 1.40-1.34 (2H, m), 1.21 (3H, s), 1.20-1.12 (2H, m), 0.93 (3H, d, J=6.4), 0.91 (3H, t, J=7.3), 0.72 (3H, s).
[0357] .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.1, 170.6, 170.4, 170.0, 128.6, 72.2, 56.3, 55.2, 52.6, 52.4, 50.1, 49.4, 44.2, 42.6, 39.1, 38.3, 37.5, 35.6, 35.1, 34.4, 34.1, 28.0, 26.3, 23.6, 20.9, 19.7, 18.1, 12.8, 11.8.
E. Synthesis of (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic Acid Dimethyl Ester
[0358] ##STR00072##
[0359] A solution of (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-cholanen-23-carboxy-24-oic acid dimethyl ester (3.5 g) in DMF (10.5 mL, 3 vol) and MeCN (21 mL, 6 vol) was purged with argon×3 and cooled to −15° C. 5% Pd on CaCO.sub.3 was added in one portion and the flask then purged with hydrogen×3 and stirred for 18 h. The flask was purged with argon×3 times and the suspension filtered through a Whatman® GF/B grade filter pad (glass fiber pore size 1 μm) and the cake washed with EtOAc (2×50 mL). The filtrate was washed with water (2×50 mL) and 5% aq. NaCl (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography gave (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic acid dimethyl ester (1.77 g, 51%).
[0360] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.75 (3H, s), 3.73 (3H, s), 3.70 (1H, s), 3.48 (1H, dd, J=11.0, 4.0), 3.35 (1H, dd, J=15.5, 13.6), 2.36 (1H, td, J=14.2, 4.8), 2.19 (1H, m), 2.14-2.08 (2H, m), 2.02-1.90 (4H, m), 1.81 (1H, dd, J=13.3, 4.5), 1.70-1.62 (2H, m), 1.54-1.34 (11H, m), 1.26-1.11 (2H, m), 1.04 (3H, s), 0.95 (3H, d, J=6.4), 0.94 (3H, d, J=7.0), 0.70 (3H, s).
[0361] .sup.13C NMR (176 MHz, CDCl.sub.3): δ=213.7, 170.4, 170.1, 72.1, 56.4, 52.6, 52.4, 50.2, 49.8, 49.4, 47.0, 46.7, 42.8, 39.5, 37.7, 36.3, 36.0, 35.7, 35.2, 34.4, 34.1, 28.1, 27.7, 24.4, 23.8, 20.8, 18.2, 13.9, 11.8.
F. Synthesis of (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic Acid Dimethyl Ester
[0362] ##STR00073##
[0363] To a solution of (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic acid dimethyl ester (1.77 g) in DCM (45 mL, 25 vol) under argon was added DMP (1.83 g, 1.2 eq) in 4 portions at 5 min intervals. After 30 mins the mixture was partitioned between EtOAc (50 mL) and 10% aq. Na.sub.2S.sub.2O.sub.3/2% aq. NaHCO.sub.3 and stirred for 1 h. The aqueous phase was extracted with EtOAc (50 mL) and the combined organic phases washed with 1M aq. NaOH (50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography gave (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (1.54 g, 87%) as a white crystalline solid.
[0364] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.75 (3H, s), 3.73 (3H, s), 3.47 (1H, dd, J=10.9, 4.0), 2.42 (1H, t, J=11.4), 2.31-2.17 (5H, m), 2.05 (1H, m), 2.01-1.93 (2H, m), 1.89-1.78 (5H, m), 1.67-1.62 (1H, m), 1.58-1.46 (5H, m), 1.39-1.15 (5H, m), 1.14 (3H, s), 0.94 (3H, d, J=6.4), 0.85 (3H, t, J=7.4), 0.71 (3H, s).
[0365] .sup.13C NMR (176 MHz, CDCl.sub.3): δ=214.6, 211.6, 170.4, 170.0, 57.2, 55.5, 52.6, 52.4, 50.3, 49.4, 48.5, 47.3, 44.9, 43.6, 43.2, 39.2, 35.8, 35.3, 35.1, 34.9, 34.3, 28.1, 24.6, 23.8, 23.5, 21.7, 18.2, 12.6, 12.2.
G. Synthesis of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic Acid Dimethyl Ester
[0366] ##STR00074##
[0367] To (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (1.46 g) in MeOH (36 mL, 25 vol) under argon was added NaOMe (324 mg, 2 eq) and the solution stirred at 40° C. for 16 hours. AcOH (5 mL) was added dropwise and the solution stirred for 5 minutes. The solution was taken up in EtOAc (100 mL) and washed with 5% aq. NaCl (2×100 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography gave (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (0.45 g, 31%).
[0368] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.75 (3H, s), 3.73 (3H, s), 3.47 (1H, dd, J=11.0, 4.0), 2.74 (1H, dd, J=11.0, 6.6), 2.47 (1H, t, J=11.3), 2.29-2.16 (5H, m), 2.09-1.96 (3H, m), 1.89-1.80 (2H, m), 1.72-1.46 (6H, m), 1.39-1.34 (1H, m), 1.33 (3H, s), 1.32-1.23 (2H, m), 1.21-1.13 (2H, m), 1.10-1.07 (1H, m), 0.99-0.95 (1H, m), 0.94 (3H, d, J=6.5), 0.81 (3H, t, J=7.4), 0.68 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.1, 210.5, 170.3, 170.0, 55.3, 52.6, 52.4, 52.3, 52.2, 49.9, 49.34, 48.8, 43.7, 42.7, 38.8, 38.3, 36.6, 35.9, 35.4, 35.1, 34.2, 28.2, 24.5, 22.9, 22.2, 18.6, 18.2, 12.1, 11.8.
H. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-23-carboxy-24-oic Acid Dimethyl Ester
[0369] ##STR00075##
[0370] To a suspension of NaBH.sub.4 (27 mg, 1 eq) in IPA (2.3 mL) at −20° C. was added a solution of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (350 mg) in EtOAc (2.3 mL, 6.5 vol) over 10 mins. After 30 mins 0.7M H.sub.2SO.sub.4 (2.5 mL) was added dropwise over 10 mins and the solution allowed to warm to 18° C. The solution was diluted with EtOAc (50 mL) and the organic phase washed with water (3×50 mL) and 5% aq. NaCl (50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography gave (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-23-carboxy-24-oic acid dimethyl ester (298 mg, 85%)
[0371] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.74 (3H, s), 3.72 (3H, s), 3.52 (1H, m), 3.47 (1H, dd, J=11.0, 4.0), 2.69 (1H, dd, J=12.8, 5.9), 2.34 (1H, t, J=11.3), 2.21-2.16 (2H, m), 1.99-1.94 (2H, m), 1.85-1.68 (7H, m), 1.50-1.43 (4H, m), 1.37-1.23 (5H, m), 1.21 (3H, s), 1.20-1.10 (4H, m), 0.92 (3H, d, J=6.5), 0.80 (3H, t, J=7.4), 0.64 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.8, 170.4, 170.0, 71.1, 55.3, 52.6, 52.4, 52.0, 50.7, 49.9, 49.4, 49.0, 43.7, 42.7, 39.0, 35.7, 35.1, 34.3, 34.2, 31.8, 29.8, 28.3, 24.6, 23.5, 21.8, 18.8, 18.2, 12.0, 12.0.
I. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic Acid Dimethyl Ester
[0372] ##STR00076##
[0373] To a solution of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-23-carboxy-24-oic acid dimethyl ester (200 mg) in THF (20 mL, 100 vol) and water (5 mL, 25 vol) at 0° C. was added NaBH.sub.4 (154 mg, 10 eq) in 3 portions. The solution was stirred for one h, allowing to warm to 18° C. MeOH/water (10 mL, 1:1) was added dropwise and the organic solvent removed in vacuo. To the aqueous solution was added 2M aq. HCl (20 mL) dropwise. The aqueous solution was extracted with EtOAc (2×30 mL) and the combined organic phases washed with 5% aq. NaHCO.sub.3 (30 mL) and water (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid dimethyl ester (90 mg, 45%).
[0374] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.75 (3H, s), 3.72 (3H, s), 3.48 (1H, dd, J=11.0, 4.0), 3.69 (1H, bs), 3.40 (1H, m), 2.18 (1H, m), 1.97-1.93 (2H, m), 1.85-1.75 (4H, m), 1.73-1.57 (4H, m), 1.51-1.11 (18H, m), 1.00 (1H, td, J=14.3, 3.4), 0.93 (3H, d, J=6.5), 0.90 (3H, t, J=7.3), 0.64 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=170.5, 170.1, 72.3, 70.9, 56.3, 52.6, 52.4, 50.5, 49.4, 45.2, 42.8, 41.2, 40.0, 39.6, 35.6, 35.5, 35.2, 34.4, 34.0, 33.2, 30.6, 28.2, 23.7, 23.2, 22.2, 20.7, 18.2, 11.8, 11.7.
J. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic Acid
[0375] ##STR00077##
[0376] To a solution of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid dimethyl ester (70 mg) in IPA (2 mL, 28 vol) was added 0.5 M aqueous NaOH (2 mL, 28 vol) and the mixture stirred at 60° C. for 2 h. The organic solvent was removed in vacuo and the aqueous solution adjusted to pH1 with 2M aq. H.sub.2SO.sub.4. EtOAc (20 mL) was added and the mixture stirred for 5 mins. The aqueous phase was re-extracted with EtOAc (10 mL). The combined organic phases were washed with 5% aq. NaCl (2×10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid as a white solid (54 mg, 81%).
[0377] .sup.1H NMR (700 MHz, d-6 Acetone): δ=3.58 (1H, bs), 3.32 (1H, dd, J=11.1, 3.6), 3.18 (1H, m), 2.03 (1H, m), 1.90-1.62 (6H, m), 1.57 (1H, m), 1.48-1.31 (8H, m), 1.28-1.13 (6H, m), 1.11-1.05 (3H, m), 0.98 (3H, m), 0.87 (3H, d, J=6.1), 0.85 (1H, m) 0.79 (3H, s), 0.75 (3H, t, J=7.3), 0.74 (3H, 5); .sup.13C NMR (176 MHz, d-6 Acetone): δ=171.7, 171.3, 72.5, 70.4, 57.5, 51.4, 46.7, 43.4, 42.6, 41.3, 40.7, 36.7, 36.3, 36.2, 35.3, 34.6, 34.0, 31.5, 30.6, 29.0, 24.3, 23.7, 23.2, 21.6, 18.7, 12.3, 12.1.
K. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic Acid
[0378] ##STR00078##
[0379] (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid (25 mg) was taken up in xylene (1.25 mL, 50 vol) and pyridine (250 μL, 10 vol) and the solution heated to reflux for 90 mins. The cooled solution was diluted with EtOAc (20 mL) and washed with 1M aq. HCl (3×10 mL). The organic phase was washed with water (3×10 mL), 5% aq. NaCl (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated. Purification by column chromatography gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid as a white solid (19 mg, 82%).
[0380] .sup.1H and .sup.13C NMR were consistent with an authentic sample of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid.
L. Synthesis of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic Acid
[0381] ##STR00079##
[0382] To a solution of (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (100 mg) in IPA (1 mL, 10 vol) was added 0.5 M aqueous NaOH (1 mL, 10 vol) and the mixture stirred at 60° C. for 2 h. The organic solvent was removed in vacuo and the aqueous solution adjusted to pH1 with 2M aqueous H2SO.sub.4. EtOAc (10 mL) was added and the mixture stirred 5 mins. The aqueous phase was re-extracted with EtOAc (10 mL). The combined organic phases were washed with 5% aq. NaCl (2×10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid (100 mg, quant.) as a white solid.
[0383] .sup.1H NMR (500 MHz, CDCl.sub.3): δ=3.51 (1H, m), 2.76 (1H, m), 2.49 (1H, t, J=11.1), 2.34-1.80 (14H, m), 1.71-1.43 (7H, m), 1.33 (3H, s), 1.23-1.04 (3H, m), 0.98 (3H, d, J=6.1), 0.94 (1H, m), 0.80 (3H, d, J=7.3), 0.69 (3H, s).
M. Synthesis of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-24-oic Acid
[0384] ##STR00080##
[0385] (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid (80 mg) was taken up in xylene (4 mL, 50 vol) and pyridine (800 μL, 10 vol) and the solution heated to reflux for 90 mins. The cooled solution was diluted with EtOAc (25 mL) and washed with 1M aq. HCl (3×10 mL). The organic phase was washed with water (3×10 mL), 5% aq. NaCl (10 mL), dried over Na.sub.2SO.sub.4 and filtered. Purification by column chromatography gave (5β, 6α)-6-ethyl-3,7-dioxo-cholan-24-oic acid as a white solid (60 mg, 83%).
[0386] .sup.1H and .sup.13C NMR were consistent with an authentic sample of the target compound, prepared as described in Example 16 of WO 2016/079520.
[0387] Although the compound of general formula (XXI) was prepared from a compound of general formula (IF) by conversion of the malonate side chain of the compound of general formula (IF) to a carboxylic acid group, a person of skill in the art will appreciate that the conversion of the malonate to carboxylic acid could take place at an earlier stage of the synthesis as described in steps M and N above, and that the carboxylic acid group could, if necessary, be protected, for example as an ester.
Example 4—Preparation of an Analogue of a Compound of General Formula (I) and a Compound of General Formula (XXI) Via Compounds of General Formula (I) with Nitrile Side Chain (Including Side Chain Extension)
[0388] Scheme 5 shows the conversion of a compound of general formula (II) in which —YR.sup.4 is CH.sub.2OH conversion to a compound of general formula (II) in which —YR.sup.4 is —CH.sub.2CH.sub.2—CN and subsequently to a compound of general formula (XXI) in which —YR.sup.4a is CH.sub.2CH.sub.2C(O)OH. The reaction proceeds via compounds of general formulae (IA), (IB), (IC) and (IE) in which —YR.sup.4 is —CH.sub.2CH.sub.2—CN. The compound of general formula (IE) is then converted to a 3-OH analogue and the side chain is then converted to —CH.sub.2CH.sub.2—C(O)OH.
##STR00081##
A. Synthesis of (20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and (20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene
[0389] ##STR00082##
[0390] To a solution of (20S)-20-bromomethyl-4-pregnen-3-one (1.00 g, 2.59 mmol) and ethylene glycol (2.0 mL, 36.25 mmol) in toluene (30 mL) was added pTSA.H.sub.2O (9.86 mg, 0.05 mmol) and the mixture was heated to reflux using a Dean Stark apparatus for 5 h. The reaction mixture was allowed to cool to room temperature before being poured onto 5% aq. NaHCO.sub.3 (30 mL). The layers were separated and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (2×30 mL). The combined organics were dried over sodium sulfate and were concentrated under reduced pressure. The residue was used in the next step without purification. A sample was purified by column chromatography (heptane/EtOAc) to give a mixture of (20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and (20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene in 68% yield (the ratio of Δ.sup.5:Δ.sup.4 was approximately 3.6:1). δH (700 MHz, CDCl.sub.3); 5.35 (0.8H, dt, J=4.4, 2.2), 5.23 (0.2H, s), 4.02-3.96 (4H, m, CH.sub.2O), 3.51 (0.8H, dd, J 9.7, 2.7), 3.51-3.49 (0.2H, m), 3.34 (0.8H, dd, J 9.7, 6.0), 3.33 (0.2H, dd, J 9.7, 6.1), 2.56 (0.8H, dq, J 14.1, 2.9), 2.20 (0.2H, td, J 13.9, 4.9, 1.8), 2.12 (0.8H, dd, J 14.2, 2.9), 2.05 (0.2H, ddd, J 14.0, 4.2, 2.4), 1.99-1.93 (2H, m), 1.91-1.83 (1H, m), 1.81-1.75 (2H, m), 1.74-1.62 (4H, m), 1.60 (0.8H, s), 1.561.51 (1H, m), 1.50-1.41 (2H, m), 1.37-1.25 (3H, m), 1.21 (1H, td, J 6.5, 4.2), 1.17-1.04 (3H, m), 1.09 (3H, d, J 6.4), 1.03 (3H, s), 1.01-0.84 (0.8H, m), 0.71 (2.4H, s), 0.70 (0.6H, s); δC (176 MHz, CDCl.sub.3); 151.6, 140.2, 122.1, 119.65, 109.5, 106.2, 64.6, 64.5, 64.2, 64.2, 56.4, 55.7, 53.8, 53.7, 53.7, 49.6, 43.6, 43.5, 42.5, 42.4, 41.8, 39.5, 39.5, 37.9, 37.8, 37.4, 36.6, 36.3, 35.8, 34.9, 32.4, 32.1, 31.9, 31.9, 31.7, 31.1, 30.0, 27.6, 27.6, 24.2, 24.1, 21.0, 18.9, 18.7, 18.6, 17.6, 12.3, 12.2.
B. Synthesis of 3,3-ethylenedioxy-4-choleno-24-nitrile and 3,3-Ethylenedioxy-5-choleno-24-nitrile
[0391] ##STR00083##
Procedure A
[0392] A solution containing MeCN (26.0 mg, 0.63 mmol) in THF (1.85 mL) was cooled to −78° C. under argon and nBuLi (0.32 mL, 2 M in cyclohexane, 0.63 mmol) was charged dropwise over 2 min. To this mixture, a solution containing (20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and (20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene (185 mg, 0.423 mmol) in THF (2.15 mL) was charged dropwise over 30 min. The reaction mixture was allowed to warm to 0° C. over 4 h, cooled to −78° C. and quenched with 10% aq. NH.sub.4Cl (3 mL). The reaction mixture was diluted with EtOAc (20 mL) and 10% aq. NH.sub.4Cl (20 mL) and the organic phase was separated. The aqueous phase was extracted with EtOAc (20 mL), and the combined organic phases were washed with 5% aq. NaCl (20 mL), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using heptane: EtOAc (5:1) as the eluent. A fraction containing 3,3-ethylenedioxy-4-choleno-24-nitrile and 3,3-ethylenedioxy-5-choleno-24-nitrile was obtained in 49% yield (the ratio of Δ.sup.5:Δ.sup.4 was approximately 7:1). δH (700 MHz, CDCl.sub.3); 5.35 (0.9H, dt, J 4.5, 2.2), 5.2 (0.1H, br s), 4.02-3.86 (4H, m), 2.56 (0.9H, dq, J 14.2, 2.9), 2.39-2.34 (0.1H, m), 2.34 (0.9H, ddd, J 16.9, 8.6, 5.1), 2.27 (0.9H, dt, J 16.8, 8.4), 2.27 (0.1H, dt, J 16.8, 8.4), 2.20 (0.1H, td, J 13.9, 5.0, 1.8), 2.12 (0.9H, dd, J 14.2, 3.0), 2.05 (0.1H, ddd, J 13.8, 4.4, 2.2), 2.01-1.95 (2H, m), 1.87-1.75 (4H, m), 1.73-1.70 (0.3H, m), 1.69-1.59 (3.4H, m), 1.58-1.52 (2H, m), 1.50-1.43 (2H, m), 1.39-1.25 (4.6H, m), 1.18 (1H, td, J 6.5, 4.2), 1.14-0.99 (4H, m), 1.03 (3H, s), 0.96 (2.7H, d, J 6.6), 0.94 (0.3H, d, J 6.7), 0.88 (0.9H, t, J 14.3), 0.70 (2.7H, s), 0.70 (0.3H, s); δC (176 MHz, CDCl.sub.3); 151.6, 140.1, 122.1, 120.2, 119.6, 109.5, 106.2, 64.6, 64.4, 64.2, 56.7, 56.0, 55.5, 55.5, 53.8, 49.6, 42.6, 42.5, 41.8, 39.8, 39.7, 37.4, 36.6, 36.3, 35.7, 35.2, 35.2, 34.9, 32.4, 32.1, 31.9, 31.9, 31.7, 31.6, 31.5, 31.1, 30.0, 29.7, 28.1, 28.1, 24.2, 24.1, 22.7, 21.0, 18.9, 17.9, 17.9, 17.6, 14.3, 14.2, 14.1, 12.0, 11.9.
Procedure B
[0393] A solution of MeCN (2.06 mL, 39.43 mmol) in THF (34 mL) was charged dropwise over 1.2 h to a solution of nBuLi (19.72 mL, 2 M in cyclohexane, 39.43 mmol) in THF (69 mL) at −60° C. under argon. To the resulting white suspension, a solution containing (20S)-20-bromomethyl-3, 3-ethylenedioxy-4-pregnene and (20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene (6.9 g, 15.77 mmol) in THF (69 mL) was charged dropwise over 1.2 h. The thick suspension that formed was warmed to 0° C. over 15 min and water (69 mL) was charged dropwise. The layers were separated and the aqueous phase was extracted with EtOAc (2×100 mL). The combined organic phases were washed with 5% aq. NaCl (2×100 mL) and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using a gradient of EtOAc in heptane as the eluent. A fraction containing 3,3-ethylenedioxy-4-choleno-24-nitrile and 3,3-ethylenedioxy-5-choleno-24-nitrile was obtained which also contained the product from double-alkylation of MeCN (mass 3.88 g).
C. Synthesis of 3-oxo-4-choleno-24-nitrile
[0394] ##STR00084##
[0395] To a solution of 3,3-ethylenedioxy-4-choleno-24-nitrile and 3,3-ethylenedioxy-5-choleno-24-nitrile (3.75 g, 9.43 mmol) in EtOH (75 mL) was added a solution of H.sub.2SO.sub.4 (1 mL, conc, 18.86 mmol) in water (7.5 mL). The reaction mixture was heated at reflux for 30 min and cooled to room temperature. A white solid was removed by filtration and the filter-cake was washed with EtOH (2×20 mL). Pyridine (3 mL) was added to the combined wash and filtrate and the mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (100 mL), washed with 1 M aq. H.sub.2SO.sub.4 (100 mL), 5% aq. NaHCO.sub.3 (100 mL), 5% aq. NaCl (2×100 mL), dried over sodium sulfate and was concentrated under reduced pressure to give the desired product (2.36 g). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.72 (1H, s, C4-CH), 2.45-2.25 (6H, m), 2.04-2.00 (2H, m), 1.89-1.82 (3H, m), 1.69 (1H, td, J 7.0, 4.6), 1.67-1.62 (1H, m), 1.59-1.51 (3H, m), 1.44 (1H, qd, J 13.1, 4.0), 1.39-1.25 (3H, m), 1.20-1.10 (3H, m), 1.18 (3H, s), 1.05-0.99 (2H, m), 0.96 (3H, d, J 6.6), 0.95-0.91 (1H, m), 0.73 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.6 (C═O), 171.4 (C═CH), 123.8 (C═CH), 120.2 (CN), 55.8, 55.5, 53.7, 42.6, 39.6, 38.6, 35.7, 35.6, 35.1, 34.0, 32.9, 32.0, 31.5, 28.1, 24.1, 21.0, 17.9, 17.4, 14.3, 12.0
D. Synthesis of 3-oxo-4,6-choladieno-24-nitrile
[0396] ##STR00085##
[0397] To a solution of 3-oxo-4-choleno-24-nitrile (2.25 g, 0.64 mmol) in toluene (2.25 mL) and AcOH (6.75 mL) was added chloranil (1.72 g, 0.70 mmol). The mixture was heated at 100° C. for 45 min and was then allow to cool to room temperature. The mixture was filtered, washing with AcOH:toluene (3:1, 20 mL) and the combined filtrates were concentrated under reduced pressure. The residue was concentrated from toluene (3×40 mL) and acetone (3×40 mL) and was then dissolved in acetone (6.75 mL). The solution was charged to an aqueous solution of NaOH (22.5 mL, 3% w/v) and the sticky solid that formed was collected by filtration and washed with water: acetone (2×20 mL, 2:1). The solid was purified by chromatography on silica gel using a gradient of EtOAc in heptane as the eluent to give the desired product as a yellow solid (1.33 g, 59% yield). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.13 (1H, d, J 11.0), 6.10 (1H, dd, J 9.8, 2.3), 5.67 (1H, s), 2.57 (1H, ddd, J 17.9, 14.5, 5.4), 2.45-2.41 (1H, m), 2.39 (1H, ddd, J 17.0, 8.3, 5.1), 2.29 (1H, dt, J 16.8, 8.4), 2.20 (1H, t, J 10.6), 2.05 (1H, dt, J 12.9, 3.4), 2.00 (1H, ddd, J 13.2, 5.3, 2.0), 1.95-1.89 (1H, m), 1.88-1.80 (2H, m), 1.71 (1H, td, J 9.7, 1.3), 1.62-1.54 (2H, m), 1.44 (1H, qd, J 9.7, 1.3), 1.41-1.34 (2H, m), 1.30 (1H, ddd, J 24.0, 11.7, 5.8), 1.25-1.19 (3H, m), 1.17 (1H, q, J 9.5), 1.11 (3H, s), 0.97 (3H, d, J 6.7), 0.78 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.6, 163.8, 141.1, 127.9, 123.6, 120.1, 55.4, 53.4, 50.6, 43.6, 39.5, 37.7, 36.0, 35.2, 34.0, 33.9, 31.4, 28.1, 23.7, 20.6, 17.9, 16.3, 14.4, 11.9
E. Synthesis of (6α, 7α)-6,7-epoxy-3-oxo-4-choleno-24-nitrile
[0398] ##STR00086##
[0399] A solution of 3-oxo-4,6-choladieno-24-nitrile (1.25 g, 3.56 mmol) in EtOAc (2.5 mL) and HFIP (5 mL) under argon was cooled to 10° C. MTO (8.9 mg, 0.036 mmol), 3-methylpyrazole (0.017 mL, 0.213 mmol) and UHP (0.37 g, 3.91 mmol) were charged and the mixture was stirred for 2 h. Further portions of MTO (8.9 mg, 0.036 mmol), 3-methylpyrazole (0.017 mL, 0.213 mmol) and UHP (67 mg, 0.71 mmol) were charged and the mixture was stirred overnight at 10° C. The reaction was quenched by addition of 5% aq. NaHSO.sub.3 (15 mL) was charged and the mixture was extracted with EtOAc (20 mL). The aqueous phase was separated and extracted with EtOAc (20 mL). The combined organic phases were washed with 5% aq. NaCl (20 mL) and were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using a gradient of EtOAc in heptane as the eluent to give the desired product (0.92 g, 70%). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.11 (1H, s), 3.46 (1H, d, J 3.7), 3.34 (1H, d, J 3.6), 2.55 (1H, ddd, J 18.1, 14.3, 5.5), 2.47-2.44 (1H, m), 2.41-2.37 (1H, ddd, J 16.9, 8.3, 5.0), 2.30 (1H, dt, J 16.8, 8.4), 2.01 (1H, dt, J 12.9, 3.3), 1.98-1.83 (5H, m), 1.71 (1H, td, J 6.9, 5.2), 1.61-1.56 (1H, m), 1.52 (1H, dq, J 12.7, 3.6), 1.46 (1H, ddd, J 12.4, 11.4, 7.0), 1.41-1.26 (5H, m), 1.22-1.17 (2H, m), 1.10 (3H, s), 0.97 (3H, d, J 6.6), 0.76 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=198.3, 162.6, 131.1, 120.1, 55.3, 54.6, 52.6, 51.3, 43.2, 50.6, 39.3, 35.6, 35.1, 34.6, 34.1, 33.9, 31.4, 28.2, 23.6, 19.9, 17.8, 17.2, 14.4, 11.8
F. Synthesis of (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile
[0400] ##STR00087##
[0401] A solution of 0.5 M ZnCl.sub.2 in THF (4.65 mL, 2.33 mmol) was cooled to −15° C. and a solution of 1M EtMgBr in TBME (4.65 mL, 4.65 mmol) was charged dropwise over 1 h. A solution of (6α, 7α)-6,7-epoxy-3-oxo-4-choleno-24-nitrile (0.95 g, 2.58 mmol) in THF (4.75 mL) was charged to the resulting mixture over 30 mins. Further portions of 1 M EtMgBr in TBME (4.65 mL, 4.65 mmol and 2.33 mL, 2.33 mmol) were charged after 15 and 20 mins respectively. The reaction mixture was quenched by addition of a sat. aq. NH.sub.4Cl (2 mL), filtered and the filter-cake washed with TBME (20 mL). The filtrate was washed with sat. aq. NH.sub.4Cl (3×20 mL), 5% w/v aq. NaCl (2×20 mL) and concentrated. The residue was purified by column chromatography using EtOAc in heptane to give (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile in 37% yield. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.78 (1H, s), 3.73 (1H, s), 2.48 (1H, ddd, J=17.5, 15.1, 4.9), 2.40-2.36 (2H, m), 2.32-2.26 (2H, m), 2.04-2.00 (2H, m), 1.94-1.89 (1H, m), 1.87-1.83 (1H, m), 1.81-1.73 (2H, m), 1.70 (1H, td, J=11.3, 2.1), 1.64-1.42 (8H, m), 1.40-1.33 (2H, m), 1.27-1.13 (3H, m), 1.22 (3H, s), 0.97 (3H, d, J=6.6), 0.92 (3H, t, J=7.4), 0.76 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.1, 170.4, 128.7, 120.1, 72.2, 55.5, 55.3, 50.1, 44.3, 42.6, 39.2, 38.3, 37.5, 35.6, 35.2, 34.1, 31.5, 28.0, 26.3, 23.6, 20.9, 19.7, 17.8, 14.3, 12.8, 11.9
G. Synthesis of (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile
[0402] ##STR00088##
[0403] To a solution of (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile (350 mg, 0.88 mmol) in DMF (2.1 mL) under argon, was charged Pd/C (83 mg, 10% Pd, 45% in H.sub.2O). The reaction vessel was purged with H2 and stirred under H2 overnight. The Pd/C was removed by filtration through a PTFE syringe filter and the filter rinsed with TBME (6×2 mL). The filtrate was washed with 5% w/vaq. NaCl (2×10 mL). The aqueous phase was extracted with TBME and the combined organic phases were concentrated to an oily residue. Purification of the residue by column chromatography using EtOAc in heptane to afford (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile in 74% yield. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.71 (1H, br, s), 3.34 (1H, dd, J=15.5, 13.4), 2.41-2.33 (2H, m), 2.30 (1H, dt, J=16.8, 8.4), 2.15-2.09 (2H, m), 2.02 (1H, dt, J=12.8, 3.5), 1.98 (1H, dd, J=11.9, 4.6), 1.94-1.89 (2H, m), 1.88-1.83 (1H, m), 1.82 (1H, dd, J=13.4, 4.6), 1.71-1.67 (1H, m), 1.65 (1H, td, J=5.6, 2.8), 1.60-1.14 (17H, m), 1.05 (3H, s), 0.98 (3H, d), 0.94 (3H, t, J=7.2), 0.88 (1H, t, J=7.1), 0.73 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=213.5, 120.2, 72.1, 55.6, 50.2, 49.9, 47.0, 46.7, 42.8, 39.5, 37.7, 36.3, 36.0, 35.7, 35.2, 34.2, 31.5, 28.1, 27.7, 24.4, 23.8, 20.8, 17.9, 14.3, 13.9, 11.8
H. Synthesis of (5β, 6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile
[0404] ##STR00089##
[0405] To a solution of (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile (245 mg, 0.61 mmol) in DCM (6.13 mL) under argon, was added DMP (312 mg, 0.74 mmol) in two portions, 5 min apart. The resulting pink suspension was stirred for 30 mins and quenched by addition of 10% w/v aq. Na.sub.2S.sub.2O.sub.3:2% w/v aq. NaHCO.sub.3 (5 mL). The aqueous phase was extracted with TBME (3×20 mL) and the combined organic phases were washed with 5% w/v aq. NaCl (20 mL) and concentrated. The residue was purified by column chromatography using EtOAc in heptane to afford (5β, 6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile in 88% yield. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=2.45 (1H, t, J=11.4), 2.38 (1H, ddd, J=16.9, 8.2, 5.1), 2.31-2.20 (5H, m), 2.06 (1H, dt, J=12.9, 3.4), 1.99 (1H, quintet, J=4.7), 1.92-1.78 (7H, m), 1.65 (1H, ddd, J=14.4, 9.9, 4.6), 1.60-1.53 (4H, m), 1.52-1.47 (1H, m), 1.40-1.29 (2H, m), 1.25-1.14 (3H, m), 1.16 (3H, s), 0.98 (3H, d, J=6.6), 0.84 (3H, t, J=7.4), 0.74 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=214.5, 211.5, 120.1, 57.4, 54.8, 50.1, 48.6, 47.2, 44.8, 43.7, 43.2, 39.1, 35.8, 35.3, 35.1, 35.0, 31.4, 28.2, 24.6, 23.9, 23.6, 21.7, 18.0, 14.3, 12.7, 12.3
I. Synthesis of (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile
[0406] ##STR00090##
[0407] To a suspension of NaBH.sub.4 (19 mg, 0.50 mmol) in IPA (0.8 mL) cooled to −20° C. was charged a solution of (5β, 6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile (200 mg, 0.50 mmol) in EtOAc (1.3 mL) dropwise over 13 mins. A solution of 0.5 M H2SO.sub.4 (0.5 mL) in water (0.8 mL) was charged slowly and the reaction mixture was stirred over 15 min and diluted with water (10 mL). The mixture was extracted with EtOAc (3×10 mL) and the combined organic phases were washed with 5% w/v aq. NaCl (3×10 mL) and concentrated. The residue was purified by column chromatography using EtOAc in heptane to afford (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile in 73% yield. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.59-3.55 (1H, m), 2.57 (1H, dd, J, 11.9, 10.8), 2.38 (1H, ddd, J 16.9, 8.4, 5.0), 2.28 (1H, dt, J 16.8, 8.4), 2.20-2.16 (1H, m), 2.00-1.94 (2H, m), 1.93-1.83 (3H, m), 1.81-1.72 (3H, m), 1.70-1.64 (3H), 1.57-0.53 (1H, m), 1.52-1.43 (4H, m), 1.39-1.34 (1H, m), 1.32-1.25 (2H, m), 1.22 (3H, s), 1.19-1.11 (4H, m), 0.96 (3H, d, J 6.6), 0.95-0.90 (1H, m), 0.85 (3H, t, J 7.3), 0.69 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=215.3, 120.1, 70.6, 62.1, 54.6, 49.6, 48.7, 45.5, 42.9, 42.6, 39.8, 38.8, 35.6, 35.4, 35.0, 31.5, 29.6, 28.2, 26.6, 26.0, 24.9, 21.4, 18.0, 14.3, 13.1, 12.2
J. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-24-oic acid
[0408] ##STR00091##
[0409] A mixture of (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile (130 mg, 0.33 mmol) in MeOH (6 mL), water (6 mL) and KOH (1.8 g, 32.14 mmol) was heated at reflux over 7 h, stirred at ambient for 16 h, then heated at reflux for a further 4 h. The reaction mixture was cooled to ambient temperature and acidified to pH1 with 6M HCl. The mixture was extracted with EtOAc (3×20 mL) and the combined organic phases were washed with 5% w/v aq. NaCl (20 mL) and concentrated to give crude (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-24-oic acid in 82% yield. .sup.1H and .sup.13C NMR matched those of an authentic sample.
[0410] The product of step J, can be converted to a compound of general formula (XXI) in which R.sup.4′ is C(O)OH by reduction, for example using sodium borohydride.
[0411] As a person of skill in the art would appreciate, the synthetic route shown in Scheme 5 could be adapted by conversion of the nitrile group to a carboxylic acid at an earlier stage followed, if necessary, by protection of the carboxylic acid group, for example as an ester.
Example 5—Preparation of an Analogue of a Compound of General Formula (I) and a Compound of General Formula (XXI) Via Compounds of General Formula (I) with Nitrile Side Chain (not Including Side Chain Extension)
[0412] Scheme 6 shows an alternative route in which the side chain is not extended.
##STR00092##
A. Synthesis of (20R)-cyanomethyl-4-pregnen-3-one
[0413] ##STR00093##
[0414] To a suspension of (20S)-20-bromomethyl-4-pregnen-3-one (15 g, 38.1 mmol) in DMF (225 mL) was added potassium cyanide (7.5 g, 114 mmol). The suspension was stirred at 80° C. for 41 h before cooling to room temperature. EtOAc (250 mL) and water (500 mL) were added and the layers were separated. The aqueous layer was extracted with EtOAc (2×250 mL) and the combined organic phases were washed with 5% aq. NaCl (250 mL) and were concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (heptane/EtOAc) to afford the desired product (9.7 g, 75%) as a white solid. δH (700 MHz, CDCl.sub.3); 5.73 (1H, s, C4-CH), 2.45-2.32 (4H, m), 2.27 (1H, ddd, J=14.6, 4.2, 2.7), 2.24 (1H, dd, J=16.8, 7.1), 2.04-1.99 (2H, m), 1.89-1.78 (3H, m), 1.72-1.65 (2H, m), 1.57-1.51 (2H, m), 1.43 (1H, qd, J=13.2, 4.0), 1.31-1.16 (4H, m), 1.18 (3H, s), 1.17 (3H, d, J=6.7), 1.11-1.01 (2H, m), 0.94 (1H, ddd, J=12.3, 10.7, 4.1), 0.74 (3H, 5); δC (176 MHz, CDCl.sub.3); 199.5, 171.2, 123.9, 118.9, 55.7, 54.7, 53.6, 42.5, 39.2, 38.5, 35.7, 35.6, 34.0, 33.6, 32.8, 31.9, 28.0, 24.8, 24.1, 20.9, 19.3, 17.4, 12.1.
B. Synthesis of (20R)-cyanomethyl-4,6-pregnadien-3-one
[0415] ##STR00094##
[0416] To a suspenstion of (20R)-cyanomethyl-4-pregnen-3-one (9.1 g, 26.8 mmol) in toluene (36 mL) and acetic acid (0.15 mL) was added p-chloranil (7.2 g, 39.5 mmol). The mixture was heated at reflux for 90 minutes before allowing to cool to room temperature. The suspension was filtered, washing with toluene (25 mL). The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (heptane/EtOAc). The material was then dissolved in acetone (35 mL) and methanol (23 mL) and 0.5 M aq. NaOH (200 mL) was added dropwise. Water (100 mL) was added and the resulting solid was filtered, washing with water (2×50 mL) and 2:1 acetone:water (2×20 mL). The solid was dried in vacuo to afford the desired product (5.4 g, 60%) as a pale brown solid. δH (700 MHz, CDCl.sub.3); 6.11 (2H, s), 5.67 (1H, s), 2.57 (1H, ddd, J=18.0, 14.4, 5.4), 2.45-2.42 (1H, m), 2.37 (1H, dd, J=16.7, 3.7), 2.25 (1H, dd, J=16.7, 7.2), 2.01 (1H, t, J=10.4), 2.03 (1H, dt, J=12.8, 3.3), 2.00 (1H, ddd, J=13.2, 5.4, 2.1), 1.96-1.91 (1H, m), 1.88-1.81 (1H, m), 1.74-1.70 (1H, m), 1.58 (1H, dq, J=13.4, 3.6), 1.44 (1H, qd, J=4.4, 3.9), 1.36-1.20 (7H, m), 1.18 (3H, d, J=6.7), 1.11 (3H, s), 0.79 (3H, s); δC (176 MHz, CDCl.sub.3); 199.6, 163.67, 140.8, 128.1, 123.7, 118.8, 54.6, 53.2, 50.5, 43.5, 39.1, 37.6, 36.0, 33.9, 33.9, 33.5, 28.0, 24.8, 23.6, 20.6, 19.3, 16.3, 12.0.
C. Epoxidation of (20R)-20-(1-cyanomethyl)-pregna-4,6-dien-3-one to form (6α, 7α, 20R)-20-(1-cyanomethyl)-6,7-epoxy-pregn-4-en-3-one
[0417] ##STR00095##
[0418] (20R)-Cyanomethyl-4,6-pregnadien-3-one (5.1 g, 15.1 mmol) was dissolved in HFIP (20 mL) and EtOAc (10 mL) and was cooled to 10° C. MTO (38 mg, 1 mol %), 3-methylpyrazole (73 μL, 6 mol %) and UHP (1.6 g, 16.6 mmol) were added and the mixture stirred at 10° C. After 4 h, MTO (38 mg, 1 mol %), 3-methylpyrazole (73 μL, 6 mol %) and UHP (0.28 g, 3.0 mmol) were added and the mixture stirred at 10° C. After a further 17 h, MTO (38 mg, 1 mol %), 3-methylpyrazole (73 μL, 6 mol %) and UHP (0.28 g, 3.0 mmol) were added and the mixture stirred at 10° C. After a further 72 h the mixture was quenched with 5% aq. sodium bisulfite (20 mL). The mixture was diluted with EtOAc (80 mL), 5% aq. sodium bisulfite (50 mL) and 5% aq. sodium chloride (50 mL). The aqueous phase was extracted with EtOAc (80 mL), and the combined organics washed with 5% aq. sodium chloride (50 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography on silica gel (heptane-EtOAc) to give the desired product (3.9 g, 73%) as an off-white solid. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.11 (1H, s, C4-CH), 3.46 (1H, d, J=3.9, C6-CH), 3.33 (1H, d, J=3.8, C7-CH), 2.55 (1H, ddd, J=5.6, 14.2, 18.1, C2-CH.sub.aH.sub.b), 2.48-2.45 (1H, m, C2-CH.sub.aH.sub.b), 2.39 (1H, dd, J=3.8, 16.7, C22-CH.sub.aH.sub.b), 2.23 (1H, dd, J=7.6, 16.8, C22-CH.sub.aH.sub.b), 2.01-1.91 (4H, m, C1-CH.sub.aH.sub.b, C12-CH.sub.aH.sub.b, C15-CH.sub.aH.sub.b, C16-CH.sub.aH.sub.b), 1.88 (1H, td, J=10.9, 1.3, C8-CH), 1.84-1.80 (1H, m, C20-CH), 1.72 (1H, td, J=5.2, 13.9, C1-CH.sub.aH.sub.b), 1.56-1.49 (2H, m, C11-CH.sub.aH.sub.b, C14-CH), 1.38-1.21 (6H, m, C9-CH, C11-CH.sub.aH.sub.b, C12-CH.sub.aH.sub.b, C15-CH.sub.aH.sub.b, C16-CH.sub.aH.sub.b, C17-CH), 1.18 (3H, d, J=6.8, C21-CH.sub.3), 1.10 (3H, s, C19-CH.sub.3), 0.77 (3H, s, C18-CH.sub.3); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=198.3, 162.5, 131.2, 118.9, 54.6, 54.5, 52.5, 51.2, 43.2, 40.5, 38.9, 35.5, 34.6, 34.1, 33.8, 33.7, 28.2, 24.8, 23.6, 19.8, 19.3, 17.2, 11.9.
D. Synthesis of (6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one
[0419] ##STR00096##
[0420] THF (17 mL) was charged to the reaction vessel, followed by 0.5 M zinc chloride in THF (16.8 mL), and the mixture cooled to −15° C. 1M Ethylmagnesium bromide in TBME (16.8 mL) was added dropwise over ca. 1 h, maintaining the temperature <−7° C. Copper (I) chloride (92 mg, 0.93 mmol) was charged to the reaction mixture. (20R)-Cyanomethyl-6,7-α-epoxy-4-pregnen-3-one (3.3 g, 9.3 mmol) was dissolved in THF (19 mL) and charged dropwise to the reaction mixture, maintaining the temperature <−7° C. The mixture was stirred at −15° C. upon complete addition. After 1 h a second portion of 1M ethylmagnesium bromide in TBME (17 mL) was added dropwise. The mixture was stirred at −15° C. After a further 30 min the mixture was quenched with sat. aq. ammonium chloride (3 mL) and allowed to warm to 15° C. The precipitate was removed by filtration and rinsed with TBME (50 mL). The filtrate was washed with sat. aq. ammonium chloride (3×50 mL) and 5% aq. sodium chloride (25 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford (6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one (3.2 g, 89%) as an off-white solid which was used without further purification.
[0421] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.78 (1H, s), 3.73 (1H, t, J=1.6), 2.48 (1H, ddd, J=17.5, 15.0, 4.9), 2.40-2.36 (2H, m), 2.31 (1H, ddd, J=8.7, 6.9, 1.9), 2.23 (1H, dd, J=16.7, 7.4), 2.03 (1H, ddd, J=13.4, 5.1, 2.3), 1.99 (1H, dt, J=12.7, 3.4), 1.95-1.90 (1H, m), 1.83-1.76 (3H, m), 1.70 (1H, td, J=5.7, 2.1), 1.63-1.44 (6H, m), 1.37-1.16 (5H, m), 1.22 (3H, s), 1.18 (3H, d, J=6.7), 0.92 (3H, t, J=7.4), 0.76 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.1, 170.3, 128.7, 118.9, 72.1, 55.3, 54.8, 50.0, 44.2, 42.6, 38.9, 38.3, 37.5, 35.6, 34.1, 33.6, 28.0, 26.3, 24.8, 23.6, 20.8, 19.7, 19.3, 12.8, 11.9.
E. Synthesis of (5β, 6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one
[0422] ##STR00097##
[0423] (6β, 7α, 20R)-Cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one (3.1 g, 8.1 mmol) was dissolved in DMF (54.5 mL) and 10% Pd/C charged (0.79 g of a 45% dispersion in water). The mixture was degassed and filled with hydrogen. After 18 h 30 min the mixture was degassed and filled with argon, filtered and rinsed with TBME (3×60 mL). The filtrate was re-filtered and rinsed with TBME (2×50 mL). The filtrate was washed with 5% aq. sodium chloride (100 mL), and the aqueous phase re-extracted with TBME (100 mL). The combined organic phases were washed with 5% aq. sodium chloride (2×100 mL) and concentrated. The residue was purified by flash chromatography (heptane-EtOAc) to afford (5β, 6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one (2.5 g, 80%) as an off-white solid.
[0424] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.69 (1H, s), 3.38 (1H, dd, J 15.5, 13.4), 2.39-2.34 (2H, m), 2.25 (1H, dd, J=16.7, 7.4), 2.14-2.08 (2H, m), 2.04-1.98 (2H, m), 1.94-1.90 (2H, m), 1.83-1.80 (2H, m), 1.76-1.74 (1H, m), 1.64 (1H, td, J=11.2, 2.7), 1.60-1.54 (2H, m), 1.51-1.40 (4H, m), 1.38-1.25 (4H, m), 1.21-1.15 (1H, m), 1.18 (3H, d, J=6.7), 1.05 (3H, s), 0.94 (3H, t, J=7.1), 0.74 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=213.7, 118.9, 71.7, 54.9, 50.0, 49.9, 47.0, 46.7, 42.7, 39.1, 37.7, 36.3, 35.9, 35.7, 34.0, 33.6, 28.1, 27.6, 24.8, 24.4, 23.7, 20.7, 19.3, 13.9, 11.9.
F. Synthesis of (5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one
[0425] ##STR00098##
[0426] (5β, 6β, 7α, 20R)-Cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one (2.4 g, 6.3 mmol) was dissolved in DCM (60.5 mL) and cooled to 0° C. Dess-Martin periodinane (DMP, 4.8 g, 11.3 mmol) was added over 1 minute. The reaction mixture was stirred at 0° C. After 1 h a second portion of DMP (1.6 g, 3.8 mmol) was added. After 2 h a third portion of DMP (1.6 g, 3.8 mmol) was added. After 3 h a fourth portion of DMP (0.5 g, 1.3 mmol) was added. After 3 h 45 min the mixture was diluted with 10% aq. Na.sub.2S2O.sub.3/5% aq. NaHCO.sub.3 (120 mL) and TBME (90 mL) and stirred vigorously. The phases were separated and the aqueous phase re-extracted with TBME (60 mL). The combined organic phases were concentrated and purified by flash chromatography (heptane-EtOAc) to afford (5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one (1.8 g, 75%) as an off-white solid.
[0427] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=2.43 (1H, t, J=11.4), 2.38 (1H, dd, J=16.7, 3.6), 2.30-2.20 (5H, m), 2.04 (1H, dt, J=12.7, 3.1), 2.01 (1H, dt, J=9.4, 4.7), 1.94-1.76 (7H, m), 1.66 (1H, ddd, J=14.3, 9.7, 4.3), 1.58-1.52 (4H, m), 1.33-1.21 (4H, m), 1.18 (3H, d, J=6.7), 1.15 (3H, s), 0.85 (3H, t, J=7.4), 0.75 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=214.4, 211.5, 118.8, 57.1, 54.0, 50.1, 48.4, 47.2, 44.7, 43.6, 43.2, 38.8, 35.8, 35.2, 34.9, 33.5, 28.1, 24.8, 24.5, 23.7, 23.4, 21.6, 19.3, 12.6, 12.3.
G. Synthesis of (3α, 5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregnane
[0428] ##STR00099##
[0429] Sodium borohydride (20 mg, 0.52 mmol) was suspended in isopropanol (0.8 mL) and cooled to −20° C. (5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one (200 mg, 0.52 mmol) was dissolved in ethyl acetate (1.7 mL) and TBME (1.2 mL) and added dropwise to the cold borohydride suspension. The mixture was stirred at −20° C. for 45 min, then quenched by addition of 0.7 M sulfuric acid (1.4 mL) and allowed to warm to 18° C. The mixture was diluted with water (10 mL) and TBME (10 mL) and the phases separated. The aqueous phase was re-extracted with TBME (10 mL) and the combined organic extracts washed with 5% aq. sodium chloride (10 mL). The organic phase was concentrated and purified by flash chromatography to afford (3α, 5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregnane (113 mg, 56%, containing 10% 3β-OH) as a pale yellow syrup.
[0430] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.00-3.99 (0.1H, m, H-3.sub.3β-OH, 3.68-3.53 (0.9H, m, H-3.sub.3α-OH, 2.57 (1H, dd, J=11.6, 11.1), 2.38 (1H, dd, J=16.7, 3.7), 2.23-2.20 (2H, m), 1.99-1.87 (5H, m), 1.83-1.64 (6H, m), 1.55-1.45 (3H, m), 1.31-1.18 (7H, m), 1.22 (3H, s), 1.17 (3H, d, J=6.7), 0.99-0.93 (1H, m), 0.84 (3H, t, J=7.3), 0.70 (3H. s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=215.3 (3α-C═O). 119.0 (CN), 70.5, 62.1, 54.0, 49.6, 48.7, 45.5, 42.8, 42.6, 39.8, 38.5, 35.6, 35.4, 33.6, 29.5, 28.2, 26.6, 26.0, 24.8, 24.8, 21.3, 19.4, 13.1, 12.2.
H. Synthesis of (3α, 5β, 6α)-6-ethyl-7-oxo-24-nor-lithocholic acid
[0431] ##STR00100##
[0432] (3α, 5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregnane (65 mg, 0.17 mmol) was dissolved in methanol (3 mL) and 30% w/v potassium hydroxide solution (3 mL) and heated to reflux for 4 days. The mixture was cooled in an ice bath and 6M hydrochloric acid added to pH 8 (2 mL). Ethyl acetate (10 mL) was added, followed by 6M HCl to pH 1 (0.5 mL). The mixture was allowed to warm to 18° C. and the phases separated. The organic phase was washed with 5% aq. sodium chloride (20 mL) and concentrated to afford (3α, 5β, 6α)-6-ethyl-7-oxo-24-nor-lithocholic acid (69 mg, quantitative) as a pale yellow syrup. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.56-3.52 (1H, m), 2.69 (1H, q, J=6.2), 2.48 (1H, dd, J=15.0, 3.3), 2.36 (1H, t, J=11.3), 2.22-2.17 (1H, m), 2.05-2.02 (1H, m), 1.99 (1H, dt, J=12.8, 3.3), 1.94-1.87 (2H, m), 1.84-1.69 (6H, m), 1.51-1.44 (3H, m), 1.32-1.09 (6H, m), 1.22 (3H, s), 1.03 (3H, d, J=6.5), 0.98-0.92 (1H, ddd, J=24.4, 12.3, 6.3), 0.86 (1H, q, J=12.6), 0.80 (3H, t, J=7.4), 0.69 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.9, 178.8, 71.2, 54.8, 52.0, 50.7, 49.9, 49.0, 43.7, 72.7, 41.2, 38.9, 35.7, 34.2, 33.5, 31.7, 29.8, 28.4, 24.6, 23.5, 21.8, 19.6, 18.8, 12.1, 12.0.
[0433] Alternatively, the product of step H can be converted to a compound of general formula (XXI) in which R.sup.4′ is C(O)OH by reduction, for example with sodium borohydride.
[0434] As a person of skill in the art would appreciate, the synthetic route shown in Scheme 5 could be adapted by conversion of the nitrile group to a carboxylic acid at an earlier stage followed, if necessary, by protection of the carboxylic acid group, for example as an ester.
Example 6—Preparation of a Compound of General Formula (IF) with an Aldehyde Side Chain
[0435] Scheme 7 illustrates a method for converting a compound of general formula (II) with an aldehyde side chain to a compound of general formula (IF) with an aldehyde side chain. The first step of the method is to protect the aldehyde as a dioxolane group. The compound of general formula (II) is then converted sequentially to compounds of general formulae (IA), (IB), (IC), (ID), (IE) and (IF) using the reagents shown in Scheme 7, still with the aldehyde protected. The protection is then removed by treatment with acid.
##STR00101## ##STR00102##
A. Synthesis of (6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one
[0436] ##STR00103##
[0437] A solution of 0.5M ZnCl.sub.2 in THF (3.1 mL) and THF (4 vol, 4 mL) was cooled to −15° C. and a solution of 1M EtMgBr in TBME (4.7 mL) was added dropwise over 10 mins, maintaining the temperature below −12° C. CuCl (13 mg, 0.13 mmol) was then charged in one portion followed by the dropwise addition of a solution of (6α, 7α, 20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregna-4-en-3-one from Example 1F (1.0 g, 2.6 mmol) in THF (8 vol, 8 mL) over 16 mins, maintaining the temperature below −12° C. The reaction was stirred at −15° C. for 40 mins (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain), warmed to ambient temperature and quenched by the dropwise addition of sat. aq. NH.sub.4Cl (2.5 vol, 2.5 mL). The reaction mixture was then diluted with EtOAc (50 mL) and washed with sat. aq. NH.sub.4Cl (2×50 mL) and water (2×50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatography gave (6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one as an off white crystalline solid (1.06 g). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.78 (1H, s), 4.85 (1H, d, J=2.0), 3.94 (2H, m), 3.89 (2H, m), 3.74 (1H, m), 2.46 (1H, m), 2.37 (1H, m), 2.31 (1H, m), 2.06-1.93 (3H, m), 1.85-1.68 (4H, m), 1.59 (3H, s), 1.58-1.25 (6H, m), 1.25 (1H, m), 1.22 (3H, s), 1.18 (1H, m), 0.95 (3H, d, J=6.7), 0.91 (3H, t, J=7.4), 0.75 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.1, 170.4, 128.7, 106.0, 72.3, 65.2, 65.1, 55.1, 52.3, 49.6, 44.4, 42.9, 39.3, 39.1, 38.3, 37.5, 35.7, 34.1, 27.3, 26.4, 23.9, 20.9, 19.7, 12.8, 11.7, 11.6.
B. Synthesis of (5β, 6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one
[0438] ##STR00104##
[0439] 5% Pd on CaCO.sub.3 (90 mg, 0.2 mass eq) was charged to a flask under argon, followed by a solution of (6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one (450 mg, 1.1 mmol) in DMF (3 vol, 2.25 mL) and MeCN (6 vol, 84.5 mL). The flask was purged with argon, then and stirred at ambient temperature. After 24 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) the reaction mixture was purged with argon and then filtered through a PTFE 0.45 μm filter. The filter was washed with EtOAc (2×25 mL). The organic phase was washed with water (3×25 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatography gave (5β, 6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one as an off white crystalline solid (167 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.85 (1H, d, J=2.0), 3.95 (2H, m), 3.85 (2H, m), 3.70 (1H, s), 3.37 (1H, dd, J=13.5, 15.5), 2.37 (1H, m), 2.11 (2H, m), 2.04-1.91 (4H, m), 1.81 (2H, m), 1.62-1.65 (3H, m), 1.55-1.40 (8H, m), 1.31-1.25 (2H, m), 1.18 (1H, m), 1.05 (3H, s), 0.95 (6H, m), 0.72 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=213.8, 106.0, 72.0, 65.2, 65.0, 52.4, 49.7, 49.6, 47.0, 46.8, 43.0, 39.3, 37.7, 36.3, 36.1, 35.8, 34.1, 31.9, 27.7, 27.4, 24.4, 24.0, 22.7, 20.8, 13.9, 11.6.
C. Synthesis of (5β, 6β, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione
[0440] ##STR00105##
[0441] To a solution of (5β, 6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one (110 mg, 0.25 mmol) in CH.sub.2Cl.sub.2 (25 vol, 2.75 mL) under argon was added Dess-Martin periodinane (127 mg, 0.3 mmol). After 30 minutes (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) the reaction mixture was diluted with EtOAc and 10% Na.sub.2S2O.sub.3/2% NaHCO.sub.3 and stirred for 1 h. The phases were separated and the aqueous extracted with EtOAc (10 mL). The combined organic phases were washed with 1M aq. NaOH (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. to give crude (5β, 6β, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione as a white solid (104 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.85 (1H, d, J=2.0), 3.94 (2H, m), 3.84 (2H, m), 2.42 (1H, t, J=11.4), 2.32-2.19 (4H, m), 2.06 (1H, m), 2.02-1.75 (8H, m), 1.65 (1H, m), 1.59-1.39 (6H, m), 1.29-1.17 (2H, m), 1.15 (3H, s), 0.94 (3H, d, J=6.7), 0.84 (3H, t, J=7.3), 0.73 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=214.6, 211.6, 105.9, 65.2, 65.0, 57.1, 51.5, 49.8, 48.4, 47.4, 44.9, 43.6, 43.4, 39.2, 39.0, 35.8, 35.3, 34.9, 27.4, 24.8, 23.8, 23.4, 21.7, 12.6, 12.0, 11.7.
D. Synthesis of (5β, 6α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione
[0442] ##STR00106##
[0443] A solution of (5β, 6β, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione (100 mg, 0.3 mmol) in MeOH (20 vol) was warmed to 50° C. and aq. 0.5M NaOH (0.65 mmol) was added. After 16 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) the reaction was diluted with EtOAc (10 mL), washed with water (2×10 mL) and then 5% aq. NaCl (1×10 mL). The combined organic phases were washed with 1M aq. NaOH (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. to give (5β, 6α, 20S)-20-(ethylenedioxyethyl)-6-ethyl-pregna-3,7-dione as a clear oil (80 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.85 (1H, d, J=2.0), 3.93 (2H, m), 3.84 (2H, m), 2.74 (1H, q, J=4.6), 2.47 (1H, t, J=11.3), 2.30-2.16 (4H, m), 2.10-2.02 (3H, m), 1.98 (1H, m), 1.91-1.79 (3H, m), 1.72 (5H, m), 1.47-1.37 (2H, m), 1.33 (3H, s), 1.23 (1H, m), 1.07 (1H, m), 0.98 (1H, m), 0.94 (3H, d, J=6.7), 0.81 (3H, t, J=7.4), 0.71 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.0, 210.6, 105.9, 65.2, 65.0, 52.3, 52.2, 51.3, 50.0, 48.4, 43.7, 42.9, 39.1, 38.7, 38.3, 36.7, 35.9, 35.5, 27.5, 24.7, 22.9, 22.2, 18.6, 11.9, 11.8, 11.7.
E. Synthesis of (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-20-(ethylenedioxymethyl)-pregna-7-one
[0444] ##STR00107##
[0445] NaBH.sub.4 (80 mg, 0.2 mmol) in IPA (1.6 mL) was cooled to −15° C. (5β, 6α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione (80 mg, 0.2 mmol) in EtOAc (1.6 mL) was added dropwise over 10 mins. After 30 mins (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) the reaction was warmed to ambient temperature and quenched by the addition of 0.7M aq. H.sub.2SO.sub.4 (7 vol) dropwise over 5 mins. The reaction mixture was diluted with EtOAc (10 mL) and the organic phase was washed with water (3×5 mL) and 5% aq. NaCl (1×5 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. to give (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-20-(ethylenedioxymethyl)-pregna-7-one as a clear oil (60 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.85 (1H, d, J=1.9), 3.93 (2H, m), 3.84 (2H, m), 3.52 (1H, m), 2.69 (1H, dd, J=5.7, 12.9), 2.21 (1H, m), 2.0-1.92 (2H, m), 1.86-1.67 (8H, m), 1.51-1.34 (6H, m), 1.25 (2H, m), 1.21 (3H, s), 1.20-1.10 (3H, m), 0.93 (3H, d, J=6.7), 0.88 (1H, m), 0.80 (3H, t, J=7.4), 1.66 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.7, 106.0, 71.2, 65.3, 65.0, 52.0, 51.3, 50.7, 50.0, 48.5, 43.7, 43.0, 39.2, 38.8, 35.7, 34.3, 31.8, 29.9, 27.6, 24.9, 23.5, 21.9, 18.8, 12.0, 11.9, 11.7;
F. Synthesis of (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-20-(ethylenedioxymethyl)-pregnane
[0446] ##STR00108##
[0447] To a solution (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-20-(ethylenedioxymethyl)-pregna-7-one (60 mg, 0.14 mmol) in THF (5 mL) and water (1.25 mL) at 0° C., was added NaBH.sub.4 (53 mg, 1.4 mmol) in one portion. After 2 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) the reaction was allowed to warm up to ambient temperature and was quenched by the addition of 1:1 MeOH:H.sub.2O (2 mL), followed by 2M aq. H.sub.2SO.sub.4 (1 mL) dropwise over 5 mins. The reaction mixture was diluted with EtOAc (20 mL) and washed with water (3×20 mL). The aqueous phase was extracted with EtOAc (20 mL) and the combined organic phases washed with 5% aq. NaCl (1×5 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. to give (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-20-(ethylenedioxymethyl)-pregnan as a clear oil (58 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.85 (1H, d, J=2.0), 3.94 (2H, m), 3.84 (2H, m), 3.40 (1H, m), 2.00-1.91 (2H, m), 1.80-1.75 (5H, m), 1.70-1.63 (2H, m), 1.61-1.56 (1H, m), 1.53-1.12 (15H, m), 1.01 (1H, m), 0.94 (3H, d, J=6.7), 0.90 (5H, m), 0.67 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=104.9, 71.2, 69.7, 64.1, 63.9, 51.1, 48.8, 44.1, 41.9, 40.0, 39.0, 38.3, 38.2, 34.4, 34.3, 32.8, 32.1, 29.5, 26.3, 22.8, 22.0, 21.1, 19.6, 10.5, 10.4, 10.4.
G. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al
[0448] ##STR00109##
[0449] (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-20-(ethylenedioxymethyl)-pregnan (58 mg, 0.14 mmol) in MeCN (1 mL, 17 vol), H.sub.2O (0.29 mL, 5 vol) and TFA (0.29 mL, 5 vol) was heated to reflux. After 2 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Anisaldehyde stain) the reaction mixture was poured onto 5% aq. NaHCO.sub.3 (30 mL) and diluted with CH.sub.2Cl.sub.2 (10 mL). After stirring for 15 minutes the phases were separated and the aqueous phase extracted with CH.sub.2Cl.sub.2 (2×100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. to give (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al as a mixture of C.sub.20 epimers as a clear oil (51 mg). NMR data matches an authentic sample of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al.
[0450] The compound of general formula (IF) with the aldehyde side chain can then be converted to a compound of general formula (XXI) in which —YR.sup.4a is C(O)OH by oxidation using any appropriate method. In one such method, the aldehyde could be directly oxidised to the acid using a Jones reaction or KMnO.sub.4. Alternatively, if chain extension is required, an olefination reaction followed by saponification will provide a compound in which R.sup.4a is C(O)OH but in which Y has been extended as shown in Example 7.
Example 7—Preparation of a Compound of General Formula (XXI) Via Compounds of General Formula (I) with OH and Aldehyde Side Chain
[0451] Scheme 8 below shows a method for the conversion of a compound of general formula (II) in which —YR.sup.4 is —CH.sub.2OH to a compound of general formula (XXI) in which —YR.sup.4a is CH.sub.2CH.sub.2C(O)OH
##STR00110## ##STR00111##
A. Synthesis of (6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-4-en-3-one
[0452] ##STR00112##
[0453] A solution of 0.5 M ZnCl.sub.2 in THF (20.2 mL) was charged to a reaction vessel under argon followed by THF (4 vol, 26 mL) and cooled to −15° C. A solution of 1 M EtMgBr in TBME (27 mL) was charged over 10 mins whilst maintaining the temperature below −12° C. CuCl (84 mg, 0.84 mmol) was then charged in one portion. (6α, 7α, 20S)-6,7-epoxy-20-acetoxymethyl-pregna-4-en-3-one (6.5 g, 16.8 mmol) in THF (8 vol, 16 mL) was charged to the reaction vessel over 16 mins while maintaining the temperature below −12° C. and the reaction warmed to ambient temperature and stirred for 90 mins. The reaction mixture was quenched by the dropwise addition of sat. aq. NH.sub.4Cl (2.5 vol, 17 mL). The reaction mixture was filtered and the filtrate washed with sat. aq. NH.sub.4Cl (2×50 mL) and 5% aq. NaCl (2×50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. The crude (6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-4-en-3-one (6.7 g) was taken on to the next step with no further purification. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=5.77 (1H, s), 4.07 (1H, dd, J=10.6, 3.1), 3.79 (1H, dd, J=10.6, 7.4), 3.74 (1H, s), 2.47 (1H, m), 2.37 (1H, m), 2.32 (1H, t, J=8.1), 2.05 (3H, s), 2.04-1.98 (3H, m), 1.90-1.65 (5H, m), 1.60-1.35 (7H, m), 1.30-1.15 (6H, m), 1.02 (3H, d, J=6.6), 0.91 (3H, t, J=7.3), 0.76 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=199.2, 171.4, 170.9, 128.5, 72.1, 69.4, 55.3, 52.6, 49.9, 44.2, 42.6, 39.0, 38.3, 37.4, 35.8, 35.6, 34.1, 27.6, 26.3, 23.7, 21.0, 20.8, 19.7, 17.1, 12.8, 11.9.
B. Synthesis of (5β, 6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-3-one
[0454] ##STR00113##
[0455] 5% Pd/CaCO.sub.3 (274 mg, 0.2 mass eq) was charged to a flask under argon. (6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-4-en-3-one (1.37 g, 3.3 mmol) in DMF (3 vol, 4.1 mL) was charged followed by MeCN (6 vol, 8.2 mL). The flask was purged with argon, purged with hydrogen and stirred at RT. After 24 h, the reaction mixture was purged with argon and filtered through Whatman® GF/B grade filter pad (glass fiber pore size 1 μm) filter pad. The solids were washed with EtOAc (2×25 mL). The filtrate was then washed with H.sub.2O (3×30 mL), dried over Na.sub.2SO.sub.4 and concentrated in-vacuo at 40° C. Purification by column chromatography gave (5β, 6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-3-one as an off white crystalline solid (0.96 g, 69%). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.08 (1H, dd, J=10.7, 3.4), 3.79 (1H, dd, J=10.7, 7.3), 3.71 (1H, s), 3.36 (1H, dd, J=15.5, 13.5), 2.36 (1H, td, J=14.1, 4.6), 2.11 (1H, m), 2.06 (3H, s), 2.03-1.10 (21H, m), 1.05 (3H, s), 1.03 (3H, d, J=6.6), 0.94 (3H, t, J=7.1), 0.73 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=213.7, 171.4, 72.0, 69.5, 52.7, 49.9, 49.8, 47.0, 46.7, 42.8, 39.3, 37.7, 36.3, 36.0, 35.8, 35.7, 34.2, 27.7, 27.6, 24.4, 23.9, 21.0, 20.8, 17.2, 13.9, 11.8.
C. Synthesis of (5β, 6β, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol acetate [or (5β, 6β, 20S)-20-acetoxymethyl-6-ethyl-pregna-3,7-dione]
[0456] ##STR00114##
[0457] (5β, 6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-3-one (3.31 g, 7.9 mmol) was dissolved in CH.sub.2Cl.sub.2 (25 vol, 83 mL) under argon and cooled to 0° C. Dess Martin periodane (4.0 g, 9.5 mmol) was charged in portions over 5 mins. After 20 mins the reaction was quenched by the addition of 10% aq. Na.sub.2SO.sub.4/2% aq. NaHCO.sub.3 (20 mL) and the mixture stirred for 20 mins. The solution was diluted with EtOAc (100 mL) and H.sub.2O (100 mL). The aqueous layer was separated and extracted with EtOAc (100 mL). The combined organic layers were washed with 1M aq. NaOH (50 mL), then 5% aq. NaCl (50 mL) and the resulting cloudy solution passed through a silica plug and washed with EtOAc (2×100 mL). Concentration in-vacuo at 40° C. followed by purification by column chromatography gave (5β, 6β, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol acetate as a white crystalline solid (2.39 g, 73%). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.08 (1H, dd, J=10.7, 3.4), 3.79 (1H, dd, J=10.7, 7.4), 2.44 (1H, t, J=11.4), 2.31-2.19 (4H, m), 2.05 (3H, s), 2.00 (1H, m), 1.92-1.71 (6H, m), 1.65 (1H, m), 1.59-1.47 (3H, m), 1.39-1.17 (7H, m), 1.16 (3H, s), 1.03 (3H, d, J=6.7), 0.85 (3H, t, J=7.4), 0.75 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=214.6, 211.6, 171.3, 69.4, 57.3, 52.0, 50.0, 48.5, 47.3, 44.9, 43.6, 43.2, 39.0, 35.8, 35.7, 35.3, 35.0, 27.7, 24.7, 23.8, 23.5, 21.7, 21.0, 17.2, 12.6, 12.2.
D. Synthesis of (5β, 6α, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol [or (5β, 6a, 20S)-6-ethyl-20-hydroxymethyl-pregna-3,7-dione]
[0458] ##STR00115##
[0459] To a suspension of (5β, 6β, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol acetate (1.77 g, 4.2 mmol) in EtOH (12 vol, 21.5 mL) at 50° C. was added dropwise 0.5M aq. NaOH (18.9 mL, 9.45 mmol). The reaction was heated at 50° C. for 16 h, then cooled to ambient temperature, diluted with H.sub.2O (50 mL) and extracted with EtOAc (50 mL). The phases were separated and the aqueous phase extracted with EtOAc (2×50 mL). The combined organic phases were washed with 5% aq. NaCl (2×50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatography gave (5β, 6α, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol as a white crystalline solid (1.35 g, 86%). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.64 (1H, dd, J=10.4, 2.9), 3.37 (1H, dd, J=10.3, 7.1), 2.69 (1H, m), 2.47 (1H, t, J=11.3), 2.30-2.16 (5H, m), 2.10-2.03 (2H, m), 1.94-1.80 (3H, m), 1.72-1.49 (6H, m), 1.43 (1H, br.$), 1.33 (3H, s), 1.32-1.17 (3H, m), 1.06 (3H, d, J=6.7), 0.98 (1H, m), 0.81 (3H, t, J=7.4), 0.71 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.1, 210.6, 67.8, 52.4, 52.2, 51.5, 50.0, 48.7, 43.7, 42.7, 38.8, 38.6, 38.3, 36.7, 35.9, 35.5, 27.9, 24.7, 22.9, 22.3, 18.6, 16.8, 12.2, 11.8.
E. Synthesis of (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23, 24-dinor-cholane-22-ol [or (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-20-hydroxymethyl-pregna-7-one]
[0460] ##STR00116##
[0461] NaBH.sub.4 (136 mg, 3.6 mmol) in IPA (6.5 vol, 9 mL) was cooled to −15° C., then a solution of (5β, 6α, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol (1.35 g, 0.3.6 mmol) in EtOAc (6.5 vol, 9 mL) was added dropwise over 10 mins. After 20 mins the reaction was warmed to ambient temperature and quenched by the dropwise addition of 0.7M aq. H.sub.2SO.sub.4 (7 vol, 9.45 mL) over 10 mins. The reaction mixture was diluted with EtOAc (50 mL) and the organic phase washed with H.sub.2O (3×50 mL) and 5% aq. NaCl (50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatography and concentration in-vacuo at 40° C. gave (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol as a white crystalline solid (0.83 g, 61%). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.64 (1H, dd, J=10.5, 3.2), 3.53 (1H, m), 3.35 (1H, dd, J=10.4, 7.1), 2.69 (1H, m), 2.35 (1H, t, J=11.2), 2.20 (1H, m), 2.00 (1H, m), 1.92-1.67 (8H, m), 1.57-1.43 (3H, m), 1.34-1.23 (2H, m), 1.23 (3H, s), 1.21-1.10 (4H, m), 1.04 (3H, d, J=6.6), 0.98-0.83 (2H, m), 0.80 (3H, t, J=7.4), 0.67 (3H, s); .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.9, 71.2, 67.9, 52.0, 51.6, 50.7, 50.0, 48.8, 43.7, 42.8, 38.9, 38.7, 35.7, 34.3, 31.8, 29.6, 27.9, 24.8, 23.5, 21.9, 18.8, 16.8, 12.1, 12.0.
F. Synthesis of (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-ol (or (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-20-hydroxymethyl-pregnan)
[0462] ##STR00117##
[0463] (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol (0.83 g, 2.2 mmol) in THF (30 mL) and water (7.5 mL) was cooled to 0° C. and NaBH.sub.4 (830 mg, 22 mmol) added in 4 portions over 15 mins. After 2 h the reaction was warmed to room temperature and quenched by the addition of 1:1 MeOH:H.sub.2O (15 mL) followed by the dropwise addition of 2M aq. H2SO.sub.4 (11 mL) over 10 mins. The reaction mixture was diluted with EtOAc (100 mL) and washed with H.sub.2O (100 mL). The aqueous phase was extracted with EtOAc (3×100 mL) and the combined organic phases were washed with 5% aq. NaCl (3×100 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. to give (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-ol as a white solid (0.53 g, 64%). .sup.1H NMR (700 MHz, MeOD): δ=3.64 (1H, s), 3.57 (1H, dd, J=10.6, 3.1), 3.30 (1H, m), 3.23 (1H, dd, J=10.5, 7.4), 2.00 (1H, m), 1.90-1.70 (6H, m), 1.59 (1H, m), 1.57-1.44 (6H, m), 1.42-1.27 (5H, m), 1.21 (2H, m), 1.13 (1H, m), 1.04 (3H, d, J=6.6), 1.00 (1H, m), 0.91 (3H, s), 0.90 (3H, t, J=7.7), 0.71 (3H, s); .sup.13C NMR (176 MHz, MeOD): δ=71.7, 69.7, 66.5, 52.5, 50.0, 45.5, 42.3, 41.7, 40.1, 39.5, 38.8, 35.3, 35.1, 33.1, 32.9, 29.8, 27.5, 23.2, 22.3, 22.0, 20.5, 15.9, 10.9, 10.6.
G. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al
[0464] ##STR00118##
[0465] (3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-ol (421 mg, 1.11 mmol) in DMF (50 vol, 20 mL) was cooled to 0° C. Dess Martin periodinane (473 mg, 1.12 mmol) was charged in portions. After 2.5 h (TLC, eluant 7:3 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain), the reaction was quenched by the addition of 10% aq. NaHSO.sub.3/2% aq. NaHCO.sub.3 (5 mL) and the mixture stirred for 10 mins. The mixture was diluted with EtOAc (100 mL) and 5% NaCl (5 mL). The aqueous layer was extracted with EtOAc (50 mL). The combined organic phases were washed with 2M aq. NaOH (50 mL) and 5% aq. NaCl (4×50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatograph gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al as a 3:1 mixture with (5β, 6α, 7α)-6-ethyl-7-hydroxy-7-oxo-23,24-dinor-cholane-22-al (white foam, 230 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=9.56 (1H, d, J=3.4), 3.71 (1H, br. s), 3.44-3.36 (1H, m), 2.38-2.33 (1H, m), 1.94-1.86 (2H, m), 1.83-1.81 (2H, m), 1.80-1.78 (2H, m), 1.74-1.36 (10H, m), 1.34-1.18 (8H, m), 1.14 (3H, d, J=6.8), 0.91 (3H, s), 0.88 (3H, t, J=7.07), 0.71 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=205.1, 72.3, 70.9, 51.0, 49.9, 49.5, 45.1, 43.3, 41.2, 40.0, 39.3, 35.6, 35.5, 34.0, 33.4, 30.6, 27.1, 24.1, 23.1, 22.2, 20.7, 13.5, 12.2, 11.6.
H. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic Acid Ethyl Ester
[0466] ##STR00119##
[0467] The HWE reagent was prepared by dropwise addition of TEPA (262 μL, 1.32 mmol) to NaOEt (91 mg, 1.3 mmol) in CH.sub.2Cl.sub.2 (2 mL) at 0° C. Thr reaction mixture was added dropwise over 10 minutes to a solution of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al (199 mg, 0.528 mmol) in CH.sub.2Cl.sub.2 (4 mL) at 0° C. The reaction was warmed to ambient temperature and stirred for 1 hour (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain). The mixture was diluted with H.sub.2O (20 mL) and CH.sub.2Cl.sub.2 (15 mL). The aqueous layer was separated and extracted with CH.sub.2Cl.sub.2 (3×20 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated. Purification by column chromatography gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic acid ethyl ester as a white foam (158 mg). The isolated product is a 4:1 mixture of the desired (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic acid ethyl ester and (5β, 6α, 7α)-6-ethyl-7-dihydroxy-3-oxo-22-cholen-24-oic acid ethyl ester. .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.83 (1H, dd, J=9.0, 15.6), 5.73 (1H, d, J=15.3), 4.17 (2H, q, J=7.1), 3.69 (1H, m), 3.40 (1H, m), 2.30-2.25 (1H, m), 1.92 (1H, m), 1.85-1.76 (2H, m), 1.76-1.62 (5H, m), 1.59 (1H, m), 1.54-1.34 (7H, m), 1.29 (3H, t, J=7.1), 1.33-1.23 (6H, m), 1.09 (3H, d, J=6.6), 0.90 (3H, s), 0.90 (3H, t, J=7.4), 0.68 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=167.1, 154.7, 119.0, 72.3, 70.8, 60.1, 54.9, 50.4, 45.2, 43.0, 41.0, 40.1, 39.8, 39.5, 35.6, 35.5, 34.0, 33.3, 30.6, 28.2, 23.7, 23.1, 22.2, 20.7, 19.3, 14.3, 12.1, 11.7.
I. (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic Acid Ethyl Ester
[0468] ##STR00120##
[0469] 10% Palladium on Carbon (79 mg) was charged to a flask under argon. A solution of (3α, 513, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic acid ethyl ester (135 mg, 0.312 mmol) in EtOAc (51 vol, 7.0 mL) was charged and purged with H2. After 70 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Anisaldehyde stain) the reaction mixture was filtered through a 0.45 μm PTFE filter and the filter washed with EtOAc (10 mL). Concentration in-vacuo at 40° C. gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid ethyl ester (134 mg) as a 4:1 mixture with (5β, 6α, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester. .sup.1H NMR (500 MHz, CDCl.sub.3): δ=4.13 (2H, q, J=7.2), 3.46-3.37 (1H, m), 2.41-2.32 (1H, m), 2.28-2.19 (1H, m), 1.89-1.76 (6H, m), 1.76-1.57 (5H, m), 1.54-1.34 (12H, m), 1.27 (3H, t, J=7.1), 1.25-1.12 (4H, m), 0.98-0.88 (9H, m), 0.68 (3H, s). .sup.13C NMR (126 MHz, CDCl.sub.3): δ=167.1, 154.7, 119.0, 72.3, 70.8, 60.1, 54.9, 50.4, 45.2, 43.0, 41.0, 40.1, 39.8, 39.5, 35.6, 35.5, 34.0, 33.3, 30.6, 28.2, 23.7, 23.1, 22.2, 20.7, 19.3, 14.3, 12.1, 11.7.
J. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic Acid (Obeticholic Acid)
[0470] ##STR00121##
[0471] To (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid ethyl ester (118 mg, 0.272 mmol) in EtOH (34 vol, 4 mL) at 50° C., was added 0.5M aq. NaOH (1.2 mL, 0.61 mmol) dropwise. The reaction mixture was stirred at 50° C. for 2.5 h (TLC, eluent 1:1 EtOAc:Heptane; visualized with Cerium Ammonium Molybdate stain) and then 0.5M aq. NaOH (1 mL, 0.5 mmol) was added. After 1 h, the reaction was quenched with 3M aq. HCl (2 mL). The aqueous phase was separated and extracted with EtOAc (3×15 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatography gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid (108 mg, white foam) as a 4:1 mixture with (5β, 6α, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid. NMR data was consistent with an authentic sample of OCA.
[0472] Alternatively, the product of step E can be converted to (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid via a 7-oxo intermediate with an aldehyde substituent on the side chain.
K. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-al
[0473] ##STR00122##
[0474] To a solution of (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol (0.5 g, 1.33 mmol) in CH.sub.2Cl.sub.2 (100 vol, 50 mL) at 0° C. under argon was charged in portions Dess Martin periodane (564 mg, 1.33 mmol) over 20 mins. After 2.5 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) the reaction was quenched by the addition of 10% aq. Na.sub.2SO.sub.4/2% NaHCO.sub.3 (10 mL) and the mixture stirred for 10 mins. The solution was diluted with EtOAc (100 mL) and H.sub.2O (100 mL). The aqueous layer was separated and extracted with EtOAc (100 mL). The combined organic layers were washed with 1M aq. NaOH (50 mL), 5% aq. NaCl (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentration in-vacuo at 40° C. Purification by column chromatography gave (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-al as an opaque oil (229 mg) along with recovered starting material (144 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=9.57 (1H, d, J=3.4), 3.54 (1H, m), 2.69 (1H, dd, J=5.7, 13.0), 2.35 (2H, m), 2.26 (2H, m), 1.97-1.90 (2H, m), 1.85-1.68 (7H, m), 1.55-1.46 (4H, m), 1.41-1.34 (1H, m), 1.23 (3H, s), 1.22-1.15 (3H, m), 1.12 (3H, d, J=6.9), 1.01 (1H, m), 0.87 (1H, m), 0.81 (3H, t, J=7.4), 0.70 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.4, 205.0, 71.1, 52.0, 50.6, 50.3, 49.8, 49.2, 48.4, 43.7, 43.2, 38.7, 35.7, 34.3, 31.8, 29.8, 27.1, 25.0, 23.5, 21.8, 18.8, 13.6, 12.5, 12.0
L. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-22-cholen-24-oic acid ethyl ester
[0475] ##STR00123##
[0476] To a suspension of NaOEt (43 mg, 0.63 mmol) in CH.sub.2Cl.sub.2 (0.8 mL) at 0° C. was added dropwise TEPA and the solution warmed to ambient temperature. The TEPA/NaOEt mixture was then added dropwise to a solution of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-23, 24-dinor-cholane-22-al (195 mg, 0.52 mmol) in CH.sub.2Cl.sub.2 (4 mL) at 0° C. over 10 mins. The reaction was stirred at 0° C. for 1 h and then ambient temperature for 1 h. The reaction mixture was then re-cooled to 0° C. and a further aliquot of the TEPA/NaOEt mixture added. The reaction was stirred at 0° C. and after 0.5 h (TLC, eluant 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain) H.sub.2O (3 vol, 0.6 mL) was added and the reaction mixture warmed to ambient temperature. The solution diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with H.sub.2O (10 mL). The aqueous layer was separated and extracted with CH.sub.2Cl.sub.2 (10 mL). The combined organic layers were washed with 5% aq. NaCl (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated. Purification by column chromatography gave (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-22-cholen-24-oic acid ethyl ester as an opaque oil (130 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=6.82 (1H, dd, J=9.0, 15.5), 5.72 (1H, d, J=15.5), 4.17 (2H, q, J=7.1), 3.53 (1H, m), 2.69 (1H, dd, J=5.8, 13.0), 2.36 (1H, t, J=11.3), 2.26 (1H, m), 2.17 (1H, m), 1.85-1.68 (9H, m), 1.47 (3H, m), 1.28 (3H, t, J=7.3), 1.27-1.23 (3H, m), 1.22 (3H, s), 1.20-1.10 (3H, m), 1.08 (3H, d, J=6.7), 0.97-0.83 (2H, m), 0.80 (3H, t, J=7.4), 0.68 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.6, 167.1, 154.5, 119.1, 71.1, 60.2, 54.0, 52.0, 50.6, 49.9, 48.9, 43.7, 42.9, 39.5, 38.8, 35.7, 34.3, 31.8, 29.9, 28.2, 24.7, 23.5, 21.8, 19.4, 18.8, 14.3, 12.4, 12.0.
M. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-24-oic acid ethyl ester
[0477] ##STR00124##
[0478] 10% Palladium on Carbon (53 mg) was charged to a flask under argon. (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-22-cholen-24-oic acid ethyl ester (107 mg, 0.24 mmol) dissolved in EtOAc (5.4 mL) was charged and the reaction purged with argon and then H2. After 16 h at ambient temperature (TLC, eluant 1:1 EtOAc: Heptane; visualized with Anisaldehyde stain) the reaction mixture was filtered through a 0.45 μm PTFE filter and the filter washed with EtOAc (10 mL). Concentration in-vacuo at 40° C. gave (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid ethyl ester as a clear oil (86 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.12 (2H, ddd, J=1.5, 7.1, 14.2), 3.53 (1H, m), 2.69 (1H, dd, J=5.7, 13.0), 2.35 (2H, m), 2.20 (2H, m), 2.00-1.90 (2H, m), 1.85-1.66 (9H, m), 1.50-1.39 (4H, m), 1.36-1.29 (2H, m), 1.25 (3H, t, J=7.1), 1.21 (3H, s), 1.20-1.08 (4H, m), 0.92 (3H, d, J=6.6), 0.90-0.82 (2H, m), 0.80 (3H, t, J=7.4), 0.65 (3H, s). .sup.13C NMR (176 MHz, CDCl.sub.3): δ=212.8, 174.3, 71.2, 60.2, 54.8, 52.0, 50.7, 49.9, 49.0, 43.7, 42.7, 39.0, 35.7, 35.2, 34.3, 31.8, 31.3, 31.0, 29.9, 28.3, 24.6, 23.5, 21.9, 18.8, 18.4, 14.3, 12.1, 12.0.
N. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid
[0479] ##STR00125##
[0480] To a solution of (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxoy-cholan-24-oic acid ethyl ester (73 mg, 0.16 mmol) in EtOH (1 mL) at 50° C. was added dropwise 0.5M aq. NaOH (0.72 mL, 0.36 mmol). The reaction was heated at 50° C. for 1 h (TLC, eluent 1:1 EtOAc: Heptane; visualized with Cerium Ammonium Molybdate stain), quenched by the addition of 2M aq. HCl (1 mL) and then diluted with H.sub.2O (10 mL) and EtOAc (10 mL). The phases were separated and the aqueous phase extracted with EtOAc (2×10 mL). The combined organic phases were washed with 5% aq. NaCl (2×10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in-vacuo at 40° C. Purification by column chromatography gave (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid as an oil (54 mg). .sup.1H NMR (700 MHz, CDCl.sub.3): δ=3.53 (1H, m), 2.69 (1H, dd, J=6.1, 12.9), 2.37 (2H, m), 2.25 (1H, m), 2.18 (1H, m), 2.0-1.89 (2H, m), 1.85-1.68 (7H, m), 1.50-1.40 (4H, m), 1.38-1.23 (5H, m), 1.22 (3H, s), 1.20-1.09 (4H, m), 0.93 (3H, d, J=6.6), 0.91-0.83 (2H, m), 0.80 (3H, t, J=7.4), 0.65 (3H, s).sup.13C NMR (176 MHz, CDCl.sub.3): δ=213.0, 179.6, 71.2, 54.8, 52.0, 50.7, 49.9, 49.0, 43.7, 42.7, 39.0, 35.7, 35.2, 34.3, 31.7, 31.0, 30.8, 29.8, 28.3, 24.6, 23.5, 21.9, 18.8, 18.4, 12.1, 12.0.
[0481] Examples 1 to 7 illustrate how the various side chains YR.sup.4 of the compounds of general formula (I) can be interconverted and how the compounds of general formula (IA), (IB), (IC), (ID), (IE) and (IF) can be converted to the required products of general formula (XXI).