5.BETA.-6-ALKYL-7-HYDROXY-3-ONE STEROIDS AS INTERMEDIATES FOR THE PRODUCTION OF STEROIDAL FXR MODULATORS

Abstract

The invention relates to compounds of formula (I): wherein R.sub.1, R.sub.2, Y, R.sub.4 and R.sub.5 are as defined herein. The compounds are intermediates in the synthesis of synthetic bile acids.

##STR00001##

Claims

1. A compound of general formula (I): ##STR00048## wherein: R.sup.1 is C.sub.1-4 alkyl optionally substituted with one or more substituents selected from halo, OR.sup.6 or NR.sup.6R.sup.7; where each of R.sup.6 and R.sup.7 is independently selected from H or C.sub.1-4 alkyl; R.sup.2 is H, halo or OH or a protected OH; Y.sup.1 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.3; each R.sup.3 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 or C.sub.1-4 alkyl; and R.sup.4 is C(O)OR.sup.10, OC(O)R.sup.10, C(O)NR.sup.10R.sup.11, OR.sup.10, OSi(R.sup.13).sub.3, S(O)R.sup.10, SO.sub.2R.sup.10, OSO.sub.2R.sup.10, SO.sub.3R.sup.10, or OSO.sub.3R.sup.10; 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, C.sub.2-20 alkynyl, —O—C.sub.1-20 alkyl, —O—C.sub.2-20 alkenyl or —O—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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2, or a 6- to 14-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, SR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or 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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; or d. a polyethylene glycol residue; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with halo, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; each R.sup.13 is independently a. C.sub.1-20 alkyl, C.sub.2-20 alkenyl or C.sub.2-20 alkynyl optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2, a 6- to 14-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; or b. 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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; R.sup.5 is H or OH or a protected OH; or a salt or an isotopic variant thereof.

2. A compound according to claim 1 wherein, independently or in any combination: Y is an alkylene linker group having from 1 to 8 carbon atoms and optionally substituted with one or more groups R.sup.3, wherein R.sup.3 is as defined in claim 1; R.sup.5 is H or OH.

3. A compound according to claim 2 wherein, independently or in any combination: R.sup.1 is ethyl; and/or R.sup.2 is H; and/or Y.sup.1 is a bond, —CH.sub.2— or —CH.sub.2CH.sub.2—, and/or R.sup.4 is C(O)OR.sup.10, where R.sup.10 is H, C.sub.1-6 alkyl or benzyl; and/or R.sup.5 is H.

4. A compound according to claim 1 which is (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid or the C.sub.1-6 alkyl and benzyl esters thereof and salts thereof, especially the methyl and ethyl esters.

5. A process for the preparation of a compound of general formula (I) ##STR00049## wherein: R.sup.1 is C.sub.1-4 alkyl optionally substituted with one or more substituents selected from halo, OR.sup.6 or NR.sup.6R.sup.7; where each of R.sup.6 and R.sup.7 is independently selected from H or C.sub.1-4 alkyl; R.sup.2 is H, halo or OH or a protected OH; Y.sup.1 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.3; each R.sup.3 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 or C.sub.1-4 alkyl; and R.sup.4 is C(O)OR.sup.10, OC(O)R.sup.10, C(O)NR.sup.10R.sup.11, OR.sup.10, OSi(R.sup.13).sub.3, S(O)R.sup.10, SO.sub.2R.sup.10, OSO.sub.2R.sup.10, SO.sub.3R.sup.10, or OSO.sub.3R.sup.10; 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, C.sub.2-20 alkynyl, —O—C.sub.1-20 alkyl, —O—C.sub.2-20 alkenyl or —O—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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2, or a 6- to 14-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, SR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or 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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; or d) a polyethylene glycol residue; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with halo, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; each R.sup.13 is independently a) C.sub.1-20 alkyl, C.sub.2-20 alkenyl or C.sub.2-20 alkynyl optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2, a 6- to 14-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; or b) 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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; R.sup.5 is H or OH or a protected OH; said process comprising either: A) reducing a compound of general formula (II): ##STR00050## wherein 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.3; or B) converting a compound of general formula (I) to another compound of general formula (I).

6. A process according to claim 5 wherein the reduction is carried out by catalytic hydrogenation.

7. A process according to claim 6 wherein the catalytic hydrogenation is carried out using a catalyst selected from a palladium/carbon, palladium/calcium carbonate, palladium/aluminium oxide, platinum/palladium or Raney nickel catalyst.

8. A process according to claim 6 wherein the catalyst is a palladium/carbon or palladium/calcium carbonate catalyst.

9. A process according to claim 8 wherein, in the catalyst the palladium is present in an amount of 5-10% by weight with respect to the weight of the matrix (where the matrix is the carbon, calcium carbonate etc).

10. A process according to claim 6 wherein the hydrogenation is carried out in a solvent selected from alcoholic solvents such as methanol, ethanol or isopropanol; ethyl acetate; pyridine; acetic acid; cyclopentyl methyl ether (CPME) or N,N-dimethylformamide (DMF), any of which may optionally be mixed with a co-solvent such as acetone or water and/or a base such as triethylamine.

11. A process according to claim 10 wherein the solvent is methanol, ethanol or DMF, particularly methanol or DMF.

12. A process according to claim 11 wherein the solvent is methanol optionally comprising a substoichiometric amount of a base such as trimethylamine.

13. A process according to claim 12, wherein the reaction is conducted at a temperature of about −30 to 25°.

14. A process according to claim 11 wherein the solvent is DMS optionally mixed with a co-solvent such as acetone, TBME, THF, acetonitrile or acetone/water and optionally comprising a substoichiometric amount, of a base such as triethylamine.

15. A process according to claim 12 wherein the reaction is conducted at a temperature of −30 to 0° C.

16. A process for the preparation of a compound of general formula (XIX): ##STR00051## wherein R.sup.1 is C.sub.1-4 alkyl optionally substituted with one or more substituents selected from halo, OR.sup.6 or NR.sup.6R.sup.7; where each of R.sup.6 and R.sup.7 is independently selected from H or C.sub.1-4 alkyl; Y.sup.1 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.3; each R.sup.3 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 or C.sub.1-4 alkyl; and R.sup.4 is C(O)OR.sup.10, OC(O)R.sup.10, C(O)NR.sup.10R.sup.11, OR.sup.10, OSi(R.sup.13).sub.3, S(O)R.sup.10, SO.sub.2R.sup.10, OSO.sub.2R.sup.10, SO.sub.3R.sup.10, or OSO.sub.3R.sup.10; 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, C.sub.2-20 alkynyl, —O—C.sub.1-20 alkyl, —O—C.sub.2-20 alkenyl or —O—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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2, or a 6- to 14-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, SR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or 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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; or d) a polyethylene glycol residue; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with halo, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; each R.sup.13 is independently a) C.sub.1-20 alkyl, C.sub.2-20 alkenyl or C.sub.2-20 alkynyl optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, OR.sup.19, SR.sup.19, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2, a 6- to 14-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; or b) 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, SO.sub.2R.sup.19, SO.sub.3R.sup.19 or N(R.sup.19).sub.2; each R.sup.19 is independently selected from H, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; R.sup.2 is H, halo or OH; R.sup.5a is H or OH; the process comprising: (i) oxidation of the compound of general formula (I) ##STR00052## wherein: R.sup.2 is H, halo or OH or a protected OH; and R.sup.5 is H or OH or a protected OH; using a suitable oxidizing agent to give a compound of general formula (XX): ##STR00053## wherein R.sup.2 is H, halo or OH or a protected OH; and R.sup.5 is H or OH or a protected OH; and (ii) epimerisation of the compound of general formula (XX) to give a compound of general formula (XXI): ##STR00054## wherein R.sup.2 is H, halo or OH or a protected OH group which is stable under basic conditions; and R.sup.5b is H or OH or a protected OH group which is stable under basic conditions; and (iii) reduction of the compound of general formula (XXI) using a suitable reducing agent and, where R.sup.2 and/or R.sup.5b is a protected OH, removal of the protecting group(s), to give a compound of general formula (XIX), wherein removal of the protecting group can take place before or after the reduction; and optionally (iv) conversion of a compound of general formula (XIX) to another compound of general formula (XIX).

17. A process according to claim 16 wherein, in step (i) the oxidation reaction is carried out using a Dess-Martin periodinane (1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol); a hypochlorite, for example sodium hypochlorite, under acidic conditions; a Jones reaction using sodium dichromate or, more usually, chromic trioxide in dilute sulfuric acid; or TEMPO ((2,2,6,6-Tetramethyl-piperidin-1-yl)oxy) or a derivative thereof.

18. A process according to claim 16 wherein, in the epimerization reaction of step (ii), the compound of general formula (XX) is dissolved in an alcoholic solvent, optionally mixed with water and contacted with a base, for example sodium or potassium hydroxide or a sodium or potassium alkoxide, typically an ethoxide.

19. A process according to claim 18 wherein, in the compound of general formula (XX), R.sup.4 is C(O)OR.sup.10, the base is sodium or potassium hydroxide and the epimerization is accompanied by hydrolysis to give a compound of general formula (XXI) in which R.sup.4 is C(O)OH; and/or in the compound of general formula (XX), R.sup.2 and/or R.sup.5 is a group OC(O)OR.sup.14, where R.sup.14 is C.sub.1-6 alkyl or benzyl; and wherein the epimerisation step yields a compound of general formula (XXI) in which R.sup.2 and/or R.sup.5b is OH; or in the compound of general formula (XX), R.sup.2 and/or R.sup.5 is a protected OH which is stable under basic conditions and the process further comprises the step of removing the protecting group before or after step (iii).

20. A process according to claim 16 wherein, in step (iii), the reducing agent is a hydride such as sodium borohydride.

21. A process according to claim 16 for the preparation of a compound of general formula (XVIII) wherein R.sup.1 is ethyl, R.sup.2 and R.sup.5a are both H, Y.sup.1 is —CH.sub.2CH.sub.2—, and R.sup.4 is C(O)OH.

Description

EXAMPLES 1 TO 4—SYNTHESIS OF (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester from Stigmasterol

Example 1—Synthesis of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid ethyl ester

[0201] ##STR00030##

[0202] The starting material, (22E)-3-oxo-4,22-choladien-24-oic acid ethyl ester, was prepared from stigmasterol according to the method described by Uekawa et al in Biosci, Biotechnol, Biochem., 2004, 68, 1332-1337.

[0203] (22E)-3-oxo-4,22-choladien-24-oic acid ethyl ester (1.00 kg, 2.509 mol; 1 eq) was charged to a reaction vessel, followed by AcOH (3 vol, 3.0 L) and toluene (1 vol, 1.0 L) with stirring. Chloranil (0.68 kg, 2.766 mol; 1.1 eq) was then charged and the reaction mixture heated to 100° C. and maintained at this temperature for 1-2 h (IPC by TLC on silica, eluent 3:7 EtOAc:Heptane; Starting Material: R.sub.f 0.50, Product: R.sub.f 0.46; visualise with anisaldehyde stain). The mixture was then cooled in an ice/water bath to 10° C. and the resulting solid was filtered off. The filter-cake was washed with premixed 3:1 AcOH:Toluene (4×0.5 vol) at 5° C.±4° C. and the filtrate concentrated in vacuo at up to 70° C. The residue was dissolved in acetone (3 vol), then 3% w/w aq. NaOH (10 vol) was charged dropwise with stirring, maintaining the temperature below 30° C. (exothermic). The resulting suspension was cooled to 10-15° C. and stirred for 30 mins. The solids were collected by filtration and the filter cake was washed with premixed 1:1 acetone:water (1×2 vol then 3×1 vol). The filter cake (tan solid) was dried under vacuum at 70-75° C., 672 g (68% yield). Characterisation of the compound agrees with the data published in the literature.

Example 2—(6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester

[0204] ##STR00031##

[0205] To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid ethyl ester (58.0 g, 146.3 mmol) in EtOAc (1.0 L) at reflux was added 80% MMPP (magnesium bis(monoperoxyphthalate) hexahydrate, 197.0 g, ca. 318.6 mmol) in four equal portions at 30 min intervals. The suspension was vigorously stirred at reflux for 5 h and at ambient temperature for a further 16 h. The reaction was then heated to reflux and stirred for an additional 6 h. The mixture was cooled to ca. 50° C. and the solids were filtered and rinsed with hot EtOAc (200 mL). The filtrate was subsequently washed with 20% aq. NaHSO.sub.3 (100 mL), 1M aq. NaOH (100 mL then 200 mL) and 10% aq. NaCl (250 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue (yellow solid) was crystallised from minimum volume of EtOAc at 60° C. to give the epoxide product as off white/pale yellow crystals (25.7 g, 43% yield, prisms). Characterisation of the compound agrees with the data published in the literature.

Example 3—Synthesis of (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester

[0206] ##STR00032##

Method 1:

[0207] To a suspension of Cul (1.40 g, 7.35 mmol) in diethyl ether (10 mL), cooled to −78° C. under an argon blanket was charged EtLi (28.8 mL, 14.4 mmol, 0.5 M solution in benzene/cyclohexane). The thick white suspension formed was allowed to warm to 0° C., stirred for 5 mins (forming a dark solution) and cooled to −78° C. A solution of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (1.00 g, 2.42 mmol) in diethyl ether/THF (24 mL, 3:1) was prepared and charged to the vessel containing the organocuprate. THF (1 mL) was used to rinse the vessel that contained the solution of the epoxide and this was also charged to the organocuprate. The reaction mixture was allowed to warm to −4° C. over 30 mins after which time the reaction was complete by TLC (silica, 1:1 EtOAc:heptane). After a further 30 mins of stirring at c.a. −4° C. a solution of aq. sat. NH.sub.4Cl was charged and the mixture was stirred over 30 mins. The mixture was transferred to a separating funnel and the aqueous phase was removed, along with solid material present at the interface. The organic phase was washed with 5 wt % aq NaHCO.sub.3 (2×50 mL.) and water (1×50 mL). TBME (50 mL) was used to extract the original aqueous phase from the reaction and the combined washes. The combined organic phases were concentrated and the residue was purified by chromatography using silica (25 g) as the stationary phase (gradient elution with 0-30% EtOAc in heptane) to give (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (0.63 g, 59%).

[0208] .sup.1H NMR (400 MHz, CDCl.sub.3): δ=6.82 (1H, dd, J=15.6, 8.9, C22H), 5.75 (1H, s, C4H), 5.74 (1H, d, J=15.6, C23H), 4.17 (2H, q, J=7.1, OCH.sub.2CH.sub.3), 3.72 (1H, br s, C7H), 2.52-2.25 (5H, m), 2.05-1.98 (2H, m), 1.82-1.10 (23H, m), 0.91 (3H, t, J=7.4, CH.sub.3), 0.77 (3H, s, CH.sub.3). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=199.2, 171.2, 167.1, 154.5, 128.4, 119.0, 71.9, 60.1, 55.3, 54.9, 49.9, 44.3, 42.7, 39.6, 39.1, 38.3, 37.4, 35.6, 34.0, 28.0, 26.3, 23.6, 20.8, 19.7, 19.2, 14.2, 12.8, 12.0; (IR) v.sub.max(cm.sup.−1): 3467, 2939, 2870, 1716, 1651, 1457, 1268, 1229, 1034; HRMS (ESI-TOF) m/z: (M+H).sup.+ calcd for C.sub.28H.sub.43O.sub.4 443.3161; found: 443.3156. mp=59.4-62.9° C.

Method 2

[0209] ZnCl.sub.2 (32.84 g, 240.9 mmol) was dried under vacuum with slow stirring at 180° C. for 2 h. The flask was cooled to room temperature under an argon atmosphere and the residue was dissolved in THF (520 mL) and transferred via cannula into a three neck reaction flask equipped with mechanical stirrer and temperature probe. The solution was cooled in an ice bath to 0-3° C. and a 3M solution of EtMgBr in Et.sub.2O (80 mL, 240.0 mmol) was added dropwise over 20 mins, maintaining the internal temperature below 10° C. Formation of a white precipitate (active zincate species) was observed after addition of ca. 1/3 of the Grignard solution. The mixture was stirred for 1.2 h at 0° C. before a solution of the epoxide (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (43.0 g, 104.2 mmol) in THF (300 mL) was added dropwise, maintaining the internal temperature below 10° C. Solid CuCl (1.03 g, 0.104 mmol) was then added in two equal portions with vigorous stirring. After 10 mins the cooling bath was removed and stirring continued at ambient temperature for an additional 1.2 h. The reaction was quenched by dropwise addition of sat. aq. NH.sub.4Cl (800 mL) at <15° C. and stirred for 0.5 h. The mixture was filtered and the solid rinsed with TBME (150 mL). The phases were separated and the aqueous phase extracted with TBME 2×250 mL. The combined organic extracts were washed with 10% aq. NaCl (2×200 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give 43.7 g of the crude (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester as a yellow foam.

Method 3

[0210] To a solution of ZnCl.sub.2 in THF (0.5 M, 8.7 mL, 4.85 mmol, 0.9 eq) was charged anhydrous THF (8.0 mL) and the contents then cooled to −25° C. A solution of EtMgBr in TBME (1.0 M, 8.7 mL, 8.70 mmol, 1.8 eq) was added over 30 mins and the mixture stirred for 45 mins at −25° C. Solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added in one portion and a solution of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (2.0 g, 4.85 mmol) in THF (8.0 mL) was added dropwise over 30 mins. The remaining solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added half way through the addition of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester. The reaction was stirred for 1 h at −25° C., (TLC 1:1 Heptane:EtOAc, visualised by UV and developed using Ceric Ammonium Molybdate stain) and then additional of EtMgBr in TBME (1.0 M, 2.9 mL, 2.91 mmol, 0.6 eq) was added over 10 mins. The reaction was stirred for 0.5 h at −25° C. and then quenched by the addition of sat. aq. NH.sub.4Cl (5 mL), maintaining the temperature below −5° C. The inorganic salts were filtered off, rinsed with TBME and the filtrate phases were separated. The aqueous layer extracted with TBME and then the combined organic extracts were washed with sat. aq. NH.sub.4Cl (3×5 mL) and 10% brine (3×6 mL). The organic phase was concentrated in vacuo at 40° C. to give crude (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester as a yellow foam (1.91 g).

Method 4

[0211] To a solution of ZnCl.sub.2 in THF (0.5 M, 8.7 mL, 4.85 mmol, 0.9 eq) was charged anhydrous THF (8.0 mL) and the contents then heated to 40° C. A solution of EtMgBr in TBME (1.0 M, 8.7 mL, 8.70 mmol, 1.8 eq) was added over 30 mins and the mixture stirred for 45 mins at 40° C. Solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added in one portion and a solution of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (2.0 g, 4.85 mmol) in THF (8.0 mL) was added dropwise over 30 mins. The remaining solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added half way through the addition of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester. The reaction was stirred for 1 h at 40° C., (TLC 1:1 Heptane:EtOAc, visualised by UV and developed using Ceric Ammonium Molybdate stain) and then quenched by the dropwise addition of sat. aq. NH.sub.4Cl (5 mL). The inorganic salts were filtered off, rinsed with TBME and the filtrate phases were separated. The aqueous layer was extracted with TBME and then the combined organic extracts were washed with sat. aq. NH.sub.4Cl (3×5 mL) and 10% brine (3×6 mL). The organic phase was concentrated in vacuo at 40° C. to give crude (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester as a yellow foam (2.08 g).

Method 5

[0212] To a solution of ZnCl.sub.2 in THF (0.5 M, 8.7 mL, 4.85 mmol, 0.9 eq) was charged anhydrous THF (8.0 mL) and the contents then cooled to −15° C. A solution of EtMgBr in THF (1.0 M, 8.7 mL, 8.70 mmol, 1.8 eq) was added over 30 mins and the mixture stirred for 45 mins at −15° C. Solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added in one portion and a solution of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (2.0 g, 4.85 mmol) in THF (8.0 mL) was added dropwise over 30 mins. The remaining solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added half way through the addition of (6α,7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester. The reaction stirred for 1 h at −15° C., (TLC 1:1 Heptane:EtOAc, visualised by UV and developed using Ceric Ammonium Molybdate stain) and then additional EtMgBr in THF (1.0 M, 4.35 mL, 4.36 mmol, 0.9 eq) was added over 15 mins and then quenched by the dropwise addition of sat. aq. NH.sub.4Cl (5 mL). The inorganic salts were filtered off, rinsed with TBME and the filtrate phases were separated. The aqueous phase was extracted with TBME and then the combined organic extracts were washed with sat. aq. NH.sub.4Cl (3×5 mL) and 10% brine (3×6 mL). The organic phase was concentrated in vacuo at 40° C. to give crude (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester as a yellow foam (1.94 g).

Example 4—Synthesis of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester

[0213] ##STR00033##

Method 1

[0214] To a suspension of 10 wt. % Pd/C (50% wet, 20 mg, 8.6 mol %) in DMF (2 mL) was added a solution of (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (50 mg, 0.11 mmol) in DMF (3 mL) and the reaction mixture was cooled to 0° C. The flask was evacuated then filled with hydrogen three times with vigorous stirring. After 3 h the flask was evacuated then filled with argon and the mixture filtered via syringe filter. The mixture was partitioned between TBME (30 mL) and H.sub.2O (20 mL). The organic phase was dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The crude product (50 mg) was a 14:1 mixture of 5β to 5α isomers (analysed by .sup.1H NMR) of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester, yield 92%.

[0215] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.12 (2H, q, J=7.1, OCH.sub.2CH.sub.3), 3.71 (1H, br s, C7H), 3.34 (1H, dd, J=15.5, 13.6, C4H), 2.39-2.32 (2H, m), 2.24-2.20 (1H, m), 2.14-2.09 (2H, m), 2.03-1.91 (4H, m), 1.83-1.79 (2H, m), 1.68-1.63 (2H, m), 1.58 (1H, s), 1.55-1.12 (19H, m), 1.04 (3H, s), 0.95-0.93 (6H, m), 0.88 (1H, J=7.0), 0.71 (3H, s). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=213.5, 174.2, 72.1, 60.2, 55.9, 50.2, 49.8, 47.0, 46.7, 42.7, 39.5, 37.7, 36.3, 36.0, 35.7, 35.3, 34.2, 31.3, 31.0, 28.1, 27.7, 24.4, 23.8, 20.8, 18.3, 14.2, 13.9, 11.8. (IR) v.sub.max(cm.sup.−1): 3514, 2939, 2870, 1710, 1462, 1377, 1159, 1099, 1032; HRMS (ESI-TOF) m/z: (M—H.sub.2O+H).sup.+ calcd for C.sub.28H.sub.45O.sub.3 429.3369; found: 429.3363.

Method 2

[0216] (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (20.0 g) was dissolved in DMF (400 mL) and added under argon to solid 10 wt. % Pd/C (50% wet, 10.0 g). The mixture was cooled in an ice-salt bath to approximately −15° C. and the flask was evacuated then filled with hydrogen three times with vigorous stirring. The mixture was stirred under an atmosphere of hydrogen for 6 h then the flask was evacuated, filled with argon and filtered through a pad of celite. The catalyst was rinsed with 400 mL of TBME. The filtrate was washed with 10% aq. NaCl (400 mL) and the aqueous phase extracted with TBME (400 mL). The combined organic phases were washed with 10% aq. NaCl (3×200 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give crude (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester (20.0 g, ca. 28:1 5Hβ:5Hα ratio) as pale yellow oil.

Method 3

[0217] 10% Pd/C was charged to a stainless steel jacketed reaction vessel under an argon atmosphere; DMF was added (20 mL), followed by a solution of crude (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester from Example 3 (approximately 72.6 mmol) in DMF (130 mL). The reaction mixture was cooled to −25° C. (over approximately 40 mins) with vigorous stirring (1200 rpm). The reaction vessel was evacuated and charged with hydrogen (10-12 bar) three times. The mixture was stirred for 16 h under an atmosphere of hydrogen (10-12 bar). The vessel was evacuated, purged with argon and warmed to 20° C. with stirring. TLC of the reaction mixture (1:1 Heptane:EtOAc, developed using Ceric Ammonium Molybdate or vanillin dip, Rf values: starting material=0.42, product=0.67) indicated complete consumption of the starting material. The suspension was diluted with CH.sub.3CN (120 mL) and H.sub.2O (30 mL) and the suspension filtered via a double GFA filter paper and the filter cake rinsed with CH.sub.3CN (60 mL). The mixture was telescoped to the next step without further purification. The mixture contained approximately 5% of the 5H-α isomer.

Optimisation

[0218] The hydrogenation reaction of this example proceeds via the intermediate shown below and produces both the required 5Hβ compound and its 5Hα isomer. A solvent and catalyst screen was carried out to determine reaction conditions which led to the highest yield and the highest ratios of 5Hβ isomer to 5Hα isomer.

##STR00034##

[0219] The solvent screen was performed using 10 wt. % Pd/C catalyst and the reactions were run at room temperature under atmospheric pressure of hydrogen. The reaction run in MeOH in the presence of NEt.sub.3 was more selective than the one run in neat MeOH, whilst the addition of 10% of H.sub.2O decreased the 5βH selectivity. The reaction in DMF provided the best β:α ratio. The reaction in pyridine gave poor conversion to the required product with mainly starting material and intermediate present in the mixture.

TABLE-US-00001 Solvent 5H β:α ratio A MeOH 4:1 B MeOH:H.sub.2O 2:1 C MeOH:NEt.sub.3 7:1 D EtOH 3:1 E IPA 2:1 F EtOAc 2:1 G Pyridine 2:1 H AcOH 1:1 I CPME 1:1 J DMF 9:1

[0220] Reactions in DMF and MeOH were tested at a range of temperatures. For reactions run in DMF temperature has substantial impact on selectivity (the selectivity decreases with increasing temperature), while little difference was observed for reactions in MeOH.

[0221] Reactions in DMF and MeOH were tested at a range of commercially available 5 and 10 wt. % Pd catalysts, on carbon, calcium carbonate, barium sulfate and aluminium oxide support.

[0222] The reactions were run in 10 volumes of solvent at −15° C. under atmospheric pressure of hydrogen gas. For reactions run in DMF pressure has lower impact on the selectivity than the temperature. The effect of dilution on the selectivity is negligible.

Examples 5 TO 14—Synthesis of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester from Deoxycholic Acid

Example 5—Synthesis of (3α,5β)-3-acetoxy-12-oxo-cholan-24-oic acid methyl ester

[0223] ##STR00035##

[0224] To a solution of deoxycholic acid (500 g, 1.27 mol) in MeOH (1.5 L) was charged H.sub.2SO.sub.4 (0.68 mL, 12.7 mmol) and the reaction heated to 64° C. until complete. The reaction was cooled to 55° C. and pyridine (2.06 mL, 25.4 mmol) was charged. MeOH (800 mL) was removed by distillation and the reaction cooled to 50° C. EtOAc (500 mL) was charged and the distillation continued. This co-evaporation was repeated until the MeOH content was <0.5%. The reaction was cooled to 40° C. and EtOAc (1.0 L) was charged followed by Pyridine (134 mL, 1.65 mol) and DMAP (1.1 g, 8.89 mmol). Acetic anhydride (150 mL, 1.58 mmol) was added dropwise and the reaction vessel stirred at 40° C. until complete. The reaction was cooled to 22° C. and 2M aq. H.sub.2SO.sub.4 (1500 mL) added maintaining the temperature below 25° C. The aqueous phase was removed and the organic phase washed with water (1.2 L), sat. aq. NaHCO.sub.3 solution (1.2 L×2) and water (1.2 L). AcOH (1.0 L) was charged to the organic layer, followed by NaBr (6.6 g, 63.5 mmol). Aq. 16.4% NaOCl solution (958 mL, 2.54 mol) was charged dropwise maintaining the reaction temperature below 25° C. The reaction was stirred until complete, then cooled to 10° C. and stirred for 90 mins. The resulting solids were collected by filtration, washed with water (3×500 mL) and the filter cake dried under vacuum at 40° C. The solids were crystallised from MeOH (10 vol) to give (3α,5β)-3-acetoxy-12-oxo-cholan-24-oic acid methyl ester as an off white solid (268 g).

Example 6—Synthesis of (3α,5β)-3-acetoxy-cholan-24-oic acid methyl ester

[0225] ##STR00036##

[0226] (3α,5β)-3-acetoxy-12-oxo-cholan-24-oic acid methyl ester (268 g, 0.6 mol) was charged to the reaction vessel under argon, followed by AcOH (1.8 L). Tosyl hydrazide (190 g, 1.02 mol) was then added maintaining the reaction temperature at 25° C. The reaction was stirred until complete and then NaBH.sub.4 (113.5 g, 3.00 mol) was charged portion-wise maintaining the temperature below 25° C. The reaction mixture was stirred until complete and then quenched by the dropwise addition of water (1.34 L) maintaining the temperature below 25° C. The reaction mixture was stirred for 30 mins, the resulting solids collected by filtration, washed with water (3×270 mL) and the solid dried under vacuum at 40° C. The solids were crystallised from MeOH (3 vol) to give (3α,5β)-3-acetoxy-cholan-24-oic acid methyl ester as an off white solid (214.5 g).

Example 7—Synthesis of (3α,5β)-3-hydroxy-cholan-24-oic acid (Lithocholic Acid)

[0227] ##STR00037##

[0228] To a solution of (3α,5β)-3-acetoxy-cholan-24-oic acid methyl ester (214.5 g, 0.50 mol) in IPA (536 mL) was charged water (536 mL) and 50% w/w NaOH (99 g, 1.24 mol). The reaction was heated to 50° C. and stirred until complete. 2M H.sub.2SO.sub.4 was charged slowly with vigorous stirring until pH 2-3 was obtained and then the reaction cooled to 20° C. The resulting solids were collected by filtration, washed with water (3×215 mL) and the resultant solid dried under vacuum at 40° C. to give (3α,5β)-3-hydroxy-cholan-24-oic acid (176.53 g)

Example 8—Synthesis of (5β)-3-oxocholan-24-oic acid ethyl ester

[0229] ##STR00038##

[0230] To a solution of (3α,5β)-3-hydroxy-cholan-24-oic acid (10 g, 26.5 mmol) in EtOH (50 mL) was charged H.sub.2SO.sub.4 96% (14 μL, 0.27 mmol) and the reaction mixture then heated to reflux for 16 h. Pyridine was then charged, the mixture stirred for 30 mins and concentrated in vacuo at 40° C. The residue was dissolved in EtOAc (30 mL) and AcOH (10 mL) and NaBr (136 mg, 1.33 mmol) was then charged. The solution was cooled to 5° C. and NaOCl 9% (27 mL, 39.8 mmol) was charged dropwise maintaining the temperature below 10° C. The resulting suspension was warmed to ambient temperature and stirred for 1 h. The reaction mixture was cooled to 0° C. for 10 mins, the solids collected by filtration and washed with water (3×3 vol). The resultant solid was dried under vacuum at 40° C. to give (5β)-3-oxocholan-24-oic acid ethyl ester (7.83 g).

Example 9—Synthesis of (4α,5β)-3-oxo-4-bromo-cholan-24-oic acid ethyl ester

[0231] ##STR00039##

[0232] To a solution of (5β)-3-oxocholan-24-oic acid ethyl ester (8.0 g, 19.9 mmol) in AcOH (84 mL) was added Br.sub.2 in AcOH (16 mL, 21.9 mmol) dropwise over 15 mins. The reaction mixture was stirred for 10 mins, then diluted with EtOAc (250 mL), washed with water (2×200 mL) and concentrated in vacuo at 40° C. The crude material was purified by column chromatography (30% Heptane:EtOAc) and concentrated in vacuo at 40° C. to give (4α,5β)-3-oxo-4-bromo-cholan-24-oic acid ethyl ester as a pale crystalline solid (7.49 g).

Example 10—Synthesis of (5β)-3-oxo-4-cholene-24-oic acid ethyl ester

[0233] ##STR00040##

[0234] To a solution of (4α,5β)-3-oxo-4-bromo-cholan-24-oic acid ethyl ester (4.0 g, 8.33 mmol) in DMF (40 mL) was charged Li.sub.2CO.sub.3 (4.0 g, 1 mass eq) and LiBr (2.0 g, 0.5 mass eq). The mixture was heated to 150° C. for 2 h then allowed to cool to ambient temperature and poured onto a mixture of water and ice (200 g, 50 volumes) and AcOH (8 mL). The resulting suspension was stirred for 15 mins, the solids collected by filtration and then purified by column chromatography (30% Heptane:EtOAc) to give 3-oxo-4-cholene-24-oic acid ethyl ester as a pale crystalline solid (1.68 g).

Example 11—Synthesis of 3-oxo-4,6-choladien-24-oic acid ethyl ester

[0235] ##STR00041##

[0236] 3-oxo-4-cholene-24-oic acid ethyl ester (2.23 g, 5.57 mmol) was charged to a reaction vessel, followed by AcOH (6.7 mL) and toluene (2.23 mL). Chloranil (1.5 g, 6.13 mmol) was charged and the reaction mixture heated to 100° C. for 2 h (IPC by TLC, 3:7 EtOAc:Heptane; visualized with Anisaldehyde stain). The reaction mixture was cooled to 10° C. for 10 mins and the resulting solid removed by filtration. The filter cake was washed with DCM (9 vol) and the resulting filtrate then concentrated in vacuo at 40° C. The residue was dissolved in acetone (9 vol) then 3% w/w aq. NaOH (27 vol) was added dropwise maintaining the temperature below 30° C. The resulting mixture was cooled in an ice bath for 10 mins and the solids collected by filtration. The filter cake was washed with water (2×9 vol) and acetone:water 2:1 (4 vol). Purification by column chromatography (0-30% Heptane:EtOAc) gave 3-oxo-4,6-choladien-24-oic acid ethyl ester as a pale crystalline solid (1.45 g)

Example 12—Synthesis of (6α,7α)-6,7-epoxy-3-oxo-4-chola-ene-24-oic acid ethyl ester

[0237] ##STR00042##

[0238] 3-oxo-4,6-choladien-24-oic acid ethyl ester (1.37 g, 4.27 mmol) was charged to a reaction vessel, followed by BHT (23 mg, 0.13 mmol), EtOAc (11 mL) and water (3.4 mL) with stirring. The solution was heated to 80° C. and then a solution of mCPBA 70% (1.5 g, 7.51 mmol) in EtOAc (7.5 mL) was added dropwise over 15 mins. The reaction mixture was stirred at 70° C. for 2 h (IPC by TLC, 3:7 EtOAc:Heptane; visualized with Anisaldehyde stain), cooled to ambient temperature and then washed with 1M aq.NaOH (2×20 mL) followed by 10% aq. NaS.sub.2O.sub.3: 2% NaHCO.sub.3 (3×20 mL). The organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuo at 40° C. The crude solids were crystalized from EtOAc (3 vol) at 60° C. to give an off white solid which was dried under vacuum at 40° C. to give (6α,7α)-6,7-epoxy-3-oxo-4-chola-ene-24-oic acid ethyl ester (0.90 g).

Example 13—Synthesis of (6β,7α)-6-ethyl-7-hydroxy-3-oxo-4-cholen-24-oic acid ethyl ester

[0239] ##STR00043##

[0240] ZnCl.sub.2 (600 mg, 4.25 mmol) was charged to a reaction vessel and dried under vacuum at 180° C. for 1 h. The reaction vessel was cooled to ambient temperature, THF (15 mL) charged and the contents of the reaction vessel cooled to 3° C. A solution of 3M EtMgBr in Et.sub.2O (1.5 mL, 4.25 mmol) was charged to the reaction vessel over 40 mins maintaining the temperature below 5° C. The reaction mixture was then stirred for 1 h. (6α,7α)-6,7-epoxy-3-oxo-4-chola-ene-24-oic acid ethyl ester (0.80 g, 1.93 mmol) in THF (6 mL) was charged to the reaction vessel over 40 mins, maintaining the temperature below 5° C. CuCl (20 mg, 0.19 mmol) was charged in one portion and the reaction stirred at ambient temperature for 16 h (IPC by TLC, 3:7 EtOAc:Heptane; visualized with Anisaldehyde stain). The reaction mixture was cooled in an ice bath and sat. aq.NH.sub.4Cl was added dropwise, maintaining the temperature below 10° C. The reaction mixture was filtered and the filter cake washed with TBME (12.5 vol). The organic phase of the filtrate was separated and the aqueous phase extracted with TBME (2×12.5 vol). The combined organic phases were washed with 5% NaCl (3×12.5 vol) and concentrated in vacuo at 40° C.

Example 14—Synthesis of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester

[0241] ##STR00044##

[0242] 10% Pd/C (70 mg) was charged to a reaction vessel under an argon atmosphere followed by the crude material from Example 13 in DMF (14.6 mL). The mixture was cooled to −10° C. and the reaction vessel was evacuated then filled with hydrogen three times with vigorous stirring. The mixture was stirred under an atmosphere of hydrogen for 24 h while maintaining the temperature at −10° C. (IPC by TLC, eluent 1:1 EtOAc:Heptane; visualized with Anisaldehyde stain) then the flask was evacuated, filled with argon and filtered through a pad of celite and rinsed with DMF (7 mL). 10% Pd/C (70 mg) was recharged to the reaction vessel under an argon atmosphere followed by the DMF reaction mixture. The mixture was cooled to approximately −10° C. and the reaction vessel was evacuated then filled with hydrogen three times with vigorous stirring. The mixture was stirred under an atmosphere of hydrogen for 24 h at −10° C. (IPC by TLC, 1:1 EtOAc:Heptane; visualized with Anisaldehyde stain) then the flask was evacuated, filled with argon and filtered through a pad of celite and washed with TBME (62.5 vol, 50 mL). The filtrate was washed with 10% aq. NaCl (4×25 vol), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo at 40° C. Purification by column chromatography (SiO.sub.2, 0-30% Heptane:EtOAc) gave (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester (0.17 g). The product was identical to the material obtained from plant origin (6β,7α,22E)-6-ethyl-7-hydroxy-3-oxo-4,22-choladien-24-oic acid ethyl ester (see Example 4).

Examples 15 to 17—Conversion of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester to (3α,5β,6α,7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid

Example 15—Synthesis of (6β,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid ethyl ester

[0243] ##STR00045##

Method 1

[0244] A solution of Jones's reagent prepared from CrO.sub.3 (1.10 g, 11 mmol) in H.sub.2SO.sub.4 (1.4 mL) and made to 5 mL with water was charged dropwise to a solution of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester (0.18 g, 0.40 mmol) in acetone (10 mL) until an orange colour persisted. The reaction mixture was quenched with IPA (1 mL), filtered through a 0.45 μm nylon syringe filter and the filter was washed with acetone (10 mL). The combined filtrate and wash was concentrated, the residue was dissolved in EtOAc (20 mL) and washed with water (2×10 mL). The aqueous phase was extracted with EtOAc (20 mL), the combined EtOAc phases were concentrated and the residue was dissolved and concentrated from toluene (20 mL) then acetone (20 mL) to give a clear oil containing (6β,5β,7α)-6-ethyl-7-hydroxy-3,7-dioxo-cholan-24-oic acid ethyl ester (185 mg).

[0245] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=4.12 (2H, q, J=7.1), 2.42 (1H, t, J=11.4), 2.38-2.17 (6H, m), 2.09-1.74 (9H, m), 1.68-1.11 (17H, m), 0.93 (3H, d, J=6.5), 0.85 (3H, t, J=7.4), 0.72 (3H, s). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=214.5, 211.4, 174.0, 60.1, 57.1, 55.1, 50.3, 48.4, 47.3, 44.9, 43.6, 43.1, 39.2, 35.8, 35.2 (x2), 34.9, 31.3, 30.9, 28.1, 24.6, 23.7, 23.4, 21.7, 18.3, 14.2, 12.6, 12.2. (IR) v.sub.max(cm.sup.−1): 2950, 2872, 1709, 1461, 1377, 1304, 1250, 1177, 1097, 1034; HRMS (ESI-TOF) m/z: (M+H).sup.+ calcd for C.sub.28H.sub.45O.sub.4 445.3318; found: 445.3312;

Method 2

[0246] To a solution of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester (41.0 g crude mass) in anhydrous CH.sub.2Cl.sub.2 (600 mL) at 0° C. was added solid DMP (34.0 g, 80.2 mmol) portion-wise over 20 mins (exothermic). The mixture was stirred at 0-5° C. for 2 h, then a further portion of DMP (4.0 g, 9.4 mmol) was added and reaction stirred at 0-5° C. for 1 h. The mixture was filtered through a GFA filter and the solid rinsed with CH.sub.2Cl.sub.2 (50 mL), the filtrate was stirred vigorously with 10% aq. Na.sub.2S.sub.2O.sub.3 and 2% aq. NaHCO.sub.3 (100 mL) for 20 mins. The phases were separated and the aq. extracted with CH.sub.2Cl.sub.2 (2×100 mL). The combined organic extracts were washed with 1M NaOH (100 mL). The mixture was diluted with CH.sub.2Cl.sub.2 (300 mL) and phases separated. The organic layer was concentrated under reduced pressure and the residue (cloudy brown oil) was dissolved in TBME (600 mL) and washed with 1M NaOH (100 mL) and NaCl (3×100 mL). The organic phase was concentrated in vacuo to give a dark yellow runny oil, crude mass 38.1 g. The oil was dissolved in EtOH (400 mL) and stirred with activated charcoal (10 g) at 50° C., the mixture was then filtered, the charcoal rinsed with EtOH (200 mL) and the filtrate concentrated in vacuo to give (6β,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid ethyl ester as a yellow oil (35.9 g).

Method 3

[0247] A solution of (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester (218 mmol) in DMF (450 ml), CH.sub.3CN (540 mL) and H.sub.2O (90 mL) was charged into a 2 L vessel and cooled to 9° C., then AcOH (180 mL) was charged, followed by NaBr (4.1 g). A solution of sodium hypochlorite (˜10.5% w/v, 450 mL) was added dropwise over 1.5 h, maintaining the internal temperature at 5-6° C., then the mixture was stirred for 5 h at 7° C. TLC of the reaction mixture indicated complete consumption of the starting material (IPC by TLC, eluent EtOAc/heptane 3:7, Rf for (6β,5β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid ethyl ester=0.34; (6β,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid ethyl ester=0.45). A solution of aq. 10% w/v Na.sub.2SO.sub.3 (360 mL) was charged dropwise with vigorous stirring, maintaining the internal temperature at 8-10° C., then H.sub.2O (270 mL) was added dropwise and the mixture stirred at 5° C. for 16 h. The solid was filtered and washed with H.sub.2O (720 mL). The solid was then dissolved in TBME (1.1 L) and subsequently washed with an aq. NaHCO.sub.3 (300 mL) and 10% brine (300 mL). The organic phase was then stirred with activated charcoal (10 g) for 20 mins at 40° C., treated with anhydrous MgSO.sub.4 (5 g) and filtered via GFA filter paper, the filter cake was rinsed with TBME (50 mL) and the filtrate concentrated in vacuo to give (6β,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid ethyl ester as light brown oil which solidifies on standing (82.7 g).

Example 16—Synthesis of (6α,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid

[0248] ##STR00046##

[0249] Into a 500 mL flask was charged 0.5 vol of 0.5 M NaOH (9 mL) followed by (6β,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid ethyl ester from Example 15 (18.00 g, 1 eq) and then IPA (180 mL, 10 vol) (the initial NaOH charge was to avoid the possibility of C3-ketal formation). The mixture was warmed to 60±2° C. and held until a solution was obtained (10-15 mins). The remaining 0.5 M NaOH solution (171 mL, 9.5 vol) was charged over 20 mins and then the reaction was stirred for a further 3.5 h at 60±2° C. The IPA was removed under vacuum at 60° C. and then 2M HCl (8 mL) charged to pH 9. EtOAc was charged (90 mL, 5 vol) followed by 2M HCl (54 mL) to pH 1. Vigorous mixing was followed by phase separation. The aqueous phase was back extracted with additional EtOAc (90 mL, 5 vol) and then the combined organic phases were washed with water (54 mL, 3 vol), followed by three portions of 10% aq. NaCl (3×54 mL, 3×3 vol). The organic phase was treated with activated charcoal (100 mesh powder, 3.37 g, ˜0.20 mass eq) for 12 mins and then filtered through GF/B. Concentration at 50° C. in vacuo gave (6α,5β)-3,7-dioxo-6-ethyl-cholan-24-oic acid as a light yellow foam in quantitative yield.

[0250] .sup.1H NMR (700 MHz, CDCl.sub.3): δ=2.74 (1H, dd, J=12.8, 5.4), 2.47 (1H, t, J=12.5), 2.43-0.90 (32H, m), 0.81 (3H, t, J=7.4), 0.70 (3H, s). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=212.1, 210.6, 179.4, 54.9, 52.4, 52.3, 50.0, 48.9, 43.7, 42.7, 38.9, 38.3, 36.7, 36.0, 35.5, 35.2, 30.9, 30.7, 28.2, 24.6, 22.9, 22.3, 18.6, 18.3, 12.1, 11.8. (IR) v.sub.max(cm.sup.−1): 2939, 2873, 1706, 1458, 1382, 1284.8. HRMS (ESI-TOF) m/z: (M+H).sup.+ calcd for C.sub.26H.sub.41O.sub.4 417.3005; found: 417.2997; mp=71.2-75.9° C.

Example 17—Synthesis of (3α,5β,6α,7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid

[0251] ##STR00047##

[0252] To a solution of crude (6α,5β)-6-ethyl-3,7-dioxo-cholan-24-oic acid (21.7 g crude mass) in H.sub.2O (260 mL) and 50% NaOH (15.2 mL) at 90° C. was added, dropwise, a solution of NaBH.sub.4 (4.4 g, 116.3 mmol) in aq. NaOH (prepared from 25 mL of H.sub.2O and 0.8 mL 50% NaOH). The mixture was heated to reflux and stirred for 3 h. The mixture was then cooled to 60° C. and a 2M solution of HCl (200 mL) added dropwise with vigorous stirring. nBuOAc (100 mL) was then charged to the reaction flask and the mixture stirred for a further 20 mins. The phases were separated and the aqueous phase (pH=1/2) extracted with nBuOAc (100 mL). The combined organic phases were washed with 2M HCl (50 mL) and 10% aq. NaCl (100 mL). The organic solvent was distilled off under reduced pressure at 70-80° C. The residue (dense oil) was dissolved in nBuOAc (60 mL) at 70° C. and allowed to gradually cool to room temperature, then stored at 6° C. for 2 h. The solid was collected via filtration, rinsed with cold nBuOAc (20 mL), then dried under vacuum at 70° C. for 5 h to give (3α,5β,6α,7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid as a white solid (8.2 g).