METHOD FOR HOMOGENIZING BILE ACID DERIVATIVES

Abstract

The present invention relates to a process for producing bile acid derivatives having a protected hydroxyl group in the 3 position comprising contacting a bile acid derivative having an unprotected 3-alpha-hydroxyl group with a specific lipase. The present invention further relates to a bile acid derivative obtained or obtainable by the process, to the use of the bile acid derivative obtained or obtainable by the process for producing lithocholic acid and also to a process for producing lithocholic acid and to lithocholic obtained by the process. The invention further relates to the use of lithocholic acid obtained or obtainable by the process for producing ursodeoxycholic acid or ursodeoxycholic acid derivatives.

Claims

1. A process for producing bile acid derivatives having a protected hydroxyl group in the 3 position comprising: i) providing a first composition comprising at least one bile acid derivative of general formula I: ##STR00031## wherein the radical R.sup.1 is selected from the group consisting of C1- to C30-alkyl group, C1- to C30-alkenyl group, C1- to C30-alkynyl group, C5- to C12-cycloalkyl group and C5- to C12-aryl group, wherein the ring B of the bile acid derivative of general formula I has one or two further hydroxyl group(s) at position 6 or at positions 6 and 7 respectively; and wherein none of the rings A, C and D has further hydroxyl groups; ii) contacting the first composition comprising at least one bile acid derivative of general formula I from i) with a compound R.sup.2—X, wherein R.sup.2 is a —C(═O)—C1- to C30-alkyl group and X is selected from the group consisting of hydroxyl group, —O—C1- to C20-alkyl group, —O—C1- to C20-alkenyl group, —O—C1- to C20-alkynyl group; thiol group, —S—C1- to C20-alkyl group, amine group, —NHR.sup.3 group, —NR.sup.3R.sup.4 group, wherein R.sup.3 and R.sup.4 are each independently a C1- to C20-alkyl group, halogen atom and —O—(C═O)—R.sup.5 group, wherein R.sup.5 is a C1- to C30-alkyl group; and a lipase selected from the group consisting of lipase B from Candida antarctica of SEQ ID no. 1, lipase 1 from Diutina rugosa of SEQ ID no. 2, lipase 2 from Diutina rugosa of SEQ ID no. 3, lipase 3 from Diutina rugosa of SEQ ID no. 4, lipase 4 from Diutina rugosa of SEQ ID no. 5, lipase 5 from Diutina rugosa of SEQ ID no. 6, lipase from Rhizopus niveus of SEQ ID no. 7, lipase from Aspergillus niger of SEQ ID no. 8 and lipase from Penicillium camemberti FM 013 of SEQ ID no. 9 or a homologous enzyme having a sequence identity of at least 65% with one of the sequences of SEQ ID no. 1 to SEQ ID no. 9 and having the same function as the lipase of SEQ ID no. 1 to SEQ ID no. 9, to obtain a second composition comprising at least one bile acid derivative of general formula II: ##STR00032## wherein the radical R.sup.1 is as defined at i) for formula I and the radical R.sup.2 is as defined at ii), wherein the ring B of the bile acid derivative of general formula II has one or two further hydroxyl group(s) at position 6 or at positions 6 and 7 respectively; and wherein none of the rings A, C and D has further hydroxyl groups.

2. The process as claimed in claim 1, wherein the radical R.sup.1 is selected from the group consisting of C1- to C18-alkyl group, C5- to C7-cycloalkyl group and phenyl group, more preferably of C1- to C5-alkyl group, more preferably is an unbranched C1- to C3-alkyl radical, more preferably a methyl radical.

3. The process as claimed in claim 1, wherein X is selected from the group consisting of hydroxyl group, —O—C1- to C20-alkyl group, —O—C1- to C20-alkenyl group, —O—C1- to C20-alkynyl group, thiol group, —S—C1- to C20-alkyl group, amine group, —NHR.sup.3 group and —NR.sup.3R.sup.4 group, wherein R.sup.3 and R.sup.4 are each independently a C1- to C20-alkyl group, preferably from the group consisting of hydroxyl group, —O—C1- to C20-alkyl group and —O—C1- to C20-alkenyl group.

4. The process as claimed in claim 1, wherein the radical R.sup.2 is an unbranched —C(═O)—C1- to C18-alkyl group, preferably an unbranched —C(═O)—C1- to C5-alkyl group, more preferably —C(═O)—CH.sub.3.

5. The process as claimed in claim 1, wherein the lipase employed in ii) is lipase B from Candida antarctica of SEQ ID no. 1 or a homologous enzyme having a sequence identity of at least 65% with the sequence of SEQ ID no. 1 and having the same function as the lipase B from Candida antarctica of SEQ ID no. 1.

6. The process as claimed in claim 1, wherein the homologous enzyme has a sequence identity of at least 80%, preferably of at least 90%, more preferably of at least 95%, more preferably of at least 98%, with the sequence of SEQ ID no. 1 to SEQ ID no. 9 and the same function as the lipase.

7. The process as claimed in claim 1, wherein the ring B of the bile acid derivative of general formula I and of the bile acid derivative of general formula II has one or two further alpha-hydroxyl group(s) at position 6 or at positions 6 and 7 respectively.

8. The process as claimed in claim 1, wherein the bile acid derivative of general formula I is selected from the group consisting of R.sup.1 esters of hyodeoxycholic acid (HDCA), hyocholic acid (HCA) and mixtures of R.sup.1 esters of hyodeoxycholic acid (HDCA) and hyocholic acid (HCA), wherein R.sup.1 is as defined in claim 1 or 2.

9. A bile acid derivative of general formula II obtained or obtainable by a process as claimed in claim 1.

10. A bile acid derivative of general formula II, ##STR00033## wherein the radical R.sup.1 is selected from the group consisting of C1- to C30-alkyl group, C1- to C30-alkenyl group, C1- to C30-alkynyl group, C5- to C12-cycloalkyl group and C5- to C12-aryl group; and the radical R.sup.2 is a —C(═O)—C1- to C30-alkyl group, wherein the ring B of the bile acid derivative of general formula II has one or two further hydroxyl group(s) at position 6 or at positions 6 and 7 respectively; and wherein none of the rings A, C and D has further hydroxyl groups.

11. The bile acid derivative as claimed in claim 10 having the formula IIb oder IIc: ##STR00034## wherein the radical R.sup.1 is in each case selected from the group consisting of C1- to C30-alkyl group, C1- to C30-alkenyl group, C1- to C30-alkynyl group, C5- to C12-cycloalkyl group and C5- to C12-aryl group, preferably from the group consisting of C1- to C18-alkyl group, C5- to C7-cycloalkyl group and phenyl group, more preferably of C1- to C5-alkyl group, more preferably is an unbranched C1- to C3-alkyl radical, more preferably a methyl radical; and the radical R.sup.2 is a —C(═O)—C1- to C30-alkyl group, preferably an unbranched —C(═O)—C1- to C18-alkyl group, more preferably an unbranched —C(═O)—C1- to C5-alkyl group, more preferably —C(═O)—CH3.

12. The use of a bile acid derivative of general formula II, preferably a bile acid derivative of general formula II obtained or obtainable by the process as claimed in claim 1, ##STR00035## wherein the radical R.sup.1 and the radical R.sup.2 are as defined in claim 1; wherein the ring B of the bile acid derivative of general formula II has one or two further hydroxyl group(s) at position 6 or at positions 6 and 7 respectively; and wherein none of the rings A, C and D has further hydroxyl groups, for producing lithocholic acid.

13. A process for producing lithocholic acid comprising i) providing a first composition comprising at least one bile acid derivative of general formula I: ##STR00036## wherein the radical R.sup.1 is selected from the group consisting of C1- to C30-alkyl group, C1- to C30-alkynyl group, C1- to C30-alkenyl group, C5- to C12-cycloalkyl group and C5- to C12-aryl group, wherein the ring B of the bile acid derivative of general formula I has one or two further hydroxyl group(s) at position 6 or at positions 6 and 7 respectively; and wherein none of the rings A, C and D has further hydroxyl groups; ii) contacting the first composition comprising at least one bile acid derivative of general formula I from i) with a compound R.sup.2—X, wherein R.sup.2 is a —C(═O)—C1- to C30-alkyl group and X is selected from the group consisting of hydroxyl group, —O—C1- to C20-alkyl group, —O—C1- to C20-alkenyl group, —O—C1- to C20-alkynyl group, thiol group, —S—C1- to C20-alkyl group, amine group, —NHR.sup.3 group, —NR.sup.3R.sup.4 group, wherein R.sup.3 and R.sup.4 are each independently a C1- to C20-alkyl group, halogen atom and —O—(C═O)—R.sup.5 group, wherein R.sup.5 is a C1- to C20-alkyl group; and a lipase selected from the group consisting of SEQ ID no. 1 to SEQ ID no. 9 or a homologous enzyme having a sequence identity of at least 65% with the sequence of SEQ ID no. 1 to SEQ ID no. 9 and having the same function as the lipase of SEQ ID no. 1 to SEQ ID no. 9 to obtain a second composition comprising at least one bile acid derivative of general formula II: ##STR00037## wherein the radical R.sup.1 is as defined at i) for formula I and the radical R.sup.2 is as defined at ii), wherein the ring B of the bile acid derivative of general formula II has one or two further hydroxyl group(s) at position 6 or at positions 6 and 7 respectively; and wherein none of the rings A, C and D has further hydroxyl groups; iii) conversion of the bile acid derivative of general formula II obtained from ii) into lithocholic acid.

14. A process for producing lithocholic acid comprising a) providing a composition comprising a bile acid derivative of general formula IIb, preferably obtained or obtainable by the process as claimed in claim 1, ##STR00038## wherein the radical R.sup.1 is selected from the group consisting of C1- to C30-alkyl group, C1- to C30-alkenyl group, C1- to C30-alkynyl group, C5- to C12-cycloalkyl group and C5- to C12-aryl group; the radical R.sup.2 is a —C(═O)—C1- to C30-alkyl group, wherein the ring B of the bile acid derivative of general formula I has a further hydroxyl group at position 6; and wherein none of the rings A, C and D has further hydroxyl groups; b) contacting the composition comprising a bile acid derivative of general formula IIb from a) with an oxidant or a C1- to C10-alkylthiol, preferably propanethiol, to convert the at least one hydroxyl group in B and/or D into an ═O group or an —S—C1- to C10-alkyl group, preferably an ═O group or an —S-propyl group to obtain a bile acid derivative of general formula IIIb, ##STR00039## wherein the radical R.sup.1 and the radical R.sup.2 are as defined in general formula II and the ring B has at least one ═O group or an —S—C1- to C10-alkyl group, preferably an ═O group or an —S-propyl group, at position 6; c) contacting the bile acid derivative of general formula IIIb from b) with a reducing agent, optionally with additional saponification, to obtain lithocholic acid.

15. A lithocholic acid obtained or obtainable by the process as claimed in claim 13.

16. The use of the lithocholic acid obtained or obtainable by the process as claimed in claim 13 for producing hydroxylated bile acids, preferably ursodeoxycholic acid or ursodeoxycholic acid derivatives.

Description

EXAMPLES

Example 1—Production of Chenodeoxycholic Acid Methyl Ester

[0064] ##STR00017##

[0065] 0.5 kg of chenodeoxycholic acid (CDCA) were dissolved in 1.5 L of (technical grade) methanol with stirring in a double-walled glass reactor. 0.0031 L of concentrated sulfuric acid (90%) were added slowly. The temperature was then adjusted to 85° C. and the reaction stirred under reflux. After complete conversion to chenodeoxycholic acid methyl ester (CDCA-Me) the reaction solution was set to 40° C. and 0.75 litres of methanol were distillatively removed under vacuum. 2 litres of ethyl acetate (technical grade) were then added to the solution. The organic phase was washed twice with 1.5 litres of saturated sodium hydrogencarbonate solution and three times with 1.5 litres of saturated sodium chloride solution. The organic phase was subsequently concentrated to dryness under vacuum.

[0066] Yield: 0.475 kg of CDCA-Me; 95% based on employed CDCA.

Example 2—Production of 3′-Acetyl-Chenodeoxycholic Acid Methyl Ester

[0067] ##STR00018##

[0068] 0.475 kg of CDCA-Me from example 1 were dissolved in 1.16 litres of ethyl acetate (technical grade) with stirring in a double-walled glass reactor. Added thereto were 0.25 litres of vinyl acetate (>95%) and 0.0035 kg of immobilized lipase B from Candida antarctica of SEQ ID no. 1. The reaction temperature was set to 45° C. Once the reaction was complete the lipase was filtered off and the solvent concentrated to dryness under vacuum to obtain 3′-acetyl-chenodeoxycholic acid methyl ester (3′-Ac-CDCA-Me) as a solid.

[0069] Yield: 0.451 kg of 3′Ac-CDCA-Me; 95% based on employed CDCA-Me.

Example 3—Production of 3′-Acetyl-7-Oxo-Chenodeoxycholic Acid Methyl Ester

[0070] ##STR00019##

[0071] 0.451 kg of 3′Ac-CDCA-Me from example 2 were dissolved in 2.65 litres of ethyl acetate (technical grade) and 0.66 litres of glacial acetic acid. 2.65 litres of sodium hypochlorite solution (5-10% technical grade) were added to the reaction with cooling so that the reaction temperature did not exceed 20° C. Upon complete conversion to the oxo compound the aqueous phase was discharged and the organic phase washed with 0.8 litres of a 10% sodium dithionite solution. The organic phase was washed with 3.5 litres of water and subsequently dried over magnesium sulfate. The dried organic phase was concentrated to dryness under vacuum to obtain 3′-acetyl-7-oxo-chenodeoxycholic acid methyl ester (3′Ac-7-oxo-CDCA-Me) as a solid.

[0072] Yield: 0.383 kg of 3′Ac-7-oxo-CDCA-Me; 90% based on employed 3′Ac-CDCA-Me.

Example 4—Production of Lithocholic Acid

[0073] ##STR00020##

[0074] 0.383 kg of 3′-acetyl-7-oxo-chenodeoxycholic acid methyl ester (3′Ac-7-oxo-CDCA-Me) from example 3 were suspended in 1.5 litres of ethylene glycol and 0.425 litres of water were added with stirring. 0.489 kg of solid potassium hydroxide and 4.1 litres of hydrazine hydrate were added to the reaction solution (50% in water). The reaction solution was heated to 130° C. and water and hydrazine hydrate were removed by distillation. Once distillative removal was complete the temperature was set to 195° C. and maintained for 2.5 h. A strong evolution of gas, indicating the progress of the reaction, was observed. The reaction solution was subsequently cooled to below 100° C. and 8.5 litres of a water/ice mixture was then added to the reaction and stirred vigorously. The mixture was then acidified to pH 1 with 0.638 litres of concentrated sulfuric acid. The crude product precipitated as a fine white solid and was filtered off. The crude product was washed with 0.5 litres of water and 0.5 litres of acetonitrile and then dried. The crude lithocholic acid was dissolved in 1.0 L of glacial acetic acid and slowly crystallized by addition of 1.0 litres of water. The produced lithocholic acid was filtered and dried.

[0075] Yield: 0.278 kg of lithocholic acid; 90% based on employed 3′Ac-7-oxo-CDCA-Me.

Example 5—Production of 3′-Acetyl-7-Propylthio-Chenodeoxycholic Acid Methyl Ester

[0076] ##STR00021##

[0077] 0.451 kg of 3′Ac-CDCA-Me from example 2 were dissolved in 4.5 litres of ethylene glycol is dimethyl ether (DME) and 0.337 litres of propanethiol and 0.135 litres of BF.sub.3×Et.sub.2O were added. The reaction solution was heated under reflux for 2 days. The cooled reaction solution was then washed to neutrality with sodium carbonate solution and the organic phase was concentrated to dryness under vacuum to obtain 3′-acetyl-7-propylthio-chenodeoxycholic acid methyl ester as a solid.

[0078] Yield: 0.405 kg of 3′-acetyl-7-propylthio-chenodeoxycholic acid methyl ester; 91% based on employed 3′Ac-CDCA-Me.

Example 6—Production of Lithocholic Acid

[0079] ##STR00022##

[0080] 0.405 kg of 3′-acetyl-7-propylthio-chenodeoxycholic acid methyl ester from example 5 and 1.3 kg of nickel chloride hexahydrate were dissolved in 10 litres of methanol-THF (1:1) at 0° C. 0.318 kg of sodium borohydride were added to the reaction solution in small portions of 20 g. Once addition was complete the solution was stirred for a further 30 min. The precipitate was filtered over celite and washed further with methanol-THF. The solvent was removed to dryness under vacuum to obtain crude lithocholic acid as a solid.

[0081] The crude lithocholic acid was dissolved in 1.0 L of glacial acetic acid and slowly crystallized by addition of 1.0 litres of water. The produced lithocholic acid was filtered and dried.

[0082] Yield: 0.281 kg of lithocholic acid; 70% based on employed 3′-acetyl-7-propylthio-chenodeoxycholic acid methyl ester.

Example 7—Production of Hyodeoxycholic Acid Methyl Ester

[0083] ##STR00023##

[0084] 0.5 kg of hyodeoxycholic acid (HDCA) were dissolved in 1.5 L of (technical grade) methanol with stirring in a double-walled glass reactor. 0.0031 L of concentrated sulfuric acid (98%) were added slowly. The temperature was then adjusted to 85° C. and the reaction stirred under reflux. After complete conversion to hyodeoxycholic acid methyl ester (HDCA-Me) the reaction solution was set to 40° C. and 0.75 litres of methanol were distillatively removed under vacuum. 2 Litres of ethyl acetate (technical grade) were then added to the solution. The organic phase was washed twice with 1.5 litres of saturated sodium hydrogencarbonate solution and three times with 1.5 litres of saturated sodium chloride solution. The organic phase was subsequently concentrated to dryness under vacuum.

[0085] Yield: 0.485 kg of HDCA-Me; 98% based on employed CDCA.

Example 8—Production of 3′-Acetyl-Hyodeoxycholic Acid Methyl Ester

[0086] ##STR00024##

[0087] 0.485 kg of HDCA-Me from example 7 were dissolved in 1.16 litres of ethyl acetate (technical grade) with stirring in a double-walled glass reactor. Added thereto were 0.25 litres of vinyl acetate (>95%) and 0.0035 kg of immobilized lipase B from Candida antarctica of SEQ ID no. 1. The reaction temperature was set to 45° C. Once the reaction was complete the lipase was filtered off and the solvent concentrated to dryness under vacuum to obtain 3′-acetyl-hyodeoxycholic acid methyl ester (3′-Ac-HDCA-Me) as a solid.

[0088] Yield: 0.470 kg of 3′Ac-HDCA-Me; 96% based on employed HDCA-Me.

[0089] It was surprisingly found that in the case of hyodeoxycholic acid (HDCA) only the 3-alpha-hydroxyl group on the A ring was acetylated despite the presence of an alpha-hydroxyl group in position 6 on the B-ring which would at least co-react under other acylation conditions and that the use of lipase B from Candida antarctica of SEQ ID no. 1 resulted in virtually complete acetylation of the 3-alpha-hydroxyl group on the A ring while the 6-alpha-hydroxyl group on the B ring remained as a hydroxyl group.

[0090] HCA-Me (0.47 kg) obtained from HCA (0.5 kg) analogously to example 7 was analogously acetylated with vinyl acetate and immobilized lipase B from Candida antarctica of SEQ ID no. 1 (3′Ac-HCA-Me, 0.44 kg, 94%). It was likewise found here that only the 3-alpha-hydroxyl group on the A ring was acetylated despite the presence of alpha-hydroxyl groups in positions 6 and 7 on the B-ring which would at least co-react under other acylation conditions and that the use of lipase B from Candida antarctica of SEQ ID no. 1 resulted in virtually complete acetylation of the 3-alpha-hydroxyl group on the A ring while the 6- and 7-alpha-hydroxyl group on the B-ring remained as hydroxyl groups; these results are clearly apparent from the NMR spectra.

[0091] The .sup.1H- and .sup.13C-NMR data are reported in the tables below.

TABLE-US-00002 [00025] 3-Ac-HDCA-Me [00026] 3-Ac-HCA-Me δ 13C (ppm) No. 3-Ac-HDCA-Me 3-Ac-HCA-Me 1 35.40 35.4 2 26.69 26.84 3 74.31 74.54 4 25.46 28.54 5 48.46 47.80 6 67.93 69.42 7 34.90 72.07 8 34.88 38.64 9 39.93 32.73 10 36.07 36.16 11 20.09 20.74 12 40.05 39.58 13 42.97 42.89 14 56.29 50.30 15 24.31 23.79 16 28.26 28.31 17 56.08 55.88 18 12.17 11.91 19 23.61 23.15 20 35.48 35.56 21 18.40 18.43 22 31.18 31.20 23 31.18 31.20 24 174.92 175.19 25 51.67 51.72 26 170.76 171.23 27 21.58 21.65

TABLE-US-00003 δ 1H (ppm) No. 3-Ac-HDCA-Me 3-Ac-HCA-Me 1 2 3 4.67, tt 4.50, tt 4 5 6 4.02, dt 3.80, brs 7 3.83, brt 8 9 10 11 12 13 14 15 16 17 18 0.59, s 0.61 19 0.87, s 0.88 20 21 0.89, d 0.89 22 23 2.17, 2.30 24 25 3.60 — 26 27 1.98 — tt: triplet of triplets; dt: doublet of triplets; s: singlet; d: doublet; brs: broad singlet; brt: broad triplet

Example 9—Production of 3′-Acetyl-7-Oxo-Hyodeoxycholic Acid Methyl Ester

[0092] ##STR00027##

[0093] 0.470 kg of 3′Ac-HDCA-Me from example 8 were dissolved in 2.65 litres of ethyl acetate (technical grade) and 0.66 litres of glacial acetic acid. 2.65 litres of sodium hypochlorite solution (5-10% technical grade) were added to the reaction with cooling so that the reaction temperature did not exceed 20° C. Upon complete conversion to the oxo compound the aqueous phase was discharged and the organic phase washed with 0.8 litres of a 10% sodium dithionite solution. The organic phase was washed with 3.5 litres of water and subsequently dried over magnesium sulfate. The dried organic phase was concentrated to dryness under vacuum to obtain 3′-acetyl-7-oxo-hyodeoxycholic acid methyl ester (3′Ac-7-oxo-HDCA-Me) as a solid.

[0094] Yield: 0.391 kg of 3′Ac-7-oxo-HDCA-Me; 83% based on employed 3′Ac-HDCA-Me.

Example 10—Production of Lithocholic Acid

[0095] ##STR00028##

[0096] 0.391 kg of 3′-acetyl-7-oxo-hyodeoxycholic acid methyl ester (3′Ac-7-oxo-HDCA-Me) from example 9 were suspended in 1.5 litres of ethylene glycol and 0.425 litres of water were added with stirring. 0.489 kg of solid potassium hydroxide and 4.1 litres of hydrazine hydrate were added to the reaction solution (50% in water). The reaction solution was heated to 130° C. and water and hydrazine hydrate were removed by distillation. Once distillative removal was complete the temperature was set to 195° C. and maintained for 2.5 h. A strong evolution of gas, indicating the progress of the reaction, was observed. The reaction solution was subsequently cooled to below 100° C. and 8.5 litres of a water/ice mixture was then added to the reaction and stirred vigorously. The mixture was then acidified to pH 1 with 0.638 litres of concentrated sulfuric acid. The crude product precipitated as a fine white solid and was filtered off. The crude product was washed with 0.5 litres of water and 0.5 litres of acetonitrile and then dried. The crude lithocholic acid was dissolved in 1.0 L of glacial acetic acid and slowly crystallized by addition of 1.0 litres of water. The produced lithocholic acid was filtered and dried.

[0097] Yield: 0.234 kg of lithocholic acid; 60% based on employed 3′Ac-7-oxo-HDCA-Me.

Example 11—Production of 3′-Acetyl-7-Propylthio-Hyodeoxycholic Acid Methyl Ester

[0098] ##STR00029##

[0099] 0.470 kg of 3′Ac-HDCA-Me from example 8 were dissolved in 4.5 litres of ethylene glycol dimethyl ether (DME) and 0.337 litres of propanethiol and 0.135 litres of BF.sub.3×Et.sub.2O were added. The reaction solution was heated under reflux for 2 days. The cooled reaction solution was then washed to neutrality with sodium carbonate solution and the organic phase was concentrated to dryness under vacuum to obtain 3′-acetyl-7-propylthio-hyodeoxycholic acid methyl ester as a solid.

[0100] Yield: 0.428 kg of 3′-acetyl-7-propylthio-hyodeoxycholic acid methyl ester; 91% based on employed 3′Ac-HDCA-Me.

Example 12—Production of Lithocholic Acid

[0101] ##STR00030##

[0102] 0.428 kg of 3′-acetyl-7-propylthio-hyodeoxycholic acid methyl ester from example 11 and 1.3 kg of nickel chloride hexahydrate were dissolved in 10 litres of methanol-THF (1:1) at 0° C. 0.318 kg of sodium borohydride were added to the reaction solution in small portions of 20 g. Once addition was complete the solution was stirred for a further 30 min. The precipitate was filtered over celite and washed further with methanol-THF. The solvent was removed to dryness under vacuum to obtain crude lithocholic acid as a solid.

[0103] The crude lithocholic acid was dissolved in 1.0 L of glacial acetic acid and slowly crystallized by addition of 1.0 litres of water. The produced lithocholic acid was filtered and dried.

[0104] Yield: 0.291 kg of lithocholic acid; 68% based on employed 3′-acetyl-7-propylthio-hyodeoxycholic acid methyl ester.