BILE ACID DERIVATIVE, COMPOSITION AND APPLICATION THEREOF

Abstract

Provided are a novel bile acid derivative for treating fatty liver disease, a pharmaceutical composition thereof, and a use in the preparation of a medicine for treating and improving diseases and symptoms mediated or caused by FXR or TGR5. The bile acid derivative of the present invention inhibits or delays the metabolism of bile acid by bacterial BSH/7α dehydroxylase in the intestine and greatly prolongs the effective survival time of bile acid in the intestine. The bile acid derivative and the pharmaceutical composition thereof can significantly stimulate bile acid membrane receptor TGR5, promote the secretion of glucagon-like peptide 1 from enteroendocrine cells, reduce liver fat accumulation, significantly improve liver function and increase glucose tolerance, thereby having an excellent effect on the treatment of fatty liver disease.

Claims

1. A bile acid derivative represented by the following formula (I) or a stereoisomer, a salt or an ester thereof, ##STR00047## wherein, R.sub.1 is α-OH or β-O(CH.sub.2).sub.aOH, wherein, a is 1-10, R.sub.2 is α-OH, H or CH.sub.2OH, R.sub.3 is α-OH, H, β-OH or CH.sub.3, R.sub.4 is H or CH.sub.3, R.sub.5 is α-OH or H, R.sub.6 is H or (CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00048## wherein, X is H or CH.sub.3, Y is CH.sub.3 or CH.sub.2OH, Z is COOH or SO.sub.3H, n is 0-10; or R.sub.7 is OH or —O(CH.sub.2).sub.tCH.sub.3, wherein, t is 0-3, wherein, the carbon attached to R.sub.6 methyl group can be S configuration or R configuration; in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration.

2. The bile acid derivative as defined in claim 1, wherein, R.sub.1 is α-OH, R.sub.2 is α-OH, R.sub.3 is α-OH, R.sub.4 is H.

3. The bile acid derivative as defined in claim 1, wherein, R.sub.1 is α-OH, R.sub.2 is α-OH, R.sub.3 is α-OH, R.sub.4 is H, R.sub.5 is H.

4. The bile acid derivative as defined in claim 3, wherein, R.sub.1 is α-OH, R.sub.2 is α-OH, R.sub.3 is α-OH, R.sub.4 is H, R.sub.5 is H, R.sub.6 is H.

5. The bile acid derivative as defined in claim 3, wherein, R.sub.1 is α-OH, R.sub.2 is α-OH, R.sub.3 is α-OH, R.sub.4 is H, R.sub.5 is H, R.sub.6 is (CH.sub.2).sub.bCH.sub.3 (b is 0-3), wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration.

6. The bile acid derivative as defined in claim 3, wherein, R.sub.7 is OH or —O(CH.sub.2).sub.tCH.sub.3, wherein t is 0-3, or, R.sub.7 is OH, or, R.sub.7 is —O(CH.sub.2).sub.tCH.sub.3, wherein t is 0-3, or, R.sub.7 is ##STR00049## wherein, X is H, Y is CH.sub.2OH, Z is COOH, n is 0-10: the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00050## wherein, X is H, Y is CH.sub.3, Z is SO.sub.3H, n is 0-10: the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00051## wherein, X is H, Y is CH.sub.2OH, Z is SO.sub.3H, n is 0-10: the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00052## wherein, X is H, Y is CH.sub.3, Z is COOH, n is 0-10: the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00053## wherein, X is CH.sub.3, Y is CH.sub.2OH, Z is COOH, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00054## wherein, X is CH.sub.3, Y is CH.sub.2OH, Z is SO.sub.3H, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00055## wherein, X is CH.sub.3, Y is CH.sub.3, Z is COOH, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00056## wherein, X is CH.sub.3, Y is CH.sub.2OH, Z is COOH, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00057## wherein, X is CH.sub.3, Y is CH.sub.3, Z is SO.sub.3H, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00058## wherein, X is CH.sub.3, Y is CH.sub.2OH, Z is SO.sub.3H, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00059## wherein, X is CH.sub.3, Y is CH.sub.3, Z is COOH, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration.

7-12. (canceled)

13. The bile acid derivative as defined in claim 2, wherein, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00060## wherein, X is H, Y is CH.sub.2OH, Z is COOH, n is 0-10; wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration; in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00061## wherein, X is H, Y is CH.sub.3, Z is SO.sub.3H, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00062## wherein, X is H, Y is CH.sub.2OH, Z is SO.sub.3H, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, R.sub.7 is ##STR00063## wherein, X is H, Y is CH.sub.3, Z is COOH, n is 0-10: wherein the carbon attached to R.sub.6 methyl group can be S configuration or R configuration: in the R.sub.7 substituent, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00064## wherein, X is H or CH.sub.3, Y is CH.sub.3 or CH.sub.2OH, Z is COOH or SO.sub.3H, n is 0-10, or R.sub.7 is OH or —O(CH.sub.2).sub.tCH.sub.3, wherein, t is 0-3, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is ##STR00065## wherein, X is H or CH.sub.3, Y is CH.sub.3 or CH.sub.2OH, Z is COOH or SO.sub.3H, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration, or, R.sub.7 is OH, or, R.sub.7 is —O(CH.sub.2).sub.tCH.sub.3, wherein, t is 0-3.

14-17. (canceled)

18. The bile acid derivative as defined in claim 4, wherein, R.sub.7 is ##STR00066## wherein, X is CH.sub.3, Y is CH.sub.3, Z is SO.sub.3H, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration.

19-28. (canceled)

29. The bile acid derivative as defined in claim 2, wherein, R.sub.7 is ##STR00067## wherein, X is H or CH.sub.3, Y is CH.sub.3 or CH.sub.2OH, Z is COOH or SO.sub.3H, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, or R.sub.6 is H.

30. The bile acid derivative as defined in claim 2, wherein, R.sub.7 is ##STR00068## wherein, X is H or CH.sub.3, Y is CH.sub.3 or CH.sub.2OH, Z is COOH or SO.sub.3H, n is 0-10, the carbon attached to the Y group can be S configuration or R configuration, R.sub.5 is α-OH or H.

31. A composition comprising the bile acid derivative as defined in claim 1 and an acceptable carrier.

32. The composition as defined in claim 31, wherein, the composition is an oral preparation.

33. (canceled)

34. A therapeutic method of treating and ameliorating diseases and symptoms mediated or caused by FXR or TGR5, comprising administering the bile acid derivative as defined in claim 1 to the patients having the diseases and symptoms.

35. The therapeutic method as defined in claim 34, wherein the dosage is a daily dose of 50-500 mg/kg of the patient's body weight.

36. The therapeutic method as defined in claim 35, wherein, the diseases and symptoms mediated or caused by the FXR or TGR5 include the following diseases or symptoms: liver disease, hyperlipidemia, hypercholesterolemia, obesity, metabolic syndrome, cardiovascular disease, gastrointestinal disease, atherosclerosis sclerosis and nephropathy, wherein, the liver disease includes simple fatty liver, primary biliary cirrhosis, primary sclerosing cholangitis, liver fibrosis, liver cirrhosis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease and their associated liver injuries.

37. The therapeutic method as defined in claim 35, wherein, the bile acid derivative can be administered to the patient in combination with conventional hypoglycemic and lipid-lowering drugs, the conventional hypoglycemic and lipid-lowering drugs are one or more than one selected from Liraglutide, Exenatide and Albiglutide.

38. The bile acid derivative as defined in claim 2, wherein, R.sub.7 is OH, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, or R.sub.6 is H, or, R.sub.7 is —O(CH.sub.2).sub.tCH.sub.3, wherein, t is 0-3, R.sub.6 is —(CH.sub.2).sub.bCH.sub.3, wherein, b is 0-3, or R.sub.6 is H.

39. The bile acid derivative as defined in claim 2, wherein, R.sub.7 is OH, R.sub.5 is α-OH or H, or, R.sub.7 is —O(CH.sub.2).sub.tCH.sub.3, wherein, t is 0-3, R.sub.5 is α-OH or H.

40. The composition as defined in claim 31, wherein, the bile acid derivative is effective amount, the effective amount refers to a daily dose of the composition comprising the bile acid derivative at 50-500 mg/kg of a patient's body weight; the acceptable carrier refers to a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 is a graph of the average hyocholic acid content of normal people, patients with nonalcoholic fatty liver disease, patients with nonalcoholic steatohepatitis, patients with nonalcoholic steatohepatitis-early stage liver fibrosis, patients with nonalcoholic steatohepatitis-late stage liver fibrosis and patients with nonalcoholic steatohepatitis-cirrhosis in the present disclosure.

[0062] FIG. 2 is a graph showing the significant decrease of liver triglyceride after 8 weeks of intervention with hyocholic acid, hyodeoxycholic acid and 9 kinds of synthetic bile acid derivatives of the present disclosure.

[0063] FIG. 3 is a graph showing the significant decrease of serum triglyceride after 8 weeks of intervention with hyocholic acid, hyodeoxycholic acid and 9 kinds of synthetic bile acid derivatives of the present disclosure.

[0064] FIG. 4 shows the changes of blood glucose level in mice after 1 week of intervention of hyocholic acid (HCA), hyodeoxycholic acid (HDCA) and 9 kinds of synthetic bile acid derivatives (50 mg/kg).

[0065] FIG. 5 shows that 50 μM bile acid and the derivatives thereof can effectively promote the secretion of GLP-1 in the enteroendocrine cell line NCI-H716 cells.

[0066] FIG. 6 shows that after NCI-H716 and STC-1 cells were treated with hyocholic acid and 6 kinds of bile acid derivatives and 19 kinds of other bile acid derivatives for 48 hours at 50 μM, it was found that hyocholic acid and the derivatives thereof were more effective in up-regulating GLP-1 protein expression in enteroendocrine cell line than other bile acids through the action of TGR5 and FXR.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0067] In order to make the purpose, technical solutions and effects of the present disclosure become clearer and definite, the present disclosure will be further illustrated in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present disclosure, but not to limit the present disclosure.

[0068] All reagents and materials in the preparation examples were purchased from commercial suppliers.

[0069] The codes and structural formulas of the compounds used in the examples are shown in the following table.

TABLE-US-00001 TABLE 1 Structure of synthesized hyocholic acid derivatives Code name Chemical name Structural formula ZN-1- 34-1 D-Alanine hyocholic acid [00025] ZN-1- 28-1 L-Alanine hyocholic acid [00026] ZN-1- 78-1 L-Serine hyocholic acid [00027] ZN-1- 93-1 N-methylglycine hyocholic acid [00028] ZN-1- 102-1 (S)-23-methyl hyocholic acid [00029] ZN-1- 80-1 D-Serine hyocholic acid [00030] ZN-1- 65-1 (2R)-2-((4R)-4- ((3R,6R,7S,10R,13R,17R)- 3,6,7-trihydroxy-10,13- dimethylhexadecahydro-1H- cyclopenta[a]phenanthren-17- yl)pentamido)propane-1- sulfonic acid [00031] ZN-1- 71-1 (2S)-2-((4R)-4- ((3R,6R,7S,10R,13R,17R)- 3,6,7-trihydroxy-10,13- dimethylhexadecahydro-1H- cyclopenta[a]phenanthren-17- yl)pentamido)propane-1- sulfonic acid [00032] ZN-1- 86-1 N-methyltauro hyocholic acid [00033]

Example 1: Synthesis of D-alanine hyocholic acid

[0070] ##STR00034##

[0071] The preparation method includes the following steps:

##STR00035##

[0072] Hyocholic acid (0.204 g, 0.5 mmol), D-phenylalanine benzyl ester p-toluenesulfonate (0.185 g, 0.52 mmol) and N,N-diisopropylethylamine (0.194 mg, 1.5 mmol)) were dissolved in dimethylformamide (5 ml) and stirred evenly. At room temperature, tetramethylurea hexafluorophosphate (0.209 g, 0.55 mmol) was added to the reaction solution at one time, and the reaction was carried out at this temperature for 1 h. After the completion of the reaction monitored by thin layer chromatography, water (10 ml) was added and the reaction solution was extracted twice with ethyl acetate. The organic phase was washed with 1N sodium hydroxide, 1N hydrochloric acid and saturated brine successively, and then the organic phase was dried and concentrated to obtain D-alanine hyocholic acid benzyl ester.

[0073] The obtained intermediate was dissolved in methanol (10 mL), and subjected to the catalytic hydrogenation in the presence of 25 mg 10% palladium carbon at room temperature. After the reaction, Pd/C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the crude product of D-propylamine hyocholic acid. D-propylamine hyocholic acid (0.216 g) was obtained by purification by column chromatography, and the yield of two steps was 90%. ESI-MS (m/z): 959.6 (2M+H).sup.+. .sup.1HNMR (300 MHz, DMSO): δ0.6 (s, 3H), 0.83 (s, 3H), 0.88 (d, 3H), 1.23 (d, 3H), 3.13 (m, 1H), 3.59 (m, 2H), 3.89 (s, 1H), 4.14 (t, 1H), 8.01 (d, 1H).

Example 2: Synthesis of L-alanine hyocholic acid

[0074] ##STR00036##

[0075] The procedure was the same as that in Example 1, except that D-alanine benzyl ester p-toluenesulfonate was replaced with L-alanine benzyl ester hydrochloride to obtain L-alanine hyocholic acid (0.164 g) with a yield of 86.2%. ESI-MS (m/z): 959.6 (2M+H).sup.+. .sup.1HNMR (300 MHz, DMSO): δ0.6 (s, 3H), 0.83 (s, 3H), 0.88 (d, 3H), 1.23 (d, 3H), 3.13 (m, 1H), 3.59 (m, 2H), 3.89 (s, 1H), 4.14 (t, 3H), 4.19 (m, 1H), 4.32 (m, 1H), 8.06 (d, 1H).

Example 3: Synthesis of L-serine hyocholic acid

[0076] ##STR00037##

[0077] The procedure was the same as that of Example 1, except that D-alanine benzyl ester p-toluenesulfonate was replaced with L-serine benzyl ester hydrochloride to obtain L-serine hyocholic acid (0.352 g) with a yield of 93.6%. ESI-MS (m/z): 991.6 (2M+H).sup.+. .sup.1HNMR (300 MHz, DMSO): δ0.6 (s, 3H), 0.83 (s, 3H), 0.88 (d, 3H), 3.13 (m, 2H), 3.59 (m, 4H), 3.89 (s, 1H), 4.20-4.28 (m, 2H), 4.19 (m, 1H), 4.32 (m, 1H), 8.06 (d, 1H).

Example 4: Synthesis of D-serine hyocholic acid

[0078] ##STR00038##

[0079] The procedure was the same as that of Example 1, except that D-alanine benzyl ester p-toluenesulfonate was replaced with D-serine benzyl ester hydrochloride to obtain D-serine hyocholic acid (0.334 g) with a yield of 89.2%. ESI-MS (m/z): 991.6 (2M+H).sup.+. .sup.1HNMR (300 MHz, DMSO): δ0.6 (s, 3H), 0.83 (s, 3H), 0.88 (d, 3H), 3.13 (m, 2H), 3.59 (m, 4H), 3.89 (s, 1H), 4.20-4.28 (m, 2H), 4.19 (m, 1H), 4.32 (m, 1H), 8.06 (d, 1H).

Example 5: ((2R)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido) propane-1-sulfonic acid)

[0080] ##STR00039##

[0081] The preparation method includes the following steps:

##STR00040##

[0082] Hyocholic acid (0.31 g, 0.76 mmol), (R)-2-aminopropanesulfonic acid (0.1 g, 0.77 mmol) and N,N-diisopropylethylamine (0.29 mg, 2.28 mmol) were dissolved in dimethylformamide (5 ml) and stirred evenly. At room temperature, tetramethylurea hexafluorophosphate (0.32 g, 0.84 mmol) was added to the reaction solution at one time, and the reaction was carried out at this temperature for 1 h. After the completion of the reaction monitored by thin-layer chromatography, the reaction solution was concentrated to remove dimethylformamide, then water (10 ml) was added, and the reaction solution was extracted twice with ethyl acetate. The pH value of the water phase was adjusted to 1-2 with 1N hydrochloric acid, and then the water phase was concentrated to dry to obtain the crude product. (2R)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthren-17-yl)pentanamido)propane-1-sulfonic acid (276 mg) was obtained after purification by column chromatography with a yield of 68.6%. ESI-MS (m/z): 1059.7 (2M+H).sup.+. .sup.1HNMR (300 MHz, CD3OD): δ0.69 (s, 3H), 0.95 (s, 3H), 0.99 (d, 3H), 1.32 (d, 3H), 2.71 (s, 1H), 2.8-3.1 (qd, 2H), 3.79 (m, 2H), 4.34 (m, 1H).

Example 6: ((2S)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propane-1-sulfonic acid)

[0083] ##STR00041##

[0084] The procedure was the same as that in Example 5, except that (R)-2-aminopropanesulfonic acid was replaced with (S)-2-aminopropanesulfonic acid to obtain (2S)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propane-1-sulfonic acid (292 mg) with a yield of 72.6%. ESI-MS (m/z): 1059.7 (2M+H).sup.+. .sup.1HNMR (300 MHz, CD3OD): δ0.69 (s, 3H), 0.95 (s, 3H), 0.99 (d, 3H), 1.32 (d, 3H), 2.8-3.1 (qd, 2H), 3.79 (m, 2H), 4.37 (m, 1H).

Example 7: Synthesis of N-methyltauro hyocholic acid

[0085] ##STR00042##

[0086] The procedure was the same as that in Example 5, except that (R)-2-aminopropanesulfonic acid was replaced by N-methyltaurine to obtain N-methyltauro hyocholic acid (164 mg) with a yield of 40.8%. ESI-MS (m/z): 1059.7 (2M+H).sup.+. .sup.1HNMR (300 MHz, CD3OD): δ0.69 (s, 3H), 0.93 (s, 3H), 0.99 (d, 3H), 1.3 (s, 3H), 2.70 (m, 1H), 2.93 (m, 1H), 2.95-3.12 (m, 2H), 3.13 (m, 1H), 3.76 (m, 4H).

Example 8: Synthesis of N-methylglycine hyocholic acid

[0087] ##STR00043##

[0088] The preparation method includes the following steps:

##STR00044##

[0089] Hyocholic acid (0.31 g, 0.76 mmol), N-methylglycine ethyl ester hydrochloride (0.117 g, 0.8 mmol) and N,N-diisopropylethylamine (0.29 g, 2.28 mmol) were dissolved in dimethylformamide (5 ml) and stirred evenly. At room temperature, tetramethylurea hexafluorophosphate (0.32 g, 0.836 mmol) was added at one time and the reaction was carried out at this temperature for 1 h. After the completion of the reaction monitored by thin layer chromatography, water (10 ml) was added and the reaction solution was extracted twice with ethyl acetate. The organic phase was washed with 1N sodium hydroxide, 1N hydrochloric acid and saturated brine successively, and then the organic phase was dried and concentrated to obtain N-methylglycine hyocholic acid ethyl ester as the intermediate.

[0090] The intermediate was dissolved in 10 mL of methanol/water (4/1 v/v), potassium hydroxide (66 mg) was added, and subjected to hydrolysis at room temperature. After the reaction, the reaction solution was concentrated under reduced pressure to remove the methanol solvent. The residue was diluted with water (5 ml), and adjusted to pH=1-2 with 1N hydrochloric acid. The reaction solution was extracted twice with ethyl acetate, and the organic phases were combined, dried and concentrated to obtain N-methylglycine hyocholic acid (263 mg) with a yield of 72.2%.

[0091] ESI-MS (m/z): 959.6 (2M+H).sup.+. .sup.1HNMR (300 MHz, DMSO): δ0.6 (s, 3H), 0.83 (s, 3H), 0.88 (d, 3H), 1.31 (s, 3H), 3.13 (m, 1H), 3.59 (m, 2H), 3.89 (s, 1H), 4.19 (m, 1H), 4.32 (m, 1H).

Example 9: Synthesis of (S)-23-methylhyocholic acid

[0092] ##STR00045##

[0093] The preparation method includes the following steps:

##STR00046##

[0094] Hyocholic acid (1.632 g, 4 mmol) was dissolved in methanol (30 mL), and three drops of concentrated sulfuric acid were added thereto for catalyzation and the reaction was carried out overnight at room temperature. After the completion of the reaction monitored by thin layer chromatography, the reaction solution was concentrated under reduced pressure to remove the methanol solvent. The result was dissolved in ethyl acetate, and washed with saturated sodium bicarbonate and brine successively. The organic phase was dried and concentrated to obtain hyocholic acid methyl ester (H1) (1.69 g).

[0095] Hyocholic acid methyl ester (1.69 g, 4 mmol) and 2,6-lutidine (4.29 g, 40 mmol) were dissolved in dichloromethane, under nitrogen atmosphere, the reaction solution was cooled to 0-5° C., and tert-butyl dimethylsilyl trifluoromethanesulfonate (2.8 ml) was added to the reaction solution dropwise, and the reaction was carried out at room temperature. After the completion of the reaction monitored by thin layer chromatography, the reaction solution was subjected to flash column chromatography to obtain the intermediate (H2) (3.1 g).

[0096] The intermediate H2 and HMPA (4.35 g, 24 mmol) were added to anhydrous tetrahydrofuran, stirred evenly, and cooled to −78° C. under nitrogen atmosphere. After the reaction was carried out at this temperature for 30 min, methyl iodide (5.7 g, 40 mmol) was slowly added dropwise to the reaction solution, the reaction was then carried out at this temperature for 1 h. The temperature was then naturally raised to room temperature and the reaction was carried out overnight. After the completion of reaction monitored by thin layer chromatography, the reaction was quenched with saturated ammonium chloride solution. The reaction solution was extracted twice with ethyl acetate. The organic phases were combined and washed once with saturated brine. The organic phase was dried and concentrated to obtain a residue, which was purified by column chromatography to obtain intermediate (H3) (2.24 g), the yield of three steps was 71.8%.

[0097] The intermediate (H3) was dissolved in methanol (20 mL), 4 drops of concentrated hydrochloric acid was added thereto for catalyzation, and subjected to deprotection of the TBS protecting group at room temperature. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the methanol solvent. The residue was dissolved in 10 mL of tetrahydrofuran/H.sub.2O (4:1), and sodium hydroxide (0.34 g, 8.6 mmol) was then added. The reaction was carried out at room temperature. After the completion of the hydrolysis, the reaction solution was extracted twice with ethyl acetate. The aqueous phase was adjusted to pH=1-2 with 1N hydrochloric acid, and extracted with ethyl acetate for three times. The organic phases were combined, dried and concentrated to obtain a mixture of (S)-23-methylhyocholic acid and (R)-23-methylhyocholic acid. The diastereomer was then subjection to resolution by column chromatography to obtain (S)-23-methylhyocholic acid (331 mg) with a two-step yield of 27.1%. ESI-MS (m/z): 959.6 (2M+H).sup.+. .sup.1HNMR (300 MHz, CD.sub.3OD): 0.68 (S, 3H), 0.93 (S, 3H), 0.98 (d, 3H), 1.12 (d, 3H), 2.57 (m, 1H), 3.58 (m, 1H), 3.78 (m, 2H).

Example 10: The Concentration of Hyocholic Acid Decreased Significantly in Patients with Fatty Liver Disease

[0098] The experimental samples in the present disclosure were approved by the local ethics committee and informed consent was obtained from all subjects. A total of 200 subjects were enrolled in Example 1 of the present disclosure, and the content of cholic acid, amino acid and fatty acid and other metabolites in serum samples of 25 healthy patients confirmed by liver puncture and 175 patients with fatty liver (including simple fatty amine, steatohepatitis, steatohepatitis with early liver fibrosis, steatohepatitis with late liver fibrosis, and steatohepatitis with liver cirrhosis) confirmed by liver biopsy and corresponding clinical indicators were detected by ultra-high performance liquid chromatography tandem mass spectrometry. The results showed that hyocholic acid decreased significantly in patients with fatty liver disease (FIG. 1).

Example 11

[0099] As shown in FIG. 2 and FIG. 3, hyocholic acid, hyodeoxycholic acid, and synthetic hyocholic acid derivatives (Table 1) can significantly improve the serum hyperlipidemia induced by high fat in mice. In the fat-induced obesity mouse model, hyocholic acid and hyodeoxycholic acid were administered by gavage at a dose of 50 mg/kg/day while feeding high-fat diet (HFD) for 8 weeks. It was found that the triglyceride levels of mice in the groups fed with hyocholic acid, hyodeoxycholic acid and synthetic hyocholic acid derivatives were significantly lower than those in the simple high-fat diet group after 8 weeks. The increase of hyocholic acid and hyodeoxycholic acid in mice can effectively improve the dyslipidemia in mice caused by high fat.

Example 12

[0100] Porcine bile acid series and synthetic hyocholic acid derivatives (50 mg/kg/day) were orally administered to C57BL/6J mice (Table 1). As shown in FIG. 4, after one week of intervention, the results showed that blood glucose was significantly decreased in all intervention groups.

Example 13

[0101] NCI-H716 cells were cultured and treated with 50 μM hyocholic acid and hyocholic acid derivatives, and a reported TGR5 agonist INT-777, and the levels of GLP-1 in the cell culture medium were measured. It was found that all compounds could effectively promote the release of GLP-1 (FIG. 5), and compound ZN-1-102-1 was superior to hyocholic acid and the existing TGR5 agonist INT-777 in the release of GLP-1.

Example 14

[0102] NCI-H716 and STC-1 cells were treated with 50 μM hyocholic acid, hyodeoxycholic acid, taurohyodeoxycholic acid, glycohyodeoxycholic acid, taurohyocholic acid, glycohyocholic acid and 19 kinds of other bile acids for 48 hours. It was found that hyocholic acid and the derivatives thereof were more effective than other bile acids in upregulating GLP-1 protein expression in enteroendocrine cell lines through the action of TGR5 and FXR (FIG. 6). (a) GLP-1 transcription was measured using real-time PCR, see FIG. 6(a). (b) GLP-1 secretion was measured using ELISA, see FIG. 6(b). (c) NCI-H716 and STC-1 and TGR5 knockdown cells thereof were treated with 6 kinds of hyocholic acids for 24 h and intracellular GLP-1, p-CREB and total CREB were measured using western blotting. See FIG. 6(c). (d) FXR protein concentration in nuclear and cytoplasmic fractions ofNCI-H716 cells after 24 h treatment with 50 μM chenodeoxycholic acid or 5-cholic acid in the presence and absence of hyocholic acid. See FIG. 6(d). * P<0.05, compared with the control.

[0103] It can be understood that for those skilled in the art, equivalent replacements or changes can be made according to the technical solutions and the inventive concept thereof of the present disclosure, and all these changes or replacements should belong to the protection scope of the appended claims of the present disclosure.