CN109641934B

CN109641934B - Cholic acid derivative free base, crystal form, preparation method and application thereof

Info

Publication number: CN109641934B
Application number: CN201780049733.6A
Authority: CN
Inventors: 苏熠东; 龚素娟; 包如迪
Original assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2016-09-20
Filing date: 2017-09-20
Publication date: 2021-07-02
Anticipated expiration: 2037-09-20
Also published as: WO2018054300A1; CN109641934A

Abstract

A crystalline cholic acid derivative free base, its preparation method and application are provided, the chemical name of the cholic acid derivative free base is (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione, the powder X-ray diffraction pattern comprises peaks at diffraction angles (2 theta) of 16.5 +/-0.2 degrees, 13.6 +/-0.2 degrees, 12.1 +/-0.2 degrees and 20.4 +/-0.2 degrees, and also relates to the cholic acid derivative with optical purity and a preparation method and application thereof, wherein the purity of the prepared optical isomer reaches over 90.0 percent, and the problem that the optical purity is difficult to separate in the prior art is solved. In addition, a preparation method and application of the cholic acid derivative are also provided. The preparation method overcomes the defects in the prior art, the reaction can be completed by multi-step reaction at normal temperature, the obtained product has good purity, high yield, strong process operability and greatly improved process safety, and the method is suitable for industrial application.

Description

Cholic acid derivative free base, crystal form, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a crystalline cholic acid derivative free base, a preparation method and application thereof, a substantially pure cholic acid derivative, a preparation method and application thereof, and a preparation method and application of a cholic acid derivative.

Background

Farnesoid derivative X receptors (FXR) belong to a member of the nuclear receptor superfamily of hormones, which are expressed predominantly in the liver, small intestine, kidney and adrenal gland, and less expressed in adipose tissue and heart. Farnesol was originally thought to be its ligand and was hence the name. When FXR ligand is directly bound to FXR carboxy-terminal Ligand Binding Domain (LBD), nuclear receptor spatial conformation is changed and forms heterodimer with retinoid receptor (RXR), and finally binds to target gene specific FXR DNA response element to regulate transcription of target gene, and is involved in regulation of sugar and lipid metabolism, which is an important energy regulator. The primary bile acids chenodeoxycholic acid are the most potent ligands of FXR, and the secondary bile acids lithocholic and deoxycholic acid may also activate FXR. Synthetic FXR ligands (e.g., 6-ECDCA, GW4064, etc.) also currently bind FXR several times stronger than natural ligands. The main target genes of FXR include Bile Salt Export Pump (BSEP), bile acid binding protein (IBABP), small heterodimer partner receptor (SHP), etc., and FXR regulates the expression of these genes by binding to FXR response element (FXRE) on the promoters of these genes. However, the promoter sequence of main FXR regulatory gene such as cholesterol 7 alpha hydroxylase (CYP7 alpha 1) has no typical FXR binding reaction sequence, FXR indirectly induces the expression of transcription repressing factor SHP, and then SHP and CYP7 alpha 1 promoter liver receptor homolog (LRH-1) form inhibitory complex, thereby blocking the transcription of CYP7 alpha 1 and other LRH-1 target genes. Due to its important role in bile acid and cholesterol metabolism, FXR has drawn increasing attention to its association with liver-related diseases. The search for novel FXR agonists became the focus of research for the treatment of liver diseases including cholestasis syndrome, 6-ethynylchenodeoxycholic acid (obeticholic acid) has completed phase III clinical trials as a treatment for Primary Biliary Cirrhosis (PBC), was first marketed in 2015, and the compound also showed satisfactory therapeutic effects in non-alcoholic steatohepatitis patients. FXR agonists can reduce cholestasis by stimulating both bile acid dependent bile flow by stimulating the Bile Salt Efflux Pump (BSEP) and bile acid independent bile flow by stimulating MRP 2. FXR is expected to become a new drug target for screening and treating other metabolic diseases such as cholestatic diseases and nonalcoholic steatohepatitis.

In 2016, Jiangsu Howesson corporation disclosed a class of cholic acid derivatives in PCT/CN2016/079167 (application date: 2016.04.13), wherein the chemical names of representative compounds are: (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione (compound of formula (I)) having the structure:

the compound has obvious agonism on FXR activity and is expected to be developed into a new FXR agonist, but the compound of the formula (I) disclosed in example fourteen in patent PCT/CN2016/079167 can only obtain a mixture containing another non-corresponding isomer after being extracted by ethyl acetate and purified by column chromatography, so that the compound is difficult to prepare into a pharmaceutical preparation suitable for clinical application, and the final physicochemical property of the product is further proved by experiments. Therefore, the development of a stable crystal with good solubility is urgently needed to meet the clinical development requirement of the medicine. There is also an urgent need in the art to develop a compound that has good optical purity and can be stably stored to meet the clinical development needs of pharmaceuticals.

In 2002, WO2002072598A1 discloses obeticholic acid and a preparation method thereof for the first time, and the synthetic route is as follows:

the patent takes 3 alpha-hydroxy-7-ketone-5 beta-cholane-24-acid and 3, 4-dihydropyran as raw materials, and firstly protects the 3 alpha-hydroxy; then reacts with ethyl bromide to replace ethyl at the 4-position and form ester at the same time, but the reaction of the step needs to be carried out under the condition of-70 to-80 ℃, and dangerous n-butyl lithium and carcinogen hexamethyl phosphoric acid amide (HMPA) are used as catalysts, so that the route is not suitable for industrial application.

In 2006, patent WO2006122977a2 disclosed a synthetic route to obeticholic acid as follows

The patent also takes 3 alpha-hydroxy-7-ketone-5 beta-cholane-24-acid and 3, 4-dihydropyran as raw materials, firstly, ester is formed, then 3 alpha-hydroxy is protected by TMS, then 6-carbonyl is protected, and then aldehyde group compound is reacted, but the reaction needs to be carried out under the condition of-60 to-90 ℃, and the solvent used in the reaction is boron trifluoride diethyl etherate solution; hydrolyzing with sodium hydroxide, reducing with palladium-carbon, performing configuration conversion at 95-105 ℃, and reducing with sodium borohydride at 70-105 ℃ to obtain obeticholic acid. The preparation method requires expensive trimethylchlorosilane, palladium carbon, unstable aldehyde compounds, unsafe boron trifluoride ether solution and harsh reaction temperature, such as configuration transformation and reduction reaction at high temperature, so the synthetic route disclosed by the patent is not suitable for industrial application.

In 2016, Jiangsu Howesson corporation disclosed a class of cholic acid derivatives in PCT/CN2016/079167 (application date: 2016.04.13), wherein the chemical names of representative compounds are: 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione, prepared as follows:

according to the patent, 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthrene-17-yl) propyl) thiazolidine-2, 4-dione is prepared by taking obeticholic acid as a raw material, esterification is performed firstly, then bromination and cyclization are performed to obtain a final product, a preparation method of obeticholic acid is not optimized, the same problem as that of WO2006122977A2 exists, and the obeticholic acid is not suitable for industrial production, so that the problem that a medicine preparation process suitable for application needs to be developed is urgently solved by technical personnel in the field.

Disclosure of Invention

The invention aims to provide a better medicine crystal, and the inventor intensively studies different aggregation states of the compound shown in the formula I to finally obtain a crystalline (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthrene-17-yl) propyl) thiazolidine-2, 4-diketone compound shown in the formula (I), so that the physicochemical properties of the compound shown in the formula (I) are greatly improved, and the compound can be used for treating FXR mediated diseases including cardiovascular diseases, atherosclerosis, arteriosclerosis, hypercholesterolemia, hyperlipidemia chronic hepatitis diseases, chronic liver diseases, Gastrointestinal diseases, nephropathy, cardiovascular diseases, metabolic diseases, cancer (such as colorectal cancer) or neurological signs such as stroke, and the like, has wide medical application, and is expected to be developed into a new generation of FXR agonist.

A crystalline form of (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione free base of the invention, designated as form I, having a powder X-ray diffraction pattern comprising peaks at diffraction angles (2 Θ) of 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, 20.4 ± 0.2 °; preferably further comprising peaks at diffraction angles (2 θ) of 11.5 ± 0.2 °, 9.6 ± 0.2 °, 19.6 ± 0.2 °, 15.0 ± 0.2 °, 20.0 ± 0.2 °; more preferably, the composition further comprises peaks at diffraction angles (2. theta.) of 23.7. + -. 0.2 degrees, 20.7. + -. 0.2 degrees, 23.0. + -. 0.2 degrees, 25.7. + -. 0.2 degrees, 17.9. + -. 0.2 degrees and 16.0. + -. 0.2 degrees.

As a most preferred embodiment, the crystalline form of (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione free base has an X-ray powder diffraction pattern having diffraction peak angles 2 θ (°) and peak intensities as set forth in the following table:

2θ(°)	strength%	2θ(°)	Strength%
				9.6	21.7	20.0	16.1
11.5	21.9	20.4	23.8
				12.1	25.9	20.7	13.9
13.6	94.0	21.9	6.7
				13.9	6.7	22.8	7.2
15.0	17.1	23.0	12.2
				16.0	10.4	23.7	15.8
16.5	100.0	25.7	11.7
				17.9	10.9	25.9	8.8
19.6	19.3	28.6	7.2

In another aspect of the present invention, there is provided a method for preparing a crystalline form of (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione free base, comprising the steps of:

1) dissolving or dispersing (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione free base in an organic solvent, water, or a mixed solvent of the organic solvent and the water;

2) cooling to separate out, or optionally adding an anti-solvent into a clear solution of the compound to separate out, or optionally slowly volatilizing the clear solution of the compound to separate out, or optionally adding solid of the original compound or other solid particle additives into the solution of the compound to be used as heteronuclear seed crystals to induce crystallization, or using the above methods in combination to obtain the crystal.

Preferably, the organic solvent is selected from methanol, ethanol, isopropanol, acetonitrile, acetone, ethyl acetate, isopropyl acetate, toluene, n-butanol, cyclohexane, dichloromethane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, dioxane, diethyl ether, n-heptane, n-hexane, methylethyl ketone, isooctane, pentane, dipropyl alcohol, tetrahydrofuran, dimethyltetrahydrofuran, trichloroethane, xylene, or a mixture thereof.

In a further preferred embodiment, the organic solvent is selected from ethyl acetate, isopropyl acetate, dichloromethane, n-heptane, n-hexane or a mixture thereof.

As a further preferred mode, the crystalline body obtained in step 2) of the production method has a powder X-ray diffraction pattern including peaks at diffraction angles (2 θ) of 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, 20.4 ± 0.2 °.

In a further aspect, the present invention provides a pharmaceutical composition comprising an effective amount of crystalline form of (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione free base, a pharmaceutically acceptable carrier or excipient.

In a further aspect, the invention provides the use of the crystalline form of (5R) -5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione free base, or a pharmaceutical composition as hereinbefore described, in the manufacture of a medicament for the prevention or treatment of an FXR mediated disease or condition.

Preferably, the FXR mediated disease or condition is selected from cardiovascular disease, hypercholesterolaemia, hyperlipidemic chronic hepatitis disease, chronic liver disease, gastrointestinal disease, renal disease, cerebrovascular disease, metabolic disease, or cancer.

As a further preferred embodiment, the chronic liver disease is selected from the group consisting of primary cirrhosis (PBC), brain xanthoma (CTX), Primary Sclerosing Cholecystitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of pregnancy, extra-intestinal absorption-related cholestasis (PNAC), bacterial overgrowth or sepsis cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver transplantation-related graft-versus-host disease, live donor liver transplantation regeneration, congenital liver fibrosis, common bile duct calculi, granulation liver disease, intrahepatic or extrahepatic malignancy, Sjogren's syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis, and alpha-hepatosis₁It is used for treating deficiency of membrane protease.

As a further preferred embodiment, the gastrointestinal Disease is selected from inflammatory bowel Disease (I BD), Irritable Bowel Syndrome (IBS), bacterial overgrowth, malnutrition, post-reflex colitis or minimal colitis, preferably Crohn's Disease or ulcerative enteropathy.

As a further preferred embodiment, the kidney disease is selected from diabetic nephropathy, Focal Segmental Glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis or polycystic kidney disease.

As a further preferred embodiment, the cardiovascular disease is selected from the group consisting of atherosclerosis, arteriosclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia and hypertriglyceridemia.

As a further preferred embodiment, the metabolic disease is selected from insulin resistance, type I diabetes, type II diabetes or obesity; the cerebrovascular disease is selected from stroke.

As a further preferred embodiment, the cancer is selected from colorectal cancer or liver cancer.

Another objective of the present invention is to isolate a substantially pure compound of formula (I), which can greatly improve the physicochemical properties of the compound of formula (I) and meet the needs of clinical research; can be used for treating FXR mediated diseases, including cardiovascular diseases, atherosclerosis, arteriosclerosis, hypercholesterolemia, hyperlipidemia, chronic hepatitis diseases, chronic liver diseases, gastrointestinal diseases, nephropathy, cardiovascular diseases, metabolic diseases, cancers (such as colorectal cancer) or nerve signs such as stroke, has wide medical application, and is expected to be developed into a new generation of FXR agonist.

In a first aspect, the present invention provides a substantially pure compound of formula (I), said substantially pure compound of formula (I) being a compound of formula (I) having an optical purity of up to 90.0% or more.

As a further preferred embodiment, the substantially pure compound of formula (I) means that the compound of formula (I) has an optical purity of 95.0% or more.

As a still further preferred embodiment, the substantially pure compound of formula (I) comprises no more than 10.0% impurities;

preferably, the substantially pure compound of formula (I) comprises no more than 5.0% impurities.

As a further preferred aspect, the optical purity is relative to its non-corresponding isomer of the compound of formula (II):

in a second aspect the present invention provides a process for the preparation of a substantially pure compound of formula (I) comprising the steps of:

1) dissolving a crude product of the compound shown in the formula (I) in a positive solvent with the mass-volume ratio of 1-10 times;

2) adding an anti-solvent with the volume ratio of 0.5-10 times of that of the normal solvent;

3) after the addition, continuously stirring for crystallization;

4) filtering and draining;

5) the filtrate is concentrated to dryness under reduced pressure to give the substantially pure compound of formula (I).

As a further preferred embodiment, the n-solvent is selected from a lower ester solvent, a haloalkane solvent, a lower alcohol solvent or a mixture thereof.

Preferably, the n-solvent is selected from preferably ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or mixtures thereof.

As a further preferred embodiment, the volume of the said positive solvent is 3-5 times of the mass volume ratio of the crude compound of formula (I).

As a further preferred embodiment, the antisolvent is selected from the group consisting of a lower alkane solvent, a cycloalkane solvent, a lower ether solvent, or a mixture thereof.

Preferably, the antisolvent is selected from the group consisting of n-heptane, n-hexane, petroleum ether, isopropyl ether, or mixtures thereof.

As a further preferable mode, the anti-solvent is used in a volume of 1 to 3 times the volume ratio of the normal solvent.

As a further preferable mode, the concentration in step 5) is carried out under reduced pressure until it is dry, then the distillation is carried out by using a lower alcohol solvent or a mixture thereof, and then the concentration is carried out until it is dry.

Preferably, the step 5) is carried out by ethanol, isopropanol or a mixture thereof after concentrating to dryness under reduced pressure.

As a further preferable embodiment, the substantially pure compound of formula (5) (the optical purity of the compound obtained is 90.0% or more; preferably, the resulting substantially pure compound of formula (I) has an optical purity of 95.0% or more.

In a third aspect the present invention provides a substantially pure compound of formula (a process for the preparation of the compound of the invention comprising the steps of:

1) dissolving or dispersing the crude compound of the formula (I) in a first solvent with the mass-volume ratio of 0.5-10 times;

2) adding a second solvent with the volume ratio of 0.5-10 times of that of the first solvent;

3) continuously stirring for crystallization;

4) filtration and suction drying of the filter cake yields the substantially pure compound of formula (I).

As a further preferable scheme, the amount of the first solvent in the step 1) is 2 to 7 times of the crude mass-to-volume ratio of the second compound in the formula (I).

As a further preferable mode, the amount of the second solvent in the step 2) is 0.5 to 3 times the volume ratio of the first solvent.

As a further preferred embodiment, the first solvent is selected from a lower ester solvent, a halogenated alkane solvent, a lower alcohol solvent or a mixture thereof.

Preferably, the first solvent is selected from ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or mixtures thereof.

As a further preferred embodiment, the second solvent is selected from a lower alkane solvent, a cycloalkane solvent, a lower ether solvent or a mixture thereof.

Preferably, the second solvent is selected from n-heptane, n-hexane, petroleum ether, isopropyl ether or a mixture thereof.

As a further preferable mode, the substantially pure compound of the formula (the optical purity of the resulting compound) obtained in the step 4) is 90.0% or more.

Preferably, the substantially pure compound of formula (4) obtained has an optical purity of 95.0% or more.

As a further preferred embodiment, the preparation method formula (crude product of the derivative can be pretreated by the following steps:

1) dissolving sodium hydroxide or potassium hydroxide in lower alcohol solvent with the mass volume ratio of 5-100 times, and cooling to below 5 ℃;

2) adding a raw material containing a compound of a formula (I) into the alkali liquor obtained in the step 1), cooling to below-10 ℃, and continuously stirring for reaction;

3) slowly adding an acidic substance until the pH value of the reaction solution is about 5-6 under the condition of controlling the temperature to be below-10 ℃;

4) controlling the temperature to be lower than 40 ℃, and decompressing and concentrating to remove part of the solvent;

5) adding water and a lower ester solvent into the residue, and separating;

6) concentrating the organic phase under reduced pressure to dryness to obtain the crude product of the compound shown in the formula (I).

As a further preferable mode, the amount of the lower alcohol solvent used in the step 1) is 20 to 60 times the mass-to-volume ratio of sodium hydroxide or potassium hydroxide.

As a further preferred embodiment, the lower alcohol solvent used in step 1) is selected from ethanol, isopropanol or a mixture thereof.

As a further preferred embodiment, the ester solvent used in step 5) is selected from ethyl acetate, isopropyl acetate or a mixture thereof.

In a fourth aspect, the invention provides a pharmaceutical composition comprising an effective amount of a substantially pure compound of formula (I) or a pharmaceutically acceptable carrier or excipient thereof.

A fifth aspect of the invention provides the use of a substantially pure compound of formula (invention, or a pharmaceutical composition as hereinbefore described) for the manufacture of a medicament for the prevention or treatment of an FXR mediated disease or condition.

As a further preferred embodiment, the FXR mediated disease or condition is selected from cardiovascular disease, hypercholesterolaemia, hyperlipidemic chronic hepatitis disease, chronic liver disease, gastrointestinal disease, renal disease, cerebrovascular disease, metabolic disease or cancer.

As a still further preferred embodiment, the chronic liver disease is selected from primary cirrhosis (PBC), brain xanthoma (CTX), Primary Sclerosing Cholecystitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of pregnancy, extra-intestinal absorption-associated cholestasis (PNAC), bacterial overgrowth or sepsis cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver transplantation-associated graft-versus-host disease, live donor liver transplantation regeneration, congenital liver fibrosis, common bile duct stones, granulation liver disease, intrahepatic or extrahepatic malignancy, Sjogren's syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis, or alpha 1-anticytoplasmic enzyme deficiency.

As a still further preferred embodiment, the gastrointestinal Disease is selected from inflammatory bowel Disease (I BD), Irritable Bowel Syndrome (IBS), bacterial overgrowth, malnutrition, post-reflex colitis or minimal colitis, preferably Crohn's Disease or ulcerative enteropathy.

As a still further preferred embodiment, the kidney disease is selected from diabetic nephropathy, Focal Segmental Glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis or polycystic kidney disease.

As a still further preferred embodiment, the cardiovascular disease is selected from the group consisting of atherosclerosis, arteriosclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia and hypertriglyceridemia.

As a still further preferred embodiment, the metabolic disease is selected from insulin resistance, type I diabetes, type II diabetes or obesity.

As a still further preferred embodiment, the cerebrovascular disease is selected from stroke.

As a still further preferred embodiment, the cancer is selected from colorectal cancer or liver cancer.

Compared with the prior art, the invention has the following advantages:

1. the invention solves the technical problem that only a mixture can be obtained after the product obtained in the fourteenth embodiment of the patent PCT/CN2016/079167 is extracted by ethyl acetate and purified by column chromatography.

2. The compound of the formula (I) obtained by the invention has high optical purity which can even reach more than 95 percent to the maximum, and the raw material with the optical purity is favorable for further pharmacological and toxicological research and meets the requirement of clinical research.

3. The separation and purification process adopted by the invention is simple to operate, green and environment-friendly, the used solvent is simple and easy to obtain, the yield is stable, the quality is reliable, and the industrial application is facilitated.

The invention also aims to provide a method for preparing the cholic acid derivative, which has mild reaction conditions, mature process and stable quality and is very suitable for industrial application.

In a first aspect, the present invention provides a process for the preparation of a compound of formula (III), comprising the steps of:

1) carrying out esterification reaction on the compound of the formula (IV) to obtain a compound of a formula (V);

2) reducing the double bond of the compound shown in the formula (V) to obtain a compound shown in a formula (VI);

3) isomerizing the compound of the formula (VI) to obtain a compound of a formula (VII);

4) protecting the hydroxyl of the compound of the formula (VII) to obtain a compound of a formula (VIII);

5) reducing the carbonyl of the compound shown in the formula (VIII) to obtain a compound shown in a formula (III);

the reaction formula is as follows:

wherein R is selected from C_1-4An alkyl group; pg is a hydroxyl protecting agent.

In a further preferred embodiment, R in the preparation method is preferably selected from methyl or ethyl.

As a further preferred embodiment, Pg in said preparation process is preferably selected from the group consisting of substituted or unsubstituted benzoyl, substituted or unsubstituted benzenesulfonyl, substituted or unsubstituted alkyl, substituted or unsubstituted benzyl or trialkylsilyl.

As a further preferred embodiment, Pg in said preparation is preferably as follows:

most preferably, Pg in the preparation is selected from the following structures:

the compounds of formula (III) above are useful as key intermediates in the preparation of obeticholic acid and 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione.

As a further preferable scheme, in the preparation method, the esterification reaction in the step 1) is carried out in an acid environment at the temperature of 20-35 ℃.

As a further preferable mode, in the preparation method, the esterification reaction in the step 1) is preferably carried out at a temperature of 22 ℃ to 27 ℃.

As a further preferable mode, in the preparation method, the esterification reaction in step 1) is performed under an acidic condition, and the acid may be selected from hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, methanesulfonic acid or a mixture thereof. Preferably, it is selected from hydrochloric acid or sulfuric acid.

In a further preferable embodiment, in the preparation method, sodium alkoxide is adopted for the isomerization reaction in the step 3) in an alcohol solvent, and the reaction temperature is 5-35 ℃. Preferably, the reaction temperature is from 15 ℃ to 25 ℃.

As a further preferable mode, in the preparation method, the sodium alkoxide used in the isomerization reaction in the step 3) is selected from sodium methoxide, sodium ethoxide or sodium tert-butoxide.

As a further preferable embodiment, the alcohol solvent used in the isomerization reaction in step 3) in the preparation method is selected from methanol, ethanol, isopropanol or tert-butanol.

As a further preferable scheme, in the step 5) of carbonyl reduction in the preparation method, sodium borohydride is used to reduce the carbonyl group of the compound of formula (VIII) in an alcohol solvent to obtain the compound of formula (III), and the reaction temperature is-5 ℃ to 10 ℃. Preferably, the reaction temperature is from 0 ℃ to 3 ℃.

As a further preferable mode, in the preparation method, the alcohol solvent used in the carbonyl reduction in the step 5) is selected from methanol, ethanol, isopropanol or tert-butanol.

As a further preferable embodiment, in the preparation method, after the carbonyl reduction reaction in step 5) is finished, the reaction is quenched by acetone and citric acid.

In another aspect of the present invention, an application of the preparation method in preparation of obeticholic acid is further provided, wherein when preparing obeticholic acid, the compound of formula (III) is prepared by the preparation method, and then the reaction is performed according to the following steps:

wherein, the acid is organic acid or inorganic acid.

As a further preferred embodiment, the organic acid is preferably selected from trifluoroacetic acid, trichloroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid or mixtures thereof; the inorganic acid is preferably selected from hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, or mixtures thereof.

as a further preferred embodiment, the compound of formula (III) is preferably subjected to selective acidolysis under the action of lithium hydroxide to produce a compound of formula (IX).

In another aspect, the invention provides a use of the above-mentioned preparation method in the preparation of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione, wherein the compound of formula (III) is prepared by the above-mentioned preparation method in the preparation of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione, then the reaction is carried out according to the following steps:

As a further preferable scheme, crude 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione obtained by cyclization reaction is refined and purified by adopting dichloromethane with the mass-volume ratio of 1: 15.

As a further preferable embodiment, the HPLC purity of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione obtained by purification by beating with dichloromethane can be 98% or more.

Compared with the prior art, the preparation method of the invention has the following advantages:

1. the invention takes (4R) -4- ((3R, 5R, 10R, 13R, 14R, 17R, Z) -6-ethylidene-3-hydroxy-10, 13-dimethyl-7-carbonyl hexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valeric acid as raw material, esterification is carried out in the first step until the hydrolysis reaction is not carried out before the obeticholic acid or the 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethyl hexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-diketone is prepared, the obtained ester-forming intermediates can be easily dissolved in hydrophobic organic solvents, and are beneficial to the post-treatment of each step of reaction, so that part of byproducts can be removed by washing. Therefore, the process of the invention has strong operability.

2. The defects that in the patent WO2002072598A1, 3 alpha-hydroxy-7-ketone-5 beta-cholane-24-acid and 3, 4-dihydropyran are used as raw materials, the raw materials are protected by 3 alpha-hydroxy and then react with bromoethane to replace ethyl at the 4 th position, and ester forming reaction is carried out at the temperature of-70 to-80 ℃, and n-butyl lithium and carcinogenic hexamethyl phosphoramide are used are overcome, the esterification reaction temperature can be carried out at the temperature near normal temperature, and the purity and the yield of the obtained product are very high. Therefore, the esterification reaction of the present invention is suitable for industrial application.

3. Solves the defect that 3 alpha-hydroxy-7-ketone-5 beta-cholane-24-acid and 3, 4-dihydropyran are used as raw materials in WO2006122977A2, and trimethylchlorosilane is required to be continuously used for protecting 3 alpha-hydroxy, 6-carbonyl after ester formation.

4. Overcomes the defect that the TMS protected in the patent WO2006122977A2 reacts with aldehyde compound and still needs to react at the temperature of-60 to-90 ℃, and also avoids the use of boron trifluoride diethyl etherate solution.

5. The defect that configuration conversion can be carried out only at 95-105 ℃ during configuration conversion in WO2006122977A2 is overcome, and configuration conversion reaction can be carried out by adopting sodium alkoxide in an alcohol solvent at the temperature near normal temperature.

6. The method overcomes the defect that the reaction can be carried out only under the condition of high temperature of 70-105 ℃ when the obeticholic acid is obtained by reducing sodium borohydride in the patent WO2006122977A2, and the reaction can be carried out at low temperature, so that the generation of impurities in the reaction is less. In addition, citric acid is additionally added when the reaction is finished by acetone, so that the generation of byproducts is further reduced. Therefore, the product obtained by the reaction has good purity and high yield, and the process safety is also improved.

7. The defects that during the preparation of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthrene-17-yl) propyl) thiazolidine-2, 4-diketone in the patent PCT/CN2016/079167, obeticholic acid is esterified firstly, then the final product is obtained through bromination and cyclization are overcome.

The invention also provides a purification method of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthrene-17-yl) propyl) thiazolidine-2, 4-dione.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of the crystalline form I of the free base; the abscissa is the diffraction peak angle 2 θ (°), and the ordinate is the intensity of the peak.

FIG. 2 is a thermogravimetric analysis of the crystalline form I of the free base; the abscissa is temperature (deg.C) and the ordinate is percent weight loss (%).

FIG. 3 is a differential scanning calorimetry chart of the free base of the crystalline form I compound; the abscissa is temperature (. degree. C.) and the ordinate is heat flow (W/G).

FIG. 4 is a dynamic water sorption DVS (dynamic vapor sorption) isotherm plot of the crystalline form I compound free base with the abscissa as relative humidity RH (%) and the ordinate as percent mass change (%).

Detailed Description

1. Term(s) for

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complication, commensurate with a reasonable benefit/risk ratio.

The term "substantially pure" as used herein means that in certain preferred embodiments of the present invention the compound of formula I has a crystalline structure in substantially pure form with an HPLC purity or crystalline form purity of substantially greater than 90% (inclusive), preferably greater than 95%, more preferably greater than 98%, and most preferably greater than 99.5%.

"polymorph" or "polymorph" as used herein refers to a crystalline form having the same chemical composition, but a different spatial arrangement of molecules, atoms and/or ions that make up the crystal. Although polymorphs have the same chemical composition, they differ in their packing and geometric arrangement and may exhibit different physical properties such as melting point, shape, color, density, hardness, deformability, stability, solubility, dissolution rate, and the like. The two polymorphs may be either monotropic or interconvertive, depending on their temperature-stability relationship. For a single denaturation system, the relative stability between the two solid phases remains unchanged upon temperature change. In contrast, in the reciprocal system, there is a transition temperature where the two phases are exchanged for stability ((the Theory and Origin of Polymorphism in "Polymorphism in Pharmaceutical Solids" (1999) ISBN:) -8247-0237). The phenomenon that such compounds exist in different crystal structures is called drug polymorphism.

The crystalline structures of the present invention may be prepared by a variety of methods including crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, jet spray, and the like. Techniques for crystallizing or recrystallizing a crystalline structure from a solvent mixture include solvent evaporation, lowering the temperature of the solvent mixture, seeding a supersaturated solvent mixture of the molecule and/or salt, lyophilizing the solvent mixture, adding an anti-solvent to the solvent mixture, and the like. High throughput crystallization techniques can be used to prepare crystalline structures, including polymorphs. Drug crystals, including polymorphs, methods of preparation and characterization of drug crystals are disclosed in Solid-State Chemistry of Drugs, S.R.Byrn, R.R.Pfeiffer, and J.G.Stowell, 2 nd edition, SSCI, West Lafayette, Indiana, 1999.

In addition, as is known to those skilled in the art, seed crystals are added to any crystallization mixture to facilitate crystallization. Thus, the present invention may also use seed crystals as a means of controlling the growth of a particular crystalline structure or as a means of controlling the particle size distribution of the crystalline product. Accordingly, the calculation of the amount of desired seed crystals depends on the size of the available seed crystals and the desired size of the average product particles, as described in "Programmed crystallization of batch crystallizers," J.W.Mullin and J.Nyvlt, Chemical Engineering Science, 1971, 26, 369-. In general, small sized seeds are required to effectively control the growth of crystals in the batch. Small size seeds may be produced by sieving, milling or micronization of larger crystals or by micro-crystallization of solutions it should be noted that milling or micronization of crystals cannot cause any change in crystallinity (i.e. become amorphous or become another polymorphic form) of the desired crystal structure.

Equivalent crystal structures disclosed or claimed herein may exhibit similar, but not identical analytical properties within reasonable error limits depending on experimental conditions, purity, equipment, and other variables known to those skilled in the art. Accordingly, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope and spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a non-limiting scope.

The term "room temperature" or "RT" as used herein refers to an ambient temperature of 20 to 25 deg.C (68-77 deg.F).

The term pharmaceutical composition as used herein means a mixture containing one or more compounds described herein or physiologically/pharmaceutically acceptable salts or prodrugs thereof, in admixture with other chemical components, or other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

2. Experimental Material

The reagents used in the examples of the invention were commercial technical or analytical grade reagents and the starting material of the compound of formula (I) was prepared according to example fourteen of Howesson patent PCT/CN 2016/079167.

3. Analytical method

3.1X-ray powder diffraction

One of ordinary skill in the art will recognize that X-ray powder diffraction patterns can be obtained with measurement errors that depend on the measurement conditions used. In particular, it is generally known that the intensity in an X-ray powder diffraction pattern may fluctuate depending on the material conditions used. It should be further understood that the relative intensities may also vary with experimental conditions, and accordingly, the exact intensities should not be taken into account. In addition, the measurement error of the conventional X-ray powder diffraction angle is generally about 5% or less, and such a measurement error degree should be regarded as belonging to the above-mentioned diffraction angle. Thus, it is to be understood that the crystalline structure of the present invention is not limited to a crystalline structure that provides an X-ray diffraction pattern that is exactly the same as the X-ray powder diffraction pattern depicted in the figures disclosed herein. Any crystal structure that provides an X-ray powder diffraction pattern substantially the same as those disclosed in the accompanying drawings falls within the scope of the present invention. The ability to determine an X-ray powder diffraction pattern that is substantially the same is within the ability of one of ordinary skill in the art. Other suitable standard calibrations known to those skilled in the art. However, the relative intensity may vary with crystal size and shape.

Polymorphic forms of the compounds of formula I are characterized by their X-ray powder diffraction pattern. Thus, radiation at Cu K α

The X-ray powder diffractogram was collected on a Rigaku UltimaIV X-ray powder diffractometer operating in reflection mode. The tube voltage and current magnitude were set to 40kV and 40mA acquisition scans, respectively. The sample was scanned over a2 theta range of 5.0 deg. to 45 deg. for a period of 5 minutes. All analyses were performed at room temperature, typically 20-30 ℃. The XRPD sample is prepared by placing the sample on a single crystal silicon wafer and lightly pressing the sample powder with a glass slide or equivalent to ensure that the sample surface is flat and of the appropriate height. The sample holder was then placed into a Rigaku UltimaIV instrument and X-ray powder diffraction patterns were collected using the instrument parameters described above. Bag with bagSeveral factors including the following produce differences in measurements associated with such X-ray powder diffraction analysis results: (a) errors in sample preparation (e.g., sample height), (b) instrument errors, (c) calibration differences, (d) operator errors (including those errors that occur when determining peak positions), and (e) properties of the material (e.g., preferred orientation errors). Calibration errors and sample height errors often result in a shift of all peaks in the same direction. Generally, this calibration factor will bring the measured peak position into agreement with the expected peak position and may be in the range of ± 0.2 ° of the expected 2 θ value.

3.2 thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) experiments in TA Instruments^TMIn model Q500. Samples (approximately 2-10 mg) were loaded into pre-tared platinum pans. The sample weight was accurately measured by the instrument and recorded to one thousandth of a milligram. The furnace was purged with nitrogen at 100 ml/min. Data were collected between room temperature and 300 ℃ at a 10 ℃/minute heating rate.

3.3 Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) experiments in TA Instruments^TMIn model Q200. Samples (approximately 2-10 mg) were weighed in an aluminum pan and recorded to exactly one hundredth of a mg and transferred to the DSC. The instrument was purged with nitrogen at 50 ml/min. Data were collected between room temperature and 300 ℃ at a 10 ℃/minute heating rate. Plots were made with the endothermic peak facing downward. However, one skilled in the art will note that in DSC measurements, there is a degree of variability in the measured onset and maximum temperatures, depending on the heating rate, crystal shape and purity, and other measured parameters.

The specific examples and preparation method examples provided below will further illustrate certain aspects of embodiments of the invention. The scope of the following examples shall not limit the scope of the invention in any way.

Example 1

Weighing about 15mg of the compound of formula I as a free base solid (amorphous form) in a 1.5mL vial, adding 0.5mL of dichloromethane, and stirring at room temperature to suspendAfter 24 hours, solid-liquid separation is carried out to obtain the free base crystal form I of the compound shown in the formula I. The X-ray powder diffraction pattern is shown in figure 1; the thermogravimetric analysis is shown in FIG. 2 (weight loss 0.29%), and the differential scanning calorimetry is shown in FIG. 3 (melting point 132.9 ℃); the dynamic water sorption DVS isotherm plot is shown in fig. 4, with DVS test conditions as follows: no N₂In the presence of conditions, temperature 25.0 ℃, Relative Humidity (RH): from 0% to 95% to 0%; the stability test is as follows:

1. stability in solvents

Compound 10mg was precisely weighed into a 100mL volumetric flask, acetonitrile/water (V/V, 1: 1) was added thereto, and dissolved by sonication to give a solution of 0.1 mg/mL. And (5) placing at room temperature, sampling at 0h, 3h, 6h and 24h for proper amount, detecting by liquid phase, and calculating the relative purity of 0 h. The results are as follows:

standing time (h)	Relative purity
		0	/
3	101.1％
		6	102.1％
24	100.4％

The experimental results show that: the compound crystal is basically stable in acetonitrile/water (V/V, 1: 1) for 24h without significant degradation.

2. Stability under high temperature and light

Weighing about 4mg of the compound, paralleling 6 parts, placing in a 4mL glass bottle, respectively placing under the conditions of 25 ℃/RH 75%, 40 ℃/RH 75%, 50 ℃, 80 ℃ and illumination for 7 days. The illumination experiment is followed to carry out the quality control experiment (namely, the quality control sample is followed to carry out the light-proof experiment of the powder). And taking out after standing for 7 days, adding a diluent into the powder sample to dissolve the powder sample to obtain a 1mg/mL sample solution, detecting the purity of the sample solution by a liquid phase, and calculating the relative purity of the sample solution at 0 day. The results are as follows:

the experimental result shows that the compound crystal is placed for 7 days at 25 ℃/RH 75%, 40 ℃/RH 75%, 50 ℃, 80 ℃ and under the illumination condition, has no obvious degradation, and can continuously keep stable.

The liquid phase analysis conditions were as follows:

1) instrument and apparatus

Name of instrument	Model number
		Analytical balance	Sartorius BSA224S-CW
Water purifier	Milli-Q Plus，Millipore
		High performance liquid chromatograph	Agilent1260
Pump and method of operating the same	Agilent G1311B
		Sample injector	G1329B
Column oven	G1316A
		Detector	G1315D

2) Chromatographic conditions

Example 2

Weighing about 15mg of a free base solid (amorphous) of the compound shown in the formula I into a 1.5mL small bottle, adding 0.5mL of n-heptane, suspending and stirring for 24 hours at room temperature, and then carrying out solid-liquid separation to obtain a free base crystal form I of the compound shown in the formula I, wherein an X-ray powder diffraction pattern of the crystal form I is basically consistent with that shown in figure 1.

Example 3

Weighing about 15mg of a free base solid (amorphous form) of the compound shown in the formula I into a 1.5mL small bottle, adding 0.5mL of water, suspending and stirring for 24 hours at room temperature, and then carrying out solid-liquid separation to obtain a free base crystal form I of the compound shown in the formula I, wherein an X-ray powder diffraction pattern of the free base crystal form I is basically consistent with that shown in a figure 1.

Example 4

Weighing about 50mg of a free base solid of the compound shown in the formula I, putting the free base solid into a 1.5mL small bottle, adding 0.8mL dichloromethane, stirring for 48 hours at room temperature, performing solid-liquid separation, and volatilizing overnight at room temperature in an open manner to obtain a free base crystal form I of the compound shown in the formula I, wherein an X-ray powder diffraction pattern of the crystal form I is basically consistent with that shown in figure 1.

The terminology and specific embodiments in the process for the preparation of cholic acid derivatives of the present invention:

“C_1-4alkyl "refers to straight and branched alkyl groups comprising 1 to 4 carbon atoms, alkyl refers to a saturated aliphatic hydrocarbon group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, and the like.

"alcoholic solvent" means an alkane compound having a hydroxyl group in the molecule, such as methanol, ethanol, isopropanol.

The structure of the compounds of the invention is determined by Nuclear Magnetic Resonance (NMR) or/and liquid mass chromatography (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated methanol (CD)₃OD) and deuterated chloroform (CDCl)₃) Internal standard is Tetramethylsilane (TMS).

LC-MS was measured using an Agilent 1200 Infinity Series Mass spectrometer. HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

The thin layer chromatography silica gel plate adopts a tobacco yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification adopted by TLC is 0.15 mm-0.20 mm, and the specification adopted by the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm. The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

The starting materials in the examples of the present invention are known and commercially available, or may be synthesized using or according to methods known in the art.

All reactions of the present invention are carried out under a dry nitrogen or argon atmosphere with continuous magnetic stirring, and the solvent is a dry solvent, unless otherwise specified.

Example 5

The method comprises the following steps: preparation of methyl (4R) -4- ((3R, 5R, 10R, 13R, 14R, 17R, Z) -6-ethylene-3-hydroxy-10, 13-dimethyl-7-carbonylhexahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

Adding 50.0g (0.12mol) of (4R) -4- ((3R, 5R, 10R, 13R, 14R, 17R, Z) -6-ethylene-3-hydroxy-10, 13-dimethyl-7-carbonyl hexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) pentanoic acid and 500ml of methanol into a reaction bottle, dropwise adding 1.5g (0.015mol) of concentrated sulfuric acid at the temperature of 22-27 ℃, continuously stirring for reaction overnight after dropwise adding, analyzing by TLC that the raw materials basically react, dropwise adding 10ml of saturated sodium bicarbonate solution at the temperature of 15-25 ℃, decompressing and concentrating to remove most of the methanol, adding 500ml of ethyl acetate and 200ml of saturated sodium bicarbonate solution, stirring for 20min, separating, washing the organic layer once by 120ml of saturated sodium chloride solution, drying by anhydrous sodium sulfate, decompressing and concentrating to dryness to obtain 50.2g of foamed solid product (yield: 97.3%, HPLC: 98.6%). The HPLC analysis method is as follows:

■ mobile phase A: water + 0.05% TFA

■ mobile phase B: ACN + 0.04% TFA

■ column: agilent XDB C-185 um 4.6 x 150mm

■Posttime：5min

■ column temperature: 25 deg.C

■ flow rate: 1ml/min

■ detection wavelength: 205 nm; 214 nm; 254 nm.

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	95	5
13	5	95
			16	5	95

Step two: preparation of methyl (4R) -4- ((3R,5S, 6S, 10S,13R, 14R, 17R) -6-ethyl-3-hydroxy-10, 13-dimethyl-7-carbonylhexahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

Methyl (4R) -4- ((3R, 5R, 10R, 13R, 14R, 17R, Z) -6-ethylene-3-hydroxy-10, 13-dimethyl-7-carbonyl hexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate 49g (0.114mol), 4.9g 10% palladium-carbon and 250ml ethanol are added into a reaction flask, hydrogen gas replacement is carried out for 3 times, stirring is carried out at 15-25 ℃ overnight under hydrogen atmosphere, the raw materials are completely reacted, filtration is carried out, the filtrate is concentrated to dryness under reduced pressure, and 46.3g of foamy solid product is obtained by adding methanol and carrying out distillation (yield: 94.1%).

Step three: preparation of methyl (4R) -4- ((3R,5S,6R, 10S,13R, 14R, 17R) -6-ethyl-3-hydroxy-10, 13-dimethyl-7-carbonylhexahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

Adding 40g (0.092mol) of methyl (4R) -4- ((3R,5S, 6S, 10S,13R, 14R, 17R) -6-ethyl-3-hydroxy-10, 13-dimethyl-7-carbonyl hexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate and 200ml of methanol into a reaction bottle, and cooling to 0-5 ℃; adding 200ml of methanol into the other reaction bottle, controlling the temperature to be below 25 ℃, adding 25.0g (0.462mol) of sodium methoxide in batches, stirring the solution until the solution is clear, dropwise adding the sodium methoxide solution into the first reaction bottle at the temperature of 0-5 ℃, naturally heating to 15-25 ℃ after dropwise adding, stirring overnight, and detecting that the raw materials are completely reacted by TLC (TLC: petroleum ether: ethyl acetate: 1, phosphomolybdic acid is developed). And adding 120ml of methanol into one reaction bottle, cooling to 0-5 ℃, dropwise adding 27.2g (0.277mol) of concentrated sulfuric acid, dropwise adding a sulfuric acid solution into the first reaction bottle at 15-25 ℃, naturally heating to 15-25 ℃ after dropwise adding, stirring overnight, and detecting complete conversion of hydrolysis impurities by TLC (TLC: petroleum ether: ethyl acetate: 1, phosphomolybdic acid is developed). And (2) dropwise adding 120ml of saturated sodium bicarbonate solution at the temperature of 15-25 ℃, adjusting the pH of the reaction solution to 7-8, concentrating under reduced pressure to remove methanol, adding 400ml of ethyl acetate, stirring, separating liquid, washing the organic phase with 120ml of saturated sodium bicarbonate solution and 120ml of saturated saline solution respectively, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, adding tetrahydrofuran, and distilling to obtain 40.8g of a foamy solid product (yield: 100%, HPLC: 99.7%).

Step four: preparation of (3R,5S,6R, 10S,13R, 14R, 17R) -6-ethyl-17- ((R) -5-methoxy-5-carbonylpentan-2-yl) -10, 13-dimethyl-7-carbonylhexadecahydro-1H-cyclopenta [ a ] phenanthren-3-yl-4-nitrobenzoate

Adding 40g (0.092mol) of methyl (4R) -4- ((3R,5S,6R, 10S,13R, 14R, 17R) -6-ethyl-3-hydroxy-10, 13-dimethyl-7-carbonyl hexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate and 400ml of tetrahydrofuran into a reaction bottle, stirring uniformly, adding 29.9g (0.231mol) of diisopropylethylamine and 1.13g (0.009mol) of 4-dimethylaminopyridine, cooling the mixed solution to 0-5 ℃, adding 34.3g (0.185mol) of paranitrobenzoyl chloride in batches at 0-5 ℃, continuing to stir for 30min, heating to 45-55 ℃, stirring for 2-4H, detecting by TLC that the raw materials completely react (TLC: petroleum ether: ethyl acetate is 1:1, phosphomolybdic acid color development). Cooling to 0-5 ℃, dropwise adding 200mL of water, stirring for 20min, then adding methyl tert-butyl ether (200mL), stirring for liquid separation, washing the organic phase with 200mL of hydrochloric acid (1mol/L), 160mL of saturated sodium bicarbonate and saturated salt solution respectively, drying with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, adding methyl tert-butyl ether, and distilling to dryness. 160ml of methyl tert-butyl ether is added into the residue, the mixture is heated and refluxed for 1 hour, the temperature is reduced to 40-50 ℃, 320ml of n-heptane is added dropwise, the temperature is naturally reduced to room temperature after the dropwise addition, the mixture is stirred overnight, the filtration is carried out, and 48.3g of a white-like solid product is obtained after a filter cake is dried (the yield is 89.8 percent, and the HPLC is 98.8 percent). The HPLC analysis method is as follows:

■ mobile phase A: water + 0.05% TFA

■ mobile phase B: ACN + 0.04% TFA

■ column: agilent Zorbax SB C-185 um 4.6 x 150mm

■Posttime：5.0min

■ column temperature: 40 deg.C

■ flow rate: 1ml/min

■ detection wavelength: 205 nm; 214 nm; 254 nm; 225nm

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	20	80
5	5	95
			16	5	95

¹HNMR：(CDCl3，400MHz)δ0.67(s，3H)，0.82(t，J＝7.6Hz，3H)，0.93(d，J＝6.4Hz，3H)，1.09-1.19(m，4H)，1.26-1.56(m，12H)，1.71-2.02(m，9H)，2.16-2.26(m，2H)，2.32-2.43(m，2H)，2.74-2.79(m，1H)，3.67(s，3H)，4.91-4.98(m，1H)，8.16-8.18(m，2H)，8.26-8.28(m，2H)。

The structure of the impurities is resolved as follows:

¹HNMR：(DMSO-d6，400MHz)δ0.62(s，3H)，0.78(t，J＝7.2Hz，3H)，0.87-1.94(m，31H)，2.18-2.24(m，1H)，2.29-2.35(m，1H)，2.86-2.93(m，1H)，3.58(s，3H)，3.81(d，J＝8.4Hz，1H)，4.81-4.88(m，1H)，8.18-8.20(m，2H)，8.33-8.35(m，2H)。

step five: preparation of (3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-7-hydroxy-17- ((R) -5-methoxy-5-carbonylpentan-2-yl) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-3-yl-4-nitrobenzoate

Adding 5g (8.59mmol) of (3R,5S,6R, 10S,13R, 14R, 17R) -6-ethyl-17- ((R) -5-methoxy-5-carbonylpentane-2-yl) -10, 13-dimethyl-7-carbonylhexadecahydro-1H-cyclopenta [ a ] phenanthrene-3-yl-4-nitrobenzoate and 30ml of tetrahydrofuran into a reaction bottle under the protection of nitrogen, stirring to dissolve, adding 30ml of methanol, cooling to 0-2 ℃, adding 0.325g (8.359mmol) of sodium borohydride, continuously stirring and reacting for 8 hours at the temperature of 0 ℃, detecting the reaction completion of the raw materials by TLC, dropping 3.2ml of acetone within 5min, stirring for 30min after dropping, then adding 1.65g (8.59mmol) of anhydrous citric acid, continuously stirring for 5min, 25ml of methyl tert-butyl ether and 20ml of water were added. The organic solvent was removed under reduced pressure, 50ml of methyl tert-butyl ether was added for extraction, the organic phase was washed successively with 20ml of water and 10ml of saturated brine each time, dried over anhydrous sodium sulfate, the methyl tert-butyl ether was removed under reduced pressure, and distilled to dryness with isopropanol. Adding 40ml of isopropanol into the residue, heating to 80 ℃, refluxing, naturally cooling to 60 ℃, gradually precipitating solids (if no precipitation exists, 20mg of seed crystal can be added), keeping the temperature at 60 ℃, stirring for 1 hour, naturally cooling to 20 ℃ after about 3 hours, continuously cooling to 0 ℃, stirring for 30min, filtering, and drying the filter cake at 40 ℃ in vacuum for 3 hours to obtain 4.33g of white solid product (yield: 86.7%, HPLC: 99.2%).

¹HNMR：(DMSO-d6，400MHz)δ0.62(s，3H)，0.82-1.93(m，33H)，2.16-2.37(m，3H)，3.53(s，1H)，3.58(s，3H)，4.17(d，J＝5.2Hz，1H)，4.70-4.76(m，1H)，8.17-8.19(m，2H)，8.32-8.35(m，2H)。

The structure of the impurities is resolved as follows:

example 6

Adding 250mg (0.43mmol) of (3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-7-hydroxy-17- ((R) -5-methoxy-5-carbonylpentane-2-yl) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-3-yl-4-nitrobenzoate into a reaction bottle under the protection of nitrogen, then sequentially adding 2ml of methanol, 1ml of tetrahydrofuran, 0.5ml of water and 51mg (1.28mmol) of sodium hydroxide, continuing stirring and reacting for 16 hours at the temperature of 25 ℃, removing the solvent under reduced pressure, adding diluted hydrochloric acid to adjust the pH to 3-4, adding 20ml of ethyl acetate, separating an ethyl acetate layer, washing an organic phase with 10ml of an aqueous sodium bicarbonate solution for 2 times respectively, the solvent was removed under reduced pressure to give 161mg (yield 89.4%) of a white foam.

Example 7

The method comprises the following steps: preparation of methyl (4R) -4- ((3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

(3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-7-hydroxy-17- ((R) -5-methoxy-5-carbonylpentan-2-yl) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] is added to the reaction flask]4.33g (7.42mmol) of phenanthrene-3-yl-4-nitrobenzoate and 26ml of tetrahydrofuran are stirred and dissolved, cooled to 0-5 ℃, and LiOH.H is dripped₂And (3) dripping O (0.58g, 13.8mmol) in methanol (13ml) for about 10min, controlling the temperature to be 0-5 ℃, stirring and reacting for 1h, naturally heating to 18-22 ℃, continuously stirring and reacting for 5-8 h, and basically completely detecting raw materials by TLC. The reaction mixture was added dropwise to 50ml of a saturated aqueous ammonium chloride solution precooled to 0 ℃, the solvent was distilled off under reduced pressure, 50ml of ethyl acetate was added to the residue, liquid separation was performed after stirring, the organic phase was washed once with 50ml of a 4% aqueous potassium phosphate solution and once with 15ml of a saturated aqueous sodium chloride solution in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain 2.9g of a foamy product (yield: 90.1%, HPLC: 100%).

Step two: preparation of obeticholic acid

Under nitrogen protection, 2.9g (6.68mmol) of methyl (4R) -4- ((3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate was added to a reaction flask, then 10ml of methanol, 5ml of tetrahydrofuran, 5ml of water, and 0.54g (13.36mmol) of sodium hydroxide were sequentially added thereto, the reaction was further stirred at a temperature of 25 ℃ for 5 hours, the solvent was removed under reduced pressure, diluted hydrochloric acid was added to adjust the pH to 3 to 4, 30ml of ethyl acetate was added, an ethyl acetate layer was separated, the organic phase was washed with 10ml of saturated sodium chloride, and the solvent was removed under reduced pressure to obtain 2.8g of a white foamy solid (yield 100%).

Example 8

The method comprises the following steps: preparation of methyl (4R) -2-bromo-4- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

144.3g (1.426mol) of diisopropylamine is dissolved in 1000ml of tetrahydrofuran, cooled to-50 ℃, added with 495ml of n-butyl lithium n-hexane solution (2.4mol/L, 1.188mol) dropwise at the temperature of-50 ℃ to-40 ℃ for about 30min, and then kept warm and stirred for 1 h. 102.9g (237.7mmol) of methyl (4R) -4- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate are dissolved in 500ml of tetrahydrofuran. And (3) dropping the mixture into the n-butyllithium solution at the temperature of between 60 ℃ below zero and 50 ℃ below zero for about 50min, wherein white viscous solids appear in the dropping process. The temperature is controlled between minus 70 ℃ and minus 65 ℃ and the stirring reaction is continued for 1 hour. 154.9g (1.426mol) of trimethylchlorosilane is added dropwise at the temperature of-70 ℃ to-60 ℃ for about 15min, and then the mixture is stirred and reacted for 3h at the temperature of-60 ℃ to-50 ℃. TLC detection raw material basically finishes the reaction. Adding 129.6g (728.2mmol) of N-bromosuccinimide in batches, naturally heating to 22-27 ℃ after the addition is finished, keeping the temperature, stirring and reacting for 22 hours, and detecting by TLC that the raw materials basically react completely. Cooling the reaction mixture to 0 ℃, dropwise adding 1000ml of saturated sodium bicarbonate solution at the temperature of 10 ℃, stirring, separating liquid, concentrating the organic phase to about 500ml, extracting the aqueous phase by 500ml of methyl tert-butyl ether, combining the organic phase concentrated solution and the methyl tert-butyl ether extract, washing once by 1000ml of water, adding 250ml of 1mol/L hydrochloric acid into the organic phase, stirring for 5 hours at room temperature, separating liquid, washing the organic phase once by 1500ml of 20% sodium sulfite solution, washing twice by 1000ml of 2 saturated sodium bicarbonate solution, drying by anhydrous sodium sulfate, filtering, and concentrating to dryness to obtain 134.8g of brown solid (viscous crude product yield: 100%, HPLC: 99.5%, including dibromo epimers). Directly putting into the next reaction.

Step two: preparation of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione

134.8g (237.7mmol) of crude methyl (4R) -2-bromo-4- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate obtained in the above reaction and 54.3g (713.1mmol) of thiourea were dissolved in 1000ml of ethanol. The mixture was heated to 70 ℃ and stirred for 5 h. TLC detection raw material reaction is complete. 1200ml of 2mol/L hydrochloric acid was added to the reaction solution, and the reaction was stirred for 30 hours while controlling the temperature at 72 ℃ and cooled to room temperature (oil may precipitate). The ethanol was concentrated off under reduced pressure, the residue was extracted with 1200ml of ethyl acetate, the layers were separated, the organic phase was washed twice with 1200ml of 2 water, dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to give 133.9g of a brown foamy solid (crude HPLC: 85.4%).

Refining: 15.0g of the brown foamy solid obtained above was taken, 150ml of dichloromethane was added, slurried with stirring for 1 hour, filtered, the filter cake was washed with a small amount of precooled dichloromethane and dried under vacuum to give 7.9g of an off-white solid (HPLC: 99.2%, containing two diastereomers).

The terms and embodiments of the present invention's optical isomer compounds are as follows:

"optical purity" is a measure of the amount of one enantiomer in an optically active sample in excess of the other, and in the present invention refers primarily to the proportion of the compound of formula (I) in a mixture comprising the compound of formula (I) and the compound of formula (II).

The "lower alkane solvent" refers to a liquid alkane containing 5 to 10 carbon atoms at normal temperature, such as n-hexane and n-heptane.

"cycloalkane solvent" means a saturated hydrocarbon containing an alicyclic structure and being liquid at ordinary temperature, for example, cyclopentane, cyclohexane.

The "halogenated alkane solvent" means an alkane containing a halogen atom which is liquid at ordinary temperature, for example, methylene chloride, chloroform.

The "lower alcohol solvent" refers to an alkane compound having a hydroxyl group in the molecule and having a carbon number of less than 12, such as methanol, ethanol, isopropanol.

The "lower ester solvent" refers to an ester compound containing a small number of carbon atoms and being in a liquid state at ordinary temperature, preferably an ester compound produced by reacting an acid having less than 4 carbon atoms with an alcohol having less than 4 carbon atoms, such as ethyl acetate, methyl acetate, isopropyl acetate.

The "lower ether solvent" is an ether compound comprising two hydrocarbon groups having a small number of carbon atoms, preferably an ether compound comprising two hydrocarbon groups having less than 4 carbon atoms, such as diethyl ether, isopropyl ether and methyl isopropyl ether.

Example 9 (preparation of starting Material)

The first step is as follows: preparation of methyl (4R) -4- ((3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

Obeticholic acid 100.0g (237.7mmol) and concentrated sulfuric acid (0.25g) were dissolved in 500ml of methanol. Heat to reflux and continue to stir the reaction for 16 h. After TLC detection reaction, the reaction liquid is concentrated to dryness, 600ml of ethyl acetate and 400ml of saturated sodium bicarbonate solution are added into the residue, the mixture is fully stirred and then stands for layering, the organic phase is washed once by 400ml of saturated sodium chloride solution, dried by anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, 200ml of toluene is added to dissolve the residue, and then concentrated to dryness under reduced pressure to obtain light brown foam solid 102.9g, which is directly put into the next reaction.

The second step is that: preparation of methyl (4R) -2-bromo-4- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate

144.3g (1.426mol, 6.0eq.) of diisopropylamine is dissolved in 1000ml of tetrahydrofuran, cooled to-50 ℃, and 495ml (1.188mol, 2.4M n-hexane solution, 5.0eq.) of n-butyllithium solution is added dropwise at the temperature of-50 ℃ to-40 ℃ for about 30 minutes, and stirring is continued for 1 hour at the temperature.

102.9g (calculated by 237.7mmol of the theoretical amount in the step) of methyl (4R) -4- ((3R,5S,6R,7R,10S,13R, 14R, 17R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate prepared in the step is dissolved in 500ml of tetrahydrofuran, and the mixture is dripped into the reaction solution, the temperature is controlled to be between-60 ℃ and-50 ℃, the dripping is finished for about 50 minutes, and white viscous solid appears in the reaction. The reaction is continued to be stirred for 1 hour and 1 hour at the temperature of between 70 ℃ below zero and 65 ℃ below zero. And (3) dropwise adding 154.9g (1.426mol, 6.0eq.) of trimethylchlorosilane (TMSCl) at the temperature of-70 ℃ to-60 ℃ for about 15 minutes, continuously stirring and reacting for 3 hours at the temperature of-60 ℃ to-50 ℃, and detecting that the raw materials basically react by TLC.

129.6g (728.2mmol, 3.06eq.) of N-bromosuccinimide (NBS) is added in portions for about 30 to 45 minutes, and the reaction temperature is increased from-60 ℃ to-40 ℃. Then, the reaction mixture was allowed to naturally warm to room temperature (22 ℃ C.) and stirred for 24 hours. TLC detects that the raw material basically finishes the reaction, the reaction liquid is cooled to 0 ℃, and 1000ml of saturated sodium bicarbonate solution is dripped under the condition of controlling the temperature to be less than 10 ℃. After stirring, the phases were separated, the organic phase was concentrated to remove part of the solvent (about 500ml), the aqueous phase was extracted with 500ml of methyl tert-butyl ether (MTBE), the organic phase concentrate and the methyl tert-butyl ether extract were combined, and the combined solution was washed once with 1000ml of water.

250ml of 1mol/L hydrochloric acid was added to the above-mentioned combined solution, and the reaction was stirred at room temperature (22 ℃ C.) for 5 hours. The organic phase is separated, washed once with 1500ml of 20% sodium sulfite solution, then washed twice with 1000ml of sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to dryness to obtain a viscous brown solid 134.8g, which is directly put into the next reaction.

The third step: preparation of 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione

134.8g of crude methyl (4R) -2-bromo-4- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) valerate prepared in the above step (calculated as 237.7mmol from the above theoretical amount) and 54.3g of thiourea (713.1mmol, 3.0eq.) were dissolved in 1000ml of ethanol. The reaction was stirred for 5 hours while heating to 70 ℃. TLC detection raw material basically finishes the reaction. 1200ml of 2mol/L hydrochloric acid is added, the reaction is continued for 30 hours under controlled temperature of 72 ℃, most of the ethanol solvent is concentrated under reduced pressure, the mixture is extracted with 1200ml of ethyl acetate, the organic phase is washed twice with 1000ml of water respectively, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to dryness to obtain 133.9g of crude product (HPLC (comprises the compound of the formula (II) and the compound of the formula (I): 85.5 percent, wherein the compound of the formula (II): the compound of the formula (I): 24.2 percent to 61.3 percent is approximately equal to 2: 5) as a brown foamed solid.

An additional 200.0g of obeticholic acid was added and reacted according to the first to third steps described above, the experimental results obtained for the preparation were as follows:

crude 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione (406.5g) from the preceding two batches were combined and chromatographed on silica gel (eluent petroleum ether: ethyl acetate: tetrahydrofuran ═ 3: 1) to give 350.1g of a brown foamy solid (HPLC (comprising compound of formula (II) and compound of formula (I): 85.7%, where compound of formula (II): compound of formula (I): 24.8%: 62.9%: 2: 5).

The first purification method comprises the following steps: 15.0g of the brown foamed solid was taken, thoroughly slurried with 150ml of dichloromethane for 2 hours, filtered, the filter cake was washed with dichloromethane and dried to give 7.9g of an off-white solid (HPLC (comprising the compound of formula (II) and the compound of formula (I): 99.2%, wherein: the compound of formula (II): the compound of formula (I): 24.7%: 74.5%: 1: 3).

And a second purification method: the filtrate from purification method one was concentrated to dryness and combined with 335.1g of the solid remaining as a brown foamy solid above, 1500ml of dichloromethane were added and the solution was stirred first (which became cloudy after continued stirring). 1200ml of n-heptane were added dropwise, the solid was washed out and stirring was continued at room temperature for 16 hours. The filter cake was filtered and dried to give 240.0g (HPLC (comprising compound of formula (II) and compound of formula (I)) of an off-white solid 97.0% where 27.6% compound of formula (II): 69.4% compound of formula (I) ≈ 2: 5).

Example 10 (conversion reaction)

Sodium hydroxide 32.2g (805mmol) was dissolved in 1920ml ethanol and cooled to 5 ℃. 240.0g of the off-white solid obtained in example 9 was added thereto, and the mixture was cooled to-20 ℃ and stirred to react for 19 hours. 4mol/L hydrochloric acid is dripped at the temperature of minus 20 ℃ until the pH value of the reaction solution is about 5 to 6, most ethanol is concentrated under reduced pressure at the temperature of below 40 ℃, 1000ml of water and 1500ml of ethyl acetate are added into the residue, and the mixture is stirred and separated. The organic phase is washed once with 1000ml of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure to dryness to give 270.0g of a brown foamy solid (HPLC (comprising compound of formula (II) and compound of formula (I): 95.9%, wherein: compound of formula (II): compound of formula (I): 13.4%: 82.5%: 0.8: 5%).

Example 11 (purification)

270.0g of the brown foamy solid prepared in example 10 was dissolved in 960ml of ethyl acetate, and then 960ml of n-heptane was added dropwise thereto, followed by stirring at room temperature for 48 hours. Filtration, washing of the filter cake with a mixed solvent (150ml of petroleum ether +150ml of ethyl acetate), drying of the filter cake to give 24.8g of an off-white solid (HPLC (comprising the compound of formula (I): 65.7%: 34.3%: 9.6: 5), concentration of the filtrate under reduced pressure to dryness, distillation with 100ml of ethanol, and finally concentration to dryness to give 245g of a brown foamed solid (HPLC (comprising the compound of formula (II): 100.0%), wherein the compound of formula (II): 7.4%: 88.8%: 0.42: 5).

245g of the above-mentioned brown foam solid are dissolved in 400ml of ethanol and 1720ml of dichloromethane and 2150ml of n-heptane are added, after 5 minutes the mixture becomes cloudy. Stirring was continued at room temperature for 17 hours. The mixture was filtered, and the filter cake was washed with 100ml of dichloromethane and 300ml of n-heptane, then with 700ml of n-heptane, and dried under vacuum at 80 ℃ for 6 hours to give 143.2g of a white powdery solid. (HPLC (comprising compound of formula (II) and compound of formula (I): 99.7%, wherein: compound of formula (II): compound of formula (I): 2.7%: 97.0%), identified by HNMR and LC-MS, was compound of formula (I) with retention time of 8.53min and compound of formula (II) with retention time of 7.57 min).

¹HNMR(DMSO-d₆，400MHz)δ：12.04(s，1H)，4.57-4.64(m，1H)，4.286-4.297(d，1H)，4.050-4.063(d，1H)，3.49(s，1H)，3.10-3.16(m，1H)，0.81-1.93(m，34H)，0.62(s，3H)。

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A process for the preparation of a substantially pure compound of formula (i) comprising the steps of:

1) dissolving a crude compound of a formula (I) in a positive solvent with the mass-volume ratio of 1-10 times;

3) after the addition, continuously stirring for crystallization;

4) filtering and draining;

5) concentrating the filtrate under reduced pressure to dryness to obtain substantially pure compound of formula (I);

the crude compound of formula (I) is: compounds comprising formula (I) and formula (II);

the normal solvent is selected from ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or a mixture thereof;

the volume of the positive solvent is 3-5 times of the mass-volume ratio of the crude product of the compound shown in the formula (I);

the antisolvent is selected from n-heptane, n-hexane, petroleum ether, isopropyl ether or mixture thereof;

the volume of the anti-solvent is 1-3 times of the volume ratio of the positive solvent;

step 5) concentrating under reduced pressure to dryness, then carrying out distillation by using ethanol, isopropanol or a mixture thereof, and then concentrating to dryness;

the substantially pure compound of formula (i) obtained in step 5) has an optical purity of 90.0% or more, said optical purity being relative to the non-corresponding isomer of formula (ii).

2. A process for the preparation of a substantially pure compound of formula (i) comprising the steps of:

3) continuously stirring for crystallization;

4) filtering and pumping, drying the filter cake to obtain the substantially pure compound of the formula (I);

the amount of the first solvent in the step 1) is 2-7 times of the mass-volume ratio of the crude product of the compound shown in the formula (I);

the amount of the second solvent in the step 2) is 0.5 to 3 times of the volume ratio of the first solvent;

the first solvent is selected from ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or mixtures thereof;

the second solvent is selected from n-heptane, n-hexane, petroleum ether, isopropyl ether or their mixture;

the substantially pure compound of formula (i) obtained in step 4) has an optical purity of 90.0% or more, said optical purity being relative to the non-corresponding isomer of formula (ii).

3. The process according to claim 1, wherein the substantially pure compound of formula (I) obtained in step 5) has an optical purity of 95.0% or more.

4. A process for the preparation of a substantially pure compound of formula (i) according to any one of claims 1 to 3, wherein the crude compound of formula (i) is optionally pre-treated by:

5) adding water and a lower ester solvent into the residue, and separating;

6) concentrating the organic phase under reduced pressure to dryness to obtain a crude product of the compound shown in the formula (I);

the amount of the lower alcohol solvent used in the step 1) is 20-60 times of the mass-volume ratio of the sodium hydroxide or the potassium hydroxide;

the lower alcohol solvent used in the step 1) is ethanol, isopropanol or a mixture thereof;

the ester solvent used in the step 5) is ethyl acetate, isopropyl acetate or a mixture thereof.

5. A method for preparing 5- ((2R) -2- ((3R,5S,6R,7R,10S,13R) -6-ethyl-3, 7-dihydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) propyl) thiazolidine-2, 4-dione, comprising the steps of:

，

wherein:

r is selected from methyl or ethyl;

pg is selected from the following structures:

，

the esterification reaction of step 1) is at 22^oC～27^oC is carried out in an acid environment;

the acid is selected from hydrochloric acid or sulfuric acid;

the isomerization reaction of the step 3) adopts sodium alkoxide to react in an alcohol solvent, and the reaction temperature is 15 DEG^oC～25^oC; the sodium alkoxide is selected from sodium methoxide, sodium ethoxide or sodium tert-butoxide, and the alcoholic solvent is selected from methanol, ethanol, isopropanol or tert-butanol;

in the step 5), carbonyl reduction is carried out on the carbonyl of the compound shown in the formula (VIII) in an alcohol solvent by sodium borohydride, so as to obtain the compound shown in the formula (III), and the reaction temperature is 0^oC～3^oC; the alcohol solvent is selected from methanol, ethanol, isopropanol or tert-butanol, and after the reaction is finished, acetone and citric acid are adopted for quenching reaction;

further comprising the steps of:

，

selectively carrying out acidolysis on the compound of the formula (III) under the action of lithium hydroxide to generate a compound of a formula (IX);

pulping, refining and purifying a crude product formula (XI) obtained by the reaction by adopting dichloromethane with the mass-volume ratio of 1: 15; the HPLC purity of the compound of the formula (XI) obtained by pulping and purifying the dichloromethane can reach more than 98 percent.