WO2018054300A1 - Cholic acid derivative free alkali, crystalline form, preparation methods therefor and applications thereof - Google Patents

Abstract

Provided is a crystalline form cholic acid derivative free alkali, a preparation method therefor and an application. The chemical name of the cholic acid derivative free alkali 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, and the power X-ray diffraction pattern thereof comprises peaks at diffraction angles (2θ) of 16.5±0.2°, 13.6±0.2°, 12.1±0.2°, and 20.4±0.2°. Also provided are an optically pure cholic acid derivative, a preparation method therefor and an application thereof. The purity of the obtained optical isomer reaches 90.0% or more, and the problem in the prior art of being difficult to obtain the optical purity by means of separation is resolved. In addition, also provided a method for preparing a cholic acid derivative and an application thereof. The preparation method overcomes the defect existing in the prior art; a multi-step reaction can be implemented at room temperature; the obtained product has good purity, high yield, and strong process operability; the process safety is greatly improved; the preparation 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, in particular to a crystalline cholic acid derivative free base, a preparation method and application thereof, and a substantially pure bile acid derivative, a preparation method and application thereof, and a cholic acid Process for the preparation of derivatives and their use.

Background technique

The farnesyl ester derivative X receptor (FXR) is a member of the hormone nuclear receptor superfamily, which is mainly expressed 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 named after it. When the FXR ligand binds directly to the FXR carboxy terminal ligand binding region (LBD), the nuclear receptor spatial conformation changes and forms a heterodimer with the retinoid receptor (RXR), and finally with the target gene specific The binding of FXR DNA response elements to regulate the transcription of target genes and participate in the regulation of sugar and lipid metabolism is an important energy regulator. Primary bile acid chenodeoxycholic acid is the most potent ligand for FXR, and secondary bile acid bile acid and deoxycholic acid can also activate FXR. There are also synthetic FXR ligands (such as 6-ECDCA, GW4064, etc.) which bind to FXR several times more strongly than natural ligands. The main target genes of FXR include bile salt export pump (BSEP), bile acid binding protein (IBABP) and small heterodimeric chaperone receptor (SHP), etc., FXR and FXR response elements on these gene promoters (FXRE) Binding to regulate the expression of these genes. However, there is no typical FXR binding reaction sequence in the promoter sequence of major FXR regulatory genes such as cholesterol 7α hydroxylase (CYP7α1), and FXR is indirectly through the induction of transcriptional repressor SHP expression, and then SHP is homologous to the CYP7α1 promoter liver receptor. (LRH-1) forms an inhibitory complex that blocks CYP7α1 and other LRH-1 target gene transcription. Due to the important role of FXR in bile acid and cholesterol metabolism, this will increasingly attract attention to liver-related diseases. The search for new FXR agonists has become a research hotspot for the treatment of liver diseases including cholestasis syndrome. 6-acetylene chenodeoxycholic acid (obeyolic acid) has been completed as a treatment for primary biliary cirrhosis (PBC). The trial, which will be available as early as 2015, and the compound also shows satisfactory therapeutic effects in patients with nonalcoholic steatohepatitis. FXR agonists can both reduce bile acid-dependent bile flow by stimulating the bile salt output pump (BSEP) and also stimulate MRP2 to increase non-biliary acid-dependent bile flow to reduce cholestasis. FXR is expected to be a new drug target for screening and treating other metabolic diseases including cholestatic diseases and nonalcoholic steatohepatitis.

In 2016, Jiangsu Haosen Company disclosed a class of cholic acid derivatives in the patent PCT/CN2016/079167 (application date: 2016.04.13), in which the chemical name of the representative compound is: (5R)-5-((2R) -2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopentadienyl[a Phenanthrene-17-yl)propyl)thiazolidine-2,4-dione (compound of formula (I)), the structure is as follows:

This compound has a significant agonistic effect on FXR activity and is expected to be developed as a novel FXR agonist, however, the compound of formula (I) disclosed in Example 14 of the patent PCT/CN2016/079167 is extracted with ethyl acetate and After purification by column chromatography, only a mixture containing another diastereomer can be obtained, which is difficult to prepare into a pharmaceutical preparation suitable for clinical application, and the final physicochemical properties of the product are further confirmed by experiments. Therefore, there is an urgent need to develop a stable, well-dissolved crystal to meet the needs of clinical development of drugs. There is also an urgent need in the art to develop a compound that is optically pure and stable in its ability to meet the needs of clinical development of drugs.

In 2002, the patent WO2002072598A1 first disclosed the oleic acid and its preparation method. The synthetic route is as follows:

The patent uses 3α-hydroxy-7-keto-5β-cholane-24-acid and 3,4-dihydropyran as raw materials to first protect 3α-hydroxyl; then react with ethyl bromide to replace at position 4 Ethyl, which is esterified at the same time, but the reaction needs to be carried out at -70 to -80 °C, and the dangerous n-butyllithium and the carcinogen hexamethylphosphoramide (HMPA) are used as catalysts. This route is not suitable for industrial applications.

In 2006, the patent WO2006122977A2 discloses a synthetic route for preparing oleic acid as follows.

The patent also uses 3α-hydroxy-7-keto-5β-cholane-24-acid and 3,4-dihydropyran as raw materials, first esters, and then protects 3α-hydroxyl by TMS, and then protects 6 positions. The carbonyl group is then reacted with the aldehyde group compound, but the reaction is carried out at -60 to -90 ° C. The solvent used in the reaction is a boron trifluoride diethyl ether solution; after hydrolysis with sodium hydroxide, it is reduced with palladium carbon, and then The configuration was switched at 95-105 ° C, and finally reduced with borohydride at 70-105 ° C to obtain oleic acid. The preparation method requires expensive trimethylchlorosilane, palladium carbon, unstable aldehyde compound, unsafe boron trifluoride diethyl ether solution, and harsh reaction temperature, for example, configuration conversion and reduction at high temperature. The reaction, therefore, the synthetic route disclosed in this patent is also not suitable for industrial applications.

In 2016, Jiangsu Haosen Company disclosed a class of cholic acid derivatives in the patent PCT/CN2016/079167 (application date: 2016.04.13), wherein the chemical name of the representative compound is: 5-((2R)-2-( (3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17 -yl)propyl)thiazolidine-2,4-dione, prepared as follows:

The patent uses oleic acid as raw material to prepare 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13- When dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione First esterification, then bromination, ring formation to obtain the final product, and no optimization method for the preparation of oleic acid, the same problem as WO2006122977A2, is not suitable for industrial production, so the development of a suitable pharmaceutical preparation process is also in the field The problem that technicians urgently need to solve.

Summary of the invention

One of the objects of the present invention is to provide a better pharmaceutical crystal. The inventors have intensively studied the different aggregation states of the compound of the formula I, and finally obtained a crystalline form (5R)-5-((2R)-2- ((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadehydro-1H-cyclopenta[a]phenanthrene- 17-yl)propyl)thiazolidine-2,4-dione of formula (I) greatly improves the physicochemical properties of the compound of formula (I) and is useful in the treatment of FXR-mediated diseases, including cardiovascular diseases, arteries Atherosclerosis, arteriosclerosis, hypercholesterolemia, hyperlipidemia, chronic hepatitis disease, chronic liver disease, gastrointestinal disease, kidney disease, cardiovascular disease, metabolic disease, cancer (such as colorectal cancer) or nerve signs such as stroke, etc. Wide range of medical applications, is expected to develop into a new generation of FXR agonists.

Crystalline type (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl) Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base, the crystalline free base designated as Form I, a powder thereof The X-ray diffraction pattern includes peaks at diffraction angles (2 theta) at 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, 20.4 ± 0.2 °; preferably also included at 11.5 ± 0.2 °, 9.6 ± 0.2 °, 19.6 ±0.2°, 15.0±0.2°, a peak at a diffraction angle (2θ) of 20.0±0.2°; more preferably further included at 23.7±0.2°, 20.7±0.2°, 23.0±0.2°, 25.7±0.2°, 17.9± Peaks at 0.2° and 16.0 ± 0.2° diffraction angle (2θ).

As a most preferred embodiment, the crystalline form (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy- 10,13-Dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base, X-ray powder diffraction pattern diffraction peak The angle 2θ (°) and peak intensity are as follows:

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

Another aspect of the present invention provides a crystalline form of (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy- Process for the preparation of 10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base, characterized by The following steps:

1) (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base is dissolved or dispersed in an organic solvent, water, or a mixture of an organic solvent and water. In solvent

2) precipitation precipitation, or optionally adding anti-solvent to the compound clarification solution, or optionally slowly evaporating the clear solution of the compound, or optionally adding the original compound solid or other solid particle additive to the compound solution as a heteronuclear The crystals are induced to crystallize, or the crystals described above are obtained in combination using the above method.

Preferably, the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, acetone, ethyl acetate, isopropyl acetate, toluene, n-butanol, cyclohexane, dichloromethane, and dimethyl. Formamide, dimethylacetamide, dimethyl sulfoxide, dioxane, diethyl ether, n-heptane, n-hexane, methyl ethyl ketone, isooctane, pentane, dipropanol, tetrahydrofuran, dimethyltetrahydrofuran, three Ethyl chloride, xylene or a mixture thereof.

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

As a further preferred embodiment, the powder X-ray diffraction pattern of the crystal obtained in the step 2) of the preparation method includes a diffraction angle (2θ) at 16.5±0.2°, 13.6±0.2°, 12.1±0.2°, and 20.4±0.2°. The peak at the place.

According to still another aspect of the present invention, there is provided a pharmaceutical composition comprising an effective amount of crystalline (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)) -6-ethyl-3,7-dihydroxy-10,13-dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4- Diketone free base, a pharmaceutically acceptable carrier or excipient thereof.

In a further aspect of the invention there is provided the crystalline form (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy) -10,13-Dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base, or the aforementioned pharmaceutical composition is prepared Use in medicines for preventing or treating FXR-mediated diseases or conditions.

Preferably, the FXR mediated disease or condition is selected from the group consisting of cardiovascular disease, hypercholesterolemia, hyperlipidemia chronic hepatitis disease, chronic liver disease, gastrointestinal disease, kidney disease, cerebrovascular disease, metabolic disease or cancer.

As a further preferred embodiment, the chronic liver disease is selected from the group consisting of primary sclerosis (PBC), cerebral xanthoma (CTX), primary sclerosing cholecystitis (PSC), drug-induced cholestasis, and intrahepatic gestation Cholestasis, parenteral absorption-related cholestasis (PNAC), bacterial overgrowth or sepsis cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fat Hepatitis (NASH), liver graft-related graft-versus-host disease, live donor liver transplant regeneration, congenital liver fibrosis, common bile duct stones, granulomatous liver disease, intrahepatic or extraneous malignancy, Sjogren syndrome, sarcoidosis , Wilson's disease, Gaucher's disease, hemochromatosis or alpha ₁ anti-membrane protease deficiency.

As a further preferred embodiment, the gastrointestinal disease is selected from the group consisting of inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), bacterial overgrowth, nutritional malabsorption, post-reflex colitis or microcolitis. The inflammatory bowel disease is preferably Crohn's Disease or Crohn's disease.

As a further preferred embodiment, the kidney disease is selected from the group consisting of diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulitis, chronic allograft 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 or hypertriglyceridemia.

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

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

Another object 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; and can be used for the treatment of FXR-mediated diseases, including cardiovascular Disease, atherosclerosis, arteriosclerosis, hypercholesterolemia, hyperlipidemia, chronic hepatitis disease, chronic liver disease, gastrointestinal disease, kidney disease, cardiovascular disease, metabolic disease, cancer (such as colorectal cancer) or nerve signs such as stroke The disease, with its wide range of medical applications, is expected to be developed into a new generation of FXR agonists.

A first aspect of the invention provides a substantially pure compound of formula (I) which means that the compound of formula (I) has an optical purity of greater than 90.0%.

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) contains no more than 10.0% impurities;

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

As a still further preferred embodiment, the optical purity refers to the compound of the formula (II) relative to its non-isomer:

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

1) The crude compound of the formula (I) is dissolved in a positive solvent having a mass to volume ratio of 1-10 times;

2) adding an anti-solvent having a positive solvent volume ratio of 0.5-10 times;

3) After adding, continue to stir and crystallize;

4) Filter and drain;

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

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

Preferably, the positive solvent is selected from the group consisting of preferably ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or a mixture thereof.

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

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

Preferably, the anti-solvent is selected from the group consisting of n-heptane, n-hexane, petroleum ether, isopropyl ether or a mixture thereof.

As a still further preferred embodiment, the anti-solvent is used in a volume of from 1 to 3 times the volume ratio of the positive solvent.

As a still further preferred embodiment, step 5) is concentrated to dryness under reduced pressure, then distilled with a lower alcohol solvent or a mixture thereof, and then concentrated to dryness.

Preferably, step 5) is concentrated under reduced pressure to dryness and then entrained with ethanol, isopropanol or a mixture thereof for distillation.

As a still further preferred embodiment, the substantially pure formula obtained in the step 5) (the optical purity of the obtained compound is 90.0% or more; preferably, the substantially pure formula obtained is obtained; the optical purity of the compound is 95.0% or more.

The third aspect of the present invention provides a substantially pure formula (the preparation method of the present compound, comprising the following steps:

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

2) adding a second solvent having a first solvent volume ratio of 0.5-10 times;

3) continue to stir and crystallize;

4) Filtration and drying, the filter cake is dried to give a substantially pure compound of formula (I).

As a further preferred embodiment, the amount of the first solvent in the step 1) is 2 to 7 times the mass to volume ratio of the crude compound in the formula (1).

As a further preferred embodiment, the amount of the second solvent in the step 2) is the first solvent volume ratio of 0.5-3. Times.

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

Preferably, the first solvent is selected from the group consisting of ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or a mixture thereof.

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

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

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

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

As a further preferred embodiment, the above preparation method formula (for the crude compound can be pretreated by the following steps:

1) dissolving sodium hydroxide or potassium hydroxide in a mass-to-volume ratio of 5-100 times lower alcohol solvent, and then cooling to below 5 ° C;

2) adding the raw material containing the compound of the formula (I) to the alkali solution obtained in the step 1), and cooling the mixture to below -10 ° C to continue the stirring reaction;

3) slowly add acidic substances below -10 ° C to the pH of the reaction solution about 5-6;

4) concentrating part of the solvent under reduced pressure under temperature control below 40 ° C;

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

6) The organic phase is concentrated under reduced pressure to dryness to give a crude compound of formula (I).

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

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

A fourth aspect of 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 present invention provides the use of the aforementioned substantially pure formula (the compound of the invention, or the aforementioned pharmaceutical composition, for the preparation of a medicament for preventing or treating an FXR-mediated disease or condition.

As a further preferred embodiment, the FXR-mediated disease or condition is selected from the group consisting of cardiovascular disease, hypercholesterolemia, hyperlipidemia, chronic hepatitis disease, chronic liver disease, gastrointestinal disease, kidney disease, cerebrovascular disease, metabolic disease or cancer. .

As a still further preferred aspect, the chronic liver disease is selected from the group consisting of primary sclerosis (PBC), cerebral xanthoma (CTX), primary sclerosing cholecystitis (PSC), drug-induced cholestasis, and gestational liver Internal cholestasis, extraintestinal absorption-related cholestasis (PNAC), bacterial overgrowth or sepsis cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic Fatty hepatitis (NASH), liver graft-related graft-versus-host disease, live donor liver transplant regeneration, congenital liver fibrosis, common bile duct stones, granulomatous liver disease, intrahepatic or extraneous malignancy, Sjogren syndrome, nodules Disease, Wilson's disease, Gaucher's disease, hemochromatosis or alpha 1 anti-membrane protease deficiency.

As a still further preferred embodiment, the gastrointestinal disease is selected from the group consisting of inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), bacterial overgrowth, malabsorption of malnutrition, post-reflex colitis or microcolitis. Inflammatory bowel disease is preferably Crohn's Disease or ulcerative bowel disease.

As a still further preferred embodiment, the kidney disease is selected from the group consisting of diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulitis, chronic allograft 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 or hypertriglyceridemia.

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

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

As a still further preferred aspect, the cancer is selected from the group consisting of colorectal cancer or liver cancer.

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

1. The present invention solves the technical problem that 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 and can reach up to 95%, and the optical purity raw material is beneficial to further pharmacological and toxicological research, and is more in line with the needs of clinical research.

3. The separation and purification process adopted by the invention is simple in operation, green and environmentally friendly, and the solvent used is simple and easy to obtain, the yield is stable, the quality is reliable, and it is beneficial to industrial applications.

Another object of the present invention is to provide a process for preparing a cholic acid derivative which has mild reaction conditions, mature process and stable quality, and is very suitable for industrial applications.

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

1) esterification of a compound of formula (IV) to give a compound of formula (V);

2) a compound of formula (V) is reduced by a double bond to give a compound of formula (VI);

3) isomerization of a compound of formula (VI) to give a compound of formula (VII);

4) protecting the compound of formula (VII) with a hydroxy group to give a compound of formula (VIII);

5) Reduction of the carbonyl group of the compound of formula (VIII) to give a compound of formula (III);

The reaction formula is as follows:

Wherein R is selected from C _1-4 alkyl; Pg is a hydroxy protecting agent.

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

As a further preferred embodiment, Pg in the preparation method is preferably a substituted or unsubstituted benzoyl group, a substituted or unsubstituted benzenesulfonyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted benzyl group or a trialkyl silicon group. .

As a still further preferred embodiment, the Pg in the preparation method is preferably from the following structure:

As a most preferred embodiment, the Pg in the preparation method is selected from the following structures:

The above compound of the formula (III) can be used as a preparation of oleic acid and 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy- A key intermediate for 10,13-dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione.

As a further preferred embodiment, the esterification reaction of the step 1) in the production method is carried out in an acidic environment at a temperature of from 20 ° C to 35 ° C.

As a still further preferred embodiment, the esterification reaction of the step 1) in the production method is preferably carried out at a temperature of from 22 ° C to 27 ° C.

As a still further preferred embodiment, the esterification reaction of the step 1) in the preparation method is carried out under acidic conditions, and the acid may be selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, and hydroiodic acid. , methanesulfonic acid or a mixture thereof. Preferably, it is selected from hydrochloric acid or sulfuric acid.

As a further preferred embodiment, the isomerization reaction of the step 3) in the production method is carried out by using sodium alkoxide in an alcohol solvent at a reaction temperature of 5 ° C to 35 ° C. Preferably, the reaction temperature is from 15 ° C to 25 ° C.

As a still further preferred embodiment, the sodium alkoxide used in the isomerization reaction of the step 3) in the preparation method is selected from the group consisting of sodium methoxide, sodium ethoxide or sodium t-butoxide.

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

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

As a still further preferred embodiment, the alcohol solvent used in the step 5) reduction of the carbonyl group in the preparation method is selected from the group consisting of methanol, ethanol, isopropanol or tert-butanol.

As a still further preferred embodiment, in the preparation method, after the completion of the carbonyl reduction reaction in the step 5), the reaction is quenched with acetone and citric acid.

Another aspect of the present invention provides a preparation method of the above preparation method for preparing oleic acid. In the preparation of oleic acid, a compound of the formula (III) is prepared by the above preparation method, and then the reaction is carried out as follows. :

Wherein the acid is an organic acid or an inorganic acid.

As a further preferred embodiment, the organic acid is preferably selected from the group consisting of trifluoroacetic acid, trichloroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid or a mixture thereof; Hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or a mixture thereof.

Another aspect of the present invention provides a preparation method of the above preparation method for preparing oleic acid. In the preparation of oleic acid, a compound of the formula (III) is prepared by the above preparation method, and then the reaction is carried out as follows. :

As a further preferred embodiment, the compound of the formula (III) is preferably selectively acid-reacted under the action of lithium hydroxide to form a compound of the formula (IX).

Another aspect of the present invention provides a process for 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-dione, in the preparation of 5-(( 2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopentadiene [a]phenanthroline-yl)propyl)thiazolidine-2,4-dione is prepared by the above preparation method to obtain a compound of the formula (III), and then the reaction is carried out as follows:

As a further preferred embodiment, the compound of the formula (III) is preferably selectively acid-reacted under the action of lithium hydroxide to form a compound of the formula (IX).

As a still further preferred embodiment, 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10, obtained by the ring formation reaction, 13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthroline-17-yl)propyl)thiazolidine-2,4-dione crude product using methylene chloride with a mass ratio of 1:15 Beating and purifying.

As a still further preferred embodiment, the compound of the formula (XI) 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3) obtained by purifying with dichloromethane is purified. , 7-dihydroxy-10,13-dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione can be obtained by HPLC purity 98% or more.

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

1. The present invention is (4R)-4-((3R,5R,10R,13R,14R,17R,Z)-6-ethylidene-3-hydroxy-10,13-dimethyl-7-carbonyl-10- Hexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoic acid is used as a raw material, and the esterification reaction is carried out in the first step until the preparation of oleic acid or 5-((2R)-2 -((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadehydro-1H-cyclopenta[a]phenanthrene The -17-yl)propyl)thiazolidine-2,4-dione has not undergone a hydrolysis reaction before, and the obtained ester-forming intermediates are easily soluble in a hydrophobic organic solvent, which is advantageous for post-treatment of each reaction. Part of the by-products can be removed by washing with water. Therefore, the process of the present invention is highly operable.

2. The patent WO2002072598A1 is solved by using 3α-hydroxy-7-keto-5β-cholane-24-acid and 3,4-dihydropyran as raw materials, and is protected by 3α-hydroxyl and reacted with ethyl bromide at 4 positions. The simultaneous esterification reaction of the substituted ethyl group needs to be carried out at -70 to -80 ° C, and the defect of n-butyl lithium and the carcinogen hexamethylphosphoramide is required, and the esterification reaction temperature of the present invention can be carried out at around normal temperature. The purity and yield of the obtained product are very high. Therefore, the esterification reaction of the present invention is suitable for industrial applications.

3. Solved the patent WO2006122977A2 with 3α-hydroxy-7-keto-5β-cholane-24-acid and 3,4-dihydropyran as raw materials. After ester formation, it is necessary to continuously use trimethylchlorosilane to protect 3α- Hydroxyl, a defect at the 6-carbonyl group.

4. Overcoming the defect of the reaction of the patent WO2006122977A2 after the TMS protection and the aldehyde-based compound still need to be reacted at -60 to -90 ° C, and the boron trifluoride diethyl ether solution is also avoided.

5. Overcoming the defect that the configuration conversion needs to be carried out at 95-105 ° C in the configuration conversion of WO2006122977A2, and the configuration conversion reaction can be carried out by using sodium alkoxide in an alcohol solvent at a normal temperature.

6. Overcoming the defect that the patent WO2006122977A2 needs to be carried out under the high temperature condition of 70-105 ° C when the urethane acid is reduced by the sodium borohydride. The invention can be carried out at a low temperature to make the reaction less. Further, when the reaction was terminated with acetone at the completion of the reaction, citric acid was additionally added to further reduce the generation of by-products. Therefore, the product obtained by the reaction in this step has good purity, high yield, and improved process safety.

7. Overcome the preparation of 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10, in the patent PCT/CN2016/079167, 13-Dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione needs to be esterified first, then By bromination and ring formation, the final product is deficient, and the invention directly adopts the compound of the formula (III), which does not need to be hydrolyzed into the oleic acid and re-esterification process step, which greatly saves energy consumption and reduces environmental stress.

The present invention also provides a 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl group) Purification method of hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione, the purification method of the invention is simple in operation, and the obtained product has high purity and good quality. It is conducive to the development of follow-up drugs.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an X-ray powder diffraction pattern of a crystalline form of a free base of the compound of Formula I; the abscissa is the diffraction peak angle 2? (?), and the ordinate is the intensity of the peak.

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

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

4 is a dynamic water adsorption DVS (Dynamic Vapour Sorption) isotherm diagram of a crystalline form of the compound free base, the abscissa is the relative humidity RH (%), and the ordinate is the mass change percentage (%).

detailed description

1. Terminology

As used herein, the term "pharmaceutically acceptable" means that it is suitable for contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problematic complications, and reasonable benefits/risks. Compounds, materials, compositions and/or dosage forms that are more commensurate.

The term "substantially pure" as used herein means that in certain preferred embodiments of the invention the crystalline structure of the compound of formula I is in substantially pure form, and the HPLC purity or crystal form purity is substantially above 90% (inclusive), preferably above 95%. More preferably, it is 98% or more, and most preferably 99.5% or more.

As used herein, "polymorph" or "polymorph" refers to a crystal form having the same chemical composition but constituting different spatial arrangements of molecules, atoms and/or ions of 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 similar properties. Depending on their temperature-stability relationship, the two polymorphs may be mono- or trans-denatured. For a single denatured system, the relative stability between the two solid phases remains constant as the temperature changes. In contrast, in a tautomeric system, there is a transition temperature in which the stability of the two phases is reversed (Theory and Origin of Polymorphism in "Polymorphism in Pharmaceutical Solids" (1999) ISBN:) -8247-0237). The phenomenon in which such compounds exist in different crystal structures is called a drug polymorphism phenomenon.

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

Additionally, as is known to those skilled in the art, seed crystals are added to any of the crystallization mixtures to promote 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 seed crystals required depends on the size of the available seed crystals, as described in "Programmed cooling of batch crystallizers," JW Mullin and J. Nyvlt, Chemical Engineering Science, 1971, 26, 369-377. The desired size of the average product particles. In general, small size seed crystals are required to effectively control the growth of crystals in the batch. Small size seed crystals can be produced by sieving, grinding or micronizing of larger crystals or by microcrystallization of solution. It should be noted that grinding or micronization of crystals does not cause any change in the crystallinity of the desired crystal structure (ie, becomes no Shape or become another polymorph).

The crystal structures of the crystal structures disclosed or claimed herein may exhibit similar but not identical analytical characteristics within reasonable margins based on test conditions, purity, equipment, and other constant variables known to those skilled in the art. Accordingly, it is apparent to those skilled in the art that various modifications and changes 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 in view of this disclosure. Applicants desire that the specification and examples be considered as illustrative and not limiting.

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

The term pharmaceutical composition as used herein denotes a mixture containing one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical components, or other components such as physiology/pharmaceuticals. Acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

2, experimental materials

The reagents used in the examples of the present invention are commercially available industrial grade or analytical grade reagents, and the starting materials of the selected compound of formula (I) are prepared according to Example 14 of Hausen Patent PCT/CN2016/079167.

3. Analysis method

3.1, X-ray powder diffraction

One of ordinary skill in the art will recognize that the X-ray powder diffraction pattern can be obtained under measurement error that depends on the measurement conditions used. In particular, it is generally known that the intensity in the 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 strength should not be taken into account. In addition, the measurement error of the conventional X-ray powder diffraction angle is usually about 5% or less, and such measurement error degree should be regarded as belonging to the above diffraction angle. Accordingly, it is to be understood that the crystal structure of the present invention is not limited to a crystal structure that provides an X-ray diffraction pattern identical to the X-ray powder diffraction pattern depicted in the drawings disclosed herein. Any crystal structure that provides substantially the same X-ray powder diffraction pattern as those disclosed in the drawings is within the scope of the invention. The ability to determine that the X-ray powder diffraction pattern is substantially the same is within the abilities 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.

The polymorphic forms of the compounds of formula I are characterized by their X-ray powder diffraction pattern. Therefore, in Cu Kα radiation

The X-ray powder diffraction pattern was acquired on a Rigaku Ultima IV X-ray powder diffractometer operating in a reflective manner. The tube voltage and current quantities were set to 40kV and 40mA acquisition scans, respectively. The sample was scanned for a period of 5 minutes in the range of 2θ from 5.0° to 45°. All analyses were performed at room temperature, typically between 20 ° C and 30 ° C. The XRPD sample is prepared by placing the sample on a single crystal silicon wafer, and gently pressing the sample powder with a glass slide or equivalent to ensure that the sample surface is flat and has an appropriate height. The sample holder was then placed in a Rigaku Ultima IV instrument and an X-ray powder diffraction pattern was acquired using the instrument parameters described above. Measurement differences associated with such X-ray powder diffraction analysis results are produced by a variety of factors including: (a) errors in sample preparation (eg, sample height), (b) instrument error, (c) calibration differences, ( d) operator error (including those that occur when determining peak position), and (e) properties of the substance (eg, preferred orientation error). Calibration errors and sample height errors often result in displacement of all peaks in the same direction. In general, this calibration factor will align the measured peak position to the expected peak position and may be in the range of the expected 2θ value ± 0.2°.

3.2, thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) experiments were performed in a TA Instruments ^TM model Q500. A sample (approximately 2-10 mg) was placed in a pre-weighed platinum pan. The sample weight was accurately measured by the instrument and recorded to a thousand of a milligram. The furnace was purged with nitrogen at 100 ml/min. Data were collected at room temperature to 300 ° C at a heating rate of 10 ° C / min.

3.3, Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) experiments were performed in a TA Instruments ^TM model Q200. Samples (approximately 2-10 mg) were weighed in an aluminum pan and accurately recorded to one hundredth of a milligram and transferred to DSC. The instrument was purged with nitrogen at 50 ml/min. Data was collected at a heating rate of 10 ° C / min between room temperature and 300 ° C. Draw with the endothermic peak facing down. However, those skilled in the art will appreciate that in DSC measurements, the measured onset temperature and maximum temperature have some degree of variability based on heating rate, crystal shape and purity, and other measured parameters.

Specific embodiments and methods of preparation provided below will further illustrate specific aspects of embodiments of the invention. The scope of the following examples is not intended to limit the scope of the invention in any way.

Example 1

Weigh about 15mg of the compound of formula I free base solid (amorphous) in a 1.5mL vial, add 0.5mL of dichloromethane, stir at room temperature for 24 hours, solid-liquid separation, to obtain the free base crystal form of the compound of formula I I. The X-ray powder diffraction pattern is shown in Figure 1; the thermogravimetric analysis chart is shown in Figure 2 (loss of weight 0.29%), the differential scanning calorimetry diagram is shown in Figure 3 (melting point 132.9 ° C); dynamic water adsorption DVS isotherm As shown in Figure 4, the DVS test conditions are as follows: without N _{2 in the} presence of temperature, 25.0 ° C, relative humidity (RH): from 0% to 95% to 0%; stability test is as follows:

1, stability in the solvent

10 mg of the compound was accurately weighed and placed in a 100 mL volumetric flask, and acetonitrile/water (V/V, 1:1) was added thereto, and solubilized by ultrasonication to obtain a solution of 0.1 mg/mL. At room temperature, the appropriate amount was taken at 0h, 3h, 6h, 24h, liquid phase detection, and the relative purity with 0h was calculated. The results are as follows:

Placement time (h)	Relative purity
0	/
3	101.1%
6	102.1%
twenty four	100.4%

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

2, stability under high temperature and light

About 4 mg of the compound was weighed, and 6 parts in parallel were placed in a 4 mL glass bottle and placed at 25 ° C / RH 75%, 40 ° C / RH 75%, 50 ° C, 80 ° C and light, and left for 7 days. The light experiment was carried out along with the quality control experiment (that is, the quality control sample was carried out with the powder in the dark). After 7 days of storage, the powder sample was dissolved in a diluent to obtain a sample solution of 1 mg/mL, and the purity was measured in a liquid phase, and the relative purity at 0 days was calculated. The results are as follows:

Placement condition	Placement time	purity(%)	Relative purity (%)
0 days	/	98.9	/
25°C/RH75%	7 days	99.0	100.1
40°C/RH75%	7 days	98.2	99.3
50 ° C	7 days	98.8	99.9

80 ° C

7 days

98.8

99.9

The experimental results show that the crystal of the compound is placed at 25 ° C / RH 75%, 40 ° C / RH 75%, 50 ° C, 80 ° C and light conditions for 7 days, no significant degradation, and can remain stable.

The liquid phase analysis conditions are as follows:

1), instruments and equipment

equipment name	model
Analytical Balances	Sartorius BSA224S-CW
Pure water machine	Milli-Q Plus, Millipore
High performance liquid chromatography	Agilent1260
Pump	Agilent G1311B
Sampler	G1329B
Column thermostat	G1316A
Detector	G1315D

2), chromatographic conditions

Example 2

Weigh about 15 mg of the compound of formula I free base solid (amorphous) in a 1.5 mL vial, add 0.5 mL of n-heptane, and stir at room temperature for 24 hours, then solid-liquid separation to obtain the free base crystal form of the compound of formula I. I, its X-ray powder diffraction pattern is basically the same as FIG.

Example 3

Weigh about 15mg of the compound of formula I free base solid (amorphous) in a 1.5mL vial, add 0.5mL of water, stir at room temperature for 24 hours, solid-liquid separation, to obtain the free base crystal form I of the compound of formula I, Its X-ray powder diffraction pattern is basically the same as FIG.

Example 4

Weigh about 50 mg of the free base solid of the compound of formula I in a 1.5 mL vial, add 0.8 mL of dichloromethane, stir at room temperature for 48 hours, solid-liquid separation, and open at room temperature overnight to obtain the free base crystal of the compound of formula I. Type I, whose X-ray powder diffraction pattern is substantially identical to Figure 1.

Terms and specific embodiments in the preparation method of the cholic acid derivative of the invention:

"C _1-4 alkyl" means a straight-chain alkyl group having 1 to 4 carbon atoms and a branched alkyl group, and the alkyl group means a saturated aliphatic hydrocarbon group such as methyl group, ethyl group, n-propyl group or the like. Propyl, n-butyl, isobutyl, tert-butyl, sec-butyl and the like.

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

The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS). The NMR chemical shift (δ) is given in parts per million (ppm). The NMR was measured by a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ). The deuterated methanol (CD ₃ OD) and deuterated chloroform (CDCl ₃ ) were labeled as the top four. Silane (TMS).

LC-MS was determined by LC-MS using an Agilent 1200 Infinity Series mass spectrometer. The HPLC was measured using an Agilent 1200 DAD high pressure liquid chromatograph (Sunfire C18 150 x 4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 x 4.6 mm column).

The thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate. The specification for TLC is 0.15mm~0.20mm, and the specification for separation and purification of thin layer chromatography is 0.4mm~0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as a carrier.

Starting materials in the examples of the invention are known and commercially available or can be synthesized or synthesized according to methods known in the art.

Unless otherwise stated, all reactions of the present invention were carried out under continuous magnetic stirring under a dry nitrogen or argon atmosphere, and the solvent was a dry solvent.

Example 5

Step 1: methyl (4R)-4-((3R,5R,10R,13R,14R,17R,Z)-6-ethylidene-3-hydroxy-10,13-dimethyl-7-carbonyl-10- Preparation of hexahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate

Add (4R)-4-((3R,5R,10R,13R,14R,17R,Z)-6-ethylidene-3-hydroxy-10,13-dimethyl-7-carbonyl-10- to the reaction flask 50.0 g (0.12 mol) of hexahydro-1H-cyclopenta[a]phenanthroline-17-yl)pentanoic acid and 500 ml of methanol, and 1.5 g (0.015 mol) of concentrated sulfuric acid was added dropwise at 22 ° C to 27 ° C. After stirring, the reaction was continued overnight, and the basic reaction of the starting material was completed by TLC. The temperature was adjusted to 15 ° C to 25 ° C, 10 ml of saturated sodium hydrogencarbonate solution was added dropwise, and concentrated under reduced pressure to remove most of the methanol, and 500 ml of ethyl acetate and 200 ml of saturated sodium hydrogencarbonate solution were added. After stirring for 20 min, the organic layer was washed with 120 ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to afford to afford 50.2 g (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-18 5um 4.6x150mm

■Posttime: 5min

■ Column temperature: 25 ° 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 2: Methyl (4R)-4-((3R,5S,6S,10S,13R,14R,17R)-6-ethyl-3-hydroxy-10,13-dimethyl-7-carbonyl Preparation of hydrogen-1H-cyclopenta[a]phenanthrene-17-yl)valerate

To the reaction flask was added methyl (4R)-4-((3R,5R,10R,13R,14R,17R,Z)-6-ethylidene-3-hydroxy-10,13-dimethyl-7- Carbonyl hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate 49g (0.114 mol), 4.9 g of 10% palladium on carbon and 250 ml of ethanol, replaced with hydrogen three times, under hydrogen atmosphere, in After stirring at 15 to 25 ° C overnight, the reaction of the starting material was complete, filtered, and the filtrate was concentrated to dryness under reduced pressure, and then distilled to afford 46.3 g (yield: 94.1%).

Step 3: Methyl (4R)-4-((3R,5S,6R,10S,13R,14R,17R)-6-ethyl-3-hydroxy-10,13-dimethyl-7-carbonyl Preparation of hydrogen-1H-cyclopenta[a]phenanthrene-17-yl)valerate

Add methyl (4R)-4-((3R,5S,6S,10S,13R,14R,17R)-6-ethyl-3-hydroxy-10,13-dimethyl-7-carbonyl to the reaction flask Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate 40g (0.092mol) and 200ml of methanol, cooled to 0 ~ 5 ° C; take another reaction flask to add 200ml methanol, temperature control 25 ° C or less, sodium methoxide 25.0g (0.462mol) was added in portions, the solution was stirred until clarification, sodium methoxide solution was added dropwise to the first reaction bottle at 0 ~ 5 ° C, and the temperature was naturally increased to 15 ~ 25 ° C After stirring overnight, the reaction of the starting material was completely completed by TLC (TLC: petroleum ether: ethyl acetate = 1:1, phosphomolybdic acid color). Then add a reaction flask to add 120ml of methanol, reduce the temperature to 0-5 ° C, add 27.2g (0.277mol) of concentrated sulfuric acid, add the sulfuric acid solution to the first reaction bottle at 15 ~ 25 ° C, and then naturally heat up After stirring overnight at 15 to 25 ° C, TLC detected complete conversion of the hydrolyzed impurities (TLC: petroleum ether: ethyl acetate = 1:1, phosphomolybdic acid color development). 120ml of saturated sodium bicarbonate solution was added dropwise at a temperature of 15 ° C ~ 25 ° C, the pH of the above reaction solution was adjusted to 7 ~ 8, concentrated under reduced pressure to remove methanol, added 400 ml of ethyl acetate, stirred and separated, the organic phase was used sequentially 120 ml of saturated sodium hydrogencarbonate solution and 120 ml of saturated brine were washed once, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure, and then distilled to afford 40.8 g (yield: 100%, HPLC: 99.7%). .

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

Add methyl (4R)-4-((3R,5S,6R,10S,13R,14R,17R)-6-ethyl-3-hydroxy-10,13-dimethyl-7-carbonyl to the reaction flask Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate 40g (0.092mol), 400ml of tetrahydrofuran, after stirring, add 29.9g (0.231mol) of diisopropylethylamine and 4-Dimethylaminopyridine 1.13 g (0.009 mol), the mixture was cooled to 0-5 ° C, and 34.3 g (0.185 mol) of p-nitrobenzoyl chloride was added in portions at 0 to 5 ° C. After 30 min, the temperature was raised to 45-55 ° C for 2-4 hours, and the reaction of the starting material was completed by TLC (TLC: petroleum ether: ethyl acetate = 1:1, phosphomolybdic acid color). The temperature was lowered to 0 to 5 ° C, 200 ml of water was added dropwise, and after stirring for 20 min, methyl tert-butyl ether (200 mL) was added thereto, and the mixture was stirred. The organic phase was successively treated with 200 ml of hydrochloric acid (1 mol/L), 160 ml of saturated sodium hydrogencarbonate, and saturated. The brine was washed once, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. 160 ml of methyl tert-butyl ether was added to the residue, and the mixture was heated under reflux for 1 hour, and the temperature was lowered to 40 °. At 50 ° C, 320 ml of n-heptane was added dropwise, and the mixture was cooled to room temperature and stirred overnight. The mixture was filtered and dried to give 48.3 g (yield: 89.8%, HPLC: 98.8%). 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‐18 5um 4.6x150mm

■Posttime: 5.0min

■ Column temperature: 40 ° C

■Flow rate: 1ml/min

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

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

¹ H NMR: (CDCl 3 , 400 MHz) δ 0.67 (s, 3H), 0.82 (t, J = 7.6 Hz, 3H), 0.93 (d, J = 6.4 Hz, 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 impurity structure is resolved as follows:

¹ H NMR: (DMSO-d6, 400 MHz) δ 0.62 (s, 3H), 0.78 (t, J = 7.2 Hz, 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 = 8.4 Hz, 1H), 4.81-4.88 (m, 1H), 8.18-8.20 (m , 2H), 8.33 - 8.35 (m, 2H).

Step 5: (3R, 5S, 6R, 7R, 10S, 13R, 14R, 17R)-6-ethyl-7-hydroxy-17-((R)-5-methoxy-5-carbonylpentane-2 Of -10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthr-3-yl-4-nitrobenzoate

Under the protection of nitrogen, (3R, 5S, 6R, 10S, 13R, 14R, 17R)-6-ethyl-17-((R)-5-methoxy-5-carbonylpentane-2 was added to the reaction flask. -yl)-10,13-dimethyl-7-carbonylhexadehydro-1H-cyclopenta[a]phenanthr-3-yl-4-nitrobenzoate 5g (8.59 mmol) and 30 ml of tetrahydrofuran The mixture was stirred and stirred, and 30 ml of methanol was added thereto, and the mixture was cooled to 0 to 2 ° C, and sodium borohydride (0.325 g, 8.359 mmol) was added thereto, and the reaction was further stirred at a temperature of 0 ° C for 8 hours. The reaction of the starting material by TLC was completed, and 3.2 ml was dropped within 5 minutes. Acetone was stirred for 30 min, then 1.65 g (8.59 mmol) of anhydrous citric acid was added, stirring was continued for 5 min, and 25 ml of methyl tert-butyl ether and 20 ml of water were added. The organic solvent was removed under reduced pressure, and extracted with 50 ml of methyl t-butyl ether. The ml water and 10 ml of saturated brine were each washed once, dried over anhydrous sodium sulfate, and the methyl tert-butyl ether was removed under reduced pressure, and distilled to dryness with isopropyl alcohol. Add 40ml of isopropanol to the residue, heat to 80 ° C reflux, naturally cool to 60 ° C, the solid gradually precipitated (if not precipitated, can add seed crystal 20mg), stir at 60 ° C for 1 hour, then naturally cool, about 3 After the hour, the temperature was lowered to 20 ° C, and the mixture was further cooled to 0 ° C, stirred for 30 min, filtered, and the filter cake was dried under vacuum at 40 ° C for 3 hours to obtain 4.33 g of a white solid product (yield: 86.7%, HPLC: 99.2%).

¹ H NMR: (DMSO-d6, 400 MHz) δ 0.62 (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 = 5.2 Hz, 1H), 4.70 - 4.76 (m, 1H), 8.17 - 8.19 (m, 2H), 8.32 - 8.35 (m, 2H).

The impurity structure is resolved as follows:

Under the protection of nitrogen, (3R, 5S, 6R, 7R, 10S, 13R, 14R, 17R)-6-ethyl-7-hydroxy-17-((R)-5-methoxy-5 was added to the reaction flask. -carbonylpentane-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthr-3-yl-4-nitrobenzoate 250 mg (0.43 mmol), Then, 2 ml of methanol, 1 ml of tetrahydrofuran, 0.5 ml of water, and 51 mg of sodium hydroxide (1.28 mmol) were successively added, and the reaction was further stirred at a controlled temperature of 25 ° C for 16 hours, and the solvent was removed under reduced pressure, and diluted with hydrochloric acid to adjust to pH = 3-4. The ethyl acetate layer was separated, and the organic layer was washed twice with 10 ml of sodium hydrogen carbonate aqueous solution, and the solvent was evaporated to give 161 mg (yield: 89.4%).

Example 7

Step 1: methyl (4R)-4-((3R,5S,6R,7R,10S,13R,14R,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-10- Preparation of hexahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate

Add (3R,5S,6R,7R,10S,13R,14R,17R)-6-ethyl-7-hydroxy-17-((R)-5-methoxy-5-carbonylpentane to the reaction flask 2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthr-3-yl-4-nitrobenzoate 4.33 g (7.42 mmol) and 26 ml of tetrahydrofuran, The mixture was stirred and dissolved, cooled to 0 to 5 ° C, and a solution of LiOH.H ₂ O (0.58 g, 13.8 mmol) in methanol (13 ml) was added dropwise thereto, and the mixture was stirred for about 10 minutes, and the reaction was stirred at 0 to 5 ° C for 1 hour, and the temperature was naturally raised to The reaction was stirred at 18 to 22 ° C for 5 to 8 hours, and the raw materials were substantially completely detected by TLC. The reaction liquid was added to 50 ml of a saturated aqueous solution of ammonium chloride precooled to 0 ° C, and the solvent was evaporated under reduced pressure. 50 ml of ethyl acetate was added to the residue, and the mixture was stirred and separated, and the organic phase was sequentially applied with 50 ml of 4% potassium phosphate. The aqueous solution and the saturated aqueous solution of 15 ml of brine were washed once, dried over anhydrous sodium sulfate, and evaporated to dryness to give 2.9 g (yield: 90.1%, HPLC: 100%).

Step 2: Preparation of oleic acid

Under nitrogen, add methyl (4R)-4-((3R,5S,6R,7R,10S,13R,14R,17R)-6-ethyl-3,7-dihydroxy-10 to the reaction flask. 13-Dimethylhexadehydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate 2.9 g (6.68 mmol), then 10 ml of methanol, 5 ml of tetrahydrofuran, 5 ml of water, sodium hydroxide 0.54g (13.36mmol), the reaction was stirred at 25 ° C for 5 hours, the solvent was removed under reduced pressure, and the mixture was diluted with hydrochloric acid to adjust to pH = 3 to 4, ethyl acetate (30 ml) was added, and the ethyl acetate layer was separated. After washing with 10 ml of saturated sodium chloride, the solvent was evaporated under reduced pressure to yield 2.8 g (yield: 100%).

Example 8

Step 1: methyl (4R)-2-bromo-4-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-10- Preparation of hexahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate

144.3 g (1.426 mol) of diisopropylamine was dissolved in 1000 ml of tetrahydrofuran, cooled to -50 ° C, and 495 ml of n-hexane solution of n-butyllithium (2.4 mol/L, 1.188) was added dropwise at a temperature of -50 ° C to -40 ° C. Mol), after about 30 minutes, continue to incubate for 1 h. Methyl (4R)-4-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclo 102.9 g (237.7 mmol) of pentadieno[a]phenanthrene-17-yl)valerate was dissolved in 500 ml of tetrahydrofuran. The temperature was controlled from -60 ° C to -50 ° C, and the solution was added dropwise to the n-butyllithium solution. After about 50 minutes, the white viscous solid appeared in the dropping process. The reaction was further stirred at a temperature of -70 ° C to -65 ° C for 1 h. 154.9 g (1.426 mol) of trimethylchlorosilane was added dropwise at a temperature of -70 ° C to -60 ° C, and the addition was completed in about 15 minutes, and the reaction was further stirred at a temperature of -60 ° C to -50 ° C for 3 hours. The basic reaction of the TLC detection of the raw materials is completed. 129.6g (728.2mmol) of N-bromosuccinimide was added in portions, and the natural temperature was raised to 22-27 °C. The reaction was stirred for 22 h, and the basic reaction of the starting material was confirmed by TLC. The reaction mixture was cooled to 0 ° C, 1000 ml of saturated sodium bicarbonate solution was added dropwise at a temperature control temperature of 10 ° C, stirred, and the organic phase was concentrated to about 500 ml, and the aqueous phase was extracted with 500 ml of methyl t-butyl ether, and the organic phase was combined. The concentrate and the methyl tert-butyl ether extract are washed once with 1000 ml of water, 250 ml of 1 mol/L hydrochloric acid is added to the above organic phase, stirred at room temperature for 5 h, and the organic phase is washed once with 1500 ml of 20% sodium sulfite solution, and then 1000 ml. *2 Saturated twice with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered and concentrated to dryness to afford 134.8 g of viscous brown solid (yield: 100%, HPLC: 99.5%, containing dibromo body). Directly invest in the next step.

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

Methyl (4R)-2-bromo-4-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-II obtained by the reaction of the previous step 134.8 g (237.7 mmol) of crude methylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate and 54.3 g (713.1 mmol) of thiourea were dissolved in 1000 ml of ethanol. The mixture was heated to 70 ° C and stirred for 5 h. The TLC detects the reaction of the starting material completely. 1200 ml of 2 mol/L hydrochloric acid was added to the reaction liquid, and the reaction was further stirred at a controlled temperature of 72 ° C for 30 hours, and cooled to room temperature (oil may be precipitated). The ethanol was concentrated under reduced pressure. EtOAc (EtOAc:EtOAc:EtOAc. %).

Refining: 15.0 g of the brown foam solid obtained above was added, 150 ml of dichloromethane was added, and the mixture was stirred for 1 hour, filtered, and the filter cake was washed with a small amount of pre-cooled dichloromethane, and dried in vacuo to give 7.9 g of white solid (HPLC: 99. %, containing two non-isomers).

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

"Optical purity" is a measure of the amount of one enantiomer in an optically active sample over the other enantiomer. In the present invention, it is primarily meant that the compound of formula (I) comprises a compound of formula (I) and (II) Proportion in the compound mixture.

The "lower alkane solvent" means a liquid alkane having 5 to 10 carbon atoms at normal temperature, such as n-hexane or n-heptane.

The "cycloalkane solvent" means a saturated hydrocarbon having an alicyclic structure and being liquid at normal temperature, such as cyclopentane or cyclohexane.

The "halogenated alkane solvent" means a halogen atom-containing alkane which is liquid at normal temperature, such as dichloromethane or chloroform.

The "lower alcohol solvent" means an alkane compound having a hydroxyl group in the molecule and having less than 12 carbon atoms, such as methanol, ethanol, or isopropanol.

The "lower ester solvent" means an ester compound having a small number of carbon atoms and a liquid state at a normal temperature, preferably an ester compound of an acid having less than 4 carbon atoms and an alcohol having less than 4 carbon atoms, such as acetic acid B. Ester, methyl acetate, isopropyl acetate.

"Lower ether solvent" means an ether compound formed by two hydrocarbon groups having a small number of carbon atoms, preferably an ether compound having two hydrocarbon groups of less than 4 carbon atoms, such as diethyl ether, diisopropyl ether, methyl isopropyl. ether.

Example 9 (raw material preparation)

First step: methyl (4R)-4-((3R,5S,6R,7R,10S,13R,14R,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl Preparation of Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate

100.0 g (237.7 mmol) of oleic acid and concentrated sulfuric acid (0.25 g) were dissolved in 500 ml of methanol. Heat to reflux and continue stirring for 16 h. After completion of the reaction by TLC, the reaction mixture was concentrated to dryness. To the residue, 600 ml of ethyl acetate and 400 ml of saturated sodium hydrogen carbonate solution were added, and the mixture was stirred well, and the mixture was allowed to stand, and the organic phase was washed once with 400 ml of saturated sodium chloride solution. The residue was dried over anhydrous sodium sulfate, filtered and evaporated to dryness.

Step 2: Methyl (4R)-2-bromo-4-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl Preparation of Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate

144.3 g (1.426 mol, 6.0 eq.) of diisopropylamine was dissolved in 1000 ml of tetrahydrofuran, cooled to -50 ° C, and the temperature was changed from -50 ° C to -40 ° C dropwise to add 495 ml of n-butyllithium solution (1.188 mol, 2.4 M. The alkane solution, 5.0 eq.), was added over about 30 minutes and stirring was continued for 1 hour.

The methyl (4R)-4-((3R,5S,6R,7R,10S,13R,14R,17R)-6-ethyl-3,7-dihydroxy-10,13-di which was prepared in the previous step. 106.9 g of methylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate (calculated in the above theoretical amount of 237.7 mmol) dissolved in 500 ml of tetrahydrofuran, dropped into the above reaction solution, and temperature-controlled -60 ° C ~ -50 ° C, about 50 minutes after the completion of the addition, a white viscous solid appeared in the reaction. The reaction was stirred at -70 ° C to -65 ° C for 1 hour and 1 h. 154.9 g (1.426 mol, 6.0 eq.) of trimethylchlorosilane (TMSCl) was added dropwise at a temperature of -70 ° C to -60 ° C. The addition was completed in about 15 minutes, and the stirring reaction was continued at -60 ° C to -50 ° C. After 3 hours, TLC detected the basic reaction of the raw materials.

129.6 g (728.2 mmol, 3.06 eq.) of N-bromosuccinimide (NBS) was added in portions over a period of about 30-45 minutes, and the reaction temperature was raised from -60 °C to -40 °C. The reaction was then naturally raised to room temperature (22 ° C) and the reaction was continued for 24 hours. The basic reaction of the TLC detection raw material is completed, and the reaction liquid is cooled to 0 ° C, and the temperature control is less than 1000 ml of a saturated sodium hydrogencarbonate solution was added dropwise at 10 °C. After stirring, the liquid was separated, and the organic phase was concentrated to remove some solvent (about 500 ml). The aqueous phase was extracted with 500 ml of methyl tert-butyl ether (MTBE), and the organic phase concentrate and methyl t-butyl ether extract were combined with 1000 ml of water. Wash the combined solution once.

250 ml of 1 mol/L hydrochloric acid was added to the above combined solution, and the reaction was stirred at room temperature (22 ° C) for 5 hours. The organic phase was separated, and the organic phase was washed once with 1500 ml of 20% sodium sulfite solution, and then washed twice with 1000 ml of sodium hydrogencarbonate solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to give a pale brown solid. Directly invest in the next step.

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

Methyl (4R)-2-bromo-4-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-di prepared by the above procedure Methylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)valerate 134.8 g crude (calculated by theoretical calculation of 237.7 mmol) and thiourea 54.3 g (713.1 mmol, 3.0 eq. ) dissolved in 1000 ml of ethanol. The mixture was heated to 70 ° C and stirred for 5 hours. The basic reaction of the TLC detection of the raw materials is completed. After adding 1200 ml of 2 mol/L hydrochloric acid, the reaction was stirred at a temperature of 72 ° C for 30 hours, and most of the ethanol solvent was concentrated under reduced pressure, and extracted with 1200 ml of ethyl acetate. The organic phase was washed twice with 1000 ml of water, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to dryness to give a brown solid, 133.9 g of crude product (HPLC (comprising compound of formula (II) and compound of formula (I)): 85.5%, wherein: compound of formula (II): compound of formula (I) = 24.2 %: 61.3% ≈ 2:5).

Another 200.0 g of oleic acid was charged. According to the above first to third reaction, the experimental results were as follows:

Combining the previous two batches of 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-10- Hexahydro-1H-cyclopenta[a]phenanthroline-17-yl)propyl)thiazolidine-2,4-dione (406.5 g), silica gel column chromatography (eluent petroleum ether: acetic acid Ethyl ester: tetrahydrofuran = 3:1:1) 350.1 g of brown foam solid (HPLC (comprising compound of formula (II) and compound of formula (I)): 85.7%, wherein: compound of formula (II): compound of formula (I) =24.8%: 62.9% ≈ 2:5).

Purification method 1: Take 15.0 g of the above brown foam solid, fully beaten with 150 ml of dichloromethane for 2 hours, filter, filter cake washed with dichloromethane, and dried to give a white solid 7.9 g (HPLC (II) Compound and compound of formula (I): 99.2%, wherein: compound of formula (II): compound of formula (I) = 24.7%: 74.5% by weight ≈ 1:3).

Purification method 2: The filtrate in the purification method 1 was concentrated to dryness, and combined with the remaining 335.1 g of the solid of the above brown foam solid, and 1500 ml of dichloromethane was added thereto, and the mixture was firstly stirred and dissolved (it became cloudy after further stirring). 1200 ml of n-heptane was added dropwise, the solid was washed out, and stirring was continued for 16 hours at room temperature. The filter cake was filtered and dried to give a white solid 240.0 g (HPLC (comprising compound of formula (II) and compound of formula (I)): 97.0%, wherein: compound of formula (II): compound of formula (I) = 27.6%: 69.4% %≈2:5).

Example 10 (conversion reaction)

32.2 g (805 mmol) of sodium hydroxide was dissolved in 1920 ml of ethanol and cooled to 5 °C. 240.0 g of the off-white solid obtained in Example 9 was added, and the mixture was cooled to -20 ° C and stirred for 19 hours. 4 mol / L hydrochloric acid was added dropwise at -20 ° C until the pH of the reaction solution was about 5-6, and most of the ethanol was concentrated under reduced pressure at a temperature lower than 40 ° C. 1000 ml of water and 1500 ml of ethyl acetate were added to the residue, and stirred. Liquid separation. The organic phase was washed once with 1000 ml of aq. Wherein: a compound of the formula (II): a compound of the formula (I) = 13.4%: 82.5% by weight ≈ 0.8: 5).

Example 11 (refining purification)

270.0 g of the brown foam solid obtained in Example 10 was dissolved in 960 ml of ethyl acetate, and then 960 ml of n-heptane was added dropwise, and the mixture was stirred at room temperature for 48 hours. Filtration, the filter cake was washed with a mixed solvent (150 ml of petroleum ether + 150 ml of ethyl acetate), and the filter cake was dried to obtain 24.8 g of an off-white solid (HPLC (comprising formula (comprising compound and compound): 100.0%, wherein: Formula (wherein the compound: compound (65.7%: 34.3% ≈ 9.6:5). The filtrate was concentrated to dryness under reduced pressure, then distilled with 100 ml of ethanol, and finally concentrated to dry brown foam solid 245 g (HPLC (II) Compound and compound of formula (I): 100.0%, wherein: compound of formula (II): compound of formula (I) = 7.4%: 88.8% ≈ 0.42: 5).

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

¹ H NMR (DMSO-d ₆ , 400 MHz) 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).

It should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art The technical solutions are modified or equivalent, without departing from the spirit and scope of the invention, and are intended to be included within the scope of the appended claims.

Claims (10)

1. A crystalline form of (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base characterized by a powder X-ray diffraction pattern comprising 16.5 ± a peak at a diffraction angle (2θ) of 0.2°, 13.6±0.2°, 12.1±0.2°, 20.4±0.2°;

   Preferably, the powder X-ray diffraction pattern further comprises a peak at a diffraction angle (2θ) of 11.5±0.2°, 9.6±0.2°, 19.6±0.2°, 15.0±0.2°, 20.0±0.2°;

   Preferably, the powder X-ray diffraction pattern further comprises peaks at 23.7±0.2°, 20.7±0.2°, 23.0±0.2°, 25.7±0.2°, 17.9±0.2° and 16.0±0.2° diffraction angle (2θ). .
2. A crystalline form of (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl A method for preparing a hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base, comprising the steps of:

   1) (5R)-5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl Hexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione free base is dissolved or dispersed in an organic solvent, water, or a mixture of an organic solvent and water. In solvent

   2) precipitating crystals, or optionally adding an anti-solvent to the compound clear solution, or optionally slowly evaporating the clear solution of the compound, or optionally adding the original compound solid or other solid particle additive to the compound solution as a heteronuclear Seed crystal-induced crystallization, or a combination of the above methods to obtain the crystal body;

   Preferably, the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, acetone, ethyl acetate, isopropyl acetate, toluene, n-butanol, cyclohexane, dichloromethane, dimethylformamide. , dimethylacetamide, dimethyl sulfoxide, dioxane, diethyl ether, n-heptane, n-hexane, methyl ethyl ketone, isooctane, pentane, dipropanol, tetrahydrofuran, dimethyltetrahydrofuran, trichloroethane Alkane, xylene or a mixture thereof; more preferably ethyl acetate, isopropyl acetate, dichloromethane, n-heptane, n-hexane or a mixture thereof;

   Preferably, the crystal powder X-ray diffraction pattern obtained in the step 2) comprises a peak at a diffraction angle (2θ) of 16.5±0.2°, 13.6±0.2°, 12.1±0.2°, and 20.4±0.2°;

   Further preferably, a pharmaceutical composition comprising an effective amount of the crystalline form (5R)-5-((2R)-2-((3R,5S,6R,7R) described in claim 1) ,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine a 2,4-dione free base, a pharmaceutically acceptable carrier or excipient;

   Further preferably, 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-dione free base, or the pharmaceutical composition described therein for preparation for prevention Or for use in a medicament for treating a FXR-mediated disease or condition, preferably, the FXR-mediated disease or condition is selected from the group consisting of cardiovascular disease, hypercholesterolemia, hyperlipidemia, chronic hepatitis disease, chronic liver disease, gastrointestinal disease, Kidney disease, cerebrovascular disease, metabolic disease or cancer; more preferably, chronic liver disease is selected from the group consisting of primary sclerosis (PBC), cerebral xanthoma (CTX), primary sclerosing cholecystitis (PSC), drug-induced Cholestatic stagnation, intrahepatic cholestasis of pregnancy, parenteral absorption-related cholestasis (PNAC), bacterial overgrowth or sepsis cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease ( NAFLD), nonalcoholic steatohepatitis (NASH), liver graft-related graft-versus-host disease, live donor liver transplant regeneration, congenital liver fibrosis, common bile duct stones, meat Liver disease, liver cancer, or outer, Sjogren's syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis, or an anti-α ₁ protease deficiency film; gastrointestinal disease is selected from inflammatory bowel disease (I BD), Irritable bowel syndrome (IBS), bacterial overgrowth, malnutrition, post-reflex colitis or microcolitis, inflammatory bowel disease is preferably Crohn's Disease or ulcerative bowel disease; kidney disease is selected from Diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulonephritis, chronic allograft glomerulopathy, chronic interstitial nephritis or polycystic kidney disease; cardiovascular disease selected from Atherosclerosis, arteriosclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia or hypertriglyceridemia; metabolic diseases selected from insulin resistance, type I diabetes, type II diabetes or obesity; cerebrovascular disease selected from Stroke; cancer is selected from colorectal cancer or liver cancer.
3. A substantially pure compound of formula (I), characterized in that said substantially pure compound of formula (I) means that the optical purity of the compound of formula (I) is above 90.0%, preferably, said substantially pure The compound of the formula (I) means that the compound of the formula (I) has an optical purity of 95.0% or more; preferably, the substantially pure compound of the formula (I) contains no more than 10.0%, more preferably no more than 5.0%;

   Preferably, the optical purity refers to the compound of formula (II) relative to its non-isomer:

   

   Further preferably, a pharmaceutical composition comprising an effective amount of a substantially pure compound of formula (I) or a pharmaceutically acceptable carrier or excipient thereof;

   Further preferred, substantially pure compound of formula (I), or a pharmaceutical composition thereof, for use in the manufacture of a medicament for the prevention or treatment of a FXR-mediated disease or condition; preferably, an FXR-mediated disease or condition From cardiovascular disease, hypercholesterolemia, hyperlipidemia, chronic hepatitis disease, chronic liver disease, gastrointestinal disease, kidney disease, cerebrovascular disease, metabolic disease or cancer; more preferably, chronic liver disease is selected from primary sclerosis (PBC) , cerebral xanthoma (CTX), primary sclerosing cholecystitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral absorption-related cholestasis (PNAC), bacterial overgrowth or pus Bile cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver transplantation-related graft-versus-host disease, live donor liver Transplantation regeneration, congenital liver fibrosis, common bile duct stones, granulomatous liver disease, intrahepatic or external malignant swelling Tumor, Sjogren syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis or alpha 1 anti-membrane protease deficiency; gastrointestinal disorders selected from inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), Bacterial overgrowth, malnutrition malabsorption, post-reflex colitis or microcolitis, inflammatory bowel disease is preferably Crohn's Disease or ulcerative bowel disease; nephropathy is selected from diabetic nephropathy, focal segmental kidney Small ball sclerosis (FSGS), hypertensive nephropathy, chronic glomerulonephritis, chronic allograft glomerulopathy, chronic interstitial nephritis or polycystic kidney disease; cardiovascular disease selected from atherosclerosis, arteriosclerosis, atherosclerosis Sclerotherapy, dyslipidemia, hypercholesterolemia or hypertriglyceridemia; metabolic diseases selected from insulin resistance, type I diabetes, type II diabetes or obesity; cerebrovascular disease selected from stroke; cancer selected from colorectal cancer or Liver cancer.
4. A substantially pure process for the preparation of a compound of formula (I), characterized by comprising the steps of:

   1) The crude compound of the formula (I) is dissolved in a positive solvent having a mass to volume ratio of 1-10 times;

   2) adding an anti-solvent having a positive solvent volume ratio of 0.5-10 times;

   3) After adding, continue to stir and crystallize;

   4) Filter and drain;

   5) The filtrate is concentrated to dryness under reduced pressure to give a substantially pure compound of formula (I);

   Preferably, the positive solvent is selected from the group consisting of a lower ester solvent, a halogenated alkane solvent, a lower alcohol solvent or a mixture thereof; more preferably ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or a mixture thereof;

   Further preferably, the positive solvent is used in a volume of 3-5 times the mass to volume ratio of the crude compound of the formula (I);

   Preferably, the anti-solvent is selected from the group consisting of a lower alkane solvent, a cycloalkane solvent, a lower ether solvent or a mixture thereof; more preferably n-heptane, n-hexane, petroleum ether, isopropyl ether or a mixture thereof;

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

   Preferably, step 5) is concentrated to dryness under reduced pressure, then distilled with a lower alcohol solvent or a mixture thereof, and then concentrated to dryness; more preferably ethanol, isopropanol or a mixture thereof;

   Further preferably, the substantially pure compound of formula (I) obtained in step 5) has an optical purity of 90.0% or more; more preferably, the substantially pure compound of formula (I) has an optical purity of 95.0% or more.
5. A substantially pure process for the preparation of a compound of formula (I), characterized by comprising the steps of:

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

   2) adding a second solvent having a first solvent volume ratio of 0.5-10 times;

   3) continue to stir and crystallize;

   4) filtration and drying, the filter cake is dried to obtain a substantially pure compound of formula (I);

   Preferably, the amount of the first solvent in the step 1) is 2-7 times the mass-to-volume ratio of the crude compound of the formula (I); the amount of the second solvent in the step 2) is 0.5-3 times the volume ratio of the first solvent;

   Preferably, the first solvent is selected from the group consisting of a lower ester solvent, a halogenated alkane solvent, a lower alcohol solvent or a mixture thereof, more preferably ethyl acetate, isopropyl acetate, ethanol, isopropanol, dichloromethane or a mixture thereof; The second solvent is selected from the group consisting of a lower alkane solvent, a cycloalkane solvent, a lower ether solvent or a mixture thereof, more preferably n-heptane, n-hexane, petroleum ether, isopropyl ether or a mixture thereof;

   Further preferably, the substantially pure compound of formula (I) obtained in step 4) has an optical purity of 90.0% or more; more preferably, the substantially pure compound of formula (I) has an optical purity of 95.0% or more.
6. Process for the preparation of a substantially pure compound of formula (I) according to any one of claims 4-5, characterized in that the crude compound of formula (I) is optionally pretreated by the following steps:

   1) dissolving sodium hydroxide or potassium hydroxide in a mass-to-volume ratio of 5-100 times lower alcohol solvent, and then cooling to below 5 ° C;

   2) adding the raw material containing the compound of the formula (I) to the alkali solution obtained in the step 1), and cooling the mixture to below -10 ° C to continue the stirring reaction;

   3) slowly add acidic substances below -10 ° C to the pH of the reaction solution about 5-6;

   4) concentrating part of the solvent under reduced pressure under temperature control below 40 ° C;

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

   6) The organic phase is concentrated under reduced pressure to dryness to obtain a crude compound of formula (I);

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

   More preferably, the lower alcohol solvent used in step 1) is preferably ethanol, isopropanol or a mixture thereof; the ester solvent used in step 5) is preferably ethyl acetate, isopropyl acetate or a mixture thereof.
7. A method for preparing a compound of formula (III), comprising the steps of:

   1) esterification of a compound of formula (IV) to give a compound of formula (V);

   2) a compound of formula (V) is reduced by a double bond to give a compound of formula (VI);

   3) isomerization of a compound of formula (VI) to give a compound of formula (VII);

   4) protecting the compound of formula (VII) with a hydroxy group to give a compound of formula (VIII);

   5) Reduction of the carbonyl group of the compound of formula (VIII) to give a compound of formula (III);

   The reaction formula is as follows:

   

   Wherein R is selected from C _1-4 alkyl; Pg is a hydroxy protecting group;

   Preferably, R is selected from methyl or ethyl;

   Preferably, Pg is selected from a substituted or unsubstituted benzoyl group, a substituted or unsubstituted benzenesulfonyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted benzyl group or a trialkylsilyl group, and more preferably has the following structure:

   

   Most preferred

   

   Preferably, the esterification reaction of the step 1) is carried out in an acidic environment at a temperature of from 20 ° C to 35 ° C, more preferably at a temperature of from 22 ° C to 27 ° C; further preferably, the acid is selected from the group consisting of Hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, methanesulfonic acid or a mixture thereof; more preferably hydrochloric acid or sulfuric acid;

   Preferably, the isomerization reaction of the step 3) is carried out by using sodium alkoxide in an alcohol solvent at a reaction temperature of 5 ° C to 35 ° C; more preferably, the sodium alkoxide is selected from the group consisting of sodium methoxide, sodium ethoxide or tert-butyl. a sodium alcohol, the alcohol solvent is selected from the group consisting of methanol, ethanol, isopropanol or tert-butanol, the reaction temperature is 15 ° C ~ 25 ° C;

   Preferably, the step 5) carbonyl reduction uses sodium borohydride to reduce the carbonyl group of the compound of the formula (VIII) in an alcohol solvent to obtain a compound of the formula (III) at a reaction temperature of -5 ° C to 10 ° C; more preferably, The alcohol solvent is selected from the group consisting of methanol, ethanol, isopropanol or tert-butanol, and the reaction temperature is 0 ° C to 3 ° C; further preferably, after the step 5) the carbonyl reduction reaction is finished, the reaction is quenched with acetone and citric acid. .
8. A method for preparing oleic acid, characterized in that, according to the preparation method of claim 7, further comprising the following steps:

   

   Wherein, the acid is an organic acid or an inorganic acid; the organic acid is preferably selected from trifluoroacetic acid, trichloroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid or a mixture thereof; From hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or a mixture thereof.
9. A method for preparing oleic acid, characterized in that, according to the preparation method of claim 7, further comprising the following steps:

   

   Preferably, the compound of formula (III) is selectively acidolyzed by the action of lithium hydroxide to form a compound of formula (IX).
10. 5-((2R)-2-((3R,5S,6R,7R,10S,13R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexadecahydrol-1H a process for producing cyclopenta[a]phenanthrene-17-yl)propyl)thiazolidine-2,4-dione, which is characterized in that, furthermore, on the basis of the preparation method according to claim 7, further Including the following steps:

    

    Preferably, the compound of formula (III) is selectively acidolyzed by lithium hydroxide to form a compound of formula (IX);

    Preferably, the crude product of formula (XI) obtained by the reaction is purified by pulping and purifying with methylene chloride having a mass ratio of 1:15; more preferably, the compound of formula (XI) obtained by purifying with dichloromethane is purified to have a purity of 98%. the above.

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