CN109641934A

CN109641934A - Chlolic acid derivatives free alkali, crystal form and its preparation method and application

Info

Publication number: CN109641934A
Application number: CN201780049733.6A
Authority: CN
Inventors: 苏熠东; 龚素娟; 包如迪
Original assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2016-09-20
Filing date: 2017-09-20
Publication date: 2019-04-16
Anticipated expiration: 2037-09-20
Also published as: WO2018054300A1; CN109641934B

Abstract

Provide a kind of crystal type chlolic acid derivatives free alkali, preparation method and application, the free-basing scientific name of chlolic acid derivatives is known as (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- sodium catchol disulfonate 0, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, 4- diketone, its x-ray diffractogram of powder includes being located at 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, peak at 20.4 ± 0.2 ° of the angle of diffraction (2 θ), further relate to optically pure chlolic acid derivatives and its preparation method and application, the purity of optical isomer is prepared up to 90.0% or more, it solves and is difficult to separate in the prior art The problem of obtaining optical purity.In addition, additionally providing a kind of preparation method and applications of chlolic acid derivatives.The preparation method overcomes defect existing in the prior art, and multistep reaction can be completed reaction at normal temperature, and products therefrom purity is good, high income, and process operability is strong, and process safety is also largely increased, and is suitble to industrial applications.

Description

Chlolic acid derivatives free alkali, crystal form and its preparation method and application

Technical field

The invention belongs to technical field of pharmaceuticals, more particularly to a kind of crystal type chlolic acid derivatives free alkali, preparation method and application, it further include a kind of substantially pure chlolic acid derivatives and its preparation method and application and a kind of preparation method and applications of chlolic acid derivatives.

Background technique

Method Buddhist nun's ester derivant X receptor (FXR) belongs to a member of hormone nuclear receptor superfamily, mainly expresses in liver, small intestine, kidney and adrenal gland, the less expression in adipose tissue and heart.It is initially considered that farnesol is its ligand, and thus gains the name.After FXR ligand and FXR carboxyl terminal ligand binding domain (LBD) are bound directly, nuclear receptor space conformation changes and forms heterodimer with retinoid receptor (RXR), the transcription of target gene is finally adjusted in conjunction with the specific FXR DNA response element of target gene, the regulation for participating in glucose-lipid metabolism, is important energy regulator.Primary bile acid chenodesoxycholic acid is the most effective ligand of FXR, and secondary bile acid lithocholic acid and deoxycholic acid can also activate FXR.There are also synthesis FXR ligand (such as 6-ECDCA, GW4064) at present, with FXR binding force several times stronger than native ligand.The major target gene of FXR includes Bile salt export pump (BSEP), cholic acid binding protein (IBABP) and small heterodimer companion receptor (SHP) etc., and FXR passes through the expression for combining to regulate and control these genes with the FXR response element (FXRE) on these gene promoters.But without typical case's FXR association reaction sequence in the promoter sequence of the main FXR controlling gene of such as cholesterol 7α-hdroxylase (CYP7 α 1), FXR is then indirectly by inducible transcription inhibiting factor SHP expression, then SHP and 1 promoter liver receptor homolog object (LRH-1) of CYP7 α form inhibition compound, so that CYP7 α 1 and other LRH-1 target genes be blocked to transcribe.Due to important function of the FXR in bile acid and cholesterol metabolic, this will make itself and liver related disease relationship of increasing concern.The novel agonist of FXR is found into the research hotspot of the treatment liver diseases such as including cholestasis syndrome, 6- acetylene chenodesoxycholic acid (shellfish cholic acid difficult to understand) has completed III clinical trial phase as treatment primary biliary cirrhosis (PBC), it will be listed earliest in 2015, and the compound also shows satisfactory therapeutic effect in Nonalcoholic Steatohepatitis.FXR agonist can both increase bile acid dependence gallbladder stream by stimulation Bile salt export pump (BSEP) and or MRP2 stimulated to increase non-bile acid dependence gallbladder stream to mitigate cholestasis.FXR is expected to become the new drug target that screening treatment includes other metabolic diseases such as cholestatic disease and nonalcoholic fatty liver disease.

2016, Jiangsu Hao Sen company discloses a kind of chlolic acid derivatives in patent PCT/CN2016/079167 (applying date: 2016.04.13), wherein representative compound chemical name are as follows: (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2,4- diketone (formula (I) compound), structure is as follows:

The compound has significantly agonism to FXR activity, it is expected to develop into a kind of new FXR agonist, but, since patent PCT/CN2016/079167 formula disclosed in embodiment 14 (I) compound is extracted through ethyl acetate and still can only obtain the mixture comprising another non-corresponding isomers after column chromatographic purifying, it is difficult to be prepared into the pharmaceutical preparation of suitable clinical application, the final physico-chemical attributes of product also need to be tested further proof.Therefore, the needs of clinical drug exploitation are met there is an urgent need to develop a kind of stable, crystalline solid that dissolubility is good.The field there is a perceived need to develop that a kind of optical purity is good, can stablize the compound for saving the needs that can satisfy clinical drug exploitation.

2002, patent WO2002072598A1 made public for the first time shellfish cholic acid difficult to understand and preparation method thereof, and synthetic route is as follows:

The patent is raw material with 3-5 β of Alpha-hydroxy-7- ketone-cholane-24- acid and 3,4- dihydropyran, first protects 3 Alpha-hydroxies；Then it is reacted with bromoethane in 4 upper substitution ethyls, simultaneously at ester, but step reaction needs to react under the conditions of -70~-80 DEG C, and it to use than relatively hazardous n-BuLi, and carcinogen hexamethyl-phosphoramide (HMPA) makees catalyst, therefore, which is not suitable for industrial applications.

2006, patent WO2006122977A2 disclosed a kind of following synthetic route for preparing shellfish cholic acid difficult to understand

The patent is equally with 3-5 β of Alpha-hydroxy-7- ketone-cholane-24- acid and 3; 4- dihydropyran is raw material; first at ester; then 3 Alpha-hydroxies are protected with TMS; 6 carbonyls are protected again; then it is reacted again with aldehyde compound, but reaction needs are reacted under the conditions of-60~-90 DEG C, reaction solvent for use is boron trifluoride ether solution；It is restored again with palladium carbon after sodium hydroxide hydrolysis, then in 95~105 DEG C of progress configuration conversions, is finally restored under the conditions of 70~105 DEG C with sodium borohydride and obtain shellfish cholic acid difficult to understand.The preparation method needs to use expensive trim,ethylchlorosilane, palladium carbon, unstable aldehyde compound, unsafe boron trifluoride ether solution, and harsh reaction temperature, such as configuration conversion and reduction reaction are carried out at high temperature, therefore, the synthetic route of the patent disclosure is also not suitable for industrial applications.

2016, Jiangsu Hao Sen company discloses a kind of chlolic acid derivatives in patent PCT/CN2016/079167 (applying date: 2016.04.13), wherein representative compound chemical name are as follows: 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2,4- diketone, the preparation method is as follows:

The patent prepares 5- ((2R) -2- ((3R by raw material of shellfish cholic acid difficult to understand, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine when want First it is esterified, then final product, and the preparation method without optimizing shellfish cholic acid difficult to understand are obtained through bromo, cyclization, there is a problem of with WO2006122977A2 same, be not suitable for industrialized production, therefore develop and the pharmaceutical preparation of application is suitble to be also those skilled in the art institute urgent problem.

Summary of the invention

One of the objects of the present invention is to provide a kind of better medicine crystals, different coherent conditions of the inventor by further investigation compound of formula I, a kind of crystal type (5R) -5- ((2R) -2- ((3R is finally obtained, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, bis- keto-acid of 4- (I) compound, substantially improve the physicochemical property of formula (I) compound, it can be used to treat the disease that treatment FXR is mediated, including cardiovascular disease, atherosclerosis, artery sclerosis, hypercholesterolemia, hyperlipidemia chronic liver disease, chronic liver disease, gastrointestinal disease, nephrosis, cardiovascular disease, metabolic disease, cancer (example Such as colorectal cancer) or neural sign such as apoplexy disease, there is extensive medical application, be expected to exploitation into FXR agonist of new generation.

Crystal type (5R) -5- ((2R) -2- ((3R of the present invention, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2,4- diketone free alkali, the crystal type free alkali is appointed as crystal form I, its x-ray diffractogram of powder includes being located at 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, the peak at 20.4 ± 0.2 ° of the angle of diffraction (2 θ)；It is also preferable to include the peaks at 11.5 ± 0.2 °, 9.6 ± 0.2 °, 19.6 ± 0.2 °, 15.0 ± 0.2 °, 20.0 ± 0.2 ° of the angle of diffraction (2 θ)；It more preferably further include the peak 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 ° angles of diffraction (2 θ).

As most preferred scheme, crystal type (5R) -5- ((2R)-the 2- ((3R, 5S, 6R, 7R, 10S, 13R) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethyl -3,7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2,4- diketone free alkali, 2 θ (°) of X-ray powder diffraction figure diffraction maximum angle and peak intensity are as in the table below:

2θ(°)	Intensity %	2θ(°)	Intensity %
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 kind of crystal type (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2, the preparation method of 4- diketone free alkali, it is characterized by comprising the following steps:

1) by (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine free alkali is dissolved or dispersed in the in the mixed solvent of organic solvent, water or organic solvent and water；

2) cooling is precipitated, or anti-solvent is added optionally into compound clear solution and is precipitated, or the clear solution for the compound that optionally slowly volatilizees is precipitated, or former compound solid or other solid particle additives are added as heteronuclear crystal seed optionally into the compound solution and induce crystallization, or the above method is used in combination and obtains the crystalline solid.

As a preferred option, the organic solvent is selected from or mixtures thereof methanol, ethyl alcohol, isopropanol, acetonitrile, acetone, ethyl acetate, isopropyl acetate, toluene, n-butanol, hexamethylene, methylene chloride, dimethylformamide, dimethyl acetamide, dimethyl sulfoxide, dioxane, ether, normal heptane, n-hexane, methyl ethyl ketone, isooctane, pentane, two propyl alcohol, tetrahydrofuran, dimethyl-tetrahydrofuran, trichloroethanes, dimethylbenzene.

As further preferred scheme, the organic solvent is selected from or mixtures thereof ethyl acetate, isopropyl acetate, methylene chloride, normal heptane, n-hexane.

As further preferred scheme, its x-ray diffractogram of powder of the preparation method step 2) crystalline solid obtained includes the peak at 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, 20.4 ± 0.2 ° of the angle of diffraction (2 θ).

Further aspect of the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes a effective amount of crystal type (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethyl -3,7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2,4- diketone free alkali, pharmaceutically acceptable carrier or excipient.

Further aspect of the present invention provides the crystal type (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) application of thiazolidine free alkali or foregoing pharmaceutical composition in the drug for preparing the disease or situation for preventing or treating FXR mediation.

As a preferred option, the disease or situation that the FXR is mediated are selected from cardiovascular disease, hypercholesterolemia, hyperlipidemia chronic liver disease, chronic liver disease, gastrointestinal disease, nephrosis, cranial vascular disease, metabolic disease or cancer.

As further preferred scheme, the chronic liver disease is selected from primary hardening (PBC), the dirty property xanthomatosis (CTX) of brain, primary sclerosing cholangitis (PSC), drug induced cholestasia, intrahepatic cholestasis of pregnancy, parenteral absorption associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NASH), the anti-host disease of liver transfer operation related Graft, live donor liver transfer operation regeneration, first Nature liver fibrosis, choledocholithiasis, granular hepatopathy, malignant tumour, Sjogren syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis or α in or beyond liver₁Primary antibody membrane proteolytic enzyme deficiency disease.

As further preferred scheme, the gastrointestinal disease is selected from that inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), bacterial overgrowth, nutrient absorption are bad, reflection postcolon is scorching or microscopic colitis, the preferred Crow grace disease of inflammatory bowel disease (Crohn ' s Disease) or routed characteristic of disease enteropathy.

As further preferred scheme, the nephrosis is selected from diabetic nephropathy, Focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulus inflammation, chronic transplant glomerulopathy, arteriosclerotic kidney or polycystic kidney disease.

As further preferred scheme, the cardiovascular disease is selected from arteriosclerosis, artery sclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia or hypertriglyceridemia.

As further preferred scheme, the metabolic disease is selected from insulin resistance, type-1 diabetes mellitus, type-2 diabetes mellitus or obesity；Cranial vascular disease is selected from apoplexy.

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

Another object of the present invention is to isolate a kind of substantially pure formula (I) compound, the physicochemical property of formula (I) compound can be substantially improved, clinical research needs are met；It can be used to treat the disease of FXR mediation, including diseases such as cardiovascular disease, atherosclerosis, artery sclerosis, hypercholesterolemia, hyperlipidemia chronic liver disease, chronic liver disease, gastrointestinal disease, nephrosis, cardiovascular disease, metabolic disease, cancer (such as colorectal cancer) or neural sign such as apoplexy, with extensive medical application, exploitation is expected into FXR agonist of new generation.

First aspect present invention provides a kind of substantially pure formula (I) compound, and described substantially pure formula (I) compound refers to that the optical purity of formula (I) compound reaches 90.0% or more.

As further preferred scheme, described substantially pure formula (I) compound refers to that the optical purity of formula (I) compound reaches 95.0% or more.

As scheme still more preferably, the impurity that described substantially pure formula (I) compound includes is no more than 10.0%；

Preferably, the impurity that described substantially pure formula (I) compound includes is no more than 5.0%.

As scheme still more preferably, the optical purity refers to for its non-corresponding isomers formula (II) compound:

Second aspect of the present invention provides a kind of preparation method of substantially pure formula (I) compound, includes the following steps:

1) formula (I) crude compound is dissolved in the positive solvent that mass volume ratio is 1-10 times；

2) positive 0.5-10 times of solvent volume ratio of anti-solvent is added；

3) it finishes, continues stirring and crystallizing；

4) filtering is drained；

5) filtrate decompression is concentrated to dryness to obtain substantially pure formula (I) compound.

As further preferred scheme, the positive solvent is selected from or mixtures thereof rudimentary esters solvent, halogenated alkane solvents, lower alcohols solvent.

Preferably, the positive solvent is selected from or mixtures thereof ethyl acetate, isopropyl acetate, ethyl alcohol, isopropanol, methylene chloride.

As scheme still more preferably, volume used in the positive solvent is 3-5 times of formula (I) crude compound mass volume ratio.

As further preferred scheme, the anti-solvent is selected from or mixtures thereof lower paraffin hydrocarbon solvent, cyclic alkane solvents, rudimentary ether solvent.

Preferably, the anti-solvent is selected from or mixtures thereof normal heptane, n-hexane, petroleum ether, isopropyl ether.

As scheme still more preferably, volume used in the anti-solvent is positive 1-3 times of solvent volume ratio.

As scheme still more preferably, step 5) is distilled after being concentrated to dryness with the entrainment of or mixtures thereof lower alcohols solvent, is then further concentrated to dry.

Preferably, with the entrainment distillation of or mixtures thereof ethyl alcohol, isopropanol after step 5) is concentrated to dryness.

As scheme still more preferably, (optical purity of gained compound is 90.0% or more to substantially pure formula obtained by step 5)；Substantially pure formula obtained by it is preferred that (on；The optical purity of compound is 95.0% or more.

Third aspect present invention provides a kind of substantially pure formula, and (preparation method of compound, includes the following steps: in sheet

1) being dissolved in or be scattered in mass volume ratio for formula (I) crude compound is in 0.5-10 times of the first solvent；

2) the second solvent of the first 0.5-10 times of solvent volume ratio is added；

3) continue stirring and crystallizing；

4) filtering is drained, and substantially pure formula (I) compound is obtained after filtration cakes torrefaction.

As further preferred scheme, in step 1) amount of the first solvent be formula (in 2-7 times of crude compound mass volume ratio.

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

As further preferred scheme, first solvent is selected from or mixtures thereof rudimentary esters solvent, halogenated alkane solvents, lower alcohols solvent.

Preferably, first solvent is selected from or mixtures thereof ethyl acetate, isopropyl acetate, ethyl alcohol, isopropanol, methylene chloride.

As further preferred scheme, second solvent is selected from or mixtures thereof lower paraffin hydrocarbon solvent, cyclic alkane solvents, rudimentary ether solvent.

Preferably, second solvent is selected from or mixtures thereof normal heptane, n-hexane, petroleum ether, isopropyl ether.

As scheme still more preferably, (optical purity for obtaining based compound is 90.0% or more to substantially pure formula obtained by step 4).

Preferably, (optical purity of gained compound is 95.0% or more to formula substantially pure obtained by step 4).

As further preferred scheme, aforementioned preparation process formula (for that can be pre-processed into crude compound through following steps:

1) sodium hydroxide or potassium hydroxide are dissolved in 5-100 times of lower alcohols solvent of mass volume ratio, are subsequently cooled to 5 DEG C or less；

2) it will be added in lye obtained by step 1) comprising the raw material of formula (I) compound, and be cooled to -10 DEG C or less and continue to be stirred to react；

3) acidic materials are slowly added to reacting liquid pH value about 5-6 for -10 DEG C of temperature control or less；

4) it is concentrated under reduced pressure partial solvent at temperature control is lower than 40 DEG C；

5) water and rudimentary esters solvent, liquid separation are added in residue；

6) organic phase is concentrated to dryness to obtain formula (I) crude compound.

As scheme still more preferably, the amount of lower alcohols solvent used in step 1) is 20-60 times of sodium hydroxide or potassium hydroxide quality volume ratio.

As scheme still more preferably, lower alcohols solvent used in step 1) is selected from or mixtures thereof ethyl alcohol, isopropanol.

As scheme still more preferably, esters solvent used in step 5) is selected from or mixtures thereof ethyl acetate, isopropyl acetate.

Fourth aspect present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes a effective amount of substantially pure formula (I) compound or its pharmaceutically acceptable carrier or excipient.

Fifth aspect present invention provides a kind of aforementioned substantially pure formula (application of invention compound or foregoing pharmaceutical composition in preparation for preventing or treating in the disease of FXR mediation or the drug of situation.

As further preferred scheme, the disease or situation that the FXR is mediated are selected from cardiovascular disease, hypercholesterolemia, hyperlipidemia chronic liver disease, chronic liver disease, gastrointestinal disease, nephrosis, cranial vascular disease, metabolic disease or cancer.

As scheme still more preferably, the chronic liver disease is selected from primary hardening (PBC), the dirty property xanthomatosis (CTX) of brain, primary sclerosing cholangitis (PSC), drug induced cholestasia, intrahepatic cholestasis of pregnancy, parenteral absorption associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NASH), the anti-host disease of liver transfer operation related Graft, live donor liver transfer operation regeneration, congenital hepatic fibrosis, choledocholithiasis, granular hepatopathy, malignant tumour in or beyond liver, Sjogren syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis Or 1 primary antibody membrane proteolytic enzyme deficiency disease of α.

As scheme still more preferably, the gastrointestinal disease is selected from that inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), bacterial overgrowth, nutrient absorption are bad, reflection postcolon is scorching or microscopic colitis, the preferred Crow grace disease of inflammatory bowel disease (Crohn ' s Disease) or routed characteristic of disease enteropathy.

As scheme still more preferably, the nephrosis is selected from diabetic nephropathy, Focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulus inflammation, chronic transplant glomerulopathy, arteriosclerotic kidney or polycystic kidney disease.

As scheme still more preferably, the cardiovascular disease is selected from arteriosclerosis, artery sclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia or hypertriglyceridemia.

As scheme still more preferably, the metabolic disease is selected from insulin resistance, type-1 diabetes mellitus, type-2 diabetes mellitus or obesity.

As scheme still more preferably, the cranial vascular disease is selected from apoplexy.

As scheme still more preferably, the cancer is selected from colorectal cancer or liver cancer.

Compared with prior art, present invention has the advantage that

1, the present invention solves in patent PCT/CN2016/079167 embodiment 14 products therefrom through ethyl acetate extracts and still can only obtain mixture after column chromatographic purifying the technical issues of.

2, present invention gained formula (I) compound light purity is high, highest can even reach 95% or more, and the raw material of the optical purity is conducive to further pharmacological toxicology research, is more in line with the needs of clinical research.

3, process for separating and purifying of the present invention is easy to operate, environmentally protective, and solvent for use is simple, is easy to get, and stable yield, reliable in quality are conducive to industrial application.

Another object of the present invention also resides in the method that offer prepares chlolic acid derivatives, and this method reaction condition is mild, technical maturity, and quality is stablized, and is very suitable for industrial application.

First aspect present invention provides a kind of preparation method of formula (III) compound, includes the following steps:

1) formula (IV) compound esterification obtains formula (V) compound；

2) formula (V) compound double bond restores to obtain formula (VI) compound；

3) formula (VI) compound isomerizate obtains formula (VII) compound；

4) formula (VII) compound hydroxyl protection obtains formula (VIII) compound；

5) formula (VIII) compound carbonyl reduction obtains formula (III) compound；

Reaction equation is as follows:

Wherein, R is selected from C_1-4Alkyl；Pg is hydroxy-protecting agent.

As further preferred scheme, R preferably is selected from methyl or ethyl in the preparation method.

As further preferred scheme, Pg preferably is selected from benzoyl, benzenesulfonyl substituted or unsubstituted, alkyl substituted or unsubstituted, benzyl substituted or unsubstituted or trialkyl silyl substituted or unsubstituted in the preparation method.

As scheme still more preferably, Pg preferably is selected from such as flowering structure in the preparation method:

As most preferred scheme, Pg is selected from such as flowering structure in the preparation method:

Aforementioned formula (III) compound can be used as preparation shellfish cholic acid difficult to understand and 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine key intermediate.

As further preferred scheme, in the preparation method esterification of step 1) 20 DEG C~35 DEG C at a temperature of carry out in acidic environment.

As scheme still more preferably, in the preparation method esterification of step 1) preferably 22 DEG C~27 DEG C at a temperature of carry out.

As scheme still more preferably, the esterification of step 1) carries out in acid condition in the preparation method, and the acid can be selected from or mixtures thereof hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, methanesulfonic acid.Preferably, hydrochloric acid or sulfuric acid are selected from.

As further preferred scheme, the isomerizationization reaction of step 3) is reacted in alcohols solvent using sodium alkoxide in the preparation method, and reaction temperature is 5 DEG C~35 DEG C.Preferably, reaction temperature is 15 DEG C~25 DEG C.

As scheme still more preferably, the sodium alkoxide that the isomerizationization reaction of step 3) uses in the preparation method is selected from sodium methoxide, sodium ethoxide or sodium tert-butoxide.

As scheme still more preferably, the alcohols solvent that the isomerizationization reaction of step 3) uses in the preparation method is selected from methanol, ethyl alcohol, isopropanol or the tert-butyl alcohol.

As further preferred scheme, the carbonyl reduction of formula (VIII) compound is obtained formula (III) compound in alcohols solvent using sodium borohydride by step 5) carbonyl reduction in the preparation method, and reaction temperature is -5 DEG C~10 DEG C.Preferably, reaction temperature is 0 DEG C~3 DEG C.

As scheme still more preferably, the alcohols solvent that step 5) carbonyl reduction uses in the preparation method is selected from methanol, ethyl alcohol, isopropanol or the tert-butyl alcohol.

As scheme still more preferably, in the preparation method step 5) carbonyl reduction after reaction, using acetone and citric acid quenching reaction.

Another aspect of the present invention also provides a kind of preparation method above-mentioned and is preparing the application in shellfish cholic acid difficult to understand, and formula (III) compound is prepared preparing shellfish cholic acid Shi Xianjing aforementioned preparation process difficult to understand, is then reacted as follows again:

Wherein, the acid is organic acid or inorganic acid.

As further preferred scheme, the organic acid preferably is selected from or mixtures thereof trifluoroacetic acid, trichloroacetic acid, Loprazolam, trifluoromethanesulfonic acid, p-methyl benzenesulfonic acid, formic acid, acetic acid；The inorganic acid preferably is selected from or mixtures thereof hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid.

As further preferred scheme, selective acidolysis generates formula (IX) compound to formula (III) compound preferably under lithium hydroxide effect.

Another aspect of the present invention also provides a kind of preparation method above-mentioned in preparation 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, application in 4- diketone, in preparation 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, formula (III) compound is prepared in 4- diketone Shi Xianjing aforementioned preparation process, then it is reacted as follows again:

As scheme still more preferably, 5- ((2R)-the 2- ((3R that annulation obtains, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2,4- diketone crude product use mass volume ratio for 1:15 methylene chloride mashing purification purification.

As scheme still more preferably, formula (XI) compound 5- ((2R)-the 2- ((3R obtained through methylene chloride mashing purification, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2, the HPLC purity of 4- diketone can reach 98% or more.

Preparation method of the invention compared with prior art, has the advantage that

1, the present invention is with (4R) -4- ((3R, 5R, 10R, 13R, 14R, 17R, Z) -6- ethylidene -3- hydroxyl -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl -7- carbonyl) valeric acid be raw material, the first step is with regard to first carrying out esterification, until shellfish cholic acid or 5- ((2R) -2- ((3R difficult to understand is prepared, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, reaction is not all hydrolyzed before 4- diketone, it is obtained to be respectively soluble in hydrophobic organic solvent at ester intermediate, be conducive to the post-processing of each step reaction, so that portion Divide by-product that can wash removing.Therefore, present invention process strong operability.

2, patent WO2002072598A1 is solved with 3-5 β of Alpha-hydroxy-7- ketone-cholane-24- acid and 3; 4- dihydropyran is raw material; it reacts after the protection of 3 Alpha-hydroxies with bromoethane and is carried out under the conditions of 4 upper substitution ethyls are needed at ester reaction at-70~-80 DEG C simultaneously; and need to use the defect of n-BuLi and carcinogen hexamethyl-phosphoramide; esterification reaction temperature of the present invention can carry out near room temperature, and products therefrom purity, yield are very high.Therefore, esterification of the present invention is suitble to industrial applications.

3, patent WO2006122977A2 is solved with 3-5 β of Alpha-hydroxy-7- ketone-cholane-24- acid and 3,4- dihydropyran is raw material, protects 3 Alpha-hydroxies, the defect of 6-carbonyl at needing continuously to use trim,ethylchlorosilane after ester.

4, it overcomes in patent WO2006122977A2 and is reacted after TMS is protected with aldehyde compound, it is still necessary to which the defect reacted under the conditions of -60~-90 DEG C also avoids using boron trifluoride ether solution.

5, the defect for needing just to can be carried out configuration conversion in patent WO2006122977A2 at 95~105 DEG C in configuration conversion is overcome, the present invention can be carried out configuration conversion reaction in alcohols solvent using sodium alkoxide near room temperature.

6, the defect that needs just can be carried out under 70~105 DEG C of hot conditions when obtaining shellfish cholic acid difficult to understand with sodium borohydride reduction in patent WO2006122977A2 is overcome, the present invention can be reacted using low temperature, so that impurity generates less in reaction.Citric acid is separately added when in addition, terminating and react with acetone in end of reaction, further reduces the generation of by-product.Therefore, products therefrom purity is good, high income for the reaction of this step, and process safety is also improved.

7, preparation 5- ((2R) -2- ((3R in patent PCT/CN2016/079167 is overcome, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, it needs when 4- diketone to be first esterified shellfish cholic acid difficult to understand, then obtains final product through bromo, cyclization again and obtain defect, the present invention directlys adopt formula (III) compound, the processing step for not needing first to be hydrolyzed into shellfish cholic acid difficult to understand, resterification, is greatly saved energy consumption, mitigates environmental pressure.

The present invention also provides a kind of 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethyl -3,7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2, the purification process of 4- diketone, purification process of the present invention is easy to operate, and products obtained therefrom is with high purity, high-quality, is conducive to Subsequent pharmacological exploitation.

Detailed description of the invention

Fig. 1 is the X-ray powder diffraction figure of crystalline form Compound I free alkali；Abscissa is 2 θ (°) of diffraction maximum angle, and ordinate is the intensity at peak.

Fig. 2 is the thermogravimetric analysis figure of crystalline form Compound I free alkali；Abscissa is temperature (DEG C), and ordinate is percent weight loss (%).

Fig. 3 is the differential scanning calorimetry figure of crystalline form Compound I free alkali；Abscissa is temperature (DEG C), and ordinate is hot-fluid (W/G).

Fig. 4 is that the Dynamic Water of crystalline form Compound I free alkali adsorbs DVS (Dynamic Vapour Sorption) isollaothermic chart, and abscissa is relative humidity RH (%), and ordinate is mass change percentage (%).

Specific embodiment

1, term

The term as used herein " pharmaceutically acceptable ", which refers to, to be suitble to contact with the tissue of human and animal in the range of appropriate medical care judges, without excessive toxicity, stimulation, allergic reaction or other problems complication, with it is reasonable be benefited/Hazard ratio those of matches compound, material, composition and/or dosage form.

The term as used herein " substantially pure " refers to that in the crystalline texture of certain preferred embodiments compounds of formula I of the invention be substantially pure form, HPLC purity or crystal form purity are substantially in 90% or more (including this number), it is preferred that 95% or more, more preferable 98% or more, most preferably 99.5% or more.

" polymorphic " or " polymorph " used herein refer to identical chemical composition, but constitute the crystal form of the different spaces arrangement of the molecule of the crystal, atom and/or ion.Although polymorph chemical composition having the same, but their accumulation and geometry arrangement are different, and different physical properties may be shown, as fusing point, shape, color, density, hardness, can deformation behavior, stability, solubility, dissolution rate and similarity.According to their temperature-stabilization sexual intercourse, two kinds of polymorphs can be monotropy or enantiotropic.For monotropy system, in temperature change, the relative stability between two kinds of solid phases is remained unchanged.On the contrary, in enantiotropy system, there are a transition temperatures, exchange ((Theory and Origin of Polymorphism in " Polymorphism in Pharmaceutical Solids " (1999) ISBN :) -8247-0237) in the stability of this two kinds of phases.This compound with different crystal structure there are the phenomenon that be referred to as polymorph in pharmaceuticals phenomenon.

Crystalline texture of the invention can be prepared by various methods, including crystallize or recrystallize from suitable solvent, distillation, from growth in molten mass, from it is another it is mutually solid state transformed, crystallization and jet stream are spraying etc. from supercritical fluid.The technology that crystalline texture is crystallized or recrystallized from solvent mixture, the seeding of the supersaturated solvent mixture including solvent evaporation, the temperature for reducing solvent mixture, the molecule and/or salt, anti-solvent etc. are added into solvent mixture at freezing solvent mixture.High-throughput crystallization technique preparation crystalline texture, including polymorph can be used.Medicine crystal, including polymorph, the preparation method and characterization of medicine crystal are disclosed in Solid-State Chemistry of Drugs, S.R.Byrn, R.R.Pfeiffer and J.G.Stowell, second edition, SSCI, in West Lafayette, Indiana, 1999.

In addition, as it is known by the man skilled in the art, crystal seed is added in any crystalline mixture to promote to crystallize.Therefore, mode of the crystal seed as the mode or the size distribution as control crystallized product for controlling specific crystalline texture growth also can be used in the present invention.Correspondingly, such as " Programmed cooling of batch crystallizers; " J.W.Mullin and J.Nyvlt, Chemical Engineering Science, 1971, described in 26,369-377, the calculating of required crystal seed amount depends on to obtain the size of crystal seed and the required size of average product particles.In general, needing the crystal seed of small size effectively to control the growth of crystal in this batch.Small size crystal seed can be generated by the screening of larger crystal, grinding or micronization or by the micro-crystallization of solution it should be noted that the grinding or micronization of crystal cannot cause any change (become amorphous or become another polymorphic) of the crystallinity of required crystal structure.

The crystal structure for the crystal structure equity that the present invention is disclosed or claimed may show similar but not exactly the same analytical characteristics with other often several variables well known by persons skilled in the art according to experimental condition, purity, equipment within the scope of reasonable error.Correspondingly, it should be apparent to those skilled in the art that can without departing substantially from scope and spirit of the present invention various modification can be adapted in the present invention and change.On the basis of considering specification and the practice of present invention disclosed herein, other embodiments of the present invention is that those skilled in the art are obvious.Applicant expects that the description and embodiments are considered as illustratively, rather than limit its range.

The term as used herein " room temperature " or " RT " refer to the environment temperature of 20 to 25 DEG C (68-77 °F).

The term as used herein pharmaceutical composition indicates the mixture containing one or more compounds described herein or its physiologically/pharmaceutically acceptable salt or pro-drug and other chemical constituents, with or other components such as physiology/pharmaceutically acceptable carrier and excipient.The purpose of pharmaceutical composition is the administration promoted to organism, plays bioactivity in turn conducive to the absorption of active constituent.

2, experimental material

Agents useful for same is commercially available technical grade or analytical grade reagent in the embodiment of the present invention, and selected formula (I) raw materials of compound is to be prepared according to the gloomy patent PCT/CN2016/079167 embodiment 14 of person of outstanding talent.

3, analysis method

3.1, X-ray powder diffraction

Ordinary skill will recognize that X-ray powder diffraction figure can obtain under measurement error, which depends on measuring condition used.Particularly, usually known is that the intensity in X-ray powder diffraction figure may be with material therefor conditional fluctuation.It will be further understood that relative intensity may also become with experiment condition, and correspondingly, definite intensity should not be counted into consideration.In addition, the measurement error at traditional X-ray powder diffraction angle is typically about 5% or lower, and this measurement error degree should be considered as belonging to the above-mentioned angle of diffraction.It will consequently be understood that crystal structure of the invention is not limited to provide the crystal structure with the identical X-ray diffractogram of X-ray powder diffraction figure drawn in attached drawing disclosed herein.Any crystal structure of those essentially identical X-ray powder diffraction figures disclosed in offer and attached drawing is all fallen within the scope of the present invention.Determine the essentially identical ability of X-ray powder diffraction figure in the limit of power of those of ordinary skill in the art.Other suitable standard calibrations well known by persons skilled in the art.But relative intensity may become with crystal size and shape.

Compound of formula I polymorphic is characterized with their X-ray powder diffraction figure.Therefore, in Cu K α radiation On the Rigaku UltimaIV x-ray powder diffraction instrument operated in reflection, the X-ray powder diffraction figure is acquired.Tube voltage and the magnitude of current are respectively set to 40kV and 40mA acquisition scans.The 5 minutes time of sample is scanned within the scope of 5.0 ° to 45 ° of 2 θ.In usually 20 DEG C -30 DEG C of all analyses of implementation at room temperature.The preparation of XRPD sample gently presses sample powder to ensure that sample surfaces are flat, and have height appropriate by the way that sample to be placed on monocrystalline silicon piece with sheet glass or equivalent.Then sample branch is put into Rigaku UltimaIV instrument, and acquires X-ray powder diffraction figure using above-described instrument parameter.By including that many factors below generate measurement difference relevant to this kind of X-ray powder diffraction analysis result: (a) error in sample preparation object (such as height of specimen), (b) instrument error, (c) difference is calibrated, (d) personal error (those of appearance error when being included in measurement peak position), and (e) property (such as preferred orientation error) of substance.Calibration error and sample height errors frequently result in displacement of all peaks in the same direction.In general, this calibration factor will make the peak position of measurement consistent with expected peak position and can be in the range of value ± 0.2 ° expected 2 θ.

3.2, thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) is tested in TA Instruments^TMIt is carried out in model Q500.By (about 2-10 milligrams) of sample in the platinum disk to tare in advance.Pass through instrument precise measurement example weight and records to one thousandth milligram (a thousand of a milligram).The furnace nitrogen is purged with 100 ml/mins.Data are collected with 10 DEG C/min of rates of heat addition between 300 DEG C in room temperature.

3.3, differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) is tested in TA Instruments^TMIt is carried out in model Q200.Sample (about 2-10 milligrams) is weighed in aluminium dish and is accurately recorded to 1 percent milligrams, and is transferred in DSC.The instrument nitrogen is purged with 50 ml/mins.It is received in room temperature between 300 DEG C with 10 DEG C/min of rates of heat addition Collect data.It draws in endothermic peak situation directed downwardly.But skilled person will note that, according to the rate of heat addition, crystal shape and purity and other measurement parameters, the start temperature and maximum temperature of actual measurement have a degree of changeability in dsc measurement.

The particular aspects of embodiment of the present invention will be further illustrated in specific embodiment and preparation method example presented below.The range of the following example will not limit the scope of the invention in any way.

Embodiment 1

About 15mg compound of formula I free alkali solid (unformed) is weighed in 1.5mL bottle, 0.5mL methylene chloride is added, at room temperature, after suspending stirring 24 hours, is separated by solid-liquid separation, obtains compound of formula I free alkali crystal form I.Its X-ray powder diffraction figure is as shown in Figure 1；As shown in Figure 2 (weightlessness 0.29%), differential scanning calorimetry figure is as shown in Figure 3 (132.9 DEG C of fusing point) for thermogravimetric analysis figure；Dynamic Water adsorbs DVS isollaothermic chart as shown in figure 4, DVS test condition is as follows: without N₂Under existence condition, 25.0 DEG C of temperature, relative humidity (RH): again to 0% from 0% to 95%；Stability test is as follows:

1, the stability in solvent

Accurate Weigh Compound 10mg is placed in 100mL volumetric flask, is added acetonitrile/water (V/V, 1:1), and ultrasound makes to dissolve, and obtains the solution of 0.1mg/mL.Be placed at room temperature, in 0h, 3h, 6h, for 24 hours when sampling it is appropriate, liquid phase detection calculates the relative purity with 0h.As a result as follows:

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

The results showed that the compound crystal in acetonitrile/water (V/V, 1:1) significantly degrade for 24 hours by kept stable, nothing.

2, the stability under high temperature, illumination

Weigh Compound about 4mg, is placed in 4mL vial by parallel 6 parts, be respectively placed in 25 DEG C/RH75%, 40 DEG C/RH75%, 50 DEG C, under 80 DEG C and illumination condition, place 7 days.Illumination experiment retinue carries out Quality Control experiment (i.e. quality-control sample retinue carries out powder and is protected from light experiment).It is taken out after placing 7 days, powder sample adds diluent to dissolve to obtain the sample solution of 1mg/mL, and liquid phase detects its purity, relative purity when calculating with 0 day.As a result as follows:

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

80℃

7 days

98.8

99.9

The experimental results showed that the compound crystal under 25 DEG C/RH75%, 40 DEG C/RH75%, 50 DEG C, 80 DEG C and illumination condition, is placed 7 days, without significant degradation, stabilization Z can be continuously maintained.

Liquid phase analysis condition is as follows:

1), instrument and equipment

Instrument title	Model
Assay balance	Sartorius BSA224S-CW
Water purification machine	Milli-Q Plus, Millipore
High performance liquid chromatograph	Agilent1260
Pump	Agilent G1311B
Sample injector	G1329B
Column oven	G1316A
Detector	G1315D

2), chromatographic condition

Embodiment 2

About 15mg compound of formula I free alkali solid (unformed) is weighed in 1.5mL bottle, 0.5mL normal heptane is added, at room temperature, after suspending stirring 24 hours, it is separated by solid-liquid separation, obtains compound of formula I free alkali crystal form I, X-ray powder diffraction figure is substantially consistent with Fig. 1.

Embodiment 3

About 15mg compound of formula I free alkali solid (unformed) is weighed in 1.5mL bottle, 0.5mL water is added, at room temperature, after suspending stirring 24 hours, it is separated by solid-liquid separation, obtains compound of formula I free alkali crystal form I, X-ray powder diffraction figure is substantially consistent with Fig. 1.

Embodiment 4

About 50mg compound of formula I free alkali solid is weighed in 1.5mL bottle, 0.8mL methylene chloride is added, at room temperature, it after stirring 48 hours, is separated by solid-liquid separation, the volatilization of room temperature opening is overnight, compound of formula I free alkali crystal form I is obtained, X-ray powder diffraction figure is substantially consistent with Fig. 1.

Term and specific embodiment in chlolic acid derivatives preparation method of the present invention:

“C_1-4Alkyl " refers to that straight chained alkyl and containg branched alkyl radical including 1 to 4 carbon atom, alkyl refer to aliphatic hydrocarbon group of saturation, such as methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, tert-butyl, sec-butyl etc..

" alcohols solvent " refers to the alkane compound referred in molecule containing hydroxyl, such as methanol, ethyl alcohol, isopropanol.

The compound of the present invention structure is by nuclear magnetic resonance (NMR) or/and LC-MS chromatography (LC-MS) come what is determined.Nmr chemical displacement (δ) is provided with the unit of hundred a ten thousandths (ppm).The measurement of NMR is to use Bruker AVANCE-400 nuclear magnetic resonance spectrometer, and measurement solvent is deuterated dimethyl sulfoxide (DMSO-d₆), deuterated methanol (CD₃) and deuterated chloroform (CDCl OD₃) in be designated as tetramethylsilane (TMS).

The measurement of LC-MS chromatography LC-MS Agilent 1200Infinity Series mass spectrograph.The measurement of HPLC uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire 150 × 4.6mm of C18 chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini 150 × 4.6mm of C18 chromatographic column).

Tlc silica gel plate uses Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica gel plate, and the specification that TLC is used is 0.15mm~0.20mm, and the specification that thin-layer chromatography isolates and purifies product use is 0.4mm~0.5mm.Column chromatography is generally carrier using 200~300 mesh silica gel of Yantai Huanghai Sea silica gel.

Starting material in the embodiment of the present invention is known and can be commercially available, or can use or synthesize according to methods known in the art.

In the case where no specified otherwise, all reactions of the invention carry out under drying nitrogen or argon atmospher under continuous magnetic agitation, and solvent is dry solvent.

Embodiment 5

Step 1: the preparation of methyl (4R) -4- (ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of (3R, 5R, 10R, 13R, 14R, 17R, Z) -6- ethylidene -3- hydroxyl -10,13- dimethyl -7- carbonyl) valerate

(4R) -4- ((3R is added into reaction flask, 5R, 10R, 13R, 14R, 17R, Z) -6- ethylidene -3- hydroxyl -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl -7- carbonyl) valeric acid 50.0g (0.12mol) and 500ml methanol, concentrated sulfuric acid 1.5g (0.015mol) is added dropwise at 22 DEG C~27 DEG C, drop Bi Jixu is stirred to react overnight, TLC analysis raw material fundamental reaction finishes, 15 DEG C~25 DEG C dropwise addition 10ml saturated sodium bicarbonate solutions of temperature control, most of methanol is removed in reduced pressure, 500ml ethyl acetate and 200ml saturated sodium bicarbonate solution is added, stir 20min, liquid separation, organic layer 120ml saturated sodium chloride solution It washes once, anhydrous sodium sulfate is dry, is concentrated to dryness to obtain blister solid product 50.2g (yield: 97.3%, HPLC:98.6%).HPLC analysis method is as follows:

■ mobile phase A: water+0.05%TFA

■ Mobile phase B: ACN+0.04%TFA

■ pillar: Agilent XDB C-18 5um 4.6x150mm

■ Posttime:5min

■ column temperature: 25 DEG C

■ flow velocity: 1ml/min

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

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

Step 2: the preparation of methyl (4R) -4- (ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of (3R, 5S, 6S, 10S, 13R, 14R, 17R) -6- ethyl -3- hydroxyl -10,13- dimethyl -7- carbonyl) valerate

Methyl (4R) -4- ((3R is added into reaction flask, 5R, 10R, 13R, 14R, 17R, Z) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethylidene -3- hydroxyl -10,13- dimethyl -7- carbonyl) valerate 49g (0.114mol), 10% palladium charcoal of 4.9g and 250ml ethyl alcohol, hydrogen displacement 3 times, under nitrogen atmosphere, it is stirred overnight, raw material fully reacting, filters at 15~25 DEG C, filtrate decompression is concentrated to dryness, and methanol entrainment distillation is added and obtains steeping last shape solid product 46.3g (yield: 94.1%).

Step 3: the preparation of methyl (4R) -4- (ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of (3R, 5S, 6R, 10S, 13R, 14R, 17R) -6- ethyl -3- hydroxyl -10,13- dimethyl -7- carbonyl) valerate

Methyl (4R) -4- ((3R is added into reaction flask, 5S, 6S, 10S, 13R, 14R, 17R) -6- ethyl -3- hydroxyl -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl -7- carbonyl) valerate 40g (0.092mol) and 200ml methanol, it is cooled to 0~5 DEG C；Take another reaction flask that 200ml methanol is added, 25 DEG C of temperature control or less, sodium methoxide 25.0g (0.462mol) is added portionwise, agitating solution is extremely clarified, sodium methoxide solution is added dropwise in first reaction flask at 0~5 DEG C, drop, which finishes, to be warmed naturally to 15~25 DEG C and is stirred overnight, and TLC detection raw material fully reacting finishes (TLC: petroleum ether: ethyl acetate=1:1, phosphomolybdic acid colour developing).Take a reaction flask that 120ml methanol is added again, it is cooled to 0-5 DEG C, it is added dropwise concentrated sulfuric acid 27.2g (0.277mol), sulfuric acid solution is added dropwise in first reaction flask at 15~25 DEG C, drop, which finishes, to be warmed naturally to 15~25 DEG C again and is stirred overnight, TLC detects hydrolysis impurity conversion (TLC: petroleum ether: ethyl acetate=1:1, phosphomolybdic acid colour developing) completely.120ml saturated sodium bicarbonate solution is added dropwise at 15 DEG C~25 DEG C of temperature control, above-mentioned reaction solution pH is adjusted to 7~8, methanol is removed in reduced pressure, 400ml ethyl acetate, liquid separation after stirring is added, organic phase successively respectively washed once with 120ml saturated sodium bicarbonate solution, 120ml saturated salt solution, anhydrous sodium sulfate is dry, it is concentrated to dryness, tetrahydrofuran entrainment distillation is added and obtains foaming solid product 40.8g (yield: 100%, HPLC:99.7%).

Step 4: (3R, 5S, 6R, 10S, 13R, 14R, 17R) the preparation of -6- ethyl -17- ((R) -5- methoxyl group -5- carbonyl pentane -2- base) -10,13- dimethyl -7- carbonyl ten hexahydro -1H- cyclopenta [a] phenanthrene -3- base -4- nitrobenzene acid esters

Methyl (4R) -4- ((3R is added into reaction flask, 5S, 6R, 10S, 13R, 14R, 17R) -6- ethyl -3- hydroxyl -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl -7- carbonyl) valerate 40g (0.092mol), 400ml tetrahydrofuran, diisopropylethylamine 29.9g (0.231mol) and 4-dimethylaminopyridine 1.13g (0.009mol) are added after stirring evenly, mixed liquor is cooled to 0-5 DEG C, paranitrobenzoyl chloride 34.3g (0.185mol) is added portionwise at 0~5 DEG C, it adds and continues to be stirred to react 30min, then heat to 45~55 DEG C of stirrings 2-4 hours, TLC detects raw material reaction (TLC: petroleum ether: ethyl acetate=1:1, phosphomolybdic acid colour developing) completely.It is cooled to 0~5 DEG C, 200ml water is added dropwise, methyl tertiary butyl ether(MTBE) (200mL) is added after stirring 20min, stir liquid separation, organic phase successively respectively washed once with 200ml hydrochloric acid (1mol/L), 160ml saturated sodium bicarbonate, saturated salt solution, anhydrous sodium sulfate is dry, is concentrated to dryness, and methyl tertiary butyl ether(MTBE) entrainment is added and distills to dry.160ml methyl tertiary butyl ether(MTBE) is added in residue, is heated to reflux 1 hour, it is cooled to 40~ 50 DEG C, 320ml normal heptane is added dropwise, drop, which finishes, to be naturally cooling to room temperature and be stirred overnight, and is filtered, is obtained off-white powder product 48.3g (yield: 89.8%, HPLC:98.8%) after filtration cakes torrefaction.HPLC analysis method is as follows:

■ mobile phase A: water+0.05%TFA

■ Mobile phase B: ACN+0.04%TFA

■ pillar: Agilent Zorbax SB C-18 5um 4.6x150mm

■ Posttime:5.0min

■ column temperature: 40 DEG C

■ flow velocity: 1ml/min

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

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

¹HNMR:(CDCl3,400MHz) δ 0.67 (s, 3H), 0.82 (t, J=7.6Hz, 3H), 0.93 (d, J=6.4Hz, 3H), (1.09-1.19 m, 4H), 1.26-1.56 (m, 12H), 1.71-2.02 (m, 9H), (2.16-2.26 m, 2H), 2.32-2.43 (m, 2H), 2.74-2.79 (m, 1H), (3.67 s, 3H), 4.91-4.98 (m, 1H), 8.16-8.18 (m, 2H), 8.26-8.28 (m, 2H).

Impurity structure elucidation is as follows:

¹HNMR:(DMSO-d6,400MHz) δ 0.62 (s, 3H), 0.78 (t, J=7.2Hz, 3H), 0.87-1.94 (m, 31H), 2.18-2.24 (m, 1H), 2.29-2.35 (m, 1H), 2.86-2.93 (m, 1H), 3.58 (s, 3H), (3.81 d, J=8.4Hz, 1H), 4.81-4.88 (m, 1H), 8.18-8.20 (m, 2H), 8.33-8.35 (m, 2H).

Step 5: (3R, 5S, 6R, 7R, 10S, 13R, 14R, 17R) -6- ethyl -7- hydroxyl -17- ((R) -5- methoxyl group -5- carbonyl pentane -2- base) -10,13- dimethyl ten hexahydro -1H- cyclopenta [a] phenanthrene -3- base -4- nitrobenzene acid esters preparation

Under nitrogen protection, (3R is added in reaction flask, 5S, 6R, 10S, 13R, 14R, 17R) -6- ethyl -17- ((R) -5- methoxyl group -5- carbonyl pentane -2- base) -10, 13- dimethyl -7- carbonyl ten hexahydro -1H- cyclopenta [a] phenanthrene -3- base -4- nitrobenzene acid esters 5g (8.59mmol) and 30ml tetrahydrofuran, stir dissolved clarification, 30ml methanol is added, it is cooled to 0~2 DEG C, it is added sodium borohydride 0.325g (8.359mmol), continue to be stirred to react 8 hours at 0 DEG C of temperature control, TLC detects raw material end of reaction, 3.2ml acetone is instilled in 5min, drop finishes stirring 30min, then anhydrous citric acid 1.65g (8.59mmol is added ), continue to stir 5min, 25ml methyl tertiary butyl ether(MTBE) and 20ml water is added.Organic solvent is removed under reduced pressure, the extraction of 50ml methyl tertiary butyl ether(MTBE) is added, organic phase successively uses 20 Ml water and 10ml saturated salt solution respectively washed once, and anhydrous sodium sulfate is dry, and methyl tertiary butyl ether(MTBE) is removed under reduced pressure, and be distilled with isopropanol entrainment to dry.40ml isopropanol is added in residue, 80 DEG C of reflux are heated to, are naturally cooling to 60 DEG C, solid is gradually precipitated (if not being precipitated, crystal seed 20mg can be added), 60 DEG C insulated and stirred 1 hour, then Temperature fall was down to 20 DEG C after about 3 hours, continue cool to 0 DEG C, it is filtered after stirring 30min, 40 DEG C of filter cake vacuum drying obtain white solid product 4.33g (yield: 86.7%, HPLC:99.2%) in 3 hours.

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

Impurity structure elucidation is as follows:

Under nitrogen protection, (3R is added in reaction flask, 5S, 6R, 7R, 10S, 13R, 14R, 17R) -6- ethyl -7- hydroxyl -17- ((R) -5- methoxyl group -5- carbonyl pentane -2- base) -10, 13- dimethyl ten hexahydro -1H- cyclopenta [a] phenanthrene -3- base -4- nitrobenzene acid esters 250mg (0.43mmol), then 2ml methanol is sequentially added, 1ml tetrahydrofuran, 0.5ml water, sodium hydroxide 51mg (1.28mmol), continue to be stirred to react 16 hours at 25 DEG C of temperature control, solvent is removed under reduced pressure, dilute hydrochloric acid is added and is adjusted to pH=3~4, 20ml ethyl acetate is added, separate ethyl acetate layer, organic phase is washed 2 times with 10ml sodium bicarbonate aqueous solution respectively , solvent is removed under reduced pressure and obtains white foam 161mg (yield 89.4%).

Embodiment 7

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

(3R will be added in reaction flask, 5S, 6R, 7R, 10S, 13R, 14R, 17R) -6- ethyl -7- hydroxyl -17- ((R) -5- methoxyl group -5- carbonyl pentane -2- base) -10,13- dimethyl ten hexahydro -1H- cyclopenta [a] phenanthrene -3- base -4- nitrobenzene acid esters 4.33g (7.42mmol) and 26ml tetrahydrofuran, stirring and dissolving is cooled to 0~5 DEG C, and LiOH.H is added dropwise₂Methanol (13ml) solution of O (0.58g, 13.8mmol), about 10min are dripped off, are stirred to react 1h at 0~5 DEG C of temperature control, warm naturally to 18~22 DEG C, continue to be stirred to react 5~8h, TLC detects raw material substantially completely.Reaction solution is added dropwise to 50ml to be cooled in advance in 0 DEG C of saturated aqueous ammonium chloride, decompression boils off solvent, 50ml ethyl acetate is added in residue, liquid separation after stirring, organic phase successively respectively washed once with 4% aqueous potassium phosphate solution of 50ml and saturation 15ml saturated salt solution, anhydrous sodium sulfate is dry, is concentrated to dryness to obtain foam-like product 2.9g (yield: 90.1%, HPLC:100%).

Step 2: the preparation of shellfish cholic acid difficult to understand

Under nitrogen protection, methyl (4R) -4- ((3R is added into reaction flask, 5S, 6R, 7R, 10S, 13R, 14R, 17R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) valerate 2.9g (6.68mmol), then 10ml methanol is sequentially added, 5ml tetrahydrofuran, 5ml water, sodium hydroxide 0.54g (13.36mmol), continue to be stirred to react 5 hours at 25 DEG C of temperature control, solvent is removed under reduced pressure, dilute hydrochloric acid is added and is adjusted to pH=3~4, 30ml ethyl acetate is added, separate ethyl acetate layer, organic phase is washed with 10ml saturated sodium-chloride, solvent is removed under reduced pressure and obtains white foam solid 2.8g (yield 100%).

Embodiment 8

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

Diisopropylamine 144.3g (1.426mol) is dissolved in 1000ml tetrahydrofuran, it is cooled to the hexane solution (2.4mol/L, 1.188mol) that 495ml n-BuLi is added dropwise at -50 DEG C, -50 DEG C~-40 DEG C of temperature control, about 30min is added, and continues insulated and stirred 1h.By methyl (4R) -4- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) valerate 102.9g (237.7mmol) is dissolved in 500ml tetrahydrofuran.It is instilled in n-butyllithium solution at -60 DEG C~-50 DEG C of temperature control, about 50min is added, and thick white solid occurs in dropwise addition process.Continue to be stirred to react 1h at -70 DEG C~-65 DEG C of temperature control.Trim,ethylchlorosilane 154.9g (1.426mol) is added dropwise at -70 DEG C~-60 DEG C of temperature control, about 15min is added, then continues to be stirred to react 3h at -60 DEG C~-50 DEG C of temperature control.TLC detection raw material fundamental reaction finishes.N- bromo-succinimide 129.6g (728.2mmol) is added portionwise, finishes and warms naturally to 22~27 DEG C and protect Temperature is stirred to react 22h, and TLC detection raw material fundamental reaction finishes.Reaction mixture is cooled to 0 DEG C, 1000ml saturated sodium bicarbonate solution is added dropwise at 10 DEG C of temperature control, liquid separation after stirring, organic phase is concentrated into about 500ml, water phase is extracted with 500ml methyl tertiary butyl ether(MTBE), merge organic phase concentrate and methyl tertiary butyl ether(MTBE) extract liquor, it is primary with 1000ml washing, 250ml 1mol/L hydrochloric acid is added in above-mentioned organic phase, 5h is stirred at room temperature, liquid separation, organic phase washed once with 20% sodium sulfite solution of 1500ml, it is washed twice again with 1000ml*2 saturated sodium bicarbonate solution, anhydrous sodium sulfate is dry, filtering, it is concentrated to dryness to obtain viscous brownish solid 134.8g (crude yield: 100%, HPLC:99.5%, include two bromo-derivative epimers).It direct plunges into and reacts in next step.

Step 2: 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) propyl) thiazolidine preparation

Bromo- the 4- ((3R of methyl (4R) -2- that upper step is reacted, 5S, 6R, 7R, 10S, 13R) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethyl -3,7- dihydroxy -10,13- dimethyl) valerate crude product 134.8g (237.7mmol) and thiocarbamide 54.3g (713.1mmol) be dissolved in 1000ml ethyl alcohol.Mixed liquor is heated to 70 DEG C and is stirred to react 5h.TLC detects raw material fully reacting.1200ml 2mol/L hydrochloric acid is added into reaction solution, continues to be stirred to react 30h at 72 DEG C of temperature control, is cooled to room temperature and (grease may be precipitated).It is concentrated under reduced pressure ethyl alcohol, residue is extracted with 1200ml ethyl acetate, liquid separation, and organic phase is washed twice with 1200ml*2, and anhydrous sodium sulfate is dry, is concentrated to dryness to obtain brown colored foams solid 133.9g (crude product HPLC:85.4%).

Purification: taking brown colored foams solid 15.0g obtained above, is added 150ml methylene chloride, and stirring to pulp 1 hour, filtering, filter cake is washed with the methylene chloride being pre-chilled on a small quantity, and off-white powder 7.9g is obtained after vacuum drying (HPLC:99.2% includes two non-corresponding isomers).

The term and specific embodiment of optical isomer compound of the present invention are as follows:

" optical purity (optical purity) " is to measure the measurement that an enantiomer in optical activity sample is more than the amount of another enantiomer, is primarily referred to as formula (I) compound in the present invention comprising the ratio in formula (I) compound and formula (II) compound mixture.

" lower paraffin hydrocarbon solvent " refers under room temperature, the liquid alkane containing 5~10 carbon atoms, such as n-hexane, normal heptane.

The saturated hydrocarbons that " cyclic alkane solvents " refer to containing alicyclic structure and are in a liquid state at normal temperature, such as pentamethylene, hexamethylene.

" halogenated alkane solvents " refer to the alkane containing halogen atom being in a liquid state under room temperature, such as methylene chloride, chloroform.

" lower alcohols solvent " refers to the alkane compound that the carbon atom number in molecule containing hydroxyl is lower than 12, such as methanol, ethyl alcohol, isopropanol.

" rudimentary esters solvent " refers to that carbon atom quantity lacks the ester type compound being in a liquid state under room temperature, and the acid of preferably shorter than 4 carbon atoms reacts the ester type compound generated, such as ethyl acetate, methyl acetate, isopropyl acetate with the alcohol lower than 4 carbon atoms.

" rudimentary ether solvent " refers to the ether compound that two few alkyl of carbon atom quantity are formed, the ether compound that preferably two alkyl are lower than 4 carbon atoms, such as ether, isopropyl ether, methyl isopropyl ether.

Embodiment 9 (raw material preparation)

Shellfish cholic acid 100.0g (237.7mmol) difficult to understand and the concentrated sulfuric acid (0.25g) are dissolved in 500ml methanol.It is heated to flowing back and continues to be stirred to react 16h.TLC detects end of reaction, reaction solution is concentrated to dryness, 600ml ethyl acetate and 400ml saturated sodium bicarbonate solution are added in residue, rear stratification is sufficiently stirred, organic phase washed once with 400ml saturated sodium chloride solution, anhydrous sodium sulfate is dry, filtering, is concentrated to dryness, and 200ml toluene dissolution residual substance is added, it is concentrated to dryness to obtain light brown foam solid 102.9g again, direct plunges into and reacts in next step.

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

By diisopropylamine 144.3g (1.426mol, it 6.0eq.) is dissolved in 1000ml tetrahydrofuran, it is cooled to -50 DEG C, -50 DEG C~-40 DEG C dropwise addition n-butyllithium solution 495ml (1.188mol of temperature control, 2.4M hexane solution, 5.0eq.), it adds within about 30 minutes, continues temperature control stirring 1 hour.

Methyl (4R) -4- ((3R, 5S, the 6R that upper step is prepared, 7R, 10S, 13R, 14R, 17R) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethyl -3,7- dihydroxy -10,13- dimethyl) valerate 102.9g (above step theoretical amount 237.7mmol calculate) is dissolved in 500ml tetrahydrofuran, it instills in above-mentioned reaction solution, it -60 DEG C~-50 DEG C of temperature control, is added dropwise within about 50 minutes, occurs thick white solid in reaction.Continue to be stirred to react 1 hour 1h at -70 DEG C~-65 DEG C.Trim,ethylchlorosilane (TMSCl) 154.9g (1.426mol, 6.0eq.) is added dropwise at -70 DEG C~-60 DEG C of temperature control, is added dropwise within about 15 minutes, continues to be stirred to react 3 hours at -60 DEG C~-50 DEG C, TLC detection raw material fundamental reaction finishes.

N- bromo-succinimide (NBS) 129.6g (728.2mmol, 3.06eq.) is added portionwise, adds within about 30-45 minutes, reaction temperature is raised to -40 DEG C from -60 DEG C.Then room temperature (22 DEG C) is raised to naturally to continue to be stirred to react 24 hours.TLC detection raw material fundamental reaction finishes, and reaction solution is cooled to 0 DEG C, temperature control is less than 1000ml saturated sodium bicarbonate solution is added dropwise at 10 DEG C.Partial solvent (surplus about 500ml) is fallen in liquid separation after stirring, organic phase concentration, and water phase is extracted with 500ml methyl tertiary butyl ether(MTBE) (MTBE), merges organic phase concentrate and methyl tertiary butyl ether(MTBE) extract liquor, with amalgamation liquid of 1000ml water washing.

Into above-mentioned amalgamation liquid plus 1mol/L hydrochloric acid 250ml, room temperature (22 DEG C) are stirred to react 5 hours.Organic phase is separated, organic phase washed once with 20% sodium sulfite solution of 1500ml, then be washed twice respectively with 1000ml sodium bicarbonate solution, anhydrous sodium sulfate is dry, filtering, filtrate decompression are concentrated to dryness to obtain viscous brownish solid 134.8g, direct plunge into and react in next step.

Step 3: 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) propyl) thiazolidine preparation

Bromo- the 4- ((3R of methyl (4R) -2- that upper step is prepared, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) valerate 134.8g crude product (the above step theoretical amount calculates 237.7mmol and calculates) and thiocarbamide 54.3g (713.1mmol, 3.0eq.) be dissolved in 1000ml ethyl alcohol.70 DEG C are heated to be stirred to react 5 hours.TLC detection raw material fundamental reaction finishes.1200ml 2mol/L hydrochloric acid is added, 72 DEG C of temperature control are continued to be stirred to react 30 hours, it is concentrated under reduced pressure most of alcohol solvent, it is extracted with 1200ml ethyl acetate, organic phase uses 1000ml water washing twice respectively, anhydrous sodium sulfate is dry, filtering, filtrate decompression is concentrated to dryness to obtain brown colored foams solid 133.9g crude product (HPLC (comprising formula (II) compound and formula (I) compound): 85.5%,, in which: formula (II) compound: formula (I) compound=24.2%:61.3% ≈ 2:5).

Separately feed intake 200.0g Austria shellfish cholic acid, and according to above-mentioned first to three-step reaction, the experimental result being prepared is as follows:

Merge 5- ((2R)-the 2- ((3R of two batch reaction of front, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, 4- diketone crude product (406.5g), silica gel column chromatography (eluent is petroleum ether: ethyl acetate: tetrahydrofuran=3:1:1) obtains brown colored foams solid 350.1g (HPLC (comprising formula (II) compound and formula (I) compound): 85.7%, wherein: formula (II) compound: formula (I) compound=24.8%:62.9% ≈ 2:5).

Purification process one: taking above-mentioned brown colored foams solid 15.0g, be sufficiently beaten 2 hours with 150ml methylene chloride, and filtering, filter cake is washed with methylene chloride, and off-white powder 7.9g is obtained after drying, and (HPLC (includes formula (II) compound and formula (I) compound): 99.2%, in which: formula (II) compound: formula (I) compound=24.7%:74.5%% ≈ 1:3).

Purification process two: the filtrate in purification process one is concentrated to dryness, and is merged with the remaining 335.1g solid of above-mentioned brown colored foams solid, and 1500ml methylene chloride is added, and first stirs dissolved clarification (will become cloudy after continuing stirring).1200ml normal heptane is added dropwise, washes out solid, room temperature continues stirring 16 hours.Cake filtration dries to obtain off-white powder 240.0g (HPLC (comprising formula (II) compound and formula (I) compound): 97.0%, in which: formula (II) compound: formula (I) compound=27.6%:69.4%% ≈ 2:5).

Embodiment 10 (conversion reaction)

Sodium hydroxide 32.2g (805mmol) is dissolved in 1920ml ethyl alcohol, is cooled to 5 DEG C.9 gained off-white powder 240.0g of embodiment is added, is cooled to -20 DEG C and is stirred to react 19 hours.4mol/L hydrochloric acid is added dropwise at -20 DEG C to reacting liquid pH value about 5-6, temperature control, which is lower than at 40 DEG C, is concentrated under reduced pressure most of ethyl alcohol, and 1000ml water and 1500ml ethyl acetate are added in residue, stirs liquid separation.Organic phase washed once with 1000ml saturated common salt aqueous solution, anhydrous sodium sulfate is dry, filtering, filtrate decompression is concentrated to dryness to obtain brown colored foams solid 270.0g (HPLC (comprising formula (II) compound and formula (I) compound): 95.9%, in which: formula (II) compound: formula (I) compound=13.4%:82.5%% ≈ 0.8:5).

Embodiment 11 (polishing purification)

The brown colored foams solid 270.0g that embodiment 10 is prepared is dissolved in 960ml ethyl acetate, 960ml normal heptane is then added dropwise, is added dropwise and is stirred at room temperature 48 hours.Filtering, filter cake is washed with mixed solvent (150ml petroleum ether+150ml ethyl acetate), filter cake dry 24.8g off-white powder (HPLC is (comprising formula (comprising compound and formula (chelate compound): 100.0%, in which: formula (wherein compound: formula (chelate compound=65.7%:34.3% ≈ 9.6:5).Filtrate decompression is concentrated to dryness, again with the entrainment distillation of 100ml ethyl alcohol, finally it is concentrated to dryness to obtain brown colored foams solid 245g (HPLC (comprising formula (II) compound and formula (I) compound): 100.0%, in which: formula (II) compound: formula (I) compound=7.4%:88.8% ≈ 0.42:5).

Above-mentioned 245g brown colored foams solid is dissolved in 400ml ethyl alcohol, 1720ml methylene chloride and 2150ml normal heptane is successively added, mixed liquor will become cloudy after five minutes.Continue stirring 17 hours at room temperature.Filtering, filter cake 100ml methylene chloride and 300ml normal heptane mixing, washing filter cake, then 700ml normal heptane filter wash cake is used, it drains, for filter cake 80, lower vacuum drying obtains white powdery solids 143.2g in 6 hours.(HPLC (includes formula (II) compound and formula (I) compound): 99.7%, in which: formula (II) compound: formula (I) compound=2.7%:97.0%), identify that retention time is formula (I) compound in 8.53min, and retention time is formula (II) compound in 7.57min through HNMR and LC-MS).

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

Finally it should be noted that, it is of the invention the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although being described the invention in detail referring to preferred embodiment, those skilled in the art should understand that, it can be with modification or equivalent replacement of the invented technical scheme, without departing from the spirit and scope of the technical solution of the present invention, should all cover in scope of the presently claimed invention.

Claims

A kind of crystal type (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2,4- diketone free alkali, it is characterised in that, its x-ray diffractogram of powder includes being located at 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, the peak at 20.4 ± 0.2 ° of the angle of diffraction (2 θ)；

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

Preferably, x-ray diffractogram of powder further comprises the peak 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 ° angles of diffraction (2 θ).
A kind of crystal type (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2, the preparation method of 4- diketone free alkali, it is characterized by comprising the following steps:

1) by (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine free alkali is dissolved or dispersed in the in the mixed solvent of organic solvent, water or organic solvent and water；

2) it precipitates crystal, or anti-solvent is added optionally into compound clear solution and is precipitated, or the clear solution for the compound that optionally slowly volatilizees is precipitated, or former compound solid or other solid particle additives are added as heteronuclear crystal seed optionally into the compound solution and induce crystallization, or the above method is used in combination and obtains the crystalline solid；

Preferably, the organic solvent is selected from or mixtures thereof methanol, ethyl alcohol, isopropanol, acetonitrile, acetone, ethyl acetate, isopropyl acetate, toluene, n-butanol, hexamethylene, methylene chloride, dimethylformamide, dimethyl acetamide, dimethyl sulfoxide, dioxane, ether, normal heptane, n-hexane, methyl ethyl ketone, isooctane, pentane, two propyl alcohol, tetrahydrofuran, dimethyl-tetrahydrofuran, trichloroethanes, dimethylbenzene；Or mixtures thereof more preferable ethyl acetate, isopropyl acetate, methylene chloride, normal heptane, n-hexane；

Preferably, step 2) crystalline solid x-ray diffractogram of powder obtained includes the peak at 16.5 ± 0.2 °, 13.6 ± 0.2 °, 12.1 ± 0.2 °, 20.4 ± 0.2 ° of the angle of diffraction (2 θ)；

Further preferred, a kind of pharmaceutical composition, described pharmaceutical composition include crystal type (5R) -5- ((2R) -2- ((3R, 5S described in a effective amount of claim 1,6R, 7R, 10S, 13R) -6- ethyl -3,7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2,4- diketone free alkali, pharmaceutically acceptable carrier or excipient；

Further preferred, crystal type (5R) -5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) -6- ethyl -3, 7- dihydroxy -10, ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of 13- dimethyl) propyl) thiazolidine -2, 4- diketone free alkali, or application of the pharmaceutical composition in the drug for preparing the disease or situation for preventing or treating FXR mediation, preferably, the disease or situation that FXR is mediated are selected from cardiovascular disease, hypercholesterolemia, hyperlipidemia chronic liver disease, chronic liver disease, gastrointestinal disease, nephrosis, cranial vascular disease, metabolic disease or cancer； More preferably, chronic liver disease is selected from primary hardening (PBC), the dirty property xanthomatosis (CTX) of brain, primary sclerosing cholangitis (PSC), drug induced cholestasia, intrahepatic cholestasis of pregnancy, parenteral absorption associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NASH), the anti-host disease of liver transfer operation related Graft, live donor liver transfer operation regeneration, congenital hepatic fibrosis, choledocholithiasis, granular hepatopathy, malignant tumour in or beyond liver, Sjogren syndrome, sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis or α₁Primary antibody membrane proteolytic enzyme deficiency disease；Gastrointestinal disease is selected from that inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), bacterial overgrowth, nutrient absorption are bad, reflection postcolon is scorching or microscopic colitis, the preferred Crow grace disease of inflammatory bowel disease (Crohn ' s Disease) or routed characteristic of disease enteropathy；Nephrosis is selected from diabetic nephropathy, Focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulus inflammation, chronic transplant glomerulopathy, arteriosclerotic kidney or polycystic kidney disease；Cardiovascular disease is selected from arteriosclerosis, artery sclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia or hypertriglyceridemia；Metabolic disease is selected from insulin resistance, type-1 diabetes mellitus, type-2 diabetes mellitus or obesity；Cranial vascular disease is selected from apoplexy；Cancer is selected from colorectal cancer or liver cancer.
A kind of substantially pure formula (I) compound, it is characterized in that, described substantially pure formula (I) compound refers to that the optical purity of formula (I) compound reaches 90.0% or more, preferably, described substantially pure formula (I) compound refers to that the optical purity of formula (I) compound reaches 95.0% or more；Preferably, the impurity that described substantially pure formula (I) compound includes is no more than 10.0%, more preferably no more than 5.0%；

Preferably, the optical purity refers to for its non-corresponding isomers formula (II) compound:

It is further preferred that a kind of pharmaceutical composition, includes a effective amount of substantially pure formula (I) compound or its pharmaceutically acceptable carrier or excipient；

It is further preferred that substantially pure formula (I) compound or its pharmaceutical composition are in preparation for preventing or treating the application in the disease of FXR mediation or the drug of situation；Preferably, the disease or situation that FXR is mediated are selected from cardiovascular disease, hypercholesterolemia, hyperlipidemia chronic liver disease, chronic liver disease, gastrointestinal disease, nephrosis, cranial vascular disease, metabolic disease or cancer；More preferably, chronic liver disease is selected from primary hardening (PBC), the dirty property xanthomatosis (CTX) of brain, primary sclerosing cholangitis (PSC), drug induced cholestasia, intrahepatic cholestasis of pregnancy, parenteral absorption associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NASH), the anti-host disease of liver transfer operation related Graft, live donor liver transfer operation regeneration, congenital hepatic fibrosis, choledocholithiasis, granular hepatopathy, it is pernicious swollen in or beyond liver Tumor, Sjogren syndrome, sarcoidosis, Wilson's disease, 1 primary antibody membrane proteolytic enzyme deficiency disease of Gaucher's disease, hemochromatosis or α；Gastrointestinal disease is selected from that inflammatory bowel disease (I BD), irritable bowel syndrome (IBS), bacterial overgrowth, nutrient absorption are bad, reflection postcolon is scorching or microscopic colitis, the preferred Crow grace disease of inflammatory bowel disease (Crohn ' s Disease) or routed characteristic of disease enteropathy；Nephrosis is selected from diabetic nephropathy, Focal segmental glomerulosclerosis (FSGS), hypertensive nephropathy, chronic glomerulus inflammation, chronic transplant glomerulopathy, arteriosclerotic kidney or polycystic kidney disease；Cardiovascular disease is selected from arteriosclerosis, artery sclerosis, atherosclerosis, dyslipidemia, hypercholesterolemia or hypertriglyceridemia；Metabolic disease is selected from insulin resistance, type-1 diabetes mellitus, type-2 diabetes mellitus or obesity；Cranial vascular disease is selected from apoplexy；Cancer is selected from colorectal cancer or liver cancer.
A kind of preparation method of substantially pure formula (I) compound, which comprises the steps of:

1) formula (I) crude compound is dissolved in the positive solvent that mass volume ratio is 1-10 times；

2) positive 0.5-10 times of solvent volume ratio of anti-solvent is added；

3) it finishes, continues stirring and crystallizing；

4) filtering is drained；

5) filtrate decompression is concentrated to dryness to obtain substantially pure formula (I) compound；

Preferably, the positive solvent is selected from or mixtures thereof rudimentary esters solvent, halogenated alkane solvents, lower alcohols solvent；Or mixtures thereof more preferable ethyl acetate, isopropyl acetate, ethyl alcohol, isopropanol, methylene chloride；

It is further preferred that volume used in the positive solvent is 3-5 times of formula (I) crude compound mass volume ratio；

Preferably, the anti-solvent is selected from or mixtures thereof lower paraffin hydrocarbon solvent, cyclic alkane solvents, rudimentary ether solvent；Or mixtures thereof more preferable normal heptane, n-hexane, petroleum ether, isopropyl ether；

1-3 times of solvent volume ratio it is further preferred that volume used in the anti-solvent is positive；

Preferably, it with the entrainment distillation of or mixtures thereof lower alcohols solvent after step 5) is concentrated to dryness, is then further concentrated to dry；Or mixtures thereof more preferable ethyl alcohol, isopropanol；

It is further preferred that the optical purity of formula (I) compound substantially pure obtained by step 5) is 90.0% or more；The optical purity of more preferably substantially pure formula (I) compound of gained is 95.0% or more.
A kind of preparation method of substantially pure formula (I) compound, which comprises the steps of:

1) being dissolved in or be scattered in mass volume ratio for formula (I) crude compound is in 0.5-10 times of the first solvent；

2) the second solvent of the first 0.5-10 times of solvent volume ratio is added；

3) continue stirring and crystallizing；

4) filtering is drained, and substantially pure formula (I) compound is obtained after filtration cakes torrefaction；

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

Preferably, the first solvent is selected from or mixtures thereof rudimentary esters solvent, halogenated alkane solvents, lower alcohols solvent, or mixtures thereof more preferable ethyl acetate, isopropyl acetate, ethyl alcohol, isopropanol, methylene chloride；The Two solvents are selected from or mixtures thereof lower paraffin hydrocarbon solvent, cyclic alkane solvents, rudimentary ether solvent, or mixtures thereof more preferable normal heptane, n-hexane, petroleum ether, isopropyl ether；

It is further preferred that the optical purity of formula (I) compound substantially pure obtained by step 4) is 90.0% or more；The optical purity of more preferably substantially pure formula (I) compound of gained is 95.0% or more.
The preparation method of substantially pure formula (I) compound according to any one of claim 4-5, which is characterized in that formula (I) crude compound is optionally pre-processed through following steps:

1) sodium hydroxide or potassium hydroxide are dissolved in 5-100 times of lower alcohols solvent of mass volume ratio, are subsequently cooled to 5 DEG C or less；

2) it will be added in lye obtained by step 1) comprising the raw material of formula (I) compound, and be cooled to -10 DEG C or less and continue to be stirred to react；

3) acidic materials are slowly added to reacting liquid pH value about 5-6 for -10 DEG C of temperature control or less；

4) it is concentrated under reduced pressure partial solvent at temperature control is lower than 40 DEG C；

5) water and rudimentary esters solvent, liquid separation are added in residue；

6) organic phase is concentrated to dryness to obtain formula (I) crude compound；

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

It is furthermore preferred that or mixtures thereof lower alcohols solvent preferred alcohol, isopropanol used in step 1)；Or mixtures thereof esters solvent ethyl acetate, isopropyl acetate used in step 5).
A kind of preparation method of formula (III) compound, which comprises the steps of:

1) formula (IV) compound esterification obtains formula (V) compound；

2) formula (V) compound double bond restores to obtain formula (VI) compound；

3) formula (VI) compound isomerizate obtains formula (VII) compound；

4) formula (VII) compound hydroxyl protection obtains formula (VIII) compound；

5) formula (VIII) compound carbonyl reduction obtains formula (III) compound；

Reaction equation is as follows:

Wherein, R is selected from C_1-4Alkyl；Pg is hydroxyl protection base；

Preferably, R is selected from methyl or ethyl；

Preferably, Pg is selected from benzoyl substituted or unsubstituted, benzenesulfonyl substituted or unsubstituted, alkyl substituted or unsubstituted, benzyl substituted or unsubstituted or trialkyl silyl, more preferably such as flowering structure:

Most preferably

Preferably, the esterification of the step 1) 20 DEG C~35 DEG C at a temperature of carry out in acidic environment, more preferably 22 DEG C~27 DEG C at a temperature of carry out；It is further preferred that the acid is selected from or mixtures thereof hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, methanesulfonic acid；More preferable hydrochloric acid or sulfuric acid；

Preferably, the isomerizationization reaction of step 3) is reacted in alcohols solvent using sodium alkoxide, and reaction temperature is 5 DEG C~35 DEG C；It is furthermore preferred that the sodium alkoxide is selected from sodium methoxide, sodium ethoxide or sodium tert-butoxide, the alcohols solvent is selected from methanol, ethyl alcohol, isopropanol or the tert-butyl alcohol, and the reaction temperature is 15 DEG C~25 DEG C；

Preferably, the carbonyl reduction of formula (VIII) compound is obtained formula (III) compound in alcohols solvent using sodium borohydride by the step 5) carbonyl reduction, and reaction temperature is -5 DEG C~10 DEG C；It is furthermore preferred that the alcohols solvent is selected from methanol, ethyl alcohol, isopropanol or the tert-butyl alcohol, reaction temperature is 0 DEG C~3 DEG C；It is further preferred that the step 5) carbonyl reduction is after reaction, using acetone and citric acid quenching reaction.
A kind of preparation method of Austria's shellfish cholic acid, which is characterized in that on the basis of the preparation method described in claim 7, further comprise following steps:

Wherein, the acid is organic acid or inorganic acid；Organic acid preferably is selected from or mixtures thereof trifluoroacetic acid, trichloroacetic acid, Loprazolam, trifluoromethanesulfonic acid, p-methyl benzenesulfonic acid, formic acid, acetic acid；Inorganic acid preferably is selected from or mixtures thereof hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid.
A kind of preparation method of Austria's shellfish cholic acid, which is characterized in that on the basis of the preparation method described in claim 7, further comprise following steps:

Preferably, formula (III) compound selective acidolysis under lithium hydroxide effect generates formula (IX) compound.
A kind of 5- ((2R) -2- ((3R, 5S, 6R, 7R, 10S, 13R) ten hexahydro -1H- cyclopenta [a] phenanthrene -17- base of -6- ethyl -3,7- dihydroxy -10,13- dimethyl) propyl) thiazolidine -2, preparation method in 4- diketone, it is characterized in that, further comprising following steps on the basis of the preparation method described in claim 7:

Preferably, formula (III) compound selective acidolysis under lithium hydroxide effect generates formula (IX) compound；

Preferably, the crude product formula (XI) reacted uses mass volume ratio for the mashing purification purification of the methylene chloride of 1:15；It is furthermore preferred that formula (XI) the compound HPLC purity obtained through methylene chloride mashing purification can reach 98% or more.