CN109467585A - For preventing or treating the disease mediated cholane acid compound of FXR- - Google Patents

Abstract

The present invention relates to a kind of for preventing or treating the disease mediated cholane acid compound of FXR-.Specifically, cholane acid compound shown in disclosure of the invention formula (I) and contain the compound or its crystal form, pharmaceutically acceptable salt, prodrug, tautomer and stereoisomer, enantiomter, the pharmaceutical composition of hydrate or solvate.Compound of the present invention can be used as farnesoid X receptor agonist, and then can be used conveniently to prepare the drug for the treatment of FXR related disease (such as fatty liver).

Description

For preventing or treating the disease mediated cholane acid compound of FXR-

The application is Chinese invention application (denomination of invention: for preventing or treating the disease mediated cholanic acid chemical combination of FXR- Object；Application number: 201610409536.8；The applying date: 2016.06.12) divisional application.

Technical field

The invention belongs to field of medicaments.In particular it relates to a kind of disease mediated for preventing or treating FXR- Cholane acid compound and its pharmaceutical composition and purposes.

Background technique

Farnesoid X receptor (FXR) is orphan nuclear receptor family member, is found earliest by Forman equal to nineteen ninety-five, because of it Transcriptional activity can be enhanced by the farnesol of supraphysiologic levels and be named.Northern and in-situ study show FXR in lung, intestines, kidney, With great expression in adrenal gland.FXR forms heterodimer in conjunction with DNA with 9- cis-retinoic acid receptor (RXR).FXR/ RXR heterodimer preferentially in conjunction with the ingredient being made of the double-core receptor half site for sharing AG (G/T) TCA, is formed reversed It repeats and the single nucleosides of an ancient exorcistic ceremony separates (IR-1 die body).However, these compounds can not activate mouse and mankind FXR, so that endogenous Property FXR aglucon naturality it is also uncertain.Some abiogenous cholic acid combine under physiological concentration and activate FXR.As FXR The cholic acid of aglucon includes chenodesoxycholic acid (CDCA), deoxycholic acid (DCA), the taurine of lithocholic acid (LCA) and these cholic acid and Amion acetic acid conjugate.

Cholic acid is to form and be secreted into duodenal cholesterol metabolic product in liver, in food fat and Wei Sheng Play a significant role in the dissolution and absorption of element.Cholic acid lowers the transcription of Cytochrome P450 7a (CYP7a), coding catalysis gallbladder The enzyme of sour biosynthesis rate-limiting step.Data show that FXR is related with the inhibition that cholic acid expresses CYP7a, although accurate mechanism Still it is not known.FXR is under the regulation of respective ligand, collaboration activation factor and hormone, to a variety of enzymes and cholate of bile acid biosynthesis Carrier carries out accurate regulation.A variety of primary and secondary bile acids of discovered in recent years physiological concentration can activate FXR, for example, goose Deoxycholic aicd (chenodexycholic acid, CDCA) is a kind of common native agonist.6-ECDCA (5 β -3 α, 7 α-two - 6 α of hydroxyl-ethyl-cholanic acid) derivative as CDCA is a kind of potential efficiently FXR agonist, than two number of CDCA high Magnitude.

Primary biliary cirrhosis (PBC) is a kind of common autoimmune disease in the liver and gallbladder, is taken place mostly in 40 years old Above female middle-aged, disease incidence 0.1%.PBC is related with cholestasis, mainly by bile intake, synthesize or paracrisis Cause, can also be formed because of obstruction of biliary tract, and gradually development is small bile duct inflammation damnification in liver, eventually leads to hardening.Currently, bear goes Oxycholic acid is the only approved drug for being used to treat PBC of FDA, it can be effectively improved the level of liver anomalies biochemical indicator, and Reduce the disease incidence of liver fibrosis and cirrhosis.INT-747 is a kind of newtype drug, and research is for those to old plant medicine Object ursodesoxycholic acid does not have abundant response or intolerable patient.The drug authorizes Orphan drug qualification by U.S. FDA, if obtaining Approval must be listed, it will obtain 7 years market exclusive rights.

G protein coupled receptor 5 (TGR5) receptor is a kind of g protein coupled receptor, has been previously identified as being a kind of cell Surface receptor carries out response to bile acid (BA).In the g protein coupled receptor 5 of the mankind, ox, rabbit, rat and mouse (TGR5) between, it has been found that primary structure possessed by the TGR5 and it be to responsiveness caused by bile acid Highly conserved, and therefore prompted TGR5 that there is important physiologic function.

TGR5 is related with the cyclisation accumulation into the cell of adenosine monophosphate (cAMP), wherein the cAMP is in various differences Cell type in widely expressed.Although this membrane receptor possessed activation energy in macrophage Enough reduce the generation of pro-inflammatory cytokine, but bile acid in fat cell and myocyte caused by the TGR5 Stimulation can increase the consumption of energy.Latter effect is related to the 2 type potassium iodide gland propylhomoserins and takes off iodine enzyme (D2) CAMP dependence inducing action, this inducing action part T4 is converted to T3 by way of cause increased thyroid gland to swash (referring to Maruyama, T. et al. is in 2006 in J.Endocrinol " endocrinology magazine " 191,197-205 for the activity of element The article delivered).Accordingly, TGR5 is a kind of for carrying out the attractive targeting of the treatment of metabolic disease, wherein institute The metabolic disease stated can be, obesity, diabetes and metabolic syndrome.

Deuterated modification is a kind of strategy for having potential attraction for improving pharmacokinetic properties.Deuterium be hydrogen it is safe and stable, The isotope of non-radiation type.Compared with c h bond, the C-D key that deuterium is formed with carbon is relatively strong because vibration frequency is lower.In addition, " heavy hydrogen " pattern of drug may be more stable to degradation and maintains the longer time in vivo.Deuterium is incorporated to replace hydrogen that can improve The pharmacodynamics and pharmacokinetic profile of drug change metabolic fate, while keeping the pharmacological activity of physiological active compound And selectivity.Deuterate drug can have positive influences to safety, effect and tolerance, have excellent Research Prospects.

Summary of the invention

The object of the present invention is to provide a kind of for preventing or treating the substituted cholane disease mediated by FXR or TGR5 Acid compound and its pharmaceutical composition and purposes.

In this regard, The technical solution adopted by the invention is as follows:

In the first aspect of the present invention, cholane acid compound shown in a kind of formula (I) or its crystal form, pharmaceutically are provided Acceptable salt, hydrate or solvated compounds.

In formula:

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、 R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸And R²⁹It is independently selected from the group being made of " hydrogen (H), deuterium (D) "；

X¹、X²、X³、X⁴It is independently selected from by " hydrogen (H), deuterium (D), methyl, CH₂D、CHD₂、CD₃、CH₂CH₃、 CHDCH₃、CHDCH₂D、CHDCHD₂、CHDCD₃、CD₂CH₃、CD₂CH₂D、CD₂CHD₂、CD₂CD₃" composition group；

And its physiologically acceptable salt, prodrug, tautomer and stereoisomer, hydrate or solvate, The mixture formed including these compounds with all proportions.

As a further improvement of the present invention, R¹、R²、R³、R⁴、R⁵It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R¹⁷、R¹⁸And R²⁹It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R¹⁹、R²⁰It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸It is each independently deuterium or hydrogen.

As a further improvement of the present invention, X¹、X²、X³、X⁴One or many deuterated alkyl can be independently selected from.

It is selected in example another, R¹、R²、R³、R⁴、R⁵It is deuterium.

It is selected in example another, R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹It is deuterium.

It is selected in example another, R¹⁷、R¹⁸It is deuterium.

It is selected in example another, R¹⁹、R²⁰It is deuterium.

It is selected in example another, R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸It is deuterium.

It is selected in example another, the compound is selected from such as the following group compound or its pharmaceutically acceptable salt, but does not limit to In following compounds:

In another preferred example, deuterium isotopic content of the deuterium in deuterated position is at least greater than natural deuterium isotope and contains for reaction It measures (0.015%), is preferably greater than 30%, even more preferably greater than 50%, even more preferably greater than 75%, even more preferably greater than 95%, more preferably Ground is greater than 99%.

Specifically, R in the present invention¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、 R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸And R²⁹Deuterium isotopic content is at least 5% in each deuterated position, It is preferably greater than 10%, even more preferably greater than 15%, even more preferably greater than 20%, even more preferably greater than 25%, even more preferably greater than 30%, more It is greater than 35%, even more preferably greater than 40%, even more preferably greater than 45%, even more preferably greater than 50%, even more preferably greater than 55% goodly, more preferably Ground is greater than 60%, even more preferably greater than 65%, even more preferably greater than 70%, even more preferably greater than 75%, even more preferably greater than 80%, more preferably Greater than 85%, even more preferably greater than 90%, even more preferably greater than 95%, even more preferably greater than 99%.

In another preferred example, in formula (I) compound R, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、 R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸And R²⁹, at least one of which R is containing deuterium, more preferably Two, ground R contains deuterium, and more preferably three R contain deuterium, and more preferably four R contain deuterium, and more preferably five R contain deuterium, and more preferably six R contain deuterium, More preferably seven R contain deuterium, and more preferably eight R contain deuterium, and more preferably nine R contain deuterium, and more preferably ten R contain deuterium, more preferably 11 R Containing deuterium, more preferably 12 R contain deuterium, and more preferably 13 R contain deuterium, and more preferably 14 R contain deuterium, and more preferably 15 R contain deuterium, More preferably 16 R contain deuterium, and more preferably 17 R contain deuterium, and more preferably 18 R contain deuterium, and more preferably 19 R contain deuterium, more preferably 20, ground R contains deuterium, and more preferably 21 R contain deuterium, and more preferably 22 R contain deuterium, and more preferably 23 R contain deuterium, more Good 24 R in ground contain deuterium, and more preferably 25 R contain deuterium, and more preferably 26 R contain deuterium, and more preferably 27 R contain Deuterium, more preferably 28 R contain deuterium, and more preferably 29 R contain deuterium.

In another preferred example, the compound does not include non-deuterated compound.

In the second aspect of the present invention, a kind of method for preparing pharmaceutical composition is provided, comprising steps of will pharmaceutically Compound described in acceptable carrier and first aspect present invention or its crystal form, pharmaceutically acceptable salt, hydrate or Solvate is mixed, to form pharmaceutical composition.

In the third aspect of the invention, provide a kind of pharmaceutical composition, it contain pharmaceutically acceptable carrier and Compound described in first aspect present invention or its crystal form, pharmaceutically acceptable salt, hydrate or solvate.

The pharmaceutically acceptable carrier that can be used in pharmaceutical composition of the present invention includes but is not limited to any glidant, increasing Edulcorant, preservative, dyestuff/colorant, flavoring reinforcing agent, surfactant, wetting agent, dispersing agent, disintegrating agent, helps diluent Suspension, stabilizer, isotonic agent, solvent or emulsifier.

Pharmaceutical composition of the present invention can be configured to solid-state, semisolid, liquid or gaseous state preparation, such as tablet, pill, capsule Agent, pulvis, granule, paste, emulsion, suspending agent, solution, suppository, injection, inhalant, gelling agent, microballoon and aerosol Deng.

The classical pathway for giving pharmaceutical composition of the present invention includes but is not limited to oral, rectum, saturating mucous membrane, through enteral administration, Or part, percutaneous, sucking, parenteral, sublingual, intravaginal, it is intranasal, intraocularly, peritonaeum is interior, intramuscular, subcutaneous, intravenous administration. It is preferred that oral administration or drug administration by injection.

Pharmaceutical composition of the invention can be manufactured using method well known in the art, such as conventional mixing method, dissolution method, Granulation, dragee method processed, levigate method, emulsion process, freeze-drying etc..

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Herein, unless otherwise instructed, " halogen " refers to F, Cl, Br and I.More preferably, halogen atom is selected from F, Cl and Br.

Herein, unless otherwise instructed, " deuterated " refers to one or more hydrogen in compound or group replaced deuterium；Deuterium In generation, can be a substitution, two replace, polysubstituted or full substitution.Term " one or more deuterated " and " one or many deuterated " It is used interchangeably.

Herein, unless otherwise instructed, " non-deuterated compound " refers to ratio containing D-atom not higher than the same position of natural deuterium The compound of cellulose content (0.015%).

The invention also includes the compounds of isotope labelling, are equal to original chemical and are disclosed.This hair can be classified as The example of bright compound isotope includes hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine isotope, respectively such as²H,³H,¹³C,¹⁴C,¹⁵N,¹⁷O,¹⁸O,³¹P,³²P,³⁵S,¹⁸F and³⁶Cl.Compound or enantiomer in the present invention, diastereomer, isomers or medicine Acceptable salt or solvate on, wherein containing the isotope of above compound or other other isotope atoms all at this Within the scope of invention.Certain compound isotopically labelleds in the present invention, such as³H and¹⁴The radioactive isotope of C also wherein, It is useful in the experiment of the Tissue distribution of drug and substrate.Tritium, i.e.,³H and carbon-14, i.e.,¹⁴C, their preparation and detection are compared It is easy, is the first choice in isotope.The compound of isotope labelling can use general method, by with the isotope mark being easy to get Note reagent replaces with non isotopic reagent, can be prepared with the scheme in example.

Pharmaceutically acceptable salt includes inorganic salts and organic salt.A kind of preferred salt is that the compounds of this invention and acid are formed Salt.The acid for suitably forming salt includes but is not limited to: the inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid； Formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, The organic acids such as citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-methyl benzenesulfonic acid, benzene sulfonic acid, naphthalene sulfonic acids；And dried meat ammonia The amino acid such as acid, phenylalanine, aspartic acid, glutamic acid.Another kind of preferred salt is the salt that the compounds of this invention and alkali are formed, Such as alkali metal salt (such as sodium salt or sylvite), alkali salt (such as magnesium salts or calcium salt), ammonium salt (such as rudimentary alkanol ammonium salt And other pharmaceutically acceptable amine salt), such as methylamine salt, ethylamine salt, propylamine salt, dimethyl amine salt, trismethylamine salt, two Ethyl amine salt, triethyl amine salt, tert-butylamine salt, ethylenediamine salt, oxyethylamine salt, dihydroxy ethylamine salt, three oxyethylamine salt, Yi Jifen The amine salt not formed by morpholine, piperazine, lysine.

The compounds of this invention can have chiral centre, for example, asymmetric carbon atom.Therefore the compounds of this invention includes all vertical The racemic mixture of body isomers, including enantiomer, diastereomer and atropisomer.In addition, the compounds of this invention includes It is any or all the enrichment on asymmetric chiral atoms or fractionation optical isomer.In other words, from description show and The chiral centre being clear to is by as chiral isomer or racemic mixture offer.Racemic mixture and diastereomeric mixing Object, and be substantially free of they mapping or diastereomeric gametophyte, separation or synthesis independent optical isomer, all fall Within the scope of the present invention.By known technology, for example, the auxiliary agent of separation optically active is for example, what acid or alkali were formed Diastereomeric salt then goes back to as optically active material, and racemic mixture is separated into their single, substantial optics Pure isomer.In most cases, conceivable optical isomer is by stereospecific reaction, from desired original What the suitable stereoisomer of material started to be synthesized.

Term " solvate " refers to that the compounds of this invention and solvent molecule are coordinated the complex to form special ratios." hydration Object " refers to that the compounds of this invention and water carry out the complex of coordination formation.

Preferably, the disease or situation that the FXR or TGR5 is mediated are selected from chronic liver disease, gastrointestinal disease, nephrosis, painstaking effort Pipe disease, silt courage imbalance, metabolic disease；Preferably, the nephrosis is diabetic nephropathy；It is hard that the cardiovascular disease is selected from artery Change, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, wherein artery sclerosis is atherosclerosis.

In specific embodiments, the disease or situation that FXR or TGR5 is mediated are cardiovascular disease, Atherosclerosis Change, artery sclerosis, hypercholesterolemia, hyperlipidemia chronic liver disease, gastrointestinal disease, nephrosis, cardiovascular disease, metabolic disease, Cancer (for example, colorectal cancer), or neural sign such as apoplexy.

In specific embodiments, chronic liver disease is primary hardening, brain tendon xanthomatosis, primary sclerotic gall-bladder Inflammation, drug induced cholestasia, intrahepatic cholestasis of pregnancy, parenteral absorption associated cholestasis, bacterial overgrowth or Sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, non-wine Essence steatohepatitis, the anti-host disease of liver transfer operation related Graft, live donor liver transfer operation regeneration, congenital hepatic fibrosis, gallbladder are total Pipe calculus, granular hepatopathy, in liver-or outer malignant tumour, Sjogren syndrome, sarcoidosis, Wilson ' s disease, Gaucher ' s disease, hemochromatosis or blood pressure nephrosis, chronic glomerulus is scorching, chronic transplant glomerulopathy, chronic interstitial kidney Inflammation or polycystic kidney disease.

In specific embodiments, cardiovascular disease is arteriosclerosis, artery sclerosis, dyslipidemia, hypercholesteremia Disease or hypertriglyceridemia.

In specific embodiments, metabolic disease is insulin resistance, I type and type-2 diabetes mellitus, or fat.

Compared with prior art, the invention has the benefit that the compounds of this invention can be used for activating farnesoid X receptor, The gene expression for inhibiting cytochromes 7A1 (CYP7A1) indirectly, promotes the synthesis of cholic acid.Furthermore the compounds of this invention simultaneously can Preferably adjust g protein coupled receptor 5 (TGR5).Change metabolism of the compound in organism by this technology of deuterate, makes Compound has better pharmacokinetic parameter characteristic.In such a case, it is possible to change dosage and form durative action preparation, change Kind applicability.Compound can be improved in animal body due to its deuterium isotope effect with the hydrogen atom in deuterium substituted compound Drug concentration, to improve curative effect of medication.It can since certain metabolites are suppressed with the hydrogen atom in deuterium substituted compound The safety of compound can be improved.

Specific embodiment

The preparation method of formula I structural compounds is described more particularly below, but these specific methods are not to this hair Bright composition any restrictions.The compounds of this invention can also be optionally by various conjunctions describing in the present specification or known in the art It combines at method and is easily made, such combination can be easy to carry out by those skilled in the art in the invention.

In general, each reaction is usually in atent solvent, in room temperature to reflux temperature (such as 0 DEG C~100 in preparation flow DEG C, preferably 0 DEG C~80 DEG C) under carry out.Reaction time is usually -60 hours 0.1 hour, preferably 0.5-24 hours.

Embodiment 1 prepares 3 α, 7 α-bis-hydroxy -7-d-6 α--5 β of ethyl-cholanic acid (compound 7)

Specific synthesis step is as follows:

Step 1: the synthesis of -5 β of Alpha-hydroxy -7- ketone group-cholanic acid methyl esters (compound 2).

The two drop concentrated sulfuric acids are added drop-wise in 3-5 β of Alpha-hydroxy-7- ketone group-cholanic acid (5.00g, 12.80mmol) methanol, Back flow reaction (65 DEG C) 3hrs.It is concentrated under reduced pressure and removes methanol, concentration fluid column chromatographs to obtain 4.60g colorless solid (compound 2), receives Rate: 88.3%.

LC-MS (APCI): m/z=405.3 [M+1]⁺。

Step 2: the synthesis of -5 β of α -7 α-two-trimethylsiloxy-cholanic acid methyl esters (compound 3).

At -78 DEG C, by trim,ethylchlorosilane (7.15mL, 57.0mmol) be added drop-wise to diisopropylamino lithium (LDA 2M, 34.2mL, 68.4mmol) anhydrous tetrahydro furan (70mL) solution in, stir 30 minutes.Then by 3 Alpha-hydroxy -7- ketone groups -5 β-cholanic acid methyl esters (2.30g, 5.7mmol) anhydrous tetrahydro furan (30mL) mixture is added drop-wise in reaction solution in 15min, Reaction solution continues to be stirred to react 1.5 hours at -78 DEG C.Be added triethylamine (15mL, 103mmol), reaction solution at -78 DEG C after It is continuous to be stirred to react 1.5 hours.Reaction is warming up to -20 DEG C, the sodium bicarbonate solution (20mL) of saturation is added, is to slowly warm up to room Temperature stirs 2 hours.Organic layer separation, water phase are extracted with ethyl acetate (100mL x 3).Merge organic layer unsaturated carbonate hydrogen Sodium solution (100mL x 3), water (100mL), saturated salt solution (100mL) washing.Anhydrous sodium sulfate is dry.It is concentrated under reduced pressure organic Layer, concentration fluid column chromatograph to obtain 3.8g beige solid, yield: 100%.

The synthesis of -5 β of step 3:3 Alpha-hydroxy -6- ethylidene -7- ketone group-cholanic acid methyl esters (compound 4).

At -60 DEG C, by boron trifluoride ether (BF₃·OEt₂, 14mL, 110mmol) and it is slowly dropped to 3 α -7 α-two-front three - 5 β of base siloxy-cholanic acid methyl esters (6.15g, 11mmol) and acetaldehyde (1.5mL, 37.11mmol) anhydrous methylene chloride In (70mL) mixture.Reaction solution is stirred to react 2.5 hours at -60 DEG C, is warming up to room temperature.Use saturated sodium bicarbonate solution (50mL) quenching reaction.Organic layer separation, water phase are extracted with methylene chloride (50mL x 3).Merge organic layer saturated salt solution (50mL) washing.Anhydrous sodium sulfate is dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 1.8g grease, yield: 33.3%.

LC-MS (APCI): m/z=431.3 [M+1]⁺。

- 6 α of step 4:3 Alpha-hydroxy--5 β of ethyl -7- ketone group-cholanic acid methyl esters (compound 5) synthesis.

By Pd/C (10%, 90mg) be added to 3-5 β of Alpha-hydroxy-6- ethylidene-7- ketone group-cholanic acid methyl esters (900mg, The in the mixed solvent of anhydrous tetrahydro furan and anhydrous methanol (30mL, 1:1v/v) 2.09mmol).Hydrogen displaced air, hydrogen Under be stirred to react overnight.Diatomite filtering, is concentrated under reduced pressure, and concentration fluid column chromatographic purifying obtains 630mg light yellow oil, produces Rate: 70.0%.

LC-MS (APCI): m/z=433.3 [M+1]⁺。

- 6 α of step 5:3 Alpha-hydroxy--5 β of ethyl -7- ketone group-cholanic acid (compound 6) synthesis.

Sodium hydroxide (205mg, 3.50mmol) is added to 3-6 α of Alpha-hydroxy-- 5 β of ethyl-7- ketone group-cholanic acid methyl esters The in the mixed solvent of the first alcohol and water (20mL, 1:1v/v) of (630mg, 1.46mmol), reaction are stirred overnight, and first is removed under reduced pressure Alcohol is diluted with water (20mL), and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), and organic layer is washed with saturated common salt It washs, anhydrous sodium sulfate is dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 430mg white solid, yield: 70.0%.

LC-MS (APCI): m/z=417.3 [M-1]^-。

The synthesis of step 6:3 α, 7 α-bis-hydroxy -7-d-6 α--5 β of ethyl-cholanic acid (compound 7).

By boron deuterate sodium (195mg, 5.00mmol) under ice bath, it is added portionwise to 3-6 α of Alpha-hydroxy-ethyl-7- ketone group-5 In the deuterated methanol-d (5mL) of β-cholanic acid (210mg, 0.50mmol), reaction is stirred at room temperature overnight in reaction solution.It will reaction Liquid pours into the ice water of 20mL, and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), and organic layer is washed with saturated common salt It washs, anhydrous sodium sulfate is dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 130mg white solid, yield: 62.0%.

LC-MS (APCI): m/z=420.3 [M-1]^-。

Embodiment 2 prepares 3 α, -6 α of 7 α-bis-hydroxy-(ethyl -1-d) -5 β-cholanic acid (compound 10)

Specific synthesis step is as follows:

- 6 α of step 1:3 Alpha-hydroxy--5 β of (ethyl -1-d) -6-d-7- ketone group-cholanic acid methyl esters (compound 8) synthesis.

By Pd/C, (10%, 55% in D₂In O, 60mg) it is added to 3-5 β of Alpha-hydroxy-6- ethylidene-7- ketone group-cholanic acid The anhydrous tetrahydro furan of methyl esters (600mg, 1.39mmol) and the in the mixed solvent of deuterated methanol (20mL, 1:1v/v).Deuterium is set It ventilates, is stirred to react under deuterium overnight.Diatomite filtering, is concentrated under reduced pressure, and it is faint yellow that concentration fluid column chromatographic purifying obtains 420mg Grease, yield: 70.0%.

LC-MS (APCI): m/z=435.3 [M+1]⁺。

- 6 α of step 2:3 Alpha-hydroxy--5 β of (ethyl -1-d) -7- ketone group-cholanic acid (compound 9) synthesis.

Sodium hydroxide (120mg, 2.90mmol) is added to -5 β of -6 α of 3 Alpha-hydroxy-(ethyl -1-d) -6-d-7- ketone group - The in the mixed solvent of the first alcohol and water (20mL, 1:1v/v) of cholanic acid methyl esters (420mg, 0.97mmol), reaction are stirred overnight, subtract Pressure removes methanol, is diluted with water (20mL), and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), organic layer saturation Brine It, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 270mg white solid, produces Rate: 66.4%.

LC-MS (APCI): m/z=418.1 [M-1]^-。

The synthesis of -6 α of step 3:3 α, 7 α-bis-hydroxy-(ethyl -1-d) -5 β-cholanic acid (compound 10).

By sodium borohydride (170mg, 5.50mmol) under ice bath, it is added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1-d) -7- In the anhydrous methanol (5mL) of -5 β of ketone group-cholanic acid (230mg, 0.55mmol), reaction is stirred at room temperature overnight in reaction solution.It will Reaction solution pours into the ice water of 20mL, and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), organic layer saturated common salt Water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 70mg white solid, yield: 30.3%.

LC-MS (APCI): m/z=420.3 [M-1]^-。

Embodiment 3 prepares 3 α, -6 α of 7 α-bis-hydroxy-ethyl -6-d-5 β-cholanic acid (compound 12)

Specific synthesis step is as follows:

- 6 α of step 1:3 Alpha-hydroxy--5 β of ethyl -6-d-7- ketone group-cholanic acid (compound 11) synthesis.

Deuterium sodium oxide molybdena (120mg, 2.90mmol) is added to 3-6 α of Alpha-hydroxy-- 5 β of ethyl-7- ketone group-cholanic acid methyl esters The in the mixed solvent of the deuterated methanol and heavy water (20mL, 1:1v/v) of (420mg, 0.97mmol), reaction are stirred overnight, and decompression removes Methanol is removed, is diluted with water (20mL), after 2N HCl acidification, ethyl acetate extracts (50mL x 3), organic layer saturated common salt Water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 280mg white solid, yield: 68.8%.

LC-MS (APCI): m/z=418.1 [M-1]^-。

The synthesis of -6 α of step 2:3 α, 7 α-bis-hydroxy-(ethyl -1-d) -5 β-cholanic acid (compound 12).

By sodium borohydride (260mg, 6.60mmol) under ice bath, it is added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1-d) -7- In the anhydrous methanol (5mL) of -5 β of ketone group-cholanic acid (280mg, 0.66mmol), reaction is stirred at room temperature overnight in reaction solution.It will Reaction solution pours into the ice water of 20mL, and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), organic layer saturated common salt Water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 70mg white solid, yield: 25.2%.

LC-MS (APCI): m/z=420.3 [M-1]^-。

Embodiment 4 prepares 3 α, -6 α of 7 α-bis-hydroxy-(ethyl -1-d) -6-d-5 β-cholanic acid (compound 14)

Specific synthesis step is as follows:

- 6 α of step 1:3 Alpha-hydroxy--5 β of (ethyl -1-d) -6-d-7- ketone group-cholanic acid (compound 13) synthesis.

Deuterium sodium oxide molybdena (150mg, 3.52mmol) is added to -5 β of -6 α of 3 Alpha-hydroxy-(ethyl -1-d) -6-d-7- ketone group - The deuterated methanol of cholanic acid methyl esters (510mg, 1.17mmol) and the in the mixed solvent of heavy water (10mL, 1:1v/v), reaction stirring Overnight, methanol is removed under reduced pressure, is diluted with water (20mL), after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), organic layer With saturated common salt water washing, anhydrous sodium sulfate drying.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 490mg white Solid, yield: 95.0%.

LC-MS (APCI): m/z=419.1 [M-1]^-。

- 6 α of step 2:3 α, 7 α-bis-hydroxy-(ethyl -1-d) -6-d-5 β-cholanic acid (compound 14) synthesis.

By sodium borohydride (225mg, 6.00mmol) under ice bath, it is added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1-d) -6- In -5 β of d-7- ketone group-cholanic acid (250mg, 0.60mmol) anhydrous methanol (5mL), reaction solution, which is stirred at room temperature, to react Night.Reaction solution is poured into the ice water of 20mL, after 2N HCl acidification, ethyl acetate extracts (50mL x 3), and organic layer is used full And brine It, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 53mg white solid, Yield: 21.0%.

LC-MS (APCI): m/z=421.4 [M-1]^-。

Embodiment 5 prepares 3 α, 7 α-bis-hydroxy -7-d-6 α-(ethyl -1-d) -6-d-5 β-cholanic acid (compound 15)

Specific synthesis step is as follows:

By boron deuterate sodium (225mg, 6.00mmol) under ice bath, it is added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1-d) -6- In -5 β of d-7- ketone group-cholanic acid (250mg, 0.60mmol) deuterated methanol (5mL), reaction solution, which is stirred at room temperature, to react Night.Reaction solution is poured into the ice water of 20mL, after 2N HCl acidification, ethyl acetate extracts (50mL x 3), and organic layer is used full And brine It, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 31mg white solid, Yield: 12.3%.

LC-MS (APCI): m/z=422.4 [M-1]^-。

Embodiment 6 prepares 3 α, -6 α of 7 α-bis-hydroxy-(ethyl -1,2,2,2-d₄) -5 β-cholanic acid (compound 19)

Specific implementation step is as follows:

Step 1:3 Alpha-hydroxy -6- (ethylidene -1,2,2,2-d₄) -5 β of -7- ketone group-cholanic acid methyl esters (compound 16) Synthesis.

At -60 DEG C, by boron trifluoride ether (BF₃·OEt₂, 7.20mL, 11.4mmol) and it is slowly dropped to 3 α -7 α-two-three - 5 β of methyl silicane oxygroup-cholanic acid methyl esters (3.10g, 5.70mmol) and deuterated acetaldehyde-d₄The anhydrous dichloromethane of (1.00mL) In alkane (58mL) mixture.Reaction solution is stirred to react 2.5 hours at -60 DEG C, is warming up to room temperature.It is molten with saturated sodium bicarbonate Liquid (50mL) quenching reaction.Organic layer separation, water phase are extracted with methylene chloride (50mL x 3).Merge organic layer saturated common salt Water (50mL) washing.Anhydrous sodium sulfate is dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 1.3g grease, produces Rate: 52.5%.

LC-MS (APCI): m/z=435.5 [M+1]⁺。

- 6 α of step 2:3 Alpha-hydroxy-(ethyl -1,2,2,2-d₄) conjunction of -5 β of -7- ketone group-cholanic acid methyl esters (compound 17) At.

Pd/C (10%, 118mg) is added to 3 Alpha-hydroxy -6- (ethylidene -1,2,2,2-d₄) -5 β of -7- ketone group-cholane The anhydrous tetrahydro furan of sour methyl esters (1.18g, 2.72mmol) and the in the mixed solvent of anhydrous methanol (30mL, 1:1v/v).Hydrogen Displaced air, stirring under hydrogen reaction is overnight.Diatomite filtering, is concentrated under reduced pressure, and it is yellowish that concentration fluid column chromatographic purifying obtains 900mg Color grease, yield: 75.8%.

LC-MS (APCI): m/z=437.4 [M+1]⁺。

- 6 α of step 3:3 Alpha-hydroxy-(ethyl -1,2,2,2-d₄) synthesis of -5 β of -7- ketone group-cholanic acid (compound 18).

Sodium hydroxide (192mg, 4.80mmol) is added to -6 α of 3 Alpha-hydroxy-(ethyl -1,2,2,2-d₄) -7- ketone group -5 The in the mixed solvent of β-cholanic acid methyl esters (600mg, 1.37mmol) first alcohol and water (20mL, 1:1v/v), reaction are stirred overnight, Methanol is removed under reduced pressure, is diluted with water (20mL), after 2N HCl acidification, ethyl acetate extracts (50mL x 3), and organic layer is used full And brine It, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 360mg white solid, Yield: 62.2%.

LC-MS (APCI): m/z=421.3 [M-1]^-。

Step 4:3 α, -6 α of 7 alpha-dihydroxy-(ethyl -1,2,2,2-d₄The conjunction of -5 β of)-ethyl-cholanic acid (compound 19) At.

By sodium borohydride (160mg, 4.10mmol) under ice bath, be added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1,2,2, 2-d₄) in -5 β of -7- ketone group-cholanic acid (175mg, 0.41mmol) anhydrous methanol (5mL), reaction solution is stirred at room temperature instead It should stay overnight.Reaction solution is poured into the ice water of 20mL, after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), and organic layer is used Saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, it is solid that concentration fluid column chromatographic purifying obtains 110mg white Body, yield: 60.5%.

LC-MS (APCI): m/z=423.3 [M-1]^-。

Embodiment 7 prepares 3 α, 7 α-bis-hydroxy -7-d--6 α-(ethyl -1,2,2,2-d₄) -5 β-cholanic acid (compound 20)

Specific synthesis step is as follows:

By boron deuterate sodium (174mg, 4.10mmol) under ice bath, be added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1,2,2, 2-d₄) in -5 β of -7- ketone group-cholanic acid (175mg, 0.41mmol) deuterated methanol (5mL), reaction solution is stirred at room temperature instead It should stay overnight.Reaction solution is poured into the ice water of 20mL, after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), and organic layer is used Saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, it is solid that concentration fluid column chromatographic purifying obtains 85mg white Body, yield: 48.5%.

LC-MS (APCI): m/z=424.3 [M-1]^-。

Embodiment 8 prepares 3 α, -6 α of 7 α-bis-hydroxy-(ethyl -1,2,2,2-d₄) -6-d-5 β-cholanic acid (compound 22)

Specific synthesis step is as follows:

- 6 α of step 1:3 Alpha-hydroxy-(ethyl -1,2,2,2-d₄) conjunction of -5 β of -6-d-7- ketone group-cholanic acid (compound 21) At.

Sodium hydroxide (97mg, 2.36mmol) is added to -6 α of 3 Alpha-hydroxy-(ethyl -1,2,2,2-d₄) -7- ketone group -5 The in the mixed solvent of β-cholanic acid methyl esters (300mg, 0.68mmol) deuterated methanol and heavy water (10mL, 1:1v/v), reaction are stirred It mixes overnight, methanol is removed under reduced pressure, diluted with water (20mL), after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), organic Layer saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, it is white that concentration fluid column chromatographic purifying obtains 150mg Color solid, yield: 51.8%.

LC-MS (APCI): m/z=422.3 [M-1]^-。

- 6 α of step 2:3 α, 7 α-bis-hydroxy-(ethyl -1,2,2,2-d₄) conjunction of -6-d-5 β-cholanic acid (compound 22) At.

By sodium borohydride (134mg, 3.50mmol) under ice bath, be added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1,2,2, 2-d₄) in -5 β of -6-d-7- ketone group-cholanic acid (150mg, 0.35mmol) anhydrous methanol (5mL), reaction solution stirs at room temperature Mix reaction overnight.Reaction solution is poured into the ice water of 20mL, after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), organic Layer saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 25mg grey Solid, yield: 16.8%.

LC-MS (APCI): m/z=424.3 [M-1]^-。

Embodiment 9 prepares 3 α, 7 α-bis-hydroxy -7-d-6 α-(ethyl -1,1,2,2,2-d₅) -6-d-5 β-cholanic acid (change Close object 25)

Specific synthesis step is as follows:

- 6 α of step 1:3 Alpha-hydroxy-(ethyl -1,1,2,2,2-d₅) -5 β of -6-d-7- ketone group-cholanic acid methyl esters (compound 23) synthesis.

By Pd/C, (10%, 50% in D₂In O, 110mg) be added to 3 Alpha-hydroxy -6- (ethylidene -1,2,2,2-d₄) -7- ketone The mixing of -5 β of base-cholanic acid methyl esters (1.10g, 2.53mmol) anhydrous tetrahydro furan and deuterated methanol (30mL, 1:1v/v) is molten In agent.Deuterium displaced air is stirred to react under deuterium overnight.Diatomite filtering, is concentrated under reduced pressure, and concentration fluid column chromatographic purifying obtains 840mg colorless oil, yield: 76.0%.

LC-MS (APCI): m/z=439.4 [M+1]⁺。

- 6 α of step 2:3 Alpha-hydroxy-(ethyl -1,1,2,2,2-d₅) -5 β of -6-d-7- ketone group-cholanic acid (compound 24) Synthesis.

Deuterium sodium oxide molybdena (96mg, 2.40mmol) is added to -6 α of 3 Alpha-hydroxy-(ethyl -1,1,2,2,2-d₅)-6-d-7- The in the mixed solvent of -5 β of ketone group-cholanic acid methyl esters (300mg, 0.68mmol) deuterated methanol and heavy water (10mL, 1:1v/v), Reaction is stirred overnight, and methanol is removed under reduced pressure, and is diluted with water (20mL), and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), organic layer saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 260mg white solid, yield: 90.1%.

LC-MS (APCI): m/z=423.3 [M-1]^-。

Step 3:3 α, 7 α-bis-hydroxy -7-d-6 α-(ethyl -1,1,2,2,2-d₅) -6-d-5 β-cholanic acid (compound 25) synthesis.

By boron deuterate sodium (134mg, 3.50mmol) under ice bath, be added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1,1,2, 2,2-d₅) in -5 β of -6-d-7- ketone group-cholanic acid (150mg, 0.35mmol) deuterated methanol (5mL), reaction solution is at room temperature It is stirred to react overnight.Reaction solution is poured into the ice water of 20mL, after 2N HCl acidification, ethyl acetate extracts (50mL x 3), Organic layer saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 70mg White solid, yield: 46.8%.

LC-MS (APCI): m/z=426.3 [M-1]^-。

Embodiment 10 prepares 3 α, -6 α of 7 α-bis-hydroxy-(ethyl -1,1,2,2,2-d₅) -5 β-cholanic acid (compound 27)

Specific synthesis step is as follows:

- 6 α of step 1:3 Alpha-hydroxy-(ethyl -1,1,2,2,2-d₅) conjunction of -5 β of -7- ketone group-cholanic acid (compound 26) At.

Sodium hydroxide (85mg, 2.04mmol) is added to -6 α of 3 Alpha-hydroxy-(ethyl -1,1,2,2,2-d₅)-6-d-7- The in the mixed solvent of -5 β of ketone group-cholanic acid methyl esters (255mg, 0.58mmol) deuterated methanol and heavy water (10mL, 1:1v/v), Reaction is stirred overnight, and methanol is removed under reduced pressure, and is diluted with water (20mL), and after 2N HCl acidification, ethyl acetate extracts (50mL x 3), organic layer saturated common salt water washing, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 220mg white solid, yield: 86.2%.

LC-MS (APCI): m/z=422.3 [M-1]^-。

- 6 α of step 2:3 α, 7 α-bis-hydroxy-(ethyl -1,1,2,2,2-d₅) -5 β-cholanic acid (compound 27) synthesis.

By sodium borohydride (200mg, 5.20mmol) under ice bath, be added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1,1,2, 2,2-d₅) in -5 β of -7- ketone group-cholanic acid (220mg, 0.52mmol) anhydrous methanol (5mL), reaction solution is stirred at room temperature Reaction is overnight.Reaction solution is poured into the ice water of 20mL, after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), organic layer With saturated common salt water washing, anhydrous sodium sulfate drying.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 120mg white Solid, yield: 53.5%.

LC-MS (APCI): m/z=424.3 [M-1]^-。

Embodiment 11 prepares 3 α, 7 α-bis-hydroxy -7-d-6 α-(ethyl -1,1,2,2,2-d₅) -5 β-cholanic acid (compound 28)

Specific synthesis step is as follows:

By boron deuterate sodium (80mg, 1.90mmol) under ice bath, be added portionwise to -6 α of 3 Alpha-hydroxy-(ethyl -1,1,2,2, 2-d₅) in -5 β of -7- ketone group-cholanic acid (80mg, 0.19mmol) deuterated methanol (5mL), reaction is stirred at room temperature in reaction solution Overnight.Reaction solution is poured into the ice water of 20mL, after being acidified with 2NHCl, ethyl acetate extracts (50mL x 3), and organic layer is used full And brine It, anhydrous sodium sulfate are dry.Organic layer is concentrated under reduced pressure, concentration fluid column chromatographic purifying obtains 40mg pale yellow colored solid Body, yield: 49.5%.

LC-MS (APCI): m/z=425.4 [M-1]^-。

Biological activity test

The external activity test of method Buddhist nun's ester X receptor (FXR) and g protein coupled receptor 5 (TGR5).

The external activity test of method Buddhist nun's ester X receptor (FXR).

The activity on FXR is evaluated by fluorescence resonance energy transfer (FRET) detection, wherein the FRET To detect recruitment of the SRC-1 peptide in mankind FXR, wherein using a kind of Enzyme-linked Immunosorbent Assay without containing cell It detects (ELISA).

The external activity test of g protein coupled receptor 5 (TGR5).

The activity of luciferase is measured in Chinese hamster ovary (CHO) cell, wherein the Chinese hamster ovary celI is steady Fixed expression human G protein-coupled receptor 5 (hTGR5) or the cell are utilized a hTGR5 expression vector and one A luciferase receptor gene by cAMP responsiveness element (CRE) driving has carried out the cotransfection of moment.The specific method is as follows.

Plasmid: the National Institutes of Health (NIH) is obtained at Invitrogen (Carlsbad, CA) and is fed Newborn animal gene preservation clone MGC:40597 (being also referred to as pCMVSPORT6/hTGR5 or pTGR5) and pcDNA 3.1 (+).The pCRE-Luc luciferase reporter-plasmid of driving (cyclisation adenosine monophosphate responsiveness element) and pCMV β be from It is obtained at Clontech (Palo Alto, CA).

Transient transfection: by Chinese hamster ovary (CHO) cell with 3.5x 10⁴The density of a cells/well is placed in the training of 96 holes It supports in plate, cultivates 24 hours, and utilize the mankind (h) g protein coupled receptor 5 of 150 nanograms in each hole after this Cyclisation adenosine monophosphate (cAMP) the responsiveness element (CRE) of expression plasmid (pCMVSPORT6/hTGR5) and 100 nanograms The luciferase reporting plasmid (pCRE-Luc) of driving is transfected, wherein according to the specification of manufacturer in the transfection Use 2000 reagent of Lipofectamine (Invitrogen).After cultures in 6 hours, the life of phosphate-buffered is utilized It manages salt water (PBS) cell is carried out once washing and exchange culture medium to be that the ox blood containing 0.1% (weight/volume) is pure The DMEM of albumen (BSA).After have passed through culture in 18 hours again, cell is carried out using the various concentration of each compound Processing in 5 hours, wherein the compound is present in the new of the bovine serum albumin(BSA) (BSA) containing 0.1% (weight/volume) Among fresh DMEM.After processing, 50 microlitres of lysis buffer (25 mmoles are recycled by a freeze-thaw Tris hydrochloric acid (pH7.6), the ethylenediamine tetra-acetic acid (EDTA) of 2 mmoles, the dithiothreitol (DTT) (DTT) of 1 mmoles, 10% (volume/ Volume) glycerine and 1% (volume/volume) Triton X-100) to the cell carry out cracking and under Method described in text makes the detection of the cell progress luciferase.

Luciferase detection and beta galactosidase detection: in order to carry out luciferase detection, by 20 microlitres of cell Lysate and the micro- luciferin to rub of 100 microlitres of luciferase reaction buffer 235,265 micro- atriphos (ATP) to rub And 135 micro- coacetylase (CoA) to rub carry out mix and using CentroXS3LB960 (Berthold Technologies, Bad Wildbad, Germany) photism is measured.In order to carry out beta galactosidase detection, by 10 microlitres of cell cracking Liquid and 100 microlitres of buffer Z [disodium hydrogen phosphate of 60 mmoles, the potassium chloride of 10 mmoles, the magnesium sulfate of 1 mmoles, 50 mmoles O-Nitrophenylfluorone-β-D- the galactopyranoside (ONPG) of beta -mercaptoethanol and 0.75 mg/ml] carry out mixing and Culture in 0.5 to 3 hour is carried out at 37 DEG C.Reaction is carried out eventually by 50 microlitres of addition of stop buffer (sodium carbonate of 1M) Only and under 420 nanometers the optical density (OD) is measured.

Establish Chinese hamster ovary (CHO) cell (CHO- of expression human G protein-coupled receptor 5 that can be stable TGR5cells): using Lipofectamin 2000, utilize the hTGR5 expression plasmid (pCMVSPORT6/ of 3.8 micrograms HTGR5), the luciferase reporting plasmid (pCRE-Luc) and 0.4 that the cAMP responsiveness element (CRE) of 3.8 micrograms drives are micro- Gram neomycin resist gene expression plasmid [pcDNA3.1 (+)] Chinese hamster ovary (CHO) cell is transfected.It utilizes The G418 sulfate of 400 mcg/mls screens the transfection body and makes single clone's object is independent to be grown in In 96 well culture plates.By the processing mode of evaluation of life cycle (LCA) to the China for expressing g protein coupled receptor 5 (TGR5) Hamster Qvary (CHO) cell line is screened, and carries out the detection of luciferase after this.

50% effective concentration (EC₅₀) and efficiency measurement: with triplicate or quadruplicate under the conditions of each Mode detected.EC is determined by probit analysis₅₀Value.Efficiency is measured by following manner: for For the research of TGR5 agonist, by way of calculating the percentage composition of numerical value of 10 micro- lithocholic acids (LCA) to rub.Carry out After the conversion of algorithm, complete to average EC₅₀Calculating and to possessed by a variety of different compounds EC₅₀The comparison carried out.

Experimental result is as shown in table 1 below, illustrates that the compounds of this invention, being capable of preferably activation method Buddhist nun compared with INT-747 Ester X receptor (FXR) and g protein coupled receptor 5 (TGR5).

Effect of the 1 embodiment compound of table to FXR receptor and TGR5 receptor

Embodiment number	FXR receptor EC50(μM)	TGR5 receptor EC50(μM)
			INT-747	0.32	0.47
Compound 7	1.07	0.37
			Compound 10	0.28	0.70
Compound 12	0.24	0.42
			Compound 14	0.37	0.88
Compound 15	0.40	0.85
			Compound 19	0.21	0.45
Compound 20	0.26	0.58
			Compound 22	0.34	0.61
Compound 25	0.36	0.72
			Compound 27	0.20	0.52
Compound 28	0.19	0.46

Metabolic stability evaluation

Microsomal assay: people's hepatomicrosome: 0.5mg/mL, Xenotech；Rat liver microsomes: 0.5mg/mL, Xenotech；Coenzyme (NADPH/NADH): 1mM, Sigma Life Science；Magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

The preparation of stock solution: precision weighs a certain amount of embodiment compound, and is dissolved to 5mM respectively with DMSO.

The preparation of phosphate buffer (100mM, pH7.4): take the 0.5M potassium dihydrogen phosphate 150mL for preparing in advance and The 0.5M dipotassium hydrogen phosphate solution of 700mL mixes, then adjusts mixed liquor pH value to 7.4 with 0.5M dipotassium hydrogen phosphate solution, uses It is preceding to dilute 5 times with ultrapure water, magnesium chloride is added, phosphate buffer (100mM) is obtained, wherein potassium phosphate containing 100mM, 3.3mM Magnesium chloride, pH 7.4.

It prepares NADPH regenerative system solution and (contains 6.5mM NADP, 16.5mM G-6-P, 3U/mLG-6-P D, 3.3mM Magnesium chloride), using it is preposition in it is wet on ice.

Prepare terminate liquid: the acetonitrile containing 50ng/mL Propranolol Hydrochloride and 200ng/mL orinase (internal standard) is molten Liquid.It takes 25057.5 μ L phosphate buffers (pH7.4) into 50mL centrifuge tube, is separately added into 812.5 μ L people's hepatomicrosomes, mix It is even, obtain the hepatomicrosome dilution that protein concentration is 0.625mg/mL.Take 25057.5 μ L phosphate buffers (pH7.4) extremely In 50mL centrifuge tube, 812.5 μ L SD rat liver microsomes are separately added into, are mixed, the liver that protein concentration is 0.625mg/mL is obtained Microsome dilution.

The incubation of sample: being diluted to 0.25mM for the stock solution of respective compound with the aqueous solution containing 70% acetonitrile respectively, It is spare as working solution.It takes people's hepatomicrosome of 398 μ L or rat liver microsomes dilution that 96 holes are added respectively to be incubated in plate (N=2), it is separately added into the working solution of 2 μ L 0.25mM, mixes.

The measurement of metabolic stability: the terminate liquid of 300 μ L pre-cooling is added in every hole of 96 hole deep-well plates, is placed in ice On, as termination plate.96 holes are incubated for plate and NADPH regenerative system is placed in 37 DEG C of water baths, 100 revs/min of concussions are incubated in advance 5min.80 μ L Incubating Solutions addition termination plate is taken out from the every hole of plate is incubated for, mixes, supplements 20 μ L NADPH regenerative system solution, make For 0min sample.Again to the NADPH regenerative system solution for being incubated for 80 μ L of the every hole addition of plate, starting reaction starts timing.Correspondingization The reaction density for closing object is 1 μM, protein concentration 0.5mg/mL.When reacting 10,30,90min, 100 μ L is respectively taken to react Liquid is added in termination plate, and vortex 3min terminates reaction.Termination plate is centrifuged 10min under the conditions of 5000 × g, 4 DEG C.Take 100 μ L Supernatant is mixed to being previously added in 96 orifice plates of 100 μ L distilled water, carries out sample analysis using LC-MS/MS.

Data analysis: by LC-MS/MS system detection respective compound and interior target peak area, calculate compound with it is interior Mark peak area ratio.Slope is measured by the natural logrithm of the percentage of compound surplus and time mapping, and according to following Formula calculates t_1/2And CL_int, wherein V/M is equal to 1/ protein concentration.

Experimental result is as shown in table 2 below, the compounds of this invention all table in people's hepatomicrosome and rat liver microsomes experiment Reveal excellent metabolic stability.

2 embodiment compound liver particle metabolism of table

Pharmacokinetic Evaluation in rat

6 male Sprague-Dawley rats, 7-8 week old, weight about 210g are divided into 2 groups, every group 3, through vein or The compound (through vein 3mg/kg, taking orally 10mg/kg) of oral single dosage, compares its pharmacokinetic difference.

Rat is raised using standard feed, gives water.Test is fasted for first 16 hours.Drug is sub- with PEG400 and diformazan Sulfone dissolution.Eye socket blood sampling, the time point of blood sampling are 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 0.083 hour after administration Hour, 6 hours, 8 hours, 12 hours and 24 hours.

Rat sucks of short duration anesthesia after ether, and eye socket acquires 300 μ L sample of blood in test tube.There are 30 μ L1% heparinates in test tube Solution.Before use, test tube is stayed overnight in 60 DEG C of drying.After being completed with the latter time point blood specimen collection, rat etherization After put to death.

It after blood specimen collection, leniently overturns test tube at least 5 times, is placed on ice after guaranteeing mixing sufficiently immediately.Blood sample is 4 DEG C 5000rpm is centrifuged 5 minutes, and blood plasma is separated with red blood cell.100 μ L blood plasma are sucked out to clean plastic centrifuge tube with pipettor In, show title and the time point of compound.Blood plasma is stored in -80 DEG C before being analyzed.With in LC-MS/MS measurement blood plasma The concentration of the compounds of this invention.Pharmacokinetic parameter is based on every animal blood concentration in different time points into calculating.

Experimental result is as shown in table 3 below, relative to control compound INT-747, the oral utilization of the compounds of this invention 19 Rate increases substantially, and illustrating it in animal body has better pharmacokinetics.

The experiment of 3 pharmacokinetics in rats of table

PK parameter	INT-747	Compound 19
			Cmax(ng/mL)	1816.4	1117.2
t1/2(hr)	5.42	6.00
			AUC0-t(hr*ng/mL)	33772.7	13475.2
AUC0-∞(hr*ng/mL)	10818.3	18625.0
			MRT(hr)	11.25	16.05
F (%)	100.59	136.64

It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention, in embodiment not The experimental method of actual conditions is indicated, usually according to normal condition, or according to the normal condition proposed by manufacturer.Unless in addition saying Bright, otherwise parts and percentages are parts by weight and weight percent.

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention Protection scope.

In conclusion the present invention relates to following technical schemes:

1. the compound of logical formula (I) or its crystal form, pharmaceutically acceptable salt:

In formula:

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、 R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸And R²⁹It is independently selected from hydrogen (H) or deuterium (D)；

X¹、X²、X³、X⁴It is independently selected from hydrogen (H), deuterium (D), methyl, CH₂D、CHD₂、CD₃、CH₂CH₃、CHDCH₃、 CHDCH₂D、CHDCHD₂、CHDCD₃、CD₂CH₃、CD₂CH₂D、CD₂CHD₂、CD₂CD₃；

Or its physiologically acceptable salt, prodrug, tautomer and stereoisomer, enantiomter, hydrate Or solvate, the mixture formed including these compounds with all proportions；

Additional conditions are that the compound does not include non-deuterated compound.

2. compound as described in claim 1, wherein X¹、X²、X³、X⁴It is each independently selected from one or many deuterated Alkyl.

3. compound as described in claim 1, wherein R¹⁷、R¹⁸、R¹⁹、R²⁰It is each independently selected from hydrogen or deuterium.

4. compound as described in claim 1, selected from formula (2) to the compound of formula (97) or its is pharmaceutically acceptable Salt.

5. a kind of pharmaceutical composition, it includes compound according to claim 1 or its pharmaceutically-acceptable salts with And the pharmaceutical composition of pharmaceutically acceptable carrier.

6. pharmaceutical composition as claimed in claim 5, wherein the pharmaceutical composition is for treating, preventing or disappearing The associated disease mediated except various FXR or TGR5；Or the pharmaceutical composition is used in different therapy fields such as cancer It treated in disease, prevent disease or obstacle or slow down the disease or obstacle process.

7. compound described in any one of claim 1-4 is preparing the use in the drug for treating or preventing disease On the way, the disease is mediated by FXR or TGR5, is selected from chronic liver disease, gastrointestinal disease, nephrosis, cardiovascular disease, silt courage imbalance, Metabolic disease.

8. purposes as claimed in claim 7, wherein the chronic liver disease is primary biliary cirrhosis or non-wine Essence steatohepatitis.

9. purposes as claimed in claim 7, wherein the metabolic disease is obesity or diabetes.

Claims (9)

1. the compound of logical formula (I) or its crystal form, pharmaceutically acceptable salt:

In formula:

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、 R²⁴、R²⁵、R²⁶、R²⁷、R²⁸And R²⁹It is independently selected from hydrogen (H) or deuterium (D)；

X¹、X²、X³、X⁴It is independently selected from hydrogen (H), deuterium (D), methyl, CH₂D、CHD₂、CD₃、CH₂CH₃、CHDCH₃、 CHDCH₂D、CHDCHD₂、CHDCD₃、CD₂CH₃、CD₂CH₂D、CD₂CHD₂、CD₂CD₃；

Or its physiologically acceptable salt, prodrug, tautomer and stereoisomer, enantiomter, hydrate or molten Object, the mixture formed including these compounds with all proportions are closed in agent；

Additional conditions are that the compound does not include non-deuterated compound.

2. compound as described in claim 1, wherein X¹、X²、X³、X⁴It is each independently selected from one or many deuterated alkyl.

3. compound as described in claim 1, wherein R¹⁷、R¹⁸、R¹⁹、R²⁰It is each independently selected from hydrogen or deuterium.

4. compound as described in claim 1, compound selected from the following or its pharmaceutically acceptable salt:

5. a kind of pharmaceutical composition, it includes compound according to claim 1 or its pharmaceutically-acceptable salts and medicine The pharmaceutical composition of acceptable carriers on.

6. pharmaceutical composition as claimed in claim 5, wherein the pharmaceutical composition is each for treating, preventing or eliminating The associated disease that kind FXR or TGR5 is mediated；Or the pharmaceutical composition is used in different therapy fields such as cancer Treatment, prevention disease or obstacle slow down the disease or obstacle process.

7. compound described in any one of claim 1-4 is preparing the purposes in the drug for treating or preventing disease, The disease is mediated by FXR or TGR5, is selected from chronic liver disease, gastrointestinal disease, nephrosis, cardiovascular disease, silt courage imbalance, metabolism Disease.

8. purposes as claimed in claim 7, wherein the chronic liver disease is primary biliary cirrhosis or non-alcoholic Steatohepatitis.

9. purposes as claimed in claim 7, wherein the metabolic disease is obesity or diabetes.