WO2017152677A1 - Cholanic acid compound for preventing or treating fxr-mediated diseases - Google Patents

Abstract

The present invention relates to a cholanic acid compound for preventing or treating FXR-mediated diseases. Specifically, disclosed are a cholanic acid compound represented by formula (I) and a pharmaceutical composition containing the compound, or a crystalline form, a pharmaceutically-acceptable salt, a prodrug, a tautomer, a stereoisomer, an enantiomer, and a hydrate or a solvate thereof. The compound in the present invention can be used as a farnesol X receptor agonist and can therefore be applied to the preparation of drugs for treating FXR-related diseases (for example, fatty liver).

Description

Cholenic acid compounds for the prevention or treatment of FXR-mediated diseases Technical field

The invention belongs to the field of medicine. In particular, the present invention relates to a cyanic acid compound and a pharmaceutical composition thereof for use in the prevention or treatment of FXR-mediated diseases and uses thereof.

Background technique

Farnesol X receptor (FXR), a member of the orphan nuclear receptor family, was first discovered by Forman et al in 1995, and its transcriptional activity can be named by the increase in superphysiological concentration of farnesol. Northern and in situ analysis revealed extensive expression of FXR in the lung, intestine, kidney, and adrenal glands. FXR forms a heterodimer with the 9-cis retinoic acid receptor (RXR) to bind to DNA. The FXR/RXR heterodimer preferentially binds to a component consisting of a binuclear receptor half site that shares AG(G/T) TCA, which forms an inverted repeat and a single nucleoside separation (IR-1 motif). However, these compounds fail to activate mouse and human FXR, making the natural nature of endogenous FXR ligands uncertain. Some naturally occurring bile acids bind to and activate FXR at physiological concentrations. Cholic acids as FXR ligands include chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and taurine and glycine conjugates of these cholic acids.

Cholic acid is a cholesterol metabolite that forms in the liver and is secreted into the duodenum, which plays an important role in the dissolution and absorption of food fats and vitamins. Cholic acid downregulates the transcription of cytochrome P4507a (CYP7a), which encodes an enzyme that catalyzes the rate-limiting step of bile acid biosynthesis. The data show that FXR is associated with the inhibition of CYP7a expression by cholic acid, although the precise mechanism remains unclear. FXR regulates the various enzymes of bile acid metabolism and bile salt carriers under the control of corresponding ligands, synergistic activating factors and hormones. In recent years, various primary and secondary bile acids at physiological concentrations have been found to activate FXR. For example, chenodeoxycholic acid (CDCA) is a commonly used natural agonist. 6-ECDCA (5β-3α,7α-dihydroxy-6α-ethyl-cholanoic acid), a derivative of CDCA, is a potentially potent FXR agonist, two orders of magnitude higher than CDCA.

Primary biliary cirrhosis (PBC) is a common autoimmune hepatobiliary disease that occurs mainly in middle-aged women over the age of 40 with an incidence of 0.1%. PBC is associated with cholestasis, mainly caused by bile uptake, synthesis or secretion disorders, but also due to biliary obstruction, and gradually develop into intrahepatic small bile duct inflammation damage, which eventually leads to hardening. Prior to the INT747, ursodeoxycholic acid was the only FDA approved drug for the treatment of PBC, which effectively improved the level of abnormal liver biochemical markers and reduced the incidence of liver fibrosis and cirrhosis. INT-747 is a novel drug that is used in patients who do not respond adequately or tolerate the old standard treatment drug ursodeoxycholic acid. The drug has been granted orphan drug status by the US FDA, and if approved for marketing, it will receive a seven-year market franchise. On May 27, 2016, the FDA accelerated the approval of INT-747 (Obecholic Acid), which can be used alone to treat primary biliary cholangitis (PBC) that is intolerant to the existing standard therapy drug ursodeoxycholic acid. Patients, or in combination with ursodeoxycholic acid, were treated with PBC patients who did not respond adequately to ursodeoxycholic acid therapy.

The G protein coupled receptor 5 (TGR5) receptor is a G protein coupled receptor that has been identified as a Cell surface receptors that respond to bile acids (BA). The primary structure of the TGR5 and its responsiveness to bile acids have been found between human, bovine, rabbit, rat, and mouse G protein-coupled receptor 5 (TGR5). It is highly conserved and therefore suggests that TGR5 has important physiological functions.

TGR5 is involved in the intracellular accumulation of cyclic adenosine monophosphate (cAMP), which is widely expressed in a variety of different cell types. Although the activation of the membrane receptor described in macrophages can reduce the production of pro-inflammatory cytokines, the stimulation of TGR5 by bile acids in fat cells and muscle cells can be increased. Energy consumption. The latter effect involves cAMP-dependent induction of the type 2 potassium iodide adenine deiodinase (D2), which induces increased thyroid hormone activity by local conversion of T4 to T3 ( See Maruyama, T. et al., 2006, J. Endocrinol, Journal of Endocrinology, 191, 197-205). Accordingly, TGR5 is an attractive targeting for the treatment of metabolic diseases, which may be obesity, diabetes, and metabolic syndrome.

Deuterated modification is a potentially attractive strategy for improving the metabolic properties of drugs. Helium is a safe, stable, non-radiative isotope of hydrogen. Compared with the C-H bond, the C-D bond formed by ruthenium and carbon is stronger because of the lower vibration frequency. In addition, the "heavy hydrogen" version of the drug may be more stable to degradation and longer in the body. Incorporating hydrazine to replace hydrogen can improve the pharmacodynamics and pharmacokinetic profile of the drug, altering the metabolic fate while maintaining the pharmacological activity and selectivity of the physiologically active compound. Deuterated drugs have a positive impact on safety, efficacy and tolerance, and have excellent research prospects.

Summary of the invention

It is an object of the present invention to provide a substituted clavonic acid compound for use in the prevention or treatment of a disease mediated by FXR or TGR5, and a pharmaceutical composition thereof and use thereof.

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

In a first aspect of the invention, there is provided a cholanoic acid compound of the formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound.

In the formula:

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ And R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ and R ²⁹ are each independently selected from "hydrogen (H), hydrazine (D) ""group;

X ¹ , X ² , X ³ , X ⁴ are independently selected from the group consisting of "hydrogen (H), hydrazine (D), methyl, CH ₂ D, CHD ₂ , CD ₃ , CH ₂ CH ₃ , CHDCH ₃ , CHDCH ₂ a group consisting of D, CHDCHD ₂ , CHDCD ₃ , CD ₂ CH ₃ , CD ₂ CH ₂ D, CD ₂ CHD ₂ , CD ₂ CD ₃ ”;

And physiologically acceptable salts, prodrugs, tautomers and stereoisomers, hydrates or solvates thereof, including mixtures of these compounds in all ratios.

As a further improvement of the present invention, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹⁷ , R ¹⁸ and R ²⁹ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹⁹ and R ²⁰ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ and R ²⁸ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, X ¹ , X ² , X ³ , X ⁴ may be independently selected from alkyl groups which are deuterated one or more times.

In another alternative, R ¹ , R ² , R ³ , R ⁴ , R ⁵ are deuterium.

In another alternative, R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ are deuterium.

In another alternative, R ¹⁷ and R ¹⁸ are deuterium.

In another alternative, R ¹⁹ and R ²⁰ are deuterium.

In another alternative, R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ are 氘.

In another embodiment, the compound is selected from the group consisting of a compound or a pharmaceutically acceptable salt thereof, but is not limited to the following compounds:

In another preferred embodiment, the cerium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, and even more preferably greater than 75%. More preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ and R ^{29 are} each in the metamorphic position The isotope content is at least 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20%, more preferably more than 25%, more preferably more than 30%, more preferably more than 35%, more Preferably more than 40%, more preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably more than 70%, more preferably The ground is greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, and even more preferably greater than 99%.

In another preferred embodiment, R R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² of the compound of formula (I), R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ and R ²⁹ , at least one of R contains ruthenium, more preferably two R contains ruthenium, more preferably three R contains ruthenium, more preferably four R contains ruthenium, more preferably five R contains ruthenium, more preferably six R氘, preferably seven R 氘, more preferably eight R 氘, more preferably nine R 氘, more preferably ten R 氘, more preferably eleven R 氘, more Preferably, twelve R 氘, preferably thirteen R 氘, more preferably fourteen R 氘, more preferably fifteen R 氘, more preferably sixteen R 氘, more The seventeen Rs contain 氘, preferably 18 R, more preferably 19 R, more preferably 20 氘, more preferably 21 R More preferably twenty-two R containing bismuth, more preferably twenty-three R containing bismuth, more preferably twenty-four R containing bismuth, more preferably twenty-five R containing bismuth, more preferably twenty-six R contains 氘, better two Seventeen R-containing strontiums, more preferably twenty-eight R 氘, more preferably twenty-nine R 氘.

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

In a second aspect of the invention, there is provided a method of preparing a pharmaceutical composition comprising the steps of: pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, pharmaceutically acceptable The accepted salt, hydrate or solvate is mixed to form a pharmaceutical composition.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, a pharmaceutically acceptable salt, hydrated Or a solvate.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, any glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents A dispersing agent, a disintegrating agent, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.

The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, coagulation Glues, microspheres and aerosols.

Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Herein, "halogen" means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.

As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".

As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).

The invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. Examples of isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, respectively. , ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. a compound, or an enantiomer, a diastereomer, an isomer, or a pharmaceutically acceptable salt or solvate of the present invention, wherein an isotope or other isotopic atom containing the above compound is within the scope of the present invention . Certain isotopically-labeled compounds of the present invention, such as the radioisotopes of ³ H and ¹⁴ C, are also useful therein, and are useful in tissue distribution experiments of drugs and substrates.氚, ie ³ H and carbon-14, ie ¹⁴ C, are easier to prepare and detect and are preferred in isotopes. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral 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, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium). Salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl A base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed of morpholine, piperazine, and lysine, respectively.

The compounds of the invention may have a chiral center, for example, a chiral carbon atom. The compounds of the invention thus include racemic mixtures of all stereoisomers, including enantiomers, diastereomers and atropisomers. Additionally, the compounds of the invention include enriched or resolved optical isomers on any or all of the asymmetric chiral atoms. In other words, the chiral centers apparent from the description are provided as chiral isomers or racemic mixtures. Racemic mixtures and diastereomeric mixtures, as well as enantiomerically or diastereomeric partners which are substantially free of them, isolated or synthesized individual optical isomers, are all within the scope of the invention. The racemic mixture is separated into their individual, substantially optical, by known techniques, for example, by separation of diastereomeric salts formed with optically active auxiliaries such as acids or bases, followed by conversion to optically active materials. Pure isomers. In most cases, the desired optical isomer is synthesized by stereospecific reaction starting from the appropriate stereoisomer of the desired starting material.

The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.

Preferably, the FXR or TGR5 mediated disease or condition is selected from the group consisting of chronic liver disease, gastrointestinal disease, kidney disease, cardiovascular disease, cholestatic disorder, metabolic disease; preferably, the kidney disease is diabetic nephropathy; The disease is selected from the group consisting of arteriosclerosis, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, in which arteriosclerosis is atherosclerosis.

In a specific embodiment, the FXR or TGR5 mediated disease or condition is cardiovascular disease, atherosclerosis, arteriosclerosis, hypercholesterolemia, hyperlipidemia, chronic hepatitis disease, gastrointestinal disease, kidney disease, cardiovascular disease, metabolism Disease, cancer (eg, colorectal cancer), or signs of nerves such as stroke.

In a specific embodiment, the chronic liver disease is primary sclerosis, cerebral palsy xanthomatosis, primary sclerosing gallbladder Inflammation, drug-induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral absorption-related cholestasis, bacterial overgrowth or sepsis cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease , nonalcoholic steatohepatitis, graft-versus-host disease associated with liver transplantation, regeneration of living donor liver transplantation, congenital liver fibrosis, common bile duct stones, granulomatous liver disease, intrahepatic- or extra-malignant tumor, Sjogren syndrome, Sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis, or blood pressure nephropathy, chronic glomerulonephritis, chronic allograft glomerulopathy, chronic interstitial nephritis, or polycystic kidney disease.

In a particular embodiment, the cardiovascular disease is arteriosclerosis, arteriosclerosis, dyslipidemia, hypercholesterolemia, or hypertriglyceridemia.

In a particular embodiment, the metabolic disease is insulin resistance, type I and type II diabetes, or obesity.

Compared with the prior art, the present invention has the beneficial effects that the compound of the present invention can be used to activate the farnesoid X receptor, indirectly inhibit the gene expression of cytochrome 7A1 (CYP7A1), and promote the synthesis of cholic acid. Furthermore, the compounds of the invention are capable of better modulating G protein coupled receptor 5 (TGR5). By deuteration this technique changes the metabolism of the compound in the organism, giving the compound a better pharmacokinetic parameter characteristic. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability. Replacing a hydrogen atom in a compound with hydrazine can increase the drug concentration of the compound in an animal to improve the efficacy of the drug due to its strontium isotope effect. Substitution of a hydrogen atom in a compound with hydrazine may increase the safety of the compound due to inhibition of certain metabolites.

detailed description

The preparation of the structural compound of the formula I of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

Example 1 Preparation of 3α,7α-di-hydroxy-7-d-6β-ethyl-5β-cholanoic acid (Compound 7), the specific synthesis steps are as follows under:

Step 1: Synthesis of methyl α-hydroxy-7-keto-5β-cholanoate (Compound 2).

Two drops of concentrated sulfuric acid were added dropwise to a solution of 3?-hydroxy-7-keto-5?-cholanoic acid (5.00 g, 12.80 mmol) in methanol (12 mL). The methanol was concentrated under reduced pressure, and the residue was applied tojjjjjjjjjjj LC-MS (APCI): m / z = 405.3 [M + 1] +.

Step 2: Synthesis of methyl α-7α-di-trimethylsiloxy-5β-cholanoate (Compound 3).

Trimethylchlorosilane (7.15 mL, 57.0 mmol) was added dropwise to a solution of lithium diisopropylamide (LDA 2M, 34.2 mL, 68.4 mmol) in anhydrous tetrahydrofuran (THF, 70 mL). minute. Then a mixture of methyl 3α-hydroxy-7-keto-5β-cholanoate (2.30 g, 5.7 mmol) in anhydrous tetrahydrofuran (30 mL) was added dropwise to the reaction mixture over 15 min. The reaction was further stirred for 1.5 hours. Triethylamine (15 mL, 103 mmol) was added, and the reaction mixture was stirred at -78 ° C for further 1.5 hours. The reaction was warmed to -20 ° C, saturated aqueous sodium bicarbonate (20 mL) was then evaporated and evaporated. The organic layer was separated and the aqueous extracted with ethyl acetate (100 mL The combined organic layers were washed with a saturated aqueous solution of sodium bicarbonate (100 mL), water (100mL) Dry over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure.

Step 3: Synthesis of 3α-hydroxy-6-ethylidene-7-keto-5β-cholanoic acid methyl ester (Compound 4).

Boron trifluoride etherate (BF ₃ ·OEt ₂ , 14 mL, 110 mmol) was slowly added dropwise to methyl αα-7α-di-trimethylsiloxy-5β-cholanoate (6.15 g) at -60 °C. , 11 mmol) and a mixture of acetaldehyde (1.5 mL, 37.11 mmol) in anhydrous dichloromethane (70 mL). The reaction solution was stirred at -60 ° C for 2.5 hours and warmed to room temperature. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL). The organic layer was separated and the aqueous extracted with dichloromethane (50 mL×3). The combined organic layers were washed with brine (50 mL). Dry over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure. LC-MS (APCI): m / z = 431.3 [M + 1] +.

Step 4: Synthesis of 3α-hydroxy-6β-ethyl-7-keto-5β-cholanoic acid methyl ester (Compound 5).

Add Pd/C (10%, 90 mg) to 3α-hydroxy-6-ethylidene-7-keto-5β-cholanoic acid methyl ester (900 mg, 2.09 mmol) in anhydrous tetrahydrofuran and anhydrous methanol (30 mL) , 1:1v/v) in a mixed solvent. The air was replaced with hydrogen, and the reaction was stirred overnight under hydrogen. The mixture was filtered through Celite, and evaporated. LC-MS (APCI): m / z = 433.3 [M + 1] +.

Step 5: Synthesis of 3α-hydroxy-6β-ethyl-7-keto-5β-cholanoic acid (Compound 6).

Add sodium hydroxide (205 mg, 3.50 mmol) to 3α-hydroxy-6β-ethyl-7-keto-5β-cholanoic acid methyl ester (630 mg, 1.46 mmol) in methanol and water (20 mL, 1:1 v/ In a mixed solvent of v), the reaction was stirred overnight, EtOAc was evaporated, evaporated, evaporated, evaporated, evaporated, evaporated, evaporated dry. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 417.3 [M-1] -.

Step 6: Synthesis of 3α,7α-di-hydroxy-7-d-6β-ethyl-5β-cholanoic acid (Compound 7).

Sodium borohydride (195 mg, 5.00 mmol) was added portionwise to a deuterated methanol of 3α-hydroxy-6β-ethyl-7-keto-5β-cholanoic acid (210 mg, 0.50 mmol) in an ice bath. In d (5 mL), the reaction solution was stirred at room temperature overnight. The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 420.3 [M-1] -.

Example 2 Preparation of 3α,7α-di-hydroxy-6β-(ethyl-1-d)-5β-cholanoic acid (Compound 10), specific synthesis steps The steps are as follows:

Step 1: Synthesis of 3α-hydroxy-6β-(ethyl-1-d)-6-d-7-keto-5β-cholanoic acid methyl ester (Compound 8).

Add Pd/C (10%, 55% in D ₂ O, 60 mg) to anhydrous 3α-hydroxy-6-ethylidene-7-keto-5β-cholanoate (600 mg, 1.39 mmol) A mixed solvent of tetrahydrofuran and deuterated methanol (20 mL, 1:1 v/v). The helium was replaced with air and the reaction was stirred overnight under helium. The mixture was filtered through Celite, EtOAc evaporated. LC-MS (APCI): m / z = 435.3 [M + 1] +.

Step 2: Synthesis of 3α-hydroxy-6β-(ethyl-1-d)-7-keto-5β-cholanoic acid (Compound 9).

Add sodium hydroxide (120 mg, 2.90 mmol) to 3α-hydroxy-6β-(ethyl-1-d)-6-d-7-keto-5β-cholanoic acid methyl ester (420 mg, 0.97 mmol) In a mixed solvent of methanol and water (20 mL, 1:1 v/v), the mixture was stirred and evaporated, evaporated, evaporated, evaporated, evaporated, evaporated, evaporated Wash with saturated brine and dry over anhydrous sodium sulfate. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 418.1 [M-1] -.

Step 3: Synthesis of 3α,7α-di-hydroxy-6β-(ethyl-1-d)-5β-cholanoic acid (Compound 10).

Sodium borohydride (170 mg, 5.50 mmol) was added portionwise to 3α-hydroxy-6β-(ethyl-1-d)-7-keto-5β-cholanoic acid (230 mg, 0.55 mmol). The reaction solution was stirred at room temperature overnight under anhydrous methanol (5 mL). The reaction solution was poured into 20 mL of ice water, and then evaporated, evaporated, evaporated The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 420.3 [M-1] -.

Example 3 Preparation of 3α,7α-di-hydroxy-6β-ethyl-6-d-5β-cholanoic acid (Compound 12), specific synthesis steps as follows:

Step 1: Synthesis of 3α-hydroxy-6β-ethyl-6-d-7-keto-5β-cholanoic acid (Compound 11).

Sodium bismuth oxide (120 mg, 2.90 mmol) was added to methyl 3α-hydroxy-6β-ethyl-7-keto-5β-cholanoate (420 mg, 0.97 mmol) in deuterated methanol and heavy water (20 mL, 1: In a mixed solvent of 1v/v), the reaction was stirred overnight, EtOAc was evaporated, evaporated, evaporated, evaporated, evaporated, evaporated. Sodium is dry. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 418.1 [M-1] -.

Step 3: Synthesis of 3α,7α-di-hydroxy-6β-(ethyl-1-d)-5β-cholanoic acid (Compound 12).

Sodium borohydride (260 mg, 6.60 mmol) was added portionwise to 3α-hydroxy-6β-(ethyl-1-d)-7-keto-5β-cholanoic acid (280 mg, 0.66 mmol). The reaction solution was stirred at room temperature overnight under anhydrous methanol (5 mL). The reaction mixture was poured into 20 mL of ice water, and then evaporated, evaporated, evaporated The organic layer was concentrated under reduced pressure. LC-MS (APCI): m / z = 420.3 [M-1] -.

Example 4 Preparation of 3α,7α-di-hydroxy-6β-(ethyl-1-d)-6-d-5β-cholanoic acid (Compound 14), specifically The steps are as follows:

Step 1: Synthesis of 3α-hydroxy-6β-(ethyl-1-d)-6-d-7-keto-5β-cholanoic acid (Compound 13).

Sodium bismuth oxide (150 mg, 3.52 mmol) was added to methyl 3?-hydroxy-6?-(ethyl-1-d)-6-d-7-keto-5?-cholanoate (510 mg, 1.17 mmol) The reaction mixture was stirred with EtOAc (EtOAc) (EtOAc (EtOAc) The organic layer was washed with brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 419.1 [M-1] -.

Step 2: Synthesis of 3α,7α-di-hydroxy-6β-(ethyl-1-d)-6-d-5β-cholanoic acid (Compound 14).

Sodium borohydride (225 mg, 6.00 mmol) was added in portions to 3α-hydroxy-6β-(ethyl-1-d)-6-d-7-keto-5β-cholanoic acid (250 mg). The reaction solution was stirred at room temperature overnight under anhydrous methanol (5 mL). The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 421.4 [M-1] -.

Example 5 Preparation of 3α,7α-di-hydroxy-7-d-6β-(ethyl-1-d)-6-d-5β-cholanoic acid (Compound 15), The body synthesis steps are as follows:

Sodium borohydride (225 mg, 6.00 mmol) was added portionwise to 3α-hydroxy-6β-(ethyl-1-d)-6-d-7-keto-5β-cholanoic acid in an ice bath ( 250 mg, 0.60 mmol) of deuterated methanol (5 mL), the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 422.4 [M-1] -.

Example 6 Preparation of 3α,7α-di-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-5β-cholanoic acid (Compound 19), specific The implementation steps are as follows:

Step 1: Synthesis of 3α-hydroxy-6-(ethylidene-1,2,2,2-d ₄ )-7-keto-5β-cholanoic acid methyl ester (Compound 16).

Boron trifluoride etherate (BF ₃ ·OEt ₂ , 7.20 mL, 11.4 mmol) was slowly added dropwise to methyl 3α-7α-di-trimethylsiloxy-5β-cholanoate at -60 °C. 3.10g, 5.70mmol) and deuterated acetaldehyde -d ₄ (1.00mL) in dry dichloromethane (58 mL) mixture. The reaction solution was stirred at -60 ° C for 2.5 hours and warmed to room temperature. The reaction was quenched with saturated aqueous sodium bicarbonate (50 mL). The organic layer was separated and the aqueous extracted with dichloromethane (50 mL×3). The combined organic layers were washed with brine (50 mL). Dry over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure. LC-MS (APCI): m / z = 435.5 [M + 1] +.

Step 2: Synthesis of 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-7-keto-5β-cholanoic acid methyl ester (Compound 17).

Add Pd/C (10%, 118 mg) to 3α-hydroxy-6-(ethylidene-1,2,2,2-d ₄ )-7-keto-5β-cholanoic acid methyl ester (1.18 g) , 2.72 mmol) in a mixed solvent of anhydrous tetrahydrofuran and anhydrous methanol (30 mL, 1:1 v/v). The air was replaced with hydrogen, and the reaction was stirred overnight under hydrogen. The mixture was filtered through Celite, and evaporated. LC-MS (APCI): m / z = 437.4 [M + 1] +.

Step 3: Synthesis of 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-7-keto-5β-cholanoic acid (Compound 18).

Add sodium hydroxide (192 mg, 4.80 mmol) to 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-7-keto-5β-cholanoic acid methyl ester (600 mg, 1.37) Methyl acetate and water (20 mL, 1:1 v/v) were stirred in EtOAc EtOAc (EtOAc) The organic layer was washed with brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 421.3 [M-1] -.

Step 4: Synthesis of 3α,7α-dihydroxy-6β-(ethyl-1,2,2,2-d ₄ )-ethyl-5β-cholanoic acid (Compound 19).

Sodium borohydride (160 mg, 4.10 mmol) was added portionwise to 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-7-keto-5β-cholane in an ice bath The reaction mixture was stirred at room temperature overnight with EtOAc (EtOAc,EtOAc. The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 423.3 [M-1] -.

Example 7 Preparation of 3α,7α-di-hydroxy-7-d--6β-(ethyl-1,2,2,2-d ₄ )-5β-cholanoic acid (Compound 20), The specific synthesis steps are as follows:

Sodium borohydride (174 mg, 4.10 mmol) was added in portions to 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-7-keto-5β-biliary in an ice bath The alkanoic acid (175 mg, 0.41 mmol) in EtOAc (5 mL) was evaporated. The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 424.3 [M-1] -.

Example 8 Preparation of 3α,7α-di-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-6-d-5β-cholanoic acid (Compound 22), The specific synthesis steps are as follows:

Step 1: Synthesis of 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-6-d-7-keto-5β-cholanoic acid (Compound 21).

Add sodium hydroxide (97 mg, 2.36 mmol) to 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-7-keto-5β-cholanoic acid methyl ester (300 mg, 0.68) Methyl acetate in a mixed solvent of deuterated methanol and heavy water (10 mL, 1:1 v/v). The mixture was stirred overnight. The solvent was evaporated in vacuo, diluted with water (20 mL), EtOAc. 3) The organic layer was washed with brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 422.3 [M-1] -.

Step 2: Synthesis of 3α,7α-di-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-6-d-5β-cholanoic acid (Compound 22).

Sodium borohydride (134 mg, 3.50 mmol) was added portionwise to the 3α-hydroxy-6β-(ethyl-1,2,2,2-d ₄ )-6-d-7-keto group in an ice bath. 5β-cholanoic acid (150 mg, 0.35 mmol) in anhydrous methanol (5 mL). The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 424.3 [M-1] -.

Example 9 Preparation of 3α,7α-di-hydroxy-7-d-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-5β-cholanoic acid (Compound 25), The specific synthesis steps are as follows:

Step 1: Synthesis of 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-7-keto-5β-cholanoic acid methyl ester (Compound 23).

Add Pd/C (10%, 50% in D ₂ O, 110 mg) to 3α-hydroxy-6-(ethylidene-1,2,2,2-d ₄ )-7-keto-5β-biliary A mixture of methyl alkanoate (1.10 g, 2.53 mmol) in anhydrous tetrahydrofuran and deuterated methanol (30 mL, 1:1 v/v). The helium was replaced with air and the reaction was stirred overnight under helium. The mixture was filtered through Celite, EtOAc evaporated. LC-MS (APCI): m / z = 439.4 [M + 1] +.

Step 2: Synthesis of 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-7-keto-5β-cholanoic acid (Compound 24).

Add sodium niobate (96 mg, 2.40 mmol) to 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-7-keto-5β-cholanoic acid Methyl ester (300 mg, 0.68 mmol) in a mixed solvent of deuterated methanol and heavy water (10 mL, 1:1 v/v), and the mixture was stirred overnight. The methanol was evaporated under reduced pressure, diluted with water (20mL), acidified with 2N HCl, acetic acid The organic layer was washed with brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 423.3 [M-1] -.

Step 3: Synthesis of 3α,7α-di-hydroxy-7-d-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-5β-cholanoic acid (Compound 25) .

Sodium borohydride (134 mg, 3.50 mmol) was added in portions to 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-7- in an ice bath. The keto-5β-cholanoic acid (150 mg, 0.35 mmol) in deuterated methanol (5 mL) was stirred and stirred at room temperature overnight. The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced pressure. LC-MS (APCI): m / z = 426.3 [M-1] -.

Example 10 Preparation of 3α,7α-di-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-5β-cholanoic acid (Compound 27), The body synthesis steps are as follows:

Step 1: Synthesis of 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-7-keto-5β-cholanoic acid (Compound 26).

Add sodium hydroxide (85 mg, 2.04 mmol) to 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-6-d-7-keto-5β-cholanoic acid Methyl ester (255 mg, 0.58 mmol) in a mixed solvent of deuterated methanol and heavy water (10 mL, 1:1 v/v), and the mixture was stirred overnight. The methanol was evaporated under reduced pressure, diluted with water (20mL), acidified with 2N HCl, acetic acid The organic layer was washed with brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 422.3 [M-1] -.

Step 2: Synthesis of 3α,7α-di-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-5β-cholanoic acid (Compound 27).

Sodium borohydride (200 mg, 5.20 mmol) was added in portions to 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-7-keto-5β- in an ice bath. The reaction solution was stirred at room temperature overnight under EtOAc (EtOAc, EtOAc) The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 424.3 [M-1] -.

Example 11 Preparation of 3α,7α-di-hydroxy-7-d-6β-(ethyl-1,1,2,2,2-d ₅ )-5β-cholanoic acid (Compound 28) , the specific synthetic steps are as follows :

Sodium borohydride (80 mg, 1.90 mmol) was added in portions to 3α-hydroxy-6β-(ethyl-1,1,2,2,2-d ₅ )-7-keto-5β in an ice bath - Cyanic acid (80 mg, 0.19 mmol) in deuterated methanol (5 mL), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into 20 mL of ice water, and then evaporated and evaporated. The organic layer was concentrated under reduced vacuo. LC-MS (APCI): m / z = 425.4 [M-1] -.

Biological activity test.

In vitro activity assay of farnesoid X receptor (FXR) and G protein coupled receptor 5 (TGR5).

In vitro activity assay of farnesoid X receptor (FXR).

Activity on FXR was assessed by fluorescence resonance energy transfer (FRET) assay to detect recruitment of the SRC-1 peptide in human FXR using an enzyme that does not contain cells Combined immunosorbent assay (ELISA).

In vitro activity assay of G protein coupled receptor 5 (TGR5).

Luciferase activity is measured in Chinese hamster ovary (CHO) cells, wherein the CHO cells stably express human G protein-coupled receptor 5 (hTGR5), or the cells are utilized an hTGR5 expression vector And a luciferase receptor gene driven by the cAMP responsive element (CRE) was transiently co-transfected. The specific method is as follows.

Plasmid: The National Institutes of Health (NIH) mammalian gene-preserving clone MGC: 40597 (also known as pCMVSPORT6/hTGR5 or pTGR5) and pcDNA 3.1 (+) were obtained from Invitrogen (Carlsbad, CA). pCRE-Luc (cyclized adenosine monophosphate responsive element driven luciferase receptor plasmid) and pCMVβ were obtained from Clontech (Palo Alto, CA).

Transient transfection: Chinese hamster ovary (CHO) cells were placed in 96-well culture plates at a density of 3.5 x 10 ⁴ cells/well for 24 hours, and after that, 150 ng of humans were utilized in each well. (h) G protein coupled receptor 5 expression plasmid (pCMVSPORT6/hTGR5) and 100 ng of circularized adenosine monophosphate (cAMP) responsive element (CRE) driven luciferase reporter plasmid (pCRE-Luc) Transfection was performed in which Lipofectamine 2000 reagent (Invitrogen) was used in the transfection as described in accordance with the manufacturer's instructions. After 6 hours of incubation, the cells were washed once with phosphate buffered saline (PBS) and the medium was exchanged for DMEM containing 0.1% (w/v) bovine serum albumin (BSA). After another 18 hours of incubation, the cells were treated with different concentrations of each compound for 5 hours, wherein the compound was present in fresh 0.1% (w/v) bovine serum albumin (BSA). Among the DMEM. After treatment, 50 μl of lysis buffer (25 mmol of Tris hydrochloric acid (pH 7.6), 2 mmol of ethylenediaminetetraacetic acid (EDTA), 1 mmol of disulfide was used through a freeze-thaw cycle. Threitol (DTT), 10% (v/v) glycerol and 1% (v/v) Triton X-100) lyse the cells and render the method as described below The cells were tested for luciferase.

Luciferase assay and beta-galactosidase assay: For luciferase assay, 20 μl of cell lysate with 100 μl of luciferase buffer 235 μM of luciferin, 265 μM Adenosine triphosphate (ATP) and 135 micromolar coenzyme A (CoA) were mixed and luminescence was measured using CentroXS3LB960 (Berthold Technologies, Bad Wildbad, Germany). For beta-galactosidase assay, 10 μl of cell lysate with 100 μl of buffer Z [60 mmol of disodium hydrogen phosphate, 10 mmol of potassium chloride, 1 mmol of magnesium sulfate) 50 μM of β-mercaptoethanol and 0.75 mg/ml of o-nitrophenyl-β-D-galactopyranoside (ONPG) were mixed and cultured at 37 ° C for 0.5 to 3 hours. The reaction was terminated by the addition of 50 microliters of stop buffer (1 M sodium carbonate) and the optical density was determined at 420 nm.

Establishment of Chinese hamster ovary (CHO) cells (CHO-TGR5 cells) capable of stably expressing human G protein-coupled receptor 5: using Lipofectamin 2000, using 3.8 μg of hTGR5 expression plasmid (pCMVSPORT6/hTGR5), 3.8 μg of cAMP responsiveness A Chinese hamster ovary (CHO) cell was transfected with a CRE-driven luciferase reporter plasmid (pCRE-Luc) and 0.4 μg of a neomycin-resistant gene expression plasmid [pcDNA3.1(+)]. The transfectants were screened with 400 μg/ml of G418 sulfate and individual clones were grown independently in 96-well culture plates. The Chinese hamster ovary (CHO) cell line expressing G protein coupled receptor 5 (TGR5) was screened by life cycle assessment (LCA) treatment, after which luciferase assay was performed.

50% effective concentration (EC ₅₀₎ and the measurement efficiency: detecting in triplicate or quadruplicate manner under each condition. The EC ₅₀ value is determined by probabilistic unit analysis. The efficiency was determined by the following method: For the study of TGR5 agonist, by calculating the percentage of the value of 10 micromoles of lithocholic acid (LCA). After performing the conversion algorithm, the completion of the comparison of the calculated average EC ₅₀ and EC for various compounds ₅₀ has performed.

The experimental results are shown in Table 1 below, indicating that the compound of the present invention can better activate farnesyl ester X receptor (FXR) and G protein coupled receptor 5 (TGR5) as compared with INT-747.

Table 1 Effect of Example Compounds on FXR Receptor and TGR5 Receptor

Metabolic stability evaluation.

Microsomal experiments: human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).

Preparation of the stock solution: A certain amount of the compound of the example was accurately weighed and dissolved to 5 mM with DMSO.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

A solution of NADPH regeneration system (containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride) was prepared and placed on wet ice before use.

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

The experimental results are shown in Table 2 below. The compounds of the present invention exhibited excellent metabolic stability in both human liver microsomes and rat liver microsome experiments.

Table 2 Example compound liver particle metabolism

Pharmacokinetic evaluation in rats.

Six male Sprague-Dawley rats, 7-8 weeks old, weighing 210 g, divided into 2 groups, 3 in each group, intravenously or orally administered a single dose of compound (3 mg/kg intravenously, 10 mg/kg orally). Its pharmacokinetic differences.

Rats were fed a standard diet and given water. Fasting began 16 hours before the test. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the eyelids at a time point of 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

Rats were briefly anesthetized after inhalation of ether, and 300 μL of blood samples were collected from the eyelids in test tubes. There was 30 μL of 1% heparin salt solution in the test tube. The tubes were dried overnight at 60 ° C before use. After the blood sample collection was completed at a later time point, the rats were anesthetized with ether and sacrificed.

Immediately after the blood sample is collected, gently invert the tube at least 5 times to ensure adequate mixing and place on ice. Blood samples were centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate plasma from red blood cells. Pipette 100 μL of plasma into a clean plastic centrifuge tube to indicate the name and time point of the compound. Plasma was stored at -80 °C prior to analysis. The concentration of the compound of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

The experimental results are shown in Table 3 below. Compared with the control compound INT-747, the oral utilization rate of the compound 19 of the present invention was greatly improved, indicating that it has better pharmacokinetics in animals.

Table 3 Rat pharmacokinetic experiments

It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples are generally in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (10)

1. A farnesoid X-receptor agonist characterized by a substituted choline acid compound represented by the formula (I), or a crystalline form thereof, or a pharmaceutically acceptable salt thereof:

   

   Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ and R ²⁹ are each independently selected from "hydrogen (H), a group consisting of 氘(D)";

   X ¹ , X ² , X ³ , X ⁴ are independently selected from the group consisting of "hydrogen (H), hydrazine (D), methyl, CH ₂ D, CHD ₂ , CD ₃ , CH ₂ CH ₃ , CHDCH ₃ , CHDCH ₂ a group consisting of D, CHDCHD ₂ , CHDCD ₃ , CD ₂ CH ₃ , CD ₂ CH ₂ D, CD ₂ CHD ₂ , CD ₂ CD ₃ ”;

   And physiologically acceptable salts, prodrugs, tautomers and stereoisomers, enantiomers, hydrates or solvates thereof, including mixtures of these compounds in all ratios;

   Additionally, the compound does not include a non-deuterated compound.
2. A compound according to claim 1 wherein X ¹ , X ² , X ³ , X ⁴ are independently selected from alkyl groups which are deuterated one or more times.
3. The compound of claim 1 wherein R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ are each independently selected from hydrogen or hydrazine.
4. The compound according to claim 1, which is selected from the group consisting of a deuterated cholic acid compound or a pharmaceutically acceptable salt thereof, but is not limited to the following compounds:

   

   

   

   

   
5. A pharmaceutical composition comprising the bile acid compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, comprising the deuterated compound according to claim 1 or a pharmaceutical thereof An acceptable salt is used as an active ingredient and contains a conventional pharmaceutical carrier.
6. The pharmaceutical composition according to claim 5, wherein said pharmaceutical composition is useful for treating, preventing or eliminating various FXR or TGR5 mediated related disorders; a pharmaceutical composition comprising these compounds is used in Treatment, prevention of disease or disorder, or slowing down the progression of the disease or disorder in different therapeutic areas, such as cancer.
7. Use of a compound according to claims 1-4 for the manufacture of a medicament for the treatment or prevention of a disease mediated by FXR or TGR5, which may be selected from the group consisting of chronic liver disease, gastrointestinal disease, kidney disease, cardiovascular disease, cholestatic Disorder, metabolic disease.
8. The use according to claim 7, wherein said chronic liver disease is primary biliary cirrhosis and nonalcoholic steatohepatitis.
9. The use according to claim 7, wherein the metabolic diseases are obesity and diabetes.
10. A method of treating and/or preventing a disease mediated by various FXR or TGR5 in a subject, the method comprising administering to the subject a formula according to any one of claims 1 to 4. (I) a compound or a polymorphic form thereof, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or a solvent compound, or the pharmaceutical composition of claim 5.