CN118620018A - A steroidal FXR agonist containing an oxadiazole structure on the side chain, and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of medicines, and discloses a steroid FXR (FXR) agonist with an oxadiazole structure in a side chain, and a preparation method and application thereof, wherein the FXR agonist is a compound shown in a formula I or an isomer thereof, or pharmaceutically acceptable salt thereof or a solvent compound of the salt, or a mixture of any two or more of the compounds; the steroid derivative with the oxadiazole structure in the side chain has high FXR agonistic activity and good pharmacokinetic advantage, and can be applied to the preparation of medicines for preventing and/or treating FXR-mediated and/or related liver and gall diseases mediated by FXR.

Description

A steroidal FXR agonist containing an oxadiazole structure on the side chain and its preparation method and application

Technical Field

The invention belongs to the field of medicine, and mainly relates to a steroidal derivative FXR agonist and a preparation method and application thereof.

Background Art

Farnesoid X receptor (FXR) is an important member of the nuclear receptor superfamily. FXR is widely expressed in various organs and tissues such as the liver, heart, kidney, intestine and adrenal cortex. After being activated by natural ligands such as chenodeoxycholic acid (CDCA) and bile acid (CA) in vivo, FXR can move to the nucleus and bind to retinal X receptor (RXR) to form a heterodimer. Under the joint action of multiple coactivators, it binds to the FXR response element on DNA and regulates the expression of downstream target genes. It not only participates in bile acid metabolism, but also plays an important role in the metabolism of substances such as carbohydrates, lipids and alkanes (J. Holt et al., Genes Dev., 2003, 17, 1581-1591; T. Inagaki et al., Cell Metab., 2005, 2, 217-225). FXR ligands, as regulators of the enterohepatic system, play a key role in maintaining bile acid homeostasis, lipid homeostasis, glucose homeostasis, and amino acid metabolism. Therefore, FXR ligands are considered as potential drugs for the treatment of diseases associated with metabolic abnormalities, including diabetes, obesity, liver disease, chronic intestinal inflammation, cholestasis, and kidney disease. In particular, FXR agonists have shown significant potential in the treatment of liver diseases, which can not only correct abnormalities in intermediary metabolism and lipid accumulation, but also inhibit p53 activation induced by metabolic stress, thereby inhibiting the progression of fibrosis and reducing inflammation. In addition, FXR plays an indispensable role in regulating bile acid homeostasis, cholesterol metabolism, and hormone regulation in the body. This makes FXR agonists of great value in the treatment of biliary diseases such as primary biliary cholangitis and primary sclerosing cholangitis. Considering the key role of the kidney in bile acid secretion and reabsorption, FXR agonists provide new possibilities for the treatment of kidney diseases through their anti-inflammatory, anti-lipogenic, antioxidant, and anti-fibrotic effects.

Chenodeoxycholic acid (CDCA) is a natural agonist of FXR and shows certain efficacy in treating hepatobiliary diseases. To date, FXR agonists remain the most popular targets for treating hepatobiliary diseases. Several FXR agonists have entered the clinical research stage for the treatment of various liver diseases, including primary biliary cholangitis, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis (NASH). Drugs that have been clinically tested include obeticholic acid (OCA, trade name Ocaliva), INT-767, Cilofexor (GS-9674), TERN-101 (LY2562175), Nidufexor (LMB763), Tropifexor (LNJ-452), and EDP-305. Unfortunately, many compounds have been suspended or terminated from clinical trials due to adverse side effects, including effects on high-density and low-density lipoproteins, dermatitis, and dyslipidemia. Among them, OCA is a drug that has been proven through clinical trials to have a clear effect in treating NASH, but it ultimately did not obtain FDA approval for marketing because too many adverse reactions (itching) were found in clinical studies.

Recently, two independent research groups reported that bile acids can activate Mas-related G protein-coupled receptor X4 (MRGPRX4, an orphan member of the GPCR family), which is related to pruritus caused by bile deposition associated with liver disease (J. Meixiong et al., P Natl Acad Sci USA 2019, 116 (21), 10525-10530; H. Yu et al., Elife, 2019, 8, e48431). Deoxycholic acid (DCA) is the most potent MRGPRX4 agonist among bile acids, with an EC ₅₀ of nM. Clinically, the plasma bile acid levels of patients with pruritus due to liver disease are significantly higher than those of patients without pruritus due to liver disease, and the plasma bile acid levels of the former are significantly reduced after the pruritus is relieved; in addition, the plasma bile acid concentration of patients with pruritus due to liver disease is sufficient to activate MRGPRX4, while the plasma bile acid concentration of healthy people cannot activate MRGPRX4. These evidences indicate that bile acid and MRGPRX4 are the itch-inducing agent and receptor, respectively, that cause pruritus in cholestasis of liver disease. Therefore, it is necessary to continue to study the FXR target in depth, on the one hand to find highly active FXR agonists, and further to find FXR agonists that can reduce side effects such as itching.

Summary of the invention

In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a steroidal derivative having an oxadiazole structure on the side chain, which has high FXR agonist activity.

Another object of the present invention is to provide a method for preparing the above-mentioned steroid derivatives.

Another object of the present invention is to provide the application of the above steroid derivatives in treating hepatobiliary diseases.

The fourth object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned steroid derivatives as active ingredients.

The purpose of the present invention is achieved through the following technical solutions:

A steroidal FXR agonist containing an oxadiazole structure in the side chain, characterized in that the FXR agonist is a compound of formula I or an isomer thereof, a pharmaceutically acceptable salt of the compound of formula I and its isomer or a solvent compound of the salt, or a mixture of any two or more of the foregoing; the isomers include tautomers, cis isomers, trans isomers, (R)-enantiomers, (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, meso-isomers, and racemic-isomers.

in,

R ₁ is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl;

R is selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted benzyl, substituted or unsubstituted acylalkyl, substituted or unsubstituted acylaryl, substituted or unsubstituted sulfonylalkyl, substituted or unsubstituted sulfonylaryl, hydrogen, fluorine _, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl; R _is self-substituted alkyl, aryl, heteroaryl, benzyl, and its substituent is one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, acyl, and sulfonyl;

X is selected from CH ₂ , NH, N-NH ₂ , O, S;

Y is selected from NH, O, S;

n is selected from 0, 1 or 2;

Preferably,

_R1 is one of hydrogen, methyl, ethyl, methoxy and ethoxy;

_R2 is one of hydrogen, methyl, ethyl, phenyl, benzyl, carboxyphenyl, carboxybenzyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, and carboxypropyl;

X is NH, O, or S;

Y is S;

n is 0 or 1.

The compound of the present invention is selected from:

The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned FXR agonist and also includes one or more of auxiliary materials, diluents, carriers, and excipients.

Use of the above FXR agonist or pharmaceutical composition in the preparation of drugs for preventing and/or treating FXR-mediated and/or FXR-mediated related hepatobiliary diseases.

The FXR agonists of the present invention can be used to prevent or treat liver/biliary system diseases, including dyslipidemia, obesity, non-alcoholic fatty liver disease/inflammation, primary sclerosing cholangitis, cholestatic liver disease, fibrotic diseases, high cholesterol diseases, high triglyceride diseases, type II diabetes, primary biliary cholangitis, portal hypertension, bile acid diarrhea or diabetic insulin resistance, primary biliary cirrhosis, gallstones, non-alcoholic cirrhosis, biliary atresia, chronic liver disease, hepatitis infection, alcoholic liver disease or liver fibrosis and cardiovascular disease.

The FXR agonists of the present invention can be used in a method for preventing or treating dyslipidemia or a disease associated with dyslipidemia, which comprises administering a therapeutically effective amount of the compound of the present invention to a patient in need of such treatment.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The steroidal derivatives containing an oxadiazole structure in the side chain of the present invention have high FXR agonist activity and good pharmacokinetic advantages, especially higher absorption and distribution and lower clearance rate.

(2) Compound A6 of the present invention also has low agonist activity on MRGPRX4 and can significantly reduce the side effects of FXR agonists such as itching.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the left lobe of the liver stained with HE at different magnifications in the model group, positive control group and A6 group.

FIG. 2 is a scoring diagram of HE staining of the left lobe of the liver in the model group, positive control group and A6 group.

DETAILED DESCRIPTION

The compounds and methods of the present invention will be better understood with reference to the following examples.

Definition and Explanation

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent as long as the valence state of the particular atom is normal and the compound after the substitution is stable.

The term "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "hydroxy" refers to an -OH group.

The term "carboxy" refers to a -COOH group.

The term "cyano" refers to a -CN group.

The term "mercapto" refers to a -SH group.

The term "amino" refers to a _-NH2 group.

The term "nitro" refers to a _-NO2 group.

The term "alkoxy" refers to an -O-alkyl group.

The term "alkylamino" refers to an -NH-alkyl group.

The term "alkylsulfonyl" refers to a _-SO2 -alkyl group.

The term "alkylthio" refers to an -S-alkyl group.

The term "alkyl" refers to a hydrocarbon group of the general formula _{CnH2n +1} .

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system.

The term "excipient" generally refers to a carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.

The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc. Their preparations are well known to those skilled in the field of cosmetics or topical medicines.

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

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, prepared from a compound with a specific substituent found in the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Pharmaceutically acceptable salts can mention metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, salts formed with basic or acidic amino acids, etc.

Certain compounds of the present invention may have asymmetric carbon atoms or double bonds or triple bonds. Racemates, diastereomers, geometric isomers and individual isomers are all within the scope of the present invention.

The graphic representation of racemic, ambiscalemic and scalemic or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, the absolute configuration of a stereocenter is indicated by a wedge-shaped bond and a dashed bond. When the compounds described herein contain alkenes, double bonds or other centers of geometric asymmetry, they include E and Z geometric isomers unless otherwise specified. Likewise, all tautomeric forms are included within the scope of the present invention.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

The solvents and reagents used in the present invention are all commercially available. The present invention uses the following abbreviations: eq represents equivalent, equal amount; rt represents room temperature; DCM represents dichloromethane; ACN represents acetonitrile; PE represents petroleum ether; EA represents ethyl acetate; MeOH represents methanol; EtOH represents ethanol; CS ₂ represents carbon disulfide; EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP represents 4-dimethylaminopyridine; DIEPA represents N,N-diisopropylethylamine; TEA represents triethylamine; KOH represents potassium hydroxide; NaOH represents sodium hydroxide; K ₂ CO ₃ represents sodium carbonate; Na ₂ SO ₄ represents anhydrous sodium sulfate; NaCl represents sodium chloride; TBS represents tert-butyldimethylsilyl.

Synthetic route 1:

Reagents and reaction conditions: (a) p-TSA·H ₂ O, MeOH, 60°C; (b) TBDMS-OTf, 2,6-Lutidine, DCM, 0°C～rt; (c) N ₂ H ₄ ·H ₂ O, EtOH, 70°C; (d) KOH, CS ₂ , EtOH, rt; (e) KOH, EtOH, 78°C; (f) TBAF, THF, 60°C; (g) RX, K ₂ CO ₃ , DMF, 70°C.

Embodiment 1:

Take a 100mL three-necked round-bottom flask, add 30mL anhydrous methanol, 1.00g obeticholic acid (2.38mmol), 0.05g p-toluenesulfonic acid monohydrate (0.24mmol), and heat the reaction system to 60°C. After reacting for 3 hours, the raw material is detected by thin layer chromatography (TLC) to complete the reaction. The solvent is concentrated on a rotary evaporator, and then 50mL of ethyl acetate is added to dissolve the residue, which is washed with saturated NaHCO ₃ solution (50mL×3) and saturated NaCl solution (50mL×3) respectively. The organic phase is collected and dried with anhydrous Na ₂ SO ₄ , filtered, and the solvent is removed under reduced pressure to obtain 0.97g of white powder solid compound A1, with a yield of 96%. ¹ H NMR (400MHz, CDCl ₃ ) δ3.75–3.70(m,1H),3.68(s,3H),3.48–3.36(m,1H),2.42–2.32(m,1H),2.29–2.19(m,1H),2.00–1.79(m,6H),1.79–1.56(m,8 H),1.56–1.23(m,12H),1.23–1.11(m,3H),1.07–0.97(m,1H),0.96–0.89(m,9H),0.67(s,3H).

Embodiment 2:

Take a 100mL three-necked flask, add 0.50g intermediate A1 (1.15mmol) and 0.27g 2,6-lutidine (2,6-Lutidine, 2.53mmol) to 30mL anhydrous dichloromethane, and stir under nitrogen protection until the raw materials are completely dissolved. Place the reaction system in an ice bath, and add tert-butyldimethylsilyl trifluoromethanesulfonate (TBDMS-OTf, 0.67g, 2.53mmol) in anhydrous dichloromethane dropwise. After the addition is complete, continue to react at 0℃ for 1 hour, then slowly warm to room temperature and continue to react for 8 hours. The raw materials have been completely reacted by thin layer chromatography TLC. The reaction solution was poured into a separatory funnel and 50 mL of dichloromethane was added. The mixture was washed with saturated NaHCO ₃ solution (50 mL × 3) and saturated NaCl solution (50 mL × 3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , filtered under reduced pressure, and the solvent was concentrated. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as the stationary phase) with petroleum ether/ethyl acetate (4:1) as the eluent to obtain the intermediate compound A2, 0.37 g. Colorless oil, yield 49%. ¹ H NMR (400 MHz, CDCl ₃ )δ3.73–3.70(m,1H),3.68(s,3H),3.46–3.33(m,1H),2.44–2.31(m,1H),2.30–2.18(m,1H),2.01–1.87(m,2H),1.87–1.75(m,4H),1.73–1.65(m,1 H),1.65–1.22(m,20H),1.22–1.10(m,3H),1.03–0.97(m,1H),0.95–0.93(m,6H),0.90(s,9H),0.89(s,3H),0.88(s,9H),0.67(s,3H),0.06(s,6H) ),0.04(s,6H).

Embodiment 3:

In a 100mL round-bottom flask, add 0.50g of intermediate A2 (0.75mmol) and 30mL of anhydrous ethanol, respectively, and stir until the raw material is completely dissolved. Continue to add 5mL of 80% hydrazine hydrate solution (N2H4·H2O) to the reaction system. Heat the reaction system to 68°C and react for 8 hours. When TLC detects that the raw material has reacted completely, cool the reaction system to room temperature and remove the solvent by vacuum distillation. Add 50mL of ethyl acetate to dissolve the residue, pour it into a separatory funnel, wash it with deionized water (50mL×3) and saturated NaCl solution (50mL×3), respectively, dry the organic phase over anhydrous Na ₂ SO ₄ , filter and concentrate the solvent under reduced pressure to obtain compound intermediate A3, a white powder solid, with a yield of 90%.

Embodiment 4:

In a 100 mL round-bottom flask, 0.50 g of intermediate A3 (0.75 mmol) was dissolved in 30 mL of anhydrous ethanol, and 0.57 g of carbon disulfide (CS ₂ , 7.50 mmol) and 0.42 g of potassium hydroxide (KOH, 7.50 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction solution was filtered through a Buchner funnel, and the solvent and carbon disulfide were removed by vacuum distillation to obtain the potassium salt of intermediate A4. It was used directly in the next step without purification.

Take a 100mL round-bottom flask again, add the potassium salt of the intermediate A4, add 30mL of anhydrous ethanol and stir to dissolve, then heat the reaction system to 78°C, add 0.08g of potassium hydroxide (KOH, 1.50mmol), and continue to react at this temperature for 12 hours. TLC monitors the reaction progress, the raw materials are basically reacted, the reaction system is cooled to room temperature, and 10-20mL of dilute hydrochloric acid is added to adjust the pH value to 4-5. Pour the reaction solution into a 250mL separatory funnel, add 40mL of chloroform to extract twice, wash with deionized water (50mL×3), saturated NaCl solution (50mL×3) and dry with anhydrous sodium sulfate; the obtained organic phase is rotary evaporated to dryness to obtain the crude product of compound A6, which is redissolved in dichloromethane and adsorbed on 100-200 mesh silica gel. The above crude product is purified by column chromatography to obtain intermediate A5, 0.38g, white powder solid, and the two-step reaction yield is 71%.

Embodiment 5:

Add 0.50g of intermediate A5 (0.71mmol) and 20mL of anhydrous tetrahydrofuran to a 100mL three-necked flask and stir under nitrogen until the raw materials are completely dissolved. Add 1.70mL of tetrabutylammonium fluoride (TBAF, 1.0mol/L inTHF) through a dropping funnel. After the addition is complete, heat the reaction system to 60°C and continue stirring for 12 hours. Detect that the raw materials have reacted completely by thin layer chromatography TLC, and concentrate the solvent on a rotary evaporator. Add 50mL of ethyl acetate to dissolve the residue, wash with saturated NaHCO ₃ solution (50mL×3) and saturated NaCl solution (50mL×3), respectively, combine the organic phases and dry with anhydrous Na ₂ SO ₄ , and remove the solvent by filtration under reduced pressure. The crude product was purified by column chromatography (200-300 mesh silica gel powder as stationary phase, methanol and dichloromethane volume ratio of 1:10 as eluent) to obtain compound A6, 0.41 g, white powder solid, yield 81%, HPLC purity 98.0%. ¹ H NMR (600 MHz, DMSO-d ₆ )δ14.28(s,1H),4.30(s,1H),4.05(d,J＝5.0Hz,1H),3.52–3.47(m,1H),3.17–3.09(m,1H),2.79–2.71(m,1H),2.67–2.59(m,1H),1.94–1.88(m,1H),1.83–1.67(m,6H),1 .56–1.49(m,1H),1.49–1.34(m,8H),1.34–1.21(m,4H),1.21–1.07(m,6H),1.03–0.95(m,1H),0.94(d,J＝6.4Hz,3H),0.92–0.85(m,1H),0.84–0.8 1(m,6H),0.61(s,3H). ¹³ C NMR (151MHz, DMSO-d ₆ ) δ178.16,165.12,71.07,68.84,55.71,50.56,45.79,42.53,41.73,40.38,39.79,35.99,35.67,35.25,34.01,33.12,31. 68,30.91,28.25,23.57,23.55,22.63,22.36,20.87,18.54,12.18,12.17.HR-MS(ESI)m/z:calcd forC ₂₇ H ₄₃ N ₂ O ₃ S[MH] ^- :475.2995; found:475.2991.

Embodiment 6:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.12 g of anhydrous potassium carbonate (K ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then iodomethane (0.07 g 0.51 mmol) was added in batches, and then the reaction system was heated to 70°C and continued to react at this temperature for 12 hours. The reaction of the raw material was detected by thin layer chromatography (TLC) to determine that the reaction was complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=2:1) as eluent to obtain 76 mg of compound B1 as a white powdery solid with a yield of 38% and a HPLC purity of 97.6%. ¹ H NMR (600 MHz, CDCl ₃ )δ3.72–3.68(m,1H),3.45–3.36(m,1H),2.90–2.82(m,1H),2.77–2.71(m,1H),2.70(s,3H),2.00–1.94(m,1H),1.94–1.86(m,2H),1.86–1.75(m,4H) ),1.71–1.63(m,2H),1.61–1.59(m,1H),1.53–1.35(m,8H),1.35–1.11(m,10H),1.05–1.00(m,1H),0.99(d,J＝6.0Hz,3H),0.93–0.88(m,6H),0.66 (s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ168.53,164.72,72.31,70.88,55.65,50.52,45.20,42.79,41.20,40.04,39.61,35.55,35.51,35.38,33.99,33.24,32.56,30. 65,28.21,23.69,23.15,22.39,22.24,20.76,18.26,14.54,11.81,11.67.HR-MS(ESI)m/z:calcd for C ₂₈ H ₄₆ N ₂ O ₃ SNa[M+Na] ⁺ :513.3127; found:513.3130.

Embodiment 7:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.12 g of anhydrous potassium carbonate (K ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of acetonitrile (MeCN). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then 3-bromopropylene (0.06 g 0.51 mmol) was added in batches, and then the reaction system was heated to 60°C and continued to react at this temperature for 12 hours. The reaction of the raw materials was detected by thin layer chromatography (TLC) to determine that the reaction was complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=4:1) as eluent to obtain 96 mg of compound B2. White powder solid, yield 46%, HPLC purity 99.4%. ¹ H NMR (600MHz,CDCl ₃ )δ5.97(ddt,J=17.0,10.0,7.0Hz,1H),5.34(dq,J=16.9,1.3Hz,1H),5.19(dd,J=10.0,1.2Hz,1H),3.84(dt,J=7.0,1.2Hz,2H),3.72-3.68(m,1H),3.44-3.36(m,1H),2.89-2.81(m,1H),2.77-2.68(m,1H ),1.96(dt,J＝12.5,3.4Hz,1H),1.94–1.86(m,2H),1.85–1.74(m,4H),1.71–1.56(m,6H),1.53–1.34(m,9H),1.34–1.09(m,10H),1.04–1.00(m,1H), 0.99(d,J＝6.1Hz,3H),0.91–0.88(m,6H),0.66(s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ 167.62, 162.34, 130.81, 118.57, 71.32, 69.88, 54.63, 49.50, 44.17, 41.78, 40.16, 39.01, 38.58, 34.51, 34.49, 34.34, 34. 14,32.99,32.22,31.50,29.63,27.18,22.67,22.13,21.39,21.20,19.73,17.23,10.78,10.63.HR-MS(ESI)m/z:calcd for C ₃₀ H ₄₈ N ₂ O ₃ SNa[M+Na] ⁺ :539.3284; found:539.3293.

Embodiment 8:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.09 g of anhydrous sodium carbonate (Na ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of acetonitrile (MeCN). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then 2-bromoethanol (0.06 g 0.51 mmol) was added in batches, and then the reaction system was heated to 55°C and continued to react at this temperature for 12 hours. The reaction of the raw material was detected by TLC to be complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=1:1) as eluent to obtain 110 mg of compound B3 as a white powdery solid with a yield of 55% and a HPLC purity of 99.2%. ¹ H NMR (600 MHz, CDCl ₃ )δ3.89(t,J＝6.0Hz,2H),3.63–3.62(m,1H),3.39–3.33(m,1H),3.32(t,J＝6.0Hz,2H),2.83–2.75(m,1H),2.69–2.61(m,1H),1.92–1.86(m,1H),1.86 –1.67(m,7H),1.64–1.56(m,2H),1.55–1.48(m,1H),1.47–1.31(m,7H),1.31–1.02(m,8H),0.92(d,J＝5.8Hz,3H),0.92–0.86(m,1H),0.85–0.80(m ,6H),0.59(s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ167.75,163.33,71.23,69.84,60.15,54.52,49.49,44.21,41.75,40.24,39.01,38.59,34.54,34.49,34.34,33.91,32.81 ,32.20,31.47,29.53,27.22,22.64,22.16,21.26,21.25,19.75,17.24,10.78,10.68.HR-MS(ESI)m/z:calcd for C ₂₉ H ₄₉ N ₂ O ₄ S[M+H] ⁺ :521.3413; found:521.3420.

Embodiment 9:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.09 g of anhydrous sodium carbonate (Na ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of acetonitrile (MeCN). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then 1-fluoro-2-bromoethane (0.06 g 0.51 mmol) was added in batches, and then the reaction system was heated to 65°C and continued to react at this temperature for 12 hours. The reaction of the raw material was detected by thin layer chromatography (TLC) to determine that the reaction was complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3) respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=4:1) as eluent to obtain 112 mg of compound B4. White powder solid, yield 56%, HPLC purity 98.8%. ¹ H NMR (600MHz,CDCl ₃ )δ4.72(t,J=5.8Hz,1H),4.64(t,J=5.8Hz,1H),3.65-3.61(m,1H),3.46(dt,J=21.6,5.8Hz,2H),3.38-3.29(m,1H),2.83-2.75(m,1H),2.71-2.63(m,1H),1.90(dt,J=12.4,3.3Hz,1H),1. 86–1.80(m,2H),1.79–1.67(m,5H),1.64–1.56(m,2H),1.56–1.49(m,1H),1.48–1.28(m,8H),1.27–1.04(m,8H),0.97–0.93(m,1H),0.93(d,J＝5. 8Hz,3H),0.86–0.80(m,6H),0.59(s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ167.86,162.12,80.55,79.41,71.27,69.84,54.61,49.50,44.18,41.77,40.19,39.01,38.59,34.53,34.49,34.34,32.91 ,32.21,31.49,31.28,31.14,29.59,27.19,22.66,22.14,21.34,21.22,19.74,17.24,10.79,10.65.HR-MS(ESI)m/z:calcd for C ₂₉ H ₄₇ FN ₂ O ₃ SNa[M+Na] ⁺ :545.3189; found:545.3197.

Embodiment 10:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.09 g of anhydrous sodium carbonate (Na ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then 2-bromoacetamide (0.06 g 0.51 mmol) was added in batches, and then the reaction system was heated to 85°C and continued to react at this temperature for 12 hours. The reaction of the raw material was detected by TLC to be complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=1:4) as eluent to obtain 135 mg of compound B5 as a white powdery solid with a yield of 67% and a HPLC purity of 97.3%. ¹ H NMR (600 MHz, DMSO-d ₆ )δ7.71(s,1H),7.31(s,1H),4.29(s,1H),4.06–4.03(m,1H),3.99(s,2H),3.52–3.47(m,1H),3.17–3.09(m,1H),2.89–2.81(m,1H),2.78–2.70(m, 1H),1.91(m,1H),1.85–1.75(m,3H),1.75–1.67(m,3H),1.54–1.49(m,1H ),1.48–1.07(m,15H),1.04–0.96(m,1H),0.96–0.91(m,3H),0.88–0.80(m ,6H),0.61(s,3H). ¹³ C NMR (151MHz, DMSO-d ₆ ) δ168.64,168.31,163.23,71.06,68.85,55.77,50.57,45.80,42.54,41.73,40.53,40.00,36.31,35.99,35.68,35.31,34 .02,33.11,32.43,30.91,28.25,23.57,22.63,22.08,20.87,18.56,15.51,12.18,12.17.HR-MS(ESI)m/z:calcd for C ₂₉ H ₄₇ N ₃ O ₄ SNa[M+Na] ⁺ :556.3185; found:556.3191.

Embodiment 11:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.12 g of anhydrous potassium carbonate (K ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then 2-bromoacetic acid (0.07 g 0.51 mmol) was added in batches, and then the reaction system was heated to 75°C and continued to react at this temperature for 12 hours. The reaction of the raw material was detected by thin layer chromatography (TLC) to determine that the reaction was complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as the stationary phase) with petroleum ether/ethyl acetate (V:V=1:4) as the eluent to obtain 101 mg of compound B6. White powder solid, yield 50%, HPLC purity 98.3%. ¹ H NMR (600MHz, DMSO-d ₆ )δ12.96(s,1H),4.29(s,1H),4.10(s,2H),4.07–4.01(m,1H),3.50(s,1H),3.17–3.09(m,1H),2.90–2.82(m,1H),2.79–2.70(m,1H),1.94–1.91(m,1H),1.85–1 .67(m,6H),1.55–1.49(m,1H),1.49–1.35(m,7H),1.35–1.07(m,9H),1.03–0.96(m,1H),0.96–0.92(m,3H),0.92–0.86(m,1H),0.86–0.80(m,6H) ,0.61(s,3H). ¹³ C NMR (151MHz, DMSO-d ₆ ) δ169.32,168.78,162.90,71.07,68.85,55.77,50.56,45.80,42.54,41.73,40.53,35.99,35.68,35.30,34.57,34.02,33 .11,32.42,30.91,28.25,23.57,23.56,22.63,22.08,21.52,20.87,18.55,12.18,12.17.HR-MS(ESI)m/z:calcd for C ₂₉ H ₄₆ N ₂ O ₅ SNa[M+Na] ⁺ :557.3025; found:557.3028.

Embodiment 12:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.12 g of anhydrous potassium carbonate (K ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature until compound A6 was completely dissolved, and then 3-bromopropionic acid (0.08 g 0.51 mmol) was added in batches, and then the reaction system was heated to 70°C and continued to react at this temperature for 12 hours. The reaction of the raw material was detected by thin layer chromatography (TLC) to determine that the reaction was complete, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3), respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=1:4) as eluent to obtain 118 mg of compound B7. White powder solid, yield 59%, HPLC purity 97.4%. ¹ H NMR (600 MHz, CD ₃ OD)δ3.67–3.63(m,1H),3.43(t,J＝6.8Hz,2H),3.35–3.31(m,1H),2.95–2.88(m,1H),2.83(t,J＝6.8Hz,1H),2.82–2.75(m,1H),2.03–1.97(m,1H),1.9 6–1.71(m,7H),1.63–1.57(m,1H),1.57–1.44(m,7H),1.42–1.26(m,6H),1.26–1.08(m,3H),1.04–1.01(m,3H),1.01–0.95(m,1H),0.93–0.86(m,6 H),0.69(s,3H). ¹³ C NMR (151MHz, CD ₃ OD) δ173.24,169.17,164.28,71.80,69.78,55.69,50.25,45.55,42.39,41.74,40.14,39.60,35.37,35.26,35.24,33.59,33.12, 33.02,32.17,29.85,27.88,27.11,23.17,22.36,22.10,21.65,20.57,17.33,10.83,10.65.HR-MS(ESI)m/z:calcd for C ₃₀ H ₄₈ N ₂ O ₅ SNa[M+Na] ⁺ :571.3182; found:571.3187.

Embodiment 13:

In a 100 mL round-bottom flask, 0.20 g of compound A6 (0.42 mmol) and 0.12 g of anhydrous potassium carbonate (K ₂ CO ₃ , 0.84 mmol) were added and dissolved in 20 mL of acetonitrile (MeCN). Stir at room temperature until compound A6 was completely dissolved, and then diethyl p-toluenesulfonyloxymethylphosphonate (0.16 g 0.51 mmol) was added in batches, and then the reaction system was heated to 60°C and continued to react at this temperature for 12 hours. Thin layer chromatography TLC was used to detect that the raw material had reacted completely, and the solvent was concentrated on a rotary evaporator. 50 mL of ethyl acetate was added to dissolve the residue, and the mixture was poured into a 250 mL separatory funnel, and washed with saturated NaHCO ₃ solution (50 mL×3) and saturated NaCl solution (50 mL×3) respectively. The organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=1:5) as eluent to obtain 136 mg of compound B8. White powder solid, yield 68%, HPLC purity 97.8%. ¹ HNMR (600MHz, DMSO-d ₆ )δ4.06–4.02(m,4H),3.67(d,J=12.6Hz,2H),3.50(s,1H),3.17–3.10(m,1H),2.92–2.84(m,1H),2.81–2.72(m,1H),1.95–1.89(m,1H),1.85–1.66(m,6H),1.55–1.49(m,1H), 1.48–1.37(m,7H),1.36–1.22(m,5H),1.20(t,J＝7.0Hz,6H),1.18–1.04(m,6H),1.03–0.97(m,1H),0.95(d,J＝6.0Hz,3H),0.93–0.85(m,2H),0.85– 0.80(m,6H),0.61(s,3H). ¹³ C NMR (151MHz, DMSO-d ₆ ) δ169.22,162.17,71.06,68.85,62.87,62.83,55.80,50.56,45.79,42.54,41.73,40.53,35.99,35.67,35.29,34.02,33.11 ,32.43,30.91,28.25,25.33,24.37,23.56,22.63,22.16,21.52,20.87,18.52,16.62,16.58,12.18,12.16.HR-MS(ESI)m/z:calcd for C ₃₂ H ₅₅ N ₂ O ₆ PSNa[M+Na] ⁺ :649.3416; found:649.3408.

Embodiment 14:

In a 100mL round-bottom flask, 0.20g of compound A6 (0.42mmol) and 0.12g of anhydrous potassium carbonate (K ₂ CO ₃ , 0.84mmol) were added and dissolved in 20mL N,N-dimethylformamide (DMF). Stir at room temperature until compound A6 is completely dissolved, then add benzyl bromide (0.09g 0.51mmol) in batches, then heat the reaction system to 80°C and continue to react at this temperature for 12 hours. Thin layer chromatography TLC was used to detect that the raw material had reacted completely, and the solvent was concentrated on a rotary evaporator. 50mL of ethyl acetate was added to dissolve the residue, poured into a 250mL separatory funnel, washed with saturated NaHCO ₃ solution (50mL×3) and saturated NaCl solution (50mL×3), respectively, the organic phases were combined and dried with anhydrous Na ₂ SO ₄ , and the solvent was removed by filtration under reduced pressure. The crude product was purified by silica gel column chromatography (200-300 mesh silica gel powder as stationary phase) with petroleum ether/ethyl acetate (V:V=1:1) as eluent to obtain 63 mg of compound B9. White powder solid, yield 32%, HPLC purity 99.3%. ¹ H NMR (600MHz,CDCl ₃ )δ7.41(d,J=7.4Hz,2H),7.32(t,J=7.4Hz,2H),7.29(d,J=7.3Hz,1H),4.44(s,2H),3.69(s,1H),3.44–3.35(m,1H),2.87–2.80(m,1H),2.75–2.67(m,1H),1.98–1.95(m,1H),1.91– 1.76(m,8H),1.70–1.65(m,2H),1.60–1.57(m,1H),1.53–1.38(m,9H),1.37–1.23(m,6H),1.23–1.09(m,4H),1.03–1.00(m,1H),0.99–0.96(m,3H ),0.91–0.87(m,6H),0.65(s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ168.58,163.56,135.63,129.10,128.77,128.04,72.24,70.81,55.66,50.52,45.22,42.78,41.24,40.04,39.63,36.74,35. 59,35.51,35.37,33.91,33.23,32.53,30.61,28.22,23.67,23.18,22.38,22.27,20.77,18.28,11.83,11.70.HR-MS(ESI)m/z:calcd for C ₃₄ H ₅₀ N ₂ O ₃ SNa[M+Na] ⁺ :589.3440; found:589.3438.

Embodiment 15:

The preparation method of compound B10 refers to the operation steps described in Example 14. After purification, 134 mg of compound B10 was obtained. White powder solid, yield 67%, HPLC purity 97.4%. ¹ H NMR (600 MHz, CDCl ₃ ) δ7.29 (d, J＝7.8 Hz, 2H), 7.13 (d, J＝7.8 Hz, 2H), 4.41 (s, 2H), 3.71–3.68 (m, 1H), 3.44–3.36 (m, 1H), 2.88–2.80 (m, 1H), 2.75–2.67 (m, 1H), 2.33 (s, 3H), 1.99–1. 94(m,1H),1.94–1.75(m,7H),1.71–1.62(m,2H),1.62–1.55(m,1H),1.52–1.10(m,17H),1.04–0.99(m,1H),0.99–0.96(m,3H),0.93–0.88(m,6H), 0.66(s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ168.52,163.68,137.86,132.52,129.46,129.02,72.27,70.84,55.66,50.52,45.22,42.78,41.23,40.04,39.62,36.56,35. 57,35.52,35.38,33.95,33.24,32.54,30.63,28.21,23.68,23.17,22.39,22.26,21.17,20.77,18.27,11.82,11.69.HR-MS(ESI)m/z:calcd for C ₃₅ H ₅₂ N ₂ O ₃ SNa[M+Na] ⁺ :603.3597; found:603.3584.

Embodiment 16:

The preparation method of compound B11 refers to the operation steps described in Example 14. After purification, 70 mg of compound B11 was obtained. White powder solid, yield 35%, HPLC purity 97.2%. ¹ H NMR (600 MHz, CDCl ₃ )δ7.40–7.31(m,2H),6.88–6.79(m,2H),4.40(s,2H),3.79(s,3H),3.72–3.68(m,1H),3.45–3.36(m,1H),2.88–2.80(m,1H),2.75–2.67(m,1H),2.0 0–1.94(m,1H),1.94–1.70(m,7H),1.68–1.63(m,2H),1.63–1.56(m,1H),1.53–1.13(m,17H),1.05–1.00(m,1H),1.00–0.97(m,3H),0.92–0.88(m, 6H),0.66(s,3H). ¹³ C NMR (151MHz, CDCl ₃ ) δ168.52,163.70,159.38,130.38,127.53,114.17,72.33,70.89,55.65,55.30,50.52,45.20,42.80,41.20,40.04,39.61,36. 40,35.55,35.52,35.38,34.00,33.24,32.54,30.65,28.21,23.69,23.16,22.40,22.24,20.76,18.27,11.81,11.67.HR-MS(ESI)m/z:calcd for C ₃₅ H ₅₂ N ₂ O ₄ SNa[M+Na] ⁺ :619.3546; found:619.3533.

Embodiment 17:

The preparation method of compound B12 refers to the operation steps described in Example 14. After purification, 138 mg of compound B12 was obtained. White powder solid, yield 69%, HPLC purity 97.7%. ¹ H NMR (600 MHz, CD ₃ OD)δ7.99–7.94(m,2H),7.53–7.52(m,2H),4.49(s,2H),3.67–3.63(m,1H),3.36–3.31(m,1H),2.90–2.83(m,1H),2.77–2.71(m,1H),1.98–1.94(m ,1H),1.89–1.71(m,8H),1.63–1.57(m,1H),1.57–1.16(m,18H),1.14–1.04(m,1H),1.03–0.99(m,1H),0.98–0.95(m,3H),0.92–0.88(m,6H),0.64 (s,3H). ¹³ C NMR (151 MHz, CD ₃ OD)δ174.66,169.34,167.91,163.57,141.72,129.74,128.79,71.80,69.85,55.67,50.23,45.50,42.38,42.36,41.71,40.12,39.57,35.66,35. 40,35.25,35.23,33.11,33.01,32.25,29.87,27.91,23.18,22.44,22.11,21.74,20.59,19.41,17.38,10.93,10.90,10.71.HR-MS(ESI)m/z:calcd for C _{3 5} H ₅₀ N ₂ O ₅ SNa[M+Na] ⁺ :633.3338; found:633.3340.

Experimental Example 1 In vitro activity study of compounds (molecular level):

The time-resolved fluorescence resonance energy transfer (TR-FRET) method was used to test the binding of compounds to FXR receptor protein.

To test the ability of compounds to bind to the FXR receptor, we used Invitrogen's LanthaScreen ^TM TR-FRET FXR coactivator assay kit (#PV4833) to test the effects of compounds on the FXR ligand binding domain (FXR-LBD). The terbium (Tb)-labeled anti-glutathione-S-transferase (GST) antibody indirectly labels FXR by binding to the GST tag. The binding of the compound to FXR causes conformational changes, resulting in an increase in the affinity of FXR for the coactivator peptide. The fluorescently labeled coactivator peptide (Fluorescein-SRC2-2) and the terbium-labeled GST antibody are close to each other, resulting in an increase in the TR-FRET signal. For specific methods, please refer to the kit instructions, and the operation is as follows:

1. Complete Coregulator buffer G: Prepare Complete Coregulator buffer G by adding 1 M DTT to a concentration of 10 mM DTT.

2. Prepare 2× working solution of the compound to be tested: dilute the 3 mM compound stock solution to 100× series concentration using DMSO, and dilute the 100× series concentration to 2× using Complete Coregulator buffer G (set 10 concentrations, 3-fold dilution).

3. Prepare 4×FXR-LBD: Dilute the stock solution to the required volume using Complete Coregulator buffer G, with a concentration of 20 nM.

4. Prepare a 4×Fluorescein-SRC2-2/terbium-labeled anti-GST antibody mixture: dilute the stock solution to the required volume using Complete Coregulator buffer G. The concentration of Fluorescein-SRC2-2 is 2 μM, and the concentration of terbium-labeled anti-GST antibody is 20 nM.

5. Add 5 μL of FXR-LBD and 5 μL of Fluorescein-SRC2-2/terbium-labeled anti-GST antibody mixture to a black U-bottom 384-well plate containing 10 μL of the test compound and seal the plate with a sealing film.

6. Centrifuge at 1000 rpm for 1 minute, shake at 300 rpm for 1 minute, and incubate at room temperature in the dark for 2 hours.

7. Time-resolved fluorescence detection: TECAN Spark multifunctional microplate detection platform reads the plate, with an excitation wavelength of 340nm and emission wavelengths of 520nm and 490nm.

Result analysis:

(1) The value at 520nm divided by the value at 490nm;

(2) Calculation of excitation rate

Excitation rate = (X-Min)/(Max-Min)*100%

X represents the 520/490 value of each concentration of the compound; Max represents the 520/490 value of the reference compound; Min represents the 520/490 value of the negative control well.

GraphPad Prism 8.0 was used to analyze the data and calculate the EC ₅₀ value of the activation effect of the compound. The highest signal value of the test compound was compared with the highest signal value of the reference compound (chenodeoxycholic acid, CDCA) to obtain the activation efficacy percentage (Efficacy) of the test compound. Specific data are shown in Table 1.

Table 1 Compound detection biochemical experimental test results (TR-FRET)

Conclusion: From the above table, we can conclude that the compounds of the embodiments of the present invention tested by the time-resolved fluorescence resonance energy transfer method have obvious agonist effects on FXR activity, especially A6, B3, B6, B7, and B12 have the best effects, reaching the nM level, and the agonist efficiency is 2-3 times higher than that of natural FXR agonists.

Experimental Example 2 In vitro activity study of compounds (cell level):

FXR luciferase reporter gene assay

To further test the efficacy of the compounds in activating FXR receptors in cells, we used Promega's Bio-Glo ^™ Luciferase Assay System (#G7940) to detect the effects of the compounds on the FXR reporter gene in HEK-293 human embryonic kidney cells transiently co-transfected with GAL4RE-FXR-LBD expression plasmid and luciferase reporter gene plasmid. The specific experimental method is as follows:

1. HEK-293 cell plating and culture

After cell digestion, the cells were seeded at an appropriate density in a 96-well cell culture plate with high-glucose DMEM complete medium and cultured at 37°C and 5% CO ₂ for 24 hours.

2. Plasmid transfection into cells

(1) Lipofectamine 3000 (Invitrogen #L3000008) was used as the transfection reagent. Prepare the transfection mixture according to the following table:

Material Volume (μL/well) Opti-MEM Reduced Serum Medium 3.35 Lipofectamine 3000 0.45 P3000 (2 μL/μg DNA) 0.6 pBIND-FXR-LBD (0.5 μg/μL) 0.2 pGL4.31 (0.5 μg/μL) 0.4 Total volume 5

(2) Mix the transfection mixture thoroughly and let it stand at room temperature for 15 minutes;

(3) Add the transfection mixture to an appropriate volume of complete culture medium;

(4) Take out the 96-well cell culture plate, carefully aspirate the culture supernatant with a 5 mL syringe, and add 95 μL of transfection mixture to each well;

(5) Incubate at 37°C and 5% CO2 for 24 hours.

3. Add the compound to be tested

(1) Prepare 10 mM compound stock solution and serially dilute it 3-fold to 6 concentrations with DMSO (dimethyl sulfoxide);

(2) Take 2 μL of each concentration from the previous step and add it to 100 μL complete medium (50×);

(3) Take 5 μL of each concentration from the previous step and add it to a 96-well cell culture plate (20×);

(4) Incubate at 37°C and 5% CO2 for 16 hours.

4. Luciferase reporter gene assay

Luciferase detection was performed according to the instructions of the Bio-Glo ^™ Luciferase Assay System Kit from Promega. Bioluminescence detection: The plate was read using a BioTek Synergy H1 multi-function microplate reader.

Results Analysis

The firefly luciferase bioluminescence signal value is detected and the activation efficiency is calculated based on the value.

Excitation rate = (X-Min)/(Max-Min)*100%

X represents the signal value of each concentration of the compound; Max represents the signal value of the reference compound; Min represents the signal value of the negative control well.

The data were analyzed using GraphPad Prism 8.0, and the _EC50 values of the activation effects of the compounds were calculated.

See Table 2 below for specific data:

Table 2 Compound detection biochemical test results (Luciferase reporter assay)

Conclusion: The FXR agonist activity tested by the luciferase reporter gene expression test method shows that the compounds A6, B2, B3, B8, and B12 of the present invention have obvious agonist effects on FXR activity, and their agonist effects are 3-4 times that of CDCA.

Experimental Example 3 Pharmacokinetic study in rats:

Two experimental groups (n=3) were set up for each test compound. All compounds were dissolved in 5% DMSO+10% Solutol HS-15+85% Sailne to obtain a colorless, clear and transparent liquid.

The first group was the intravenous injection group (iv), which was injected into the rat tail vein at a dose of 2 mg/kg, and the administration volume was 2 mL/kg. Blood samples were collected from the jugular vein before administration (0 h), 2 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h after administration, respectively, and EDTA-K ₂ was used as an anticoagulant.

The second group was the oral administration group (po), which was administered orally at a dose of 10 mg/kg, with a dosing volume of 10 mL/kg. Blood samples were collected from the jugular vein before administration (0 h), 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h after administration, respectively, with EDTA-K ₂ as the anticoagulant.

The concentration of the test sample in the rat plasma sample was determined by LC-MS analysis, and the corresponding blood drug concentration-time curve was obtained, and then the PK parameters of the target compound were calculated.

Table 3 Pharmacokinetic parameters of some compounds

Conclusion: After oral administration, compound A6 showed higher drug exposure (AUC) and slower plasma clearance (CL), consistent with its longer half-life (t _1/2 ). For intravenous administration, compound B3 showed a higher AUC _(0-t) , followed by compound A6. These results further support the characteristics of compounds A6 and B3 having excellent absorption and distribution characteristics in SD rats. The high oral bioavailability of compound A6 in SD rats (F = 70.3%) indicates its excellent gastrointestinal permeability. In summary, these data indicate that compounds A6 and B3 exhibit relatively good pharmacokinetic properties in SD rats, characterized by higher absorption and distribution and lower clearance.

Experimental Example 4:

MRGPRX4 analysis of FXR agonists and compound A6

Cell treatment:

1. MRGPRX4 stably overexpressing cell lines were amplified from frozen stocks according to standard procedures.

2. Seed the cells in a total volume of 20 μL into a black-walled, clear-bottomed, Poly-D-lysine-coated 384-well microplate and incubate at 37°C for the appropriate time before testing.

Dye loading:

1. The assay was performed in 1×Dye Loading Buffer, which consisted of 1×Dye, 1×Additive A and 2.5 mM Probenecid (dissolved in HBSS/20 mM HEPES).

2. Before testing, cells were loaded with dye. The supernatant was extracted from the cells and replaced with 20 μL Dye Loading Buffer.

3. Incubate cells at 37°C for 1 hour.

Agonist form:

1. For agonist assays, cells are incubated with the sample to induce a response.

2. After dye loading, remove cells from the incubator and add 10 μL HBSS/20 mM HEPES. When performing agonist dose curves to determine the EC ₈₀ for subsequent antagonist analysis, include 3× vehicle in the buffer. Incubate cells in the dark at room temperature for 30 minutes to equilibrate the plate temperature.

3. Make an intermediate dilution of the sample in assay buffer to generate a 4× sample.

4. Measure the activity of the composite agonists on the FLIPR Tetra (MDS). Calcium mobilization was monitored for 2 minutes, and 10 μL of 4× sample in HBSS/20 mM HEPES was added to the cells 5 seconds later.

Data Analysis:

For agonist mode analysis, calculate percent activity using the following formula:

Activity percentage = 100% x (average RFU of test sample - average RFU of blank control) / (average MAX RFU of reference ligand - average RFU of blank control).

Table 4 MRGPRX4 agonist activity test results

Compound Test Method _EC50 (μM) DCA (deoxycholic acid) Calcium Flux 0.28 OCA (Obetocholic Acid) Calcium Flux 5.38 A6 Calcium Flux 11.21

The research results show that the agonist potency of compound A6 on the potential itch receptor MRGPRX4 is very weak, much lower than its natural bile acid compound DCA, and significantly lower than the OCA compound with obvious itch symptoms in clinical studies. Its _EC50 value is 40 times that of the natural MRGPRX4 agonist DCA, indicating that A6 has high safety.

Experimental Example 5:

Improvement effect of the compound of the present invention on cholestatic liver injury

Eight-week-old C57BL/6J male mice were randomly divided into five groups, including 3 mice in each normal group, 6 mice in each model group, positive control group, and test compound group. The mice in the normal group and model group were gavaged with purified water containing 5% propylene glycol and 5% polyoxy-15 hydroxystearate, and the mice in the drug group were gavaged with obeticholic acid (30 mg/kg) or compound A6 (30 mg/kg) once a day for 5 consecutive days. Four hours after the drug administration on the 5th day, the normal group was intraperitoneally injected with olive oil, and the model group and the drug administration group were intraperitoneally injected with 60 mg/kg α-naphthalene isothiocyanate (ANIT) to induce cholestatic liver injury, with an injection volume of 4 mL/kg. The last drug administration was on the 6th day. On the 7th day, the mice were fasted for 6 hours and blood was collected from the orbit under anesthesia. The mice were then killed to obtain the liver. After washing with physiological saline, the left lobe of the liver was cut and fixed in tissue fixative. The remaining liver tissues were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use. HE staining of the left lobe of the liver was performed to observe the improvement of liver injury. The staining and scoring results are shown in Figures 1 and 2.

The experimental results show that after administration to mice with cholestatic liver injury, compound A6 (30 mg/kg) achieved more significant improvements in liver cell necrosis, inflammatory cell infiltration and bleeding compared with obeticholic acid (30 mg/kg), indicating that the effect of compound A6 of the present invention in improving cholestatic liver injury is significantly better than obeticholic acid and has broader prospects for pharmaceutical development.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (8)

1. A steroid FXR agonist with an oxadiazole structure-containing side chain, which is characterized in that the FXR agonist is a compound of a formula I or an isomer thereof, or a pharmaceutically acceptable salt thereof or a solvent compound of the salt, or a mixture of any two or more of the above;

Wherein X is selected from CH ₂、NH、N-NH₂ and O, S;

y is selected from NH, O and S;

n is selected from 0,1 or 2;

R ₁ is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl;

R ₂ is selected from alkyl, aryl, heteroaryl, benzyl, hydrogen, fluoro, chloro, bromo, hydroxy, amino, mercapto, cyano, carbonyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate, phosphate.

2. The FXR agonist of claim 1, wherein R ₂ is selected from substituted alkyl, aryl, heteroaryl, benzyl, wherein the substituents are one or more of hydrogen, fluoro, chloro, bromo, hydroxy, amino, mercapto, cyano, carbonyl, acyl, sulfonyl.

3. The FXR agonist of claim 1, wherein said isomers include tautomers, cis-isomers, trans-isomers, (R) -enantiomers, (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, meso, racemates.

4. The FXR agonist of claim 1, wherein the compound of formula I is selected from:

5. A process for the preparation of a compound as claimed in claim 1 or 2 or 3 or 4, characterized in that the synthetic route is as follows:

6. A pharmaceutical composition comprising the FXR agonist of any of claims 1-4, and further comprising one or more of an adjuvant, diluent, carrier, excipient.

7. Use of an FXR agonist according to any of claims 1-4 or a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the prevention and/or treatment of FXR mediated and/or related liver and gall diseases involving FXR mediated.

8. The use according to claim 7, wherein the FXR mediated associated liver and gall disease comprises dyslipidemia, obesity, non-alcoholic fatty liver disease/inflammation, primary sclerosing cholangitis, primary biliary cholangitis, cholestatic liver disease, fibrotic disease, hypercholesteremic disease, hypertriglyceridemia, type II diabetes, portal hypertension, bile acid diarrhea or diabetic insulin resistance, primary biliary cirrhosis, gallstones, non-alcoholic cirrhosis, bile duct occlusion, chronic liver disease, hepatitis infection, alcoholic liver disease and cardiovascular disease, dyslipidemia or diseases related to dyslipidemia.