Oxadiazole-steroid compound, pharmaceutical composition and application

Abstract

The embodiment of the invention provides an oxadiazole-steroid compound, a pharmaceutical composition and application. The oxadiazole-steroid compound is a compound with a structure as shown in a formula I or pharmaceutically acceptable salt of the compound or a mixture of the compound and the pharmaceutically acceptable salt. The oxadiazole-steroid compound provided by the embodiment of the invention can effectively activate a metabolic regulation target FXR, and has excellent potential for preventing or treating MAFLD and related complications thereof. Meanwhile, the compound has no excitation effect on an MRGPRX4 receptor possibly causing pruritus while maintaining the FXR excitation activity, and a safer choice is provided for treatment of related diseases. Therefore, the oxadiazole-steroid compound has a more remarkable application prospect in preparation of the medicine for treating the metabolism-related fatty liver disease.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical technologies, and particularly relates to an oxadiazole-steroid compound, a pharmaceutical composition and uses thereof. Background Art

[0002] With the changes in modern lifestyle and dietary structure, metabolic associated fatty liver disease (MAFLD) and its related complications (such as liver fibrosis, cirrhosis and liver cancer, etc.) have become important health problems. MAFLD is the accumulation of liver fat caused by multiple factors and may further develop into serious liver diseases, which is usually closely related to metabolic syndromes such as obesity, diabetes and hyperlipidemia. Due to the complex pathogenesis of MAFLD, existing treatment options mostly focus on lifestyle changes or symptomatic treatment, but lack specific drugs targeting the pathological mechanism.

[0003] In recent years, steroid compounds have shown significant biological activities in regulating lipid metabolism, anti-inflammation, antioxidant, etc., and particularly have shown potential in the treatment of abnormal liver metabolism. For example, in the drug treatment of MAFLD, clinically, it mainly treats MAFLD by targeting anti-insulin resistance, antioxidant stress, lipid-lowering, promoting lipid metabolism and protecting the liver cell membrane, etc.; such as the use of hepatoprotective drugs such as metformin, vitamin E, farnesoid X receptor (FXR) agonist obeticholic acid (OCA), liver injury treatment drugs, etc. However, at the same time, the limitations of the above drug treatment methods are also great. On the one hand, the treatment dosage and effectiveness of the drugs still need to be further studied and confirmed; on the other hand, the pruritus side effects and hepatotoxicity of FXR agonists such as obeticholic acid also greatly limit their application in the field of MAFLD.

[0004] As a class of organic compounds containing an oxadiazole ring, oxadiazole compounds have also made remarkable progress in the field of medicinal chemistry in recent years. Oxadiazole compounds have good biocompatibility, targeting and low toxicity, and have been found to effectively act on a variety of biological targets and have a variety of pharmacological activities such as anti-tumor, anti-inflammatory and antioxidant. In particular, the potential of oxadiazole compounds in the treatment of liver diseases has been gradually recognized, and relevant studies have shown that they can relieve MAFLD and its complications by regulating lipid metabolism, inhibiting liver inflammatory reactions, improving oxidative stress, etc.

[0005] In this context, steroidal-coupled oxadiazole structure compounds are considered an innovative drug design concept. By introducing an oxadiazole ring onto the steroid, the synergistic effect of two pharmacophores may be achieved, thus achieving better effects in the treatment of MAFLD and related hepatobiliary system diseases. Such a composite structure can not only target multiple biological targets but also improve the selectivity and biological activity of the drug, providing a new treatment strategy for drug development. However, the oxadiazole-steroid compounds in the prior art also have the problem of potentially triggering MRGPRX4-mediated pruritus, which in turn causes adverse clinical reactions such as itching, as disclosed in the Chinese patent application with the publication number CN118620018A.

[0006] Therefore, this application designs and synthesizes oxadiazole-steroid compounds, aiming to enhance their pharmacodynamic effects in the treatment of metabolic-associated fatty liver disease, liver fibrosis, and other hepatobiliary system diseases, and reduce or avoid the occurrence of pruritus complications, providing a new drug development direction for drugs treating metabolic diseases. Summary of the Invention

[0007] The purpose of this application is to provide an oxadiazole-steroid compound, a pharmaceutical composition, and uses thereof.

[0008] To achieve the above object, the embodiments of this application propose the following technical solutions: In the first aspect, the embodiments of this application provide an oxadiazole-steroid compound, which is a compound having the structure shown in Formula I or a pharmaceutically acceptable salt thereof, and mixtures thereof: ; wherein, n is selected from 0, 1, 2, or 3; R1 is selected from hydrogen, fluorine, chlorine, bromine, mercapto, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl, or carboxypropyl; R2 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, or carboxypropyl; R3 is selected from alkyl, aryl, heteroaryl, benzyl, hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate group, phosphate group, or is selected from substituted alkyl, aryl, heteroaryl, benzyl, the substituents of which are one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate group, phosphate group.

[0009] As an embodiment, n is selected from 0, 1 or 2.

[0010] As an embodiment, R1 is selected from hydrogen.

[0011] As an embodiment, R2 is selected from hydrogen, methyl or ethyl.

[0012] As an embodiment, R3 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl, or is selected from substituted alkyl, aryl, heteroaryl, benzyl, the substituents of which are one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl.

[0013] As a preferred embodiment, the oxadiazole-steroid compound is selected from at least one of the following compounds or its pharmaceutically acceptable salts: 、 、 、 、 、 、 、 、 。

[0014] In a second aspect, an embodiment of the present application provides a pharmaceutical composition, which includes the oxadiazole-steroid compound described in the first aspect.

[0015] As an embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant.

[0016] In a third aspect, an embodiment of the present application provides the use of the oxadiazole-steroid compound described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a drug for preventing and / or treating related hepatobiliary diseases mediated by metabolic associated fatty liver disease.

[0017] Exemplarily, the related hepatobiliary diseases mediated by the metabolic associated fatty liver disease include dyslipidemia, obesity, metabolic associated fatty liver disease / inflammation, primary sclerosing cholangitis, primary biliary cholangitis, cholestatic liver diseases, fibrotic diseases, hypercholesterolemia diseases, hypertriglyceridemia diseases, type II diabetes, portal hypertension, bile acid diarrhea or diabetes insulin resistance, primary biliary cirrhosis, gallstones, metabolic associated cirrhosis, biliary atresia, chronic liver diseases, hepatitis infection, alcoholic liver diseases and cardiovascular diseases, lipid metabolism disorders or diseases related to lipid metabolism disorders.

[0018] The embodiments of the present application at least have the following beneficial effects: The oxadiazole-steroid compound provided by the embodiments of the present application can effectively activate the metabolic regulation target FXR, and has excellent potential for preventing or treating MAFLD and its related complications. At the same time, while maintaining the FXR agonist activity, the compound has no agonist effect on the MRGPRX4 receptor that may cause itching, and can further reduce or even avoid the adverse reaction of itching clinically, providing a safer option for the treatment of related diseases. Thus, it is indicated that the oxadiazole-steroid compound has a more significant application prospect in the preparation of drugs for metabolic associated fatty liver disease.

[0019] The additional aspects and advantages of the present application will be partially given in the following description, and these will become obvious from the following description, or be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shows the schematic diagram of the test results of blood biochemical indexes of the normal group, model group, positive control group and compound 8 group in biological evaluation example 5; Figure 2 Shows the schematic diagram of the test results of liver lipid content of the normal group, model group, positive control group and compound 8 group in biological evaluation example 5; Figure 3 Shows the schematic diagram of the test results of different magnifications of H&E staining and Sirius red staining of the left lobe of the liver of the normal group, model group, positive control group and compound 8 group in biological evaluation example 5; Figure 4 Shows the schematic diagram of the pathological analysis results of H&E staining and Sirius red staining of the left lobe of the liver of the normal group, model group, positive control group and compound 8 group in biological evaluation example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] Next, in combination with the embodiments of the present application and the accompanying drawings, the technical solutions in the embodiments will be clearly and completely described. Obviously, the embodiments described below are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0022] It should be understood that when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0023] It should also be understood that the terms used in the specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0024] First, some of the terms and raw materials involved in this embodiment will be explained below to facilitate the understanding of those skilled in the art.

[0025] The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence state of the specific atom is normal and the resulting compound is stable.

[0026] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0027] The term "hydroxyl" refers to the -OH group.

[0028] The term "carboxyl" refers to the -COOH group.

[0029] The term "cyano" refers to the -CN group.

[0030] The term "mercapto" refers to the -SH group.

[0031] The term "amino" refers to the -NH2 group.

[0032] The term "nitro" refers to the -NO2 group.

[0033] The term "alkoxy" refers to -O-alkyl.

[0034] The term "alkylamino" refers to -NH-alkyl.

[0035] The term "alkylsulfonyl" refers to -SO2-alkyl.

[0036] The term "alkylthio" means -S-alkyl.

[0037] The term "alkyl" refers to a hydrocarbon group having the general formula C n H2 n+1 and so on.

[0038] The term "aryl" refers to an aromatic ring group of a monocyclic or fused polycyclic all-carbon group having a conjugated π-electron system.

[0039] In the following examples, eq represents equivalent; rt represents room temperature; Ts represents p-toluenesulfonyl; Boc represents tert-butoxycarbonyl; DCM represents dichloromethane; MeCN represents acetonitrile; PE represents petroleum ether; EA represents ethyl acetate; MeOH represents methanol; EtOH represents ethanol; THF represents tetrahydrofuran; AcOH represents glacial acetic acid; TFA represents trifluoroacetic acid; p -TSA·H2O represents p-toluenesulfonic acid monohydrate; TEMPO represents 2,2,6,6-tetramethylpiperidine oxide; PIDA represents iodobenzene diacetate; Ts-NHNH2 represents p-toluenesulfonylhydrazine; LiOH represents lithium hydroxide; NaBH4 represents sodium borohydride; Boc-NHNH2 represents tert-butyl carbazate; C3H5KOS2 represents potassium ethyl xanthate; TBTU represents O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate; DMAP represents 4-dimethylaminopyridine; DIEPA represents N,N-diisopropylethylamine; DMF represents N,N-dimethylformamide; Na2SO4 represents anhydrous sodium sulfate; MgSO4 represents anhydrous sodium sulfate; NaCl represents sodium chloride; NaHCO3 represents sodium bicarbonate; K2CO3 represents potassium carbonate.

[0040] The oxadiazole-steroid compounds, pharmaceutical compositions and uses of the examples will be described in detail below.

[0041] First, the oxadiazole-steroid compounds of the first aspect of this example will be described.

[0042] Oxadiazole-steroid compound Those skilled in the art know that metabolic-associated fatty liver disease-mediated related hepatobiliary diseases, such as cholestatic liver diseases, are often accompanied by itching symptoms, which seriously reduce the quality of life of patients; at present, the treatment means for cholestatic pruritus are quite limited, and the basic molecular mechanism behind this symptom has not been fully understood. Recently, relevant studies have found that the G protein-coupled receptor X4 (MRGPRX4) related to MAS can act as a receptor for bile acids and bilirubin, and its role in cholestatic pruritus has been confirmed.

[0043] However, steroidal FXR agonists such as obeticholic acid are also considered to potentially trigger MRGPRX4-mediated pruritus; at the same time, the oxadiazole-steroid compounds in the prior art also have the problem of potentially triggering MRGPRX4-mediated pruritus, thereby causing adverse reactions of pruritus clinically, such as the Chinese patent application for invention with the publication number CN118620018A.

[0044] In view of this, this embodiment provides an oxadiazole-steroid compound; specifically, the oxadiazole-steroid compound is a compound having the structure shown in Formula I or a pharmaceutically acceptable salt thereof, and mixtures thereof: ; wherein, n is selected from 0, 1, 2 or 3; R1 is selected from hydrogen, fluorine, chlorine, bromine, mercapto, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl or carboxypropyl; R2 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl or carboxypropyl; R3 is selected from alkyl, aryl, heteroaryl, benzyl, hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate group, phosphate group, or is selected from substituted alkyl, aryl, heteroaryl, benzyl, and its substituents are one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate group, phosphate group.

[0045] It can be understood that compared with the oxadiazole-steroid compounds in the prior art (CN118620018A), in the oxadiazole-steroid compounds provided in this embodiment, the OH at the 3-position is changed to H or other groups, which can not only effectively activate the metabolic regulation target FXR and has excellent potential for preventing or treating MAFLD and its related complications. At the same time, while maintaining the FXR agonist activity, this compound has no agonist effect on the MRGPRX4 receptor that may cause pruritus, and can further reduce or even avoid the adverse reaction of pruritus clinically, providing a safer choice for the treatment of related diseases.

[0046] In other words, the oxadiazole-steroid compound of this embodiment can not only ensure the activity of FXR, but also no longer activate the MRGPRX4 target that causes itching; therefore, the oxadiazole-steroid compound of this embodiment can effectively avoid the occurrence of itching adverse reactions in the clinical application of traditional steroidal FXR agonists and existing oxadiazole-steroid compounds.

[0047] As a preferred embodiment, n is selected from 0, 1 or 2; R1 is selected from hydrogen; R2 is selected from hydrogen, methyl or ethyl; R3 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl, or is selected from substituted alkyl, aryl, heteroaryl, benzyl, and its substituents are one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl.

[0048] Next, the above-mentioned oxadiazole-steroid compound will be further described with specific examples.

[0049] Exemplarily, the oxadiazole-steroid compound provided in this embodiment can be selected from at least one of the following compounds or its pharmaceutically acceptable salts: , , , , , , , , .

[0050] It can be understood that the term "pharmaceutically acceptable salt" refers to the salt of the compound of this embodiment, which is prepared from the compound with specific substituents found in this embodiment and a relatively non-toxic acid or base. When the compound of this embodiment contains relatively acidic functional groups, the base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compound of this embodiment contains relatively basic functional groups, the acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Pharmaceutically acceptable salts can be 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.

[0051] Exemplarily, the pharmaceutically acceptable salts of the above-mentioned compounds provided in this embodiment can be: sodium salt, potassium salt, ammonium salt, amino acid salt, lactate, hydrochloride, phosphate, acetate, malate, citrate or aspartate, etc. This embodiment does not make specific limitations on drug salts.

[0052] Next, the preparation method of the above-mentioned oxadiazole-steroid compound will be described.

[0053] Preparation method This example also provides a preparation method of the above-mentioned oxadiazole-steroid compound. This preparation method can refer to the following synthetic route: 。

[0054] Among them, R3 in Compounds 9-15 is the corresponding group of R3 in the above Formula I compound.

[0055] Next, each step in the above synthetic route of the preparation method will be further described.

[0056] Among them, the reagents and reaction conditions used in the above steps (a)-(i) are as follows: (a) p -TSA·H2O, MeOH, 60 °C; (b) TEMPO, PIDA, MeCN, 10 °C; (c) Ts-NHNH2, THF / AcOH, 80 °C; (d) NaBH4, DCM / AcOH, 0 °C; (e) LiOH, THF / MeOH / H2O, 30 °C; (f) TBTU, Boc-NHNH2, DIEPA, THF, 50 °C; (g) TFA, DCM, 30 °C; (h) C3H5KOS2, EtOH, 100 °C; (i) RX, K2CO3, DMF, 80 °C.

[0057] Secondly, the pharmaceutical composition of the second aspect of this example will be described.

[0058] Pharmaceutical composition This example also provides a pharmaceutical composition. As described in the first aspect, based on the oxadiazole-steroid compound of this example, not only can the activity of FXR be ensured, but the MRGPRX4 target that causes itching is no longer activated; therefore, the pharmaceutical composition containing the oxadiazole-steroid compound prepared in this example can also effectively avoid the occurrence of itching adverse reactions in the clinical application of traditional steroidal FXR agonists and existing oxadiazole-steroid compounds.

[0059] Generally, in addition to the oxadiazole-steroid compound (or active ingredient or main drug), the pharmaceutical composition also includes pharmaceutically acceptable adjuvants. The adjuvants and the active ingredient together constitute a complete pharmaceutical composition.

[0060] Among them, the adjuvants can be classified according to their functions and uses, including but not limited to fillers, binders, disintegrants, lubricants, wetting agents, solvents, flavoring agents, preservatives, coating materials, coloring agents, etc.

[0061] Exemplarily, in oral preparations, excipients such as starch, dextrin, lactose, etc. are often used as fillers to increase the volume and weight of tablets, facilitate molding and dosing. At the same time, they can also improve the taste and swallowability of drugs. In injection preparations, excipients such as water for injection, physiological saline, etc. are used as solvents to dissolve or dilute the active ingredients for injection administration.

[0062] Hereinafter, the uses of the third aspect of this embodiment will be described.

[0063] Use As mentioned above, the oxadiazole-steroid compound provided in this embodiment can not only effectively activate the metabolic regulation target FXR, but also has excellent potential for preventing or treating MAFLD and its related complications. At the same time, while maintaining the FXR agonist activity, this compound has no agonist effect on the MRGPRX4 receptor that may cause itching, and can further reduce or even avoid the adverse reaction of itching clinically, providing a safer choice for the treatment of related diseases.

[0064] At the same time, the pharmaceutical composition of this embodiment also has the same or similar clinical therapeutic effects as the oxadiazole-steroid compound.

[0065] Based on this, the oxadiazole-steroid compound or pharmaceutical composition of this embodiment can be used to prepare drugs for preventing and / or treating related hepatobiliary diseases mediated by metabolic associated fatty liver disease.

[0066] Exemplarily, the related hepatobiliary diseases mediated by the above-mentioned metabolic associated fatty liver disease include dyslipidemia, obesity, metabolic associated fatty liver disease / inflammation, primary sclerosing cholangitis, primary biliary cholangitis, cholestatic liver disease, fibrotic disease, hypercholesterolemia, hypertriglyceridemia, type II diabetes, portal hypertension, bile acid diarrhea or diabetes insulin resistance, primary biliary cirrhosis, gallstones, metabolic associated cirrhosis, biliary atresia, chronic liver disease, hepatitis infection, alcoholic liver disease and cardiovascular disease, lipid metabolism disorder or diseases related to lipid metabolism disorder.

[0067] Specifically, the above-mentioned drug is a drug for activating the metabolic regulation target FXR and not activating the MRGPRX4 target, so it has higher safety performance.

[0068] As mentioned above, when the above-mentioned oxadiazole-steroid compound is made into a corresponding drug, the drug also includes a pharmaceutically acceptable carrier and / or adjuvant.

[0069] For example, for the drug described in this embodiment, the drug uses an oxadiazole-steroid compound as the active ingredient, without excluding changes in the formulation system and administration method, medicinal salts obtained by simple chemical modification and adjustment of the above oxadiazole-steroid compound, and the combination of multiple compounds, etc.

[0070] For example, in this embodiment, one or more compounds in the oxadiazole-steroid compound of this embodiment can be formulated as the active ingredient in a non-toxic, inert, and pharmaceutically acceptable carrier and / or adjuvant; the formulated drug can be administered through conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

[0071] For example, when the dosage form of the drug in this embodiment is a drug for oral administration, it contains a safe and effective amount of the oxadiazole-steroid compound and a pharmaceutically acceptable carrier and / or adjuvant. The drug for oral administration can be made into common dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, suppositories, etc.; in this embodiment, no specific limitations are imposed on the carrier and / or adjuvant, and the carrier and / or adjuvant can be adaptively adjusted according to the specific drug dosage form.

[0072] Generally, the "effective amount" of a compound (oxadiazole-steroid compound) refers to the amount sufficient to cause a target biological response. As understood by those of ordinary skill in the art, the effective amount of the compound in this embodiment can be changed according to the following factors: for example, components such as the vehicle in the drug, as well as the age, health condition of the subject, and symptoms of related hepatobiliary diseases mediated by metabolic associated fatty liver disease.

[0073] Among them, the effective amount includes a therapeutically effective amount and a prophylactically effective amount.

[0074] Unless otherwise stated, the "therapeutically effective amount" of the compound used in this embodiment is the amount sufficient to provide a benefit during the treatment of symptoms of related hepatobiliary diseases mediated by metabolic associated fatty liver disease, or the amount that minimizes the improvement or remission of one or more symptoms (manifestations) related to the state of related hepatobiliary diseases mediated by metabolic associated fatty liver disease. The "prophylactically effective amount" of the compound used in this embodiment is the amount sufficient to prevent the occurrence of related hepatobiliary diseases mediated by metabolic associated fatty liver disease, or the amount sufficient to prevent one or more symptoms related to the occurrence of the state of related hepatobiliary diseases mediated by metabolic associated fatty liver disease.

[0075] It can be understood that the drug of this embodiment can also be made into an injection. For example, the oxadiazole-steroid compound can be made into a corresponding injection with water for injection, normal saline, or glucose water under a sterile operating environment, and the above injection can be prepared by conventional methods.

[0076] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain the present application and not to limit the scope of the present application.

[0077] In the following embodiments, the materials, reagents, and instruments used, unless otherwise specified, can be obtained from commercial sources.

[0078] Example 1 The synthetic route of Example 1 is as follows: 。

[0079] The specific steps are as follows: Take a 250 mL round-bottom flask, add 50 mL of MeOH, 2.00 g of obeticholic acid (4.76 mmol), and 0.10 g p -TSA·H2O (0.48 mmol). Heat the reaction system to 60 °C and stir continuously. After reacting for 3 hours, concentrate the solvent on a rotary evaporation device, then add 50 mL of EA to dissolve the residue. Wash it with saturated NaHCO3 solution (50 mL × 3) and saturated NaCl solution (50 mL × 3) respectively, collect the organic phase, dry it with anhydrous MgSO4, filter, and remove the solvent under reduced pressure to obtain 1.94 g of a white powdery solid compound 1 with a yield of 96%.

[0080] The structural characterization data of compound 1 are as follows: 1 H NMR (400 MHz, CDCl3) δ 3.75 – 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, 8H), 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).

[0081] Example 2 The synthetic route of Example 2 is as follows: 。

[0082] The specific steps are as follows: Add compound 1 (1.09 g, 2.50 mmol) and a mixed solvent of acetonitrile and water (20 mL, MeCN:H2O = 1:1) to a 100 mL round-bottom flask, stir to dissolve, add PIDA (0.87 g, 2.70 mmol) and TEMPO (0.04 g, 0.25 mmol), react at 10 °C for 24 hours, then concentrate the reaction solution. Dissolve the residual solid in EA (50 mL), and wash it successively with saturated NaHCO3 solution (50 mL × 3) and saturated NaCl solution (50 mL × 3). Dry the organic phase with anhydrous MgSO4, filter to remove anhydrous MgSO4, collect the organic phase, distill off the solvent under reduced pressure, and purify by column chromatography to obtain 0.88 g of a white solid, which is compound 2, yield: 82%.

[0083] The structure characterization data of compound 2 are as follows: 1 H NMR (600 MHz, CDCl3) δ 3.71 – 3.68 (m, 1H), 3.68 (s, 3H), 2.37 –2.26 (m, 2H), 2.21 – 2.12 (m, 2H), 2.09 – 2.03 (m, 1H), 1.98 – 1.92 (m, 2H),1.90 – 1.80 (m, 2H), 1.75 – 1.48 (m, 6H), 1.47 – 1.23 (m, 10H), 1.21 – 1.03(m, 4H), 0.92 (s, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H),0.62 (s, 3H).

[0084] Example 3 The synthetic route of Example 3 is as follows: .

[0085] The specific steps are as follows: Dissolve compound 2 (0.50 g, 1.16 mmol) in 20 mL of anhydrous THF in a 150 mL thick-walled pressure-resistant tube, and successively add p-toluenesulfonyl hydrazide (0.32 g, 1.74 mmol) and glacial acetic acid (5 mL). Heat to 80 °C and continuously stir and react for 12 hours. Slowly cool to room temperature, concentrate under reduced pressure to remove the solvent to obtain the crude product of compound 3, and the crude product is directly used for the next reaction without purification.

[0086] Example 4 The synthetic route of Example 4 is as follows:

[0087] The specific steps are as follows: At room temperature, take a three-necked flask and dissolve compound 3 (0.50 g, 0.83 mmol) in a mixed solution of dichloromethane and glacial acetic acid (50 mL, DCM:AcOH = 3:2). Stir the solution until it becomes clear, and then cool it to below 0 °C in an ice-water bath. Slowly add NaBH4 (0.32 g, 8.33 mmol) to the flask in batches while controlling the system temperature not to exceed 5 °C. Stir the reaction mixture at room temperature for 8 hours. After the reaction is completed, quench it by slowly dropping ice water (50 mL) while maintaining the temperature below 5 °C. Separate the liquid, extract the aqueous phase with dichloromethane (50 mL × 3), and combine the organic phases. Wash with saturated NaCl solution (50 mL × 3). Dry the organic phase with anhydrous MgSO4, filter off the anhydrous MgSO4, collect the organic phase, and remove the solvent by distillation under reduced pressure to obtain the crude product of compound 4, which is directly used in the next reaction without purification.

[0088] Example 5 The synthetic route of Example 5 is as follows: 。

[0089] The specific steps are as follows: At room temperature, add compound 4 (0.20 g, 0.48 mmol) to a three-necked flask in a mixed solution of tetrahydrofuran, methanol and water (30 mL, THF:MeOH:H2O = 1:1:1). Slowly add LiOH (0.12 g, 4.80 mmol) to the flask in batches. Continue to stir the reaction at 30 °C for 12 hours. Add 10 mL of dilute hydrochloric acid to the reaction flask and adjust the pH value to 5. The precipitated white solid is filtered and washed with ether (10 mL × 3) to obtain compound 5 (0.14 g, yield 75%).

[0090] The structural characterization data of compound 5 are as follows: 11H NMR (600 MHz, CDCl3) δ 3.71 – 3.68 (m, 1H), 2.42 – 2.36 (m, 1H), 2.28 – 2.23 (m, 1H), 1.97 – 1.93 (m, 1H), 1.93 – 1.87 (m, 2H), 1.84 – 1.75 (m, 4H), 1.71 – 1.60 (m, 6H), 1.53 – 1.46 (m, 4H), 1.46 – 1.36 (m, 6H), 1.36 – 1.32 (m, 3H), 1.32 – 1.28 (m, 2H), 1.28 – 1.25 (m, 2H), 1.25 – 1.13 (m, 6H), 1.13 – 1.03 (m, 2H), 1.03 – 0.97 (m, 1H), 0.94 (d, J J = 6.4 Hz, 3H), 0.93 – 0.82 (m, 9H), 0.66 (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 177.59, 70.27, 54.75, 49.58, 45.96, 41.77, 40.36, 39.04, 38.67, 36.99, 35.32, 34.35, 32.35, 29.80, 29.72, 27.16, 26.65, 23.27, 23.00, 22.72, 21.21, 20.35, 19.76, 17.23, 10.78, 10.71. HR-MS (ESI) m / z : calcd for C 26 H 44 ClO3 [M + Cl] - : 439.2979; found: 439.3017.

[0091] Example 6 The synthetic route of Example 6 is as follows: .

[0092] The specific steps are as follows: At room temperature, dissolve compound 5 (0.10 g, 0.25 mmol) in 20 mL of anhydrous THF. Add TBTU (0.10 g, 0.30 mmol) and DIEPA (0.06 g, 0.50 mmol) to the reaction flask in sequence. Raise the temperature to 50 °C and continue stirring the reaction for 12 hours. Concentrate the reaction solution, dissolve the residual solid in EA (20 mL), and wash it with dilute hydrochloric acid solution (20 mL × 3) and saturated NaCl solution (20 mL × 3) in sequence. Dry the organic phase with anhydrous MgSO4, filter to remove anhydrous MgSO4, collect the organic phase, and remove the solvent by distillation under reduced pressure to obtain the crude product of compound 6. The crude product is directly used in the next reaction without purification.

[0093] Example 7 The synthetic route of Example 7 is as follows: 。

[0094] The specific steps are as follows: At room temperature, take a round-bottom flask and dissolve compound 6 (0.10 g, 0.19 mmol) in a mixed solution of dichloromethane and trifluoroacetic acid (10 mL, DCM:TFA = 3:2). Continue stirring the reaction at 30 °C for 12 hours. Concentrate the reaction solution, dissolve the residual solid in EA (20 mL), and wash it with saturated NaHCO3 solution (20 mL × 3) and saturated NaCl solution (20 mL × 3) in sequence. Dry the organic phase with anhydrous MgSO4, filter to remove anhydrous MgSO4, collect the organic phase, and remove the solvent by distillation under reduced pressure to obtain the crude product of compound 7. The crude product is directly used in the next reaction without purification.

[0095] Example 8 The synthetic route of Example 8 is as follows: 。

[0096] The specific steps are as follows: In a 150 mL thick-walled pressure-resistant tube, dissolve compound 7 (0.10 g, 0.24 mmol) in 20 mL of anhydrous EtOH, and add potassium ethylxanthate (0.06 g, 0.36 mmol). Heat to 100 °C and continue stirring the reaction for 12 hours. Slowly cool to room temperature, concentrate and remove the solvent under reduced pressure. Dissolve the residual solid in EA (20 mL), and wash it with dilute hydrochloric acid solution (20 mL × 3) and saturated NaCl solution (20 mL × 3) in sequence. Dry the organic phase with anhydrous MgSO4, filter to remove anhydrous MgSO4, collect the organic phase, and remove the solvent by distillation under reduced pressure. The residue is separated by column chromatography to obtain 0.10 g of white solid compound 8 with a yield of 92%.

[0097] The structural characterization data of Compound 8 are as follows: 1 H NMR (600 MHz, CDCl3) δ 10.82 (s, 1H), 3.69 – 3.62 (m, 1H), 2.70 – 2.63 (m, 1H), 2.55 – 2.48 (m, 1H), 1.91 – 1.86 (m, 1H), 1.86 – 1.76 (m, 2H), 1.75 – 1.70 (m, 2H), 1.67 – 1.59 (m, 4H), 1.47 – 1.27 (m, 11H), 1.26 – 1.04 (m, 10H), 1.00 – 0.94 (m, 1H), 0.92 (d, J J = 6.2 Hz, 3H), 0.89 – 0.86 (m, 1H), 0.85 – 0.81 (m, 6H), 0.80 – 0.71 (m, 1H), 0.60 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 177.32, 164.38, 70.47, 54.48, 49.59, 45.88, 41.81, 40.33, 38.95, 38.64, 36.97, 35.31, 34.33, 32.35, 30.73, 27.19, 26.63, 23.28, 22.97, 22.70, 21.70, 21.18, 20.34, 19.74, 17.21, 10.78, 10.69. HR-MS (ESI) m / z : calcd for C 27 H 43 N2O2S [M - H] - : 459.3045; found: 459.3066.

[0098] Example 9 The synthetic route of Example 9 is as follows: .

[0099] The specific steps are as follows: Add 0.23 g of compound 8 (0.50 mmol) and 0.14 g of anhydrous K2CO3 (1.00 mmol) into a 100 mL round-bottom flask, and dissolve them in 20 mL of DMF. Stir at room temperature until compound 8 is completely dissolved, then add methyl iodide (0.11 g, 0.75 mmol) in batches. Then heat the reaction system to 80 °C and continue the reaction at this temperature for 8 hours. Detect that the raw materials have completely reacted by thin-layer chromatography (TLC), and concentrate the solvent on a rotary evaporator. Add 30 mL of ethyl acetate to dissolve the residue, pour it into a 250 mL separatory funnel, wash it with 1M HCl solution (20 mL × 3) and saturated NaCl solution (20 mL × 3) respectively, combine the organic phases and dry them with anhydrous Na2SO4, and filter under reduced pressure to remove the solvent. The crude product is purified by silica gel column chromatography, using petroleum ether / ethyl acetate (V:V = 4:1) as the eluent to obtain 100 mg of compound 9 with a yield of 50%.

[0100] The structural characterization data of compound 9 are as follows: 1 H NMR (600 MHz, CDCl3) δ 3.67 – 3.61 (m, 1H), 2.71 (s, 3H), 2.69 –2.61 (m, 1H), 2.53 – 2.47 (m, 1H), 1.89 – 1.84 (m, 1H), 1.83 – 1.74 (m, 2H),1.73 – 1.68 (m, 2H), 1.66 – 1.58 (m, 4H), 1.45 – 1.25 (m, 11H), 1.24 – 1.02(m, 10H), 0.98 – 0.92 (m, 1H), 0.90 (d, J J = 6.2 Hz, 3H), 0.87 – 0.84 (m, 1H),0.83 – 0.79 (m, 6H), 0.78 – 0.69 (m, 1H), 0.59 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ 169.30, 164.35, 70.45, 54.46, 49.57, 45.86, 41.79, 40.31, 38.93, 38.62, 36.95, 35.29, 34.31, 32.33, 30.71, 27.17, 26.61, 23.26, 22.95, 22.68, 21.68, 21.16, 20.32, 19.72, 17.19, 14.31, 10.76, 10.67.

[0101] Example 10 The synthetic route of Example 10 is as follows: 。

[0102] This example refers to the synthetic steps described in Example 9. The difference from Example 9 is only that methyl iodide is replaced by deuterated methyl iodide. After purification, 70 mg of Compound 10 was obtained. It is a white powdery solid with a yield of 35%.

[0103] The structural characterization data of Compound 10 are as follows: 1 1H NMR (600 MHz, CDCl3) δ 3.70 – 3.64 (m, 1H), 2.71 – 2.65 (m, 1H), 2.56 – 2.50 (m, 1H), 1.92 – 1.87 (m, 1H), 1.88 – 1.78 (m, 2H), 1.76 – 1.71 (m, 2H), 1.69 – 1.60 (m, 4H), 1.48 – 1.28 (m, 11H), 1.27 – 1.05 (m, 10H), 1.01 – 0.95 (m, 1H), 0.93 (d, J J = 6.3 Hz, 3H), 0.90 – 0.87 (m, 1H), 0.86 – 0.82 (m, 6H), 0.81 – 0.72 (m, 1H), 0.61 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ169.34, 164.40, 70.49, 54.50, 49.61, 45.90, 41.83, 40.35, 38.97, 38.66, 36.99, 35.33, 34.35, 32.37, 30.75, 27.21, 26.65, 23.30, 22.99, 22.72, 21.72, 21.20, 20.36, 19.76, 17.23, 14.29, 10.80, 10.71.

[0104] Example 11 The synthetic route of Example 11 is as follows: 。

[0105] This example refers to the synthetic steps described in Example 9. The difference from Example 9 is only that methyl iodide is replaced by benzyl bromide. After purification, 153 mg of Compound 11 was obtained. It is a white powdery solid with a yield of 66%.

[0106] The structural characterization data of Compound 11 are as follows: 1 1H NMR (600 MHz, CDCl3) δ 7.38 – 7.28 (m, 4H), 7.27 – 7.22 (m, 1H), 4.45 (s, 2H), 3.68 – 3.63 (m, 1H), 2.69 – 2.62 (m, 1H), 2.54 – 2.48 (m, 1H), 1.90 – 1.85 (m, 1H), 1.85 – 1.76 (m, 2H), 1.74 – 1.69 (m, 2H), 1.67 – 1.59 (m, 4H), 1.46 – 1.26 (m, 11H), 1.25 – 1.03 (m, 10H), 0.99 – 0.93 (m, 1H), 0.91 (d, J J = 6.2 Hz, 3H), 0.88 – 0.85 (m, 1H), 0.84 – 0.80 (m, 6H), 0.79 – 0.70 (m, 1H), 0.60 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ 169.31, 164.37, 136.67, 129.33, 128.69, 127.69, 70.46, 54.47, 49.58, 45.87, 41.80, 40.32, 38.94, 38.63, 36.96, 36.11, 35.30, 34.32, 32.34, 30.72, 27.18, 26.62, 23.27, 22.96, 22.69, 21.69, 21.17, 20.33, 19.73, 17.20, 10.77, 10.68.

[0107] Example 12 The synthetic route of Example 12 is shown as follows: 。

[0108] This example refers to the synthetic steps described in Example 9. The difference from Example 9 is only that methyl iodide is replaced by 3-bromopropene. After purification, 99 mg of Compound 12 was obtained. It is a white powdery solid with a yield of 47%.

[0109] The structure characterization data of Compound 12 are as follows: 1 1H NMR (600 MHz, CDCl3) δ 5.86 (tt, J J = 16.9, 8.1 Hz, 1H), 5.28 – 5.11(m, 2H), 3.82 – 3.77 (m, 2H), 3.66 – 3.60 (m, 1H), 2.68 – 2.60 (m, 1H), 2.52 – 2.46 (m, 1H), 1.88 – 1.83 (m, 1H), 1.82 – 1.73 (m, 2H), 1.72 – 1.67 (m, 2H), 1.65 – 1.57 (m, 4H), 1.44 – 1.24 (m, 11H), 1.23 – 1.01 (m, 10H), 0.97 – 0.91 (m, 1H), 0.89 (d, J J = 6.1 Hz, 3H), 0.86 – 0.83 (m, 1H), 0.82 – 0.78 (m, 6H), 0.77 – 0.68 (m, 1H), 0.58 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ 169.28, 164.33, 131.80, 118.06, 70.44, 54.44, 49.55, 45.85, 41.78, 40.30, 38.92, 38.60, 36.94, 35.28, 34.52, 34.30, 32.32, 30.70, 27.16, 26.60, 23.25, 22.94, 22.67, 21.67, 21.15, 20.31, 19.71, 17.18, 10.75, 10.66.

[0110] Example 13 The synthetic route of Example 13 is as follows: .

[0111] This example refers to the synthetic steps described in Example 9. The difference from Example 9 is only that methyl iodide is replaced by 2-bromoacetic acid. After purification, 96 mg of Compound 13 was obtained. It is a white powdery solid with a yield of 44%.

[0112] The structural characterization data of Compound 13 are as follows: 1 1H NMR (600 MHz, CDCl3) δ 3.97 (s, 2H), 3.71 – 3.65 (m, 1H), 2.72 – 2.66 (m, 1H), 2.57 – 2.51 (m, 1H), 1.93 – 1.88 (m, 1H), 1.89 – 1.79 (m, 2H), 1.77 – 1.72 (m, 2H), 1.70 – 1.61 (m, 4H), 1.49 – 1.29 (m, 11H), 1.28 – 1.06 (m, 10H), 1.02 – 0.96 (m, 1H), 0.94 (d, J J = 6.3 Hz, 3H), 0.91 – 0.88 (m, 1H), 0.87 – 0.83 (m, 6H), 0.82 – 0.73 (m, 1H), 0.62 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ 169.35, 171.91, 164.41, 70.50, 54.51, 49.62, 45.91, 41.84, 40.36, 38.98, 38.67, 37.00, 35.34, 34.36, 33.99, 32.38, 30.76, 27.22, 26.66, 23.31, 23.00, 22.73, 21.73, 21.21, 20.37, 19.77, 17.24, 10.81, 10.72.

[0113] Example 14 The synthetic route of Example 14 is as follows: .

[0114] This example refers to the synthetic steps described in Example 9. The difference from Example 9 is only that methyl iodide is replaced by 2-bromoethanol. After purification, 127 mg of Compound 14 was obtained. It is a white powdery solid with a yield of 60%.

[0115] The structure characterization data of Compound 14 are as follows: 1 1H NMR (600 MHz, CDCl3) δ 3.89 – 3.77 (m, 2H), 3.65 – 3.59 (m, 1H), 3.42 – 3.37 (m, 2H), 2.67 – 2.59 (m, 1H), 2.51 – 2.45 (m, 1H), 1.87 – 1.82 (m, 1H), 1.81 – 1.72 (m, 2H), 1.71 – 1.66 (m, 2H), 1.64 – 1.56 (m, 4H), 1.43 – 1.23 (m, 11H), 1.22 – 1.00 (m, 10H), 0.96 – 0.90 (m, 1H), 0.88 (d, J J = 6.0 Hz, 3H), 0.85 – 0.82 (m, 1H), 0.81 – 0.77 (m, 6H), 0.76 – 0.67 (m, 1H), 0.57 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ 169.27, 164.32, 70.43, 60.39, 54.43, 49.54, 45.84, 41.77, 40.29, 38.91, 38.59, 36.93, 35.27, 34.29, 33.51, 32.31, 30.69, 27.15, 26.59, 23.24, 22.93, 22.66, 21.66, 21.14, 20.30, 19.70, 17.17, 10.74, 10.65. Example 15 The synthetic route of Example 15 is shown as follows: 。

[0116] This example refers to the synthetic steps described in Example 9. The difference from Example 9 is only that methyl iodide is replaced by 3-bromopropionic acid. After purification, 94 mg of Compound 15 was obtained. It is a white powdery solid with a yield of 42%.

[0117] The structural characterization data of Compound 15 are as follows: 1 1H NMR (600 MHz, CDCl3) δ 3.68 – 3.61 (m, 1H), 3.47 (td, J J = 8.8, 2.7Hz, 2H), 2.71 – 2.64 (m, 1H), 2.60 (t, J J = 8.9 Hz, 2H), 2.54 – 2.47 (m, 1H),1.90 – 1.85 (m, 1H), 1.85 – 1.75 (m, 2H), 1.74 – 1.68 (m, 2H), 1.66 – 1.58(m, 4H), 1.46 – 1.26 (m, 11H), 1.25 – 1.03 (m, 10H), 0.99 – 0.93 (m, 1H),0.91 (d, J J = 6.2 Hz, 3H), 0.88 – 0.85 (m, 1H), 0.84 – 0.80 (m, 6H), 0.79 –0.70 (m, 1H), 0.59 (s, 3H). 1313C NMR (151 MHz, CDCl3) δ 175.15, 169.17, 164.36, 70.45, 54.46, 49.57, 45.85, 41.80, 40.30, 38.93, 38.61, 36.94, 35.28, 34.30, 33.88, 32.32, 30.70, 27.16, 26.96, 26.60, 23.25, 22.94, 22.67, 21.67, 21.15, 20.31, 19.71, 17.18, 10.76, 10.67. The relevant properties (biological evaluation) of the compounds prepared in the above examples will be verified below.

[0118] Biological evaluation example 1 FXR biochemical experiment (testing the binding of compounds to FXR-LBD protein by TR-FRET method): To evaluate the binding ability of the compounds to the FXR receptor, the LanthaScreen ™ TR-FRET FXR coactivator detection kit from Invitrogen was used to detect the effect of the compounds on the FXR ligand-binding domain (FXR-LBD). Experimental principle: The terbium (Tb)-labeled anti-glutathione-S-transferase (GST) antibody is used to indirectly label FXR by binding to the GST tag. When the compound binds to FXR, it will change the protein conformation, thereby enhancing the affinity of FXR for the coactivator peptide. At this time, the fluorescence-labeled coactivator peptide (Fluorescein-SRC 2-2) will approach the Tb-labeled anti-GST antibody, resulting in an increase in the TR-FRET signal.

[0119] The experimental procedure is as follows: First, prepare Complete Coregulator buffer G. Add 1 M DTT to Coregulator buffer G to make its concentration reach 10 mM, thus obtaining Complete Coregulator buffer G. Then, start preparing various solutions required for the experiment. On the one hand, prepare 2× working solution of the compound to be tested. First, dilute the 3 mM compound stock solution to a 100× series of concentrations with DMSO, and then dilute the 100× series of concentrations to 2× with the prepared Complete Coregulator buffer G. A total of 10 concentrations are set, using 3-fold dilution and 2 replicates. On the other hand, prepare 4× FXR-LBD protein, dilute the stock solution to the required volume with Complete Coregulator buffer G to make its concentration 20 nM; at the same time, prepare 4× Fluorescein-SRC 2-2 / Tb-labeled anti-GST antibody mixture, and also dilute the stock solution to the required volume with Complete Coregulator buffer G to make the concentration of Fluorescein-SRC 2-2 2 μM and the concentration of Tb-labeled anti-GST antibody 20 nM. Then, perform the sample addition operation. Add 5 μL of FXR-LBD and 5 μL of Fluorescein-SRC 2-2 / Tb-labeled anti-GST antibody mixture to a black U-bottom 384-well plate containing 10 μL of the compound to be tested, and seal the plate with a sealing film. Centrifuge the plate at 1000 rpm for 1 minute, and then shake it at 300 rpm for 1 minute, and incubate it in the dark at room temperature for 2 hours. Finally, perform time-resolved fluorescence detection. Read the plate using a TECAN microplate reader with an excitation wavelength of 340 nm, and read the absorption signals at 490 / 520 nm. The value at 520 nm is divided by the value at 490 nm to calculate the agonist rate.

[0120] Agonist rate = (X - Min) / (Max - Min) * 100%.

[0121] Where X represents the 520 / 490 value of each concentration of the compound; Max represents the 520 / 490 value of the highest concentration of the reference compound chenodeoxycholic acid; Min represents the 520 / 490 value of the negative control well. Use GraphPad Prism 8.0 to analyze the data and calculate the EC 50 value.

[0122] Biological evaluation example 2 FXR cell experiment (FXR-Luc luciferase reporter gene experiment at the cell level): This experiment evaluated the activation effect of compounds on FXR based on the luciferase reporter gene system. The Promega Bio-Glo ™ Luciferase Assay System detection platform was used. By specifically inserting the FXR response element into the luciferase reporter vector and transfecting it into host cells, the change in signal intensity after compound treatment was quantitatively analyzed. During the detection process, the bioluminescence intensity generated by the luciferase catalyzing the substrate was positively correlated with the transcriptional activity of FXR.

[0123] The experimental procedure is as follows: HEK293 cells were seeded in a 96-well cell culture plate at an appropriate density and cultured at 37 °C and 5% CO2 for 24 hours. GAL4RE-FXR-LBD and the luciferase reporter gene plasmid were transfected using Lipofectamine 3000. The transfection solution was allowed to stand at room temperature for 15 minutes and then added to the cells, and the cells were cultured for another 24 hours. Compounds were prepared with DMSO (6 concentrations diluted in a 3-fold gradient, with 2 replicates set), diluted with complete medium, and then added to the cells, and the cells were cultured at 37 °C and 5% CO2 for 16 hours. Luciferase detection was performed according to the method described in the Bio-Glo ™ Luciferase Assay System kit instructions. A BioTek Synergy H1 multi-functional microplate reader was used to read the plate to obtain the bioluminescence signal values.

[0124] Calculate the activation rate (activation rate = (X - Min) / (Max - Min) * 100%), where X is the signal value at each concentration, Max is the signal value at the highest concentration of the reference compound chenodeoxycholic acid, and Min is the signal value of the negative control well. GraphPad Prism 8.0 was used to analyze the data and calculate the EC 50 value.

[0125] Please refer to Table 1 for the test results of the FXR biochemical experiment and the cell experiment. In Table 1, +++: EC 50 ≤1.0 μM; ++: 1.0 μM < EC 50 ≤10 μM; +: EC 50 > 10 μM.

[0126] Efficacy is the percentage of the activation potency of the test compound obtained by comparing the highest signal value of the test compound with the highest signal value of the reference compound chenodeoxycholic acid (CDCA). CDCA was set to 100% for normalization; A: Efficacy > 200%; B: 100% < Efficacy ≤ 200%; C: Efficacy ≤ 100%; Table 1: Test results of the FXR biochemical experiment and the cell experiment Based on the test results in Table 1, the compounds provided in this embodiment all have a significant agonistic effect on FXR activity; among them, the compounds of Example 8, Example 12, Example 13, Example 14, and Example 15 have a more significant agonistic effect.

[0127] Biological evaluation example 3 Study on MRGPRX4 activity (testing the response of compounds to the downstream signals of MRGPRX4 at the cellular level): In the experimental design, the stable expression cell model of MRGPRX4 receptor constructed by our research group before - the HEK293T / hMRGPRX4 cell line established by lentiviral vector transfection technology was selected, and the IP - ONE HTRF ® (Homogeneous Time - Resolved Fluorescence) detection kit developed by Cisbio was used to quantitatively analyze the activation effect of the test compounds on the MRGPRX4 receptor based on the principle of IP1 metabolite accumulation detection of the Gq protein - coupled receptor signaling pathway.

[0128] The experimental steps are as follows: The digested hMRGPRX4 stable transfected cells were seeded in a fully white 384 - well microplate coated with Poly - D - lysine at an appropriate density and incubated at 37 °C, 5% CO2 for an appropriate time before the test. Add 1×Stimulation Buffer (containing 10 concentrations of compounds diluted in a 3 - fold gradient, with 2 replicates) to each experimental well and incubate at 37 °C, 5% CO2 for 1 hour. Dilute IP1 - d2 and Anti - IP1 - Cryptate to the working concentration with Lysis&Detection Buffer and add them to each experimental well, then incubate at room temperature for 1 hour. Read the plate using a TECAN microplate reader, with an excitation wavelength of 340 nm, and read the absorbance signal OD value at 620 / 665 nm.

[0129] Ratio calculation: Ratio = OD 665 / OD 620 , and draw a curve according to the standard product concentration of the kit and the corresponding fluorescence ratio. Analyze the data using GraphPad Prism 8.0 and calculate the EC 50 value.

[0130] Please refer to Table 2 for the test results of the agonistic activity of the compounds on MRGPRX4. In Table 2, ***: EC 50 ≤ 10 μM; **: 10 μM < EC 50 ≤ 100 μM; *: EC 50 > 100 μM (indicating that no activation was observed for the compound within the tested concentration range); Table 2: Test results of the agonistic activity of compounds on MRGPRX4 Compound number Test method <![CDATA[EC 50 (μM)]]> Nateglinide IP-ONE *** Deoxycholic acid IP-ONE *** Obeticholic acid IP-ONE *** Example 5 IP-ONE * Example 8 IP-ONE * Example 10 IP-ONE * Example 11 IP-ONE * Example 12 IP-ONE * Example 14 IP-ONE * Combined with the test results in Table 2, it can be obtained that the compound provided in this example (with the OH at the 3-position changed to H or other groups) has no agonist effect on the potential pruritus receptor MRGPRX4 (no activation was observed within the tested concentration range), indicating that the compound of this example can not only ensure the activity of FXR, but also no longer activate the MRGPRX4 target that causes pruritus; therefore, the oxadiazole-steroid compound of this implementation can effectively avoid the occurrence of pruritus adverse reactions in the clinical application of traditional steroidal FXR agonists and existing oxadiazole-steroid compounds, and has higher safety performance.

[0131] Biological evaluation example 4 In vivo activity study (pharmacokinetics study in SD rats after single-dose administration): Taking the compound of Example 8 as an exemplary compound, the in vivo activity study was carried out below.

[0132] Two experimental groups (n = 6, half male and half female) were set for the compound of Example 8, and it was completely dissolved by the solvent formulation of 5% DMSO + 10% Solutol HS-15 + 85% Saline to obtain a colorless and clear solution.

[0133] The first group was the intravenous injection group (IV), and the drug was administered by tail vein injection at 2 mg / kg and 2 mL / kg. Blood samples (using EDTA-K2 as an anticoagulant) were collected from the jugular vein at 0 h (before dosing), 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours after dosing.

[0134] The second group was the oral administration group (PO), and the drug was administered by gavage at 5 mg / kg and 5 mL / kg. Blood samples (using EDTA-K2 as an anticoagulant) were collected from the jugular vein at 0 h (before dosing), 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 hours after dosing.

[0135] Plasma samples were analyzed using LC-MS / MS to obtain the plasma concentration-time curve, and WinNonlin software was used to calculate the pharmacokinetic parameters. The results are expressed as Mean±SD, and the specific data are shown in Table 3: Table 3: Pharmacokinetic parameters of the compound of Example 8

[0136] As shown by the test data in Table 3, after oral administration of the compound of Example 8, the blood drug concentration and exposure level in rats were relatively high, and the clearance rate was relatively low, which was consistent with a relatively long elimination half-life. In addition, the bioavailability exceeded 90%, reflecting a weak first-pass effect and prominent gastrointestinal absorption efficiency. In summary, the pharmacokinetic parameters of the compound of Example 8 indicate its excellent pharmacokinetic properties.

[0137] Biological evaluation example 5 In vivo activity study (therapeutic effect of the compound on metabolic associated steatohepatitis (MASH)): Taking the compound 8 of Example 8 as an exemplary compound, the above in vivo activity study was carried out.

[0138] SPF-grade 8-week-old male C57BL / 6J mice were selected and randomly divided into a normal control group, a model control group, an obeticholic acid treatment group (positive control), and a compound 8 group (n = 8). Except for the normal control group which was intraperitoneally injected with olive oil (4 mL / kg), the other groups were intraperitoneally injected with 5% carbon tetrachloride solution (CCl4 in olive oil, 4 mL / kg) once a week for 12 consecutive weeks. During the modeling period, the animals were fed a Western diet feed containing 41% sucrose, 21% fat, and 1.25% cholesterol (Dyets, catalog number D18061501) and a mixed sugar water (containing 23.1 g / L of D-fructose and 18.9 g / L of D-anhydrous glucose) to establish a MASH model. In the last 4 weeks of the modeling period, drug treatment was carried out. The normal control group and the model control group were gavaged with 0.5% sodium carboxymethylcellulose solution, and the obeticholic acid treatment group and the compound 8 group were gavaged with 20 mg / kg of the corresponding drug (dissolved in 0.5% sodium carboxymethylcellulose solution).

[0139] At the end of the experiment, the animals were fasted for 12 hours and then anesthetized. Blood was collected from the inferior orbital venous plexus, and then the mice were euthanized according to animal ethics standards. The liver tissues were obtained by dissection and processed. The experimental results are shown in Figures 1-4 .

[0140] Among them, Figure 1 shows the schematic diagram of the test results of blood biochemical indexes of the normal group, the model group, the positive control group, and the compound 8 group; Figure 2 shows the schematic diagram of the test results of liver lipid contents of the normal group, the model group, the positive control group, and the compound 8 group; Figure 3 shows the schematic diagram of the test results of different magnifications of H&E staining and Siriusred staining of the left lobe of the liver of the normal group, the model group, the positive control group, and the compound 8 group; Figure 4 shows the schematic diagram of the pathological analysis results of H&E staining and Sirius red staining of the left lobe of the liver of the normal group, the model group, the positive control group, and the compound 8 group.

[0141] Combined with Figures 1-4 the test results, compared with the mice in the MASH model group, the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and low-density lipoprotein (LDL) in the mice given compound 8 in this example were significantly decreased (please refer to Figure 1 ), and at the same time, the contents of triglyceride and total cholesterol in the liver tissue were significantly reduced (please refer to Figure 2 ).

[0142] Secondly, through H&E staining and Sirius red staining analysis, it was found that compound 8 could significantly reduce the activity score of metabolic associated fatty liver disease and significantly reduce the area of liver collagen deposition (please refer to Figure 3 and Figure 4 ). Therefore, the test results of the above in vivo activity studies all indicate the positive effect of compound 8 in regulating liver lipid metabolism disorders and inhibiting the fibrosis process, and it is significantly better than the positive control obeticholic acid, with a broader prospect for medicinal development.

[0143] In summary, the oxadiazole-steroid compound provided in this example can effectively activate the metabolic regulatory target FXR and has excellent potential for preventing or treating MAFLD and its related complications. At the same time, while maintaining the FXR agonist activity, this compound has no agonist effect on the MRGPRX4 receptor that may cause itching, providing a safer option for the treatment of related diseases. Thus, it is suggested that this oxadiazole-steroid compound has a more significant application prospect in the preparation of drugs for metabolic associated fatty liver disease.

[0144] The technical solutions provided in the embodiments of the present application have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present application. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present application; at the same time, for those of ordinary skill in the art, based on the embodiments of the present application, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. An oxadiazole-steroid compound, characterized in that, The oxadiazole-steroid compound is a compound having the structure shown in Formula I or a pharmaceutically acceptable salt thereof, and mixtures thereof: ； Wherein, n is selected from 0, 1, 2 or 3; R1 is selected from hydrogen, fluorine, chlorine, bromine, mercapto, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl or carboxypropyl; R2 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl or carboxypropyl; R3 is selected from alkyl, aryl, heteroaryl, benzyl, hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, carbonyl, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate group, phosphate group, or is selected from substituted alkyl, aryl, heteroaryl, benzyl, and the substituents are one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, ethoxy, propoxy, trifluoromethyl, trifluoromethoxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, aminomethyl, aminoethyl, aminopropyl, carboxymethyl, carboxyethyl, carboxypropyl, sulfonate group, phosphate group.

2. The oxadiazole-steroid compound according to claim 1, wherein n is selected from 0, 1 or 2.

3. The oxadiazole-steroid compound according to claim 1, characterized in that, R1 is selected from hydrogen.

4. The oxadiazole-steroid compound according to claim 1, characterized in that, R2 is selected from hydrogen, methyl or ethyl.

5. The oxadiazole-steroid compound according to claim 1, wherein R3 is selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl, or is selected from substituted alkyl, aryl, heteroaryl, benzyl, and the substituents are one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, mercapto, cyano, methoxy, trifluoromethyl, trifluoromethoxy, carboxymethyl, carboxyethyl.

6. The oxadiazole-steroid compound according to any one of claims 1-5, characterized in that, The oxadiazole-steroid compound is selected from at least one of the following compounds or a pharmaceutically acceptable salt thereof: 、 、 、 、 、 、 、 、 。 7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the oxadiazole-steroid compound according to any one of claims 1-6.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

9. Use of the oxadiazole-steroid compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for preventing and / or treating related hepatobiliary diseases mediated by metabolic associated fatty liver disease.

10. The use according to claim 9, characterized in that, The related hepatobiliary diseases mediated by metabolic associated fatty liver disease include dyslipidemia, obesity, metabolic associated fatty liver disease / inflammation, primary sclerosing cholangitis, primary biliary cholangitis, cholestatic liver diseases, fibrotic diseases, hypercholesterolemia diseases, hypertriglyceridemia diseases, type II diabetes, portal hypertension, bile acid diarrhea or diabetes insulin resistance, primary biliary cirrhosis, gallstones, metabolic associated cirrhosis, biliary atresia, chronic liver diseases, hepatitis infection, alcoholic liver diseases and cardiovascular diseases, dyslipidemia or diseases related to dyslipidemia.

Images