A novel pyrazine structure FXR agonist, preparation method and application

Abstract

The present invention relates to a novel pyrazine structure FXR agonist, a preparation method and application thereof, and specifically to a compound of the following general formula, a hydrate, a solvate, a pharmaceutically acceptable salt or a single isomer thereof, which has the effect of treating non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to a method for preparing a pharmaceutical compound, and in particular to a novel pyrazine structure FXR agonist, a preparation method and application thereof. Background Art

[0002] Since the discovery in 1999 that bile acids can activate FXR to produce a variety of physiological functions, selective and highly active FXR agonists have been discovered one after another. These FXR agonists can be divided into steroidal and non-steroidal types according to their structures. Steroidal types mainly include chenodeoxycholic acid (CDCA, one of the bile acids) and its derivatives and the FXR agonist MFA-1 developed by Merck; non-steroidal types include isoxazole compounds GW4064 and its analogs, Fexaramine compounds, azaindole compounds XL335 and its derivatives, benzimidazole amide compounds, pyrazolidinedione compounds, etc.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a clinical pathological syndrome characterized by fatty degeneration and fat storage in hepatocytes without a history of excessive drinking and with liver histological changes similar to those of alcoholic liver disease. It includes simple fatty liver and its evolved fatty hepatitis and cirrhosis. Today, NAFLD has become the second most serious chronic liver disease affecting human health after hepatitis B. NASH is a chronic progressive liver disease caused by accumulation of fat in the liver, which can lead to cirrhosis, liver failure and hepatocellular carcinoma. To be precise, NASH is only a stage in the development of non-alcoholic fatty liver disease (NAFLD).

[0004] Among the drugs under clinical development, obeticholic acid is a FXR agonist. It was approved by the FDA in May 2016 for the treatment of primary bile acid cirrhosis (PBC) and is also the first NASH drug to enter Phase III clinical trials. The interim analysis of the key Phase III REGENERATE study of obeticholic acid for NASH patients with grade 2-3 liver fibrosis achieved positive results.

[0005] Other FXR agonists such as PX-104 have entered Phase II clinical trials, and their main indication is also non-alcoholic fatty liver disease (NAFLD).

[0006]

[0007] In 2000, Maloney et al. reported the first highly active and selective isoxazole FXR agonist GW4064, whose extracellular activity was EC 50 =15nmol·L -1, EC at the cellular level 50 The value is 90nmol·L -1 , which can completely excite the FXR target protein; but in terms of pharmacokinetics, the oral availability at t1 / 2 = 3.5 hours is only 10%, which does not meet the conditions for drug development, so it is used as a tool compound for studying FXR function and related diseases. Subsequently, GSK, Novartis, Roche, Lilly, Phenex and other companies have modified the structure of GW4064 in order to obtain new compounds with higher activity, better oral availability and more drug properties. So far, a large number of isoxazole compounds have been successfully synthesized, but they are insufficient in terms of drugability such as activity, water solubility and oral bioavailability.

[0008]

[0009] Chinese patent CN103702719A discloses novel FXR (NR1H4) binding and activity regulating compounds, which specifically disclose the following compounds:

[0010]

[0011] And the use of these compounds in the treatment of diseases such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). These compounds are improvements based on the compounds disclosed in WO2011 / 020615, and are achieved by introducing polar hydroxyl groups on 1,3-cyclobutylene or 1,3-aziridinyl groups replacing 1,2-cyclopropylene groups. The results show that the obtained compounds maintain activity on the FXR receptor and show improved physicochemical properties, such as higher water solubility and / or membrane permeability. Better water solubility and membrane permeability lead to higher oral bioavailability. However, the activation activity of the above compounds at the farnesoid nuclear receptor FXR target is relatively low. Summary of the invention

[0012] In order to solve the above technical problems, the present invention provides a compound having a structure of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, pharmaceutically acceptable salt or a single isomer thereof:

[0013]

[0014] Wherein, R1 is selected from: halogen, -COOH,

[0015] R2 is selected from: C1-C6 hydrocarbon group, cycloalkyl group, aromatic group, substituted hydrocarbon group or substituted aromatic group;

[0016] R3 is selected from: -H or C1-C3 hydrocarbon group, cycloalkyl group, substituted hydrocarbon group;

[0017] X is C or N.

[0018] Preferably, wherein

[0019] R1 is selected from -Br;

[0020] R2 is selected from a C1-C3 hydrocarbon group, a cycloalkyl group;

[0021] R3 is selected from -CH3;

[0022] X is C or N.

[0023] More preferably, wherein

[0024] R2 is selected from

[0025] The most preferred compounds are selected from the following:

[0026]

[0027]

[0028] The present invention further provides a pharmaceutical composition comprising the compound as an active ingredient.

[0029] The pharmaceutical composition may further contain a pharmaceutically acceptable carrier as required.

[0030] The present invention further provides a method for preparing the compound, comprising the following steps:

[0031] The invention relates to use of the compound of the present invention in preparing medicine for treating non-alcoholic fatty liver.

[0032] The non-alcoholic fatty liver disease is non-alcoholic fatty liver disease.

[0033] The pharmaceutical composition of the present invention is used in preparing a drug for treating non-alcoholic fatty liver disease, preferably non-alcoholic fatty liver disease.

[0034] The compounds described in the present invention include all isomeric forms and isomeric mixtures thereof and may also exist in the form of solvates.

[0035] The pharmaceutical composition of the present invention is preferably in the form of a unit dose pharmaceutical preparation, and can be made into any pharmaceutically acceptable dosage form when being made into a pharmaceutical preparation, and these dosage forms are selected from: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, and patches. Preferably, it is in the form of an oral preparation, and most preferably, tablets and capsules.

[0036] The pharmaceutical preparation can be prepared by conventional pharmacy techniques, and the pharmaceutically acceptable carrier includes but is not limited to: mannitol, sorbitol, sorbic acid or potassium salt, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin A, vitamin C, vitamin E, vitamin D, azone, disodium EDTA, calcium sodium EDTA, carbonate, acetate, phosphate of monovalent alkali metal or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, fumaric acid, sodium chloride, potassium chloride, sodium lactate, wood Sugar alcohol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, propylene glycol, ethanol, Tween 60-80, Span-80, beeswax, lanolin, liquid paraffin, hexadecanol, gallic acid esters, agar, triethanolamine, basic amino acids, urea, allantoin, calcium carbonate, calcium bicarbonate, polyethylene glycol, cyclodextrin (such as β-cyclodextrin), phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc.

[0037] When the pharmaceutical composition of the present invention is prepared into a medicament, a unit dose of the medicament may contain 0.1-1000 mg of the pharmaceutical active substance of the present invention, and the rest is a pharmaceutically acceptable carrier, which may be 0.1-99.9% by weight of the total weight of the preparation.

[0038] The dosage and administration of the pharmaceutical composition of the present invention are determined according to the patient's condition when used.

[0039] The preferred preparation method of the present invention comprises the following steps:

[0040] 1) The synthesis method of isoxazole intermediates is as follows: benzaldehyde 1a is condensed with hydroxylamine hydrochloride under alkaline conditions to obtain benzaldehyde oxime 1b, and oxime 1b is reacted with NCS / DMF to generate chlorobenzaldehyde oxime 1c; then, a 1,3-dipolar cycloaddition reaction is performed with ketoester to obtain trisubstituted isoxazole ring 1d-1f; DIBAL-H is added under nitrogen protection to reduce the ester bond to a hydroxyl group to obtain intermediate 1-a-1-c;

[0041] 2) The isoxazole intermediate reacts with dibromopyrazine under alkaline conditions to undergo nucleophilic substitution reaction to obtain pyrazine intermediate 2-a-2-c; the temperature is controlled to be maintained at minus 78°C, and n-butyl lithium is used to perform lithium bromine exchange with the four-membered ring 2a-d for carbon-carbon bond connection to generate terminal ester products 3-a-3-g; finally, an ester hydrolysis reaction occurs under alkaline conditions to obtain the target compound TM.

[0042] Beneficial effects of the present invention: The compounds of the present invention, especially TM-01 and TM-09, have a significant effect on reducing liver triglyceride and cholesterol levels, increase mouse liver weight and liver-to-body weight ratio, reduce pathological scores and collagen deposition in the liver, and have an improving effect on non-alcoholic fatty liver disease.

[0043] The compounds of the present invention have strong agonist activity on the farnesoid nuclear receptor FXR target, and show high permeability between cell membranes. In a high-fat-fed mouse model, they have a dose-dependent effect on the weight of fat-enriched liver. It is predicted that they will have good uses in the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver disease (NASH). The beneficial effects of the present invention are illustrated by experimental data below.

[0044] Experimental Example 1: Study on the agonist activity of the compounds of the present invention on FXR receptor

[0045] Experimental steps: FXR (GST-tagged recombinant human FXR protein, manufacturer Invitroren, catalog number PV4835) and SRC-1 (steroid receptor coactivator-1) were melted on ice, and three solutions ABC were prepared with buffer: solution A, 0.4nM FXR and 30nM SRC-1; solution B, 10ug / ml Acceptor Beads; solution C, 10ug / ml Donor Beads. Add solution A to the plate, 15uL per well. Incubate at room temperature for 1 hour. Add solution B to the plate, 7.5uL per well. Incubate at room temperature for 1 hour. Add solution C to the plate, 7.5uL per well. Incubate at room temperature for 1 hour. Read the results using Envision microplate reader. The data were curve fitted using Prism 5.0 to calculate the EC 50 The results are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049]

[0050] According to the sample activity results in Table 1, TM-05, TM-07, and TM-10 with poor activity were removed, and the remaining 7 samples were selected for intracellular FXR-TR-FRET experiments.

[0051] Experimental Example 2: Intracellular FXR-TR-FRET Experiment

[0052] 1. Cell culture: a. Trypsinize the dish and seed cells in 10 ml complete trypsin at an appropriate density at 37°C. b. Culture the cells for 24 hours under 5% CO2 and humidified conditions.

[0053] 2. Cell seeding and transfection: FuGENE HD transfection reagent was used as the transfection reagent.

[0054] aPrepare the transfection mixture according to the following procedure:

[0055]

[0056] b. Tap the tube vigorously to mix the contents. Incubate the mixture at room temperature for 15 minutes.

[0057] c Trypsinize the disk and determine the cell density.

[0058] d. Dilute the cell slurry to the required volume (100 ul / well for 96-well plate) at a density of 500,000 cells / ml.

[0059] eAdd the required volume of the previously prepared transfection mix to both cell slurries, then dispense 100ul / well of the cell slurry onto the assay plate.

[0060] f Incubate the assay plate at 37°C, 5% CO2 under humidified conditions for 24 hours.

[0061] 3. Compound preparation:

[0062] a. Prepare compound stocks at 10 mM working concentration for FXR and then dilute 3-fold in 100% DMSO.

[0063] b. Add 10ul of compound to 90ul of complete culture medium.

[0064] c. Add 5ul of compound solution to each well.

[0065] d. Incubate the plate at 37°C, 5% CO2 and humidified conditions for 18 hours.

[0066] 4. Dual luciferase assay: Firefly and Renilla luciferase signals were analyzed by Promega's DualLuciferase Reporter Assay System. Envision was used as a luminometer.

[0067] 5. Calculation of results: Normalize the data values ​​by dividing the firefly signal by the Renilla signal. "F / R" stands for "firefly / Renilla". This normalization eliminates the differences in the number of cells and transfection efficiency in each well. Calculate the %Activation value. Calculate the %Activation value by the following formula.

[0068]

[0069] X is the "F / R" value for each concentration point. The minimum value is the average "F / R" value of the no compound control. The maximum value is the average "F / R" value of the reference compound control.

[0070] 6. The activity results are shown in Table 2.

[0071] Table 2: Activity results

[0072] Sample No. <![CDATA[EC 50 (μM)]]> TM-01 1.28 TM-02 3.14 TM-03 >5000 TM-04 >5000 TM-06 1.79 TM-08 1.51 TM-09 0.26 GW4064 0.30 M3 1.49

[0073] The activity of the tested compounds on FXR-TR-FRET was studied. Most of these compounds containing pyrazine structures showed better agonist activity at the cellular level compared with the reference substances GW4064 and M3.

[0074] According to the results of FXR-TR-FRET experiments, TM-03 and TM-04 were removed, and Caco-2 monolayer cell membrane transport experiments were performed on the remaining compounds.

[0075] Experimental Example 3: Caco-2 monolayer cell membrane transport experiment

[0076] The Caco-2 monolayer cell model of human colon adenocarcinoma cell line was used to study the bidirectional transport of the target compound from the apical side (AP) to the basolateral side (BL) and from the BL side to the AP side. High performance liquid chromatography was used for quantitative analysis, and the transport parameters, apparent permeability coefficient (Papp) and efflux ratio were calculated. M3 was used as the positive control and digoxin, the substrate of P-gp, was used as the reference. These five samples with high intracellular activity were selected for testing to predict the in vivo oral bioavailability of this type of pyrazine isoxazole derivatives and their affinity with P-gp.

[0077] The results are shown in Tables 3, 4 and 5.

[0078] Table 3: A-to-B apparent permeability coefficients in the Caco-2 cell model

[0079]

[0080]

[0081] Table 4: Permeabilization mass recovery in Caco-2 cell model

[0082]

[0083] Table 5 Efflux rate in Caco-2 cell model

[0084]

[0085] Note: a Excretion rate = Papp BA / Papp AB

[0086] The experimental results are shown in Table 3. The Papp values ​​of the series of pyrazine isoxazole compounds of the present invention are higher than that of the P-gp substrate digoxin (Papp A-to-B <0.04), and better than M3 (Papp A-to-B <0.15). In particular, the Papp, A-to-B values ​​of the four compounds TM-01, TM-02, TM-06 and TM-09 are >2.5×10-6cm / s, which are high permeability substrates. These data show that this type of pyrazine isoxazole compound has good membrane permeability and is predicted to be better absorbed in the body than M3.

[0087] Table 4 shows the recovery rates of these pyrazine-containing compounds after membrane permeation. The bidirectional transport of these five compounds was evaluated, and the results are shown in Table 5. From the efflux rate, it can be seen that compared with M3, these derivatives have greatly weakened the efflux rate, and their efflux rates are much lower than the reference substance digoxin (efflux rate>262.93), and it is predicted that the oral absorption in the body will also be improved accordingly.

[0088] According to the results of Experiment 3, TM-08 with poor effect was removed, and other compounds were retained for animal experiments. Regarding the control group: M3 was excluded because of its poor membrane permeation effect. GW4064 was excluded as a tool molecule in preliminary research because of its poor drugability and low oral bioavailability. M2 was also used as a control compound in the early stage. In the first protein level screening, its activity was lower than that of the compound in this patent and was excluded. Obeticholic acid is a drug currently in clinical research in the field of non-alcoholic fat and is close to approval. It was selected as a control substance for animal experiments.

[0089] Test Example 4: Efficacy test of high-fat diet (MCD) to simulate non-alcoholic fatty liver disease

[0090] Obeticholic acid is a drug currently under clinical research in the field of non-alcoholic fat. It is close to approval and was selected as a control substance for animal experiments.

[0091] 1. Experimental Animals

[0092] Animal strain: C57 / BL6, animal grade: SPF grade, sex: male, animal age: 8 weeks old, animal acceptance date: September 28, 2018, animal source: Shanghai Lingchang Biotechnology Co., Ltd., animal certificate number: SCXK (Shanghai) 2013-00182013001836799, mouse breeding environment: temperature 20-26 ° C, humidity 40-70%, 12-h day and night cycle.

[0093] 2. Experimental design and methods

[0094] 2.1 Drug configuration

[0095] 2.1.1 Solvent preparation:

[0096] 5% Solutol HS15 / normal saline: After dissolving Solutol HS15 (polyethylene glycol 15-hydroxystearate solubilizer) in a 37°C water bath, take 5 ml and dissolve it in 100 ml normal saline, stir it thoroughly and set aside.

[0097] 2.1.2 Preparation of dosing solution:

[0098] Obeticholic acid / 5% Solutol HS15 / normal saline: Accurately weigh 12 mg of obeticholic acid, add 0.15 ml of Solutol HS15 to fully dissolve it, add 2.85 ml of normal saline, vortex vigorously, and use ultrasound to assist dissolution to fully dissolve it.

[0099] TM-01 group / 5% Solutol HS15 / normal saline: Take 2.5 ml of 6 mg / ml TM-01 suspension, add 2.5 ml of 5% Solutol HS15 / normal saline, and mix thoroughly.

[0100] TM-02 group / 5% Solutol HS15 / normal saline: take 2.5 ml of 6 mg / ml TM-02 suspension, add 2.5 ml of 5% Solutol HS15 / normal saline, and mix thoroughly.

[0101] TM-06 group / 5% Solutol HS15 / normal saline: take 2.5 ml of 6 mg / ml TM-06 suspension, add 2.5 ml of 5% Solutol HS15 / normal saline, and mix thoroughly.

[0102] TM-09 group / 5% Solutol HS15 / normal saline: take 2.5 ml of 6 mg / ml TM-09 suspension, add 2.5 ml of 5% Solutol HS15 / normal saline, and mix thoroughly.

[0103] All drugs were prepared fresh before administration.

[0104] 2.2 Administration route and volume

[0105] The vehicle and the test substance were administered by gavage, with a dosing volume of 10 ml / kg;

[0106] 2.3 Experimental process, grouping and specific administration method

[0107] After 70 8-week-old C57 / BL6 mice arrived at the animal room, they were adapted to feeding. When the average weight reached 23g, the mice were randomly divided into 7 groups according to their weight and changed to model feed. The first group was given control feed MCS, and the other groups were given MCD food. At the same time, each group of mice received compound treatment as follows:

[0108] Group 1: control group: MCS, vehicle, intragastric administration once a day;

[0109] Group 2 Model group: MCD, vehicle, intragastric administration once a day;

[0110] Group 3: obeticholic acid group: MCD, 40 mpk obeticholic acid, intragastrically administered once a day;

[0111] Group 4. TM-01 group: MCD, 30 mpk TM-01, oral administration once a day;

[0112] Group 5. TM-02 group: MCD, 30 mpk TM-02, intragastric administration once a day;

[0113] Group 6. TM-06 group: MCD, 30 mpk TM-06, intragastric administration once a day;

[0114] Group 7. TM-09 group: MCD, 30 mpk TM-09, intragastric administration once a day;

[0115] 2.4 Experimental methods

[0116] During the experiment, body weight and food intake were measured weekly. After 21 days of administration, blood was collected from the tail tip using a microcapillary tube for AST and ALT determination. After 28 days of administration, the mice were terminated, blood was collected from the heart, and liver tissue was collected and weighed. One part was quickly frozen with liquid nitrogen for subsequent analysis, and the other part was fixed for pathological analysis.

[0117] 2.4.1 Blood index determination

[0118] Mouse blood was centrifuged at 5000 rpm for 10 min, and the supernatant was collected for the determination of TG (triglyceride), TC (total cholesterol), HDL (high-density lipoprotein), LDL (low-density lipoprotein), AST (aspartate aminotransferase) and ALT (alanine aminotransferase).

[0119] AST and ALT were measured three and four weeks after administration according to the kit instructions; blood lipid indicators were sent to Aidikang Medical Laboratory Co., Ltd. for testing four weeks after administration.

[0120] 2.4.2 Determination of blood cytokines

[0121] Mouse blood was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected for the detection of cytokines (mKC and MCP1). Serum was stored at -80°C before detection.

[0122] The determination of mKC and MCP1 was carried out according to the instructions of the kit.

[0123] 2.4.3 Determination of liver TC and TG

[0124] The liver was taken out from -80°C, homogenized in PBS (phosphate buffered saline), and the organic phase was extracted with chloroform and methanol. TC and TG were measured using a kit and normalized by protein amount.

[0125] 2.4.4 Tissue Collection

[0126] After 4 weeks of administration, the mice were anesthetized, blood was collected from the heart, the liver was separated and weighed, and the right lobe was quickly frozen with liquid nitrogen and stored at -80°C for liver lipid analysis. The left lobe was separated and fixed in 10% formalin for subsequent HE and Sirius Red staining.

[0127] 2.5 Result processing and data analysis

[0128] The test results were expressed as mean ± standard error (Mean ± SEM), and T-Test was used for significance analysis. Compared with the model group, * indicates p < 0.05 for significant difference, ** indicates p < 0.01 for strong significant difference, and *** p < 0.001 for extremely significant difference.

[0129] 3 Test results

[0130] 3.1 Effects of compounds on body weight and food intake of mice

[0131] After the mice were fed with MCD, their body weight decreased significantly as expected. And as time went on, their body weight continued to decrease. The body weight of each experimental group treated with the compound was also slightly lower than that of the model group (Table 6).

[0132] Table 6: Effects of compounds on mouse body weight

[0133]

[0134]

[0135] Table 7: Effects of compounds on food intake in mice

[0136]

[0137] 3.2 Effects of compounds on blood parameters

[0138] After 3 weeks of MCD and compound treatment, tail tip blood was collected for AST and ALT determination. The results showed that the ALT in the model group increased by about 3 times compared with the control group, and the AST increased by more than 2 times. The obeticholic acid group resulted in higher AST and ALT levels than the model group. The ALT and AST in the TM-01 group, TM-06 group, and TM-09 group decreased significantly compared with the model group, and the TM-02 group showed the same performance as the obeticholic acid group. (Table 8)

[0139] After 4 weeks of MCD and compound treatment, the blood lipid level of animals in the model group was slightly higher than that in the control group. Obeticholic acid treatment reduced the cholesterol content in the blood. The TM-01 group, TM-02 group, TM-06 group and TM-09 group had different effects on reducing blood lipids (Table 9).

[0140] Table 8: Changes of AST and ALT after 3 and 4 weeks of compound treatment

[0141]

[0142] Table 9: Changes in blood lipid levels after 4 weeks of compound treatment

[0143] Group TG (mmol / L) TC(mmol / L) LDL (mmol / L) HDL (mmol / L) Group 1-control 0.52±0.10* 0.77±0.09*** 2.05±0.06*** 0.34±0.03** Group 2-model+vehicle 0.60±0.03 1.12±0.08 2.90±0.04 0.60±0.01 Group 3-obeticholic acid 0.56±0.02 0.86±0.04** 2.80±0.03 0.55±0.01** Group 4-TM-01: 30mpk 0.53±0.10 0.95±0.44* 2.61±0.26* 0.59±0.11 Group 5-TM-02: 30mpk 0.57±0.06 1.25±0.14 2.93±0.12 0.61±0.02* Group 6-TM-06: 30mpk 0.59±0.02 0.97±0.16 3.01±0.09* 0.42±0.06*** Group 7-TM-09: 30mpk 0.54±0.04* 0.86±0.09* 2.52±0.07** 0.56±0.02*

[0144] 3.3 Effects of compounds on liver lipid content

[0145] After 4 weeks of MCD and compound treatment, the liver was collected to measure lipids, and the liver fat content was normalized with protein. In the model group, liver triglycerides and cholesterol were significantly higher than those in the control group. After treatment with obeticholic acid, both indicators decreased slightly. The TM-01 group, TM-02 group, TM-06 group and TM-09 group had different reducing effects on the content of liver triglycerides (liver TC) and total cholesterol (liver TG), among which the TM-01 group and TM-09 group showed significant reduction levels. (Table 10).

[0146] Table 10: Changes in liver lipid content after compound treatment

[0147] Group Liver TG (mol / g protein) liver TC(mol / g protein) Group 1-control 112.55±8.59*** 21.47±1.29*** Group 2-model+vehicle 339.42±40.24 44.94±5.50 Group 3-obeticholic acid 298.46±36.25 35.7±3.38 Group 4-TM-01: 30mpk 246.69±54.09* 34.85±8.20 Group 5-TM-02: 30mpk 307.38±26.30 41.67±5.73 Group 6-TM-06: 30mpk 303.5±46.52 38.23±5.04 Group 7-TM-09: 30mpk 231.73±18.4* 37.67±3.65*

[0148] 3.4 Effects of compounds on liver weight in mice

[0149] After 4 weeks of MCD and compound treatment, the livers were collected and weighed. The liver weight and liver / body weight ratio of the model group mice were significantly lower than those of the control group. When analyzing the results, it was unexpectedly found that such compounds had a significant effect on liver weight, among which the TM-01 group, TM-02 group, TM-06 group and TM-09 group all significantly increased the liver weight (liver weight) and liver / body weight ratio (i.e., liver / body weight) of mice. (Table 11).

[0150] Table 11: Effects of compound treatment on liver weight

[0151]

[0152] 3.5 Effects of Compounds on Liver Pathology

[0153] After fixation, mouse liver was stained with HE and Sirius Red. After staining, the whole slide was scanned. Six 20x fields of view were randomly selected. The six 20x fields of view stained with HE were comprehensively scored for pathology (i.e., HE score). The pathology scoring criteria are shown in Table 12. The six 20x fields of view stained with Sirius Red were used to calculate the ratio of the positive staining area of ​​Sirius Red to the total area (i.e., Sirius red staining area) using Image J.

[0154] Table 12: Pathological scoring criteria

[0155]

[0156]

[0157] After 4 weeks of MCD feeding, fat cells accumulated in the liver of the model group, and inflammatory cell infiltration was occasionally seen. After treatment with obeticholic acid, fat cell accumulation and inflammatory cell infiltration were more significant. Pathological scores showed that after MCD feeding, the pathological score of the model group was around 2, and the score increased after obeticholic acid treatment, while the pathological condition was alleviated to a certain extent after compound treatment (Table 13).

[0158] Table 13: Pathological scoring results

[0159]

[0160] 3.4 Conclusion: After 4 weeks of MCD feeding, mice induced an increase in AST, ALT and lipids in the blood, promoted the accumulation of fat in the liver and the formation of inflammatory foci, and modeled various indicators characteristic of non-alcoholic fatty liver disease.

[0161] The compounds provided by the present invention, especially TM-01 and TM-09, have a significant effect on reducing the levels of triglycerides and cholesterol in the liver, increase the liver weight and liver-to-body weight ratio of mice, and reduce the pathological score and collagen deposition in the liver to a certain extent. It is preliminarily determined that they have an improving effect on non-alcoholic fatty liver in animals.

[0162] Instruction Manual

[0163] Figure 1 The compound TM-1 of the present invention 1 H-NMR spectrum;

[0164] Figure 2 is the mass spectrum of compound TM-1 of the present invention;

[0165] Figure 3 The compound TM-2 of the present invention 1 H-NMR spectrum;

[0166] Figure 4 is the mass spectrum of compound TM-2 of the present invention;

[0167] Figure 5 The compound TM-3 of the present invention 1 H-NMR spectrum;

[0168] Figure 6 The compound TM-3 of the present invention 13 C-NMR spectrum;

[0169] Figure 7 is the mass spectrum of compound TM-3 of the present invention;

[0170] Figure 8 is the compound TM-4 of the present invention 1 H-NMR spectrum;

[0171] Fig. 9 is the mass spectrum of compound TM-4 of the present invention;

[0172] Fig.10 The compound TM-5 of the present invention 1 H-NMR spectrum;

[0173] Fig.11 is the mass spectrum of compound TM-5 of the present invention;

[0174] Fig.12 The compound TM-6 of the present invention 1 H-NMR spectrum;

[0175] Fig.13 The compound TM-6 of the present invention 13 C-NMR spectrum;

[0176] Fig.14 is the mass spectrum of compound TM-6 of the present invention;

[0177] Fig.15 The compound TM-7 of the present invention 1 H-NMR spectrum;

[0178] Fig.16 is the mass spectrum of compound TM-7 of the present invention;

[0179] Fig.17 The compound TM-8 of the present invention 1 H-NMR spectrum;

[0180] Fig.18 The compound TM-8 of the present invention 13 C-NMR spectrum;

[0181] Fig.19 is the mass spectrum of compound TM-8 of the present invention;

[0182] Fig. 20 The compound TM-9 of the present invention 1 H-NMR spectrum;

[0183] Fig.21 The compound TM-9 of the present invention 13 C-NMR spectrum;

[0184] Fig. 22 is the mass spectrum of compound TM-9 of the present invention;

[0185] Fig.23 The compound TM-10 of the present invention 1 H-NMR spectrum;

[0186] Fig.24 The compound TM-10 of the present invention 13 C-NMR spectrum;

[0187] Fig.25 It is the mass spectrum of the compound TM-10 of the present invention. DETAILED DESCRIPTION

[0188] The present invention is explained in detail below in conjunction with specific embodiments so that those skilled in the art can more fully understand the present invention. The specific embodiments are only used to illustrate the technical solutions of the present invention and do not limit the present invention in any way.

[0189] The synthetic route of the intermediate 1(3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-cyclopropylisoxazole) is as follows:

[0190]

[0191] The synthesis method is as follows:

[0192] 1.1 Synthesis of 2,6-dichlorobenzaldehyde oxime

[0193]

[0194] Hydroxylamine hydrochloride (11 g, 1 eq) and sodium hydroxide (6.3 g, 1.2 eq) were dissolved in water, and a solution of 2,6-dichlorobenzaldehyde (25 g, 0.14 mmol, 1.2 eq) in ethanol (200 mL) was added at room temperature. The mixture was stirred at 90°C for 1 hour. After cooling to room temperature, the ethanol was distilled off under reduced pressure, filtered, and the filter cake was washed with water (2×100 mL). After drying, 9.46 g of a white solid (2,6-dichlorobenzaldehyde oxime) was obtained with a yield of 84%.

[0195] 1.2 Synthesis of 2,6-dichloro-N-hydroxy-chlorobenzaldehyde oxime

[0196]

[0197] At 40°C, a DMF (90 mL) solution of N-chlorosuccinimide (16.08 g, 0.12 mol, 1 eq) was slowly added dropwise to a DMF (90 mL) solution of 2,6-dichlorobenzaldehyde oxime (22.8 g, 0.12 mol, 1 eq), stirred, and monitored by TLC. After the reaction was complete, the solution was cooled to room temperature and poured into ice water (200 mL). The mixture was extracted with methyl tert-butyl ether for 3 times (3×100 mL). The organic phases were combined and washed with water (3×100 mL) and saturated brine (100 mL). The ester layer was dried over anhydrous sodium sulfate and filtered. The organic solvent was removed by vacuum distillation to obtain a yellow oily crude product. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=5:1, v / v) to obtain 26 g of a white solid (2,6-dichloro-N-hydroxy-chlorobenzaldehyde oxime) with a yield of 97%.

[0198] 1.3 Synthesis of methyl 3-(2,6-dichlorophenyl)-5-cyclopropylisoxazole-4-carboxylate

[0199]

[0200] Add methyl 3-cyclopropyl-3-oxopropanoate (637.7 mg, 4.49 mmol, 1 eq) to a 100 mL reaction bottle, seal with a rubber stopper, add triethylamine (907.9 mg, 8.97 mmol, 2 eq) to the reaction bottle with a syringe, stir vigorously at room temperature for 30 min, cool the reaction solution in an ice bath below 10°C, slowly dropwise add 2,6-dichloro-N-hydroxy-chlorobenzaldehyde oxime (1.0 g, 4.49 mmol, 1 eq) in ethanol (monitor the internal temperature < 24°C) under stirring, slowly warm to room temperature, stir vigorously overnight, remove ethanol by distillation under reduced pressure, add ethyl acetate for extraction 3 times (3×100 mL), wash the organic layer with water (3×100 mL) and saturated brine (100 mL), dry over anhydrous sodium sulfate to remove the solvent, and obtain an oily crude product. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=40:1, v / v) to obtain 0.89 g of a white solid (methyl 3-(2,6-dichlorophenyl)-5-cyclopropylisoxazole-4-carboxylate) with a yield of 55%.

[0201] 1.4 Synthesis of 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-cyclopropylisoxazole

[0202]

[0203] Under nitrogen protection and ice bath conditions, a toluene solution of diisobutylaluminum hydride (4.0 mL, 6.0 mmol, 2.1 eq, 1.5 M toluene solution) was slowly added dropwise to anhydrous THF of 3-(2,6-dichlorophenyl)-5-cyclopropylisoxazole-4-carboxylic acid methyl ester (0.89 g, 2.8 mmol, 1 eq), and the mixture was heated to room temperature and stirred vigorously overnight. The reaction solution was cooled to 0°C again, methanol (2 mL) was slowly added dropwise and stirred for 10 min, and the reaction solution was poured into a 50 mL ice-water mixture to form a gel suspension. Filtered through diatomaceous earth, extracted three times with ethyl acetate (3×100 mL), the ester layers were combined, washed with water (3×100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered to remove the solvent to obtain a white solid. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=20:1, v / v) to obtain 0.45 g of a white solid (3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-cyclopropylisoxazole) in a yield of 56%.

[0204] 2. Synthesis of 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-isopropylisoxazole

[0205] 2.1 Synthesis of methyl 3-(2,6-dichlorophenyl)-5-isopropylisoxazole-4-carboxylate

[0206]

[0207] Methyl isobutyrylacetate (16.6 mL, 0.12 mol, 1 eq) was added to a 100 mL reaction bottle, which was sealed with a rubber stopper. Triethylamine (33.25 mL, 0.24 mol, 2 eq) was added to the reaction bottle with a syringe, and the mixture was vigorously stirred for 30 min at room temperature. The reaction solution was cooled in an ice bath below 10°C, and an ethanol solution of 2,6-dichloro-N-hydroxy-chlorobenzaldehyde oxime (26.6 g, 0.12 mol, 1 eq) was slowly added dropwise under stirring (the internal temperature was monitored to be <24°C), the temperature was slowly raised to room temperature, and the mixture was vigorously stirred overnight. The ethanol was removed by distillation under reduced pressure, and ethyl acetate was added for extraction 3 times (3×100 mL). The organic layer was washed with water (3×100 mL) and saturated brine (100 mL), and the solvent was removed by drying over anhydrous sodium sulfate to obtain an oily crude product. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=40:1, v / v) to obtain 21 g of white solid (methyl 3-(2,6-dichlorophenyl)-5-isopropylisoxazole-4-carboxylate) with a yield of 56%.

[0208] 2.2 Synthesis of 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-isopropylisoxazole

[0209]

[0210] Under nitrogen protection and ice bath conditions, a toluene solution of diisobutylaluminum hydride (92mL, 0.14mol, 2.1eq, 1.5M toluene solution) was slowly added dropwise to anhydrous THF of 3-(2,6-dichlorophenyl)-5-isopropylisoxazole-4-carboxylic acid methyl ester (20g, 0.06mol, 1eq), and the mixture was heated to room temperature and stirred vigorously overnight. The reaction solution was cooled to 0°C again, methanol (20mL) was slowly added dropwise and stirred for 10min, and the reaction solution was poured into a 200mL ice-water mixture to form a gel suspension. Filtered through diatomaceous earth, extracted three times with ethyl acetate (3×100mL), the ester layers were combined, washed with water (3×100mL) and saturated brine (100mL), dried over anhydrous sodium sulfate, and filtered to remove the solvent to obtain a white solid. The residue was separated and purified by silica gel column chromatography with gradient elution (PE:EA=40:1, v / v) to obtain 18 g of a white solid (3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-isopropylisoxazole) in a yield of 94%.

[0211] 3 Synthesis of 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-phenylisoxazole

[0212] 3.1 Synthesis of ethyl 3-(2,6-dichlorophenyl)-5-phenylisoxazole-4-carboxylate

[0213]

[0214] Ethyl benzoyl acetate (5.7 mL, 50 mmol, 1 eq) was added to a 100 mL reaction bottle, which was sealed with a rubber stopper. Triethylamine (13.86 mL, 100 mmol, 2 eq) was added to the reaction bottle with a syringe. The reaction was vigorously stirred for 30 min at room temperature. The reaction solution was cooled in an ice bath below 10°C. An ethanol solution of 2,6-dichloro-N-hydroxy-chlorobenzaldehyde oxime (10 g, 50 mmol, 1 eq) was slowly added dropwise under stirring (the internal temperature was monitored to be <24°C). The temperature was slowly raised to room temperature and vigorously stirred overnight. The ethanol was removed by distillation under reduced pressure. Ethyl acetate was added and extracted 3 times (3×100 mL). The organic layer was washed with water (3×100 mL) and saturated brine (100 mL), and the solvent was removed by drying over anhydrous sodium sulfate to obtain an oily crude product. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=40:1, v / v) to obtain 11.7 g of white solid (methyl 3-(2,6-dichlorophenyl)-5-phenylisoxazole-4-carboxylate) with a yield of 65%.

[0215] 3.2 Synthesis of 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-phenylisoxazole

[0216]

[0217] Under nitrogen protection and ice bath conditions, slowly drop a toluene solution of diisobutylaluminum hydride (18 mL, 27 mmol, 2.1 eq, 1.5 M toluene solution) into anhydrous THF of 3-(2,6-dichlorophenyl)-5-phenylisoxazole-4-carboxylic acid methyl ester (4.72 g, 13 mmol, 1 eq), warm to room temperature and stir vigorously overnight. Cool the reaction solution to 0°C again, slowly drop methanol (20 mL) and stir for 10 min, pour the reaction solution into 200 mL of ice-water mixture to form a gel suspension. Filter through diatomaceous earth, extract three times with ethyl acetate (3×100 mL), combine the ester layers, wash with water (3×100 mL) and saturated brine (100 mL), dry over anhydrous sodium sulfate, and filter to remove the solvent to obtain a white solid. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=20:1, v / v) to obtain 9.1 g of a white solid (3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-phenylisoxazole) with a yield of 70%.

[0218] Example 1: Synthesis route of compound TM-1

[0219]

[0220] Test steps:

[0221] 3-(3-Bromophenyl)cyclobutanone

[0222]

[0223] At -15°C, trifluoromethanesulfonic anhydride (11.6 g, 41.0 mmol) was slowly added dropwise to a solution of N,N-dimethylformamide (2.1 g, 24.6 mmol) in 1,2-dichloroethane (40 mL), and stirred at -15°C for 30 minutes. Then 3-bromostyrene (3.0 g, 16.4 mmol) and 2,4,6-trimethylpyridine (2.9 g, 24.6 mmol) were added and stirred at room temperature overnight. Water was added to quench the reaction, stirred at room temperature overnight, and dichloromethane was added to dilute and separate the organic phase. The organic phase was washed with water and saturated brine (200 ml) respectively, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated and purified by gradient elution of silica gel column chromatography (PE: EA = 15: 1, v / v) to obtain 1.3 g of yellow solid 3-(3-bromophenyl)cyclobutanone with a yield of 35%.

[0224] Methyl 3-(3-oxocyclobutyl)benzoate

[0225]

[0226] At room temperature, in a carbon monoxide balloon environment, triethylamine (2.2 g, 21.3 mmol) was added to methyl 3-(3-oxocyclobutyl)benzoate (1.6 g, 7.1 mmol) and (1,1'-bis(diphenylphosphino)ferrocene)palladium dichloride (520 mg, 0.7 mmol) in a mixed solvent of methanol (20 mL) and N,N-dimethylformamide (10 mL), heated to 55°C for 18 hours, the solvent was distilled off under reduced pressure and dissolved in ethyl acetate, washed with water, the organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated and purified by gradient elution on a silica gel column chromatography (PE:EA=3:1, v / v) to obtain 1.1 g of methyl 3-(3-oxocyclobutyl)benzoate as a yellow oily solid with a yield of 75%.

[0227] 4-(5-Bromopyrazine-2-methyleneoxy)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole

[0228]

[0229] In a 100ml round-bottom flask, put sodium hydride (4.9g, 121.6mmol), add a small amount of petroleum ether, wash the kerosene layer on the surface of sodium hydride, wash twice. Add tetrahydrofuran (30ml), cool the reaction bottle in an ice bath at 0℃, dissolve 2,5-dibromopyrazine (13.1g, 55.3mmol) in tetrahydrofuran (10ml), and add it dropwise to the round-bottom flask under stirring. After reacting for 20min, dissolve 1(5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methanol (15.7g, 55.3mmol) in tetrahydrofuran (10ml), slowly add it dropwise to the reaction bottle with a syringe, and warm it to room temperature to react for 12h. After the reaction is completed, pour the reaction solution into a 100ml ice-water mixture, then extract it with ethyl acetate (3×100ml), combine the organic phases, wash with water and saturated brine. Dry with anhydrous MgSO4. The product was separated and purified by gradient elution on a silica gel column (PE:EA=10:1, v / v) to give 20.2 g of a white solid 6 (4-(5-bromopyrazine-2-methyleneoxy)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole) with a yield of 83%.

[0230] Methyl 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate

[0231]

[0232] In a 100 ml three-necked flask, nitrogen was protected. 6 (20.2 g, 45.8 mmol) was dissolved in tetrahydrofuran (80 ml) and added to the reaction flask. Then the temperature was lowered to -78 °C and n-butyl lithium (1.6 M in hexane, 30.0 mL, 48.0 mmol) was slowly added dropwise. After stirring for 10 min, a solution of methyl 3-(3-oxocyclobutyl)benzoate 2 (9.0 g, 43.6 mmol) dissolved in tetrahydrofuran (20 ml) was slowly added dropwise. The mixture was reacted at -78 °C for 2 h and then warmed to room temperature for overnight reaction. After the reaction was completed, the reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate, and the organic phase was washed with saturated brine (200 ml), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was distilled off under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 15: 1, v / v) to obtain 5.6 g of yellow solid 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoic acid methyl ester with a yield of 19%.

[0233] 3-(3-(5-(5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxycyclobutane)benzoic acid

[0234]

[0235] Methyl 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate (5.6 g, 10.1 mmol) was dissolved in a mixed solvent of THF (10 mL) and methanol (10 mL). A solution of LiOH·H2O (1.8 g, 42.6 mmol) in water (5 ml) was added at 35°C and stirred overnight. The organic solvent was distilled off under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, and the mixture was dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the mixture was separated and purified by high pressure preparative liquid chromatography (acetonitrile: water = 3:4, v / v) to obtain 3.28 g of white solid TM-1 3-(3-(5-(5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxycyclobutane)benzoic acid, with a yield of 57%.

[0236] Figure 1 As shown, 1H-NMR (400MHz, DMSO-D6): δ12.95(s,1H),8.22-8.21(m,1H),8.12-8.11(m,1H),7.95-7.94(m,1H),7.72(d,J=2Hz,1H),7.66-7.49(m,4H),7. 48-7.37(m,1H),6.06(s,1H),5.24(s,2H),3.57-3.37(m,1H),2.94-2. 89 (m, 2H), 2.55-2.42 (m, 3H), 1.21 (d, J = 24Hz, 2H), 1.15 (d, J = 16Hz, 2H)

[0237] Figure 2 As shown, SI-MS: m / z[M+H]+: Calcd.for C 28 H 23 Cl2N3O5:551.1,Found:552.2

[0238] Example 2: Synthesis route of compound TM-2

[0239]

[0240] Synthesis steps:

[0241] Synthesis of methyl 3-(3-hydroxyazetidin-1-yl)benzoate 3b

[0242]

[0243] 3-Azetidine-3-ol hydrochloride (2.5 g. 22.9 mmol), Cs2CO3 (15.5 g, 47.7 mmol), CuI (726 mg, 3.8 mmol) and L-proline (878 mg, 7.6 mmol) were added to a solution of methyl 3-iodobenzoate 3a (5.0 g, 19.1 mmol) in DMSO-D6 (70 mL), and the mixture was heated at 90 ° C for 18 hours under an argon atmosphere. The solution was diluted with ethyl acetate and water, and the organic layer was washed three times with brine, concentrated under reduced pressure, and separated and purified by silica gel column chromatography (DCM / MeOH=10 / 1, v / v) to obtain a white solid product 3b (2.7 g, 68%).

[0244] Synthesis of methyl 3-(3-oxoazetidin-1-yl)benzoate 3

[0245]

[0246] Dimethyl sulfoxide (1.6 g, 20.3 mmol) was dissolved in dichloromethane (30 mL), oxalyl chloride (1.3 g, 10.1 mmol) was added at -78 ° C, and stirred at -78 ° C for 30 minutes, then 3-(3-hydroxyazetidin-1-yl) benzoic acid methyl ester (1.4 g, 6.8 mmol) was dissolved in dichloromethane, and slowly added to the reaction solution at -78 ° C, and the time was controlled at 30 minutes, and then stirred at -78 ° C for 30 minutes, followed by triethylamine (4.1 g, 40.5 mmol), reacted at -78 ° C for 1 hour, warmed to room temperature and reacted at room temperature for 2 hours. The reaction solution was diluted with water and extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated by suction, and separated and purified by silica gel column chromatography (PE / EA=2 / 1) to obtain a white solid product 3 (0.9 g, 65%).

[0247] Methyl 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxyazetidin-1-yl)benzoate

[0248]

[0249] In a 100 ml three-necked flask, nitrogen was protected. 6 (1.9 g, 4.4 mmol) was dissolved in tetrahydrofuran (25 ml) and added to the reaction flask. Then the temperature was lowered to -78 °C and n-butyl lithium (2.5 M in hexane, 2.6 mL, 6.6 mmol) was slowly added dropwise. After stirring for 10 min, a solution of methyl 3-(3-oxoazetidin-1-yl)benzoate 3 (0.9 g, 4.4 mmol) dissolved in tetrahydrofuran (5 ml) was slowly added dropwise. The mixture was reacted at -78 °C for 2 h and then warmed to room temperature for overnight reaction. After the reaction was completed, the reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate, and the organic phase was washed with saturated brine (200 ml), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 15: 1, v / v) to obtain 560 mg of methyl 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate as a yellow solid, with a yield of 22%.

[0250] 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxyazetidin-1-yl)benzoic acid

[0251]

[0252] Methyl 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate (270 mg, 0.5 mmol) was dissolved in a mixed solvent of THF (3 mL) and methanol (3 mL), and a solution of LiOH·H2O (60 mg, 1.5 mmol) in water (3 ml) was added at 35°C and stirred overnight. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, and the mixture was dried over anhydrous magnesium sulfate. The solvent was removed by distillation under reduced pressure, and the mixture was separated and purified by high pressure preparative liquid chromatography (acetonitrile: water = 3:4, v / v) to obtain 40 mg of 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutylbenzoic acid as a white solid with a yield of 15%.

[0253] Figure 3 As shown, 1 H-NMR (400MHz, DMSO-D6): δ8.22-8.21(m,1H),8.08-8.08(m,1H),7.59-7.51(m,4H),7.27-7.25(m,2H),7.00(s,1H),6.67 (s,1H),5.24(s,2H),4.21(d,J=16Hz,2H),3.98(d,J=16Hz,2H),2.51-2.50(m,1H),1.22-1.18(m,2H),1.15-1.12(m,2H).

[0254] Figure 4 As shown, ESI-MS: m / z[M+H]+:Calcd.for C 27 H 22 Cl2N4O5:552.1,Found:553.1 (Note, the values ​​are on the right side of the figure).

[0255] Example 3: Synthesis route of compound TM-3

[0256] 3-(3-bromophenyl)-1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)cyclobutanol

[0257]

[0258] In a 100mL three-necked flask, nitrogen was used to protect the flask. 4-(5-bromopyrazine-2-methyleneoxy)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole (1.14 g, 2.3 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) and poured into the reaction bottle. Then, ethanol and liquid nitrogen were added into a 500mL low-temperature Dewar flask to lower the temperature to -78°C. n-Butyl lithium (1.7 ml, 2.7 mmol) was slowly added dropwise. After stirring for 10 min, a solution of methyl 3-(3-oxocyclobutanone)benzoate (0.56 g, 2.5 mmol) dissolved in tetrahydrofuran (10 mL) was slowly added dropwise. The reaction was carried out at -78°C for 2 h and then the temperature was raised to room temperature for overnight reaction. After the reaction was completed, the reaction solution was slowly poured into a mixture of ice and water, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was distilled off under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 10: 1, v / v) to obtain a white solid 3-(3-bromophenyl)-(5-(5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)cyclobutane-1-ol, 567 mg, with a yield of 42%.

[0259] Figure 5 As shown, 1 H-NMR (400MHz, CDCl3): δ8.30 (1H, s), 8.06 (1H, d, J = 4Hz), 7.45 (1H, s), 7.42 (1H,d,J=1.6Hz),7.40(1H,s),7.36-7.32(2H,m,J=16Hz),7.22-7.18(2H,m, J=16Hz),5.23(2H,s),3.35-3.26(1H,m),2.99-2.93(2H,m),2.63-2.57(2H, m),2.36-2.29(1H,m),1.33-1.29(2H,d,J=24Hz),1.21-1.16(2H,d,J=16Hz);

[0260] Figure 6 As shown, 13 C-NMR(100MHz,DMSO-D6):173.0,159.6,158.4,150.9,147.1,130.0,129.8, 129.3,128.0,127.9,125.3,122.6,110.3,71.0,56.8,44.5,29.8,8.5,7.8.

[0261] Figure 7 As shown, ESI-MS: m / z[M+2+H] + :Calcd.forC 27 H 22BrCl2N3O3:585.0222,Found:588.0300

[0262] Example 4: Synthesis route of compound TM-4

[0263] 1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-(3-(methylsulfonyl)phenyl)cyclobutanol

[0264]

[0265] To a solution of 3-(3-bromophenyl)-1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)cyclobutanol (500 mg, 0.85 mmol) in DMSO-D6, sodium methanesulfinate (130 mg, 1.28 mmol), CuI (50.2 mg, 0.26 mmol), L-proline (97.9 mg, 0.85 mmol) and diisopropylethylamine (DIEA) (109.9 mg, 0.85 mmol) were added. The mixture was stirred at 95° C. overnight, then diluted with water and extracted with EA. The organic phases were combined, washed with water and dried over Na2SO4. The residue was concentrated to dryness under reduced pressure and separated and purified by high pressure preparative liquid chromatography (acetonitrile: water = 3:4, v / v) to give 324 mg of white solid 1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-(3-(methylsulfonyl)phenyl)cyclobutanol in a yield of 65%.

[0266] Figure 8 As shown, 1 H-NMR (400MHz, CDCl3): δ8.15(s,1H),7.92(s,1H),7.72-7.65(m,2H),7.42-7.40(m,2H),7.28-7.12(m,2H),5.08(s,2H ), 3.33-3.29 (m, 1H), 2.93-2.85 (m, 5H), 2.51-2.46 (m, 2H), 2.21-2.18 (m, 1H), 1.15 (d, J = 16Hz, 2H), 1.05 (d, J = 16Hz, 2H)

[0267] Fig. 9 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 28 H 25 Cl2N3O5S:585.1,Found:586.3

[0268] Example 5: Synthesis route of compound TM-5

[0269] 1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-(3-(phenylthio)phenyl)cyclobutanol

[0270]

[0271] Under argon protection, DIEA (0.44 g, 3.41 mmol), benzyl mercaptan (0.21 g, 1.7 mmol), Pd2(dba)3 (0.34 g, 0.37 mmol) and 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (0.16 g, 0.27 mmol) were added to a solution of 3-(3-bromophenyl)-1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)cyclobutanol (1.0 g, 1.7 mmol) in toluene. The mixture was then stirred at 115°C for 4 hours. The mixture was cooled to room temperature, diluted with water and extracted with EA. The organic phases were combined, washed with water and dried with Na2SO4. The residue was concentrated to dryness under reduced pressure and separated and purified by high pressure preparative liquid chromatography (acetonitrile: water = 3:4, v / v) to obtain 367 mg of white solid 1-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-(3-(phenylthio)phenyl)cyclobutanol, with a yield of 35%.

[0272] Fig.10 As shown, 1 H-NMR (400MHz, CDCl3): δ8.23(s,1H),8.01(s,1H),7.46-7.54(m,3H),7.23-7.33(m,8H),7.14(br d,J=7.6Hz,1H),5.28(s,2H),3.31-3.34(m,1H),4.85(H2O),4.58(HDO), 3.30(CD3OD),2.92-3.02(m,2H),2.41-2.50(m,2H),1.23(s,1H),1.21(br d,J=2.0Hz,2H),1.19(br d,J=2.0Hz,2H)

[0273] Fig.11 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 33 H 27 Cl2N3O3S:615.1150, Found:616.1421.

[0274] Example 6: Synthesis of Compound TM-6

[0275] Synthesis of 4-(5-bromopyrazine-2-methyleneoxy)-5-isopropyl-3-(2,6-dichlorophenyl)isoxazole

[0276]

[0277] In a 100mL round-bottom flask, put sodium hydride (60%, 0.83g, 21mmol), add a small amount of petroleum ether, wash the kerosene layer on the surface of NaH, wash twice. Add 30mL tetrahydrofuran, and cool the reaction bottle in an ice bath at 0℃. Dissolve 2,5-dibromopyrazine (833mg, 3.5mmol) in 10mL tetrahydrofuran and add it dropwise to the round-bottom flask under stirring. After reacting for 20min, dissolve 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-isopropylisoxazole (1g, 3.5mmol) in 10mL tetrahydrofuran and slowly add it dropwise to the reaction bottle through a syringe. Warm to room temperature and react for 12h. After the reaction is completed, pour the reaction solution into 100mL ice-water mixture, then extract with ethyl acetate (3×100mL), combine the organic phases and wash with water and saturated brine. Dry with anhydrous MgSO4. The product was separated and purified by silica gel column chromatography with gradient elution (PE:EA=10:1, v / v) to obtain 0.7 g of white solid 4-(5-bromopyrazine-2-methyleneoxy)-5-isopropyl-3-(2,6-dichlorophenyl)isoxazole with a yield of 53%.

[0278] Synthesis of methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate

[0279]

[0280] In a 100mL three-necked flask, nitrogen was used to protect the flask. 4-(5-bromopyrazine-2-methyleneoxy)-5-isopropyl-3-(2,6-dichlorophenyl)isoxazole (1g, 2.3mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and poured into the reaction bottle. Then, ethanol and liquid nitrogen were added into a 500mL low-temperature Dewar flask to lower the temperature to -78°C. n-Butyl lithium (1.7ml, 2.7mmol) was slowly added dropwise. After stirring for 10min, a solution of methyl 3-(3-oxocyclobutanone)benzoate (0.51g, 2.5mmol) dissolved in tetrahydrofuran (10mL) was slowly added dropwise. The reaction was carried out at -78°C for 2h and then the temperature was raised to room temperature for overnight reaction. After the reaction was completed, the reaction solution was slowly poured into a mixture of ice and water, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 10: 1, v / v) to obtain 549 mg of methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate as a white solid with a yield of 42%.

[0281] Synthesis of 3-(3-(5-(5-isopropyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxycyclobutane)benzoic acid

[0282]

[0283] Methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate (319 mg, 0.6 mmol, 1 eq) was dissolved in 20 mL of THF and a solution of LiOH·H2O (99 mg, 2.4 mmol, 4.2 eq) in water (5 mL) was stirred at 35°C overnight. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, the mixture was dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and the mixture was separated and purified by high pressure preparative liquid chromatography using a Waters XBridge C18 column (150nm*4.6nm*3.5um), acetonitrile and water as the mobile phase, the flow rate was 18mL / min, and the fractions with a gradient of 45%-75% were collected. The fractions were concentrated to remove most of the acetonitrile, and the mixture was freeze-dried with a freeze dryer to obtain 126 mg of a white powdery solid (3-(3-(5-(5-isopropyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxycyclobutane)benzoic acid) with a yield of 38%.

[0284] Fig.12 Shown 1H-NMR (400MHz, DMSO-D6): δ12.97(s,1H),8.20(d,J=1.3Hz,1H),8.09(d,J=1.3Hz ,1H),7.95(s,1H),7.79-7.77(d,J=7.7Hz,1H),7.63-7.61(m,2H),7.55-7.53(m, 2H), 7.44 (t, J = 7.7Hz, 1H), 6.09 (s, 1H), 5.19 (s, 2H), 3.61-3.54 (m, 1H), 3.42-3. 33(m,1H),2.90(td,J=8.9,2.5Hz,2H),2.47-2.42(m,2H),1.37(d,J=7.0Hz,6H);

[0285] Fig.13 As shown, 13 C-NMR(100MHz, CDCl3):176.8,171.0,159.3,158.4,150.9,145.2,131.2,129.4,1 28.7,128.4,128.2,128.1,128.0,109.1,71.1,56.8,44.5,29.9,27.0,20.9,1.0;

[0286] Fig.14 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 28 H 25 Cl2N3O5:553.1171,Found:554.1211.

[0287] Example 7: Synthesis of Compound TM-7

[0288] Synthesis of 4-(5-bromopyrazine-2-methyleneoxy)-5-phenyl-3-(2,6-dichlorophenyl)isoxazole

[0289]

[0290] In a 100mL round-bottom flask, put sodium hydride (60%, 0.83g, 21mmol), add a small amount of petroleum ether, wash the kerosene layer on the surface of NaH, wash twice. Add 30mL tetrahydrofuran, and cool the reaction bottle in an ice bath at 0℃. Dissolve 2,5-dibromopyrazine (833mg, 3.5mmol) in 10mL tetrahydrofuran and add it dropwise to the round-bottom flask under stirring. After reacting for 20min, dissolve 3-(2,6-dichlorophenyl)-4-hydroxymethyl-5-phenylisoxazole (1.12g, 3.5mmol) in 10mL tetrahydrofuran and slowly add it dropwise to the reaction bottle through a syringe. Warm to room temperature and react for 12h. After the reaction is completed, pour the reaction solution into 100mL ice-water mixture, then extract with ethyl acetate (3×100mL), combine the organic phases and wash with water and saturated brine. Dry with anhydrous MgSO4. The product was separated and purified by gradient elution on a silica gel column (PE:EA=10:1, v / v) to give a white solid (4-(5-bromopyrazine-2-methyleneoxy)-5-phenyl-3-(2,6-dichlorophenyl)isoxazole), 752 mg, with a yield of 45%.

[0291] Synthesis of methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate

[0292]

[0293] In a 100mL three-necked flask, nitrogen was used to protect the solution. 4-(5-bromopyrazine-2-methyleneoxy)-5-phenyl-3-(2,6-dichlorophenyl)isoxazole (1g, 2.1mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and poured into the reaction bottle. Then, ethanol and liquid nitrogen were added into a 500mL low-temperature Dewar flask to lower the temperature to -78°C. n-Butyl lithium solution (1.1N cyclohexane solution, 1.75ml, 2.8mmol) was added dropwise. After stirring for 10min, a solution of methyl 3-(3-oxocyclobutanone)benzoate (0.47g, 2.3mmol) dissolved in tetrahydrofuran (10mL) was slowly added dropwise. The reaction was carried out at -78°C for 2h and then the temperature was raised to room temperature for overnight reaction. After the reaction was completed, the reaction solution was slowly poured into a mixture of ice and water, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was distilled off under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 10: 1, v / v) to obtain 329 mg of methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate as a white solid with a yield of 26%.

[0294] Synthesis of 3-(3-(5-(5-phenyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxycyclobutane)benzoic acid

[0295]

[0296] Methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate (118 mg, 0.2 mmol, 1 eq) was dissolved in 20 mL of THF and a solution of LiOH·H2O (35 mg, 0.8 mmol, 4.2 eq) in water (5 mL) was stirred at 35°C overnight. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, the mixture was dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and the mixture was separated and purified by high pressure preparative liquid chromatography using a Waters X Bridge C18 column (150nm*4.6nm*3.5um), acetonitrile and water as the mobile phase, the flow rate was 18mL / min, and the fractions with a gradient of 45%-75% were collected. The fractions were concentrated to remove most of the acetonitrile, and the mixture was freeze-dried with a freeze dryer to obtain 39 mg of a white powdery solid (3-(3-(5-(5-phenyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxycyclobutane)benzoic acid) with a yield of 33%.

[0297] Fig.15 As shown, 1 H-NMR (400MHz, DMSO-D6): δ12.97(s,1H),8.15(d,J=1.3Hz,1H),8.10(d,J=1.3Hz,1H), 7.99-7.95(m,J=16Hz,3H),7.79-7.78(d,J=4Hz,1H),7.67-7 .65(m,J=8Hz,5H),7.61-7.55(m,J=8Hz,2H),7.46-7.42(m,1H),6.08(s,1 H),5.38(s,2H),5.39-3.33(m,1H),2.94-2.88(m,2H),2.47-2.42(m,2H);

[0298] Fig.16 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 31 H 23 Cl2N3O5: 587.1015, Found: 588.1062

[0299] Example 8: Synthesis of Compound TM-8

[0300]

[0301] Methyl 3-methyl-5-vinylbenzoate

[0302]

[0303] In a 100 mL round-bottom flask, methyl 3-bromobenzoate (1.12 g, 5 mmol), potassium trifluoroborate (820 mg, 6.12 mmol), PdCl2 (17.5 mg, 0.1 mmol), PPh3 (80 mg, 0.3 mmol) and Cs2CO3 (5 g, 15 mmol) were added, and then THF (18 mL) and H2O (2 mL) were added under N2. The mixture was stirred at 80 ° C for 22 h, cooled to room temperature, washed with water, dried over anhydrous magnesium sulfate, and filtered. After vacuum distillation, the product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 60: 1, v / v) to obtain 164 mg of a light pink oily liquid (methyl 3-methyl-5-vinylbenzoate) with a yield of 30%.

[0304] Methyl 3-(2,2-dichloro-3-oxocyclobutanone)-5-methylbenzoate

[0305]

[0306] Under nitrogen protection, methyl 3-methyl-5-vinylbenzoate (5.46g, 31mmol, 1eq) was dissolved in ether (150mL). Zinc powder (6g, 93mmol, 3eq) was added, and after ultrasonication for 30min, a solution of trichloroacetyl chloride (8.7mL, 77.5mmol, 2.5eq) in Et2O (50mL) was added dropwise, and ultrasonication was continued for 30 minutes. The mixture was heated to 35°C. Ultrasonication was continued for 2.5h, and after the reaction was completed, it was cooled to room temperature and quenched by slowly adding water (50mL). The mixture was poured into water and stirred for 20min, filtered, and then rinsed with Et2O. The organic layer was washed with water (250mL), saturated sodium bicarbonate (250mL) and saturated sodium chloride (250mL), dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by reduced pressure distillation to obtain a yellow oily crude product. The product was separated and purified by gradient elution on a silica gel column (PE:EA=50:1, v / v) to obtain 3.56 g of methyl 3-(2,2-dichloro-3-oxocyclobutanone)-5-methylbenzoate as a yellow oily liquid with a yield of 40%.

[0307] Methyl 3-methyl-5-(3-oxocyclobutyl)benzoate

[0308]

[0309] Mix 3-(2,2-dichloro-3-oxocyclobutanone)-5-methylbenzoic acid methyl ester (2.79g, 9.7mmol, 1eq) and zinc powder (2.54g, 38.8mmol, 4eq) in 60mL acetic acid and stir at room temperature for 1h. Then reflux in an oil bath at 80℃ for 3.5h. After the reaction is completed, cool to room temperature. Dilute the solvent acetic acid with 100mL water and extract with ether (3×40mL). After combining the organic phases, wash with saturated sodium carbonate solution (3×40mL), water (100mL), and saturated brine (100mL) in turn. Dry with a certain amount of anhydrous MgSO4. Separate and purify by silica gel column chromatography gradient elution (PE:EA=50:1, v / v) to obtain 1.38g of the compound (3-(3-oxocyclobutanone)benzoic acid methyl ester) with a yield of 65%.

[0310] Synthesis of methyl 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate

[0311]

[0312] In a 100mL three-necked flask, nitrogen was used to protect the solution. 4-(5-bromopyrazine-2-methyleneoxy)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole (1.02g, 2.3mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and poured into the reaction bottle. Then, ethanol and liquid nitrogen were added into a 500mL low-temperature Dewar flask to lower the temperature to -78°C. n-Butyl lithium (1.7ml, 2.7mmol) was slowly added dropwise. After stirring for 10min, a solution of methyl 3-methyl-5-(3-oxocyclobutyl)benzoate (0.55g, 2.5mmol) dissolved in tetrahydrofuran (10mL) was slowly added dropwise. The reaction was carried out at -78°C for 2h and then the temperature was raised to room temperature for overnight reaction. After the reaction was completed, the reaction solution was slowly poured into a mixture of ice and water, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was distilled off under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 10: 1, v / v) to obtain 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid methyl ester as a white solid with a yield of 47%.

[0313] 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid

[0314]

[0315] Methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-cyclopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)benzoate (116 mg, 0.2 mmol, 1 eq) was dissolved in 20 mL of THF and a solution of LiOH·H2O (35 mg, 0.8 mmol, 4.2 eq) in water (5 mL) was stirred at 35°C overnight. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, the mixture was dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and the mixture was separated and purified by high pressure preparative liquid chromatography using a Waters XBridge C18 column (150nm*4.6nm*3.5um), acetonitrile and water as the mobile phase, the flow rate was 18mL / min, and the fractions with a gradient of 45%-75% were collected. The fractions were concentrated to remove most of the acetonitrile, and the mixture was freeze-dried with a freeze dryer to obtain a white powdery solid 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid, 37 mg, with a yield of 33%.

[0316] Fig.17 As shown, 1 H-NMR (400MHz, DMSO-D6): δ12.90(1H,s),8.21(1H,d,J=1.2Hz),8.11(1H,d, J=1.2Hz),7.74(1H,s),7.62-7.60(3H,m),7.56-7.52(1H,m),7.37(1H,s),6 .06(1H,s),5.24(2H,s),3.38-3.29(1H,m),2.92-2.87(2H,m),2.59-2.53(1 H,m),2.46-2.41(2H,m),2.35(3H,s),1.24-1.18(2H,m),1.16-1.14(2H,m);

[0317] Fig.18 As shown, 13 C-NMR (400MHz, DMSO-D6): δ176.9,168.9,168.0,159.3,157.9,153.5,146.2,135.1, 133.4,133.0,128.9,127.6,125.1,109.9,70.9,56.3,45.5,29.8,26.5,21.3,21.1;

[0318] Fig.19 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 29 H 25Cl2N3O5:565.1171,Found:566.1234

[0319] Example 9: Synthesis of Compound TM-9

[0320] Synthesis of methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate

[0321]

[0322] In a 100mL three-necked flask, nitrogen was used to protect the flask. 4-(5-bromopyrazine-2-methyleneoxy)-5-isopropyl-3-(2,6-dichlorophenyl)isoxazole (1.02g, 2.3mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and poured into the reaction bottle. Then, ethanol and liquid nitrogen were added into a 500mL low-temperature Dewar flask to lower the temperature to -78°C. n-Butyl lithium (1.7ml, 2.7mmol) was slowly added dropwise. After stirring for 10min, a solution of methyl 3-methyl-5-(3-oxocyclobutyl)benzoate (0.55g, 2.5mmol) dissolved in tetrahydrofuran (10mL) was slowly added dropwise. The reaction was carried out at -78°C for 2h and then the temperature was raised to room temperature for overnight reaction. After the reaction was completed, the reaction solution was slowly poured into a mixture of ice and water, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was removed by distillation under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE:EA=10:1, v / v, PE is petroleum ether, EA is ethyl acetate) to obtain 643 mg of a white solid (methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate) with a yield of 48%.

[0323] Synthesis of 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid

[0324] Methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate (117 mg, 0.2 mmol, 1 eq) was dissolved in 20 mL of THF and a solution of LiOH·H2O (35 mg, 0.8 mmol, 4.2 eq) in water (5 mL) was stirred at 35°C overnight. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, the mixture was dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and the mixture was separated and purified by high pressure preparative liquid chromatography using a Waters X Bridge C18 column (150nm*4.6nm*3.5um), acetonitrile and water as the mobile phase, the flow rate was 18mL / min, and the fractions with a gradient of 45%-75% were collected. The fractions were concentrated to remove most of the acetonitrile, and the mixture was freeze-dried with a freeze dryer to obtain a white powdery solid (3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid), 46 mg, with a yield of 40%.

[0325] Fig. 20 As shown, 1 H-NMR (400MHz, DMSO-D6): δ12.88 (1H, brs), 8.20 (1H, d, J = 1.2Hz), 8.08 (1H, d, J = 1.2Hz), 7.73 (1H, s), 7.63-7.60 (3H, m), 7.56-7.52 (1H, s) ,7.37(1H,s),6.06(1H,brs),5.19(2H,s),3.61-3.54(1H,m),2.91-2.85(2H,m),2.45-2.40(2H,m),2.35(3H,s),1.38-1.36(6H,d,J=8Hz);

[0326] Fig.21 As shown, 13 C-NMR(100MHz,DMSO-D6):176.9,168.0,159.3,157.9,153.5,146.2,135.1,133 .4,133.0,128.9,127.6,125.1,109.9,70.9,56.3,45.5,29.8,26.5,21.3,21.1;

[0327] Fig. 22 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 29 H 27 Cl2N3O5:567.1328,Found:568.1412

[0328] Example 10: Synthesis of Compound TM-10

[0329] Synthesis of methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate

[0330]

[0331] In a 100mL three-necked flask, nitrogen was used to protect the solution. 4-(5-bromopyrazine-2-methyleneoxy)-5-phenyl-3-(2,6-dichlorophenyl)isoxazole (1.03g, 2.3mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and poured into the reaction bottle. Then, ethanol and liquid nitrogen were added into a 500mL low-temperature Dewar flask to lower the temperature to -78°C. n-Butyl lithium (1.7ml, 2.7mmol) was slowly added dropwise. After stirring for 10min, a solution of methyl 3-methyl-5-(3-oxocyclobutyl)benzoate (0.55g, 2.5mmol) dissolved in tetrahydrofuran (10mL) was slowly added dropwise. The reaction was carried out at -78°C for 2h and then the temperature was raised to room temperature for overnight reaction. After the reaction was completed, the reaction solution was slowly poured into a mixture of ice and water, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered, and the organic solvent was distilled off under reduced pressure. The product was separated and purified by gradient elution with silica gel column chromatography (PE: EA = 10: 1, v / v) to obtain 496 mg of a white solid (methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate) with a yield of 35%.

[0332] Synthesis of 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid

[0333]

[0334] Methyl 3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoate (123 mg, 0.2 mmol, 1 eq) was dissolved in 20 mL of THF and a solution of LiOH·H2O (35 mg, 0.8 mmol, 4.2 eq) in water (5 mL) was stirred at 35°C overnight. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, ethyl acetate was added for extraction three times, the mixture was dried over anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and the product was separated and purified by high pressure preparative liquid chromatography using a Waters XBridge C18 column (150nm*4.6nm*3.5um), acetonitrile and water as the mobile phase, the flow rate was 18mL / min, and the fractions with a gradient of 45%-75% were collected. The product was concentrated to remove most of the acetonitrile, and the product was freeze-dried with a freeze dryer to obtain a white powdery solid (3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3-hydroxycyclobutyl)-5-methylbenzoic acid), 37 mg, with a yield of 31%.

[0335] Fig.23 As shown, 1 H-NMR (400MHz, DMSO-D6): δ12.89 (1H, s), 8.14 (1H, d, J = 1.6Hz), 8.09 (1H, d, J = 1.6Hz), 7.99-7.96 (3H, m), 7.73 (1H, s), 7.67-7.65 (5H ,m),7.61-7.57(2H,m),7.37(1H,s),6.06(1H,s),5.38(2H,s),3.33-3.28(1H,m),2.91-2.86(2H,m),2.45-2.40(2H,m),2.35(3H,s);

[0336] Fig.24 As shown, 13 C-NMR (100MHz, DMSO-D6):168.4,168.0,160.4,157.8,153.7,146.2,135.2,133. 3,131.6,130.0,129.0,127.7,127.2,125.1,111.5,70.9,57.0,45.5,29.8,21.3;

[0337] Fig.25 As shown, ESI-MS: m / z[M+H]+: Calcd.for C 32 H 25 Cl2N3O5:601.1171,Found:602.1249.

Claims

1. A compound having the structure of formula (I), or a pharmaceutically acceptable salt thereof: in, R1 is -COOH; R2 is selected from: R3 is selected from: -H or -CH3; X is C or N.

2. The compound according to claim 1, characterized in that Selected from the following compounds or their pharmaceutically acceptable salts 3. A pharmaceutical composition, characterized in that The compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof is used as an active ingredient.

4. The pharmaceutical composition according to claim 3 may further contain a pharmaceutically acceptable carrier as required.

5. Use of the compound according to any one of claims 1 to 2 in the preparation of a medicament for treating non-alcoholic fatty liver disease. The use according to claim 5 , wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.

7. Use of the pharmaceutical composition according to claim 3 in preparing a medicament for treating non-alcoholic fatty liver disease.

8. The use according to claim 7, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.

Images