WO2022134146A1 - 一种新型吡嗪结构fxr激动剂、制备方法及应用 - Google Patents

一种新型吡嗪结构fxr激动剂、制备方法及应用 Download PDF

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WO2022134146A1
WO2022134146A1 PCT/CN2020/140588 CN2020140588W WO2022134146A1 WO 2022134146 A1 WO2022134146 A1 WO 2022134146A1 CN 2020140588 W CN2020140588 W CN 2020140588W WO 2022134146 A1 WO2022134146 A1 WO 2022134146A1
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dichlorophenyl
group
mmol
methyl
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French (fr)
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马维维
王国成
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江苏天士力帝益药业有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the invention relates to a preparation method of a pharmaceutical compound, in particular to a novel FXR agonist with a pyrazine structure, a preparation method and an application.
  • FXR agonists can be divided into steroids and non-steroids according to their structures.
  • the steroids are mainly chenodeoxycholine (chenodeoxycholic acid, CDCA, one of the bile acids) and its derivatives and the FXR agonist MFA-1 developed by Merck; non-steroids include isoxazoles GW4064 and its derivatives. Analogs, Fexaramine compounds, azaindole compounds XL335 and its derivatives, benzimidazolyl amide compounds, pyrazolidinedione compounds, etc.
  • Non-alcoholic fatty liver disease is a group of patients with no history of excessive alcohol consumption, liver histological changes similar to alcoholic liver disease, and a clinicopathological syndrome characterized by hepatic parenchymal steatosis and fat storage. Symptoms, including simple fatty liver and its evolution from steatohepatitis and cirrhosis. Now NAFLD has become the second type of chronic liver disease after hepatitis B that affects human health. NASH is a chronic progressive liver disease caused by the accumulation of fat in the liver, which can lead to liver cirrhosis, liver failure and hepatocellular carcinoma. To be precise, NASH is only a stage of the development of non-alcoholic fatty liver disease (NAFLD).
  • obeticholic acid is an FXR agonist. Approved by the FDA in May 2016 for the treatment of primary bile acid cirrhosis (PBC), it is also the first NASH drug to enter Phase III clinical trials. Positive results from the interim analysis of the pivotal Phase III REGENERATE study of obeticholic acid in NASH patients with grade 2-3 liver fibrosis.
  • PBC primary bile acid cirrhosis
  • FXR agonists such as PX-104 have entered Phase II clinical trials, and the main indication is also non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • These compounds are improvements on the basis of the compounds disclosed in WO2011/020615, by replacing the 1,3-cyclobutylene or 1,3-azetidinyl group of 1,2-cyclopropylene to introduce polar achieved by the hydroxyl group.
  • the results show that the resulting compound retains activity at the FXR receptor and exhibits improved physicochemical properties, such as higher water solubility and/or membrane permeability. While better water solubility and membrane permeability lead to higher oral bioavailability.
  • the above compounds have relatively low activating activity on the farnesoid nuclear receptor FXR target.
  • the present invention provides a compound having the structure of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, pharmaceutically acceptable salt or a single resolved isomer thereof :
  • R 1 is selected from: halogen, -COOH,
  • R 2 is selected from: C 1 -C 6 hydrocarbon group, cyclic hydrocarbon group, aryl group, substituted hydrocarbon group or substituted aryl group;
  • R 3 is selected from: -H or C 1 -C 3 hydrocarbyl, cyclic hydrocarbyl, and substituted hydrocarbyl;
  • X is C or N.
  • R 1 is selected from -Br
  • R 2 is selected from C 1 -C 3 hydrocarbon group, cyclic hydrocarbon group
  • R 3 is selected from -CH 3 ;
  • X is C or N.
  • R 2 is selected from
  • the present invention further provides pharmaceutical compositions containing said compounds as active ingredients.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier as required.
  • the present invention further provides a method for preparing the compound, comprising the steps of:
  • the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
  • composition of the present invention in preparing a medicine for treating non-alcoholic fatty liver disease, preferably non-alcoholic steatohepatitis.
  • the compounds of the present invention include all isomeric forms and isomeric mixtures thereof. It can also exist in the form of a solvate.
  • the pharmaceutical composition of the present invention preferably in the form of a unit dose pharmaceutical preparation, can be made into any pharmaceutically acceptable dosage form when it is 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, buccal preparations, granules, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, patch. Oral formulations are preferred, and tablets and capsules are most preferred.
  • the pharmaceutical preparation can be prepared by conventional techniques of pharmacy, and the pharmaceutically acceptable carriers include but are not limited to: mannitol, sorbitol, sorbic acid or potassium salt, sodium metabisulfite, sodium hydrogen sulfite, sodium thiosulfate, Cysteine hydrochloride, thioglycolic acid, methionine, vitamin A, vitamin C, vitamin E, vitamin D, azone, disodium EDTA, calcium sodium EDTA, monovalent alkali metal carbonate, acetate, phosphate or Its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, fumaric acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, silicon Derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone,
  • a unit dose of the medicament may contain 0.1-1000 mg of the active pharmaceutical substance of the present invention, and the rest are pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier can be 0.1-99.9% by weight of the total weight of the formulation.
  • the dosage of the pharmaceutical composition of the present invention is determined according to the condition of the patient during use.
  • the preferred preparation method of the present invention comprises the following steps:
  • the synthetic method of isoxazole intermediate is as follows: benzaldehyde 1a is condensed with hydroxylamine hydrochloride under alkaline conditions to obtain benzaldehyde oxime 1b, and oxime 1b generates chlorobenzaldehyde oxime 1c under NCS/DMF conditions; The trisubstituted isoxazole ring 1d-1f was obtained by 1,3-dipolar cycloaddition; DIBAL-H was added under nitrogen protection, and the ester bond was reduced to hydroxyl to obtain the intermediate 1-a-1-c;
  • the compounds of the present invention have obvious reducing effects on liver triglyceride and cholesterol levels, increase liver weight and liver weight ratio in mice, and reduce pathological scores and liver Collagen deposition in non-alcoholic fatty liver has improved effect.
  • the compounds of the present invention have strong agonistic activity on the farnesoid nuclear receptor FXR target, show high permeability between cell membranes, and in a high-fat feeding mouse model, have a strong effect on fat enrichment.
  • Liver weight shows a dose-dependent effect, and it is predicted to show good use in the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver (NASH).
  • metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver (NASH).
  • Test Example 1 Study on the agonistic activity of the compounds of the present invention on FXR receptors
  • FXR (it is GST-labeled recombinant human FXR protein, manufacturer Invitroren, product number PV4835) and SRC-1 (steroid receptor coactivator-1) were thawed on ice, and three solutions of ABC were prepared with buffer, A Solution, 0.4nM FXR and 30nM SRC-1; Solution B, 10ug/ml Acceptor Beads (acceptor beads); Solution C, 10ug/ml Donor Beads (donor beads). Add solution A to the plate, 15uL per well. Incubate for 1 hour at room temperature. Add solution B to the plate, 7.5uL per well. Incubate for 1 hour at room temperature. Add solution C to the plate, 7.5uL per well. Incubate for 1 hour at room temperature. Microplate reader Envision reading. Data were curve fitted with Prism 5.0 and EC50 was calculated. The results are shown in Table 1.
  • Cell culture a. Trypsinize dishes and seed cells in 10 ml complete trypsin at appropriate density at 37°C. b. Cells were cultured for 24 hours under 5% CO 2 and humid conditions.
  • cytoplasm Dilute the cytoplasm at a density of 500,000 cells/ml to the desired volume (100 ul/well for a 96-well plate).
  • Dual Luciferase Assay Firefly and Renilla luciferase signals were analyzed by Promega's Dual Luciferase Reporter Assay System. Envision is used as a photometer.
  • X is the "F/R" value for each concentration point.
  • the minimum value is the mean “F/R” value of the no compound control.
  • the maximum value is the mean “F/R” value of the reference compound control.
  • test compounds on FXR-TR-FRET were studied. Compared with the control substances GW4064 and M3, most of these compounds containing pyrazine structure showed better agonistic activity at the cellular level.
  • the human-derived colon adenocarcinoma cell line Caco-2 monolayer cell model was used to study the bidirectional transport of target compounds from the apical side (apical, AP) to the basolateral side (basolateral, BL) and from the BL side to the AP side. Quantitative analysis by high performance liquid chromatography, calculation of transport parameters, apparent permeability coefficient (Papp) and efflux ratio (efflux ratio), with M3 as the positive control and P-gp substrate digoxin as the reference The five samples with higher intracellular activity were selected for testing to predict the in vivo oral bioavailability and affinity with P-gp of the isoxazole derivatives of this pyrazine structure.
  • Papp apparent permeability coefficient
  • efflux ratio efflux ratio
  • Table 4 shows the recovery rate of these compounds containing pyrazine structure after permeation through the membrane. The two-way transport of these five compounds was evaluated, and the results are shown in Table 5. It can be seen from the efflux rate that compared with M3, these derivatives have greatly weakened the efflux. The efflux rate is much lower than that of the reference substance digoxin (efflux rate>262.93), and it is predicted that the oral absorption in the body will also be improved accordingly.
  • TM-08 with poor effect was removed, and other compounds were retained for animal experiments.
  • M3 was excluded because of its poor membrane permeation effect.
  • GW4064 is currently excluded as a tool molecule for preliminary research due to its reported poor drugability and low oral bioavailability.
  • M2 was also used as a control compound in the early stage, and its activity was lower than that of the compound in this patent in the first protein level screening and was excluded.
  • Obeticholic acid is currently a drug under clinical research in the field of non-alcoholic fat, and it is close to being approved, and is selected as a reference substance for animal testing.
  • Test Example 4 Efficacy test of high-fat feed (MCD) simulating non-alcoholic fatty liver disease
  • Obeticholic acid is currently a drug under clinical research in the field of non-alcoholic fat, and it is close to being approved, and is selected as a reference substance for animal testing.
  • Animal strain C57/BL6, animal grade: SPF, gender: male, animal age: 8 weeks old, animal acceptance date: September 28, 2018, animal source: Shanghai Lingchang Biotechnology Co., Ltd., animal certificate No.: SCXK (Shanghai) 2013-0018 2013001836799, mouse rearing environment: temperature 20-26 °C, humidity 40-70%, 12 hours day and night cycle.
  • Solutol HS15/physiological saline After Solutol HS15 (polyethylene glycol 15 hydroxystearate solubilizer) was dissolved in a water bath at 37°C, 5 ml was dissolved in 100 ml of physiological saline, fully stirred and used for later use.
  • Solutol HS15 polyethylene glycol 15 hydroxystearate solubilizer
  • Obeticholic acid/5% Solutol HS15/physiological saline Accurately weigh 12 mg of obeticholic acid, add 0.15 ml of Solutol HS15 to fully dissolve it, add 2.85 ml of physiological saline, vortex vigorously, and ultrasonically aid solubilization to fully dissolve it.
  • TM-01 group/5%Solutol HS15/physiological saline Take 2.5ml of 6mg/ml TM-01 suspension, add 2.5ml of 5%Solutol HS15/physiological saline, and mix well.
  • TM-02 group/5%Solutol HS15/physiological saline Take 2.5ml of 6mg/ml TM-02 suspension, add 2.5ml of 5%Solutol HS15/physiological saline, and mix well.
  • TM-06 group/5%Solutol HS15/physiological saline Take 2.5ml of 6mg/ml TM-06 suspension, add 2.5ml of 5%Solutol HS15/physiological saline, and mix well.
  • TM-09 group/5% Solutol HS15/physiological saline Take 2.5ml of 6mg/ml TM-09 suspension, add 2.5ml of 5% Solutol HS15/physiological saline, and mix well.
  • the vehicle and the test substance were administered by gavage, and the administration volume was 10ml/kg;
  • mice After 70 8-week-old C57/BL6 mice arrived in the animal room, they were fed adaptively. After the average body weight reached 23 g, the mice were randomly divided into 7 groups according to their body weight and replaced with model feed. The first group was given the control diet MCS, the remaining groups were given the MCD diet, and the mice in each group received compound treatment in the following manner:
  • Control group MCS, vehicle, intragastric administration once a day;
  • Group 2 model group MCD, vehicle, intragastric administration once a day;
  • Obeticholic acid group MCD, 40mpk obeticholic acid, administered by gavage once a day;
  • TM-01 group MCD, 30mpk TM-01, intragastric administration once a day;
  • TM-02 group MCD, 30mpk TM-02, intragastric administration once a day;
  • TM-06 group MCD, 30mpk TM-06, administered by gavage once a day;
  • TM-09 group MCD, 30mpk TM-09, administered by gavage once a day;
  • TG triglyceride
  • TC total cholesterol
  • HDL high density lipoprotein
  • LDL low density lipoprotein
  • AST aspartate aminotransferase
  • Determination of ALT alanine aminotransferase
  • AST and ALT after 3 weeks and 4 weeks of administration were measured according to the kit instructions; blood lipid indexes after 4 weeks of administration were sent to Adicon Medical Laboratory Co., Ltd. for detection.
  • 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 prior to assay.
  • mKC and MCP1 The determination of mKC and MCP1 was carried out according to the kit instructions.
  • liver After the liver was taken out from -80°C, it was homogenized in PBS (phosphate-buffered saline), extracted with chloroform-methanol organic phase, and then TC and TG were measured with a kit, which was normalized with the amount of protein.
  • PBS phosphate-buffered saline
  • mice were anesthetized, and blood was collected from the heart, and the liver was isolated and weighed.
  • the right lobe was quickly frozen with liquid nitrogen and stored at -80°C for liver lipid analysis.
  • the left lobe was isolated and fixed in 10% formalin for subsequent HE and Sirius Red staining.
  • test results are expressed as mean ⁇ standard error (Mean ⁇ SEM), and T-Test is used for significance analysis.
  • ***p ⁇ 0.001 is a very significant difference.
  • mice were fed with MCD After the mice were fed with MCD, the body weight dropped significantly as expected. And over time, the weight continued to drop. Compared with the model group, the body weight of each experimental group treated with the compound also decreased slightly (Table 6).
  • ALT in the model group was increased by about 3 times compared with the control group, and AST was increased by more than 2 times, and the obeticholic acid group resulted in higher AST and ALT levels than the model group.
  • ALT and AST of TM-01 group, TM-06 group and TM-09 group were significantly decreased, and the performance of TM-02 group was consistent with that of obeticholic acid group.
  • livers were collected for lipid determination, and liver lipid content was normalized with protein.
  • liver triglyceride and cholesterol were significantly higher than those in the control group.
  • both indexes decreased slightly.
  • TM-01 group, TM-02 group, TM-06 group and TM-09 group had different reducing effects on the content of liver triglyceride (liver TC) and total cholesterol (liver TG), among which TM-01 group and TM The -09 group showed significantly lower levels. (Table 10).
  • livers were harvested and weighed.
  • the liver weight and liver weight ratio of mice in the model group were significantly lower than those in the control group.
  • TM-01 group, TM-02 group, TM-06 group and TM-09 group all significantly increased the liver weight (liver weight) and liver weight of mice.
  • Liver body weight ratio ie liver/body weight). (Table 11).
  • HE and Sirius Red staining were performed. After staining, the whole slide was scanned. Randomly select 6 20x fields of view. Six 20x fields of HE staining were comprehensively scored for pathology (ie, HE score). The pathological scoring criteria are shown in Table 12. Six 20x field images of Sirius Red staining were used to calculate the ratio of Sirius Red positive staining area to total area (i.e. Sirius red staining area) using Image J.
  • adipocytes accumulated in the liver of the model group, and inflammatory cell infiltration was occasionally seen. Adipocyte accumulation and inflammatory cell infiltration were more pronounced after obeticholic acid treatment.
  • the pathological score showed that after MCD feeding, the pathological score of the model group was around 2, the score was increased after obeticholic acid treatment, and the pathological condition was alleviated to a certain extent after compound treatment (Table 13).
  • mice for 4 weeks induced the increase of AST, ALT and lipid in the blood, which promoted the accumulation of fat in the liver and the generation of inflammatory foci, modeling various indicators of non-alcoholic fatty liver disease feature.
  • the compounds provided by the present invention can obviously reduce the levels of triglyceride and cholesterol in the liver, increase the ratio of liver weight and liver weight in mice, and reduce the pathological score and liver to a certain extent. It is preliminarily judged that it has an improvement effect on non-alcoholic fatty liver in animals.
  • Fig. 1 is the 1 H-NMR spectrum of the compound TM-1 of the present invention.
  • Fig. 2 is the mass spectrum of the compound TM-1 of the present invention.
  • Fig. 3 is the 1 H-NMR spectrum of the compound TM-2 of the present invention.
  • Fig. 4 is the mass spectrum of the compound TM-2 of the present invention.
  • Fig. 5 is the 1 H-NMR spectrum of the compound TM-3 of the present invention.
  • Fig. 6 is the 13 C-NMR spectrum of the compound TM-3 of the present invention.
  • Fig. 7 is the mass spectrum of the compound TM-3 of the present invention.
  • Fig. 8 is the 1 H-NMR spectrum of the compound TM-4 of the present invention.
  • Fig. 9 is the mass spectrum of the compound TM-4 of the present invention.
  • Figure 10 is the 1 H-NMR spectrum of the compound TM-5 of the present invention.
  • Figure 11 is the mass spectrum of the compound TM-5 of the present invention.
  • Figure 12 is the 1 H-NMR spectrum of the compound TM-6 of the present invention.
  • Figure 13 is the 13 C-NMR spectrum of the compound TM-6 of the present invention.
  • Figure 14 is the mass spectrum of the compound TM-6 of the present invention.
  • Figure 15 is the 1 H-NMR spectrum of the compound TM-7 of the present invention.
  • Figure 16 is the mass spectrum of the compound TM-7 of the present invention.
  • Figure 17 is the 1 H-NMR spectrum of the compound TM-8 of the present invention.
  • Figure 18 is the 13 C-NMR spectrum of the compound TM-8 of the present invention.
  • Figure 19 is the mass spectrum of the compound TM-8 of the present invention.
  • Figure 20 is the 1 H-NMR spectrum of the compound TM-9 of the present invention.
  • Figure 21 is the 13 C-NMR spectrum of the compound TM-9 of the present invention.
  • Figure 22 is the mass spectrum of the compound TM-9 of the present invention.
  • Figure 23 is the 1 H-NMR spectrum of the compound TM-10 of the present invention.
  • Figure 24 is the 13 C-NMR spectrum of the compound TM-10 of the present invention.
  • Figure 25 is a mass spectrum of the compound TM-10 of the present invention.
  • the synthesis method is as follows:
  • reaction solution was diluted with water and extracted with ethyl acetate.
  • reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate, the organic phase was washed with saturated brine (200 ml), dried over anhydrous magnesium sulfate, filtered with suction, distilled under reduced pressure to remove the organic solvent, and subjected to silica gel column chromatography.
  • reaction solution was slowly poured into an ice-water mixture, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered with suction, distilled under reduced pressure to remove the organic solvent, and washed with a gradient of silica gel column chromatography.
  • the organic solvent was distilled off under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, extracted with ethyl acetate for three times, dried with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and separated and purified by high pressure preparative liquid chromatography, using Waters XBridge C18 column (150nm*4.6nm*3.5um), the mobile phase is acetonitrile and water, the flow rate is 18mL/min, the fractions with a gradient of 45%-75% are collected, concentrated to remove most of the acetonitrile, and freeze-dried with a freeze dryer to obtain a white powder Solid (3-(3-(5-(5-isopropyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxyl Cyclobutane) benzoic acid) 126 mg, yield 38%.
  • the organic solvent was distilled off under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, extracted with ethyl acetate for three times, dried with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and separated and purified by high pressure preparative liquid chromatography, using Waters X Bridge C18 Column (150nm*4.6nm*3.5um), the mobile phase is acetonitrile and water, the flow rate is 18mL/min, the fractions with a gradient of 45%-75% are collected, concentrated to remove most of the acetonitrile, and freeze-dried with a freeze dryer to obtain a white powder (3-(3-(5-(5-phenyl-3-(2,6-dichlorophenyl)isoxazole-4-methyleneoxy)-2-pyrazine)-1-hydroxyl cyclobutane) benzoic acid) 39 mg, yield 33%.
  • methyl 3-methyl-5-vinylbenzoate (5.46 g, 31 mmol, 1 eq) was dissolved in diethyl ether (150 mL).
  • Zinc powder (6g, 93mmol, 3eq) was added, and after sonication for 30min, a solution of trichloroacetyl chloride (8.7mL, 77.5mmol, 2.5eq) in Et2O (50mL) was added dropwise, and the sonication was continued for 30 minutes. The mixture was heated to 35°C. Continuously sonicated for 2.5 h, cooled to room temperature after the reaction was completed, and quenched by slowly adding water (50 mL).
  • the organic solvent was distilled off under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, extracted with ethyl acetate for three times, dried with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and separated and purified by high pressure preparative liquid chromatography, using Waters XBridge C18 column (150nm*4.6nm*3.5um), the mobile phase is acetonitrile and water, the flow rate is 18mL/min, the fractions with a gradient of 45%-75% are collected, concentrated to remove most of the acetonitrile, and freeze-dried with a freeze dryer to obtain a white powder Solid 3-(3-(5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)pyrazin-2-yl)-3 -Hydroxycyclobutyl)-5-methylbenzoic acid, 37 mg, 33% yield.
  • the organic solvent was distilled off under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, extracted with ethyl acetate for three times, dried with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and separated and purified by high pressure preparative liquid chromatography, using Waters X Bridge C18 Column (150nm*4.6nm*3.5um), the mobile phase is acetonitrile and water, the flow rate is 18mL/min, the fractions with a gradient of 45%-75% are collected, concentrated to remove most of the acetonitrile, and freeze-dried with a freeze dryer to obtain a white powder as solid (3-(3-(5-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)pyrazin-2-yl) -3-hydroxycyclobutyl)-5-methylbenzoic acid), 46 mg, 40% yield.
  • 13 C-NMR (100 MHz, 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;
  • reaction solution was slowly poured into an ice-water mixture, extracted with ethyl acetate, the ester layer was washed with water (100 mL), dried over anhydrous magnesium sulfate, filtered with suction, distilled under reduced pressure to remove the organic solvent, and washed with a gradient of silica gel column chromatography.
  • the organic solvent was distilled off under reduced pressure, the pH was adjusted to 5 with 1N hydrochloric acid, extracted with ethyl acetate for three times, dried with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and separated and purified by high pressure preparative liquid chromatography, using Waters XBridge C18 column (150nm*4.6nm*3.5um), the mobile phase is acetonitrile and water, the flow rate is 18mL/min, the fractions with a gradient of 45%-75% are collected, concentrated to remove most of the acetonitrile, and freeze-dried with a freeze dryer to obtain a white powder Solid (3-(3-(5-((3-(2,6-dichlorophenyl)-5-phenylisoxazol-4-yl)methoxy)pyrazin-2-yl)-3 -Hydroxycyclobutyl)-5-methylbenzoic acid), 37 mg, 31% yield.

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Abstract

一种新型吡嗪结构FXR激动剂、其制备方法及应用，具体涉及一种如下通式（I）的化合物，其水合物、溶剂化物、药学上可接受的盐或其拆分的单一异构体，其具有治疗非酒精性脂肪肝的作用。

Description

一种新型吡嗪结构FXR激动剂、制备方法及应用 技术领域
本发明涉及一种药物化合物的制备方法，特别涉及一种新型吡嗪结构FXR激动剂、制备方法及应用。
背景技术
自1999年发现胆汁酸能激活FXR产生多种生理功能以来，具有选择性和高活性的FXR激动剂相继被发现，这些FXR激动剂按结构可分为甾体类和非甾体类。甾体类主要为鹅脱氧胆碱(chenodeoxycholic acid，CDCA，胆汁酸其中一种)及其衍生物和Merck公司开发的FXR激动剂MFA-1；非甾体类包括异噁唑类化合物GW4064及其类似物，Fexaramine类化合物，氮杂并吲哚类化合物XL335及其衍生物，苯咪唑基酰胺类化合物，吡唑啶二酮类化合物等。
非酒精性脂肪性肝病(non-alcoholic fatty liver disease NAFLD)是一组无过量饮酒史，肝组织学改变与酒精性肝病相类似，以肝实质细胞脂肪变性和脂肪贮积为特征的临床病理综合征，包括单纯性脂肪肝以及由其演变的脂肪性肝炎和肝硬化。现今NAFLD已成为仅次于乙肝的影响人类健康的第二类慢性肝脏疾病。NASH是一种肝内脂肪积聚而导致的慢性进展性肝病，可导致肝硬化、肝衰竭及肝细胞癌，确切的说，NASH只是非酒精性脂肪性肝病(NAFLD)病程发展的一个阶段。
临床在研药物中，奥贝胆酸是一种FXR激动剂。在2016年5月被FDA批准，用于治疗原发性胆汁酸肝硬化(PBC)，也是第一个进入III期临床的NASH药物。奥贝胆酸针对伴有2～3级肝纤维化的NASH患者的关键III期REGENERATE研究的期中分析取得积极结果。
其他的FXR激动剂如PX-104已经进入Ⅱ期临床，主要适应症也是非酒精性脂肪肝病(NAFLD)。
Figure PCTCN2020140588-appb-000001
2000年Maloney等报道了第一个具有高活性和高选择性的异噁唑类FXR激动剂GW4064，其胞外活性为EC 50＝15nmol·L -1，在细胞水平的EC 50值为90nmol·L -1，可以完全激动FXR靶点蛋白；但在药代动力学方面，在t1/2＝3.5小时的口服利用度只有10％，不具备成药的条件，所以作为研究FXR功能和相关疾病的工具化合物。随后GSK、Novartis、Roche、Lilly、Phenex等公司分别对GW4064的结构进行了改造，以求得到活性更高，口服利用度更好，更具成药性质的新化合物。到目前为止，数目众多的异噁唑类化合物被成功合成，但在活性、水溶性及口服生物利用度等成药性方面均有不足，
Figure PCTCN2020140588-appb-000002
中国专利CN103702719A公开了新型FXR(NR1H4)结合及活性调节化合物，其中具体公开了如下化合物：
Figure PCTCN2020140588-appb-000003
以及这些化合物在治疗非酒精性脂肪肝病(NAFLD)或非酒精性脂肪性肝炎(NASH)等疾病中的应用。这些化合物是在WO2011/020615公开的化合物的基础上进行的改进，通过替换1,2-环亚丙基的1,3-环亚丁基或1,3-亚氮杂环丁烷基上引入极性羟基实现的。结果表明，所得化合物保持了在FXR受体上的活性，而且显现出改进的物化性能，例如较高的水溶性和/或膜渗透性。而更好的水溶性和膜渗透性导致更高的口服生物利用度。但是，上述化合物在法尼醇核受体FXR靶点的激活活性相对较低。
发明内容
为了解决上述技术问题，本发明提供一种具有式(I)结构的化合物，或其药学上可接受的盐，水合物、溶剂化物、药学上可接受的盐或其拆分的单一异构体：
Figure PCTCN2020140588-appb-000004
其中，R 1选自：卤素、-COOH、
Figure PCTCN2020140588-appb-000005
R 2选自：C 1-C 6的烃基、环烃基、芳香基、取代烃基或取代芳香基；
R 3选自：-H或C 1～C 3的烃基、环烃基、取代烃基；
X为C或N。
优选的，其中，
R 1选自-Br；
R 2选自C 1-C 3的烃基、环烃基；
R 3选自-CH 3；
X为C或N。
更优选的，其中，
R 2选自
Figure PCTCN2020140588-appb-000006
最优选的选自以下化合物为：
Figure PCTCN2020140588-appb-000007
Figure PCTCN2020140588-appb-000008
本发明进一步提供以所述的化合物作为活性成分的药物组合物。
所述的药物组合物，根据需要，还可含有药学上可接受的载体。
本发明进一步提供所述的化合物的制备方法，包括如下步骤：
Figure PCTCN2020140588-appb-000009
Figure PCTCN2020140588-appb-000010
本发明所述的化合物在制备治疗非酒精性脂肪肝的药物中的用途。
其中所述非酒精性脂肪肝，为非酒精性脂肪肝炎。
本发明所述药物组合物在制备治疗非酒精性脂肪肝的药物中的用途，优选非酒精性脂肪肝炎。
本发明所述化合物包括其所有异构体形式和异构体混合物的形式。也可以以溶剂化物的形式存在。
本发明的药物组合物，优选的是单位剂量的药物制剂形式，在制成药物制剂时可以制成任何可药用的剂型，这些剂型选自：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、混悬剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、贴剂。优选的是口服制剂形式，最佳优选的是片剂，胶囊剂。
可以采用制剂学常规技术制备该药物制剂，所述药学上可接受的的载体包括但不限于：甘露醇、山梨醇、山梨酸或钾盐、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素A、维生素C、维生素E、维生素D、氮酮、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、富马酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、丙二醇、乙醇、吐温60-80、司盘-80、蜂蜡、羊毛脂、液体石蜡、十六醇、没食子酸酯类、琼脂、三乙醇胺、碱性氨基酸、尿素、尿囊素、碳酸钙、碳酸氢钙、聚乙二醇、环糊精(如β-环糊精)、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
本发明的药物组合物，在制成药剂时，单位剂量的药剂可含有本发明的药物活性物质0.1-1000mg，其余为药学上可接受的载体。药学上可接受的载体以重量计可以是制剂总重量的0.1-99.9％。
本发明的药物组合物在使用时根据病人的情况确定用法用量。
本发明优选的制备方法包括以下步骤：
1)异噁唑中间体合成方法为，苯甲醛1a与盐酸羟胺在碱性条件下缩合得到苯甲醛肟1b，肟1b在NCS/DMF条件下生成氯代苯甲醛肟1c；接着与酮酸酯通过1,3-偶极环加成反应得到三取代异噁唑环1d-1f；氮气保护下加入DIBAL-H，还原酯键为羟基，得到中间体1-a-1-c；
2)异噁唑中间体与二溴吡嗪在碱性条件下发生亲核取代反应，得到吡嗪中间体2-a-2-c；控制温度保持在零下78℃，用正丁基锂进行锂溴交换与四元环2a-d进行碳-碳键的连接，生成末端酯基产物3-a-3-g，最后碱性条件下发生酯水解反应得到目标化合物TM。
本发明的有益效果：本发明的化合物尤其是TM-01和TM-09对肝脏甘油三酯和胆固醇水平有明显的降低作用，提高了小鼠肝重和肝脏体重比，降低了病理评分和肝脏中的胶原沉积，对非酒精性脂肪肝有改善效果。
本发明的化合物对法尼醇核受体FXR靶点的具有较强激动活性，并表现出了在细胞膜间的高通透性，且在高脂饲喂小鼠模型中，对脂肪富集的肝脏重量呈现剂量依赖影响，预测在肥胖、糖尿病和非酒精性脂肪肝(NASH)等代谢类疾病的治疗中表现出较好的用途，以下通过实验数据说明本发明的有益效果。
试验例一、本发明化合物对FXR受体的激动活性研究
实验步骤：FXR(是GST标记的重组人FXR蛋白，厂家Invitroren，货号PV4835)和SRC-1(类固醇受体共激活因子-1)在冰上进行融化，用缓冲液配制ABC三种溶液，A液，0.4n M FXR和30nM SRC-1；B液，10ug/ml Acceptor Beads(受体微珠)；C液，10ug/ml Donor Beads(供体微珠)。将A液加入到板中，每孔15uL。室温孵育1小时。将B液加入到板中，每孔7.5uL。室温孵育1小时。将C液加入板中，每孔7.5uL。室温孵育1小时。酶标仪Envision读数。数据用Prism 5.0进行曲线拟合，计算出EC 50。结果见表1。
表1
Figure PCTCN2020140588-appb-000011
Figure PCTCN2020140588-appb-000012
Figure PCTCN2020140588-appb-000013
根据表1中的样品活性结果，去除活性较差的去除TM-05、TM-07、TM-10，选择其余7个样品进行了细胞内FXR-TR-FRET实验。
试验例二、细胞内FXR-TR-FRET实验
1、细胞培养：a、用适当的密度在10ml完整的胰蛋白酶中胰蛋白酶消化皿和种子细胞中在37℃。b、5％的CO 2和潮湿条件下培养细胞24小时。
2、细胞接种和转染：FuGENE HD转染试剂用作转染试剂。
a根据以下流程准备转染混合物：
Figure PCTCN2020140588-appb-000014
b用力敲打试管以混合内容物。将混合物在室温下孵育15分钟。
c用胰蛋白酶消化盘并确定细胞密度。
d将细胞浆以500,000个细胞/ml的密度稀释至所需的体积(对于96孔板，为100ul/孔)。
e将所需体积的先前制备的转染混合物添加到两个细胞浆液中，然后将100ul/孔的细胞浆液分配到测定板上。
f在加湿条件下于37℃，5％CO 2下将测定板孵育24小时。
3、化合物配制：
a、准备FXR工作浓度为10mM的化合物库存，然后在100％DMSO中稀释3倍。
b、将10ul化合物添加到90ul完全培养基中。
c、向每个孔中加入5ul化合物溶液。
d、将板在37℃，5％CO 2和湿润条件下孵育18小时。
4、双重荧光素酶测定：萤火虫和海肾荧光素酶信号通过Promega的Dual Luciferase Reporter Assay System进行分析。Envision用作光度计。
5、结果计算：通过将萤火虫信号除以海肾信号来归一化数据值。“F/R”表示“萤火虫/Renilla”。这种标准化消除了每个孔中不同细胞数量和转染效率的差异。计算％Activation(活性)值。通过以下公式计算％Activation值。
Figure PCTCN2020140588-appb-000015
X是每个浓度点的“F/R”值。最小值是无化合物对照的平均“F/R”值。最大值是参考化合物对照的平均值“F/R”值。
6、活性结果见表2，
表2：活性结果
样品编号 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
对受试化合物对于FXR-TR-FRET的活性进行了研究，此类含吡嗪结构的化合物，相比对照品GW4064和M3，多数表现出了细胞水平上较好的激动活性。
根据FXR-TR-FRET实验结果，去除TM-03和TM-04，对剩余部分化合物进行Caco-2单层细胞膜转运实验。
试验例三、Caco-2单层细胞膜转运实验
利用人源性结肠腺癌细胞系Caco-2单层细胞模型研究目标化合物由绒毛面侧(apical,AP)到基底面侧(basolateral,BL)以及从BL侧到AP侧的双向转运情况，应用高效液相色谱法定量分析，计算转运参数和表观渗透系数(apparent permeabilitycoefficient,Papp)以及外排率(efflux ratio)，以M3为阳性对照，以P-gp的作用底物地高辛为参照物，选取这5个细胞内活性较高的样品进行试验，来预测这类吡嗪结构异噁唑类衍生物的体内口服生物利用度以及与P-gp的亲和作用情况。
结果见表3、4、5
表3：在Caco-2细胞模型中的A-to-B的表观渗透系数
Figure PCTCN2020140588-appb-000016
表4：在Caco-2细胞模型中的透膜质量回收率
Figure PCTCN2020140588-appb-000017
Figure PCTCN2020140588-appb-000018
表5在Caco-2细胞模型中的外排率
Figure PCTCN2020140588-appb-000019
注：a外排率＝Papp B-A/Papp A-B
实验结果如表3所示，本发明的这一系列吡嗪结构异噁唑类化合物A-to-B的Papp值都高于P-gp的底物地高辛(Papp A-to-B<0.04)，优于M3(Papp A-to-B<0.15)，特别是TM-01、TM-02，TM-06和TM-09这四个化合物的Papp，A-to-B值>2.5×10-6cm/s，属于高通透性底物。这些数据表明这类吡嗪结构异噁唑类化合物具有良好的通膜能力，预测在体内的吸收优于M3。
表4中显示的是这些含吡嗪结构化合物的透膜后的回收率情况。对这5个化合物对其双向转运情况进行评价，结果如表5所示，从外排率上可以看到相对于M3来说，这类衍生物都很大程度上减弱了外排情况，其外排率均远远小于对照品地高辛(外排率>262.93)，预测在体内的口服吸收也会相应得到提高。
根据试验例三结果，去除效果较差的TM-08，保留其他化合物进行动物实验，关于对照组：M3因为透膜效果较差，被排除。GW4064因为报道成药性较差，生物口服利用度较低，目前作为前期研究的一个工具分子，被排除。M2也是作为前期一个对照化合物，在首次蛋白水平的筛选中活性低于本专利中化合物，被排除。奥贝胆酸是目前非酒精性脂肪领域处在临床研究的药物，接近获批，选取作为动物试验的对照品。
试验例四：高脂饲料饲喂(MCD)模拟非酒精性脂肪肝的药效试验
奥贝胆酸是目前非酒精性脂肪领域处在临床研究的药物，接近获批，选取作为动物试验的对照品。
1、实验动物
动物品系：C57/BL6，动物等级：SPF级，性别：雄性，动物年龄：8周龄，动物接受日期：2018年09月28日，动物来源：上海灵畅生物科技有限公司，动物合格证号：SCXK(沪)2013-0018  2013001836799，小鼠饲养环境：温度20-26℃，湿度40-70％，12小时昼夜循环。
2、试验设计与方法
2.1药物配置
2.1.1溶媒配制：
5％Solutol HS15/生理盐水：Solutol HS15(聚乙二醇15羟硬脂酸酯增溶剂)在37℃水浴锅中溶化后，取5ml溶于100ml生理盐水中，充分搅拌后备用。
2.1.2给药溶液配制：
奥贝胆酸/5％Solutol HS15/生理盐水：精确称取12mg奥贝胆酸，加入0.15ml Solutol HS15使之充分溶解，加入2.85ml生理盐水，剧烈涡旋，超声助溶使其充分溶解。
TM-01组/5％Solutol HS15/生理盐水：取2.5ml 6mg/ml的TM-01悬浮液，加入2.5ml 5％Solutol HS15/生理盐水，充分混匀。
TM-02组/5％Solutol HS15/生理盐水：取2.5ml 6mg/ml的TM-02悬浮液，加入2.5ml 5％Solutol HS15/生理盐水，充分混匀。
TM-06组/5％Solutol HS15/生理盐水：取2.5ml 6mg/ml的TM-06悬浮液，加入2.5ml 5％Solutol HS15/生理盐水，充分混匀。
TM-09组/5％Solutol HS15/生理盐水：取2.5ml 6mg/ml的TM-09悬浮液，加入2.5ml 5％Solutol HS15/生理盐水，充分混匀。
所有药物给药前新鲜配制。
2.2给药途径及给药容积
溶媒与测试物灌胃给药，给药体积为10ml/kg；
2.3实验过程、分组和具体给药方式
70只8周龄的C57/BL6小鼠到达动物房后，进行适应喂养，待平均体重到达23g后，根据体重将小鼠随机分成7组，并更换成模型饲料。第一组给予对照饲料MCS，其余组给予MCD粮食，同时各组小鼠按以下方式接受化合物处理：
组1对照组：MCS，溶媒，每天一次灌胃给药；
组2模型组：MCD，溶媒，每天一次灌胃给药；
组3奥贝胆酸组：MCD，40mpk奥贝胆酸，每天一次灌胃给药；
组4.TM-01组：MCD，30mpk TM-01，每天一次灌胃给药；
组5.TM-02组：MCD，30mpk TM-02，每天一次灌胃给药；
组6.TM-06组：MCD，30mpk TM-06，每天一次灌胃给药；
组7.TM-09组：MCD，30mpk TM-09，每天一次灌胃给药；
2.4实验方法
在实验过程中，每周测定体重和摄食量。给药21天后，用微量毛细管进行尾尖取血进行AST，ALT测定。给药28天后，终止小鼠，心脏取血，收集肝组织，称重，一部分用液氮速冻用于后续分析，另一部分固定进行病理学分析。
2.4.1血液指标测定
小鼠血液在5000rpm离心10分钟，收集上清，用于TG(甘油三酯)，TC(总胆固醇)，HDL(高密度脂蛋白)，LDL(低密度脂蛋白)，AST(谷草转氨酶)和ALT(谷丙转氨酶)的测定。
给药三周和四周后的AST和ALT按试剂盒说明书进行测定；给药4周后的血液脂质指标送艾迪康医学检验所有限公司检测。
2.4.2血液细胞因子的测定
小鼠血液在5000rpm离心10分钟，收集上清，用于细胞因子(mKC和MCP1)的检测。检测前血清储存于-80℃。
mKC和MCP1的测定按试剂盒说明书进行。
2.4.3肝脏TC和TG的测定
肝脏从-80℃取出后，PBS(磷酸盐缓冲生理盐水)中匀浆，氯仿甲醇有机相抽提后，用试剂盒进行TC和TG的测定，用蛋白量进行归一化。
2.4.4组织收集
给药4周后，麻醉小鼠，心脏取血后，分离肝脏称重，取右侧叶用液氮速冻，保存于-80℃用于肝脏脂质分析。分离左侧叶，于10％福尔马林中固定，用于后续的HE和Sirius Red染色。
2.5结果处理和数据分析
试验结果以均数±标准误(Mean±SEM)表示，用T-Test进行显著性分析。与模型组比较，*表示p<0.05为有显著性差异，**表示p<0.01为有强显著性差异，***p<0.001为有极显著性差异。
3试验结果
3.1化合物对小鼠体重和摄食量的影响
小鼠经MCD喂养后，体重即如预期显著性下降。并且随着时间的延长，体重持续下降。经化合物处理的各实验组较模型组体重也略有下降(表6)。
表6：化合物对小鼠体重的影响
Figure PCTCN2020140588-appb-000020
表7：化合物对小鼠摄食量的影响
Figure PCTCN2020140588-appb-000021
3.2化合物对血液指标的影响
MCD和化合物处理3周后，取尾尖血进行AST和ALT测定。结果表明，模型组的ALT较对照组升高了约3倍，AST升高了2倍以上，奥贝胆酸组导致了比模型组更高的AST和ALT水平。TM-01组、TM-06组和TM-09组的ALT和AST较模型组有显著下降，TM-02组与奥贝胆酸组表现一致。(表8)
MCD和化合物处理4周后，模型组中动物血脂水平略高于对照组。奥贝胆酸处理降低了血液中的胆固醇含量。TM-01组、TM-02组、TM-06组和TM-09组对血脂有不同的降低作用(表9)。
表8：化合物处理3周和4周后AST和ALT变化
Figure PCTCN2020140588-appb-000022
表9：化合物处理4周后血脂水平变化
组别 TG(mmol/L) TC(mmol/L) LDL(mmol/L) HDL(mmol/L)
组1-control 0.52±0.10* 0.77±0.09*** 2.05±0.06*** 0.34±0.03**
组2-model+vehicle 0.60±0.03 1.12±0.08 2.90±0.04 0.60±0.01
组3-obeticholic acid 0.56±0.02 0.86±0.04** 2.80±0.03 0.55±0.01**
组4-TM-01:30mpk 0.53±0.10 0.95±0.44* 2.61±0.26* 0.59±0.11
组5-TM-02:30mpk 0.57±0.06 1.25±0.14 2.93±0.12 0.61±0.02*
组6-TM-06:30mpk 0.59±0.02 0.97±0.16 3.01±0.09* 0.42±0.06***
组7-TM-09:30mpk 0.54±0.04* 0.86±0.09* 2.52±0.07** 0.56±0.02*
3.3化合物对肝脂含量的影响
MCD和化合物处理4周后，收集肝脏测定脂质，肝脂含量用蛋白进行均一化。在模型组中，肝脏甘油三酯和胆固醇显著高于对照组。奥贝胆酸处理后，这两个指标均略有所下降。TM-01组、TM-02组、TM-06组和TM-09组对肝脏甘油三酯(liver TC)和总胆固醇的含量(liver TG)有不同的降低效果，其中TM-01组和TM-09组表现出显著降低水平。(表10)。
表10：化合物处理后肝脏脂含量变化
组别 liver TG(mol/g protein) liver TC(mol/g protein)
组1-control 112.55±8.59*** 21.47±1.29***
组2-model+vehicle 339.42±40.24 44.94±5.50
组3-obeticholic acid 298.46±36.25 35.7±3.38
组4-TM-01:30mpk 246.69±54.09* 34.85±8.20
组5-TM-02:30mpk 307.38±26.30 41.67±5.73
组6-TM-06:30mpk 303.5±46.52 38.23±5.04
组7-TM-09:30mpk 231.73±18.4* 37.67±3.65*
3.4化合物对小鼠肝重的影响
MCD和化合物处理4周后，收集肝脏，称重。模型组小鼠的肝重和肝脏体重比均显著低于对照组。分析结果时意外的发现此类化合物对肝重有明显的影响，其中TM-01组、TM-02组、TM-06组和TM-09组均显著提高了小鼠肝重(liver weight)和肝脏体重比(即liver/body weight)。(表11)。
表11：化合物处理对肝重的影响
Figure PCTCN2020140588-appb-000023
Figure PCTCN2020140588-appb-000024
3.5化合物肝脏病理的影响
小鼠肝脏固定后，进行HE和Sirius Red染色。染色结束后，全片扫描。随机选取6个20x视野。HE染色的6个20x视野综合进行病理评分(即HE score)。病理评分标准如表12。Sirius Red染色的6个20x视野图像用Image J计算Sirius Red的阳性染色面积与总面积比(即Sirius red staining area)。
表12：病理评分标准
Figure PCTCN2020140588-appb-000025
经MCD喂养4周后，模型组肝脏中有脂肪细胞堆积，间或可见炎性细胞浸润。奥贝胆酸处理后，脂肪细胞堆积和炎性细胞浸润更为显著。病理评分显示，MCD喂养后，模型组病理评分在2左右，奥贝胆酸处理后评分有所升高，而化合物处理后，病理状况有一定的减轻(表13)。
表13：病理评分结果
Figure PCTCN2020140588-appb-000026
3.4结论：MCD喂养小鼠4周后，诱导了血液中AST，ALT和脂质的上升，促使脂肪在肝脏中的堆积和炎性灶的生成，建模具有非酒精性脂肪肝的各项指标特征。
本发明提供的化合物尤其是TM-01和TM-09对肝脏甘油三酯和胆固醇水平有明显的降低作用，提高了小鼠肝重和肝脏体重比，并在一定程度上降低了病理评分和肝脏中的胶原沉积，初步判定在动物体内对非酒精性脂肪肝有改善效果。
附图说明
图1为本发明化合物TM-1的 1H-NMR谱图；
图2为本发明化合物TM-1的质谱图；
图3为本发明化合物TM-2的 1H-NMR谱图；
图4为本发明化合物TM-2的质谱图；
图5为本发明化合物TM-3的 1H-NMR谱图；
图6为本发明化合物TM-3的 13C-NMR谱图；
图7为本发明化合物TM-3的质谱图；
图8为本发明化合物TM-4的 1H-NMR谱图；
图9为本发明化合物TM-4的质谱图；
图10为本发明化合物TM-5的 1H-NMR谱图；
图11为本发明化合物TM-5的质谱图；
图12为本发明化合物TM-6的 1H-NMR谱图；
图13为本发明化合物TM-6的 13C-NMR谱图；
图14为本发明化合物TM-6的质谱图；
图15为本发明化合物TM-7的 1H-NMR谱图；
图16为本发明化合物TM-7的质谱图；
图17为本发明化合物TM-8的 1H-NMR谱图；
图18为本发明化合物TM-8的 13C-NMR谱图；
图19为本发明化合物TM-8的质谱图；
图20为本发明化合物TM-9的 1H-NMR谱图；
图21为本发明化合物TM-9的 13C-NMR谱图；
图22为本发明化合物TM-9的质谱图；
图23为本发明化合物TM-10的 1H-NMR谱图；
图24为本发明化合物TM-10的 13C-NMR谱图；
图25为本发明化合物TM-10的质谱图。
具体实施方式
以下结合具体实施例详细地解释本发明，使得本领域技术人员更全面地理解本专利。具体实施例仅用于说明本发明的技术方案，并不以任何方式限定本发明。
1(3-(2,6-二氯苯基)-4-羟甲基-5-环丙基异噁唑)中间体的合成路线如下：
Figure PCTCN2020140588-appb-000027
合成方法如下：
1.1 2,6-二氯苯甲醛肟的合成
Figure PCTCN2020140588-appb-000028
将盐酸羟氨(11g，1eq)和氢氧化钠(6.3g，1.2eq)溶于水中，室温下加入2,6-二氯苯甲醛(25g, 0.14mmol,1.2eq)的乙醇(200mL)溶液，90℃下搅拌1小时，冷却至室温后减压蒸馏除去乙醇，抽滤，水洗涤滤饼(2×100mL)，干燥后得白色固体(2,6-二氯苯甲醛肟)9.46g，收率84％。
1.2 2,6-二氯-N-羟基-氯代苯甲醛肟的合成
Figure PCTCN2020140588-appb-000029
于40℃下，将N-氯代丁二酰亚胺(16.08g,0.12mol,1eq)的DMF(90mL)溶液缓慢滴加到2,6-二氯苯甲醛肟(22.8g,0.12mol,1eq)的DMF(90mL)溶液中，搅拌，TLC监测，反应完全后冷却到室温，将溶液倒入冰水(200mL)中，甲基叔丁基醚萃取3次(3×100mL)，合并有机相后用水(3×100mL)、饱和食盐水(100mL)洗涤。无水硫酸钠干燥酯层后抽滤，减压蒸馏除去有机溶剂得黄色油状粗品，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝5:1,v/v)得白色固体(2,6-二氯-N-羟基-氯代苯甲醛肟)26g，收率97％。
1.3 3-(2,6-二氯苯基)-5-环丙基异噁唑-4-甲酸甲酯的合成
Figure PCTCN2020140588-appb-000030
将3-环丙基-3-氧代丙酸甲酯(637.7mg,4.49mmol,1eq)加入到100mL反应瓶，胶塞密闭，用针管把三乙胺(907.9mg,8.97mmol,2eq)加入到反应瓶内，室温下剧烈搅拌30min，反应液冰浴冷却低于10℃，搅拌下缓慢滴加2,6-二氯-N-羟基-氯代苯甲醛肟(1.0g,4.49mmol,1eq)的乙醇溶液(监测内温＜24℃)，缓慢升至室温，剧烈搅拌过夜，减压蒸馏除去乙醇，加入乙酸乙酯萃取3次(3×100mL)，有机层用水(3×100mL)、饱和食盐水(100mL)洗涤，无水硫酸钠干燥除去溶剂得油状粗品。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝40:1,v/v)得白色固体(3-(2,6-二氯苯基)-5-环丙基异噁唑-4-甲酸甲酯)0.89g，收率55％。
1.4 3-(2,6-二氯苯基)-4-羟甲基-5-环丙基异噁唑的合成
Figure PCTCN2020140588-appb-000031
氮气保护和冰浴条件下将二异丁基氢化铝的甲苯溶液(4.0mL,6.0mmol,2.1eq,1.5M的甲苯溶液)缓慢滴加至3-(2,6-二氯苯基)-5-环丙基异噁唑-4-甲酸甲酯(0.89g,2.8mmol,1eq)的无水THF中，升至室温剧烈搅拌过夜。重新将反应液冷却至0℃，缓慢滴加甲醇(2mL)搅拌10min，反应液倒入50mL冰水混合物中，生成凝胶状悬浮物。硅藻土过滤，用乙酸乙酯(3×100mL)萃取三次，合并酯层，用水(3×100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤除去溶剂得白色固体。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝20:1,v/v)得到白色固体(3-(2,6-二氯苯基)-4-羟甲基-5-环丙基异噁唑)0.45g，收率56％。
2、3-(2,6-二氯苯基)-4-羟甲基-5-异丙基异噁唑的合成
2.1 3-(2,6-二氯苯基)-5-异丙基异噁唑-4-甲酸甲酯的合成
Figure PCTCN2020140588-appb-000032
将异丁酰乙酸甲酯(16.6mL,0.12mol,1eq)加入到100mL反应瓶，胶塞密闭，用针管把三乙胺(33.25mL,0.24mol,2eq)加入到反应瓶内，室温下剧烈搅拌30min，反应液冰浴冷却低于10℃，搅拌下缓慢滴加2,6-二氯-N-羟基-氯代苯甲醛肟(26.6g,0.12mol,1eq)的乙醇溶液(监测内温＜24℃)，缓慢升至室温，剧烈搅拌过夜，减压蒸馏除去乙醇，加入乙酸乙酯萃取3次(3×100mL)，有机层用水(3×100mL)、饱和食盐水(100mL)洗涤，无水硫酸钠干燥除去溶剂得油状粗品。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝40:1,v/v)得白色固体(3-(2,6-二氯苯基)-5-异丙基异噁唑-4-甲酸甲酯)21g，收率56％。
2.2 3-(2,6-二氯苯基)-4-羟甲基-5-异丙基异噁唑的合成
Figure PCTCN2020140588-appb-000033
氮气保护和冰浴条件下将二异丁基氢化铝的甲苯溶液(92mL,0.14mol,2.1eq,1.5Μ的甲苯溶液)缓慢滴加至3-(2,6-二氯苯基)-5-异丙基异噁唑-4-甲酸甲酯(20g,0.06mol,1eq)的无水THF中，升至室温剧烈搅拌过夜。重新将反应液冷却至0℃，缓慢滴加甲醇(20mL)搅拌10min，反应液倒入200mL冰水混合物中，生成凝胶状悬浮物。硅藻土过滤，用乙酸乙酯(3×100mL)萃取三次，合并酯层，用水(3×100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤除去溶剂得白色固体。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝40:1,v/v)得到白色固体(3-(2,6-二氯苯基)-4-羟甲基-5-异丙基异噁唑)18g，收率94％。
3 3-(2,6-二氯苯基)-4-羟甲基-5-苯基异噁唑的合成
3.1 3-(2,6-二氯苯基)-5-苯基异噁唑-4-甲酸乙酯的合成
Figure PCTCN2020140588-appb-000034
将苯甲酰乙酸乙酯(5.7mL,50mmol,1eq)加入到100mL反应瓶，胶塞密闭，用针管把三乙胺(13.86mL,100mmol,2eq)加入到反应瓶内，室温下剧烈搅拌30min，反应液冰浴冷却低于10℃，搅拌下缓慢滴加2,6-二氯-N-羟基-氯代苯甲醛肟(10g,50mmol,1eq)的乙醇溶液(监测内温＜24℃)，缓慢升至室温，剧烈搅拌过夜，减压蒸馏除去乙醇，加入乙酸乙酯萃取3次(3×100mL)，有机层用 水(3×100mL)、饱和食盐水(100mL)洗涤，无水硫酸钠干燥除去溶剂得油状粗品。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝40:1,v/v)得白色固体(3-(2,6-二氯苯基)-5-苯基异噁唑-4-甲酸甲酯)11.7g，收率65％。
3.2 3-(2,6-二氯苯基)-4-羟甲基-5-苯基异噁唑的合成
Figure PCTCN2020140588-appb-000035
氮气保护和冰浴条件下将二异丁基氢化铝的甲苯溶液(18mL,27mmol,2.1eq,1.5M的甲苯溶液)缓慢滴加至3-(2,6-二氯苯基)-5-苯基异噁唑-4-甲酸甲酯(4.72g,13mmol,1eq)的无水THF中，升至室温剧烈搅拌过夜。重新将反应液冷却至0℃，缓慢滴加甲醇(20mL)搅拌10min，反应液倒入200mL冰水混合物中，生成凝胶状悬浮物。硅藻土过滤，用乙酸乙酯(3×100mL)萃取三次，合并酯层，用水(3×100mL)和饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤除去溶剂得白色固体。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝20:1,v/v)得到白色固体(3-(2,6-二氯苯基)-4-羟甲基-5-苯基异噁唑)9.1g，收率70％。
实施例1：化合物TM-1合成路线
Figure PCTCN2020140588-appb-000036
试验步骤：
3-(3-溴苯基)环丁酮
Figure PCTCN2020140588-appb-000037
在-15℃条件下，向N,N-二甲基甲酰胺(2.1g,24.6mmol)的1,2-二氯乙烷(40mL),溶液中，缓慢滴加三氟甲磺酸酐(11.6g,41.0mmol)，在-15℃搅拌30分钟。然后加入3-溴苯乙烯(3.0g,16.4mmol)和2,4,6-三甲基吡啶(2.9g,24.6mmol)，室温搅拌过夜。加水猝灭反应，室温搅拌过夜，加入二氯甲烷稀释分离有机相，分别用水和饱和食盐水(200ml)洗涤有机相，无水硫酸镁干燥，抽滤，减压浓缩，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝15:1,v/v)得到黄色固体3-(3-溴苯基)环丁酮1.3g,产率35％。
3-(3-氧代环丁基)苯甲酸甲酯
Figure PCTCN2020140588-appb-000038
在室温，一氧化碳气球环境中，将三乙胺(2.2g,21.3mmol)加入3-(3-氧代环丁基)苯甲酸甲酯(1.6g,7.1mmol)和(1,1'-双(二苯基膦基)二茂铁)二氯化钯(520mg,0.7mmol)在甲醇(20mL)和N,N-二甲基甲酰胺(10mL)的混合溶剂中，加热到55℃反应18小时，减压蒸馏除去溶剂并溶于乙酸乙酯中，用水洗涤，有机层用无水硫酸镁干燥，抽滤，减压浓缩，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝3:1,v/v)得到黄色油状固体3-(3-氧代环丁基)苯甲酸甲酯1.1g,产率75％。
4-(5-溴吡嗪-2-亚甲氧基)-5-环丙基-3-(2,6-二氯苯基)异噁唑
Figure PCTCN2020140588-appb-000039
在100ml的圆底烧瓶中，放入氢化钠(4.9g,121.6mmol)，加入少量石油醚，洗涤氢化钠表面的煤油层，洗涤两次。加入四氢呋喃(30ml)，反应瓶置于0℃冰浴下冷却，将2,5-二溴吡嗪(13.1g,55.3mmol)溶于四氢呋喃(10ml)，搅拌下滴加到圆底烧瓶中。反应20min后，将1(5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲醇(15.7g,55.3mmol)溶于四氢呋喃(10ml)中，针管缓慢滴加到反应瓶中，升至室温反应12h。反应完毕，将反应液倒入100ml冰水混合物中，然后用乙酸乙酯(3×100ml)萃取，合并有机相后用水、饱和食盐水洗涤。无水MgSO 4进行干燥。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体6(4-(5-溴吡嗪-2-亚甲氧基)-5-环丙基-3-(2,6-二氯苯基)异噁唑)20.2g，产率为83﹪。
3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯
Figure PCTCN2020140588-appb-000040
100ml三颈烧瓶氮气保护，将6(20.2g,45.8mmol)溶于四氢呋喃(80ml)加入反应瓶，然后将 温度降到-78℃，缓慢滴加正丁基锂(1.6M in hexane,30.0mL,48.0mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(20ml)中的3-(3-氧代环丁基)苯甲酸甲酯2(9.0g,43.6mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕后，用饱和氯化铵猝灭反应，用乙酸乙酯萃取，饱和食盐水(200ml)洗涤有机相，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝15:1,v/v)得到黄色固体3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯5.6g，产率为19﹪。
3-(3-(5-(5-环丙基-3-(2,6-二氯苯基)异噁唑-4-亚甲氧基)-2-吡嗪)-1-羟基环丁烷)苯甲酸
Figure PCTCN2020140588-appb-000041
将3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯(5.6g,10.1mmol)溶解于THF(10mL)和甲醇(10mL)混合溶剂中，35℃下加入LiOH·H 2O(1.8g,42.6mmol)的水(5ml)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，经高压制备液相色谱仪分离纯化(乙腈:水＝3:4,v/v)得白色固体TM-1 3-(3-(5-(5-环丙基-3-(2,6-二氯苯基)异噁唑-4-亚甲氧基)-2-吡嗪)-1-羟基环丁烷)苯甲酸3.28g，收率57％。
图1所示、 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)
图2所示，SI-MS:m/z[M+H]+:Calcd.for C 28H 23Cl 2N 3O 5:551.1,Found:552.2
实施例2：化合物TM-2合成路线
Figure PCTCN2020140588-appb-000042
合成步骤：
3-(3-羟基氮杂环丁烷-1-基)苯甲酸甲酯3b的合成
Figure PCTCN2020140588-appb-000043
向3-碘苯甲酸甲酯3a(5.0g,19.1mmol)在DMSO-D 6(70mL)的溶液中加入3-氮杂环环丁烷-3-醇盐酸盐(2.5g.22.9mmol)、Cs 2CO 3(15.5g,47.7mmol)、CuI(726mg,3.8mmol)和L-脯氨酸(878mg,7.6mmol)，然后将该混合物在氩气气氛下在90℃加热18小时。溶液用乙酸乙酯和水稀释，然后将有机层用盐水洗涤三次，减压浓缩，用硅胶柱层析色谱分离纯化(DCM/MeOH＝10/1,v/v)，得到白色固体产物3b(2.7g,68％)。
3-(3-氧代氮杂环丁烷-1-基)苯甲酸甲酯3的合成
Figure PCTCN2020140588-appb-000044
将二甲亚砜(1.6g,20.3mmol)溶于二氯甲烷(30mL)中，在-78℃下，加入草酰氯(1.3g,10.1mmol)，并在-78℃搅拌30分钟，然后加入3-(3-羟基氮杂环丁烷-1-基)苯甲酸甲酯(1.4g,6.8mmol)溶于二氯甲烷，在-78℃下缓慢滴加到反应液中，并控制时间在30分钟，然后在-78℃搅拌30分钟，随后加入三乙胺(4.1g,40.5mmol)，在-78℃反应1小时，升至室温并在室温下反应2小时。反应液用水稀释，并用乙酸乙酯萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤浓缩，用硅胶柱层析色谱分离纯化(PE/EA＝2/1)得到白色固体产物3(0.9g,65％)。
3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基氮杂环丁烷-1-基)苯甲酸甲酯
Figure PCTCN2020140588-appb-000045
100ml三颈烧瓶氮气保护，将6(1.9g,4.4mmol)溶于四氢呋喃(25ml)加入反应瓶，然后将温度降到-78℃，缓慢滴加正丁基锂(2.5M in hexane,2.6mL,6.6mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(5ml)中的3-(3-氧代氮杂环丁烷-1-基)苯甲酸甲酯3(0.9g,4.4mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕后，用饱和氯化铵猝灭反应，用乙酸乙酯萃取，饱和食盐水(200ml)洗涤有机相，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝15:1,v/v)得到黄色固体7 3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯560mg，产率为22﹪
3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基氮杂环丁烷-1-基)苯甲酸
Figure PCTCN2020140588-appb-000046
将3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯(270mg,0.5mmol)溶解于THF(3mL)和甲醇(3mL)混合溶剂中，35℃下加入LiOH·H 2O(60mg,1.5mmol)的水(3ml)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，经高压制备液相色谱仪分离纯化(乙腈:水＝3:4，v/v)得白色固体3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基苯甲酸)40mg，收率15％。
图3所示、 1H-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)。
图4所示、ESI-MS:m/z[M+H]+:Calcd.for C 27H 22Cl 2N 4O 5:552.1,Found:553.1(注，数值在图的右侧)。
实施例3：化合物TM-3合成路线
3-(3-溴苯基)-1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)环丁醇
Figure PCTCN2020140588-appb-000047
100mL三颈烧瓶氮气保护，将4-(5-溴吡嗪-2-亚甲氧基)-5-环丙基-3-(2,6-二氯苯基)异噁唑(1.14g，2.3mmol)溶于无水四氢呋喃(5mL)打入反应瓶，然后将乙醇与液氮加入500mL低温杜瓦瓶使温度降到-78℃，缓慢滴加正丁基锂(1.7ml,2.7mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(10mL)中的3-(3-氧代环丁酮)苯甲酸甲酯(0.56g,2.5mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕将反应液缓慢倒入冰水混合物中，用乙酸乙酯萃取，水(100mL)洗涤酯层，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1，v/v)得到白色固体3-(3-溴苯基)-(5-(5-环丙基-3-(2,6-二氯苯基)异恶唑-4-基)甲氧基)吡嗪-2-基)环丁烷-1-醇，567mg,产率为42﹪。
图5所示、 1H-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)；
图6所示、 13C-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。
图7所示、ESI-MS:m/z[M+2+H] +:Calcd.forC 27H 22BrCl 2N 3O 3:585.0222,Found:588.0300
实施例4：化合物TM-4合成路线
1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-(3-(甲基磺酰基)苯基)环丁醇
Figure PCTCN2020140588-appb-000048
向3-(3-溴苯基)-1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)环丁醇(500mg,0.85mmol)在DMSO-D6中的溶液中，加入甲烷亚磺酸钠(130mg,1.28mmol)、CuI(50.2mg,0.26mmol)、L-脯氨酸(97.9mg,0.85mmol)和二异丙基乙胺(DIEA)(109.9mg,0.85mmol)。将该混合物在95℃搅拌过夜，然后用水稀释并用EA萃取。有机相合并，用水洗涤并用Na 2SO 4干燥。减压浓缩至干经高压制备液相色谱仪分离纯化(乙腈:水＝3:4,v/v)得白色固体1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-(3-(甲基磺酰基)苯基)环丁醇324mg，收率65％。
图8所示、 1H-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)
图9所示、ESI-MS:m/z[M+H]+:Calcd.for C 28H 25Cl 2N 3O 5S:585.1,Found:586.3
实施例5：化合物TM-5合成路线
1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-(3-(苯硫基)苯基)环丁醇
Figure PCTCN2020140588-appb-000049
在氩气保护下，向3-(3-溴苯基)-1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)环丁醇(1.0g,1.7mmol)在甲苯中的溶液中加入DIEA(0.44g,3.41mmol)、苯甲硫醇(0.21g,1.7mmol)、Pd 2(dba) 3(0.34g,0.37mmol)和4,5-双二苯基膦-9,9-二甲基氧杂蒽(0.16g,0.27mmol)。然后将混合物在115℃搅拌4小时。冷却至室温，用水稀释并用EA萃取。有机相合并，用水洗涤并用Na 2SO 4干燥。减压浓缩至干经高压制备液相色谱仪分离纯化(乙腈:水＝3:4,v/v)得白色固体1-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-(3-(苯硫基)苯基)环丁醇367mg,收率35％。
图10所示、 1H-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(H 2O),4.58(HDO),3.30(CD 3OD)，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)
图11所示、ESI-MS:m/z[M+H]+:Calcd.for C 33H 27Cl 2N 3O 3S:615.1150,Found:616.1421。
实施例6：化合物TM-6的合成
4-(5-溴吡嗪-2-亚甲氧基)-5-异丙基-3-(2,6-二氯苯基)异噁唑的合成
Figure PCTCN2020140588-appb-000050
在100mL的圆底烧瓶中，放入氢化钠(60％,0.83g,21mmol)，加入少量石油醚，洗涤NaH表面的煤油层，洗涤两次。加入30mL四氢呋喃，反应瓶置于0℃冰浴下冷却。将2,5-二溴吡嗪(833mg,3.5mmol)溶于10mL四氢呋喃，搅拌下滴加到圆底烧瓶中。反应20min后，将3-(2,6-二氯苯基)-4-羟甲基-5-异丙基异噁唑(1g,3.5mmol)溶于10mL四氢呋喃中，针管缓慢滴加到反应瓶中。升至室温反应12h。反应完毕，将反应液倒入100mL冰水混合物中，然后用乙酸乙酯(3×100mL)萃取，合并有机相后用水、饱和食盐水洗涤。无水MgSO 4进行干燥。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体4-(5-溴吡嗪-2-亚甲氧基)-5-异丙基-3-(2,6-二氯苯基)异噁唑0.7g，产率为53﹪。
3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯的合成
Figure PCTCN2020140588-appb-000051
100mL三颈烧瓶氮气保护，将4-(5-溴吡嗪-2-亚甲氧基)-5-异丙基-3-(2,6-二氯苯基)异噁唑(1g,2.3mmol)溶于无水四氢呋喃(20mL)打入反应瓶，然后将乙醇与液氮加入500mL低温杜瓦瓶使温度降到-78℃，缓慢滴加正丁基锂(1.7ml,2.7mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(10mL)中的3-(3-氧代环丁酮)苯甲酸甲酯(0.51g,2.5mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕将反应液缓慢倒入冰水混合物中，用乙酸乙酯萃取，水(100mL)洗涤酯层，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯549mg,产率为42﹪。
3-(3-(5-(5-异丙基-3-(2,6-二氯苯基)异噁唑-4-亚甲氧基)-2-吡嗪)-1-羟基环丁烷)苯甲酸的合成
Figure PCTCN2020140588-appb-000052
将3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯(319mg,0.6mmol,1eq)溶解于20mL THF中，35℃下LiOH·H 2O(99mg,2.4mmol,4.2eq)的水(5mL)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，使用高压制备液相色谱仪分离纯化，采用Waters XBridge C18柱(150nm*4.6nm*3.5um)，流动相为乙腈和水，流速18mL/min，收集梯度为45％-75％的馏分，浓缩除掉大部分乙腈，用冻干机冻干得白色粉末状固体(3-(3-(5-(5-异丙基-3-(2,6-二氯苯基)异噁唑-4-亚甲氧 基)-2-吡嗪)-1-羟基环丁烷)苯甲酸)126mg,收率38％。
图12所示 1H-NMR(400MHz,DMSO-D 6):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)；
图13所示、 13C-NMR(100MHz,CDCl3):176.8,171.0,159.3,158.4,150.9,145.2,131.2,129.4,128.7,128.4,128.2,128.1,128.0,109.1,71.1,56.8,44.5,29.9,27.0,20.9,1.0；
图14所示、ESI-MS:m/z[M+H]+:Calcd.for C 28H 25Cl 2N 3O 5:553.1171,Found:554.1211.
实施例7：化合物TM-7的合成
4-(5-溴吡嗪-2-亚甲氧基)-5-苯基-3-(2,6-二氯苯基)异噁唑的合成
Figure PCTCN2020140588-appb-000053
在100mL的圆底烧瓶中，放入氢化钠(60％,0.83g,21mmol)，加入少量石油醚，洗涤NaH表面的煤油层，洗涤两次。加入30mL四氢呋喃，反应瓶置于0℃冰浴下冷却。将2,5-二溴吡嗪(833mg,3.5mmol)溶于10mL四氢呋喃，搅拌下滴加到圆底烧瓶中。反应20min后，将3-(2,6-二氯苯基)-4-羟甲基-5-苯基异噁唑(1.12g,3.5mmol)溶于10mL四氢呋喃中，针管缓慢滴加到反应瓶中。升至室温反应12h。反应完毕，将反应液倒入100mL冰水混合物中，然后用乙酸乙酯(3×100mL)萃取，合并有机相后用水、饱和食盐水洗涤。无水MgSO4进行干燥。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体(4-(5-溴吡嗪-2-亚甲氧基)-5-苯基-3-(2,6-二氯苯基)异噁唑)，752mg，产率为45﹪。
3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯的合成
Figure PCTCN2020140588-appb-000054
100mL三颈烧瓶氮气保护，将4-(5-溴吡嗪-2-亚甲氧基)-5-苯基-3-(2,6-二氯苯基)异噁唑(1g,2.1mmol)溶于无水四氢呋喃(20mL)打入反应瓶，然后将乙醇与液氮加入500mL低温杜瓦瓶使温度降到-78℃，滴加正丁基锂溶液(1.1N环己烷溶液,1.75ml,2.8mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(10mL)中的3-(3-氧代环丁酮)苯甲酸甲酯(0.47g,2.3mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕将反应液缓慢倒入冰水混合物中，用乙酸乙酯萃取，水(100mL)洗涤酯层，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯329mg，产率为26﹪。
3-(3-(5-(5-苯基-3-(2,6-二氯苯基)异噁唑-4-亚甲氧基)-2-吡嗪)-1-羟基环丁烷)苯甲酸的合成
Figure PCTCN2020140588-appb-000055
将3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯(118mg,0.2mmol,1eq)溶解于20mL THF中，35℃下LiOH·H 2O(35mg,0.8mmol,4.2eq)的水(5mL)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，使用高压制备液相色谱仪分离纯化，采用Waters X Bridge C18柱(150nm*4.6nm*3.5um)，流动相为乙腈和水，流速18mL/min，收集梯度为45％-75％的馏分，浓缩除掉大部分乙腈，用冻干机冻干得白色粉末状固体(3-(3-(5-(5-苯基-3-(2,6-二氯苯基)异噁唑-4-亚甲氧基)-2-吡嗪)-1-羟基环丁烷)苯甲酸)39mg，收率33％。
图15所示、 1H-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,1H),5.38(s,2H),5.39-3.33(m,1H),2.94-2.88(m,2H),2.47-2.42(m,2H)；
图16所示，ESI-MS:m/z[M+H]+:Calcd.for C 31H 23Cl 2N 3O 5:587.1015,Found:588.1062
实施例8：化合物TM-8的合成
Figure PCTCN2020140588-appb-000056
3-甲基-5-乙烯基苯甲酸甲酯
Figure PCTCN2020140588-appb-000057
100mL圆底烧瓶，加入3-溴苯甲酸甲酯(1.12g,5mmol)，乙烯三氟硼酸钾(820mg,6.12mmol)，PdCl2(17.5mg,0.1mmol)，PPh3(80mg,0.3mmol)和Cs 2CO 3(5g,15mmol)，然后在N2下加入THF(18mL)和H2O(2mL)。混合物在80℃下搅拌反应22h，冷却至室温后，用水洗涤、无水硫酸镁干燥、抽滤。减压蒸馏，用硅胶柱层析法梯度洗脱分离纯化(PE：EA＝60:1,v/v)得到浅粉色油状液体(3-甲基-5-乙烯基苯甲酸甲酯)164mg，产率30％。
3-(2,2-二氯-3-氧环丁酮)-5-甲基苯甲酸甲酯
Figure PCTCN2020140588-appb-000058
氮气保护下，将3-甲基-5-乙烯基苯甲酸甲酯(5.46g，31mmol,1eq)溶解于乙醚(150mL)中。加入锌粉(6g,93mmol,3eq)，超声30min后，滴加一种三氯乙酰氯(8.7mL,77.5mmol,2.5eq)的Et2O溶液(50mL)，继续超声30分钟。混合物加热到35℃。持续超声2.5h，反应完成后冷却至室温，缓慢滴加水(50mL)淬灭。混合物倒入水中搅拌20min后，过滤，再用Et 2O漂洗。有机层用水(250mL)、饱和碳酸氢钠(250mL)和饱和氯化钠(250mL)洗涤，用无水硫酸镁干燥，过滤后减压蒸馏除去溶剂得黄色油状粗品。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝50:1,v/v)得到黄色油状液体3-(2,2-二氯-3-氧环丁酮)-5-甲基苯甲酸甲酯3.56g，产率40％。
3-甲基-5-(3-氧环丁基)苯甲酸甲酯
Figure PCTCN2020140588-appb-000059
混合3-(2,2-二氯-3-氧环丁酮)-5-甲基苯甲酸甲酯(2.79g,9.7mmol,1eq)与锌粉(2.54g,38.8mmol,4eq)溶于60mL乙酸中，室温下搅拌1h。然后在油浴80℃下回流3.5h，反应完毕后冷却至室温。用100mL水稀释溶剂乙酸，用乙醚(3×40mL)萃取。合并有机相后依次用饱和碳酸钠溶液(3×40mL)、水(100mL)、饱和食盐水(100mL)洗涤。用一定量的无水MgSO 4进行干燥。硅胶柱层析法梯度洗脱分离纯化(PE：EA＝50:1,v/v)得到化合物(3-(3-氧代环丁酮)苯甲酸甲酯)1.38g，产率65％。
3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯的合成
Figure PCTCN2020140588-appb-000060
100mL三颈烧瓶氮气保护，将4-(5-溴吡嗪-2-亚甲氧基)-5-环丙基-3-(2,6-二氯苯基)异噁唑(1.02g,2.3mmol)溶于无水四氢呋喃(20mL)打入反应瓶，然后将乙醇与液氮加入500mL低温杜瓦瓶使温度降到-78℃，缓慢滴加正丁基锂(1.7ml,2.7mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(10mL)中的3-甲基-5-(3-氧环丁基)苯甲酸甲酯(0.55g,2.5mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕将反应液缓慢倒入冰水混合物中，用乙酸乙酯萃取，水(100mL)洗涤酯层，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯，产率为47﹪。
3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸
Figure PCTCN2020140588-appb-000061
将3-(3-(5-((3-(2,6-二氯苯基)-5-环丙基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)苯甲酸甲酯(116mg,0.2mmol,1eq)溶解于20mL THF中，35℃下LiOH·H 2O(35mg,0.8mmol,4.2eq)的水(5mL)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，使用高压制备液相色谱仪分离纯化，采用Waters XBridge C18柱(150nm*4.6nm*3.5um)，流动相为乙腈和水，流速18mL/min，收集梯度为45％-75％的馏分，浓缩除掉大部分乙腈，用冻干机冻干得白色粉末状固体3-(3-(5-((5-环丙基-3-(2,6-二氯苯基)异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸，37mg，收率33％。
图17所示、 1H-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(1H,m),2.46-2.41(2H,m),2.35(3H,s),1.24-1.18(2H,m),1.16-1.14(2H,m)；
图18所示、 13C-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；
图19所示、ESI-MS:m/z[M+H]+:Calcd.for C 29H 25Cl 2N 3O 5:565.1171,Found:566.1234
实施例9：化合物TM-9的合成
3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯的合成
Figure PCTCN2020140588-appb-000062
100mL三颈烧瓶氮气保护，将4-(5-溴吡嗪-2-亚甲氧基)-5-异丙基-3-(2,6-二氯苯基)异噁唑(1.02g,2.3mmol)溶于无水四氢呋喃(20mL)打入反应瓶，然后将乙醇与液氮加入500mL低温杜瓦瓶使温度降到-78℃，缓慢滴加正丁基锂(1.7ml,2.7mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(10mL)中的3-甲基-5-(3-氧环丁基)苯甲酸甲酯(0.55g,2.5mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕将反应液缓慢倒入冰水混合物中，用乙酸乙酯萃取，水(100mL)洗涤酯层，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v，PE是石油醚，EA是乙酸乙酯)得到白色固体(3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯)643mg，产率为48﹪。
3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸的合成
Figure PCTCN2020140588-appb-000063
将3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯(117mg,0.2mmol,1eq)溶解于20mL THF中，35℃下LiOH·H 2O(35mg,0.8mmol,4.2eq)的水(5mL)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，使用高压制备液相色谱仪分离纯化，采用Waters X Bridge C18柱(150nm*4.6nm*3.5um)，流动相为乙腈和水，流速18mL/min，收集梯度为45％-75％的馏分，浓缩除掉大部分乙腈，用冻干机冻干得白色粉末状固体(3-(3-(5-((3-(2,6-二氯苯基)-5-异丙基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸)，46mg，收率40％。
图20所示、 1H-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)；
图21所示、 13C-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；
图22所示、ESI-MS:m/z[M+H]+:Calcd.for C 29H 27Cl 2N 3O 5:567.1328,Found:568.1412
实施例10：化合物TM-10的合成
3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯的合成
Figure PCTCN2020140588-appb-000064
100mL三颈烧瓶氮气保护，将4-(5-溴吡嗪-2-亚甲氧基)-5-苯基-3-(2,6-二氯苯基)异噁唑(1.03g,2.3mmol)溶于无水四氢呋喃(20mL)打入反应瓶，然后将乙醇与液氮加入500mL低温杜瓦瓶使温度降到-78℃，缓慢滴加正丁基锂(1.7ml,2.7mmol)，搅拌10min后，缓慢滴加溶于四氢呋喃(10mL)中的3-甲基-5-(3-氧环丁基)苯甲酸甲酯(0.55g,2.5mmol)溶液，-78℃反应2h后升至室温反应过夜。反应完毕将反应液缓慢倒入冰水混合物中，用乙酸乙酯萃取，水(100mL)洗涤酯层，无水硫酸镁干燥，抽滤，减压蒸馏除去有机溶剂，硅胶柱层析法梯度洗脱分离纯化(PE：EA＝10:1,v/v)得到白色固体(3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异噁唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯)496mg，产率为35﹪。
3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸的合成
Figure PCTCN2020140588-appb-000065
将3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸甲酯(123mg,0.2mmol,1eq)溶解于20mL THF中，35℃下LiOH·H 2O(35mg,0.8mmol,4.2eq)的水(5mL)溶液，搅拌过夜。减压蒸馏除去有机溶剂，用1N盐酸调节pH至5，加入乙酸乙酯萃取三次，用无水硫酸镁干燥，减压蒸馏除去溶剂，使用高压制备液相色谱仪分离纯化，采用Waters XBridge C18柱(150nm*4.6nm*3.5um)，流动相为乙腈和水，流速18mL/min，收集梯度为45％-75％的馏分，浓缩除掉大部分乙腈，用冻干机冻干得白色粉末状固体(3-(3-(5-((3-(2,6-二氯苯基)-5-苯基异恶唑-4-基)甲氧基)吡嗪-2-基)-3-羟基环丁基)-5-甲基苯甲酸)，37mg，收率31％。
图23所示、 1H-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)；
图24所示、 13C-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；
图25所示、ESI-MS:m/z[M+H]+:Calcd.for C 32H 25Cl 2N 3O 5:601.1171,Found:602.1249。

Claims (10)

  1. 一种具有式(I)结构的化合物，或其药学上可接受的盐、水合物、溶剂化物、药学上可接受的盐或其拆分的单一异构体：
    Figure PCTCN2020140588-appb-100001
    其中，
    R 1选自：卤素、-COOH、
    Figure PCTCN2020140588-appb-100002
    R 2选自：C 1～C 6的烃基、环烃基、芳香基、取代烃基或取代芳香基；
    R 3选自：-H或C 1～C 3的烃基、环烃基、取代烃基；
    X为C或N。
  2. 如权利要求1所述的化合物，其特征在于，
    其中，
    R 1选自-Br；
    R 2选自C 1～C 3的烃基、环烃基；
    R 3选自-CH 3。
  3. 如权利要求1所述的化合物，其特征在于，
    其中，
    R 2选自
    Figure PCTCN2020140588-appb-100003
  4. 如权利要求1所述的化合物，其特征在于，选自以下化合物或其药学上可接受的盐
    Figure PCTCN2020140588-appb-100004
    Figure PCTCN2020140588-appb-100005
    Figure PCTCN2020140588-appb-100006
  5. 一种药物组合物，其特征在于，以权利要求1-4任一项所述的化合物或其药学上可接受的盐作为活性成分。
  6. 如权利要求5所述的药物组合物，根据需要，还可含有药学上可接受的载体。
  7. 权利要求1所述的化合物的制备方法，包括如下步骤：
    Figure PCTCN2020140588-appb-100007
  8. 权利要求1～4任一所述的化合物在制备治疗非酒精性脂肪肝的药物中的用途。
  9. 如权利要求8所述的用途，其中所述非酒精性脂肪肝，为非酒精性脂肪肝炎。
  10. 如权利要求5所述的药物组合物在制备治疗非酒精性脂肪肝的药物中的用途，优选非酒精性脂肪肝炎。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013007387A1 (en) * 2011-07-13 2013-01-17 Phenex Pharmaceuticals Ag Novel fxr (nr1h4) binding and activity modulating compounds
WO2013037482A1 (en) * 2011-09-15 2013-03-21 Phenex Pharmaceuticals Ag Farnesoid x receptor agonists for cancer treatment and prevention
WO2017201150A1 (en) * 2016-05-18 2017-11-23 Enanta Pharmaceuticals, Inc. Isoxazole analogs as fxr agonists and methods of use thereof
CN109071470A (zh) * 2016-01-26 2018-12-21 江苏豪森药业集团有限公司 Fxr激动剂及其制备方法和应用
WO2020033382A1 (en) * 2018-08-08 2020-02-13 Inorbit Therapeutics Ab Compounds useful in modulating the farnesoid x receptor and methods of making and using the same

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017128896A1 (zh) * 2016-01-26 2017-08-03 江苏豪森药业集团有限公司 Fxr激动剂及其制备方法和应用
CA2968836A1 (en) * 2016-06-13 2017-12-13 Gilead Sciences, Inc. Fxr (nr1h4) modulating compounds
SI3730487T1 (sl) * 2016-06-13 2022-08-31 Gilead Sciences, Inc. Azetidinski derivati kot modulatorji FXR (NR1H4)
CN107973790A (zh) * 2016-10-22 2018-05-01 合帕吉恩治疗公司 杂环fxr调节剂

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013007387A1 (en) * 2011-07-13 2013-01-17 Phenex Pharmaceuticals Ag Novel fxr (nr1h4) binding and activity modulating compounds
WO2013037482A1 (en) * 2011-09-15 2013-03-21 Phenex Pharmaceuticals Ag Farnesoid x receptor agonists for cancer treatment and prevention
CN109071470A (zh) * 2016-01-26 2018-12-21 江苏豪森药业集团有限公司 Fxr激动剂及其制备方法和应用
WO2017201150A1 (en) * 2016-05-18 2017-11-23 Enanta Pharmaceuticals, Inc. Isoxazole analogs as fxr agonists and methods of use thereof
WO2020033382A1 (en) * 2018-08-08 2020-02-13 Inorbit Therapeutics Ab Compounds useful in modulating the farnesoid x receptor and methods of making and using the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OLAF KINZEL, CHRISTOPH STEENECK, THOMAS SCHLüTER, ANDREAS SCHULZ, CHRISTIAN GEGE, ULRIKE HAHN, EVA HAMBRUCH, MARTIN HORNBERGE: "Novel substituted isoxazole FXR agonists with cyclopropyl, hydroxycyclobutyl and hydroxyazetidinyl linkers: Understanding and improving key determinants of pharmacological properties", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 26, no. 15, 1 August 2016 (2016-08-01), Amsterdam NL , pages 3746 - 3753, XP055745290, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2016.05.070 *

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