CN113105443B

CN113105443B - Isoxazole FXR receptor agonist as well as preparation method and medical application thereof

Info

Publication number: CN113105443B
Application number: CN202010033239.4A
Authority: CN
Inventors: 向华; 李振邦; 肖茂旭; 陶睿; 骆国顺
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2022-10-04
Anticipated expiration: 2040-01-13
Also published as: CN113105443A

Abstract

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to an isoxazole FXR receptor agonist, and a preparation method and medical application thereof. A compound of formula (I), a pharmaceutically acceptable salt thereof, an isotopic label:

the compound shown in the formula (I) and pharmaceutically acceptable salts and isotope markers thereof have FXR receptor agonist activity, and can be used for preparing medicines for treating or preventing hyperlipidemia, atherosclerosis, non-alcoholic steatohepatitis and type II diabetes.

Description

Isoxazole FXR receptor agonist, preparation method and medical application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to an isoxazole FXR receptor agonist, and a preparation method and medical application thereof.

Background

Dyslipidemia refers to an increase in Total cholesterol (Tc), triglycerides (TG) and Low-density lipoprotein cholesterol (LDL-C) and a decrease in High-density lipoprotein cholesterol (HDL-C) in plasma. LDL can be oxidized into oxLDL, excessive oxLDL is accumulated in macrophages to become foam cells, and vascular endothelial cells are simultaneously caused to die to cause inflammatory reaction, so that the formation of atherosclerotic plaques is promoted; the nascent small particle HDL receives cholesterol from macrophages in the vascular wall, transports the cholesterol to the liver after esterification, and metabolizes the cholesterol into cholate to clear the cholesterol out of the body, so that Reverse Cholesterol Transport (RCT) is achieved. Excessive lipid exchange caused by high TG can form large particle HDL containing less cholesterol and much TG, so that small particle HDL-C level with strong RCT capability is reduced, cholesterol is deposited on the inner wall of blood vessel, and atherosclerosis is increased

Farnesoid X Receptor (FXR), a member of the nuclear receptor superfamily. FXR is involved in regulating various physiological activities of the body, including bile acid metabolism, lipid metabolism, sugar metabolism, and liver protection. Based on its role in bile acid metabolism and lipid metabolism, FXR is considered to be a promising new target for the treatment of dyslipidemia. After FXR is activated, intestinal cells are induced to synthesize and secrete human Fibroblast Growth Factor (FGF) 19, after FGF19 enters liver, FGF receptor 4 on the surface of liver cells is activated, and further expression of cholesterol 7 hydroxylase 1 serving as a rate-limiting enzyme for bile acid synthesis is inhibited, and bile acid synthesis is reduced. Activating FXR can promote expression of B type I scavenger receptor, inhibit expression of sterol regulatory element binding protein 1c, induce peroxisome proliferator activated receptor alpha expression, promote reverse transfer of cholesterol into liver, reduce lipid synthesis, and promote fatty acid beta oxidation.

In the research of isoxazole FXR agonists, maloney et al screened to obtain a first non-steroidal FXR agonist GW4064 (14) with an EC50 value of 15nmol/L. But the bioavailability is low, and the stilbene group in the structure has potential toxicity, so that the further research on the stilbene derivative is limited, and currently, GW4064 is mainly used as a tool drug in FXR research. To overcome the drawbacks of GW4064, many subsequent pharmaceutical companies and researchers have synthesized a number of derivatives of GW 4064. The FXR is taken as a target, the research on the blood fat regulating medicine of the isoxazole derivatives is carried out, and the FXR has important significance for reducing the residual cardiovascular risk.

Disclosure of Invention

The invention aims to provide an isoxazole FXR receptor agonist.

The invention also aims to provide a preparation method of the isoxazole FXR receptor agonist.

It is a further object of the present invention to provide a pharmaceutical use of the compound.

A compound of formula (I), pharmaceutically acceptable salts, isotopic labels thereof:

wherein R1 and R2 are respectively and independently selected from H, CONH (CH) ₂ )nCH ₃ ，COO(CH ₂ )n CH ₃ ，O(CH ₂ )n CH ₃ ，Cl，Br，F，I，COOH，NO ₂ ，SO ₃ H，CN，

n represents 0, 1, 2, 3, 4,5, 6.

The compound shown in the formula (I), the pharmaceutically acceptable salt and the isotopic marker thereof are preferably R1 and R2 which are respectively and independently selected from the group consisting of Table H, CONH (CH) ₂ )nCH ₃ ，COO(CH ₂ )n CH ₃ ，O(CH ₂ )n CH ₃ (ii) a n preferably represents 0, 1, 2.

The compound represented by the formula (I) is further preferably selected from any one of the following:

numbering	R1、R2
		A1	-H、-H
A2	-H、-CONHCH3
		A3	-CONHCH3、-H
A4	-COOCH3、-H
		A5	-COOC2H5、-H
A6	-H、-COOCH3
		A7	-H、-COOC2H5
A8	-OCH3、-H
		A9	-H、-OCH3

The preparation method of the compound shown in the formula (I) comprises the following reaction route:

(a)NH ₂ OH·HCl,NaOH,EtOH；(b)NCS,DMF；(c)NaOCH ₃ ,CH ₃ OH,THF；(d)LiAlH4,THF；(e)CBr ₄ ,PPh ₃ DCM, N2; (f) N-Boc-4-hydroxypiperidine, 18-crown-6, (CH) ₃ ) ₃ COK,THF,N2；(g)TFA,THF；(h-i)bis(trichloromethyl)carbonate,Et ₃ N,DCM.

The compound shown in the formula (I), and the pharmaceutically acceptable salt and the isotopic marker thereof are applied to the preparation of FXR receptor agonists.

The compound shown in the formula (I), and pharmaceutically acceptable salts and isotopic markers thereof can be used for preparing medicines for treating or preventing hyperlipidemia, atherosclerosis, non-alcoholic steatohepatitis and diabetes mellitus type II.

The code of the compound in the pharmacological experiment and experimental examples is equal to the structure of the compound corresponding to the code.

Has the advantages that: as a result of the activity of the compound of the present invention, it was found that the compound synthesized in the present invention had inhibitory activity against 3T3-L1 cells at 40uM (see FIG. 1). In the effect on the expression of FXR and FXR downstream gene mRNA in HepG2 hepatoma cells, compound A4 was able to up-regulate the transcriptional levels of FXR mRNA, SHP mRNA and BSEP mRNA and down-regulate the transcriptional level of BSREBP-1c mRNA (see FIGS. 2-5). Compound A4 was able to significantly activate FXR in the FXR agonistic activity assay at a concentration of 10. Mu. Mol/L (see FIG. 6).

The action mechanism, action intensity and action time of the compound for regulating blood fat and reducing cardiovascular diseases need to be elucidated in further research.

Drawings

FIG. 1 Effect of Compounds on 3T3-L1 cell proliferative Activity

FIG. 2 Effect of Compounds on FXR mRNA transcription

FIG. 3 Effect of Compounds on SHP mRNA transcription

FIG. 4 Effect of Compounds on BSEP mRNA transcription

FIG. 5 Effect of Compounds on BSREBP-1c mRNA transcription

FIG. 6 agonistic activity of Compound A4 against FXR

Detailed Description

The claims hereof set forth the novel features of the invention with particularity. The following sets forth illustrative embodiments in which the principles of the invention may be employed. The features and advantages of the present invention may be better understood by reference to the following.

Although preferred embodiments of the present application are described herein, these embodiments are provided by way of example only. It is to be understood that variations of the embodiments of the present application described herein may also be used to practice the teachings of the present application. Those of ordinary skill in the art will appreciate that various modifications, changes, and substitutions can be made without departing from the scope of the present application. It should be understood that the scope of the various aspects of the application is defined by the claims and that methods and structures within the scope of these claims and their equivalents are intended to be covered thereby.

Some of the compounds were prepared as follows:

melting points were determined by XRC-1 micro melting point apparatus (thermometer uncorrected), IR by Fourier transform infrared spectrometer type Nicolet iS10 (KBr pellet), 1H-NMR by Bruker AV300 type (300 MHz) NMR (TMS as internal standard), and mass spectra by Agilent Q-TOF 6520 (HR-MS) and Agilent 1100LC-MSD-Trap/SL type mass spectrometer (ESI-MS), respectively.

The column chromatography silica gel is 100-200 mesh or 200-300 mesh silica gel (Qingdao ocean chemical plant), and the eluent is petroleum ether-ethyl acetate system or dichloromethane-methanol system. Thin Layer Chromatography (TLC) using GF254 thin layer chromatography plate (smoke bench Jiang You silica gel development Co., ltd.); the TLC development system is a petroleum ether-ethyl acetate system or a dichloromethane-methanol system, and a small amount of acetic acid or triethylamine is added when necessary; TLC was visualized by irradiation under a model ZF7 three-way UV analyzer (Henan Shenyi Yunhua instruments, inc.).

Example 1

Synthesis of intermediate 2-trifluoromethoxybenzaldehyde oxime (2)

Under ice bath, the H is put into ₂ NOH & HCl (2.2g, 31.7mmol) and NaOH (1.2g, 30.0mmol) were dissolved in H ₂ O, to prepare a solution a. 2-trifluoromethoxybenzaldehyde (5g, 26.3mmol) was dissolved in CH ₃ CH ₂ And in OH, dropwise adding the prepared solution A, and reacting at room temperature for 1h. Removal of CH under reduced pressure ₃ CH ₂ OH, addition of H ₂ Diluted with O, and extracted with Ethyl Acetate (EA) and H ₂ Separating by O extraction, dewatering with saturated salt solution, drying with anhydrous Na2SO4, spin-drying under reduced pressure, and directly adding into the reactor. Crude 5g, yield: 93 percent.

Example 2

Synthesis of intermediate 2-trifluoromethoxy-N-hydroxy-chlorobenzaldehyde oxime (3)

2-Trifluoromethoxybenzaldoxime (crude 5g,24.4 mmol) was dissolved in DMF, and N-chlorosuccinimide (NCS) (2.5 g,18.7 mmol) was added thereto in portions at 25 ℃ or lower, and reacted at room temperature for 3 hours. The reaction solution is extracted by EA and H2OWashing organic phase with water, removing water with saturated salt solution, and removing anhydrous Na ₂ SO ₄ Drying, rotary drying under reduced pressure, and directly adding. Crude 4.5g, yield 77.1%.

Example 3

Synthesis of intermediate 3- (2-trifluoromethoxyphenyl) -5-cyclopropylisoxazole-4-carboxylic acid methyl ester (4)

Under the ice salt bath, naOCH is added ₃ (2.64g, 48.9 mmol) in CH ₃ OH, preparing a solution B. Methyl 3-cyclopropyl-3-oxopropanoate (5.34g, 37.6mmol) was dissolved in CH ₃ In OH. Slowly adding the solution B into the solution B for more than 5min. 2-trifluoromethoxy-N-hydroxy-chlorobenzaldehyde oxime (4.5g, 18.8mmol) was dissolved in CH ₃ In OH, dropwise adding into the above mixed solution in an ice salt bath, wherein the reaction temperature is always less than-5 ℃. After 5min, the ice salt bath was removed, the temperature was raised to 35 ℃ and the reaction was carried out for 12h. By CH ₃ COOH neutralizes excess NaOCH ₃ Removal of CH under reduced pressure ₃ OH, with EA and H ₂ And (4) extracting. Removing water and anhydrous Na from saturated salt solution ₂ SO ₄ And (5) drying and spin-drying. Column chromatography purification, product 2.5g, yield: 40.7 percent. 1H NMR (DMSO-d 6, 400MHz) delta 7.65-7.60 (m, 1H, ph), 7.53 (dd, J =7.6,2Hz,1H, ph), 7.48-7.43 (m, 2H, ph), 3.68 (s, 3H, -COOCH ₃ ),2.89(m,J＝6.8Hz,1H,-CH(CH2)2),1.30(m,J＝6.8Hz,4H,-CH(CH ₂ ) ₂ )。

Example 4

Synthesis of intermediate 3- (2-trifluoromethoxyphenyl) -4-hydroxymethyl-5-cyclopropylisoxazole (5)

LiAlH4 (0.58g, 15.3 mmol) was placed in an eggplant type bottle, anhydrous THF was slowly dropped under ice bath, stirred, and mixed well. After the bubbles were reduced, methyl 3- (2-trifluoromethoxyphenyl) -5-cyclopropylisoxazole-4-carboxylate (2.5g, 7.6 mmol) was dissolved in anhydrous THF and slowly added dropwise for a reaction of 1h. Slowly drop saturated Na ₂ SO ₄ The reaction was stopped by adding 15% NaOH solution to precipitate the aluminium ions, filtered through Celite and the filter cake was washed with EA. Removing the product-containing mixture under reduced pressure, using EA and H ₂ Extracting with water, removing water from organic phase with saturated salt solution, and removing anhydrous Na ₂ SO ₄ Drying, rotary drying under reduced pressure, and directly adding. Yield: 2.1g, yield: 91.9 percent. MS M/z [ M + H ]]+＝300.1.

Example 5

Synthesis of intermediate 3- (2-trifluoromethoxyphenyl) -4-bromomethyl-5-cyclopropylisoxazole (6)

3- (2-trifluoromethoxyphenyl) -4-hydroxymethyl-5-cyclopropylisoxazole (1g, 3.34mmol) and PPh3 (1.31g, 5.0 mmol) were dissolved in anhydrous DCM under ice salt bath and stirred until completely dissolved. N is a radical of ₂ Under protection, CBr was added slowly ₄ (1.66g CBr4,5.0 mmol) in dry DCM for 1h. The reaction solution is directly decompressed and dried by spinning, and purified by column chromatography. The product is as follows: 0.6g, yield: 49.5 percent. 1H NMR (CDCl) ₃ ,400MHz)δ7.55-7.50(m,1H,Ph),7.49-7.45(m,1H,Ph),7.40-7.32(m,2H,Ph),4.27(s,2H,Ar-CH ₂ -Br),2.11-1.99(m,1H,-CH(CH2)2),1.24-1.17(m,2H,-CH(CH ₂ ) ₂ ),1.17-1.09(m,2H,-CH(CH ₂ ) ₂ ).

Example 6

Synthesis of intermediate 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethyl-N-Boc-piperidine) -5-cyclopropylisoxazole (7)

N-Boc-4-hydroxypiperidine (367mg, 1.82mmol) and 18-crown-6 (482mg, 1.82mmol) were put into a three-necked flask and dissolved in anhydrous THF. Potassium tert-butoxide (204.2mg, 1.82mmol) was dissolved in anhydrous THF, mixed well and added dropwise to a three-necked flask. Stirring for 1h under the protection of N2. 3- (2-trifluoromethoxyphenyl) -4-bromomethyl-5-cyclopropylisoxazole (600mg, 1.66mmol) was dissolved in anhydrous THF, added dropwise, and stirred overnight. Neutralizing excess potassium tert-butoxide with CH3COOH, with EA and H ₂ Extracting with saturated saline water, removing water, drying with anhydrous Na2SO4, and spin-drying. And (5) performing column chromatography purification. The product is as follows: 401mg, yield: 50.0 percent. <xnotran> 1H NMR (DMSO-d6,300MHz) δ 7.92-7.61 (m, 4H,Ph), 4.30 (s, 2H,Ar-CH 2-O-), 3.49 (m, 4H, -O-CH2-CH 2-N-), 3.37 (m, 1H, -O-CH- (CH 2) 5363 zxft 5363 (m, 1H,Ar-CH- (CH 2) 2), 1.66 (m, 4H, -O-CH2-CH 2-N-), 1.24 (m, 1H,Ar-CH- (CH 2) 2), 1.06 (-O-C- (CH 3) 3), 3242 zxft 3242 (m, 2H,Ar-CH- (CH 2) 2). </xnotran>

Example 7

Synthesis of intermediate 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropylisoxazole (8)

TFA (2ml, 26.9 mmol) was dissolved in dry DCM to make a 20% TFA solution. 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethyl-N-Boc-piperidine) -5-cyclopropylisoxazole (400mg, 0.83mmol) was dissolved in DCM and added dropwise thereto and reacted at room temperature for 1h. Saturated NaHCO ₃ Terminating the reaction with the solution, adjusting the reaction solution to be alkaline, and reacting with EA and H ₂ Extracting with water, removing water with saturated salt water, and removing anhydrous Na ₂ SO ₄ Drying, spin-drying, and directly feeding.

Example 8

Synthesis of intermediate methyl 4-aminobenzoate

P-aminobenzoic acid (1g, 7.3mmol) was dissolved in CH3OH (10 ml), and thionyl chloride (1ml, 13.7 mmol) was slowly added dropwise under ice bath. After the thionyl chloride is completely added, the ice bath is removed, the temperature is slowly raised to 50 ℃, and the reaction is carried out for 2 hours. Saturated NaHCO ₃ Stopping reaction, adjusting reaction solution to alkalinity, extracting with EA and H2O, removing water with saturated salt solution, and removing anhydrous Na ₂ SO ₄ And (5) drying and spin-drying.

Example 9

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidinphenylurea) -5-cyclopropylisoxazole (A1)

Aniline (29.2mg, 0.31mmol) was dissolved in anhydrous DCM. Triethylamine (0.1 ml) and triphosgene (46.5mg, 0.15mmol) were dissolved in DCM under ice bath, and then added dropwise to react for 1h. Excess triphosgene was removed under reduced pressure. Adding anhydrous DCM for dissolving for standby. 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropylisoxazole (120mg, 0.31mmol) was dissolved in anhydrous DCM and added dropwise, the temperature was raised to 35 ℃ and the reaction was allowed to proceed overnight. EA and H for reaction solution ₂ Extracting with saturated saline water, removing water, drying with anhydrous Na2SO4, and spin-drying. And (5) performing column chromatography purification. To obtain a compound A1, a product: 60mg, yield: 40.5 percent. An oil. 1H NMR (300MHz, chloroform-d) delta 7.77-7.46 (m, 1H), 7.46-7.38 (m, 1H), 7.37-7.21 (m, 2H), 7.09-6.98 (m, 0H), 6.57 (s, 0H), 3.61 (ddd, J =11.9,7.1,3.9Hz, 1H), 3.50 (dt, J =7.6,3.7Hz, 0H), 2.22-2.11 (m, 0H), 1.76 (td, J =7.9,3.6Hz, 1H), 1.52 (ddt, J =12.8,8.5,3.9Hz, 1H), 1.37-1.22 (m, 1H), 1.19-1.08 (m, 1H). 13C NMR (75MHz, CDCl) ₃ )δ171.9,159.5,155.0,146.9,139.1,131.8,131.2,128.8,127.0,123.1,123.1,120.8,120.0,119.3,111.6,77.5,77.1,76.7,73.5,59.0,41.4,30.5,8.1,7.6.HRMS(ESI)m/z[M+H]+502.2883,found502.1938。

Example 10

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-4-methylaminoacylphenylurea) -5-cyclopropylisoxazole (A2)

4-amino-N-methylbenzamide was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in an ice bath and added dropwise to react for 1h. Excess triphosgene was removed under reduced pressure. Adding anhydrous DCM for dissolving for standby. 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropylisoxazole was dissolved in anhydrous DCM, added dropwise, and the temperature was raised to 35 ℃ for overnight reaction. The reaction solution is extracted by EA and H2O, the saturated saline solution is dehydrated, and the anhydrous Na2SO4 is dried and spin-dried. And (5) performing column chromatography purification. Compound A2 was obtained in 62.3% yield. An oil. 1H NMR (DMSO-d6, 300MHz) delta 8.86 (s, 1H, -CONH-), 7.48-6.9 (m, 8H, ar), 4.30 (s, 2H, ar-CH) ₂ -O-),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.81(s,3H,-CONHCH ₃ ),2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ ),13C NMR(75MHz,DMSO-d6)δ172.05,158.23,155.39,154.84,147.00,139.18,131.66,131.04,128.40,126.75,122.77,120.85,119.81,111.45,77.88,77.18,76.26,74.65,73.60,59.20,41.61,30.50,8.25,7.36.HRMS(ESI)m/z[M+H]+559.2098,found 559.2153。

Example 11

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-3-methylaminoacylphenylurea) -5-cyclopropylisoxazole (A3)

3-amino-N-methylbenzamide was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in an ice bath and added dropwise to react for 1h. Excess triphosgene was removed under reduced pressure. Adding anhydrous DCM for dissolving for standby. Dissolving 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropyl isoxazole in anhydrous DCM, adding dropwise, heating to 35 ℃, and reacting overnight. The reaction solution was extracted with EA and H2O, and the saturated brine was dehydrated, dried over anhydrous Na2SO4, and spin-dried. And (5) performing column chromatography purification. Compound A3 was obtained in 63.5% yield. An oil. 1H NMR (DMSO-d 6, 300MHz) delta 8.86 (s, 1H, -CONH-), 7.48-6.9 (m, 8H, ar), 4.30 (s, 2H, ar-CH) ₂ -O-),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.81(s,3H,-CONHCH ₃ ),2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,DMSO-d6)δ172.1,159.3,157.4,154.8,147.0,139.2,131.7,131.0,128.4,126.8,122.8,120.8,119.8,111.4,77.8,77.5,76.3,74.6,73.6,59.2,41.6,30.5,8.0,7.3.HRMS(ESI)m/z[M+H]+559.2098,found 559.2150。

Example 12

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-3-methoxyacylphenylurea) -5-cyclopropylisoxazole (A4)

Methyl 3-aminobenzoate was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in ice bath and added dropwise for 1h reaction. Excess triphosgene was removed under reduced pressure. Adding anhydrous DCM for dissolving for standby. 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropylisoxazole was dissolved in anhydrous DCM, added dropwise, and the temperature was raised to 35 ℃ for overnight reaction. The reaction solution is extracted by EA and H2O, the saturated saline solution is dehydrated, and the anhydrous Na2SO4 is dried and spin-dried. And (5) performing column chromatography purification. Compound A4 was obtained in 37.5% yield. An oil. 1H NMR (DMSO-d6, 300MHz) delta 7.48-6.9 (m, 8H, ar), 4.30 (s, 2H, ar-CH) ₂ -O-),3.89(s,3H,-COOCH ₃ ),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,DMSO-d6)δ172.1,159.3,159.2,154.8,148.0,139.2,131.7,131.0,128.4,126.8,122.8,120.8,119.8,111.4,77.7,77.1,76.3,73.6,59.2,41.6,30.5,8.8,7.9.HRMS(ESI)m/z[M+H]+532.1989,found 532.2056。

Example 13

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-3-ethoxyacylphenylurea) -5-cyclopropylisoxazole (A5)

Ethyl 3-aminobenzoate was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in an ice bath and added dropwise to react for 1h. Excess triphosgene was removed under reduced pressure. Adding anhydrous DCM for dissolving for standby. Dissolving 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropyl isoxazole in anhydrous DCM, adding dropwise, heating to 35 ℃, and reacting overnight. The reaction solution is extracted by EA and H2O, the saturated saline solution is dehydrated, and the anhydrous Na2SO4 is dried and spin-dried. And (5) performing column chromatography purification. Compound A5 was obtained with a yield of 45.2%. An oil. 1H NMR (DMSO-d6, 300MHz) delta 7.48-6.9 (m, 8H, ar), 4.30 (s, 2H, ar-CH 2-O-), 4.30 (q, 2H, -COOCH ₂ CH ₃ ),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.30(t,3H,-COOCH ₂ CH ₃ ),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,CDCl ₃ )δ172.1,159.3,158.4,154.8,147.0,139.2,131.6,131.1,128.4,126.8,122.8,120.8,119.8,111.4,77.8,77.1,76.3,73.6,59.2,41.6,30.5,8.0,7.3.HRMS(ESI)m/z[M+H]+573.2095,found 573.2155。

Example 14

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-4-methoxyacylphenylurea) -5-cyclopropylisoxazole (A6)

Methyl 4-aminobenzoate was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in ice bath and added dropwise for 1h reaction. Excess triphosgene was removed under reduced pressure. Anhydrous DCM was added and dissolved for further use. 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropylisoxazole was dissolved in anhydrous DCM, added dropwise, and the temperature was raised to 35 ℃ for overnight reaction. The reaction solution was extracted with EA and H2O, and the saturated brine was dehydrated, dried over anhydrous Na2SO4, and spin-dried. And (5) performing column chromatography purification. Compound A6 was obtained with a yield of 45.6%. An oil. 1H NMR (DMSO-d 6, 300MHz) delta 7.48-6.9(m,8H,Ar),4.30(s,2H,Ar-CH ₂ -O-),3.89(s,3H,-COOCH ₃ ),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,DMSO-d6)δ172.05,159.31,158.63,154.84,147.00,139.16,131.66,131.05,128.40,126.75,122.77,120.83,119.81,111.41,77.78,77.08,76.26,73.60,59.20,41.61,30.50,7.95,7.33.HRMS(ESI)m/z[M+H]+573.2095,found 573.2153.

Example 15

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-4-ethoxyacylphenylurea) -5-cyclopropylisoxazole (A7)

Ethyl 4-aminobenzoate was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in an ice bath and added dropwise to react for 1h. Excess triphosgene was removed under reduced pressure. Anhydrous DCM was added and dissolved for further use. 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropylisoxazole was dissolved in anhydrous DCM, added dropwise, and the temperature was raised to 35 ℃ for overnight reaction. EA and H for reaction solution ₂ Extracting with saturated saline water, removing water, drying with anhydrous Na2SO4, and spin-drying. And (5) performing column chromatography purification. Compound A7 was obtained in 43.6% yield. An oil. 1H NMR (DMSO-d6, 300MHz) delta 7.48-6.9 (m, 8H, ar), 4.30 (s, 2H, ar-CH) ₂ -O-),4.30(q,2H,-COOCH ₂ CH ₃ ),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.30(t,3H,-COOCH ₂ CH ₃ ),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,DMSO-d6)δ172.1,159.3,154.8,147.0,139.2,131.7,131.0,127.5,126.7,122.9,120.4,119.9,111.4,77.8,77.3,76.2,73.6,59.2,40.6,30.5,8.5,7.6.HRMS(ESI)m/z[M+H]+573.2095,found 573.2153。

Example 16

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-4-methoxyphenylurea) -5-cyclopropylisoxazole (A8)

3-Methoxyaniline was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in an ice bath and added dropwise to react for 1h. Excess triphosgene was removed under reduced pressure. Anhydrous DCM was added and dissolved for further use. Dissolving 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropyl isoxazole in anhydrous DCM, adding dropwise, heating to 35 ℃, and reacting overnight. EA and H for reaction solution ₂ Extracting with saturated saline water, removing water, drying with anhydrous Na2SO4, and spin-drying. And (5) performing column chromatography purification. Compound A8 was obtained in 37.5% yield. An oil. 1H NMR (CDCl3, 300MHz) delta 7.48-6.9 (m, 8H, ar), 4.30 (s, 2H, ar-CH) ₂ -O-),3.61(s,3H,-OCH ₃ ),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,CDCl ₃ )δ172.1,159.7,154.8,152.0,149.2,131.7,131.0,128.4,126.8,122.8,120.4,119.9,111.9,77.8,77.1,76.3,73.6,59.2,41.6,30.5,8.0,7.3.HRMS(ESI)m/z[M+H]+532.1989,found 532.2045。

Example 17

Synthesis of 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine-3-methoxyphenylurea) -5-cyclopropylisoxazole (A9)

4-Methoxyaniline was dissolved in anhydrous DCM. Triethylamine and triphosgene were dissolved in DCM in ice bath and added dropwise for 1h reaction. Excess triphosgene was removed under reduced pressure. Anhydrous DCM was added and dissolved for further use. Dissolving 3- (2-trifluoromethoxyphenyl) -4- (4-oxymethylpiperidine) -5-cyclopropyl isoxazole in anhydrous DCM, adding dropwise, heating to 35 ℃, and reacting overnight. EA and H for reaction solution ₂ Extracting with saturated saline water, removing water, drying with anhydrous Na2SO4, and spin-drying. And (5) performing column chromatography purification. Compound A9 was obtained in 38.6% yield. An oil. 1H NMR (CDCl) ₃ ,300MHz)δ7.48-6.9(m,8H,Ar),4.30(s,2H,Ar-CH ₂ -O-),3.61(s,3H,-OCH ₃ ),3.49(m,4H,-O-CH ₂ -CH ₂ -N-),3.37(m,1H,-O-CH-(CH ₂ ) ₂ ,2.22(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.68(m,4H,-O-CH ₂ -CH ₂ -N-),1.25(m,1H,Ar-CH-(CH ₂ ) ₂ ),1.02,0.99(m,2H,Ar-CH-(CH ₂ ) ₂ )13C NMR(75MHz,CDCl ₃ )δ172.1,159.3,154.8,147.0,139.2,131.7,131.0,128.4,126.8,122.8,120.8,119.8,116.4,78.0,77.4,76.3,73.6,58.2,41.6,30.5,8.6,7.9.HRMS(ESI)m/z[M+H]+532.1989,found 532.2040。

Example 18

Experiment for testing 3T3-L1 cell proliferation by MTT method

The concentration is about 1X 10 ⁵ ml ^-1 The 3T3-L1 cells were seeded into 96-well cell culture plates in 100. Mu.l per well. Standing in a cell incubator (37 ℃,5% CO) ₂ ) After 24h, 100. Mu.l of medium containing samples at different concentrations were added to each well to give final sample concentrations of 5. Mu. Mol/L, 10. Mu. Mol/L, 20. Mu. Mol/L and 40. Mu. Mol/L, respectively, with 3 duplicate wells per concentration. Blank control was an equal volume of DMSO medium at 0.1%. After further culturing for 48 hours, 20. Mu.L of MTT solution at a concentration of 5mg/mL was added to each well and cultured for 4 hours. After 4h, carefully sucking the liquid in the wells, adding 150 μ L DMSO into each well, oscillating for 20min at low speed on an oscillator, detecting the absorbance OD value of each well at 490nm by using an enzyme-labeled detector, and calculating the cell inhibition rate.

Inhibition rate = [1- (experimental OD value-blank OD value)/(control OD value-blank OD value) ] × 100%

The results of the proliferation activity of 3T3-L1 preadipocytes are shown in FIG. 1. It can be seen that most compounds have weak inhibitory effect on cell proliferation at low concentration (5. Mu. Mol/L and 10. Mu. Mol/L), and individual compounds such as A8, etc. have effect of promoting cell proliferation at 5. Mu. Mol/L. At high concentrations (20. Mu. Mol/L and 40. Mu. Mol/L), the compound has certain inhibitory activity on cells, wherein the inhibition rate of the compound A7 on cell survival reaches more than 50% at the concentration of 40. Mu. Mol/L. Therefore, we chose the concentration of the compound to be 10. Mu. Mol/L for the next activity test.

2. Influence on expression of FXR and FXR downstream gene mRNA in HepG2 liver cancer cells

Selection of He in exponential growth phasepG2 hepatoma cells, digested with 0.25% pancreatic enzyme. After digestion is complete, the digestion is terminated with complete medium. Centrifugation, resuspension of cells with complete medium, 1X 10 plating of each well in a six-well plate ⁵ And (4) cells. After 24h, GW4064 and the target compound were added at a concentration of 10. Mu.M. After 24h of treatment, the medium was removed, and PBS was removed by washing the cells once with PBS in a six-well plate. 1ml of trizol was added, left at room temperature for 2min, and the trizol was transferred to an RNase-free EP tube. Mu.l of chloroform was added thereto, and the mixture was vigorously shaken for 15 seconds, allowed to stand at room temperature for 15min, and centrifuged (4 ℃,12000g, 15min). After centrifugation the liquid was divided into three layers, the upper colourless liquid was carefully aspirated and transferred into a new EP tube. Adding equal volume of isopropanol, turning upside down to mix well, standing at room temperature for 5min, and centrifuging (4 deg.C, 12000g, 10min). The supernatant was removed, and to the precipitate was added 1ml of 75% ethanol, gently shaken for 15s, and centrifuged (4 ℃,7500g,5 min). Carefully remove the supernatant, and the precipitate in the tube is dried by air blast standing in a clean bench for 3-5min. Dissolving in 50 μ l DEPC water, and storing in refrigerator at-80 deg.C. The extracted RNA was reverse transcribed into cDNA and the reverse transcription protocol was performed according to the reverse transcription kit instructions (takara, RR 047). The cDNA obtained by reverse transcription was reacted on Stepone plus fluorescent quantitative PCR (ABI). The product double-stranded DNA is subjected to fluorescence quantification by combining with SYBR Green I dye, and the relative expression quantity of mRNA is obtained by the formula 2- ^ΔΔ Ct is calculated.

The experimental results show that compound A4 can up-regulate the transcription levels of FXR mRNA and SHP mRNA, the up-regulated transcription level of FXR mRNA is weaker than that of GW4064 which is a positive drug, the up-regulated transcription level of SHP mRNA is equivalent to that of GW4064 which is a positive drug (figure 2-3), the up-regulated transcription level of BSEP mRNA is weaker than that of GW4064 which is a positive drug, and the down-regulated transcription level of BSREBP-1c mRNA is weaker than that of GW4064 which is a positive drug (figure 4-5).

FXR agonistic Activity assay

HepG2 cells in logarithmic growth phase were prepared as a cell suspension (cell concentration about 2X 10) ⁵ Individual cells/ml) were inoculated into 96-well plates, 37 ℃ C., 5% CO ₂ Culturing in an incubator. After 90% fusion of cells, for each well of cells, 30ng of plasmid (GAL 4-FXRLBD) +50ng of reporter plasmid pGL4.35+20ng of renilla luciferase control vector + were diluted in 25. Mu.L LOPTI-MEM medium40ng pGEM carrier DNA, gently mixed, and then placed at room temperature for about 5min; mu.L of OPTI-MEM medium was diluted to 0.35. Mu.L of liposome 3000 reagent, gently mixed, and then left at room temperature for about 5min. Mixing the two solutions, gently and uniformly blowing, and standing at room temperature for about 20min. The cells in the 96-well plate are washed 2 times with PBS, the mixed solution is added and mixed evenly, and the cell culture box is continued to culture for about 8 hours. After 8 hours, the old medium was replaced with a DMEM medium containing 0.1% DMSO or 10. Mu. Mol/L of GW4064 and Compound A4, and after further culturing for about 18 hours, the temperature between the operations was set to 22 ℃ and light was not used for luciferase activity detection. The detection steps of the dual-luciferase reporter gene system are as follows: the cells were lysed, the old cell culture was discarded, and 100. Mu.L of reporter cell lysate per well was added to fully lyse the cells. After the cells were lysed, the cells in each well were transferred to an EP tube and mixed well by centrifugation. Slowly melting the firefly luciferase detection reagent and the renilla luciferase detection buffer solution to room temperature. Renilla luciferase assay substrate (100X) was placed in an ice bath for use. Adding a renilla luciferase detection substrate (100 x) into a renilla luciferase detection buffer solution according to a ratio of 1. And (3) starting a GLO-MAX 20/20 fluorescence detector, setting detection parameters, wherein the detection interval time is 2s, and the detection time is10 s. When the firefly luciferase activity value F (firefly luciferase) is detected, 100 mu L of firefly luciferase detection reagent is added into each cell sample, after the mixture is uniformly mixed, an EP tube is immediately placed into a Berthold LB941 micropore plate type multifunctional microplate reader, and the value F is calculated. Adding 100 mul of Renilla Luciferase detection working solution, mixing uniformly, immediately placing into a Berthold LB941 micropore plate type multifunctional microplate reader, and calculating the Renilla Luciferase activity value R (Renilla Luciferase). After the determination of all samples is completed, F values and R values detected by a Berthold LB941 micropore plate type multifunctional microplate reader are used for calculating relative luciferase activity values, then the values of all drug treatment groups are normalized to the value of a negative control hole, and the obtained new ratio is counted.

The results of the experiments show that compound A4 is able to significantly activate FXR at a concentration of 10. Mu. Mol/L (FIG. 6).

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt thereof:

n represents 0, 1, 2, 3, 4,5 and 6.

2. The compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein R1, R2 are each independently selected from H, CONH (CH) ₂ )nCH ₃ ，COO(CH ₂ )nCH ₃ ，O(CH ₂ )nCH ₃ And n represents 0, 1 or 2.

3. The compound of formula (I), a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein the compound of formula (I) is selected from any one of the following:

number of R1、R2 A1 -H、-H A2 -H、-CONHCH3 A3 -CONHCH3、-H A4 -COOCH3、-H A5 -COOC2H5、-H A6 -H、-COOCH3 A7 -H、-COC2H5 A8 -OCH3、-H A9 -H、-OCH3

。

4. A process for the preparation of a compound of formula (I) as claimed in claim 1, characterized by the following reaction scheme:

(a)NH ₂ OH·HCl,NaOH,EtOH；(b)NCS,DMF；(c)NaOCH ₃ ,CH ₃ OH,THF；(d)LiAlH4,THF；(e)CBr ₄ ,PPh ₃ DCM, N2; (f) N-Boc-4-hydroxyPiperidine, 18-crown-6, (CH) ₃ ) ₃ COK,THF,N2；(g)TFA,THF；(h-i)bis(trichloromethyl)carbonate,Et ₃ N,DCM。

5. A pharmaceutical combination comprising a compound of formula (I), a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-3, and a pharmaceutically acceptable carrier.

6. Use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-3, in the preparation of an FXR receptor agonist.

7. Use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-3, in the manufacture of a medicament for the treatment or prophylaxis of hyperlipidemia, atherosclerosis, nonalcoholic steatohepatitis, type ii diabetes.