4-amino phenylacetamide derivatives and use thereof

Abstract

The application discloses a 4-amino phenylacetamide derivative and application thereof, and provides derivatives shown in the following formula (I) and formula (II). The compounds or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof are used as farnesol X receptor antagonists, and in vitro FXR antagonistic activity research, HepG2 and L02 cytotoxicity research, HepG2 cell oil red O staining experiment, HepG2 cell triglyceride content test, in vivo anti-non-alcoholic fatty liver disease activity and tissue distribution research are mainly carried out. It is proved that the compound has very strong FXR antagonistic activity, has very high intestinal selectivity, and has the potential of preparing a drug for treating non-alcoholic fatty liver disease and non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of new drug design and synthesis, and specifically relates to the preparation and application of a 4-aminophenylacetamide derivative. Technical Background

[0002] The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor primarily expressed in the liver, intestines, and kidneys. It plays a crucial role in the metabolism of bile acids, cholesterol, lipids, and glucose. FXR is closely associated not only with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), but also with various other diseases such as cholestasis, gallstones, diabetes, atherosclerosis, and cancer. Since the FDA approved obeticholic acid (OCA), a steroidal FXR agonist, as a second-line treatment for primary biliary cholangitis (PBC) in 2016, an increasing number of small molecule compounds targeting FXR agonists have been developed and entered clinical trials, such as LJN452 and MET409 (Kremoser CJ Hepatol. 2021, 75:12-15; Oseini AM, Sanyal AJ, et al. Liver Int. 2017, 37:97-103). However, reported FXR agonists generally have serious adverse reactions, including increased low-density lipoprotein cholesterol (LDL-C) and decreased high-density lipoprotein cholesterol (HDL-C) levels, and causing severe skin itching in some patients. These adverse reactions limit the clinical application of FXR agonists as drugs for the treatment of NASH.

[0003] FXR antagonists have been shown to have good effects in improving metabolic disorders and liver-related diseases. Most reported FXR antagonists are natural products or endogenous bile acid derivatives. The natural FXR antagonist, gugggulsterone (GS), was the first FXR antagonist discovered, but it belongs to a heterogeneous ligand group with poor selectivity (Urizar NL. Science, 2002, 296:1703-1706; Takanori Yamada. Adv Exp Med Biol, 2016, 929:329-361). Endogenous FXR antagonists such as ursodeoxycholic acid (UDCA) were the first marketed FXR antagonist (Sun, L. Nat Med, 2018, 24:1919-1929), approved for primary biliary cholangitis, but its potency is relatively weak (IC50). 50 =90 μM). Tauroursodeoxycholic acid (TUDCA) is a taurine conjugate of UDCA and an FXR antagonist that can inhibit FXR transcriptional activation in a dependent manner (Zangerolamo L. Life Sci, 2021, 272:119252). Naturally derived or endogenous FXR antagonists generally suffer from limited sources, low potency, poor selectivity, and difficulties in synthesis. Although some synthetic FXR antagonists have been reported (Dou X, et al. Eur J Med Chem. 2024, 269:116323), the reported FXR antagonists have limited structural types and also suffer from poor selectivity and poor drug-likeness. The biological functions of FXR vary depending on their tissue distribution. Tissue-specific FXR agonists / antagonists have high concentrations in target tissues but lower total concentrations in systemic circulation and other tissues, making them more likely to achieve good pharmacological activity and safety. Recent studies have shown that intestinal FXR antagonists have the advantage of acting primarily in the gut, avoiding the side effects of systemic release, and have significant effects on improving metabolic diseases such as NAFLD and NASH. In conclusion, the development of novel, highly active, and highly selective intestinal FXR antagonists is of great significance for the development of drugs to treat metabolic diseases such as NAFLD and NASH.

[0004] The applicant has disclosed a class of aminophenylacetamide compounds in a previous patent application (publication number: CN117285504A), in which compound V02-8 (IC) 50 =0.891 μM, compound number 8 in CN117285504A) exhibits strong FXR antagonistic activity.

[0005] The present invention aims to further modify the structure of compound VO2-8 disclosed in the patent application (CN117285504A) to synthesize a series of novel 4-aminophenylacetamide derivatives. Experimental studies have demonstrated that the 4-aminophenylacetamide derivatives have strong FXR antagonistic effects and therapeutic effects on non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0006] The structural formula of V02-8 is as follows:

[0007] Summary of the Invention

[0008] In order to expand the resources of clinical drugs, the purpose of this invention is to provide a new class of 4-aminophenylacetamide derivatives as FXR antagonists for the development of drugs for the treatment of metabolic diseases such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cholestasis, primary biliary cholangitis, and lipid metabolism disorders.

[0009] A first aspect of the present invention is to provide 4-aminophenylacetamide derivatives represented by the following general structural formulas (I) and (II).

[0010]

[0011] in:

[0012] R1 is selected from any one of the following groups:

[0013]

[0014] R2 is selected from any one of the following groups:

[0015]

[0016] In a first aspect of the present invention, the 4-aminophenylacetamide derivatives represented by general structural formulas (I) and (II) are preferably compounds 1-19, with specific structural formulas as follows:

[0017]

[0018] In a second aspect, the present invention provides a method for preparing derivatives as shown in general structural formulas (I) and (II), the preparation route of which is as follows:

[0019]

[0020] The preparation method is as follows:

[0021] Compound III reacts with compound IV under alkaline conditions to give the derivative shown in formula I, and reacts with compound V to give the derivative shown in formula II.

[0022] The synthesis of compound III has been disclosed in CN 117285504A.

[0023] Furthermore, the organic solvent used in the reaction is selected from one of anhydrous tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dichloromethane, and chloroform; the base used is triethylamine; and the catalyst used is 4-dimethylaminopyridine (DMAP).

[0024] A third aspect of the present invention provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cholestasis, primary biliary cholangitis, and lipid metabolism disorders, comprising a derivative of formula (I), a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0025] In a fourth aspect, the invention provides the use of formula (I), derivatives of formula (II), or pharmaceutically acceptable salts thereof as, or pharmaceutical compositions thereof, as FXR antagonists in the preparation of medicaments for treating farnesoid X receptor-related diseases.

[0026] Furthermore, the present invention provides the use of formula (I), derivatives of formula (II) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0027] This invention discloses the unique use of a 4-aminophenylacetamide derivative as an FXR antagonist. The study focused on in vitro FXR antagonistic activity assays, HepG2 and L02 cytotoxicity assays, HepG2 cell Oil Red O staining experiments, HepG2 cell triglyceride content assays, and in vivo anti-nonalcoholic steatohepatitis (NAFLD) activity and tissue distribution studies. The results confirm that the compound described in this invention possesses strong FXR antagonistic activity, high intestinal selectivity, and the potential to develop drugs for treating NAFLD and NAFLD. Attached Figure Description

[0028] Figure 1 The graph shows the results of the test on the effect of compound 10 on triglyceride (TG) content in HepG2 cells;

[0029] Figure 2 This is a schematic diagram of oil red staining of NASH mouse liver by compound 10;

[0030] Figure 3 This is a schematic diagram illustrating the effect of compound 10 on serum triglycerides (TG) in NASH mice;

[0031] Figure 4 This is a schematic diagram illustrating the effect of compound 10 on serum cholesterol (TC) in NASH mice;

[0032] Figure 5 This is a schematic diagram illustrating the effect of compound 10 on serum low-density lipoprotein cholesterol (LDL-C) in NASH mice;

[0033] Figure 6 This is a schematic diagram illustrating the effect of compound 10 on serum high-density lipoprotein cholesterol (HDL-C) in NASH mice;

[0034] Figure 7 This is a schematic diagram illustrating the effect of compound 10 on alanine aminotransferase (ALT) in NASH mice;

[0035] Figure 8 This is a schematic diagram illustrating the effect of compound 10 on aspartate aminotransferase (AST) in NASH mice;

[0036] Figure 9 This is a schematic diagram of the tissue distribution of compound 10. Detailed Implementation

[0037] The present invention will be further illustrated below with reference to embodiments and accompanying drawings, but these are not intended to limit the scope of protection of the present invention.

[0038] Example 1: Preparation of compounds 1-19

[0039] Preparation of ethyl benzoate 1,4-((2-(4-((4-methoxy-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate (compound 1)

[0040]

[0041] Under nitrogen protection, ethyl 4-((2-(4-(((3-methylbenzyl)amino)phenyl)acetamido)methyl)benzoate (III, 1.22 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), followed by the sequential addition of 4-dimethylaminopyridine (DMAP, 0.12 mmol), triethylamine (2.44 mmol), and 4-methoxybenzenesulfonyl chloride (1.83 mmol). The reaction was carried out at 0-5 °C for 6 h. The tetrahydrofuran was removed by vacuum distillation, and the mixture was extracted with distilled water (30 mL) and ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL), dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by vacuum distillation. Petroleum ether / ethyl acetate was used as the eluent, and the mixture was purified by column chromatography to give compound 1; yield 71.6%, white solid. 1H NMR (400MHz, DMSO-d6) δ8.62(t,J=5.9Hz,1H),7.88(d,J=8.2Hz,2H),7.58(d,J=8.9Hz, 2H),7.31(d,J=8.2Hz,2H),7.16(d,J=8.4Hz,2H),7.13(t,J=3.6Hz,2H),7.10(s,1H),7 .06(s,1H),7.02(d,J=7.0Hz,2H),6.99(d,J=6.6Hz,2H),4.70(s,2H),4.32(d,J=5.6Hz ,2H),4.31–4.26(m,2H),3.86(s,3H),3.43(s,2H),2.21(s,3H),1.30(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.39,166.03,163.14,145.49,137.85,137.69,136.76,136.05,130.10,129.99,129.82,129.60,128 .93,128.88,128.61,128.49,127.67,125.51,114.91,61.10,56.18,53.82,42.36,42.17,21.40,14.62; HRMS(m / z)(ESI):calcd for C 33 H 34 N₂NaO₆S[M+Na] + :609.2035; found:609.2037.

[0042] Preparation of ethyl benzoate (compound 2) of 2,4-((2-(4-((N-(3-methylbenzyl)-2,3-dihydrobenzofuran)-6-sulfonamide)phenyl)acetamido)methyl)benzoate:

[0043]

[0044] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 2,3-dihydrobenzofuran-6-sulfonyl chloride, with all other conditions remaining the same. Compound 2: 73.7% yield, white solid; 1H NMR (400MHz, DMSO-d6) δ8.63(t,J=5.9Hz,1H),7.88(d,J=8.3Hz,2H),7.54(d,J=1.6Hz,1H),7. 36(dd,J=8.4,2.0Hz,1H),7.31(d,J=8.3Hz,2H),7.17(d,J=8.4Hz,2H),7.12(d,J=7.5Hz,1H),7 .07(s,1H),7.06–7.01(m,3H),7.00(d,J=7.6Hz,1H),6.91(d,J=8.4Hz,1H),4.72–4.64(m,4H) ,4.31(q,J=7.1Hz,4H),3.44(s,2H),3.24(t,J=8.8Hz,2H),2.22(s,3H),1.31(t,J=7.1Hz,3H); 13 CNMR(101MHz,DMSO-d6)δ170.41,166.03,163.93,145.49,137.84,137.81,136.85,136.00,130.00,129.78,129.60,129.41,129.24,128.94 ,128.87,128.61,128.46,127.66,125.51,125.18,109.52,72.76,61.10,53.86,42.36,42.18,28.93,21.41,14.63; HRMS(m / z)(ESI):calcd for C 34 H 34 N₂NaO₆S[M+Na] + :621.2035; found:621.2036.

[0045] Preparation of ethyl benzoate (compound 3) of 3,4-((2-(4-((3-fluoro-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate:

[0046]

[0047] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 3-fluorobenzenesulfonyl chloride, with all other conditions remaining the same. Compound 3: 80.12% yield, pale yellow solid; 1H NMR (400MHz, DMSO-d6) δ8.65(t,J=5.9Hz,1H),7.89(d,J=8.2Hz,2H),7.73–7.56(m,2H),7.50(t,J=7.7Hz,2H),7.31(d,J=8.2Hz,2H),7.20(d,J=8.4 Hz,2H),7.14(t,J=7.5Hz,1H),7.09(s,1H),7.04(d,J=8.5Hz,4H),4.80(s ,2H),4.36–4.27(m,4H),3.46(s,2H),2.23(s,3H),1.31(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.36,166.03,163.47,161.00,145.48,140.51,13 7.93,137.19,136.50,136.49,132.17,132.10,129.97,129.60,128.98,128. 89,128.71,128.67,128.61,127.64,125.55,124.08,120.91,120.70,114.9 1,114.67,61.09,54.23,42.37,42.18,21.39,14.61; HRMS(m / z)(ESI):calcd for C 32 H 31 FN2NaO5S[M+Na] + :597.1834; found:597.1835.

[0048] Preparation of ethyl benzoate 4,4-((2-(4-((2-methoxy-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate (compound 4):

[0049]

[0050] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 2-methoxybenzenesulfonyl chloride, with all other conditions remaining the same. Compound 4: 73.74% yield, white solid; 1H NMR (400MHz, DMSO-d6) δ8.60(t,J=5.9Hz,1H),7.87(d,J=8.2Hz,2H),7.66–7.59(m,2H),7.29(d,J=8.1Hz,3H),7.16(dd,J=14.3,8.9Hz ,2H),7.11(s,2H),7.06–6.97(m,4H),4.95(s,2H),4.32(q,J=6.1Hz,4H),3.98(s,3H),3.39(s,2H),2.25(s,3H),1.32(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.39,166.04,157.01,145.47,138.00,137.85,137.63,135.68,135.59,131.31,129.86,129.59,128.85,128.71, 128.65,128.36,128.24,127.63,127.05,125.26,120.58,113.36,61.11,56.61,55.39,42.33,42.10,21.46,14.64; HRMS(m / z)(ESI):calcd for C 33 H 34 N₂NaO₆S[M+Na] + :609.2035; found:609.2037.

[0051] Preparation of ethyl benzoate (compound 5) of 5,4-((2-(4-((N-(3-methylbenzyl)-4-propylphenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate:

[0052]

[0053] Following the preparation method of compound 1, 4-propylbenzenesulfonyl chloride was substituted for 4-methoxybenzenesulfonyl chloride, with all other conditions remaining the same. Compound 5: Yield 78.25%, yellow gel-like substance; 1H NMR (400MHz, DMSO-d6) δ8.63(t,J=5.5Hz,1H),7.88(d,J=8.0Hz,2H),7.57(d,J=8 .0Hz,2H),7.43(d,J=8.0Hz,2H),7.31(d,J=7.9Hz,2H),7.17–7.11(m,3H),7.07–7 .6.99(m,4H),4.73(s,2H),4.32(d,J=6.7Hz,2H),3.43(s,2H),2.70–2.65(m,2H) ,2.22(s,3H),1.64(q,J=7.4Hz,2H),1.31(t,J=7.0Hz,3H),0.91(t,J=7.3Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.39,166.04,157.01,145.47,138.00,137.85,137.63,135.68,135.59,131.31,129.86,129.59,128.85,128.71, 128.65,128.36,128.24,127.63,127.05,125.26,120.58,113.36,61.11,56.61,55.39,42.33,42.10,21.46,14.64; HRMS(m / z)(ESI):calcd for C 35 H 38 N₂NaO₅S[M+Na] + :621.2396; found:621.2399.

[0054] Preparation of ethyl benzoate 6,4-((2-(4-((2,5-difluoro-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate (compound 6):

[0055]

[0056] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 2,5-difluorobenzenesulfonyl chloride, with all other conditions remaining the same. Compound 6: 76.8% yield, pale yellow solid; 1H NMR (400MHz, DMSO-d6) δ8.64(t,J=5.5Hz,1H),7.88(d,J=7.9Hz,2H),7.71–7.57(m,2H),7.46(s,1H),7.30(d,J=7.9Hz,2H),7.17(dd,J=15 .7,7.8Hz,3H),7.12–7.06(m,3H),7.04(d,J=6.8Hz,2H),4.89(s,2H),4.48–4.21(m,4H),3.44(s,2H),2.24(s,3H),1.32(t,J=7.0Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ170.32,166.03,145.46,138.00,136.65,136.58,136.51,130.09,129.59,128.95,128. 74,127.62,125.53,123.11,117.77,117.50,61.10,54.64,42.34,42.14,21.40,14.63; HRMS(m / z)(ESI):calcd for C 32 H 30 F₂N₂NaO₅S[M+Na] + :615.1741; found:615.1743.

[0057] Preparation of ethyl benzoate (compound 7) of 7,4-((2-(4-((3,5-difluoro-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate:

[0058]

[0059] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 3,5-difluorobenzenesulfonyl chloride, with all other conditions remaining the same. Compound 7: 74.13% yield, yellow gel-like substance; 1H NMR(400MHz,DMSO-d6)δ8.66(t,J=6.0Hz,1H),7.87(d,J=8.3Hz,2H),7.73(s,1H ),7.40–7.35(m,2H),7.30(d,J=8.2Hz,2H),7.20(d,J=8.4Hz,2H),7.14(t,J=7. 5Hz,1H),7.10–7.02(m,2H),7.01(d,J=7.1Hz,2H),4.82–4.79(m,2H),4.35–4.2 9(m,2H),4.29(t,J=7.1Hz,2H),3.45(s,2H),2.23(s,2H),1.30(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.39,170.36,166.03,145.50,141.55,137.98,13 7.98,137.92,136.94,136.73,136.36,130.06,129.94,129.60,129.00,128. 86,128.79,128.72,128.68,127.63,127.61,125.58,111.75,111.55,111.4 7,109.53,61.10,54.39,42.33,42.17,21.40,14.61; HRMS(m / z)(ESI):calcd for C 32 H 30 F2N2O5NaS[M+Na] + :615.1741; found:615.1745.

[0060] Preparation of 8,4-((2-(4-((3,4-difluoro-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate (compound 8):

[0061]

[0062] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 3,4-difluorobenzenesulfonyl chloride, with all other conditions remaining the same. Compound 8: Yield 68.61%, white solid; 1H NMR (400MHz, DMSO-d6) δ8.66(t,J=6.0Hz,1H),7.87(d,J=8.3Hz,2H),7.73(tt,J=9. 1,2.3Hz,1H),7.42–7.34(m,2H),7.30(d,J=8.2Hz,2H),7.20(d,J=8.4Hz,2H),7.14 (t,J=7.5Hz,1H),7.07(d,J=4.2Hz,2H),7.03(d,J=8.4Hz,2H),4.80(s,2H),4.35–4 .29(m,2H),4.29(t,J=7.1Hz,2H),3.45(s,2H),2.23(s,3H),1.30(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.40,166.04,152.43,150.86,150.76,149.96,14 5.50,137.89,137.47,136.66,136.29,130.37,129.91,129.62,128.96,128 .87,128.69,128.65,128.55,127.66,125.60,125.54,115.68,115.47,114. 93,65.38,61.10,53.98,42.35,42.17,21.40,14.61; HRMS(m / z)(ESI):calcd for C 32 H 30 F2N2O5S[M+Na] + :615.1741; found:615.1746.

[0063] Preparation of 9,4-((2-(4-((4-isopropyl-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoate (compound 9):

[0064]

[0065] Following the preparation method of compound 1, 4-isopropylbenzenesulfonyl chloride was substituted for 4-methoxybenzenesulfonyl chloride, with all other conditions remaining the same. Compound 9: Yield 79.84%, pale yellow gel-like substance; 1H NMR (400MHz, DMSO-d6) δ8.65(t,J=6.0Hz,1H),7.88(d,J=8.3Hz,2H),7.59(d,J=8 .4Hz,2H),7.48(d,J=8.5Hz,2H),7.31(d,J=8.3Hz,2H),7.17(d,J=8.5Hz,2H),7.1 2(t,J=7.5Hz,1H),7.07–6.97(m,5H),4.73(s,2H),4.34–4.26(m,4H),3.44(s,2H) ,3.01(p,J=6.9Hz,1H),2.21(s,3H),1.30(t,J=7.1Hz,3H),1.24(d,J=6.9Hz,6H); 13 C NMR(101MHz,DMSO-d6)δ170.41,166.03,154.38,145.52,137.86,137.60,136.73,136.14,136.12,129.86,129.62,128.94,128.87,128.63 ,128.60,128.52,127.97,127.71,127.67,125.53,112.64,61.10,53.85,42.35,42.17,33.86,23.91,21.41,14.62; HRMS(m / z)(ESI):calcd for C 35 H 38 N₂NaO₅S[M+Na] + :621.2399; found:621.2404.

[0066] Preparation of 10.4-((2-(4-((4-(tert-butyl)-N-(3-methylbenzyl)phenyl)sulfonylamino)phenyl)acetamido)methyl)benzoic acid (compound 10):

[0067]

[0068] Following the preparation method of compound 1, 4-tert-butylbenzenesulfonyl chloride was substituted for 4-methoxybenzenesulfonyl chloride, with all other conditions remaining the same. Compound 10: Yield: 70.56%, white solid; 1H NMR(400MHz,DMSO-d6)δ8.67(t,J=6.0Hz,1H),7.89(d,J=8.3Hz,2H),7.71–7 .55(m,4H),7.32(d,J=8.0Hz,2H),7.19(d,J=8.5Hz,2H),7.17–7.09(m,2H),7 .06(d,J=8.1Hz,2H),7.02(d,J=6.8Hz,2H),4.74(s,2H),4.32(dd,J=8.4,4. 5Hz,4H),4.31–4.27(m,2H),2.22(s,3H),1.33(s,9H),1.30(d,J=7.0Hz,3H); 13 CNMR(101MHz,DMSO-d6)δ170.42,166.03,156.60,147.79,145.80,145.51,140.79, 137.86,137.61,136.73,136.12,135.94,129.87,129.62,129.59,128.94,128.87,1 28.63,128.60,127.71,127.67,126.61,125.53,124.70,123.65,112.65,61.10,53. 85,47.06,42.36,35.40,31.26,21.55,21.41,14.65,14.62; HRMS(m / z)(ESI):calcd forC 36 H 40 N₂NaO₅S[M+Na] + :635.2556; found:635.2557.

[0069] Preparation of ethyl benzoate 11,4-((2-(4-(2,6-difluoro-N-(3-methylbenzyl)benzoylamino)phenyl)acetamido)methyl)benzoate (compound 11):

[0070]

[0071] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 2,6-difluorobenzoyl chloride, with all other conditions remaining the same. Compound 11: Yield 72.87%, white gel-like substance; 1H NMR (400MHz, DMSO-d6) δ8.60(t,J=6.0Hz,1H),7.87(d,J=8.3Hz,2H),7.33(dd,J=8.4,6.7Hz,1H),7.27(d,J=8.2Hz,2H),7.24–7.19(m,1H),7 .15–7.09(m,3H),7.07(d,J=7.2Hz,2H),7.02–6.94(m,4H),5.04(s,2H ),4.34–4.26(m,4H),3.36(s,2H),2.27(s,3H),1.32(t,J=7.1Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ170.35,168.95,166.02,145.49,141.10,140.89,138.00,137.57,135.42,130.57,130.10,129.59,129.45,128. 86,128.81,128.77,128.32,128.03,127.64,125.59,125.23,122.88,61.10,60.22,42.32,42.04,21.47,14.61; HRMS(m / z)(ESI):calcd forC 33 H 30 F₂N₂NaO₄[M+Na] + :579.2071; found:579.2075.

[0072] Preparation of ethyl benzoate 12,4-((2-(4-(3-(3-methoxy-N-(3-methylbenzyl)benzoylamino)phenyl)acetamido)methyl)benzoate (compound 12):

[0073]

[0074] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 3-methoxybenzoyl chloride, with all other conditions remaining the same. Compound 12: Yield 76.84%; pale yellow gel-like substance; 1H NMR (400MHz, DMSO-d6) δ8.60(s,1H),7.87(d,J=8.3Hz,2H),7.29(d,J=8.3Hz,2H),7.18(t,J=7.5Hz,1H),7.16–7.06(m,5H),7.03(t ,J=7.8Hz,3H),6.92–6.80(m,3H),5.04(s,2H),4.31(q,J=7.2Hz,4H),3.59(s,3H),3.38(s,2H),2.25(s,3H),1.31(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.41,169.80,166.05,158.91,145.52,141.82,137.92,137.89,134.98,129.96,129.62,129.38,128.85,128 .75,128.21,127.77,127.65,125.17,121.11,116.02,114.12,61.12,55.47,53.27,42.33,42.08,21.49,14.64; HRMS(m / z)(ESI):calcd for C 34 H 34 N₂NaO₅[M+Na] + :573.2365; found:573.2369.

[0075] Preparation of ethyl benzoate 13,4-((2-(4-(3-methyl-N-(3-methylbenzyl)benzoamide)phenyl)acetamido)methyl)benzoate (compound 13):

[0076]

[0077] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 3-methylbenzoyl chloride, with all other conditions remaining the same. Compound 13: Yield 73.22%, white gel-like substance; 1H NMR(400MHz, DMSO-d6)δ8.60(t,J=6.0Hz,1H),7.89–7.84(m,2H),7.28(d,J=8.2Hz,2H),7.22–7.16(m,2H),7.12–7.06(m,5H),7.04(t, J=6.1Hz,3H),6.99(d,J=8.3Hz,3H),5.04(s,2H),4.31(q,J=7.1Hz,4H),3.38(s,2H),2.25(s,3H),2.18(s,3H),1.31(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.43,170.22,166.04,145.53,141.87,137.98,137.92,137.59,136.60,134.91,130.60,129.91,129.61,129.50, 128.84,128.73,128.19,127.96,127.80,127.64,125.81,125.14,61.12,53.23,42.32,42.10,21.49,21.23,14.65; HRMS(m / z)(ESI):calcd for C 34 H 34 N₂NaO₄[M+Na] + :557.2416; found:557.2421.

[0078] Preparation of ethyl benzoate 14,4-((2-(4-(2-methoxy-N-(3-methylbenzyl)benzoylamino)phenyl)acetamido)methyl)benzoate (compound 14):

[0079]

[0080] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 2-methoxybenzoyl chloride, with all other conditions remaining the same. Compound 14: Yield 81.8%, white gel-like substance; 1H NMR (400MHz, DMSO-d6) δ8.56(t,J=6.0Hz,1H),7.87(d,J=8.2Hz,2H),7.27(d,J= 8.0Hz,2H),7.24–7.15(m,4H),7.12(d,J=6.8Hz,1H),7.05(d,J=7.5Hz,1H),7.00 (d,J=8.1Hz,2H),6.94(d,J=8.1Hz,2H),6.81(q,J=8.4,7.9Hz,2H),5.01(s,2H), 4.34–4.27(m,4H),3.62(s,3H),3.32(s,2H),2.29(s,3H),1.32(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.38,168.53,166.05,155.13,145.52,141.03,137.98,137.85,135.14,130.63,129.61,129.30,128.83,128.70, 128.64,128.50,128.08,127.59,127.06,124.96,120.50,111.51,61.13,55.65,52.31,42.30,42.06,21.55,14.65; HRMS(m / z)(ESI):calcd for C 34 H 34 N₂NaO₅[M+Na] + :573.2365; found:573.2369.

[0081] Preparation of ethyl benzoate 15,4-((2-(4-(3-ethoxy-N-(3-methylbenzyl)benzoamide)phenyl)acetamido)methyl)benzoate (compound 15):

[0082]

[0083] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 3-ethoxybenzoyl chloride, with all other conditions remaining the same. Compound 15: Yield 82.3%, white gel-like substance; 1H NMR (400MHz, DMSO-d6) δ8.60(t,J=6.0Hz,1H),7.88(d,J=8.3Hz,2H),7.28(dd,J=11.9,8. 5Hz,4H),7.18(t,J=7.5Hz,1H),7.14–7.09(m,3H),7.05(dd,J=14.6,7.7Hz,2H),7.01–6. 98(m,2H),6.73(d,J=8.8Hz,2H),5.04(s,2H),4.35–4.31(m,2H),4.31–4.27(m,2H),3.93 (q,J=6.9Hz,2H),3.40(s,2H),2.26(s,3H),1.32(d,J=7.1Hz,3H),1.27(d,J=7.0Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.45,169.72,166.03,159.90,145.53,142.41, 138.18,137.87,134.63,131.82,131.01,130.00,129.60,128.85,128.69 ,128.34,128.13,127.64,127.57,125.10,114.63,113.87,63.89,63.54, 61.11,53.41,42.33,42.10,21.50,14.95,14.64; HRMS(m / z)(ESI):calcd forC 35 H 36 N₂NaO₅[M+Na] + :587.2522; found:587.2527.

[0084] Preparation of ethyl benzoate 16,4-((2-(4-(N-(3-methylbenzyl)cyclobutaneformylamino)phenyl)acetamido)methyl)benzoate (compound 16):

[0085]

[0086] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with cyclobutaneformyl chloride, with all other conditions remaining the same. Compound 16: 80.8% yield, yellow gel-like substance; 1H NMR (400MHz, CDCl3) δ7.99–7.95(m,2H),7.25(d,J=4.4Hz,2H),7.23(d,J=4.3Hz ,2H),7.10(t,J=7.5Hz,2H),7.02(d,J=9.3Hz,2H),6.92(d,J=8.1Hz,2H),5.89(d ,J=6.1Hz,1H),4.81(s,2H),4.36(q,J=7.1Hz,2H),3.61(s,2H),2.27(s,3H),1. 75–1.73(m,2H),1.70(d,J=5.4Hz,2H),1.38(t,J=7.1Hz,3H),1.28–1.23(m,2H); 13 C NMR (101MHz, CDCl3) δ174.54,170.36,166.21,143.03,141.33,138.02,137.45,134.37,130.18,129.94,129.75,129.27,128.93 ,128.17,128.03,127.27,125.57,61.06,52.96,43.31,43.14,38.14,29.70,25.70,21.38,17.78,14.32; HRMS(m / z)(ESI):calcd for C 31 H 34 N₂NaO₄[M+Na] + :521.2416; found:521.2420.

[0087] Preparation of ethyl benzoate 17,4-((2-(4-(4-(4-chloro-N-(3-methylbenzyl)benzoylamino)phenyl)acetamido)methyl)benzoate (compound 17):

[0088]

[0089] Following the preparation method of compound 1, 4-chlorobenzoyl chloride was substituted for 4-methoxybenzenesulfonyl chloride, with all other conditions remaining the same. Compound 17: white solid, yield 70.4%; 1H NMR (600MHz, DMSO-d6) δ8.60(t,J=5.9Hz,1H),7.87(d,J=8.2Hz,2H),7.34(d,J=8.4Hz,2H),7.29(dd,J=8.1,5.3Hz,4H),7.18(t,J=7.5Hz,1H),7 .13–7.09(m,3H),7.07(d,J=7.7Hz,1H),7.05–7.00(m,3H),5.04(s,2H) ,4.30(q,J=7.1Hz,4H),3.39(s,2H),2.26(s,3H),1.31(t,J=7.0Hz,3H); 13 C NMR(151MHz,DMSO-d6)δ170.43,170.09,166.04,145.87,145.52,142.10,138.06,137.90,134.79,134.00,129.97,129.59,129.05,128 .87,128.71,128.17,127.69,127.65,127.59,125.12,61.11,53.42,42.34,42.09,28.28,21.49,15.48,14.64; HRMS(m / z)(ESI):calcd for C 33 H 32 ClN2O4[M+H] + :555.2051; found:555.2159.

[0090] Preparation of ethyl benzoate 18,4-((2-(4-(4-(4-fluoro-N-(3-methylbenzyl)benzoylamino)phenyl)acetamido)methyl)benzoate (compound 18):

[0091]

[0092] Following the preparation method of Compound 1, 4-fluorobenzoyl chloride was substituted for 4-methoxybenzenesulfonyl chloride, with all other conditions remaining the same. Compound 18: white solid, yield 81.3%; 1H NMR (600MHz, DMSO-d6) δ8.60(t,J=5.9Hz,1H),7.87(d,J=8.2Hz,2H),7.38(dd,J=8.4,5.6Hz,2H),7.29(d,J=8.2Hz,2H),7.18(t,J=7.5Hz,1H ),7.11(d,J=8.4Hz,3H),7.06(m,4H),7.00(d,J=8.2Hz,2H),5.04(s,2H),4.37–4.26(m,4H),3.38(s,2H),2.26(s,3H),1.31(t,J=7.1Hz,3H); 13 CNMR(151MHz,DMSO-d6)δ170.39,169.19,166.03,145.52,141.75,137.94,137.85,135.03,133.13,131.42,131.37,130.03,129.60, 128.86,128.74,128.23,127.83,127.66,125.17,115.33,115.18,61.11,53.31,42.33,42.06,21.49,14.64; HRMS(m / z)(ESI):calcd for C 33 H 31 FN₂O₄Na[M+Na] + :561.2175; found:561.2166.

[0093] Preparation of ethyl benzoate 19,4-((2-(4-(N-(3-methylbenzyl)-4-(trifluoromethyl)benzoylamino)phenyl)acetamido)methyl)benzoate (compound 19):

[0094]

[0095] Following the preparation method of compound 1, 4-methoxybenzenesulfonyl chloride was replaced with 4-(trifluoromethyl)benzoyl chloride, with all other conditions remaining the same. Compound 19: white solid, yield 75.5%; 1H NMR (600MHz, DMSO-d6) δ8.60(s,1H),7.87(d,J=8.2Hz,2H),7.60(d,J=7.6Hz,2H),7.55(d,J=7.4Hz,2H),7.28(d,J=8.1Hz,2H),7.19(t, J=7.5Hz,1H),7.13–7.07(m,4H),7.05(d,J=6.9Hz,3H),5.06(s,2H),4.44–4.24(m,4H),3.38(s,2H),2.26(s,3H),1.31(t,J=6.3Hz,3H); 13 C NMR(151MHz,DMSO-d6)δ170.34,168.94,166.02,145.49,140.89,138.00,137.57,135.42,130.09,129.59,129.46,128.85,128.78 ,128.32,128.03,127.64,125.29,125.24,125.14,61.10,53.26,42.32,42.04,31.62,30.29,21.48,14.62; HRMS(m / z)(ESI):calcd for C 34 H 31 F3N2O4Na[M+Na] + :611.2140; found:611.2134.

[0096] Example 2: FXR antagonistic activity test

[0097] This invention utilizes a dual-luciferase reporter gene assay to test the antagonistic effects of all synthesized compounds on FXR. HEK293T cells were seeded at a density of 20,000 cells / well in 96-well blank plates and grown overnight at 37°C with 5% CO2 until reaching 50%–60% confluency. The culture medium was then replaced with serum-free DMEM. Plasmids pCDNA-FXR, PGL3-FXRE-luc, PRL-TK, and Lipo transfection reagent were mixed and incubated for 15 min. The mixture was then added to 96-well plates (10 μL / well) for transfection. Six hours after transfection, the medium was replaced with DMEM containing 10% fetal bovine serum, and different concentrations of the compounds and 5 μM GW4064 (a potent FXR agonist) were added for 24 hours. After 24 hours, the culture medium was removed, and diluted lysis buffer was added to lyse the cells for 20 minutes. The cells were centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected. The FXR antagonistic activity was measured using a dual-luciferase reporter gene assay kit (UElandy) on a microplate reader. The antagonistic rate was calculated using the formula: Inhibition rate (%) = (Max - X) / (Max - Min) × 100%. Results were expressed as firefly fluorescence value / sea urchin fluorescence value. Max represents the result value after adding 5 μM GW4064, X represents the result value after adding the test compound, and Min represents the negative control result value after adding DMSO. The half-maximal inhibitory concentration (IC50) of the compound was calculated using a nonlinear regression (curve fitting) method. 50 The results are shown in Table 1.

[0098] Example 3: Cytotoxicity Detection

[0099] This invention utilizes the MTT assay to test the cytotoxicity of all synthesized compounds against HepG2 and L02 cells. HepG2 and L02 cells were uniformly seeded in 96-well plates and incubated for 18 hours until cell attachment. After cell attachment, the original culture medium was removed, and the cells were incubated for 24 hours with drug concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μM, respectively. Then, 20 μL of MTT solution was added to each well, and the cells were incubated in the dark for 4 hours. After incubation, the culture medium was aspirated, and 150 μL of DMSO was added to each well, followed by low-speed shaking to dissolve any crystals. The absorbance of each well was measured at 490 nm. Cell viability was calculated using the formula: Cell viability (%) = (OD0.05) / (OD0.05) 待测 -OD 空白 ) / (OD 对照 -OD 空白 ×100. The half-maximal inhibitory concentration (IC50) of the compound on cells was calculated using a nonlinear regression (curve fitting) method. 50 The results are shown in Table 1.

[0100] Table 1: FXR antagonistic activity and cytotoxicity results of the compounds

[0101]

[0102] Data are presented as the average of at least three independent replicates. TUDCA (tauroursodeoxycholic acid) and GS (bimethasterone) were positive control compounds.

[0103] Table 1 shows that the FXR antagonistic activities of the 19 4-aminophenylacetamide derivatives synthesized in this invention are significantly stronger than those of the positive control drug TUDCA (IC). 50 =56.20μM) and GS(IC 50 =61.83 μM). Additionally, compared to the published compound VO2-8, the IC50 value was... 50 With a concentration of 1.21 μM, compounds 8, 9, 10, 13, and 14 synthesized in this invention exhibited stronger FXR antagonistic activity. Among them, compound 10 showed the strongest FXR antagonistic activity (IC50). 50 =0.60 μM), its activity was increased by 93 times, 103 times and 2 times compared with TUDCA, GS and VO2-8, respectively, belonging to a potent FXR antagonist. In addition, the compound synthesized in this invention has essentially no cytotoxicity to HepG2 cells and L02 cells (IC50). 50 >100μM). In summary, the above data show that the compounds synthesized in this invention are highly efficient and low in toxicity in vitro.

[0104] Example 4: Determination of triglyceride (TG) content in HepG2 cells, referring to... Figure 1 .

[0105] This invention further uses free fatty acids (FFA) to induce HepG-2 cells to construct a cell steatosis model, and examines the effect of the preferred compound 10 in improving cell steatosis.

[0106] HepG2 cells in logarithmic growth phase were seeded into 6-well plates. When the cells reached 80%–90% confluence, the old culture medium was discarded, and the cells were starved for 16 h in DMEM medium containing 1% bovine serum albumin. Then, 0.6 mmol of free fatty acids (oleic acid:palmitic acid = 2:1) was added to induce a fatty degeneration model for 12 h. The cells were then cultured with different concentrations of compounds for 24 h (three replicates per compound). After compound treatment, each well was washed twice with 1 mL of PBS, followed by centrifugation at 1000 rpm for 5 min with 0.25% trypsin added. The supernatant was discarded. Lysis buffer (1% Triton X-100) was added to the cell pellet to lyse the cells for 30 min. After thorough shaking and lysis, 2.5 μL of the supernatant was mixed with 250 μL of a pre-mixed triglyceride detection working solution and incubated at 37°C for 10 min. The absorbance was measured using a microplate reader (510 nm). The triglyceride concentration was calculated using the formula: Sample concentration (mmol / g protein) = (OD200 / g protein) / (250 μL / g protein) * (OD20 ... 样品 -OD 空白 ) / (OD 标准 -OD 空白 × standard concentration (mmol / L) / protein concentration (gprotein / L).

[0107] from Figure 1 As can be seen, compared with the model group, compound 10 at different concentrations significantly reduced intracellular TG levels in HepG2 cells, and the high-dose group of compound 10 (10 μM) was more effective than the positive control group (50 μM TUDCA and 10 μM M02-8). Data are expressed as mean ± SEM. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001.

[0108] Example 5: Pharmacodynamic experiment of anti-NASH mice, referring to... Figures 2 to 8 .

[0109] This invention uses a high-fat diet and chemical induction to establish a NASH mouse model with mild to moderate liver fibrosis, in order to examine the in vivo anti-NASH activity of compound 10.

[0110] Six-week-old male C57BL / 6J mice were randomly divided into four groups: a normal group, a model group (NASH group), a positive compound VO2-8 group (10 mg / kg), a TUDCA group (50 mg / kg), and low (1 mg / kg), medium (5 mg / kg), and high (10 mg / kg) dose groups of compound 10, with 10 mice in each group. The normal group was fed a standard diet, while the other groups were fed a high-fat diet for 17 consecutive weeks. In the last four weeks, the VO2-8 group, the TUDCA group, and the high, medium, and low dose groups of the test compound 10 were administered the corresponding doses of the drug via gavage. The normal group and the model group were administered an equal volume of 0.5% CMC-Na solution via gavage, and the model group and the drug-treated groups were injected intraperitoneally with 3.5% CCl4 solution twice a week. Throughout the experimental period, the mice's mental status was observed, and their weight was recorded once a week. At the end of the experiment, the mice were fasted for 12 hours without access to water or food. Blood was collected from the abdominal aorta after anesthesia with ether, the organs were separated, washed with physiological saline, weighed, and recorded. Oil Red O staining was used to observe the hepatic steatosis in mice. Serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using a biochemical analyzer.

[0111] from Figure 2 It can be seen that, after 4 weeks of continuous administration, different concentrations of compound 10 improved hepatic steatosis in mice compared to the NASH group. Figures 3 to 6 It can be seen that, after 4 weeks of continuous administration, compared with the NASH group, different concentrations of compound 10 can reduce serum TG, TC, and LDL-C levels and increase HDL-C levels in NASH mice. The medium (5 mg / kg) and high (10 mg / kg) doses of compound 10 are also superior to the VO2-8 and TUDCA groups in reducing serum TG, TC, and LDL-C levels and increasing HDL-C levels in NASH mice. Furthermore, from... Figure 7 , Figure 8 As can be seen, compared with the NSAH group, the medium (5 mg / kg) and high (10 mg / kg) dose groups of compound 10 significantly reduced the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and their effects were also superior to the VO2-8 group and the TUDCA group. Data are expressed as mean ± SEM. Compared with the NSAH group, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0112] In summary, the above results indicate that compound 10 can effectively regulate blood lipid levels in NASH mice and improve dyslipidemia, with better effects than VO2-8 and TUDCA.

[0113] Example 6: Tissue distribution results of preferred compound 10, referring to Figure 9 .

[0114] To determine the tissue distribution of compound 10 in vivo, we conducted an oral tissue distribution study in mice. Mice were administered compound 10 (10 mg / kg) by gavage, and sacrificed at 0.5 h, 2 h, and 8 h. Plasma, liver, and small intestinal tissues were collected for compound concentration analysis. The tissue distribution results of compound 10 are shown below. Figure 9 The results showed that after oral administration of compound 10 to mice, the concentration of the compound in the small intestine was higher than that in the liver and plasma at 0.5h, 2h and 8h, which preliminarily confirmed that compound 10 can selectively accumulate in the intestine in vivo and is a potential intestinal FXR antagonist.

Claims

1. A 4-aminophenylacetamide derivative represented by general structural formula (I), characterized in that, The general structural formula (I) is as follows: Where R1 is: 、 、 、 、 、 、 、 、 。 2. The 4-aminophenylacetamide derivative as described in claim 1, characterized in that, The 4-aminophenylacetamide derivative is any one of the following compounds 1, 3-10, with the following specific structural formula: 。 3. The method for preparing 4-aminophenylacetamide derivatives as described in claim 1 or 2, characterized in that, The preparation route is as follows: ； The preparation method is as follows: Compound III was reacted with compound IV under alkaline conditions to obtain the derivative shown in formula I; The organic solvent used in the reaction is selected from one of anhydrous tetrahydrofuran, N,N-dimethylformamide, dichloromethane, and trichloromethane; The base used was triethylamine; The catalyst used was 4-dimethylaminopyridine.

4. The use of the 4-aminophenylacetamide derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as a farnesoid X receptor antagonist in the preparation of a medicament for treating farnesoid X receptor-related diseases.

5. The use of the 4-aminophenylacetamide derivative of claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating non-alcoholic fatty liver disease.

6. The use of the 4-aminophenylacetamide derivatives of claim 1 or 2, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for treating non-alcoholic steatohepatitis, liver fibrosis and cholestasis, primary biliary cholangitis, and lipid metabolism disorders.

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