Amino phenyl acetamide derivative, preparation method and application thereof

Abstract

The application discloses an amino phenyl acetic amide derivative, a preparation method thereof, and a unique use of the derivative as a farnesol X receptor antagonist. In vitro FXR antagonistic activity research, HepG2 and L02 cell toxicity research, HepG2 cell oil red O staining experiment, HepG2 cell triglyceride content test, and in vivo anti-non-alcoholic fatty liver disease activity research are mainly carried out. It is proved that the compound has very strong farnesol X receptor antagonistic activity, and has the potential to prepare a medicine 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 an aminophenylacetamide derivative, its preparation method and application. Technical Background

[0002] Nonalcoholic steatohepatitis (NASH) is a metabolic-associated fatty liver disease and a more severe form of nonalcoholic fatty liver disease (NAFLD). Its pathological manifestations include hepatic steatosis, hepatocellular carcinoma, ballooning lesions, and inflammation and damage; in severe cases, liver fibrosis may also occur. Uncontrolled NASH can progress to cirrhosis, liver failure, and ultimately liver cancer. Currently, the global prevalence of NAFLD is as high as 25%, and approximately 15-20% of NAFLD patients also have NASH. However, there are currently no effective drugs on the market to prevent or treat NASH; therefore, exploring effective treatments for NASH has broad application prospects.

[0003] The farnesoid X receptor (FXR), also known as the bile acid receptor, belongs to the nuclear receptor superfamily and is widely distributed in organs such as the liver, intestines, kidneys, and gallbladder. As a metabolic regulator, FXR plays a crucial role in the metabolism of bile acids, cholesterol, lipids, and glucose, and is considered one of the most promising targets for treating NAFLD. To date, research on FXR agonists is relatively mature, with several compounds in preclinical or clinical research stages. The most representative is obeticholic acid (OCA), a steroidal FXR agonist developed by Intercept Pharmaceuticals, which was officially approved by the US FDA in May 2016 for the treatment of primary biliary cholangitis and is currently the only marketed FXR agonist. Although several FXR agonists have been discovered, almost all of them have drawbacks such as causing itching, increasing low-density lipoprotein (LDL), and decreasing high-density lipoprotein (HDL), significantly limiting their clinical application. In recent years, research on FXR antagonists has gradually increased, and they have also shown good effects in improving metabolic disorders and liver-related diseases (Li F, Nat Commun, 2013, 4:2384; Jiang CC. Nat Commun, 2015, 6:10166.). FXR antagonists can not only increase the activity of cholesterol 7α-hydroxylase (CYP7A1) and reduce total cholesterol levels, but also reduce liver triglyceride and low-density lipoprotein levels, alleviating steatosis, inflammation, and fibrosis in NAFLD (Zhang, CJ Med Chem, 2022, 65:13452-13472;). However, most reported FXR antagonists are natural products or endogenous bile acids. For example, gugggulsterone (GS) was the first discovered natural FXR antagonist, but it is a heterogeneous ligand with poor selectivity (Urizar NL. Science, 2002, 296:1703-1706; Takanori Yamada. Adv Exp Med Biol, 2016, 929:329-361). Ursodeoxycholic acid (UDCA) was 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. LifeSci, 2021, 272:119252). Naturally derived or endogenous FXR antagonists generally suffer from limited sources, low potency, poor selectivity, and difficulties in synthesis. Therefore, finding highly active and selective FXR antagonists is of great significance for developing effective drugs to treat NASH.

[0004] In our previous application, "Farnesin X Receptor Antagonists and Their Virtual Screening Methods and Applications" (CN116130027A), our research group has screened several novel FXR receptor antagonists using a virtual screening method. Among them, compound V023-9340 (IC) possesses an aminophenylacetamide skeleton. 50 =4.31 μM (compound number 1 in CN116130027A) exhibits potent FXR antagonism. This invention aims to conduct in-depth research on the screened aminophenylacetamide FXR antagonists, further modify the structure of compound V023-9340 disclosed in CN116130027A, synthesize a series of novel aminophenylacetamide derivatives, and demonstrate through experimental studies that these aminophenylacetamide derivatives possess strong FXR antagonistic activity and therapeutic effects on non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0005] The structural formulas of myrrh sterone (GS), tauroursodeoxycholic acid (TUDCA), and VO23-9340 are as follows:

[0006] Summary of the Invention

[0007] This invention discloses a series of novel aminophenylacetamide derivatives as farnesoid X receptor antagonists for the development of drugs for the treatment of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0008] A first aspect of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof represented by formula (I).

[0009]

[0010] in:

[0011] R1 is selected from methyl, halogen, and tert-butyl;

[0012] R2 is selected from any one of the following groups: R3 is selected from any one of the following groups: In a first aspect of the present invention, the specific structural formula of the derivative represented by formula (I) is as follows:

[0013]

[0014] In a second aspect, the present invention provides a method for preparing the derivative as shown in formula (I), the preparation route of which is as follows:

[0015]

[0016] The preparation method is as follows:

[0017] Step a: Under alkaline conditions, ethyl 4-aminophenylacetate II and benzyl bromide derivative III are reacted in an organic solvent to obtain compound IV;

[0018] Step b: Compound IV is hydrolyzed under solvent and alkaline conditions, followed by an acidification reaction to prepare compound V;

[0019] Step c: Compound V and amino derivative VI undergo an amidation reaction in a suitable solvent and condensing agent to obtain compound VII;

[0020] Step d: Compound VII reacts with acyl chloride derivative VIII under alkaline conditions to obtain the derivative shown in Formula I.

[0021] Furthermore, in the preparation method, the molar ratio of ethyl 4-aminophenylacetate II to benzyl bromide derivative III in step a is 1:1.1, and the reaction temperature is 45–60 °C;

[0022] The organic solvent is selected from one of anhydrous tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dichloromethane, and trichloromethane;

[0023] The alkali is one of potassium carbonate, sodium carbonate, and triethylamine.

[0024] Furthermore, the solvent mentioned in step b is one of anhydrous ethanol, methanol, and tetrahydrofuran;

[0025] The alkali mentioned is sodium hydroxide or potassium hydroxide;

[0026] The hydrolysis reaction temperature is 20–40°C, and the reaction time is 12–24 h.

[0027] For the acidification reaction described above, those skilled in the art can make appropriate selections of the reaction conditions, preferably using a 1 mol / L hydrochloric acid solution, adjusting the pH to 1-2, and reacting at 0-5°C.

[0028] Further, in step c, the molar ratio of compound V to amino derivative VI is 1:1.5, the reaction temperature is 45–50°C, and the reaction time is 8–24 h;

[0029] The suitable solvent is selected from one of anhydrous tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dichloromethane, and trichloromethane;

[0030] The condensing agents for the amidation reaction are 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA).

[0031] Furthermore, in step d, the molar ratio of compound VII to acyl chloride derivative VIII is 1:1.5, the reaction temperature is 10–30 °C, and the reaction time is 8–16 h;

[0032] The alkali mentioned is triethylamine.

[0033] A third aspect of the present invention provides a pharmaceutical composition for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, comprising a derivative of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0034] In a fourth aspect, the invention provides the use of the derivative of formula (I) or a pharmaceutically acceptable salt thereof as, or a pharmaceutical composition thereof, as a farnesoid X receptor antagonist.

[0035] Furthermore, the present invention provides the use of the derivative of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0036] This invention discloses the unique use of aminophenylacetamide derivatives as farnesoid X receptor antagonists. The study focused on in vitro FXR antagonistic activity, HepG2 and L02 cytotoxicity studies, HepG2 cell Oil Red O staining assays, HepG2 cell triglyceride content testing, and in vivo anti-nonalcoholic steatohepatitis (NAHH) activity studies. These findings confirm that the compounds described in this invention possess strong farnesoid X receptor antagonistic activity and have the potential to be used in the preparation of drugs for treating NHA and NHA. Attached Figure Description

[0037] Figure 1 This is a graph showing the results of the triglyceride content test in HepG2 cells for compound 8;

[0038] Figure 2 This is a diagram showing the results of Oil Red O staining of HepG2 cells with compound 8;

[0039] Figure 3 This is a schematic diagram illustrating the effect of compound 8 on serum triglycerides in NASH mice;

[0040] Figure 4This is a schematic diagram illustrating the effect of compound 8 on serum cholesterol in NASH mice;

[0041] Figure 5 This is a schematic diagram illustrating the effect of compound 8 on serum low-density lipoprotein in NASH mice;

[0042] Figure 6 This is a schematic diagram illustrating the effect of compound 8 on serum high-density lipoprotein in NASH mice;

[0043] Figure 7 This is a schematic diagram illustrating the effect of compound 8 on alanine aminotransferase (ALT) in NASH mice.

[0044] Figure 8 This is a schematic diagram showing the effect of compound 8 on aspartate aminotransferase in NASH mice. Detailed Implementation

[0045] 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.

[0046] Example 1: Preparation of compounds IV-1 to IV-3

[0047] Preparation of ethyl 2-(4-((3-methylbenzyl)amino)phenyl)acetate (Ⅳ-1)

[0048]

[0049] Compound 4-aminophenylethyl acetate (II, 27.89 mmol) was added to a reaction flask, followed by anhydrous tetrahydrofuran (THF) (50 mL) and potassium carbonate (30.69 mmol). After stirring at room temperature for 10 min, compound 3-methylbenzyl bromide (30.69 mmol) was added. The reaction solution was heated to 45–60 °C and reacted for 8–16 h. After the reaction was complete, monitored by TLC (petroleum ether:ethyl acetate = 3:1, v / v), the reaction solution was allowed to stand at room temperature, poured into 100 mL of cold water, and extracted with ethyl acetate (60 mL × 3). The organic phases were combined, washed with saturated NaCl (60 mL), dried over anhydrous MgSO4, filtered, and the solvent was removed under reduced pressure. Using petroleum ether / ethyl acetate as eluent, column chromatography was performed to give compound IV-1 as a yellow solid, yield 76.4%. 1H NMR (600MHz, DMSO-d6) δ7.20–7.12(m,3H),7.02(d,J=7.4Hz,1H),6.92(d,J=8.4Hz,2H),6.51(d,J=8.4Hz,2H),6. 14(t,J=5.9Hz,1H), 4.19(d,J=6.0Hz,2H), 4.03(q,J=7.1Hz,2H), 3.41(s,2H), 2.28(s,3H), 1.16(t,J=7.1Hz,3H).

[0050] Preparation of ethyl 2-(4-((3-chlorobenzyl)amino)phenyl)acetate (IV-2)

[0051]

[0052] The preparation method for compound IV-1 was followed, except that 3-methylbenzyl bromide was replaced with 3-chlorobenzyl bromide, while all other conditions remained the same. Compound IV-2: yellow solid, yield 71.8%. 1 H NMR (600MHz, DMSO-d6) δ7.39(s,1H),7.36–7.30(m,2H),7.27(d,J=7.5Hz,1H),6.93(d,J=8.4Hz,2H),6.51(d,J=8 .5Hz, 2H), 6.26 (t, J = 6.2Hz, 1H), 4.26 (d, J = 6.2Hz, 2H), 4.03 (q, J = 7.1Hz, 2H), 3.42 (s, 2H), 1.15 (t, J = 7.1Hz, 3H).

[0053] Preparation of 2-(4-((4-(tert-butyl)benzyl)amino)phenyl)ethyl acetate (IV-3)

[0054]

[0055] Following the preparation method of compound IV-1, 3-methylbenzyl bromide was replaced with 4-tert-butylbenzyl bromide, with all other conditions remaining the same. Compound IV-3: white solid, yield 74.5%. 1 H NMR (600MHz, DMSO-d6) δ7.33(d,J=8.3Hz,1H),7.26(d,J=8.3Hz,1H),6.92(d,J=8.4Hz,1H),6.51(d,J=8.5Hz,1H),6 .11(t,J=6.0Hz,1H),4.18(d,J=6.0Hz,1H),4.03(q,J=7.1Hz,1H),3.41(s,1H),1.26(s,5H),1.15(t,J=7.1Hz,2H).

[0056] Example 2: Preparation of compounds V-1 to V-3

[0057] Preparation of 2-(4-((3-methylbenzyl)amino)phenyl)acetic acid (V-1)

[0058]

[0059] Compound 2-(4-((3-methylbenzyl)amino)phenyl)ethyl acetate (Ⅳ-1, 21.19 mmol) was added to a reaction flask, and 20 mL of anhydrous ethanol was added and stirred to dissolve. After complete dissolution, 25 mL of 1.0 mol / L NaOH ethanol solution was added dropwise. After the addition was complete, the reaction temperature was maintained at 20–40 °C, and the reaction was carried out for 12–24 h. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 1:1, v / v). After the reaction was completed, the solvent was removed by vacuum distillation, and 10 mL of water was added. The pH was adjusted to 1–2 by slowly adding 1.0 mol / L HCl under an ice-water bath. 100 mL of water was added, and the mixture was extracted with ethyl acetate (60 mL × 3). The organic phases were combined, washed with saturated NaCl (60 mL), dried over anhydrous MgSO4, filtered, and the solvent was removed by vacuum distillation. Petroleum ether / ethyl acetate was used as the eluent, and column chromatography was used to obtain compound V-1. Yellow solid, yield 83.6%, 1H NMR (600MHz, DMSO-d6) δ 12.06 (s, 1H), 7.20–7.12 (m, 3H), 7.02 (d, J = 7.4 Hz, 1H), 6.91 (d, J = 8.5 Hz, 2H), 6.50 (d, J = 8.5 Hz, 2H), 6.10 (s, 1H), 4.19 (s, 2H), 2.28 (s, 3H).

[0060] Preparation of 2-(4-((3-chlorobenzyl)amino)phenyl)acetic acid (V-2)

[0061]

[0062] Following the preparation method for compound V-1, ethyl 2-(4-((3-methylbenzyl)amino)phenyl)acetate (Ⅳ-1) was replaced with ethyl 2-(4-((3-chlorobenzyl)amino)phenyl)acetate (IV-2), with all other conditions remaining the same. Compound V-2: yellow solid, yield 85.9%. 1 H NMR(600MHz,DMSO-d6)δ12.07(s,1H),7.39(s,1H),7.36–7.29(m,2H),7.26(d,J=7.5Hz ,1H),6.92(d,J=8.4Hz,2H),6.50(d,J=8.5Hz,2H),6.23(s,1H),4.26(d,J=4.7Hz,2H).

[0063] Preparation of 2-(4-((4-(tert-butyl)benzyl)amino)phenyl)acetic acid (V-3)

[0064]

[0065] Following the preparation method for compound V-1, compound 2-(4-((3-methylbenzyl)amino)phenyl)ethyl acetate (Ⅳ-1) was replaced with compound 2-(4-((4-(tert-butyl)benzyl)amino)phenyl)ethyl acetate (IV-3), with all other conditions remaining the same. Compound V-3: white solid, yield 82.2%. 1 H NMR (600MHz, DMSO-d6) δ12.06(s,1H),7.33(d,J=8.3Hz,2H),7.26(d,J=8.2Hz,2H),6.91(d ,J=8.4Hz,2H),6.51(d,J=8.5Hz,1H),6.09(s,1H),4.19(s,1H),3.32(s,2H),1.26(s,9H).

[0066] Example 3: Preparation of compounds VII-1 to 9

[0067] Preparation of N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-1)

[0068]

[0069] Compound 2-(4-((3-methylbenzyl)amino)phenyl)acetic acid (V-1, 3.36 mmol) was added to a reaction flask, followed by anhydrous THF (20 mL), HATU (4.37 mmol), and DIPEA (6.72 mmol). After activation at room temperature for 30 min, compound benzo[d][1,3]dioxono-5-methylamine (5.04 mmol) was added, and the mixture was heated to 45–50 °C and reacted for 8–24 h. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 1:1, v / v). After the reaction was complete, the solvent was removed under reduced pressure, 80 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated NaCl (50 mL), dried over anhydrous MgSO4, filtered, and the solvent was removed under reduced pressure using petroleum ether / ethyl acetate as eluent. Column chromatography was then performed to give compound VII-1 as a brown solid, in 81.2% yield. 1H NMR(600MHz, DMSO-d6)δ8.26(t,J=5.8Hz,1H),7.20–7.16(m,2H),7.13(d,J=7.6H z,1H),7.02(d,J=7.4Hz,1H),6.93(d,J=8.4Hz,2H),6.80(d,J=7.9Hz,1H),6.76(d ,J=1.3Hz,1H),6.70–6.65(m,1H),6.49(d,J=8.5Hz,2H),6.06(t,J=6.0Hz,1H),5. 96(s,2H),4.19(d,J=6.0Hz,2H),4.13(d,J=5.9Hz,2H),3.24(s,2H),2.27(s,3H).

[0070] Preparation of N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2)

[0071]

[0072] Following the preparation method for compound VII-1, compound 2-(4-((3-methylbenzyl)amino)phenyl)acetic acid (V-1) was replaced with compound 2-(4-((3-chlorobenzyl)amino)phenyl)acetic acid (V-2), with all other conditions remaining the same. Compound VII-2: yellow solid, yield 84.4%. 1 H NMR(600MHz,DMSO-d6)δ8.27(t,J=5.8Hz,1H),7.39(s,1H),7.36–7.29(m,2H),7 .26(dd,J=7.5,1.6Hz,1H),6.94(d,J=8.4Hz,2H),6.80(d,J=7.9Hz,1H),6.76(d ,J=1.4Hz,1H),6.67(dd,J=7.9,1.4Hz,1H),6.49(d,J=8.5Hz,2H),6.19(t,J=6. 2Hz, 1H), 5.96 (s, 2H), 4.26 (d, J = 6.2Hz, 2H), 4.13 (d, J = 5.9Hz, 2H), 3.24 (s, 2H).

[0073] Preparation of N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((4-(tert-butyl)benzyl)amino)phenyl)acetamide (VII-3)

[0074]

[0075] Following the preparation method for compound VII-1, compound 2-(4-((3-methylbenzyl)amino)phenyl)acetic acid (V-1) was replaced with compound 2-(4-((4-(tert-butyl)benzyl)amino)phenyl)acetic acid (V-3), with all other conditions remaining the same. Compound VII-3: white solid, yield 74.4%. 1 H NMR(600MHz,DMSO-d6)δ8.26(t,J=5.8Hz,1H),7.32(d,J=8.3Hz,2H),7.26(d, J=8.2Hz,2H),6.93(d,J=8.4Hz,2H),6.80(d,J=7.9Hz,1H),6.76(d,J=1.1Hz,1 H),6.68(d,J=7.9Hz,1H),6.50(d,J=8.5Hz,2H),6.04(t,J=6.0Hz,1H),5.96( s, 2H), 4.18 (d, J = 6.0Hz, 2H), 4.13 (d, J = 5.9Hz, 2H), 3.24 (s, 2H), 1.26 (s, 9H).

[0076] Preparation of 2-(4-((3-methylbenzyl)amino)phenyl)-N-(naphth-2-ylmethyl)acetamide (VII-4)

[0077]

[0078] Following the preparation method for compound VII-1, benzo[d][1,3]dioxono-5-methylamine was replaced with naphthyl-2-methylamine, with all other conditions remaining the same. Compound VII-4: white solid, yield 74.4%. 1 H NMR(600MHz,DMSO-d6)δ8.44(t,J=5.9Hz,1H),7.88–7.85(m,1H),7.83(d,J=8.4Hz,1H),7.80 –7.76(m,1H),7.63(s,1H),7.51–7.44(m,2H),7.36(dd,J=8.4,1.5Hz,1H),7.21–7.16(m,2H) ,7.14(d,J=7.6Hz,1H),7.02(d,J=7.4Hz,1H),6.99(d,J=8.4Hz,2H),6.52(d,J=8.5Hz,2H),6 .09(t,J=6.0Hz,1H),4.40(d,J=5.9Hz,2H),4.21(d,J=6.0Hz,2H),3.31(s,2H),2.27(s,3H).

[0079] Preparation of N-([1,1'-biphenyl]-4-ylmethyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-5)

[0080]

[0081] Following the preparation method for compound VII-1, benzo[d][1,3]dioxono-5-methylamine was replaced with [1,1'-biphenyl]-4-methylamine, with all other conditions remaining the same. Compound VII-5: white solid, yield 85.8%. 1 H NMR (600MHz, DMSO-d6) δ8.37(t,J=5.9Hz,1H),7.63(d,J=7.2Hz,2H),7.58(d,J=8.2Hz,2H) ,7.45(t,J=7.7Hz,2H),7.35(t,J=7.4Hz,1H),7.29(d,J=8.1Hz,2H),7.20–7.14(m,2H),7. 13(d,J=7.6Hz,1H),7.01(d,J=7.4Hz,1H),6.96(d,J=8.4Hz,2H),6.51(d,J=8.4Hz,2H),6. 07(t,J=6.0Hz,1H),4.27(d,J=5.9Hz,2H),4.20(d,J=5.8Hz,2H),3.28(s,2H),2.27(s,3H).

[0082] Preparation of N-(4-methylbenzyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-6)

[0083]

[0084] Following the preparation method for compound VII-1, benzo[d][1,3]dioxono-5-methylamine was replaced with p-toluenemethylamine, with all other conditions remaining the same. Compound VII-6: white solid, yield 73.5%. 1 H NMR (600MHz, DMSO-d6) δ8.27(t,J=5.8Hz,1H),7.20–7.16(m,2H),7.13(d,J=7.6Hz,1H),7.08(s,4H),7.02(d,J=7.4Hz,1H),6.94( d,J=8.4Hz,2H),6.49(d,J=8.5Hz,2H),6.06(t,J=6.0Hz,1H),4.18(dd,J=9.6,6.0Hz,4H),3.24(s,2H),2.27(s,3H),2.26(s,3H).

[0085] Preparation of N-(4-chlorobenzyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-7)

[0086]

[0087] Following the preparation method for compound VII-1, benzo[d][1,3]dioxono-5-methylamine was replaced with (4-chlorophenyl)methylamine, with all other conditions remaining the same. Compound VII-7: yellow solid, yield 81.5%. 1 H NMR (600MHz, DMSO-d6) δ8.36(t,J=5.9Hz,1H),7.32(dd,J=6.3,4.5Hz,2H),7.23–7.15(m,4H),7.13(d,J=7.6Hz,1H),7.02(d,J=7.4 Hz, 1H), 6.94 (d, J = 8.4Hz, 2H), 6.50 (d, J = 8.5Hz, 2H), 6.08 (t, J = 6.0Hz, 1H), 4.20 (dd, J = 8.9, 6.1Hz, 4H), 3.26 (s, 2H), 2.27 (s, 3H).

[0088] Preparation of N-(4-isopropylbenzyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-8)

[0089]

[0090] Following the preparation method of compound VII-1, benzo[d][1,3]dioxono-5-methylamine was replaced with (4-isopropylphenyl)methylamine, with all other conditions remaining the same. Compound VII-8: white solid, yield 82.6%. 1 H NMR (600MHz, DMSO-d6) δ8.27(t,J=5.8Hz,1H),7.20–7.16(m,3H),7.15–7.10(m,4H),7.02(d,J=7.4Hz,1H),6.94(d,J=8.4Hz,2H),6.49 (d,J=8.5Hz,2H),6.07(t,J=6.0Hz,1H),4.19(dd,J=8.6,6.1Hz,4H),3.24(s,2H),2.8–2.81(m,1H),2.27(s,3H),1.17(d,J=6.9Hz,6H).

[0091] Preparation of ethyl 4-((2-(4-((3-methylbenzyl)amino)phenyl)acetamido)methyl)benzoate (VII-9)

[0092]

[0093] Following the preparation method of compound VII-1, benzo[d][1,3]dioxono-5-methylamine was replaced with ethyl 4-(aminomethyl)benzoate, with all other conditions remaining the same. Compound VII-9: yellow solid, yield 71.3%. 1 H NMR (600MHz, DMSO-d6) δ8.43(t,J=5.9Hz,1H),7.88(d,J=8.2Hz,2H),7.32(d,J =8.2Hz,2H),7.20–7.15(m,2H),7.13(d,J=7.6Hz,1H),7.02(d,J=7.4Hz,1H),6 .95(d,J=8.4Hz,2H),6.51(d,J=8.5Hz,2H),6.08(t,J=6.0Hz,1H),4.32–4.28( m, 4H), 4.20 (d, J = 6.0Hz, 2H), 3.28 (s, 2H), 2.27 (s, 3H), 1.31 (t, J = 7.1Hz, 3H).

[0094] Example 4: Preparation of compounds 1-14

[0095] 1. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-N-(3-chlorobenzyl)cyclopentanecarboxamide (compound 1)

[0096]

[0097] Under nitrogen protection, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2, 1.22 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), followed by the sequential addition of 4-dimethylaminopyridine (0.12 mmol), triethylamine (2.44 mmol), and cyclopentylformyl chloride (VIII-1, 1.83 mmol). The reaction was carried out at low temperature for 6 h. The tetrahydrofuran was removed by vacuum distillation, followed by the addition of distilled water (30 mL) and extraction with 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 column chromatography was performed to give compound 1. The yield was 78.6%, and the compound was a yellow gel. 1H NMR(600MHz,DMSO-d6)δ8.50(t,J=5.7Hz,1H),7.35–7.25(m,4H),7.21(s,1H),7 .13(d,J=7.3Hz,1H),7.08(d,J=8.2Hz,2H),6.80(d,J=7.9Hz,1H),6.75(d,J=1. 3Hz,1H),6.73–6.65(m,1H),5.96(s,2H),4.82(s,2H),4.16(d,J=5.8Hz,2H),3. 46(s,2H),2.58–2.51(m,1H),1.68(m,2H),1.65–1.54(m,4H),1.42–1.31(m,2H); 13 CNMR(151MHz,DMSO-d6)δ176.19,170.14,147.70,146.51,140.99,136.47,133.70,133.40,130.66,128.36,128. 05,127.50,126.90,120.84,108.36,101.27,52.23,42.46,42.18,41.73,31.12,26.27.HR-MS(m / z)(ESI):calcd for C 29 H 30 ClN2O4[M+H] + :505.1894; found:505.1895.

[0098] 2. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-N-(4-(tert-butyl)benzyl)cyclopentaneformamide (compound 2):

[0099]

[0100] Following the preparation method for compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((4-(tert-butyl)benzyl)amino)phenyl)acetamide (VII-3) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), with all other conditions remaining the same. Compound 2: 79.7% yield, white solid. 1H NMR(600MHz,DMSO-d6)δ8.50(t,J=5.6Hz,1H),7.31–7.28(m,4H),7.08(d,J=7 .8Hz,4H),6.80(d,J=7.9Hz,1H),6.76(d,J=1.2Hz,1H),6.68(dd,J=7.9,1.5Hz ,1H),5.96(s,2H),4.78(s,2H),4.16(d,J=5.8Hz,2H),3.45(s,2H),2.61–2.50 (m,1H),1.71–1.65(m,2H),1.64–1.51(m,4H),1.37–1.34(m,2H),1.25(s,9H); 13 C NMR(151MHz,DMSO-d6)δ175.91,170.18,149.73,147.70,146.51,141.40,136.25,135.32,133.71,130.51,128.38,127 .82,125.50,120.83,108.36,101.27,52.52,42.46,42.18,41.75,34.63,31.62,31.16,26.29.HR-MS(m / z)(ESI):calcd for C 33 H 39 N₂O₄[M+H] + :527.2910; found:527.2903.

[0101] 3. Preparation of N-(3-methylbenzyl)-N-(4-(2-((naphthyl-2-ylmethyl)amino)-2-oxoethyl)phenyl)cyclopentaneformamide (compound 3):

[0102]

[0103] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2) was replaced with 2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-4), with all other conditions remaining the same. Compound 3: 86.12% yield, white solid. 1H NMR (400MHz, DMSO-d6) δ8.76 (t, J=5.7Hz, 1H), 7.92–7.80 (m, 3H), 7.71 (s, 1H), 7.54–7. 47(m,2H),7.43–7.37(m,1H),7.33(d,J=8.1Hz,2H),7.19(t,J=7.5Hz,1H),7.12(d,J=8. 1Hz,2H),7.05(d,J=7.5Hz,1H),6.97(d,J=10.2Hz,2H),4.82(s,2H),4.46(d,J=5.8Hz, 2H),3.54(s,2H),2.61–2.55(m,1H),2.26(s,3H),1.79–1.55(m,6H),1.42–1.33(m,2H); 13 C NMR (126MHz, DMSO-d6) δ175.93,170.38,141.23,138.24,137.81,137.45,136.32,133.30,132.51,130.51,128.80,128.67,128.47,128.31,12 8.10,127.97,127.87,126.65,126.27,126.12,125.66,125.22,52.64,42.76,42.28,41.73,31.15,26.29,21.47.HR-MS(m / z)(ESI):calcdfor C 33 H 35 N₂O₂[M+H] + :491.2699; found:491.2707.

[0104] 4. Preparation of N-(4-(2-(([1,1'-biphenyl]-4-ylmethyl)amino)-2-oxoethyl)phenyl)-N-(3-methylbenzyl)cyclopentaneformamide (compound 4):

[0105]

[0106] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-5) was substituted for N-([1,1'-biphenyl]-4-ylmethyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-5), with all other conditions remaining the same. Compound 4: 77.04% yield, white solid. 1H NMR (400MHz, DMSO-d6) δ8.68(t,J=5.7Hz,1H),7.65(d,J=7.4Hz,2H),7.60(d,J=8.1Hz,2H) ,7.48(t,J=7.6Hz,2H),7.38(t,J=7.3Hz,1H),7.31(d,J=7.8Hz,4H),7.18(t,J=7.5Hz,1H), 7.10(d,J=8.0Hz,2H),7.04(d,J=7.5Hz,1H),7.00–6.94(m,2H),4.82(s,2H),4.33(d,J=5.8 Hz,2H),3.52(s,2H),2.59–2.55(m,1H),2.26(s,3H),1.75–1.52(m,6H),1.47–1.26(m,2H); 13 C NMR (126MHz, DMSO-d6) δ175.92,170.30,141.20,140.39,139.23,139.10,138.24,137.81,136.30,130.50,129.37,128.81,128.66, 128.47,128.25,128.09,127.81,127.03,125.23,52.62,42.33,42.23,41.72,38.71,31.14,26.29,21.47.HR-MS(m / z)(ESI):calcd for C 35 H 37 N₂O₂[M+H] + :517.2855; found:517.2866.

[0107] 5. Preparation of N-(3-methylbenzyl)-N-(4-(2-((4-methylbenzyl)amino)-2-oxoethyl)phenyl)cyclopentanecarboxamide (compound 5):

[0108]

[0109] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-6) was substituted for N-(4-methylbenzyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-6), with all other conditions remaining the same. Compound 5: Yield 68.25%, white gel. 1H NMR (400MHz, DMSO-d6) δ8.58(t,J=5.6Hz,1H),7.29(d,J=8.0Hz,2H),7.19(t,J=7.5Hz,1H),7.12–7.03(m,7H),7.02–6.93( m,2H),4.82(s,2H),4.23(d,J=5.8Hz,2H),3.48(s,2H),2.58(m,1H),2.27(d,J=3.9Hz,6H),1.80–1.54(m,6H),1.39(s,2H); 13 C NMR (126MHz, DMSO-d6) δ175.91,170.18,141.17,138.23,137.81,136.78,136.34,136.24,130.47,129.22,128.81,128 .67,128.44,128.10,127.58,125.24,52.61,42.33,42.23,41.73,31.15,26.29,21.48,21.10.HR-MS(m / z)(ESI):calcd for C 30 H 35 N₂O₂[M+H] + :455.2699; found:455.2703.

[0110] 6. Preparation of N-(4-(2-((4-chlorobenzyl)amino)-2-oxoethyl)phenyl)-N-(3-methylbenzyl)cyclopentaneformamide (compound 6):

[0111]

[0112] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-7) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-7), with all other conditions remaining the same. Compound 6: 76.8% yield, yellow gelatinous substance. 1H NMR (400MHz, DMSO-d6) δ8.67(t,J=5.7Hz,1H),7.35(d,J=8.3Hz,2H),7.29(d,J=8.0Hz,2H),7.16–7.24(m,3H),7.04–7.12(m,3H),7.0 1–6.93(m,2H),4.82(s,2H),4.27(d,J=5.9Hz,2H),3.49(s,2H),2.63–2.54(m,1H),2.27(s,3H),1.81–1.54(m,6H),1.39–1.28(m,2H); 13 C NMR (126MHz, DMSO-d6) δ175.91,170.37,141.20 138.96,138.22,137.81,136.19,131.75,130.48,129.43,128.80,128.67 128.61,128.47,128.1,125.23,52.60,42.18,41.91,41.73,31.14,26.29,21.48.HR-MS(m / z)(ESI):calcd for C 29 H 32 ClN2O2[M+H] + :475.2152; found:475.2162.

[0113] 7. Preparation of N-(4-(2-((4-isopropylbenzyl)amino)-2-oxoethyl)phenyl)-N-(3-methylbenzyl)cyclopentaneformamide (compound 7):

[0114]

[0115] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-8) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-8), with all other conditions remaining the same. Compound 7: Yield 79.13%, white gel-like substance. 1H NMR (600MHz, DMSO-d6) δ8.51(t,J=5.6Hz,1H),7.26(d,J=8.1Hz,2H),7.19–7.00(m,8H),6.98–6.90(m,2H),4.79(s,2H),4.21(d,J=5. 8Hz,2H),3.45(s,2H),2.86–2.81(m,1H),2.61–2.50(m,1H),2.24(s,3H),1.79–1.48(m,6H),1.51–1.29(m,2H),1.17(d,J=6.9Hz,6H); 13 C NMR(151MHz,DMSO-d6)δ175.92,170.16,147.43,141.20,138.27,137.81,137.17,136.35,130.46,128.83,128.66,128.45 ,128.10,127.73,126.58,125.26,52.65,42.44,42.22,41.76,33.58,31.14,26.30,24.38,21.48.HR-MS(m / z)(ESI):calcd for C 32 H 39 N₂O₂[M+H] + :483.3012; found:483.3018.

[0116] Preparation of ethyl benzoate (compound 8) of 8,4-((2-(4-(N-(3-methylbenzyl)cyclopentanecarbamate)phenyl)acetamido)methyl)benzoate:

[0117]

[0118] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamido)acetamide (VII-2) was replaced with ethyl 4-((2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-9), with all other conditions remaining the same. Compound 8: Yield 71.61%, yellow gelatinous substance. 1H NMR(600MHz,DMSO-d6)δ8.67(t,J=5.8Hz,1H),7.86(d,J=8.1Hz,2H),7.31–7.28(m,4H),7 .16(t,J=7.5Hz,1H),7.07(d,J=8.0Hz,2H),7.03(d,J=7.5Hz,1H),6.98–6.91(m,2H),4.7 9(s,2H),4.34(d,J=5.9Hz,2H),4.30(q,J=7.1Hz,2H),3.49(s,2H),2.58–2.52(m,1H),2. 24(s,3H),1.72–1.68(m,2H),1.65–1.53(m,4H),1.41–1.33(m,2H),1.31(t,J=7.1Hz,3H); 13 CNMR(151MHz,DMSO-d6)δ175.91,170.50,166.02,145.55,141.25,138.25,137.82,136.20,130.46,129.56,128.88,128.85,12 8.66,128.52,128.11,127.62,125.29,61.10,52.64,42.34,42.27,41.78,31.13,26.29,21.46,14.63.HR-MS(m / z)(ESI):calcd for C 32 H 37 N₂O₄[M+H] + :513.2753; found:513.2751.

[0119] 9. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-N-(3-methylbenzyl)cyclohexanecarboxamide (compound 9):

[0120]

[0121] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-(((3-methylbenzyl)amino)phenyl)acetamide (VII-1) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), and cyclohexylformyl chloride (VIII-2) was substituted for cyclopentylformyl chloride (VIII-1), with all other conditions remaining the same. Compound 9: Yield 69.84%, white gel-like substance. 1H NMR (600MHz, DMSO-d6) δ8.49(t,J=5.7Hz,1H),7.26(d,J=8.1Hz,2H),7.16(t,J=7.5Hz,1H),7.06(d,J=8.2H z,2H),7.02(d,J=7.5Hz,1H),6.98–6.89(m,2H),6.80(d,J=7.9Hz,1H),6.76(d,J=1.3Hz,1H),6.68(dd,J=7. 9,1.5Hz,1H),5.96(s,2H),4.76(s,2H),4.16(d,J=5.8Hz,2H),3.46(s,2H),2.24(s,3H),2.17(t,J=10.8Hz, 1H),1.63(t,J=14.7Hz,4H),1.51(d,J=12.4Hz,1H),1.48–1.35(m,2H),1.11(q,J=12.8Hz,1H),0.87(m,2H); 13 C NMR(151MHz,DMSO-d6)δ175.42,170.17,147.70,146.51,141.17,138.25,137.81,136.32,133.71,130.53,128.70,128 .10,125.15,120.85,108.37,101.27,52.44,42.47,42.19,41.08,29.58,25.77,25.50,21.47.HR-MS(m / z)(ESI):calcd for C 31 H 35 N₂O₄[M+H] + :499.2597; found:499.2598.

[0122] 10. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-N-(3-methylbenzyl)benzamide (compound 10):

[0123]

[0124] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-(((3-methylbenzyl)amino)phenyl)acetamide (VII-1) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), and cyclopentylformyl chloride (VIII-1) was substituted for benzoyl chloride (VIII-3), with all other conditions remaining the same. Compound 10: Yield: 80.56%, white solid, melting point 51.3-54.6℃. 1 H NMR (400MHz, DMSO-d6) δ8.46(t,J=5.7Hz,1H),7.35–7.20(m,6H),7.13–7.02(m,5H),7.01(d,J=8.1Hz,2H),6.82(d,J=7. 9Hz,1H),6.76(s,1H),6.68(d,J=7.9Hz,1H),6.00(s,2H),5.07(s,2H),4.15(d,J=5.8Hz,2H),3.36(s,2H),2.29(s,3H); 13 C NMR(126MHz,DMSO-d6)δ170.17,170.09,147.67,146.49,141.79,137.94,136.68,134.99,133.66,129.99,129.94,128.76,12 8.68,128.26,128.20,127.74,125.09,120.84,108.43,108.31,101.27,53.25,42.40,42.02,21.50.HR-MS(m / z)(ESI):calcd for C 31 H 29 N₂O₄[M+H] + :493.2127; found:493.2132.

[0125] 11. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-2-methyl-N-(3-methylbenzyl)benzamide (compound 11):

[0126]

[0127] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-(((3-methylbenzyl)amino)phenyl)acetamide (VII-1) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), and cyclopentylformyl chloride (VIII-1) was substituted for 2-methylbenzoyl chloride (VIII-4), with all other conditions remaining the same. Compound 11: Yield 79.87%, white solid, melting point 50.8-52.5℃. 1 H NMR (600MHz, DMSO-d6) δ8.38(s,1H),7.19(d,J=7.0Hz,1H),7.15–6.91(m,10H),6.79(d,J=7.9Hz,1H),6.73(s,1H ),6.64(d,J=7.6Hz,1H),5.97(s,2H),5.05(s,2H),4.11(d,J=5.3Hz,2H),3.29(s,2H),2.30(s,3H),2.27(s,3H); 13 C NMR(151MHz,DMSO-d6)δ170.39,169.97,147.63,146.45,140.74,137.92,137.10,135.23,134.57,133.59,130.38,129.70,128.85,1 28.74,128.23,127.68,125.40,125.16,120.80,108.37,108.28,101.22,52.32,42.39,41.93,21.45,19.53.HR-MS(m / z)(ESI):calcd for C 32 H 31 N₂O₄[M+H] + :507.2284; found:507.2291.

[0128] 12. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-4-methyl-N-(3-methylbenzyl)benzamide (compound 12):

[0129]

[0130] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-1) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), and cyclopentylformyl chloride (VIII-1) was substituted for 4-methylbenzoyl chloride (VIII-5), with all other conditions remaining the same. Compound 12: Yield 73.84%; white solid, melting point 51.2-53.3℃. 1 H NMR(600MHz,DMSO-d6)δ8.38(s,1H),7.19(d,J=7.0Hz,1H),7.15–6.90(m,11H),6.79(d,J=7.9Hz,1H),6.73(s,1H ),6.64(d,J=7.6Hz,1H),5.97(s,2H),5.05(s,2H),4.11(d,J=5.3Hz,2H),3.29(s,2H),2.30(s,3H),2.28(s,3H); 13 C NMR (151MHz, DMSO-d6) δ170.07,147.63,146.45,142.01,139.70,138.01,137.85,134.83,133.69,133.63,129.93,128.92,128.76,128. 68,128.63,128.12,127.58,125.04,120.79,108.37,108.27,101.22,53.34,42.39,41.99,40.38,21.46,21.26.HR-MS(m / z)(ESI):calcd for C 32 H 31 N₂O₄[M+H] + :507.2284; found:507.2297.

[0131] 13. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-4-ethyl-N-(3-methylbenzyl)benzamide (compound 13):

[0132]

[0133] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-1) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), and cyclopentylformyl chloride (VIII-1) was substituted for 4-ethylbenzoyl chloride (VIII-5), with all other conditions remaining the same. Compound 13: Yield 79.22%, white gel. 1 H NMR(600MHz,DMSO-d6)δ8.41(t,J=5.8Hz,1H),7.24(d,J=8.0Hz,1H),7.18(t ,J=7.5Hz,1H),7.11–7.02(m,5H),6.98(d,J=8.2Hz,1H),6.79(d,J=7.9Hz,1H ),6.74(s,1H),6.65(d,J=7.9Hz,1H),5.96(s,2H),5.03(s,2H),4.12(d,J=5. 8Hz,2H),3.34(s,1H),2.57–2.47(m,4H),2.26(s,2H),1.10(t,J=7.6Hz,2H); 13 C NMR(151MHz,DMSO-d6)δ170.06,147.63,146.44,145.83,142.02,138.02,137.85,134.84,133.92,133.62,129.93,129.00,128.68,1 28.62,128.12,127.56,125.03,120.78,108.37,108.27,101.22,53.38,42.39,42.00,28.25,21.46,15.46.HR-MS(m / z)(ESI):calcd for C 33 H 33 N₂O₄[M+H] + :521.2440; found:521.2457.

[0134] 14. Preparation of N-(4-(2-((benzo[d][1,3]dioxono-5-ylmethyl)amino)-2-oxoethyl)phenyl)-4-chloro-N-(3-methylbenzyl)benzamide (compound 14):

[0135]

[0136] Following the preparation method of compound 1, N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-methylbenzyl)amino)phenyl)acetamide (VII-1) was substituted for N-(benzo[d][1,3]dioxono-5-ylmethyl)-2-(4-((3-chlorobenzyl)amino)phenyl)acetamide (VII-2), and cyclopentylformyl chloride (VIII-1) was substituted for 4-chlorobenzoyl chloride (VIII-6), with all other conditions remaining the same. Compound 14: Yield 82.8%, white gel. 1 H NMR (400MHz, DMSO-d6) δ8.48(t,J=5.7Hz,1H),7.37–7.30(m,4H),7.21(t,J=7.5Hz,1H),7.15–6.99(m,7H),6.82(d,J=7. 9Hz,1H),6.76(s,1H),6.68(d,J=7.9Hz,1H),6.00(s,2H),5.07(s,2H),4.15(d,J=5.7Hz,2H),3.37(s,2H),2.29(s,3H); 13 CNMR(126MHz,DMSO-d6)δ170.07,169.11,147.67,146.49,141.49,137.95,137.74,135.51,135.27,134.63,133.66,130.70,130.0 7,128.74,128.38,128.25,127.79,125.14,120.82,108.42,108.30,101.27,53.27,42.41,42.01,21.48.HR-MS(m / z)(ESI):calcd for C 31 H 28 ClN2O4[M+H] + :527.1738; found:527.1746.

[0137] Example 5: FXR antagonistic activity test

[0138] 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 were added for 24 hours of treatment. 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 13000 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 / kidney fluorescence value. Max represents the positive control result value with 5 μM GW4064 compound added, X represents the result value with the test compound added, and Min represents the negative control result value with DMSO added. The results are shown in Table 1.

[0139] Example 6: Cytotoxicity Detection

[0140] 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. The original culture medium was then 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 results are shown in Table 1.

[0141] Table 1: FXR antagonistic activity and cytotoxicity results of the compounds

[0142]

[0143] Data are presented as the average of at least three independent replicates. TUDCA (tauroursodeoxycholic acid) and GS (bimethasterone) were positive control compounds.

[0144] Table 1 shows that the FXR antagonistic activity of the 14 aminophenylacetamide derivatives synthesized in this invention is significantly stronger than that of the positive control drugs TUDCA and GS. Furthermore, compared with the IC50 of the previously reported compound V023-9340... 50 With a concentration of 4.31 μM, derivatives 6, 7, 8, 9, 12, 13, and 14 synthesized in this invention exhibited stronger FXR antagonistic activity. Among them, compound 8 showed the strongest FXR antagonistic activity (IC50). 50 =0.891 μM), its activity was 63-fold, 69-fold, and 4.8-fold higher than that of TUDCA, GS, and VO23-9340, respectively, classifying it as a potent FXR antagonist. Furthermore, the compound synthesized in this invention showed virtually no cytotoxicity (IC50) to HepG2 and L02 cells. 50 >100μM). In summary, the above data demonstrate that the compounds synthesized in this invention are characterized by high efficiency and low toxicity.

[0145] Example 7: Determination of triglyceride (TG) content in HepG2 cells, referring to... Figure 1 .

[0146] The present invention further determined the effect of preferred compound 8 on TG content in HepG2 cells.

[0147] 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 (g protein / L).

[0148] from Figure 1 As can be seen, compared with the model group, compound 8 at different concentrations significantly reduced the intracellular TG content in HepG2 cells, and the effect was better than the positive control TUDCA group and the VO23-9340 group. Data are expressed as mean ± SEM. Compared with the NASH group, *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001.

[0149] Example 8: Oil Red O staining experiment, refer to Figure 2 .

[0150] This invention further uses free fatty acids (FFA) to induce HepG-2 cells to construct a cell steatosis model and examines the effect of compound 8 in improving cell steatosis.

[0151] HepG2 cells 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 hours 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 hours. After successful modeling, the cells were cultured with different concentrations of the compound for 24 hours. The culture medium was then removed from the wells, the cells were washed twice with PBS, fixed with Oil Red O fixative for 25 minutes, washed twice with distilled water, immersed in 60% isopropanol for 3 minutes, and stained with Oil Red O staining solution for 30 minutes. After staining, the cells were rinsed with 60% isopropanol for 30 seconds, and then each well was kept moist with 1 mL of distilled water. The cells were observed under an inverted microscope.

[0152] from Figure 2 As can be seen, compared with the model group, intervention with different concentrations of compound 8 (10 μM, 30 μM) significantly improved the red lipid droplets, especially the 30 μM compound 8, which showed better improvement in fatty degeneration than the 30 μM V023-9340 and 50 μM TUDCA. These data indicate that compound 8 can significantly improve FFA-induced fatty degeneration in HepG2 cells in vitro.

[0153] Example 9: Pharmacodynamic experiment of anti-NASH mice, referring to... Figures 3 to 8 .

[0154] This invention uses a high-fat diet and chemical induction method. A high-fat diet combined with weekly intraperitoneal injection of CCl4 aggravates liver damage in mice, thereby accelerating the development of fibrosis and effectively shortening the model period. A NASH mouse model with mild to moderate fibrosis was established to examine the in vivo anti-NASH activity of compound 8.

[0155] Six-week-old male C57BL / 6J mice were randomly divided into three groups: a normal group, a model group (NASH group), a positive compound V023-9340 group (30 mg / kg), a TUDCA group (50 mg / kg), and low (5 mg / kg), medium (10 mg / kg), and high (20 mg / kg) dose groups of compound 8, 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 V023-9340 group, the TUDCA group, and the high, medium, and low dose groups of the test compound 8 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, while the model group and the drug-treated groups were intraperitoneally injected twice a week with 3.5% CCl4 solution. Throughout the experimental period, the mice's mental status was observed, and their weight was recorded weekly. At the end of the experiment, mice were fasted for 12 hours without access to water or food. Blood was collected from the abdominal aorta after anesthesia with ether, organs were separated, washed with physiological saline, weighed, and recorded. Serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using a biochemical analyzer.

[0156] from Figures 3 to 8 It can be seen that, after 4 weeks of continuous administration, compared with the NASH group, different concentrations of compound 8 could reduce serum TG, TC, and LDL levels and increase HDL levels in NASH mice. The medium (10 mg / kg) and high (20 mg / kg) doses of compound 8 were also superior to the VO23-9340 and TUDCA groups in reducing serum TG, TC, and LDL levels and increasing HDL levels in NASH mice. Furthermore, compared with the NSAH group, the medium (10 mg / kg) and high (20 mg / kg) doses of compound 8 significantly reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and their effects were also superior to the VO23-9340 and TUDCA groups. Data are expressed as mean ± SEM. Compared with the NASH group, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. In summary, the above results indicate that compound 8 can effectively regulate blood lipid levels in NASH mice and improve dyslipidemia, with better effects than VO23-9340 and TUDCA.

Claims

1. An aminophenylacetamide derivative with the following structural formula, characterized in that, The structural formula is: ； The derivative shown in Formula 8 is ethyl 4-((2-(4-(N-(3-methylbenzyl)cyclopentanecarbamate)phenyl)acetamido)methyl)benzoate.

2. The method for preparing the aminophenylacetamide derivative as described in claim 1, characterized in that, Includes the following steps: Step a: In an organic solvent, ethyl 4-aminophenylacetate is reacted with 3-methylbenzyl bromide under alkaline conditions to give ethyl 2-(4-((3-methylbenzyl)amino)phenyl)acetate; Step b: Hydrolyze the product obtained in step a under solvent and alkaline conditions, and then acidify it to obtain 2-(4-((3-methylbenzyl)amino)phenyl)acetic acid; Step c: The product obtained in step b is reacted with ethyl 4-(aminomethyl)benzoate in a suitable solvent and with the condensing agents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine to undergo an amidation reaction, yielding ethyl 4-((2-(4-((3-methylbenzyl)amino)phenyl)acetamido)methyl)benzoate; Step d: The product obtained in step c is reacted with cyclopentylformyl chloride under alkaline conditions to obtain the derivative shown in Formula 8 of claim 1.

3. The method for preparing the aminophenylacetamide derivative as described in claim 2, characterized in that: In step a, the molar ratio of ethyl 4-aminophenylacetate to 3-methylbenzyl bromide is 1:1.1, and the reaction temperature is 45-60°C. The hydrolysis reaction in step b is carried out at a temperature of 20-40°C for 12-24 hours, and the acidification reaction is carried out by adjusting the pH to 1-2 with 1 mol / L hydrochloric acid. In step c, the molar ratio of 2-(4-((3-methylbenzyl)amino)phenyl)acetic acid to ethyl 4-(aminomethyl)benzoate is 1:1.5, the reaction temperature is 45-50°C, and the reaction time is 8-24h. The appropriate solvent is anhydrous tetrahydrofuran; In step d, the molar ratio of ethyl 4-((2-(4-(((3-methylbenzyl)amino)phenyl)acetamido)methyl)benzoate to cyclopentylformyl chloride is 1:1.5, the reaction temperature is 10-30°C, and the reaction time is 8-16h.

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

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

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