CN117164554A - FXR agonist and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and relates to a toolA compound having FXR agonistic activity, or a pharmaceutically acceptable salt thereof, which is represented by formula (I):wherein ring A represents a benzene ring, a cyclohexene ring, a cyclohexane ring, and a bicyclo [2.2.1]]Heptane, bicyclo [2.2.2]Octane, bicyclo [2.2.1]Hept-2-ene or bicyclo [2.2.2]Oct-2-ene; r is R ₁ 、R ₂ 、R ₃ 、R ₄ Independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, nitro, amino, hydroxy, carboxyl, benzyloxycarbonyl, or trifluoromethyl; r is R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ Independently selected from hydrogen, halogen, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxycarbonyl, or trifluoromethyl; r is R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ Independently selected from hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy. The invention also discloses a preparation method of the compound, a pharmaceutical composition and application thereof in preparing medicines for treating or preventing fatty liver, liver cirrhosis, diabetic complications, cardiovascular diseases, gastrointestinal diseases and other related diseases mediated by FXR receptors.

Description

A class of FXR agonists and their applications

Technical Field

The present invention belongs to the field of medical technology and relates to a class of compounds having FXR agonist activity or pharmaceutically acceptable salts thereof, a preparation method thereof and use thereof in drugs for treating and/or preventing non-alcoholic fatty liver disease, primary biliary cirrhosis, lipid metabolism disorders, diabetic complications, malignant tumors and other related diseases mediated by FXR receptors.

Background Art

Farnesoid X receptor (FXR) is a distinctive member of the metabolic subfamily of the nuclear receptor superfamily. It is a transcription factor expressed in multiple tissues such as the liver, intestine, adipose tissue, and kidney. FXR has a typical nuclear receptor structure, including a highly conserved DNA binding domain (DBD) at the N-terminus, a ligand binding domain (LBD) at the C-terminus that allows receptor dimerization, an N-terminal ligand-independent transcriptional activation domain (AF1), a C-terminal ligand-dependent transcriptional activation domain (AF1), and a hinge region. After activation, FXR can form heterodimers with retinol X receptor (RXR) and induce the expression of its target gene SHP, which in turn leads to transcriptional inhibition of CYP7A1 and LRH-1. FXR can also stimulate the synthesis of FGF-19 and inhibit the expression of CYP7A1 and CYP8B1 through the FGFR4 pathway in hepatocytes. The FXR/SHP and FXR/FGF19/FGFR4 pathways constitute the main negative regulators of bile acid synthesis and play an important role in regulating bile acid levels in the enterohepatic circulation. In addition, FXR is directly or indirectly involved in several important metabolic pathways in the body, such as regulating glucose and lipid metabolism. Therefore, the activation or inhibition of FXR plays an important role in metabolic homeostasis.

Steroids are the main ligands of FXR, among which chenodeoxycholic acid (CDCA) is the most effective endogenous agonist of FXR. Based on the physiological effects of FXR activation, FXR agonists are expected to treat a variety of metabolic diseases, such as cholestasis, liver fibrosis, inflammatory bowel disease, type 2 diabetes, atherosclerosis, and erectile dysfunction. Ursodeoxycholic acid (UDCA) is a FXR agonist approved by the FDA for the treatment of primary biliary cirrhosis (PBC) and is widely used to treat a variety of chronic cholestatic diseases. Obeticholic acid (OCA) is another bile acid FXR agonist approved for PBC. Phase III clinical trials of OCA have also been conducted for non-alcoholic steatohepatitis. However, due to its serious adverse reactions such as itching at effective doses, increased total cholesterol and low-density lipoprotein cholesterol levels, and reduced low-density lipoprotein cholesterol levels, many bile acid FXR agonists have been discontinued in preclinical or clinical trials. Therefore, the development of new FXR receptor agonists is expected to meet clinical needs.

Summary of the invention

This patent provides compounds with novel molecular structures, their pharmaceutically acceptable salts, their esters or their stereoisomers and their preparation methods. This series of compounds can effectively stimulate FXR receptors, and provide new solutions for the prevention and treatment of major human diseases such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, primary biliary cirrhosis, primary sclerosing cholangitis, lipid metabolism disorders, diabetic complications and malignant tumors.

In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

In the first aspect, a compound as shown in formula (I) is provided:

in:

Ring A may be a benzene ring, a cyclohexene ring, a cyclohexane ring, a bicyclo[2.2.1]heptane, a bicyclo[2.2.2]octane, a bicyclo[2.2.1]hept-2-ene or a bicyclo[2.2.2]oct-2-ene;

R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, nitro, amino, hydroxy, carboxyl, benzyloxycarbonyl, or trifluoromethyl;

R ₅ , R ₆ , R ₇ , R ₈ , R ₉ are independently selected from hydrogen, halogen, hydroxy, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxycarbonyl or trifluoromethyl;

R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are independently selected from hydrogen, halogen, C1-C4 alkyl and C1-C4 alkoxy.

Preferably, the compound is a compound represented by formula (II):

Wherein, R ₅ , R ₆ , R ₇ , R ₈ , R _{9 , R 10 ,} R ₁₁ , R ₁₂ and R ₁₃ in formula (II) are defined as in formula (I).

Preferably, the compound is a compound represented by formula (III):

Wherein, R ₅ , R ₆ , R ₇ , R ₈ , R _{9 , R 10 ,} R ₁₁ , R ₁₂ and R ₁₃ in formula (III) are defined as in formula (I).

Preferably, the compound is a compound represented by formula (IV):

Wherein, R ₅ , R ₆ , R ₇ , R ₈ , R _{9 , R 10 ,} R ₁₁ , R ₁₂ and R ₁₃ in formula (IV) are defined as in formula (I).

Preferably, the compound provided by the present invention is:

In a second aspect, the present invention provides a method for preparing a compound, comprising the following steps:

wherein, the definitions of ring A and R ₁₀ , R ₁₁ , R ₁₂ , and R ₁₃ in formula (V), (VI), (X), and (XI) are the same as those of the compounds according to any one of claims 1 to 5; and R 1 , R ₂ , R ₃ , and R ₄ in (VII), (VIII ₎ , (IX), (X), and (XI) are independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, nitro, benzyloxycarbonyl, or trifluoromethyl.

(1) At -25°C, an appropriate amount of anhydrous sodium sulfate, 1 equivalent of commercially available compound V and 1.2 equivalents of acetaldehyde were added to a round-bottom flask equipped with a magnetic stirrer. The reaction solution was stirred for 1 hour, and then 0.05 equivalent of (11bS)-2,6-bis(4-chlorophenyl)-4-hydroxy-8,9,10,11,12,13,14,15-octahydronaphthalene[2,1-d:1',2'-f][1,3,2]dioxaphosphine 4-oxide and 1 equivalent of O-benzyl-N-vinylcarbamate were added. The reaction solution was stirred at room temperature for 2 hours, separated by silica gel column chromatography, and recrystallized from n-hexane/ethyl acetate to obtain intermediate VI.

(2) 1 equivalent of the intermediate VII of the commercially available compound was placed in a round-bottom flask equipped with a magnetic stirrer, and 1 equivalent of L-alanine and glacial acetic acid were added. The mixture was stirred at room temperature for 2 h, and the intermediate VIII was separated by column chromatography.

(3) 1 equivalent of intermediate VIII was placed in a round-bottom flask equipped with a magnetic stirrer, and 1.5 equivalents of oxalyl chloride, dichloromethane and 2-3 drops of DMF were added. The mixture was stirred at room temperature for 0.5 h and the solvent was dried to obtain intermediate IX.

(4) 1 equivalent of intermediate VI was placed in a round-bottom flask equipped with a magnetic stirrer, and dichloromethane, 1.3 equivalents of DIPEA and 1.2 equivalents of intermediate IX were added. The mixture was stirred at room temperature for 3 h, and intermediate X was separated by column chromatography.

(5) 1 equivalent of intermediate X was placed in a round-bottom flask equipped with a magnetic stirrer, and acetonitrile, 3 equivalents of trimethylsilyl chloride and 3 equivalents of sodium iodide were added. The mixture was stirred at room temperature for 1 h, washed with cyclohexane 5 times, the solvent was spin-dried, extracted with ethyl acetate, washed once with saturated sodium carbonate solution, sodium thiosulfate solution and saturated brine respectively, and the solvent was spin-dried to obtain intermediate XI.

wherein, the definitions of ring A and R ₅ , R 6 , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ in formula (XI) and (XIII ₎ are the same as those of the compounds described in any one of claims 1 to 5; R ₁ , R ₂ , R ₃ and R ₄ in formula (XI) are independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, nitro, benzyloxycarbonyl or trifluoromethyl; formula (XIII)

wherein R ₁ , R ₂ , R ₃ and R ₄ are independently selected from nitro, benzyloxy and benzyloxycarbonyl.

(6) 1 equivalent of intermediate XI was placed in a round-bottom flask equipped with a magnetic stirrer, and 2 equivalents of commercially available compound XII and 2 equivalents of anhydrous copper acetate were added. DMF and 5 equivalents of pyridine were added, and the mixture was stirred at room temperature for 12 h. The target compound I was separated by column chromatography.

(7) 1 equivalent of intermediate XIII was placed in a round-bottom flask equipped with a magnetic stirrer, and 2 equivalents of commercially available compound XII and 2 equivalents of anhydrous copper acetate were added. DMF and 5 equivalents of pyridine were added, and the mixture was stirred at room temperature for 12 h. The mixture was filtered through celite, and the reaction solution was spun dry and then dissolved in tetrahydrofuran. 0.1 equivalent of palladium-carbon catalyst was added, and the mixture was stirred at room temperature for 2 h under hydrogen. The target product I was separated by column chromatography.

In a third aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

In a fourth aspect, the present invention provides use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a farnesoid X receptor agonist.

In a fifth aspect, the present invention provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof for treating and/or preventing FXR-mediated diseases and related diseases, including fatty liver, cirrhosis, hepatitis, liver failure, cholestasis, cholelithiasis, bile acid disorders, atherosclerosis, arteriosclerosis, fibrosis-related diseases, thrombosis, myocardial infarction, stroke, clinical complications of type 1 or type 2 diabetes, hyperproliferative diseases and inflammatory bowel disease.

Preferably, the fatty liver is selected from alcoholic fatty liver and non-alcoholic fatty liver; the cirrhosis is selected from primary biliary cirrhosis and primary bile duct cirrhosis; the hepatitis is selected from hyperlipidemia chronic hepatitis, chronic hepatitis, non-viral hepatitis, alcoholic fatty liver and non-alcoholic fatty liver; the cholestasis is selected from benign intrahepatic cholestasis, progressive familial intrahepatic cholestasis, extrahepatic cholestasis, drug-induced cholestasis, pregnancy cholestasis, cholestasis associated with gastrointestinal nutrition and extrahepatic cholestasis; the diabetic complications are selected from diabetic nephropathy, diabetic neuropathy and diabetic retinopathy; the tumor disease is selected from hepatocellular carcinoma, colon adenoma, polyposis, colon adenocarcinoma, breast cancer, pancreatic cancer, esophageal cancer and other forms of gastrointestinal and liver tumor diseases.

Compared with the prior art, the present invention has the following beneficial effects:

The experiment discovered for the first time a new type of FXR agonist that can inhibit the transcriptional activity of the downstream molecule SREBP, providing a new solution for the prevention and treatment of major human diseases such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, primary biliary cirrhosis, primary sclerosing cholangitis, lipid metabolism disorders, diabetic complications and malignant tumors. Because this structural type is different from the reported FXR inhibitors, it has the value of in-depth research and the potential for clinical application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Dose-effect curves and _EC50 of compounds stimulating FXR

The results showed that the EC50 values of T1 (XJ02862-S2), T2 (QT-05-09), T3 (QT-05-11), T4 (QT-05-22), T5 (QT-05-19), T6 (QT-05-13), T7 (QT-05-14), T10 (QT-08-21), T11 (QT-08-22), T12 (QT-08-23) and T13 (QT-08-24) were 0.99, 0.14, 1.30, 2.87, 0.10, 0.92, 0.18, 1.25, _1.24 , 1.00 and 1.12 μM, respectively.

Figure 2: Effects of compound T1 on serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in high-fat-fed C57 obese mice

The results showed that T1 could significantly reduce the levels of TC and LDL-C in the serum of C57BL/6J obese mice fed with high-fat diet.

Figure 3: Effect of compound T1 on total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the liver of high-fat fed C57 obese mice. The results showed that T1 (50 mg/kg, 100 mg/kg) had the effect of lowering total cholesterol in the liver; T1 (50 mg/kg) had a tendency to lower liver low-density lipoprotein cholesterol, and T1 (100 mg/kg) significantly lowered liver low-density lipoprotein cholesterol; T1 (50 mg/kg) had a tendency to lower serum alanine aminotransferase, and T1 (100 mg/kg) significantly lowered serum alanine aminotransferase.

Figure 4: Effect of compound T1 on blood glucose in high-fat fed C57 obese mice

The results showed that T1 (50 mg/kg) had a tendency to lower fasting blood glucose, and T1 (100 mg/kg) had a significant effect in lowering fasting blood glucose; T1 (50 mg/kg, 100 mg/kg) had the effect of lowering the area under the blood glucose curve (AUC) after insulin administration, that is, it could increase the sensitivity of peripheral tissues to insulin.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific examples, but the examples are only used to illustrate the present invention, rather than to limit the present invention. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

1. Preparation and structural identification of new compounds

Example 1: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T1)

At -25 °C, aniline (3.64 mL, 1 eq), 2 g of anhydrous sodium sulfate and 40 mL of ultra-dry dichloromethane were added to a round-bottom flask, followed by acetaldehyde (2.68 mL, 1.2 eq). The reaction mixture was stirred for 1 h and then (11bS)-2,6-bis(4-chlorophenyl)-4-hydroxy-8,9,10,11,12,13,14,15-octahydronaphthalene [2,1-d :1',2'-f][1,3,2]dioxaphosphine 4-oxide (1.16 g, 0.05 eq), followed by the addition of O-benzyl-N-vinylcarbamate (7.08 g, 1 eq) dissolved in 40 mL of ultra-dry dichloromethane. The reaction solution was stirred at room temperature for 2 h, and the solvent was removed by rotary evaporation. The reaction was separated by silica gel column chromatography and then recrystallized from n-hexane/ethyl acetate to obtain a white intermediate 1 (7.67 g, yield: 65%).

1,2-cyclohexanedicarboxylic anhydride (7.71 g, 1 eq), L-alanine (4.45 g, 1 eq) and 30 mL of glacial acetic acid were added to a round-bottom flask, and the reaction solution was stirred at 120 ° C for 2 h. The reaction solution was cooled to room temperature, poured into ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain white intermediate 2 (10.1 g, yield: 90%).

To a round-bottom flask, add intermediate 2 (1.71 g, 1 eq) and 40 mL of ultra-dry dichloromethane, add oxalyl chloride (964 μL, 1.5 eq), and then add 2-3 drops of DMF. The reaction solution is stirred at room temperature for 0.5 h, and the solvent is removed by rotary evaporation to obtain white intermediate 3 (1.57 g, yield: 85%).

To a round-bottom flask, intermediate 1 (1.50 g, 1 eq), DIPEA (1.15 mL, 1.3 eq) and 15 mL of ultra-dry dichloromethane were added, followed by the addition of intermediate 3 (1.57 g, 1.2 eq) dissolved in 10 mL of ultra-dry dichloromethane. The reaction solution was stirred at room temperature for 3 h, washed with 1 M hydrochloric acid, saturated sodium bicarbonate and saturated brine, respectively, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain white intermediate 4 (1.78 g, yield: 70%).

To a round-bottom flask, intermediate 4 (1.78 g, 1 eq), anhydrous sodium iodide (1.59 g, 3 eq) and 30 mL of ultra-dry acetonitrile were added, followed by the addition of trimethylsilyl chloride (1.35 mL, 3 eq). The reaction solution was stirred at room temperature for 2 h, washed with n-hexane 5 times, and the solvent was removed by rotary evaporation. The mixture was extracted with ethyl acetate and saturated sodium carbonate solution. The organic phase was washed with sodium thiosulfate solution and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain white foamy intermediate 5 (1.11 g, yield: 85%).

To a round-bottom flask were added intermediate 5 (1.11 g, 1 eq), phenylboronic acid (0.73 g, 2 eq), anhydrous copper acetate (0.82 g, 1.5 eq), pyridine (1.21 mL, 5 eq) and 30 mL of ultra-dry DMF. The reaction solution was stirred at room temperature for 12 h, filtered with celite, extracted with ethyl acetate and water, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain the target product 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T1) (562 mg, yield: 42%).

White solid. ESI-MS (m/z): 446.57[M+H] ⁺ . ¹ H NMR (500MHz, Chloroform-d) δ7.52 (d, J = 7.8Hz, 1H), 7.36 (d, J = 7.5Hz, 2H),7.27–7.24(m,1H),7.19(t,J＝7.7Hz,2H),6.76(t,J＝7.3Hz,1H),6.60(d,J＝8.0Hz,2H),5.50( q,J＝7.6Hz,1H),4.84(q,J＝7.8Hz,1H),4.19(dd,J＝11.5,5.6Hz,1H),3.85(d, J＝6.0Hz,1H),2.90(dq,J＝14.4,7.1Hz,2H),2.69(ddd,J＝12.8,8.9,4.4Hz,1H),1.96–1.88(m,3H),1.85(q ,J＝6.9,6.0Hz,1H),1.56(t,J＝5.9Hz,1H),1.46(qd,J＝9.1,6.2,5.4Hz,3H),1.33(d,J＝7.5Hz,3H) ,1.26–1.19(m,1H),1.12(d,J=6.4Hz,3H).

Example 2: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-3a,4,7,7a-hexahydro-1H-4,7-methyleneisoindolyl-1,3(2H)-dione (T2)

Using nadic anhydride as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 456.34[M+H] ⁺ . ¹ H NMR (400MHz, Chloroform-d) δ7.50 (dd, J＝7.7, 1.8Hz, 1H), 7.35 (t, J＝7.2 Hz,2H),7.27(d,J＝1.2Hz,1H),7.24–7.15(m,2H),6.77(t,J＝7.3Hz,1H),6.60(d,J＝7.9Hz,2H), 6.35–6.27(m,2H),5.51(q,J＝7.5Hz,1H),4.83(ddt,J ＝14.9,8.5,6.3Hz,1H),4.18(dd,J＝12.1,4.4Hz,1H),3.34(ddq,J＝16.5,2.6,1.3Hz,2H),2.73–2.66(m,2H), 1.83(d,J＝9.9Hz,1H),1.54(dt,J＝10.0,1.6Hz,1H),1.34–1.25(m,4H),1.25–1.16(m,1H),1.13(d,J＝ 6.4Hz,3H).

Example 3: 5,6-difluoro-2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)isoindolyl-1,3-dione (T3)

Using 4,5-difluorophthalic anhydride as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS(m/z):476.36[M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.71–7.65(m,2H),7.55(d,J＝7.9Hz,1H), 7.40(d,J＝7.6Hz,2H),7.29(s,1H),7.20(d,J＝7.4Hz,2H),6.78(t,J＝7.3Hz,1H),6.63(d,J＝7.8 Hz,2H),5.69( p,J＝6.6,5.7Hz,1H),4.88–4.79(m,1H),4.21(dd,J＝12.7,4.6Hz,1H),2.70(ddd,J＝14.0,9.3,4.8Hz,1H) ,1.46(d,J＝7.7Hz,3H), 1.27(dt,J＝12.1,6.0Hz,1H), 1.13(d,J＝6.8Hz,3H).

Example 4: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-5-trifluoromethylisoindolyl-1,3-dione (T4)

Using 5-(trifluoromethyl)isobenzofuran-1,3-dione as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 508.22 [M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ8.14(s,1H),8.01(s,2H),7.57(d,J＝7.5Hz,1H),7.41(dd,J＝12.3,7.0Hz,2H),7.31(s,1H),7.21(t,J＝8.9Hz,2H),6.78(t,J＝7.0Hz, 1H),6.63(d,J＝7.6Hz,2H),5.74(s,1H),4.84(d,J＝7.4Hz,1H),4.22(d,J＝1 2.0Hz,1H),2.71(s,1H),1.51–1.46(m,3H),1.28–1.25(m,1H),1.16–1.11( m,3H).

Example 5: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-4,7-ethylideneisoindolyl-1,3(2H)-dione (T5)

4-Oxatricyclo[5.2.2.0,2,6]undecane-3,5-dione was used as the raw material and the synthesis method was the same as in Example 1. White solid. ESI-MS (m/z): 472.37 [M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.56(d,J＝7.9Hz,1H),7.37(d,J＝7.5Hz,2H),7.27(s,1H),7.19(t,J＝7.7Hz,2H),6.76(t,J＝7.4Hz,1H),6.60(d,J＝8.0Hz,2H),5.56( q,J＝7.4Hz,1H),4.87(q,J＝8.0Hz,1H),4.20(dd,J＝12.0,6.2Hz,1H),3.83(d,J＝6.7Hz,1H) ,2.84(s,2H),2.69(ddd,J＝12.7,8.9,4.3Hz,1H),2.21(d,J＝8.7Hz,2H),1.88(d,J＝14.8Hz,1H),1.74(s,1H),1.70(d,J＝16.7Hz,1H),1.64(d,J＝10.6Hz ,2H),1.58(s,1H),1.49–1.46(m,1H),1.35(d,J＝7.6Hz,3H),1.29–1.19(m,2H),1.13(d,J＝6.4Hz,3H).

Example 6: Benzyl 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylate (T6)

Using 1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxylic acid benzyl ester as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 574.25 [M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.66–7.56(m,3H),7.47(s,2H),7.40–7.35(m,5H),7.20(dd,J＝8.1,3.7Hz,4H),6.77(t,J＝7.9Hz,1H),6.63(d,J＝8.0Hz,2H),5.70(s ,1H),5.35(s,2H),4.89–4.81(m,1H),4.21(s,1H),3.85(s,1H),2.74–2.65(m,1H),1.48(d,J＝7.7Hz,3H),1.22(s,1H),1.13(d,J＝6.5Hz,3H).

Example 7: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T7)

In a hydrogen atmosphere, compound T6 (500 mg) was added to a round-bottom flask, and then palladium carbon catalyst (50 mg) was added. The reaction solution was stirred at room temperature for 2 h, filtered through celite, and separated by silica gel column chromatography to obtain the target product 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T7) (340 mg, yield: 85%).

White solid. ESI-MS(m/z):484.20[M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ8.46(s,1H),8.39(s,1H),7.93(s,1H), 7.61(s,1H),7.40(d,J＝8.4Hz,2H),7.30(s,1H),7.20(s,2H),6.77(d,J＝7.8Hz,1H),6.62(s,2H ),5.75(d,J＝9.4Hz,1H),4.88(s,1H),4.23(d,J＝12.9Hz,1H),2.70(s,1H),1.50(s,3H),1.26(s ,1H),1.16–1.12(m,3H).

Example 8: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-4-nitroisoindolyl-1,3-dione (T8)

Using 3-nitrophthalic anhydride as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 485.17 [M+Na] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ8.13(dd,J＝12.9,7.8Hz,2H),7.92(t,J＝7.8Hz,1H),7.58(d,J＝7.8Hz,1H),7.39(d,J＝7.5Hz,2H),7.32(d,J＝10.1Hz,1H),7.19(s,2H) ,6.78(t,J＝7.3Hz,1H),6.62(d,J＝8.0Hz,2H),5.76(q,J＝7.6Hz,1H),4.83(q,J＝7.7Hz,1H),4.21(dd,J＝12.1,4.2Hz,1H),2.70(ddd,J＝12.8,8.8,4.3Hz,1 H),1.48(d,J＝

7.6Hz, 3H), 1.26 (q, J = 12.2Hz, 1H), 1.13 (d, J = 6.4Hz, 3H).

Example 9: 4-amino-2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)isoindolyl-1,3-dione (T9)

In a hydrogen atmosphere, compound T8 (350 mg) was added to a round-bottom flask, and then palladium carbon catalyst (35 mg) was added. The reaction solution was stirred at room temperature for 2 h, filtered with celite, and separated by silica gel column chromatography to obtain the target product 4-amino-2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)isoindolyl-1,3-dione (T9) (280 mg, yield: 88%).

White solid. ESI-MS (m/z): 455.26 [M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.59(d,J＝7.8Hz,1H),7.40(t,J＝8.5Hz,3H),7.23–7.16(m,4H),6.84(d,J＝8.3Hz,1H),6.80–6.75(m,1H),6.64(d,J＝8.0Hz,2H),5. 67(d,J＝8.0Hz,1H),4.85(s,1H),4.21(dd,J＝12.4,4.2Hz,1H),2.72–2.65(m,1H),1.46(d,J＝7.6Hz,3H),1.25(s,1H),1.14(d,J＝6.4Hz,3H).

Example 10: 2-((S)-1-((2S,4R)-2-methyl-4-(3-trifluoromethylphenylamino)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T10)

Using 3-trifluoromethylphenylboronic acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 514.22[M+H] ⁺ . ¹ H NMR (500MHz, Chloroform-d) δ7.54 (dd, J＝8.0, 4.0Hz, 1H), 7.38 (ddt, J＝10.5 ,7.6,2.6Hz,1H),7.29(d,J＝5.7Hz,3H),7.00(t,J＝5.9Hz,1H),6.83–6.79(m,1H),6.72(d,J＝8.9Hz ,1H),5.50(dq,J＝11.7,7.4,5.4Hz,1H),4.87(dt,J＝14.0,7.3Hz, 1H),4.24–4.16(m,1H),2.92(tt,J＝11.7,5.2Hz,2H),2.70(ddt,J＝13.0,8.8,4.2Hz,1H),1.97–1.89(m,3H) ,1.84(d,J＝13.8Hz,1H),1.59–1.54(m,1H),1.52–1.42(m,3H),1.33(dd,J＝8.0,3.9Hz,3H),1.30–1.23(m ,1H),1.14(t,J=5.4Hz,3H).

Example 11: 2-((S)-1-((2S,4R)-4-(3-hydroxyphenylamino)-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindole-1,3(2H)-dione (T11)

Using 3-hydroxyphenylboronic acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 462.25 [M+H] ⁺ . ¹ H NMR (500MHz, Chloroform-d) δ7.51 (d, J = 7.8 Hz, 1H), 7.40–7.31 (m, 2H), 7.25 (s, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.27 (d, J = 7.9 Hz, 1H), 6.22 (d, J = 8.2 Hz, 1H), 6.17 (s, 1H), 5.48 (q, J = 7.5 Hz, 1H), 4.81 (s, 1H), 4. 16(dd,J＝12.1,4.4Hz,1H),3.48(q,J＝7.0Hz,1H),2.93–2.86(m,2H),2.70–2.62(m,1H),1.90(d,J＝5.6Hz,3H),1.88(s,1H),1.46(s,4H),1.31(d,J＝7 .6Hz,3H),1.21(d,J＝7.0Hz,1H),1.11(d,J＝6.4Hz,3H).

Example 12: 2-((S)-1-((2S,4R)-4-(3-methoxyphenyl)-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T12)

Using 3-methoxyphenylboronic acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 476.27 [M+H] ⁺ . ¹ H NMR (500 MHz, Chloroform-d) δ7.51 (d, J = 7.7 Hz, 1H), 7.35 (dd, J = 13.9, 7.5 Hz, 2H), 7.21 (s, 1H), 7.08 (s, 1H), 6.33 (d, J = 8.4 Hz, 1H), 6.22 (t, J = 11.1 Hz, 1H), 6.15 (s, 1H), 5.49 (q, J = 8.4, 8.0 Hz, 1H), 4.83 ( s,1H),4.21–4.14(m,1H),3.72(d,J＝1.5Hz,3H),2.96–2.86(m,2H),2.68(ddd,J＝13.3,9.2,4.2Hz,1H),1.90(s,4H),1.45(d,J＝14.9Hz,4H),1.33(d,J ＝7.6Hz,3H),1.28–1.23(m,1H),1.15–1.09(m,3H).

Example 13: 2-((S)-1-((2S,4R)-2-methyl-4-(m-toluamino)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T13)

Using 3-methoxyphenylboronic acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS(m/z):460.37[M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.52(d,J＝7.8Hz,1H),7.40–7.33(m,2H), 7.21(s,1H),7.08(t,J＝7.8Hz,1H),6.60(d,J＝7.5Hz,1H),6.46(s,1H),6.42(d,J＝7.7Hz,1H), 5.50(q,J＝7.6Hz,1H),4.83(dd,J＝14.6,7.8Hz,1H), 4.19(dd,J＝12.3,4.4Hz,1H),2.90(dq,J＝14.6,7.3Hz,2H),2.68(ddd,J＝12.9,8.8,4.3Hz,1H),2.27(s,3H) ,1.94–1.88(m,3H),1.85(s,1H),1.51–1.43(m,4H),1.33(d,J＝7.6Hz,3H),1.28(s,1H),1.12(d,J =6.4Hz,3H).

Example 14: 2-((S)-1-((2S,4R)-4-(4-fluorophenylamino)-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T14)

Using 4-fluorophenylboric acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS(m/z):464.25[M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.52(d,J＝7.7Hz,1H),7.39–7.31(m,2H), 7.28(d,J＝4.7Hz,1H),6.90(td,J＝8.7,1.9Hz,2H),6.53(dd,J＝8.8,4.3Hz,2H),5.53–5.45(m,1H),4.83 (td,J＝8.6,4.2Hz,1H),4.11(dd,J＝12.2 ,4.3Hz,1H),2.91(dq,J＝12.4,6.7Hz,2H),2.67(ddd,J＝12.7,8.6,4.1Hz,1H),1.96–1.87(m,3H),1.83(dd, J＝12.7,6.5Hz,1H),1.55–1.42(m,4H),1.31(d,J＝7.6Hz,3H),1.26–1.19(m,1H),1.12(dd,J＝6.3,1.7Hz ,3H).

Example 15: 2-((S)-1-((2S,4R)-4-(3-fluorophenylamino)-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)hexahydro-1H-isoindolyl-1,3(2H)-dione (T15)

Using 3-fluorophenylboric acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS(m/z):464.23[M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.53(d,J＝7.9Hz,1H),7.39–7.29(m,2H), 7.25(s,1H),7.11(q,J＝7.8Hz,1H),6.48–6.40(m,1H),6.36(dt,J＝7.3,3.6Hz,1H),6.30–6.23(m,1H) ,5.49(q,J＝7.5Hz,1H),4.84(dp,J＝12.9,6.8,6.4Hz,1H),4.15(dd, J＝12.3,4.6Hz,1H),2.90(dq,J＝14.6,7.7,7.2Hz,2H),2.68(ddd,J＝12.9,8.8,4.6Hz,1H),1.90(q,J＝6.7, 6.1Hz,3H),1.84(s,1H),1.58–1.51(m,1H),1.45(q,J＝9.4,6.9Hz,3H),1.33(d,J＝7.8Hz,3H),1.28– 1.19(m,1H),1.12(d,J＝6.6Hz,3H).

Example 16: 2-((S)-1-((2S,4R)-2-methyl-4-(3,4,5-trifluoromethylphenylamino)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T16)

Using 3,4,5-trifluorophenylboric acid as raw material, the synthesis method is the same as Example 7.

White solid. ESI-MS (m/z): 538.15 [M+H] ⁺ . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ8.38 (d, J=7.7 Hz, 1H), 8.27 (s, 1H), 7.97 (d, J=7.7 Hz, 1H), 7.52 (d, J=7.8 Hz, 1H), 7.45 (t, J=7.6 Hz, 1H), 7.35 (t, J=7.7 Hz, 1H), 7.27 (d, J=7.7 Hz, 1H), 6.63 (d, J=8.2 Hz,1H),6.51–6.48(m,1H),5.55(q,J＝7.5Hz,1H),4.65(q,J＝7.1Hz,1H),4.26(d,J＝9.5Hz,1H),2.63(s,1H),1.36(d,J＝7.6Hz,3H),1.23(s,1H),1.03( d,J＝6.3Hz,3H).

Example 17: 2-((S)-1-((2S,4R)-6-fluoro-2-methyl-4-(m-toluamino)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T17)

Using 4-fluoroaniline and 3-methylphenylboronic acid as raw materials, the synthesis method is the same as Example 7.

White solid. ESI-MS (m/z): 516.19 [M+H] ⁺ . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.34 (d, J = 7.7 Hz, 1H), 8.25 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.55 (dd, J = 8.9, 4.8 Hz, 1H), 7.29 (t, J = 8.9 Hz, 1H), 7.04–6.95 (m, 2H), 6.48 (s, 1H), 6.43 (dd, J = 13.8, 7 .7Hz,2H),6.08(d,J＝7.8Hz,1H),5.47(q,J＝7.6Hz,1H),4.66(s,1H),2.65(s,1H),2.19(s,3H),1.40(d,J＝7.5Hz,3H),1.17(d,J＝10.0Hz,1H),1.02(d, J＝6.4Hz,3H).

Example 18: 2-((S)-1-((2S,4R)-7-fluoro-2-methyl-4-(m-toluinyl)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T18)

Using 3-fluoroaniline and 3-methylphenylboronic acid as raw materials, the synthesis method is the same as Example 7.

White solid. ESI-MS (m/z): 516.21 [M+H] ⁺ . ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.35(d,J＝7.6Hz,1H),8.29(s,1H),7.84(d,J＝7.6Hz,1H),7.40(dd,J＝9.7,2.4Hz,1H),7.25(t,J＝7.3Hz,1H),7.22–7.15(m,1H),7.01(t,J＝7.8Hz,1H) ,6.49–6.39(m,3H),6.12(d,J＝7.3Hz,1H),5.55(s,1H),4.66(s,1H),4.10(s,1H),2.66(s,1H),2.19(s,3H),1.45(d,J＝7.5Hz,3H),1.07(d,J＝6.4Hz ,3H).

Example 19: 2-((S)-1-((2S,4R)-8-fluoro-2-methyl-4-(m-toluinyl)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T19)

Using 2-fluoroaniline and 3-methylphenylboronic acid as raw materials, the synthesis method is the same as Example 7.

White solid. ESI-MS (m/z): 516.21 [M+H] ⁺ . ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.33(d,J＝7.9Hz,1H),8.26(s,1H),7.81(d,J＝7.6Hz,1H),7.36(s,1H),7.07(s,1H),6.99(d,J＝8.6Hz,1H),6.44–6.37(m,3H),6.18(d,J＝7.3Hz,1H) ,5.21(d,J＝7.8Hz,1H),4.72(s,1H),4.11(s,1H),2.69(s,1H),2.17(s,3H),1.36(d,J＝7.6Hz,3H),1.23(s,1H),1.02(d,J＝6.4Hz,3H).

Example 20: 4-Benzyloxy-2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)isoindolyl-1,3-dione (T20)

Using 4-benzyloxyisobenzofuran-1,3-dione as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 546.23 [M+H] ⁺ . ¹ H NMR(500MHz,Chloroform-d)δ7.66–7.56(m,3H),7.47(s,2H),7.40–7.35(m,5H),7.20(dd,J＝8.1,3.7Hz,4H),6.77(t,J＝7.9Hz,1H),6.63(d,J＝8.0Hz,2H),5.70(s ,1H),5.35(s,2H),4.89–4.81(m,1H),4.21(s,1H),3.85(s,1H),2.74–2.65(m,1H),1.48(d,J＝7.7Hz,3H),1.22(s,1H),1.13(d,J＝6.5Hz,3H).

Example 21: 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T21)

In a hydrogen atmosphere, compound T20 (480 mg) was added to a round-bottom flask, and then palladium carbon catalyst (48 mg) was added. The reaction solution was stirred at room temperature for 2 h, filtered with celite, and separated by silica gel column chromatography to obtain the target product 2-((S)-1-((2S,4R)-2-methyl-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T7) (344 mg, yield: 86%).

White solid. ESI-MS (m/z): 456.23 [M+H] ⁺ . ¹ H NMR (500 MHz, Chloroform-d) δ7.75 (s, 1H), 7.62–7.54 (m, 2H), 7.40 (q, J = 6.1, 4.5 Hz, 3H), 7.30 (d, J = 7.5 Hz, 1H), 7.22–7.17 (m, 2H), 6.78 (t, J = 7.3 Hz, 1H), 6.62 (d, J = 7.9 Hz ,2H),5.68(q,J＝7.6Hz,1H),4.88–4.81(m,1H),4.21(d,J＝11.9Hz,1H),2.70(ddd,J＝12.6,8.8,4.5Hz,1H),1.47(d,J＝7.5Hz,3H),1.32–1.21(m,2H),1 .14(d,J＝6.3Hz,3H).

Example 22: 2-((S)-1-((2S,4R)-2-methyl 1-4-phenylamino-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-3a,4,7,7a-tetrahydro-1H-isoindolyl-1,3(2H)-dione (T22)

Tetrahydrophthalic anhydride was used as the raw material, and the synthesis method was the same as that in Example 1.

White solid. ESI-MS (m/z): 444.22[M+H] ⁺ . ¹ H NMR (400MHz, Chloroform-d) δ7.50 (d, J = 7.7Hz, 1H), 7.35 (d, J = 7.5Hz, 2H),7.26–7.15(m,3H),6.76(t,J＝7.4Hz,1H),6.59(d,J＝7.9Hz,2H),5.92(t,J＝3.4Hz,2H),5.50( q,J＝7.5Hz,1H),4.83(q,J＝7.7Hz,1H),4.17(d d,J＝12.1,4.3Hz,1H),3.84(s,1H),3.19–3.06(m,2H),2.68(ddd,J＝12.5,8.7,4.2Hz,1H),2.63–2.55(m, 2H),2.30(td,J＝14.4,13.4,6.7Hz,2H),1.30(d,J＝7.6Hz,3H),1.21(d,J＝12.1Hz,1H),1.12(d,J＝6.4 Hz,3H).

Example 23: 2-((S)-1-((2S,4R)-6-methoxy-2-methyl-4-(m-toluinyl)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindole-5-carboxylic acid (T23)

Using p-methoxyaniline and m-methylphenylboronic acid as raw materials, the synthesis method is the same as Example 7.

White solid. ESI-MS (m/z): 528.21[M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.37 (d, J = 7.7Hz, 2H), 8.26 (s, 2H), 7.93 (d,J＝7.7Hz,2H),7.42(d,J＝8.5Hz,2H),7.00(t,J＝7.9Hz,4H),6.78(d,J＝2.9Hz,2H),6.47(s ,2H),6.41(t,J＝5.8Hz,4H),6.02(d,J＝7.6Hz,2H),5.51(q,J ＝7.5Hz,2H),4.64(q,J＝7.6Hz,2H),4.09(t,J＝7.1Hz,2H),3.72(s,6H),2.62(ddd,J＝12.9,8.7,4.0Hz ,2H),2.18(s,6H),1.38(d,J＝7.4Hz,5H),1.23(s,1H),1.19–1.07(m,2H),1.00(d,J＝6.3Hz,6H) .

Example 24: 2-((S)-1-((2S,4R)-2,6-dimethyl-4-(m-toluamino)-3,4-dihydroquinolin-1(2H)-yl)-1-propionyl-2-yl)-1,3-dioxoisoindolyl-5-carboxylic acid (T24)

Using p-methylaniline and m-methylphenylboric acid as raw materials, the synthesis method is the same as Example 7.

White solid. ESI-MS (m/z): 512.21[M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.36 (d, J = 7.7Hz, 1H), 8.26 (s, 1H), 7.91 (d,J＝7.7Hz,1H),7.39(d,J＝7.9Hz,1H),7.23(d,J＝7.9Hz,1H),7.08(s,1H),6.99(t,J＝7.7Hz ,1H),6.47(s,1H),6.40(t,J＝8.3Hz,2H),6.02(d,J＝7.3Hz,1H ),5.53(q,J＝7.5Hz,1H),4.62(q,J＝7.7Hz,1H),4.12–4.01(m,1H),2.61(ddd,J＝12.9,8.7,4.2Hz,1H) ,2.29(s,3H),2.18(s,3H),1.38(d,J＝7.5Hz,3H),1.14(q,J＝12.0Hz,1H),1.00(d,J＝6.3Hz,3H) .

Example 25: Methyl 4-(((2S,4R)-1-((2S)-2-(1,3-dioxooctahydro-2H-isoindol-2-yl)propanoyl-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)amino)benzoate (T25)

Using 4-methoxycarbonylphenylboronic acid as raw material, the synthesis method is the same as that in Example 1.

White solid. ESI-MS (m/z): 504.24 [M+H] ⁺ . ¹ H NMR (400 MHz, Chloroform-d) δ

7.92–7.85(m,2H),7.54(d,J＝7.8Hz,1H),7.37(dt,J＝8.6,4.8Hz,1H),7.25(d,J＝4.3Hz,2H),6.56(d ,J＝8.3Hz,2H),5.49(q,J＝7.5Hz,1H),4.86(q,J＝7.6Hz,1H),4.26(dd,J＝12.0,4.3Hz,1H),3.85(d ,J＝1.5Hz,3H ),2.91(h,J＝7.3Hz,2H),2.70(td,J＝10.9,8.8,4.3Hz,1H),1.92(p,J＝7.1,5.8Hz,3H),1.86(s,1H) ,1.58–1.52(m,1H),1.46(q,J＝7.4,6.7Hz,3H),1.33(d,J＝7.5Hz,3H),1.30–1.24(m,1H),1.13(d,J =6.3Hz,3H).

2. Biological Activity Assay

Experimental Example 1: Preliminary screening of farnesoid X receptor (FXR) activity

1. Methods: Genetic engineering technology was used to construct the reporter gene plasmid system of PCMX-Gal4-FXRLBD and Peak12-Gal4UAS-Luci. 293T cells were transiently transfected with the PCMX-Gal4-FXRLBD/Peak12-Gal4UAS-luci gene plasmid system, and different concentrations of compounds (including positive drug OCA or blank control DMSO) were added to detect the fluorescence intensity of the reporter gene luciferase. The ratio of the fluorescence intensity of the reporter gene luciferase after compound treatment to the fluorescence intensity after DMSO treatment (fluorescence ratio) reflects the agonist intensity of the compound on FXR. In order to eliminate the differences in different batches of experiments, the fluorescence ratio of the positive drug OCA in the same batch was set to 100%, and the fluorescence ratio of DMSO (no FXR agonist activity) was set to 0. The activity of the tested compound was expressed as the percentage of activity relative to OCA. Compounds with an activity percentage greater than 0 all have FXR agonist activity.

2. The agonist activity of the compounds on FXR at a concentration of 10 μM is shown in Table 1.

Experimental Example 2: EC ₅₀ determination of compounds agonistically activating FXR transcriptional activity

1. Methods: 293T cells were transfected with reporter gene plasmid system, and different concentrations of FXR test compounds were added. The enzyme activity of reporter gene luciferase was used to reflect the activation activity of FXR. The EC ₅₀ of the compound was obtained based on the numerical value by analyzing the data using GraphPadPrism 6 software.

2. The dose-effect curves and EC ₅₀ of the compounds for FXR stimulation are shown in Figure 1. The results showed that the EC 50 values of T1 (XJ02862-S2), T2 (QT-05-09), T3 (QT-05-11), T4 (QT-05-22), T5 (QT-05-19), T6 (QT-05-13), T7 (QT-05-14), T10 (QT-08-21), T11 (QT-08-22), T12 (QT-08-23) and T13 (QT-08-24) were 0.99, 0.14, 1.30, 2.87, 0.10, 0.92, 0.18, 1.25, 1.24, _1.00 , 1.12 μM, respectively.

Experimental Example 3: Effects of Compound T1 on Serum Total Cholesterol (TC) and Low-density Lipoprotein Cholesterol (LDL-C) in C57 Obese Mice Fed with High Fat

1. Methods: Male 11-week-old C57BL/6J mice weighing 28-32g were fed a high-fat diet for 12 weeks. Ten mice of the same age were fed a normal diet as a normal control group (Normal). The mice fed a high-fat diet weighed about 45g after 12 weeks. They were randomly divided into three groups according to body weight, random blood glucose, fasting blood glucose, 40-min blood glucose reduction rate, serum triglyceride content, and total cholesterol content, namely, a model group (Control), a low-dose T1 group (50 mg/kg), and a high-dose T1 group (100 mg/kg). T1 was fully ground, 0.5% CMC-Na was fully dissolved, and the volume was fixed. The drug was administered orally once a day. Blood was collected from the tail tip in the third week of administration to determine the TC and LDL-C levels in serum.

2. The experimental results are shown in Figure 2. T1 can significantly reduce the levels of TC and LDL-C in the serum of high-fat-fed C57BL/6J obese mice.

Experimental Example 4: Effects of Compound T1 on Total Cholesterol (TC), Low-density Lipoprotein Cholesterol (LDL-C) and Alanine Aminotransferase (ALT) in the Liver of C57 Obese Mice Fed with High Fat

1. Methods: Male 11-week-old C57BL/6J mice weighing 28-32g were fed with a high-fat diet for 12 weeks. Ten mice of the same age were fed with a normal diet as a normal control group (Normal). The mice fed with a high-fat diet weighed about 45g after 12 weeks and were randomly divided into three groups, namely, a model group (Control), a low-dose T1 group (50mg/kg), and a high-dose T1 group (100mg/kg). T1 was fully ground, dissolved in 0.5% CMC-Na, and fixed to volume. The mice were gavaged once a day. After 6 weeks of gavage, the animals were killed, and blood and liver were collected to determine the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the liver and alanine aminotransferase (ALT) in the serum.

2. The experimental results are shown in Figure 3. T1 (50 mg/kg, 100 mg/kg) has the effect of lowering total cholesterol in the liver; T1 (50 mg/kg) has a tendency to lower liver low-density lipoprotein cholesterol, and T1 (100 mg/kg) significantly lowers liver low-density lipoprotein cholesterol; T1 (50 mg/kg) has a tendency to lower serum alanine aminotransferase, and T1 (100 mg/kg) significantly lowers serum alanine aminotransferase.

Experimental Example 5: Effect of Compound T1 on Blood Glucose in High-Fat-Fed C57 Obese Mice

1. Methods: Male 11-week-old C57BL/6J mice weighing 28-32g were fed a high-fat diet for 12 weeks. Ten mice of the same age were fed a normal diet as a normal control group (Normal). The mice fed a high-fat diet weighed about 45g after 12 weeks and were divided into groups. T1 was fully ground, 0.5% CMC-Na was fully dissolved and fixed to volume, and administered orally once a day. Blood was collected from the tail tip for 3 weeks to measure fasting blood sugar and blood sugar levels 40 minutes and 90 minutes after subcutaneous injection of insulin (insulin tolerance test).

2. The experimental results are shown in Figure 4. T1 (50 mg/kg) has a tendency to reduce fasting blood glucose, and T1 (100 mg/kg) has a significant effect on reducing fasting blood glucose; T1 (50 mg/kg, 100 mg/kg) has the effect of reducing the area under the blood glucose curve (AUC) after insulin administration, that is, it can increase the sensitivity of peripheral tissues to insulin.

Table 1: Agonist activity of representative compounds on farnesoid X receptor at a concentration of 10 μM (activity percentage relative to positive drug OCA)

Claims (11)

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in: Ring A can be benzene ring, cyclohexene ring, cyclohexane ring, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[2.2.1]hept-2-ene or bicyclo[2.2 .2]oct-2-ene; R ₁ , R ₂ , R ₃ and R ₄ are independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, nitro, amino, hydroxyl, carboxyl, benzyloxycarbonyl or trifluoromethyl; R ₅ , R ₆ , R ₇ , R ₈ , R ₉ are independently selected from hydrogen, halogen, hydroxyl, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxycarbonyl or trifluoromethyl ; R ₁₀ , R ₁₁ , R ₁₂ , and R ₁₃ are independently selected from hydrogen, halogen, C1-C4 alkyl, and C1-C4 alkoxy. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that the compound is a compound of formula (II): Wherein, the definitions of R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ in formula (II) are as defined in formula (I) of claim 1. 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (III): Wherein, the definitions of R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ in formula (III) are as defined in formula (I) of claim 1. 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (IV): Wherein, the definitions of R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ in formula (IV) are as defined in formula (I) of claim 1. 5. The compound or pharmaceutically acceptable salt thereof according to claim 1, 2, 3 or 4, characterized in that the compound is selected from the following group: 6. The preparation method of the compound according to any one of claims 1-4, characterized in that it includes the following steps: (1) Commercially available compound V reacts with acetaldehyde and O-benzyl-N-vinyl carbamate to form compound VI through an enantioselective three-component Povarov reaction; compound VII is condensed with L-alanine to form compound VIII , and then undergo chlorination to form compound IX; compound VI and compound IX are condensed to form compound X, and deprotection gives compound XI; Wherein, the definitions of R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ in ring A and formulas (V), (VI), (X) and (XI) are the same as the definitions of the compounds described in any one of claims 1 to 5. , R ₁ , R ₂ , R ₃ and R ₄ in (VII), (VIII), (IX), (X) and (XI) are independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, Nitro, benzyloxycarbonyl or trifluoromethyl; (2) Chan-Evans-Lam cross-coupling of XI and commercially available compound Deprotection or hydrogenation generates the corresponding compound (I) according to claim 1; Among them, in Ring A and formulas (XI) and (XIII), R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are as defined in claims 1 to 5 Definition of a compound described in paragraph 1; in formula (XI), R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from hydrogen, halogen, C1-C4 alkoxy, benzyloxy, nitro, and benzyloxycarbonyl Or trifluoromethyl; in formula (XIII), R ₁ , R ₂ , R ₃ and R ₄ are independently selected from nitro, benzyloxy and benzyloxycarbonyl. 7. A pharmaceutical composition, characterized in that it includes the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. 8. The pharmaceutical composition according to claim 7, characterized in that the pharmaceutical composition is selected from the group consisting of injections, tablets, pills, capsules, suspensions or emulsions. 9. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a farnesoid X receptor agonist. 10. The use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the treatment and/or prevention of FXR-mediated diseases and related diseases, including fatty liver, Cirrhosis, hepatitis, liver failure, cholestasis, gallstone disease, bile acid disorders, hypercholesterolemia, atherosclerosis, arteriosclerosis, fibrosis-related diseases, thrombosis, myocardial infarction, stroke, type 1 or type 2 diabetes of clinical complications, hyperproliferative diseases, neoplastic diseases, and inflammatory bowel disease. 11. The application according to claim 10, characterized in that the fatty liver is selected from alcoholic fatty liver and non-alcoholic fatty liver; the liver cirrhosis is selected from primary biliary cirrhosis and primary cholangiohepatitis. Cirrhosis, the hepatitis is selected from the group consisting of hyperlipidemic chronic hepatitis disease, chronic hepatitis, non-viral hepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis; the cholestasis is selected from the group consisting of benign intrahepatic cholestasis, progressive familial intrahepatic cholestasis Cholestasis, extrahepatic cholestasis, drug-induced cholestasis, cholestasis of pregnancy, cholestasis associated with gastrointestinal nutrition, extrahepatic cholestasis; the type 1 or type 2 diabetes complications are selected from diabetic nephropathy, Diabetic neuropathy, diabetic retinopathy; the neoplastic disease is selected from the group consisting of hepatocellular carcinoma, colon adenoma, polyposis, colon adenocarcinoma, breast cancer, pancreatic cancer, esophageal cancer and other forms of gastrointestinal and liver tumors disease.