Preparation methods of polysubstituted phenyl Nur77 receptor modulators and their applications in antifibrosis

Abstract

This invention relates to methods for preparing polysubstituted phenyl Nur77 receptor modulators and their applications in anti-fibrosis, involving trisubstituted phenyl derivatives and providing a class of polysubstituted phenyl derivatives with novel structures. Methods for preparing polysubstituted phenyl derivatives represented by structural formulas I, II, and III are provided. The invention also provides the use of a class of polysubstituted phenyl derivatives with novel structures as Nur77 receptor modulators, which can improve liver injury, reduce collagen deposition, lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and improve fibrosis by modulating the activity of Nur77-related signaling pathways.

Description

Technical Field

[0001] This invention relates to the field of polysubstituted phenyl derivatives, and more particularly to a method for preparing polysubstituted phenyl Nur77 receptor modulators and their application in antifibrosis. Background Technology

[0002] Liver fibrosis, primarily caused by viral or metabolic chronic liver diseases, is a significant health concern. Many chronic factors contribute to the progression of liver fibrosis, including persistent viral infections (such as hepatitis B and hepatitis C viruses), toxic injury, chronic excessive alcohol consumption, non-alcoholic fatty liver disease (NAFLD) / non-alcoholic lipohepatitis (NASH), autoimmune liver diseases, and various metabolic and genetic disorders. Fibrosis is associated with the progression of liver disease and is a key risk factor for cirrhosis and hepatocellular carcinoma (HCC). Activation of hepatic stellate cells (HSCs) is considered a critical event in the development of liver fibrosis. Following liver injury, dormant HSCs are activated and transform into myofibroblasts, leading to excessive secretion of extracellular matrix (ECM) components. This process ultimately results in excessive ECM deposition within the liver. This excessive ECM accumulation triggers abnormal wound healing responses. Recent scientific advances have revolutionized our understanding of the mechanisms of liver fibrosis and demonstrated that it can be reversed by eliminating or eradicating pathogens (e.g., by controlling or curing viral infections). However, the reversal process often unfolds too slowly or infrequently to prevent life-threatening complications, especially in the later stages of fibrosis. Despite numerous efforts, currently available antifibrotic drug candidates have shown limited efficacy in clinical trials, and no treatment for liver fibrosis has been approved to date. There is an urgent need to develop new antifibrotic compounds with novel drug targets.

[0003] Nuclear receptor 77 (Nur77, also known as TR3 or NR4A1) belongs to the steroid / thyroid hormone receptor superfamily and plays diverse roles in various physiological activities. Nur77 plays a crucial role in various fibrotic and tissue remodeling diseases, including skin, lung, liver, and kidney fibrosis. It is a potential drug target for antifibrotic therapy. In liver tissue with CCl4-induced liver fibrosis, cellular levels of Nur77 are significantly reduced. Nur77 deficiency leads to the progression of liver fibrosis in mice. TGF-β1 (transforming growth factor β1) is known to participate in liver fibrosis through the Smad3 signaling pathway. Recent studies have shown that aberrant activation of the AKT signaling pathway in TGF-β1-driven liver fibrosis is a SMAD-independent pathway. Activation of the AKT signaling pathway in TGF-β1-treated LX2 cells (human hepatic stellate cells) promotes Nur77 degradation, leading to activation of hepatic stellate cells (HSCs). Therefore, stabilizing Nur77 or increasing its expression is a strategy for preventing liver fibrosis, and the development of Nur77-targeted anti-fibrotic drugs also has great potential to solve multi-organ fibrosis. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide a method for preparing polysubstituted phenyl Nur77 receptor modulators and their application in antifibrosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The primary objective of this invention is to provide a polysubstituted phenyl Nur77 receptor modulator with a novel structure, comprising compounds with at least one of the following structural formulas:

[0007]

[0008] Wherein, R1 represents H, F, Cl, Br, OH, OMe; R2 represents a straight-chain alkyl group with a length of 6 to 13, preferably n-octyl alkyl and n-nonyl alkyl; R3 represents H, Me, Et.

[0009] The polysubstituted phenyl Nur77 receptor modulators are as follows:

[0010] 1-(3,4,5-Trihydroxyphenyl)-1-decanone (A1), 1-(3,4,5-Trihydroxyphenyl)-1-decanone oxime (A2)

[0011]

[0012] 1-(3,4,5-trihydroxyphenyl)dec-1-one O-methyl oxime (A3), 1-(3,5-dibromo-4-hydroxyphenyl)-1-decone (A4),

[0013]

[0014] 1-(3,5-dihydroxy-4-methylphenyl)-1-decanone (A5), 1-(3,5-dihydroxy-4-bromophenyl)dec-1-one O-ethyl oxime (A6),

[0015]

[0016] 3,4,5-Trihydroxy-N-octylbenzamide (A7), 3,4,5-Trihydroxy-N-methyl-N-octylbenzamide (A8)

[0017]

[0018] 3,4,5-Trihydroxy-N-ethyl-N-octylbenzamide (A9), 4-Fluoro-3-hydroxy-N-methyl-N-octylbenzamide (A10)

[0019] 4-Fluoro-3-hydroxy-N-nonylbenzamide (A11), 4-Fluoro-3-hydroxy-N-decylbenzamide (A12)

[0020]

[0021] 3-Fluoro-4-hydroxy-N-methyl-N-octylbenzamide (A13), 3-Fluoro-4-hydroxy-N-nonylbenzamide (A14)

[0022]

[0023] 1-(3,5-Dihydroxy-4-bromophenyl)-1-decanone (A15).

[0024]

[0025] A second objective of this invention is to provide a method for preparing polysubstituted phenyl Nur77 receptor modulators.

[0026]

[0027] Route 1

[0028] 1) Under ice bath conditions, add raw material 1a to DMF, then add NaH, stir in ice bath, add BnCl, then heat and stir. After extraction with ethyl acetate / water, evaporate and concentrate the organic phase to obtain crude product, which is then directly hydrolyzed with methanol / water solution of sodium hydroxide to finally obtain intermediate 2a.

[0029] 2) Intermediate 2a is condensed under HATU / DIEA conditions to obtain intermediate 3a;

[0030] 3) Intermediate 3a reacts with the corresponding alkyl Grignard reagent to give intermediate 4a;

[0031] 4) The benzyl protecting group was removed under hydrogen / palladium on carbon conditions to give the final product A1;

[0032] 5) The final product A1 was refluxed with O-substituted hydroxylamine in ethanol to obtain the final products A2 and A3.

[0033]

[0034] Route 2

[0035] 1) React raw material 1b with iodomethane at room temperature under potassium carbonate / DMF conditions to obtain intermediate 2b;

[0036] 2) Intermediate 2b is condensed under HATU / DIEA conditions to obtain intermediate 3b;

[0037] 3) Intermediate 3b reacts with the corresponding alkyl Grignard reagent to give intermediate 4b;

[0038] 4) The methyl protecting group was removed from toluene with aluminum trichloride to give the final product A4.

[0039]

[0040] Route 3

[0041] 1) The raw material 1c was condensed with the corresponding alcohol under EDCI / HOBt / triethylamine conditions to obtain the final product A7.

[0042] A third object of the present invention is to provide a pharmaceutical composition comprising the polysubstituted phenyl Nur77 receptor modulator or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0043] A fourth objective of this invention is to provide the use of the aforementioned polysubstituted phenyl Nur77 receptor modulator or its pharmaceutical salt, polycrystalline mixture, or pharmaceutical composition in the preparation of medicaments for the prevention or treatment of diseases associated with the orphan nuclear receptor Nur77, particularly in the preparation of medicaments for the prevention or treatment of diseases such as fibrosis.

[0044] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0045] The multi-substituted phenyl Nur77 receptor modulators of this invention can improve liver damage, reduce collagen deposition, and lower serum alanine aminotransferase and aspartate aminotransferase levels by regulating the activity of Nur77-related signaling pathways, thereby being used for the treatment and prevention of various diseases in humans and animals, such as fibrosis. Attached Figure Description

[0046] Figure 1 This describes the induction of α-SMA transcriptional activity by A-series compounds.

[0047] Figure 2 This is a schematic diagram of the crystal structure of the A8 compound and the Nur77-LBD complex.

[0048] Figure 3 The diagram shows the binding of compound A8 to Nur77-LBD using isothermal calorimetric titration.

[0049] Figure 4 To investigate the effect of compound A8 on Nur77 expression levels in LX2 cells.

[0050] Figure 5 Morphology and α-SMA distribution of LX2 cells treated with compound A8.

[0051] Figure 6 The results show the effects of compound A8 on the expression levels of TGF-β1-induced Nur77 and α-SMA in LX2 cells.

[0052] Figure 7 To assess liver fibrosis in mice using H&E.

[0053] Figure 8 Sirius red staining of mouse liver sections.

[0054] Figure 9 The distribution level of α-SMA protein in liver tissue by immunofluorescence staining is a marker of HSC activation.

[0055] Figure 10 Serum ALT and AST levels were measured by ELISA.

[0056] Figure 11 To examine the lungs, kidneys, and spleen of a mouse model using H&E staining. Detailed Implementation

[0057] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0058] Example 1: Preparation of 1-(3,4,5-trihydroxyphenyl)-1-decanone (A1), 1-(3,4,5-trihydroxyphenyl)-1-decanone oxime (A2), and 1-(3,4,5-trihydroxyphenyl)dec-1-one O-methyl oxime (A3).

[0059]

[0060] 3,4,5-Trihydroxybenzoic acid 1a (10 g, 58.8 mmol) was added dropwise to a suspension of NaH (18.8 g, 470.3 mmol, 60%) in DMF (150 mL) at 0 °C under nitrogen atmosphere for at least 30 minutes. The mixture was stirred at room temperature for 13 hours, then heated to 40 °C and stirred for another 5 hours. The complete disappearance of compound 1a was detected by TLC (petroleum ether / ethyl acetate = 2 / 1). The reaction mixture was quenched with water (300 mL) and then extracted with dichloromethane (200 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to give a brown oil. The oil was added to a NaOH solution (5 M, 200 mL, MeOH / H2O = 1 / 1). The reaction was stirred at 55 °C for 5 hours, then quenched with water (50 mL), and the solution was acidified with 2 M HCl to pH = 3. The mixture was stirred for 30 minutes. After filtration and oven drying, the precipitate yielded 16.60 g of brown solid 3,4,5-tris(benzyloxy)benzoic acid (2a). The purity was 90.1%, and it could be used in the next step without further purification.

[0061] To a DMF (6 mL) solution of compound 2a (6 g, 13.6 mmol), DIEA (3.56 mL, 20.5 mmol), HATU (6.42 g, 16.9 mmol), and N,O-dimethylhydroxylamine (1.77 g, 17.7 mmol) were added sequentially. The reaction was stirred at room temperature for 2 hours, and then diluted with water (120 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with water (50 mL × 2) and saturated brine (30 mL), and dried over anhydrous sodium sulfate to give a yellow oily crude product, which was purified by silica gel column chromatography (eluting with petroleum ether / ethyl acetate = 5 / 1) to give 4.68 g of a yellow solid 3,4,5-tris(benzyloxy)-N-methoxy-N-methylbenzamide (3a), with a yield of 71% and a purity of 95.3%. 1 H NMR(600MHz,DMSO-d6)d ppm 3.20(s,3H)3.47(s,3H)5.02(s,2H)5.17(s,4H)7.00(s,2H)7.25-7.50(m,15H).LCMS m / z:484.1[M+H] + .

[0062] At 0℃, to C9H 19Compound 3a (0.40 g, 0.8 mmol, dissolved in 1 mL THF) was added dropwise to a suspension of MgBr (1.16 g, 5.0 mmol, 1 M in THF). The reaction was stirred at room temperature for 30 min, and the complete disappearance of compound 3a was monitored by TLC (petroleum ether / ethyl acetate = 2 / 1). The reaction was quenched with an aqueous solution of ammonium chloride (10 mL) and extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate to give a white crude solid. The crude solid was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 elution) to give 0.56 g of a yellow oil, 1-(3,4,5-tris(benzyloxy)phenyl)-1-decanone (4a), in 100% yield and with a purity of 95.1%. 1 H NMR(600MHz,DMSO-d6)d ppm0.85-0.87(m,3H)1.25-1.29(m,12H)1.56(t,J=6.94Hz,2H)2.96(s,2H)5.05(s,2H)5.22(s,4H)7.26-7.32(m,3H)7.32-7.50(m,14H).LCMS m / z:551.3[M+H] + .

[0063] Under hydrogen atmosphere, 50 mg Pd / C was added to a THF (3 mL) solution of compound 4a (0.20 g, 0.4 mmol). The reaction was stirred at 50 °C for 15 hours. The mixture was filtered through silica gel, and the solid was washed with ethyl acetate (5 mL × 3). The filtrate was concentrated. The crude product was purified by silica gel column chromatography (elution with petroleum ether / ethyl acetate = 2 / 1) to give 0.06 g of brown solid 1-(3,4,5-trihydroxyphenyl)-1-decanone (A1), with a yield of 59% and a purity of 95.0%. 1 H NMR(600MHz,DMSO-d6)dppm 0.82-0.90(m,3H)1.19-1.32(m,12H)1.55(t,J=6.94Hz,2H)2.79(t,J=7.27Hz,2H)6.93(s,2H)8.99(s,1H)9.17-9.27(m,2H).LCMS m / z:281.1[M+H] + .

[0064] O-substituted hydroxylamine hydrochloride (2.1 mmol) was added to a solution of compound A1 (0.1 g, 0.4 mmol) in EtOH (15 mL). The reaction was refluxed for 2 hours, concentrated, and diluted with water (50 mL). The solution was acidified to pH 6 with 1 M HCl. The mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate to give the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give the corresponding compound.

[0065] 1-(3,4,5-trihydroxyphenyl)-1-decanone oxime (A2), R=H, brown solid, 100% yield, 95.3% purity. 1 H NMR(600MHz,DMSO-d6)d ppm 0.83-0.88(m,3H)1.23-1.29(m,12H)1.36-1.44(m,2H)2.52-2.56(m,2H)6.58(s,2H)8.26(s,1H)8.88(s,2H)10.62-10.75(m,1H).LCMS m / z:297.2[M+H] + .

[0066] 1-(3,4,5-trihydroxyphenyl)dec-1-one O-methyl oxime (A3), R=Me, brown solid, 100% yield, 95.5% purity. 1 H NMR(600MHz,DMSO-d6)d ppm 0.83-0.87(m,3H)1.20-1.29(m,12H)1.37-1.45(m,2H)2.52-2.56(m,2H)3 .82(s,3H)6.60(s,2H)8.40(s,1H)8.91-9.00(m,2H).LCMSm / z:310.2[M+H] + .

[0067] Example 2: 1-(3,5-dibromo-4-hydroxyphenyl)-1-decanone (A4)

[0068]

[0069] 3,5-Dibromo-4-hydroxybenzoic acid (1b, 0.3 g, 1.0 mmol) was added to a solution of K₂CO₃ (0.28 g, 2.0 mmol) in DMF (5 mL). The reaction was stirred at room temperature for 3 hours and quenched with water (20 mL). The mixture was extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to give a yellow solid. This solid was added to MeOH (5.5 mL) and LiOH (4 M in H₂O, 1.5 mL). The reaction was stirred at room temperature for 1 hour and then concentrated. The crude product was diluted with water (5 mL). The solution was acidified to pH 6 with 1 M HCl, and the mixture was extracted with DCM (10 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate to give 0.27 g of a white solid, 3,5-dibromo-4-methoxybenzoic acid (2b), in 86% yield and 95.3% purity. 1 H NMR(600MHz,DMSO-d6)d ppm 3.87(s,3H)8.11(s,2H).LCMS m / z:311.2[M+H] + .

[0070] To a DCM (5 mL) solution of compound 2b (0.25 g, 0.8 mmol), DIEA (3.56 mL, 2.0 mmol), EDCI (0.23 g, 1.2 mmol), and HOBt (0.16 g, 1.2 mmol) were added sequentially. The reaction was stirred at room temperature for 1 hour. N,O-dimethylhydroxylamine (0.12 g, 1.2 mmol) was added, and the reaction was stirred at room temperature for 2 hours. The mixture was diluted with water (20 mL). The mixture was washed sequentially with 1 M HCl (5 mL) and 0.5 M NaOH (5 mL), and extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate to give a white crude solid. Purified by silica gel column chromatography (elution with petroleum ether / ethyl acetate = 5 / 1), 0.16 g of orange oily substance 3,5-dibromo-N,4-dimethoxy-N-methylbenzamide (3b) was obtained with a yield of 55% and a purity of 95.6%. 1 H NMR(600MHz,DMSO-d6)d ppm 3.26(s,3H)3.57(s,3H)3.85(s,3H)7.87(s,2H).LCMS m / z:354.2[M+H] + .

[0071] At 0℃, to C9H 193b (0.47 g, 1.3 mmol, in 10 mL THF) was added dropwise to a suspension of MgBr (1.51 g, 5.0 mmol, 1 M in THF). The reaction was stirred at room temperature for 30 min. The reaction was quenched with an aqueous solution of ammonium chloride (10 mL) and extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate to give a yellow oily crude product. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 elution) gave 0.37 g of a colorless oily product 1-(3,5-dibromo-4-methoxyphenyl)-1-decanone (4b), in 66% yield and 95.4% purity. 1 LCMS m / z:421.4[M+H] + .

[0072] Anhydrous aluminum chloride (0.48 g, 3.6 mmol) was added to a toluene (10 mL) solution of compound 4b (0.3 g, 0.7 mmol). The reaction was stirred at room temperature for 2 h and quenched with 6 M HCl (10 mL). The mixture was stirred at room temperature for 10 min and extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate to give the crude product. The crude product was purified by silica gel column chromatography (eluting with petroleum ether / ethyl acetate = 5 / 1) to give 0.28 g of brown solid 1-(3,5-dibromo-4-hydroxyphenyl)-1-decanone (A4) in 97% yield and 95.2% purity. 1 H NMR(600MHz, DMSO-d6)d ppm 0.81-0.91(m,3H)1.18-1.32(m,12H)1.56(t,J=7.06Hz,2H)2.96(t,J=7.24Hz,2H)8.09(s,2H).LCMS m / z:407.1[M+H] + .

[0073] Example 3: 1-(3,5-dihydroxy-4-methylphenyl)-1-decanone (A5)

[0074] 1-(3,5-dihydroxy-4-methylbenzoic acid)-1-decanone (A5) was obtained as a starting material using a method similar to that in Example 2. It was a brown solid with a yield of 73% and a purity of 95.1%. 1H NMR(600MHz,DMSO-d6)d ppm 0.87(dt,J=10.98,7.22Hz,3H)1.18-1.35(m,12H)1.57(br.s.,2H)1.94-2.04(m,3H)2.81(t,J=7.27Hz,2H)6.90(s,2H)9.45(s,2H).LCMS m / z:279.1[M+H] + .

[0075] Example 4: 1-(3,5-dihydroxy-4-bromophenyl)dec-1-one O-ethyl oxime (A6)

[0076] Starting with 3,5-dihydroxy-4-bromobenzoic acid, a ketone compound was obtained by a method similar to that in Example 2. Finally, it was reacted with hydroxylamine using a method similar to that in Example 1 to give 1-(3,5-dihydroxy-4-bromophenyl)dec-1-one O-ethyl oxime (A6). A brown oil was obtained with a yield of 73% and a purity of 95.1%. 1 H NMR(600MHz,DMSO-d6)d ppm 0.78-0.89(m,3H)1.15-1.29(m,15H)1.39-1.49(m,2H)2.54-2.64(m,2H)4.12(q,J=7.03Hz,2H)6.73(s,2H)10.09(s,2H).LCMS m / z:389.3[M+H] + .

[0077] Example 5: 3,4,5-Trihydroxy-N-octylbenzamide (A7)

[0078]

[0079] EDCI (0.12 g, 0.65 mmol), HOBt (0.09 g, 0.65 mmol), and triethylamine (0.1 mL, 0.71 mmol) were added sequentially to a DMF (1.5 mL) solution of 3,4,5-trihydroxybenzoic acid (0.1 g, 0.59 mmol), and the reaction was stirred at room temperature for 10 min. Octylamine (0.12 mL, 0.76 mmol) was then added. The reaction mixture was stirred at room temperature for 16 h. The mixture was diluted with water (10 mL), and extracted with ethyl acetate (3 mL × 3). The combined organic phases were washed with saturated brine (5 mL) and dried over anhydrous sodium sulfate to give a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 2) to give 86 mg of a white solid, 3,4,5-trihydroxy-N-octylbenzamide (A7), in 52% yield and 95.4% purity. 1H NMR(600MHz,CHLOROFORM-d)d ppm 0.84-0.90(m,3H)1.04-1.41(m,10H)1.48-1.68(m,2H)3.20-3.54(m,2H)6.33-6.44(m,2H).LCMS m / z:282.1[M+H] + .

[0080] Example 6: A8~A14

[0081] Using the corresponding polysubstituted benzoic acid and amine as raw materials, the corresponding amide compounds were obtained by a method similar to that in Example 5.

[0082] 3,4,5-Trihydroxy-N-methyl-N-octylbenzamide (A8) is produced from 3,4,5-trihydroxybenzoic acid and N-methyloctyl-1-amine. It is a yellow oil with a yield of 17% and a purity of 95.1%. 1 LCMS m / z:296.1[M+H] + .

[0083] 3,4,5-Trihydroxy-N-ethyl-N-octylbenzamide (A9). Starting materials are 3,4,5-trihydroxybenzoic acid and N-ethyloctyl-1-amine. It is a brown oil with a yield of 45% and a purity of 96.1%. 1 H NMR(600MHz,DMSO-d6)d ppm0.79-0.90(m,3H)1.06(t,J=6.44Hz,3H)1.13-1.32(m,10H)1.50(br.s.,2H)3.25(br.s.,4H)6.20-6.29(m,2H)8.34(s,1H)9.02(s,2H).LCMS m / z:310.2[M+H] + .

[0084] 4-Fluoro-3-hydroxy-N-methyl-N-octylbenzamide (A10). Starting materials are 4-fluoro-3-hydroxybenzoic acid and N-methyloctyl-1-amine. It is a white solid with a yield of 48% and a purity of 96.0%. 1H NMR(600MHz,CHLOROFORM-d)dppm0.80-0.95(m,3H)1.03-1.70(m,12H)2.86-3.11(m,3H )3.23(t,J=7.34Hz,1H)3.51(t,J=7.15Hz,1H)6.79(br.s.,1H)6.95-7.11(m,2H).LCMS m / z:282.1[M+H] + .

[0085] 4-Fluoro-3-hydroxy-N-nonylbenzamide (A11). Starting materials are 4-fluoro-3-hydroxybenzoic acid and n-nonylamine. It is a white solid with a yield of 52% and a purity of 95.0%. 1 H NMR(600MHz,CHLOROFORM-d)d ppm 0.89(t,J=6.97Hz,3H)1.23-1.32(m,8H)1.61(br.s.,6H)3.43(q,J=6.79Hz,2H)7.08-7.15(m,1H)7.23(s,1H)7.44-7.48(m,1H).LCMS m / z:282.2[M+H] + .

[0086] 4-Fluoro-3-hydroxy-N-decylbenzamide (A12). Starting materials are 4-fluoro-3-hydroxybenzoic acid and n-decylamine. It is a white solid with a yield of 49% and a purity of 95.2%. 1 H NMR(600MHz,CHLOROFORM-d)d ppm 0.88(t,J=6.88Hz,3H)1.24-1.73(m,18H)3.44(q,J=6.66Hz,2H)6.11(br.s.,1H)7.06-7.12(m,1H)7.18-7.24(m,1H)7.59-7.67(m,1H).LCMS m / z:296.2[M+H] + .

[0087] 3-Fluoro-4-hydroxy-N-methyl-N-octylbenzamide (A13). Starting materials are 3-fluoro-4-hydroxybenzoic acid and N-methyloctyl-1-amine. It is a white solid with a yield of 51% and a purity of 95.1%. 1H NMR(600MHz,CHLOROFORM-d)dppm0.87(br.s.,3H)1.08-1.72(m,12H)2.94-3.11(m, 3H)3.20-3.58(m,2H)6.86(t,J=8.34Hz,1H)6.98(br.s.,1H)7.07(br.s.,1H).LCMS m / z:282.2[M+H] + .

[0088] 3-Fluoro-4-hydroxy-N-nonylbenzamide (A14). Starting materials are 3-fluoro-4-hydroxybenzoic acid and n-nonylamine. It is a white solid with a yield of 41% and a purity of 95.3%. 1 H NMR(600MHz,CHLOROFORM-d)d ppm 0.89(t,J=6.88Hz,3H)1.22-1.44(m,14H)3.43(q,J=6.42Hz,2H)7.04(t,J=8.44Hz,1H)7.43(d,J=8.07Hz,1H)7.58(d,J=11.19Hz,1H).LCMS m / z:282.1[M+H] + .

[0089] Example 7: 1-(3,5-dihydroxy-4-bromophenyl)-1-decanone (A15)

[0090] Starting with 3,5-dihydroxy-4-bromobenzoic acid, 1-(3,5-dihydroxy-4-bromophenyl)-1-decanone (A15) was obtained by a method similar to that in Example 2. It was a brown solid with a yield of 61% and a purity of 95.1%. 1 H NMR(600MHz,DMSO-d6)d ppm 0.83-0.89(m,3H)1.22-1.30(m,12H)1.57(d,J=7.27Hz,2H)2.85(s,2H)6.98(s,2H)10.36(s,2H).LCMS m / z:344.2[M+H] + .

[0091] Example 8: Experiment on the transcriptional activation of α-SMA by some of the A-series compounds of the present invention in LX2 cells.

[0092] A luciferase reporter gene assay was used to analyze the effects of series A compounds on α-SMA transcriptional activity, a key biomarker for hepatic stellate cell activation. The luciferase assay was performed using a system from Promega according to the manufacturer's instructions. First, LX2 cells stably transfected with PGL3CAGA(12)-α-SMA were cultured in 96-well plates (100 μL / well) at a density of 15,000 cells / well for 24 hours in DMEM medium containing 10% fetal bovine serum albumin. Cells were then treated with or without the compound and / or TGF-β1 (5 ng / mL) for 24 hours. Fluorescence signal intensity was measured using a luciferase assay system from Promega.

[0093] Figure 1 The experiment demonstrated the effect of A-series compounds on α-SMA transcriptional activation using a dual luciferase reporter gene system. The results showed that A-series molecules could inhibit the transcriptional activity of α-SMA. The control group consisted of no TGF-β1 and no compounds.

[0094] Example 9: Compound A8 directly binds to Nur77 and promotes its expression level.

[0095] Protein samples were obtained from HSC-T6 cells, LX2 cells, and mouse liver tissue using RIPA lysis buffer (Sigma) and a mixture containing protease inhibitor PMSF and phosphatase inhibitor. Protein samples were collected by centrifugation at 12,000 rpm for 15 minutes at 4°C. Samples were separated on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Immobilion-p, Darmstadt). The membrane was immersed in 5% skim milk for 2 hours and incubated overnight at 4°C with primary antibody. The PVDF membrane was washed three times with 1×TBST and incubated for 2 hours at room temperature with anti-rabbit or anti-mouse HRP-conjugated secondary antibody. Proteins were visualized using an enhanced chemiluminescence (ECL) kit. Protein bands were quantified using ImageJ software (NIH, Bethesda, Maryland).

[0096] By using X-ray crystallography and in vitro binding experiments (isothermal calorimetric titration), it was directly demonstrated that compound A8 can directly bind to Nur77-LBD. Figure 2 The crystal structure diagram of the A8 and Nur77-LBD complex is shown, where Mol I and Mol II represent molecules 1 and 2 of the Nur77-LBD dimer, respectively. Figure 3 To determine the binding force between A8 and Nur77-LBD using isothermal calorimetric titration, the binding constant Kd was 3.71 μM. Figure 3 In Figure A, the enthalpy change of the binding reaction is represented. Figure 3Figure B shows the fitting curve based on energy changes; Figure 4 This demonstrates that A8 effectively activates the expression level of Nur77 in LX2 cells, as detected using Western blot. Figure 4 Figure A shows the results of Western blotting. Figure 4 Figure B in the middle is a quantitative chart calculated based on the results.

[0097] Example 9: Compound A8 inhibits the activation of hepatic stellate cells

[0098] LX2 cells (human hepatic stellate cells) were seeded in 24-well plates and cultured in MDEM medium containing 10% serum for 24 hours. Cells were then treated with compound A8 (7.5 μM and 15 μM) with or without TGF-β1 (5 ng / mL) for 24 hours. Cell morphology was observed using a confocal microscope (Zeiss). For immunofluorescence staining, cells were blocked with 5% BSA for 1 hour and then incubated overnight at 4°C with a primary antibody against α-SMA (1:200). After washing three times with 1xPBS, cells were incubated with a secondary fluorescent antibody at room temperature for 1 hour. Cell nuclei were stained with DAPI (Beyotime Biotechnology Co.) and images were acquired using a scanning confocal microscope (Carl Zeiss).

[0099] The effect of A8 on LX2 cell morphology is as follows: Figure 5 As shown, the results indicate that A8 can effectively inhibit the increase in α-SMA levels induced by TGF-β1 in LX2 cells, among which... Figure 5 The "control" represents the control group without the compound and without TGF-β1 treatment. Figure 5 The A8 compound indicates that it can inhibit the TGF-β1-induced transformation of LX2 cells into elongated spindle-shaped cells. Figure 5 The results from B indicate that compound A8 can effectively inhibit TGF-β1-induced 3D spheroidization in LX2 cells. Figure 5 The C-value indicates that compound A8 effectively inhibits TGF-β1-induced α-SMA (red) expression in LX2 cells. Simultaneously, Western blot results show that in LX2 cells, compound A8 effectively promotes Nur77 expression and inhibits α-SMA expression. The results are as follows... Figure 6 As shown, this effectively inhibited the activation of LX2 cells.

[0100] Example 10: Compound A8 improves liver fibrosis in mice

[0101] The therapeutic potential of compound A8 for liver fibrosis was evaluated using a mouse model. C57 mice were administered carbon tetrachloride (CCl4) to induce liver injury. Four weeks after CCl4 introduction, C57 mice were treated with compound A8 at a dose of 20 mg / kg once daily for 4 weeks. Western blot analysis showed that in a CCl4-induced liver fibrosis model, compound A8 downregulated the levels of α-SMA, p-AKT, and AKT, while upregulating the level of Nur77 (…). Figure 6 These data are consistent with cell-based assays and demonstrate that compound A8 upregulates Nur77, attenuates HSC activation, and improves liver fibrosis by inhibiting AKT activation.

[0102] In addition, histological analysis was performed on the liver tissue of the experimental mice. In the H&E staining analysis, the control group (untreated) exhibited typical liver tissue structure, such as... Figure 7 The A group showed severe liver damage, including structural damage and extensive bleeding, such as... Figure 7 In contrast, compound A8 treatment significantly reduced the pathological changes of CCl4-induced tissue damage, such as... Figure 7 In the histological analysis of collagen accumulation using Picro Sirius red staining, control was the untreated control group. Figure 8 In mice treated with CCl4, collagen levels increased, leading to the formation of large fibrous septa and surrounding collagen fiber bundles, such as... Figure 8 In contrast, compound A8 significantly reduced CCl4-induced collagen fiber deposition, such as... Figure 8 Immunofluorescence staining results of α-SMA in liver tissue (C) are as follows: Figure 9 "control" refers to the untreated control group, such as... Figure 9 Treatment with compound A8 significantly reduced the fluorescence intensity of CCl4-induced α-SMA, as shown in the following results. Figure 9 Results B and C are shown in the table. Furthermore, compared to untreated mice, mice treated with compound A8 had significantly lower serum alanine aminotransferase (ALT) levels. Figure 10 (A) and aspartate aminotransferase (AST) Figure 10 The levels of B12 (B) were significantly reduced, with the control group being the untreated group. These results indicate that treatment with compound A8 significantly improved CCl4-induced liver fibrosis in mice.

[0103] Histological analysis using H&E staining also showed that compound A8 ameliorated CCl4-induced organ damage in the lungs, kidneys, and spleen of experimental mice, as indicated by the results. Figure 11 This indicates that compound A8 has no significant toxicity to major organs, where control represents the untreated control group.

Claims

1. The application of polysubstituted phenyl Nur77 receptor modulators, characterized in that, The polysubstituted phenyl Nur77 receptor modulators include compound A8 with the following structural formula: ， The polysubstituted phenyl Nur77 receptor modulator is used to prepare drugs for treating antifibrotic diseases.

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