Ror gamma covalent inhibition compound and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a ROR gamma covalent inhibition compound and a preparation method and application thereof. Experiments prove that the compound can be covalently combined with ROR gamma protein, has good selective specificity for ROR gamma, can significantly inhibit ROR gamma, and can prevent and treat diseases related to ROR gamma. In addition, the inhibition of ROR gamma can reduce the expression of an androgen receptor AR gene in cells and the AR protein level, induce the apoptosis of cancer cells, inhibit the growth and metastasis of cancer cells, and finally achieve the effect of preventing and treating cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a RORγ covalent inhibition compound and a preparation method and application thereof. BACKGROUND

[0002] The nuclear receptor RORs (Retinoic acid receptor-related orphan nuclear receptors) family includes three subtypes: RORα, RORβ, RORγ. Among them, RORγ promotes the differentiation of effector cells Th17 and secretes inflammatory factors IL-17, therefore, inhibiting RORγ can effectively inhibit the differentiation of Th17 cells and reduce the occurrence of inflammatory reactions. Chronic inflammation is a major inducer of tumors: for example, there is high expression of RORγ in the tumor microenvironment of gastric cancer patients, accompanied by an increase in the number of Th17 cells, an increase in the expression level of genes such as IL-17, and a close relationship with the malignant degree of tumor. It can be seen that RORγ is involved in the immunopathological process of gastric cancer (Oncol Lett. 2016; 12: 897-903). Reya team found that RORγ is up-regulated during the progression of pancreatic cancer, and inhibiting RORγ can effectively inhibit the growth of pancreatic cancer (Cell. 2019; 177 (3), 572-586e22.). It can be seen that RORγ is not only an important target for treating inflammation and autoimmune diseases, but also a new target for anti-tumor drug development. There are multiple candidate compounds targeting RORγ in the clinical I / II phase research stage, but the RORγ inhibitors developed at present are all non-covalent reversible inhibitors, and the anti-cancer activity is not outstanding, and there are shortcomings such as short action time with the target, poor drug efficacy, and easy drug resistance. SUMMARY

[0003] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the existing RORγ-targeted compounds, such as poor anti-cancer activity, short action time with the target, poor drug efficacy, and easy drug resistance, and to provide a RORγ covalent inhibition compound with long action time with the target, strong drug efficacy, and not easy to be drug resistant, and with significant inhibitory effect on cancer.

[0004] The purpose of the present application is to provide a preparation method of the RORγ covalent inhibition compound.

[0005] Another purpose of the present application is to provide an application of the RORγ covalent inhibition compound.

[0006] Still another purpose of the present application is to provide a RORγ covalent inhibitor.

[0007] The above purposes of the present application are achieved by the following technical solutions:

[0008] A RORy covalent inhibition compound having the structure of Formula (I):

[0009]

[0010] wherein n is an integer from 0 to 1; X, Y, Z, W are independently selected from C or N, when W is N, there is no substituent R4; L is R1is

[0011] R a , R b , R c are each independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C3-C6cycloalkyl, or R a and R b form a C3-C6cycloalkyl with the carbon atom to which they are attached; R d , R e , R f are each independently selected from hydrogen, halogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C3-C6cycloalkyl; the substitution means at least one site is substituted with halogen, C1-C6alkyl, C1-C6alkoxy, C3-C8cycloalkyl, C1-C6alkylamino; R2, R3, R4are each independently selected from hydrogen, halogen, C1-C6alkyl, halogenated C1-C6alkyl, C1-C6alkoxy, C1-C6alkylamino, C3-C6cycloalkyl, C3-C6heterocycloalkyl; R5is selected from hydrogen, cyano, hydroxyl, nitro, substituted or unsubstituted sulfone, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy; the substitution means at least 1 site is substituted with halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, C1-C6alkyl, C1-C6alkoxy, C 1-6 alkylamino.

[0012] Preferably, n is an integer from 0 to 1; X is C, W, Y, Z are independently selected from C or N, when W is N, there is no substituent R4; L is R1is R a , R b , R c are each independently selected from hydrogen, C1-C6alkyl, cyano, halogen, or R a and R b form a C3-C6cycloalkyl with the carbon atom to which they are attached; R d , R e, R f each independently selected from hydrogen, halogen, substituted or non-substituted C1-C6 alkyl, the substitution means at least one site is substituted with C1-C6 alkoxy; R2 is hydrogen, R3, R4 are each independently selected from hydrogen, halogen, C1-C6 alkyl, halo C1-C6 alkyl; R5 is selected from hydrogen, cyano, nitro, sulfone group, C1-C6 alkyl substituted sulfone group, C1-C6 alkyl, C1-C6 alkoxy.

[0013] More preferably, n is an integer from 0 to 1; X is C, W, Y, Z are independently selected from C or N, when W is N, there is no substituent R4; L is R1 is R a , R b , R c each independently selected from hydrogen, C1-C3 alkyl, cyano, halogen, or R a and R b form a cyclopropyl ring with the carbon atom to which they are attached; R d , R e , R f each independently selected from hydrogen, halogen, substituted or non-substituted C1-C3 alkyl, the substitution means at least one site is substituted with C1-C3 alkoxy; R2 is hydrogen, R3, R4 are each independently selected from hydrogen, halogen, C1-C3 alkyl, halo C1-C3 alkyl; R5 is selected from hydrogen, cyano, nitro, sulfone group, C1-C3 alkyl substituted sulfone group, C1-C3 alkyl, C1-C3 alkoxy.

[0014] Further, the RORγ covalent inhibitory compound can also be a pharmaceutically acceptable salt or solvate thereof.

[0015] In addition, the present application also provides a preparation method of the RORγ covalent inhibitory compound, which specifically comprises the following steps:

[0016]

[0017] SI1, compound 1-1 is reacted with under the condition of catalyst, inorganic base, Suzuki reaction occurs to obtain compound 1-2;

[0018] SI2, compound 1-2 is subjected to condensation reaction with R1-COOH, or subjected to nucleophilic substitution reaction with R1-COCl to obtain compound 1-3;

[0019] SI3: compound 1-3 is subjected to reduction reaction to obtain compound 1-4;

[0020] SI4: compound 1-4 is subjected to condensation reaction with or subjected to condensation reaction with After the nucleophilic substitution reaction, the target product (I) is obtained;

[0021] wherein n, X, Y, Z, W, L, R1, R2, R3, R4, R5 are defined as above.

[0022] Further, in step SI1, the catalyst is selected from one or more of 1,1-bis(diphenylphosphino)ferrocene palladium dichloride or its dichloromethane complex, bisbenzaldehyde palladium; the inorganic base is selected from one or more of cesium carbonate, potassium carbonate, potassium phosphate; the catalyst ratio is 0.05-0.1 equivalent; the temperature of the Suzuki reaction is 100-110℃, and the reaction time is preferably 1-16h, and the reaction solvent is dioxane / water or acetonitrile / water (volume ratio 2:1-5:1). In steps SI2, SI4, the condensation agent of the condensation reaction is selected from one or more of HATU, EDCI, CDI, PyBop, and the organic base is selected from triethylamine or DIPEA; the temperature of the nucleophilic substitution reaction is -10-10℃. In step SI3, the reducing agent of the reduction reaction is selected from one or more of iron powder, palladium on carbon, or Raney nickel / hydrogen.

[0023] In addition, the present application also provides a preparation method of the RORγ covalent inhibition compound, which specifically comprises the following steps:

[0024]

[0025] SII1, compound 2-1 is reacted with After the condensation reaction, compound 2-2 is obtained;

[0026] SII2: Compound 2-2 is reacted with Suzuki reaction occurs in the presence of a catalyst and an inorganic base to obtain compound 2-3;

[0027] SII3: Compound 2-3 is subjected to a reduction reaction to obtain compound 2-4;

[0028] SII4: Compound 2-4 is subjected to a condensation reaction with R1-COOH, or a nucleophilic substitution reaction with R1-COCl to obtain the target product (I);

[0029] wherein n, X, Y, Z, W, L, R1, R2, R3, R4, R5 are defined as above.

[0030] Further, in step SII1, the condensing agent of the condensation reaction is selected from one or more of HATU, EDCI, CDI, and the organic base is selected from triethylamine or DIPEA. In step SII2, the catalyst is selected from one or more of 1,1-bis(diphenylphosphino)ferrocene palladium dichloride or its dichloromethane complex, bisbenzaldehyde palladium; the inorganic base is selected from one or more of cesium carbonate, potassium carbonate, potassium phosphate; the catalyst ratio is 0.05-0.1 equivalent; the temperature of the Suzuki reaction is 100-110°C, the reaction time is preferably 1-16h, and the reaction solvent is dioxane / water or acetonitrile / water (volume ratio 2:1-5:1). In step SII3, the reducing agent of the reduction reaction is selected from one or more of iron powder, palladium on carbon, or Raney nickel / hydrogen. In step SII4, the condensing agent of the condensation reaction is selected from one or more of HATU, EDCI, CDI, and the organic base is selected from triethylamine or DIPEA; the temperature of the nucleophilic substitution reaction is -10-10°C.

[0031] There is a cysteine Cys320 in the binding pocket inside the RORγ ligand binding domain, which is a target for covalent binding inhibitors.

[0032] In addition, the present application also claims the use of the RORγ covalent inhibition compound in the preparation of RORγ inhibitors.

[0033] Further, the use of the RORγ covalent inhibition compound in the preparation of drugs for preventing and treating diseases related to RORγ is protected.

[0034] Preferably, the diseases related to RORγ are inflammation, autoimmune diseases, viral infections, tumors or cancers, and immune deficiency disorders, etc.

[0035] In addition, based on the significant anti-tumor effect of the RORγ covalent inhibition compound of the present application, it can inhibit tumor growth and metastasis, and therefore, the use of the RORγ covalent inhibition compound in the preparation of drugs for preventing and treating tumors is also claimed.

[0036] Preferably, the tumor is one or several of a cancer or a benign tumor. The cancer can be pancreatic cancer, breast cancer, lung cancer, bone cancer, stomach cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, brain cancer, pituitary adenoma, epidermoid carcinoma, T-cell lymphoma, chronic and acute leukemia, large intestine cancer, kidney cancer, esophagus cancer, breast cancer, cervical cancer, bladder cancer, fibrosarcoma, esophagus cancer, bladder cancer, hematopoietic system cancer, lymphoma, medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, melanoma, oligodendroglioma, glioblastoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, multiple myeloma (AML), mantle cell lymphoma, triple negative breast cancer, non-small cell lung cancer.

[0037] The RORy covalent inhibition compound of the present application is proved by experiments to significantly reduce the expression of androgen receptor AR and androgen receptor variable splicing body AR-V in castration-resistant prostate cancer cells (LNCaP, 22Rv1 and C4-2B), inhibit cell proliferation, and reduce cell drug resistance.

[0038] In addition, the present application also claims a RORy covalent inhibitor containing the RORy covalent inhibition compound.

[0039] The present application has the following beneficial effects:

[0040] The present application provides a RORy covalent inhibition compound, which is proved by experiments to be able to covalently bind to RORy protein, has good selectivity to RORy, can significantly inhibit RORy, and prevent and treat diseases related to RORy. In addition, inhibiting RORy can reduce the expression of AR gene and the level of AR protein in cells, induce apoptosis of cancer cells, inhibit growth and metastasis of cancer cells, and ultimately achieve the effect of preventing and treating cancer. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure is a predicted binding mode diagram of GSK805 and compound 22 docking to the LBD binding domain of RORy.

[0042] Figure 2 Figure is a mass spectrum diagram of the covalent binding of compound 22 to RORy.

[0043] Figure 3 Figure is a statistical diagram of the results of plate cloning experiment of compound 22.

[0044] Figure 4 Figure is a statistical diagram of the results of apoptosis experiment of prostate cancer cells induced by compound 22.

[0045] Figure 5Statistical chart of experimental results for the influence of compound 22 on the expression of downstream target genes.

[0046] Figure 6 Statistical chart of experimental results for the inhibition of androgen receptor expression by compound 22.

[0047] Figure 7 Statistical chart of experimental results for the anti-tumor effect of compound 22 in a 22Rv1 nude mouse xenograft model. DETAILED DESCRIPTION

[0048] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The examples do not imply any limitation on the scope of the application, as the present application comprehends any and all combinations of features described. The reagents, methods, and apparatuses employed in the present application are those that are conventionally used in the art, unless otherwise specified.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 Preparation of N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)acrylamide (Compound 1)

[0051] The synthetic route for preparing the compound 1 is as follows:

[0052]

[0053] Specifically comprising the following steps:

[0054] S1, Preparation of N-(4-bromo-3-methylphenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (Compound 1a): 4-bromo-3-methylaniline (3.7 g, 19.7 mmol), 4-ethylsulfonylphenylacetic acid (3.0 g, 13.1 mmol), HATU (10.0 g, 26.2 mmol), and N,N-diisopropylethylamine (7 mL, 39.3 mmol) were dissolved in an appropriate amount of N,N-dimethylformamide, and stirred at room temperature overnight. After the reaction was completed, ethyl acetate and water were extracted, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain a crude product. Column chromatography yielded 4.7 g of white solid with a yield of 90.4%. 1 H NMR (400 MHz, CDC13) δ 7.85 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.45 - 7.41 (m, 2H), 7.18 (dd, J = 8.6, 2.2 Hz, 1H), 3.77 (s, 2H), 3.12 (q, J = 7.4 Hz, 2H), 2.35 (s, 3H), 1.29 (t, J = 7.4 Hz, 3H). LCMS: m / z (M+H + ): 396.02.

[0055] Preparation of S2, 2-(4-(ethylsulfonyl)phenyl)-N-(2-methyl-2'-nitro-[1,1'-biphenyl]-4- yl)acetamide (Compound 1b): Compound 1a (4.0 g, 10.1 mmol), p-nitrobenzene boronic acid (3.37 g, 20.2 mmol), chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (0.37 g, 0.51 mmol) and potassium phosphate (4.3 g, 20.2 mmol) were dissolved in an appropriate amount of tetrahydrofuran / water (1 / 2) and reacted under microwave at 100 °C for 1 hour under nitrogen protection. After the reaction was completed, ethyl acetate and water were used for extraction, and anhydrous sodium sulfate was used for drying. The solvent was removed by rotary evaporation to obtain a crude product. Column chromatography was used to obtain 3.03 g of brown oil with a yield of 68.5%. 1 H NMR (500 MHz, CDC13) δ 7.97 (d, J = 8.0 Hz, 1H), 7.87 - 7.83 (m, 3H), 7.62 (t, J = 6.8 Hz, 1H), 7.52 (d, J = 6.5 Hz, 3H), 7.43 (s, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.27 (d, J = 9.2 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 3.77 (s, 2H), 3.11 (d, J = 7.4 Hz, 2H), 2.04 (s, 3H), 1.27 (d, J = 5.5 Hz, 3H). LCMS: m / z (M+H + ): 439.12.

[0056] Preparation of S3, N-(2'-amino-2-methyl-[1,1'-biphenyl]-4-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (Compound 1c): Compound 1b (3.0 g, 6.8 mmol), iron powder (2.7 g, 47.6 mmol), ammonium chloride (3.6 g, 68 mmol) were dissolved in an appropriate amount of ethanol / water (2 / 1) and refluxed at 90 °C. After the reaction was completed, the mixture was filtered while hot, and the solvent was removed by rotary evaporation to obtain a crude product. Column chromatography was used to obtain 2.4 g of white solid with a yield of 85.5%. 1H NMR (500MHz, DMSO-d6) δ10.28(s,1H),7.86(d,J=8.1Hz,2H),7.62(d,J=8.1Hz,2H),7.54(s,1H),7.48(d,J=8.2Hz,1H),7.04(t,J=8.9Hz,2H),6.82( d,J=7.3Hz,1H),6.73(d,J=8.0Hz,1H),6.61(t,J=7.3Hz,1H),3.81(s,2H) ,3.27(q,J=7.3Hz,2H),2.05(s,3H),1.10(t,J=7.3Hz,3H).LCMS:m / z(M+H + ):409.15.

[0057] Preparation of S4, N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamyl)-2'-methyl-[1,1'-biphenyl]-2-yl)acrylamide (compound 1): Compound 1c (100 mg, 0.25 mmol) was dissolved in an appropriate amount of anhydrous dichloromethane. Triethylamine (139.2 μL, 1.0 mmol) was added under ice bath conditions, followed by slow dropwise addition of acryloyl chloride (20.3 μL, 0.25 mmol). The mixture was stirred for 20 minutes and then stirred at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. Column chromatography yielded 75.3 mg of a white solid, with a yield of 66.5%. 1 H NMR (500MHz, DMSO-d6) δ10.26(s,1H),8.98(s,1H),7.86(d,J=8.3Hz,2H),7.66(d,J=7.9Hz,1H),7.62(d,J=8. 3Hz,2H),7.51(d,J=1.8Hz,1H),7.46(dd,J=8.3,1.9Hz,1H),7.38–7.33(m,1H),7.26–7.21(m,1H),7.15(dd,J =7.6,1.4Hz,1H),7.02(d,J=8.3Hz,1H),6.31(dd,J=17.0,10.2Hz,1H),6.11(dd,J=17.0,2.1Hz,1H),5.60(dd ,J=10.2,2.1Hz,1H),3.81(s,2H),3.28(q,J=7.4Hz,2H),1.98(s,3H),1.10(t,J=7.4Hz,3H).HRMS(ESI)calcd for C 26 H 26 N₂O₄S[M+Na] + ]:485.1505; found485.1506.

[0058] Example 2 Preparation of 1-cyano-N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)cyclopropane-1-carboxamide (compound 2)

[0059]

[0060] Compound 1c (100 mg, 0.25 mmol), 1-cyano-1-cyclopropanecarboxylic acid (42 mg, 0.38 mmol), HATU (186 mg, 0.50 mmol), and N,N-diisopropylethylamine (128 μL, 0.75 mmol) were dissolved in an appropriate amount of N,N-dimethylformamide and stirred overnight at room temperature. After the reaction was completed, the mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. Column chromatography gave 88.7 mg of a white solid, with a yield of 70.8%. 1 HNMR(500MHz,DMSO-d6)δ10.34(s,1H),9.03(s,1H),7.86(d,J=8.1Hz,2H),7.62(t,J=8.8 Hz,3H),7.55(s,1H),7.49(d,J=8.2Hz,1H),7.37(t,J=7.6Hz,1H),7.28(t,J=7.4Hz,1H), 7.18(d,J=7.4Hz,1H),7.04(d,J=8.3Hz,1H),3.83(s,2H),3.28(q,J=7.3Hz,2H),2.04(s, 3H),1.51(d,J=3.2Hz,2H),1.34(d,J=3.3Hz,2H),1.10(t,J=7.3Hz,3H).HRMS(ESI)calcd for C 28 H 27 N3O4S[M+Na + ]:524.1613; found 524.1614.

[0061] Example 3 Preparation of 2-cyano-N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)-2-methylpropionamide (compound 3)

[0062]

[0063] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with 2-cyano-2-methylpropionic acid. The other required raw materials, reagents and preparation methods were the same as in Example 2, and a white solid was obtained. 1H NMR (500MHz, DMSO-d6) δ10.27(s,1H),9.30(s,1H),7.85(d,J=8.2Hz,2H),7.62( d,J=8.2Hz,2H),7.49(s,1H),7.43(d,J=8.3Hz,1H),7.39(d,J=3.4Hz,2H),7.35 –7.30(m,1H),7.20(d,J=7.4Hz,1H),7.00(d,J=8.3Hz,1H),3.81(s,2H),3.28(q ,J=7.3Hz,2H),2.03(s,3H),1.33(s,6H),1.10(t,J=7.3Hz,3H).HRMS(ESI)calcd for C 28 H 29 N3O4S[M+Na + ]:526.1711; found 526.1711.

[0064] Example 4: Preparation of N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamyl)-2'-methyl-[1,1'-biphenyl]-2-yl)methacrylamide (compound 4)

[0065]

[0066] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with methacrylic acid, and the other required raw materials, reagents and preparation methods were the same as in Example 2, resulting in a white solid. 1 H NMR (500MHz, DMSO-d6) δ10.28(s,1H),8.64(s,1H),7.86(d,J=8.3Hz,2H),7.66(d,J=7.9Hz,1H) ,7.62(d,J=8.2Hz,2H),7.53(s,1H),7.46(dd,J=8.3,1.6Hz,1H),7.37(t,J=7.0Hz,1H),7.26(t, J=7.1Hz,1H),7.18(dd,J=7.5,1.1Hz,1H),7.06(d,J=8.3Hz,1H),5.43(s,1H),5.30(s,1H),3.81 (s,2H),3.28(q,J=7.3Hz,2H),2.02(s,3H),1.73(s,3H),1.11(t,J=7.3Hz,3H).HRMS(ESI)calcd forC 27 H 28 N₂O₄S[M+Na] + ]:499.1662; found 499.1661.

[0067] Example 5: Preparation of (E)-N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)but-2-enamide (compound 5)

[0068]

[0069] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with crotonic acid, and the other required raw materials, reagents and preparation methods were the same as in Example 2, resulting in a white solid. 1 H NMR (500MHz, DMSO-d6) δ10.27(s,1H),8.69(d,J=31.3Hz,1H),7.86(d,J=8.2Hz,2H),7.66(d,J=7.4Hz,1H ),7.62(d,J=8.2Hz,2H),7.51(d,J=5.7Hz,1H),7.46(d,J=8.2Hz,1H),7.33(t,J=7.7Hz,1H),7.20(t,J=7. 4Hz,1H),7.12(d,J=7.6Hz,1H),7.02–7.00(m,1H),6.71–6.60(m,1H),5.99(d,J=15.3Hz,1H),3.81(s,2H ),3.28(d,J=7.4Hz,2H),1.97(s,3H),1.76(dd,J=6.8,1.1Hz,3H),1.09(d,J=7.4Hz,3H).HRMS(ESI)calcd for C 27 H 28 N₂O₄S[M+Na] + ]:499.1662; found 499.1662.

[0070] Example 6: Preparation of N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)-2-fluoroacrylamide (compound 6)

[0071]

[0072] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with 2-fluoroacrylic acid. The other required raw materials, reagents and preparation methods were the same as in Example 2, and a white solid was obtained. 1H NMR(500MHz,DMSO-d6)δ7.86(d,J=8.3Hz,2H),7.65–7.60(m,3H),7.53(s,1H),7. 45(d,J=8.3Hz,1H),7.40(t,J=7.7Hz,1H),7.34–7.29(m,1H),7.21(dd,J=7.6,1.1 Hz,1H),7.04(dd,J=8.3,1.8Hz,1H),5.58–5.45(m,1H),5.29–5.23(m,1H),3.81( s,2H),3.28(q,J=7.3Hz,2H),2.02(s,3H),1.10(t,J=7.4Hz,3H).HRMS(ESI)calcd for C 26 H 25 N₂O₄FS[M+Na] + ]:503.1411; found 503.1412.

[0073] Example 7 Preparation of (E)-N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)-4-methoxybut-2-enamide (compound 7)

[0074]

[0075] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with 4-methoxycrotonic acid. The other required raw materials, reagents and preparation methods were the same as in Example 2, and a white solid was obtained. 1 H NMR (500MHz, DMSO-d6) δ10.31(s,1H),8.19(s,1H),7.91(d,J=8.1Hz,1H),7.86(d,J=8.3Hz,2H),7.63(d,J =8.2Hz,2H),7.57(d,J=1.2Hz,1H),7.52–7.48(m,1H),7.35–7.30(m,1H),7.17(t,J=7.4Hz,1H),7.11(d,J =7.4Hz,1H),7.03(d,J=8.2Hz,1H),5.90(d,J=6.1Hz,1H),4.30(dd,J=13.6,7.4Hz,1H),3.82(s,2H),3.40 (s,3H),3.28(q,J=7.3Hz,2H),2.83(d,J=7.4Hz,2H),1.97(s,3H),1.10(t,J=7.4Hz,3H).HRMS(ESI)calcd for C 28 H 30 N₂O₅S[M+Na]+ ]:529.1768; found 527.1767.

[0076] Example 8 Preparation of 2-cyano-N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamyl)-2'-methyl-[1,1'-biphenyl]-2-yl)acetamide (compound 8)

[0077]

[0078] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with cyanoacetic acid, and the other required raw materials, reagents and preparation methods were the same as in Example 2, resulting in a white solid. 1 H NMR (500MHz, DMSO-d6) δ10.30(s,1H),9.25(s,1H),7.87(d,J=8.3Hz,2H),7.63(d,J= 8.3Hz,3H),7.54(d,J=1.7Hz,1H),7.49(dd,J=8.3,1.8Hz,1H),7.39–7.34(m,1H),7.2 6(t,J=7.4Hz,1H),7.18–7.12(m,1H),7.02(d,J=8.3Hz,1H),3.82(s,2H),3.69(d,J=3 .0Hz,2H),3.28(q,J=7.3Hz,2H),2.00(s,3H),1.10(t,J=7.4Hz,3H).HRMS(ESI)calcd for C 26 H 25 N3O4S[M+Na + ]:498.1458; found 498.1458.

[0079] Example 9 Preparation of 2-chloro-N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamyl)-2'-methyl-[1,1'-biphenyl]-2-yl)acetamide (compound 9)

[0080]

[0081] In Example 2, 1-cyano-1-cyclopropanecarboxylic acid was replaced with chloroacetic acid, and the other required raw materials, reagents and preparation methods were the same as in Example 2, resulting in a white solid. 1H NMR (500MHz, DMSO-d6) δ10.30(s,1H),9.05(s,1H),7.87(d,J=8.3Hz,2H),7.74(d,J=8.1Hz ,1H),7.63(d,J=8.3Hz,2H),7.55(s,1H),7.50(dd,J=8.3,1.7Hz,1H),7.40–7.35(m,1H),7. 25(t,J=7.4Hz,1H),7.16(dd,J=7.5,1.1Hz,1H),7.04(d,J=8.3Hz,1H),4.12(d,J=3.3Hz,2 H),3.82(s,2H),3.28(q,J=7.3Hz,2H),2.00(s,3H),1.10(t,J=7.4Hz,3H).HRMS(ESI)calcd for C 25 H 25 ClN2O4S[M+Na + ]:507.1116; found 507.1116.

[0082] Example 10: Preparation of N-(2'-methyl-4'-(2-(2-nitrophenyl)acetamide)-[1,1'-biphenyl]-2-yl)acrylamide (compound 10)

[0083] The synthetic route for preparing compound 10 is as follows:

[0084]

[0085] Specifically, the following steps are included:

[0086] Preparation of S1, 2'-methyl-4'-nitro-[1,1'-biphenyl]-2-amine (compound 10a): 2-bromo-5-nitrotoluene (3 g, 13.9 mmol), 2-aminophenylboronic acid (2.85 g, 20.8 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (1.13 g, 1.39 mmol), and potassium carbonate (5.75 g, 41.7 mmol) were dissolved in an appropriate amount of 1,4-dioxane / water (2 / 1), and refluxed at 110 °C under nitrogen protection. After the reaction was complete, the mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. Column chromatography yielded 2.75 g of a yellow solid, with a yield of 86.9%. 1H NMR (500MHz, DMSO-d6) δ8.19(d,J=2.3Hz,1H),8.09(dd,J=8.3,2.4Hz,1H),7.39(d,J=8.4Hz,1H),7.13–7.08(m,1 H),6.86(dd,J=7.5,1.4Hz,1H),6.76(d,J=7.5Hz,1H),6.66–6.61(m,1H),4.69(s,2H),2.23(s,3H).LCMS:m / z(M+H + ):229.09.

[0087] Preparation of S2, N-(2'-methyl-4'-nitro-[1,1'-biphenyl]-2-yl)acrylamide (compound 10b): Compound 10a (3.05 g, 13.4 mmol) was dissolved in an appropriate amount of anhydrous dichloromethane, and triethylamine (7.44 mL, 53.6 mmol) was added under ice bath conditions. Then, acryloyl chloride (1.2 mL, 14.7 mmol) was slowly added dropwise, and the mixture was stirred for 20 minutes and then stirred at room temperature. After the reaction was completed, the mixture was extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. Column chromatography yielded 2.36 g of a white solid, with a yield of 62.6%. 1 H NMR(400MHz,DMSO-d6)δ9.32(s,1H),8.18(d,J=2.2Hz,1H),8.08(dd,J=8.4,2 .4Hz,1H),7.64(d,J=8.0Hz,1H),7.44(dd,J=11.1,4.4Hz,1H),7.33(dd,J=17 .7,7.9Hz,2H),7.23(d,J=6.5Hz,1H),6.26(dd,J=17.0,10.1Hz,1H),6.09(dd ,J=17.0,2.0Hz,1H),5.61(dd,J=10.1,2.0Hz,1H),2.16(s,3H).LCMS:m / z(M+H + ):283.10.

[0088] Preparation of S3, N-(4'-amino-2'-methyl-[1,1'-biphenyl]-2-yl)acrylamide (compound 10c): Compound 10b (2.2 g, 7.8 mmol), iron powder (3.1 g, 54.6 mmol), and ammonium chloride (4.13 g, 78 mmol) were dissolved in an appropriate amount of ethanol / water (2 / 1) and refluxed at 90 °C. After the reaction was completed, the mixture was filtered while hot, and the solvent was evaporated to obtain the crude product. Column chromatography yielded 1.65 g of a yellow oil, with a yield of 83.9%. 1H NMR(500MHz,DMSO-d6)δ8.77(s,1H),7.70(d,J=7.8Hz,1H),7.32–7.25(m,1H),7.17 (t,J=7.4Hz,1H),7.10(dd,J=7.6,1.4Hz,1H),6.74(d,J=8.1Hz,1H),6.46(d,J=1.9 Hz, 1H), 6.43 (dd, J=8.1, 2.1Hz, 1H), 6.34 (dd, J=17.0, 10.2Hz, 1H), 6.12 (dd, J=17. 0,2.0Hz,1H),5.62(dd,J=10.2,2.0Hz,1H),5.04(s,2H),1.87(s,3H).LCMS:m / z(M+H + ):253.13.

[0089] Preparation of S4, N-(2'-methyl-4'-(2-(2-nitrophenyl)acetamide)-[1,1'-biphenyl]-2-yl)acrylamide (compound 10): Compound 10c (100 mg, 0.40 mmol), o-nitrophenylacetic acid (108 mg, 0.60 mmol), HATU (302 mg, 0.80 mmol), and N,N-diisopropylethylamine (0.21 mL, 1.2 mmol) were dissolved in an appropriate amount of N,N-dimethylformamide and stirred overnight at room temperature. After the reaction was completed, the mixture was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. Column chromatography yielded 157.5 mg of a white solid, with a yield of 95.6%. 1 H NMR (500MHz, DMSO-d6) δ10.24(s,1H),9.00(s,1H),8.06(d,J=8.1Hz,1H),7.74–7.70(m,1H),7.67(d ,J=7.8Hz,1H),7.60–7.55(m,2H),7.48(d,J=1.4Hz,1H),7.44–7.40(m,1H),7.37–7.33(m,1H),7.23( t,J=7.4Hz,1H),7.15(dd,J=7.5,1.1Hz,1H),7.01(d,J=8.3Hz,1H),6.33(dd,J=17.0,10.2Hz,1H),6 .13(dd,J=17.0,1.9Hz,1H),5.61(dd,J=10.2,2.0Hz,1H),4.14(s,2H),1.98(s,3H).HRMS(ESI)calcd for C 24 H 21 N3O4[M+Na + ]:438.1424; found 438.1419.

[0090] Example 11: Preparation of N-(2'-methyl-4'-(2-(4-(methylsulfonyl)phenyl)acetamido)-[1,1'-biphenyl]-2-yl)acrylamide (compound 11)

[0091]

[0092] In Example 10, o-nitrophenylacetic acid was replaced with 4-methanesulfonylphenylacetic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a yellow solid was obtained. 1 H NMR (500MHz, DMSO-d6) δ10.27(s,1H),9.01(s,1H),7.89(t,J=8.6Hz,2H),7.65(d,J=7.5Hz,1H),7 .61(d,J=8.2Hz,2H),7.49(s,1H),7.46(d,J=8.1Hz,1H),7.35(t,J=7.6Hz,1H),7.24(t,J=7.4Hz, 1H),7.15(d,J=7.2Hz,1H),7.02(d,J=8.2Hz,1H),6.31(dd,J=16.9,10.2Hz,1H),6.11(dd,J=17.0 ,1.4Hz,1H),5.61(dd,J=10.2,1.6Hz,1H),3.80(s,2H),3.21(s,3H),1.98(s,3H).HRMS(ESI)calcd forC 25 H 24 N₂O₄S[M+Na] + ]:471.1349; found 471.1352.

[0093] Example 12: Preparation of N-(2'-acrylamido-2-methyl-[1,1'-biphenyl]-4-yl)-2-methylpyrimidine-5-carboxamide (compound 12)

[0094]

[0095] In Example 10, o-nitrophenylacetic acid was replaced with 2-methyl-5-pyrimidinecarboxylic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a pale yellow solid was obtained. 1H NMR(500MHz,DMSO-d6)δ10.50(s,1H),9.18(s,2H),9.08(s,1H),7.68–7.62(m ,3H),7.40–7.35(m,1H),7.26(t,J=7.3Hz,1H),7.19(dd,J=7.5,1.1Hz,1H),7 .10(d,J=8.1Hz,1H),6.33(dd,J=17.0,10.2Hz,1H),6.13(dd,J=17.0,1.9Hz, 1H),5.62(dd,J=10.2,2.0Hz,1H),2.72(s,3H),2.04(s,3H).HRMS(ESI)calcd forC 22 H 20 N4O2[M+Na + ]:395.1478; found 395.1476.

[0096] Example 13: Preparation of N-(4'-(2-(4-cyanophenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)acrylamide (compound 13)

[0097]

[0098] In Example 10, o-nitrophenylacetic acid was replaced with 4-cyano-phenylacetic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1 H NMR (500MHz, DMSO-d6) δ10.26(s,1H),8.99(s,1H),7.81(d,J=8.2Hz,2H),7.66(d,J=7.6Hz,1H ),7.55(d,J=8.1Hz,2H),7.50(s,1H),7.46(d,J=8.3Hz,1H),7.35(t,J=7.7Hz,1H),7.24(t,J=7 .4Hz,1H),7.15(d,J=7.5Hz,1H),7.02(d,J=8.2Hz,1H),6.31(dd,J=17.0,10.2Hz,1H),6.12(d d,J=17.0,1.8Hz,1H),5.61(dd,J=10.2,1.9Hz,1H),3.78(s,2H),1.98(s,3H).HRMS(ESI)calcd for C 25 H 21 N3O2[M+Na + ]:418.1526; found418.1522.

[0099] Example 14 Preparation of N-(2'-acrylamido-2-methyl-[1,1'-biphenyl]-4-yl)-4-(methylsulfonyl)benzamide (compound 14)

[0100]

[0101] In Example 10, o-nitrophenylacetic acid was replaced with 4-methanesulfonylbenzoic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a yellow solid was obtained. 1 H NMR (500MHz, DMSO-d6) δ10.50(s,1H),9.08(s,1H),8.20(d,J=8.3Hz,2H),8.11(d,J=8.3 Hz,2H),7.71(s,1H),7.67(d,J=6.3Hz,2H),7.38(t,J=7.2Hz,1H),7.27(t,J=7.3Hz,1H), 7.20(d,J=7.3Hz,1H),7.10(d,J=8.2Hz,1H),6.34(dd,J=17.0,10.2Hz,1H),6.14(dd,J=1 7.0,1.6Hz,1H),5.63(dd,J=10.2,1.7Hz,1H),3.30(s,3H),2.04(s,3H).HRMS(ESI)calcd for C 24 H 22 N₂O₄S[M+Na] + ]:457.1192; found457.1196.

[0102] Example 15: Preparation of N-(2'-acrylamido-2-methyl-[1,1'-biphenyl]-4-yl)-6-methoxynicotinamide (compound 15)

[0103]

[0104] In Example 10, o-nitrophenylacetic acid was replaced with 6-methoxynicotinic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1H NMR (400MHz, DMSO-d6) δ10.23(s,1H),9.04(s,1H),8.80(d,J=2.3Hz,1H),8.25(dd,J=8.7,2 .4Hz,1H),7.69–7.62(m,3H),7.37(t,J=7.7Hz,1H),7.26(t,J=7.4Hz,1H),7.19(d,J=6.9Hz ,1H),7.07(d,J=8.2Hz,1H),6.96(d,J=8.7Hz,1H),6.33(dd,J=17.0,10.1Hz,1H),6.13(dd, J=17.0,1.9Hz,1H),5.62(dd,J=10.1,1.9Hz,1H),3.95(s,3H),2.03(s,3H).HRMS(ESI)calcd for C 23 H 21 N3O3[M+Na + ]:410.1475; found 410.1472.

[0105] Example 16: Preparation of N-(2'-acrylamido-2-methyl-[1,1'-biphenyl]-4-yl)-3-cyanobenzamide (compound 16)

[0106]

[0107] In Example 10, o-nitrophenylacetic acid was replaced with 3-cyanobenzoic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1 H NMR (500MHz, DMSO-d6) δ10.24(s,1H),9.00(s,1H),7.80(s,1H),7.75(d,J=7.7Hz,1H),7.68(t,J=10 .1Hz,2H),7.56(t,J=7.8Hz,1H),7.51(s,1H),7.47(d,J=8.3Hz,1H),7.35(dd,J=11.2,4.1Hz,1H),7 .24(t,J=7.4Hz,1H),7.15(d,J=6.8Hz,1H),7.02(d,J=8.2Hz,1H),6.31(dd,J=17.0,10.2Hz,1H),6. 12(dd,J=17.0,1.8Hz,1H),5.61(dd,J=10.2,1.8Hz,1H),3.76(s,2H),1.99(s,3H).HRMS(ESI)calcd forC 25 H 21 N3O2[M+Na +]:418.1526; found 418.1425.

[0108] Example 17 Preparation of N-(4'-(2-(3-cyanophenyl)acetamido)-2'-methyl-[1,1'-biphenyl]-2-yl)acrylamide (compound 17)

[0109]

[0110] In Example 10, o-nitrophenylacetic acid was replaced with 3-cyanophenylacetic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1 H NMR(500MHz,DMSO-d6)δ10.43(s,1H),9.10(s,1H),8.43(s,1H),8.27(d,J=7.9Hz,1H),8.0 8(d,J=7.6Hz,1H),7.77(t,J=7.8Hz,1H),7.71–7.64(m,3H),7.38(t,J=7.4Hz,1H),7.27(t ,J=7.3Hz,1H),7.20(d,J=7.3Hz,1H),7.10(d,J=8.2Hz,1H),6.34(dd,J=16.9,10.2Hz,1H) ,6.14(dd,J=17.1,1.4Hz,1H),5.63(dd,J=10.1,1.5Hz,1H),2.04(s,3H).HRMS(ESI)calcd for C 24 H 19 N3O2[M+Na + ]:404.1369; found404.1365.

[0111] Example 18 Preparation of N-(2'-acrylamido-2-methyl-[1,1'-biphenyl]-4-yl)-5-(methanesulfonyl)pyridine amide (compound 18)

[0112]

[0113] In Example 10, o-nitrophenylacetic acid was replaced with 5-methylsulfonyl-2-pyridinecarboxylic acid. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a yellow solid was obtained. 1H NMR (500MHz, DMSO-d6) δ10.84(s,1H),9.22(d,J=1.7Hz,1H),9.11(s,1H),8.60(dd,J=8.2,2.1Hz,1H ),8.40(d,J=8.2Hz,1H),7.87(s,1H),7.81(d,J=8.2Hz,1H),7.67(d,J=7.8Hz,1H),7.40–7.36(m,1H) ,7.26(t,J=7.4Hz,1H),7.20(d,J=6.7Hz,1H),7.11(d,J=8.3Hz,1H),6.34(dd,J=17.0,10.2Hz,1H),6 .14(dd,J=17.0,1.7Hz,1H),5.62(dd,J=10.2,1.8Hz,1H),3.44(s,3H),2.05(s,3H).HRMS(ESI)calcd for C 23 H 21 N3O4S[M+Na + ]:458.1145; found 458.1138.

[0114] Example 19: Preparation of N-(2'-methyl-4'-(((4-nitrophenyl)methyl)sulfonamide)-[1,1'-biphenyl]-2-yl)acrylamide (compound 19)

[0115]

[0116] Compound 10c (100 mg, 0.40 mmol) was dissolved in an appropriate amount of anhydrous dichloromethane. Triethylamine (55.7 μL, 1.2 mmol) was added under ice bath conditions, followed by slow dropwise addition of p-nitrobenzenesulfonyl chloride (103.8 mg, 0.44 mmol). The mixture was stirred for 20 minutes and then stirred at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane and water, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. Column chromatography yielded 138.2 mg of a white solid, with a yield of 77.2%. 1H NMR(500MHz,DMSO-d6)δ9.97(s,1H),9.07(s,1H),8.24(d,J=8.6Hz,2H),7.67(d,J=7 .8Hz,1H),7.58(d,J=8.6Hz,2H),7.38(t,J=7.3Hz,1H),7.26(t,J=7.4Hz,1H),7.17( d,J=7.1Hz,1H),7.08–7.04(m,3H),6.33(dd,J=17.0,10.2Hz,1H),6.14(dd,J=17.0, 1.6Hz,1H),5.63(dd,J=10.2,1.7Hz,1H),4.72(s,2H),1.99(s,3H).HRMS(ESI)calcd for C 23 H 21 N3O5S[M+Na + ]:474.1094; found 474.1089.

[0117] Example 20: Preparation of N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-[1,1'-biphenyl]-2-yl)acrylamide (compound 20)

[0118]

[0119] In Example 10, 2-bromo-5-nitrotoluene was replaced with 4-nitroiodobenzene. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1 H NMR(400MHz,DMSO-d6)δ10.39(s,1H),9.43(s,1H),7.87(d,J=8.3Hz,2H),7 .64(t,J=8.6Hz,4H),7.53(d,J=7.6Hz,1H),7.37–7.27(m,5H),6.35(dd,J=1 7.0,10.1Hz,1H),6.16(dd,J=17.0,1.7Hz,1H),5.66(dd,J=10.2,1.6Hz,1H),3.83(s,2H),3.28(q,J=7.3Hz,2H),1.11(t,J=7.4Hz,3H).HRMS(ESI)calcd for C 25 H 24 N₂O₄S[M+Na] + ]:471.1349; found471.1348.

[0120] Example 21: Preparation of N-(2-(5-(2-(4-(ethylsulfonyl)phenyl)acetamido)pyridin-2-yl)phenyl)acrylamide (compound 21)

[0121]

[0122] In Example 10, 2-bromo-5-nitrotoluene was replaced with 2-bromo-5-nitropyridine. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1 H NMR (400MHz, DMSO-d6) δ12.08(s,1H),10.71(s,1H),8.99(d,J=2.3Hz,1H),8.36(d,J=8.1Hz,1H),8 .17(dd,J=8.8,2.4Hz,1H),7.89(dd,J=8.2,5.8Hz,3H),7.79(d,J=7.5Hz,1H),7.65(d,J=8.2Hz,2H) ,7.41(t,J=7.7Hz,1H),7.22(t,J=7.5Hz,1H),6.43(dd,J=17.0,10.2Hz,1H),6.23(d,J=17.0Hz,1H) ,5.77(d,J=10.3Hz,1H),3.90(s,2H),3.29(q,J=7.3Hz,2H),1.11(t,J=7.3Hz,3H).HRMS(ESI)calcd for C 24 H 23 N3O4S[M+Na + ]:472.1301; found 472.1302.

[0123] Example 22: Preparation of N-(2'-chloro-4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-[1,1'-biphenyl]-2-yl)acrylamide (compound 22)

[0124]

[0125] In Example 10, 2-bromo-5-nitrotoluene was replaced with 1-bromo-2-chloro-4-nitrobenzene. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1H NMR(500MHz,DMSO-d6)δ10.51(s,1H),9.15(s,1H),7.91–7.84(m,3H),7.69(d,J=8.1Hz,1 H),7.62(d,J=8.0Hz,2H),7.50(dd,J=8.4,2.2Hz,1H),7.39(td,J=7.7,1.8Hz,1H),7.27– 7.19(m,3H),6.34(dd,J=17.0,10.2Hz,1H),6.12(dd,J=17.1,2.1Hz,1H),5.61(dd,J=10. 1,2.1Hz,1H),3.84(s,2H),3.28(q,J=7.4Hz,2H),1.11(t,J=7.3Hz,3H).HRMS(ESI)calcd for C 25 H 23 ClN2O4S[M+Na + ]:505.0959; found505.0958.

[0126] Example 23 Preparation of N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamyl)-2'-fluoro-[1,1'-biphenyl]-2-yl)acrylamide (compound 23)

[0127]

[0128] In Example 10, 2-bromo-5-nitrotoluene was replaced with 1-bromo-2-fluoro-4-nitrobenzene. The other required raw materials, reagents and preparation methods were the same as in Example 10, and a white solid was obtained. 1 H NMR (400MHz, DMSO-d6) δ10.55(s,1H),9.34(s,1H),7.87(d,J=8.3Hz,2H),7.63(d,J=8.2H z,4H),7.43–7.39(m,1H),7.36(dd,J=8.6,2.0Hz,1H),7.28(d,J=7.4Hz,2H),7.24(d,J=8. 5Hz,1H),6.34(dd,J=17.0,10.2Hz,1H),6.14(dd,J=17.0,1.9Hz,1H),5.64(dd,J=10.2,1. 9Hz,1H),3.85(s,2H),3.28(dd,J=7.3,3.8Hz,2H),1.11(t,J=6.5Hz,3H).HRMS(ESI)calcd for C 25 H 23 FN2O4S[M+Na +]:489.1255; found 489.1254.

[0129] Example 24 Preparation of N-(4'-(2-(4-(ethylsulfonyl)phenyl)acetamido)-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 24)

[0130]

[0131] Replace 2-bromo-5-nitrotoluene in Example 10 with 2-bromo-5-nitrotrifluorotoluene, and use the same raw materials, reagents and preparation methods as in Example 10 to obtain a white solid. 1 H NMR (400MHz, DMSO-d6) δ10.55(s,1H),9.17(s,1H),7.92(d,J=1.9Hz,1H),7.88(d,J=8.3Hz,2H),7.8 3(d,J=8.3Hz,1H),7.64(d,J=8.3Hz,2H),7.53(dd,J=8.4,2.0Hz,1H),7.48(d,J=8.2Hz,1H),7.42–7. 36(m,1H),7.25–7.21(m,2H),6.36(dd,J=17.0,10.2Hz,1H),6.14(dd,J=17.0,2.0Hz,1H),5.62(dd, J=10.1,2.0Hz,1H),3.86(s,2H),3.28(dd,J=7.3,4.2Hz,3H),1.12(t,J=6.6Hz,3H).HRMS(ESI)calcd for C 26 H 23 F3N2O4S[M+Na + ]:539.1223; found 539.1223.

[0132] Example 25: Preparation of N-(4'-(2-(4-(methanesulfonyl)phenyl)acetamido)-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 25)

[0133] The synthetic route for preparing compound 25 is as follows:

[0134]

[0135] Specifically, the following steps are included:

[0136] Preparation of S1, 4'-nitro-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-amine (compound 25a): Replace 2-bromo-5-nitrotoluene in Example 10 with 2-bromo-5-nitrotrifluorotoluene. The other required raw materials, reagents and preparation methods are the same as in step S1 of Example 10, and a yellow oily substance is obtained. 1 H NMR (400MHz, DMSO) δ8.18(dd,J=9.6,2.1Hz,1H),8.14(dd,J=8.4,2.3Hz,1H),7.67(t,J=7.9Hz,1H),7.14(t, J=7.2Hz,1H),6.99(d,J=7.5Hz,1H),6.79(d,J=8.1Hz,1H),6.65(t,J=7.4Hz,1H),4.98(s,2H).LCMS:m / z(M+H + ):283.06.

[0137] S2. Preparation of N-(4'-nitro-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 25b): Replace compound 10a in Example 10 with compound 25a. The other required raw materials, reagents and preparation methods are the same as in step S2 of Example 10, and a yellow oily substance is obtained. 1 H NMR(500MHz,DMSO)δ9.19(s,1H),8.55(d,J=2.3Hz,1H),8.52(dd,J=8.4,2.3 Hz,1H),7.76(d,J=8.1Hz,1H),7.62(d,J=8.4Hz,1H),7.52–7.46(m,1H),7.3 0(td,J=7.6,0.9Hz,1H),7.22(d,J=7.5Hz,1H),6.29(dd,J=17.0,10.2Hz,1H ),6.12(dd,J=17.0,2.1Hz,1H),5.63(dd,J=10.1,2.1Hz,1H).LCMS:m / z(M+H + ):337.05.

[0138] Preparation of S3, N-(4'-amino-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 25c): Replace compound 10b in Example 10 with compound 25b. The other required raw materials, reagents and preparation methods are the same as in step S3 of Example 10, to obtain a yellow oily substance. 1H NMR (400MHz, CDCl3) δ8.35(s,1H),7.42–7.33(m,1H),7.15(dd,J=17.3,7.0Hz,3H),7.04(d,J=8.2Hz,1H),6.81(d,J=2.3Hz,1H),6.64( dd,J=8.2,2.3Hz,1H),6.24(d,J=16.9Hz,1H),6.03(dd,J=16.7,10.4Hz,1H),5.64(dd,J=10.3,0.7Hz,1H),3.93(s,2H).LCMS:m / z(M+H + ):207.10.

[0139] Preparation of S4, N-(4'-(2-(4-(methylsulfonyl)phenyl)acetamido)-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 25): Replace compound 10c in Example 10 with compound 25c. The other required raw materials, reagents and preparation methods are the same as in step S4 of Example 10, and a white solid is obtained. 1 H NMR (500MHz, DMSO-d6) δ10.67(s,1H),8.94(s,1H),8.17(d,J=2.3Hz,1H),7.92(d,J =7.9Hz,2H),7.82–7.72(m,2H),7.63(d,J=8.0Hz,2H),7.42–7.36(m,1H),7.22(t,J= 7.7Hz,2H),7.15(d,J=7.7Hz,1H),6.34(dd,J=17.0,10.1Hz,1H),6.12(dd,J=17.1, 2.1Hz,1H),5.60(dd,J=10.2,2.1Hz,1H),3.86(s,2H),3.21(s,3H).HRMS(ESI)calcd for C 25 H 21 F3N2O4S[M+Na + ]:525.1066; found 525.1066.

[0140] Example 26 Preparation of N-(4'-(2-(2-nitrophenyl)acetamido)-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 26)

[0141]

[0142] Replace 4-methanesulfonylphenylacetic acid with o-nitrophenylacetic acid in Example 25, and use the same raw materials, reagents and preparation methods as in Example 25 to obtain a white solid.1 H NMR (500MHz, DMSO-d6) δ10.72 (s, 1H), 8.95 (s, 1H), 8.16 (d, J = 2.2Hz, 1H), 8.10 (dd, J=8.2,1.3Hz,1H),7.79–7.72(m,3H),7.63–7.57(m,2H),7.39(td,J=7.8,1.6Hz,1H ),7.22(t,J=7.9Hz,2H),7.16(d,J=7.7Hz,1H),6.37(dd,J=17.0,10.2Hz,1H),6.14 (dd,J=17.0,2.1Hz,1H),5.61(dd,J=10.1,2.1Hz,1H),4.20(s,2H).HRMS(ESI)calcd for C 24 H 18 F3N3O4[M+Na + ]:492.1142; found 492.1138.

[0143] Example 27 Preparation of N-(4'-(2-(3-cyanophenyl)acetamido)-2'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)acrylamide (compound 27)

[0144]

[0145] Replace 4-methanesulfonylphenylacetic acid in Example 25 with 3-cyanophenylacetic acid, and use the same raw materials, reagents and preparation methods as in Example 25 to obtain a white solid. 1 H NMR (500MHz, DMSO-d6) δ10.71(s,1H),8.95(s,1H),8.19(s,1H),7.85–7.70(m,5H),7.57(t,J=7.8Hz,1H),7.40(t,J=7.7Hz,1H),7.23(d,J=8 .0Hz,2H),7.15(d,J=7.7Hz,1H),6.35(dd,J=17.0,10.1Hz,1H),6.13(d,J=16.9Hz,1H),5.60(d,J=10.3Hz,1H),3.83(s,2H).HRMS(ESI)calcd for C 25 H 18 F3N3O2[M+Na + ]:472.1243; found 472.1242.

[0146] Example 28 Preparation of N-(2'-acrylamido-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-3-cyanobenzamide (compound 28)

[0147]

[0148] In Example 25, 4-methanesulfonylphenylacetic acid was replaced with 3-cyanobenzoic acid. The other required raw materials, reagents and preparation methods were the same as in Example 25, and a white solid was obtained. 1 H NMR (500MHz, DMSO-d6) δ10.80(s,1H),9.04(s,1H),8.47(s,1H),8.34(d,J=2.2Hz,1H),8.30(d,J=8.0H z,1H),8.11(d,J=7.8Hz,1H),8.07(dd,J=8.5,2.2Hz,1H),7.80(t,J=7.8Hz,1H),7.75(d,J=8.1Hz,1H) ,7.42(td,J=7.8,1.7Hz,1H),7.31(d,J=8.4Hz,1H),7.25(t,J=7.5Hz,1H),7.19(d,J=7.6Hz,1H),6.37 (dd,J=17.0,10.2Hz,1H),6.14(dd,J=17.0,2.2Hz,1H),5.62(dd,J=10.2,2.2Hz,1H).HRMS(ESI)calcd for C 24 H 16 F3N3O2[M+Na + ]:458.1087; found 458.1093.

[0149] Example 29 Preparation of N-(2'-acrylamido-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-6-methoxynicotinamide (compound 29)

[0150]

[0151] In Example 25, 4-methanesulfonylphenylacetic acid was replaced with 6-methoxynicotinic acid. The other required raw materials, reagents and preparation methods were the same as in Example 25, and a white solid was obtained. 1H NMR (500MHz, DMSO-d6) δ10.70(s,1H),9.03(s,1H),8.87(d,J=2.5Hz,1H),8.37(d,J=2.2Hz,1H) ,8.31(dd,J=8.7,2.5Hz,1H),8.07(dd,J=8.4,2.2Hz,1H),7.75(d,J=8.1Hz,1H),7.41(t,J=7.7H z,1H),7.30–7.22(m,2H),7.19(d,J=7.6Hz,1H),6.99(d,J=8.7Hz,1H),6.38(dd,J=17.0,10.2H z,1H),6.14(dd,J=17.1,2.1Hz,1H),5.62(dd,J=10.1,2.2Hz,1H),3.96(s,3H).HRMS(ESI)calcd for C 23 H 18 F3N3O3[M+Na + ]:464.1192; found 464.1192.

[0152] Example 30: Preparation of N-(2'-acrylamido-2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-methylpyrimidin-5-carboxamide (compound 30)

[0153]

[0154] Replace 4-methanesulfonylphenylacetic acid in Example 25 with 2-methyl-5-pyrimidinecarboxylic acid, and use the same raw materials, reagents and preparation methods as in Example 25 to obtain a white solid. 1 H NMR (400MHz, DMSO-d6) δ10.87(s,1H),9.22(s,2H),9.02(s,1H),8.31(d,J=2.2Hz,1H) ,8.03(dd,J=8.4,2.2Hz,1H),7.74(d,J=8.1Hz,1H),7.45–7.39(m,1H),7.31(d,J=8.4H z,1H),7.25(t,J=7.5Hz,1H),7.19(d,J=7.6Hz,1H),6.36(dd,J=17.0,10.1Hz,1H),6. 14(dd,J=17.0,2.2Hz,1H),5.61(dd,J=10.1,2.2Hz,1H),2.73(s,3H).HRMS(ESI)calcd for C 22 H 17 F3N4O2[M+Na +]:449.1196; found449.1195.

[0155] Example 31: Preparation of N-(2',6'-dichloro-4'-(2-(4-(methanesulfonyl)phenyl)acetamido)-[1,1'-biphenyl]-2-yl)acrylamide (compound 31)

[0156] The synthetic route for preparing compound 31 is as follows:

[0157]

[0158] Specifically, the following steps are included:

[0159] Preparation of S1, 2'-6'-dichloro-4'-nitro-[1,1'-biphenyl]-2-amine (compound 31a): 2-bromo-1,3-dichloro-5-nitrobenzene (1 g, 3.5 mmol), pinacol ester of 2-aminophenylboronic acid (851 mg, 3.9 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (229 mg, 0.28 mmol), and cesium carbonate (1.5 g, 4.6 mmol) were dissolved in an appropriate amount of acetonitrile / water (3 / 1). The mixture was reacted under nitrogen protection at 100 °C with microwave for 1 h. After the reaction was complete, the solvent was evaporated, and column chromatography yielded 837 mg of a yellow oil, with a yield of 83.7%. 1 H NMR(400MHz, DMSO-d6)δ8.39(s,2H),7.13(ddd,J=8.4,7.2,1.6Hz,1H),6.82(dd,J=7.6,1.6 Hz,1H),6.75(dd,J=8.1,1.1Hz,1H),6.63(td,J=7.4,1.1Hz,1H),4.87(s,2H).LCMS:m / z(M+H + ):284.05.

[0160] S2. Preparation of N-(2',6'-dichloro-4'-nitro-[1,1'-biphenyl]-2-yl)acrylamide (compound 31b): Replace compound 10a in Example 10 with compound 31a. The other required raw materials, reagents and preparation methods are the same as in step S2 of Example 10, and a yellow solid is obtained. 1H NMR (400MHz, DMSO-d6) δ9.30(s,1H),8.42(s,2H),7.87(d,J=8.2Hz,1H),7.49(td,J=7.8,1.7Hz,1H),7.32(td,J=7.5,1.2Hz,1H),7 .22(dd,J=7.7,1.6Hz,1H),6.35(dd,J=17.0,10.1Hz,1H),6.15(dd,J=17.0,2.2Hz,1H),5.65(dd,J=10.1,2.2Hz,1H).LCMS:m / z(M+H + ):338.09.

[0161] Preparation of S3, N-(2',6'-dichloro-4'-amino-[1,1'-biphenyl]-2-yl)acrylamide (compound 31c): Replace compound 10b in Example 10 with compound 31b. The other required raw materials, reagents and preparation methods are the same as in step S3 of Example 10, and a white solid is obtained. 1 H NMR(400MHz, DMSO-d6)δ8.88(s,1H),7.87(d,J=8.2Hz,1H),7.37–7.30(m,1H),7.17(t,J=7.5Hz,1H),7.08(dd,J=7.6,1.6Hz,1H), 6.68(s,2H),6.48(dd,J=17.0,10.1Hz,1H),6.15(dd,J=17.1,2.2Hz,1H),5.72(s,2H),5.62(dd,J=10.2,2.2Hz,1H).LCMS:m / z(M+H + ):307.17.

[0162] Preparation of S4, N-(2',6'-dichloro-4'-(2-(4-(methanesulfonyl)phenyl)acetamido)-[1,1'-biphenyl]-2-yl)acrylamide (compound 31): Replace compound 10c in Example 10 with compound 31c. The other required raw materials, reagents and preparation methods are the same as in step 4 of Example 10, and a pale yellow solid is obtained. 1H NMR(500MHz,DMSO-d6)δ10.65(s,1H),9.07(s,1H),7.87(d,J=8.0Hz,3H),7.80(s,2H),7 .63(d,J=8.0Hz,2H),7.41(td,J=7.9,1.6Hz,1H),7.24(t,J=7.5Hz,1H),7.14(dd,J=7.7, 1.6Hz, 1H), 6.42 (dd, J=17.0, 10.2Hz, 1H), 6.15 (dd, J=17.0, 2.2Hz, 1H), 5.63 (dd, J=10. 1,2.2Hz,1H),3.86(s,2H),3.29(q,J=7.3Hz,2H),1.11(t,J=7.3Hz,3H).HRMS(ESI)calcd for C 25 H 22 Cl2N2O4S[M+Na + ]:539.0570; found 539.0570.

[0163] Experiment Example 1: Covalent Verification Experiment

[0164] 1. To facilitate more intuitive covalent verification, the inventors designed and synthesized a non-covalent control compound 32 of compound 22. The two have similar physicochemical properties, but compound 32 does not have an electrophilic active group at a suitable distance from cysteine ​​Cys320, and therefore does not have the ability to covalently bind.

[0165]

[0166] Compound 32 was prepared by the following process: replacing acryloyl chloride in Example 22 with an equimolar amount of propionyl chloride, and using the same raw materials, reagents and preparation methods as in Example 22, to obtain a white solid. 1 H NMR (400MHz, DMSO-d6) δ10.51(s,1H),8.87(s,1H),7.94–7.83(m,3H),7.62(t,J=8.5Hz,3H),7.52(d,J=8.5Hz,1H),7.36(t,J=7.6Hz ,1H),7.22(d,J=8.7Hz,3H),3.85(s,2H),3.29(q,J=7.4Hz,2H),2.12(q,J=7.6Hz,2H),1.11(t,J=7.3Hz,3H),0.93(t,J=7.6Hz,3H). 13C NMR(101MHz,DMSO-d)δ172.45,169.08,142.32,139.94,137.39,136.46,132.91,132.63,132.56,131.12,1 30.69,128.55,128.35,125.91,125.17,119.82,118.03,49.71,43.43,29.40,10.23,7.61.HRMS(ESI)calcd for C 25 H 25 ClN2O4S[M+Na + ]:507.1116; found 507.1116.

[0167] 2. Molecular docking experiment

[0168] Molecular docking simulation experiments were conducted. AutoDock software was used to simulate docking GSK805 (yellow) and compound 22 (yellow) into the LBD binding domain (PDB code: 5NU1) of RORγ, respectively. The binding modes were obtained using PyMOL version 2.4.1. (See attached diagram). Figure 1 It can be seen that the distance between the cysteine ​​residue Cys320 at the ortho position of the benzene ring of GSK805 and the LBD binding domain of RORγ is... This design is suitable for adding covalent warheads to design covalent inhibitors. Compound 22 can enter this binding pocket and covalently bind to cysteine ​​(Cys320) to form a CS bond, further verifying the covalent nature. Furthermore, the ortho- and posterior positions of the benzene ring in compounds of formula (I) all contain electrophilic active groups, i.e., covalent warheads, and therefore can all undergo Michael addition reactions with cysteine ​​residues near the binding pocket of the target protein to form covalent bonds, achieving the purpose of covalent binding.

[0169] 3. Mass spectrometry experiment

[0170] The molecular weight of the RORγ protein with this amino acid sequence is known to be 28 kDa. Compound 22 was incubated with the RORγ ligand-binding domain protein, and analysis was performed using electrospray ionization time-of-flight mass spectrometry. The results are shown below. Figure 2 The results showed that a new peak appeared near 28.515 kDa after small molecule treatment. The molecular weight difference between this peak (28997 Da) and that of RORγ protein (28515 Da) was 482 Da, which was consistent with the molecular weight of compound 22, proving that compound 22 covalently binds to RORγ.

[0171] Experiment Example 2: Compound Inhibits Prostate Cancer Cell Activity

[0172] Cell viability was determined using the CCK8 assay to test the inhibitory activity of the compound against androgen receptor-positive prostate cancer cells (C4-2B). The experimental method was as follows: 1500 cells were uniformly seeded in 96-well plates. After 24 hours of cell culture, the compound prepared in the previous example and the positive control GSK805 (Wuhan WuXi AppTec New Drug Development Co., Ltd., batch number: EW14069-3-P1) were sequentially diluted 1 / 2 times to different concentrations, and 50 μL of culture medium was added to each well. After 4 days of cell culture, 10 μL of CCK8 reagent was added, and the cells were incubated in an incubator for 1–2 hours. The absorbance was measured at 450 nm, and the in vitro IC50 of the compound was calculated using GraphPad Prism 7 software. 50 The values ​​and experimental results are shown in Table 1.

[0173] Table 1. Inhibitory activity of compounds against prostate cancer cells.

[0174]

[0175]

[0176] As shown in the table, the non-covalent control compound 32 exhibited inhibitory activity >20 μM against prostate cancer cells C4-2B, while the corresponding covalent compound 22 showed even better inhibitory activity. The data obtained above indicate that the compounds of this invention possess certain anti-drug-resistant prostate cancer activity, and most compounds showed significantly better inhibitory activity against cells than the positive control GSK805, particularly compounds 1, 20, 22, 23, 24, and 31. Furthermore, the compounds of this invention have advantages such as good drug-likeness and suitable physical properties, making them promising lead compounds for future research.

[0177] Experimental Example 3: Receptor Selectivity Test

[0178] Using compounds 1, 24, 22, and 31 as examples, selectivity experiments were conducted on each receptor subtype of RORα, RORγ, RXRγ, and ERRγ. Experimental methods:

[0179] 293T cells were distributed at 10 per well. 4 Cells were seeded at a density of 1000 g / well in 96-well plates. After 24 h of culture, 10 μL of a mixture containing the following plasmids was added to each well for co-transfection: Lipofectamine 2000, pBD-RORγ-LBD or pBD-RORα-LBD or pCMV-RXRγ-LBD or pCMV-ERRγ-LBD, pFR-luci, pcDNA-PGC-1a plasmid, and Renilla luciferase plasmid as an internal control. 24 h after transfection, 50 μL of the above-mentioned culture medium containing the corresponding compound concentration was added to each well. After 48 h of culture, subsequent activity assays were performed using the Promega dual-luciferase reporter gene assay system. The results are shown in Table 2.

[0180] Table 2. Selectivity of compounds for RORγ

[0181]

[0182]

[0183] As can be seen from the table, the compounds of this invention have significant inhibitory activity against RORγ, and have almost no inhibitory activity against other subtypes RORα, RXRγ, and ERRγ (>20 μM); indicating that the compounds have good selectivity for RORγ and are a class of inhibitors that specifically target RORγ.

[0184] Plate cloning experiment of compound 22 in Experiment Example 4

[0185] Prostate cancer cells 22Rv1 and C4-2B were seeded at 500 cells per well in six-well plates. After 24 hours of culture, different concentrations (0 μM, 1.25 μM, 2.5 μM, 5 μM) of compound 22 were added. The culture medium was then replaced with fresh compound 22 at different concentrations every three days. After 10–12 days of culture, the cells were fixed with 4% paraformaldehyde and stained with crystal violet solution in the dark. Cell colony counts were performed, and the experiment was repeated three times.

[0186] Experimental results are as follows Figure 3 As shown, compound 22 significantly inhibited clonal colony formation of prostate cancer cells in a concentration-dependent manner. Compared with the non-covalent control compound 32, covalent compound 22 produced significantly fewer clonal colonies in both prostate cancer cell lines (C4-2B and 22Rv1), and its inhibitory effect on the survival of both prostate cancer cell lines (C4-2B and 22Rv1) was more significant than that of the previously reported compound GSK805.

[0187] Experimental Example 5: Compound 22 induces apoptosis in prostate cancer cells.

[0188] Prostate cancer cells 22Rv1 and C4-2B were detected at a concentration of 1.5 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 1000 mcg in six-well plates. After culturing the cells for 24 h, different concentrations (0 μM, 1.25 μM, 2.5 μM, 5 μM) of compound 22 were added. After incubation for 48 h, the cells were collected in a white plate, and cell collection medium and Caspase-Glo 3 / 7 reaction solution were added. The mixture was shaken and mixed for 30 min. The results were measured using a GLOMAX fluorescent microplate reader. The remaining cell collection medium was used to measure cell protein concentration. The results were calculated as follows: Caspase-3 / 7 activity = GLOMAX fluorescent microplate reader reading value / cell protein concentration.

[0189] Experimental results are as follows Figure 4As shown, compared with the control group, the activity of apoptosis-related Caspase 3 / 7 in the treatment group was significantly increased in a dose-dependent manner, indicating that compound 22 can induce apoptosis in prostate cancer cells, and the apoptosis-inducing effect is more significant than that of the previously reported compound GSK805 in prostate cancer cells. In contrast, the non-covalent control compound 32 had almost no apoptosis-inducing effect on either type of prostate cancer cell.

[0190] Effect of compound 22 on downstream target gene expression in Experiment Example 6 (Quantitative Real-Time PCR)

[0191] Cells were treated with compound 22 for 48 h, then lysed using Trizol, followed by chloroform addition, vortexing for 15 s, incubating at room temperature for 10 min, and centrifuging at 12000 rpm for 15 min at 4 °C. The supernatant was transferred to a new centrifuge tube, isopropanol was added and gently mixed, and the mixture was incubated at room temperature for 10 min. The tube was then centrifuged at 12000 rpm for 15 min at 4 °C, the supernatant was discarded, and 75% pre-chilled ethanol solution was added and gently mixed. The tube was then centrifuged at 12000 rpm for 15 min at 4 °C, the supernatant was discarded, and the cells were air-dried to obtain RNA precipitate. The RNA was dissolved in DEPC water, and the concentration was determined using Nanodrop. Template DNA was obtained by reverse transcription. A 10 μL sample system was prepared using the DNA template (2 μg), primers, and 2X SYBR, and quantitative real-time PCR was performed. The results were statistically analyzed. Primers for quantitative real-time PCR are listed in Table 3.

[0192] Table 3 Primers for Quantitative Real-Time PCR

[0193] Gene Primer sequence AR-FL F: 5'-AGGCCAAGTGATGCCAGAAC-3' AR-V7 F: 5'-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3' KLK2 F: 5'-CAACATCTGGAGGGGAAAGGG-3' KLK3 / PSA F: 5'-GGA AAT GAC CAG GCC AAG AC-3' NKX3.1 F: 5'-CCA TAC CTG TAC TGC GTG GG-3' β-Actin F: 5'-GAGAAAATCTGGCACCACACC-3'

[0194] Experimental results are as follows Figure 5 As shown, compound 22 significantly inhibited the mRNA expression of AR and its mutant AR-V7 in prostate cancer cells in a concentration-dependent manner, and also inhibited the gene expression of AR target genes KLK3, KLK2, and NKX3-1; indicating that compound 22 can inhibit the gene expression of AR and its mutant AR-V7 in prostate cancer cells, thereby inhibiting the AR signaling pathway and reducing cellular drug resistance. In contrast, the non-covalent control compound 32 had no significant effect on the AR signaling pathway in prostate cancer cells, and compound 22 showed a more significant inhibitory effect on the AR signaling pathway in prostate cancer cells than the previously reported compound GSK805.

[0195] Example 7: Compound 22 inhibits androgen receptor expression.

[0196] Cells were lysed with RIPA lysis buffer, and the supernatant was collected. Protein concentration was quantified by BCA assay. After denaturation by boiling with 5× loading buffer, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane (PVDF membrane), blocked with skim milk powder at room temperature for 1 h, washed 3 times with TBS-T solution, and incubated overnight at 4°C with AR and GAPDH primary antibodies. The next day, the cells were incubated with rabbit secondary antibody at room temperature for 1 h, and the protein bands were visualized using a imaging system.

[0197] Experimental results are as follows Figure 6 As shown, compound 22 significantly inhibited the protein expression of androgen receptor AR and its variant AR-V7 in prostate cancer cells in a concentration-dependent manner. Compared with the non-covalent control compound 32, covalent compound 22 showed more significant inhibition of the protein expression of AR and its variant AR-V7 in prostate cancer cells, and had a stronger inhibitory effect on the AR signaling pathway in prostate cancer cells than the previously reported compound GSK805.

[0198] Example 8: Antitumor efficacy of compound 22 in the 22Rv1 nude mouse xenograft model

[0199] Each nude mouse was subcutaneously injected with 5×10 [units of something] on both sides of its thigh. 6 22Rv1 cells, when the tumor volume reaches 50 mm. 3 At that time, 28 nude mice were randomly divided into 4 groups; the formula for calculating tumor volume was major axis × minor axis. 2 / 2; The group and administration settings are as follows: The control group received 100 μL of placebo intraperitoneally daily; the administration group received 100 μL of the compound solution intraperitoneally daily; tumor size and mouse weight were recorded every 3 days; once the tumor volume in the placebo group exceeded 1000 mm², the administration was stopped. 3 The mice were euthanized and then tested.

[0200] Experimental results are as follows Figure 7 As shown, compound 22 can significantly reduce the volume and weight of tumors (prostate cancer) in mice, indicating that compound 22 can inhibit the growth and proliferation of tumors in mice (and the higher the concentration, the more obvious the inhibitory effect); and compound 22 has little effect on mouse body weight and the morphology of major organs, indicating good safety.

[0201] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A RORγ covalent inhibitory compound, characterized in that, The RORγ covalent inhibitory compound has the structure of formula (I): Where n is an integer from 0 to 1; X, Y, Z, and W are independently selected from C or N, and when W is N, there is no substituent R4; L is R1 is R a For halogens, R b R c Each is independently selected from hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl, or R a and R b It forms a C3-C6 cycloalkyl group with the attached carbon atom; R d R e R f Each is independently selected from hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl; the substitution means that at least one site is substituted by the following substituents: halogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C1-C6 alkylamino; R2, R3, and R4 are each independently selected from hydrogen, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, C 1- C6 alkylamino, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl; R5 is selected from hydrogen, cyano, hydroxyl, nitro, substituted or unsubstituted sulfone, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; the substitution refers to at least one site being substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, C1-C6 alkyl, C1-C6 alkoxy, C 1-6 Alkylamino.

2. The RORγ covalent inhibitory compound according to claim 1, characterized in that, n is an integer from 0 to 1; X is C, W, Y, and Z are independently selected from C or N, and when W is N, there is no substituent R4; L is R1 is R a For halogens, R b R c Each is independently selected from hydrogen, C1-C6 alkyl, halogen, or R a and R b It forms a C3-C6 cycloalkyl group with the attached carbon atom; R d R e R f Each is independently selected from hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl groups, wherein the substitution means that at least one site is substituted by a C1-C6 alkoxy group; R2 is hydrogen, and R3 and R4 are each independently selected from hydrogen, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl; R5 is selected from hydrogen, cyano, nitro, sulfone, C1-C6 alkyl-substituted sulfone, C1-C6 alkyl, and C1-C6 alkoxy.

3. The RORγ covalent inhibitory compound according to claim 2, characterized in that, n is an integer from 0 to 1; X is C, W, Y, and Z are independently selected from C or N, and when W is N, there is no substituent R4; L is R1 is R a For halogens, R b R c Each is independently selected from hydrogen, C1-C3 alkyl, halogen, or R a and R b It forms a cyclopropyl group with the attached carbon atom; R d R e R f Each is independently selected from hydrogen, halogen, substituted or unsubstituted C1-C3 alkyl groups, wherein the substitution means that at least one site is substituted by a C1-C3 alkoxy group; R2 is hydrogen, and R3 and R4 are each independently selected from hydrogen, halogen, C1-C3 alkyl, and halo-C1-C3 alkyl; R5 is selected from hydrogen, cyano, nitro, sulfone, C1-C3 alkyl-substituted sulfone, C1-C3 alkyl, and C1-C3 alkoxy.

4. The RORγ covalent inhibitory compound according to any one of claims 1 to 3, characterized in that, The RORγ covalent inhibitory compound may also be a pharmaceutically acceptable salt thereof.

5. A method for preparing the RORγ covalent inhibitory compound according to any one of claims 1 to 3, characterized in that, Specifically, the following steps are included: SI1, Compound 1-1 and The Suzuki reaction occurred in the presence of a catalyst and an inorganic base to give compounds 1-2. SI2, compounds 1-2 react with R1-OH via a condensation reaction, or react with R1-Cl via a nucleophilic substitution reaction, to give compounds 1-3; SI3: Compounds 1-3 undergo a reduction reaction to yield compounds 1-4; SI4: Compounds 1-4 and After condensation reaction, or with The target product (I) was obtained through a nucleophilic substitution reaction; The definitions of n, X, Y, Z, W, L, R1, R2, R3, R4, and R5 are consistent with those of any one of claims 1 to 3.

6. The method for preparing the RORγ covalent inhibition compound according to claim 1, characterized in that, Specifically, the following steps are included: SII1, Compound 2-1 and After a condensation reaction, compound 2-2 was obtained; SII2: Compound 2-2 and The Suzuki reaction occurred in the presence of a catalyst and an inorganic base to give compounds 2-3. SII3: Compound 2-3 undergoes a reduction reaction to yield compound 2-4; SII4: Compound 2-4 reacts with R1-OH via a condensation reaction or with R1-Cl via a nucleophilic substitution reaction to obtain the target product (I); The definitions of n, X, Y, Z, W, L, R1, R2, R3, R4, and R5 are consistent with those of any one of claims 1 to 3.

7. The use of the RORγ covalent inhibitory compound of claim 1 in the preparation of RORγ inhibitors.

8. The use of the RORγ covalent inhibitory compound of claim 1 in the preparation of drugs for the prevention and treatment of RORγ-related diseases.

9. The use of the RORγ covalent inhibitory compound of claim 1 in the preparation of antitumor drugs.

10. A RORγ covalent inhibitor, characterized in that, Contains the RORγ covalent inhibitory compound according to any one of claims 1 to 4.

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