BENZIDINE COMPOUND AND APPLICATION THEREOF

Abstract

The present application relates to a benzidine compound and an application thereof, the benzidine compound having the structure as shown in formula I below. Further provided in the present application are a pharmaceutically acceptable salt, isomer, racemate, prodrug co-crystal complex, hydrate, and solvate of the compound, as well as an application thereof in the preparation of a drug for the treatment or prevention of RORγ-regulated diseases; more importantly, such a compound can also be used in the preparation of a drug for the treatment of inflammation, immune diseases, cancer and neurological diseases.

Claims

1. A benzidine compound, having a structure as shown in Formula I: ##STR00016## wherein X is ##STR00017## R.sub.1 is selected from substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl; R.sub.2 is selected from H, halogen, substituted or unsubstituted alkyl, cyano or —COH(CF.sub.3).sub.2; and R.sub.3 and R.sub.4 are independently selected from H, halogen or substituted or unsubstituted alkyl.

2. The benzidine compound according to claim 1, wherein R.sub.1 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted benzyl or substituted or unsubstituted benzyl-(C1-C6) alkyl.

3. The benzidine compound according to claim 1, wherein R.sub.2 is selected from H, substituted or unsubstituted C1-C6 alkyl or —COH(CF.sub.3).sub.2.

4. The benzidine compound according to claim 2, wherein in the substituted or unsubstituted benzyl or the substituted or unsubstituted benzyl-(C1-C6) alkyl, the substituent of benzyl is selected from nitro, amido, methylsulfonyl, ethylsulfonyl, N-methylamido, carbomethoxy, carboxyl, trifluoromethyl, amino, phenyl or cyclohexyl.

5. The benzidine compound according to claim 1, wherein the benzidine compound has the structure as shown in Formula II or Formula III: ##STR00018## wherein R is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted benzyl, or substituted or unsubstituted benzyl-(C1-C6) alkyl, wherein the substituent of benzyl is selected from nitro, amido, methylsulfonyl, ethylsulfonyl, N-methylamido, carbomethoxy, carboxyl or amino; and R′ is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted benzyl, or substituted or unsubstituted benzyl-(C1-C6) alkyl, wherein the substituent of benzyl is selected from nitro, amido, methylsulfonyl, ethylsulfonyl, N-methylamido, carbomethoxy, carboxyl or amino.

6. The benzidine compound according to claim 5, wherein R′ is selected from substituted or unsubstituted benzyl, wherein the substituent of benzyl is selected from amino, nitro, methylsulfonyl or ethylsulfonyl.

7. The benzidine compound according to claim 1, wherein the benzidine compound is any one or a combination of at least two of the following compounds: N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-(methylsulfonyl)phenyl)acetamide; 2-(4-(ethylsulfonyl)phenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,11,1′-biphenyl]-4-yl)acetamide; N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-(nitrophenyl)acetamide; N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(2-(nitrophenyl)acetamide; 2-(4-aminophenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)acetamide; 2-(2-aminophenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)acetamide; and N-(2′-fluoro-4′-(1,1,1,3,3,3 -hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)aniline.

8. A pharmaceutically acceptable salt, isomer, racemate, prodrug co-crystalline complex or solvate of the benzidine compound according to claim 1.

9. Use of the benzidine compound according to claim 1 in the preparation of a RORγ receptor antagonist.

10. Use of the benzidine compound according to claim 1 in the preparation of a drug for the treatment of inflammation, an immune disease, cancer or a neurological disease.

11. The use according to claim 10, wherein the immune disease is encephalomyelitis, collagen-induced arthritis, multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn's disease or asthma.

12. The use according to claim 10, wherein the cancer is prostate cancer, breast cancer, colon cancer, acute leukemia, non-small cell lung cancer, osteosarcoma, lung cancer, cervical cancer or renal cancer.

13. The benzidine compound according to claim 3, wherein R.sub.2 is selected from —COH(CF.sub.3).sub.2.

14. The benzidine compound according to claim 6, wherein R′ is selected from ##STR00019##

Description

DETAILED DESCRIPTION

[0047] To further elaborate on the technical means adopted and the effects achieved in the present application, the technical solutions of the present application are further described below in conjunction with the preferred examples of the present application, but the present application is not limited to the scope of the examples.

EXAMPLE 1

[0048] The present example provides a benzidine compound: N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-(methylsulfonyl)phenyl)acetamide, and the preparation method thereof includes the steps described below.

(1) Preparation of 2-(4-amino-3-fluorophenyl)-1,1,1,3,3,3-hexafluoro-2-propanol having a structure as shown in the following formula:

##STR00005##

[0049] Difluoroaniline (6.0 g, 54 mmol), hexafluoroacetone trihydrate (12.5 g, 56.7 mmol), and p-toluenesulfonic acid (0.85 g, 5.4 mmol) were placed in a pressure vessel. After the pressure vessel was vacuumized, the mixture was heated to 90° C. under argon protection and reacted overnight. The reaction product was cooled to room temperature, then washed once with saturated sodium bicarbonate, and extracted three times with ethyl acetate (3×50 mL). The organic layers were combined, washed once with saturated sodium chloride, and dried with anhydrous sodium sulfate, and the organic phase was subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=10:1) to obtain 4.45 g of white solid (with a yield of 90%). Characterization results of MS (ESI) mass spectrometry: calculated 277.14, found 278.0.

[0050] (2) Preparation of 2-(4-iodo-3-fluorphenyl)-1,1,1,3,3,3-hexafluoro-2-propanol having a structure as shown in the following formula:

##STR00006##

[0051] The compound 2-(4-amino-3-fluorophenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (4.45 g, 16.1 mmol) was dissolved in DMF (50 mL). Concentrated HCl (18 mL, 73 mmol) was added to the solution at 0° C. and stirred for 5 minutes, and then, sodium nitrite (1.7 g, 24 mmol) aqueous solution (20 mL) added to the solution. The reaction mixture was stirred at 0° C. for 30 minutes, then potassium iodide (4.0 g, 24 mmol) was added portion-wise, and the reaction mixture was stirred at room temperature overnight. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=50:1) to obtain 6.2 g of product (with a yield of 90%). Characterization results of nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.31 (d, J=8.4 Hz, 1H), 7.48 (d, J=1.6 Hz, 9.2 Hz, 1H), 8.05 (dd, J=6.8 Hz, 8.4 Hz, 1H), 9.07 (s, 1H).

[0052] (3) Preparation of tert-butyl ester-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)carbamic acid having a structure as shown in the following formula:

##STR00007##

[0053] The compound 2-(4-iodo-3-fluorphenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (6.2 g, 16 mmol) was dissolved in 1,4-dioxane (100 mL) and water (20 mL). 4-(N-BOC-amino)phenylboronic acid (4.2 g, 17.6 mmol), potassium carbonate (6.6 g, 48 mmol), and Pd(PPh.sub.3).sub.4 (0.9 g, 0.78 mmol) were added to the solution, and the solution was refluxed overnight under argon protection. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=20:1) to obtain 5.4 g of product (with a yield of 74%). Characterization results of nuclear magnetic resonance: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.54-7.44 (m, 6H), 7.40 (d, J=8.4 Hz, 1H), 6.57 (s, 1H), 3.82 (s, 1H), 1.53 (d, J=3.2 Hz, 9H).

[0054] (4) Preparation of 2-(4′-amino-2-fluoro-[1,1′-biphenyl]-4-yl-1,1,1,3,3,3-hexafluoro-2-propanol having a structure as shown in the following formula:

##STR00008##

[0055] The compound tert-butyl ester-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)carbamic acid (5.35 g, 11.8 mmol) was dissolved with 50 mL of DCM, trifluoroacetic acid (7 mL, 96 mmol) was dropped at 0° C., and the solution was reacted at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, the mixture was concentrated under reduced pressure and then recrystallized in petroleum ether/ethyl acetate to obtain 3.8 g of product (with a yield of 91%). Characterization results of nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.91 (s, 1H), 7.61 (t, J=8.4 Hz, 1H), 7.50-7.47 (m, 2H), 7.30 (d, J=7.2 Hz, 2H), 6.65 (d, J=8.4 Hz, 2H), 5.41 (s, 2H).

[0056] The compound tert-butyl ester-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)carbamic acid (5.35 g, 11.8 mmol) was dissolved with 50 mL of DCM, trifluoroacetic acid (7 mL, 96 mmol) was dropped at 0° C., and the solution was reacted at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, the mixture was concentrated under reduced pressure and then recrystallized in petroleum ether/ethyl acetate to obtain 3.8 g of product (with a yield of 91%). Characterization results of nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.91 (s, 1H), 7.61 (t, J=8.4 Hz, 1H), 7.50-7.47 (m, 2H), 7.30 (d, J=7.2 Hz, 2H), 6.65 (d, J=8.4 Hz, 2H), 5.41 (s, 2H).

[0057] (5) Preparation of N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-(methylsulfonyl)phenyl)acetamide having a structure as shown in the following formula:

##STR00009##

[0058] The compound 2-(4-(methylsulfonyl)phenyl)acetic acid (66 mg, 0.31 mmol), diisopropylethylamine (0.5 mL), and HATU (646.4 mg, 1.70 mmol) were dissolved in 20 mL of DCM. The reaction mixture was stirred at room temperature for 5 minutes, then the compound 2-(4′-amino-2-fluoro-[1,1′-biphenyl]-4-yl-1,1,1,3,3,3-hexafluoro-2-propanol (100 mg, 0.28 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=5:1) to obtain 73 mg of white solid (with a yield of 43%).

[0059] The characterization results of H nuclear magnetic resonance were: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.43 (s, 1H), 9.00 (s, 1H), 7.89 (d, J=8.4 Hz, 2H), 7.77-7.66 (m, 3H), 7.66-7.32 (m, 6H), 3.83 (s, 2H), 3.19 (s, 3H). Characterization results of C nuclear magnetic resonance: .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 168.4, 159.7, 157.7, 141.8, 139.3, 139.2, 131.6, 130.9, 130.2, 129.7, 129.6, 129.3, 129.3, 128.6, 127.0, 123.2, 119.2, 115.0, 114.8, 43.6, 43.0.

[0060] Characterization results of MS (ESI) mass spectrometry for C.sub.24H.sub.18F.sub.7NO.sub.4S ([M−1].sup.−): calculated 549.46, found: 547.9. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 7.26 min, 99.51%.

EXAMPLE 2

[0061] The present example provides a benzidine compound: 2-(4-(ethylsulfonyl)phenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3 -hexafluoro-2-hydroxypropan-2-yl)-[1,1 1,1′-biphenyl]-4-yl)acetamide, and the preparation method thereof includes the steps described below.

[0062] Steps (1) to (4) were consistent with steps (1) to (4) in Example 1, and then step (5) was performed.

[0063] (5) Preparation of 2-(4-(ethylsulfonyl)phenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1 1,1′-biphenyl]-4-yl)acetamide having a structure as shown in the following formula:

##STR00010##

[0064] The compound 2-(4-(ethylsulfonyl)phenyl)acetic acid (70.7 mg, 0.31 mmol), diisopropylethylamine (0.5 mL), and HATU (646.4 mg, 1.70 mmol) were dissolved in 20 mL of DCM. The reaction mixture was stirred at room temperature for 5 minutes, then the compound 2-(4′-amino-2-fluoro-[1,1′-biphenyl]-4-yl-1,1,1,3,3,3-hexafluoro-2-propanol (100 mg, 0.28 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=3:1) to obtain 78.9 mg of white solid (with a yield of 50%).

[0065] Characterization results of H nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.44 (s, 1H), 9.00 (s, 1H), 7.85 (d, J=8.4 Hz, 2H), 7.76-7.65 (m, 3H), 7.62 (d, J=8.4 Hz, 2H), 7.59-7.48 (m, 4H), 3.82 (d, J=12.4 Hz, 2H), 3.27 (q, J=14.8, 7.2 Hz, 2H), 1.09 (t, J=7.2 Hz, 3H). Characterization results of C nuclear magnetic resonance: .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 168.90, 160.15, 158.19, 142.50, 139.75, 137.35, 132.20, 131.36, 130.69, 130.16, 129.82, 129.79, 129.14, 128.32, 123.67, 119.70, 115.51, 115.31, 49.72, 43.43, 7.61.

[0066] Characterization results of MS (ESI) mass spectrometry for C.sub.25H.sub.20F.sub.7NO4S([M−1].sup.−): calculated 563.49, found 562.2. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 7.92 min, 99.71%.

EXAMPLE 3

[0067] The present example provides a benzidine compound: N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-nitrophenyl)acetamide, and the preparation method thereof includes the steps described below.

[0068] Steps (1) to (4) were consistent with steps (1) to (4) in Example 1, and then step (5) was performed.

[0069] (5) Preparation of N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-nitrophenyl)acetamide having a structure as shown in the following formula:

##STR00011##

[0070] The compound 2-(4-nitrophenyl)acetic acid (56.2 mg, 0.31 mmol), diisopropylethylamine (0.5 mL), and HATU (646.4 mg, 1.70 mmol) were dissolved in 20 mL of DCM. The reaction mixture was stirred at room temperature for 5 minutes, then the compound 2-(4′-amino-2-fluoro-[1,1′-biphenyl]-4-yl-1,1,1,3,3,3-hexafluoro-2-propanol (100 mg, 0.28 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=3:1) to obtain 67.9 mg of white solid (with a yield of 47%).

[0071] Characterization results of H nuclear magnetic resonance: .sup.1H NMR (400 MHz, CDC1.sub.3) δ 8.25 (d, J=8.0 Hz, 2H), 7.61-7.45 (m, 8H), 7.35 (s, 1H), 4.29 (s, 1H), 3.85 (s, 2H). Characterization results of C nuclear magnetic resonance: .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 168.17, 164.56, 159.64, 157.68, 146.40, 143.82, 139.21, 131.70, 130.83, 130.57, 129.66, 129.32, 129.30, 128.66, 123.35, 123.16, 119.21, 115.01, 114.81, 38.20.

[0072] Characterization results of MS (ESI) mass spectrometry for C.sub.23H.sub.15F.sub.7N.sub.2O.sub.4([M+1].sup.+): calculated 516.37, found 517.0. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 12.23 min, purity 96.63%.

EXAMPLE 4

[0073] The present example provides a benzidine compound:

[0074] N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(2-nitrophenyl)acetamide, and the preparation method thereof includes the steps described below.

[0075] Steps (1) to (4) were consistent with steps (1) to (4) in Example 1, and then step (5) was performed.

[0076] (5) Preparation of N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(2-nitrophenyl)acetamide having a structure as shown in the following formula:

##STR00012##

[0077] The compound 2-(2-nitrophenyl)acetic acid (56.2 mg, 0.31 mmol), diisopropylethylamine (0.5 mL), and HATU (646.4 mg, 1.70 mmol) were dissolved in 20 mL of DCM. The reaction mixture was stirred at room temperature for 5 minutes, then the compound 2-(4′-amino-2-fluoro-[1,1′-biphenyl]-4-yl-1,1,1,3,3,3-hexafluoro-2-propanol (100 mg, 0.28 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=4:1) to obtain 47.7 mg of white solid (with a yield of 33%).

[0078] Characterization results of H nuclear magnetic resonance: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.09 (d, J=8.4 Hz, 1H), 7.94 (s, 1H), 7.66 (t, J=7.2 Hz, 1H), 7.53 (qd, J=16.4, 8.4 Hz, 9H), 4.26 (s, 1H), 4.03 (s, 2H). Characterization results of C nuclear magnetic resonance: .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 167.82, 159.65, 157.69, 149.03, 139.33, 133.73, 133.52, 131.69, 130.84, 130.50, 129.70, 129.60, 129.31, 129.29, 128.41, 124.55, 123.86, 123.16, 121.56, 119.07, 115.01, 114.80, 40.73.

[0079] Characterization results of MS (ESI) mass spectrometry for C.sub.23H.sub.15F.sub.7N.sub.2O.sub.4([M+1].sup.+): calculated 516.37, found 517.0. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 9.83 min, purity 99.45%.

EXAMPLE 5

[0080] The present example provides a benzidine compound: 2-(4-aminophenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)acetamide, and the preparation method thereof includes the steps described below.

##STR00013##

[0081] The compound N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(4-nitrophenyl)acetamide (80 mg, 0.15 mmol) and 10% palladium on carbon (water content of about 55%) (15 mg) were added to methanol (10 mL) as a solvent and reacted under hydrogen at room temperature for 5 hours. After the reaction finished, the mixture was subjected to suction filtration with the aid of Celite, and the filtrate was concentrated to obtain 62 mg of product (with a yield of 85%).

[0082] Characterization results of H nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.20 (s, 1H), 9.00 (s, 1H), 7.82-7.62 (m, 3H), 7.62-7.41 (m, 4H), 6.99 (d, J=8.4 Hz, 2H), 6.55-6.41 (m, 2H), 4.94 (s, 2H), 3.45 (s, 2H). Characterization results of C nuclear magnetic resonance: .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 170.17, 157.68, 147.22, 139.62, 130.81, 129.74, 129.63, 129.42, 129.22, 129.20, 123.84, 123.13, 122.66, 119.06, 114.99, 114.77, 113.84, 99.49, 42.69.

[0083] Characterization results of MS (ESI) mass spectrometry for C.sub.23H.sub.17F.sub.7N.sub.2O.sub.2 ([M−1].sup.−): calculated 486.39, found 485.0. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 7.47 min, purity 99.17%.

EXAMPLE 6

[0084] The present example provides a benzidine compound: 2-(2-aminophenyl)-N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)acetamide, and the preparation method thereof includes the steps described below.

##STR00014##

[0085] The compound N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)-2-(2-nitrophenyl)acetamide (80 mg, 0.15 mmol) and 10% palladium on carbon (water content of about 55%) (15 mg) were added to methanol (10 mL) as a solvent and reacted under hydrogen at room temperature for 5 hours. After the reaction finished, the mixture was subjected to suction filtration with the aid of Celite, and the filtrate was concentrated to obtain a crude product. The crude product was separated by silica gel column chromatography (PE: EA=2:1, v/v) to obtain a target compound as a white solid (64.2 mg, with a yield of 88%).

[0086] Characterization results of H nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.31 (s, 1H), 9.00 (s, 1H), 7.76 -7.65 (m, 3H), 7.61-7.49 (m, 4H), 7.06 (dd, J=7.5, 1.3 Hz, 1H), 6.95 (td, J=7.9, 1.5 Hz, 1H), 6.67 (dd, J=7.9, 1.0 Hz, 1H), 6.54 (td, J=7.4, 1.1 Hz, 1H), 5.09 (s, 2H), 3.52 (s, 2H).

[0087] Characterization results of MS (ESI) mass spectrometry for C.sub.23H.sub.17F.sub.7N.sub.2O.sub.2 ([M−1].sup.−): calculated 486.39, found 485.0. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 7.44 min, purity 98.86%.

EXAMPLE 7

[0088] The present example provides a benzidine compound: N-(2′-fluoro-4′-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1′-biphenyl]-4-yl)aniline, and the preparation method thereof includes the steps described below.

##STR00015##

[0089] Steps (1) to (4) were consistent with steps (1) to (4) in Example 1, and then step (5) was performed.

[0090] The compound n-pentanoic acid (31.7 mg, 0.31 mmol), diisopropylethylamine (0.5 mL), and HATU (646.4 mg, 1.70 mmol) were dissolved in 20 mL of DCM. The reaction mixture was stirred at room temperature for 5 minutes, then the compound 2-(4′-amino-2-fluoro-[1,1′-biphenyl]-4-yl-1,1,1,3,3,3-hexafluoro-2-propanol (100 mg, 0.28 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. After the reaction finished, water was added, and the reaction product was extracted with ethyl acetate (3×50 mL). The organic phase was washed once with saturated saline solution, dried with anhydrous sodium sulfate, and subjected to rotary evaporation under vacuum. The crude product was separated by a silica gel separation column (PE:EA=8:1) to obtain 31.7 mg of white solid (with a yield of 72%).

[0091] Characterization results of H nuclear magnetic resonance: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.07 (s, 1H), 9.13 (s, 1H), 7.71-7.66 (m, 3H), 7.57-7.50 (m, 4H), 2.32 (t, J=7.2 Hz, 2H), 1.59-1.53 (m, 2H), 1.36-1.27 (m, 2H), 0.88 (t, J=7.2 Hz, 3H). Characterization results of C nuclear magnetic resonance: .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 170.20, 159.65, 157.69, 139.48, 130.84, 129.78, 129.68, 129.20, 129.17, 128.24, 123.15, 119.17, 115.00, 114.79, 49.62, 30.84, 29.57.

[0092] Characterization results of MS (ESI) mass spectrometry for C.sub.20H.sub.18F.sub.7NO.sub.2 ([M+1].sup.+): calculated 437.36, found 438.1. Characterization results of liquid chromatography using methanol:H.sub.2O (80:20) as mobile phase: peak appearance time 16.78 min, purity 99.95%.

EXAMPLE 8

[0093] In vitro activity test: in this example, the inhibitory activity of the compounds of the present application on RORγ protein was detected by AlphaScreen detection technology.

[0094] Experimental materials: target protein RORγ with the final concentration of 200 nM; experimental buffer (10×) MOPS (500 mM) PH 7.4, CHAPS (0.5 mM), NaF (500 mM), and

[0095] BSA (1 mg/ml); donor microbeads in the kit with the final concentration of 5 μg/mL, and acceptor microbeads with the final concentration of 5 μg/mL; co-agonist of RORγ, short peptide bSRC1-4(Biotin-QKPTSGPQTPQAQQKSLLQQLLTE), with the final concentration of 50 nM. 150 μL of reaction system: RORγ 15 μL, experimental buffer 15 μL, deionized water 60 μL, small molecule compound 15 μL, donor microbeads 15 μL, and acceptor microbeads 15 μL; positive inhibitors T0901317 and UA.

[0096] Experimental method: The protein, co-agonist (b-SRC1-4), 10× AlphaScreen buffer, and ultra-pure water were prepared into a mixed solution with the final volumes of 15 μL, 15 μL, 15 μL, and 60 μL, respectively (the final concentration ratio of protein to co-agonist is 200:50 nM). 105 μL of the mixed solution was added to each sample to be tested in a 96-well transparent plate. If the single-point inhibition rate of the compound was tested, the compound was diluted to the final concentration of 50 μM, and 15 μL of the diluted compound was added to each sample. If the IC.sub.50 value of the compound was tested, the compound was doubling diluted (to 200 to 0.075 μM), and 15 μL of the diluted compound was added to each sample (generally, in order to save manpower and material resources, a batch of new compounds were subjected to single-point preliminary screening, and then compounds with an inhibition rate of about 50% were tested for IC.sub.50 curves). The donor microbeads and acceptor microbeads in the final concentration of 5000 g/mL should be prepared to 5 g/mL, and in the green light environment, 30 μL of the mixed solution of the two microbeads was added to each well. The mixture was centrifuged at room temperature at 1000 rpm for 1 minute to make the system fully mixed. After being wrapped in tin foil, the mixture was incubated in the dark for 1.5 hours. After that, the mixture was transferred to a 384-well white opaque plate and put into an EnSpire Alpha 2390 multifunctional microplate reader to detect the inhibitory activity of the compound.

[0097] Experimental result: The inhibitory activity data of the compounds prepared in Examples 1 to 7 of the present application on RORγ protein are shown in Table 1.

TABLE-US-00001 TABLE 1 No. Name IC.sub.50 (μM) 1 Compound prepared in Example 1 2.28 ± 0.05 2 Compound prepared in Example 2 0.75 ± 0.09 3 Compound prepared in Example 3 4.85 ± 0.04 4 Compound prepared in Example 4 3.46 ± 1.32 5 Compound prepared in Example 5 2.68 6 Compound prepared in Example 6 5.17 7 Compound prepared in Example 7 10.19 ± 1.61 8 Positive inhibitor T0901317 2.50 9 Positive inhibitor UA 9.41

[0098] The experimental result shows that the compounds of the present application have a very good inhibitory effect on RORγ protein, and especially, the inhibitory activity of compounds prepared in Examples 1 and 2 on RORγ protein is better than the inhibitory activity of the control drug 10901317.

EXAMPLE 9

[0099] In vitro activity test: in this example, the inhibitory activity of the compounds of the present application of RORγ protein was verified by Luciferase detection technology on the cellular cell level.

[0100] Experimental materials: human renal epithelial cell line 293T cells; DMEM medium containing 10% fetal bovine serum; 96-well plate transparent plate; double reporter gene detection kit; Opti-MEM reagent; Lipo-fectamine 2000 transfection reagent; recombinant plasmid: Gal4-RORγLBD: 25 ng, RORE_Luc: 25 ng, pG5-luc, and Renilla; positive inhibitors: 10901317 and UA.

[0101] Experimental method: Human renal epithelial cell line 293T cells were cultured in DMEM medium containing 10% fetal bovine serum. The cells were cultured in a 96-well plate one day before transfection, with a cell density of 1.5×10.sup.4 cells/well. After 24 hours of adherent growth, the cells were subjected to transient transfection by using the method of double reporter gene co-transfection with the transfection reagent of Lipo-fectamine 2000, where the transfection reagent and plasmids were diluted with Opti-MEM reagent, respectively. Ga14-RORγLBD was 25 ng per well; PG5-luc gene was 25 ng per well; and Renilla was 5 ng per well. After 24 hours of co-transfection, different concentrations of compounds were added. After 24 hours of incubation, Luciferase double reporter gene detection kit was used to detect luminescence signals. Each sample had 3 duplicate wells. The readings were detected by multifunctional detection microplate reader (excitation wavelength of 680 nm, emission wavelength of 520 nm to 620 nm), and the IC.sub.50 value (half inhibitory concentration) was calculated by software.

[0102] Experimental result: The inhibitory activity of compounds 1 to 7 of the present application on RORγ protein on the cellular level is shown in Table 2.

TABLE-US-00002 TABLE 2 No. Name IC.sub.50 (μM) 1 Compound prepared in Example 1 0.12 ± 0.03 2 Compound prepared in Example 2 0.03 ± 0.01 3 Compound prepared in Example 3 1.49 ± 0.5 4 Compound prepared in Example 4 0.19 ± 0.02 5 Compound prepared in Example 5 4.16 ± 2.26 6 Compound prepared in Example 6 0.96 ± 0.46 7 Compound prepared in Example 7 4.17 ± 2.10 8 Positive inhibitor T0901317 1.7 ± 0.05 9 Positive inhibitor UA 0.68 ± 0.1

[0103] The experimental result shows that through the detection of RORγ protein on the cellular level, the compounds of the present application have a very good inhibitory effect, and especially the inhibitory activity of compounds prepared in Examples 1, 2, and 4 on RORγ protein is better than the inhibitory activity of the control drug UA. The inhibitory activity of the compound prepared in Example 3 on RORγ protein is almost equivalent to the inhibitory activity of the control drug T0901317.

EXAMPLE 10

[0104] In vitro activity test: in this example, the inhibitory activity of the compounds of the present application on RORγ protein was detected by TSA detection technology.

[0105] Experimental materials: hRORγ protein, fluorescent dye Sypro Orange, 10p buffer, ultrapure water, and HSP-96 well reaction plates.

[0106] Experimental method: The diluted protein (with the final concentration of 10 μM), fluorescent dye (with the final concentration of 5×), 10× TSA buffer, and double distilled water were prepared into a mixed solution with final component volumes of 1 μL, 1 μL, 1 μL, and 2 μL, receptively. The mixed solution was transferred to an HSP-96 well reaction plate, 5 μL each well. The ligand, i.e., small molecule compound, was added, 5 μL per well, with the final concentration of 200 M. The mixture was centrifuged at room temperature at 1000 rpm for 1 minute to make the system fully mixed. The mixture was placed on an ice bath in low light for more than 30 minutes, and after that, put into RT-PCR instrument for testing. The temperature was set from 30° C. to 80° C., and the mixture was detected every time the temperature was raised by 0.5° C. and every 5 seconds.

[0107] Experimental result: The influence data of the compounds prepared in Examples 1 to 7 of the present application on the activity of RORγ protein are shown in Table 3.

TABLE-US-00003 TABLE 3 No. Name Δ.sub.Tm (° C.) 1 Compound prepared in Example 1 7.8 2 Compound prepared in Example 2 10.1 3 Compound prepared in Example 3 6 4 Compound prepared in Example 4 4.2 5 Compound prepared in Example 5 1.5 6 Compound prepared in Example 6 1.5 7 Compound prepared in Example 7 6.3 8 Positive inhibitor T0901317 7.7 9 Positive inhibitor UA 7.0

[0108] The experimental result shows that the compounds of the present application have a very good stabilizing effect on RORγ protein, and especially, the stabilizing effects of compounds prepared in Examples 1 and 2 on RORγ protein are better than the stabilizing effect of the control drug T0901317.

EXAMPLE 11

[0109] In this example, the specificity of the compounds prepared in Examples 1, 2, and 4 for RORγ and homologous proteins thereof in cells was evaluated.

[0110] Experimental materials: human renal epithelial cell line 293T cells; DMEM medium containing 10% fetal bovine serum; 96-well plate transparent plate; double reporter gene detection kit; Opti-MEM reagent; Lipo-fectamine 2000 transfection reagent; and recombinant plasmid: Ga14-RORγLBD: 25 ng, RORE_Luc: 25 ng, pG5-luc, and Renilla; positive inhibitor: T0901317.

[0111] Experimental method: Human renal epithelial cell line 293T cells were cultured in DMEM medium containing 10% fetal bovine serum. The cells were cultured in a 96-well plate one day before transfection, with a cell density of 1.5×10.sup.4 cells/well. After 24 hours of adherent growth, the cells were subjected to transient transfection by using the method of double reporter gene co-transfection with the transfection reagent of Lipo-fectamine 2000, and the transfection reagent and plasmids were diluted with Opti-MEM reagent, respectively. Gal4-RORγLBD was 25 ng per well; PG5-luc gene was 25 ng per well; and Renilla was 5 ng per well. After 24 hours of co-transfection, different concentrations of compounds were added. After 24 hours of incubation, Luciferase double reporter gene detection kit was used to detect luminescence signals. Each sample had 3 duplicate wells. The IC.sub.50 value (half inhibitory concentration) was calculated by software.

[0112] Experimental result: The specificity characterization data of the compounds prepared in Examples 1, 2, and 4 of the present application on RORγ and homologous proteins thereof in cells are shown in Table 4.

TABLE-US-00004 TABLE 4 IC.sub.50 (μM) Example RORγ RORα LXRα FXR Example 1 0.12 ± 0.03 NA NA NA Example 2 0.03 ± 0.01 NA NA NA Example 4 0.19 ± 0.02 7.57 ± 0.01 NA NA

[0113] The experimental result shows that the compounds of the present application have specific selectivity to RORγ, especially the compounds prepared in Examples 1 and 2 have better selectivity (NA in the table indicates no inhibitory effect), and the compound prepared in Example 4 has an inhibitory rate of 37% on RORα.

EXAMPLE 12

[0114] In this example, the binding activity of the compounds of the present application to proteins was detected by using ITC detection technology.

[0115] Experimental materials: the instrument for detecting heat change: ITC200 (Microcal, produced by GE Healthcare Company); buffer solution used by the dilution reagent: 50 mM of HEPES, 150 mM of NaCl, 0.5 mM of TCEP, and pH 7.5.

[0116] Experimental method: All experiments were performed at 25° C. while the ITC buffer (50 mM of HEPES, 150 mM of NaCl, 0.5 mM of TCEP, and pH 7.5) was stirred at 1000 rpm. The titration of RORγ injection protein of all ligands was performed at an initial injection of 0.5 μL, followed by 20 identical 2 μL phase injections, with each injection lasting 4 seconds, at an injection interval of 180 seconds. The stock solutions of ligands and RORγ ligand protein were diluted with the ITC buffer to a concentration of 30 μM for the compounds and a concentration of 300 μM for the protein before titration. The final concentration of DMSO in the reaction buffer was less than 0.25% of the total volume.

[0117] In all cases, the best fit value of stoichiometry (n), enthalpy change (H), and binding constant (Kd) was obtained in a single binding site model (n=1) by using a nonlinear least squares algorithm. The thermodynamic parameter was then calculated by using the equation ΔG=ΔH−TΔS=−RT ln K, wherein ΔG, ΔH, ΔS, T, and R were the free energy change, enthalpy change, entropy change, experimental temperature, and gas constant, respectively. The data were collected and processed by MicroCal™ Origin 7 software.

[0118] Experimental result: The experimental data of the binding activity of the compounds prepared in Examples 1, 2, and 4 of the present application to the protein are shown in Table 5.

TABLE-US-00005 TABLE 5 K.sub.d (μM) Example ITC Example 1 0.56 Example 2 0.38 Example 4 0.78

[0119] The experimental result shows that the compounds of the present application have a good binding effect on RORγ.

EXAMPLE 13

[0120] In this example, the inhibitory effects of the compounds prepared in Examples 1, 2, and 4, the RORγ antagonist SR.sub.2211, and the drug enzalutamide on different cell lines were evaluated.

[0121] Experimental materials: fluorescence signal detection instrument: EnSpire Alpha 2390 multifunctional microplate reader (produced by Perkin Elmer Company); 384-well bottom permeable microwell plate; Cell-Titer GLO luminescent reagent; cancer cells to be tested; medium and fetal bovine serum required for cell culture.

[0122] Experimental method: 500 to 1000 cells to be tested per well in 20 μL of medium were seeded in a 384-well clear-bottom microwell plate (the actual number of cells was related to cell cycle and cell volume). 12 hours later, 10 μL of culture medium containing the compound (the concentration of the compound ranged from 5 nM to 100 nM) was added to each well. After incubation with the compound for 72 to 144 hours, Cell-Titer GLO reagent 25-T was added to each well, and the plate was shaken for 20 minutes to lyse the cells. After incubation for 10 minutes, the cells were centrifuged for 1 minute, the signal value of luminescence 384 was measured. The inhibition curve was fitted by GraphPad Prism software to obtain IC.sub.50.

[0123] Experimental result: The inhibitory data of the compounds prepared in Examples 1 and 2 of the present application, the RORγ antagonist SR.sub.2211, and the drug enzalutamide on the following tumor cell lines are shown in Table 6 and Table 7.

TABLE-US-00006 TABLE 6 IC.sub.50 (μM) Compound Compound prepared prepared Cell line in Example 1 in Example 2 SR2211 LNCaP (AR-positive prostate cancer) 11.10 ± 0.42 5.83 ± 1.39 6.79 ± 1.18 22Rv1 (AR-positive prostate cancer) 18.21 ± 0.99 14.47 ± 1.10  6.42 ± 1.03 C4-2B (AR-positive prostate cancer) 13.82 10.57 10.06 VcaP (AR-positive prostate cancer) 12.82 13.04 37.64 Du145 (AR-negative prostate cancer) 19.11 11.51 ~10 (~50%) PC-3 (AR-negative prostate cancer) 15.99 15.03 NA MCF-7 (ER + breast cancer) 13.35 10.45 ~9.982 (42%) Hs578T (triple negative breast cancer) 18.44 11.86 ~10 (~50%) MDA-MB-231 (triple negative breast ~34.72 ~80 NA cancer) HT-29 (colon cancer) 14.61 6.07 >50 MV-4-11 (acute leukemia) 4.824 9.476 11.47 A549 (non-small cell lung cancer) 21.97 7.08 ~10 (55%) U2OS (osteosarcoma) 34.54 15.92 ~10 (52%) H1975 (lung cancer cells) 24.04 15.96 30.68 (41%) Hela (cervical cancer cells) 31.38 25.09 NA 293T (human renal epithelial cell 15.34 6.22 ~80 (67%) line transfected with adenovirus E1A gene, expressing SV40 large T antigen) Cos7 (African green monkey kidney 40.01 29.04 >100 cells) HFL-1 (lung fibroblasts) 30.02 ~31.93 >50 HL-7702 (liver epithelial cells) ~34.56 ~50 NA

TABLE-US-00007 TABLE 7 IC.sub.50 (μM) Compound prepared Cell line in Example 4 Enzalutamide LNCaP (AR-positive prostate cancer) 5.14 ± 0.36 42.37 ± 2.37  22Rv1 (AR-positive prostate cancer) 9.00 ± 0.33 36.66 ± 4.21  C4-2B (AR-positive prostate cancer) 9.20 ± 0.25 23.56 ± 0.61  Du145 (AR-negative prostate cancer) 28.43 ± 0.89  44.70 ± 0.93  PC-3 (AR-negative prostate cancer) 11.14 ± 1.78  53.38 ± 0.47 

[0124] The experimental result shows that the compounds of the present application have different degrees of inhibitory effects on prostate cancer, breast cancer, colon cancer, acute leukemia, lung cancer, osteosarcoma, and cervical cancer cells (NA in the table indicates no inhibitory effect; the values labeled by ˜ are estimates; the values in brackets are inhibition rates).

EXAMPLE 14

[0125] In this example, the pharmacokinetics of the compounds prepared in Examples 1, 2, and 4 were evaluated.

[0126] MATERIALS: Pharmacokinetic analysis was performed by Medicilon Corporation in Shanghai. Twelve Sprague and Dawley rats were provided by Super-B&K laboratory animal Corp. Ltd in Shanghai.

[0127] Experimental method: The compound was dissolved in DMSO:PEG 400:20% HP-400 (5:40:55, v:v:v) to serve as a stock solution (0.4 mg/ml, intravenous injection; 1 mg/ml, oral administration). The stock solution was administrated orally to three SD rats at a dose of 10 mg/kg, and administrated intravenously to three SD rats at a single dose of 2 mg/kg. Blood was collected from the jugular vein before oral administration and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral administration. Blood was collected from the jugular vein before intravenous injection and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intravenous injection. About 200 μL of blood sample was collected into a heparinized tube and immediately centrifuged at 8000 rpm for 6 minutes, and the resulting plasma was stored at −80° C. until analysis.

[0128] Experimental result: The pharmacokinetic data of the compounds prepared in Examples 1, 2, and 4 are shown in Table 8.

TABLE-US-00008 TABLE 8 Compound prepared in Compound prepared in Compound prepared Example 1 Example 2 in Example 4 Intravenous Oral Intravenous Oral Intravenous Oral Parameter injection administration injection administration injection administration C.sub.max  979.69 ± 53.17 1424.92 ± 160.53 817.70 ± 18.81 1200.06 ± 181.71 838.81 ± 38.11  721.41 ± 120.36 (μg/L) T.sub.max (h)   0.08 ± 0.00  5.33 ± 2.31  0.14 ± 0.10  6.00 ± 2.00  0.19 ± 0.10  8.00 ± 0.00 AUC.sub.(0-t)  7660.10 ± 329.15 21644.93 ± 3949.88 6105.31 ± 718.96 18828.77 ± 3082.62 3334.56 ± 495.08 3217.09 ± 781.06 (μg/L .Math. h) AUC.sub.(0-∞) 10351.63 ± 664.07 24481.36 ± 5806.64 7398.03 ± 756.99 19577.04 ± 1878.59  6443.71 ± 1719.19 — (μg/L .Math. h) T.sub.1/2 (h)  12.71 ± 1.21  8.67 ± 0.05  9.98 ± 0.88  7.32 ± 1.08  7.67 ± 2.36 — Cl  193.73 ± 12.28 — 272.26 ± 28.23 — 326.67 ± 92.57 — (mL/h/kg) Vz  3543.81 ± 270.89 — 3934.90 ± 693.46 — 3424.67 ± 594.43 — (mg/kg) F (%) — 51.91 — 59.07 — 19

[0129] The experimental result shows that the compounds of the present application have good pharmacokinetic properties, and the oral bioavailability of compounds 1 and 2 is better than the oral bioavailability of compound 4 (- in the table indicates no measured values).

[0130] The applicant has stated that although the benzidine compound and the application thereof in the present application are described through the embodiments described above, the present application is not limited to the embodiments described above, which means that implementation of the present application does not necessarily depend on the embodiments described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.

[0131] Though the preferred embodiments of the present application have been described above in detail, the present application is not limited to details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application without departing from the scope of the present application. These simple modifications are all within the protection scope of the present application.

[0132] In addition, it is to be noted that if not in collision, the specific technical features described in the above specific embodiments may be combined in any suitable manner. In order to avoid unnecessary repetition, the present application does not further specify any of various possible combination manners.