THIOPHENE DERIVATIVES AS XANTHINE OXIDASE INHIBITORS AND APPLICATION THEREOF

Abstract

A class of xanthine oxidase (XO) inhibitors, and application thereof in the preparation of drugs for treating XO-related diseases. Specifically disclosed is a compound represented by formula (I) and a pharmaceutically acceptable salt thereof.

##STR00001##

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, ##STR00066## wherein, each R.sub.1 is independently selected from H, halogen, OH, NH.sub.2, CN, C.sub.1-3 alkyl, and C.sub.1-3 alkoxy, the C.sub.1-3 alkyl and the C.sub.1-3 alkoxy are optionally substituted with 1, 2 or 3 R.sub.a; n is selected from 0, 1, 2, 3, and 4; R.sub.a is selected from H, F, Cl, Br, I, OH, and NH.sub.2; R.sub.2 is selected from H, halogen, OH, NH.sub.2, and CN; ring A is selected from C.sub.5-6 cycloalkyl and 5-6 membered heterocycloalkyl; and the 5-6 membered heterocycloalkyl contains 1, 2, 3 or 4 heteroatoms or heteroatomic groups independently selected from —NH—, —O— and N.

2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, each R.sub.1 is independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, and CH.sub.3O; and the CH.sub.3, the CH.sub.3CH.sub.2 and the CH.sub.3O are optionally substituted with 1, 2 or 3 R.sub.a.

3. The compound or the pharmaceutically acceptable salt thereof according to claim 2, wherein, each R.sub.1 is independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, and CF.sub.3.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, ring A is selected from cyclopentyl, cyclohexyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, dioxanyl, and piperidyl.

5. The compound or the pharmaceutically acceptable salt thereof according to claim 4, wherein, ring A is selected from cyclopentyl, cyclohexyl, tetrahydrofuranyl and dioxanyl.

6. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein, the structural unit ##STR00067## is selected from ##STR00068## ##STR00069## ##STR00070## ##STR00071## .

7. The compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein, the structural unit ##STR00072## is selected from ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## .

8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from ##STR00078## ##STR00079## wherein, R.sub.1, n and R.sub.2 are as defined in claim 1; and E.sub.1, E.sub.2, and E.sub.3 are each independently selected from CH.sub.2 and O.

9. A compound represented by the following formula or a pharmaceutically acceptable salt thereof, ##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084## .

10. A method of treating a disease mediated by a xanthine oxidase in a subject, which comprises administering to the subject the compound or the pharmaceutically acceptable salt thereof according to claim 1.

11. The method according to claim 10, wherein the diseases isegouty arthritis or hyperuricemia.

12. A method of treating a disease mediated by a xanthine oxidase in a subject, which comprises administering to the subject the compound or the pharmaceutically acceptable salt thereof according to claim 9.

13. The method according to claim 12, wherein the disease is gouty arthritis or hyperuricemia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0081] The present disclosure is described in detail below by means of examples. However, it is not intended that these examples have any disadvantageous limitations to the present disclosure. The present disclosure has been described in detail herein, and the embodiments are also disclosed herein. It will be apparent to those skilled in the art that various changes and modifications may be made to the embodiments disclosed herein without departing from the spirit and scope disclosed herein.

Example 1: Preparation of Compound 1

[0082] ##STR00061##

Step 1: Synthesis of Compound 1-2

[0083] Compound 1-1 (1.0 g, 3.46 mmol) was dissolved in a mixed solvent of methanol (5 mL) and water (5 mL), and then sodium hydroxide (276.62 mg, 6.92 mmol) was added. The resulting reaction solution was stirred at 55° C. for 4 hours. The methanol was removed by rotary-evaporated, and the residue was adjusted to pH 2-3 with 2 M hydrochloric acid. A large amount of solid was precipitated. The solid was collected by filtration to give a crude product, which was added to ethyl acetate/petroleum ether (V/V=1:1, 3 mL) and the mixture was stirred at room temperature for 10 minutes. The solid was collected by filtration, and dried under vacuum at 45° C. for 30 minutes to give the compound 1-2. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ: 3.06-3.00 (m, 2H), 2.59-2.52 (m, 2H), 1.81-1.68 (M, 4H); MS (ESI): m/z 260.9 [M+H].sup.+.

Step 2: Synthesis of Compound 1-3

[0084] Compound 1-2 (390 mg, 1.49 mmol) was dissolved in dichloromethane (2 mL), and then carbonyldiimidazole (290.60 mg, 1.79 mmol) was added. The resulting reaction solution was stirred at 25° C. for 1 hour. The reaction solution was then poured into aqueous ammonia (1.54 g, 7.47 mmol, 1.69 mL, 17% content), and then stirred vigorously for 20 minutes. The solvent was evaporated to give the crude product, and then purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~35%) to give the compound 1-3..sup.1H NMR: (400 MHz, CDCl.sub.3) δ: 5.56 (brs, 1H), 2.99-2.94 (m, 2H), 2.59-2.53 (m, 2H), 1.81-1.74 (m, 4H); MS (ESI): m/z 259.6 [M+H].sup.+.

Step 3: Synthesis of Compound 1-4

[0085] Compound 1-3 (271 mg, 1.04 mmol) was dissolved in N,N-dimethylformamide (1 mL), and the resulting solution was cooled to 0° C., and then cyanuric chloride (230.52 mg, 1.25 mmol) was added to afford the final reaction solution. The ice bath was removed, and the reaction solution was stirred at room temperature of 25° C. for 1 hour. The reaction solution was diluted with ethyl acetate (20 mL), and then washed with water (3 mL×3). The organic phase was dried with an appropriate amount of anhydrous sodium sulfate, then filtrated and the solvent was removed under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~3%) to give the compound 1-4.

Step 4: Synthesis of Compound 1-5

[0086] Compound 1-4 (82 mg, 338.65 .Math.mol), boronic acid 1-4A (106.67 mg, 507.98 .Math.mol) and potassium carbonate (93.61 mg, 677.31 .Math.mol) were dissolved in a mixed solvent of dioxane (2 mL) and water (0.4 mL), and then 1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl.sub.2) (24.78 mg, 33.87 .Math.mol) was added. After gas in the system was fully replaced by nitrogen, the reaction solution was placed in an oil bath at 110° C. and stirred for 18 hours. The solvent was evaporated to give the crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~20%) to give compound 1-5. MS (ESI): m/z 327.9 [M+H].sup.+.

Step 5: Synthesis of Compound 1-6

[0087] Compound 1-5 (40 mg, 122.18 .Math.mol) was dissolved in anhydrous dichloromethane (0.5 mL) and cooled to 0° C. with an ice bath, and then boron tribromide (61.22 mg, 244.35 .Math.mol, 23.54 .Math.L) was added under nitrogen. The obtained reaction solution was stirred at 25° C. for 2 hours. Water (0.5 mL) was added to quench the reaction in an ice-water bath, and then ethyl acetate (10 mL) was added and stirred to dissolution. The obtained solution was washed with water (2 mL×2), and the organic phase was evaporated to give the crude compound 1-6. The crude product was used directly in the next step.

Step 6: Synthesis of Compound 1

[0088] Compound 1-6 (37.83 mg, 120.71 .Math.mol) was dissolved in tetrahydrofuran (1 mL) and water (1 mL), and then lithium hydroxide monohydrate (15.20 mg, 362.12 .Math.mol) was added. The resulting reaction solution was stirred at 25° C. for 15 hours. The solvent was removed under reduced pressure, then the residue was adjusted to pH 2-3 with 1 M hydrochloric acid. The crude product was purified by preparative HPLC (chromatographic column: Venusil ASB Phenyl 150*30 mm*5 .Math.m; mobile phase: [Water (0.05%HCI)-ACN]; ACN%: 65%-95%, 9 min) to give Compound 1.

[0089] .sup.1H NMR: (400 MHz, MeOD-d.sub.4) δ: 7.96 (d, J= 8.0 Hz, 1H), 7.13-7.05 (m, 2H), 2.90 (t, J=6.4 Hz, 2H), 2.83 (t, J= 6.4 Hz, 2H), 1.93-1.77 (m, 4H). MS (ESI): m/z 299.9 [M+H].sup.+.

Example 2: Preparation of Compound 2

[0090] ##STR00062##

Step 1: Synthesis of Compound 2-2

[0091] Compound 2-1 (2.5 g, 10.15 mmol) was dissolved in methanol (10 mL), and water (10 mL) and sodium hydroxide (1.62 g, 40.61 mmol) were added. The resulting reaction solution was placed in an oil bath at 40° C. and stirred for 2 hours. The reaction solution was concentrated under reduced pressure to half volume, and water (5 mL) was added to the residue. 6 M hydrochloric acid was used to adjust pH=2~3 under stirring, and a large amount of white solid was precipitated. The solid was collected by filtration and dried under vacuum at 50° C. for 3 hours to give the compound 2-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.28 (s, 1H), 3.30 (t, J=7.0 Hz, 2H), 3.22 (t, J=14.3 Hz, 2H), 2.25 (tt, J=6.8, 13.4 Hz, 2H).

Step 2: Synthesis of Compound 2-3

[0092] Compound 2-2 (500 mg, 2.29 mmol) was dissolved in dichloromethane (5 mL), and then carbonyldiimidazole (445.83 mg, 2.75 mmol) was added. The resulting reaction solution was stirred under nitrogen for 1 hour, and then poured into vigorously stirred ammonia water (2.87 g, 22.91 mmol, 3.15 mL, content 28%) in tetrahydrofuran (5 mL). The reaction solution was stirred for 30 minutes, concentrated under reduced pressure at 25° C., and the residue was extracted with ethyl acetate (20 ml×3). The organic phases were combined, and rotary-evaporated to give a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~45%) to give the compound 2-3. .sup.1H NMR (400 MHz, CDCl.sub.3 ) δ: 7.10 (s, 1H), 5.58 (br s, 2H), 3.28 (t, J=6.9 Hz, 2H), 3.21 (t, J=14.4 Hz, 2H), 2.24 (tt, J=6.9, 13.4 Hz, 2H).

Step 3: Synthesis of Compound 2-4

[0093] Compound 2-3 (320 mg, 1.47 mmol) was dissolved in DMF (3 mL), and cooled to 0° C. Then cyanuric chloride (298.81 mg, 1.62 mmol) was added, and the reaction solution was stirred under nitrogen for 2 hours (a large amount of white solid was precipitated during this time). The reaction solution was diluted with ethyl acetate (50 mL), then washed with water (10 mL×3) and saturated brine (10 mL), dried with an appropriate amount of anhydrous sodium sulfate, filtrated and the solvent was removed under reduced pressure to give the crude compound 2-4, which was used directly in the next step. .sup.1H NMR: (400 MHz, CDCl.sub.3) δ: 7.25 (s, 1H), 3.21 (t, J=14.3 Hz, 2H), 3.09 (t, J=6.9 Hz, 2H), 2.28 (tt, J=6.8, 13.2 Hz, 2H).

Step 4: Synthesis of Compound 2-5

[0094] Compound 2-4 (290 mg, 1.46 mmol) was dissolved in acetic acid (2 mL), and then liquid bromine (348.94 mg, 2.18 mmol, 112.56 .Math.L) was added. The reaction solution was stirred at 25° C. for 15 hours. The reaction solution was removed under reduced pressure, and ethyl acetate (30 mL) was added to the residue. The mixture was then adjusted to pH 7-8 with saturated sodium carbonate. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (30 mL). The combined organic phases were concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~5%) to give the compound 2-5. .sup.1H NMR: (400 MHz, CDCL.sub.3) δ: 3.10-2.99 m, 4H), 2.32-2.19 (m, 2H).

Step 5: Synthesis of Compound 2-6

[0095] Compound 2-5 (140 mg, 503.39 .Math.mol), boronate 2-5A (178.39 mg, 553.73 .Math.mol), and potassium carbonate (139.14 mg, 1.01 mmol) were dissolved in dioxane (3 mL) and water (0.6 mL), and then 1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl.sub.2) (36.83 mg, 50.34 .Math.mol) was added. The reaction solution was heated to 105° C. under nitrogen and stirred for 15 hours. The reaction solution was removed under reduced pressure to give a crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~25%) to give the compound 2-6. .sup.1H NMR: (400 MHz, CHCl.sub.3) δ: 7.87 (d, J=8.0 Hz, 1H), 7.28 (d, J=1.6 Hz, 1H), 7.10 (dd, J=1.6, 8.0 Hz, 1H), 5.0 (s, 2H), 3.3 (s, 3H), 3.55(s, 3H), 3.23 (t, J=14.4 Hz, 2H), 3.13 (t, J=6.8 Hz, 2H), 2.39-2.24 (m, 2H).

Step 6: Synthesis of Compound 2-7

[0096] Compound 2-6 (105 mg, 266.90 .Math.mol) was dissolved in tetrahydrofuran (2 mL), and then aqueous lithium hydroxide monohydrate (2 M, 533.80 .Math.L) was added. The resulting reaction solution was stirred at 25° C. for 15 hours. Tetrahydrofuran was removed from the reaction solution under reduced pressure at 40° C., and the residue was adjusted to pH 2-3 with 2 M hydrochloric acid. A large amount of solid was precipitated. Ethyl acetate (50 mL) was added and the mixture was stirred. Ethyl acetate was separated out, and the mixture was rotary-evaporated to dryness to give the Compound 2-7. The crude product was used directly in the next step.

Step 7: Synthesis of Compound 2

[0097] Compound 2-7 (105 mg, 276.77 .Math.mol) was dissolved in methanol (1 mL), and then hydrochloric acid (60.55 mg, 1.66 mmol, 59.36 .Math.L) was added. The reaction solution became cloudy, and the reaction was stirred at 25° C. for 3 hours. The reaction solution was rotated-evaporated to dryness at 40° C., and the obtained residue was purified by preparative HPLC (chromatographic column: Venusil ASB Phenyl 150*30 mm*5 .Math.m; mobile phase: [water (0.05% HCl)-ACN]; ACN%: 60%-90%, 9 min) to give the compound 2.

[0098] .sup.1H NMR (400 MHz, MeOD-d.sub.4) δ: 8.00 (d, J=8.0 Hz, 1H), 7.13 - 7.04 (m, 2H), 3.35-3.32 (m, 2H), 3.12 (t, J=7.2 Hz, 2H), 2.45-2.30 (m, 2H); MS (ESI) m/z: 334.02 [M-H].sup.-.

Example 3: Preparation of Compound 3

[0099] ##STR00063##

Step 1: Synthesis of Compound 3-2

[0100] Compound 3-1 (15.01 g, 82.36 mmol) was dissolved in N,N-dimethylformamide (80 mL), and then N-bromosuccinimide (23.46 g, 131.78 mmol) was added. The mixture was stirred at 25° C. for 12 h. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (60 mL), washed with water (20 mL) and saturated brine (15 mL), and dried over anhydrous sodium sulfate, and filtrated, and the filtrate was evaporated under reduced pressure to remove solvent. Then the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~15%) to give the compound 3-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ : 3.76 (s, 3H), 2.91 (t, J=7.2 Hz, 2H), 2.56 - 2.50 (m, 2H), 2.38 - 2.30 (m, 2H). MS (ESI): m/z 260.8 [M+H] .sup.+.

Step 2: Synthesis of Compound 3-3

[0101] Compound 3-2 (1.98 g, 7.58 mmol) was dissolved in a mixed solution of methanol (10 mL) and water (10 mL), and then sodium hydroxide (606.54 mg, 15.16 mmol) was added. The reaction mixture was stirred at 50° C. for 1 hour. After the reaction was completed, the solvent was removed under reduced pressure. Water (20 mL) was added to the residue, followed by washing with ethyl acetate (10 mL). The aqueous phase was adjusted to pH 4~5 with hydrochloric acid, and a large amount of gray-yellow solid was precipitated. After filtration, the filter cake was washed with water (10 mL), and then dried in vacuum to give the compound 3-3. .sup.1H NMR (400 MHz, DMSO-d6) δ: 13.09 (brs, 1H), 2.90 (t, J=7.2 Hz, 2H), 2.58 - 2.53 (m, 2H), 2.39-2.33 (m, 2H); MS (ESI): m/z 246.8 [M+H] .sup.+.

Step 3: Synthesis of Compound 3-4

[0102] Compound 3-3 (1.57 g, 6.36 mmol) was dissolved in dichloromethane (10 mL), and carbonyldiimidazole (1.24 g, 7.63 mmol) was added. The reaction solution was stirred at 25° C. for 1.5 hours under nitrogen, and then poured into a stirred solution of aqueous ammonia (3 M, 21.19 mL) in tetrahydrofuran. The mixture was stirred for another 0.5 h. The solvent was removed under reduced pressure, and then ethyl acetate (20 mL) was added. The resulting mixture was washed with water (10 mL) and then saturated brine (5 mL), and the organic layer was dried over anhydrous sodium sulfate, filtrated and the filtrate was evaporated to remove solvent under reduced pressure to give a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~50%) to give the compound 3-4. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.60-6.90 (m, 2H), 2.93 (t, J=8.0 Hz, 2H), 2.57 - 2.55 (m, 2H), 2.39-2.29 (m, 2H). MS (ESI): m/z 247.9 [M+H] .sup.+.

Step 4: Synthesis of Compound 3-5

[0103] Compound 3-4 (550 mg, 2.23 mmol) was dissolved in N,N-dimethylformamide (6 mL), and cyanuric chloride (412.09 mg, 2.23 mmol) was added at 0° C. The reaction solution was warmed to 25° C., and stirred under nitrogen for 2 hours. A large amount of white solid was precipitated. The reaction solution was diluted with methyl tert-butyl ether (40 mL), then washed with water (10 mL) and saturated brine (5 mL). After the organic phase was dried over anhydrous sodium sulfate, filtrated and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~20%) to give the compound 3-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 2.82 (t, J=7.2 Hz, 2H), 2.59 - 2.52 (m, 2H), 2.44 - 2.35 (m, 2H).

Step 5: Synthesis of Compound 3-6

[0104] Compound 3-5 (150 mg, 657.58 .Math.mol), boronate 3-5A (233.03 mg, 723.34 .Math.mol) and 1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd (dppf)Cl.sub.2) (48.12 mg, 65.76 .Math.mol) were put into the reaction flask, followed by potassium carbonate (181.76 mg, 1.32 mmol). Then, a mixture of water (0.6 mL) and dioxane (3 mL) was added, and the reaction solution was placed in an oil bath at 105° C. under nitrogen for 12 hours. The solvent was removed under reduced pressure to give a crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0~20%) to give the compound 3-6. .sup.1H NMR (400 MHz,DMSO-d.sub.6) δ: 7.82 (d, J=8.0 Hz, 1H), 7.48 (s, 1H), 7.37-7.32 (m, 1H), 5.35 (s, 2H), 3.87 (s, 3H), 3.52 (s, 3H), 3.02 (t, J=8.0 Hz, 2H), 2.89 (t, J=8.0 Hz, 2H), 2.61-2.51 (m, 2H).

Step 6: Synthesis of Compound 3-7

[0105] Compound 3-6 (168 mg, 489.23 .Math.mol) was dissolved in tetrahydrofuran (5 mL), and then lithium hydroxide (2 M, 1.47 mL) was added. The reaction was stirred at 23° C. for 2 hours. The pH was adjusted to 4~5 with 2 M hydrochloric acid, and then tetrahydrofuran was removed under reduced pressure. Ethyl acetate (15 mL) was added to the residue, and the mixture was washed with water (5 mL) and saturated brine (5 mL). The organic phase was dried with an appropriate amount of anhydrous sodium sulfate, filtrated and the solvent was removed under reduced pressure to give the crude compound 3-7, which was used directly in the next step. MS (ESI): m/z 329.9 [M+H] .sup.+.

Step 7: Synthesis of Compound 3

[0106] Compound 3-7 (160 mg, 485.78 .Math.mol) was dissolved in methanol (2 mL), and hydrochloric acid (49.20 mg, 485.78 .Math.mol, 48.23 .Math.L, 36% pure) was added. After the reaction mixture was stirred at 23° C. for 3 hours, the solvent was removed under reduced pressure to give a crude product, which was purified by preparative HPLC (chromatographic column: Venusil ASB Phenyl 150*30 mm*5 .Math.m; mobile phase: [water (0.05%HCI)-ACN]; ACN%: 55%-85%, 9 min) to give compound 3. .sup.1H NMR (400 MHz, MeOD-d.sub.4) δ: 7.97 - 7.92 (m, 1H), 7.45-7.21 (m, 2H), 3.01 (t, J=7.2 Hz, 2H), 2.89 (t, J=7.2 Hz, 2H), 2.65-2.51 (m, 2H); MS (ESI): m/z 286.0 [M+H] .sup.+.

Example 4: Preparation of Compound 4

[0107] ##STR00064##

Step 1: Synthesis of Compound 4-2

[0108] Compound 4-1 (1 g, 7.03 mmol) was dissolved in tetrahydrofuran (20 mL), and n-butyllithium (2.5 M, 3.10 mL) was added dropwise at -78° C. under nitrogen. After the mixture was stirred at this temperature for 30 minutes, N,N-dimethylformamide (950.00 mg, 13.00 mmol, 1.00 mL) was added dropwise. Then the mixture was heated to 23° C. for 1 h. Hydrochloric acid was added to adjust the pH to 2~3, and a solid was precipitated in the solution. The precipitated solid was filtered, and the filter cake was washed with 5 mL of water, and then dried under vacuum to give the compound 4-2. .sup.1H NMR (400 MHz,CDCl.sub.3) δ: 9.94 (s, 1H) 6.82 (s, 1 H) 4.37 - 4.41 (m, 2 H) 4.28 - 4.31 (m, 2 H).

Step 2: Synthesis of Compound 4-3

[0109] Compound 4-2 (200 mg, 1.18 mmol) was dissolved in DMF (3 mL), and N-bromosuccinimide (250.99 mg, 1.41 mmol) was added, then the resulting reaction solution was stirred at 23° C. for 48 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was diluted with ethyl acetate (20 mL), and washed with water (3 mL*2) and saturated brine (2 mL), and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure again to give a crude product, which was purified by column chromatography (ethyl acetate/petroleum ether=0~20%) to give the compound 4-3. .sup.1H NMR (400 MHz,CDCl.sub.3) δ: 9.85 (s, 1 H), 4.45-4.30 (m, 4 H); LCMS m/z= 246.9 [M+H] .sup.+.

Step 3: Synthesis of Compound 4-4

[0110] Compound 4-3 (215 mg, 863.17 .Math.mol) was dissolved in ethanol (4 mL), and aqueous hydroxylamine solution (50%, 114.04 mg, 1.73 mmol) was added, then the reaction solution was refluxed at 90° C. for 2 hours. After the reaction was completed, the solvent was directly removed under reduced pressure. Acetonitrile (4 mL) was added, and the solvent was removed again to give the compound 4-4. MS (ESI): m/z 263.9 [M+H] .sup.+

Step 4: Synthesis of Compound 4-5

[0111] Compound 4-4 (130 mg, 492.24 .Math.mol) was dissolved in acetonitrile (5 mL), and thionyl chloride (234.25 mg, 1.97 mmol, 142.84 .Math.L) was added under nitrogen. The mixture was heated to reflux at 90° C. for 4 hours. The solvent was removed under reduced pressure and the residue was purified by column chromatography (ethyl acetate/petroleum ether=0~30%) to give the compound 4-5. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 4.32 - 4.41 (m, 4H); MS (ESI): m/z 247.0 [M+H] .sup.+.

Step 5: Synthesis of Compound 4

[0112] Compound 4-5 (110 mg, 447.01 .Math.mol), 4-5A (138.71 mg, 491.71 .Math.mol) and potassium carbonate (123.56 mg, 894.01 .Math.mol) were dissolved in dioxane (2 mL)/water (0.4 mL), and then Pd(dppf)Cl.sub.2 (32.71 mg, 44.70 .Math.mol) was added under nitrogen. The resulting reaction solution was heated to 105° C. for 15 hours. The reaction solution was concentrated, and trifluoroacetic acid (2 mL) was added. The reaction solution was stirred at room temperature for 1 hour and concentrated, and the residue was dissolved in dimethyl sulfoxide (3 mL) and purified by preparative HPLC (column: Agela ASB 150*25 mm*5 .Math.m; mobile phase: [water (0.05%HCI)-ACN]; Acetonitrile %: 46%-76%, 9 min) to give compound 4. .sup.1H NMR (400 MHz, DMSO_d.sub.6) δ: 7.86 (d, J= 8.4 Hz, 1H), 7.34-7.24 (m, 2H), 4.49 (d, J= 6.4 Hz, 4H). MS (ESI): m/z 302.0 [M-H].sup.-.

Example 5: Preparation of Compound 5

[0113] ##STR00065##

Step 1: Synthesis of Compound 5-2

[0114] Compound 5-1 (5 g, 21.09 mmol), 1,2-dibromoethane (31.70 g, 168.73 mmol, 12.73 mL) and potassium carbonate (11.66 g, 84.36 mmol) were dissolved in N,N-dimethylformamide (50 mL), and the mixture was heated to 85° C. for 4 hours. The reaction solution was concentrated, and 100 mL of ethyl acetate was added. The mixture was stirred for 10 minutes and filtrated, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (ethyl acetate/petroleum ether=0~2%) to give the compound 5-2. .sup.1H NMR (400 MHz,CDCl.sub.3) δ: 7.41 (s, 1H) 4.49 (t, J= 6.8 Hz, 2H), 3.89 (s, 3H), 3.70 (t, J= 6.8 Hz, 2H).

Step 2: Synthesis of Compound 5-3

[0115] Compound 5-2 (6.5 g, 18.89 mmol) was dissolved in tetrahydrofuran (60 mL), and the solution was stirred and cooled to -78° C. Then n-butyllithium (2.5 M, 7.56 mL) was added dropwise. Then the reaction mixture was stirred at this temperature for 2 hours. The reaction was quenched by saturated ammonium chloride solution (20 mL) and water (30 mL), and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (50 mL), and the combined organic phases were concentrated, the crude product was purified by column chromatography (ethyl acetate/petroleum ether=0~35%) to give the compound 5-3. .sup.1H NMR (400 MHz,CDCl.sub.3) δ: 6.95 (s, 1H), 5.10 (t, J= 8.4 Hz, 2H), 3.86 (s, 3H), 3.04 (t, J= 8.4 Hz, 2H).

Step 3: Synthesis of Compound 5-4

[0116] Compound 5-3 (620 mg, 3.37 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then N-bromosuccinimide (898.56 mg, 5.05 mmol) was added. The resulting reaction solution was stirred at 25° C. for 24 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (50 mL). The solution was washed with saturated sodium bisulfite (10 mL) and saturated brine (10 mL). The organic phase was concentrated, the crude product was purified by column chromatography (ethyl acetate/petroleum ether=0∼25 %) to give the compound 5-4. MS (ESI): m/z 262.9 [M+H].sup.+.

Step 4: Synthesis of Compound 5-5

[0117] Compound 5-4 (318 mg, 1.21 mmol) was dissolved in methanol (2 mL), and then sodium hydroxide solution (2 M, 1.21 mL) was added. The reaction solution was stirred at 45° C. for 2 hours. The reaction solution was concentrated, and water (2 mL) was added to the residue. The pH was adjusted to about 2-3 with 6 M hydrochloric acid, and a large amount of precipitation was precipitated. After stirring for 10 minutes, it was collected by filtration, and the filter cake was dried in vacuum at 45° C. for 2 hours to give the compound 5-5. MS (ESI): m/z 248.9 [M+H]+.

Step 5: Synthesis of Compound 5-6

[0118] Compound 5-5 (250 mg, 1.00 mmol) was dissolved in dichloromethane (3 mL), and then carbonyldiimidazole (244.12 mg, 1.51 mmol) was added. The resulting reaction solution was stirred under nitrogen for 1 hour. Then the reaction solution was poured into ammonia water (1.30 g, 10.04 mmol, 1.43 mL, 27% concentration) in tetrahydrofuran solution (5 mL), and the reaction was stirred for 30 minutes. The reaction solution was concentrated, and the residue was dissolved in ethyl acetate (50 mL). The solution was washed with water (10 mL) and saturated brine (10 mL). The organic phase was dried with an appropriate amount of anhydrous sodium sulfate, and filtrated, and the filtrate was concentrated under reduced pressure to give the crude compound 5-6, which was used directly in the next step. MS (ESI): m/z 249.9 [M+H].sup.+.

Step 6: Synthesis of Compound 5-7

[0119] Compound 5-6 (230 mg, 927.06 .Math.mol) was dissolved in N,N-dimethylformamide (3 mL), and then cyanuric chloride (256.44 mg, 1.39 mmol) was added. The resulting reaction solution was stirred at 25° C. for 2 hours. The reaction solution was diluted with ethyl acetate (80 mL), and then washed with water (20 mL) and saturated brine (20 mL). The organic phase was dried with an appropriate amount of anhydrous sodium sulfate, and filtrated, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (ethyl acetate/petroleum ether=0~15%) to give the compound 5-7. MS (ESI): m/z 252.2 [M+Na] .sup.+.

Step 7: Synthesis of Compound 5-8

[0120] Compound 5-7 (130 mg, 565.02 .Math.mol), 5-7A (308.70 mg, 847.53 .Math.mol) and potassium carbonate (195.22 mg, 1.41 mmol) were dissolved in dioxane (1.5 mL) and water (0.3 mL), and then Pd(dppf)Cl.sub.2 (82.69 mg, 113.00 .Math.mol) was added under nitrogen. The resulting reaction solution was heated to 110° C. for 15 hours. The reaction solution was concentrated, and the residue was purified by column chromatography (ethyl acetate/petroleum ether=0~25%) to give the compound 5-8. MS (ESI): m/z 388.1 [M+H] .sup.+.

Step 8: Synthesis of Compound 5

[0121] Compound 5-8 (140 mg, 361.34 .Math.mol) was dissolved in dichloromethane (0.5 mL), and then trifluoroacetic acid (412.01 mg, 3.61 mmol, 267.54 .Math.L) was added. The resulting reaction solution was stirred at 25° C. for 2 hours. The reaction solution was concentrated and the residue was dissolved in N,N-dimethylformamide (5 mL). The solution was purified by preparation HPLC (chromatographic column: Venusil ASB Phenyl 150*30 mm*5 .Math.m; mobile phase: [water ( 0.05% HCl)-ACN]; acetonitrile %: 45%-75%, 9 min) to give the compound 5. .sup.1H NMR (400 MHz, CD.sub.3OD) δ: 7.96 (d, J=8.8 Hz, 1H), 7.20 - 7.05 (m, 2H), 5.20 (t, J=8.0 Hz, 2H), 3.39 (t, J=8.0 Hz, 2H). MS (ESI): m/z 286.0 [M-1].sup.-.

Biological Test Data

Assay Example 1: Xanthine Oxidase Inhibitory Activity Test

1.1 Purpose of the Assay

[0122] Compounds were evaluated for the level of inhibiting xanthine oxidase activity.

1.2 Reagent

[0123] The main reagents used in this study included xanthine (Sigma, Cat. No. X4002-1G, Lot: SLBB5664V) and xanthine oxidase (Sigma, Cat. No. X4376-5UN, Lot: SLBQ1518V).

1.3 Instrument

[0124] The main instrument used in this study is a multi-function microplate reader.

1.4 Method of the Assay

[0125] 1) 50 .Math.L of Dulbecco’s Phosphate Buffered Saline (DPBS) was added to background control wells of compounds and positive control wells (HPE: 100% inhibitory activity).

[0126] 2) 2 U/mL of xanthine oxidase was diluted with DPBS to 0.04 U/mL, and 50 .Math.L xanthine oxidase was added to the compound wells and negative control wells (ZPE: 0% inhibitory activity) according to the assay layout in Figure 1.

[0127] 3) The compounds were serially 3-fold diluted with DMSO for 8 points, then the compound was diluted with DPBS, and 50 .Math.L of the mix was added to each well in triple wells. 50 .Math.L of DPBS was added to each well of positive control wells (HPE: 100% inhibitory activity) and negative control wells (ZPE: 0% inhibitory activity).

[0128] 4) 200 mM xanthine was diluted to 300 .Math.M with DPBS. 100 .Math.L of xanthine was added to each well according to the assay layout in Figure 1. The mixture was pre-treated at room temperature for 30 minutes. The final concentration of xanthine oxidase in each well was 0.01 U/mL, and the final concentration of DMSO in each well was 0.5%. Positive control wells (HPE: 100% inhibitory activity) contained xanthine but no xanthine oxidase, and negative control wells (ZPE: 0% inhibitory activity) contained xanthine and xanthine oxidase. Compound background control wells contained various concentrations of the compound and xanthine but without xanthine oxidase.

[0129] 5) Detecting the absorbance value at 290 nm with a spectrophotometer.

[0130] 6) Data analysis: calculating the inhibition of xanthine oxidase in each well according to the following equation:

[00001]$\text{Inhibition}\%=\left(1-\frac{{\text{OD}}_{\text{test}\text{sample}}-{\text{OD}}_{\text{compound}\text{control}}}{{\text{OD}}_{\text{ZPE}}-{\text{OD}}_{\text{HPE}}}\right)\ast 100\%$

[0131] * OD.sub.test.sub.sample is the optical density value of the compound activity test well, containing the compound, xanthine and xanthine oxidase; [0132] OD.sub.compound.sub.control is the background optical density value of the compound to be tested at different concentrations, containing the compound and xanthine, without xanthine oxidase; [0133] OD.sub.ZPE: the mean value of optical density of the negative control wells, containing 0.5% DMSO, xanthine and xanthine oxidase; [0134] OD.sub.HPE is the mean value of optical density of the positive control wells, containing 0.5% DMSO and xanthine, without xanthine oxidase.

[0135] 7) GraphPad Prism software was used to perform log(agonist) vs. response -- Variable slope nonlinear fitting analysis on the inhibition data (inhibition %) of the compound to give the IC.sub.50 value of the compound. The fitting formula: Y=Bottom + (Top-Bottom)/(1+10^((LogIC.sub.50-X)*HillSlope))

1.5 Assay Results

[0136] TABLE-US-00001 Results of xanthine oxidase inhibitory activity test of the compounds Compound Number XO IC.sub.50 (nM) Compound 1 25.0 Compound 2 20.7 Compound 3 25.9 Compound 4 22.8 Compound 5 24.0

[0137] The assay results show that the compounds have good xanthine oxidase inhibitory activity.

Assay Example 2: Inhibitory Activity Test of the Compounds on Uric Acid Uptake

1. Purpose of the Assay

[0138] In this study, human Urat1 gene stably transfected cell lines were used to evaluate the inhibitory activity of the test compounds on uric acid uptake.

2. Materials of the Assay

2.1 Cell Lines

[0139] The human Urat1 gene stably transfected cell line was constructed by WuXi AppTec. Human Urat1 gene stably transfected cell line (Urat1-MDCK) is MDCK cells transfected with human Urat1 gene and obtained by G418 screening. Urat1-MDCK cells were cultured in MEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 .Math.g/ml streptomycin, 2 mM L-glutamine and 1% non-essential amino acids, and 250 .Math.g/ml G418.

2.2 Reagent

[0140] The main reagent used in this study included .sup.14C-uric acid (ARC, Cat. No. ARC-0513, Lot No. 200122).

2.3 Instrument

[0141] The main instrument used in this study was a liquid scintillation analyzer (Perkin Elmer, Tri-Carb 4910TR).

3. Method of the Assay

3.1 Cell Plating

[0142] 3.1.1 The Urat1-MDCK cells cultured in the T150 cell culture flask were digested with 0.25% trypsin, then adjusted to a suspension with 200,000 cells/ml with fresh culture medium.

[0143] 3.1.2 Cells were seeded into a 48-well cell culture plate at 0.5 ml per well at a final cell density of 100,000 cells/well.

[0144] 3.1.3 The cell culture plate was incubated in a 37° C., 5% CO.sub.2 incubator overnight.

[0145] 3.2 Treatment with the compounds and detection

[0146] 3.2.1 A serially 5-fold dilution of the compounds in DMSO was made for 4 points, and the diluted concentration was 200 times the final assay concentration. Compounds were then diluted 10-fold in HBSS buffer.

[0147] 3.2.2 10 mM concentrated stock solution of .sup.14C-uric acid was diluted to 1 mM with HBSS buffer.

[0148] 3.2.3 After the cell culture plate was incubated overnight, the cell culture medium was removed from the plate, and the cells were washed three times with HBSS buffer followed by adding 90 .Math.l of HBSS buffer to each well.

[0149] 3.2.4 5 .Math.l of diluted compound was added to each well, and the cells were incubated in a 37° C., 5% CO.sub.2 incubator for 20 minutes. The final concentration of DMSO in each well was 0.5%. The test compound (10 .Math.M) was used as a 100% inhibition control, and 0.5% DMSO was used as a 0% inhibition control.

[0150] 3.2.5 5 .Math.l per well diluted .sup.14C-uric acid was added to the cell plate, and the final concentration of uric acid in each well was 50 .Math.M. The cells were incubated in a 37° C., 5% CO.sub.2 incubator for 15 minutes. Cells were then washed 3 times with pre-chilled HBSS buffer.

[0151] 3.2.6 150 .Math.l 0.1 M NaOH was added to each well to lyse cells for 10 minutes.

[0152] 3.2.7 The cell lysate was collected into liquid scintillation detection vial, and 2 ml of scintillation fluid was added to each vial for testing.

[0153] 3.2.8 The .sup.14C content of sample in each tube was detected with a liquid scintillation analyzer.

[0154] 3.2.9 Data analysis:

[00002]$\text{Inhibition}\%=\left(HC-CPD\right)/\left(HC-LC\right)\times 100\%\text{*}$

[0155] * CPD is the radioactive signal value of the compound well; [0156] HC is the mean value of the radioactive signal from the 0% inhibition control wells; [0157] LC is the mean value of radioactive signal from the 100% inhibition control wells.

[0158] 3.2.10 Using GraphPad Prism software, the nonlinear regression log(inhibitor) vs. response -- Variable slope method was adopted to fit the dose-response curve according to the following formula, and the IC.sub.50 and IC.sub.90 values of the compounds were obtained.

[0159] Y=Bottom + (Top-Bottom)/(1+10^((LogIC.sub.50-X)*HillSlope))

4. Assay Results

[0160] TABLE-US-00002 Inhibitory activity of the compounds on uric acid uptake Compound Number IC.sub.50 (.Math. M) Compound 2 2.24 Compound 3 4.05 Compound 4 36.88 Compound 5 45.45

[0161] Conclusion: Assay results show that the compounds of the present disclosure have a good inhibitory activity on uric acid uptake

Assay Example 3: Hepatocytes Metabolic Stability (HMS) Study

1. Purpose of the Assay

[0162] To test the metabolic stability of the test sample in human and rat hepatocytes.

2. Materials of the Assay

[0163] 2.1 Test compound (10 mM), control substance: 7-Ethoxycoumarin (30 mM), 7-Hydroxycoumarin (control substance, 30 mM)

TABLE-US-00003 Cell Hepatocyte cell viability Supplier Cat No. Rat hepatocytes 85% BioreclamationIVTM00005 Human hepatocytes 84% Bioreclamation IVTX008001

2.3 Buffer System:

[0164] Thawing medium: Williams’ medium E containing 5% fetal bovine serum, 30% Percoll solution and other auxiliary materials [0165] Incubation medium: Williams medium E (without phenol red) containing 2 mM L-glutamine and 25 mM hydroxyethylpiperazine ethane sulfonic acid. [0166] Stop solution: 200 ng/mL of tosylbutyramide and labetalol in acetonitrile as internal standards. [0167] Dilution solution: Ultrapure water.

3. Method of the Assay

[0168] 1) The exact amount of positive control compound was dissolved in dimethyl sulfoxide (DMSO) to give a 30 mM solution

[0169] 2) 10 mM test compound and 30 mM positive control compound were diluted to 1 mM and 3 mM with DMSO in 96-well plates.

[0170] 3) 1 mM test compound and 3 mM positive control compound was diluted into 100 .Math.M and 300 .Math.M quantification solutions with acetonitrile.

[0171] 4) The cryopreserved cells were thawed, dissociated and suspended in culture medium, and then diluted to 0.5 × 10.sup.6 cells/mL with pre-warmed culture medium.

[0172] 5) 198 .Math.L of pre-warmed cell suspension was added to a 96-well plate.

[0173] 6) 100 .Math.L of stop solution (acetonitrile containing 200 ng/mL tosylbutyramide and 200 ng/mL labetalol as internal standard) was transferred in a set of pre-labeled 96-well plates.

[0174] 7) 2 .Math.L of 100 .Math.M test compound or 300 .Math.M positive control quantitation solution was added in duplicate to each well of the 96-well plate.

[0175] 8) The T0 samples were mixed to achieve uniform suspension over about 1 min, and then 20 .Math.L of each sample was immediately transferred into wells containing 100 .Math.L of ice-cold stop solution followed by mixing.

[0176] 9) All plates were incubated at 37° C. in 5% CO.sub.2 in a 95% humidified incubator, with constant shaking at approximately 600 rpm for reaction.

[0177] 10) At 15, 30, 60 and 90 min, samples were mixed, and then 20 .Math.L of each sample was transferred to wells containing 100 .Math.L of ice-cold stop solution at each time point and mixed.

[0178] 11) Medium control (MC) sample plates (labeled as T0-MC and T90-MC) were prepared at T0 and T90 by adding the same components to each well except for the cell suspension. Final concentration table was obtained.

[0179] 12) At each respective time point, the reaction was stopped by removing the plate from the incubator and mixing with 100 .Math.L of ice-cold stop solution.

[0180] 13) Immediately vortexing the plate on a plate shaker at 500 rpm for 10 minutes. Then, all sample plates were centrifuged at 3220 x g at 4° C. for 20 min.

[0181] 14) After centrifugation, 35 .Math.L/well of the supernatant from the sample plate was transferred to another set of pre-labeled 96-well plates containing 70 .Math.L of ultrapure water according to the plate diagram.

[0182] 15) The assay plates were sealed and stored at 4° C. until LC-MS-MS analysis.

[0183] The residual ratio of the test compound and the control compound was obtained by the following formula:

[00003]$\text{Residual}\text{ratio}\left(\%\right)=\frac{\begin{array}{c}\text{Peak}\text{area}\text{ratio}\text{of}\text{compound}\text{to}\text{internal}\\ \text{standard}\text{at}\text{any}\text{time}\text{point}\end{array}}{\begin{array}{c}\text{Peak}\text{area}\text{ratio}\text{of}\text{compound}\text{to}\text{internal}\\ \text{standard}\text{at}0\text{minutes}\end{array}}\times 100\%$

[0184] The elimination rate constant k of the test compound and the control compound in hepatocytes was calculated by plotting the logarithm of the residual ratio vs time, and the half-life (T.sub.½) and in vitro intrinsic clearance rate (CL.sub.int) were obtained by using the elimination rate k. The formula as follows: [0185] Tl.sub./2 = 0.693 / k [0186] CL.sub.int.sub.(hep) = k / Cells per ml (million cells/mL) [0187] CL.sub.int.sub.(liver) = CL.sub.int.sub.(hep) × Liver weight to weight ratio × number of hepatocytes per gram of liver

[0188] The parameters of the species in the formula were as follows:

TABLE-US-00004 Species Liver weight to weight ratio (g/kg) Hepatic blood flow (Q.sub.h) (mL/min/kg) Number of hepatocytes (cells per gram of liver) Mouse 88 90.0 135×10.sup.6 Rat 40 55.2 117×10.sup.6 Dog 32 30.9 215×10.sup.6 Monkey 30 43.6 120×10.sup.6 Human 20 20.7 139×10.sup.6

4. Assay Results

[0189] The results are shown in Table 3.

TABLE-US-00005 Intrinsic clearance rates of the compounds in human and rat liver Compound Intrinsic hepatic clearance (mL/min/Kg) Number Human Rat Compound 2 31.8 188.4 Compound 3 48.2 1097.9

[0190] Conclusion: Compound 2 and compound 3 are both at moderate clearance in human hepatocytes and at high clearance in rat hepatocytes.

Assay Example 4. Membrane Permeability MDR1 Test

1. Purpose of the Assay:

[0191] MDR1-MDCK II cells are Madin-Darby canine kidney cells transfected with human MDR1 gene, which can stably express high P-gp. The aim of this study was to test the bidirectional permeability of compounds across the MDR1-MDCK II cell model and to assess whether they are transported by efflux.

2. Cell Culture:

[0192] MDR1-MDCK II cells (obtained from Piet Borst, Netherlands Cancer Institute) were seeded at a density of 2.5 × 10.sup.5 cells/mL onto polyethylene membrane (PET) in a 96-well insert for 4-7 days until forming fusion cell monolayer.

3. Method of the Assay

[0193] Test compounds were diluted in transport buffer (HBSS, 10 mM Hepes with DMSO, pH 7.4) to a concentration of 2 .Math.M (DMSO <1%) and applied on the apical or basolateral side of the cell monolayer. The compound to be tested was tested in duplicate in the direction of from A to B or from B to A, digoxin was tested at 10 .Math.M in the direction of from A to B or from B to A, while nadolol and metoprolol were tested at 2 .Math.M from A to B. Plates were incubated in a CO.sub.2 incubator at 37±1° C. in a saturated humidity of 5% CO.sub.2 for 2.5 hours without shaking. In addition, the efflux ratio of each compound was determined, and for the test and reference compounds quantitation was conducted. Analysis was carried out by LC/MS/MS according to the peak area ratio of analyte/IS. After the transport assay, the Lucifer Yellow exclusion assay was used to determine integrity of the cell monolayer. The buffer was removed from the apical and basolateral compartments, then 75 .Math.L of 100 .Math.M luciferin yellow was added to the transport buffer, and 250 .Math.L of the transport buffer was added to the apical and basolateral compartments, respectively. The plate was incubated at 37° C., 5% CO.sub.2 and saturated humidity for 30 minutes without shaking. After 30 minutes of incubation, 20 .Math.L of luciferin yellow sample was withdrawn from the apical, followed by the addition of 60 .Math.L of transport buffer. An 80 .Math.L luciferin yellow sample was then collected basolaterally. Relative fluorescence units (RFU) of luciferin yellow were measured at 425/528 nm (excitation/emission) with an Envision microplate reader.

4. Data Calculation

[0194] The apparent permeability coefficient (P.sub.app, cm/s), efflux rate and recovery rate were calculated using the following formulae.

[0195] The apparent permeability coefficient (P.sub.app, cm/s) was calculated using the following formula:

[00004]${\text{P}}_{\text{app}}=\left({\text{dC}}_{\text{r}}/{\text{d}}_{\text{t}}\right)\times {\text{V}}_{\text{r}}/\left(\text{A}\times {\text{C}}_0\right)$

dC.sub.r/d.sub.t is the cumulative concentration of the compound at the receiver end over unit time (.Math.M/s); V.sub.r is the volume of the receiver end solution (0.075 mL and 0.250 mL for the apical and basal ends, respectively); Ais the relative surface area (0.0804 cm.sup.2) of the cell monolayer; C.sub.0 is the initial concentration (nM) of the test substance at the administration end or the peak area ratio of the reference substance.

[0196] The efflux ratio was calculated using the following formula:

[00005]$\text{Efflux}\text{ratio}={\text{P}}_{\text{app}}\left(\text{BA}\right)/{\text{P}}_{\text{app}}\left(\text{AB}\right)$

[0197] The recovery rate was calculated using the following formula:

[00006]$\%\text{Recovery}=100\times \left[\left({\text{V}}_{\text{r}}\times {\text{C}}_{\text{r}}\right)+\left({\text{V}}_{\text{d}}\times {\text{C}}_{\text{d}}\right)\right]/\left({\text{V}}_{\text{d}}\times {\text{C}}_0\right)$

C.sub.0 is the initial concentration (nM) of the test substance at the administration end or the peak area ratio of the reference substance; V.sub.d is the volume of the administration end (0.075 mL for the apical side and 0.250 mL for the basal side); C.sub.d and C.sub.r are the final concentration (nM) of the test substance at the administration end and the receiver end or the peak area ratio of the reference substance.

[0198] The percentage of Lucifer Yellow in the basolateral pores was calculated using the following formula:

[00007]$\begin{array}{l}\%LuciferYellow=\\ \frac{V_{Basolateral}\times RF{U}_{Basolateral}}{V_{Apical}\times RF{U}_{Apical}+V_{Basolateral}\times RF{U}_{Basolateral}}\times 100\end{array}$

[0199] wherein RFUApical and RFUBasolateral are the relative fluorescence unit values of Lucifer Yellow in the apical and basolateral pores, respectively; VApical and VBasolateral are the volumes of the apical and basolateral pores (0.075 mL and 0.25 mL), respectively. % Lucifer Yellow should be less than 2.

5. Assay Results

[0200] The results are shown in Table 4.

TABLE-US-00006 Data of membrane permeability of the compounds to MDR1 cell Compound Number P.sub.app (AB) (10.sup.-6 cm/s) P.sub.app (BA) (10.sup.-6 cm/s) Efflux ratio Compound 2 26.42 6.63 0.25 Compound 3 38.63 10.99 0.28

[0201] Conclusion: Compound 2 and compound 3 both have high permeability.

Assay Example 5. Cytochrome P450 Isozyme Inhibitory Activity Test

1. Purpose of the Assay

[0202] The inhibitory activities of test compounds against different subtypes of human cytochrome P450 isoenzymes were determined.

2. Method of the Assay

[0203] Preparing test compounds, standard inhibitor (100× final concentration) and mixed substrate working solutions; Microsomes (purchased from Coming Inc) frozen in -80° C. freezer was taken out and thawed. 20 .Math.L of the test compound and standard inhibitor solution were added to the corresponding wells, and at the same time 20 .Math.L of the corresponding solvent was added to the no inhibitor control wells (NIC) and blank control wells (Blank); then 20 .Math.L of mixed substrate solution was added to the corresponding wells, except for Blank wells (adding 20 .Math.L of Phosphate Buffer (PB) to Blank wells); the human liver microsome solution was prepared (marking the date after use and putting back in the refrigerator immediately), then 158 .Math.L of human liver microsome solution was immediately added to all wells; the above sample plate was pre-incubated in a 37° C. water bath, and then coenzyme factor (NADPH) solution was prepared; 10 minutes later, 20 .Math.L of NADPH solution was added to all wells. After the sample plate was shaken well, it was incubated in a 37° C. water bath for 10 minutes; at the corresponding time point, 400 .Math.L of cold acetonitrile solution (internal standard: 200 ng/mL tolbutamide and labetalol) was added to stop the reaction; After the sample plate was mixed well, it was centrifuged at 4000 rpm for 20 minutes to precipitate proteins; 200 .Math.L of supernatant was taken out and added to 100 .Math.L of water, the mix was shaked well, and then subjected to LC/MS/MS for detection.

3. Assay Results

[0204] The results are shown in Table 5.

TABLE-US-00007 IC.sub.50 values of compounds for P450 isoenzyme inhibition Compound Number Cytochrome P450 isoenzyme IC.sub.50 (.Math.M) CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4-M Compound 2 >50 18.1 >50 >50 >50 Compound 3 >50 14.8 >50 >50 >50

[0205] Conclusion: The tested compounds have very low inhibitory activity on CYP1A2, CYP2C19, CYP2D6 and CYP3A4-M, and have moderate inhibitory activity on CYP2C9.

Assay Example 6: Pharmacokinetics in SD Rats

1. Purpose of the Assay:

[0206] Pharmacokinetics of test compounds in SD rats

2. Materials of the Assay:

[0207] Sprague Dawley Rat (male, 180-350 g, 6-10 weeks old, Charles River Laboratories, Beijing)

3. Method of the Assay:

[0208] Compound 2 was mixed with 5% DMSO/10% Solutol/85% water, and the mixture was stirred and vortexed to prepare a clear solution at 0.6 mg/mL for the administration of the injection group, and was filtered by a microporous membrane for use. Compound 2 was mixed with 5% DMSO/10% Solutol/85% water, and the mixture was stirred and vortexed to prepare a clear solution at 1 mg/mL for oral administration. Six male SD rats were divided into 2 groups. Animals in group 1 were administered intravenously in a single dose of 3 mg/kg, the vehicle was 5% DMSO/10% Solutol/85% water, and the administration volume was 5 mL/kg. Animals in the second group were administered with the test compound 2 by oral gavage in a single dose of 10 mg/kg, the oral vehicle was 5% DMSO/10% Solutol/85% water, and the oral volume was 10 mL/kg. Whole blood was collected at 0 (the gavage group only), 0.083 (intravenous only), 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration. Whole blood was centrifuged at 3200 g at 4° C. for 10 min to obtain plasma. The concentrations of Compound 2 and uric acid (the oral gavage group only) in plasma were determined by LC/MS/MS method, and the pharmacokinetic parameters, such as peak concentration, Time to peak, clearance rate, half-life, area under the curve, bioavailability, etc were calculated by Phoenix WinNonlin software.

[0209] Assay results are as follows in Table 6:

TABLE-US-00008 Pharmacokinetic data of compound 2 in rats 2.89 mpk Intravenous injection C.sub.0 (.Math.M) T.sub.½ (hr) Vd.sub.ss (L/kg) Cl (mL/min/kg) AUC.sub.0-inf (.Math.M.hr) Compound 2 96.6 3.74 0.32 3.59 41.8 8.58 mpk Oral C.sub.max (.Math.M) T.sub.max (hr) T.sub.½ (hr) AUC.sub.0-inf (.Math.M.hr) Bioavailability (%) Compound 2 27.5 0.5 2.47 86.6 62.1

[0210] Conclusion: Compound 2 has good pharmacokinetic properties and high oral bioavailability. wherein, C.sub.0 is the initial concentration, T.sub.½ is the elimination half-life, Vd.sub.ss is the steady-state apparent volume of distribution, Cl is the total clearance, and AUC.sub.0-last is the area under the plasma concentration-time curve from time 0 to the last quantifiable time point, AUC.sub.0-inf is the area under the plasma concentration-time curve from time 0 to extrapolated infinity, C.sub.max is the peak concentration, and T.sub.max is the time to peak.