SPIRO COMPOUND SERVING AS ERK INHIBITOR, AND APPLICATION THEREOF

Abstract

A Spiro compound serving as an ERK inhibitor, and an application thereof in preparing a drug for treating an ERK-related disease. The present invention specifically relates to a compound represented by formula (III) or a pharmaceutically acceptable salt thereof.

##STR00001##

Claims

1. A compound of formula (III) or a pharmaceutically acceptable salt thereof, ##STR00038## wherein n is 0 or 1; m is 1 or 2; ring A is ##STR00039## T.sub.1, T.sub.2 and T.sub.3 are each independently selected from N and CH; E.sub.1 is O, S or NH; R.sub.1 is selected from H and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R.sub.a; R.sub.2 and R.sub.3 are each independently selected from H, F, Cl, Br, I, OH, CN, NH.sub.2 and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R.sub.b; R.sub.4 is H; R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are each independently selected from H, F, Cl, Br, I, OH, CN, NH.sub.2 and C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R.sub.c; and R.sub.a, R.sub.b and R.sub.c are each independently selected from F, Cl, Br, I, OH, CN and NH.sub.2.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is selected from H and CH.sub.3, wherein the CH.sub.3 is optionally substituted by 1, 2 or 3 R.sub.a.

3. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein R.sub.1 is CH.sub.3.

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

5. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R.sub.2 and R.sub.3 are each independently selected from H and CH.sub.3.

6. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are each independently selected from H, F, Cl, Br, I, OH, CN, NH.sub.2, CH.sub.3 and —CH.sub.2—CH.sub.3, wherein the CH.sub.3 and —CH.sub.2—CH.sub.3 are optionally substituted by 1, 2 or 3 R.

7. The compound or pharmaceutically acceptable salt thereof according to claim 6, wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are each independently selected from H, F, Cl, Br, I, OH, CN and NH.sub.2.

8. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the structural moiety ##STR00040##

9. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is ##STR00041##

10. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is ##STR00042## wherein m, n, E.sub.1, T.sub.1, T.sub.2 and T.sub.3 are as defined in claim 1; R.sub.1 is as defined in claim 1; R.sub.2 and R.sub.3 are as defined in claim 1; R.sub.4 is as defined in claim 1; and R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are as defined in claim 1.

11. The compound or pharmaceutically acceptable salt thereof according to claim 10, wherein the compound is ##STR00043## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, E.sub.1, T.sub.1, T.sub.2 and T.sub.3 are as defined in claim 10.

12. The compound or pharmaceutically acceptable salt thereof according to claim 10, wherein the compound is ##STR00044## wherein R.sub.11, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, T.sub.1, T.sub.2 and T.sub.3 are as defined in claim 10.

13. The compound or pharmaceutically acceptable salt thereof according to claim 12, wherein the compound is ##STR00045## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are as defined in claim 12.

14. A compound represented by the following formula or a pharmaceutically acceptable salt thereof, ##STR00046## ##STR00047##

15. The compound or pharmaceutically acceptable salt thereof according to claim 14, wherein the compound is ##STR00048##

16. A method of treating a disease related to ERK in a subject, comprising administering to a subject in need thereof Use of the compound or pharmaceutically acceptable salt thereof according to claim 1.

17. A method of treating a disease related to ERK in a subject, comprising administering to a subject in need thereof the compound or pharmaceutically acceptable salt thereof according to claim 14.

18. A method of treating a disease related to ERK in a subject, comprising administering to a subject in need thereof the compound or pharmaceutically acceptable salt thereof according to claim 15.

19. A medicament, comprising the compound or pharmaceutically acceptable salt thereof according to claim 1.

20. A medicament, comprising the compound or pharmaceutically acceptable salt thereof according to claim 14.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1: Tumor growth curve of human colon cancer HCT.sub.116 in model animal after administration of solvent and WX007 respectively;

[0096] FIG. 2: Rate of weight change (%) in model animal of human colon cancer HCT.sub.116 during the administration.

DETAILED DESCRIPTION OF THE INVENTION

[0097] 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 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.

Reference Example 1: Fragment A-1

[0098] ##STR00021##

[0099] Step 1: synthesis of compound A-1-2

[0100] To a pre-dried single-necked flask was added a solution of sodium acetate (4.64 g, 56.60 mmol, 5 eq), potassium monopersulfate (13.92 g, 22.64 mmol, 2 eq) and water (47 mL). The mixture was cooled to 0° C. A solution of A-1-1 (4.7 g, 11.32 mmol, 1 eq), solvent tetrahydrofuran (47 mL) and methanol (47 mL) was added dropwise and the mixture was stirred at 0° C. for 1 hour. Then the mixture was stirred in an oil bath at 29° C. for 15 hours. After completion of the reaction, the reaction solution was poured into water (200 mL), and the aqueous phase was extracted with ethyl acetate (50 mL×3). The organic phases were combined, and the combined organic phase was sequentially washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was collected and concentrated under reduced pressure to give a residue. The residue was purified by flash column chromatography to give A-1-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 8.67 (d, J=4.9 Hz, 1H), 7.64 (d, J=4.9 Hz, 1H), 3.37 (s, 3H), 1.63−1.53 (m, 6H), 1.39−1.30 (m, 6H), 1.26−1.12 (m, 6H), 0.90 (t, J=7.3 Hz, 9H).

[0101] Step 2: synthesis of compound A-1.

[0102] To a reaction flask were added A-1-2 (3.9 g, 8.72 mmol, 1 eq), A-1-3 (1.02 g, 10.46 mmol, 1.2 eq) and tetrahydrofuran (117 mL). The atmosphere was replaced with nitrogen gas, and then lithium hexamethyldisilazide (1 M, 18.31 mL, 2.1 eq) was added dropwise at −35° C. The mixture solution was reacted at −35° C. for 10 minutes. After completion of the reaction, the reaction solution was quenched with saturated aqueous ammonium chloride solution (100 mL), and extracted with ethyl acetate (100 mL×2) and dichloromethane (100 mL). The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness to give a crude product. The crude product was purified by column chromatography to give A-1. .sup.1H NMR (400 MHz, CDCl.sub.3) δ ppm 8.17 (d, J=4.85 Hz, 1 H), 7.46 (d, J=1.76 Hz, 1 H), 6.91 (d, J=4.63 Hz, 1 H), 6.60 (s, 1 H), 6.32 (d, J=1.98 Hz, 1 H), 3.79 (s, 3 H), 1.52-1.61 (m, 6 H), 1.28-1.40 (m, 6 H), 1.03-1.20 (m, 6 H), 0.89 (t, J=7.28 Hz, 9 H).

Example 1

[0103] ##STR00022##

[0104] Route of synthesis:

##STR00023## ##STR00024##

[0105] Step 1: synthesis of WX001-3

[0106] To a reaction flask were added WX001-1 (5 g, 24.03 mmol, 1 eq) and tetrahydrofuran (200 mL). The atmosphere was replaced with nitrogen gas, and the mixture was cooled to −78° C., and lithium diisopropylamide (2 mol/L, 28.84 mL, 2.4 eq) and tetramethylethylenediamine (4.19 g, 36.05 mmol, 5.44 mL, 1.5 eq) were slowly added dropwise. The mixture was stirred at −78° C. for 0.5 hours, and WX001-2 (3.10 g, 36.05 mmol, 1.5 eq) was then added. The mixture was reacted at −78° C. for 2 hours. After completion of the reaction, the reaction solution was quenched with saturated aqueous ammonium chloride solution (250 mL), adjusted to a pH of 2-3 with 2 mol/L hydrochloric acid, and extracted with ethyl acetate (100 mL*3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump at 45° C. to give WX001-3.

[0107] Step 2: synthesis of WX001-4

[0108] To a reaction flask were added WX001-3(1 g, 3.40 mmol, 1 eq) and acetonitrile (10 mL). The atmosphere was replaced with nitrogen gas, and then the mixture was cooled to 0° C. Boron trifluoride etherate (579.06 mg, 4.08 mmol, 503.53 μL, 1.2 eq) was slowly added dropwise. The mixture solution was reacted at 20° C. for 2 hours, then heated to 50° C. and reacted for another 16 hours, and then heated to 60° C. and reacted for another 8 hours. After completion of the reaction, the reaction solution was diluted with water (20 mL), and extracted with ethyl acetate (20 mL*3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was purified by column chromatography to give WX001-4. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 4.18 (d, J=8.6 Hz, 1H), 4.06-4.13 (m, 2H), 3.76 (d, J=8.6 Hz, 1H), 2.76-2.87 (m, 1H), 2.52 (s, 3H), 2.16 (dt, J=12.9, 7.5 Hz, 1H).

[0109] Step 3: synthesis of WX001-5

[0110] To a reaction flask were added WX001-4 (250 mg, 788.25 μmol, 1 eq), hydrochloric acid (2 mol/L, 1.97 mL, 5 eq) and ethanol (2 mL). The mixture solution was reacted at 70° C. for 16 hours. After completion of the reaction, the reaction solution was diluted with water (2 mL), and extracted with ethyl acetate (5 mL*3). The organic phase was washed with saturated brine (2 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-5. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 9.23 (br s, 1H), 3.97-4.14 (m, 2H), 3.84-3.95 (m, 1H), 3.76 (br d, J=8.7 Hz, 1H), 2.17-2.33 (m, 1H).

[0111] Step 4: synthesis of WX001-7

[0112] To a reaction flask were added WX001-5 (150 mg, 545.21 μmol, 1 eq) and N,N-dimethylformamide (2 mL). The atmosphere was replaced with nitrogen gas, and then the mixture was cooled to 0° C. Sodium hydride (26.17 mg, 654.25 μmol, 60% purity, 1.2 eq) was added. The mixture was stirred for 0.5 hours, and WX001-6 (134.44 mg, 654.25 μmol, 85.63 μL, 1.2 eq) was then added. The mixture was slowly warmed to 20° C. and reacted for 0.5 hours. After completion of the reaction, the reaction solution was diluted with water (20 mL), and extracted with ethyl acetate (10 mL*3). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-7. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 7.30-7.39 (m, 3H), 7.23-7.29 (m, 1H), 4.62-4.79 (m, 2H), 4.02-4.11 (m, 1H), 3.95 (q, J=8.4 Hz, 1H), 3.72-3.82 (m, 2H), 2.30-2.38 (m, 2H).

[0113] Step 5: synthesis of WX001-8

[0114] To a reaction flask were added WX001-7 (100 mg, 250.19 μmol, 1 eq), A-1 (127.76 mg, 275.21 μmol, 1.1 eq) and toluene (2 mL). The atmosphere was replaced with nitrogen gas, and tetrakis(triphenylphosphine)palladium (57.82 mg, 50.04 μmol, 0.2 eq) was then added. The mixture solution was reacted at 125° C. for 14 hours. After completion of the reaction, the reaction solution was directly rotary evaporated to dryness to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-8.

[0115] Step 6: synthesis of WX001A or WX001B

[0116] WX001-8 was chirally resolved by supercritical fluid chromatography (separation condition: chromatographic column: DAICEL CHIRALCEL OJ (250*30 mm i.d. 10 μm); mobile phase: A was CO.sub.2, B was ethanol (0.1% NH.sub.3H.sub.2O), B %=50%; flow rate: 70 mL/min) to give WX001A or WX001B. The retention time of WX001A was 1.782 minutes and the retention time of WX001B was 1.969 minutes.

Example 2

[0117] ##STR00025##

[0118] Route of synthesis:

##STR00026##

[0119] Step 1: synthesis of WX002-2

[0120] To a reaction flask were added WX001-1 (5 g, 24.03 mmol, 1 eq) and tetrahydrofuran (250 mL) under nitrogen gas, and lithium diisopropylamide (2 M, 28.84 mL, 2.4 eq) and tetramethylethylenediamine (4.19 g, 36.05 mmol, 5.44 mL, 1.5 eq) were slowly added at −78° C. The mixture was reacted at −78° C. for 0.5 hours, and a solution of WX002-1 (4.81 g, 48.07 mmol, 4.42 mL, 2 eq) in tetrahydrofuran (10 mL) was then added. The mixture was reacted at −78° C. for 2 hours. After completion of the reaction, the reaction solution was slowly poured into 100 mL of saturated aqueous ammonium chloride solution at 0° C., adjusted to a pH of about 3-4 with hydrochloric acid (2 M), and extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated brine (200 mL*3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump at 45° C. to give WX002-2.

[0121] Step 2: synthesis of WX002-3

[0122] To a reaction flask were added WX002-2 (1 g, 3.25 mmol, 1 eq) and acetonitrile (20 mL). The atmosphere was replaced with nitrogen gas, and then boron trifluoride etherate (552.70 mg, 3.89 mmol, 480.61 μL, 1.2 eq) was added. The mixture solution was reacted at 60° C. for 16 hours. After completion of the reaction, saturated aqueous sodium bicarbonate solution (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated brine (30 mL*3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was slurried with ethyl acetate (10 mL) to give WX002-3.

[0123] Step 3: synthesis of WX002-4

[0124] To a dry reaction flask were added WX002-3 (260 mg, 785.06 μmol, 1 eq), hydrochloric acid (2 M, 4 mL, 10.19 eq) and ethanol (6 mL). The mixture was reacted at 50° C. for 16 hours, and then heated to 70° C. and reacted for 4 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL*3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was slurried with ethyl acetate (10 mL) to give WX002-4. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.32 (s, 1H), 3.73-3.79 (m, 2H), 3.56-3.63 (m, 2H), 2.27-2.34 (m, 1H), 1.86-1.91 (m, 2H), 1.80-1.82 (m, 1H).

[0125] Step 4: synthesis of WX002-5

[0126] To a dry reaction flask were added WX002-4 (50 mg, 172.92 μmol, 1 eq) and N,N-dimethylformamide (2 mL). The atmosphere was replaced with nitrogen gas, and then sodium hydride (10.37 mg, 259.38 μmol, 60% purity, 1.5 eq) was added at 0° C. The mixture was reacted at 0° C. for 0.5 hours, and WX001-6 (35.53 mg, 172.92 μmol, 22.63 μL, 1 eq) was then added. The reaction solution was slowly warmed to 25° C. and reacted for another 1.5 hours. After completion of the reaction, the reaction solution was added to 30 mL of water, and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with saturated brine (50 mL*3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX002-5. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 7.42 (s, 1H), 7.29-7.38 (m, 3H), 4.77 (s, 2H), 4.03 (br dd, J=12.3, 4.1 Hz, 2H), 3.45 (br t, J=12.0 Hz, 2H), 2.17-2.25 (m, 4.8 Hz, 2H), 1.38 (br d, J=13.0 Hz, 2H).

[0127] Step 5: synthesis of WX002

[0128] To a reaction flask were added WX002-5 (50 mg, 120.86 μmol, 1 eq), A-1 (65.66 mg, 120.86 μmol, 1 eq) and toluene (1 mL), and the atmosphere was replaced with nitrogen gas. The mixture was heated to 125° C., and tetrakis(triphenylphosphine)palladium (27.93 mg, 24.17 μmol, 0.2 eq) was then slowly added. The mixture was reacted at 125° C. for 48 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was purified by high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile: 28%-58%, 8 min) to give WX002.

Example 3

[0129] ##STR00027##

[0130] Route of synthesis:

##STR00028##

[0131] Step 1: synthesis of WX003-1

[0132] To a pre-dried reaction flask were added WX002-5 (100 mg, 241.71 μmol, 1 eq), A-1-2 (108.10 mg, 241.71 μmol, 1 eq) and toluene (2 mL), and the atmosphere was replaced with nitrogen gas. Tetrakis(triphenylphosphine)palladium (55.86 mg, 48.34 μmol, 0.2 eq) was added at 125° C., and the mixture was reacted with stirring for 48 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure with a water pump at 45° C. to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX003-1. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.29 (d, J=5.1 Hz, 1H), 8.48 (d, J=5.1 Hz, 1H), 7.45 (s, 1H), 7.31-7.39 (m, 3H), 4.83 (s, 2H), 4.07 (m, J=12.3, 4.4 Hz, 2H), 3.64 (m, J=12.0 Hz, 2H), 3.54 (s, 3H), 2.24-2.31 (m, 2H), 1.43 (m, J=12.9 Hz, 2H).

[0133] Step 2: synthesis of WX003

[0134] To a pre-dried reaction flask were added WX003-1 (50 mg, 101.84 μmol, 1 eq), tetrahydropyran-4-amine (10.30 mg, 101.84 μmol, 1 eq) and dimethyl sulfoxide (1 mL). The mixture was reacted with stirring at 100° C. for 16 hours. After completion of the reaction, the reaction solution was purified by high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile: 32%-62%, 8 min) to give WX003.

Example 4

[0135] ##STR00029##

[0136] Route of synthesis:

##STR00030## ##STR00031##

[0137] Step 1: synthesis of WX004-1

[0138] To a reaction flask were added WX001-1 (10 g, 48.07 mmol, 1 eq) and tetrahydrofuran (200 mL). The atmosphere was replaced with nitrogen gas, and a solution of borane-tetrahydrofuran complex (1 M, 144.21 mL, 3 eq) was then slowly added dropwise under nitrogen gas. The mixture was reacted at 25° C. for 5 hours. After completion of the reaction, methanol (100 mL) was slowly added dropwise to the reaction solution under nitrogen gas. The mixture was stirred at 25° C. for 16 hours, and then rotary evaporated to dryness at 40° C. to give a crude product. The crude product was purified by column chromatography to give WX004-1. .sup.1H NMR (CDCl.sub.3, 400 MHz): δ (ppm) 7.47 (s, 1H), 4.52 (d, J=1.0 Hz, 2H).

[0139] Step 2: synthesis of WX004-3

[0140] To a reaction flask were added WX004-1 (7.86 g, 40.51 mmol, 1 eq) and tetrahydrofuran (78.6 mL), and the atmosphere was replaced with nitrogen gas. The mixture was cooled to −78° C. Lithium diisopropylamide (2 M, 48.61 mL, 2.4 eq) was slowly added, and tetramethylethylenediamine (7.06 g, 60.76 mmol, 9.17 mL, 1.5 eq) was then added. The mixture was reacted at −78° C. for 0.5 hours, and a mixed solution of WX004-2 (10.65 g, 60.76 mmol, 1.5 eq) and tetrahydrofuran (10 mL) was then added. The mixture was reacted at −78° C. for 1 hour. After completion of the reaction, the reaction solution was quenched with saturated aqueous ammonium chloride solution (100 mL), and extracted with ethyl acetate (50 mL*3). The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness at 45° C. to give a crude product. The crude product was purified by column chromatography to give WX004-3. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 6.39 (s, 1H), 5.42 (t, J=5.6 Hz, 1H), 4.84-5.03 (m, 4H), 4.38 (d, J=5.6 Hz, 2H), 1.12 (s, 9H).

[0141] Step 3: synthesis of WX004-4

[0142] To a reaction flask were added tetrahydrofuran (56 mL), WX004-3 (5.6 g, 11.07 mmol, 73% purity, 1 eq) and tributylphosphine (4.48 g, 22.14 mmol, 5.46 mL, 2 eq). After completion of the dissolution, the atmosphere was replaced with nitrogen gas. The mixture was cooled to 0° C., and diisopropyl azodicarboxylate (4.48 g, 22.14 mmol, 4.30 mL, 2 eq) was slowly added. The mixture was slowly warmed to 20° C. and reacted for 2 hours. After completion of the reaction, water (60 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL*3). The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness at 45° C. to give a crude product. The crude product was purified by column chromatography to give WX004-4. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 5.30 (d, J=7.6 Hz, 1H), 4.75-4.87 (m, 3H), 4.61 (d, J=12.8 Hz, 1H), 4.22 (d, J=12.8 Hz, 1H), 1.27 (s, 9H).

[0143] Step 4: synthesis of WX004-5

[0144] To a reaction flask were added WX004-4 (500 mg, 1.42 mmol, 1 eq), tetrahydrofuran (8.3 mL), water (1.6 mL) and iodine (72.25 mg, 284.67 μmol, 57.34 μL, 0.2 eq). The atmosphere was replaced with nitrogen gas, and the mixture was reacted at 30° C. for 16 hours. After completion of the reaction, the reaction solution containing WX004-5 was obtained and directly used in the next reaction.

[0145] Step 5: synthesis of WX004-6

[0146] To the reaction solution containing WX004-5 obtained in Step 4 were sequentially added tetrahydrofuran (8.3 mL), water (1.6 mL), di-tert-butyl carbonate (465.00 mg, 2.13 mmol, 489.47 μL, 1.5 eq) and sodium carbonate (301.10 mg, 2.84 mmol, 2 eq), and the mixture was reacted at 25° C. for 4 hours. After completion of the reaction, the reaction solution was quenched with water (10 mL), and extracted with ethyl acetate (5 mL*3). The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness at 45° C. to give a crude product. The crude product was purified by column chromatography to give WX004-6. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 5.46 (br d, J=5.8 Hz, 1H), 5.32 (br d, J=6.3 Hz, 1H), 4.64 (br d, J=6.3 Hz, 1H), 4.55 (br d, J=5.8 Hz, 1H), 4.49 (br d, J=9.5 Hz, 2H), 1.47-1.57 (m, 9H).

[0147] Step 6: synthesis of WX004-7

[0148] To a dry reaction flask were added WX004-6 (150 mg, 431.99 μmol, 1 eq), chromium trioxide (86.39 mg, 863.99 μmol, 32.00 μL, 2 eq) and acetic acid (3 mL), and the mixture was reacted at 25° C. for 12 hours. After completion of the reaction, the reaction solution was diluted with water (3 mL), and extracted three times with dichloromethane (5mL). The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump to give a crude product. The crude product was purified by column chromatography to give WX004-7.

[0149] Step 7: synthesis of WX004-8

[0150] To a dry reaction flask were added WX004-7 (70 mg, 193.79 μmol, 1 eq), dichloromethane (1 mL) and trifluoroacetic acid (287.47 mg, 2.52 mmol, 186.67 μL, 13.01 eq), and the mixture was reacted at 25° C. for 1 hour. After completion of the reaction, the reaction solution was directly concentrated to give a crude product, and the crude product was purified by thin layer chromatography on silica gel plate to give WX004-8. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.59(br, s, 1H), 4.89 (s, 4H).

[0151] Step 8: synthesis of WX004-10

[0152] To a dry reaction flask were added WX004-8 (45 mg, 172.35 μmol, 1 eq), N,N-dimethylformamide (2mL), cesium carbonate (84.23 mg, 258.53 μmol, 1.5 eq) and WX004-9 (39.09 mg, 206.82 μmol, 25.39 μL, 1.2 eq). The atmosphere was replaced with nitrogen gas, and the mixture was reacted at 25° C. for 12 hours. After completion of the reaction, the reaction solution was diluted with water (2 mL), and extracted three times with dichloromethane (2mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump to give a crude product, and the crude product was purified by thin layer chromatography on silica gel plate to give WX004-10.

[0153] Step 9: synthesis of WX004-10

[0154] To a dry reaction flask were added WX004-10 (45 mg, 121.88 μmol, 1 eq), A-1 (62.24 mg, 134.07 μmol, 1.1 eq) and toluene (1 mL). The atmosphere was replaced with nitrogen gas, and tetrakis(triphenylphosphine)palladium (28.17 mg, 24.38 μmol, 0.2 eq) was then added. The mixture was heated to 125° C. and reacted for 16 hours. After completion of the reaction, the reaction solution was directly concentrated to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate, and then purified by high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [H.sub.2O (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile %: 25%-45%, 8 min) to give WX004.

Example 5

[0155] ##STR00032##

[0156] Route of synthesis:

##STR00033##

[0157] Step 1: synthesis of WX005-1

[0158] To a dry reaction flask were added WX004-8 (300 mg, 1.15 mmol, 1 eq), N,N-dimethylformamide (6mL), cesium carbonate (561.55 mg, 1.72 mmol, 1.5 eq) and WX001-6 (283.32 mg, 1.38 mmol, 180.46 μL, 1.2 eq). The atmosphere was replaced with nitrogen gas, and the mixture was reacted at 25° C. for 16 hours. After completion of the reaction, the reaction solution was diluted with water (10 mL), and extracted three times with ethyl acetate (20 mL). The organic phases were combined, washed with saturated brine (20 mL*5), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump to give a crude product. The crude product was purified by column chromatography to give WX005-1. .sup.1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.42 (s, 1H), 7.32-7.40 (m, 2H), 7.26-7.32 (m, 1H), 4.98 (s, 2H), 4.79-4.89 (m, 4H)

[0159] Step 2: synthesis of WX005

[0160] To a dry reaction flask were added WX005-1 (30 mg, 77.79 μmol, 1 eq), A-1 (39.72 mg, 85.57 μmol, 1.1 eq) and toluene (1 mL). The atmosphere was replaced with nitrogen gas, and tetrakis(triphenylphosphine)palladium (17.98 mg, 15.56 μmol, 0.2 eq) was then added. The mixture was heated to 125° C. and reacted for 16 hours. Additional tetrakis(triphenylphosphine)palladium (8.99 mg, 7.78 μmol, 0.1 eq) was added, and the mixture was reacted at 125° C. for 3 hours. After completion of the reaction, the reaction solution was directly concentrated to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate, and then purified by high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [water (10mM ammonium bicarbonate)-acetonitrile]; acetonitrile %: 25%-55%, 8 min) to give WX005.

Example 6

[0161] ##STR00034##

[0162] Route of synthesis:

##STR00035##

[0163] Step 1: synthesis of WX006-2

[0164] To a dry reaction flask were added WX004-10 (105 mg, 284.39 μmol, 1 eq), tetrahydrofuran (1 mL) and zinc chloride (0.7 M, 406.27 μL, 1 eq). The atmosphere was replaced with nitrogen gas, and n-butyl lithium (2.5 M, 170.64 μL, 1.5 eq) was added at −30° C. The mixture was stirred at 25° C. for 1 hour, and then cooled to −30° C. A solution of B-1 (75.68 mg, 284.39 μmol, 1 eq) and tetrakis(triphenylphosphine)palladium (16.43 mg, 14.22 μmol, 0.05 eq) in tetrahydrofuran (0.5 mL) was added, and the mixture was heated to 60° C. and reacted for another 16 hours. After completion of the reaction, 2mL of saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (5mL*3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump to give a crude product. The crude product was purified by slurrying with 2 mL of methyl tert-butyl ether to give WX006-2. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 8.77 (s, 1H), 7.37-7.42 (m, 1H), 7.20 (br d, J=8.6 Hz, 3H), 5.04 (s, 2H), 4.94 (d, J =7.5 Hz, 2H), 4.86 (d, J=7.2 Hz, 2H), 2.64 (s, 3H), 2.62 (s, 3H).

[0165] Step 2: synthesis of WX006-3

[0166] To a dry reaction flask were added WX006-2 (20 mg, 46.67 μmol, 1 eq) and dichloromethane (1 mL). The atmosphere was replaced with nitrogen gas, and then m-chloroperoxybenzoic acid (30.20 mg, 140.02 μmol, 80% purity, 3 eq) was added at 0° C. The mixture was slowly warmed to 25° C. and reacted for 3 hours. After completion of the reaction, saturated sodium thiosulfate solution (10 mL) was added to the reaction solution until the color of starch potassium iodide test paper was not changed to blue. The mixture was diluted with dichloromethane (10 mL). The layers were separated. The organic phase was then collected, and the collected organic phase was washed respectively with 10mL of saturated sodium bicarbonate solution and 10 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX006-3. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.20 (s, 1H), 7.37-7.45 (m, 1H), 7.21 (br d, J=8.0 Hz, 2H), 7.09-7.15 (m, 1H), 5.05(s, 2H), 4.93-4.96 (m, 2H), 4.89-4.91 (m, 2H), 3.51 (s, 3H), 2.82 (s, 3H).

[0167] Step 3: synthesis of WX006

[0168] To a dry reaction flask were added WX006-3 (28 mg, 60.80 μmol, 1 eq), A-1-3 (11.81 mg, 121.61 μmol, 2 eq), dichloromethane (1 mL) and tetrahydrofuran (1 mL). The atmosphere was replaced with nitrogen gas, and lithium hexamethyldisilazide (1 M, 127.69 μL, 2.1 eq) was added at −30° C. The mixture was reacted at 25° C. for 1 hour. After completion of the reaction, 1 mL of water was added to the reaction solution. The organic solvent in the reaction solution was rotary evaporated, and solids were precipitated. The mixture was filtered, and the solids were collected to give a crude product. The crude product was purified by high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*25 mm*5 μm; mobile phase: [H.sub.2O (10 mM ammonium bicarbonate)-acetonitrile]; acetonitrile%: 25%-60%, 10min) to give WX006.

Example 7

[0169] ##STR00036##

[0170] Route of synthesis:

##STR00037##

[0171] Step 1: synthesis of WX007-1

[0172] To a dry reaction flask were added WX005-1 (100 mg, 259.29 μmol, 1 eq), zinc chloride (0.7 M, 370.42 μL, 1 eq) and tetrahydrofuran (1.5 mL). The atmosphere was replaced with nitrogen gas, and then the mixture was cooled to −30° C. n-Butyl lithium (2.5 M, 155.58 μL, 1.5 eq) was added, and the mixture was reacted at 20° C. for 1 hour. The mixture was then cooled to −30° C. A solution of tetrakis(triphenylphosphine)palladium (14.98 mg, 12.96 μmol, 0.05 eq) and B-1 (69.00 mg, 259.29 μmol, 1 eq) in tetrahydrofuran (0.5 mL) was slowly added dropwise, and the mixture was reacted at 60° C. for 15 hours. After completion of the reaction, the reaction solution was quenched with 5 mL of saturated ammonium chloride solution, and extracted three times with ethyl acetate (10 mL). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure with a water pump to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX007-1. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ(ppm) 8.78 (s, 1H), 7.45 (s, 1H), 7.30-7.42 (m, 3H), 5.03 (s, 2H), 4.91-4.96 (m, 2H), 4.83-4.89 (m, 2H), 2.59-2.71 (m, 6H).

[0173] Step 2: synthesis of WX007-2

[0174] To a dry reaction flask were added WX007-1 (40 mg, 89.90 μmol, 1 eq) and dichloromethane (1 mL). The atmosphere was replaced with nitrogen gas, and then m-chloroperoxybenzoic acid (58.18 mg, 269.69 μmol, 80% purity, 3 eq) was added at 0° C. The mixture was slowly warmed to 25° C. and reacted for 3 hours. After completion of the reaction, saturated sodium thiosulfate solution (10 mL) was added to the reaction solution until the color of starch KI test paper was not changed to blue. The mixture solution was diluted with dichloromethane (10 mL). The layers were separated. The organic phase was then collected, and the collected organic phase was respectively washed with 10 mL of saturated sodium bicarbonate solution and 10 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX007-2.

[0175] Step 3: synthesis of WX007

[0176] To a dry reaction flask were added WX007-2 (35 mg, 73.38 μmol, 1 eq), A-1-3 (14.97 mg, 154.10 μmol, 2.1 eq), dichloromethane (0.5 mL) and tetrahydrofuran (0.5 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0° C., and lithium hexamethyldisilazide (1 M, 146.76 μL, 2 eq) was added dropwise. The mixture was reacted at 0° C. for 0.5 hours and reacted at 25° C. for another 1 hour. After completion of the reaction, the reaction solution was quenched with 10 mL of water, and extracted with 20 mL of dichloromethane. The layers were separated. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (3*20 mL). The organic phases were combined, and the combined organic phase was sequentially washed with saturated brine (3*20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue. The crude product was purified by high performance liquid chromatography (chromatographic column: Phenomenex Gemini-NX C18 75*30 mm*3 μm; mobile phase: [H.sub.2O (10mM ammonium bicarbonate)-acetonitrile]; acetonitrile %: 35%-55%, 8 min) to give WX007.

[0177] The data of .sup.1H NMR spectrum and mass spectrum of each example were shown in Table 1.

TABLE-US-00001 TABLE 1 Example Compound NMR MS m/z 1 WX001A .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 9.81 (br s, 494.2 1H), 8.70 (d, J = 4.9 Hz, 1H), 7.55 (d, J = 5.0 Hz, 1H), [M + H].sup.+ 7.25-7.45 (m, 5H), 6.32 (s, 1H), 4.67-4.83 (m, 2H), 3.99-4.13 (m, 2H), 3.82-3.88 (m, 1H), 3.76-3.81 (m, 1H), 3.72 (s, 3H), 2.35-2.45 (m, 2H). WX001B .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 9.80 (br s, 494.1 1H), 8.69 (d, J = 4.9 Hz, 1H), 7.55 (d, J = 4.9 Hz, 1H), [M + H].sup.+ 7.25-7.46 (m, 5H), 6.32 (d, J = 1.6 Hz, 1H), 4.67-4.84 (m, 2H), 3.98-4.14 (m, 2H), 3.83-3.88 (m, 1H), 3.76-3.81 (m, 1H), 3.72 (s, 3H), 2.33-2.45 (m, 2H). 2 WX002 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.81 (s, 1H), 508.3 8.71 (d, J = 4.9 Hz, 1H), 7.58 (d, J = 5.0 Hz, 1H), 7.44 [M + 1].sup.+ (s, 1H), 7.42 (d, J = 1.9 Hz, 1H), 7.30-7.40 (m, 3H), 6.34 (d, J = 1.5 Hz, 1H), 4.81 (s, 2H), 4.07 (br dd, J = 12.1, 4.0 Hz, 2H), 3.74 (s, 3H), 3.57 (br t, J = 12.0 Hz, 2H), 2.23-2.31 (m 4.5 Hz, 2H), 1.40 (br d, J = 12.7 Hz, 2H). 3 WX003 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 8.54 (br d, 512.3 J = 5.0 Hz, 1H), 7.51-7.69 (m, 1H), 7.44 (s, 1H), [M + 1].sup.+ 7.30-7.39 (m, 3H), 7.28 (d, J = 4.9 Hz, 1H), 4.80 (s, 2H), 4.06 (br dd, J = 12.9, 3.9 Hz, 3H), 3.90 (br d, J = 11.0 Hz, 2H), 3.53-3.64 (m, 2H), 3.37-3.51 (m, 2H), 2.22-2.29 (m, 4.6 Hz, 2H), 1.82-1.94 (m, 2H), 1.51-1.63 (m, 2H), 1.40 (br d, J = 12.7 Hz, 2H). 4 WX004 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.84 (br s, 464.0 1H), 8.70 (d, J = 5.1 Hz, 1H), 7.55 (d, J = 4.9 Hz, 1H), [M + 1].sup.+ 7.36-7.44 (m, 2H), 7.15-7.21 (m, 2H), 7.06-7.15 (m, 1H), 6.35 (s, 1H), 5.03 (s, 2H), 4.89-4.95 (m, 2H), 4.83-4.88 (m, 2H), 3.74 (s, 3H). 5 WX005 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ(ppm) 9.84 (br s, 480.1 1H), 8.71 (d, J = 4.9 Hz, 1H), 7.55 (d, J = 5.0 Hz, 1H), [M + 1].sup.+ 7.29-7.46 (m, 5H), 6.35 (d, J = 1.5 Hz, 1H), 5.02 (s, 2H), 4.89-4.94 (m, 2H), 4.84-4.89 (m, 2H), 3.74 (s, 3H). 6 WX006 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ (ppm) 9.68 (s, 1H), 478.1 8.62 (s, 1H), 7.37-7.43 (m, 2H), 7.16-7.21 (m, 2H), [M + 1].sup.+ 7.08-7.15 (m, 1H), 6.36 (d, J = 1.8 Hz, 1H), 5.03 (s, 2H), 4.94 (d, J = 7.5 Hz, 2H), 4.83 (d, J = 7.3 Hz, 2H), 3.73 (s, 3H), 2.58 (s, 3H). 7 WX007 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ(ppm) 9.66 (s, 1H), 494.1 8.62 (s, 1H), 7.28-7.46 (m, 5H), 6.36 (s, 1H), 5.02 (s, [M + 1].sup.+ 2H), 4.94 (d, J = 7.3 Hz, 2H), 4.83 (d, J = 7.3 Hz, 2H), 3.73 (s, 3H), 2.59 (s, 3H).

Assay Example 1. Assay of In Vitro Kinase Activity:

[0178] 1. Purpose of the assay:

[0179] The ability of compounds to inhibit ERK2 kinase activity was measured.

[0180] 2. Assay buffer:

[0181] 20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg/mL bovine serum albumin (BSA), 0.1 mM Na.sub.3VO.sub.4, 2 mM dithiothreitol (DTT), 1% DMSO.

[0182] 3. Processing of compound:

[0183] The assay compound was dissolved in 100% DMSO to prepare a stock solution of specific concentration. The compound was serially diluted in DMSO solution using Integra Viaflo Assist smart pipette.

[0184] 4. Method of the assay

[0185] 1) The substrate MBP was prepared in freshly prepared reaction buffer;

[0186] 2) ERK2 kinase was added to the above-mentioned MBP solution and mixed gently;

[0187] 3) The compound dissolved in 100% DMSO was added to the kinase reaction system using ultrasound technology (Echo550; nanoliter range), and the mixture was incubated at room temperature for 20 minutes;

[0188] 4) .sup.33P-ATP (specific concentration of 10 μCi/μL) was added to the reaction system, and the reaction was started at this time;

[0189] 5) The mixture was incubated at room temperature for 2 hours;

[0190] 6) The amount of radioactivity was detected by filter-binding method;

[0191] 7) ERK2 kinase activity was calculated as the ratio of the remaining kinase activity in the assay sample to the kinase activity of the control group (treated by DMSO). Curve was fitted using Prism (GraphPad software) and IC.sub.50 values were calculated.

[0192] 5. The assay results were shown in Table 2:

TABLE-US-00002 TABLE 2 Results of kinase activity assay in vitro ERK2 Compound IC.sub.50 (nM) WX001A 0.31 WX001B 0.32 WX002 0.4 WX003 0.29 WX004 1.1 WX005 0.36 WX006 0.94 WX007 0.48

[0193] Conclusion: The compounds of the present disclosure exhibit excellent activity of inhibiting ERK2 kinase.

Assay Example 2. In Vitro Cell Proliferation Inhibition Assay:

[0194] 1. Purpose of the assay:

[0195] The ability of compounds to inhibit the proliferation of HT.sub.29 tumor cells was measured.

[0196] 2. Processing of compound:

[0197] The assay compound was dissolved in 100% DMSO to prepare 10 mM stock solution.

[0198] 3. Method and step of the assay

[0199] 1) UV light of a biological safety cabin was turned on, and 30 minutes were counted down;

[0200] 2) In a 37° C. water bath, RPMI1640 medium and trypsin were preheated;

[0201] 3) After completion of the UV irradiation, the biological safety cabin was opened. The preheated medium, trypsin and phosphate buffered saline (PBS), etc. were wiped with alcohol and placed in the biological safety cabin;

[0202] 4) HT.sub.29 cells were removed from the incubator, and the old medium was removed in biological safety cabin. 10 ml of PBS was added. The mixture was shaken gently, and then PBS was removed;

[0203] 5) 1.5 ml of preheated 0.25% trypsin was added. The culture vessel was shaken horizontally so that the trypsin evenly covered the cells at the bottom, and placed in an incubator for 2 minutes;

[0204] 6) Cell digestion was stopped with complete medium, and the cell suspension was pipetted to homogeneity and counted;

[0205] 7) According to the result of cell counting, the density of cell suspension was adjusted to 1500 cells per well, and the cell suspension was seeded at 50 μl per well;

[0206] 8) The stock solution of compounds was serially diluted in DMSO solution, and compounds were added to cell plate using Tecan;

[0207] 9) The compound-added cell plate and CellTiterGlo were equilibrated at room temperature, and 25 microliters of CellTiterGlo was then added to each well. The cell plate was shaken for 1-2 minutes and then allowed to stand for 10 minutes. The signal value was then detected. The data were analyzed using XL-Fit, and the IC.sub.50 of each compound was calculated.

[0208] 4. The assay results were shown in Table 3:

TABLE-US-00003 TABLE 3 Results of cell activity assay in vitro HT29 Compound IC.sub.50 (nM) WX001A 21 WX001B 26 WX002 73 WX003 69 WX004 125 WX005 49 WX006 14 WX007 11

[0209] Conclusion: The compounds of the present disclosure exhibit excellent activity of inhibiting the proliferation of HT.sub.29 cells.

Assay Example 3. Assay of In Vivo DMPK:

[0210] In vivo DMPK assay in mouse

[0211] 1. Purpose of the assay:

[0212] Female BALB/c mice were used as assay animals to determine the blood concentration of compounds and evaluate the pharmacokinetic behavior after a single administration.

[0213] 2. Procedure of the assay:

[0214] Eight healthy adult female BALB/c mice were selected, wherein 4 mice were in the intravenous injection group and 4 mice were in the oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, vortexed and sonicated to prepare a clear solution of 0.5 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, vortexed and sonicated to prepare a solution of 0.3 mg/mL. Mice were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

Note: DMSO: dimethyl sulfoxide; HP-β-CD: hydroxypropyl-β-cyclodextrin.

[0215] 3. The assay results were shown in Table 4:

TABLE-US-00004 TABLE 4 Results of PK assay of the compounds Oral Cl C.sub.max DNAUC Vd.sub.ss (mL/min/ T.sub.1/2 Compound (nM) F % (nM .Math. h/mpk) (L/kg) kg) (h) WX006 1400 70% 1356 1.7 17.2 1.4 WX007 595 46% 1086 1.7 14.4 1.3 Note: C.sub.max is maximum concentration; F % is oral bioavailability; DNAUC is AUC.sub.PO/Dose, AUC.sub.PO is oral exposure, and Dose is drug dose; Vd.sub.ss is distribution volume; Cl is clearance rate; and T.sub.1/2 is half-life.

[0216] Conclusion: The compounds of the present disclosure exhibit excellent oral exposure and bioavailability.

Assay Example 4. Assay of In Vivo Efficacy of Subcutaneous Xenograft Tumor of Human Colon Cancer HCT-116 Cells in BALB/c Nude Mouse Model:

[0217] 1. Purpose of the assay:

[0218] The anti-tumor effect of WX007 was evaluated using a subcutaneous xenograft tumor model of human colon cancer HCT-116 cells in nude mouse.

[0219] 2. Assay animal:

[0220] Species: mouse

[0221] Strain: BALB/c nude mouse

[0222] Age: 6-8 weeks old

[0223] Gender: female

[0224] Weight: 17-23 grams

[0225] Vendor: Laboratory Animal Management Department, Shanghai Institute for Biomedical and Pharmaceutical Technologies

[0226] Animal certificate number: 20180006020214

[0227] 3. Assay procedure:

[0228] 1) Assay cells and culture: Human colon cancer HCT-116 cells were cultured in monolayer in vitro. The culture conditions were McCoy's 5a medium plus 10% fetal bovine serum, and a 5% CO.sub.2 incubator at 37° C. Routine digestion with trypsin-ethylene diamine tetraacetic acid was performed three times a week for passage. When the cell saturation was 80%-90% and the amount reached the requirement, the cells were harvested, counted, and inoculated;

[0229] 2) Tumor tissue inoculation and grouping: 0.2 mL (5×10.sup.6) of HCT-116 cells were subcutaneously inoculated into the right armpit of each mouse. When the average tumor volume reached 149 mm.sup.3, the animals were randomly divided into two groups and the administration was started. The grouping and administration schedule of the assay were shown in Table 5;

TABLE-US-00005 TABLE 5 Grouping and administration schedule of assay animals Adminis- Route and Number of Dosage tration frequency of Group animals Drug (mg/kg) cycle administration 1 6 Solvent — 18 days Oral control administration (Vehicle) (PO), once daily (QD) 2 6 WX007 15 18 days Oral administration (PO), once daily (QD)

[0230] 3) Daily observation of assay animals: The development of this assay protocol and any modifications were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC). The use and welfare of assay animals were carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were monitored daily for health and death. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behavior such as behavioral activities, food and water intake (visual inspection only), weight changes (weight measurements twice a week), appearance signs or other abnormalities. Animal deaths and side effects in each group were recorded based on the number of animals in each group.

[0231] 4) Formulation of assay compound

[0232] a) Vehicle group: corn oil.

[0233] b) Assay compound group: A quantitative amount of the assay compound was weighed in a formulation bottle. A corresponding volume of corn oil was added and then the mixture was vortexed to obtain a clear solution. The compound was formulated once a week.

[0234] 5) Tumor measurement and assay indicator:

[0235] a) Tumor diameter was measured twice a week with a vernier caliper. The calculation formula of tumor volume was: TV=½×a×b.sup.2, wherein a and b represent the long and short diameters of tumor, respectively;

[0236] b) The tumor-inhibitory efficacy of the compound was evaluated by TGI (%).TGI (%) reflected the inhibition rate of tumor growth. TGI (%) was calculated as follows: TGI (%) ={[1−(average tumor volume at the end of administration of a treatment group−average tumor volume at the beginning of administration of the treatment group)]/(average tumor volume at the end of treatment in a solvent control group−average tumor volume at the beginning of treatment in the solvent control group)}×100%.

[0237] 4. Assay results:

[0238] 1) As shown in Table 6 and FIG. 1, in the subcutaneous xenograft tumor model of human colon cancer HCT-116 cells in nude mouse, when administered orally to the 18th day, WX007 15 mg/kg in the administration group had a significant inhibitory effect on tumor growth with a TGI of 62%.

[0239] 2) The body weight of assay animals was used as a reference index for indirect determination of drug toxicity. As shown in FIG. 2, when administered to the 18th day, the body weight of all animals in the solvent control group and WX007 15 mg/kg administration group did not decrease significantly, and there was no morbidity or death.

TABLE-US-00006 TABLE 6 Results of in vivo efficacy assay in mouse HCT116 model Group Drug TGI 2 WX007 (15 mg/kg, PO, QD) 62%

[0240] Conclusion: The compounds of the present disclosure can significantly inhibit the growth of tumor. During the administration, the body weight of animals is not observed to decrease significantly, and the tolerance is good.