CRYSTAL FORM OF C-MET/AXL INHIBITOR

Abstract

A crystal form and a salt form of a uracil compound acting as a c-MET/AXL inhibitor and a preparation method therefor, specifically relating to the crystal form and the salt form of the compound shown in formula (I), and also comprising an application of the crystal form and the salt form in the preparation of drugs for the treatment of tumours.

##STR00001##

Claims

1. A crystal form A or crystal form B of a compound represented by formula (I), ##STR00011## wherein the crystal form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 9.37°±0.20°, 17.17°±0.20°, and 18.89°±0.20°; wherein the crystal form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 9.19±0.20°, 12.34±0.20° and 16.45±0.20°.

2. The crystal form A or the crystal form B as defined in claim 1, wherein the crystal form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 9.37°±0.20°, 10.37±0.20°, 12.92±0.20°, 17.17±0.20°, 18.89±0.20°, 19.82±0.20°, 22.09±0.20° and 24.48±0.20°; or, the crystal form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 9.19±0.20°, 12.34±0.20°, 16.45±0.20°, 16.88±0.20°, 18.95±0.20°, 21.34±0.20°, 22.39±0.20° and 24.34±0.20°, or, the crystal form A has a differential scanning calorimetry curve having an endothermic peak with onset at 206.05° C.±3° C.; or, the crystal form A has a thermogravimetric analysis curve having a weight loss of 0.07730% occurred at 158.11° C.±3° C., and a weight loss of 1.0628% occurred at 203.86° C.±3° C.; or, the crystal form B has a differential scanning calorimetry curve having endothermic peaks with onset at 136.23° C.±3° C. and 206.26±3° C.; or, the crystal form B has a thermogravimetric analysis curve having a weight loss of 7.912% occurred at 136.32° C.±3° C., and a weight loss of 9.993% occurred at 198.78° C.±3° C.

3. The crystal form A or the crystal form B as defined in claim 2, wherein the crystal form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 8.10°, 9.37°, 10.37°, 10.92°, 12.92°, 14.11°, 14.67°, 15.21°, 15.85°, 16.21°, 16.66°, 17.17°, 17.64°, 18.89°, 19.18°, 19.82°, 20.74°, 21.30°, 22.09°, 22.91°, 23.90°, 24.48°, 25.56°, 25.92°, 26.29°, 27.04°, 27.39°, 28.32°, 29.27°, 29.86°, 30.57°, 31.34°, 32.16°, 32.62°, 33.27°, 33.79°, 34.45°, 34.75°, 36.80° and 39.33°; or, the crystal form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.35°, 9.19°, 10.00°, 12.34°, 12.74°, 13.57°, 16.55°, 16.88°, 17.40°, 17.80°, 18.28°, 18.95°, 19.60°, 20.19°, 21.34°, 21.69°, 22.39°, 23.33°, 23.68°, 24.34°, 24.73°, 25.56°, 26.35°, 26.94°, 27.69°, 28.36°, 29.03°, 29.35°, 30.06°, 30.55°, 31.12°, 33.19°, 33.86°, 34.10°, 36.01° and 36.66°; or, the crystal form A has a differential scanning calorimetry curve pattern as shown in FIG. 2; or, the crystal form A has a thermogravimetric analysis curve pattern as shown in FIG. 3; or, the crystal form B has a differential scanning calorimetry curve pattern as shown in FIG. 5; or, the crystal form B has a thermogravimetric analysis curve pattern as shown in FIG. 6.

4-14. (canceled)

15. A compound represented by formula (II), (III) or (IV), ##STR00012##

16. A crystal form C of the compound represented by formula (II) as defined in claim 15, wherein the crystal form C has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 4.22±0.20°, 14.91±0.20° and 20.75±0.20°.

17. The crystal form C as defined in claim 16, wherein the crystal form C has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 4.22±0.20°, 10.23±0.20°, 14.34±0.20°, 14.91±0.20°, 19.27±0.20°, 19.94±0.20°, 20.75±0.20°, 23.51±0.20°, 28.38±0.20°, 29.03±0.20° and 29.50±0.20°; or, the crystal form C has a differential scanning calorimetry curve having an endothermic peak with onset at 220.74° C.±3° C.; or, the crystal form C has a thermogravimetric analysis pattern having a weight loss of 0.004784% occurred at 159.80° C.±3° C.

18. The crystal form C as defined in claim 17, wherein the crystal form C has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 4.22°, 7.18°, 8.26°, 10.23°, 13.47°, 14.34°, 14.91°, 15.68°, 16.06°, 16.48°, 17.07°, 17.67°, 18.12°, 18.65°, 19.27°, 19.94°, 20.35°, 20.75°, 21.55°, 22.23°, 22.48°, 23.51°, 24.73°, 25.34°, 26.07°, 26.35°, 26.94°, 27.25°, 27.63°, 28.38°, 29.03°, 29.50°, 29.96°, 30.57°, 31.24°, 32.03°, 32.91°, 33.59°, 34.32°, 34.94°, 35.79°, 37.69° and 38.28°; or, the crystal form C has a differential scanning calorimetry curve pattern as shown in FIG. 8; or, the crystal form C has a thermogravimetric analysis curve pattern as shown in FIG. 9.

19-23. (canceled)

24. A crystal form D of the compound represented by formula (III) as defined in claim 15, wherein the crystal form D has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.49±0.20°, 9.64±0.20° and 19.23±0.20°.

25. The crystal form D as defined in claim 24, wherein the crystal form D has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.49±0.20°, 9.64±0.20°, 18.75±0.20°, 19.23±0.20°, 20.93±0.20°, 21.55±0.20° and 22.17±0.20°; or, the crystal form D has a differential scanning calorimetry curve having an endothermic peak with onset at 223.59° C.±3° C.; or, the crystal form D has a thermogravimetric analysis curve having a weight loss of 0.3850% occurred at 150.12° C.±3° C.

26. The crystal form D as defined in claim 25, wherein the crystal form D has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.49°, 7.89°, 8.50°, 9.17°, 9.64°, 11.20°, 11.67°, 12.28°, 14.93°, 15.40°, 17.35°, 18.75°, 19.23°, 20.93°, 21.55°, 22.17°, 23.31°, 24.12°, 24.88°, 25.58°, 27.53°, 26.53°, and 31.10°; or, the crystal form D has a differential scanning calorimetry curve pattern as shown in FIG. 11; or, the crystal form D has a thermogravimetric analysis curve pattern as shown in FIG. 12.

27-31. (canceled)

32. A crystal form E of the compound represented by formula (IV) as defined in claim 15, wherein the crystal form E has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.94±0.20°, 10.00±0.20° and 11.73±0.20°.

33. The crystal form E as defined in claim 32, wherein the crystal form E has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 5.85±0.20°, 6.94±0.20°, 10.00±0.20°, 11.73±0.20°, 15.82±0.20°, 17.10±0.20°, 20.39±0.20° and 23.74±0.20°; or, the crystal form E has a differential scanning calorimetry curve having exothermic peaks with onset at 67.18° C.±3° C. and 203.17° C.±3° C., and endothermic peaks with onset at 181.72° C.±3° C. and 201.40° C.±3° C.; or, the crystal form E has a thermogravimetric analysis curve having a weight loss of 0.5018% occurred at 52.80° C.±3° C., a weight loss of 4.4958% occurred at 173.60° C.±3° C., and a weight loss of 5.8808% occurred at 210.40° C.±3° C.

34. The crystal form E as defined in claim 33, wherein the crystal form E has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 5.85°, 6.94°, 10.00°, 11.73°, 13.83°, 14.41°, 15.82°, 16.38°, 17.10°, 17.47°, 18.06°, 18.95°, 20.00°, 20.39°, 20.88°, 22.25°, 23.74°, 24.91°, 25.48°, 26.39°, 27.57°, 29.86°, 30.49°, 32.62°, 35.79° and 37.14°; or, the crystal form E has a differential scanning calorimetry curve as shown in FIG. 14; or, the crystal form E has a thermogravimetric analysis curve pattern as shown in FIG. 15.

35-38. (canceled)

39. A method for treating cancer in a subject in need thereof, comprising administering the crystal form A or the crystal form B as defined in claim 1 to the subject.

40. A method for treating cancer in a subject in need thereof, comprising administering the compound as defined in claim 15 to the subject.

41. A method for treating cancer in a subject in need thereof, comprising administering the crystal form C as defined in claim 16 to the subject.

42. A method for treating cancer in a subject in need thereof, comprising administering the crystal form D as defined in claim 24 to the subject.

43. A method for treating cancer in a subject in need thereof, comprising administering the crystal form E as defined in claim 32 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1 is the XRPD pattern of the crystal form A of the compound represented by formula (I).

[0109] FIG. 2 is the DSC pattern of the crystal form A of the compound represented by formula (I).

[0110] FIG. 3 is the TGA pattern of the crystal form A of the compound represented by formula (I).

[0111] FIG. 4 is the XRPD pattern of the crystal form B of the compound represented by formula (I).

[0112] FIG. 5 is the DSC pattern of the crystal form B of the compound represented by formula (I).

[0113] FIG. 6 is the TGA pattern of the crystal form B of the compound represented by formula (I).

[0114] FIG. 7 is the XRPD pattern of the crystal form C of the compound represented by formula (II).

[0115] FIG. 8 is the DSC pattern of the crystal form C of the compound represented by formula (II).

[0116] FIG. 9 is the TGA pattern of the crystal form C of the compound represented by formula (II).

[0117] FIG. 10 is the XRPD pattern of the crystal form D of the compound represented by formula (III).

[0118] FIG. 11 is the DSC pattern of the crystal form D of the compound represented by formula (III).

[0119] FIG. 12 is the TGA pattern of the crystal form D of the compound represented by formula (III).

[0120] FIG. 13 is the XRPD pattern of the crystal form E of the compound represented by formula (IV).

[0121] FIG. 14 is the DSC pattern of the crystal form E of the compound represented by formula (IV).

[0122] FIG. 15 is the TGA pattern of the crystal form E of the compound represented by formula (IV).

DETAILED DESCRIPTION OF THE EMBODIMENT

[0123] For better understanding of the content of the present disclosure, the present disclosure is described in detail through the embodiments, but the embodiments do not mean any limitation on the present disclosure.

[0124] For better understanding of the content of the present disclosure, the present disclosure is described in detail through the embodiments, but the embodiments do not mean any limitation on the present disclosure.

Embodiment 1: Preparation of the Crystal Form A of the Compound Represented by Formula (I)

[0125] ##STR00006## ##STR00007##

[0126] Preparation of 1-C:

[0127] Under nitrogen protection, DIPEA (202.56 g, 1.57 mol) and compound 1-B (251.66 g, 1.34 mol) were added to a solution of compound 1-A (201.93 g, 1.47 mol) in toluene (2 L) with stirring. The reaction mixture was reacted at 100° C. for 16 hours. The reaction mixture was naturally cooled to room temperature and stirred for 16 hours, filtered, and the filter cake was collected to obtain intermediate 1-C. LCMS (ESI) m/z: 347.0 [M+Na].sup.+, .sup.1HNMR (400 MHz, DMSO-d6) δ ppm 1.26 (dt, J=12.26, 7.08 Hz, 6H) 4.15 (q, J=7.09 Hz, 2H) 4.24 (q, J=7.13 Hz, 2H) 7.12-7.26 (m, 2H) 7.44-7.59 (m, 2H) 8.47 (d, J=12.23 Hz, 1H) 10.40 (s, 1H) 10.57 (br d, J=12.47 Hz, 1H).

[0128] Preparation of 1-D:

[0129] Under nitrogen protection, potassium carbonate (169.94 g, 1.23 mol) was added to a solution of intermediate 1-C (197.73 g, 0.61 mol) in ethanol (1 L). The reaction mixture was reacted at 75° C. for 2 hours, and bromomethylcyclopropane (166.63 g, 1.23 mol) was added thereto. The reaction mixture was reacted at 75° C. for 16 hours, and water (1 L) was added thereto. The reaction mixture was reacted at 75° C. for 16 hours. The reaction mixture was concentrated under reduced pressure to remove ethanol, the residue was extracted with ethyl acetate (500 mL*2), the aqueous phase was collected. 12M hydrochloric acid was added to adjust the pH to 1, then the mixture was filtered, and the filter cake was collected and dried to obtain intermediate 1-D. LCMS (ESI) m/z: 304.9 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 0.37-0.45 (m, 2H) 0.50-0.60 (m, 2H) 1.07-1.35 (m, 1H) 3.80 (d, J=7.21 Hz, 2H) 7.23-7.51 (m, 4H) 8.83 (s, 1H) 12.63 (br s, 1H).

[0130] Preparation of 1-G:

[0131] Under nitrogen protection, a solution of compound 1-E (50.09 g, 0.45 mol), compound 1-F (77.47 g, 0.49 mol) and potassium carbonate (66.06 g, 0.48 mol) in acetonitrile (250 mL) was heated to 50° C. and reacted for 16 hours. 750 mL of water was added, the reaction mixture was stirred at room temperature for 48 hours, then filtered, and the filter cake was collected and dried to obtain intermediate 1-G. LCMS (ESI) m/z: 250.0 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 6.00 (d, J=2.32 Hz, 1H) 6.10 (s, 2H) 6.26 (dd, J=5.75, 2.32 Hz, 1H) 7.45-7.59 (m, 1H) 7.90 (d, J=5.75 Hz, 1H) 8.11-8.24 (m, 1H) 8.39 (dd, J=10.45, 2.75 Hz, 1H).

[0132] Preparation of 1-H:

[0133] Under nitrogen protection, phenyl chloroformate (51 mL, 0.40 mol) was added dropwise to a mixture of compound 1-G (50.35 g, 0.20 mol) and DIPEA (105 mL, 0.61 mol). After the reaction mixture was reacted at 0° C. for 3 hours, 300 mL of dimethylamine tetrahydrofuran solution (2 moles per liter) was added, and the reaction mixture was reacted at 50° C. for 16 hours. The reaction mixture was cooled to room temperature and stirred for 16 hours, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain intermediate 1-H. LCMS (ESI) m/z: 321.0 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 2.91 (s, 6H) 6.75 (dd, J=5.69, 2.38 Hz, 1H) 7.53 (d, J=2.32 Hz, 1H) 7.56-7.63 (m, 1H) 8.16-8.24 (m, 2H) 8.43 (dd, J=10.52, 2.69 Hz, 1H) 9.07 (s, 1H)).

[0134] Preparation of 1-I:

[0135] Under nitrogen protection, iron powder (13.08 g, 234.2 mmol) was added to a mixture of compound 1-H (15 g, 46.8 mmol), acetic acid (14.06 g, 234.12 mmol), THF (150 mL) and water (30 mL), and the reaction mixture was reacted for 16 hours at room temperature. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure. The residue was dissolved by adding 500 mL of ethyl acetate and then washed with saturated saline (300 mL*2). The organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain intermediate 1-I. LCMS (ESI) m/z: 291.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 2.91 (s, 6H) 6.75 (dd, J=5.69, 2.38 Hz, 1H) 7.53 (d, J=2.32 Hz, 1H) 7.56-7.63 (m, 1H) 8.16-8.24 (m, 2H) 8.43 (dd, J=10.52, 2.69 Hz, 1H) 9.07 (s, 1H)).

[0136] Preparation of the Crystal Form A of the Compound Represented by Formula (I):

[0137] Under nitrogen protection, TBTU (6.33 g, 19.7 mmol) was added to a solution of compound 1-D (5.01 g, 16.4 mmol) and DIPEA (6.37 g, 49.3 mmol) in DMF (50 mL), and after the mixture was stirred for 0.5 hours, compound 1-I (5.03 g, 17.2 mmol) was added thereto, and the reaction mixture was reacted at room temperature for 16 hours. Then 50 mL of water was added dropwise to the reaction reaction, the mixture was stirred for 2 hours at room temperature and filtered, and the filter cake was collected, dried, and recrystallized by ethyl acetate to obtain compound represented by formula (I), which was detected by XRPD (FIG. 1) as crystal form A of the compound represented by formula (I). LCMS (ESI) m/z: 577.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 0.39-0.48 (m, 2H) 0.52-0.61 (m, 2H) 1.18-1.33 (m, 1H) 2.89 (s, 6H) 3.86 (d, J=7.21 Hz, 2H) 6.61 (dd, J=5.75, 2.45 Hz, 1H) 7.29-7.41 (m, 4H) 7.41-7.47 (m, 2H) 7.49 (dd, J=8.86, 1.28 Hz, 1H) 7.97 (dd, J=12.96, 2.45 Hz, 1H) 8.12 (d, J=5.75 Hz, 1H) 8.81-9.01 (m, 2H) 11.01 (s, 1H).

Embodiment 2: Preparation of the Crystal Form B of the Compound Represented by Formula (I)

[0138] 100 mg of crystal form A of the compound represented by formula (I) was weighed and added to a 40 mL vial, then 2 mL of acetone was added thereto. The sample was placed on a magnetic stirrer (40° C.) and stirred for 16 hours, filtered, and the obtained solid was dried at 40° C. under vacuum, which was detected by XRPD (FIG. 4) as crystal form B of the compound represented by formula (I).

Embodiment 3: Preparation of the Crystal Form C of the Compound Represented by Formula (II)

[0139] ##STR00008##

[0140] 1 g of crystal form A of the compound represented by formula (I) was weighted and added to a 40 mL vial, then 20 mL of THF was added thereto. The obtained sample was placed on a magnetic stirrer (40° C.) and stirred for 10 min, and then an appropriate amount of p-toluenesulfonic acid (the molar ratio of the compound represented by formula (I) to p-toluenesulfonic acid was 1:1.05, added after diluting with THF) was slowly added thereto, and the reaction mixture was dissolved until clear. The sample was placed on a magnetic stirrer (40° C.) and was stirred for 16 hours. A white solid was precipitated from the reaction mixture. Then the mixture was filtered, and the obtained solid was dried at 40° C. overnight in a vacuum drying oven to obtain compound represented by formula (II), which was detected by XRPD (FIG. 7) as crystal form C of the compound represented by formula (II). .sup.1H NMR (400 MHz, DMSO-d6) δ=11.07 (s, 1H), 10.07 (br s, 1H), 8.92 (s, 1H), 8.28 (d, J=6.8 Hz, 1H), 8.07 (dd, J=2.4, 12.9 Hz, 1H), 7.60 (dd, J=1.5, 8.9 Hz, 1H), 7.52-7.41 (m, 5H), 7.41-7.33 (m, 2H), 7.18-7.08 (m, 3H), 7.04 (d, J=2.1 Hz, 1H), 3.87 (d, J=7.2 Hz, 2H), 2.97 (s, 6H), 2.29 (s, 3H), 1.32-1.18 (m, 1H), 0.63-0.52 (m, 2H), 0.49-0.39 (m, 2H).

Embodiment 4: Preparation of the Crystal Form D of the Compound Represented by Formula (III)

[0141] ##STR00009##

[0142] 1 g of crystal form A of the compound represented by formula (I) was weighted and added to a 40 mL vial, then 20 mL of THF was added thereto. The obtained sample was placed on a magnetic stirrer (40° C.) and stirred for 10 min, and then an appropriate amount of methanesulfonic acid (the molar ratio of the compound represented by formula (I) to methanesulfonic acid was 1:1.05, added after diluting with THF) was slowly added, and the reaction mixture was dissolved until clear. The sample was placed on a magnetic stirrer (40° C.) and was stirred for 16 hours. A white solid was precipitated from the reaction mixture. Then the mixture was filtered, and the obtained solid was dried at 40° C. overnight in a vacuum drying oven to obtain compound represented by formula (III), which was detected by XRPD (FIG. 10) as crystal form D of the compound represented by formula (III). .sup.1H NMR (400 MHz, DMSO-d6) δ=11.07 (s, 1H), 10.09 (br s, 1H), 8.92 (s, 1H), 8.28 (d, J=7.0 Hz, 1H), 8.07 (dd, J=2.4, 12.8 Hz, 1H), 7.60 (dd, J=1.4, 9.0 Hz, 1H), 7.53-7.41 (m, 3H), 7.41-7.31 (m, 2H), 7.14 (br d, J=6.4 Hz, 1H), 7.04 (d, J=2.2 Hz, 1H), 3.87 (d, J=7.1 Hz, 2H), 2.98 (s, 6H), 2.31 (s, 3H), 1.34-1.18 (m, 1H), 0.63-0.53 (m, 2H), 0.49-0.38 (m, 2H).

Embodiment 5: Preparation of the Crystal Form E of the Compound Represented by Formula (IV)

[0143] ##STR00010##

[0144] 1 g of crystal form A of the compound represented by formula (I) was weighted and added to a 40 mL vial, then 20 mL of THF was added thereto. The obtained sample was placed on a magnetic stirrer (40° C.) and stirred for 10 min, and then an appropriate amount of hydrochloric acid (the molar ratio of the compound represented by formula (I) to hydrochloric acid was 1:1.05, added after diluting with THF) was slowly added, and the reaction mixture was dissolved until clear. The sample was placed on a magnetic stirrer (40° C.) and was stirred for 16 hours. A white solid was precipitated from the reaction mixture. Then the mixture was filtered, and the obtained solid was dried at 40° C. overnight in a vacuum drying oven to obtain compound represented by formula (IV), which was detected by XRPD (FIG. 13) as crystal form E of the compound represented by formula (IV). .sup.1H NMR (400 MHz, DMSO-d6) δ=11.07 (s, 1H), 10.25 (br s, 1H), 8.92 (s, 1H), 8.28 (d, J=6.8 Hz, 1H), 8.06 (dd, J=2.4, 12.9 Hz, 1H), 7.64-7.54 (m, 1H), 7.51-7.41 (m, 3H), 7.41-7.33 (m, 2H), 7.22 (d, J=2.4 Hz, 1H), 7.09 (br d, J=5.3 Hz, 1H), 3.87 (d, J=7.1 Hz, 2H), 2.98 (s, 6H), 1.34-1.16 (m, 1H), 0.67-0.51 (m, 2H), 0.48-0.37 (m, 2H).

Embodiment 6: Study on the Hygroscopicity

[0145] About 10 to 15 mg of the sample was subjected to DVS detection, and the test results was as shown in Table 8

TABLE-US-00008 TABLE 8 Table of hygroscopic information Compound Hygroscopic weight gain at 25/80% RH Crystal form A of the compound 0.2801% represented by formula (I) Crystal form C of the compound 0.999% represented by formula (II)

[0146] Conclusion: The crystal form A of the compound represented by formula (I) and the crystal form C of the compound represented by formula (II) are slightly hygroscopic.

Embodiment 7: Enzymatic Activity Test of the Compound Represented by Formula (I)

[0147] Reagents and Consumables:

[0148] Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA (bovine serum albumin), 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT (dithiothreitol), 1% DMSO and corresponding cofactors

[0149] Preparation of the Compound:

[0150] Test compound and reference compound were diluted with 100% DMSO to 0.33 μM, then fully automated microplate pretreatment system ECHO was used for a 3-fold dilution with 10 concentration gradients.

[0151] Reaction Operation:

[0152] 1) The substrate was dissolved in freshly prepared buffer,

[0153] 2) The required cofactors was added to the buffer,

[0154] 3) The enzyme was added to the above solution and the mixture was mix well,

[0155] 4) The test sample solution was added and incubated for 20 min at room temperature,

[0156] 5) .sup.33p-ATP was added to the reaction mixture and then incubated at room temperature for 2 hours,

[0157] 6) Radiation signal was detected,

[0158] 7) The results were analyzed with GraphPad prism software.

[0159] Experimental Result:

[0160] As shown in Table 9.

TABLE-US-00009 TABLE 9 The IC.sub.50 value of the compound represented by formula (I) on the inhibition of kinase activity Test compound AXL IC.sub.50 (nM) c-MET IC.sub.50 (nM) Crystal form A of the compound 4.41 2.01 represented by formula (I)

[0161] The experimental result shows that the compound represented by formula (I) has strong inhibitory activity on c-MET and AXL enzyme.

Embodiment 8: Cell Proliferation Inhibition Experiment of the Compound Represented by Formula (I)

[0162] Reagents and Consumables:

[0163] 1) Cell culture: DMEM medium, fetal bovine serum, DPBS

[0164] 2) Cell line: MKN45 gastric cancer cell line

[0165] 3) Detection reagent: live cell detection kit CellTiter-Glo

[0166] 4) Other major consumables and reagents: compound dilution plate, intermediate plate, test plate, DMSO

[0167] Experimental Principle:

[0168] The content of ATP directly reflects the number of cells and their status, and the number of live cells can be detected by quantitative determination of ATP. The Live Cell Assay Kit contains fluorogenic luciferase and its substrate. Through the involvement of ATP, luciferase can catalyze the substrate and emit a stable optical signal, and the content of ATP in the cell can be measured by detecting the intensity of the signal. The light signal is directly proportional to the amount of ATP in the cell, and ATP is positively related to the number of living cells, so that the cell proliferation can be detected. The test plate was analyzed by Envision of PE company.

[0169] Experimental Method:

[0170] 1. Preparation of the Cell Plates

[0171] MKN45 cells were seeded separately into 384-well plates with each of the well containing 200 cells. The cell plates were placed and incubated in a carbon dioxide incubator overnight.

[0172] 2. Preparation of the Compound

[0173] Echo (automatic microplate pretreatment system) was used for 5-fold dilution and 9 concentrations were prepared, double duplicate wells assay was set up.

[0174] 3. Treatment of Cells with the Compound

[0175] The compound was transferred to the cell plates at a starting concentration of 10 μM. The cell plates were incubated in a carbon dioxide incubator for 3 days.

[0176] 4. Detection

[0177] The Promegaer CellTiter-Glo reagent was added to the cell plates and the plates were incubated at room temperature for 10 minutes until the luminescence signal was stable. Reading was performed with a PerkinElmer Envision multi-label analyzer.

[0178] Experimental Result:

[0179] As shown in Table 10.

TABLE-US-00010 TABLE 10 The IC.sub.50 value of the Crystal form A of the compound represented by formula (I) on cell proliferation inhibition Cell name IC.sub.50 (nM) MKN45 cells 7.64

[0180] The result of the experiment shows that the crystal form A of the compound represented by formula (I) has good inhibitory activity on MKN45 cell.

Embodiment 9: In Vivo Pharmacodynamic Studies of the Compound Represented by Formula (I)

[0181] Cell Culture:

[0182] Human gastric cancer HS 746T cells were cultured in a single layer in vitro. The culturing condition was DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin in 37° C., 5% CO.sub.2 incubator. Digestion and passage treatment with trypsin-EDTA was carried out twice a week. When the cell saturation was 80%-90% and the number reached the required level, the cells were collected, counted and seeded.

[0183] Animal:

[0184] BALB/c nude mice, male. 6-8 weeks old, weighting 18-22 g.

[0185] Tumor Inoculation:

[0186] 0.2 mL (2×10.sup.6, cells:Matrigel=1:1) HS 746T cells were inoculated subcutaneously on the right back of each mouse. The drug was administered in groups when the average tumor volume reached approximately 100-150 mm.sup.3.

[0187] Experimental index: The experimental index was whether the tumor growth was inhibited, delayed or cured. The diameters of the tumor were measured twice a week using a vernier caliper. The formula for calculating the tumor volume is V=0.5a×b.sup.2, a and b represent the long and short diameters of the tumor respectively. The antitumor effect (TGI) of the compound was evaluated by T-C (days) and T/C (%).

[0188] Experimental results: As shown in Table 11.

TABLE-US-00011 TABLE 11 Evaluation of anti-tumor efficacy of test drug on human Hs746t gastric cancer cell xenograft tumor model (Calculated based on the tumor volume on the 21st day after administration) Tumor volume (mm.sup.3).sup.a T/C TGI Group (20th day) (%) (%) P vaule.sup.b Blank 2537 ± 425 — — — BMS777607 1872 ± 355 27.61% 73.58 <0.001 LY2801653  88 ± 13 101.62% 3.45 Compound represented by formula (I)  4 ± 2 104.82% 0.15 <0.001 Note: .sup.aaverage value ± SEM; .sup.bp value was calculated based on the tumor volume.

[0189] Conclusion: The compound represented by formula (I) shows better tumor inhibitory effect than BMS777607 and LY2801653 in the pharmacodynamic experiment on Hs746t gastric cancer cell xenograft tumor model.