CRYSTAL FORMS OF PYRIDOPYRAZOLE COMPOUNDS AND PREPARATION METHOD THEREFOR

Abstract

Provided are crystal forms of compounds represented by formula(II)-formula (VIII) and formula (I)-formula (VIII-1), a preparation method therefor and an application of the compounds and crystal forms in the preparation of a drug for treating a related disease.

##STR00001##

Claims

1. A compound selected from a compound of Formulas (II) to (VIII), ##STR00026## ##STR00027## ##STR00028## wherein n.sub.2, n.sub.3, n.sub.4, n.sub.5, n.sub.6, n.sub.7 and n.sub.8 are selected from 0.8-1.5.

2. The compound according to claim 1, wherein n.sub.2, n.sub.3, n.sub.4, n.sub.5, n.sub.6, n.sub.7 and n.sub.8 are each independently selected from the group consisting of: 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 and 1.5.

3. The compound according to claim 2, wherein selected from the group consisting of: ##STR00029## ##STR00030##

4. A crystal form A of the compound of Formula (I), wherein an X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the 2θ angles of: 16.30±0.20°, 21.69±0.20° and 24.63±0.20° ##STR00031##

5. The crystal form A according to claim 4, wherein the X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the 2θangles of: 14.88±0.20°, 15.51±0.20°, 16.30±0.20°, 18.49±0.20°, 19.16±0.20°, 19.70±0.20°, 21.69±0.20° and 24.63±0.20°.

6. The crystal form A according to claim 5, wherein the X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the 2θangles of: 7.79±0.20°, 9.58±020°, 12.61±0.20°, 14.88±0.20°, 15.51±0.20°, 16.30±0.20°, 18.49±0.20°, 19.16±0.20°, 19.70±0.20°, 21.69±0.20° and 24.63±0.20°.

7. The crystal form A according to claim 4, wherein the XRPD pattern of the crystal form A is as shown in FIG. 1.

8. The crystal form A according, to claim 4, wherein a differential scanning calorimetry curve of the crystal form A has a starting point of an endothermic peak at 188.7±2° C.

9. The crystal form A according to claim 8, wherein a DSC thermogram of the crystal form A is as shown in FIG. 2.

10. The crystal form A according to claim 4, wherein a thermogravimetric analysis curve of the crystal form A shows a weight loss of 1.20% at 180.0±3° C.

11. The crystal form A according to claim 10, wherein a TGA spectrum of the crystal form A is as shown in FIG. 3.

12. A method of treating solid tumor in a subject in need thereof, comprising administering an effective amount of at least one of the compounds of Formulas (II) to (VIII) according to claim 1 to the subject, wherein the solid tumor refers to an RET kinase-associated solid tumor.

13. The method according to claim 12, wherein the solid tumor refers to an RET kinase-associated solid tumor.

14. The crystal form A according to claim 5, wherein a differential scanning calorimetry curve of the crystal form A has a starting point of an endothermic peak at 188.7±2° C.

15. The crystal form A according to claim 6, wherein a differential scanning calorimetry curve of the crystal form A has a starting point of an endothermic peak at 188.7±2° C.

16. The crystal form A according to claim 5, wherein a thermogravimetric analysis curve of the crystal form A shows a weight loss of 1.20% at 180.0±3° C.

17. The crystal form A according to claim 6, wherein a thermogravimetric analysis curve of the crystal form A shows a weight loss of 1.20% at 180.0±3° C.

18. A method of treating solid tumor in a subject in need thereof, comprising administering an effective amount of the crystal form A of the compound of Formula (I) according to claim 4 to the subject.

19. The method according to claim 18, wherein the solid tumor refers to an RET kinase-associated solid tumor.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0184] FIG. 1 is a Cu-Kα-radiated XRPD pattern of crystal form A of the compound of formula (I).

[0185] FIG. 2 is a DSC thermogram of crystal form A of the compound of formula (I).

[0186] FIG. 3 is a TGA spectrum of crystal form A of the compound of formula (I).

[0187] FIG. 4 is a Cu-Kα-radiated XRPD pattern of Cu-Kα radiation of crystal form B of the compound of formula (I).

[0188] FIG. 5 is a Cu-Kα-radiated XRPD pattern of crystal form C of the compound of formula (I).

[0189] FIG. 6 is a DSC thermogram of crystal form C of the compound of formula (I).

[0190] FIG. 7 is a TGA spectrum of crystal form C of the compound of formula (I).

[0191] FIG. 8 is a Cu-Kα-radiated XRPD pattern of crystal form D of the compound of formula (I).

[0192] FIG. 9 is a Cu-Kα-radiated XRPD pattern of crystal form E of the compound of formula (I).

[0193] FIG. 10 is a DSC thermogram of crystal form E of the compound of formula (I).

[0194] FIG. 11 is a TGA spectrum of crystal form E of the compound of formula (I).

[0195] FIG. 12 is a Cu-Kα-radiated XRPD pattern of crystal form F of the compound of formula (I).

[0196] FIG. 13 is a Cu-Kα-radiated XRPD pattern of crystal form G of the compound of formula (II-1).

[0197] FIG. 14 is a TGA spectrum of crystal form G of the compound of formula (II-1).

[0198] FIG. 15 is a Cu-Kα-radiated XRPD pattern of crystal form H of the compound of formula (II-1).

[0199] FIG. 16 is a TGA spectrum of crystal form H of the compound of formula (II-1).

[0200] FIG. 17 is a Cu-Kα-radiated XRPD pattern of crystal form I of the compound of formula (III-1).

[0201] FIG. 18 is a DSC thermogram of crystal form I of the compound of formula (III-1).

[0202] FIG. 19 is a TGA spectrum of crystal form I of the compound of formula (III-1).

[0203] FIG. 20 is a Cu-Kα-radiated XRPD pattern of crystal form J of the compound of formula (IV-1).

[0204] FIG. 21 is a TGA spectrum of crystal form J of the compound of formula (IV-1).

[0205] FIG. 22 is a Cu-Kα-radiated XRPD pattern of crystal form K of the compound of formula (V-1).

[0206] FIG. 23 is a TGA spectrum of crystal form K of the compound of formula (V-1).

[0207] FIG. 24 is a Cu-Kα-radiated XRPD pattern of crystal form L of the compound of formula (V-1).

[0208] FIG. 25 is a TGA spectrum of crystal form L of the compound of formula (V-1).

[0209] FIG. 26 is a Cu-Kα-radiated XRPD pattern of crystal form M of the compound of formula (VI-1).

[0210] FIG. 27 is a TGA spectrum of crystal form M of the compound of formula (VI-1).

[0211] FIG. 28 is a Cu-Kα-radiated XRPD pattern of crystal form N of the compound of formula (VII-1).

[0212] FIG. 29 is a TGA spectrum of crystal form N of the compound of formula (VII-1).

[0213] FIG. 30 is a Cu-Kα-radiated XRPD pattern of crystal form 0 of the compound of formula (VII-1).

[0214] FIG. 31 is a TGA spectrum of crystal form 0 of the compound of formula (VII-1).

[0215] FIG. 32 is a Cu-Kα-radiated XRPD pattern of crystal form P of the compound of formula (VII-1).

[0216] FIG. 33 is a TGA spectrum of crystal form P of the compound of formula (VII-1).

[0217] FIG. 34 is a Cu-Kα-radiated XRPD pattern of crystal form Q of the compound of formula (VIII-1).

[0218] FIG. 35 is a TGA spectrum of crystal form Q of the compound of formula (VIII-1).

[0219] FIG. 36 is a Cu-Kα-radiated XRPD pattern of crystal form R of the compound of formula (VIII-1).

[0220] FIG. 37 is a TGA spectrum of crystal form R of the compound of formula (VIII-1).

[0221] FIG. 38 is a DVS plot of crystal form A of the compound of formula (I).

[0222] FIG. 39 is a Cu-Kα-radiated XRPD pattern of crystal form A of the compound of formula (I).

DETAILED DESCRIPTION

[0223] In order to better understand the content of the present invention, the present invention will be further illustrated below in conjunction with specific examples, and the specific embodiments are not intended to limit the content of the present invention.

Example 1: Preparation of Compound of Formula (I) and Trifluoroacetate Thereof

[0224] ##STR00017## ##STR00018##

Step 1

[0225] 2,5-dibromopyrazine (4 g, 16.82 mmol) and 6-tert-butyloxycarbonyl-3,6-diazabicyclo[3.1.1]-heptane (4.00 g, 20.18 mmol) were dissolved in N-methylpyrrolidone (50 mL), diisopropylethylamine (6.52 g, 50.45 mmol, 8.79 mL) was added, and the mixture was stirred at 100° C. for 16 hours. 60 mL of water was added, extraction was carried out with ethyl acetate (100 mL×3), the organic phases were combined, washed with water (150 mL×5) and a saturated sodium chloride solution (150 mL×1), and dried over anhydrous sodium sulfate, and finally, the solvent was dried off by spinning to obtain a crude product. The crude product was purified by an automated column chromatography (petroleum ether:ethyl acetate=4:1) to obtain compound 1.

[0226] LCMS (ESI) m/z: 354.9 [M+1].sup.+, 356.9 [M+3].sup.+;

[0227] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.15 (d, J=1.2 Hz, 1H), 7.77 (s, 1H), 4.28-4.31 (m, 2H), 3.90-4.12 (m, 2H), 3.42 (d, J=12.0 Hz, 2H), 2.64-2.72 (m, 1H), 1.50 (d, J=12.4 Hz, 1H), 1.38 (s, 9H).

Step 2

[0228] Compound 1 (3 g, 8.45 mmol) was dissolved in ethyl acetate (15 mL), hydrogen chloride/ethyl acetate (4 M, 20 mL) was added, and the mixture was stirred at 16° C. for 3 hours. The solvent was dried off by spinning to obtain crude product 2, which was directly subjected to the next step of reaction without purification.

[0229] LCMS (ESI) m/z: 254.9 [M+1].sup.+, 256.9 [M+3].sup.+.

Step 3

[0230] Compound 2 (2.45 g, 8.40 mmol) and 6-methoxy-3-pyridylaldehyde (2.30 g, 16.81 mmol) were added to DCM (50 mL), sodium borohydride acetate (5.34 g, 25.21 mmol) was then added, and the mixture was stirred at 16° C. for 1.5 hours. The reaction liquid became clear. 50 mL of water was added to the reaction liquid, extraction was carried out with dichloromethane (50 mL×3), the organic phases were combined, washed with a saturated sodium chloride solution (100 mL×1), and dried over anhydrous sodium sulfate, and finally, the solvent was dried off by spinning to obtain a crude product. The crude product was purified by an automated column chromatography (petroleum ether:ethyl acetate=1:3 to dichloromethane:methanol=10:1) to obtain compound 3.

[0231] LCMS (ESI) m/z: 376.0 [M+1].sup.+, 378.0 [M+3].sup.+.

Step 4

[0232] Compound 3 (1.8 g, 4.78 mmol) and bis(pinacolato)diboron (1.82 g, 7.18 mmol) were dissolved in 1,4-dioxane (15 mL), and [1,1′-bis(diphenylphosphino)ferrocene]dichl oropalladium(II) (350.05 mg, 478.40 μmol) and potassium acetate (1.41 g, 14.35 mmol) were added, and the mixture was stirred at 80° C. for 16 hours under nitrogen protect ion. Some dehalogenation by-products were obtained in the reaction. The reaction liquid was directly filtered and washed with ethyl acetate twice, and the filtrate was spin-dried to obtain crude product 4, which was directly used for the next step of reaction.

[0233] LCMS (ESI) m/z: 342.1 [M+1].sup.+.

Step 5

[0234] 3,6-Dihydro-2H-pyran-4-boronic acid pinacol ester (1.5 g, 7.14 mmol), compound 5 (1.80 g, 7.14 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (261.23 mg, 357.01 μmol) and potassium phosphate (4.55 g, 21.42 mmol) were added together to 1,4-dioxane (12 mL) and water (6 mL), then heated to 100° C. by a microwave synthesizer under nitrogen protection and stirred for 30 minutes. 20 mL of water and 20 mL of ethyl acetate were added to the reaction liquid for extraction. After liquid separation, the aqueous phase was then extracted with 20 mL of ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by flash column chromatography on silica gel (petroleum ether/ethyl acetate=1/1) to obtain compound 6.

[0235] LCMS (ESI) m/z: 255.9 [M+1].sup.+.

Step 6

[0236] Pyridine hydrochloride (4.53 g, 39.17 mmol) was added to compound 6 (1 g, 3.92 mmol), then heated to 180° C. by microwave under nitrogen protection and stirred for 20 minutes. A saturated aqueous sodium bicarbonate solution was added to the reaction liquid until the pH value was 7. 50 mL of ethyl acetate was then added for extraction. After liquid separation, the aqueous phase was then extracted with 50 mL of ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain crude product 7, which was directly used in the next step without further purification.

Step 7

[0237] Compound 7 (460 mg, 1.91 mmol), N-phenyl-bis(trifluoromethanesulfonyl)imide (1.02 g, 2.86 mmol) and diisopropylethylamine (739.31 mg, 5.72 mmol) were added together to N,N-dimethylformamide (10 mL) and then stirred under nitrogen protection at 10-20° C. for 16 hours. The reaction liquid was directly added to 50 mL of water, 20 mL of ethyl acetate was then added for extraction. After liquid separation, the aqueous phase was then extracted with 20 mL of ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain crude product 8.

Step 8

[0238] Compound 8 (560 mg, 1.50 mmol), compound 4 (511.79 mg, 1.50 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (54.88 mg, 75.01 μmol) and potassium phosphate (955.27 mg, 4.50 mmol) were added together to 1,4-dioxane (12 mL) and water (6 mL), then heated to 90° C. by a microwave synthesizer under nitrogen protection and reacted with stirred for 0.5 hours. 20 mL of water and 20 mL of ethyl acetate were added to the reaction liquid. After liquid separation, the aqueous phase was then extracted with 20 mL of ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and rotary evaporation was carried out to obtain crude product of Formula (I). The crude product was purified by a preparative chromatographic column (YMC-Triart Prep C18 150×40 mm×7 μm; mobile phase: [water (0.1% TFA)-ACN]; acetonitrile: 30-40%, 10 min) to obtain a trifluoroacetate of the compound of Formula (I). The trifluoroacetate of the compound of Formula (I) was added to a sodium bicarbonate solution and extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the compound of Formula (I).

[0239] LCMS (ESI) m/z: 521.1 [M+1]+;

[0240] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.81-8.80 (m, 1H), 8.71-8.65 (m, 1H), 8.48-8.45 (m, 1H), 8.40-8.30 (m, 2H), 8.00-7.92 (m, 1H), 7.90-7.92 (m, 1H), 6.95-6.92 (m, 2H), 6.53 (s, 1H), 4.74-4.71 (m, 2H), 4.63-4.61 (d, J=8 Hz, 1H), 4.39-4.37 (m, 2H), 4.43-4.30 (m, 3H), 4.10-4.06 (m, 1H), 4.00-3.96 (m, 6H), 3.65-3.62 (m, 1H), 2.62 (s, 2H), 2.25-2.21 (m, 1H).

Example 2: Preparation of Crystal Form a of the Compound of Formula (I)

[0241] The crude product of Formula (I) was mixed with silica gel and then purified by an automated column chromatography (dichloromethane:methanol=20:1) to obtain the compound of Formula (I), 200 mL of methanol (40 folds) was added to 5 g of the compound of Formula (I) and pulped for 16 hours overnight, and after solid precipitation, filtration was carried out to obtain crystal form A of the compound of Formula (I). The XRPD pattern thereof was as shown in FIG. 1, the DSC thermogram thereof was as shown in FIG. 2, and a TGA spectrum thereof was as shown in FIG. 3.

[0242] About 400 g of the crude product of Formula (I) was added to about 6 L of methanol, stirred at 20-30° C. for 88-96 hours, the reaction liquid was filtered, and the filter cake was rinsed with methanol (0.5 L) and dried in vacuum for 16-24 hours. 5.78 L of purified water was added to the obtained solid, the temperature was controlled at 90-100° C., the mixture was stirred for 24-48 hours and cooled to 20-30° C., and the reaction liquid was filtered and dried in vacuum for 40-96 hours to obtain crystal form A. The XRPD pattern thereof was as shown in FIG. 39.

Example 3: Preparation of Crystal Form B of the Compound of Formula (I)

[0243] The compound of Formula (I) (20.9 mg) was added to 1.0 mL of 1,4-dioxane and stirred at room temperature to obtain a suspension, and after centrifugal separation and drying, a solid, i.e., crystal form B of the compound of Formula (I) was obtained. The XRPD pattern thereof was as shown in FIG. 4.

Example 4: Preparation of Crystal Form C of the Compound of Formula (I)

[0244] Crystal form B of the compound of Formula (I) was placed in open containers at room temperature overnight to obtain a solid, i.e., crystal form C of the compound of Formula (I). The XRPD pattern thereof was as shown in FIG. 5, the DSC thermogram thereof was as shown in FIG. 6, and the TGA spectrum thereof was as shown in FIG. 7.

Example 5: Preparation of Crystal Form D of the Compound of Formula (I)

[0245] Crystal form A of the compound of Formula (I) (20.6 mg) was added to 0.5 mL of 1,4-dioxane/n-butanol (volume ratio 1:1) and stirred at room temperature for 5 days to form a suspension, and after centrifugal separation and drying, a solid, i.e., crystal form D of the compound of Formula (I) was obtained. The XRPD pattern thereof was as shown in FIG. 8.

Example 6: Preparation of Crystal Form E of the Compound of Formula (I)

[0246] The compound of Formula (I) (20.6 mg) was added to 2.0 mL of acetone and stirred at 50° C. for 1 hour to obtain a suspension, the suspension was filtered to obtain a clear solution, the temperature was reduced from 50° C. to 5° C. within 40 hours to precipitate out a small amount of solid precipitate, the solution was then transferred to −20° C. and a solid was obtained after 6 days, and after centrifugal separation and drying, crystal form E of the compound of Formula (I) was obtained. The XRPD pattern thereof was as shown in FIG. 9, the DSC thermogram thereof was as shown in FIG. 10, and the TGA spectrum thereof was as shown in FIG. 11.

Example 7: Preparation of Crystal Form F of the Compound of Formula (I)

[0247] The compound of Formula (I) (20.5 mg) was added to 2.0 mL of dimethyl tetrahydrofuran and stirred at 50° C. for 1 hour to obtain a suspension, the suspension was filtered to obtain a clear solution, the temperature was reduced from 50° C. to 5° C. within 40 hours to precipitate out a small amount of solid, the solution was then transferred to −20° C. and a solid was obtained after 6 days, and after centrifugal separation and drying, crystal form F of the compound of Formula (I) was obtained. The XRPD pattern thereof was as shown in FIG. 12.

Example 8: Preparation of Crystal Form G of the Compound of Formula (II-1)

[0248] ##STR00019##

[0249] The compound of Formula (I) (19.9 mg) was added to 0.5 mL of ethanol/water (volume ratio 9:1) containing 4.8 mg of maleic acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form G of the compound of Formula (II-1). The XRPD pattern thereof was as shown in FIG. 13 and the TGA spectrum thereof was as shown in FIG. 14.

Example 9: Preparation of Crystal Form H of the Compound of Formula (II-1)

[0250] The compound of Formula (I) (20.7 mg) was added to 0.5 mL of acetone containing 4.8 mg of maleic acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form H of the compound of Formula (II-1). The XRPD pattern thereof was as shown in FIG. 15 and the TGA spectrum thereof was as shown in FIG. 16.

Example 10: Preparation of Crystal Form I of the Compound of Formula (III-1)

[0251] ##STR00020##

[0252] The compound of Formula (I) (20.5 mg) was added to 0.5 mL of ethanol/water (volume ratio 9:1) containing 8.6 mg of mucic acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form I of the compound of Formula (III-1). The XRPD pattern thereof was as shown in FIG. 17, the DSC thermogram thereof was as shown in FIG. 18, and the TGA spectrum thereof was as shown in FIG. 19.

Example 11: Preparation of Crystal Form J of the Compound of Formula (IV-1)

[0253] ##STR00021##

[0254] The compound of Formula (I) (19.1 mg) was added to 0.5 mL of ethanol/water (volume ratio 9:1) containing 5.8 mg of tartaric acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form J of the compound of Formula (IV-1). The XRPD pattern thereof was as shown in FIG. 20 and the TGA spectrum thereof was as shown in FIG. 21.

Example 12: Preparation of Crystal Form K of the Compound of Formula (V-1)

[0255] ##STR00022##

[0256] The compound of Formula (I) (20.4 mg) was added to 0.5 mL of ethanol/water (volume ratio 9:1) containing 4.4 mg of fumaric acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form K of the compound of Formula (V-1). The XRPD pattern thereof was as shown in FIG. 22 and the TGA spectrum thereof was as shown in FIG. 23.

Example 13: Preparation of Crystal Form L of the Compound of Formula (V-1)

[0257] The compound of Formula (I) (20.7 mg) was added to 0.5 mL of acetone containing 4.6 mg of fumaric acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form L of the compound of Formula (V-1). The XRPD pattern thereof was as shown in FIG. 24 and the TGA spectrum thereof was as shown in FIG. 25.

Example 14: Preparation of Crystal Form M of the Compound of Formula (VI-1)

[0258] ##STR00023##

[0259] The compound of Formula (I) (19.4 mg) was added to 0.5 mL of acetone containing 7.4 mg of citric acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form M of the compound of Formula (VI-1). The XRPD pattern thereof was as shown in FIG. 26 and the TGA spectrum thereof was as shown in FIG. 27.

Example 15: Preparation of Crystal Form N of the Compound of Formula (VII-1)

[0260] ##STR00024##

[0261] The compound of Formula (I) (19.9 mg) was added to 0.5 mL of ethanol/water (volume ratio 9:1) containing 5.0 mg of oxalic acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form N of compound of Formula (VII-1). The XRPD pattern thereof was as shown in FIG. 28 and the TGA spectrum thereof was as shown in FIG. 29.

Example 16: Preparation of Crystal Form O of the Compound of Formula (VII-1)

[0262] The compound of Formula (I) (19.8 mg) was added to 0.5 mL of acetone containing 5.5 mg of oxalic acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form O of the compound of Formula (VII-1). The XRPD pattern thereof was as shown in FIG. 30 and the TGA spectrum thereof was as shown in FIG. 31.

Example 17: Preparation of Crystal Form P of the Compound of Formula (VII-1)

[0263] The compound of Formula (I) (19.3 mg) was added to 0.5 mL of ethyl acetate containing 5.1 mg of oxalic acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form P of the compound of Formula (VII-1). The XRPD pattern thereof was as shown in FIG. 32 and the TGA spectrum thereof was as shown in FIG. 33.

Example 18: Preparation of Crystal Form Q of the Compound of Formula (VIII-1)

[0264] ##STR00025##

[0265] The compound of Formula (I) (19.3 mg) was added to 0.5 mL of ethanol/water (volume ratio 9:1) containing 3.9 mg of phosphoric acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form Q of compound of Formula (VIII-1). The XRPD pattern thereof was as shown in FIG. 34 and the TGA spectrum thereof was as shown in FIG. 35.

Example 19: Preparation of Crystal Form R of the Compound of Formula (VIII-1)

[0266] The compound of Formula (I) (19.7 mg) was added to 0.5 mL of acetone containing 4.4 mg of phosphoric acid, stirred at room temperature for 2 days to form a suspension, and after centrifugation, evacuation was performed on the solid in vacuum at room temperature for 1 hour to obtain a solid, i.e., crystal form R of the compound of Formula (VIII-1). The XRPD pattern thereof was as shown in FIG. 36 and the TGA spectrum thereof was as shown in FIG. 37.

Example 20: Hygroscopicity Study on Crystal Form a of the Compound of Formula (I)

[0267] Experimental Material:

[0268] SMS DVS Intrinsic Dynamic Vapor Sorption Instrument

[0269] Experimental Method:

[0270] 10-15 mg of crystal form A of the compound of Formula (I) was taken and placed in a DVS sample tray for testing.

[0271] Experimental Results:

[0272] A DVS plot of crystal form A of the compound of Formula (I) was as shown in FIG. 38, ΔW=0.8%.

[0273] Experimental Conclusion:

[0274] The hygroscopic weight gain of crystal form A of the compound of Formula (I) was 0.8% at 25° C. and 80% RH, i.e., being slightly hygroscopic.

Example 21: Solid Stability Experiment of Crystal Form a of the Compound of Formula (I)

[0275] According to the Guidelines for the Stability Testing of Raw Materials and Preparations (General Chapter No. 9001 part of Volume IV of Chinese Pharmacopoeia 2015 Edition), the stability of crystal form A of the compound of Formula (I) was investigated under conditions of high temperature (60° C., open) and high humidity (room temperature/relative humidity 92.5%, open).

[0276] 15 mg of crystal form A of the compound of Formula (I) was weighed out, placed at the bottom of a glass sample flask, spread out into a thin layer. For samples placed under high temperature and high humidity, use aluminum foil to seal the bottle, and prick some holes on the aluminum foil to ensure that the sample can fully contact with the ambient air. The samples placed under different conditions were sampled for XRPD detection on Days 5 and 10, and the test results were compared with the initial test results on Day 0. The test results were shown in Table 19 below:

TABLE-US-00021 TABLE 19 Solid stability experiment results of crystal form A of the compound of Formula (I) Test conditions Time point Crystal form — Day 0 Crystal form A High temperature (60° C., open) Day 5 Crystal form A Day 10 Crystal form A High humidity (room temperature/ Day 5 Crystal form A relative humidity 92.5%, open) Day 10 Crystal form A

[0277] Conclusion: The crystal form A of the compound of Formula (I) had good stability under conditions of high temperature and high humidity.

Example 22: Solid Stability Study on Crystal Form a of the Compound of Formula (I)

[0278] According to the Guidelines for the Stability Testing of Raw Materials and Preparations (General Chapter No. 9001 of Volume IV of Chinese Pharmacopoeia 2015 Edition), the stability of crystal form A of the compound of Formula (I) was investigated under long-term experimental conditions. Approximately 10 mg of crystal form A of the compound of Formula (I) was weighed out, placed at the bottom of a glass sample flask, spread into a thin layer and sealed with an aluminum foil, the aluminum foil was pierced with small holes. The flasks were placed under 40° C./75% RH conditions for 3 months or under 25° C./60% RH conditions for 3 months, then sampled for XRPD detection, and the detection results were compared with the initial test results of Day 0. The results were as shown in Table 20. The crystal form A of the compound of Formula (I) had no crystal form change under all stability conditions.

[0279] The experimental results were shown in Table 20 below:

TABLE-US-00022 TABLE 20 Solid stability experiment results of crystal form A of the compound of Formula (I) Test conditions Condition of taking points Crystal form Initial crystal form A / Crystal form A 40° C./75% RH 3 months Crystal form A 25° C./60% RH 3 months Crystal form A

[0280] Experimental conclusion: The crystal form A of the compound of Formula (I) had good stability.

Biological Test Data:

Experiment 1: In Vitro Enzyme Activity Test of the Compound of the Present Invention

Experimental Purpose

[0281] The enzyme activity was experimentally detected by Z'-LYTE™ kinase test, and the inhibitory effect of the compound on RET and RET (V804M) kinase was evaluated with the IC.sub.50 value of the compound as an indicator.

[0282] Experimental Method

[0283] The concentration of the compound used for the RET and RET (V804M) kinase test was diluted by a factor of 3, giving 10 concentrations from 3 μM to 0.152 nM. The content of DMSO in the detection reaction was 1%.

[0284] Reagents:

[0285] Basic reaction buffer, 20 mM hydroxyethyl piperazine-ethanesulfonic acid (Hepes) (pH 7.5) buffer, 10 mM MgCl.sub.2, 1 mM ethylene glycol bis(aminoethyl ether)tetraacetic acid (EGTA), 0.02% polyoxyethylene dodecyl ether (Brij35), 0.02 mg/mL bovine serum protein, 0.1 mM Na.sub.3VO.sub.4, 2 mM dithiothreitol (DTT) and 1% DMSO.

Compound:

[0286] The compound to be tested was dissolved in 100% DMSO system and diluted to 10 mM for use. Integra Viaflo Assist was used for solution dilution.

Reaction Process of Generic Enzyme:

[0287] Test conditions: The concentration of RET enzyme was 3 μM, the concentration of the peptide substrate CHKtide was 1000 μM, and the concentration of ATP was 20 μM; and the concentration of RET (V804M) enzyme was 80 μM, the concentration of substrate peptide was 1000 μM, and the concentration of ATP was 30 μM.

[0288] Reaction process: A kinase/polypeptide solution was prepared according to the test conditions. Compound solutions of different concentrations were added, incubation was carried out at room temperature for 20 minutes, 33P-ATP at the corresponding concentration was added, and incubation was carried out at room temperature for 120 minutes. Radioactivity was detected by filter-binding method.

Reaction Detection:

[0289] Phosphoric acid with a concentration of 0.5% was added to the kinase reaction solution to stop the reaction, and Envision instrument was used for plate reading.

Data Analysis

[0290] The data were converted into phosphorylation rate and inhibition rate, and the IC.sub.50 data of the compound was obtained by parameter curve fitting (GraphPad Software).

The Experimental Results were Shown in Table 21:

TABLE-US-00023 TABLE 21 IC.sub.50 test results of kinase activity of the compound of the present invention RET enzyme RET V804M Test article IC.sub.50 (nM) IC.sub.50 (nM) Trifluoroacetate of the 0.72 5.86 compound of Formula (I)

[0291] Experimental conclusion: The compound of the present invention had excellent inhibitory activity on RET and its mutant RET V804M and would have excellent therapeutic effects on patients with abnormal RET tumors.

Experiment 2: Pharmacokinetic Evaluation of the Compound of the Present Invention

[0292] Experimental procedure: A 0.1 mg/mL clear solution of the test compound in the corresponding solvent medium (see Table 22) was injected into female Balb/c mice (fasting overnight, 7-9 weeks old) via tail vein at a dose of 0.2 mg/kg. About 30 μL of blood was collected from jugular vein or tail vein at 0.0833, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 24 h after intravenous administration. 0.2 mg/mL of the test compound suspended in the corresponding solvent medium (see Table 22) was given to female Balb/c mice (fasting overnight, 7-9 weeks old) by gavage at a dose of 2 mg/kg. The experimental conditions were detailed in Table 22. At 0.0833, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 h after oral administration, about 30 μL of blood was collected from the jugular vein or tail vein of the female Balb/c mice. The blood was placed in an anticoagulant tube in which EDTA-K2 had been added, and plasma was separated by centrifugation. The plasma concentration was determined by LC-MS/MS method, and the relative pharmacokinetic parameters were calculated using WinNonlin™ Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software by non-compartment model linear logarithmic trapezoidal method. The experimental results were as shown in Table 23.

TABLE-US-00024 TABLE 22 Experimental conditions for pharmacokinetics in mice IV (injection) PO (oral) Dose Solvent medium Dose Solvent medium Trifluoroacetate 0.2 mg/kg 0.1 mg/mL 2 mg/kg 0.2 mg/mL of the compound 5% DMSO + 10% 5% DMSO + 10% of Formula (I) polyethylene glycol-15 polyethylene glycol-15 hydroxystearate hydroxystearate (Solutol) + 85% H.sub.2O (Solutol) + 85% H.sub.2O clear solution clear solution

TABLE-US-00025 TABLE 23 Experimental results of pharmacokinetics in mice IV 0.2 mg/kg P0 Cl 2 mg/kg (mL/min/ V.sub.dss T.sub.1/2 AUC.sub.0-last C.sub.max T.sub.max AUC.sub.0-last F Dose kg) (L/kg) (h) (nM .Math. h) (nM) (h) (nM .Math. h) (%) Trifluoro- 2.3 0.5 2.6 2794 5340 2 26926 96.4 acetate of the compound of Formula (I) Note: Plasma clearance (Cl), apparent steady-state distribution volume (V.sub.dss), elimination half-life (T.sub.1/2), area under plasma concentration curve from 0 to the last quantifiable time point (AUC.sub.0-last), bioavailability (F), peak concentration (C.sub.max) and peak time T.sub.max.

[0293] Conclusion: From the experimental results, the compounds of the present invention all exhibited a low clearance, a low distribution volume, a relatively long half-life and excellent drug exposure after intravenous administration. For oral administration, the compounds of the present invention all exhibited relatively a short T.sub.max, excellent oral absorption exposure and overall showed excellent oral absorption bioavailability.

Experiment 3: Analysis of Tumor Growth Inhibition (TGI)

[0294] Ba/F3-CCDC6-RET cell strain was cultured using 1640 medium (Biological Industries)+10% fetal bovine serum (BI)+1% double antibody (Penicillin Streptomycin solution, Coring, USA) at 37° C. with 5% CO.sub.2 and subcultured twice a week. When the cell saturation is 80-90%, cells were collected, counted, and inoculated subcutaneously into the right axillary of BALB/c nude female mice (6-8 weeks). After the inoculation was completed, the tumor growth status was observed day by day. When the average tumor volume reached about 165.77 mm.sup.3, the mice were randomly divided into groups, with 6 mice per group, and administration was started.

[0295] The health status and death of the animals were detected daily. Routine examinations included tumor growth, activity, diet, weight, eyes, hair and other abnormal behaviors of the animals, and the tumor volume and weight were measured twice a week (Tuesday and Friday).

[0296] The inhibitory effect of the compound on tumor growth was evaluated by the relationship between tumor volume and time. The tumor volume was measured by vernier caliper, with the formula being TV=0.5 a×b.sup.2, wherein, “a” was the long diameter of the tumor and “b” was the short diameter of the tumor. TGI was calculated by the difference between the median tumor volume of the mice in the solvent group and the median tumor volume of the mice in the drug group, expressed as the percentage of the median tumor volume in the solvent control group,

[0297] it was calculated by the following formula:

TGI(TGI(%)=[1−(T.sub.23−T.sub.0)/(V.sub.23−V.sub.0)]×100)

[0298] Unless otherwise specified, the data were expressed as mean±standard error (Mean±SE), and one way ANOVA test method was used to determine whether there was significant difference between the tumor volume in the treatment group and the tumor volume in the control group. P<0.05 referred to significant difference. 5% DMSO+10% polyethylene glycol-15 hydroxystearate (Solutol)+85% H.sub.2O was used as negative control. The experimental results were as shown in Table 24.

TABLE-US-00026 TABLE 24 Experimental results of antitumor activity in mice Ba/F3-CCDC6-RET TGI % (tumor volume cell xenograft on Day 23 after P Test article tumor model administration) value Trifluoroacetate 10 mg/kg.sup.(D 0-D 13)/ 98 <0.001 of the compound 5 mg/kg.sup.(D 14-D 17)/ of Formula (I) 2.5 mg/kg.sup.(D 18-D 28) (BID) Note: BID: twice a day; QD: once a day; and TGI %: tumor growth inhibition rate.

[0299] Conclusion: The compound of the present invention exhibited excellent tumor growth inhibition effect in the tumor model Ba/F3-CCDC6-RET.