CRYSTAL FORM OF PYRIDINYLPHENYL COMPOUND AND PREPARATION METHOD THEREFOR

Abstract

A crystal form of a pyridinylphenyl compound and a preparation method therefor. Use of the crystal form in the preparation of a medicament of treating related diseases is also comprised.

Claims

1-10. (canceled)

11. A crystal form B of a compound of formula (II), wherein the crystal form B has an X-ray powder diffraction pattern comprising characteristic diffraction peaks at the following 2θ angles: 8.231±0.200°, 17.058±0.200° and 18.955±0.200°, ##STR00017##

12. The crystal form B according to claim 11, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 8.231±0.200°, 17.058±0.200°, 18.955±0.200°, 21.712±0.200° and 25.678±0.200°.

13. The crystal form B according to claim 12, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 8.231±0.200°, 12.068±0.200°, 16.505±0.200°, 17.058±0.200°, 18.955±0.200°, 21.712±0.200°, 24.242±0.200° and 25.678±0.200°.

14. The crystal form B according to claim 13, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 7.083±0.200°, 8.231±0.200°, 11.294±0.200°, 12.068±0.200°, 14.091±0.200°, 16.505±0.200°, 17.058±0.200°, 18.955±0.200°, 21.712±0.200°, 24.242±0.200°, 25.678±0.200° and 30.869±0.200°.

15. The crystal form B according to claim 14, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 7.083°, 8.231°, 11.294°, 12.068°, 14.091°, 14.806°, 16.505°, 17.058°, 18.202°, 18.955°, 21.712°, 22.182°, 23.903°, 24.242°, 24.653°, 25.3500, 25.678°, 26.270°, 27.001°, 27.658°, 29.052°, 29.721°, 30.869°, 34.644° and 35.009°.

16. The crystal form B according to claim 15, wherein the XRPD pattern thereof is as shown in FIG. 3.

17. The crystal form B according to claim 11, wherein the differential scanning calorimetry curve thereof has endothermic peaks with an onset at 101.7±3.0° C. and 158.7±3.0° C., respectively; or, the thermogravimetric analysis curve thereof has a weight loss of 5.477% at 120.00° C.±3.0° C.

18. The crystal form B according to claim 17, wherein the DSC spectrum thereof is as shown in FIG. 4; or, the TGA spectrum thereof is as shown in FIG. 5.

19-65. (canceled)

66. A method for treating dry eye in a subject in need thereof, comprising administering to the subject an effective amount of the crystal form B according to claim 11.

67. A compound of formula (II), ##STR00018##

68. A compound, of formula (III), formula (IV) or formula (V): ##STR00019## m is selected from 0 to 1, preferably 0, 0.25, 0.5 or 1; or, ##STR00020## n is selected from 0 to 1, preferably 0, 0.25, 0.5 or 1; or, ##STR00021##

69. The compound according to claim 68, wherein the compound of formula (III) is the compound of formula (III-1), ##STR00022## or, the compound of formula (IV) is the compound of formula (IV-1), ##STR00023##

70. A crystal form, wherein the crystal form is a crystal form A of a compound of formula (I), wherein the crystal form A has an X-ray powder diffraction pattern comprising characteristic diffraction peaks at the following 2θ angles: 7.14±0.20°, 12.46±0.20° and 18.87±0.20°, ##STR00024## or, a crystal form C of a compound of formula (I), wherein the crystal form C has an X-ray powder diffraction pattern comprising characteristic diffraction peaks at the following 2θ angles: 12.871±0.200°, 17.488±0.200° and 19.079±0.200°, ##STR00025## or, a crystal form D of the compound of formula (III-1) according to claim 69, wherein the crystal form D has an X-ray powder diffraction pattern comprising characteristic diffraction peaks at the following 2θ angles: 6.600±0.200°, 17.775±0.200° and 19.138±0.200°, ##STR00026## or, a crystal form E of the compound of formula (IV-1) according to claim 69, wherein the crystal form E has an X-ray powder diffraction pattern comprising characteristic diffraction peaks at the following 2θ angles: 18.097±0.200°, 18.691±0.200° and 20.693±0.200°, ##STR00027## or, a crystal form F of a compound of formula (V), wherein the crystal form F has an X-ray powder diffraction pattern comprising characteristic diffraction peaks at the following 2θ angles: 14.012±0.200°, 16.936±0.200° and 17.424±0.200°, ##STR00028##

71. The crystal form according to claim 70, wherein the X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.14±0.20°, 12.46±0.20°, 15.99±0.20°, 17.06±0.20°, 18.87±0.20°, 20.25±0.20°, 21.41±0.20° and 25.00±0.20°; or, the X-ray powder diffraction pattern of the crystal form C has characteristic diffraction peaks at the following 2θ angles: 12.871±0.200°, 17.488±0.200°, 18.403±0.200°, 19.079±0.200° and 20.853±0.200°; or, the X-ray powder diffraction pattern of the crystal form D has characteristic diffraction peaks at the following 2θ angles: 6.600±0.200°, 13.178±0.200°, 17.775±0.200°, 19.138±0.200° and 25.798±0.200°; or, the X-ray powder diffraction pattern of the crystal form E has characteristic diffraction peaks at the following 2θ angles: 17.465±0.200°, 18.097±0.200°, 18.691±0.200°, 19.179±0.200° and 20.693±0.200°; or, the X-ray powder diffraction pattern of the crystal form F has characteristic diffraction peaks at the following 2θ angles: 14.012±0.200°, 16.936±0.200°, 17.424±0.200°, 17.954±0.200° and 22.043±0.200°.

72. The crystal form according to claim 71, wherein the X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.14±0.20°, 10.11±0.20°, 12.46±0.20°, 14.35±0.20°, 15.99±0.20°, 17.06±0.20°, 18.87±0.20°, 20.25±0.20°, 21.41±0.20° and 25.00±0.20°; or, the X-ray powder diffraction pattern of the crystal form C has characteristic diffraction peaks at the following 2θ angles: 12.871±0.200°, 13.274±0.200°, 14.294±0.200°, 17.488±0.200°, 18.403±0.200°, 19.079±0.200°, 20.853±0.200° and 21.468±0.200°; or, the X-ray powder diffraction pattern of the crystal form D has characteristic diffraction peaks at the following 2θ angles: 6.600±0.200°, 13.178±0.200°, 17.303±0.200°, 17.775±0.200°, 18.667±0.200°, 19.138±0.200°, 21.245±0.200° and 25.798±0.200°; or, the X-ray powder diffraction pattern of the crystal form E has characteristic diffraction peaks at the following 2θ angles: 6.675±0.200°, 16.119±0.200°, 17.465±0.200°, 18.097±0.200°, 18.691±0.200°, 19.179±0.200°, 20.693±0.200° and 26.658±0.200°; or, the X-ray powder diffraction pattern of the crystal form F has characteristic diffraction peaks at the following 2θ angles: 12.567±0.200°, 14.012±0.200°, 15.666±0.200°, 16.936±0.200°, 17.424±0.200°, 17.954±0.200°, 20.717±0.200° and 22.043±0.200°.

73. The crystal form according to claim 72, wherein the X-ray powder diffraction pattern of the crystal form C has characteristic diffraction peaks at the following 2θ angles: 10.105±0.200°, 12.871±0.200°, 13.274±0.200°, 14.294±0.200°, 17.488±0.200°, 18.403±0.200°, 19.079±0.200°, 20.853±0.200°, 21.468±0.200°, 22.647±0.200°, 23.977±0.200° and 24.409±0.200°; or, the X-ray powder diffraction pattern of the crystal form D has characteristic diffraction peaks at the following 2θ angles: 6.600±0.200°, 11.004.200°, 13.178±0.200°, 15.521±0.200°, 16.592±0.200°, 17.303±0.200°, 17.775±0.200°, 18.667±0.200°, 19.138±0.200°, 21.245±0.200°, 25.798±0.200° and 27.353±0.200°; or, the X-ray powder diffraction pattern of the crystal form E has characteristic diffraction peaks at the following 2θ angles: 6.675±0.200°, 8.741±0.200°, 11.391±0.200°, 13.762±0.200°, 16.119±0.200°, 17.465±0.200°, 18.097±0.200°, 18.691±0.200°, 19.179±0.200°, 20.693±0.200°, 23.386±0.200° and 26.658±0.200°; or, the X-ray powder diffraction pattern of the crystal form F has characteristic diffraction peaks at the following 2θ angles: 6.104±0.200°, 6.104±0.200°, 12.567±0.200°, 14.012±0.200°, 15.666±0.200°, 16.936±0.200°, 17.424±0.200°, 17.954±0.200°, 20.717±0.200°, 22.043±0.200° and 25.436±0.200°.

74. The crystal form according to claim 73, wherein the X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.142°, 8.234°, 10.112°, 11.302°, 12.056°, 12.377°, 12.456°, 14.086°, 14.347°, 15.993°, 16.5300, 16.834°, 17.057°, 17.482°, 18.868°, 20.249°, 21.413°, 22.599°, 22.721°, 23.750°, 23.941°, 24.191°, 24.763°, 24.995°, 25.897°, 27.931°, 29.566°, 30.442°, 31.373°, 31.582°, 32.162°, 32.936°, 33.830°, 34.537°, 34.774°, 35.312°, 36.107°, 36.401° and 37.575°; or, the X-ray powder diffraction pattern of the crystal form C has characteristic diffraction peaks at the following 2θ angles: 9.128°, 10.105°, 12.871°, 13.274°, 13.933°, 14.294°, 16.420°, 17.488°, 18.403°, 19.079°, 20.853°, 21.468°, 22.647°, 23.042°, 23.507°, 23.977°, 24.409°, 24.798°, 25.861°, 26.309°, 28.698°, 30.293° and 37.464°; or, the X-ray powder diffraction pattern of the crystal form D has characteristic diffraction peaks at the following 2θ angles: 6.600°, 8.659°, 9.558°, 11.004°, 12.463°, 13.178°, 13.728°, 14.871°, 15.521°, 16.592°, 17.303°, 17.775°, 18.379°, 18.667°, 19.138°, 20.572°, 21.245°, 21.989°, 22.247°, 22.561°, 23.940°, 25.087°, 25.798°, 26.686°, 27.353°, 28.044°, 28.859°, 29.645°, 30.375°, 32.351°, 34.603°, 35.456° and 39.550°; or, the X-ray powder diffraction pattern of the crystal form E has characteristic diffraction peaks at the following 2θ angles: 6.675°, 8.741°, 9.365°, 11.391°, 12.205°, 13.310°, 13.762°, 15.302°, 16.119°, 16.407°, 17.465°, 18.097°, 18.691°, 19.179°, 20.693°, 21.329°, 21.860°, 22.674°, 23.386°, 24.819°, 25.311°, 25.757°, 26.658°, 26.980°, 29.110°, 29.540°, 30.536°, 31.639°, 33.068°, 33.972°, 36.724° and 38.646°, or, the X-ray powder diffraction pattern of the crystal form F has characteristic diffraction peaks at the following 2θ angles: 6.104°, 9.825°, 12.567°, 14.012°, 15.666°, 16.936°, 17.424°, 17.954°, 19.298°, 20.717°, 21.106°, 22.043°, 25.436°, 26.230°, 26.94° and 29.721°.

75. The crystal form according to claim 74, wherein the XRPD pattern of the crystal form A is as shown in FIG. 1; or, the XRPD pattern of the crystal form C is as shown in FIG. 6; or, the XRPD pattern of the crystal form D is as shown in FIG. 8; or, the XRPD pattern of the crystal form E is as shown in FIG. 10; or, the XRPD pattern of the crystal form F is as shown in FIG. 12.

76. The crystal form according to claim 70, wherein the differential scanning calorimetry curve of the crystal form A has endothermic peaks with an onset at 81.03±3.0° C., 113.62±3.0° C. and 151.37±3.0° C., respectively; preferably, the DSC spectrum of the crystal form A is as shown in FIG. 2; or, the differential scanning calorimetry curve of the crystal form C has an endothermic peak with an onset at 157.36±3.0° C.; preferably, the DSC spectrum thereof is as shown in FIG. 7; or, the differential scanning calorimetry curve of the crystal form D has endothermic peaks with an onset at 116.98±3.0° C., 133.04±3.0° C. and 154.86±3.0° C., respectively; preferably, the DSC spectrum thereof is as shown in FIG. 9; or, the differential scanning calorimetry curve of the crystal form E has endothermic peaks with an onset at 106.62±3.0° C., 134.32±3.0° C. and 158.33±3.0° C., respectively; preferably, the DSC spectrum of the crystal form E is as shown in FIG. 11; or, the differential scanning calorimetry curve of the crystal form F has endothermic peaks with an onset at 83.44±3.0° C. and 154.65±3.0° C., respectively; preferably, the DSC spectrum of the crystal form F is as shown in FIG. 13.

77. The crystal form according to claim 70, wherein the thermogravimetric analysis curve of the crystal form A has a weight loss of 2.046% at 96.01° C.±3.0° C., and a weight loss of 7.708% at 163.66° C.±3.0° C.; preferably, the thermogravimetric analysis spectrum of the crystal form A is as shown in FIG. 2; or, the thermogravimetric analysis curve of the crystal form C has a weight loss of 1.589% at 120.00° C.±3.0° C.; preferably, the TGA spectrum of the crystal form C is as shown in FIG. 7; or, the thermogravimetric analysis curve of the crystal form D has a weight loss of 6.939% at 150.00° C.±3.0° C.; preferably, the TGA spectrum of the crystal form D is as shown in FIG. 9; or, the thermogravimetric analysis curve of the crystal form E has a weight loss of 6.673% at 150.00° C.±3.0° C.; preferably, the TGA spectrum of the crystal form E is as shown in FIG. 11; or, the thermogravimetric analysis curve of the crystal form F has a weight loss of 2.972% at 120.00° C.±3.0° C.; preferably, the TGA spectrum of the crystal form F is as shown in FIG. 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0100] FIG. 1 is the Cu-Kα radiation XRPD pattern of the crystal form A of the compound of formula (I).

[0101] FIG. 2 is the DSC and TGA spectrum of the crystal form A of the compound of formula (I).

[0102] FIG. 3 is the Cu-Kα radiation XRPD pattern of the crystal form B of the compound of formula (II).

[0103] FIG. 4 is the DSC spectrum of the crystal form B of the compound of formula (II).

[0104] FIG. 5 is a TGA spectrum of the crystal form B of the compound of formula (II).

[0105] FIG. 6 is the Cu-Kα radiation XRPD pattern of the crystal form C of the compound of formula (I).

[0106] FIG. 7 is the DSC and TGA spectrum of the crystal form C of the compound of formula (I).

[0107] FIG. 8 is the Cu-Kα radiation XRPD pattern of the crystal form D of the compound of formula (III-1).

[0108] FIG. 9 is the DSC and TGA spectrum of the crystal form D of the compound of formula (III-1).

[0109] FIG. 10 is the Cu-Kα radiation XRPD pattern of the crystal form E of the compound of formula (IV-1).

[0110] FIG. 11 is the DSC and TGA spectrum of the crystal form E of the compound of formula (IV-1).

[0111] FIG. 12 is the Cu-Kα radiation XRPD pattern of the crystal form F of the compound of formula (V).

[0112] FIG. 13 is the DSC and TGA spectrum of the crystal form F of the compound of formula (V).

[0113] FIG. 14 is a three-dimensional structural ellipsoid plot of the crystal form B of the compound of formula (II).

[0114] FIG. 15 is the nuclear magnetic spectrum of the crystal form D of the compound of formula (III-1).

[0115] FIG. 16 is the nuclear magnetic spectrum of the crystal form E of the compound of formula (IV-1).

[0116] FIG. 17 shows the tear secretion amount of animals in the scopolamine dry eye model of mice.

[0117] FIG. 18 shows the fluorescent staining of the cornea of animals in the scopolamine dry eye model of mice.

[0118] FIG. 19 shows the tear secretion amount of animals in the hyperosmolar dry eye model of rats.

[0119] FIG. 20 shows the fluorescent staining of the cornea of animals in the hyperosmolar dry eye model of rat.

[0120] FIG. 21 shows the tear film breakup time of animals in the hyperosmolar dry eye model of rats.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0121] In order to better understand the content of the present disclosure, it is further described in conjunction with specific examples, but the specific implementation is not a limitation to the content of the present disclosure.

Example 1: Preparation of Crystal Form B of the Compound of Formula (II)

[0122] ##STR00010##

[0123] Synthetic Route:

##STR00011##

Step 1: Preparation of Compound 2

[0124] To toluene (500 mL) was added compound 1 (30 g, 130.4 mmol, 1 eq), bis(pinacolato)diboron (66.23 g, 260.80 mmol, 2 eq), [1,1-bis(diphenylphosphino)ferrocene]-dichloropalladium (II) dichloromethane complex (5.32 g, 6.52 mmol, 0.1 eq) and potassium acetate (25.60 g, 260.80 mmol, 2 eq). After replaced with nitrogen three times, the reaction solution was stirred at 110° C. for 15 hours. After the reaction was completed, the reaction solution was filtered through celite, and the filtrate was concentrated, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=0 to 100:6) to obtain compound 2.

[0125] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.84 (d, J=8.0 Hz, 1H), 7.06 (s, 1H), 7.04 (d, J=8.0 Hz, 1H), 5.65 (brs, 2H), 3.87 (s, 3H), 1.35 (s, 12H).

Step 2: Preparation of Compound 4

[0126] After compound 3 (100 g, 460.79 mmol, 1 eq) was dissolved in anhydrous ethanol (1 L), concentrated sulfuric acid (225.97 g, 2.30 mol, 122.81 mL, 5 eq) and anhydrous sodium sulfate Na.sub.2SO.sub.4 (65.45 g, 460.79 mmol, 46.75 mL, 1 eq) were added, and the reaction solution was stirred at 85° C. for 48 hours. After the reaction was completed, the reaction solution was cooled to room temperature. To the reaction solution was added dropwise saturated aqueous sodium bicarbonate solution (1 L), and a large amount of solid was formed. After filtration, the filter cake was washed with water (500 mL), and the obtained solid was dried in vacuum to obtain compound 4.

[0127] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.10 (d, J=1.8 Hz, 1H), 7.26 (s, 1H), 4.47 (q, J=7.1 Hz, 2H), 1.46 (t, J=7.2 Hz, 3H).

Step 3: Preparation of Compound 5

[0128] Compound 4 (70.00 g, 285.63 mmol, 1 eq) was dissolved in tetrahydrofuran (1 L) and then cooled to −78° C. under nitrogen protection, and to the reaction solution was slowly added dropwise methyllithium (1.6 M, 892.59 mL, 5 eq). The reaction solution was stirred at −78° C. for 3 hours. After the reaction was completed, water (100 mL) was slowly added dropwise to quench the reaction. When warmed to room temperature, the reaction solution was diluted with saturated aqueous solution of ammonium chloride (500 mL) and extracted with ethyl acetate (500 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was slurried with n-heptane (500 mL), filtered and dried to obtain compound 5.

[0129] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.86 (d, J=1.9 Hz, 1H), 6.98 (d, J=1.9 Hz, 1H), 4.57 (br s, 2H), 1.57 (s, 6H).

Step 4: Preparation of Compound 6

[0130] To dioxane (300 mL) and water (75 mL) was added compound 5 (10 g, 43.27 mmol, 1 eq), compound 2 (23.98 g, 86.55 mmol, 2 eq), [1,1-bis(diphenylphosphino)ferrocene]-dichloropalladium (II) dichloromethane complex (1.77 g, 2.16 mmol, 0.05 eq) and cesium carbonate (28.20 g, 86.55 mmol, 2 eq). After replaced with nitrogen three times, the reaction solution was stirred at 80° C. for 5 hours. After the reaction was completed, the reaction solution was concentrated, and the residue was purified by column chromatography (petroleum ether: tetrahydrofuran=0 to 100:40) to obtain crude compound 6. The crude product was heated to 80° C. with tetrahydrofuran (4 mL/g), then cooled to recrystallize. The solution was stirred at 25° C. for 15 hours, filtered, and the filter cake was dried to obtain compound 6.

[0131] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.94 (d, J=2.0 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.17 (d, J=2.0 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 7.77-7.75 (m, 3H), 5.69 (s, 2H), 5.50 (s, 1H), 3.81 (s, 3H), 1.52 (s, 6H).

Step 5: Preparation of the Compound of Formula (I)

[0132] Compound 6 (8.78 g, 29.14 mmol, 1 eq) was dissolved in tetrahydrofuran (80 mL), and cooled to 0° C. under nitrogen protection. To the reaction solution was added dropwise methylmagnesium bromide (3 M, 97.12 mL, 10 eq), and the reaction solution was stirred at 0° C. for 1 hour. After completion of the reaction, saturated aqueous solution of ammonium chloride (400 mL) was slowly added to quench the reaction, then the reaction solution was extracted with ethyl acetate (400 mL*2). The organic phase was concentrated under reduced pressure, and the residue, i.e. the crude product was purified by dichloromethane (3 mL/g) at 25° C., filtered and dried to obtain the product.

[0133] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.88 (d, J=1.8 Hz, 1H), 7.15-7.01 (m, 2H), 6.82 (d, J=1.6 Hz, 1H), 6.69 (dd, J=1.5, 8.0 Hz, 1H), 5.59 (br s, 2H), 5.51 (br s, 2H), 5.44 (s, 1H), 5.23 (s, 1H), 1.51 (d, J=3.6 Hz, 12H).

[0134] 11.9 g of the above product was added into a round-bottomed flask, and 150 mL of methyl tert-butyl ether was added. The above sample was stirred at 50° C. for 12 hours, then cooled down to 25° C. and stirred for 4 hours. After filtration and drying, a solid was obtain. It was detected as the crystal form A by XRPD.

[0135] About 50 mg of the crystal form A was added into a 2.0 mL glass vial, and an appropriate amount of solvent or solvent mixture was added to make it into a suspension. After a magnetic stir bar was added, the above sample was placed on a magnetic heating stirrer (25° C./50° C.) and stirred for a week, and after centrifugation, the obtained solid sample was placed in a vacuum oven at 40° C. and dried overnight to obtain the crystal form B of the compound of formula (II).

TABLE-US-00008 No. Solvent Volume Ratio Crystal Form 1 Water / Crystal Form B 2 Acetonitrile/Water 3/1 Crystal Form B 3 Methanol/Water 3/1 Crystal Form B 4 Tetrahydrofuran/Water 3/1 Crystal Form B 5 Ethanol/Water 3/1 Crystal Form B 6 Isopropanol/Water 3/1 Crystal Form B

Example 2: Confirmation of the Structure of the Compound of Formula (II)

[0136] ##STR00012##

[0137] Process of cultivation of single crystal: About 5 mg crystal form B of the compound of formula (II) was dissolved in 3 mL of dichloromethane/methanol (2:1) at room temperature. The sample solution was placed in a 4 mL semi-sealed sample vial and evaporated slowly at room temperature. Colorless transparent crystals were obtained on the 5th day. The crystal size used for diffraction experiments was: 0.20×0.18×0.15 mm.sup.3.

[0138] Single crystal diffraction instrument: Bruker D8 venture

[0139] The information of the measured compound was: molecular formula C.sub.17H.sub.25N.sub.3O.sub.3, crystal system Monoclinic, space group C2/c, unit cell parameters a=20.6603(14) Å, b=15.6554(14) Å, c=21.0124(16) Å, β=90.01°, α=γ=90°, volume V=6796.4(9) Å.sup.3. FIG. 14 was a three-dimensional structural ellipsoid plot of the crystal form B of the compound of formula (II). At the same time, the crystal form of the single crystal was measured and confirmed to be crystal form B.

Example 3: Preparation of Each Crystal Form

[0140] ##STR00013##

[0141] About 50 mg of the crystal form A was added into a 2.0 mL glass vial, and an appropriate amount of methanol or ethanol was added to make it into a suspension. After a magnetic stir bar was added, the above sample was placed on a magnetic heating stirrer (50° C.) and stirred for a week, and after centrifugation, the obtained solid sample was placed in a vacuum oven at 40° C. and dried overnight to obtain the crystal form C of the compound of formula (I).

##STR00014##

[0142] About 50 mg of the crystal form A was added into a 2.0 mL glass vial, and an appropriate amount of ethyl acetate was added to make it into a suspension. After a magnetic stir bar was added, the above sample was placed on a magnetic heating stirrer (25° C./50° C.) and stirred for a week, and after centrifugation, the obtained solid sample was placed in a vacuum oven at 40° C. and dried overnight to obtain the crystal form D of the compound of formula (III-1). NMR data were shown in FIG. 15.

##STR00015##

[0143] About 50 mg of the crystal form A was added into a 2.0 mL glass vial, and an appropriate amount of acetonitrile was added to make it into a suspension. After a magnetic stir bar was added, the above sample was placed on a magnetic heating stirrer (25° C./50° C.) and stirred for a week, and after centrifugation, the obtained solid sample was placed in a vacuum oven at 40° C. and dried overnight to obtain the crystal form E of the compound of formula (IV-1). NMR data were shown in FIG. 16.

##STR00016##

[0144] About 50 mg of the crystal form A was added into a 2.0 mL glass vial, and an appropriate amount of dichloromethane was added to make it into a suspension. After a magnetic stir bar was added, the above sample was placed on a magnetic heating stirrer (25° C.) and stirred for a week, and after centrifugation, the obtained solid sample was placed in a vacuum oven at 40° C. and dried overnight to obtain the crystal form F of the compound of formula (V).

Example 4: Pre-Stability Experiment of the Crystal Form B of the Compound of Formula (II)

[0145] About 50 mg of the crystal form B of the compound of formula (II) was accurately weighed, placed in a dry and clean glass bottle, spread into a thin layer as the tested sample, respectively, and placed under the test condition of influencing factors (40° C., 25° C./92.5% RH, light, light control) and accelerated condition (40° C./75% RH and 60° C./75% RH). The samples were fully exposed. Samples under the test condition of 40° C., 25° C./92.5% RH, light, light control were taken at 5 days and 10 days for analysis, and samples under the accelerated condition were taken at 1 month, 2 months and 3 months for analysis.

TABLE-US-00009 Experimental Conditions Sampling Time Crystal Form 40° C. (open) 10 days Crystal Form B 1 month Crystal Form B 25° C./92.5% RH (open) 5 days Crystal Form B 10 days Crystal Form B Samples under light (visible light 10 days Crystal Form B intensity 5000 lux and ultraviolet intensity 90 W/cm.sup.2, open) light control (10 days) Crystal Form B 40° C./75% RH (open) 1 month Crystal Form B 2 month Crystal Form B 3 month Crystal Form B 60° C./75% RH (open) 1 month Crystal Form B 2 month Crystal Form B 3 month Crystal Form B

[0146] Experimental conclusion: The crystal form B of the compound of formula (II) was placed under the test condition of the influencing factor (40° C., 25° C./92.5% RH, light, light control) for 10 days, 40° C. for 1 month, long-term accelerated condition (40° C./75% RH and 60° C./75% RH) for 3 months, and the crystal form was stable.

Example 5: 25° C. Competition Experiment of Water Activity of the Crystal Form B of the Compound of Formula (II) and Crystal Form C of the Compound of Formula (I)

[0147] About 15 mg of the crystal form C of the compound of formula (I) was weighed respectively, and an appropriate amount of solvent system was added at room temperature to form a saturated solution. If the solution was clear, the compound was continued to add until the solution was saturated. The solution was filtered into the liquid phase vial through a 0.45 μm nylon needle filter, and then to the liquid phase vial was added almost equal amounts of the crystal form B of the compound of formula (II) and crystal form C of the compound of formula (I) to make a suspension liquid. After addition of a magnetic stir bar, the above suspension sample was placed on a constant temperature mixer (25° C., 700 rpm) for shaking.

TABLE-US-00010 Solvent Time for Suspension (Ethanol:Water) Water Activity and Beating (Days) XRPD Pure Ethanol 0 3 Crystal Form C 5 Crystal Form C 95:5 0.3 3 Crystal Form B 5 Crystal Form B 90:10 0.5 3 Crystal Form B 5 Crystal Form B 80:20 0.7 3 Crystal Form B 5 Crystal Form B 50:50 0.9 3 Crystal Form B 5 Crystal Form B Pure Water 1.0 3 Crystal Form B 5 Crystal Form B

[0148] The results showed that: the crystal form B of the compound of formula (II) and the crystal form C of the compound of formula (I) were suspended and beaten for 5 days under the condition of water activity of 0, and the crystal form C of the compound of formula (I) was obtained. However, with the increase of water activity, after 5 days of suspension and beating, the crystal form obtained were all the crystal form B of the compound of formula (II). And the products obtained with water activity of 0.9 and 1.0 were characterized after drying, and the two were almost the same. Therefore, the crystal form B of the compound of formula (II) was more stable than the crystal form C of the compound of formula (I).

Biological Test Data

Example 1: Experimental Study on the Effect of Eye Drops of the Crystal Form B of the Compound of Formula (II) on Dry Eye Models of Mice

[0149] Experimental Purpose:

[0150] The C57BL/6 dry eye model of mice was induced by subcutaneous injection of scopolamine hydrobromide solution in lower limbs to investigate the therapeutic effect of the eye drops of the crystal form B of the compound of formula (II) on the model.

[0151] Experiment Procedure:

[0152] According to the tear secretion amount, the animals were randomly and evenly divided into 5 groups, which were negative control group (normal saline), model control group (vehicle), low (1 mg/mL), medium (2.5 mg/mL) and high (5 mg/mL) concentration groups for the crystal form B of the compound of formula (II), with 8 animals in each group, all female. The grouping day was recorded as DO. After divided into groups, Animals in each group were subjected to modeling, eye drop administration, scoring of corneal fluorescent staining, tear secretion measurement, etc., according to the experimental settings.

[0153] Experimental Results:

[0154] The solution of the crystal form B of the compound of formula (II) (1 mg/mL, 2.5 mg/mL, 5 mg/mL) had good therapeutic effect on the dry eye model of mice induced by scopolamine hydrobromide solution, which mainly improved tear secretion amount and corneal damage of mouse in dry eye model. Combining the tear secretion amount (Table 7, FIG. 17) and the scoring of corneal fluorescent staining (Table 8, FIG. 18), it was concluded that eye drops of the crystal form B of the compound of the formula (II) had the best therapeutic effect at a concentration of 5 mg/mL.

TABLE-US-00011 TABLE 7 Effect of the solution of the crystal form B of the compound of formula (II) on tear secretion amount of mice in the dry eye models (x ± S) Wetted length of phenol red cotton thread (mm) Group D0 D7 D12 Negative control group (G1) 4.33 ± 1.78 4.10 ± 1.12 4.07 ± 0.66 Model control group (G2) 4.41 ± 1.29 1.70 ± 0.30##** 1.97 ± 0.59##** 1 mg/mL group (G3) of the crystal form 4.54 ± 1.84 2.13 ± 0.69##**& 2.72 ± 0.91##**&& B of the compound of formula II 2.5 mg/mL group (G4) of the crystal 4.30 ± 1.08 2.14 ± 0.69##**& 2.58 ± 0.82##**& form B of the compound of formula II 5 mg/mL group (G5) of the crystal form 4.28 ± 1.41 2.73 ± 0.48##**&& 2.85 ± 0.57##**&& B of the compound of formula II Note: Compared with D0, #P < 0.05; ##P < 0.01; compared with G1 group, *P < 0.05; **P < 0.01; compared with G2 group, &P < 0.05; &&P < 0.01.

TABLE-US-00012 TABLE 8 Effect of the solution of the crystal form B of the compound of formula (II) on the scoring of cornea fluorescent staining of mice in the dry eye models (x ± S) Scoring of fluorescent staining of the cornea Group D0 D7 D12 Negative control group (G1) 0.13 ± 0.34 0.50 ± 1.03 0.63 ± 0.96# Model control group (G2) 0.63 ± 1.09 4.19 ± 2.07##** 3.63 ± 1.82##** 1 mg/mL group (G3) of the crystal form B 0.50 ± 0.97 2.56 ± 1.50##**& 2.31 ± 1.35##**& of the compound of formula II 2.5 mg/mL group (G4) of the crystal form 0.31 ± 0.87 2.88 ± 2.53##** 2.56 ± 1.55##** B of the compound of formula II 5 mg/mL group (G5) of the crystal form B 0.25 ± 0.58 2.38 ± 2.09##**& 2.38 ± 1.20##**& of the compound of formula II Note: Compared with D0, #P < 0.05; ##P < 0.01; compared with G1 group, *P < 0.05; **P < 0.01; compared with G2 group, &P < 0.05; &&P < 0.01.

[0155] Experimental Results:

[0156] The eye drops of the crystal form B of the compound of formula (II) had good therapeutic effect on the dry eye model of mice induced by scopolamine hydrobromide solution, which mainly improved tear secretion amount and corneal damage of mouse in dry eye model.

Example 2: Experimental Study on the Effect of Eye Drops of the Crystal Form B of the Compound of Formula (II) on Hyperosmola Dry Eye Models of Rats

[0157] 20 female SD rats qualified for the adaptability observation were subjected to the scoring of fluorescent staining of the cornea and measurement of tear secretion amount of both eyes for the animals, and animals with abnormalities of problematic fluorescent staining of the cornea and significant differences in tear secretion amount were excluded.

[0158] A pipette was used to draw 20 μL of sodium chloride solution (osmotic pressure of 500 mOsmol/L), which was dropped into the conjunctival sac of both eyes of the animal, 5 times/day, 20 μL/time, with an interval of about 2 hours between each time, for 28 consecutive days. After dropping the solution, the animal's eyelids were passively closed for about 90 s. On the 14th day of the modeling period, the animals were subjected to scoring of fluorescent staining of the cornea of both eyes and measurement of tear secretion amount, and the data were compared with the basic data, and the animals with significant differences in tear secretion amount of both eyes were selected for grouping. Animals were randomly and evenly divided into 4 groups according to the average of tear secretion amount of both eyes, namely the model control group, low and high concentration groups (1 mg/mL, 5 mg/mL) of the crystal form B of the compound of formula (II), with 4 animals in each group and 8 eyes, and the grouping day was recorded as DO.

[0159] Animals in each group were administered eye drops at D1, 10 μL/eye/time, 4 times/day, with an interval of about 3 hours between administrations, for a total of 14 days and weighed once a week during the administration period. About 30 minutes after the second administration on D7 and D14 respectively, the tear secretion amount of both eyes was measured, and the scoring of fluorescent staining of the cornea was performed about 30 minutes after the third administration on D7 and D14. The animals were euthanized by carbon dioxide inhalation after the measurement of D14 indicators.

[0160] Experimental results: See Tables 9 and 10 and FIGS. 19, 20 and 21.

TABLE-US-00013 TABLE 9 Effect of the solution of the crystal form B of the compound of formula (II) on tear secretion amount in rats of the dry eye model (x ± S) Wetted length of phenol red cotton thread (mm) Group D0 D14 D21 Model control group 12.31 ± 3.22 7.98 ± 2.53 7.05 ± 2.08 (G1) 1 mg/mL group (G2) of 13.78 ± 5.25 8.12 ± 2.81 9.24 ± 4.07 the crystal form B of the compound of formula II 5 mg/mL group (G3) of 13.87 ± 4.45 7.16 ± 2.77  11.37 ± 3.26** the crystal form B of the compound of formula II Note: Compared with G1 group, *P < 0.05; **P < 0.01.

TABLE-US-00014 TABLE 10 Effect of the solution of the crystal form B of the compound of formula (II) on the assessment of fluorescein staining of the cornea in mice of the dry eye model (x ± S) Scoring of fluorescent staining of the cornea Group D0 D14 D21 Model control group (G1) 0.38 ± 0.52 2.88 ± 2.47 3.63 ± 2.97 1 mg/mL group (G2) of the 0.00 ± 0.00 4.75 ± 3.73 3.00 ± 2.78 crystal form B of the compound of formula II 5 mg/mL group (G3) of the 0.13 ± 0.35 3.88 ± 2.36 1.75 ± 1.49 crystal form B of the compound of formula II Note: Compared with G1 group, *P < 0.05; **P < 0.01.

[0161] Experimental conclusion: The eye drops of the crystal form B of the compound of formula (II) had good therapeutic effect on the dry eye model of rat induced by hypertonic sodium chloride solution, which mainly improved tear secretion amount, corneal damage and tear film breakup time of rat in dry eye model.