Crystalline sulfamide compound

Abstract

The present application relates to a crystalline sulfamide compound, and in particular relates to a crystalline (S)—N—((S)-1-(2-chlorphenyl)-2-((3,3-difluorocyclobutyl)amido)-2oxoethyl)-2-(4-cyanopyridin-2-base)-N-(3-fluorophenyl)-isothiazolidine-3-formamide 1,1-dioxide, and a preparation method therefor, a crystalline composition, a pharmaceutical composition and uses thereof. An X-ray powder diffraction spectrum of a crystalline hydrate of formula II of the present application has diffraction peaks at positions of about 14.40°, 20.28°, 20.94°, 22.02°, and 24.46°, represented by 2θ. The crystalline hydrate of formula II of the present application has good IDH1 inhibitory activity and performs high stability, and therefore has advantages in physical property, safety and metabolic stability, and has high medicine value. ##STR00001##

Claims

1. A crystalline form of a monohydrate of (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide represented by formula II, ##STR00010## wherein the X-ray powder diffraction spectrum thereof has diffraction peaks at 14.40°±0.2°, 20.28°±0.2°, 20.94°±0.2°, 22.02°±0.2° and 24.46°±0.2°, represented by 2θ values.

2. The crystalline form of the monohydrate of formula II according to claim 1, wherein the X-ray powder diffraction spectrum thereof has diffraction peaks at 9.12°±0.2°, 13.32°±0.2°, 14.40°±0.2°, 15.64°±0.2°, 16.46°±0.2°, 20.28°±0.2°, 20.94°±0.2°, 22.02°±0.2°, 22.98°±0.2°, 24.46°±0.2° and 29.34°±0.2°, represented by 2θ values.

3. The crystalline form of the monohydrate of formula II according to claim 2, wherein the X-ray powder diffraction spectrum thereof has diffraction peaks at 5.52°±0.2°, 9.12°±0.2°, 10.30°±0.2°, 10.48°±0.2°, 11.96°±0.2°, 13.32°±0.2°, 14.40°±0.2°, 14.90°±0.2°, 15.64°±0.2°, 16.46°±0.2°, 17.28°±0.2°, 17.58°±0.2°, 18.60°±0.2°, 19.14°±0.2°, 19.32°±0.2°, 20.28°±0.2°, 20.94°±0.2°, 21.20°±0.2°, 22.02°±0.2°, 22.98°±0.2°, 23.52°±0.2°, 24.46°±0.2°, 25.74°±0.2°, 26.06°±0.2°, 26.74°±0.2°, 27.32°±0.2°, 27.98°±0.2°, 28.40°±0.2°, 28.90°±0.2°, 29.34°±0.2°, 30.36°±0.2°, 31.00°±0.2°, 31.74°±0.2°, 32.22°±0.2°, 32.82°±0.2°, 33.32°±0.2° and 37.84°±0.2°, represented by 2θ values.

4. The crystalline form of the monohydrate of formula II according to claim 1, wherein the differential scanning calorimetry (DSC) measurement pattern thereof has an onset point at 186° C.±5° C.

5. A crystal composition, characterized in that the crystalline form of the monohydrate of formula II according to claim 1 represents 50% or more of the weight of the crystal composition.

6. A pharmaceutical composition, comprising the crystalline form of the monohydrate of formula II according to claim 1.

7. A method for treating IDH1 mutation-induced cancer, comprising administering a therapeutically effective amount of the crystalline form of the monohydrate of formula II according to claim 1 to a subject in need thereof.

8. A method of preparing the crystalline form of the monohydrate of formula II according to claim 1, comprising: (1) dissolving a compound of formula I in an organic solvent, and stirring till the solution is clear, wherein the organic solvent is selected from one or more of methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, acetone, tetrahydrofuran, acetonitrile, dichloromethane, or ethyl acetate; (2) adding water to the solution obtained in step (1); and (3) cooling down the solution to crystallization, filtering and drying, ##STR00011##

9. The method according to claim 8, wherein in step (1), the organic solvent is selected from one or more mixed solvents of methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, acetone, tetrahydrofuran, acetonitrile, dichloromethane, or ethyl acetate.

10. The method according to claim 8, wherein in step (1), the molar volume ratio of the compound of formula I to the organic solvent is 1 mmol: 2-20 mL.

11. The method according to claim 8, wherein in step (1), the temperature for dissolving the compound of formula I in the organic solvent is 20° C. to 100° C.

12. The method according to claim 8, wherein the molar volume ratio of the compound of formula I in step (1) to the water in step (2) is 1 mmol: 0.01-5 mL.

13. A crystalline form of (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide represented by formula I, ##STR00012## wherein the X-ray powder diffraction spectrum thereof has diffraction peaks at 8.64°±0.2°, 9.34°±0.2°, 20.72°±0.2°, 21.30°±0.2° and 24.02°±0.2°, represented by 2θ values.

14. The crystalline form of the compound of formula I according to claim 13, wherein the X-ray powder diffraction spectrum thereof has diffraction peaks at 8.64°±0.2°, 9.34°±0.2°, 14.62°±0.2°, 19.66°±0.2°, 20.04°±0.2°, 20.46°±0.2°, 20.72°±0.2°, 21.30°±0.2°, 22.46°±0.2°, 24.02°±0.2° and 27.42°±0.2°, represented by 2θ values.

15. The crystalline form of the compound of formula I according to claim 14, wherein the X-ray powder diffraction spectrum thereof has diffraction peaks at 8.64°±0.2°, 9.34°±0.2°, 11.18°±0.2°, 12.80°±0.2°, 13.68°±0.2°, 14.62°±0.2°, 15.18°±0.2°, 15.58°±0.2°, 16.36°±0.2°, 17.04°±0.2°, 17.60°±0.2°, 18.14°±0.2°, 18.40°±0.2°, 18.88°±0.2°, 19.66°±0.2°, 20.04°±0.2°, 20.46°±0.2°, 20.72°±0.2°, 21.30°±0.2°, 22.16°±0.2°, 22.46°±0.2°, 22.92°±0.2°, 23.16°±0.2°, 24.02°±0.2°, 24.32°±0.2°, 24.92°±0.2°, 25.14°±0.2°, 25.48°±0.2°, 25.92°±0.2°, 26.30°±0.2°, 27.42°±0.2°, 27.84°±0.2°, 28.46°±0.2° 30.16°±0.2°, 30.98°±0.2° and 33.18°±0.2°, represented by 2θ values.

16. The crystalline form of the compound of formula I according to claim 13, wherein the differential scanning calorimetry (DSC) measurement pattern thereof has an onset point at 103° C.±5° C.

17. A method for preparing the crystalline form of the (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide of formula I according to claim 13, comprising: (1) dissolving the (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl)-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide of formula I in an anhydrous organic solvent, stirring till the solution is clear, and added with 4A molecular sieve to drying, wherein the anhydrous organic solvent is selected from one or more of dichloromethane, isopropanol, n-hexane, ethyl acetate, or methyl tert-butyl ether; (2) filtering under nitrogen protection, and cooling down the filtrate to crystallization; and (3) filtering under nitrogen protection and drying ##STR00013##

18. A crystal composition, wherein the crystalline form of the compound of formula I according to claim 13 represents 50% or more of the weight of the crystal composition.

19. A pharmaceutical composition comprising the crystalline form of the compound of formula I according to claim 13.

20. A method for treating IDH1 mutation-induced cancer, comprising administering a therapeutically effective amount of the crystalline form of the compound of formula I according to claim 13 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an X-ray powder diffraction pattern (XRPD) for the crystal of the monohydrate of formula II in Example 5.

(2) FIG. 2 is a differential scanning calorimetry (DSC) curve for the crystal of the monohydrate of formula II in Example 5.

(3) FIG. 3 is a thermogravimetric analysis (TGA) pattern for the crystal of the monohydrate of formula II in Example 5.

(4) FIG. 4 is an X-ray powder diffraction pattern (XRPD) for the crystal of the compound of formula I in Example 7.

(5) FIG. 5 is a differential scanning calorimetry (DSC) curve for the crystal of the compound of formula I in Example 7.

(6) FIG. 6 is a thermogravimetric analysis (TGA) pattern for the crystal of the compound of formula I in Example 7.

(7) FIG. 7 is an X-ray powder diffraction pattern (XRPD) for the amorphous form in Example 9.

(8) FIG. 8 is a differential scanning calorimetry (DSC) curve for the amorphous form in Example 9.

(9) FIG. 9 is an X-ray powder diffraction pattern calculated from the single crystal in Example 4.

DETAILED DESCRIPTION

(10) The following specific examples are intended to enable those skilled in the art to more clearly understand and implement the present application. They should not be considered as limiting the scope of the application, but are merely illustrations and typical representatives of the application. Those skilled in the art will understand that there are other synthetic routes for preparing the compounds of the present application, and the following are provided as non-limiting examples.

Example 1

Preparation of the Compound of Formula I

(11) Step A: dimethyl L-homocysteinate dihydrochloride

(12) ##STR00004##

(13) Under stirring in an ice bath, thionyl chloride (10.64 g, 89.4 mmol) was added dropwise into a suspension of L-homocysteine (8.0 g, 29.8 mmol) in methanol. The solution was gradually clear. After the addition was completed, the reaction solution was stirred for 10 min, followed by removing the ice bath, and stirred again at room temperature overnight. The solvent was removed, so as to give dimethyl L-homocysteinate dihydrochloride (10.6 g).

(14) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=8.79 (s, 6H), 3.75 (s, 6H), 2.95-2.80 (m, 4H), 2.52-2.47 (m, 2H), 2.20-2.10 (m, 4H).

(15) Step B: methyl (S)-2-amino-4-chlorosulfonylbutyrate hydrochloride

(16) ##STR00005##

(17) Under stirring in an ice bath, chlorine gas was introduced into a mixed solution of dimethyl L-homocysteinate dihydrochloride (10.6 g, 28.8 mmol) in ethanol (40 mL) and chloroform (80 mL) for 20 minutes, generating a white solid. The reaction solution was filtered and washed with chloroform, to give methyl (S)-2-amino-4-chlorosulfonylbutyrate hydrochloride (7.5 g).

(18) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=13.46 (s, 1H), 8.57 (s, 2H), 3.66 (s, 3H), 3.18-2.95 (m, 2H), 2.52-2.45 (m, 1H), 2.22-1.97 (m, 2H).

(19) Step C: methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide

(20) ##STR00006##

(21) Under stirring in an ice-salt bath, a solution of triethylamine in chloroform was added dropwise into a suspension of methyl (S)-2-amino-4-chlorosulfonylbutyrate hydrochloride (4.5 g, 17.85 mmol) in chloroform. After the addition was completed, the ice-salt bath was removed. It was stirred at room temperature overnight and the solvent was removed. Then it was filtered through diatomite and washed with ethyl acetate. The solvent was removed to give a light yellow oil, namely methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide (3.2 g).

(22) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=4.98 (s, 1H), 4.21 (dd, J=8.3, 4.6 Hz, 1H), 3.84 (s, 3H), 3.30-3.11 (m, 1H), 3.09-2.90 (m, 1H), 2.90-2.73 (m, 1H), 2.60 (ddd, J=18.4, 8.9, 4.7 Hz, 1H).

(23) Step D: methyl (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylate 1,1-dioxide

(24) ##STR00007##

(25) Methyl (S)-isothiazolidine-3-carboxylate 1,1-dioxide (200 mg, 1.11 mmol), 2-bromo-4-cyanopyridine (204 mg, 1.11 mmol), cuprous iodide (105 mg, 0.55 mmol), N,N′-dimethylethylenediamine (98 mg, 1.11 mmol) and cesium carbonate (723 mg, 2.22 mmol) were added into a sealed tube reactor, dioxane (8 mL) was added thereto, nitrogen gas was introduced thereto for 5 min and the tube was sealed. They were reacted overnight at 80° C. After the starting materials were consumed, the solvent was removed and then separation by column chromatography (petroleum ether: ethyl acetate=1:1) was performed, to give the title compound methyl (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylate 1,1-dioxide (230 mg).

(26) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.40 (dd, J=5.2, 0.8 Hz, 1H), 7.69 (t, J=1.0 Hz, 1H), 7.19 (dd, J=5.2, 1.0 Hz, 1H), 5.01 (dd, J=8.0, 3.6 Hz, 1H), 3.78 (s, 3H), 3.64-3.55 (m, 1H), 3.48-3.42 (m, 1H), 2.95-2.84 (m, 1H), 2.65-2.52 (m, 1H).

(27) Step E: (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide

(28) ##STR00008##

(29) Under stirring in an ice bath, a suspension of lithium hydroxide was added dropwise into a solution of methyl (S)-2-(4-cyanopyridin-2-yl) isothiazolidine-3-carboxylate 1,1-dioxide (116 mg, 0.41 mmol) in methanol-tetrahydrofuran, reacting overnight. After the reaction was finished, it was diluted with 10 mL water, and extracted with ethyl acetate to remove impurities. The aqueous phase was added dropwise with 1N hydrochloric acid to make the pH thereof less than 5, and then extracted with ethyl acetate. The solvent was removed to give (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide (103 mg).

(30) .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ=13.5 (s, 1H), 8.54 (d, J=5.0, 1H), 7.51 (dd, J=3.74, 4.76 Hz, 1H), 7.45 (s, 1H), 4.95-4.90 (m, 1H), 3.75-3.60 (m, 2H), 2.85-2.72 (m, 1H), 2.46-2.38 (m, 1H).

(31) Step F: (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide

(32) ##STR00009##

(33) At room temperature, 3-amino-5-Fluorouridine (57 mg, 0.508 mmol) and o-chlorobenzaldehyde (72 mg, 0.512 mmol) were dissolved in methanol, and stirred for 30 min. (S)-2-(4-cyanopyridin-2-yl)isothiazolidine-3-carboxylic acid 1,1-dioxide (136 mg, 0.508 mmol) was then added into the mixed solution, stirred for 10 min, then added with 1,1-difluoro-3-isocyanocyclobutane (prepared according to the method described in patent CN103097340, 60 mg, 0.508 mmol), and stirred overnight. The solvent was removed and the residue was separated by thin layer chromatography, to give the title compound (S)—N—((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-2-(4-cyanopyridin-2-yl-N-(3-fluorophenyl)-isothiazolidine-3-carboxamide 1,1-dioxide (the compound of formula I).

(34) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ=8.46 (m, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.63 (s, 1H), 7.22-6.84 (m, 8H), 6.47 (d, J=3.6, 1H), 6.08 (s, 1H), 4.82 (d, J=6.1 Hz, 1H), 4.33 (m, 1H), 3.68-3.60 (m, 1H), 3.40-3.28 (m, 1H), 3.10-2.98 (m, 2H), 2.68-2.38 (m, 4H).

(35) m/z=618 [M+H].sup.+.

Example 2

Preparation of Single Crystal of the Crystal of the Monohydrate of Formula II

(36) 1.0 g of the compound of formula I prepared in Example 1 was added to 2.5 mL of anhydrous methanol, stirred till the solution was clear, and filtered through the membrane. 2 mL of the above-mentioned filtrate was taken and added with 0.2 mL of water, and the solution was allowed to stand at room temperature and the crystal slowly precipitated, which is the single crystal of the crystal of the monohydrate of formula II.

Example 3

Cell Parameters of the Monohydrate Crystal of Formula II

(37) The crystallographic data and atomic coordinates of the crystal of the monohydrate of formula II are shown in Tables 3, 4 and 5:

(38) TABLE-US-00003 TABLE 3 Crystal data and structure refinement Empirical formula C.sub.28H.sub.25ClF.sub.3N.sub.5O.sub.5S formula weight 636.04 Temperature 173(2)K Wavelength 1.54178 Å crystal system orthorhombic system Space group P21 21 21 cell parameters a = 8.7606(6) Å b = 10.1371(7) Å c = 32.183(2) Å α = 90 deg. β = 90 deg. γ = 90 deg. Cell volume 2858.0(3) Å.sup.3 Z 4 Calculated density 1.478 Mg/m.sup.3 Absorption correction parameter 2.466 mm.sup.−1 F(000) 2679 Crystal size 0.05 × 0.04 × 0.03 mm Angle range for data collection 5.157 deg. to 66.672 deg. hkl index range for collection −10 <= h <= 10, −12 <= k <= 10, −38 <= | <= 38 Reflection data collection/unique 26530/5023 [R(int) = 0.0278] data completeness to theta = 99.40% 66.672 Refinement method F.sup.2 full-matrix least-squares method Data/restraints/parameters 5023/2/400 Goodness-of-fit on F.sup.2 1.072 Final R indices [I > 2 sigma(I)] R1 = 0.0255, wR2 = 0.0680 R indices (all data) R1 = 0.0258, wR2 = 0.0682 Absolute configuration parameters 0.031(3) Maximum difference between peak 0.334 and −0.264 e .Math. Å.sup.−3 and hole

(39) TABLE-US-00004 TABLE 4 Atomic coordinates (×10.sup.4) and equivalent isotropic displacement parameters (Å.sup.2 × 10.sup.3) x y z U (eq) Cl(1) 6119(1) 5636(1) 6560(1) 46(1) S(1) 2239(1) 3898(1) 4898(1) 25(1) F(1) 10806(2)  −1693(2)  6727(1) 69(1) O(1)  812(2) 4211(2) 4704(1) 36(1) N(1) 3025(2) 6374(2) 5789(1) 31(1) C(1) 2225(3) 2294(2) 5116(1) 29(1) F(2) 9426(2) −2883(2)  6319(1) 63(1) O(2) 3590(2) 4158(2) 4660(1) 36(1) N(2) 2324(2) 4673(2) 5356(1) 26(1) C(2) 1571(3) 2503(2) 5551(1) 29(1) F(3)  364(2) 1191(2) 7207(1) 58(1) O(3) 4966(2) 3581(2) 5647(1) 31(1) N(3) 3999(2) 3147(2) 6284(1) 24(1) C(3) 2261(2) 3800(2) 5716(1) 24(1) O(4) 4775(2)  431(2) 6358(1) 34(1) N(4) 7281(2)  954(2) 6298(1) 31(1) C(4) 3873(2) 3547(2) 5879(1) 23(1) O(5) 3977(2) 1508(2) 3803(1) 45(1) N(5) 3480(3) 9717(2) 4504(1) 48(1) C(5) 2731(2) 5989(2) 5399(1) 26(1) C(6) 2798(3) 6845(2) 5057(1) 31(1) C(7) 3232(3) 8133(2) 5132(1) 33(1) C(8) 3542(3) 8561(2) 5537(1) 37(1) C(9) 3411(3) 7639(2) 5848(1) 38(1) C(10) 3371(3) 9024(2) 4782(1) 38(1) C(11) 5534(2) 2735(2) 6417(1) 25(1) C(12) 5801(2) 1253(2) 6349(1) 27(1) C(13) 7795(3) −391(2) 6264(1) 33(1) C(14) 9500(3) −583(3) 6154(1) 41(1) C(15) 9548(3) −1642(3)  6482(1) 40(1) C(16) 8083(3) −1153(3)  6676(1) 41(1) C(17) 2780(2) 3266(2) 6582(1) 26(1) C(18) 2275(3) 4508(2) 6705(1) 32(1) C(19) 1116(3) 4613(3) 6999(1) 40(1) C(20)  470(3) 3494(3) 7171(1) 40(1) C(21)  999(3) 2287(3) 7043(1) 37(1) C(22) 2141(3) 2132(2) 6751(1) 30(1) C(23) 5867(2) 3143(2) 6862(1) 28(1) C(24) 5906(3) 2245(2) 7187(1) 32(1) C(25) 6184(3) 2648(3) 7593(1) 41(1) C(26) 6436(3) 3966(3) 7677(1) 45(1) C(27) 6435(3) 4877(3) 7359(1) 40(1) C(28) 6143(3) 4460(2) 6957(1) 33(1)

(40) TABLE-US-00005 TABLE 5 Hydrogen atomic coordinates (×10.sup.4) and isotropic displacement parameters (Å.sup.2 × 10.sup.3) x y z U(eq) H(1)  4300(400)  1100(400) 3610(80) 1500(500) H(1A) 1571 1692 4952 35 H(1B) 3269 1925 5130 35 H(2) 3780(60)  910(40) 3965(14) 120(20) H(2A) 1851 1757 5735 35 H(2B)  444 2566 5539 35 H(3) 1601 4186 5939 29 H(4) 7880(40) 1540(30) 6269(9)  35(7) H(6) 2554 6551 4785 37 H(8) 3828 9447 5594 45 H(9) 3609 7918 6125 45 H(11) 6281 3212 6237 30 H(13) 7123 −908 6073 40 H(14A) 9678 −911 5868 49 H(14B) 10149   192 6215 49 H(16A) 8234 −579 6922 49 H(16B) 7328 −1856  6734 49 H(18) 2717 5279 6588 39 H(19)  767 5459 7083 48 H(20) −319 3560 7373 49 H(22) 2478 1280 6669 36 H(24) 5740 1337 7131 39 H(25) 6199 2019 7812 49 H(26) 6612 4246 7954 54 H(27) 6633 5781 7415 48

Example 4

Single-Crystal Calculation X-Ray Powder Diffraction Data for the Crystal of the Monohydrate of Formula II

(41) 1. Calculation software: Mercury 3.8 (Build RC2); wavelength: 1.54056.

(42) 2. X-ray powder diffraction data:

(43) The peak positions and intensity of the characteristic peak of the X-ray powder diffraction spectrum calculated from the single crystal of the crystal of the monohydrate of formula II are shown in Table 6:

(44) TABLE-US-00006 TABLE 6 Nos. 2θ (degree) relative intensity (I/I.sub.0) 1 5.48 16 2 9.14 62 3 10.30 9 4 12.00 6 5 13.34 26 6 14.44 100 7 14.94 10 8 15.70 24 9 16.52 34 10 17.70 9 11 18.70 7 12 19.20 21 13 19.38 15 14 20.42 42 15 21.00 61 16 21.30 31 17 21.90 23 18 22.08 52 19 22.26 22 20 23.08 31 21 23.60 6 22 24.56 35 23 24.74 23 24 25.88 8 25 26.22 19 26 27.46 10 27 27.68 7 28 28.14 8 29 29.08 12 30 29.50 21 31 30.56 12 32 31.26 9 33 31.86 10 34 32.36 8 35 32.96 5 36 33.58 7 37 36.40 7 38 38.02 12

Example 5

Preparation of the Crystal of the Monohydrate of Formula II

(45) 1.0 g of the compound of formula I prepared in Example 1 was added to 15 mL of methanol, stirred at room temperature till the solution was clear, and then added with 2 mL of water. The solution was cooled down to 0-5° C. with stirring, for crystallization. The crystal was filtered, and dried under reduced pressure at 40° C., to give 0.5 g of the crystal of the monohydrate of formula II.

Example 6

Preparation of the Crystal of the Monohydrate of Formula II

(46) 17 g of the compound of formula I prepared in Example 1 was added to 75 mL of anhydrous ethanol, heated to 60° C. with stirring till the solution was clear, and then added with 1.5 mL of water. The solution was cooled down to 0-5° C. with stirring, for crystallization. The crystal was filtered, and dried under reduced pressure at 40° C., to give 15.8 g of the crystal of the monohydrate of formula II.

Example 7

Preparation of the Crystal of the Compound of Formula I

(47) 1.0 g of the compound of formula I prepared in Example 1 was added to 5 mL of anhydrous dichloromethane, and stirred at room temperature till the solution was clear. The solution was then added with 1 g of 4A molecular sieve, and dried with stirring for 2 hours. The mixture was filtered under nitrogen protection. The filtrate was concentrated under reduced pressure at room temperature to remove a half volume of the solvent, transferred to −10° C., and stirred under nitrogen protection to crystallization, followed by filtration. The filter cake was dried under reduced pressure at 40° C., to give the crystal of the compound of formula I.

Example 8

Preparation of the Crystal of the Compound of Formula I

(48) 1.5 g of the compound of formula I prepared in Example 1 was added to 10 mL of anhydrous isopropanol, and heated to 60° C. with stirring till the solution was clear. The solution was then added with 2 g of 4A molecular sieve, and dried with stirring for 2 hours. The mixture was filtered under nitrogen protection. The filtrate was sealed, then naturally cooled down to room temperature and stirred to crystallization under nitrogen protection, followed by filtration. The filter cake was dried under reduced pressure at 40° C., to give the crystal of the compound of formula I.

Example 9

Amorphous Form of the Compound of Formula I

(49) 1.5 g of the compound of formula I prepared in Example 1 was added to 10 mL of anhydrous dichloromethane, and stirred at room temperature till the solution was clear. The solution was concentrated under reduced pressure to give a solid. The solid was then dried under reduced pressure at 40° C., to give an amorphous form of the compound of formula I.

Example 10

Amorphous Form of the Compound of Formula I

(50) 1 g of the compound of formula I prepared in Example 1 was added to a mixed solvent of anhydrous ethyl acetate (1 mL) and anhydrous dichloromethane (8 mL), and stirred at room temperature till the solution was clear. The solution was then concentrated under reduced pressure to give a solid. Subsequently, the solid was dried under reduced pressure at 40° C., to give an amorphous form of the compound of formula I.

Test Example 1

Stability Testing for the Crystal of the Monohydrate of Formula II

(51) In accordance with “Stability Testing of New Drug Substances and Products” in ICH Q1A and “Guidelines for the Stability Testing of Drug Substances and Preparations” in Pharmacopoeia of China, fourth part, 2015 edition, 9001, the stability influencing factors were tested for the crystal of the monohydrate formula II, including high temperature, high humidity and light tests. The results are shown in Tables 7, 8 and 9:

(52) TABLE-US-00007 TABLE 7 Stability results of high temperature test High temperature test High temperature test (40° C.) (60° C.) Item 0 (day) 5 (day) 10 (day) 5 (day) 10 (day) Appearance White White White White White powder powder powder powder powder Total impurities 0.38 0.45 0.47 0.48 0.47 (%) Enantiomers (%) 0.01 0.01 0.01 ND 0.01 Total of 0.05 0.05 0.05 0.05 0.05 diastereoisomers (%) Water (%) 3.10 2.69 2.45 2.67 2.51 Note: ND means not detected.

(53) TABLE-US-00008 TABLE 8 Stability results of high humidity test High humidity test High humidity test (75% RH) (92.5% RH) Item 0 (day) 5 (day) 10 (day) 5 (day) 10 (day) Appearance White White White White White powder powder powder powder powder Total impurities 0.38 0.44 0.50 0.49 0.49 (%) Enantiomers (%) 0.01 0.01 0.01 0.01 0.01 Total of 0.05 0.05 0.05 0.04 0.05 diastereoisomers (%) Water (%) 3.10 2.69 2.53 2.72 2.42

(54) TABLE-US-00009 TABLE 9 Stability results of light test Lighting shading Item 0 (day) 5 (day) 10 (day) 5 (day) 10 (day) Appearance White White White White White powder powder powder powder powder Total impurities 0.38 0.56 0.63 0.49 0.47 (%) Enantiomers (%) 0.01 0.01 0.01 0.01 0.01 Total of 0.05 0.05 0.05 0.05 0.05 diastereoisomers (%) Water (%) 3.10 2.72 2.47 2.73 2.55

Test Example 2

Stability Testing of the Crystal of the Compound of Formula I

(55) In accordance with “Stability Testing of New Drug Substances and Products” in ICH Q1A and “Guidelines for the Stability Testing of Drug Substances and Preparations” in Pharmacopoeia of China, fourth part, 2015 edition, 9001, the stability influencing factors were tested for the crystal of the compound of formula I, including high temperature and high humidity tests. The results are shown in Tables 10 and 11:

(56) TABLE-US-00010 TABLE 10 Stability results of high temperature test High temperature test High temperature test (40 °C.) (60° C.) Item 0 (day) 5 (day) 12 (day) 5 (day) 12 (day) Appearance White White White White White powder powder powder powder powder Total impurities 0.39 0.42 0.37 0.63 0.54 (%) Water (%) 0.45 0.85 0.85 0.59 0.73

(57) TABLE-US-00011 TABLE 11 Stability results of high humidity test High humidity test High humidity test (75% RH) (92.5% RH) Item 0 (day) 5 (day) 12 (day) 5 (day) 12 (day) Appearance White White White White White powder powder powder powder powder Total impurities 0.39 0.43 0.37 0.47 0.37 (%) Water (%) 0.45 0.94 1.16 1.58 1.75

Test Example 3

Stability Testing of Amorphous Form of the Compound of Formula I

(58) In accordance with “Stability Testing of New Drug Substances and Products” in ICH Q1A and “Guidelines for the Stability Testing of Drug Substances and Preparations” in Pharmacopoeia of China, fourth part, 2015 edition, 9001, the stability influencing factors were tested for the amorphous form of the compound of formula I, including high temperature and high humidity tests. The results are shown in Tables 12 and 13:

(59) TABLE-US-00012 TABLE 12 Stability results of high temperature test High temperature test (40° C.) High temperature test (60° C.) Item 0 (day) 5 (day) 10 (day) 30 (day) 5 (day) 10 (day) 30 (day) Appearance White White White White White White White powder powder powder powder powder powder powder Total 0.28 0.25 0.28 0.26 0.31 0.27 0.28 impurities (%) Water (%) 1.4 1.1 1.0 0.8 0.8 0.6 0.5

(60) TABLE-US-00013 TABLE 13 Stability results of high humidity test High humidity test (75% RH) High humidity test (92.5% RH) Item 0 (day) 5 (day) 10 (day) 30 (day) 5 (day) 10 (day) 30 (day) Appearance White White White White White White White powder powder powder powder powder powder powder Total 0.28 0.30 0.30 0.27 0.30 0.29 0.26 impurities (%) Water (%) 1.4 1.7 1.8 1.8 2.1 2.3 2.4

Test Example 4

Bioactivity Experiments

(61) Enzyme Assay:

(62) Resazurin is a traditional redox dye, and after a redox reaction, it can be reduced from a blue resazurin without fluorescence to a pink fluorescent substance, resorufin, which can be measured and quantified with relative fluorescence unit (RFU) of fluorophotometer (Ex=530-570 nm, Em=590-620 nm). At present, resazurin is widely used for determining the viability of bacteria, cells, etc., and the enzyme activity detection of oxidoreductase. We detected the decrease of cofactor NADPH to determine the inhibitory activity of a compound against IDH1m and detected the generation of cofactor NADPH to determine the inhibitory activity of a compound against IDH WT. The compound was pre-incubated with IDH1m and NADPH, and then the reaction was initiated by adding α-KG and performed for certain time under a linear condition. Then, diaphorase (lipoamide dehydrogenase) and the corresponding substrate resazurin were added thereto for detection. Lipoamide dehydrogenase terminated the IDH1m reaction by decreasing the available cofactor NADPH, which oxidized NADPH to NADP, and reduced resazurin to high fluorescent resorufin. The amount of the remaining cofactor NADPH after a specific reaction time was quantified via an easily detectable fluorophore.

(63) The compound was pre-incubated with IDH-WT and NADP, and then the reaction was initiated by adding isocitric acid, diaphorase (lipoamide dehydrogenase) and the corresponding substrate resazurin, and performed for certain time under a linear condition, followed by detecting the amount of fluorescent substance. NADP was reduced to NADPH in this experiment, and the latter reduced resazurin to high fluorescent resorufin under the action of lipoamide dehydrogenase. The amount of the generated cofactor NADPH after a specific reaction time was quantified via a detectable fluorophore, so as to calculate the inhibitory effect of the compound on IDH-WT.

(64) The specific operation was as follows: 2.5 μl of the compound diluted in a 3-fold gradient was added to a 384-well plate, followed by adding 5 μl of the reaction buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10 mM MgCl.sub.2; 0.4 mg/mL BSA (Bovine Serum Albumin) and 2 mM DTT (dithiothreitol)) containing 40 nM IDH1 (R132H/R132C) and 20 μM NADPH. Then, the above test mixture was incubated at 23° C. for 16 hours, and then 2.5 μl of the reaction buffer containing 4 mM α-KG was added to initiate the reaction. After they were incubated for 60 minutes at room temperature, 5 μl of the termination mixture (0.4 U/ml diaphorase and 20 μM resazurin) formulated with the reaction buffer was added to convert resazurin to resorufin, so as to measure the amount of the remaining NADPH. After incubating at 23° C. for 10 minutes, fluorescence values were determined through Flexstation 3 at Ex535/Em595. The enzyme activity of the compound was respectively determined at 12 concentrations, and the data were calculated using the software GraFit6.0 (Erithacus Software) to obtain the IC.sub.50 value of the compound.

(65) 2-HG determination:

(66) In the presence of 2-HG, phosphoglycerate dehydrogenase PHGDH can reduce NAD to NADPH, and the latter may be quantitatively determined by lipoamide dehydrogenase and the substrate thereof, resazurin.

(67) HT-1080 cell is a human fibrosarcoma cell line with an IDH1 mutation (R132C). U87 cell is a human glioblastoma cell line with an IDH1 mutation (R132H). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 0.1 mg/mL streptomycin.

(68) Cells was digested with trypsin, and inoculated into a 6-well plate at a density of 5×10.sup.5, and cultured overnight in an incubator at 37° C. Next day, the test compound was added (the final concentration of DMSO is 0.1%) thereto and cultured for another 24 hours. Culture medium of each sample was sucked out and centrifuged at 1000 rpm for 10 min. The supernatant was sucked out to detect the content of 2-HG therein. Additionally, cells were washed with PBS (phosphate buffered saline), digested with trypsin and collected. After the collected cells were washed with PBS for one time, the determination of intracellular 2-HG content was performed.

(69) The method for determining the intracellular 2-HG was as follows: cells were re-suspended with 300 μL reaction buffer (40 mM Tris-HCl, pH 8.5; 150 mM NaCl) and disrupted by ultrasonication. They were centrifuged for 10 min at 12,000 rpm and 4° C. to remove insoluble substances. 25 μL supernatant was sucked out to determine the protein concentration by a BCA kit. Another 200 μL supernatant was transferred to a new group of centrifuge tubes, each of which was added with 4 μL of 3 M HCl, placed at room temperature for 5 min and centrifuged at 12,000 rpm for 5 min at room temperature. 100 μL supernatant was sucked out and transferred to a 96-well “V” bottom plate, and 3.6 μL of 2 M Tris base (tromethamine) was added to each well, it was placed at room temperature for 5 min and centrifuged at 12,000 rpm for 2 min. The pH was approximately equal to 8.0 through detection by pH test paper.

(70) Preparation of standard curve of 2-HG: 2-HG stock solution was diluted to 500 μM with reaction buffer, and then 200 μL was taken therefrom for a 2-fold gradient dilution, 10 concentrations in total. The following operations were same as described above, including the steps for acid treatment and alkali neutralization.

(71) The aforementioned samples, the test cell samples or standard samples, were diluted in 5 folds, and then 5 μL of each sample was taken therefrom and added to a 384-well plate. 10 μL of the detection mixture (8 μM PHGDH (phosphoglycerate dehydrogenase); 0.5 mM NAD; 0.1 U/mL diaphorase and 10 μM resazurin) was added to each well, and they were reacted for 60 min at 23° C. Fluorescence values were determined through Flexstation 3 at Ex535/Em595.

(72) The measured fluorescence values were compared after being corrected with the protein concentrations of the corresponding samples.

(73) The method for determining extracellular 2-HG was as follows: 500 μL of each culture medium supernatant was taken. 10 μL of 3 M HCl was added into each tube and placed for 5 min at room temperature. Then, 18 μL of 2 M Tris base was added into each tube and placed for 5 min at room temperature. It was centrifuged at 12,000 rpm for 2 min. The pH was approximately equal to 8.0 detected by pH test paper. Preparation of standard curve of 2-HG: 2-HG stock solution was diluted to 500 μM with complete medium, and then 500 μL was taken therefrom for a 2-fold gradient dilution, 10 concentrations in total. The following operations were same as described above, including the steps for acid treatment and alkali neutralization. The aforementioned samples, the test culture supernatant samples or standard samples, were diluted in 5 folds, and then 5 μL was taken therefrom and added to a 384-well plate. 10 μL of the detection mixture (8 μM PHGDH; 0.5 mM NAD; 0.1 U/mL diaphorase and 10 μM resazurin) was added to each well and reacted for 60 min at 23° C. Fluorescence values were determined through Flexstation 3 at Ex535/Em595.

(74) The compound of formula I was analyzed according to the biological methods herein, and the results are as follows:

(75) The inhibitory activities (IC.sub.50) of the compound of formula I against IDH1 mutants (R132H and R132C) are shown in Table 14.

(76) TABLE-US-00014 TABLE 14 Compound IDH1(R132H) IC.sub.50 (nM) IDH1(R132C) IC.sub.50 (nM) Compound of formula <20 <20 I

Test Example 3

Pharmacokinetic Experiments

(77) Male SD rats were from Beijing Vital River Laboratory Animal Technology Co., Ltd., and divided into groups (3 rats per group). The rats were intragastrically administered with the test sample suspension (5 mg/kg) via a single peroral administration, respectively. The animals were fasted overnight before this study. The fasting time period was from 10 hours before administration to 4 hours after administration. Blood samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. After the rats were anesthetized with isoflurane using an anesthesia machine for small animal, and then 0.3 mL whole blood samples were taken from the fundus venous plexus. The blood samples were placed in heparin anticoagulant tubes, and centrifuged for 5 min at 4° C. and 4000 rpm. The plasma was transferred to centrifuge tubes, and stored at −80° C. till analysis. The samples in plasma were extracted through protein precipitation. The liquid extract was analyzed by LC-MS/MS, wherein HPLC conditions were as follows: flow rate 0.4 mL/min; mobile phase A: water/formic acid (99.9/0.1, v/v); mobile phase B: acetonitrile/formic acid (99.9/0.1, v/v); injection volume: 5 μL; column temperature: RT; autosampler temperature: RT; run time: 2.5 min.

(78) Pharmacokinetic data of the compound of formula I is shown in Table 15:

(79) TABLE-US-00015 TABLE 15 The compound of Formula I Gender of rata male Oral dose (mg/kg) 5 T.sub.1/2(hr) 10.7 Tmax(hr) 4.0 Cmax(ng/mL) 556 AUC.sub.INF_.sub.obs(hr*ng/mL) 10567 Formulation of dosage forms 0.5% MC, 0.2% Tween80