Entinostat-containing compound, crystal form of compound thereof, and preparation method therefor and pharmaceutical composition thereof

Abstract

The present invention relates to a compound formed by entinostat as shown in formula (I) and acidic counterion. Compared with the known solid form of entinostat, the compound involved has advantages in terms of solubility, stability, etc. The present invention also relates to a crystalline form of the compound and a preparation method therefor, a pharmaceutical composition thereof and the use thereof in the preparation of a drug for preventing and/or treating a disease associated with differentiation or proliferation. ##STR00001##

Claims

1. A crystalline Compound A of formula (II): ##STR00007## wherein the X-ray powder diffraction pattern of the crystalline Compound A, expressed as 2θ angles, has the following characteristic peaks: 3.9°±0.2°, 13.4°±0.2°, 16.4°±0.2°, and 18.8°±0.2°.

2. The crystalline Compound A according to claim 1, wherein the X-ray powder diffraction pattern of the crystalline Compound A, expressed as 2θ angles, has the following characteristic peaks: 11.5°±0.2°, 15.3°±0.2°, 18.3°±0.2°, and 19.7°±0.2°.

3. The crystalline Compound A according to claim 2, wherein the X-ray powder diffraction pattern of the crystalline Compound A, expressed as 2θ angles, has the following characteristic peaks: 7.7°±0.2°, 10.9°±0.2°, 22.8°±0.2° and 26.2°±0.2°.

4. A method of preparing the crystalline Compound A according to claim 1, the method comprising: (a) dissolving entinostat and fumaric acid respectively in a solvent to form a solution of entinostat and a solution of fumaric acid, then mixing the solution of entinostat and the solution of fumaric acid and stirring for crystallization, separating precipitated crystals, and drying to obtain the crystalline Compound A; wherein: the solvent is an alcohol or a ketone; the mass-volume ratio of entinostat to the solvent in the solution of entinostat ranges from 10 mg/mL to 100 mg/mL; the molar ratio of entinostat to fumaric acid ranges from 1:0.5 to 1:1.0; the duration for stirring is from 1 to 3 days; and the operation is performed at 10° C. to 40° C.; or (b) forming a solution of the mixture of entinostat and fumaric acid in a solvent, then drying the solvent by volatilization to obtain the crystalline Compound A; wherein: the solvent is an alcohol or a ketone; the mass-volume ratio of entinostat to the solvent ranges from 5 to 50 mg/mL; the molar ratio of entinostat to fumaric acid ranges from 1:0.5 to 1:1.0; and the volatilization is performed at 10° C. to 40° C.

5. The method according to claim 4, wherein the solvent is ethanol.

6. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline Compound A according to claim 1, and at least one pharmaceutically acceptable carrier or additive.

7. A crystalline Compound B of formula (III): ##STR00008## wherein the X-ray powder diffraction pattern of the crystalline Compound B, expressed as 2θ angles, has the following characteristic peaks: 9.8°±0.2°, 15.9°±0.2°, 19.7°±0.2°, and 23 0.2°±0.2°.

8. The crystalline Compound B according to claim 7, wherein the X-ray powder diffraction pattern of the crystalline Compound B, expressed as 2θ angles, has the following characteristic peaks: 14.1°±0.2°, 14.8°±0.2°, 17.7°±0.2°, and 18.9°±0.2°.

9. The crystalline Compound B according to claim 8, wherein the X-ray powder diffraction pattern of the crystalline Compound B, expressed as 2θ angles, has the following characteristic peaks: 14.5°±0.2°, 21.4°±0.2°, 23.8°±0.2°, and 24.8°±0.2°.

10. A method of preparing the crystalline Compound B according to claim 7, the method comprising: (a) dissolving entinostat and maleic acid respectively in a solvent to form a solution of entinostat and a solution of maleic acid, then mixing the solution of entinostat and the solution of maleic acid and stirring for crystallization, separating precipitated crystals, and drying to obtain the crystalline Compound B; wherein: the solvent is an alcohol or a ketone; the mass-volume ratio of entinostat to the solvent in the solution of entinostat ranges from 10 mg/mL to 100 mg/mL; the molar ratio of entinostat to maleic acid ranges from 1:1.0 to 1:1.5; the duration for stirring is from 1 to 3 days; and the operation is performed at room temperature; or (b) forming a solution of the mixture of entinostat and maleic acid in a solvent, then drying the solvent by volatilization to obtain the crystalline Compound B; wherein: the solvent is an alcohol or a ketone; the mass-volume ratio of entinostat to the solvent ranges from 5 to 50 mg/mL; the molar ratio of entinostat to maleic acid ranges from 1:1.0 to 1:1.5; and the volatilization is performed at room temperature.

11. The method according to claim 10, wherein the solvent is ethanol.

12. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline Compound B according to claim 7, and at least one pharmaceutically acceptable carrier or additive.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is the XPRD pattern of entinostat Form A prepared with reference to U.S. Pat. No. 6,174,905B1.

(2) FIG. 2 is the XPRD pattern of entinostat Form B prepared with reference to CN102137846B.

(3) FIG. 3 is the XPRD pattern of entinostat Form C prepared with reference to CN102137846B.

(4) FIG. 4 is the XRPD pattern of Crystalline Form of Compound A of the present invention.

(5) FIG. 5 is the XRPD pattern of Crystalline Form of Compound B of the present invention.

(6) FIG. 6 is the XRPD pattern of Crystalline Form of Compound C of the present invention.

(7) FIG. 7 is the XRPD pattern of Crystalline Form of Compound D of the present invention.

(8) FIG. 8 is the DSC thermogram of Crystalline Form of Compound A of the present invention.

(9) FIG. 9 is the DSC thermogram of Crystalline Form of Compound B of the present invention.

(10) FIG. 10 is the TGA thermogram of Crystalline Form of Compound A of the present invention.

(11) FIG. 11 is the TGA thermogram of Crystalline Form of Compound B of the present invention.

(12) FIG. 12 is the IR pattern of Crystalline Form of Compound A of the present invention.

(13) FIG. 13 is the IR pattern of Crystalline Form of Compound B of the present invention.

SPECIFIC IMPLEMENTATIONS

(14) The following examples will help to further understand the present invention, but are not intended to limit the contents of the present invention.

(15) Instruments and Characterization Methods:

(16) X-ray powder diffraction (XRPD): performed on Bruker D8 Advance diffractometer. The scan range 3-40° 20, step size 0.02020, and speed 0.2 s/step.

(17) Differential thermal analysis (DSC) data were collected on TA Instruments Q200 DSC. The heating rate is 10° C./min under the protection of nitrogen purge.

(18) Thermogravimetric analysis (TGA) data were collected on TA Instruments Q500 TGA, using High Resolution™, with the heating rate of 10° C./min under the protection of nitrogen purge.

(19) .sup.1H Nuclear magnetic resonance spectrum (.sup.1H-NMR): data were collected on BrukerAvance II DMX 400 MHZ nuclear magnetic resonance spectrometer. The sample was dissolved using deuterated reagent.

(20) Infrared spectral analysis (IR) data were collected on Bruker Tensor 27, using diffuse reflection (KBr) and ATR.

(21) HPLC data were collected on Ultimate 3000 under the following conditions: column, YMC-Pack ODS-AQ (4.6*250 mm*5 μm); column temperature, 40° C.; flow rate, 1.5 mL/min; detection wavelength, 254 nm; injection volume 3 uL; operation time, 40 min; mobile phase, a mixture of acetonitrile: 10 mM monopotassium phosphate=1:2.

(22) Unless particularly specified, all reagents used in the Examples were commercially available.

(23) Unless particularly specified, all Examples were operated at room temperature.

Preparation Example 1

(24) Entinostat was prepared by referencing the methods described in example 48 of patent document U.S. Pat. No. 6,174,905B1.

(25) HPLC: a maximum single impurity of 1.42% at the RRT of 1.403.

(26) The XRPD pattern is shown in FIG. 1, which is consistent with the diffraction pattern of entinostat Form A in patent document CN102137846B.

Preparation Example 2

(27) Entinostat Form B and entinostat Form C were prepared by referencing the methods described in example 1 of patent document CN102137846B.

(28) The XRPD pattern of entinostat Form B is shown in FIG. 2.

(29) HPLC: a maximum single impurity of 1.43% at the RRT of 1.403.

(30) The XRPD pattern of entinostat Form C is shown in FIG. 3.

(31) There is a maximum single impurity of 1.42% at the RRT of 1.403.

Example 1

(32) Ethanol (8 mL) was added to entinostat (80 mg) to form a solution, slowly dripping in 1 mL of fumaric acid solution (0.5 molar equivalent, in ethanol) to it. The mixture was stirred to crystallize for 2 days at room temperature, centrifuged, and dried under vacuum at room temperature to obtain Compound A (78 mg). Yield: 84%.

(33) .sup.1H-NMR (500 MHz, Methanol-d4) δ ppm: 8.58 (d, J=2.3 Hz, 1H), 8.50 (dd, J=4.9, 1.7 Hz, 1H), 7.94 (d, J=8.0 Hz, 2H), 7.89 (dt, J=7.9, 2.0 Hz, 1H), 7.50-7.35 (m, 3H), 7.22-7.16 (m, 1H), 7.09 (td, J=7.7, 1.5 Hz, 1H), 6.92 (dd, J=8.0, 1.5 Hz, 1H), 6.79 (td, J=7.6, 1.4 Hz, 1H), 6.75 (s, 1H), 5.18 (s, 2H), 4.39 (d, J=5.4 Hz, 2H), indicating that the molar ratio of entinostat to fumaric acid in Compound A is 1:0.5.

Example 2

(34) Ethanol (2 mL) was added to entinostat (60 mg), and was heated moderately to form a solution, slowly dripping in 1 mL of fumaric acid solution (0.6 molar equivalent, in ethanol) to it. The mixture was stirred to crystallize for 3 days at room temperature, centrifuged, and dried under vacuum at room temperature to obtain Compound A (60 mg). Yield: 87%.

(35) Its XRPD pattern is shown in FIG. 4, showing the compound is Crystalline Compound A.

(36) Its DSC thermogram is shown in FIG. 8, showing a melting point of 166° C.

(37) Its TGA thermogram is shown in FIG. 10, showing a weight loss of 0.3% before 100° C. It is anhydrous, with a decomposition temperature at 160° C.

Example 3

(38) Acetone (3 mL) was added to entinostat (45 mg) to form a solution, slowly dripping in 1 mL of fumaric acid solution (0.8 molar equivalent, in acetone) to it. The mixture was stirred to crystallize for 1 day at 10° C., centrifuged, and dried under vacuum at room temperature to obtain Compound A (39 mg). Yield: 75%.

Example 4

(39) Methanol (0.5 mL) and acetone (0.5 mL) were added to entinostat (100 mg), and was heated moderately to form a solution, then slowly dripping in 1 mL of fumaric acid solution (1 molar equivalent, in methanol) to it. The mixture was stirred to crystallize for 3 days at 40° C., centrifuged, and dried under vacuum at room temperature to obtain Compound A (75 mg). Yield: 65%.

Example 5

(40) Compound A can also be obtained by replacing the solvents in Example 4 with the following solvents in the table below.

(41) TABLE-US-00001 Experiment Number Solvent 1 Solvent 2 Experiment 1 methanol ethanol Experiment 2 ethanol butanone Experiment 3 trifluoroethanol acetone Experiment 4 isopropanol acetone Experiment 5 n-propanol acetone Experiment 6 sec-butanol isopropanol Experiment 7 n-butanol butanone

Example 6

(42) Ethanol (5 mL) was added to a mixture of entinostat (25 mg) and fumaric acid (3.9 mg) to form a solution. The solution was volatilized until solvent dryness at room temperature to obtain Compound A (26 mg). Yield: 90%.

Example 7

(43) Ethanol (10 mL) was added to a mixture of entinostat (200 mg) and fumaric acid (37.0 mg), and was heated moderately to form a solution. The solution was volatilized until solvent dryness at room temperature to obtain Compound A (220 mg). Yield: 95%.

Example 8

(44) Methanol (1 mL) was added to a mixture of entinostat (50 mg) and fumaric acid (15.4 mg) to form a solution. The solution was volatilized until solvent dryness at 40° C. to obtain Compound A (48 mg). Yield: 83%.

Example 9

(45) Methanol (1.5 mL) and acetone (1.5 mL) were added to a mixture of entinostat (90 mg) and fumaric acid (25.0 mg) to form a solution. The solution was volatilized until solvent dryness at 10° C. to obtain Compound A (85 mg). Yield: 82%.

Example 10

(46) The Compound A can also be obtained by replacing the solvents in Example 9 with the following solvents in the table below.

(47) TABLE-US-00002 Experiment Number Solvent 1 Solvent 2 Experiment 1 methanol isopropanol Experiment 2 ethanol trifluoroethanol Experiment 3 trifluoroethanol butanone Experiment 4 isopropanol acetone Experiment 5 n-propanol acetone Experiment 6 sec-butanol ethanol Experiment 7 n-butanol acetone

(48) The samples prepared in Examples 3 to 10 had the same or similar XRPD patterns (not shown), DSC thermograms (not shown) and TGA thermograms (not shown) as the sample in Example 2. It indicates that the samples in Examples 3 to 10 and the sample in Example 2 are the same compounds.

Example 11

(49) Ethanol (10 mL) was added to entinostat (100 mg) to form a solution, then slowly dripping in 2 mL of maleic acid solution (1 molar equivalent, in ethanol) to it. The mixture was stirred to crystallize for 2 days at room temperature, centrifuged, and dried under vacuum at room temperature to obtain Compound B (108 mg). Yield: 83%.

(50) .sup.1H-NMR (500 MHz, Methanol-d4) δ ppm: 8.65 (d, J=2.1 Hz, 1H), 8.56 (dd, J=5.2, 1.6 Hz, 1H), 8.03 (dt, J=7.9, 1.9 Hz, 1H), 7.98 (d, J=8.0 Hz, 2H), 7.58 (dd, J=7.9, 5.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 2H), 7.32-7.24 (m, 1H), 7.20 (td, J=7.7, 1.5 Hz, 1H), 7.07 (dd, J=8.0, 1.4 Hz, 1H), 6.99 (td, J=7.6, 1.5 Hz, 1H), 6.30 (s, 2H), 5.23 (s, 2H), 4.40 (s, 2H), indicating that the molar ratio of entinostat to maleic acid in Compound B is 1:1.

Example 12

(51) Ethanol (10 mL) was added to entinostat (300 mg) and was heated moderately to form a solution, then slowly dripping in 6 mL of maleic acid solution (1.2 molar equivalent, in ethanol) to it. The mixture was stirred to crystallize for 3 days at room temperature, centrifuged, and dried under vacuum at room temperature to obtain Compound B (349 mg). Yield: 89%.

(52) Its XRPD pattern is shown in FIG. 5, showing that the compound is Crystalline Compound B.

(53) Its DSC thermogram is shown in FIG. 9, showing a melting point of 129° C.

(54) Its TGA thermogram is shown in FIG. 11, showing a weight loss of 0.3% before 100° C. It is anhydrous, with a decomposition temperature at 125° C.

Example 13

(55) Butanone (1 mL) was added to entinostat (15 mg) to form a solution, then slowly dripping in 0.5 mL of maleic acid solution (1.3 molar equivalent, in acetone) to it. The mixture was stirred to crystallize for 1 day at 10° C., centrifuged, and dried under vacuum at room temperature to obtain Compound B (15 mg). Yield: 76%.

Example 14

(56) Ethanol (0.5 mL) and acetone (0.5 mL) were added to entinostat (100 mg) and heated moderately to form a solution, then slowly dripping in 2 mL of maleic acid solution (1.5 molar equivalent, in ethanol) to it. The mixture was stirred to crystallize for 3 days at 40° C., centrifuged, and dried under vacuum at room temperature to obtain Compound B (99 mg). Yield: 76%.

Example 15

(57) The Compound B can be obtained by replacing the solvents in Example 14 with the following solvents in the table below.

(58) TABLE-US-00003 Experiment Number Solvent 1 Solvent 2 Experiment 1 methanol — Experiment 2 ethanol butanone Experiment 3 trifluoroethanol — Experiment 4 isopropanol ethanol Experiment 5 n-propanol butanone Experiment 6 sec-butanol trifluoroethanol Experiment 7 n-butanol butanone

Example 16

(59) Ethanol (40 mL) was added to a mixture of entinostat (200 mg) and maleic acid (61.7 mg) to form a solution. The solution was volatilized until solvent dryness at room temperature to obtain Compound B (245 mg). Yield: 94%.

Example 17

(60) Ethanol (10 mL) was added to a mixture of entinostat (200 mg) and maleic acid (74.0 mg), and heated moderately to form a solution. The solution was volatilized until solvent dryness at room temperature to obtain Compound B (230 mg). Yield: 88%.

Example 18

(61) Trifluoroethanol (2 mL) was added to a mixture of entinostat (100 mg) and maleic acid (46.3 mg), and it was heated moderately to form a solution. The solution was volatilized until solvent dryness at 10° C. to obtain Compound B (107 mg). Yield: 82%.

Example 19

(62) Ethanol (1.5 mL) and acetone (1.5 mL) were added to a mixture of entinostat (120 mg) and maleic acid (55.5 mg), and it was heated moderately to form a solution. The solution was volatilized until solvent dryness at 40° C. to obtain Compound B (125 mg). Yield: 80%.

Example 20

(63) The Compound B can be obtained by replacing the solvents in Example 19 with the following solvents in the table below.

(64) TABLE-US-00004 Experiment Number Solvent 1 Solvent 2 Experiment 1 methanol — Experiment 2 ethanol methanol Experiment 3 trifluoroethanol acetone Experiment 4 acetone — Experiment 5 n-propanol isopropanol Experiment 6 sec-butanol methanol Experiment 7 n-butanol acetone

(65) The samples prepared in Examples 13 to 20 had the same or similar XRPD patterns (not shown), DSC thermograms (not shown) and TGA thermograms (not shown) as the sample in Example 12. It indicates that the samples in Examples 13 to 20 and the sample in Example 12 are the same compounds.

Example 21

(66) Tetrahydrofuran (4 mL) was added to a mixture of entinostat (100 mg) and succinic acid (15.7 mg) to form a solution. The solution was volatilized until solvent dryness at room temperature to obtain Compound C (90 mg). Yield: 78%.

(67) Its XRPD pattern shown in FIG. 6, indicates that the compound is Crystalline Compound C.

Example 22

(68) Acetone (5 mL) was added to entinostat (80 mg) to form a solution, then slowly dripping in 1 mL of adipic acid solution (1.25 molar equivalent, in acetone) to it. The mixture was stirred to crystallize for 1 day at room temperature, centrifuged, and dried under vacuum at room temperature to obtain Compound D (86 mg). Yield: 77%.

(69) Its XRPD pattern is shown in FIG. 7, showing that the compound is Crystalline Compound D.

Example 23

(70) TABLE-US-00005 Component Dosage (mg) Compound A (active ingredient basis) or 4 Compound B (active ingredient basis) starch 407  magnesium stearate 3 Total 414 

(71) Compound A or Compound B, starch, and magnesium stearate were blended, sifted through 20 mesh sieve, and then filled into the hard capsule in an amount of 414 mg.

Example 24

(72) TABLE-US-00006 Component Dosage (mg) Compound A (active ingredient basis) or 8 Compound B (active ingredient basis) microcrystalline cellulose 200  silica colloid 10  magnesium stearate 5 Total 223 

(73) The above components were blended and compressed into tablets, and each tablet weighed 223 mg.

Comparative Example 1

(74) Entinostat, Compound A and Compound B in the present invention were tested for the apparent water solubility, and the operations were as follows:

(75) Placed 3 mg of sample in a 100 mL Erlenmeyer flask in a water bath at 25° C.±2° C., added water by doubling, stirred at about 200 rpm/min, checked visually if the solution was clear. The interval between each addition of water was 3 minutes, and the maximum amount of water to add was 60 mL.

(76) The results shown in the table below indicate that the water solubility of the Compound A and Compound B in the present invention is better than entinostat in the prior art.

(77) TABLE-US-00007 Compound Solubility (mg/mL) Compound A 0.13 Compound B 1.01 entinostat <0.05

Comparative Example 2

(78) Appropriate amounts of Crystalline Form of Compound A and Crystalline Form of Compound B in the present invention were used respectively for the phase stability test. The conditions were room temperature and high temperature (50° C.). The results are shown in the following table below:

(79) TABLE-US-00008 Crystal Form of Crystal Form of Conditions Days Compound A Compound B Room temperature 7 Crystalline form Crystalline form unchanged unchanged 14 Crystalline form Crystalline form unchanged unchanged High temperature 7 Crystalline form Crystalline form unchanged unchanged 14 Crystalline form Crystalline form unchanged unchanged

(80) The results show that the phases of Crystalline Form of Compound A and Crystalline Form of Compound B in the present invention are unchanged after having been stored at room temperature and high temperature for 14 days, demonstrating good crystal stability.

Comparative Example 3

(81) 1. Appropriate amounts of entinostat and Compound A in the present invention were used respectively for HPLC analysis, the purity of the compounds and the maximum single impurity (RRT=1.403) were calculated.

(82) 2. Chemical stability tests of entinostat and Compound A in the present invention were performed. The conditions were as follows: room temperature, high temperature (40° C.), high humidity (75% RH), and acceleration (40° C.-75% RH), and the storage time is 14 days. Using HPLC analysis, the change of maximum single impurity (RRT=1.403), the change of the main ingredient level and the change of total impurities number were studied. The results are shown in the following table below:

(83) TABLE-US-00009 Entinostat Compound A Number Number Main of Main of Condi- RRT = 1.403 ingredient impurities RRT = 1.403 ingredient impurities tion Days (%) (%) (≥0.04%) (%) (%) (≥0.04%) Room 0 1.42 98.4 4 0.12 99.9 0 temperature Room 14 1.42 98.4 4 0.13 99.9 0 temperature High 14 1.44 98.4 5 0.11 99.9 0 temperature High 14 1.43 98.4 5 0.11 99.9 0 temperature Speeded-up 14 1.41 98.4 5 0.13 99.9 0

(84) The results show that:

(85) 1. Compared with entinostat, Compound A of the present invention has significantly reduced level of maximum single impurity (RRT=1.403), and has fewer impurities and higher purity.

(86) 2. After having been stored for 14 days under the conditions of room temperature, high temperature, high humidity and acceleration, the impurity at RRT=1.403, the main ingredient and the number of impurities of both entinostat and Compound A of the present invention remained essentially unchanged.

(87) The above results show that compared with entinostat, Compound A of the present invention has lower impurity level at RRT=1.403, less number of impurities, higher chemical purity and excellent chemical stability under the conditions of room temperature, high temperature, high humidity and acceleration, respectively.

Comparative Example 4

(88) 50 mg of entinostat from Preparation Example 1 and 50 mg of Compound B of the present invention were respectively stirred in 1.3 ml of methanol, heated to dissolve, after 1 hour, cooled to 4° C. for recrystallization, and were analyzed by HPLC. Experimental results: after recrystallization, the impurity level at RRT=1.403 in the entinostat sample was 1.42%, and the purity of the entinostat sample was 98.4%, both were the same as before recrystallization. The impurity level of RRT=1.403 in Compound B decreased from 1.53% before recrystallization to 1.27% after recrystallization, and its purity increased from 98.3% before recrystallization to 98.5% after recrystallization. The results show that Compound B of the present invention is easier to purify by recrystallization using organic solvents than the known entinostat.

(89) The described above are only specific embodiments for illustrating the present invention, but without limiting it to that. Any changes or alternations, without creative work, made by those skilled in the art within the technical scope disclosed by the present invention, should fall within the scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection defined in the claims.