Crystal forms of a 9-aminomethyl substituted tetracycline compound and a preparation method thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and crystal forms of a 9-aminomethyl substituted tetracycline compound and a process for preparing the same. More specifically, the present invention relates to crystal forms of the compound represented by formula (1), a process for preparing crystal forms of the compound represented by formula (1) and use of said crystal forms in manufacture of medicament for treating and/or preventing an infection disease caused by tetracycline-sensitive bacteria and/or tetracycline-resistant bacteria. ##STR00001##

Claims

1. Crystal forms of a compound represented by formula (1), (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide, ##STR00004## which are characterized by having X-ray powder diffraction patterns comprising the following characteristic peaks expressed by 2 degree, when measured using Cu-Ka radiation: Crystal form I: 10.60.2, 13.30.2, 15.90.2, 24.00.2; Crystal form II: 10.20.2, 15.90.2, 17.90.2, 24.10.2; Crystal form VII: 11.70.2, 15.10.2, 15.90.2, 17.90.2.

2. Crystal forms of the compound represented by formula (1) of claim 1, which are characterized by having X-ray powder diffraction patterns comprising the following characteristic peaks expressed by 2 degree, when measured using Cu-Ka radiation: Crystal form I: 9.00.2, 10.60.2, 13.30.2, 15.90.2, 23.60.2, 24.00.2; Crystal form II: 9.30.2, 10.20.2, 14.00.2, 15.90.2, 17.90.2, 24.10.2; Crystal form VII: 11.70.2, 15.10.2, 15.90.2, 17.90.2, 20.10.2, 21.80.2.

3. Crystal forms of the compound represented by formula (1) of claim 1, which are characterized by having X-ray powder diffraction patterns comprising the following characteristic peaks expressed by 2 degree, when measured using Cu-Ka radiation: Crystal form I: 9.00.2, 10.60.2, 13.30.2, 14.30.2, 15.90.2, 18.00.2, 20.00.2, 21.30.2, 23.60.2, 24.00.2; Crystal form II: 9.30.2, 10.20.2, 14.00.2, 15.90.2, 17.90.2, 20.80.2, 23.00.2, 24.10.2, 24.80.2, 27.70.2; Crystal form VII: 11.00.2, 11.70.2, 13.80.2, 14.30.2, 15.10.2,15.90.2, 17.90.2, 20.10.2, 21.80.2, 25.60.2.

4. Crystal forms of the compound represented by formula (1) of claim 1, which are characterized in that: Crystal form I has an X-ray powder diffraction pattern substantially as shown in FIG. 1; Crystal form II has an X-ray powder diffraction pattern substantially as shown in FIG. 3; Crystal form VII has an X-ray powder diffraction pattern substantially as shown in FIG. 10.

5. Crystal forms of the compound represented by formula (1) of claim 1, which are characterized in that: Crystal form I has an endothermic peak in the range of 180-220 C. in its differential scanning calorimetry curve; Crystal form II has an endothermic peak in the range of 195-215 C. in its differential scanning calorimetry curve; Crystal form VII has an endothermic peak in the range of 165-205 C. in its differential scanning calorimetry curve.

6. A process for preparing crystal forms I, II, and VII of the compound represented by formula (1) according to claim 1, wherein: the compound represented by formula (1) is placed in anhydrous acetonitrile for slurry-washing or in tetrahydrofuran for stirring, a solid separates from the mixture, and crystal form I is obtained through filtration; the compound represented by formula (1) is placed in a lower alcohol, ethyl acetate, acetone, or a mixed solution of methanol and water in a ratio of 1:1-9:1 (v/v) for slurry-washing, or dissolved in tetrahydrofuran under an atmosphere of isopropanol or n-heptane, a solid separates from the mixture, and crystal form II is obtained through filtration; the compound represented by formula (1) is placed in a mixed solution of acetonitrile and water in a ratio of 1:1-9:1 (v/v) for slurry-washing, filtered, and dried in vacuum to produce crystal form VII.

7. A pharmaceutical composition, wherein said pharmaceutical composition contains crystal form I, II, VII of the compound represented by formula (1) or a combination thereof according to claim 1, and pharmaceutically acceptable carrier(s).

8. The pharmaceutical composition of claim 7, wherein said crystal form has a content of not lower than 94.5%.

9. A pharmaceutical formulation containing crystal form I, II, VII of the compound represented by formula (1) or a combination thereof according to claim 1, and one or more pharmaceutically acceptable carriers and/or diluents, which is in any pharmaceutically acceptable dosage form.

10. A method for treating a disease caused by tetracycline-sensitive bacteria and/or tetracycline-resistant bacteria, comprising: administering to a recipient in need thereof, an amount of a medicament comprising the crystal form of the compound represented by formula (1) of claim 1, a pharmaceutically acceptable salt, solvate thereof, wherein said disease caused by tetracycline-sensitive bacteria and/or tetracycline-resistant bacteria is selected from infection, cancer, diabetes and other diseases that have been found to be treatable by other tetracycline compounds, wherein said crystal form is selected from crystal form I, II, VII or a combination thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: the XRPD spectrum for crystal form I of the compound represented by formula (1);

(2) FIG. 2: the DSC curve for crystal form I of the compound represented by formula (1);

(3) FIG. 3: the XRPD spectrum for crystal form II of the compound represented by formula (1);

(4) FIG. 4: the DSC curve for crystal form II of the compound represented by formula (1);

(5) FIG. 5: the XRPD spectrum for crystal form III of the compound represented by formula (1);

(6) FIG. 6: the DSC curve for crystal form III of the compound represented by formula (1);

(7) FIG. 7: the XRPD spectrum for crystal form IV of the compound represented by formula (1);

(8) FIG. 8: the XRPD spectrum for crystal form V of the compound represented by formula (1);

(9) FIG. 9: the XRPD spectrum for crystal form VI of the compound represented by formula (1);

(10) FIG. 10: the XRPD spectrum for crystal form VII of the compound represented by formula (1);

(11) FIG. 11: the DSC curve for crystal form VII of the compound represented by formula (1);

(12) FIG. 12: the XRPD spectrum for crystal form VIII of the compound represented by formula (1);

(13) FIG. 13: the XRPD spectrum for amorphous form of the compound represented by formula (1);

(14) FIG. 14: the conversion relation among crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, crystal form VII, crystal form VIII and amorphous form of the compound represented by formula (1), wherein:

(15) 1. Slurry-washing in methanol or slurry-washing in a mixed solution of methanol and water (1:1-9:1, v/v);

(16) 2. Slurry-washing in acetonitrile or tetrahydrofuran;

(17) 3. Slurry-washing in a mixed solution of acetonitrile and water in a ratio of 1:1-4:1;

(18) 4. Slurry-washing in a mixed solution of acetonitrile and water in a ratio 9:1;

(19) 5. Slurry-washing in isopropyl acetate;

(20) 6. Crystallization with slow volatilization of chloroform solution or under the induction of polyvinyl acetate, polyphenylene sulfide, 1,3-dimethylimidazoline mesylate or 1,3-dimethylimidazoline perchlorate in the chloroform solution,

(21) 7. Slow volatilization of a mixed solution of tetrahydrofuran and isopropyl acetate (1:1, v/v); and

(22) 8. Drying under heat.

(23) The slurry-washing according to the present invention means washing a large amount of solid with a small amount of solvent under stirring.

(24) The lower alcohol according to the present invention means an alcohol containing 1-6 carbon atoms, including methanol, ethanol, propanol, isopropanol, butanol, iso-butanol, n-butanol, pentanol, hexanol and the like.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

(25) The present invention will be described in details by the following embodiments in form of Examples. However, it should be understood that the scope of the present invention is not limited by the following Examples, and all of the technical solutions accomplished based on the above contents of the present invention belong to the scope of the present invention.

Example 1: Preparation of an Amorphous Form of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

(26) According to the preparation process disclosed in the patent application WO2013013505A1, 8 g of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide was synthesized. The content of the crude product was 84.8%.

(27) 8 g of the crude product was dissolved in 1.5 L of water. Trifluoroacetic acid was added to adjust the pH to 2-4. After dissolution, ammonia water was added to adjust the pH to 6.5-7.5. After loading, the preparative separation was carried out with (1TFA/water)/acetonitrile (6%) system. The product-containing fraction was collected. The aqueous phase was adjusted with an aqueous sodium hydroxide solution to the pH of 7.5-8.5. The aqueous phase was kept at a temperature of 25-30 C., and extracted with dichloromethane for three times. The dichloromethane phases were combined. The combined dichloromethane phase was concentrated, and dropwise added to n-heptane. The mixture was cooled to a temperature of 0-5 C., stirred for 10-30 min, and filtered by suction to produce 3.17 g of the compound (having a content of 87.7% and a purity of 96.2%).

(28) Molecular formula: C.sub.29H.sub.36N.sub.4O.sub.7

(29) .sup.1H-NMR (CD.sub.3OD, 400 MHz) : 8.48 (s, 1H), 4.54 (brs, 2H), 4.22 (s, 1H), 3.65 (brs, 4H), 3.46 (m, 1H), 3.35 (s, 6H), 3.25 (m, 1H), 3.10 (s, 3H), 3.05 (s, 1H), 3.00 (s, 3H), 2.60 (m, 1H), 2.37 (m, 1H), 1.87-1.97 (m, 2H), 1.68 (m, 1H), 1.07 (m, 1H), 0.78 (m, 1H).

(30) In order to prepare the amorphous form of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide having a high content, the following processes were attempted:

(31) Crystal form II of the compound represented by formula (1) (having a content of 95.6%) was dissolved in an appropriate amount of dichloromethane, and dropwise added to the n-heptane system at a temperature of 0-5 C. The mixture was stirred for 30 min, and filtered by suction. The resulting solid was dried in vacuum to produce the amorphous form of the compound represented by formula (1) (having a content of 93.3%).

(32) Crystal form II of the compound represented by formula (1) (having a content of 97.0%) was dissolved in an appropriate amount of dichloromethane, and dropwise added to the n-heptane system at a temperature of 0-5 C. The mixture was stirred for 30 min, and filtered by suction. The resulting solid was dried in vacuum to produce the amorphous form of the compound represented by formula (1) (having a content of 93.9%).

(33) Crystal form II of the compound represented by formula (1) (having a content of 98.4%) was dissolved in an appropriate amount of dichloromethane, and dropwise added to the n-heptane system at a temperature of 0-5 C. The mixture was stirred for 30 min, and filtered by suction. The resulting solid was dried in vacuum to produce the amorphous form of the compound represented by formula (1) (having a content of 97.1%).

(34) The solid was characterized with XPRD as an amorphous form, as shown in FIG. 13.

Example 2: Preparation of Crystal Form I of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

(35) 0.5 g of the amorphous form of the compound represented by formula (1) prepared in Example 1 (having a content of 87.7%, and a purity of 96.2%) was placed in 5 mL of acetonitrile or 2 mL of THF. The mixture was heated to 55 C. and stirred for 0.5 h, and filtered. The obtained solid was placed in a vacuum drying oven and dried in vacuum at 50 C. for 12 h to produce crystal form I having a content of 97.0%, and a purity of 98.2%.

(36) Crystal form I had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 1, in which the main parameters were as follows:

(37) TABLE-US-00001 2 angles () d value () Strength (%) 9.025765 9.79798 38.74 10.586120 8.35704 100.00 13.306510 6.65401 43.61 14.277980 6.20339 31.16 15.716330 5.63875 55.34 15.935210 5.56179 53.43 17.646390 5.02613 19.96 18.039520 4.91747 31.42 19.985140 4.44292 30.34 21.261890 4.17892 28.80 21.452190 3.96000 28.54 23.618520 3.76702 35.07 23.966900 3.71305 58.77 24.727390 3.60055 24.17 26.599820 3.35120 4.21 27.485250 3.24522 6.23 28.370430 3.14595 4.91 31.601690 2.83126 2.64 32.827100 2.72832 6.25 35.731580 2.51293 8.09 36.550010 2.45852 4.56 39.294720 2.29288 6.57

(38) Crystal form I of the compound represented by formula (1) had a DSC curve as shown in FIG. 2.

Example 3: Preparation of Crystal Form II of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

(39) Method 1: 1.0 g of the amorphous form of the compound represented by formula (1) prepared in Example 1 (having a content of 87.7%, and a purity of 96.2%) was dissolved in 10 mL of methanol. The mixture was stirred at 55 C. for 1-2 h, and filtered. The obtained solid was placed in a vacuum drying oven, and dried in vacuum at 50 C. for 12 h to produce crystal form II having a content of 94.5%, and a purity of 96.4%.

(40) Method 2: 1.0 g of the amorphous form of the compound represented by formula (1) prepared in Example 1 (having a content of 87.7%, and a purity of 96.2%), after several purifications, was dissolved in 10 mL of methanol. The mixture was stirred at 55 C. for 1-2 h, and filtered. The obtained solid was placed in a vacuum drying oven, and dried in vacuum at 50 C. for 12 h to produce crystal form II having a content of 98.4%.

(41) Method 3: 15 mg of the obtained compound represented by formula (1) was placed in a 3 mL vial. The vial was placed in a 20 mL carboy. The carboy contains 4 mL of methanol. After being placed at room temperature for 10 days, the solid was crystal form II.

(42) Method 4: 15 mg of the obtained compound represented by formula (1) was placed in a 3 mL vial and dissolved in 0.6 mL of THF. The vial was placed in a 20 mL carboy. The carboy contains 4 mL of n-heptane. After being placed at room temperature for 4 days, a solid separated from the mixture. The mixture was filtered to produce crystal form II.

(43) Crystal form II had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 3, in which the main parameters were as follows:

(44) TABLE-US-00002 2 angles () d value () Strength (%) 9.260124 9.55053 32.36 10.219830 8.65573 100.00 13.957680 6.34502 30.22 15.906220 5.57186 67.00 17.867450 4.96444 33.45 20.822590 4.26608 21.68 23.047770 3.85900 22.73 24.083840 3.69528 40.93 24.817680 3.58766 14.67 27.666960 3.22432 4.69 34.791430 2.57865 4.46 36.987580 2.43043 2.36

Example 4: Preparation of Crystal Form III of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (as Acetonitrile Solvate)

(45) Method 1: 500 mg of the amorphous form of the compound represented by formula (1) prepared in Example 1 (having a content of 87.7%, and a purity of 96.2%) was placed in a mixed solution of acetonitrile and water (1:1). The mixture was stirred at 5 C. for 2 h, and filtered to produce crystal form III having a content of 90.1%, and a purity of 97.7%. The data for investigating the stability was mentioned as above.

(46) Method 2: 16.1 mg of the amorphous form of the compound represented by formula (1) prepared in Example 1 (having a content of 97.1%) was placed in a mixed solution of 0.15 mL of acetonitrile and 0.15 mL of water. The mixture was stirred at 5 C. for 2 h, and filtered to produce crystal form III.

(47) Crystal form III had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 5, and a DSC curve as shown in FIG. 6, in which the main parameters were as follows:

(48) TABLE-US-00003 2 angles () d value () Strength (%) 8.687313 10.17893 27.52 9.534775 9.27603 57.43 10.907640 8.11142 39.04 11.659660 7.58989 100.00 12.693860 6.97375 48.44 13.549430 6.53526 43.82 14.368000 6.16473 17.17 15.326080 5.78144 21.57 16.603930 5.33926 64.78 18.088170 4.90436 18.77 19.776820 4.48924 44.02 20.496670 4.33318 76.31 22.142080 4.01475 53.89 23.105800 3.84944 27.66 23.494860 3.78657 41.85 23.965170 3.71331 21.43 24.828240 3.58615 5.35 25.543780 3.48730 27.49 26.341220 3.38351 24.53 27.295890 3.26730 51.68 27.651590 3.22607 47.77 30.106550 2.96837 11.96 32.005070 2.79649 17.76 33.610130 2.66653 8.05 35.313900 2.54169 18.90 36.611660 2.45452 14.25 37.342910 2.40812 15.87

Example 5: Preparation of Crystal Form IV of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

(49) 15 mg of the amorphous form of the compound represented by formula (1) prepared in Example 1 was placed in a mixed solution of 0.9 mL of acetonitrile and 0.1 mL of water. The mixture was stirred at 80 C. for 2 h, and filtered to produce crystal form IV. Crystal form IV was unstable, resulting in a bad repeatability.

(50) Crystal form IV had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 7, in which the main parameters were as follows:

(51) TABLE-US-00004 2 angles () d value () Strength (%) 6.822 12.94699 100.0 7.168 12.32169 6.5 9.013 9.80423 8.3 9.995 8.84303 31.0 11.112 7.95586 39.2 11.779 7.50736 16.7 12.338 7.16824 9.3 13.037 6.78547 5.3 13.887 6.37191 16.8 14.859 5.95719 13.8 15.143 5.84613 7.1 15.535 5.69940 14.6 16.580 5.34236 7.8 17.601 5.03492 3.9 18.177 4.87656 12.4 18.867 4.69985 7.8 19.100 4.64303 6.7 19.843 4.47079 13.6 20.082 4.41808 20.8 20.453 4.33877 18.9 20.862 4.25457 4.9 21.472 4.13512 25.6 22.332 3.97776 7.1 22.592 3.93260 9.6 22.887 3.88253 8.9 23.369 3.80350 7.3 23.770 3.74034 8.1 24.069 3.69447 7.8 24.488 3.63213 7.0 25.576 3.48007 4.4 26.576 3.35140 7.0 27.561 3.23374 5.7 28.205 3.16138 4.6 28.526 3.12656 5.2 29.936 2.98239 7.5 30.269 2.95039 5.8 30.752 2.90511 4.9 31.668 2.82316 4.5 32.258 2.77281 4.7 32.645 2.74087 4.2 33.946 2.63876 4.7 35.371 2.53563 4.4 38.014 2.36515 4.7 39.120 2.30080 4.3 40.111 2.24622 4.6 43.500 2.07876 4.9 44.106 2.05161 4.1

Example 6: Preparation of Crystal Form V of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (as Isopropyl Acetate Solvate)

(52) Method 1: 14.8 mg of the compound represented by formula (1) prepared in Example 1 was dissolved in 0.7 mL of THF and 0.7 mL of isopropyl acetate. The solvent slowly volatilized at 5 C. or at room temperature, and a solid separated from the mixture. The mixture was filtered to produce crystal form V.

(53) Method 2: 15 mg of the compound represented by formula (1) prepared in Example 1 was dissolved in 1.0 mL of THF. Then 4 mL of isopropyl acetate was dropwise added, and a solid separated from the mixture. The mixture was filtered to produce the crystal form.

(54) Crystal form V had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 8, in which the main parameters were as follows:

(55) TABLE-US-00005 2 angles () d value () Strength (%) 4.727485 18.69238 4.86 7.095715 12.45813 16.14 9.727849 9.09236 100.00 11.846180 7.47080 17.17 16.491390 5.37545 14.44 17.883960 4.95989 56.77 19.183180 4.62681 53.32 22.061500 4.02924 39.42 23.092320 3.85166 15.52 23.754860 3.74571 45.79 24.895740 3.57658 11.30 37.480630 2.39958 4.29

Example 7: Preparation of Crystal Form VI of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (as Chloroform Solvate)

(56) Method 1: 15 mg of the compound represented by formula (1) prepared in Example 1 was dissolved in 0.5 mL of chloroform. The solvent slowly volatilized at room temperature, and a solid separated from the mixture to produce crystal form VI.

(57) Method 2: 16.1 mg of the compound represented by formula (1) prepared in Example 1 was placed in a 3 mL vial, and dissolved in 0.8 mL of chloroform. The vial was placed in a 20 mL carboy, and the carboy contained 4 mL of isopropanol or n-heptane. The mixture was placed at room temperature for 4 days. A solid separated from the mixture to produce crystal form VI.

(58) Method 3: 15 mg of crystal form II of the compound represented by formula (1) prepared in the example was placed in a 3 mL vial, and the vial was placed in a 20 mL carboy. The carboy contained 4 mL of chloroform. The mixture was placed at room temperature for 10 days, and the resulting solid was crystal form VI.

(59) Method 4: 15 mg of the compound represented by formula (1) was placed in a 3 mL vial. 0.5 mL of chloroform was added until dissolved. Then 2.98 mg of polyphenylene sulfide was added. The solvent slowly volatilized at room temperature, and a solid separated from the mixture to produce crystal form VI.

(60) Method 5: 15 mg of the compound represented by formula (1) was placed in a 3 mL vial. 0.5 mL of chloroform was added until dissolved. Then 3 mg of 1,3-dimethylimidazoline mesylate was added. The solvent slowly volatilized at room temperature, and a solid separated from the mixture to produce crystal form VI.

(61) Crystal form VI had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 9, in which the main parameters were as follows:

(62) TABLE-US-00006 2 angles () d value () Strength (%) 7.692313 11.49320 58.12 15.457590 5.73255 100.00 15.909050 5.57087 43.32 17.196630 5.15655 34.40 17.976590 4.93454 29.91 19.542360 4.54257 56.79 20.646680 4.30203 32.34 21.224630 4.18618 40.47 21.974070 4.04507 28.99 29.474920 3.03053 13.31

Example 8: Preparation of Crystal Form VII of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

(63) Method 1: 16.1 mg of the amorphous form of the compound represented by formula (1) prepared in Example 1 was placed in a mixed solution of 0.15 mL of acetonitrile and 0.15 mL of water. The mixture was stirred at 5 C. for 2 h, filtered, and dried in vacuum to produce crystal form VII.

(64) Method 2: 15 mg of crystal form II of the compound represented by formula (1) prepared in the Examples was placed in a mixed solution of 0.8 mL of acetonitrile and 0.2 mL of water (ACN/H.sub.2O, V/V, 4:1). The mixture was reacted at room temperature, filtered, dried in vacuum at 50 C. for 2 h to produce a solid as crystal form VII.

(65) Method 3: crystal form III of the compound represented by formula (1) prepared in the Examples was dried in vacuum to produce crystal form VII.

(66) Crystal form VII had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 10, in which the main parameters were as follows:

(67) TABLE-US-00007 2 angles () d value () Strength (%) 11.008970 8.03698 20.09 11.728880 7.54525 62.33 13.794880 6.41953 30.30 14.308280 6.19032 30.51 15.145890 5.84981 69.50 15.873440 5.58329 47.53 17.911120 4.95243 100.00 20.133590 4.41049 32.48 21.828890 4.07164 34.86 24.512310 3.63165 12.97 25.622960 3.47670 14.08 26.747930 3.33298 13.74 37.385250 2.40549 4.92

Example 9: Preparation of Crystal Form VIII of (4S,4aS,5aR,12aS)-9-(3-azabicyclo[3.1.0]hexan-3-ylmethyl)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (as Isopropyl Acetate Solvate)

(68) 15 mg of the amorphous form of the compound represented by formula (1) prepared in Example 1 was placed in 1 mL of isopropyl acetate. The mixture was stirred at 5 C. for 2 h, filtered to produce a solid as crystal form VIII.

(69) Crystal form VIII had an X-ray powder diffraction (XRPD) spectrum as shown in FIG. 12, in which the main parameters were as follows:

(70) TABLE-US-00008 2 angles () d value () Strength (%) 4.819580 18.33540 11.85 9.734293 9.08635 100.00 14.642940 6.04959 2.19 19.602850 4.52869 6.36 21.998290 4.04067 0.70 24.522330 3.63019 0.51

(71) Hereinafter, the beneficial effects of the present compound will be further discussed through the stability assays of the amorphous form and the crystal forms. However, it should not be understood that the present compound only has the following beneficial effects.

(72) Assay 1: Stability for Amorphous Form and Crystal Forms of the Present Compound

(73) Tested sample: amorphous form, crystal form I, crystal form II, and crystal form VII of the compound represented by formula (1) were prepared according to Examples;

(74) Test Conditions for Investigating the Influencing Factors:

(75) High temperature tests: amorphous form, crystal form I, crystal form II, and crystal form VII of the compound represented by formula (1) were laid on a dry and clean watch glass, and kept at 60 C.5 C. for 14 days. Samples were taken respectively on Day 7 and Day 14. The content and the purity of the compound represented by formula (1) were measured, and compared with those of the sample taken on Day 0.

(76) High humidity test: amorphous form, crystal form I, crystal form II, and crystal form VII of the compound represented by formula (1) were laid on a dry and clean watch glass, and kept at 25 C.2 C., RH75%5%, 40 C.2 C., RH75%5% for 14 days. Samples were taken respectively on Day 7 and Day 14. The content and the purity of the compound represented by formula (1) were measured, and compared with those of the sample taken on Day 0.

(77) Illumination test: amorphous form, crystal form I, crystal form II, and crystal form VII of the compound represented by formula (1) were laid on a dry and clean watch glass, and kept at an illuminance of 4500 Lx500 Lx in an illumination box for 14 days. Samples were taken respectively on Day 7 and Day 14. The content and the purity of the compound represented by formula (1) were measured, and compared with those of the sample taken on Day 0.

(78) Content: it was measured by using an external standard method in accordance with the High Performance Liquid Chromatography in Chinese Pharmacopoeia, Appendix V D, Edition 2010.

(79) Purity: it was measured by using an area normalization method in accordance with the High Performance Liquid Chromatography in Chinese Pharmacopoeia, Appendix V D, Edition 2010.

(80) TABLE-US-00009 TABLE 1 The investigation results of the influencing factor tests for amorphous form and crystal forms of the compound represented by formula (1) Amorphous form Crystal form I Crystal form II Crystal form VII Test Purity Content Purity Content Purity Content Purity Content Test conditions Day (%) (%) (%) (%) (%) (%) (%) (%) / Day 0 98.0 97.1 98.2 97.0 96.4 94.5 97.4 95.8 High temperature 60 C. Day 7 95.8 94.5 97.9 97.7 96.1 95.8 96.8 95.2 Day 14 94.3 90.5 97.7 97.3 96.0 96.0 96.2 93.6 25 C./75% 5% RH Day 7 97.1 98.7 98.1 97.2 96.3 95.9 97.1 96.1 Day 14 96.7 93.3 98.1 98.2 96.2 96.1 97.2 95.8 40 C./75% 5% RH Day 7 94.9 94.7 98.0 97.4 96.2 96.5 96.4 94.6 Day 14 92.9 89.6 97.8 97.5 96.1 96.1 95.7 94.5 Illumination Day 14 92.9 85.0 98.1 97.8 96.2 96.1 96.6 95.7 45001x 5001x

(81) It could be clear from the investigation results that the content and the purity of amorphous form of the compound represented by formula (1) changed very much at the conditions of high temperature, high humidity and illumination, showing the amorphous form of the compound represented by formula (1) was unstable. However, the content and the purity of crystal forms I, II, VII of the compound represented by formula (1) changed very little at the conditions of high temperature, high humidity and illumination, showing crystal forms I, II, VII of the compound represented by formula (1) had a relatively high stability that was suitable for drug manufacture, storage and transport and was favorable for ensuring the validity and the safety in the drug use.

(82) Assay 2: Stability for Crystal Form II and Crystal Form IV of the Present Compound

(83) Tested sample: crystal form II and crystal form IV of the compound represented by formula (1) were prepared according to Examples;

(84) Test Conditions for Investigating the Influencing Factors:

(85) High temperature tests: crystal form II and crystal form IV of the compound represented by formula (1) were laid on a dry and clean watch glass, and kept at 60 C.5 C. for 15 days. Samples were taken respectively on Day 5 and Day 15. The content and the purity of the compound represented by formula (1) were measured, and compared with those of the sample taken on Day 0.

(86) High humidity test: crystal form II and crystal form IV of the compound represented by formula (1) were laid on a dry and clean watch glass, and kept at 25 C.2 C., RH92.5%5%, 25 C.2 C., RH75%5%, 40 C.2 C., RH75%5% for 15 days. Samples were taken respectively on Day 5 and Day 15. The content and the purity of the compound represented by formula (1) were measured, and compared with those of the sample taken on Day 0.

(87) Illumination test: crystal form II and crystal form IV of the compound represented by formula (1) were laid on a dry and clean watch glass, and kept at an illuminance of 4500 Lx500 Lx in an illumination box for 15 days. Samples were taken respectively on Day 5 and Day 15. The content and the purity of the compound represented by formula (1) were measured, and compared with those of the sample taken on Day 0.

(88) Content: it was measured by using an external standard method in accordance with the High Performance Liquid Chromatography in Chinese Pharmacopoeia, Appendix V D, Edition 2010.

(89) Purity: it was measured by using an area normalization method in accordance with the High Performance Liquid Chromatography in Chinese Pharmacopoeia, Appendix V D, Edition 2010.

(90) Test results were shown in Table 2.

(91) TABLE-US-00010 TABLE 2 The investigation results of the influencing factor tests for crystal forms of the compound represented by formula (1) Crystal form II Crystal form IV Test conditions Day Content % Content % / 0 98.4 97.0 60 C. 15 97.0 88.6 4500 Lx 500 Lx 15 97.8 74.6 25 C./RH 75% 5% 15 98.3 93.1 25 C./RH 92.5% 5% 15 96.7 90.3 40 C./RH 75% 5% 15 98.0 74.6

(92) It could be clear from the investigation results that the content of the compound represented by formula (1) changed very much, when crystal form IV of the compound represented by formula (1) was in the conditions of high temperature, high humidity and illumination, while the content of the compound represented by formula (1) changed very little, when crystal form II of the compound represented by formula (1) was in the conditions of high temperature, high humidity and illumination, showing crystal form II of the compound represented by formula (1) had higher stability than crystal form IV, and it was suitable for drug manufacture, storage and transport and favorable for ensuring the validity and the safety in the drug use.