SALTS AND CRYSTAL FORMS OF DIAZA-BENZOFLUORANTHRENE COMPOUNDS

Abstract

The present invention discloses to a hydrochloride salt, a citrate salt, a phosphate salt or a sulfate salt of compound 1, the crystal forms of the aforementioned salts, and a preparation method thereof. The present invention also relates their use in the preparation of a medicament for the treatment of cerebral apoplexy or epilepsy.

##STR00001##

Claims

1. A hydrochloride salt, a citrate salt, a phosphate salt, or a sulfate salt of compound 1: ##STR00011##

2. The hydrochloride salt, the citrate salt, the phosphate salt, or the sulfate salt of compound 1 according to claim 1, selected from: ##STR00012##

3. A crystal form A of a compound of formula (I) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at 2 angles of 9.140.2, 10.430.2, 11.380.2, 12.540.2, 13.860.2, 19.040.2, 19.360.2, 21.000.2.

4. The crystal form A of the compound of formula (I) according to claim 3, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table 1.

5. A crystal form B of a compound of formula (I) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form B has characteristic diffraction peaks at 2 angles of 9.170.2, 11.750.2, 12.160.2, 12.670.2, 15.140.2, 17.810.2, 20.540.2, 22.340.2.

6. The crystal form B of the compound of formula (I) according to claim 5, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-2.

7. A crystal form C of a compound of formula (II) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form C has characteristic diffraction peaks at 2 angles of 14.040.2, 16.280.2, 16.700.2, 17.730.2, 18.180.2, 20.290.2, 23.400.2, 25.950.2.

8. The crystal form C of the compound of formula (II) according to claim 7, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-3.

9. A crystal form D of a compound of formula (III) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form D has characteristic diffraction peaks at 2 angles of 4.470.2, 9.800.2, 10.670.2, 13.050.2, 16.300.2, 16.800.2, 17.650.2, 17.820.2.

10. The crystal form D of the compound of formula (III) according to claim 9, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-4.

11. A crystal form E of a compound of formula (IV) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form E has characteristic diffraction peaks at 2 angles of 4.710.2, 12.300.2, 16.260.2, 16.780.2, 19.800.2, 23.700.2, 25.650.2, 26.220.2.

12. The crystal form E of the compound of formula (IV) according to claim 11, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-5.

13. A crystal form F of a compound of formula (IV) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form F has characteristic diffraction peaks at 2 angles of 5.790.2, 9.750.2, 14.030.2, 15.670.2, 17.460.2, 18.860.2, 20.420.2, 20.990.2.

14. The crystal form F of the compound of formula (IV) according to claim 13, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-6.

15. A crystal form G of a compound of formula (V) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form G has characteristic diffraction peaks at 2 angles of 4.590.2, 12.240.2, 15.930.2, 16.660.2, 18.460.2, 19.720.2, 22.100.2, 23.560.2.

16. The crystal form G of the compound of formula (V) according to claim 15, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-7.

17. A crystal form H of a compound of formula (V) according to claim 2, wherein an X-ray powder diffraction pattern of the crystal form H has characteristic diffraction peaks at 2 angles of 5.850.2, 8.800.2, 9.870.2, 12.470.2, 14.060.2, 17.620.2, 18.700.2, 20.580.2.

18. The crystal form H of the compound of formula (V) according to claim 17, wherein analytical data of the X-ray powder diffraction pattern thereof is shown in Table-8.

19. A method for preparing the crystal form according to claim 3, including contacting a free base with an acid, washing, and drying.

20. A method for treating cerebral apoplexy or epilepsy in a subject, comprising administering the compound according to claim 1 to the subject.

21. A method for preparing the crystal form according to claim 4, including contacting a free base with an acid, washing, and drying.

22. A method for preparing the crystal form according to claim 5, including contacting a free base with an acid, washing, and drying.

23. A method for preparing the crystal form according to claim 6, including contacting a free base with an acid, washing, and drying.

24. A method for preparing the crystal form according to claim 7, including contacting a free base with an acid, washing, and drying.

25. A method for preparing the crystal form according to claim 8, including contacting a free base with an acid, washing, and drying.

26. A method for preparing the crystal form according to claim 9, including contacting a free base with an acid, washing, and drying.

27. A method for preparing the crystal form according to claim 10, including contacting a free base with an acid, washing, and drying.

28. A method for preparing the crystal form according to claim 11, including contacting a free base with an acid, washing, and drying.

29. A method for preparing the crystal form according to claim 12, including contacting a free base with an acid, washing, and drying.

30. A method for preparing the crystal form according to claim 13, including contacting a free base with an acid, washing, and drying.

31. A method for preparing the crystal form according to claim 14, including contacting a free base with an acid, washing, and drying.

32. A method for preparing the crystal form according to claim 15, including contacting a free base with an acid, washing, and drying.

33. A method for preparing the crystal form according to claim 16, including contacting a free base with an acid, washing, and drying.

34. A method for preparing the crystal form according to claim 17, including contacting a free base with an acid, washing, and drying.

35. A method for preparing the crystal form according to claim 18, including contacting a free base with an acid, washing, and drying.

36. A method for treating cerebral apoplexy or epilepsy in a subject, comprising administering the crystal form according to claim 3 to the subject.

37. U A method for treating cerebral apoplexy or epilepsy in a subject, comprising administering the crystal form according to claim 4 to the subject.

38. A method for treating cerebral apoplexy or epilepsy in a subject, comprising administering the crystal form according to claim 5 to the subject.

39. A method for treating cerebral apoplexy or epilepsy in a subject, comprising administering the crystal form according to claim 6 to the subject.

40. A method for treating cerebral apoplexy or epilepsy in a subject, comprising administering the crystal form according to claim 7 to the subject.

Description

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

[0037] The invention is described in detail below by the embodiments, but these embodiments are not intended to limit the invention. The present invention has been described in detail herein; the embodiments of the present invention are disclosed herein. It would be obvious for the person skilled in the art to make various modifications and changes to the embodiments of the present invention without departing from the spirit and scope of the invention.

Reference Embodiment 1the Preparation of Compound 1

[0038] ##STR00005##

[0039] To a solution of (4.sup.1S,13aS)-13a-ethyl-2,3,4.sup.1,5,6,13a-hexahydro-1H-indolo[3,2,1-de]pyrido[3, 2,1-ij][1,5]naphthyridine-12-carboxylic acid (14 g, 43.4 mmol), 1-hydroxybenzotriazole (300 mg, 2.17 mmol) and triethylamine (31 mL, 217 mmol) in N,N-dimethylformamide (200 mL), 0-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (14.6 g, 45.6 mmol) and N-hydroxyacetamidine hydrochloride (5.28 g, 47.8 mmol) were added respectively, and the reaction mixture was stirred at room temperature overnight. Brine was added to the reaction mixture, then the mixture obtained was filtered, and the filtrate was diluted with water and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, components of low boiling point were evaporated off. The remaining crude product in N,N-dimethylformamide was directly heated under microwave to 160 C. and reacted for 50 min. The crude product was purified by alkaline preparative High Performance Liquid Chromatography to obtain the target compound (4.0 g, yield: 25%).

[0040] .sup.1H NMR (CDCl.sub.3, 400 MHz) ppm 7.46 (d, J=6.8 Hz, 1H), 7.13-7.06 (m, 2H), 6.73 (d, J=8.0 Hz, 1H), 6.08 (s, 1H), 4.23 (s, 1H), 3.38-3.34 (m, 2H), 3.29-3.28 (m, 2H), 2.65-2.63 (m, 2H), 2.55-2.51 (m, 1H), 2.51 (s, 3H), 1.97-1.92 (m, 2H), 1.59-1.55 (m, 2H), 1.45-1.41 (m, 1H), 1.11-1.10 (m, 1H), 1.00 (t, J=7.2 Hz, 3H).

Embodiment 1Preparation of the Compound of Formula (I) and Crystal Forms Thereof

[0041] ##STR00006##

[0042] Preparation of the Compound of Formula (I)

[0043] Compound 1 (15.00 g, 41.61 mmol, 1.00 Eq.) was placed in a 500 mL three-neck flask. 150 mL of ethyl acetate and 15 mL of dichloromethane were added, the reaction system was replaced three times with nitrogen. 1N HCl/EA (60 mL) was added dropwise to the reaction liquid. The reaction mixture was stirred at 25 C. for 30 minutes, which led to the appearance of a large amount of white solid; the white solid was then filtrated. The filter cake was washed once with 50 mL of ethyl acetate. The filter cake was dried to give a white product (15.00 g, 37.79 mmol, 90.82%). .sup.1H NMR (400 MHz, CDCl.sub.3)=7.52 (dd, J=1.8 Hz, 1H), 7.24-7.22 (m, 2H), 6.78 (dd, J=4.0 Hz, 1H), 6.14 (s, 1H), 4.78 (s, 1H), 3.83-3.66 (m, 2H), 3.32-3.01 (m, 4H), 2.55 (s, 3H), 2.33-2.25 (m, 3H), 1.81-1.68 (m, 1H), 1.28-1.27 (m, 1H), 1.12 (t, J=8.0 Hz, 3H).

[0044] The Preparation of Crystal Form A:

[0045] Approximately 50 mg of the compound of formula (I) was added to methanol (1.5 mL). The suspension was stirred at 40 C. for three days. The remaining solid was centrifuged (10 min at 14,000 rpm) to separate and was dried overnight in a vacuum oven at 40 C. to give crystal form A.

[0046] The Preparation of Crystal Form B:

[0047] Crystal form B was prepared using the preparation method of crystal form A, except that methanol was replaced with acetone to obtain crystal form B.

Embodiment 2Preparation of the Compound of Formula (II) and Crystal Form C Thereof

[0048] ##STR00007##

[0049] Compound 1 (2.00 g, 5.55 mmol, 1.00 eq) and citric acid (1.17 g, 6.11 mmol, 1.10 eq) were added to a 100 mL three-neck flask; 30 mL of ethanol was also added, and the reaction system was replaced three times with nitrogen. The reaction temperature was raised to 85-95 C. When the internal temperature reached 45-60 C., the reaction solution became clear. When the internal temperature reached 60 C. or above, turbidity began to appear. The reaction temperature was kept at 85-95 C. and the reaction mixture was stirred for 30 minutes, which lead to the appearance of a large amount of white solid. Heating was stopped and the internal temperature was lowered to 20-30 C., followed by filtering. The filter cake was washed once with 200 mL of ethanol. The filter cake was dried to give a white product (2.50 g, 4.56 mmol, 82.21%) which is crystal form C.

[0050] .sup.1H NMR (400 MHz, MeOD) ppm 7.50-7.70 (m, 1H), 7.06-7.30 (m, 2H), 6.64-6.84 (m, 1H), 6.21 (s, 1H), 3.69-3.76 (m, 2H), 2.94-3.13 (m, 2H), 2.87 (dd, J=15.56, 1.00 Hz, 2H), 2.77 (d, J=15.31 Hz, 2H), 2.52 (s, 3H), 1.89-2.15 (m, 3H)), 1.11 (t, J=7.40 Hz, 3H).

Embodiment 3Preparation of the Compound of Formula (III) and Crystal Form D Thereof

[0051] ##STR00008##

[0052] Preparation of the Compound of Formula (III):

[0053] Compound 1 (1.00 g, 2.77 mmol, 1.00 eq.) was placed in a 100 mL three-neck flask; 15 mL of ethanol was also added, and the reaction system was replaced three times with nitrogen. Phosphoric acid (319.36 mg, 2.77 mmol, 1.00 eq.) was added dropwise to the reaction liquid. The reaction temperature was raised to 60 C., the reaction mixture was kept at 60 C. and stirred for 30 minutes, which resulted in the appearance of a large amount of white solid. Heating was stopped; when the internal temperature was lowered to 20-30 C., the reaction mixture was filtered. The filter cake was washed once with 20 mL of ethanol. The filter cake was dried to give a white product (1.20 g, 2.61 mmol, 94.30%). .sup.1H NMR (400 MHz, MeOD)=7.57 (d, J=6.8 Hz, 1H), 7.17 (t, J=6.0 Hz, 2H), 6.72 (d, J=7.5 Hz, 1H), 6.20 (s, 1H), 3.75 (d, J=6.3 Hz, 2H), 3.23 (d, J=15.1 Hz, 2H), 3.14-2.96 (m, 2H), 2.50 (s, 3H), 2.14-1.96 (m, 3H), 1.83-1.64 (m, 2H), 1.28-1.21 (m, 1H), 1.10 (t, J=7.3 Hz, 3H).

[0054] Preparation of Crystal Form D:

[0055] Approximately 30 mg of the compound of formula (III) was added to ethanol (0.5 mL) and was stirred at 40 C. for three days. The remaining compound was centrifuged (10 min at 14,000 rpm) to separate and was dried overnight in a vacuum oven at 40 C. to produce a dry solid, which is crystal form D.

Embodiment 4Preparation of the Compound of Formula (IV) and Crystal Forms Thereof

[0056] ##STR00009##

[0057] Preparation of the Compound of Formula (IV):

[0058] Compound 1 (1.00 g, 2.77 mmol, 1.00 eq.) was added to a 100 mL three-neck flask, 15 mL of ethyl acetate and 3 mL of dichloromethane were also added; the reaction system was replaced three times with nitrogen. 1 mL of sulfuric acid (272.10 mg, 2.77 mmol, 1.00 eq.) diluted with water was added dropwise to the reaction liquid. The reaction temperature was kept at 25 C. and stirred for 30 minutes, which led to the appearance of a large amount of white solid. The white solid was filtered, the filter cake was washed once with 10 mL of ethyl acetate. The filter cake was dried to give a white product (1.10 g, 2.40 mmol, 86.61%). .sup.1H NMR (400 MHz, MeOD)=7.65-7.53 (m, 1H), 7.25-7.12 (m, 2H), 6.79-6.68 (m, 1H), 6.19 (s, 1H), 5.02 (s, 1H), 3.90-3.74 (m, 2H), 3.34 (d, J=12.3 Hz, 1H), 3.26-3.04 (m, 3H), 2.48 (s, 3H), 2.06-1.89 (m, 3H), 1.81-1.67 (m, 2H), 1.29-1.16 (m, 1H), 1.10 (t, J=7.4 Hz, 3H).

[0059] Preparation of Crystal Form E:

[0060] About 30 mg of the compound of formula (IV) was added to the solvent IPA: H.sub.2O=1:9 (0.5 mL) and stirred at 40 C. for three days. The residual compound was centrifuged (10 min at 14,000 rpm) to separate and was dried overnight in a vacuum oven at 40 C. to obtain a dry solid which is crystal form E.

[0061] Preparation of Crystal Form F:

[0062] Crystal form F was prepared using the preparation method for crystal form E, except that the solvent IPA:H.sub.2O=1:9 was replaced with ethanol to obtain crystal form E.

Embodiment 5Preparation of the Compound of Formula (V) and Crystal Forms Thereof

[0063] ##STR00010##

[0064] Preparation of the Compound of Formula (V):

[0065] Compound 1 (1.00 g, 2.77 mmol, 1.00 eq.) was added to a 100 mL three-neck flask, 15 mL of ethyl acetate and 3 mL of dichloromethane were also added; the reaction system was replaced three times with nitrogen. 1 mL of sulfuric acid (135.84 mg, 1.39 mmol, 0.50 eq.) diluted with water was added dropwise to the reaction liquid. The reaction temperature was kept at 25 C. and the reaction mixture was stirred for 30 minutes, which led to the appearance of a large amount of white solid. The white solid was filtered; the filter cake was washed once with 10 mL of ethyl acetate. The filter cake was dried to give a white product (500.00 mg, 1.09 mmol, 39.37%). .sup.1H NMR (400 MHz, MeOD)=7.59 (dd, J=2.4, 4.6 Hz, 1H), 7.27-7.08 (m, 2H), 6.73 (d, J=7.5 Hz, 1H), 6.29-6.13 (m, 1H), 5.06 (d, J=14.8 Hz, 1H), 3.93-3.73 (m, 2H), 3.47-3.31 (m, 1H), 3.28-3.02 (m, 3H), 2.53-2.41 (m, 3H), 2.11-1.88 (m, 3H), 1.77 (d, J=4.8 Hz, 2H), 1.25 (d, J=10.3 Hz, 1H), 1.15-1.02 (m, 3H).

[0066] Preparation of Crystal Form G:

[0067] Approximately 30 mg of the compound of formula (V) was added to a solvent (0.5 mL). The reaction mixture was stirred at 40 C. for three days. Centrifugation (10 min at 14,000 rpm) was performed to separate the residual solid compound; the separated compound was dried under vacuum at 40 C. overnight. A dry solid is obtained which is crystal form G.

[0068] Preparation of Crystal Form H:

[0069] Crystal form H was prepared using the preparation method of crystal form G, except that the solvent IPA:H.sub.2O=1:9 was replaced with EtOAc to obtain crystal form H.

Experimental Example 1: The In Vitro Detection of Phosphodiesterase (PDE)

[0070] Principle of the Experiment:

[0071] The assay measures PDE1A enzyme activity based on fluorescence polarization detection of AMP/GMP production. The principle of the reaction is to replace the binding of AMP/GMP to its antibody by AlexaFluor 633 labeled AMP/GMP.

[0072] Experimental Reagents:

[0073] Reaction buffer: 10 mM Tris-HCl, pH=7.5, 5 mM magnesium chloride, 0.01% Brij 35, 1 mM DTT, and 1% DMSO;

[0074] Enzyme substrate: 1M cAMP or cGMP (Ca.sup.2-calmodulin as a cofactor of PDE1A);

[0075] Detection Reagents:

[0076] Transcreener AMP2/GMP2 antibody;

[0077] AMP2/GMP2 AlexaFluor 633 marker.

[0078] Experimental Procedures and Methods:

[0079] 1. Human enzyme to be tested (purchased from SignalChem) and the substrate were diluted with a freshly prepared reaction buffer.

[0080] 2. An enzyme solution (concentration: 3 pM) was added to the wells of a reaction plate.

[0081] 3. Echo 550 was employed to add a number of 100% DMSO compound solutions to the wells of the reaction plate at required concentrations; said wells of the reaction plate contain the enzyme solution. The reaction plate was then incubated at room temperature for 10 minutes.

[0082] 4. The substrate solution was added to the wells of the reaction plate containing the enzyme and the compound solution to initiate the reaction.

[0083] 5. The reaction plate was incubated for 1 hour at room temperature with shaking.

[0084] 6. A detection mixture (a stop buffer in the tracer and the antibody) was added to stop the enzymatic reaction; the reaction plate was incubated for 90 minutes with shaking.

[0085] 7. The following devices were used for detection: EnVision (PerkinElmer), Cy5FP Ex FP 620, Em S-pol 688/P-pol 688, FP mirror D658fp/D688; fluorescence polarization was detected using Ex/Em 620/688.

[0086] Data Analysis:

[0087] Enzymatic activity corresponding to the FP signal was retrieved in an AMP/GMP standard curve using DMSO as a control in an Excel table. The enzymatic activity was converted to nM product concentration. An analysis was performed using GraphPad Prism and IC.sub.50 values were calculated.

[0088] The experimental results are shown in Table 1:

TABLE-US-00009 TABLE 1 IC.sub.50 values measured by PDE1 detection Tested compound PDE1 Compound 1 1 uM < B 20 uM