CRYSTAL FORM E OF BULLEYACONITINE A, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

Provided is a crystal form E of bulleyaconitine A and a preparation method for the crystal form E of bulleyaconitine A. An X-ray powder diffraction spectrum of the crystal form measured by Cu-Kα-ray is as shown in FIG. 1. The crystal form E of bulleyaconitine A is prepared by adding a mixed solution of alcohol and water to bulleyaconitine A, stirring to obtain a suspended solid, and centrifugally collecting the solid. The alcohol is methanol, ethanol or n-butanol. The preparation process is simple, and the obtained crystal form has high purity and is characterized by XRD, DSC, TGA, and .sup.1HNMR to be determined as the crystal form E. The obtained bulleyaconitine A crystal is an anhydrous crystal form, and stability test results show that the crystal has good light, humidity and heat stability.

Claims

1. A crystalline form E of bulleyaconitine A, wherein its X-ray powder diffraction spectrum shows obvious characteristic absorption peaks at 2θ values of 7.8±0.2, 9.4±0.2, 11.5±0.2, 12.4±0.2, 13.2±0.2, 13.8±0.2, 14.8±0.2, 16.6±0.2, 18.8±0.2, 19.3±0.2, 22.1±0.2, and 33.6±0.2.

2. The crystalline form E of bulleyaconitine A according to claim 1, wherein its thermogravimetric analysis graph shows a weight loss of 0.3% when heated to 150° C.

3. The crystalline form E of bulleyaconitine A according to claim 1, wherein its differential scanning calorimetry analysis graph shows an endothermic peak at 160-164° C.

4. The crystalline form E of bulleyaconitine A according to claim 1, wherein its hydrogen nuclear magnetic resonance spectrum is shown in FIG. 3.

5. A preparation method of the crystalline form E of bulleyaconitine A according to claim 1, comprising adding a mixed solution of alcohol and water to bulleyaconitine A, stirring to obtain a suspended solid, and centrifugally collecting the solid; wherein the alcohol is methanol, ethanol or n-butanol.

6. The preparation method of the crystalline form E of bulleyaconitine A according to claim 5, wherein the volume ratio of alcohol to water in the mixed solution of alcohol and water is 10:1-1:10.

7. The preparation method of the crystalline form E of bulleyaconitine A according to claim 5, wherein, in mg/ml, the mass-volume ratio of the bulleyaconitine A to the mixed solution of alcohol and water is 3:1-1000:1.

8. The preparation method of the crystalline form E of bulleyaconitine A according to claim 5, wherein the stirring time is at least 0.5 hours.

9. The preparation method of the crystalline form E of bulleyaconitine A according to claim 5, wherein the stirring temperature is 0° C.-50° C.

10. A method for preventing and/or treating rheumatoid arthritis, osteoarthritis, myofibrositis, pain in neck and shoulder, pain in lower extremities and waist, or cancerous pain, comprising administering a therapeutically effective amount of the crystalline form E of bulleyaconitine A according to claim 1 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the examples of the present disclosure or in the prior art, the drawings used in the examples or the prior art will be briefly introduced below.

[0027] FIG. 1 XRPD graph of the crystalline form E of bulleyaconitine A;

[0028] FIG. 2 TGA/DSC graph of the crystalline form E of bulleyaconitine A;

[0029] FIG. 3 .sup.1H NMR spectrum of the crystalline form E of bulleyaconitine A;

[0030] FIG. 4 PLM graph of the crystalline form E of bulleyaconitine A;

[0031] FIG. 5 DVS graph of the crystalline form E of bulleyaconitine A;

[0032] FIG. 6 XRPD comparison graph before and after DVS test of the crystalline form E of bulleyaconitine A;

[0033] FIG. 7 XRPD comparison graph before and after stability evaluation of the crystalline form E of bulleyaconitine A.

DETAILED DESCRIPTION

[0034] Hereinafter, the technical solutions in embodiments of the present disclosure will be described clearly and completely in conjunction with examples of the present disclosure. It is apparent that the described examples are merely part of the present disclosure rather than all. Based on the examples in the present disclosure, all other examples obtained by those of ordinary skill in the art without creative work are within the scope of the present disclosure.

[0035] The present disclosure will be illustrated in detail in combination with specific examples below in order to further understand the present disclosure. In the following examples, unless otherwise specified, the test method is usually implemented in accordance with conventional conditions or conditions recommended by the manufacturer.

Test Parameters

[0036] The XRPD patterns were collected on PANalytacal Empyrean and X' Pert3 X-ray powder diffraction analyzers. The scanning parameters are shown in Table 1.

TABLE-US-00001 TABLE 1 XRPD test parameters Parameters Reflection mode Transmission mode Instrument model Empyrean X′ Pert3 X′ Pert3 X′ Pert3 X-ray Cu, kα, Kα1 (Å): 1.540598, Kα2 (Å): 1.544426; Kα2/Kα1 intensity ratio: 0.50 X-ray tube setting 45 kV, 40 mA Divergence slit Automatic Fixed ⅛° Fixed ⅙° Fixed ½° Scanning mode Continuous Scanning range (°2Theta) 3~40 Scanning time per step (s) 17.8 46.7 33.02 Scanning step size (°2Theta) 0.0167 0.0263 0.0167 Test time 5 min 30 s 5 min 4 s 10 min 11 s

Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC)

[0037] TGA and DSC graphs were collected on TA Q5000 TGA/TA Discovery TGA5500 thermogravimetric analyzer and TA Q2000 DSC/TA Discovery DSC2500 differential scanning calorimeter, respectively. Table 2 lists the test parameters.

TABLE-US-00002 TABLE 2 TGA and DSC test parameters Parameters TGA DSC Method Linear heating Linear heating Sample pan Aluminum pan, open Aluminum pan, gland Temperature range Room temperature- 25° C.- End temperature set End temperature set Scanning rate (° C./min) 10 10 Protective gas Nitrogen Nitrogen

Dynamic Vapor Sorption (DVS)

[0038] The dynamic vapor sorption (DVS) graph was collected on DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25° C. was corrected by the deliquescent point of LiCl, Mg(NO.sub.3).sub.2 and KCl. DVS test parameters are listed in Table 3.

TABLE-US-00003 TABLE 3 DVS test parameters Parameter Set value Temperature 25° C. Sample size 10-20 mg Protective gas and flow N.sub.2, 200 ml/min rate dm/dt 0.002%/min Minimum dm/dt balance 10 min time Maximum balance time 180 min RH range 0% RH-90% RH-0% RH RH gradient 10%(0% RH-90% RH, 90% RH-0% RH) 5%(90% RH-95% RH, 95% RH-90% RH)

Liquid NMR

[0039] The liquid NMR spectra were collected on Bruker 400M NMR spectrometer, with DMSO-d6 as the solvent.

Example 1. Preparation and Identification of Crystalline Form E of Bulleyaconitine A

[0040] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.5 ml of n-butanol-water (1:1) was added, and stirred for 2 hours at 5° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD, TGA/DSC and .sup.1HNMR tests.

[0041] The XRPD results show that there are obvious characteristic absorption peaks at the diffraction angle (2θ angle) of 7.6±0.2, 9.4±0.2, 11.3±0.2, 12.4±0.2, 13.4±0.2, 13.9±0.2, 14.8±0.2, 16.8±0.2, 18.8±0.2, 19.4±0.2, 22.2±0.2, and 33.1±0.2. The TGA/DSC results show that when the temperature rises to 150° C., the weight loss is 0.3%, and the DSC graph shows a sharp endothermic peak at 160.9° C. (initial temperature), which may be caused by melting. Combined with the TGA weight loss, it is speculated that the thermal signal appearing above 200° C. on the DSC is caused by the decomposition of the sample. .sup.1HNMR results show that there is no obvious solvent residue in the sample. The PLM results show a composition of irregular small particles.

[0042] It was identified as crystalline form E, anhydrous crystal.

[0043] For the sample, the XRPD graph is shown in FIG. 1, the TGA/DSC characterization result graph is shown in FIG. 2, the .sup.1HNMR graph is shown in FIG. 3, and the PLM result graph is shown in FIG. 4.

Example 2. Preparation of Crystalline Form E of Bulleyaconitine A

[0044] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.5 ml of n-butanol-water (1:10) was added, and stirred for 0.5 hours at 25° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 163.9° C.

Example 3. Preparation of Crystalline Form E of Bulleyaconitine A

[0045] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.3 ml of n-butanol-water (1:1) was added, and stirred for 3 hours at 50° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 160° C.

Example 4. Preparation of Crystalline Form E of Bulleyaconitine A

[0046] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.3 ml of n-butanol-water (1:10) was added, and stirred for 1 hours at 25° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 164° C.

Example 5. Preparation of Crystalline Form E of Bulleyaconitine A

[0047] 1500 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 15 ml of n-butanol-water (5:1) was added, and stirred for 5 hours at 15° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 162.9° C.

Example 6. Preparation of Crystalline Form E of Bulleyaconitine A

[0048] 100 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 1 ml of n-butanol-water (1:8) was added, and stirred for 10 hours at 5° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 161.5° C.

Example 7. Preparation of Crystalline Form E of Bulleyaconitine A

[0049] 1000 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 1 ml of n-butanol-water (10:1) was added, and stirred for 24 hours at 0° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 163.1° C.

Example 8. Preparation of Crystalline Form E of Bulleyaconitine A

[0050] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.5 ml of methanol-water (1:10) was added, and stirred for 0.5 hours at 25° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 161.7° C.

Example 9. Preparation of Crystalline Form E of Bulleyaconitine A

[0051] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.3 ml of methanol-water (1:1) was added, and stirred for 3 hours at 50° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 162.6° C.

Example 10. Preparation of Crystalline Form E of Bulleyaconitine A

[0052] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.3 ml of methanol-water (1:10) was added, and stirred for 1 hours at 25° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 161.8° C.

Example 11. Preparation of Crystalline Form E of Bulleyaconitine A

[0053] 1500 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 15 ml of methanol-water (5:1) was added, and stirred for 5 hours at 15° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 163.98° C.

Example 12. Preparation of Crystalline Form E of Bulleyaconitine A

[0054] 100 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 1 ml of methanol-water (1:8) was added, and stirred for 10 hours at 5° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 161.9° C.

Example 13. Preparation of Crystalline Form E of Bulleyaconitine A

[0055] 1000 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 1 ml of methanol-water (10:1) was added, and stirred for 24 hours at 0° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 160.2° C.

Example 14. Preparation of Crystalline Form E of Bulleyaconitine A

[0056] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.5 ml of ethanol-water (1:10) was added, and stirred for 0.5 hours at 25° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 160.8° C.

Example 15. Preparation of Crystalline Form E of Bulleyaconitine A

[0057] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.3 ml of ethanol-water (1:1) was added, and stirred for 3 hours at 50° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 163° C.

Example 16. Preparation of Crystalline Form E of Bulleyaconitine A

[0058] 15 mg of bulleyaconitine A was weighed out and placed in a 3 ml vial. Then 0.3 ml of ethanol-water (1:10) was added, and stirred for 1 hours at 25° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 162° C.

Example 17. Preparation of Crystalline Form E of Bulleyaconitine A

[0059] 1500 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 15 ml of ethanol-water (5:1) was added, and stirred for 5 hours at 15° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 163.5° C.

Example 18. Preparation of Crystalline Form E of Bulleyaconitine A

[0060] 100 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 1 ml of ethanol-water (1:8) was added, and stirred for 10 hours at 5° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 162.4° C.

Example 19. Preparation of Crystalline Form E of Bulleyaconitine A

[0061] 1000 mg of bulleyaconitine A was weighed out and placed in a beaker. Then 1 ml of ethanol-water (10:1) was added, and stirred for 24 hours at 0° C. Centrifugation was performed to separate and obtain a solid. The solid was subjected to XRPD test, and the results are consistent with FIG. 1 that it was determined to be the crystalline form E of bulleyaconitine A; the DSC graph shows an endothermic peak of 161.6° C.

Example 20. Stability Test of Crystalline Form E of Bulleyaconitine A

1) DVS Characterization of Crystalline Form E

[0062] In order to evaluate the hygroscopicity and stability of anhydrous crystalline form E under different humidity conditions, DVS and XRPD tests were performed on crystalline form E samples at a constant temperature of 25° C.

[0063] The crystalline form E continued to slowly adsorb water as the humidity increased. When the humidity reached 80% RH, 0.12% of water was adsorbed, indicating that the sample has no hygroscopicity. The XRPD characterization results show that the crystalline form of the crystalline form E sample before and after the DVS test did not change. It can be seen from the XRPD comparison results that the crystalline form of the sample did not change after the DVS test.

[0064] The DVS graph of the crystalline form E is shown in FIG. 5, and the XRPD comparison graph of the crystalline form E before and after the DVS test is shown in FIG. 6.

2) Evaluation of the Solid State Stability of Crystalline Form E

[0065] In order to evaluate the solid state stability of crystalline form E, an appropriate amount of samples was weigh out and placed in an open place at 25° C./60% RH and 40° C./75% RH for 1 week and 1 month, respectively, and placed in a sealed place at 80° C. for 24 hours. XRPD and HPLC characterization of the placed samples were performed to detect the changes of crystalline form and chemical purity.

[0066] The HPLC results are shown in Table 4 that the chemical purity of the sample has not changed under the selected test conditions; and the XRPD results show that the crystalline form of the sample has not changed under the selected test conditions.

TABLE-US-00004 TABLE 4 Summary of stability data of crystalline form E Crystalline HPLC purity form Area % of Final crystalline (Sample No.) Conditions % Control form Crystalline 80° C., 24 hours 99.34 99.9 Crystalline form E 25° C./ 1 week 99.48 100.0 form E 60% RH 1 month 99.53 100.1 40° C./ 1 week 99.49 100.0 75% RH 1 month 99.47 100.0

[0067] In conclusion, the crystalline form E has good physical and chemical stability.

[0068] The XRPD comparison graph of the crystalline form E before and after the stability evaluation is shown in FIG. 7.