CN112209926A

CN112209926A - Polymorphic substance of pyronaridine phosphate and preparation method and application thereof

Info

Publication number: CN112209926A
Application number: CN201910632108.5A
Authority: CN
Inventors: 王锦玉; 张东; 孙鹏; 杨岚; 仝燕
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-07-12
Filing date: 2019-07-12
Publication date: 2021-01-12

Abstract

Disclosed are polymorphs of pyronaridine phosphate, methods of preparing the crystalline forms, and pharmaceutical formulations comprising the crystalline forms, as well as uses of the crystalline forms as medicaments for treating diseases associated with malaria, tuberculosis, and cancer. Particularly, the anhydrous crystal form B has good physical stability and low hygroscopicity, is convenient to store, and can avoid the risk of crystal transformation in the process of drug development and generation.

Description

Polymorphic substance of pyronaridine phosphate and preparation method and application thereof

Technical Field

The application relates to a polymorph of pyronaridine phosphate, a preparation method and application thereof, belonging to the field of medicines.

Background

Malaria is a contagious disease transmitted by the bites of the anopheles by plasmodium, and is prevalent mainly in tropical and subtropical zones, and secondly in temperate zones, especially in poor africa and southeast asia, with a statistically 3-5 million cases per year, and with millions of cases dying from malaria. Clinically, the medicine is characterized by periodically and regularly attacking chills, hyperpyrexia, sweating and abating fever, anemia and splenomegaly. Especially, chloroquine-resistant malaria is generated and widely spread, and great difficulty is brought to the malaria prevention and treatment work.

Pyronaridine (malaridine), marketed as "malarial", code "7351", is a novel, chemically synthesized antimalarial drug developed in 1970 by the institute of parasitosis, the national academy of preventive medicine sciences, used in the treatment of malaria in china and southeast asia. The pyronaridine belongs to the benzonaphthyridine class, has a chemical structure similar to that of chloroquine, is a killing agent aiming at an erythrocyte inner-stage schizont, is commonly used as the pyronaridine phosphate, is suitable for various types of antimalarial treatment, has good curative effect even on chloroquine-resistant malignant malaria and cerebral malaria patients, and has small toxic and side effects.

A periodical: killing activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug resistant Plasmodium falciparum gametophytes in vitro [ J]Foreign medicine (Parasitisis Manual), 2001(01):32-33, discusses that pyronaridine phosphate has a strong killing effect on merozoites in the erythrocytic stage of Plasmodium. A periodical: : the EU improved antisense pyrolidine show and synthesis with rifampicin, targeting RNAPLymerase [ J]The function of pyronaridine phosphate as an adjuvant for Tuberculosis to inhibit the RNA polymerase of mycobacterium Tuberculosis is discussed in Tuberculosis (Edinburgh, Scotland),2018,112. The literature: pyronaridine exits post cytotoxin on human breast and biochemical cancer cells through induction of apoptosis [ J]PloS one,2018,13(11) discusses the anticancer effect of pyronaridine phosphate, which can continuously induce phosphatidylserine externalization, mitochondrial depolarization, and DNA cleavage. A periodical: pyronaridine reversal of human breast cancer MCF-7/ADM cell drug resistance and mechanism discussion [ J]J. Med.Sci.Zhongnan, 2017,45(04):346-Thereby promoting apoptosis and reversing drug resistance of MCF-7 cells. A periodical: pyronaridine reversal of multidrug resistance in tumors and mechanism of action (English) [ J ]]Acta Pharmacological Sinica,2002(06):66-72, which discusses pyronaridine vs mdr1⁺The human leukemia cell and the drug-resistant cell breast cancer multidrug-resistant (MDR) cell lines K562/A02 and MCF-7/ADR have growth inhibition effect; the low-toxic-dose pyronaridine remarkably enhances the cytotoxic and apoptosis-inducing effects of adriamycin on drug-resistant cells, increases the accumulation of adriamycin in the drug-resistant cells and reduces the efflux of rhodamine (Rh123), and RT-PCR results show that the pyronaridine has no down-regulation effect on MDR1 gene, can be used as a third-generation P-glycoprotein (P-gp) inhibitor, and can generate a strong reversal MDR effect by down-regulating the function of a P-gp drug efflux pump.

However, pyronaridine phosphate includes polymorphs, amorphous substances, and its crystal form has not been studied in the prior art and has not been determined for pharmaceutical applications. Therefore, there is a need in the art to develop polymorphic forms of pyronaridine and the use of the polymorphic forms in medicine.

Disclosure of Invention

In order to solve the problems, the polymorphic substances of the pyronaridine phosphate, the preparation method and the application thereof are provided. The application prepares various crystal forms of the pyronaridine phosphate, identifies and evaluates the prepared pyronaridine phosphate crystal forms, and provides the pyronaridine phosphate new crystal form suitable for pharmaceutical research and industrial production.

According to one aspect of the present application, there is provided a hydrate form a of the phosphate salt of the compound of formula i having at least one of the following:

1) the hydrate form A has at least the following peaks in an X-ray powder diffraction pattern expressed by 2 theta angle by using Cu-Kalpha radiation: 7.97 +/-0.2 degrees, 8.74 +/-0.2 degrees, 18.04 +/-0.2 degrees and 23.89 +/-0.2 degrees;

2) the hydrate crystal form A has endothermic peaks at 106.7 +/-2 ℃, 198.1 +/-2 ℃ and 225.3 +/-2 ℃ measured by differential scanning calorimetry; and

3) the hydrate form a has a heat loss of about 3.1 ± 1 wt% at 150 ℃ as measured by thermogravimetric analysis;

alternatively, the hydrate form a has an exothermic peak at 204.9 ± 2 ℃ as measured by differential scanning calorimetry.

Optionally, the hydrate form a further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 11.69 +/-0.2 degrees, 14.16 +/-0.2 degrees, 18.89 +/-0.2 degrees, 21.45 +/-0.2 degrees, 22.82 +/-0.2 degrees and 25.79 +/-0.2 degrees.

Preferably, the IC-tested phosphate content of the hydrate form a is 37.5 ± 0.5 wt%.

Preferably, the hydrate form a has an onset temperature of an exothermic peak of 204.9 ± 2 ℃ as measured by differential scanning calorimetry.

According to another aspect of the present application, there is provided a process for preparing hydrate form a as described in any one of the above, the process comprising:

dissolving phosphate of the compound shown in the formula I in water, adding ethanol, and separating hydrate crystal form A of the phosphate of the compound shown in the formula I.

According to one aspect of the present application, there is provided an anhydrous crystalline form B of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

1) the anhydrous crystalline form B has at least peaks at the following positions in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation: 9.46 +/-0.2 degrees, 11.09 +/-0.2 degrees, 18.04 +/-0.2 degrees, 18.29 +/-0.2 degrees, 20.57 +/-0.2 degrees, 22.75 +/-0.2 degrees and 24.13 +/-0.2 degrees;

2) the anhydrous crystal form B has an endothermic peak at 234.8 +/-2 ℃ as measured by differential scanning calorimetry; and

3) the anhydrous crystalline form B has a heat loss of about 2.4 ± 1 wt% at 210 ℃ as measured by thermogravimetric analysis;

optionally, the anhydrous crystalline form B further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 14.21 +/-0.2 degrees, 17.67 +/-0.2 degrees, 19.03 +/-0.2 degrees, 23.82 +/-0.2 degrees, 24.91 +/-0.2 degrees and 29.19 +/-0.2 degrees.

Optionally, the anhydrous crystalline form B further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 13.24 +/-0.2 degrees, 13.85 +/-0.2 degrees, 21.37 +/-0.2 degrees, 26.60 +/-0.2 degrees, 28.20 +/-0.2 degrees and 28.70 +/-0.2 degrees.

Preferably, the phosphate content of the anhydrous crystalline form B by IC test is 34.2 ± 0.5 wt%.

Preferably, the anhydrous crystalline form B has an onset temperature of the melting endotherm of 229.7 ± 2 ℃ as measured by differential scanning calorimetry.

According to another aspect of the present application, there is provided a method of preparing the anhydrous crystalline form B described in any one of the above, comprising:

dissolving phosphate of a compound shown in a formula I in water, adding ethanol, and separating hydrate crystal form A of the phosphate of the compound shown in the formula I;

dispersing the hydrate crystal form A in a solvent I, stirring at room temperature, and separating an anhydrous crystal form B; or

Dispersing the hydrate crystal form A in a solvent II, stirring at 40-60 ℃, and separating an anhydrous crystal form B;

wherein, the solvent I comprises methanol or methanol and halogenated alkane;

the solvent II comprises one of ethanol, N-methyl pyrrolidone and dimethylformamide and isopropyl acetate.

Preferably, the halogenated alkane is selected from at least one of dichloromethane, dichloroethane and chloroform.

Preferably, the solvent I comprises 30-70% by volume of methanol. More preferably, the solvent i comprises 50% by volume of methanol.

Preferably, the solvent II comprises 30-70% by volume of isopropyl acetate. More preferably, the solvent II comprises 50% by volume of isopropyl acetate.

According to another aspect of the present application, there is provided an anhydrous crystalline form E of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

1) the anhydrous crystalline form E has at least peaks at the following positions in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation: 6.47 +/-0.2 degrees, 9.03 +/-0.2 degrees, 9.27 +/-0.2 degrees, 14.30 +/-0.2 degrees, 14.64 +/-0.2 degrees, 20.71 +/-0.2 degrees, 23.67 +/-0.2 degrees and 27.44 +/-0.2 degrees;

2) the anhydrous crystal form E has an endothermic peak at 228.4 +/-2 ℃ as measured by differential scanning calorimetry; and

3) the anhydrous crystalline form E has a heat loss of about 6.0 ± 1 wt% at 200 ℃ as measured by thermogravimetric analysis;

optionally, the anhydrous crystalline form E further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 18.10 + -0.2 deg., 19.47 + -0.2 deg. and 21.29 + -0.2 deg..

Optionally, the anhydrous crystalline form E further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 11.52 +/-0.2 degrees, 16.60 +/-0.2 degrees, 22.88 +/-0.2 degrees, 25.36 +/-0.2 degrees, 26.69 +/-0.2 degrees and 32.22 +/-0.2 degrees.

Preferably, the phosphate content of the anhydrous crystalline form E by IC test is 33.7 ± 0.5 wt%.

Preferably, the anhydrous crystalline form E has an onset temperature of the melting endotherm of 214.4 ± 2 ℃ as measured by differential scanning calorimetry.

According to another aspect of the present application, there is provided a method for preparing anhydrous crystalline form E as described in any one of the above, comprising:

and heating the hydrate crystal form A to the temperature of 180-230 ℃, and then cooling to obtain an anhydrous crystal form E.

Preferably, the hydrate crystal form a is heated to 200 ℃ and then cooled to obtain an anhydrous crystal form E.

According to another aspect of the present application, there is provided an anhydrous crystalline form G of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

1) the anhydrous crystalline form G has at least peaks at the following positions in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation: 8.90 +/-0.2 degrees, 9.44 +/-0.2 degrees, 13.56 +/-0.2 degrees, 17.84 +/-0.2 degrees, 19.93 +/-0.2 degrees, 20.98 +/-0.2 degrees, 23.80 +/-0.2 degrees, 25.91 +/-0.2 degrees and 26.71 +/-0.2 degrees;

optionally, the anhydrous crystalline form G further comprises one or more peaks at positions selected from the group consisting of: 6.66 +/-0.2 degrees, 8.34 +/-0.2 degrees, 11.41 +/-0.2 degrees and 16.02 +/-0.2 degrees.

According to another aspect of the present application, there is provided a method for preparing any of the above anhydrous crystalline forms G, comprising:

heating the hydrate crystal form A to 140 ℃ in an inert gas environment, and cooling to 25-35 ℃ to obtain an anhydrous crystal form G.

Preferably, the hydrate crystal form A is heated to 120 ℃ in an inert gas environment and then cooled to 30 ℃, and the anhydrous crystal form G is prepared under the protection of the inert gas.

According to another aspect of the present application there is provided a hydrate form D of the phosphate salt of the compound of formula i, characterized in that it has at least one of the following:

1) the hydrate form D has at least the following peaks in an X-ray powder diffraction pattern expressed by 2 theta angle by using Cu-Kalpha radiation: 13.35 +/-0.2 degrees and 17.82 +/-0.2 degrees;

2) the hydrate crystal form D has endothermic peaks at 96.8 +/-2 ℃, 194.1 +/-2 ℃ and 230.4 +/-2 ℃ measured by differential scanning calorimetry; and

3) the hydrate form D has a heat loss of about 3.7 ± 1 wt% at 150 ℃ as measured by thermogravimetric analysis;

optionally, the hydrate form D further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 8.83 +/-0.2 degrees, 9.07 +/-0.2 degrees, 11.04 +/-0.2 degrees, 20.51 +/-0.2 degrees, 20.82 +/-0.2 degrees, 21.26 +/-0.2 degrees or 25.99 +/-0.2 degrees.

Optionally, the hydrate form D further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 4.46 +/-0.2 degrees, 6.30 +/-0.2 degrees, 11.86 +/-0.2 degrees, 14.34 +/-0.2 degrees, 16.44 +/-0.2 degrees, 22.36 +/-0.2 degrees, 23.40 +/-0.2 degrees and 23.98 +/-0.2 degrees.

Preferably, the IC-tested phosphate content of the hydrate form D is 36.6 ± 0.5 wt%.

Preferably, the hydrate form D has an onset temperature of the melting endotherm of 217.9 ± 2 ℃ as measured by differential scanning calorimetry.

According to another aspect of the present application, there is provided a method of preparing hydrate form D as described in any one of the above, comprising:

and volatilizing the aqueous solution of the hydrate crystal form A at room temperature to obtain a hydrate crystal form D.

According to another aspect of the present application, there is provided a hydrate crystalline form F of the phosphate salt of the compound of formula i, characterized in that it has at least one of the following:

1) the hydrate form F has at least peaks at the following positions in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation: 7.89 +/-0.2 degrees, 16.04 +/-0.2 degrees and 20.06 +/-0.2 degrees;

2) the hydrate crystal form F has an endothermic peak at 86.1 +/-2 ℃, 195.0 +/-2 ℃ and 228.2 +/-2 ℃ as measured by differential scanning calorimetry; and

3) the hydrate crystalline form F has a heat loss of about 4.6 ± 1 wt% at 150 ℃ as measured by thermogravimetric analysis;

optionally, the hydrate form F further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 12.03 +/-0.2 degrees, 13.27 +/-0.2 degrees, 17.73 +/-0.2 degrees, 18.13 +/-0.2 degrees, 20.97 +/-0.2 degrees, 23.45 +/-0.2 degrees, 24.05 +/-0.2 degrees, 27.03 +/-0.2 degrees and 29.47 +/-0.2 degrees.

Optionally, the hydrate form F further comprises one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 10.59 +/-0.2 degrees, 22.30 +/-0.2 degrees, 23.00 +/-0.2 degrees, 25.93 +/-0.2 degrees, 31.44 +/-0.2 degrees, 33.94 +/-0.2 degrees, 35.00 +/-0.2 degrees and 37.65 +/-0.2 degrees.

Preferably, the hydrate form F has an onset temperature of the melting endotherm of 217.9 ± 2 ℃ as measured by differential scanning calorimetry.

According to another aspect of the present application, there is provided a method of preparing hydrate form F as described in any one of the above, comprising:

carrying out gas-solid diffusion on the hydrate crystal form A in a steam environment to prepare a hydrate crystal form F; or

And (3) after dissolving in a solvent III of tetrahydrofuran and water, performing gas-liquid diffusion in an acetone atmosphere to obtain a hydrate crystal form F.

Preferably, the solvent III comprises tetrahydrofuran and water in a volume ratio of 1.5-2.5: 1. More preferably, the solvent iii comprises tetrahydrofuran and water in a volume ratio of 2: 1.

Optionally, the preparation method of the hydrate form a comprises the following steps:

1) mixing and stirring phosphate of a compound shown in a formula I with water in a mass ratio of 1:5-7, heating to 48-52 ℃, and stirring for at least 1 h;

2) adding ethanol with the mass ratio of 1:6-9 to the phosphate of the compound shown in the formula I, keeping the temperature at 48-52 ℃ for at least 20min, cooling to 15-25 ℃ in a cold water bath, and stirring for at least 1 h;

3) and (4) performing centrifugal separation to obtain a crude hydrate crystal form A.

Preferably, the preparation method of the hydrate crystal form A further comprises the following steps:

4) mixing the crude hydrate crystal form A prepared in the step 3), water and activated carbon according to the mass ratio of 1:4-8:0.04-0.06 to dissolve the crude hydrate crystal form A, and stirring at 0-5 ℃ for at least 1 h;

5) filtering the product of the step 4) in a vacuum state;

6) adding the filtered product of the step 5) into a reaction kettle in a mass ratio of 1:6-9 of ethanol, keeping the temperature at 48-52 ℃ for at least 20min, cooling to 15-25 ℃ in a cold water bath, and stirring for at least 1 h;

7) washing with ethanol, and drying to obtain hydrate crystal form A.

As an embodiment, the method for preparing hydrate form a comprises the steps of:

1) 1.56kg of phosphate of the compound of formula I and 9.36kg of water are stirred and mixed in a 50L reaction tank, dissolved at 48-50 ℃ and stirred for 1 h;

2) adding 11.6kg of absolute ethyl alcohol, keeping the temperature at 48-52 ℃ for 30min, cooling to 15-25 ℃ in a cold water bath, and stirring for 1 h;

3) putting the system into a centrifuge, and centrifuging until no liquid drops flow out;

4) adding 9.36kg of water into a 50L reaction tank, adding the centrifugal solid and 78g of activated carbon while stirring, heating to 48-52 ℃, dissolving, cooling to 0-5 ℃, and stirring for 1 h;

5) filtering the product obtained in the step 4) in a vacuum state, filtering for the first time by filter paper, and filtering for the second time by a 0.45um microporous filter membrane;

6) transferring the filtered product in the step 5) into a crystallizing tank, raising the temperature to 48-52 ℃, adding 11.6kg of absolute ethyl alcohol, preserving the temperature for 30min, cooling the temperature to 15-25 ℃ in a cold water bath, and stirring for 1 h;

7) putting the product obtained in the step 6) into a centrifugal machine, centrifuging, washing the centrifuged solid with 1kg of absolute ethyl alcohol, and centrifuging until no liquid drops flow out; and (4) drying the solid obtained by centrifugation in a vacuum oven at 45-50 ℃ for 8h to obtain 870g of yellow solid.

According to another aspect of the present application, there is provided a pharmaceutical formulation characterized in that it comprises: at least one of the hydrate crystal form A, the hydrate crystal form A prepared by the method, the anhydrous crystal form B prepared by the method, the anhydrous crystal form E prepared by the method, the anhydrous crystal form G prepared by the method, the hydrate crystal form D prepared by the method, the hydrate crystal form F and the hydrate crystal form F prepared by the method, and a pharmaceutically acceptable carrier and/or a diluent.

Optionally, the pharmaceutical formulation comprises at least one of artemisinin, artemether, arteether, artesunate and dihydroartemisinin, and pyronaridine.

According to another aspect of the application there is provided the use of the above pharmaceutical formulation as a medicament for the treatment of malaria, tuberculosis and cancer.

Optionally, the cancer comprises breast cancer.

In the present application, XRPD is an abbreviation for X-ray powder diffraction pattern; DSC is an abbreviation for differential scanning calorimetry; TGA is an abbreviation for thermogravimetric analysis; IC is an abbreviation for ion chromatography; DVS is an abbreviation for dynamic moisture adsorption; PLM is a polarizing microscope; HPLC is high performance liquid chromatography; RH is an abbreviation for humidity.

In the present application, the room temperature is 20 ℃ to 30 ℃. Preferably, the room temperature is 20 ℃ to 25 ℃.

In the present application, the phosphate of the compound of formula i is referred to as pyronaridine phosphate, and the formula: c₂₉H₃₂ClN₅O₂·4H₃PO₄。

In the present application, the position of the endothermic peak refers to the position of the peak of the endothermic peak.

In the present application, the phosphate salts of the compounds of formula I are prepared according to methods known in the art.

Benefits of the present application include, but are not limited to:

1. the application provides a polymorphic substance of pyronaridine phosphate, a preparation method and application thereof, identifies and evaluates the prepared crystal form, and provides a new crystal form suitable for pharmaceutical research and industrial production.

2. The anhydrous crystal form B of the phosphate of the compound shown in the formula I has good physical stability and low hygroscopicity, is convenient to store, and can avoid the risk of crystal transformation in the process of drug development and generation.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is an XRPD pattern, as measured in the X' Pert-3 reflectance mode, of form A as an example of the present application, related to hydrate.

Fig. 2 is a TGA/DSC diagram relating to form a of the hydrate according to the examples of the present application, in which the ordinate represents mass percent (%), the abscissa represents time-temperature (° c) of the complex, and in which the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c).

FIG. 3 is an XRPD pattern, as measured by the X' Pert-3 reflectance mode, of form B anhydrous according to the examples herein.

Fig. 4 is a TGA/DSC graph relating to anhydrous crystalline form B of the examples herein, in which the ordinate represents percent by mass (%), the abscissa represents time-temperature (° c) of the complex, and in which the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c).

FIG. 5 is an XRPD pattern, as measured in the X' Pert-3 reflectance mode, of crystalline anhydrate form E of the examples herein.

Fig. 6 is a TGA/DSC graph relating to the anhydrous crystalline form E of the examples of the present application, in which the ordinate represents mass percent (%), the abscissa represents time-temperature (° c) of the complex, and in which the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c).

FIG. 7 is an XRPD pattern, as measured in the Empyrean reflex mode, for form G anhydrous as contemplated by the examples herein.

Figure 8 is an XRPD pattern of form D of the hydrate related to example of the application, tested in X' Pert-3 reflectance mode.

Fig. 9 is a TGA/DSC graph relating to form D of the hydrate according to the examples of the present application, in which the ordinate represents mass percent (%), the abscissa represents time-temperature (° c) of the complex, and in which the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c).

Figure 10 is an XRPD pattern of form F, related to hydrate, tested in the X' Pert-3 reflection mode, of the examples herein.

Fig. 11 is a TGA/DSC plot of hydrate form F according to an example of the present application, wherein the ordinate represents mass percent (%), the abscissa represents time-temperature (° c) on the complex scale, and the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c) on the DSC plot.

FIG. 12 is an XRPD overlay of a product prepared according to the test of example 6 herein, tested in the X' Pert-3 reflectance mode.

FIG. 13 is an XRPD overlay of a product prepared according to the test of example 7 herein, tested in the X' Pert-3 reflectance mode.

FIG. 14 is an XRPD overlay of a product prepared according to the test of example 7 herein, tested in the X' Pert-3 reflectance mode.

FIG. 15 is an XRPD overlay of a product prepared according to the test of example 8 herein, tested in the X' Pert-3 reflectance mode.

Fig. 16 is a DVS overlay of crystalline anhydrate form B according to an embodiment of the present application.

FIG. 17 is an XRPD overlay of a product prepared according to the test of example 9 herein, tested in the X' Pert-3 reflectance mode.

Fig. 18 is a PLM overlay relating to anhydrous crystalline form B in examples of the present application.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise indicated, the starting materials and catalysts in the examples of the present application are commercially available and the phosphate salts of the compounds of formula i, namely pyronaridine phosphate, are prepared by methods of preparation known in the art, for example: chenchang, synthesis of pyronaridine phosphate, a new antimalarial drug [ J ] pharmaceutical industry, 1981(9), 12-13; chemical synthesis of pyrrolizidine phosphate as one new antimalarial medicine [ J ] Zhejiang chemical industry 1979(04): 15-20.

The analysis method in the examples of the present application is as follows:

powder X-ray diffraction was carried out on an X-ray powder diffractometer of Empyrean type from the company PANALYTIC, the Netherlands and X' Pert3, the test conditions being as shown in Table 1.

TABLE 1

Thermogravimetric analysis was performed on a model TAQ5000/Discovery 5500 thermogravimetric analyzer from TA, USA, under the following test conditions: the temperature is in the range of room temperature to 350 ℃, the scanning speed is 10 ℃/min, and the protective gas is nitrogen.

Calorimetric analysis of samples was carried out in a differential scanning calorimeter model TAQ5000/Discovery 5500, TA, USA, under the following test conditions: the temperature is in the range of room temperature to 350 ℃, the scanning speed is 10 ℃/min, and the protective gas is nitrogen.

The samples were subjected to a dynamic moisture sorption curve test in a dynamic moisture sorption instrument of SMS corporation, england. Dynamic water adsorption curves were collected on DVS Intrasic in SMS (surface measurement systems). At a relative humidity of 25 deg.C using LiCl, Mg (NO)₃)₂And deliquescence point correction of KCl. The test conditions were: 10-20mg, at 25 ℃, with a protective gas and flow of nitrogen 200mL/min, an RH range of 0% RH-95% RH-0% RH, a gradient of 10% (0% RH-90% RH)/5% (90% RH-95% RH).

The sample was taken with a microscope using an Axio scope, Al polarizing microscope from Carl Zeiss Germany.

The product purity was analyzed by HPLC, model 1260, from Agilent, USA, under the conditions shown in Table 2.

TABLE 2

The content of the sample was measured by ion chromatography, ThermoFisher ICS-1100, a thermoelectric company, USA, and the test conditions are shown in Table 3.

TABLE 3

Example 1 hydrate form a

The preparation method of the hydrate crystal form A of the phosphate of 2-methoxy-7-10- [ (3 ', 5 ' -bistetrahydropyrrole methylene) -4 ' -hydroxy phenylpropylamino ] benzo [ b ]1, 5-naphthyridine shown as the formula I comprises the following steps:

The XRPD pattern of hydrate form a is shown in fig. 1, and the peak data is shown in table 4.

TABLE 4

The TGA/DSC of hydrate form a is shown in fig. 2, wherein the ordinate represents mass percent (%) and the abscissa represents time-temperature (deg.c) in the complex, and the ordinate represents thermal power (W/g) and the abscissa represents temperature (deg.c) in the DSC chart. Line 1 in figure 2 represents TGA results, indicating that there is a 3.1% weight loss of hydrate form a upon heating to 150 ℃; line 2 in fig. 2 represents DSC results, showing that the sample has an endothermic peak at 106.7 ℃ (peak temperature), either due to dehydration or solvent; 198.1 ℃ (peak temperature) has endothermic peak and 204.9 ℃ (peak temperature) has exothermic peak, supposedly the sample melts and transforms into anhydrous crystal form E; the presence of an endothermic peak at 217.3 deg.C (peak temperature) is presumed to be caused by melting of the sample. IC testing of hydrate form a showed that the sample contained 37.5% phosphate.

In order to research the thermal signal of the hydrate crystal form A in DSC, the temperature-variable XRPD test is carried out on the hydrate crystal form A, and the result shows that the hydrate crystal form A sample is N₂Heating under protection toChanging into an anhydrous crystal form G at 120 ℃, and cooling to 30 ℃ to obtain the anhydrous crystal form G; heating the hydrate crystal form A to 120 ℃, then cooling to room temperature and exposing in the environment to obtain the hydrate crystal form A, judging that the hydrate crystal form A is a hydrate, dehydrating to obtain an anhydrous crystal form G, and re-absorbing water in the environment to obtain the hydrate crystal form A.

Example 2 Anhydrous crystalline form B

A first method for preparing anhydrous crystalline form B of the phosphate salt of 2-methoxy-7-10- [ (3 ', 5 ' -bistetrahydropyrrolidinomethyl) -4 ' -hydroxyphenylpropylamino ] benzo [ B ]1, 5-naphthyridine of formula I comprises: the compound is obtained by suspending and stirring hydrate crystal form A in methanol (MeOH for short) solvent at room temperature.

A second method of making anhydrous crystalline form B comprises: by mixing hydrate crystal form A and a mixture with the volume ratio of 1:1 MeOH and dichloromethane (DCM for short) at room temperature,

a third method of preparing anhydrous crystalline form B comprises: suspending and stirring the mixture in a solvent II with isopropyl acetate (IPAc) according to the volume ratio of 1:1 in ethanol (EtOH), N-methylpyrrolidone (NMP) and Dimethylformamide (DMF) at 50 ℃, and the like.

The XRPD pattern of anhydrous form B is shown in fig. 3, and the peak data is shown in table 5.

TABLE 5

The TGA/DSC of the anhydrous crystalline form B is shown in FIG. 4, wherein the ordinate represents mass percent (%) and the abscissa represents time-temperature (. degree. C.) in the complex, and the ordinate represents thermal power (W/g) and the abscissa represents temperature (. degree. C.) in the DSC chart. The TGA results are represented by line 3 in figure 4, indicating that the anhydrous crystalline form B sample had a weight loss of 2.4% when heated to 210 ℃; the DSC results are represented by line 4 in figure 4, showing that the presence of an endothermic peak at 229.7 ℃ (onset temperature) in the sample is presumed to be due to sample melting. The sample of the anhydrous crystal form B is subjected to an IC test, and the result shows that the sample contains 34.2% of phosphate radical.

In order to research the weight loss of the anhydrous crystal form B before melting, the anhydrous crystal form B sample is subjected to variable temperature XRPD test, and the result shows that the anhydrous crystal form B sample is N₂And heating to 200 ℃ and cooling to 30 ℃ under protection, wherein the crystal form is not changed, and the anhydrous crystal form B is an anhydrous crystal form.

Example 3 Anhydrous crystalline form E

A preparation method of an anhydrous crystal form E of phosphate of 2-methoxy-7-10- [ (3 ', 5 ' -bistetrahydropyrrole methylene) -4 ' -hydroxy phenylpropylamino ] benzo [ b ]1, 5-naphthyridine shown as a formula I comprises the following steps: heating a hydrate crystal form A sample to 210 ℃ and cooling to room temperature to obtain the hydrate crystal form A.

The XRPD pattern of anhydrous crystalline form E is shown in fig. 5, and the peak data is shown in table 6.

TABLE 6

The TGA/DSC of the anhydrous crystalline form E is shown in FIG. 6, wherein the ordinate represents mass percent (%) and the abscissa represents time-temperature (. degree. C.) in the complex, and the ordinate represents thermal power (W/g) and the abscissa represents temperature (. degree. C.) in the DSC chart. The TGA results are represented by line 5 in fig. 6, indicating a 6.0% weight loss of the anhydrous crystalline form E sample upon heating to 200 ℃, and by line 6 in fig. 6 representing DSC results, indicating that the presence of an endothermic peak at 104.6 ℃ (peak temperature) of the sample is presumed to be due to dehydration of the sample, and the presence of an endothermic peak at 214.4 ℃ (onset temperature) is presumed to be due to melting of the sample. The sample of the anhydrous crystal form E is subjected to an IC test, and the result shows that the sample contains 33.7 percent of phosphate radical.

In order to research whether an endothermic signal in DSC is to remove crystal water, a temperature-variable XRPD test is carried out on the anhydrous crystal form E sample, and the result shows that the anhydrous crystal form E sample is in N₂And heating to 120 ℃ under protection and cooling to ensure that the crystal form is not changed, so that the anhydrous crystal form E is an anhydrous crystal form.

Example 3 Anhydrous crystalline form G

2-methoxy-7-10- [ (3 ', 5 ' -bistetrahydropyrrole methylene) -4 ' -hydroxy phenylpropylamino group shown in formula I]Benzo [ b ]]Anhydrous crystalline forms of phosphate of 1, 5-naphthyridineThe preparation method of G comprises the following steps: by hydrate form A sample at N₂Heating to 120 ℃ under protection to obtain a product under N₂And the crystal form G still keeps the anhydrous crystal form after being cooled to 30 ℃ under protection, and the anhydrous crystal form G is converted into a hydrate crystal form A after being exposed to the environment.

The XRPD pattern of anhydrous crystalline form G is shown in fig. 7 and the peak data is shown in table 7, anhydrous crystalline form G being anhydrous crystalline form.

TABLE 7

Example 4 hydrate form D

2-methoxy-7-10- [ (3 ', 5 ' -bistetrahydropyrrole methylene) -4 ' -hydroxy phenylpropylamino group shown in formula I]Benzo [ b ]]A first method of preparing hydrate form D of the phosphate salt of 1, 5-naphthyridine comprises: hydrate crystal form A sample is placed in H₂And volatilizing the O solution at room temperature to obtain the product.

The XRPD pattern of hydrate form D is shown in figure 8 and the peak data is shown in table 8.

TABLE 8

The TGA/DSC of hydrate form D is shown in fig. 9, in which the ordinate represents mass percent (%), the abscissa represents time-temperature (° c) on the complex coordinate, and in which the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c) on the DSC chart. Line 7 in figure 9 represents TGA, indicating a 3.7% weight loss of hydrate form D upon heating to 150 ℃; the 8-line in fig. 9 represents DSC, and the results show that the presence of endothermic peaks at 96.8 ℃ and 194.1 ℃ (peak temperature) in the sample is presumed to be due to dehydration, and the presence of endothermic peaks at 217.9 ℃ (onset temperature) is presumed to be due to melting of the sample. IC testing of hydrate form D showed that the sample contained 36.6% phosphate.

In order to research endothermic signals before melting in DSC, temperature-variable XRPD test is carried out on the hydrate crystal form D, and the result shows that the hydrate crystal form D is in N₂Heating to 110 ℃ and 200 ℃ under protection to obtain the anhydrous crystal form E, cooling to 30 ℃ to obtain the anhydrous crystal form E, and obtaining the hydrate crystal form D sample as a hydrate.

Example 5 hydrate form F

2-methoxy-7-10- [ (3 ', 5 ' -bistetrahydropyrrole methylene) -4 ' -hydroxy phenylpropylamino group shown in formula I]Benzo [ b ]]A first method of preparing hydrate form F of the phosphate salt of 1, 5-naphthyridine comprises: sample hydrate crystal form A in H₂Gas-solid diffusion in O environment.

The second preparation method comprises the following steps: dissolving in a solvent III of tetrahydrofuran and water with the volume ratio of 2:1, and performing gas-liquid diffusion in an acetone atmosphere to obtain a hydrate crystal form F. The XRPD pattern of hydrate form F is shown in fig. 10 and the peak data is shown in table 9.

TABLE 9

The TGA/DSC of hydrate form F is shown in fig. 11, in which the ordinate represents mass percent (%), the abscissa represents time-temperature (° c) on the complex coordinate, and in which the ordinate represents thermal power (W/g) and the abscissa represents temperature (° c) on the DSC chart. The 9 line in fig. 11 represents TGA, indicating a 4.6% weight loss of hydrate form F upon heating to 150 ℃; the 10 line in fig. 11 represents DSC, and shows that the presence of endothermic peaks at 86.1 ℃ and 195.0 ℃ (peak temperature) in the sample is presumably due to dehydration, and the presence of endothermic peaks at 217.9 ℃ (onset temperature) is presumably due to melting of the sample.

In order to research an endothermic signal before melting in DSC, XRPD is tested after the hydrate crystal form F is heated to 100 ℃ and 200 ℃, and the result shows that the hydrate crystal form F obtains an anhydrous crystal form E after being heated to 100 ℃ and 200 ℃, and the hydrate crystal form F is a hydrate.

Example 6 transformation of Anhydrous Crystal form B with Anhydrous Crystal form E

In order to study the stability relationship between the anhydrous crystalline form B and the anhydrous crystalline form E, suspension competition tests of anhydrous crystalline form samples in acetonitrile (ACN for short) and isopropanol (IPA for short) at room temperature and 50 ℃ were set, as shown in table 10, and the specific steps were as follows:

1) preparing a nearly saturated solution of the anhydrous crystal form B in different solvents at a specified temperature;

2) respectively adding equal mass of anhydrous crystal form B and E samples (about 6 mg each) into 0.5 ml of near-saturated solution to form suspension;

3) suspension stirring at room temperature and 50 ℃ for about 6 days (800 rpm);

4) the solid was isolated and tested for XRPD.

Watch 10

According to the XRPD comparison in fig. 12, line 11 in fig. 12 represents the XRPD curve of anhydrous form B, and

lines

12, 13, 14 and 15 in fig. 12 represent the XRPD results of

tests

12, 13, 14 and 15 thereof, respectively, all of which finally convert to anhydrous form B, indicating that anhydrous form B is thermodynamically more stable over the range of room temperature to 50 ℃ than anhydrous form E.

Example 7 transition of Anhydrous form B and hydrate form A, D

In order to research the transformation relation between the anhydrous crystal form B and the hydrate crystal form A, D, the anhydrous crystal form B, the hydrate crystal form A and the hydrate crystal form D are set at different volume ratios ACN/H at room temperature₂Suspension competition tests under different water activities (aw) in the O system are summarized in Table 11, and the specific steps are as follows:

1) preparing a near-saturated solution of a hydrate crystal form A sample in different water activity systems at room temperature;

2) respectively adding anhydrous crystal form B, hydrate crystal form A and hydrate crystal form D samples (about 5 mg each) with equal mass into 0.5 ml of near-saturated solution to form suspension;

3) suspension stirring at room temperature for about 4 days (-800 rpm);

4) the solid was isolated and tested for XRPD.

TABLE 11

Based on the comparison of the XRPD patterns in FIGS. 13 and 14, line 16 in FIG. 13 represents the XRPD pattern for crystalline anhydrate form B and lines 17-20 represent the XRPD pattern results for the products of runs 17-20, respectively; line 21 in figure 14 represents the XRPD curve of hydrate form D and lines 22, 23 represent the XRPD curve results of

trials

22, 23 respectively. Fig. 13 and 14 show that the anhydrous crystal form B is obtained in suspension competition experiments between 0 and 0.6 of water activity; obtaining a hydrate crystal form D with water activity of 0.8-1.0.

Example 8 solid state stability of Anhydrous crystalline form B

To evaluate the solid state stability of form B anhydrous, appropriate amounts of samples were weighed and placed in a closed environment at 80 ℃ for 1 day and in an open environment at 25 ℃/60% RH and 40 ℃/75% RH for one week, respectively, and the solid samples in different conditions were evaluated for physical and chemical stability by XRPD and HPLC tests, respectively, and the evaluation results are summarized in table 12 and fig. 15.

TABLE 12

Based on the XRPD comparison in FIG. 15, line 24 in FIG. 15 represents the anhydrous form B and lines 25-27 represent the XRPD curve results for runs 25-27, respectively. Solid state stability studies show that anhydrous form B remains physically stable under all three conditions, but has a slight decrease in chemical purity at 25 ℃/60% RH and 40 ℃/75% RH for one week.

Example 9 hygroscopicity of Anhydrous crystalline form B

The hygroscopicity of the anhydrous crystalline form B was evaluated by a dynamic moisture sorption test (DVS) at 25 ℃, and the result is shown in fig. 16. The results show that the weight gain of the anhydrous crystal form B from 0% RH to 80% RH is about 2.6%, indicating that the anhydrous crystal form B has hygroscopicity; the XRPD detection result of the anhydrous crystal form B before DVS test is shown in figure 17, wherein a line 30 represents the XRPD curve of the anhydrous crystal form B before DVS, a line 31 represents the XRPD curve of the anhydrous crystal form B after DVS, and the comparison result shows that the crystal form of the anhydrous crystal form B before and after DVS is not changed.

EXAMPLE 10 morphology of Anhydrous Crystal form B

The sample morphology of the anhydrous crystal form B was evaluated by PLM test, and the result is shown in fig. 18, where the anhydrous crystal form B sample was irregular particles and partially agglomerated.

The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.

Claims

1. A hydrate form a of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

2. hydrate form a according to claim 1, further comprising one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 11.69 +/-0.2 degrees, 14.16 +/-0.2 degrees, 18.89 +/-0.2 degrees, 21.45 +/-0.2 degrees, 22.82 +/-0.2 degrees and 25.79 +/-0.2 degrees.

3. A process for preparing the hydrate form a of claim 1 or 2, comprising:

4. An anhydrous crystalline form B of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

5. anhydrous crystalline form B according to claim 4, characterized in that it further comprises one or more peaks in its X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-Ka radiation, at positions selected from: 14.21 +/-0.2 degrees, 17.67 +/-0.2 degrees, 19.03 +/-0.2 degrees, 23.82 +/-0.2 degrees, 24.91 +/-0.2 degrees and 29.19 +/-0.2 degrees.

6. Anhydrous crystalline form B according to claim 4, characterized in that it further comprises one or more peaks in its X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-Ka radiation, at positions selected from: 13.24 +/-0.2 degrees, 13.85 +/-0.2 degrees, 21.37 +/-0.2 degrees, 26.60 +/-0.2 degrees, 28.20 +/-0.2 degrees and 28.70 +/-0.2 degrees.

7. A method of preparing the anhydrous crystalline form B of any one of claims 4 to 6, comprising:

wherein, the solvent I comprises methanol or methanol and halogenated alkane;

8. An anhydrous crystalline form E of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

9. anhydrous crystalline form E according to claim 8, characterized in that it further comprises one or more peaks in its X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-Ka radiation, at positions selected from: 18.10 + -0.2 deg., 19.47 + -0.2 deg. and 21.29 + -0.2 deg..

10. Anhydrous crystalline form E according to claim 8, characterized in that it further comprises one or more peaks in its X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-Ka radiation, at positions selected from: 11.52 +/-0.2 degrees, 16.60 +/-0.2 degrees, 22.88 +/-0.2 degrees, 25.36 +/-0.2 degrees, 26.69 +/-0.2 degrees and 32.22 +/-0.2 degrees.

11. A method of preparing the anhydrous crystalline form E of any one of claims 8 to 10, comprising:

12. An anhydrous crystalline form G of the phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

13. the anhydrous crystalline form G according to claim 12, characterized in that it further comprises one or more peaks in the X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 6.66 +/-0.2 degrees, 8.34 +/-0.2 degrees, 11.41 +/-0.2 degrees and 16.02 +/-0.2 degrees.

14. A process for preparing the anhydrous crystalline form G of claim 12 or 13, comprising:

15. A hydrate crystalline form D of a phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

16. hydrate form D according to claim 15, further comprising one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 8.83 +/-0.2 degrees, 9.07 +/-0.2 degrees, 11.04 +/-0.2 degrees, 20.51 +/-0.2 degrees, 20.82 +/-0.2 degrees, 21.26 +/-0.2 degrees or 25.99 +/-0.2 degrees.

17. Hydrate form D according to claim 15, further comprising one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 4.46 +/-0.2 degrees, 6.30 +/-0.2 degrees, 11.86 +/-0.2 degrees, 14.34 +/-0.2 degrees, 16.44 +/-0.2 degrees, 22.36 +/-0.2 degrees, 23.40 +/-0.2 degrees and 23.98 +/-0.2 degrees.

18. A method of preparing the hydrate form D according to any one of claims 15 to 17, comprising:

19. A hydrate crystalline form F of a phosphate salt of a compound of formula i, characterized in that it has at least one of the following:

20. the hydrate crystalline form F according to claim 19, further comprising one or more peaks in an X-ray powder diffraction pattern expressed in degrees 2 Θ using Cu-ka radiation at positions selected from: 12.03 +/-0.2 degrees, 13.27 +/-0.2 degrees, 17.73 +/-0.2 degrees, 18.13 +/-0.2 degrees, 20.97 +/-0.2 degrees, 23.45 +/-0.2 degrees, 24.05 +/-0.2 degrees, 27.03 +/-0.2 degrees and 29.47 +/-0.2 degrees.

21. A method of preparing the hydrate crystalline form F of claim 19 or 20, comprising:

22. Pharmaceutical formulation, characterized in that it comprises: at least one of the hydrated crystalline form a of claim 1 or 2, the anhydrous crystalline form B prepared by the process of claim 3, the anhydrous crystalline form B of any one of claims 4 to 6, the anhydrous crystalline form B prepared by the process of claim 7, the anhydrous crystalline form E of any one of claims 8 to 10, the anhydrous crystalline form E prepared by the process of claim 11, the anhydrous crystalline form G of claim 12 or 13, the anhydrous crystalline form G prepared by the process of claim 14, the hydrate crystalline form D of any one of claims 15 to 17, the hydrate crystalline form D prepared by the process of claim 18, the hydrate crystalline form F of claim 19 or 20, and the hydrate crystalline form F prepared by the process of claim 21, and a pharmaceutically acceptable carrier and/or diluent.

23. The pharmaceutical formulation as claimed in claim 22, which comprises at least one of artemisinin, artemether, arteether, artesunate and dihydroartemisinin, and pyronaridine.

24. Use of a pharmaceutical formulation according to claim 22 or 23 as a medicament for the treatment of malaria, tuberculosis and cancer.