Azacitidine crystal form D

Abstract

The present invention belongs to the technical field of crystal drug molecules, and specifically provides an azacitidine crystal form D. The azacitidine crystal form D prepared by the present invention uses Cu-Kα radiation, and an X-ray diffraction spectrum represented by 2θ has characteristic peaks at 5.51±0.2°, 11.32±0.2°, 12.45±0.2°, 22.21±0.2°, and 28.77±0.2°. The yield of the azacitidine crystal form D prepared by the present invention is greater than 90%, and the purity is higher than 99.82%. Moreover, the azacitidine crystal form D prepared by the present invention has higher solubility in different dissolution media than existing crystal forms, and the preparation process of the present invention is simple and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of crystal drug molecules, and in particular to a crystal form of azacitidine. Background Art

[0002] In recent years, studies have found that different drug crystal forms can change the physical and chemical properties (density, hardness, solubility, stability, optical and electrical properties, etc.), dissolution rate, biological effects, etc. Therefore, studying drug crystal forms has important practical value in medicine. Crystalline drug molecules include polymorphs, hydrates, solvates and salts of drug molecules. Through the method of drug crystallization, not only can the crystallographic parameters of crystal drug molecules be clarified, but also the types and numbers of solvent molecules in the crystal form (such as crystal water molecules) can be determined, which plays a very important role in understanding and mastering the spatial arrangement and physical and chemical properties of drug molecules.

[0003] Azacitidine is a white or off-white powder. Its chemical name is 4-amino-1-(β-D-ribofuranosyl)-1,3,5-triazine-2(1H)-one, and its molecular formula is C8H 12 N4O5, molecular weight is 244.2, CAS number is 320-67-2, and the structure is as follows:

[0004]

[0005] Azacitidine, with the chemical name 1-(β-D-ribofuranosyl)-4-amino-1,3,5-triazine-2(1H)-one, is a DNA methyltransferase inhibitor developed by Pharmion Corporation in the United States. It was first launched in the United States in July 2004 under the trade name Vidaza. Its mechanism of action is that azacitidine binds to DNA molecules after phosphorylation, and DNA methyltransferase reacts with azacitidine to form a covalently bound product. The activity of DNA methyltransferase is inhibited and degraded, causing a decrease in the DNA methylation level in tumor tissues, demethylation of hypermethylated tumor suppressor genes, and restoration of gene expression, thereby inhibiting tumor cells. It is mainly used clinically to treat myelodysplastic syndrome (MDS) and acute non-lymphocytic leukemia, and can also be used to treat breast cancer, melanoma, and colon cancer.

[0006] Patent WO2004082619 discloses eight crystal forms of azacitidine, which are crystal forms I to VIII, among which the most suitable crystal form for use as a raw material is crystal form I, and patent WO2004082822 discloses a method for preparing azacitidine crystal form I, which uses a dipolar aprotic solvent such as DMSO, DMF, NMIP, etc. as a good solvent to dissolve azacitidine, and adds an antisolvent to change the polarity of the solution to promote the precipitation of azacitidine crystals. Since azacitidine has multiple crystal forms, in order to more stably prepare azacitidine crystal form I, patent document US2004186284 improves the preparation method of azacitidine crystal form I, which is to prepare azacitidine crystal form I in a preferred polar aprotic solvent as the main solvent and a preferred C 2-5 Recrystallization in a co-solvent of alcohol or aliphatic ketone or alkyl. However, the improved method of US2004186284 still has some problems, such as unsatisfactory impurity control and high solvent residue. Patent CN103450303A discloses a method for preparing azacitidine crystal form A and crystal form B.

[0007] These azacitidine crystal forms only have X-ray powder diffraction data or simple DSC characterization, without clear crystallographic main parameters and exact atomic spatial positions, which makes it quite difficult to understand and master the spatial arrangement and physical and chemical properties of drug molecules. The known azacitidine crystal forms cannot meet the requirements of drug preparations in terms of purity and solubility, so more crystal forms need to be developed. On the one hand, more azacitidine crystal forms are provided for drug applications, and on the other hand, more economically efficient azacitidine crystal forms are also needed. Summary of the invention

[0008] In view of the deficiencies in the prior art, the first object of the present invention is to provide an azacitidine crystalline form D, which has a clear crystal form and number of methanol, exact main crystallographic parameters and atomic spatial positions; another object of the present invention is to provide a method for preparing the azacitidine crystalline form D.

[0009] The specific technical solutions of the present invention are as follows:

[0010] An azacitidine crystal form D, using Cu-Kα radiation, has an X-ray diffraction spectrum represented by 2θ with characteristic peaks at 5.51±0.2°, 11.32±0.2°, 12.45±0.2°, 22.21±0.2°, and 28.77±0.2°.

[0011] Preferably, the azacitidine form D uses Cu-Kα radiation, and the X-ray diffraction spectrum expressed in 2θ has characteristic peaks at 5.51±0.2°, 11.32±0.2°, 12.45±0.2°, 15.97±0.2°, 20.54±0.2°, 22.21±0.2°, 22.83±0.2°, 25.73±0.2°, 28.77±0.2°, and 31.10±0.2°.

[0012] Preferably, the azacitidine crystalline form D, when irradiated with Cu-Kα, has characteristic peaks as follows: Figure 3 The X-ray powder diffraction pattern is shown.

[0013] Preferably, the crystallographic parameters of the azacitidine crystal form D are: monoclinic system, space group P21 / c, molecular weight 277.24; unit cell parameters are: α=90.00°,β=99.105(2)(3)°,γ=90.00°,unit cell volume

[0014] The preparation method of the above-mentioned azacitidine crystal form D comprises the following steps:

[0015] Azacitidine is added to a mixed solvent of an aprotic solvent and methanol, heated and stirred, and the solvent is filtered to evaporate to obtain azacitidine methanol solvate.

[0016] Preferably, the mass-to-volume ratio of azacitidine to the mixed solvent is 0.55-0.85:1, wherein the mass is measured in mg and the volume is measured in ml.

[0017] Further preferably, the mass-to-volume ratio of azacitidine to the mixed solvent is 0.6-0.7:1, wherein the mass is measured in mg and the volume is measured in ml.

[0018] Preferably, the aprotic solvent is selected from one of acetone, acetonitrile, dichloromethane, chloroform and tetrahydrofuran.

[0019] Preferably, the volume percentage of methanol in the mixed solvent is 90.9% to 100%.

[0020] Preferably, the heating and dissolving temperature is 50-60°C.

[0021] Confirmation of the structure of azacitidine crystal form D

[0022] Crystals that meet the specifications were selected from the prepared crystalline samples, and their X-ray crystal data were collected on a Japanese Rigaku XtaLAB Synergy instrument. The test temperature was 293 (2) K, the voltage was 50 kV, the current was 1 mA, and CuKa radiation was used. The data were collected in ω scanning mode and Lp correction was performed. The structure was analyzed by direct method, and all non-hydrogen atoms were found by difference Fourier method. All hydrogen atoms on carbon and nitrogen were obtained by theoretical hydrogenation, and the structure was refined by least square method.

[0023] The crystallographic data obtained by testing and analyzing the azacitidine hydrate crystal prepared by the present invention are shown in Table 1: its crystallographic parameters are: monoclinic system, space group is P21 / c, molecular weight is 277.24; unit cell parameters are: α=90.00°,β=99.105(2)(3)°,γ=90.00°,unit cell volume ORTEP diagram of the azacitidine crystalline form of the present invention ( Figure 1 ) shows that each azacitidine molecule is crystallized with one methanol molecule, and it can be confirmed that this crystal form is azacitidine methanol complex, and the H6A of the hydroxyl group of the methanol solvent molecule is connected to the azacitidine hydroxyl group O4 through a hydrogen bond. The stacking diagram of the azacitidine crystal form of the present invention ( Figure 2 ) showed that the methanol complex of azacitidine formed a stacking structure through hydrogen bonding, π-π stacking and other interactions.

[0024] Table 1 Main crystallographic data of azacitidine form D

[0025]

[0026]

[0027] The X-ray powder diffraction test instrument and test conditions involved in the present invention are as follows: X-ray powder diffractometer: PANalytical EMPYREAN; Cu-Kα; sample stage: flat plate; incident light path: BBHD; diffraction light path: PLXCEL; voltage 45kv, current 40mA; divergence slit: 1 / 4; anti-scattering slit: 1; Soller slit: 0.04rad; step size: 0.5s; scanning range: 3-50°.

[0028] According to the above crystallographic data, the corresponding characteristic peaks in the X-ray powder diffraction pattern (Cu-Kα) are shown in Figure 3 And Table 2.

[0029] Table 2 PXRD peaks of azacitidine crystal

[0030]

[0031]

[0032] The TGA / DSC thermal analysis test conditions for the azacitidine crystal form D of the present invention are: Mettler-Toledo TGA / DSC thermal analyzer (model: TGA / DSC3+), dynamic temperature section: 30-300°C, heating rate: 10°C / min, program section gas N2, flow rate: 50ml / min, crucible: aluminum crucible 40μl. Figure 4 As shown, the azacitidine crystal form D has two endothermic peaks detected by TGA / DSC, ranging from 92.7 to 133.05°C and 222.67 to 237.56°C, respectively, and the corresponding endothermic peak values ​​are 120.10°C and 237.11°C. The endothermic peak of 92.7 to 133.05°C is the endothermic peak of losing methanol molecules, and the corresponding weight loss is 9.5807%. One methanol molecule is lost, so the azacitidine crystal form D is an azacitidine methanol complex containing one methanol molecule, and the result is consistent with the X single crystal data.

[0033] All samples prepared in the examples have the same crystallographic parameters, X-ray powder diffraction patterns and differential scanning calorimetry (DSC / TGA) curves.

[0034] Beneficial effects of the present invention:

[0035] 1. The preparation method of azacitidine crystal form D of the present invention is simple, the obtained crystal has a regular crystal form, uniform particle size, good stability, a clear crystal form and number of methanol, clear main crystallographic parameters and exact atomic spatial position, and is suitable for large-scale promotion and application.

[0036] 2. High yield and high purity. The yield is greater than 90% and the purity is higher than 99.82%.

[0037] 3. Good solubility. The azacitidine crystal form D prepared by the present invention has higher solubility in different dissolution media than the existing crystal forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : ORTEP diagram of azacitidine crystal form.

[0039] Figure 2 : Azacitidine crystal stacking diagram.

[0040] Figure 3 : X-ray powder diffraction pattern of azacitidine crystal form.

[0041] Figure 4 : Differential scanning calorimetry (DSC / TGA) curve of azacitidine crystal. DETAILED DESCRIPTION

[0042] The present invention is further described below by way of examples. It should be correctly understood that the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention, so simple improvements to the present invention based on the method of the present invention are within the scope of protection claimed by the present invention.

[0043] Materials used in the experiments: Azacitidine can be purchased or prepared according to existing technologies; other materials used in the experiments without indicating the source and specification are all commercially available analytical grade or chemically pure.

[0044] Example 1

[0045] Azacitidine (21.45 mg) was added to 33 ml of methanol solution, heated to 55°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 98.3% and a purity of 99.92%.

[0046] Example 2

[0047] Azacitidine (189.72 mg) was added to a mixed solvent of 6.2 ml acetone and 310 ml methanol, heated to 60°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 98.2% and a purity of 99.91%.

[0048] Example 3

[0049] Azacitidine (90.9 mg) was added to a mixed solvent of 6.2 ml acetonitrile and 123.2 ml methanol, heated to 60°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 97.9% and a purity of 99.90%.

[0050] Example 4

[0051] Azacitidine (28.05 mg) was added to a mixed solvent of 3.3 ml of dichloromethane and 50 ml of methanol, heated to 50°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 97.5% and a purity of 99.88%.

[0052] Example 5

[0053] Azacitidine (102 mg) was added to a mixed solvent of 10.9 ml chloroform and 109.1 ml methanol, heated to 55°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 95.4% and a purity of 99.86%.

[0054] Example 6

[0055] Azacitidine (60 mg) was added to 150 ml of methanol solution, heated to 50°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 91.2% and a purity of 99.84%.

[0056] Example 7

[0057] Azacitidine (25.2 mg) was added to a mixed solvent of 3 ml tetrahydrofuran and 25 ml methanol, heated to 45°C and stirred to dissolve, and the mixture was kept warm and stirred for 0.5 h. The mixture was filtered while hot, pierced and evaporated slowly to obtain azacitidine form D with a yield of 90.0% and a purity of 99.82%.

[0058] Comparative Example 1

[0059] Under nitrogen protection, add 200g of anhydrous methanol and 5.0g of 30% sodium methoxide methanol solution to a 500ml reaction bottle, start stirring, add 15.5g of p-chlorobenzoyl azacitidine, and after a large amount of solid precipitates in the system, stir and react for 2h, filter, slurry the filter cake with 60g of anhydrous methanol for 1h, filter and collect the filter cake. Place the filter cake on a plate in a vacuum dryer, control the temperature at 60°C, and dry for 25h to obtain 5.2g of azacitidine hemi-methanolate, with a yield of 72.2% and a purity of 96.82%.

[0060] Comparative Example 2

[0061] 10.0 g of azacitidine form III prepared according to U.S. Patent US20040186065 was added to 10000 ml of methanol and heated to dissolve, crystallized overnight at 0-8°C, filtered, and the filter cake was vacuum dried at 65-70°C to obtain 8.0 g of azacitidine type A crystals with a yield of 80% and a purity of 99.15%.

[0062] Comparative Example 3

[0063] 10.0 g of azacitidine form IV prepared according to U.S. Patent US20040186065 was added to 10000 ml of methanol and heated to dissolve. After concentration until a large amount of crystals precipitated, the mixture was crystallized at 0-8°C overnight, filtered, and the filter cake was vacuum dried at 65-70°C to obtain 7.9 g of azacitidine type B crystals with a yield of 79% and a purity of 99.23%.

[0064] Comparative Example 4

[0065] 5.0 g of crude azacitidine was added to 25 ml of N,N-dimethylformamide, dissolved at 50-60°C, filtered, 200 ml of anhydrous ethanol was added to the filtrate, stirred and cooled to room temperature, stirred at 0-5°C for 1 hour for crystallization. The obtained solid was added to 150 ml of anhydrous ethanol, stirred at room temperature for 2 hours, filtered, and dried to obtain 4.0 g of azacitidine crystal form I, with a yield of 80% and a purity of 97.91%.

[0066] Comparative Example 5

[0067] 83.0 g of crude azacitidine obtained in Example 1 of CN102850418A was dissolved in 692 ml of water preheated to 60°C, 1.0 g of activated carbon was added, stirred for 2 minutes, filtered while hot, 3458 ml of methanol preheated to 65°C was added to the filtrate while hot, cooled to 40°C, a trace amount of seed crystals was added, the temperature was slowly cooled to below 10°C, and kept at 0-10°C for 13 hours. Vacuum filtration was performed, and the filter cake was vacuum dried at room temperature to constant weight to obtain about 48.0 g of dry product.

[0068] 48.0 g of the obtained crude product was dissolved in 692 ml of water preheated to 75°C, 1.0 g of activated carbon was added, stirred for 2 minutes, filtered while hot, 3458 ml of methanol preheated to 60°C was added to the filtrate while hot, cooled to 40°C, a trace amount of seed crystals was added, the temperature was slowly cooled to below 10°C, and kept at 0-10°C for 13 hours. Vacuum filtered, the filter cake was vacuum dried at room temperature to constant weight, and 35.0 g of dry product was obtained.

[0069] The obtained 35.0 g crude product was dissolved in 25.2 L methanol by reflux, cooled to room temperature naturally, and allowed to stand for 3 days for crystallization. The filter cake was then filtered and vacuum dried at room temperature to obtain 30.0 g of white needle-shaped crystalline powder.

[0070] The obtained 30.0 g crude product was dissolved in 24 L of methanol by reflux, cooled to room temperature naturally, and allowed to stand for 3 days for crystallization. The filter cake was filtered and vacuum dried at room temperature to obtain 25.0 g of white needle-shaped crystalline powder.

[0071] 5.0 g of the obtained 25.0 g crystalline powder was taken and dried in vacuum at 150° C. for 30 minutes to obtain 4.5 g of white powder with a yield of 90% and a purity of 99.46%.

[0072] Solubility test

[0073] The excess azacitidine crystals of Examples 1 to 7 and Comparative Examples 1 to 5 were accurately weighed and placed in small vials. Water (pH 7.0) and pH 7.4 PBS were added to prepare saturated azacitidine solutions. The solutions were shaken to dissolve and filtered. The solubility was calculated by measuring the absorbance at a wavelength of 280 nm using the UV-Vis spectrophotometry method (General Rule 0401). The results are shown in Table 3.

[0074] Table 3 Solubility of different azacitidine crystal forms in different media

[0075]

[0076] After testing, all azacitidine crystal forms D prepared by the scheme of the present invention have similar solubility effects. As can be seen from Table 3, the solubility of azacitidine prepared by the scheme of the present invention in different pH solutions is higher than that of the crystal forms of Comparative Examples 1 to 5. The azacitidine crystal form D prepared by the present invention has higher solubility than the existing crystal forms.

Claims

1. A crystalline form D of azacitidine, It is characterized in that The azacitidine crystal form D, using Cu-Kα radiation, has characteristic peaks consistent with the X-ray powder diffraction pattern shown in FIG3 .

2. The azacitidine crystalline form D according to claim 1, It is characterized in that The crystallographic parameters of the azacitidine form D are: monoclinic system, space group P2 1 / c; the unit cell parameters are: a = 5.12460(10) Å, b = 7.78420(10) Å, c =15.3455(3) Å, α = 90.00°, β = 99.105(2) °, γ = 90.00°, unit cell volume V =604.433(19)Å 3 .

3. A method for preparing the azacitidine crystalline form D according to any one of claims 1 to 2, It is characterized in that The specific preparation steps include: Azacitidine is added to a mixed solvent of an aprotic solvent and methanol, heated and stirred, and the solvent is filtered to evaporate to obtain an azacitidine methanol solvate; wherein the mass-to-volume ratio of azacitidine to the mixed solvent is 0.55-0.85:1, wherein the mass is measured in mg and the volume is measured in ml; the aprotic solvent is selected from one of acetone, acetonitrile, dichloromethane, chloroform, and tetrahydrofuran; the volume percentage of methanol in the mixed solvent is 90.9%-100%; and the heating and dissolving temperature is 50-60°C.

4. The method for preparing the azacitidine crystalline form D according to claim 3, It is characterized in that The mass-to-volume ratio of azacitidine to the mixed solvent is 0.6-0.7:1, wherein the mass is measured in mg and the volume is measured in ml.

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