CN110225918B

CN110225918B - Preparation method of azacitidine crystal form

Info

Publication number: CN110225918B
Application number: CN201880008521.8A
Authority: CN
Inventors: 林青; 何雷; 余俊
Original assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd
Current assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd
Priority date: 2017-12-29
Filing date: 2018-12-29
Publication date: 2022-10-11
Anticipated expiration: 2038-12-29
Also published as: CN110225918A; WO2019129260A1; CN109988207B; CN109988207A

Abstract

The invention relates to a preparation method of a azacitidine crystal form. The invention relates to a preparation method of a azacitidine crystal form I, which uses DMSO as a solvent and uses a mixed alcohol solvent as an anti-solvent, and the prepared crystal form is single, has good purity, no mixed crystal phenomenon and low solvent residue, and is suitable for industrial application and production.

Description

Preparation method of azacitidine crystal form

Technical Field

The invention relates to the field of medicine preparation, and in particular relates to a method for preparing a azacitidine crystal form I.

Background

Azacitidine is also known as 5-azacytidine, chemical name: 1- (. Beta. -D-ribofuranosyl) -4-amino-1, 3, 5-triazin-2 (1H) -one. Azacitidine was approved by FDA in us for the first drug to be marketed in 2004, and approved by eu in 2008 for the first drug to be marketed in 12 months, and became the first therapeutic drug in europe that could significantly prolong the overall survival of intermediate-2 and high-risk MDS and AML patients, and this drug was marketed in 2011 in 3 months in japan. Azacitidine has a rare disease medication status in Europe, america and Japan.

Azacitidine is a 5-aza analogue of cytidine and belongs to a class of epigenetic antineoplastics known as hypomethylated drugs. Aberrant DNA methylation inactivates key genes that regulate normal cell growth, differentiation, and apoptosis, and is involved in the development and progression of tumors. Azacitidine is effective in the treatment of MDS mainly due to its DNA hypomethylation activity and direct cytotoxic effects on abnormal hematopoietic cells in the bone marrow. Azacitidine is a DNA methyltransferase inhibitor that does not significantly inhibit DNA synthesis at concentrations that maximally inhibit DNA methylation.

The structural formula of azacitidine is shown as a formula I:

patent WO2004082619 discloses 8 crystal forms of azacitidine, namely crystal forms I to VIII, wherein the crystal form most suitable as a raw material drug is the crystal form I, the characteristic peaks of X-ray diffraction of the crystal form I are 12.1, 13.0, 14.4, 16.5, 18.6, 19.0, 20.2, 21.3, 23.0, 23.9, 26.9, 27.1, 29.3, 29.6, 30.4 and 32.1, X-ray diffraction patterns are shown in figure 1 in WO2004082619, and patent WO2004082822 discloses a preparation method of the azacitidine crystal form I, namely dipolar aprotic solvents DMSO, DMF, NMP and the like are used as good solvents to dissolve the azacitidine, and an anti-solvent is added to change the polarity of the solution so as to precipitate azacitidine crystals. The anti-solvent used comprises 7 kinds of chemical reagents such as alcohols (particularly C2-C5 alcohols), esters, ethers, alkanes, substituted alkanes, ketones, nitriles and the like, and the total number of the chemical reagents is 43. The patent specifically states that the use of isopropanol and acetonitrile leads to the stable formation of form I. This patent covers almost all conventional chemical reagents and common experimental methods. The inventor of the invention proves through experiments that the product obtained by adopting the method provided by the patent WO2004082822 has the following defects: ester solvents, ether solvents, alkane solvents, substituted alkane solvents and ketone solvents are used as anti-solvents, and the crystal form of the obtained product is mixed crystal, which brings difficulty to the quality control of the medicine; secondly, the method comprises the following steps: acetonitrile is used as an anti-solvent to obtain a crystal form I product, but the problem of solvent residue is serious, one is more than 1000ppm, and the limit of acetonitrile specified by pharmacopoeia is 410ppm; thirdly, the method comprises the following steps: the adoption of alcohols as anti-solvents has the double problems of crystal form purity and solvent residue. In conclusion, the patent WO2004082822 only provides the preparation method for preparing the azacitidine crystal form I, but the method can not produce qualified medicines in the actual medicine production process.

Disclosure of Invention

Aiming at the defects, the invention carries out systematic research and provides a process for preparing the azacitidine crystal form I with low solvent residue and high crystal purity.

The process adopts DMSO as a solvent, and adopts a mixed solvent containing at least two solvents as an anti-solvent. The mixed solvent consists of a first solvent and a second solvent, wherein the first solvent is an aliphatic alcohol with the carbon atom number more than 4, preferably an aliphatic alcohol with the carbon atom number more than 6, and particularly preferably an aliphatic alcohol with the carbon atom number of 6-8; the second solvent is methanol, ethanol and the solvent containing methanol and ethanol.

The invention provides an operation method for preparing azacitidine crystal form I with low solvent residue and high crystal purity, which comprises the following steps:

(1) dissolving a azacitidine sample by DMSO under the heating condition,

(2) slowly dripping the anti-solvent into the mixture,

(3) cooling and crystallizing to obtain crystal form I product, wherein XRD spectrogram is shown in figure 1.

According to the preparation process of the invention, the heating temperature is selected to be more than 40-190 ℃, preferably more than 60-120 ℃, and most preferably 80-90 ℃.

According to the preparation process of the invention, the azacitidine sample is dissolved by using a proper amount of DMSO, and the volume of the added anti-solvent is more than 50% of the volume of the DMSO. Preferably, the volume ratio of DMSO to the anti-solvent is 1:1-20, more preferably 1:5-20.

According to the preparation process of the invention, the anti-solvent consists of a first solvent and a second solvent, wherein the first solvent is an alcohol with the carbon number more than 4, preferably an alcohol with the carbon number of 5-8, most preferably a straight-chain alcohol with the carbon number of 6-7, including n-hexanol, n-heptanol and n-octanol.

The second solvent is methanol, ethanol, or organic solvent containing methanol and ethanol, preferably methanol and ethanol, and most preferably methanol; the volume proportion of the second solvent in the mixed solvent is 30-80%, preferably 40-70%, and most preferably 50-60%.

When the second solvent is methanol, the volume ratio of the second solvent to the mixed solvent is 20 to 60%, preferably 40 to 60%, and more preferably 50 to 60%.

When the second solvent is ethanol, the volume ratio of the second solvent in the mixed solvent is 60-80%.

The mixed solvent refers to the total volume of the anti-solvent, i.e., the sum of the volumes of the first type of solvent and the second type of solvent.

According to the preparation process of the invention, the anti-solvent is added and the temperature is reduced to below 40 ℃, preferably 0-40 ℃, and more preferably room temperature.

According to the preparation process of the invention, the sample is dried in vacuum, the drying temperature is generally 60-120 ℃, preferably 70-100 ℃, and most preferably 80-90 ℃.

Azacitidine crystal form I prepared according to the preparation method can be used for treating myelodysplastic syndrome.

The invention researches the solubility of azacitidine and tests the solubility of azacitidine in the following solvents: alcohol solvents including C1-C8 aliphatic alcohols such as methanol, ethanol, ethylene glycol, isopropanol, n-propanol, 1, 3-propanediol, n-butanol, t-butanol, n-pentanol, 2-pentanol, cyclopentanol, n-hexanol, cyclohexanol, n-heptanol, n-octanol, and the like, and aromatic alcohol solvents such as benzyl alcohol; the ester solvent comprises ethyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, tert-butyl acetate, n-pentyl acetate, isoamyl acetate, methyl propionate, ethyl propionate and the like; the alkane solvent comprises n-pentane, n-hexane, cyclohexane, n-heptane, n-octane, etc.; the substituted alkane solvent comprises dichloromethane, chloroform, chlorobenzene, nitromethane and the like; the ketone solvent comprises acetone, butanone, 2-pentanone, cyclopentanone, etc.; the ether solvent comprises diethyl ether, isopropyl ether, n-butyl ether, methyl tert-butyl ether, anisole, ethylene glycol methyl ether, ethylene glycol dimethyl ether, etc.; cyclic ether type solvents such as tetrahydrofuran, 1, 4-dioxane, 2-methyltetrahydrofuran, etc.; cyano-containing solvents such as acetonitrile, propionitrile, butyronitrile, and the like. The results show that azacitidine is hardly soluble in these solvents, and these solvents are not good solvents for crystallization and are only suitable as poor solvents. This study also tested the solubility of azacitidine in dipolar aprotic solvents including dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), N-Dimethylacetamide (DMA) and N-methylpyrrolidone (NMP) at 20 ℃, and the results are shown below:

solvent(s)	Solubility in water	Pharmacopoeia solvent classification
			DMF	0.04g/ml	II
DMA	0.05g/ml	II
			NMP	0.09g/ml	II
DMSO	0.43g/ml	III

In terms of solubility, the solubility in DMSO is 4 to 10 times that of the other three solvents, and in terms of solvent classification, DMF is a group II solvent, the solvent residue limit is required to be 880ppm, DMA is a group II solvent, the solvent residue limit is required to be 1090ppm, NMP is a group II solvent, the solvent residue limit is required to be 530ppm, DMSO is a group III solvent, and the solvent residue limit is required to be 5000ppm. The results show that DMSO is an ideal solvent for azacitidine crystallization.

The invention takes DMSO as a solvent to screen a poor solvent, research results show that ethers, cyclic ethers, esters, alkanes, substituted alkanes and ketones are difficult to control as anti-solvent crystallization processes, and the obtained crystal form of a sample is generally a mixture of various crystal forms, so that the solvents cannot be adopted in the industrial production of azacitidine bulk drugs.

According to the invention, solvents containing cyano groups such as acetonitrile, propionitrile and butyronitrile are researched, and the three solvents can be used as anti-solvents to stably obtain a crystal form I sample, but the propionitrile and the butyronitrile are not used as organic solvents industrially, and the solvent residue is seriously exceeded as described above in the production of acetonitrile.

The invention researches a crystallization method by using an alcohol solvent as an anti-solvent. Research contents comprise the crystal form purity of a product and solvent residues of the product, and research results show that the crystal form purity of the product has a great relationship with the number of carbon atoms contained in adopted alcohol, higher alcohol is adopted as an anti-solvent to obtain a pure crystal form I of azacitidine, lower alcohol is adopted as an anti-solvent to obtain azacitidine which is a mixture of the crystal form I and other crystal forms, specifically, when the number of carbon atoms in an alcohol molecule is more than 2, the crystal form of the azacitidine obtained by crystallization is the crystal form I, such as propanol and alcohol with more carbon atoms; when the carbon atoms of the alcohol molecules are 1 and 2, such as methanol and ethanol, the azacitidine obtained by crystallization is a mixture of the crystal form I and other crystal forms; on the other hand, the pure crystal form I can be obtained by crystallization by using a higher alcohol containing more than 2 carbon atoms as an anti-solvent, but the defect that the residual amount of DMSO in the product is very high exists, and the research focuses on the result of crystallization by using alcohol containing 1-8 carbon atoms as the anti-solvent, wherein the residual DMSO ratio of a sample is lower and is about 1000ppm when methanol is used as the anti-solvent for crystallization, and the residual DMSO solvent ratio is as high as 18000ppm when n-octanol is used as the anti-solvent, as shown in figure 7.

Aiming at the condition that the crystal form purity and the solvent residue cannot be considered, two or more poor solvents are creatively adopted as the anti-solvent in the research, the first poor solvent is selected from C4-C8 fatty alcohol and has the function of promoting the generation of a sample of the crystal form I, and the second poor solvent has the function of reducing the solvent residue of DMSO in the sample. Through a large number of experimental screens, the confirmed solvents comprise ethyl formate, methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, tert-butyl acetate, n-pentyl acetate, isoamyl acetate, methyl propionate, ethyl propionate and the like; the alkane solvent comprises n-pentane, n-hexane, cyclohexane, n-heptane, n-octane, etc.; the substituted alkane solvent comprises dichloromethane, chloroform, chlorobenzene, nitromethane and the like; the ketone solvent comprises acetone, butanone, 2-pentanone, cyclopentanone, etc.; the ether solvent comprises diethyl ether, isopropyl ether, n-butyl ether, methyl tert-butyl ether, anisole, ethylene glycol methyl ether, ethylene glycol dimethyl ether, etc.; cyclic ether solvents such as tetrahydrofuran, 1, 4-dioxane, 2-methyltetrahydrofuran, etc. do not effectively remove the DMSO solvent residue in the product; the effect of removing DMSO solvent residue in the product by using lower aliphatic alcohol, such as methanol, ethanol and the like, is obvious, but mixed crystals can be generated due to excessive addition, the DMSO solvent residue of the product can be removed to a certain extent by using isopropanol as a second anti-solvent, but the DMSO content in the product is still very high, and the requirement of pharmacopoeia cannot be met.

Table one: product crystal form condition of methanol as second solvent

Table two: product crystal form condition of ethanol as second solvent

Table three: product DMSO residue with methanol as a second type of solvent

Table four: product DMSO residue with ethanol as a second type of solvent

Drawings

FIG. 1: an XRD pattern of azacitidine crystalline form I.

FIG. 2: an XRD pattern of azacitidine form III.

FIG. 3: XRD pattern of azacitidine crystal form IV.

FIG. 4: an XRD pattern of a mixture of azacitidine form I and form III.

FIG. 5: XRD patterns of mixtures of azacitidine crystal forms I, IV, V and VII.

FIG. 6: XRD patterns of mixtures of azacitidine crystal forms I, III, IV, V and VII.

FIG. 7: different alcohol solvents are used as the solvent residue of azacitidine as an anti-solvent.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1:

mixing 1g of azacitidine with 5ml of DMSO, heating to 75 ℃, dissolving the sample completely, slowly adding 30ml of a mixed solvent consisting of methanol/n-hexanol = 1:1, cooling to room temperature, stirring for 1h, filtering, and vacuum-drying the sample at 80 ℃ for 24h to obtain 0.8g of a crystal form I sample, wherein the solvent residue of the DMSO is 830ppm, the purity is more than 99.9%, and an XRD (X-ray diffraction) pattern is shown in figure 1.

Example 2:

10g azacitidine and 40mL DMSO are mixed, heated to 90 ℃ to dissolve a sample, 200mL of mixed solvent consisting of methanol/n-heptanol = 1:1 is slowly added, the temperature is reduced to room temperature, stirring is continued for 1h, filtering is carried out, the sample is dried for 24h at 70 ℃, 9.1g of crystal form I sample is obtained, 1100ppm of DMSO solvent residue is obtained, the purity is more than 99.9%, and the XRD pattern is basically consistent with that of figure 1.

Example 3:

1g of azacitidine is mixed with 8ml of DMSO, the mixture is heated to 65 ℃ to completely dissolve the sample, 80ml of mixed solution consisting of ethanol/n-octanol = 2: 8 is slowly added, the temperature is reduced to room temperature, the stirring is continued for 1h, the filtration is carried out, the vacuum drying is carried out for 20h at 75 ℃ to obtain 088g of a crystal I sample, the DMSO solvent residue is 5722ppm, and the XRD pattern is basically consistent with that of the attached figure 1.

Example 4:

mixing 1g of azacitidine and 6ml of DMSO, heating to 70 ℃ to dissolve a sample, slowly adding 100ml of a mixed solvent consisting of methanol/n-octanol = 3: 2, cooling to room temperature, continuously stirring for 1h, filtering, and vacuum drying for 26h at 70 ℃ to obtain 084g of a crystal I sample, wherein the DMSO solvent residue is 1208ppm, the purity is greater than 99.9%, and an XRD pattern is basically consistent with that of figure 1.

Example 5:

mixing 1g of azacitidine with 7ml of DMSO, heating to 70 ℃ to completely dissolve solids, slowly adding 40ml of a mixed solvent with ethanol/n-heptanol = 2: 8, cooling to room temperature, continuing stirring for 1h, filtering, drying the sample in vacuum at 70 ℃ for 22h to obtain 0.9g of a crystal form I sample, and measuring the DMSO solvent residue to be 6423ppm, wherein the XRD pattern is basically consistent with that of figure 1.

Example 6:

mixing 1g of azacitidine with 7ml of DMSO, heating to 75 ℃ to dissolve the solid, slowly adding 40ml of methanol/n-hexanol = 4: 1 mixed solution, cooling to room temperature, stirring for 8h, and filtering to obtain the solid which is the mixed crystal of the crystal form I and the crystal form III. The XRD pattern is shown in figure 4.

Example 7:

mixing 1g of azacitidine with 7ml of DMSO, heating to 75 ℃ to dissolve the solid, slowly adding 40ml of methanol, cooling to room temperature, stirring for 8h, and filtering to obtain the solid which is the mixed crystal of crystal forms I, IV, V and VII. The XRD pattern is shown in FIG. 5.

Example 8:

mixing 1g azacitidine with 7ml DMSO, heating to 75 ℃ to dissolve the solid, slowly adding 40ml of ethanol/n-butanol = 9: 1 mixed solution, cooling to room temperature, stirring for 24h, and filtering to obtain the solid which is the mixed crystal of crystal forms I, III, IV, V and VII. The XRD pattern is shown in FIG. 6.

Example 9:

mixing 1g azacitidine with 7ml DMSO, heating to 75 ℃ to dissolve the solid, slowly adding 45ml isopropanol, cooling to room temperature, stirring for 3h, filtering, vacuum drying at 75 ℃ for 24h to obtain 0.85g crystal form I sample, measuring the solvent residue 6549ppm, and the XRD pattern is basically consistent with that of figure 1.

Example 10:

mixing 1g of azacitidine with 7ml of DMSO, heating to 75 ℃ to dissolve the solid, slowly adding 45ml of n-hexanol, cooling to room temperature, continuously stirring for 3h, filtering, and vacuum drying at 75 ℃ for 24h to obtain 0.87g of a crystal form I sample, wherein the DMSO solvent residue is 16153ppm, and the XRD pattern is basically consistent with that of the attached figure 1.

Example 11:

mixing 1g of azacitidine with 7ml of DMSO, heating to 75 ℃ to dissolve the solid, slowly adding 45ml of n-octanol, cooling to room temperature, continuously stirring for 3h, filtering, and vacuum drying at 75 ℃ for 24h to obtain 0.87g of a crystal form I sample, wherein the DMSO solvent residue is 18376ppm, and the XRD pattern is basically consistent with that of the attached figure 1.

Claims

1. A method for preparing azacitidine crystal form I, comprising the steps of:

1) Dissolving a azacitidine sample with DMSO under a heating condition;

2) Slowly dropwise adding an anti-solvent, wherein the anti-solvent consists of a first solvent and a second solvent, the first solvent is selected from C4-C8 alcohol, the second solvent is methanol, and the volume proportion of the second solvent in the mixed solvent is 40-60%;

3) Cooling and crystallizing;

4) Filtering and drying to obtain the crystal form I.

2. The method for preparing azacitidine crystal form I according to claim 1, wherein the second solvent accounts for 50-60% of the mixed solvent by volume.

3. The process of claim 1, wherein the heating temperature is selected to be greater than 40 ℃.

4. The process according to claim 1, wherein the heating temperature is selected to be greater than 60 ℃.

5. The preparation method of azacitidine crystal form I according to claim 1, wherein the heating temperature is selected from 80 to 90 ℃.

6. The process of claim 1, wherein the volume of anti-solvent added is greater than 50% of the volume of DMSO.

7. The method of claim 6, wherein the volume ratio of DMSO to the anti-solvent is 1:1-20.

8. The method of claim 6, wherein the volume ratio of DMSO to the anti-solvent is 1:5-20.

9. The process of claim 1, wherein the first solvent is a C6 to C8 alcohol.

10. The method of claim 1, wherein the first solvent is a C6-C7 alcohol.

11. The preparation method of azacitidine crystal form I according to claim 1, wherein the volume ratio of the second solvent to the mixed solvent is 50-60%.

12. The process of claim 1, wherein the first solvent is n-hexanol or n-heptanol.

13. The process for preparing azacitidine crystal form I as claimed in claim 1, wherein an anti-solvent is added to reduce the temperature to below 40 ℃.

14. The process for preparing azacitidine crystal form I as claimed in claim 1, wherein the temperature of the mixture is lowered to 0-40 ℃ by adding an anti-solvent.

15. The process for preparing azacitidine crystal form I according to claim 1, further comprising adding an anti-solvent to cool to room temperature.

16. The preparation method of azacitidine crystal form I as claimed in claim 1, wherein the sample is vacuum dried at 60-120 deg.C.

17. The preparation method of azacitidine crystal form I as claimed in claim 1, wherein the sample is vacuum dried at 70-100 ℃.

18. The method for preparing azacitidine crystal form I according to claim 1, wherein the sample is dried in vacuum at 80-90 ℃.