A method for purifying azacitidine

Abstract

The application relates to the field of medicine synthesis, in particular to a refining method of azacitidine. Crude azacitidine is added into a refining solvent to be stirred and dissolved, a crystallization solvent is added dropwise to be stirred, after crystals are separated out, temperature is reduced to be stirred to separate out crystals, and then filtration is carried out; the filter cake is washed with the crystallization solvent, and high-purity single crystal azacitidine is obtained; the refining solvents used in the application are low-toxicity solvents, the operation is simple and safe, and the problem of excessive residue is avoided; the crystallization solvents are low-boiling-point solvents, which are beneficial to recycling and utilization of the crystallization solvents and cost saving. Finally, single crystal products are obtained, the problem caused by different crystal types is avoided, the yield is improved, the operation is simplified, and the method is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for purifying azacitidine. Background Technology

[0002] Azacitidine, chemically named 1-(β-D-ribofuranosyl)-4-amino-1,3,5-triazine-2(1H)-one, is a DNA methyltransferase inhibitor developed by Pharmion Laboratories. It was first marketed in the United States in July 2004 under the brand name Vidaza. Its mechanism of action involves phosphorylation of azacitidine, which binds to the DNA molecule. DNA methyltransferases then undergo methylation with azacitidine, forming a covalently bound product. This inhibits and degrades the activity of DNA methyltransferases, leading to a decrease in DNA methylation levels in tumor tissue. Hypermethylated tumor suppressor genes are demethylated, allowing gene expression to resume and thus inhibiting tumor cells. Clinically, it is primarily used to treat acute non-lymphocytic leukemia associated with myelodysplastic syndromes (MDS), and can also be used to treat breast cancer, melanoma, and colorectal cancer.

[0003] Numerous reports have been published on the synthesis of azacitidine. Recent improvements to the synthesis methods mainly involve replacing hexamethyldisilazane with N,O-bis-trimethylsilyltrifluoroacetamide in the synthesis of intermediate I, or replacing ammonium sulfate with a quaternary ammonium salt phase transfer catalyst. In the synthesis of intermediate II, trifluoromethanesulfonic acid or trimethylsilyltrifluoromethanesulfonate is used instead of the Lewis acid tin tetrachloride as the catalyst. Purification of azacitidine uses N-methylpyrrolidone and cyclohexane. Examples of such methods include those described in patents US9266863B2, CN201010504343.3, US8212021B2, US7038038, and CN201210231079.

[0004] Previous purification methods for azacitidine have not only resulted in low yields, high levels of single impurities, and long processing times, but also often used highly toxic solvents such as N-methylpyrrolidone or dimethyl sulfoxide. These solvents pose significant health risks to operators and can easily lead to excessive solvent residues in the final azacitidine product. Therefore, finding a simple, safe, and effective purification method for azacitidine is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a new purification method for azacitidine, addressing the aforementioned deficiencies in existing processes.

[0006] Specifically, the present invention is achieved through the following technical solution:

[0007] A method for purifying azacitidine involves dissolving crude azacitidine in a refining solvent at a temperature of 5–30°C, preferably 20°C; adding a crystallization solvent dropwise while stirring at a temperature of -5–30°C, preferably 0°C, for a crystallization time of 0.5–5 h, preferably 3 h; maintaining the crystallization temperature while stirring to continue crystallization; filtering; and washing the filter cake with the crystallization solvent to obtain high-purity, single-crystal azacitidine; the mass-to-volume ratio of crude azacitidine to the refining solvent is 1:1–10 g / ml, preferably 1:5 g / ml.

[0008] The mass-to-volume ratio of crude azacitidine to the crystallization solvent is 1:5-20 g / ml, preferably 1:10 g / ml.

[0009] Preferably, in the purification method of azacitidine, the purification solvent is N,N-dimethylformamide.

[0010] In a preferred method for purifying azacitidine, the crystallization solvent is selected from one or more of methanol, ethanol, acetone, and isopropanol, with methanol being preferred.

[0011] Compared with the prior art, the present invention achieves the following technical effects:

[0012] (1) After dissolving in a refining solvent, a crystallization solvent is added for recrystallization to obtain azacitidine refined product. The refining yield is above 98.9%. This method can increase the purity of azacitidine from 90% to above 99.8%, and ultimately stabilize the content of single impurities below 0.1%.

[0013] (2) All refined solvents used are low-toxicity solvents, which are not only simple and safe to operate, but also avoid the problem of excessive solvent residue.

[0014] (3) The methanol and ethanol crystallization solvents used are both low-boiling-point solvents, which not only avoids the problem of excessive residual solvents such as DMSO, but also facilitates the recycling of crystallization solvents and saves costs.

[0015] (4) The present invention ultimately obtains a single crystal form product, avoiding the troubles that may be caused by different crystal forms, and improves the yield, simplifies the entire process operation, and the operation method is simple and suitable for industrial production. Detailed Implementation

[0016] The beneficial effects of the present invention will be further described through the following embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the present invention. Obvious improvements and modifications made to the present invention by those skilled in the art are also within the scope of protection of the present invention.

[0017] Example 1

[0018] 10 g of crude azacitidine and 10 mL of N,N-dimethylformamide were added to a three-necked flask and stirred at 5 °C until the solid dissolved. 25 mL of methanol was slowly added dropwise, causing the liquid to become turbid. Crystallization continued for 1 hour. After crystallization was complete, another 25 mL of methanol was added dropwise. The reaction mixture was then placed in an ice-water bath at -5 °C and stirred to crystallize for 0.5 hours. The mixture was filtered, the filter cake was washed with methanol, and dried to obtain pure azacitidine with a purity of 99.96% and a yield of 99.3%. DSC analysis confirmed the presence of a single-crystal product, and the residual solvent test met the quality standards.

[0019] Example 2

[0020] 10 g of crude azacitidine and 100 mL of N,N-dimethylformamide were added to a three-necked flask and stirred at 30 °C until the solid dissolved. 100 mL of ethanol was slowly added dropwise, causing the liquid to become cloudy. Crystallization continued for 1 hour. After crystallization was complete, another 100 mL of ethanol was added dropwise. The reaction mixture was then placed in a 30 °C water bath and stirred to crystallize for 5 hours. The crystals were filtered, the filter cake was washed with methanol, and dried to obtain pure azacitidine with a purity of 99.87% and a yield of 98.9%. DSC analysis confirmed the presence of single-crystal products, and the residual solvent test met the quality standards.

[0021] Example 3

[0022] 10 g of crude azacitidine and 50 mL of N,N-dimethylformamide were added to a three-necked flask and stirred at 20 °C until the solid dissolved. 50 mL of methanol was slowly added dropwise, causing the liquid to become cloudy. Crystallization continued for 1 hour. After crystallization was complete, another 50 mL of methanol was added dropwise. The reaction mixture was then placed in an ice-water bath at 0 °C and stirred to precipitate crystals for 3 hours. The crystals were filtered, the filter cake was washed with methanol, and dried to obtain pure azacitidine with a purity of 99.99% and a yield of 99.8%. DSC analysis confirmed the presence of single-crystal products, and the residual solvent test met the quality standards.

[0023] Example 4

[0024] 10 g of crude azacitidine and 50 mL of N,N-dimethylformamide were added to a three-necked flask and stirred at 20 °C until the solid dissolved. 50 mL of methanol was slowly added dropwise, causing the liquid to become cloudy. Crystallization continued for 1 hour. After crystallization was complete, another 50 mL of methanol was added dropwise. The reaction mixture was then placed in a 40 °C water bath and stirred to precipitate crystals for 3 hours. The crystals were filtered, the filter cake was washed with methanol, and dried to obtain pure azacitidine with a purity of 99.82% and a yield of 94.6%. DSC analysis confirmed the presence of single-crystal products, and the residual solvent test met the quality standards.

[0025] Example 5

[0026] 10 g of crude azacitidine and 50 mL of N,N-dimethylformamide were added to a three-necked flask and stirred at 20 °C until the solid dissolved. 50 mL of methanol was slowly added dropwise, causing the liquid to become cloudy. Crystallization continued for 1 hour. After crystallization was complete, another 50 mL of methanol was added dropwise. The reaction solution was then placed in a -10 °C water bath and stirred to precipitate crystals for 3 hours. The crystals were filtered, the filter cake was washed with methanol, and dried to obtain pure azacitidine with a purity of 99.78% and a yield of 95.8%. DSC analysis confirmed the presence of single-crystal products, and the residual solvent content met the quality standards.

[0027] Example 6

[0028] 10 g of crude azacitidine and 100 mL of N-methylpyrrolidone were added to a three-necked flask and stirred until the solid dissolved. Since complete dissolution was not achieved, the temperature was raised to 80 °C and stirred until dissolved. 200 mL of methanol was slowly added dropwise. After the addition was complete, the reaction mixture was placed in an ice-water bath at -5 to 0 °C and stirred to allow crystallization for 2–3 hours. The mixture was then filtered, the filter cake was washed with methanol, and dried to obtain purified azacitidine. The purity was 99.69%, and the yield was 93.8%. DSC analysis showed that the product was amorphous, and the residual solvent test did not meet the quality standards.

[0029] Example 7

[0030] 10 g of crude azacitidine and 100 mL of dimethyl sulfoxide were added to a three-necked flask and stirred until the solid dissolved. 100 mL of methanol was slowly added dropwise. After the addition was complete, the reaction mixture was placed in an ice-water bath at -5 to 0 °C and stirred to induce crystallization for 2–3 hours. The mixture was filtered, the filter cake was washed with methanol, and dried to obtain purified azacitidine. The purity was 99.61%, and the yield was 92.6%. DSC analysis showed that the product was amorphous, and the residue test did not meet the quality standards.

Claims

1. A method of purifying azacitidine, characterized in that, The azacitidine crude product is added into N,N-dimethylformamide to be stirred and dissolved, the dissolving temperature is 20℃, methanol is added dropwise to be stirred, the crystallization temperature is 0℃, after the crystals are precipitated, the crystallization temperature is maintained to be stirred, the crystallization time is 3h, and the filter cake is washed with methanol to obtain high-purity single crystal form azacitidine; the mass-volume ratio of the azacitidine crude product and N,N-dimethylformamide is 1:5g / ml; the mass-volume ratio of the azacitidine crude product and methanol is 1:10g / ml.