阅读说明：本技术 一种合成地西他滨的方法 (Method for synthesizing decitabine ) 是由 杨西宁 李涛 卫涛 邢善涛 刘亚利 马冠军 张志强 于 2019-10-27 设计创作，主要内容包括：本发明公开了一种合成地西他滨的方法,属于有机化学中核苷合成领域。其反应步骤如下：以5-氮杂胞嘧啶和2’-脱氧鸟苷为原料,在N-脱氧核糖转移酶(NTD)的作用下发生碱基交换得到地西他滨粗品,采用羧酸乙烯酯作为酰化试剂能够直接在水中实现粗品地西他滨的酰化再经过碱解得到高纯地西他滨成品。整个过程仅需三步,解决了易水解粗品地西他滨采用生物发酵法水溶液中提取问题,避免使用昂贵的1-氯-2-脱氧核糖等试剂,降低了原料成本。(The invention discloses a method for synthesizing decitabine, belonging to the field of nucleoside synthesis in organic chemistry. The reaction steps are as follows: 5-azacytosine and 2' -deoxyguanosine are used as raw materials, base exchange is carried out under the action of N-deoxyribotransferase (NTD) to obtain a decitabine crude product, carboxylic acid vinyl ester is used as an acylation reagent, and the acylation of the decitabine crude product can be directly realized in water, and then the decitabine crude product is subjected to alkaline hydrolysis to obtain a high-purity decitabine finished product. The whole process only needs three steps, the problem of extracting the crude decitabine easy to hydrolyze from the aqueous solution by adopting a biological fermentation method is solved, expensive reagents such as 1-chloro-2-deoxyribose are avoided, and the raw material cost is reduced.)

1. A method for synthesizing decitabine, which is characterized by comprising the following steps expressed by a reaction equation:

2. the method for synthesizing decitabine as claimed in claim 1, specifically comprising the steps of:

mixing 5-azacytosine, 2' -deoxyguanosine and water, adding a catalytic amount of N-deoxyribotransferase for constant-temperature reaction until the content of a product reaches more than or equal to 40%, cooling and filtering, wherein the filtrate is a decitabine aqueous solution;

secondly, adding the decitabine aqueous solution into vinyl carboxylate and inorganic base for reaction, extracting and separating, concentrating and crystallizing to obtain diacyl decitabine;

and thirdly, mixing diacyl decitabine with an alcohol solvent, adding alkali for deacetylation, and refining the alcohol solvent to obtain the decitabine.

3. The method of synthesizing decitabine as defined in claim 2, wherein: the vinyl carboxylate is selected from vinyl formate, vinyl acetate or vinyl propionate.

4. The method of synthesizing decitabine as defined in claim 2, wherein: the inorganic base is selected from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, cesium carbonate or lithium carbonate.

5. The method of synthesizing decitabine as defined in claim 2, wherein: the alcohol solvent used in the third step is selected from methanol, ethanol or isopropanol.

6. The method of synthesizing decitabine as defined in claim 2, wherein: in the third step, the alkali is selected from ammonia methanol, ammonia ethanol, sodium hydroxide or potassium hydroxide.

7. The method of synthesizing decitabine as defined in claim 2, wherein: in the first step, the reaction is carried out at a constant temperature of 30-32 ℃, and the temperature is reduced and the reaction is filtered to remove guanine and unreacted 5-azacytosine.

8. The method of synthesizing decitabine as claimed in claim 2 or 5, wherein: and refining by adopting an absolute ethyl alcohol solvent in the third step.

Technical Field

The invention belongs to the field of organic chemistry, relates to the synthesis of pyrimidine nucleoside, and particularly relates to a method for synthesizing decitabine.

Background

Decitabine, chemical name: 1- (β -D-2-deoxyribose) -4-amino-1, 3, 5-triazin-2 (1H) -one, CAS No.: 2353-33-5, molecular formula C₈H₁₂N₄O₄The compound is an important antiviral drug, and the methods reported in the literature at present comprise the following steps:

pliml et al in 1964 first reported a synthesis method of decitabine, comprising the steps of using silyl ether to protect 5-azacytosine and chlorodeoxyribose, condensing under the catalysis of stannic chloride, and carrying out ammonolysis to obtain the product decitabine.

A. pai ski la et al reported that 1-chloro-3, 5-diacetoxy-2-D-ribose was used as the raw material, condensed with silyl ether protected 5-azacytosine, and then alkaline hydrolyzed to obtain decitabine of a single configuration.

Be aza Hattar et al reported that 1-methoxy-2-deoxy-D ribose as raw material was condensed with 5-azacytosine protected by Fmoc and silyl ether under catalysis of stannic chloride, and hydrolyzed to obtain decitabine.

In the above methods, all the processes are carried out by a condensation method, and a metal catalyst such as tin tetrachloride is required, so that certain pollution is inevitably brought about in the post-treatment. With the improvement of environmental requirements and the development of green chemistry, the synthesis of decitabine by using biological enzymes is a promising industrial method.

Disclosure of Invention

In order to overcome the defects, the invention discloses a process route for synthesizing decitabine by adopting a biological enzyme method. 5-azacytosine and 2' -deoxyguanosine are used as raw materials, base exchange is carried out under the action of alkali transferase to obtain a decitabine crude product, carboxylic acid vinyl ester is used as an acylation reagent, and the acylation of the decitabine crude product can be directly realized in water, and then a high-purity decitabine finished product is obtained through alkaline hydrolysis. The whole process only needs three steps, the problem of extracting the crude decitabine easy to hydrolyze from the aqueous solution by adopting a biological fermentation method is solved, expensive reagents such as 1-chloro-2-deoxyribose are avoided, and the raw material cost is reduced.

The method for synthesizing decitabine adopted by the invention is completed by three steps, and the reaction equation is as follows:

the method for synthesizing decitabine by adopting a biological enzyme method comprises the following steps:

mixing 5-azacytosine, 2' -deoxyguanosine and water, adding a catalytic amount of bio-enzyme for constant-temperature reaction until the content of a product reaches more than or equal to 40%, cooling and filtering, wherein the filtrate is a decitabine aqueous solution; wherein the biological enzyme is N-deoxyribose transferase, NTD for short, specifically Klebsiella melcescens, and the number of the enzyme-producing strain is as follows: strain accession number of DUR 201505001: CGMCC 16111. (refer to the applicant's published patent, application No. 201811103551.5)

Secondly, adding the aqueous solution of decitabine into vinyl carboxylate and inorganic base, reacting to obtain acetylated decitabine, extracting, separating, concentrating and crystallizing to obtain diacetyl decitabine;

and thirdly, mixing diacetyl decitabine with an alcohol solvent, adding alkali for deacetylation, cooling, performing suction filtration, and refining the alcohol solvent to obtain the decitabine.

Further, in the above technical solution, the vinyl carboxylate is selected from vinyl formate, vinyl acetate or vinyl propionate.

Further, in the above technical solution, the inorganic base is selected from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, cesium carbonate or lithium carbonate.

Further, in the above technical scheme, the molar ratio of 5-N cytosine to deoxyguanosine in the first reaction step is 1: 1.04.

Further, in the above technical scheme, the alcohol solvent used in the third step is selected from methanol, ethanol or isopropanol.

Further, in the above technical solution, the alkali in the third step is selected from the group consisting of ammonia methanol, ammonia ethanol, sodium hydroxide and potassium hydroxide.

Further, in the technical scheme, in the first step, the constant temperature reaction is 30 ℃, and the temperature is reduced and the filtration is carried out to filter off guanine and unreacted 5-azacytosine.

Further, in the above technical scheme, anhydrous ethanol is adopted for refining in the third step.

The core of the invention lies in how to effectively extract and separate the easily hydrolyzed crude decitabine from the aqueous solution after the biological fermentation. The technical problem is effectively solved by adopting a method of acetylization and derivatization and then hydrolysis.

The invention has the beneficial effects that:

1. the reaction route is novel, the condensation of 5-azacytosine and 1-chloro-2-deoxy-diacetyl ribose under the catalysis of stannic chloride is avoided, and the steps are reduced.

2. The use of organic solvent is reduced, the generation of solid waste and liquid waste is reduced, and the environmental protection cost is reduced.

3. Compared with the traditional process, the process is simple to operate, stable in yield in kilogram-level verification, good in reproducibility and capable of providing reliable data support for large-scale production.

The specific embodiment is as follows: