阅读说明：本技术 一种头孢呋辛钠甲氧亚胺异构体的制备方法 (Preparation method of cefuroxime sodium methoxyimino isomer ) 是由 贾永兵 谭胜连 文青 唐康 张旭途 刘佳 于 2019-09-27 设计创作，主要内容包括：本发明涉及一种头孢呋辛钠甲氧亚胺异构体的制备方法,属于医药企业药物质量研究、质量控制和安全性研究,它公开了步骤(1)：在非质子性、质子性溶剂中,头孢呋辛酯在紫外光催化作用下产生头孢呋辛酯甲氧亚胺异构体；步骤(2)：将步骤(1)得到的头孢呋辛酯甲氧异构体通过反相色谱纯化,然后萃取,干燥,浓缩即得头孢呋辛酯甲氧异构体纯干品；步骤(3)将步骤(2)中得到的头孢呋辛酯甲氧异构体通过酯酶水解得头孢呋辛钠甲氧亚胺异构体。本发明,反应操作简单,条件温和,产物纯度高,所得的头孢呋辛钠甲氧亚胺异构体可作为标准品应用在头孢呋辛酯、头孢呋辛钠原料药和制剂的质量控制。(The invention relates to a preparation method of cefuroxime sodium methoxyimino isomer, belonging to the medicine quality research, quality control and safety research of pharmaceutical enterprises and disclosing the step (1): in an aprotic and protic solvent, cefuroxime axetil generates cefuroxime axetil methoxyimino isomer under the catalysis of ultraviolet light; step (2): purifying the cefuroxime axetil methoxy isomer obtained in the step (1) by reverse phase chromatography, then extracting, drying and concentrating to obtain a pure dry product of the cefuroxime axetil methoxy isomer; and (3) hydrolyzing the cefuroxime axetil methoxyisomer obtained in the step (2) by esterase to obtain a cefuroxime sodium methoxyimine isomer. The invention has simple reaction operation, mild condition and high product purity, and the obtained cefuroxime sodium methoxyimino isomer can be used as a standard substance for the quality control of cefuroxime axetil, cefuroxime sodium raw material medicines and preparations.)

1. A preparation method of cefuroxime sodium methoxyimino isomer comprises the following steps:

step (1): dissolving cefuroxime axetil in one of aprotic and protic solvents to prepare a cefuroxime axetil solution with a concentration of less than or equal to 0.5g/ml, and then placing the solution under an ultraviolet lamp for 2 to 8 hours to obtain a mixture containing 25 to 50 percent of cefuroxime axetil methoxy isomer and cefuroxime axetil;

step (2): purifying the mixture of cefuroxime axetil and the methoxyimino isomer thereof obtained in the step (1) by reversed phase liquid chromatography, wherein the purification chromatographic condition is that water/methanol is 40/60, the flow rate is 40ml/min, the ultraviolet detection wavelength is 230nm, collecting the corresponding fraction, then extracting, drying and rotary evaporating to obtain a pure dry product of cefuroxime axetil methoxyimino isomer;

and (3): dissolving the cefuroxime axetil methoxyimino isomer obtained in the step (2) in a mixed solution of an aprotic solvent, a protic solvent and water, adding 0.5-2 times of immobilized acetylde-esterification enzyme for hydrolysis, reacting for 7-24 hours, and controlling the pH value to be within the range of 6.5-8.5.

2. The method according to claim 1, wherein the aprotic solvent is ethyl acetate, tetrahydrofuran, dioxane, acetonitrile, dichloromethane or acetone, and the protic solvent is methanol, ethanol, isopropanol, t-butanol, ethylene glycol, propylene glycol or glycerol.

3. The method for preparing cefuroxime sodium methoxyimine isomer according to claim 1, wherein the concentration of cefuroxime axetil in step (1) is less than or equal to 0.5 g/ml.

4. The method for preparing cefuroxime sodium methoxyimine isomer according to claim 1, wherein the UV irradiation time in step (1) is 2-8 hours.

5. The method according to claim 1, wherein in step (1), the cefuroxime axetil isomer in the mixture of cefuroxime axetil and cefuroxime axetil-methoxyimine isomer accounts for 25% -50%.

6. The method for preparing cefuroxime sodium methoxyimine isomer according to claim 1, wherein the reverse phase chromatography in step (2) is performed under water/acetonitrile 50/50 and at a detection wavelength of 230 nm.

7. The method according to claim 1, wherein the solvent used in the extraction in step (2) is ethyl acetate, dichloromethane, chloroform or carbon tetrachloride.

8. The method for preparing cefuroxime sodium methoxyimine isomer according to claim 1, wherein in the step (3), the aprotic solvent is acetone or acetonitrile, the protic solvent is methanol or ethanol, and the ratio of the aprotic solvent to the protic solvent to water is 1.2:1-4: 1.

9. The process according to claim 1, wherein in step (3), the amount of immobilized acetyl-deacetylase is 0.5-2 times and the pH is controlled to be 6.5-8.5.

10. The method for preparing cefuroxime sodium methoxyimine isomer according to claim 1, wherein the hydrolysis time in step (3) is 7-24 hours.

Technical Field

The invention belongs to the technical field of medicines, and relates to a preparation method of cefuroxime sodium methoxyimino isomer.

Technical Field

Cefuroxime is a cephalosporin antibiotic developed by Kuran Smith company, is firstly marketed in the United states in 1988 and expires in 1996, and is taken as the second generation oral cephalosporin, so that the medicine has strong lipid solubility and good oral absorption; cefuroxime axetil is an esterified cefuroxime, which is hydrolyzed in vivo to release cefuroxime and exert its antibacterial activity; the latter has wide antibacterial spectrum, strong antibacterial effect and stability to lactamase. Cefuroxime axetil has very low antibacterial activity, and after being absorbed orally for 3-4 min, it is hydrolyzed fast by non-specific esterase in intestinal mucosa and portal circulation to release cefuroxime and exert its antibacterial action, so that the antibacterial spectrum and antibacterial activity of cefuroxime axetil are the same as those of cefuroxime. The traditional Chinese medicine composition is mainly used for treating mild and moderate respiratory tract infection, urogenital system infection, skin and soft tissue infection, gonorrhea and the like caused by sensitive bacteria clinically.

Because the isomerism of the methoxyimine in the side chain at the 7-position is biased towards the furan ring end, the cefuroxime sodium methoxyimine isomer has far-ranging potency compared to cefuroxime sodium. Therefore, the cefuroxime sodium methoxyimino isomer is required to be controlled in the process of producing the bulk drugs and the preparations. In the quality research of cefuroxime sodium and cefuroxime axetil, the methoximine isomer is taken as the medicine impurity for control research.

Disclosure of Invention

The invention aims to provide a method for preparing cefuroxime sodium methoxyimino isomer, which is simple to operate and mild in condition. Firstly, cefuroxime axetil is destroyed by ultraviolet to generate cefuroxime axetil methoxyimino isomer, then pure cefuroxime axetil methoxyimino isomer is obtained by preparation and purification, then immobilized acetyl esterase is used for hydrolysis to obtain pure cefuroxime sodium methoxyimino isomer, and pure dry cefuroxime sodium methoxyimino isomer can be obtained by precipitation, filtration and drying.

For cephalosporin antibiotic preparations with iminooxime structural fragments, trans-isomers with the content of 0.1-2% can be mostly generated under ultraviolet illumination. The 2% trans isomer is a concept of high impurity level in quality control, but if it is used for preparing a dry product with pure trans isomer, the 2% trans isomer is a concept of low content level, and if it is purified and enriched from the 2% level, it takes a lot of manpower and material resources, which is obviously not a good method.

According to the invention, researches show that when cefuroxime sodium and acid are in a solid state, the content of the methoxyimine isomer generated by ultraviolet damage is very low and is below 2%. If cefuroxime sodium is prepared into an aqueous solution, although the amount of the methoxyimine isomer generated by ultraviolet damage can reach about 10%, other unknown impurities can be generated at the same time, and the separation and purification are not facilitated. Meanwhile, the acid is not stable, so that the obtained cefuroxime acid methoxyimino isomer is damaged and is not beneficial to storage in a solution state. The fraction of the methyloxy cefuroxime acid isomer purified by the preparation of the liquid phase can be obtained only as a dry product by freeze-drying and is faced with the problem of removing the buffer salts in the mobile phase.

In order to solve the above problems, the present invention provides a method for preparing cefuroxime axetil methoxyimine isomers from cefuroxime axetil and then preparing cefuroxime sodium methoxyimine isomers.

According to the invention, researches show that under the ultraviolet irradiation, the content of the methoxyimino isomer generated by the cefuroxime axetil and the cefuroxime sodium solid powder is very low, only about 2%, and the time consumption is long, and needs 5-7 days. The cefuroxime sodium is unstable in aqueous solution, and can generate a methoxyimine isomer by ultraviolet irradiation damage, and only the cefuroxime axetil is dissolved and dispersed in an organic solvent, the 25-50% methoxyimine isomer can be generated in a short time, and other impurities are few, so that the subsequent separation and purification operation is convenient.

According to the invention, researches show that the concentration of cefuroxime axetil in the aprotic and protic solvents has an influence on the effect of generating the methoxyimine isomer under the damage of ultraviolet irradiation, and when the concentration is more than 0.5g/ml, the content of the methoxyimine isomer is sharply reduced and is only about 5-15%. When the concentration is 0.1g/ml, the content of the methoxyimine isomer generated by illumination is the highest, about 50 percent.

According to the invention, researches show that aprotic solvents such as ethyl acetate, tetrahydrofuran, dioxane, acetonitrile, dichloromethane and acetone and protic solvents such as methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, propylene glycol and glycerol can be used for dissolving cefuroxime axetil to generate methoxyimino isomers with high purity level. The content of cefuroxime axetil methoxyimine isomer generated by light damage in the dichloromethane system is the minimum in the above solvent. The purity of cefuroxime axetil methoxyimino isomer produced by the light damage of ethylene glycol, propylene glycol and glycerol system is 45-50%.

The invention finds that the illumination time needs to be proper through research. The time is too short, the cefuroxime axetil raw material is not damaged enough, and the produced methoxyimino isomer is less. For a long time, the cefuroxime axetil and the methoxim isomer are destroyed to generate many other impurities, which makes the separation and purification difficult. When a certain amount of the methoxyimine isomer is generated, the illumination time is prolonged without increasing, but the generated methoxyimine isomer is destroyed due to the prolonged time.

The invention adopts a reversed phase C18 column to separate and purify cefuroxime axetil and the mixture of the methoxyimino isomers thereof, the chromatographic condition is that water/acetonitrile is 50/50, an ultraviolet detector is adopted, the flow rate is 40ml/min, and the wavelength is 230 nm. The organic phase proportion is more than 50%, the peak of the furoctyl methoxyimino isomer and the main peak of the furoctyl are overlapped, the organic phase proportion is less than 40%, the separation degree of the peak of the furoctyl methoxyimino isomer and the main peak of the furoctyl is good, but the peaks are flat, the number of the tower plates is low, more fractions are collected, and the concentration of the methoxyimino isomer is low. And other impurities with insufficient ultraviolet absorption can be conveniently observed by adopting the wavelength of 230 nm. If the chromatographic separation condition adopts a buffer salt system such as acetate, phosphate and the like, partial degradation of the target methoxyimine isomer can be caused in the process of fraction enrichment and storage, and the purity of the finished product is reduced.

The invention hydrolyzes cefuroxime axetil methoxyimino isomer by immobilized acetyl esterase to obtain cefuroxime sodium methoxyimino isomer. In the hydrolysis process, both the lipid solubility of cefuroxime axetil and the hydrophilic property of esterase are required to be considered. So that acetone, acetonitrile, methanol and ethanol with water intersolubility are selected. The ratio of aprotic solvent to protic solvent is low, and cefuroxime axetil methoxyimino isomers are separated out, which affects the hydrolysis efficiency. The ratio of aprotic solvent to protic solvent is high, the hydrolysis ability of esterase is reduced, and the hydrolysis efficiency is affected. The ratio of aprotic, protic solvent to water is therefore from 1.2/1 to 10/1.

The invention carries out hydrolysis on cefuroxime axetil methoxyimino to obtain cefuroxime sodium methoxyimino isomer by immobilized acetyl esterase. The pH value will be reduced continuously with the progress of hydrolysis, and after the pH value is reduced to a certain degree, the hydrolysis capability of esterase will be greatly reduced. During hydrolysis, the pH value cannot be too high, and other impurities can be generated due to too high pH value, so that the product purity is influenced. So that the pH range is controlled within the range of 5.8-9.5 during the hydrolysis process.

The preparation of cefuroxime sodium methoxyimino isomer comprises the following three steps:

step (1): preparing 0.1g/ml of cefuroxime axetil isopropanol solution, and then placing the solution in an ultraviolet box for illumination for 6 hours to obtain a mixture of cefuroxime axetil methoxyimino isomer and furoctyl ester with the purity of 43 percent;

step (2): separating and purifying the methoxyimine isomer mixture obtained in the step (1) by a reversed phase C18 column, wherein the chromatographic conditions of purification are that water/acetonitrile is 50/50, the flow rate is 40ml/min, and the ultraviolet detection wavelength is 230 nm. Collecting target fractions, enriching and collecting, extracting with ethyl acetate, drying with anhydrous sodium sulfate, filtering, and rotary-steaming to obtain cefuroxime axetil methoxyimino isomer with purity of 99%.

And (3): dissolving the pure dry cefuroxime axetil methoxyimino isomer obtained in the step (2) with 1.2/1 of acetone aqueous solution, adding 1 time of immobilized acetyldelipase, controlling the pH value within the range of 6.5-8.5, filtering to remove esterase after hydrolysis is finished, adding 20 times of acetone into the filtrate, and separating out cefuroxime sodium methoxyimino isomer.

In another processing mode of the step (3), after the esterase is removed, acetone is removed by concentration under reduced pressure, and acid is added to adjust the pH value to be below 3, so that cefuroxime acid methoxyimino isomer is separated out.

The preparation method of cefuroxime axetil methoxyimine isomer in the step (1) is characterized in that the aprotic solvent used for preparing the cefuroxime axetil solution in the step (1) is ethyl acetate, tetrahydrofuran, dioxane, acetonitrile, dichloromethane or acetone, and the protic solvent is one of methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, propylene glycol and glycerol; the concentration of the cefuroxime axetil organic solution is less than or equal to 0.5g/ml, the ultraviolet illumination time is 2-8 hours, and the purity of the cefuroxime axetil methoxyimino isomer is 25-50 percent

The purification preparation condition in the step (2) is reversed phase C18 column, the mobile phase is water/acetonitrile 50/50, the flow rate is 40ml/min, and the ultraviolet detection wavelength is 230 nm. And in the enrichment fraction treatment, the extraction solvent is ethyl acetate, dichloromethane, chloroform and carbon tetrachloride.

The aprotic solvent in the step (3) is acetone or acetonitrile, and the protic solvent is methanol or ethanol; the ratio of the aprotic, protic solvent to water is 1.2/1-10/; the dosage of the immobilized acetyl-deacetylase is 0.5-2 times; the hydrolysis time is 7-24 hours; the pH control range is 5.8-9.5.

The invention has the advantages that: the preparation method of the cefuroxime sodium methoxyimino isomer is simple to operate, mild in condition and low in cost, and the obtained cefuroxime sodium methoxyimino isomer is high in purity and can be used as an impurity reference substance for quality control research of cefuroxime axetil, sodium raw material medicines and preparations. Compared with the purchased reference substance, the method has great cost advantage. Compared with other literature reports, the ultraviolet irradiation damage method of the invention can produce the cefuroxime axetil with the highest purity of the isomeric cefuroxime axetil, thereby greatly saving the cost for producing the pure cefuroxime axetil isomer in one gram. The preparation method for preparing the cefuroxime sodium methoxyimino isomer from the cefuroxime axetil methoxyimino isomer overcomes the difficulties that the existing preparation method for preparing the cefuroxime sodium methoxyimino isomer has low efficiency and large separation difficulty, and a sample is unstable in the preparation process, and overcomes the difficulty that a special freeze dryer is needed to be used for obtaining a pure dry product of the cefuroxime sodium methoxyimino isomer.

Detailed Description