CN115558696A

CN115558696A - Method for preparing cefixime by enzyme method

Info

Publication number: CN115558696A
Application number: CN202211142470.2A
Authority: CN
Inventors: 刘宏飞; 白飞; 幸华龙; 冯涛; 王维毅; 王世珍
Original assignee: Sinopharm Weiqida Pharmaceutical Co Ltd
Current assignee: Sinopharm Weiqida Pharmaceutical Co Ltd
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2023-01-03

Abstract

The invention belongs to the technical field of pharmacy, and relates to a method for preparing cefixime by using an enzyme method. The method comprises the following steps: in water, in the presence of hydrolase, carrying out enzymolysis reaction on cefixime alkyl ester at the pH value of 7-8.5 to obtain cefixime. According to the method for preparing cefixime by using the enzyme method, the reaction conditions become milder, the generation of impurities in the reaction process is effectively controlled, and the quality and yield of cefixime products are improved.

Description

Method for preparing cefixime by enzyme method

Technical Field

The invention belongs to the technical field of pharmacy, and relates to a method for preparing cefixime by an enzymatic method.

Background

Cefixime is the third generation developed by Tanzuzer pharmaceutical Co., ltdOral cephalosporin which is highly stable against beta lactamase and has high affinity with penicillin-binding protein, can block the synthesis of bacterial cell walls, and rapidly dissolves and dies bacteria. Has good antibacterial activity against Streptococcus pyogenes, pneumococcus, streptococcus agalactiae, gonococcus, bacillus influenzae, moraxella catarrhalis, escherichia coli, and Bacillus pneumoniae. The chemical name is: (6R,7R) -7- [ [ (z) -2- (2-amino-4-thiazolyl) -2- [ (carboxymethoxy) imino]Acetyl group]Amino group]-3-vinyl-8-oxo-5-thia-1-azabicyclo [4.2.0]Oct-2-ene-2-carboxylic acid trihydrate of the formula C ₁₆ H ₁₅ N ₅ O ₇ S ₂ ·3H ₂ O, structural formula as follows:

the preparation method of cefixime reported in the literature at present is summarized as follows:

chinese patent document CN103467495A discloses a preparation method of cefixime, specifically, 7-amino-3-vinyl cephalosporanic acid (7-AVCA) and active ester are subjected to acylation reaction to obtain cefixime methyl ester solution; extracting the cefixime methyl ester solution by water, adding active carbon, sodium hydrosulfite and ethylene diamine tetraacetic acid into the water phase, uniformly stirring, decoloring for 0.15-1 hour, and filtering to obtain a cefixime methyl ester water solution; adding an organic solvent b into the cefixime methyl ester aqueous solution, quickly adding an inorganic alkali solution at the temperature of between 10 ℃ below zero and 30 ℃ for hydrolysis reaction, hydrolyzing for 5 to 30 minutes, adding a hydrochloric acid solution for crystallization, adjusting the pH value to between 4 and 6, adding activated carbon and ethylene diamine tetraacetic acid for decolorization, adjusting the pH value of filtrate to between 2.0 and 3.5 with hydrochloric acid after filtration for crystal growth for 2 to 4 hours, and filtering, washing and drying to obtain the cefixime compound with the yield of about 90 percent. The organic solvent b is hydrophilic organic solvent such as acetone, methanol, ethanol, isopropanol, etc.

Chinese patent CN104193765B discloses a synthesis method of cefixime, which comprises carrying out amidation reaction to obtain cefixime methyl ester; and hydrolyzing the cefixime methyl ester under the action of inorganic base to obtain the cefixime. Wherein, the organic alcohol solvent is adopted to replace low-boiling-point acetone in the crystallization process of the product, and the isobutanol solvent is mainly adopted to help to increase the solubility of cefixime impurities in water and reduce the introduction of the impurities into the product; meanwhile, the loss of the product along with the mother liquor after centrifugation is reduced, the purity of the product is further improved, the content of the final product reaches over 99.4 percent, but the weight yield of the product calculated by 7-AVCA is only 180-200 percent.

Chinese patent document CN104447796A discloses an improved method for preparing cefixime, which comprises preparing cefixime methyl ester by amidation reaction, and hydrolyzing cefixime methyl ester with sodium hydroxide to obtain cefixime trihydrate. Adding pre-frozen 20% sodium hydroxide solution into cefixime methyl ester at a fast flow rate, hydrolyzing for 15min at 0 ℃, dropwise adding 4N hydrochloric acid at the fast flow rate to adjust the pH value to 5.5, adding activated carbon and sodium hydrosulfite, decolorizing at room temperature, filtering, combining filtrate and washing liquor, heating to 30-35 ℃, adjusting the pH value to 2.0-2.5 by using dilute hydrochloric acid to crystallize, filtering, washing to neutrality by using deionized water, and drying in vacuum to obtain cefixime trihydrate, wherein the yield is 83% and the purity is 99.6% (HPLC area normalization method). The scheme is completely carried out in an aqueous solution system, the yield of the final product is only 83 percent, the yield is relatively low, and meanwhile, a large amount of salt substances are generated in a crystallization system due to the use of high-concentration alkali liquor, and a large amount of deionized water is needed for washing to be neutral to ensure that no salt substances remain.

Aiming at the phenomenon that the cefixime primary crystallization product content is relatively low, a plurality of re-refining processes are developed at present, comprising the following steps: decolorizing crystallization method, sodium salt-forming purification method, tert-octanoate-forming method, dicyclohexylamine salt-forming method, etc., and decolorizing crystallization method and sodium salt-forming purification method are commonly used in production. For example, chinese patent document CN113968874A discloses a method for refining cefixime, specifically, a cefixime crude product is mixed with water at 0 ℃ to 10 ℃, and then alkali is added to dissolve the cefixime crude product, so as to obtain a clear solution; adjusting the pH of the solution to 4.5-6.5 by acid, and then carrying out activated carbon decolorization treatment; first crystallization: adding water and acetone into the cefixime solution decolored by the active carbon at the temperature of between 25 and 35 ℃, gradually adding acid while stirring to separate out the cefixime until the solution is in a fog shape, and growing the crystal for 30 to 240 minutes; then, acetone is added into the mixture, and crystal growth is continued for 30 to 60 minutes; second crystallization: after crystal growth at 25-35 ℃, acid is gradually added into the first crystallization liquid under stirring until the pH value is 2.5-3.0, and then crystal growth is carried out for 30-240 minutes; then, acetone is added into the mixture, and crystal growth is continued for 30 to 60 minutes; third crystallization: after crystal growth at 25-35 ℃, acid is gradually added into the second crystallization liquid under stirring until the pH value is 1.8-2.2, and then crystal growth is carried out for 30-240 minutes; then, acetone is added into the mixture, and crystal growth is continued for 30 to 60 minutes; and then, filtering, washing and drying the third crystallization liquid to obtain the cefixime crystal, wherein the yield is between 90 and 93 percent.

In addition, in order to reduce the degradation of products in a strong alkaline environment, a two-phase hydrolysis method and a high-solubility solvent impurity removal method are also developed. Patent document GB2330141A reports the hydrolysis of cefixime methyl ester to cefixime in a system of dichloromethane and water by the action of potassium carbonate and phase transfer catalyst tetrabutylammonium bromide. Patent document GB2330140A reports the hydrolysis of cefixime methyl ester to cefixime in a system of DMF water catalyzed by potassium carbonate. The application of the two-phase organic solvent and the high-solubility solvent DMF not only greatly reduces the yield of the product, but also causes adverse effect on the quality index of the product.

Because the cephalosporin compounds have the characteristic of easy degradation under the conditions of strong acid and strong base, the existing reported process has the problem that the product quality and the yield cannot be considered in the industrial production process, and production enterprises often reduce the yield of products in order to improve the product quality and even improve the product quality by adopting a mode of refining for many times. Meanwhile, the control difficulty of production process parameters is high due to the reasons, and because the hydrolysis of cefixime methyl ester is carried out under the strong alkali condition, the relationship between the residue of cefixime methyl ester and the degradation of products needs to be strictly controlled, and the reaction temperature, time, concentration and the like need to be accurately controlled, so that very high requirements are provided for production equipment and production process detection equipment.

Disclosure of Invention

The present invention has been made in view of the above problems, and the present inventors have innovatively proposed a scheme for preparing cefixime by an enzymatic method, i.e., using cefixime alkyl ester as a raw material, hydrolyzing an ester bond by the catalytic action of a hydrolytic enzyme, and then adjusting acid to crystallize to obtain a cefixime product. Therefore, the present invention aims to provide a method for preparing cefixime by an enzymatic method.

According to the invention, the method for preparing cefixime by using the enzyme method comprises the following steps: and (3) carrying out enzymolysis reaction on cefixime alkyl ester in water in the presence of hydrolase at the pH of 7-8.5 to obtain cefixime.

The process for preparing cefixime by the enzymatic method of the present invention is described in more detail below.

In the invention, the inventor innovatively proposes that cefixime is prepared by an enzyme method, wherein the hydrolase is thermolysin (Protease) produced by Geobacillus stearothermophilus in bacillus (Bacillus sp.), and the enzyme activity is generally 20-300U/mg. The thermolysin is commercially available, for example, from Tianye enzyme preparation (Jiangsu) Co. In order to facilitate the recycling of thermolysin, immobilized thermolysin is preferred. In the enzymolysis reaction of cefixime alkyl ester, the enzyme activity of the reaction liquid is generally controlled to 10 relative to every 1kg of cefixime alkyl ester ⁶ ～10 ⁹ U, preferably 10 ⁷ ～10 ⁸ U。

The cefixime alkyl ester may be a cefixime alkyl ester of the structure,

wherein R is C1-C6 alkyl, preferably C1-C5 alkyl, more preferably C1-C4 alkyl, most preferably C1-C3 alkyl, and can be selected from any one of methyl, ethyl, n-propyl and isopropyl.

The cefixime alkyl ester can be obtained by acylation reaction of 7-amino-3-vinyl cephalosporanic acid (7-AVCA) and cefixime side chain acid active ester. As a specific example, CN104193765B reports the preparation of cefixime methylester, wherein the obtained cefixime methylester wet mass can be directly used for enzymatic hydrolysis. This patent document CN104193765B is incorporated herein by reference. In the enzymolysis reaction, the cefixime alkyl ester is added in an amount which enables the concentration of the cefixime alkyl ester in the reaction liquid to be 8-20 wt%.

The enzymolysis reaction is carried out at 0-30 ℃, preferably 10-20 ℃; the pH is controlled to be 7-8.5, preferably 7-8 by alkali liquor, wherein the alkali liquor is selected from one or two of diethylamine, triethylamine, N-diisopropylethylamine, potassium carbonate aqueous solution, potassium bicarbonate aqueous solution, sodium carbonate aqueous solution and sodium bicarbonate aqueous solution, and is preferably sodium bicarbonate aqueous solution.

In the presence of hydrolase, carrying out enzymolysis reaction on cefixime alkyl ester at the pH value of 7-8.5 to generate cefixime. During the enzymatic hydrolysis reaction, the reaction is performed by HPLC until the cefixime alkyl ester residue is acceptable, for example, the area percentage of cefixime alkyl ester is less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%.

After the enzymolysis reaction is finished, the pH value of the enzymolysis reaction liquid is adjusted to 3.8-2 by acid, so that the cefixime is crystallized and separated out. For immobilized thermolysin, immobilized thermolysin can be filtered, and the pH of the filtered enzymolysis reaction solution is adjusted to 3.8-2 by acid, so that cefixime is crystallized and separated out.

More specifically, the pH of the enzymolysis reaction solution or the filtered enzymolysis reaction solution is adjusted to 7-6 by acid, then active carbon is added into the enzymolysis reaction solution to stir and decolor for 10-30 min, and the active carbon is filtered out to obtain decolored enzymolysis reaction solution; adjusting the pH value to be between 3.8 and 3.6 by using acid, adding seed crystals, wherein the addition amount of the seed crystals is 0.5 to 1 percent of the weight of the cefixime alkyl ester, then continuously adding the acid to adjust the pH value to be between 3.2 and 3.0, and stirring for growing crystals; and continuously adding acid to adjust the pH value to 2.2-2.0, growing crystals, then carrying out solid-liquid separation, washing the solid with water, and drying to obtain the cefixime.

Wherein the acid is one or two selected from hydrochloric acid, sulfuric acid, nitric acid, formic acid and acetic acid, and hydrochloric acid is preferred, and 3M hydrochloric acid is further preferred.

Advantageous effects

The invention innovatively provides a scheme for preparing cefixime by an enzyme method, and the implementation of the scheme realizes double promotion of the quality and yield of cefixime products, and breaks through the dilemma that the cefixime product and the cefixime product cannot be obtained at present.

According to the method for preparing cefixime by using the enzyme method, the reaction conditions become milder, and the generation of impurities in the reaction process is effectively controlled. Wherein, the pH value of the reaction is adjusted from strong basicity to weak basicity, thereby greatly inhibiting the degradation of the product and essentially improving the quality and the yield of the product; the reaction temperature is increased from the original temperature below 0 ℃ to room temperature, so that the consumption of production energy is effectively saved; the reaction time is effectively controlled by controlling the enzyme amount, and compared with the original short-time high-precision control, the enzyme amount is adjusted to be common control, so that the production process is smoother, and the requirements on production equipment are reduced.

Detailed Description

The process for the preparation of cefixime of the present invention is further illustrated in detail by the following examples, without limiting the scope of the invention to the following examples, which are given for illustrative purposes only and are not to be construed as limiting the invention in any way.

Example 1

Adding 6.5kg of water into a reactor, controlling the temperature to be 5-10 ℃, adding 0.1kg of thermolysin freeze-dried powder (from Tianye enzyme preparation (Jiangsu) Co., ltd.) with the enzyme activity of 200U/mg and 1.5kg of cefixime methyl ester wet material (the drying loss is 28%), uniformly stirring, adding 6wt% of sodium bicarbonate solution to adjust the pH of the material liquid to be 7.9, and continuously controlling the temperature to be 5-10 ℃ to stir and react until the cefixime methyl ester residue is qualified. After the reaction was complete, 3mol/L HCl was added to neutralize to pH 6.2.

And (3) putting the reaction liquid from the reactor into a decoloring tank, adding 25g of activated carbon, and stirring and decoloring for 20min. After the decolorization is finished, the carbon is removed by filtration, 200mL of water is used for washing the carbon, and the filtrate and the washing liquid are combined and transferred to a crystallizing tank. Quickly adding 1mol/L HCl to adjust the pH value to 3.6, adding 6g of seed crystal, stirring uniformly, continuously adding 1mol/L HCl to adjust the pH value to 3.2, growing the crystal for 30min, after the crystal growing is finished, continuously adding 1mol/L HCl to adjust the pH value to 2.1, and growing the crystal for 30min. After the crystal growth is finished, solid-liquid separation is carried out, the solid is washed by water and then dried in vacuum, and 1.15kg of cefixime is obtained, the yield is 98.2 percent, and the content is 99.4 percent.

Example 2

Adding 7.5kg of water into a reactor, controlling the temperature to be 15-17 ℃, adding 1.5kg of immobilized thermolysin (from Tianye enzyme preparation (Jiangsu) Co., ltd.) with the enzyme activity of 60U/mg and 1.3kg of cefixime methyl ester wet material (the drying weight loss is 23%), stirring uniformly, adding 6wt% of sodium bicarbonate solution to adjust the pH value of the material liquid to be 7.3, and continuously controlling the temperature to be 15-17 ℃, stirring and reacting until the cefixime methyl ester residue is qualified. After the reaction, the immobilized thermolysin was separated with a 80 mesh sieve, and 3mol/L HCl was added to the feed solution to neutralize to pH 6.7.

And (3) putting the reaction liquid from the reactor into a decoloring tank, adding 25g of activated carbon, and stirring and decoloring for 20min. After the decolorization is finished, the carbon is removed by filtration, 100mL of water is used for washing the carbon, and the filtrate and the washing liquid are combined and transferred to a crystallizing tank. Quickly adding 1mol/L HCl to adjust the pH value to 3.7, adding 7g of seed crystal, stirring uniformly, continuously adding 1mol/L HCl to adjust the pH value to 3.1, growing the crystal for 30min, continuously adding 1mol/L HCl to adjust the pH value to 2.0 after the crystal growth is finished, and growing the crystal for 30min. After the crystal growth is finished, solid-liquid separation is carried out, the solid is washed by water and then dried in vacuum, and 1.06kg of cefixime is obtained, the yield is 97.6 percent, and the content is 99.6 percent.

Comparative example

10kg of aqueous solution with 5 percent of cefixime methyl ester concentration and 1.4kg of sodium hydroxide solution with 15 percent are mixed at the temperature of 0.5-1 ℃, and rapidly stirred and hydrolyzed for 6-8min. 3N hydrochloric acid is dripped at a fast flow rate to adjust the pH value to 6.7 after the hydrolysis is finished; adding 12g of active carbon, stirring and decoloring for 20min, filtering and removing the carbon after decoloring, washing the carbon with 100mL of water, combining the filtrate and the washing liquid, and transferring to a crystallizing tank. Quickly adding 1mol/L HCl to adjust the pH value to 3.7, adding 3g of seed crystals, stirring uniformly, continuously adding 1mol/L HCl to adjust the pH value to 3.1, growing crystals for 30min, continuously adding 1mol/L HCl to adjust the pH value to 2.0 after the crystal growth is finished, and growing crystals for 30min. And after the crystal growth is finished, carrying out solid-liquid separation, washing the solid with water, and carrying out vacuum drying to obtain 488g of cefixime, wherein the yield is 90%, and the content is 99.1%.

Claims

1. A process for the enzymatic preparation of cefixime comprising: and (3) carrying out enzymolysis reaction on cefixime alkyl ester in water in the presence of hydrolase at the pH of 7-8.5 to obtain cefixime.

2. The enzymatic process of producing cefixime according to claim 1, wherein the hydrolytic enzyme is thermolysin produced by Geobacillus stearothermophilus in Bacillus sp.

3. The enzymatic process for the preparation of cefixime according to claim 1 or 2, wherein the hydrolase is immobilized thermolysin.

4. The enzymatic process for preparing cefixime according to claim 1, wherein the cefixime alkyl ester is a cefixime alkyl ester having the following structure,

in the formula, R is C1-C6 alkyl.

5. The enzymatic process for preparing cefixime according to claim 4, wherein R is any one selected from the group consisting of methyl, ethyl, n-propyl and isopropyl, and preferably the cefixime alkyl ester is cefixime methyl ester.

6. The enzymatic process for the preparation of cefixime according to any one of claims 1-5, wherein the enzymatic activity of the reaction solution is generally controlled to 10 per 1kg of cefixime alkyl ester in the enzymatic hydrolysis of cefixime alkyl ester ⁶ ～10 ⁹ U, preferably 10 ⁷ ～10 ⁸ U; the cefixime alkyl ester is added in an amount such that the concentration thereof in the reaction solution is 8 to 20wt%.

7. The enzymatic process for preparing cefixime according to claim 1, wherein the enzymatic hydrolysis is carried out at 0-30 ℃; the pH is controlled to be 7-8.5 by adopting alkali liquor, and the alkali liquor is selected from one or two of diethylamine, triethylamine, N-diisopropylethylamine, a potassium carbonate aqueous solution, a potassium bicarbonate aqueous solution, a sodium carbonate aqueous solution and a sodium bicarbonate aqueous solution.

8. The enzymatic process of claim 1 for preparing cefixime, wherein the reaction is carried out by HPLC during the enzymatic hydrolysis until the cefixime alkyl ester remains until the area percentage of cefixime alkyl ester is less than 0.5%.

9. The enzymatic process of claim 1 for preparing cefixime, wherein after the completion of the enzymatic hydrolysis reaction, the solution of the enzymatic hydrolysis reaction is adjusted to pH 3.8-2 with an acid to crystallize cefixime.

10. The method for preparing cefixime by the enzymatic method according to claim 1, which is characterized in that the enzymatic hydrolysis reaction liquid or the filtered enzymatic hydrolysis reaction liquid is adjusted to pH 7-6 by acid, then activated carbon is added into the enzymatic hydrolysis reaction liquid for stirring and decoloring for 10-30 min, and the activated carbon is filtered to obtain decolored enzymatic hydrolysis reaction liquid; adjusting the pH value to be between 3.8 and 3.6 by using acid, adding seed crystals, wherein the addition amount of the seed crystals is 0.5 to 1 percent of the weight of the cefixime alkyl ester, then continuously adding the acid to adjust the pH value to be between 3.2 and 3.0, and stirring for growing crystals; and continuously adding acid to adjust the pH value to 2.2-2.0, growing crystals, then carrying out solid-liquid separation, washing the solid with water, and drying to obtain the cefixime.