Process for the enzymatic preparation of cefixime

Abstract

The application 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: performing an enzymatic hydrolysis reaction on cefixime alkyl ester in water in the presence of a hydrolytic enzyme at a pH of 7-8.5 to obtain cefixime. According to the method for preparing cefixime by using the enzyme method, the reaction condition becomes milder, the generation of impurities in the reaction process is effectively controlled, and the product quality and yield of cefixime are both improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for preparing cefixime using an enzymatic method. Background Technology

[0002] Cefixime is a third-generation oral cephalosporin developed by Fujisawa Pharmaceutical Co., Ltd. of Japan. It is highly stable against β-lactamases and has a high affinity for penicillin-binding proteins, inhibiting bacterial cell wall synthesis and leading to rapid bacterial lysis and death. It exhibits good antibacterial activity against most Enterobacteriaceae bacteria, including Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, and Klebsiella pneumoniae. Its chemical name is (6R,7R)-7-[[(z)-2-(2-amino-4-thiazolyl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-vinyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate, with the chemical formula C. 16 H 15 N5O7S2·3H2O, structural formula as follows:

[0003]

[0004] The following is a summary of the methods for preparing cefixime reported in the current literature:

[0005] Chinese patent document CN103467495A discloses a method for preparing cefixime. Specifically, 7-amino-3-vinylcephalosporanic acid (7-AVCA) is acylated with an active ester to obtain a cefixime methyl ester solution. The cefixime methyl ester solution is extracted with water, and activated carbon, sodium hydrosulfite, and disodium EDTA are added to the aqueous phase and stirred evenly for decolorization. The decolorization time is 0.15-1 hour, and the solution is filtered to obtain a cefixime methyl ester aqueous solution. Organic solvent b is added to the above cefixime methyl ester aqueous solution, and an inorganic alkali solution is rapidly added at a temperature of -10℃ to 30℃ to carry out a hydrolysis reaction for 5-30 minutes. Then, hydrochloric acid solution is added to induce crystallization, and the pH is adjusted to 4-6. Activated carbon and EDTA are added for decolorization, and the filtrate is filtered and the pH is adjusted to 4-6 with hydrochloric acid. The cefixime compound is obtained by crystallizing at 2.0-3.5°C for 2-4 hours, followed by filtration, washing, and drying, with a yield of approximately 90%. The organic solvent b used is a hydrophilic organic solvent such as acetone, methanol, ethanol, or isopropanol.

[0006] Chinese patent CN104193765B discloses a method for synthesizing cefixime, which involves an amidation reaction to obtain cefixime methyl ester; cefixime methyl ester is then hydrolyzed under the action of an inorganic base to obtain cefixime. In the product crystallization process, an organic alcohol solvent is used instead of low-boiling-point acetone, with isobutanol being the primary solvent. This helps increase the solubility of cefixime impurities in water, reducing the introduction of impurities into the product; it also reduces the loss of product with the mother liquor after centrifugation, further improving the product purity. The final product content reaches over 99.4%, but the product weight yield based on 7-AVCA is only 180-200%.

[0007] Chinese patent document CN104447796A discloses an improved method for preparing cefixime. Cefixime methyl ester is prepared by amidation reaction, and then hydrolyzed with sodium hydroxide to obtain cefixime trihydrate. Specifically, cefixime methyl ester is added at a fast flow rate to a pre-frozen 20% sodium hydroxide solution and hydrolyzed at 0°C for 15 min. The pH is adjusted to 5.5 by adding 4N hydrochloric acid dropwise at a fast flow rate. Activated carbon and sodium hydrosulfite are added, and the mixture is decolorized at room temperature. After filtration, the filtrate and washings are combined, heated to 30–35°C, and the pH is adjusted to 2.0–2.5 with dilute hydrochloric acid to induce crystallization. The crystals are then filtered, washed with deionized water until neutral, and vacuum dried to obtain cefixime trihydrate with a yield of 83% and a purity of 99.6% (HPLC area normalization method). The process was carried out entirely in an aqueous solution system, and the final product yield was only 83%, which was relatively low. At the same time, the use of high-concentration alkaline solution produced a large amount of salt in the crystallization system, which required washing with a large amount of deionized water until neutral to ensure that no salt residue remained.

[0008] In response to the frequent occurrence of low content in cefixime primary crystallization products, many refining processes have been developed, including: decolorization crystallization, sodium salt purification, tert-octanoate formation, and dicyclohexylamine salt formation. The decolorization crystallization and sodium salt purification methods are commonly used in production. For example, Chinese patent document CN113968874A discloses a method for purifying cefixime. Specifically, crude cefixime is mixed with water at 0℃~10℃, and then alkali is added to dissolve it, obtaining a clear solution. The pH of the clear solution is adjusted to 4.5~6.5 using acid, followed by activated carbon decolorization. First crystallization: at 25℃~35℃, water and acetone are added to the activated carbon-decolorized cefixime solution. Under stirring, acid is gradually added to precipitate cefixime until the solution becomes misty, and crystallization is carried out for 30~240 minutes. Then, acetone is added to continue crystallization for 30~60 minutes. Second crystallization... At 25℃~35℃, after crystallization, acid is gradually added to the first crystallization solution with stirring until the pH value is 2.5~3.0, and then crystallization is continued for 30~240 minutes; then acetone is added to the solution, and crystallization continues for 30~60 minutes; for the third crystallization: at 25℃~35℃, after crystallization, acid is gradually added to the second crystallization solution with stirring until the pH value is 1.8~2.2, and then crystallization is continued for 30~240 minutes; then acetone is added to the solution, and crystallization continues for 30~60 minutes; then the third crystallization solution is filtered, washed, and dried to obtain cefixime crystals with a yield of 90~93%.

[0009] Furthermore, to reduce the degradation of the product by the strongly alkaline environment, two-phase hydrolysis and high-solubility solvent purification methods have been developed. Patent document GB2330141A reports the hydrolysis of cefixime methyl ester in a dichloromethane-water system using potassium carbonate and the phase-transfer catalyst tetrabutylammonium bromide to produce cefixime. Patent document GB2330140A also reports the hydrolysis of cefixime methyl ester in a DMF-water system using potassium carbonate as a catalyst. The application of two-phase organic solvents and the high-solubility solvent DMF not only significantly reduces the product yield but also adversely affects some of the product's quality indicators.

[0010] Because cephalosporin compounds are easily degraded under strong acid and alkali conditions, current reported processes for industrial production face a trade-off between product quality and yield. Manufacturers often reduce yield to improve product quality, sometimes even employing multiple refining processes. This also makes controlling process parameters difficult. Hydrolyzing cefixime methyl ester under strong alkaline conditions requires strict control over the relationship between cefixime methyl ester residue and product degradation, necessitating precise control of reaction temperature, time, and concentration. This places extremely high demands on production equipment and process monitoring equipment. Summary of the Invention

[0011] In view of the above-mentioned problems, this invention was made. The inventors have innovatively proposed a method for the enzymatic preparation of cefixime, namely, using cefixime alkyl esters as raw materials, hydrolyzing the ester bonds through the catalytic action of hydrolytic enzymes, and then adjusting the acid and crystallizing to obtain the cefixime product. Therefore, the purpose of this invention is to provide a method for the enzymatic preparation of cefixime.

[0012] According to the present invention, the method for preparing cefixime by enzymatic method includes: in water, in the presence of a hydrolytic enzyme, at a pH of 7-8.5, performing an enzymatic hydrolysis reaction of cefixime alkyl ester to obtain cefixime.

[0013] The method for preparing cefixime using an enzymatic method according to the present invention is described in more detail below.

[0014] In this invention, the inventors innovatively propose an enzymatic method for preparing cefixime. The hydrolytic enzyme is a thermophilic protease produced by *Geobacillus stearothermophilus*, a type of *Bacillus* sp., with an enzyme activity typically ranging from 20 to 300 U / mg. This thermophilic protease is commercially available, for example, from Amano Enzyme Preparations (Jiangsu) Co., Ltd. To facilitate the reusability of the thermophilic protease, immobilized thermophilic protease is preferred. In the enzymatic hydrolysis of cefixime alkyl esters, the enzyme activity of the reaction solution is generally controlled at 10 U / mg relative to 1 kg of cefixime alkyl esters. 6 ~10 9 U, preferred 10 7 ~10 8 U.

[0015] The cefixime alkyl ester may be a cefixime alkyl ester with the following structure.

[0016]

[0017] In the formula, R is a C1-C6 alkyl group, preferably a C1-C5 alkyl group, more preferably a C1-C4 alkyl group, and most preferably a C1-C3 alkyl group, for example, it can be selected from any one of methyl, ethyl, n-propyl and isopropyl.

[0018] The cefixime alkyl ester can be obtained by acylation of 7-amino-3-vinylcephalosporanic acid (7-AVCA) with the active ester of the cefixime side chain acid. As a specific example, CN104193765B reports the preparation of cefixime methyl ester, wherein the obtained wet cefixime methyl ester can be directly used for enzymatic hydrolysis. This patent document CN104193765B is incorporated herein by reference. In the enzymatic hydrolysis reaction, the amount of cefixime alkyl ester added is such that its concentration in the reaction solution is 8 wt% to 20 wt%.

[0019] The enzymatic hydrolysis reaction is carried out at 0℃ to 30℃, preferably 10℃ to 20℃; the pH is controlled at 7 to 8.5 using an alkaline solution, preferably 7 to 8, and the alkaline solution is selected from one or two of diethylamine, triethylamine, N,N-diisopropylethylamine, potassium carbonate aqueous solution, potassium bicarbonate aqueous solution, sodium carbonate aqueous solution, and sodium bicarbonate aqueous solution, with sodium bicarbonate aqueous solution being preferred.

[0020] In the presence of hydrolytic enzymes at a pH of 7–8.5, cefixime alkyl esters undergo enzymatic hydrolysis to produce cefixime. During the enzymatic hydrolysis process, the reaction is monitored by HPLC until the residual cefixime alkyl esters meet acceptable standards, for example, the area percentage of cefixime alkyl esters is less than 2%, or less than 1%, or less than 0.5%, or less than 0.1%.

[0021] After the enzymatic hydrolysis reaction is complete, the pH of the hydrolysis solution is adjusted to 3.8–2 with acid to induce cefixime crystallization. For the use of immobilized thermophilic protease, the immobilized thermophilic protease can be filtered out, and the pH of the filtered hydrolysis solution is adjusted to 3.8–2 with acid to induce cefixime crystallization.

[0022] More specifically, the pH of the enzymatic hydrolysis solution or the filtered enzymatic hydrolysis solution is adjusted to 7-6 with acid, and then activated carbon is added and stirred for decolorization for 10-30 minutes. The activated carbon is filtered out to obtain the decolorized enzymatic hydrolysis solution. The pH is adjusted to 3.8-3.6 with acid, and seed crystals are added. The amount of seed crystals added is 0.5%-1% of the weight of cefixime alkyl ester. Then, acid is added to adjust the pH to 3.2-3.0 and stirred to grow crystals. Then, acid is added to adjust the pH to 2.2-2.0 and crystals are grown. Then, solid-liquid separation is performed, and the solid is washed with water and dried to obtain cefixime.

[0023] The acid is selected from one or two of hydrochloric acid, sulfuric acid, nitric acid, formic acid and acetic acid, with hydrochloric acid being preferred, and 3M hydrochloric acid being even more preferred.

[0024] Beneficial effects

[0025] The inventors have innovatively proposed an enzymatic method for preparing cefixime, which has achieved a dual improvement in the quality and yield of cefixime products, breaking through the current dilemma that the two cannot be achieved simultaneously.

[0026] The enzymatic method for preparing cefixime of this invention provides milder reaction conditions and effectively controls the generation of impurities during the reaction process. Specifically, adjusting the reaction pH from strongly alkaline to weakly alkaline significantly inhibits product degradation, fundamentally improving product quality and yield. The reaction temperature has been increased from below 0°C to room temperature, effectively saving energy consumption. Furthermore, by controlling the amount of enzyme added, the reaction time has been effectively controlled, moving from the previous short-time, high-precision control to a more moderate control, resulting in a smoother production process and reduced requirements for production equipment. Detailed Implementation

[0027] The following examples provide a more detailed description of the preparation method of cefixime according to the present invention. The scope of protection of the present invention is not limited to the following examples. These examples are listed for illustrative purposes only and do not limit the present invention in any way.

[0028] Example 1

[0029] In the reactor, add 6.5 kg of water, maintain the temperature at 5-10℃, add 0.1 kg of lyophilized thermophilic protease powder (from Amano Enzyme Preparations (Jiangsu) Co., Ltd.) with an enzyme activity of 200 U / mg, and 1.5 kg of wet cefixime methyl ester (loss on drying 28%). Stir well, add 6 wt% sodium bicarbonate solution to adjust the pH of the solution to 7.9, and continue stirring at 5-10℃ until the cefixime methyl ester residue is within acceptable limits. After the reaction is complete, add 3 mol / L HCl to neutralize to a pH of 6.2.

[0030] The above reaction solution was transferred from the reactor to a decolorization tank, and 25g of activated carbon was added. The mixture was stirred and decolorized for 20 minutes. After decolorization, the carbon was removed by filtration, and the carbon was washed with 200mL of water. The filtrate and washings were combined and transferred to a crystallization tank. 1mol / L HCl was quickly added to adjust the pH to 3.6, and 6g of seed crystals were added. After stirring evenly, 1mol / L HCl was added again to adjust the pH to 3.2, and crystals were cultured for 30 minutes. After crystallization, 1mol / L HCl was added again to adjust the pH to 2.1, and crystals were cultured for another 30 minutes. After crystallization, solid-liquid separation was performed. The solid was washed with water and vacuum dried to obtain 1.15kg of cefixime, with a yield of 98.2% and a purity of 99.4%.

[0031] Example 2

[0032] In the reactor, 7.5 kg of water was added, and the temperature was controlled at 15-17℃. 1.5 kg of immobilized thermophilic protease (from Amano Enzyme Preparations (Jiangsu) Co., Ltd.) with an enzyme activity of 60 U / mg was added, along with 1.3 kg of wet cefixime methyl ester (with a drying loss of 23%). The mixture was stirred thoroughly, and a 6 wt% sodium bicarbonate solution was added to adjust the pH of the solution to 7.3. The reaction was continued at 15-17℃ with stirring until the cefixime methyl ester residue met the acceptable level. After the reaction was complete, the immobilized thermophilic protease was separated using an 80-mesh sieve, and 3 mol / L HCl was added to the solution to neutralize it to a pH of 6.7.

[0033] The above reaction solution was transferred from the reactor to a decolorization tank, and 25g of activated carbon was added. The mixture was stirred and decolorized for 20 minutes. After decolorization, the carbon was removed by filtration, and the carbon was washed with 100mL of water. The filtrate and washings were combined and transferred to a crystallization tank. 1mol / L HCl was quickly added to adjust the pH to 3.7, and 7g of seed crystals were added. After stirring evenly, 1mol / L HCl was added again to adjust the pH to 3.1, and crystallization was continued for 30 minutes. After crystallization, 1mol / L HCl was added again to adjust the pH to 2.0, and crystallization was continued for another 30 minutes. After crystallization, solid-liquid separation was performed. The solid was washed with water and vacuum dried to obtain 1.06kg of cefixime, with a yield of 97.6% and a purity of 99.6%.

[0034] Comparative example

[0035] At 0.5-1℃, 10 kg of a 5% aqueous solution of cefixime methyl ester was mixed with 1.4 kg of a 15% sodium hydroxide solution and hydrolyzed by rapid stirring for 6-8 min. After hydrolysis, 3N hydrochloric acid was added dropwise at a rapid flow rate to adjust the pH to 6.7. 12 g of activated carbon was added, and the mixture was stirred for decolorization for 20 min. After decolorization, the carbon was removed by filtration, and the carbon was washed with 100 mL of water. The filtrate and washings were combined and transferred to a crystallizer. 1 mol / L HCl was added rapidly to adjust the pH to 3.7, 3 g of seed crystals were added, and the mixture was stirred evenly. 1 mol / L HCl was added again to adjust the pH to 3.1, and crystallization was carried out for 30 min. After crystallization, 1 mol / L HCl was added again to adjust the pH to 2.0, and crystallization was carried out for another 30 min. After crystallization, solid-liquid separation was performed. The solid was washed with water and vacuum dried to obtain 488 g of cefixime, with a yield of 90% and a purity of 99.1%.

Claims

1. A method for preparing cefixime using an enzymatic method, comprising: In water, in the presence of hydrolytic enzymes, at a pH of 7–8.5, alkyl cefixime undergoes enzymatic hydrolysis to yield cefixime. The hydrolytic enzyme is a thermophilic protease produced by Bacillus stearothermophilus. The alkyl cefixime ester is cefixime methyl ester. In the enzymatic hydrolysis of cefixime alkyl esters, the enzyme activity of the reaction solution was controlled at 10 relative to 1 kg of cefixime alkyl esters. 6 ~10 9 U; The enzymatic hydrolysis reaction is carried out at 0℃~30℃.

2. The method for preparing cefixime by enzymatic method according to claim 1, characterized in that, The hydrolytic enzyme is an immobilized thermophilic bacterial protease.

3. The method for preparing cefixime by enzymatic method according to claim 1, characterized in that, In the enzymatic hydrolysis of cefixime alkyl esters, the enzyme activity of the reaction solution was controlled at 10 relative to 1 kg of cefixime alkyl esters. 7 ~10 8 U; The amount of cefixime alkyl ester added is such that its concentration in the reaction solution is 8wt% to 20wt%.

4. The method for preparing cefixime by enzymatic method according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out at 10℃ to 20℃; the pH is controlled at 7 to 8.5 using an alkaline solution, which is selected from one or two of diethylamine, triethylamine, N,N-diisopropylethylamine, potassium carbonate aqueous solution, potassium bicarbonate aqueous solution, sodium carbonate aqueous solution, and sodium bicarbonate aqueous solution.

5. The method for preparing cefixime by enzymatic method according to claim 1, characterized in that, During the enzymatic hydrolysis process, HPLC was used to detect the reaction progress until the percentage of cefixime alkyl ester residue was less than 0.5% of the total area.

6. The method for preparing cefixime by enzymatic method according to claim 1, characterized in that, After the enzymatic hydrolysis reaction is completed, the pH of the enzymatic hydrolysis solution is adjusted to 3.8-2 with acid to induce cefixime crystallization.

7. The method for preparing cefixime by enzymatic method according to claim 1, characterized in that, The pH of the enzymatic hydrolysis solution or the filtered enzymatic hydrolysis solution is adjusted to 7-6 with acid. Then, activated carbon is added and the solution is stirred for decolorization for 10-30 minutes. The activated carbon is filtered out to obtain the decolorized enzymatic hydrolysis solution. The pH is adjusted to 3.8-3.6 with acid, and seed crystals are added. The amount of seed crystals added is 0.5%-1% of the weight of cefixime alkyl ester. Then, acid is added to adjust the pH to 3.2-3.0 and stirred to grow crystals. Then, acid is added to adjust the pH to 2.2-2.0 and crystals are grown. Then, solid-liquid separation is performed. The solid is washed with water and dried to obtain cefixime.

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