A process for the preparation of cefazolin acid

Abstract

The application relates to the technical field of medicines and chemical industry, and particularly discloses a preparation method of cefazolin acid. The preparation method of the cefazolin acid comprises the following steps: continuously refluxing reaction of tetrazole acetic acid and alcohol under the condition of 78-79 DEG C and strong acid catalyst, on-line drying of collected distillate in the refluxing reaction process, refluxing into a reaction system, vacuum distillation of reaction liquid after reaction, and obtaining tetrazole acetic acid ethyl ester suspension; under the condition of a solvent and pH of 6.3-6.5, condensation reaction of TDA and the tetrazole acetic acid ethyl ester suspension is carried out by using immobilized acylase as a catalyst, and cefazolin acid is obtained. The preparation method provided by the application optimizes a process route, adopts a combination mode of continuous azeotropic refluxing reaction and on-line drying, improves the preparation quality of tetrazole acetic acid ethyl ester, further uses immobilized acylase as a catalyst to prepare cefazolin acid, and the product quality of the cefazolin acid is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and chemical technology, and in particular to a method for preparing cefazolin. Background Technology

[0002] Cefazolin, a semi-synthetic cephalosporin antibiotic, has a broad antibacterial spectrum and is widely used clinically. It is one of the first-line drugs for the prevention of primary infections in surgical procedures and plays an important role in the treatment of various infections, including respiratory tract, urinary tract, and sepsis. Market demand remains strong. Currently, the industrial preparation of cefazolin is still mainly based on chemical methods, but this process has many technical challenges that urgently need to be addressed, severely restricting product quality and production efficiency.

[0003] Existing chemical preparation processes suffer from problems such as lengthy production cycles and harsh operating conditions. They not only require the use of various toxic and harmful substances in large quantities, but also necessitate maintaining a low-temperature reaction environment of -70 to -40°C, significantly increasing energy consumption and production costs. They also pose high safety risks and environmental pressures. Furthermore, the existing process has a complex reaction pathway, which easily generates various byproducts, resulting in a wide variety and high content of impurities in the product. This not only affects the purity and efficacy of cefazolin but also increases the difficulty of subsequent separation and purification, leading to low product yields and making it difficult to meet the demands of large-scale industrial production and high quality.

[0004] Ethyl tetrazolium is a key side chain in the preparation of cefazolin, and the quality of its preparation process directly affects the overall preparation efficiency and product quality of cefazolin. Currently, the conventional method for preparing ethyl tetrazolium involves reacting tetrazolium acetic acid with ethanol under strong acid catalysis, requiring 3-5 intermittent cycles of "reaction-distillation-alcohol addition-reaction". This process is time-consuming, energy-intensive, produces a dark-colored reaction solution, exhibits numerous side reactions, and consumes a large amount of ethanol, increasing production costs and easily introducing impurities, further affecting the purity and yield of subsequent cefazolin synthesis.

[0005] In summary, existing cefazolin preparation processes are complex, time-consuming, energy-intensive, and environmentally unfriendly, resulting in poor product quality. Furthermore, the preparation processes for key intermediates also have numerous drawbacks, failing to meet the demands of current industrialized green production and high-quality products. Therefore, developing a simpler, more efficient, energy-efficient, environmentally friendly, and safe method for cefazolin preparation that can significantly improve product quality has become a pressing technical problem to be solved in this field. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing cefazolin. The method for preparing cefazolin provided by the present invention optimizes the process route and solves the problems of complex process flow, long reaction time, high energy consumption, high environmental pressure, and poor product quality in existing cefazolin preparation processes.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a method for preparing cefazolin, comprising the following steps: S1. Tetrazazole acetic acid and alcohol are subjected to continuous reflux reaction at 78~79℃ under strong acid catalyst conditions. During the continuous reflux reaction, the collected distillate is dried online and then refluxed back into the reaction system. After the reaction is completed, the reaction solution is distilled under reduced pressure to obtain a tetrazazole acetic acid suspension. S2. Under solvent and pH conditions of 6.3-6.5, TDA and the ethyl tetrazolium ethyl acetate suspension are subjected to a condensation reaction using an immobilized acylase as a catalyst to obtain cefazolin acid.

[0008] Compared to existing technologies, the cefazolin preparation method provided by this invention involves a reflux reaction of tetrazolium acetic acid and alcohol. The alcohol and water generated during the reflux reaction are evaporated via azeotropic distillation to form steam. The steam is further liquefied into distillate, and the collected distillate is dried to remove water. This ensures anhydrous conditions in the reaction mixture. Furthermore, the reflux of the distillate back into the reaction system enables continuous preparation of ethyl tetrazolium acetate. This continuous reflux reaction significantly improves the purity and quality of ethyl tetrazolium acetate, thereby facilitating further improvement in the content and preparation quality of cefazolin.

[0009] This invention further utilizes immobilized acylase as a catalyst to catalyze the condensation reaction of TDA and ethyl tetrazolium ethyl acetate suspension. The specific catalyst significantly improves the purity of cefazolin acid, enhances the transmittance of cefazolin acid, and improves the preparation quality of cefazolin acid.

[0010] Preferably, in S1, the alcohol is anhydrous ethanol.

[0011] Preferably, in S1, the strong acid catalyst is concentrated sulfuric acid.

[0012] More preferably, the concentration of the concentrated sulfuric acid is ≥98%.

[0013] Preferably, in S1, the mass ratio of tetrazolium acetic acid to the strong acid catalyst is 12.8:(14~16).

[0014] Preferably, in S1, the mass-to-volume ratio of tetrazolium acetic acid and alcohol is 12.8 g: (550~700) mL.

[0015] Preferably, in S1, the desiccant used for online drying includes anhydrous sodium sulfate and molecular sieve.

[0016] More preferably, the mass ratio of tetrazolium acetic acid to anhydrous sodium sulfate is 12.8:(4~5).

[0017] More preferably, the mass ratio of tetrazolium acetic acid to molecular sieve is 12.8:(2~3).

[0018] This invention further specifies the desiccants used in online drying and the amount of each desiccant. Under specific conditions, the water in the distillate can be completely removed, ensuring anhydrous conditions in the reaction solution and further improving the preparation quality of cefazolin.

[0019] More preferably, the molecular sieve is a 3A molecular sieve.

[0020] It should be further noted that the distillate is refluxed into the reaction system until the reaction is complete.

[0021] Preferably, in S1, the specific conditions for vacuum distillation include: performing vacuum distillation on the reaction system after the reaction is completed; when the liquid becomes viscous, adding water and continuing vacuum concentration at -0.098 MPa and 30°C to collect the remaining ethanol, thereby obtaining the tetrazolium ethyl acetate suspension.

[0022] For example, the mass ratio of tetrazolium acetic acid to water is 12.8:(19~21).

[0023] Preferably, in S1, the mass fraction of ethyl tetrazolium ethyl acetate in the ethyl tetrazolium ethyl acetate suspension is 43%~50%.

[0024] Preferably, in S1, the endpoint of the continuous reflux reaction is when the content of tetrazolium acetic acid in the reaction system is ≤2%.

[0025] Preferably, in S1, the content of anhydrous ethanol in the tetrazolium ethyl acetate suspension is ≤5%.

[0026] Preferably, in S2, the temperature of the condensation reaction is 15~20℃.

[0027] Preferably, in S2, the TDA is 7-amino-3-[([5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]cephalosporin.

[0028] Preferably, in S2, the endpoint of the condensation reaction is that the content of TDA in the reaction system is <3%.

[0029] Preferably, in S2, the immobilized acylase is an immobilized penicillin acylase.

[0030] More preferably, in S2, the immobilized acylase is Novozyme PGA-450.

[0031] Through numerous experiments, the inventors discovered that when immobilized penicillin acylase is selected as the catalyst for the condensation reaction, the purity of cefazolin acid can be significantly improved, the transmittance of cefazolin acid can be improved, and thus the preparation quality of cefazolin acid can be enhanced.

[0032] Preferably, in S2, the mass ratio of TDA to immobilized acylase is 32.5:(35~40).

[0033] Preferably, the mass ratio of TDA to tetrazolium acetic acid is 32.5:(12.8~13.0).

[0034] Preferably, in S2, the solvent is water.

[0035] More preferably, in S2, the mass ratio of TDA to solvent is 32.5:(780~820).

[0036] Preferably, in S2, ammonia is used to adjust the pH to 6.3-6.5.

[0037] Preferably, in step S2, after the condensation reaction is completed, the following post-processing step is further included: Step 1: After the reaction is completed, cool the reaction solution to 0~2℃, adjust the pH to 6.5~7.0, separate the immobilized acylase, and obtain the reaction mother solution; Step 2: Add activated carbon to the reaction mother liquor, filter, and obtain the mother liquor to be crystallized; Step 3: Crystallize the mother liquor to be crystallized at 15~20℃ and pH 2~2.5, filter, wash, and dry to obtain cefazolin acid.

[0038] In a more preferred embodiment, after adjusting the pH to 6.5-7.0 in step 1, stirring for 10-12 minutes is required.

[0039] More preferably, in step 2, the mass ratio of activated carbon to TDA is 32.5:(1.8~2.2).

[0040] In a further preferred embodiment, after adding activated carbon in step 2, it is necessary to stir for 30-35 minutes and then filter.

[0041] More preferably, in step 3, the pH is adjusted to 2-2.5 using a sulfuric acid solution with a mass concentration of 30%-50%.

[0042] The method for preparing cefazolin provided by this invention has a simple process flow and is easy to operate. It can significantly shorten the reaction cycle and reduce production energy consumption, making it more suitable for industrial-scale production.

[0043] The cefazolin acid prepared by this invention has high purity and low impurity content, and the product quality is stable and controllable, effectively improving the quality and safety of the final product. At the same time, this method optimizes the traditional preparation route, overcomes many shortcomings of the existing process, and provides a new idea and reliable technical support for the efficient, green and low-cost preparation of cefazolin acid. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0046] Example 1 This embodiment provides a method for preparing cefazolin, comprising the following steps: S1. Add 12.8g of tetrazolium acetic acid to 600mL of anhydrous ethanol, then slowly add 15g of anhydrous concentrated sulfuric acid, heat to 79℃, and reflux until the content of tetrazolium acetic acid in the reaction system is ≤2%. During the reflux reaction, use 4.5g of anhydrous sodium sulfate and 2.5g of 3A molecular sieve to dry and collect the distillate, and then reflux it into the reaction system until the reaction is complete. S2. After the reaction is completed, the reaction system is subjected to vacuum distillation to remove the ethanol. Then, 20 mL of water is added and stirred evenly. The ethanol is collected under vacuum conditions of -0.098 MPa and the temperature is controlled at ≤30℃. Vacuum collection is stopped when the anhydrous ethanol content is ≤5%, and a tetrazolium ethyl acetate suspension with a mass fraction of 45% is obtained. S3. Add 32.5g TDA to 800mL of water and stir for 10min. Adjust the pH to 6.5 by adding 20wt% ammonia at 20℃. Then add 35g of immobilized penicillin acylase Novozyme PGA-450, along with a suspension of 20wt% ammonia and ethyl tetrazolium. The process takes 30min. After the tetrazolium ethyl acetate suspension was added, the reaction continued, and the pH of the reaction solution was maintained at 6.3-6.5. The reaction endpoint was set at TDA residue of less than 3%, and the reaction time was 160 min. The reaction system was then cooled to 0-2℃, and 20 wt% ammonia was added to adjust the pH to 6.8. The mixture was stirred for 10 min, and the immobilized penicillin acylase was separated to obtain the reaction mother solution. Add 2g of activated carbon to the mother liquor, stir for 30min, filter, and obtain the crystallization solution at 0~2℃. Add 50wt% sulfuric acid to the solution to be crystallized to induce crystallization. The final pH of the crystallization is 2. The temperature is controlled at 15~20℃ during the crystallization process. After filtration, washing and drying, cefazolin acid is obtained.

[0047] Example 2 This embodiment provides a method for preparing cefazolin, comprising the following steps: S1. Add 12.8g of tetrazolium acetic acid to 700mL of anhydrous ethanol, then slowly add 14g of anhydrous concentrated sulfuric acid, heat to 79℃, and reflux until the content of tetrazolium acetic acid in the reaction system is ≤2%. During the reflux reaction, use 4g of anhydrous sodium sulfate and 2g of 3A molecular sieve to dry the collected distillate, and then reflux it into the reaction system until the reaction is complete. S2. After the reaction is complete, the reaction system is subjected to vacuum distillation to remove the ethanol. Then, 19 mL of water is added and stirred evenly. The ethanol is collected under vacuum conditions of -0.098 MPa and the temperature is controlled at ≤30℃. Vacuum collection is stopped when the anhydrous ethanol content is ≤5%, and a tetrazolium ethyl acetate suspension with a mass fraction of 48% is obtained. S3. Add 32.5g TDA to 780mL of water and stir for 10min. Adjust the pH to 6.3 by adding 20wt% ammonia at 15℃. Then add 40g of immobilized penicillin acylase Novozyme PGA-450, along with a suspension of 20wt% ammonia and ethyl tetrazolium. The process takes 30min. After the tetrazolium ethyl acetate suspension was added, the reaction continued, and the pH of the reaction solution was maintained at 6.3-6.5. The reaction endpoint was set at TDA residue of less than 3%, and the reaction time was 180 min. The reaction system was then cooled to 0-2℃, and 20 wt% ammonia was added to adjust the pH to 7. The mixture was stirred for 12 min, and the immobilized penicillin acylase Novozyme PGA-450 was separated to obtain the reaction mother solution. Add 2.2g of activated carbon to the mother liquor, stir for 33min, filter to obtain the crystallization solution, and the temperature is 0~2℃; Add 40wt% sulfuric acid to the solution to be crystallized to induce crystallization. The final pH of the crystallization is 2.5. The temperature is controlled at 15~20℃ during the crystallization process. After filtration, washing and drying, cefazolin acid is obtained.

[0048] Example 3 This embodiment provides a method for preparing cefazolin, comprising the following steps: S1. Add 12.8g of tetrazolium acetic acid to 600mL of anhydrous ethanol, then slowly add 16g of anhydrous concentrated sulfuric acid, heat to 79℃, and reflux until the content of tetrazolium acetic acid in the reaction system is ≤2%. During the reflux reaction, use 5g of anhydrous sodium sulfate and 3g of 3A molecular sieve to dry the collected distillate, and then reflux it into the reaction system until the reaction is complete. S2. After the reaction is complete, the reaction system is subjected to vacuum distillation to remove the ethanol. Then, 20 mL of water is added and stirred evenly. Ethanol is collected under vacuum conditions of -0.098 MPa and temperature ≤30℃. Vacuum collection is stopped when the anhydrous ethanol content is ≤5%, and a tetrazolium ethyl acetate suspension with a mass fraction of 44% is obtained. S3. Add 32.5g TDA to 820mL of water and stir for 10min. Adjust the pH to 6.5 by adding 20wt% ammonia at 20℃. Then add 38g of immobilized penicillin acylase Novozyme PGA-450, along with a suspension of 20wt% ammonia and ethyl tetrazolium. The process takes 30min. After the tetrazolium ethyl acetate suspension was added, the reaction continued, and the pH of the reaction solution was maintained at 6.3-6.5. The reaction endpoint was set at TDA residue of less than 3%, and the reaction time was 170 min. The reaction system was then cooled to 0-2℃, and 20 wt% ammonia was added to adjust the pH to 6.5. The mixture was stirred for 12 min, and the immobilized penicillin acylase Novozyme PGA-450 was separated to obtain the reaction mother solution. Add 2g of activated carbon to the mother liquor, stir for 35min, filter, and obtain the crystallization solution at 0~2℃. Add 40wt% sulfuric acid to the solution to be crystallized to induce crystallization. The final pH of the crystallization is 2. The temperature is controlled at 15~20℃ during the crystallization process. After filtration, washing and drying, cefazolin acid is obtained.

[0049] Example 4 This embodiment provides a method for preparing cefazolin, which differs from Example 1 in that, in S3, the immobilized penicillin acylase Novozyme PGA-450 is replaced with an equal amount of synthetic immobilized penicillin acylase (SIPA-V); The other components and steps are the same as in Example 1, and will not be repeated here.

[0050] Example 5 This embodiment provides a method for preparing cefazolin, which differs from Example 1 in that, in S1, the 3A molecular sieve is replaced with an equal amount of anhydrous sodium sulfate; The other components and steps are the same as in Example 1, and will not be repeated here.

[0051] Comparative Example 1 This comparative example provides a method for preparing cefazolin, which differs from Example 1 in that: in S3, the pH value of the condensation reaction is 6; Specifically, the steps include the following: S1. Add 12.8g of tetrazolium acetic acid to 600mL of anhydrous ethanol, then slowly add 15g of anhydrous concentrated sulfuric acid, heat to 79℃, and reflux until the content of tetrazolium acetic acid in the reaction system is ≤2%. During the reflux reaction, use 4.5g of anhydrous sodium sulfate and 2.5g of 3A molecular sieve to dry and collect the distillate, and then reflux it into the reaction system until the reaction is complete. S2. After the reaction is completed, the reaction system is subjected to vacuum distillation to remove the ethanol. Then, 20 mL of water is added and stirred evenly. The ethanol is collected under vacuum conditions of -0.098 MPa and the temperature is controlled at ≤30℃. Vacuum collection is stopped when the anhydrous ethanol content is ≤5%, and a tetrazolium ethyl acetate suspension with a mass fraction of 45% is obtained. S3. Add 32.5g TDA to 800mL of water and stir for 10min. Adjust the pH to 6 by adding 20wt% ammonia at 20℃. Then add 35g of immobilized penicillin acylase Novozyme PGA-450, along with a suspension of 20wt% ammonia and ethyl tetrazolium. The process takes 30min. After the tetrazolium ethyl acetate suspension was added, the reaction continued, and the pH of the reaction solution was maintained at 6 until the endpoint was reached. The reaction time was about 280 min. The reaction system was cooled to 0~2℃, and 20wt% ammonia was added to adjust the pH to 6.8. The mixture was stirred for 10 min to separate the immobilized penicillin acylase Novozyme PGA-450 and obtain the reaction mother solution. Add 2g of activated carbon to the mother liquor, stir for 30min, filter, and obtain the crystallization solution at 0~2℃. Add 50wt% sulfuric acid to the solution to be crystallized to induce crystallization. The final pH of the crystallization is 2. The temperature is controlled at 15~20℃ during the crystallization process. After filtration, washing and drying, cefazolin acid is obtained.

[0052] Comparative Example 2 This comparative example provides a method for preparing cefazolin, which differs from Example 1 in that: in S3, the pH value of the condensation reaction is 7; Specifically, the steps include the following: S1. Add 12.8g of tetrazolium acetic acid to 600mL of anhydrous ethanol, then slowly add 15g of anhydrous concentrated sulfuric acid, heat to 79℃, and reflux until the content of tetrazolium acetic acid in the reaction system is ≤2%. During the reflux reaction, use 4.5g of anhydrous sodium sulfate and 2.5g of 3A molecular sieve to dry and collect the distillate, and then reflux it into the reaction system until the reaction is complete. S2. After the reaction is completed, the reaction system is subjected to vacuum distillation to remove the ethanol. Then, 20 mL of water is added and stirred evenly. The ethanol is collected under vacuum conditions of -0.098 MPa and the temperature is controlled at ≤30℃. Vacuum collection is stopped when the anhydrous ethanol content is ≤5%, and a tetrazolium ethyl acetate suspension with a mass fraction of 45% is obtained. S3. Add 32.5g TDA to 800mL of water and stir for 10min. Adjust the pH to 7 by adding 20wt% ammonia at 20℃. Then add 35g of immobilized penicillin acylase Novozyme PGA-450, along with a suspension of 20wt% ammonia and ethyl tetrazolium. The process takes 30min. After the tetrazolium ethyl acetate suspension was added, the reaction continued, and the pH of the reaction solution was maintained at 7. The reaction was carried out until the endpoint, and the reaction time was 100 min. The reaction system was cooled to 0~2℃, and 20wt% ammonia was added to adjust the pH to 6.8. The mixture was stirred for 10 min, and the immobilized penicillin acylase Novozyme PGA-450 was separated to obtain the reaction mother solution. Add 2g of activated carbon to the mother liquor, stir for 30min, filter, and obtain the crystallization solution at 0~2℃. Add 50wt% sulfuric acid to the solution to be crystallized to induce crystallization. The final pH of the crystallization is 2. The temperature is controlled at 15~20℃ during the crystallization process. After filtration, washing and drying, cefazolin acid is obtained.

[0053] Comparative Example 3 This comparative example provides a method for preparing cefazolin, which differs from Example 1 in that: in S3, the immobilized penicillin acylase Novozyme PGA-450 is replaced with an equal amount of lipase FLG-LIP-1; The other components and steps are the same as in Example 1, and will not be repeated here.

[0054] Comparative Example 4 This comparative example provides a method for preparing cefazolin, which differs from Example 1 in that: cefazolin is prepared by a chemical method; Specifically, the steps include the following: Add 300 mL of anhydrous dichloromethane to the reaction vessel, then add 32.5 g of TDA and 13 g of tetramethylguanidine. Control the temperature at ≤-5℃ during the process. After the reaction is complete, cool the mixture to -45℃ to obtain solution 1. In another reaction vessel, add 130 mL of anhydrous dichloromethane, 12.8 g of tetrazolium acetic acid, and 11 g of triethylamine, maintaining the temperature ≤ -5℃. Add 0.65 g of 2,6-dimethylpyridine and 30 g of pentanoyl chloride. Control the solution temperature at -32℃. After the reaction is complete, cool to -45℃ to obtain solution 2. Solution 1 was added to solution 2 to carry out a condensation reaction at a controlled temperature of -32℃ until the residual TDA was ≤2.0%. Solution 3 was obtained. Add 300 mL of water to the hydrolysis tank, maintain the temperature at 20 °C, add solution 3, stir for 15 min, adjust the pH of the hydrolysate to 6.0 with triethylamine, stir for 15 min, let stand for 30 min, and separate the phases. Add 220 mL of water to the dichloromethane phase, adjust the pH to 6.0 with triethylamine, stir for 15 min, let stand for 30 min, separate the phases, and combine the aqueous phases with the remaining liquid in the hydrolysis tank.

[0055] Add 2g of disodium ethylenediaminetetraacetate, 3g of sodium metabisulfite, and 7g of activated carbon to the hydrolysis tank, decolorize for 30 minutes, filter, and obtain a liquid with crystals; Add 300 mL of acetonitrile to the crystallization solution and maintain the temperature at 20 °C. Add 20% hydrochloric acid to adjust the pH to 3.5 and stir to grow crystals for 30 min. Then adjust the pH to 1.5 with hydrochloric acid and grow crystals for 30 min. Filter, wash, and dry to obtain cefazolin acid.

[0056] Comparative Example 5 This comparative example provides a method for preparing cefazolin, which specifically includes the following steps: S1. Add 12.8g of tetrazolium acetic acid to 450mL of anhydrous ethanol, then slowly add 15g of anhydrous concentrated sulfuric acid, heat to 79℃, reflux for 2 hours, then vacuum dry the ethanol, then add another 450mL of anhydrous ethanol, heat to 79℃, reflux for 2 hours, then vacuum dry the ethanol. Repeat this process 3-5 times until the content of tetrazolium acetic acid in the reaction system is ≤2%, at which point the reaction ends. S2. After the reaction is completed, the reaction system is subjected to vacuum distillation to remove the ethanol. Then, 20 mL of water is added and stirred evenly. The ethanol is collected under vacuum conditions of -0.098 MPa and the temperature is controlled at ≤30℃. Vacuum collection is stopped when the anhydrous ethanol content is ≤5%, and a tetrazolium ethyl acetate suspension with a mass fraction of 45% is obtained. S3. Add 32.5g TDA to 800mL of water and stir for 10min. Adjust the pH to 6.5 by adding 20wt% ammonia at 20℃. Then add 35g of immobilized penicillin acylase Novozyme PGA-450, along with a suspension of 20wt% ammonia and ethyl tetrazolium. The process takes 30min. After the tetrazolium ethyl acetate suspension was added, the reaction continued, and the pH of the reaction solution was maintained at 6.3-6.5. The reaction endpoint was set at TDA residue of less than 3%, and the reaction time was 160 min. The reaction system was then cooled to 0-2℃, and 20 wt% ammonia was added to adjust the pH to 6.8. The mixture was stirred for 10 min, and the immobilized penicillin acylase was separated to obtain the reaction mother solution. Add 2g of activated carbon to the mother liquor, stir for 30min, filter, and obtain the crystallization solution at 0~2℃. Add 50wt% sulfuric acid to the solution to be crystallized to induce crystallization. The final pH of the crystallization is 2. The temperature is controlled at 15~20℃ during the crystallization process. After filtration, washing and drying, cefazolin acid is obtained.

[0057] Example of effect The content, transmittance, polymer content, and TDA residue of cefazolin prepared in the embodiments and comparative examples of the present invention were tested, and the specific test results are shown in Table 1.

[0058] The content of cefazolin was determined by liquid chromatography. Required reagents: Anhydrous disodium hydrogen phosphate: analytical grade; citric acid: analytical grade; acetonitrile: chromatographic grade Solution preparation: Mobile phase: (1.33g disodium hydrogen phosphate and 1.12g citric acid, dissolved in water and diluted to 1000ml) - acetonitrile (88:12) as the mobile phase; Test method: Chromatographic conditions: Column: C18 Detection wavelength: UV, 254nm Injection volume: 10 μL Test solution: Weigh 4 mg of this product accurately, place it in a 50 ml volumetric flask, add the mobile phase to dissolve and measure to the mark, shake well to prepare the test solution; Reference solution: Weigh 4 mg of cefazolin reference standard accurately, place it in a 50 ml volumetric flask, add the mobile phase to dissolve and measure to the mark, shake well to obtain the reference solution; Measurement method: Accurately inject 10 μL of each of the test solution and reference solution into the liquid chromatograph and record the chromatograms. Calculate the C content in the test solution based on peak area using the external standard method. 14 H 14The content of N8O4S3.

[0059] The transmittance of cefazolin was determined using a spectrophotometer method: Instrument: Ultraviolet-Vis Spectrophotometer Electronic balance Reagent: 2.5% sodium bicarbonate solution Procedure: Dissolve 5.0g of this product in 50mL of 2.5% sodium bicarbonate solution, and perform the ultraviolet-visible spectrophotometry method, measuring the transmittance of the solution at a wavelength of 400nm.

[0060] Polymer content of cefazolin acid by liquid chromatography: Required reagents: Disodium hydrogen phosphate: analytical grade Sodium dihydrogen phosphate: analytical grade; Solution preparation: Mobile phase A: 0.1 mol / L phosphate buffer at pH 7.0 [0.1 mol / L disodium hydrogen phosphate solution - 0.1 mol / L sodium dihydrogen phosphate solution (61:39)] Mobile phase B: Water Test method: Chromatographic conditions Column: Packed with dextran gel G-10 Flow rate: 1.5 mL / min Detection wavelength: 254nm Injection volume: 200 μL Reference solution: Accurately weigh 12.5 mg of cefazolin reference standard, place it in a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, shake well, accurately measure 2 mL of this solution, place it in a 50 mL volumetric flask, add mobile phase to quantitatively dilute to the mark, shake well, and prepare a 10 pg / mL solution.

[0061] Test solution: Accurately weigh 1 g of the test sample, place it in a 50 mL volumetric flask, add an appropriate amount of mobile phase to dissolve it, dilute to the mark with mobile phase, shake well, and inject the sample immediately.

[0062] Determination Procedure: Accurately inject 200 μL of the test solution into the liquid chromatograph using mobile phase A, and record the chromatogram. Separately, accurately inject 200 μL of the reference solution into the liquid chromatograph using mobile phase B, and record the chromatogram. Calculate the content of cefazolin polymer as cefazolin using the external standard method based on peak area.

[0063] TDA residue was determined by liquid chromatography: Chromatographic conditions Instrument: High-performance liquid chromatograph with ultraviolet detector Column: C18 Detection wavelength: UV254nm Flow rate: 1.5 mL / min Column temperature: 35℃ Solution preparation: mobile phase Take 1.33g of anhydrous disodium hydrogen phosphate and 1.12g of citric acid, dissolve them in water and dilute to 1000mL of acetonitrile (90:10) to obtain the product; pH 7.0 phosphate solution Weigh 1.4g of anhydrous disodium hydrogen phosphate and dissolve it in 1000ml of purified water. Adjust the pH to 7.0 with phosphoric acid to obtain the final product. Reference solution Accurately weigh approximately 25 mg of TDA reference standard into a 100 mL volumetric flask, add an appropriate amount of pH 7.0 phosphate buffer to dissolve, dilute the phosphate solution to the mark, accurately transfer 0.5 mL to 100 mL, bring the mobile phase to volume and mix well; the solution is then ready. Test solution Add 1 drop of the reactant to a 50 mL volumetric flask, dissolve it in the mobile phase and bring the volume to 50 mL, then inject the sample. Measurement process Take 20 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and calculate the content of TDA in the reaction solution.

[0064] Table 1

[0065] As shown in Table 1, the ethyl tetrazolium prepared by continuous reflux reaction in Examples 1-5 was used for the enzymatic preparation of cefazolin. Due to the short preparation cycle and short high temperature time of ethyl tetrazolium, the ethyl tetrazolium was of high quality and had few impurities, which in turn made the condensation reaction more complete. The resulting cefazolin had a high content, high transmittance, and few polymer impurities.

[0066] Comparative Example 1 lowered the pH value of the condensation reaction, which inhibited the catalytic activity of the enzyme during the condensation reaction, resulting in a long condensation reaction time and incomplete reaction, high TDA residue, and thus affecting the quality of cefazolin.

[0067] Comparative Example 2 increased the pH value of the condensation reaction, which enhanced both the enzyme's synthetic and hydrolytic activities. The increased synthetic activity accelerated the reaction rate and shortened the reaction time; however, the increased hydrolytic activity led to the hydrolysis of ethyl tetrazolium and cefazolinate, resulting in insufficient ethyl tetrazolium. Consequently, the reaction was incomplete, leading to high TDA residue and affecting product quality.

[0068] Comparative Example 3, which used an immobilized acylate, did not catalyze the reaction.

[0069] Comparative Example 4 uses a chemical method to prepare cefazolin, which is a long process with complex reactions, uses a variety of raw materials in large quantities, and uses a large amount of organic solvents. The reaction is carried out at a low temperature of around -40°C, which has the disadvantages of high energy consumption, high pollution, and poor quality.

[0070] Comparative Example 5 uses a discontinuous reflux reaction to prepare ethyl tetrazolium. This method has a long reflux reaction time, large solvent consumption, and high energy consumption. Furthermore, the long high temperature and time lead to an increase in side reactions and a low yield, resulting in poor quality and insufficient quantity of ethyl tetrazolium. The residual TDA from the condensation reaction is high, and there are many side reactions, which affects the quality of cefazolin.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing cefazolin, characterized in that, Includes the following steps: S1. Tetrazazole acetic acid and alcohol are subjected to continuous reflux reaction at 78~79℃ under strong acid catalyst conditions. During the continuous reflux reaction, the collected distillate is dried online and then refluxed back into the reaction system. After the reaction is completed, the reaction solution is distilled under reduced pressure to obtain a tetrazazole acetic acid suspension. S2. Under solvent and pH conditions of 6.3-6.5, TDA and the ethyl tetrazolium ethyl acetate suspension are subjected to a condensation reaction using an immobilized acylase as a catalyst to obtain cefazolin acid.

2. The method for preparing cefazolin as described in claim 1, characterized in that, In S1, the mass ratio of tetrazolium acetic acid to the strong acid catalyst is 12.8:(14~16). In S1, the alcohol is anhydrous ethanol; In S1, the strong acid catalyst is concentrated sulfuric acid.

3. The method for preparing cefazolin as described in claim 1, characterized in that, In S1, the mass-to-volume ratio of tetrazolium acetic acid and alcohol is 12.8 g: (550~700) mL.

4. The method for preparing cefazolin as described in claim 1, characterized in that, In S1, the desiccant used in the online drying includes anhydrous sodium sulfate and molecular sieves.

5. The method for preparing cefazolin as described in claim 4, characterized in that, The mass ratio of tetrazolium acetic acid to anhydrous sodium sulfate is 12.8:(4~5). The mass ratio of tetrazolium acetic acid to molecular sieve is 12.8:(2~3).

6. The method for preparing cefazolin as described in claim 1, characterized in that, In S1, the mass fraction of ethyl tetrazolium in the ethyl tetrazolium suspension is 43%~50%; In S1, the endpoint of the continuous reflux reaction is when the content of tetrazolium acetic acid in the reaction system is ≤2%; In S1, the content of anhydrous ethanol in the tetrazolium ethyl acetate suspension is ≤5%.

7. The method for preparing cefazolin as described in claim 1, characterized in that, In S2, the temperature of the condensation reaction is 15~20℃; In S2, the endpoint of the condensation reaction is when the content of TDA in the reaction system is <3%.

8. The method for preparing cefazolin as described in claim 1, characterized in that, In S2, the immobilized acylase is an immobilized penicillin acylase.

9. The method for preparing cefazolin as described in claim 1, characterized in that, In S2, the mass ratio of TDA to immobilized acylase is 32.5:(35~40). The mass ratio of TDA to tetrazolium acetic acid is 32.5:(12.8~13.0). In S2, the solvent is water; In S2, the mass ratio of TDA to solvent is 32.5:(780~820).

10. The method for preparing cefazolin as described in claim 1, characterized in that, In S2, after the condensation reaction is completed, the following post-processing steps are also included: Step 1: After the reaction is completed, cool the reaction solution to 0~2℃, adjust the pH to 6.5~7.0, separate the immobilized acylase, and obtain the reaction mother solution; Step 2: Add activated carbon to the reaction mother liquor, filter, and obtain the mother liquor to be crystallized; Step 3: Crystallize the mother liquor to be crystallized at 15~20℃ and pH 2~2.5, filter, wash, and dry to obtain cefazolin acid.

Images