Method for synthesizing mother nucleus of cefvimicin by enzyme method
The method of synthesizing the parent nucleus of cefvecin by enzymatic methods solves the problem that the preparation method in the prior art requires ultra-low temperature conditions and anhydrous and anaerobic operation, and achieves efficient and environmentally friendly synthesis under mild conditions, which is suitable for industrial production.
Patent Information
- Application Number
- CN202311011514.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-11
- Publication Date
- 2025-02-18
AI Technical Summary
The existing preparation method of cefvecin parent nucleus requires ultra-low temperature conditions and anhydrous and anaerobic operation, resulting in high energy consumption, cumbersome operation and low yield in industrial production. At the same time, it generates highly corrosive hydrogen chloride, which has a greater impact on the equipment and the environment. Require.
The method of synthesizing the cefvecin parent nucleus by enzymatic method was used to deprotection of the biprotection parent nucleus Sm, and the immobilized penicillin G acylase was used to remove the amine group protection to obtain the cefvecin parent nucleus. The method is carried out under mild conditions, with simple process, easy equipment matching, and environmentally friendly.
It has achieved efficient synthesis of cefvecin parent core under mild conditions, with a total molar yield of 75-85%, a product purity of more than 96%, environmentally friendly process, equipment friendly, and suitable for industrial production.
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Figure CN119464405A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pharmaceutical synthesis, and in particular relates to a method for synthesizing a cefuroxime nucleus by enzymatic method. Background Art
[0002] Cefovecin was originally developed by SmithKline Bee-cham as a semi-synthetic third-generation cephalosporin. In the mid-1990s, Pfizer Animal Health developed cefovecin sodium salt, i.e., cefovecin sodium, which enabled cefovecin to be produced on a commercial scale. The drug was first approved for use in the treatment of skin infections in cats and dogs in the European Union in 2006, but was not approved for use in the United States for the treatment of skin and soft tissue infections in cats and dogs until 2008.
[0003]
[0004] The synthesis of cefuroxime sodium is difficult, and its key intermediate is the cefuroxime nucleus, the structure of which is as follows:
[0005]
[0006] Currently, the only method for preparing cefuroxime nucleus is the chloramine method:
[0007] That is, the double-protected intermediate Sm is first chlorinated under ultra-low temperature conditions of phosphorus pentachloride, and then the amino protection is removed through the intermediate state of chloroimide and imidate. In the second step, the carboxyl protection is removed with a sulfite salt to obtain the cefuroxime mother nucleus. The route is as follows:
[0008]
[0009] Although this method is a commonly used method for the industrial production of cephalosporin APIs, it requires ultra-low temperature conditions and anhydrous and oxygen-free operations. The industrial energy consumption is extremely high and the operation is cumbersome, and the yield is generally not high. In addition, the reaction generates a large amount of hydrogen chloride, which is highly corrosive, and has high requirements for production equipment, waste solvent recovery, and wastewater treatment.
[0010] Therefore, a more gentle and environmentally friendly method for synthesizing the cefuroxime nucleus is urgently needed. Summary of the invention
[0011] The invention aims to provide a green method for synthesizing cefuroxime nucleus by enzymatic method, which has simple process, mild conditions, easy equipment matching, is environmentally friendly, has high yield, light product color and is suitable for industrial production.
[0012] In order to solve the above technical problems, the technical solution adopted by the present invention is:
[0013] A green method for synthesizing cefuroxime nucleus by enzymatic method comprises the following steps:
[0014] The further improvement of the technical solution of the present invention is:
[0015] A green method for synthesizing cefuroxime nucleus by enzymatic method comprises the following two steps:
[0016] Step 1: The double protected mother core Sm is deprotected by removing the carboxyl protecting group to obtain an aqueous solution of the salt of the intermediate A;
[0017] Step 2: The intermediate A is enzymatically deprotected from the amino group to obtain the cefuroxime nucleus.
[0018]
[0019] The specific steps are as follows:
[0020] Step 1: Add the starting material Sm, acetone and water into the reaction flask, stir and mix, and adjust the pH to neutral with ammonia water;
[0021] Step 2: slowly add a mixture of sulfite and water to the mixed solution in batches, keep the mixture warm after the addition is complete, adjust the pH with ammonia water to keep it neutral, and monitor the progress of the Sm reaction until the Sm reaction is complete;
[0022] Step 3: After the reaction of Sm is complete, acetone is removed by concentration, MIBK is added, and extraction is performed with an alkaline aqueous solution. The aqueous phases are combined and washed with MIBK. The aqueous phases are then distilled under high vacuum and reduced pressure at room temperature to remove part of the water, thereby obtaining an aqueous solution of intermediate A;
[0023] Step 4: add sulfuric acid solution to the aqueous solution of intermediate A obtained in the previous step under stirring to adjust the pH, then add boric acid-borax buffer, keep warm, add immobilized penicillin G acylase, start the reaction, and simultaneously add ammonia solution to adjust the pH of the reaction solution, monitor the reaction progress of intermediate A, until the reaction is complete;
[0024] Step 5: After the reaction is completed, filter, rinse the filter cake with water and store the recovered enzyme in a buffer solution, adjust the pH of the mother liquor to acidic with hydrochloric acid solution, grow crystals after a large amount of solids are precipitated, filter, wash and drain;
[0025] Step 6: Dry under reduced pressure to constant weight to obtain an off-white solid.
[0026] The further improvement of the technical solution of the present invention is that: in step one, the pH is adjusted to 7±0.5, in step two, the pH is adjusted to 7±0.5, and the insulation temperature in step two is 40-45°C.
[0027] A further improvement of the technical solution of the present invention is that the sulfite salt in step 2 includes sodium dithionite, sodium bisulfite, and sodium pyrosulfite.
[0028] A further improvement of the technical solution of the present invention is that the alkaline aqueous solution in step 3 is selected from a mixture of one or more of a sodium bicarbonate aqueous solution, a potassium bicarbonate aqueous solution, and a triethylamine aqueous solution, and the aqueous phases are combined and then washed with MIBK for 1-2 times.
[0029] The technical solution of the present invention is further improved in that: the concentration of the sulfuric acid solution in step 4 is 1-20%, the pH is adjusted to 7.5-8.5 with sulfuric acid, the pH of the boric acid-borax buffer is 7.5-8.5, the insulation temperature is 30±5°C, the mass concentration of the ammonia solution is 5%, and the pH is adjusted to 7.5-8.5 with the ammonia solution.
[0030] A further improvement of the technical solution of the present invention is that the mass concentration of the hydrochloric acid solution in step five is 5-37%, preferably 15-20%, and the hydrochloric acid solution is used to adjust the pH to 3.2±0.3.
[0031] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:
[0032] The process reaction conditions for preparing the cefuroxime core in the present application are mild, avoiding the use of harsh process conditions (ultra-low temperature conditions, anhydrous and oxygen-free operation, extremely high industrial energy consumption and complicated operation); and the process yield of the present application is relatively high, with a total molar yield of 75-85% based on Sm, and a product purity of more than 96%.
[0033] The process of this application is green and environmentally friendly, does not require harsh conditions such as anhydrous and oxygen-free, ultra-low temperature, does not produce highly corrosive substances, is friendly to production equipment, waste solvents are easy to recycle, and wastewater treatment requirements are not high. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the liquid phase spectrum of the cefuroxime core product obtained in Example 1 of the present invention;
[0035] Figure 2 It is the liquid phase spectrum of the cefuroxime mother core obtained as a product in the comparative example of the present invention. DETAILED DESCRIPTION
[0036] The present invention is further described in detail below in conjunction with embodiments:
[0037] Example 1
[0038] Section 1: Preparation of Intermediate A
[0039] Acetone (90 ml) and water (30 ml) were added into the reaction flask and stirred to mix.
[0040] The starting material Sm (15 g) was added into the reaction flask under stirring, and the pH was adjusted to 7 with aqueous ammonia.
[0041] Then, a mixture of sodium hypochlorite (19.95 g) and water (45 ml) was slowly added to the mixed solution in batches. After the addition, the mixture was kept warm at 40-45°C. At the same time, the pH was adjusted to 7 with ammonia water. The progress of the Sm reaction was monitored by TLC until the Sm reaction was complete.
[0042] After the reaction, acetone was removed by concentration, MIBK (100 ml) was added, and the mixture was extracted twice with 7% sodium bicarbonate (100 ml in total). The aqueous phases were combined, and washed once more with MIBK. The aqueous phase was then distilled under high vacuum at room temperature for 60 min. The aqueous solution of intermediate A was directly used for the next step.
[0043] Section 2: Preparation of cefuroxime core
[0044] The aqueous solution of intermediate A obtained in the previous step was added with 10% sulfuric acid under stirring to adjust the pH to 7.8-8.2, and then a boric acid-borax buffer solution with a pH of 8 was added, and the temperature was kept at 30±2°C, and immobilized penicillin G acylase was added to start the reaction, and 5% ammonia water was added to adjust the pH of the reaction solution to 7.8-8.2. The reaction was complete when the pH no longer changed.
[0045] After the reaction is completed, filter, rinse the filter cake with water and keep the recovered enzyme in buffer, adjust the mother liquor to pH = 3.2 ± 0.3 with 17% hydrochloric acid, grow the crystals for 0.5h after a large amount of solid precipitates, filter, wash with purified water, wash with acetone and drain.
[0046] The product was dried under reduced pressure at 35-40°C to constant weight to obtain 6.15 g of an off-white solid.
[0047] Based on SM as the starting material, the two-step molar yield was 79.42%.
[0048] Example 2
[0049] Section 1: Preparation of Intermediate A
[0050] Acetone (90 ml) and water (30 ml) were added into the reaction flask and stirred to mix.
[0051] The starting material Sm (15 g) was added into the reaction flask under stirring, and the pH was adjusted to 7.1 with aqueous ammonia.
[0052] Then, a mixture of hydrosulfite (19.95 g) and water (45 ml) was slowly added to the mixed solution in batches. After the addition was completed, the mixture was kept warm at 40-45°C. At the same time, the pH was adjusted to 7.1 with ammonia water. The progress of the Sm reaction was monitored by TLC until the reaction was complete.
[0053] After the reaction, acetone was removed by concentration, MIBK (100 ml) was added, and the mixture was extracted twice with 5% potassium bicarbonate (100 ml in total). The aqueous phases were combined, and washed once more with MIBK. The aqueous phase was then distilled under high vacuum at room temperature for 60 min. The aqueous solution of intermediate A was directly used for the next step.
[0054] Section 2: Preparation of cefuroxime core
[0055] The aqueous solution of intermediate A obtained in the previous step was added with 10% sulfuric acid under stirring to adjust the pH to 7.8-8.2, and then a boric acid-borax buffer solution with a pH of 8 was added, and the temperature was kept at 33±2°C, and immobilized penicillin G acylase was added to start the reaction, and 5% ammonia water was added to adjust the pH of the reaction solution to 7.8-8.2. The reaction was complete when the pH no longer changed.
[0056] After the reaction is completed, filter, rinse the filter cake with water and keep the recovered enzyme in buffer, adjust the mother liquor to pH = 3.2 ± 0.3 with 17% hydrochloric acid, grow the crystals for 0.5h after a large amount of solid precipitates, filter, wash with purified water, wash with acetone and drain.
[0057] The product was dried under reduced pressure at 35-40°C to constant weight to obtain 5.90 g of an off-white solid.
[0058] Based on SM as the starting material, the two-step molar yield is 76.19%
[0059] Example 3
[0060] Section 1: Preparation of Intermediate A
[0061] Acetone (90 ml) and water (30 ml) were added into the reaction flask and stirred to mix.
[0062] The starting material Sm (15 g) was added into the reaction flask under stirring, and the pH was adjusted to 7 with aqueous ammonia.
[0063] Then, a mixture of hydrosulfite (19.95 g) and water (45 ml) was slowly added to the mixed solution in batches. After the addition, the mixture was kept warm at 40-45°C. At the same time, the pH was adjusted to 7 with ammonia water. The progress of the Sm reaction was monitored by TLC.
[0064] After the reaction, acetone was removed by concentration, MIBK (100 ml) was added, and the mixture was extracted twice with 7% sodium bicarbonate (100 ml in total). The aqueous phases were combined, and washed once more with MIBK. The aqueous phase was then distilled under high vacuum at room temperature for 60 min. The aqueous solution of intermediate A was directly used for the next step.
[0065] Section 2: Preparation of cefuroxime core
[0066] The aqueous solution of intermediate A obtained in the previous step was added with 10% sulfuric acid under stirring to adjust the pH to 8.0-8.4, and then a boric acid-borax buffer solution with a pH of 8.2 was added, and the temperature was kept at 30±2°C, and immobilized penicillin G acylase was added to start the reaction, and 5% ammonia water was added to adjust the pH of the reaction solution to 8.0-8.4. The reaction was complete when the pH no longer changed.
[0067] After the reaction is completed, filter, rinse the filter cake with water and keep the recovered enzyme in buffer, adjust the mother liquor to pH = 3.2 ± 0.3 with 17% hydrochloric acid, grow the crystals for 0.5h after a large amount of solid precipitates, filter, wash with purified water, wash with acetone and drain.
[0068] The product was dried under reduced pressure at 35-40°C to constant weight to obtain 6.21 g of an off-white solid.
[0069] Based on SM as the starting material, the two-step molar yield is 80.19%
[0070] Comparative Example (Imidochloride Method)
[0071] SM (15 g) was added to dichloromethane (150 ml), cooled to -40°C, then phosphorus pentachloride (104.11 g) was added dropwise, and a dichloromethane solution (5 ml) containing 2-methylpyridine (1.22 ml) was added while maintaining the temperature between -40°C and 30°C. The mixture was stirred for 1 hour, and then isopropanol (52 ml) was added. The reaction mixture was heated to 22°C, filtered and dried to obtain intermediate A (5.8 g, yield 45.8%).
[0072] The intermediate B (5.0 g), water (7.5 ml), and acetone (22.5 ml) obtained in the previous step were combined to form a slurry. The pH of the slurry was adjusted to 7 with aqueous ammonia. Sodium bisulfite (8 g, suspension) in water (10 ml) was added to the resulting solution. The pH of the resulting solution was adjusted to 7 with aqueous ammonia while maintaining the temperature between 40°C and 45°C. After stirring at 45°C for 1 hour, the pH was adjusted to 3.5 again with a saline solution. Filter and dry to obtain a khaki to light yellow substance (2.51 g, yield 82.1%)
[0073] Taking SM as the starting material, the two-step molar yield is 37.6% (=45.8%*82.1%)
[0074] The cefuroxime nucleus obtained in Example 1 and the comparative example was tested by HPLC. The test results are as follows: Figure 1 , 2As shown, it can be seen that the retention time of the main component of Example 1 is 4.429, and the peak area % is 96.19%, and the retention time of the main component of the comparative example is 4.412, and the peak area % is 88.63%. It can be clearly seen from the figure that the product obtained in the comparative example has a lot of impurity peaks and poor product purity. By comparing the appearance of the products, the products obtained in the comparative example are darker in color, and are khaki to light yellow solids, while the products obtained in the examples are all off-white solids.
[0075] Experiment Name Molar yield purity color Comparative Example 37.6％ 88.63％ Earthy yellow to light yellow substance Example 1 79.42 96.19％ Off-white solid Example 2 76.19％ 96.78％ Off-white solid Example 3 80.19％ 96.06％ Off-white solid
Claims
1. A method for enzymatically synthesizing cefuroxime nucleus, characterized in that It includes the following two sections: Step 1: The double-protected mother core Sm is deprotected by removing the carboxyl protecting group to obtain an aqueous solution of the salt of the intermediate A; Step 2: The intermediate A is enzymatically deprotected from the amino group to obtain the cefuroxime nucleus.
2. The method for enzymatically synthesizing cefuroxime nucleus according to claim 1, characterized in that The following steps are involved: Step 1: Add the starting material Sm, acetone and water into the reaction flask, stir and mix, and adjust the pH to neutral with ammonia water; Step 2: slowly add a mixture of sulfite and water to the mixed solution in batches, keep the mixture warm after the addition is complete, adjust the pH with ammonia water to keep it neutral, and monitor the progress of the Sm reaction until the Sm reaction is complete; Step 3: After the reaction of Sm is complete, acetone is removed by concentration, MIBK is added, and extraction is performed with an alkaline aqueous solution. The aqueous phases are combined and washed with MIBK. The aqueous phases are then distilled under high vacuum and reduced pressure at room temperature to remove part of the water, thereby obtaining an aqueous solution of intermediate A; Step 4: add sulfuric acid solution to the aqueous solution of intermediate A obtained in the previous step under stirring to adjust the pH, then add boric acid-borax buffer, keep warm, add immobilized penicillin G acylase, start the reaction, and simultaneously add ammonia solution to adjust the pH of the reaction solution, monitor the reaction progress of intermediate A, until the reaction is complete; Step 5: After the reaction is completed, filter, wash the filter cake with water and store the recovered enzyme in a buffer solution, adjust the pH of the mother liquor to acidic with hydrochloric acid solution, grow crystals after a large amount of solids are precipitated, filter, wash and drain; Step 6: Dry under reduced pressure to constant weight to obtain an off-white solid.
3. A method for enzymatically synthesizing cefuroxime nucleus according to claim 2, characterized in that: In step 1, the pH is adjusted to 7±0.5, in step 2, the pH is adjusted to 7±0.5, and in step 2, the insulation temperature is 40-45°C.
4. A method for enzymatically synthesizing cefuroxime nucleus according to claim 2, characterized in that: The sulfite salts in step 2 include sodium dithionite, sodium bisulfite, and sodium pyrosulfite.
5. The method for enzymatically synthesizing cefuroxime nucleus according to claim 2, characterized in that: In step 3, the alkaline aqueous solution is selected from a mixture of one or more of a sodium bicarbonate aqueous solution, a potassium bicarbonate aqueous solution, and a triethylamine aqueous solution. The aqueous phases are combined and then washed with MIBK for 1-2 times.
6. The method for enzymatically synthesizing cefuroxime nucleus according to claim 2, characterized in that: In step 4, the concentration of the sulfuric acid solution is 1-20%, and the pH is adjusted to 7.5-8.5 with sulfuric acid. The pH of the boric acid-borax buffer is 7.5-8.
5. The insulation temperature is 30±5°C. The mass concentration of the ammonia solution is 5%, and the pH is adjusted to 7.5-8.5 with the ammonia solution.
7. A method for enzymatically synthesizing cefuroxime nucleus according to claim 2, characterized in that: In step 5, the mass concentration of the hydrochloric acid solution is 15-20%, and the hydrochloric acid solution is used to adjust the pH to 3.2±0.3.