Biosynthesis method of ezetimibe intermediate
Through biological enzyme catalytic technology, the stereoselective synthesis of ZT-5 is achieved in the synthesis of Ezemelb intermediates, solving the problem of by-products and impurities in the prior art, improving yield and purity, and reducing production costs and environmental pollution.
Patent Information
- Application Number
- CN202411703471.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-02-28
AI Technical Summary
The existing chemical synthesis route of Ezemelbow intermediates has the generation of by-products and impurities. The synthesis process is complex and the equipment requirements are high, resulting in high production costs and environmental pollution.
Using biological enzyme catalytic technology, the stereoselective synthesis of the erzemebu intermediate ZT-5 is achieved by screening specific enzyme species, and the conditions for the use of enzymes are optimized to improve the yield and purity of the product.
It significantly improves the selectivity and efficiency of the reaction, reduces the generation of by-products, improves the yield and purity of the product, reduces production costs, and reduces environmental pollution.
Smart Images
Figure CN119530314A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field related to the synthesis of pharmaceutical intermediates, and in particular to a biosynthetic method for an ezetimibe intermediate. Background Art
[0002] Ezetimibe is the first selective cholesterol absorption inhibitor developed by Schering-Plough and Merck. Schering-Plough was acquired by Merck in 2009. The FDA approved Ezetrol tablets launched by Schering-Plough and Vytorin tablets launched by Merck in 2002 and 2004, respectively. Unlike statins, which inhibit cholesterol synthesis in the liver, ezetimibe, as the first selective cholesterol absorption inhibitor, works by selectively inhibiting the absorption of cholesterol in the intestine. The following is a common synthesis route: The above route uses glutaric anhydride and fluorobenzene as raw materials, and obtains the corresponding product through Friedel-Crafts reaction, and then acylates and amidates with chiral auxiliary (S)-4-phenyl-2-oxazolidinone, followed by CBS / BH3 asymmetric reduction, condenses with N-(4-fluorophenyl)-4-hydroxybenzylideneamine under titanium tetrachloride conditions, and then closes the ring under tetrabutylammonium fluoride TBAF and N,O-bistrimethylsilaneacetamide BSA conditions, and finally hydrolyzes and deprotects to obtain ezetimibe. The advantages of this route are that the raw materials are relatively easy to obtain, the synthesis route has a certain degree of maturity, the product quality is controllable to a certain extent, and the yield is relatively high. However, its disadvantages are also obvious. Some by-products and impurities may be produced during the synthesis process, which require strict separation and purification. In addition, the conditions of some synthesis steps are relatively harsh and require high equipment. Summary of the invention
[0003] In view of the above-mentioned shortcomings, the present invention aims to provide a biosynthetic method of an ezetimibe intermediate. In the process of synthesizing the ezetimibe intermediate ZT-5, the bioenzyme catalysis technology is applied to significantly improve the selectivity and efficiency of the reaction. By screening specific enzyme species, the stereoselective synthesis of ZT-5 is achieved, thereby effectively reducing the generation of by-products. By optimizing the conditions for the use of the enzyme, such as temperature, pH value and substrate concentration, the yield and purity of the product are further improved. The bioenzyme method also has the advantages of mild reaction conditions and environmental friendliness, providing a new idea for green chemical synthesis. These research results not only provide a new method for the synthesis of the ezetimibe intermediate ZT-5, but also provide a reference for the biocatalytic synthesis of other complex molecules.
[0004] The technical solution of the present invention is as follows: A biosynthetic method for an ezetimibe intermediate comprises using compound I as a starting material, and synthesizing an ezetimibe intermediate compound II by carbonyl reductase catalysis in the presence of a coenzyme, a coenzyme cycle enzyme, a cosolvent, and a buffer. The reaction is shown below: A biosynthetic method for an ezetimibe intermediate comprises the following steps: adding compound I into a reaction bottle, adding a solvent to dissolve the compound I to obtain a solution of the compound I, dissolving a carbonyl reductase, a coenzyme, and a coenzyme cycle enzyme in a buffer to obtain an enzyme buffer solution, adding the enzyme buffer solution to the solution of the compound I, keeping the temperature to react until the reaction is completed, separating the product, and obtaining compound II, i.e., an ezetimibe intermediate compound ZT-5; The compound I is the intermediate compound ZT-4 of ezetimibe; the carbonyl reductase-producing strain is EscherichiacoliTM1908, and the strain collection number is CCTCCNO: M2019714; The carbonyl reductase producing strain described in the present invention is Escherichia coli TM1908 (EscherichiacoliTM1908), which has been deposited in the China Center for Type Culture Collection (CCTCC) on September 11, 2019, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code: 430072, Registration Number CCTC CNO: M2019714, Classification Name: Escherichia coli TM1908 (Escherichiaco liTM1908).
[0005] The preparation method of the carbonyl reductase of the present invention is as follows: inoculate EscherichiacoliTM1908 in 10 ml LB liquid culture medium, the inoculation amount is 1%, the culture medium contains 1 mg kanamycin, and culture at 37°C, 220 rpm shaking table for 8 hours to obtain seed liquid; The above seed liquid was inoculated into the fermentation enzyme production medium with an inoculation amount of 0.5-2%. Kanamycin with a final concentration of 12 mg / 100 mL was added to the fermentation medium. The culture was shaken at 37°C and 220 rpm until OD600 was 0.6-0.8, and IPTG with a final concentration of 0.5 mM was added. The culture was induced at 18°C for 24 h. After the fermentation broth was induced, the cells were collected by centrifugation at 12000 rpm for 3 min at 4°C. The cells were washed and resuspended in 0.2 M PBbuffer with pH 7.0, and then lyophilized after ultrasonic disruption to obtain lyophilized carbonyl reductase enzyme powder.
[0006] The catalytic reaction equation is shown below:
[0007] Furthermore, the solvent is isopropanol.
[0008] Furthermore, the buffer is PBS buffer or HEPES buffer with a concentration of 0.2-0.25 M and a pH value of 7.1-7.3.
[0009] Furthermore, the volume ratio of the solution of compound I to the enzyme buffer is 1:5-10.
[0010] Furthermore, the mass concentration ratio of the carbonyl reductase to compound I in the reaction system is 1:10-15; Furthermore, the coenzyme cycle enzyme is isopropanol dehydrogenase, glucose dehydrogenase or formate dehydrogenase.
[0011] Furthermore, the mass concentration of the compound I in the system is 10-15%.
[0012] Furthermore, the coenzyme is NADPH, and the mass ratio of the added amount of NADPH to the lyophilized powder of carbonyl reductase is 1:80-500.
[0013] Furthermore, when the compound I is subjected to the carbonyl reductase catalytic reaction, the reaction temperature is maintained at 25-35°C.
[0014] Furthermore, the mass ratio of the coenzyme to the coenzyme cycle enzyme in the reaction system is 1:200-300.
[0015] The beneficial effects of the present invention are: 1. The biosynthesis method adopted by the present invention effectively solves the problems of large-scale use of chemical catalysts and complex synthesis process of ezetimibe intermediates in the prior art, and provides a novel and simple synthesis route; 2. The biosynthesis method adopted by the present invention can reduce the use of chemical catalysts, thereby reducing production costs, while reducing pollution caused by chemical reagents, and at the same time, the reaction selectivity is good, so that the product yield is high, the product purity is high, the raw materials are cheap and easy to obtain, and it is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The following is a schematic diagram of a conventional reaction flow of the synthesis route of ezetimibe; Figure 2 It is a schematic diagram of the reaction route flow chart of the present invention. DETAILED DESCRIPTION
[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0018] Combination Figure 1 to Figure 2 Shown: The above seed liquid was inoculated into the fermentation enzyme production medium with an inoculation amount of 1%. Kanamycin with a final concentration of 10 mg / 100 mL was added to the fermentation medium. The culture was shaken at 37°C and 220 rpm until OD600 was 0.6-0.8, and IPTG with a final concentration of 0.5 mM was added. The culture was induced at 18°C for 24 h. After the fermentation broth was induced, the cells were collected by centrifugation at 12000 rpm for 3 min at 4°C. The cells were washed and resuspended in 0.2 M PBbuffer with pH 7.0, and then lyophilized after ultrasonic disruption to obtain lyophilized carbonyl reductase enzyme powder.
[0019] In a reaction bottle, add 2g of compound I, add 20ml of solvent isopropanol to dissolve, to obtain a solution of compound I, dissolve 2g of carbonyl reductase lyophilized powder, 25mg of coenzyme NADPH, and 5g of coenzyme cycle enzyme isopropanol dehydrogenase in 100ml PBS buffer (concentration is 0.2M, pH value is 7.1-7.3) to obtain an enzyme buffer solution, add the enzyme buffer solution to the solution of compound I, and stir at 25-35°C and 300rpm for 16-18h, terminate the reaction, extract with ethyl acetate to obtain the product compound I, and obtain 1.87g of compound II, i.e., the intermediate compound ZT-5 of ezetimibe, with a yield of 93.5%, an optical purity of 98%, and an ee value of 99.8%.
[0020] In a reaction bottle, add 2g of compound I, add 20ml of solvent isopropanol to dissolve, to obtain a solution of compound I, dissolve 2g of carbonyl reductase lyophilized powder, 4mg of coenzyme NADPH, and 0.8g of coenzyme cycle enzyme isopropanol dehydrogenase in 100ml of PBS buffer (concentration of 0.2M, pH value of 7.1-7.3) to obtain an enzyme buffer solution, add the enzyme buffer solution to the solution of compound I, stir and react at 25-35°C and 300rpm for 16-18h, terminate the reaction, extract with ethyl acetate to obtain the product compound I, and obtain 1.85g of compound II, i.e., the intermediate compound ZT-5 of ezetimibe, with a yield of 92.5%, an optical purity of 98%, and an ee value of 99.7%.
[0021] In a reaction bottle, add 2g of compound I, add 20ml of solvent isopropanol to dissolve, to obtain a solution of compound I, dissolve 2g of carbonyl reductase lyophilized powder, 4mg of coenzyme NADPH, and 1.2g of coenzyme cycle enzyme isopropanol dehydrogenase in 100ml PBS buffer (concentration is 0.2M, pH value is 7.1-7.3) to obtain an enzyme buffer solution, add the enzyme buffer solution to the solution of compound I, and stir at 25-35°C and 300rpm for 16-18h, terminate the reaction, extract with ethyl acetate, and obtain the product compound I. After liquid phase detection, 1.86g of compound II, i.e., the intermediate compound ZT-5 of ezetimibe, is obtained with a yield of 93.0%, an optical purity of 98%, and an ee value of 99.6%.
[0022] In a reaction bottle, add 2g of compound I, add 20ml of solvent isopropanol to dissolve, to obtain a solution of compound I, dissolve 2g of carbonyl reductase lyophilized powder, 4mg of coenzyme NADPH, and 1.2g of coenzyme cycle enzyme isopropanol dehydrogenase in 200ml PBS buffer (concentration is 0.2M, pH value is 7.1-7.3) to obtain an enzyme buffer solution, add the enzyme buffer solution to the solution of compound I, and stir at 25-35°C and 300rpm for 16-18h, terminate the reaction, extract with ethyl acetate, and obtain the product compound I. After liquid phase detection, 1.84g of compound II, i.e., the intermediate compound ZT-5 of ezetimibe, is obtained with a yield of 92.0%, an optical purity of 98%, and an ee value of 99.7%.
[0023] In a reaction bottle, add 2g of compound I, add 20ml of solvent isopropanol to dissolve, to obtain a solution of compound I, dissolve 2g of carbonyl reductase lyophilized powder, 4mg of coenzyme NADPH, and 1.2g of coenzyme cycle enzyme isopropanol dehydrogenase in 100ml of HEPES buffer (concentration of 0.2M, pH value of 7.1-7.3) to obtain an enzyme buffer solution, add the enzyme buffer solution to the solution of compound I, and stir at 25-35°C and 300rpm for 16-18h, terminate the reaction, extract with ethyl acetate, and obtain the product compound I. After liquid phase detection, 1.82g of compound II, i.e., the intermediate compound ZT-5 of ezetimibe, is obtained with a yield of 91.0%, an optical purity of 98%, and an ee value of 99.6%.
[0024] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.
[0025] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A biosynthetic method for an ezetimibe intermediate, characterized in that The method comprises the following steps: adding compound I into a reaction bottle, adding a solvent to dissolve the compound I to obtain a solution of the compound I, dissolving carbonyl reductase, coenzyme, and coenzyme cycle enzyme in a buffer to obtain an enzyme buffer solution, adding the enzyme buffer solution to the solution of the compound I, keeping the temperature to react until the reaction is completed, separating the product, and obtaining compound II, i.e., ezetimibe intermediate compound ZT-5; The compound I is the intermediate compound ZT-4 of ezetimibe; the carbonyl reductase-producing strain is EscherichiacoliTM1908, and the strain collection number is CCTCCNO: M2019714; The reaction equation is as follows: 。 2. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The solvent is isopropanol.
3. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The buffer is PBS buffer or HEPES buffer with a concentration of 0.2-0.25 M and a pH value of 7.1-7.
3.
4. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The volume ratio of the solution of compound I to the enzyme buffer is 1:5-10.
5. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The mass concentration ratio of the carbonyl reductase to the compound I in the reaction system is 1:10-15.
6. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The coenzyme cycle enzyme is isopropanol dehydrogenase, glucose dehydrogenase or formate dehydrogenase.
7. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The mass concentration of the compound I in the system is 10-20%.
8. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The coenzyme is NADPH, and the mass ratio of the added amount of NADPH to the lyophilized powder of carbonyl reductase is 1:80-500.
9. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: When the compound I is subjected to the carbonyl reductase catalytic reaction, the reaction temperature is maintained at 25-35°C.
10. The biosynthetic method of an ezetimibe intermediate according to claim 1, characterized in that: The mass ratio of the coenzyme to the coenzyme cycle enzyme in the reaction system is 1:200-300.