Biosynthesis method of carbonyl reductase and remegapam intermediate

Abstract

The invention relates to the technical field of medical intermediates, in particular to a biosynthesis method of carbonyl reductase and a remegapam intermediate. The amino acid sequence of the carbonyl reductase is shown as SEQ ID NO: 1, the carbonyl reductase can be used for synthesizing a remegapam intermediate, and the specific method is as follows: a compound II is taken as a substrate, and in the presence of the carbonyl reductase, a coenzyme, an enzyme circulating hydrogen donor, a coenzyme circulating enzyme, a cosolvent and a buffer solution, a biological catalytic reaction is performed to generate the remegapam intermediate, namely a compound I; the synthesis method disclosed by the invention is mild in reaction condition, simple and convenient to operate, environment-friendly, low in cost, few in by-products and high in ee value of the product, and efficient synthesis of the remegapam intermediate is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical intermediates, and specifically to a carbonyl reductase and a method for the biosynthesis of rimegepant intermediates. Background Art

[0002] Migraine is a chronic neurovascular disease. Migraine mostly starts in children and adolescence, reaches the peak of incidence in young and middle-aged people, is more common in women, and the ratio of male to female patients is about 1:2 - 3, often with a genetic background.

[0003] Rimegepant is the first small molecule CGRP receptor antagonist with a fast orally disintegrating tablet dosage form (ODT), which treats the root cause of migraine by blocking the CGRP receptor. On February 27, 2020, Biohaven announced that the US FDA approved the company's CGRP receptor inhibitor Nurtec ODT orally disintegrating tablet for the treatment of acute migraine in adults.

[0004] (R)-9-Hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one (Compound I) is a key intermediate in the synthesis route of rimegepant. Therefore, the preparation method of this intermediate has become the focus in the research field of rimegepant.

[0005]

[0006] The main method reported for synthesizing Compound I is to first synthesize Compound II and then further resolve it to obtain Compound I. Patent CN113717103A discloses a preparation method of a ketone compound, which uses a chemical reduction method. Under the action of a rhodium complex catalyst, dichloromethane is used as a solvent and formic acid is used as a hydrogen source to achieve the stereoselective reduction of Compound II. Then, after crystallization and purification with tert-butyl acetate and n-hexane, the ee value of Compound I is about 99.4%. However, the precious metal catalyst used in this method is expensive, difficult to obtain and difficult to recycle and reuse, and industrial production cannot be achieved.

[0007]

[0008] Patent CN114805206A also discloses an industrial preparation method of a rimegepant intermediate with high optical purity. This method also uses Compound II as a raw material and undergoes asymmetric hydrogenation under the action of a Rh-(S-Binap)(COD)BF 4 metal catalyst to obtain a chiral mixture, and then uses chiral camphorsulfonic acid as a resolving agent to perform chiral resolution on the mixed product to obtain Compound I with a single configuration. This method also uses a precious metal catalyst and requires three-step reactions to complete. The entire route process is relatively cumbersome and the yield is not high.

[0009]

[0010] Therefore, there is an urgent need in the art to develop a method for preparing a remegipam intermediate compound I that is low-cost, environmentally friendly, and suitable for industrial production. Summary of the invention

[0011] In view of the above-mentioned deficiencies in the prior art, the present invention provides a carbonyl reductase and a biosynthetic method of a remeditated intermediate which is simple to operate, environmentally friendly and easy to industrialize.

[0012] To achieve the above object, the present invention provides the following technical solutions:

[0013] A carbonyl reductase, whose amino acid sequence is shown in SEQ ID NO: 1.

[0014] The preparation method of the carbonyl reductase comprises the following steps:

[0015] 1) Transformation: The plasmid containing the target gene is transformed into E. coli, and the target gene is amplified by the reproduction and amplification of E. coli. After the plasmid is added to the competent E. coli, it is plated and cultured overnight;

[0016] 2) Seed preservation: Pick a single clone and culture it for 6-8 hours, add 30% sterilized glycerol, and store the strain at -80°C;

[0017] 3) Shake flask culture: Take the preserved bacteria, inoculate them into the shake flask culture medium, and culture them until they are qualified;

[0018] 4) Seed tank culture: inoculate the seed solution from the shake flask into the first-level seed tank and culture until qualified;

[0019] 5) Fermentation tank culture: The cultured seed solution is transferred to a fermentation tank for culture, inducing the synthesis of the target protein, and cultured until qualified;

[0020] 6) Extraction and packaging: The bacterial solution is concentrated, crushed and then packaged.

[0021] A biosynthetic method for a remgipam intermediate comprises: using compound II as a substrate, in the presence of the above-mentioned carbonyl reductase, coenzyme, enzyme cycle hydrogen donor, coenzyme cycle enzyme, cosolvent and buffer, a biocatalytic reaction is carried out to generate a remgipam intermediate, namely compound I; the specific synthesis route is as follows:

[0022]

[0023] Furthermore, the coenzyme is NADP+ or NADPH.

[0024] Furthermore, the coenzyme cycle enzyme is glucose dehydrogenase or isopropanol dehydrogenase.

[0025] Further, the coenzyme cycle hydrogen donor is glucose or isopropanol.

[0026] Further, the cosolvent is any one of isopropanol, DMSO or ethanol.

[0027] Further, the buffer solution is any one of PB buffer solution, phosphate buffer solution or Tris-HCl buffer solution, and the pH is 6.0 - 8.0.

[0028] Further, the mass ratio of the compound II to the carbonyl reductase is 1:0.05 - 1.

[0029] Further, the mass-to-volume ratio of the compound II to the cosolvent is 1 g:10 - 50 mL.

[0030] Further, the reaction temperature is 20 - 30 °C.

[0031] Compared with the prior art, the beneficial effects of the carbonyl reductase and the biosynthetic method of the remimazolam intermediate of the present invention are as follows:

[0032] Compared with the existing method for synthesizing the compound I by a pure chemical route, the biosynthetic method of the present invention does not require extreme catalytic environments such as high temperature and high pressure, and also reduces the use of chemical reagents harmful to humans and the environment, reduces the generation of waste, and is environmentally friendly; more importantly, the enzyme has excellent stereoselectivity, can effectively improve the yield and purity of the product, and has excellent industrialization potential. Detailed implementation manners

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] Example 1 Preparation of carbonyl reductase

[0035] 1) Transformation: The plasmid containing the target gene (the sequence is as shown in SEQ ID NO: 1) is transformed into Escherichia coli, and the target gene is amplified through the reproduction and amplification of Escherichia coli. After adding the plasmid to the Escherichia coli competent cells, it is plated and cultured overnight.

[0036] 2) Preservation of seeds: Pick monoclonal colonies and culture them for 6 - 8 h, add 30% sterilized glycerol, and store the bacterial strains at -80 °C.

[0037] 3) Shake flask culture: Take the stored bacterial strains, inoculate them into the shake flask medium, and culture until qualified.

[0038] 4) Seed tank culture: The shake flask seed liquid is inoculated into the first-stage seed tank and cultured until qualified.

[0039] 5) Fermentation tank culture: The well-cultured seed liquid is transferred to the fermentation tank for culture to induce the synthesis of the target protein and cultured until qualified.

[0040] 6) Extraction and packaging: The bacterial liquid is concentrated, broken, and then packaged.

[0041] Example 2 Preparation of Compound I

[0042]

[0043] Add 30 mL of PB buffer (pH 7.5) to the reaction flask, and dissolve 2 g of the carbonyl reductase obtained in Example 1, 20 mg of NADP+, 60 mg of glucose dehydrogenase enzyme powder, and 12 g of glucose in sequence. Dissolve 10 g of substrate compound II in 200 mL of isopropanol, add it to the reactor, stir and react at 25 °C, detect by HPLC. After the reaction is completed, compound I is obtained with a conversion rate of 98.5%, a purity of 99.5%, and an ee value of 99.7%.

[0044] Example 3 Preparation of Compound I

[0045]

[0046] Add 30 mL of phosphate buffer (pH 7) to the reaction flask, and dissolve 1 g of the carbonyl reductase obtained in Example 1, 20 mg of NADPH, 60 mg of glucose dehydrogenase enzyme powder, and 12 g of glucose in sequence. Dissolve 10 g of substrate compound II in 400 mL of isopropanol, add it to the reactor, stir and react at 30 °C, detect by HPLC. After the reaction is completed, compound I is obtained with a conversion rate of 96.8%, a purity of 99.2%, and an ee value of 99.6%.

[0047] Example 4 Preparation of Compound I

[0048]

[0049] Add 30 mL of PB buffer (pH 7.5) to the reaction flask, and dissolve 3 g of the carbonyl reductase obtained in Example 1, 20 mg of NADP+, 60 mg of isopropanol dehydrogenase enzyme powder, and 12 g of isopropanol in sequence. Dissolve 10 g of substrate compound II in 200 mL of ethanol, add it to the reactor, stir and react at 20 °C, detect by HPLC. After the reaction is completed, compound I is obtained with a conversion rate of 95.1%, a purity of 99.1%, and an ee value of 99.4%.

[0050] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A carbonyl reductase, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the carbonyl reductase according to claim 1, characterized in that: The following steps are involved: 1) Transformation: The plasmid containing the target gene is transformed into E. coli, and the target gene is amplified by the reproduction and amplification of E. coli. After the plasmid is added to the competent E. coli, it is plated and cultured overnight; 2) Seed preservation: Pick a single clone and culture it for 6-8 hours, add 30% sterilized glycerol, and store the strain at -80°C; 3) Shake flask culture: Take the preserved bacteria, inoculate them into the shake flask culture medium, and culture them until they are qualified; 4) Seed tank culture: inoculate the seed solution from the shake flask into the first-level seed tank and culture until qualified; 5) Fermentation tank culture: The cultured seed solution is transferred to a fermentation tank for culture, inducing the synthesis of the target protein, and cultured until qualified; 6) Extraction and packaging: The bacterial solution is concentrated, crushed and then packaged.

3. A biosynthetic method of a remegipam intermediate, characterized in that: Compound II is used as a substrate, and in the presence of the carbonyl reductase, coenzyme, enzyme cycle hydrogen donor, coenzyme cycle enzyme, cosolvent and buffer according to claim 1, a biocatalytic reaction is carried out to generate a remegipam intermediate, namely compound I; the specific synthetic route is as follows:

4. The biosynthetic method of the remegipan intermediate according to claim 3, characterized in that: The coenzyme is NADP+ or NADPH.

5. The biosynthetic method of the Remigipam intermediate according to claim 3, characterized in that: The coenzyme cycle enzyme is glucose dehydrogenase or isopropanol dehydrogenase.

6. The biosynthetic method of the Remigipam intermediate according to claim 3, characterized in that: The coenzyme cycle hydrogen donor is glucose or isopropanol.

7. The biosynthetic method of the remegipam intermediate according to claim 3, characterized in that: The co-solvent is any one of isopropanol, DMSO or ethanol.

8. The biosynthetic method of the Remigipam intermediate according to claim 3, characterized in that: The buffer is any one of PB buffer, phosphate buffer or Tris-HCl buffer, and the pH value is 6.0-8.

0.

9. The biosynthetic method of the Remigipam intermediate according to claim 3, characterized in that: The mass ratio of the compound II to the carbonyl reductase is 1:0.05-1; the mass volume ratio of the compound II to the co-solvent is 1 g:10-50 mL.

10. The biosynthetic method of the Remigipam intermediate according to claim 3, characterized in that: The reaction temperature is 20-30°C.

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