Enzymatic synthesis method of transaminase and remegapam intermediate

Abstract

The invention relates to the technical field of medical intermediates, in particular to an enzymatic synthesis method of transaminase and remegapam intermediates. The amino acid sequence of the transaminase is shown as SEQ ID NO: 1, the transaminase can be used for synthesizing a remegapam intermediate, and the specific method comprises the following steps: by taking a compound IV as a starting raw material, in the presence of a buffer solution, a cosolvent, a coenzyme PLP and an amino donor, carrying out catalytic conversion by the transaminase to obtain a compound V; the preparation of the remegapam intermediate compound V is realized by utilizing transaminase, and the preparation method is simple and convenient to operate, mild in reaction condition, green, environment-friendly, high in yield, good in stereoselectivity, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical intermediates, and specifically relates to an enzyme-catalyzed synthesis method of a transaminase and a remegipan intermediate. Background Art

[0002] Migraine is a chronic neurovascular disease, currently the third most common disease in the world, and the economic burden caused by the disease ranks second among all brain diseases. It is the main cause of disability. The global population suffering from migraine accounts for more than 13% of the total population. Migraine often starts in childhood and adolescence, with the peak incidence in middle-aged and young people. It is more common in women, with a male-female ratio of about 1:2-3, and often has a genetic background.

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

[0004] Compound V is a key intermediate in the synthesis route of Remigipam. Therefore, the preparation method of this intermediate has become the focus of Remigipam research.

[0005]

[0006] Patent WO2011046997A discloses that compound IV is used as a raw material to generate compound V through reduction, chlorination, azide and other reactions (the synthesis route is as follows). This route has many reaction steps and is cumbersome. A large number of chiral impurities are introduced during the synthesis process, resulting in low quality of the final product. In addition, highly toxic and explosive chemicals such as sodium azide are used in the process, which brings great EHS risks to industrial production.

[0007]

[0008] The patent also introduces another preparation method for the intermediate compound V-1 of Remgipam (the synthetic route is as follows), which uses compound IV as a raw material, uses tetraisopropoxytitanium as a catalyst to generate an imine intermediate, and then uses palladium to catalyze hydrogenation to amino groups. The carbonyl group is converted into amino groups through an amination reaction. The product yield obtained in this process is low, and the subsequent processing steps are complicated, which seriously affects the yield of the final product Remgipam.

[0009]

[0010] Patent CN116768938A reports a method for preparing compound V (synthesis route is as follows):2+ In the presence of EDTA / EDTA catalyst, a mixture of compound V is obtained by asymmetric reduction amination, and then dextrorotatory camphorsulfonic acid is used as a chiral resolution agent to prepare a single-configuration compound V. However, the entire preparation process is cumbersome, and the purity and yield of the obtained compound V are low.

[0011]

[0012] Therefore, there is an urgent need in the art to develop a method for preparing a remegipam intermediate compound V that is simple in process, environmentally friendly, and suitable for industrial production. Summary of the invention

[0013] The first object of the present invention is to provide a transaminase.

[0014] The second object of the present invention is to provide an enzyme-catalyzed synthesis method of a remegipam intermediate.

[0015] To achieve the above object, the present invention provides the following technical solutions:

[0016] A transaminase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0017] The preparation method of the transaminase is:

[0018] 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;

[0019] 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;

[0020] 3) Shake flask culture: Take the preserved bacteria, inoculate them into the shake flask culture medium, and culture them until they are qualified;

[0021] 4) Seed tank culture: inoculate the seed solution from the shake flask into the first-level seed tank and culture until qualified;

[0022] 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;

[0023] 6) Extraction and packaging: The bacterial solution is concentrated, crushed and then packaged.

[0024] An enzymatic synthesis method of a remegipam intermediate, specifically: using compound IV as a starting material, in the presence of a buffer, a cosolvent, a coenzyme PLP (pyridoxal phosphate), and an amino donor, converting it into compound V by transaminase catalysis, wherein the reaction route is:

[0025]

[0026] Furthermore, the co-solvent is selected from any one of methanol, ethanol, butyl acetate or DMSO.

[0027] Furthermore, the amino donor is selected from one or more of alanine, triethanolamine, isopropylamine, propylamine, ethylamine, tryptophan, butylamine or α-aminoglutaric acid.

[0028] Furthermore, the buffer is any one of phosphate buffer, Tris-HCl buffer, isopropylamine hydrochloride buffer or triethanolamine hydrochloride buffer.

[0029] Furthermore, the mass ratio of compound IV to transaminase is 1:1-10.

[0030] Furthermore, the mass ratio of the transaminase to the coenzyme PLP is 5 to 30:1.

[0031] Furthermore, the molar ratio of the compound IV to the amino donor is 1:10-30.

[0032] Furthermore, the reaction temperature is 30-50°C.

[0033] Compared with the prior art, the enzymatic synthesis method of the transaminase and the intermediate of Remigipam of the present invention has the following beneficial effects:

[0034] The invention obtains a transaminase for catalytic conversion of compound IV, and the transaminase realizes efficient synthesis of remegipam intermediate compound V. The synthesis method is simple to operate, has mild reaction conditions, is green and environmentally friendly, has high yield, good stereoselectivity, low cost, and is suitable for industrial production. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0036] Example 1 Preparation of transaminase

[0037] 1) Transformation: The plasmid containing the target gene (sequence shown in SEQ ID NO: 1) 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.

[0038] 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.

[0039] 3) Shake flask culture: Take the preserved bacterial strain, inoculate it into the shake flask culture medium, and culture it until it is qualified.

[0040] 4) Seed tank culture: Inoculate the seed liquid from the shake flask into the first-level seed tank and culture until it is qualified.

[0041] 5) Fermentation tank culture: The cultured seed liquid is transferred to a fermentation tank for culture to induce the synthesis of the target protein and culture until it is qualified.

[0042] 6) Extraction and packaging: The bacterial solution is concentrated, crushed and then packaged.

[0043] Example 2 Synthesis of Compound V

[0044]

[0045] 300 mL DMSO, 600 mL Tris-HCl buffer, compound IV (20 g, 0.045 mol), isopropylamine (53.2 g, 0.9 mol) and pyridoxal phosphate (5.3 g) were added to the reactor, the reaction temperature was controlled at 40°C, transaminase (80 g) was added, and the pH of the reaction system was maintained at 8.5-9.0 with 10% sodium hydroxide solution until the reaction was complete.

[0046] After the reaction, concentrated hydrochloric acid was added dropwise to adjust the pH of the reaction solution to 1-2, sodium chloride was added, suction was filtered, and the filter cake was washed with dilute hydrochloric acid (0.1M). The pH of the filtrate was adjusted to 8.5-9.0 with 10% sodium hydroxide solution, and then extracted with ethyl acetate, concentrated under reduced pressure, and then methyl tert-butyl ether was added, cooled to 5-10°C, solids were precipitated, filtered, and dried to obtain compound V with a yield of 95.9%, a purity of 99.8%, and an ee value of 99.97%.

[0047] Example 3 Synthesis of Compound V

[0048]

[0049] 300 mL DMSO, 600 mL phosphate buffer, compound IV (20 g, 0.045 mol), isopropylamine (39.9 g, 0.675 mol) and pyridoxal phosphate (6.5 g) were added to the reactor, the reaction temperature was controlled at 35°C, transaminase (130 g) was added, and the pH of the reaction system was maintained at 8.5-9.0 with 10% sodium hydroxide solution until the reaction was complete.

[0050] After the reaction, concentrated hydrochloric acid was added dropwise to adjust the pH of the reaction solution to 1-2, sodium chloride was added, suction was filtered, and the filter cake was washed with dilute hydrochloric acid (0.1M). The pH of the filtrate was adjusted to 8.5-9.0 with 10% sodium hydroxide solution, and then extracted with ethyl acetate, concentrated under reduced pressure, and then methyl tert-butyl ether was added, the temperature was lowered to 5-10°C, solids were precipitated, filtered, and dried to obtain compound V with a yield of 93.1%, a purity of 99.6%, and an ee value of 99.94%.

[0051] Example 4 Synthesis of Compound V

[0052]

[0053] 300 mL of methanol, 600 mL of triethanolamine hydrochloride buffer, compound IV (20 g, 0.045 mol), isopropylamine (66.5 g, 1.125 mol) and pyridoxal phosphate (4 g) were added to the reactor, the reaction temperature was controlled at 45°C, transaminase (60 g) was added, and the pH of the reaction system was maintained at 8.5-9.0 with 10% sodium hydroxide solution until the reaction was complete.

[0054] After the reaction is completed, concentrated hydrochloric acid is added dropwise to adjust the pH of the reaction solution to 1-2, sodium chloride is added, suction is filtered, and the filter cake is washed with dilute hydrochloric acid (0.1M). The pH of the filtrate is adjusted to 8.5-9.0 with 10% sodium hydroxide solution, and then extracted with ethyl acetate, concentrated under reduced pressure, and then methyl tert-butyl ether is added, the temperature is lowered to 5-10°C, a solid is precipitated, filtered, and dried to obtain compound V with a yield of 90.7%, a purity of 99.5%, and an ee value of 99.92%.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

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

1.

2. The method for preparing the transaminase 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. An enzyme-catalyzed synthesis method of a remegipam intermediate, characterized in that: Compound IV is used as a starting material, and in the presence of a buffer, a cosolvent, a coenzyme PLP, and an amino donor, it is converted into compound V by the transaminase of claim 1, and the reaction route is:

4. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The co-solvent is selected from any one of methanol, ethanol, butyl acetate or DMSO.

5. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The amino donor is selected from one or more of alanine, triethanolamine, isopropylamine, propylamine, ethylamine, tryptophan, butylamine or α-aminoglutaric acid.

6. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The buffer is any one of phosphate buffer, Tris-HCl buffer, isopropylamine hydrochloride buffer or triethanolamine hydrochloride buffer.

7. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The mass ratio of the compound IV to the transaminase is 1:1-10.

8. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The mass ratio of the transaminase to the coenzyme PLP is 5 to 30:

1.

9. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The molar ratio of the compound IV to the amino donor is 1:10-30.

10. The enzymatic synthesis method of the Remigipam intermediate according to claim 3, characterized in that: The reaction temperature is 30-50°C.

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