Method for preparing zalvizepam chiral intermediate by enzyme method

Abstract

The invention discloses a method for preparing a zalvizepam chiral intermediate by an enzyme method. According to the method disclosed by the invention, a compound II is used as a substrate and is converted into a compound I under the catalytic action of transaminase. According to the method disclosed by the invention, the concentration of a conversion substrate can reach 50g / L, the conversion rate reaches 98.5%, and the ee value of the product reaches 99.9%. The method disclosed by the invention is simple to operate and good in selectivity, and can be used for industrial production.

Description

Technical Field:

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to a method for enzymatically preparing a chiral intermediate of zavegepant. Background Art:

[0002] Zavegepant was developed by Bristol-Myers Squibb Company and was launched in the United States on March 9, 2023, under the trade name Zavzpret. It is mainly used for the treatment of acute migraine in adults with or without aura. Its chemical structural formula is shown as follows:

[0003]

[0004] Bristol-Myers Squibb Company disclosed a preparation method of zavegepant in the literature Organic Process Research & Development, 2012, 16(12), 1953 - 1966. The route is as shown in Scheme 1. This method uses compound 1 as the starting material, first reacts with tert-butyl nitrite to construct an indazole ring, and then obtains compound II through reactions such as halogen-metal exchange, Erlenmeyer- ring opening, hydrolysis, etc. Then, nearly 30 g / L of compound II is transformed for 72 hours under the action of transaminase to obtain compound I. The yield of this enzymatic reaction step is 77%, and the ee value is 99%. Subsequently, compound I undergoes multiple steps of reaction to generate zavegepant.

[0005]

[0006]

[0007] It can be seen from Scheme 1 reported by the original research company that (R)-2-amino-3-(7-methyl-1H-indazol-5-yl)propanoic acid (compound I, CAS is 1061710-38-0) is the key chiral intermediate for preparing zavegepant.

[0008] Patent WO2022172829A1 discloses a method for preparing compound I by multi-enzyme catalysis. The route is as shown in Scheme 2. In this method, compound 3 is obtained through 2 steps of reaction to get the enzymatic substrate compound 10, and then undergoes hydrolysis reaction under the action of allantoinase, decarbamoylase, allantoin racemase, etc. to generate compound I. This reaction involves 3 enzymes, requires a relatively large amount of cells, with a yield of 92.4%, a conversion rate of 96.7%, and an ee value of 99.0%.

[0009]

[0010] Therefore, we need to develop a method for preparing the chiral intermediate (Compound I) of zavilgipam, which is simple to operate, has a high yield, low cost, and is easy to industrialize, and then prepare zavilgipam. Summary of the Invention:

[0011] The object of the present invention is to provide a method for preparing the chiral intermediate I of zavilgipam, which is simple to operate, low in cost, and easy to industrialize, in view of the deficiencies of the prior art.

[0012] The technical solution adopted by the present invention is as shown in Scheme 3:

[0013]

[0014] The present invention provides a method for preparing the chiral intermediate I of zavilgipam by an enzymatic method, which specifically includes the following steps: using Compound II as a substrate and converting it into Compound I under the catalytic action of a transaminase.

[0015] Further, the transaminase is derived from Sciscionella sp., and the NCBI accession number of the amino acid sequence is WP_031466213.1.

[0016] Further, the amino acid sequence and nucleotide sequence of the transaminase are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0017] Further, the transaminase participates in the catalytic reaction in the form of transaminase enzyme powder, transaminase enzyme supernatant, transaminase homogenate, cells containing transaminase, immobilized cells, immobilized enzyme, etc., and preferably transaminase cells.

[0018] Further, the transaminase expression receptor strain is selected from Escherichia coli, yeast, Streptomyces, or Bacillus subtilis, and preferably Escherichia coli.

[0019] Further, isopropylamine needs to be added to the reaction, and the molar ratio of isopropylamine to Compound II is 1:1 to 7:1, preferably 3:1.

[0020] Further, the pH of the reaction needs to be controlled at 6.0 to 11.0, preferably pH 8.0 to 9.0.

[0021] The beneficial effect of the present invention is that the present invention discloses a method for preparing the chiral intermediate I of zavilgipam by an enzymatic method. The transaminase used in this method can selectively convert Compound II into Compound I. The conversion substrate concentration can reach 50 g / L, the conversion rate reaches 98.5%, and the ee value of the product reaches 99.9%. This method is simple to operate, has high atom economy, high yield, and can be used for industrial production. Brief Description of the Drawings

[0022] Figure 1 Expression Protein Map of Example 2 SS-ATA

[0023] Figure 2 HPLC Chromatogram of Enzymatic Catalyzed Transformation in Example 4

[0024] Figure 3 Chiral HPLC Chromatogram of Example 4 Detailed Implementation Manner

[0025] The technical content of the present invention will be further elaborated below in conjunction with specific embodiments, with the aim of better understanding the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0026] Example 1 Scanning of ATA Enzyme Library

[0027] The enzymes in the transaminase enzyme library of Shangke Biopharmaceuticals (Shanghai) Co., Ltd. were used to screen the reaction of the substrate. The parameters of the reaction system are shown in Table 1. The amino donor (±)-phenethylamine and buffer PBS were sequentially added to the reaction kettle, and the pH value was adjusted to 9.0 with 6M HCl. Then, the substrate compound II (dissolved in DMSO), coenzyme PLP, and transaminase cells were sequentially added, and the oscillating reaction was carried out at 30 °C. After 24 hours of reaction, the reaction was terminated, and the sample was taken for TLC plate analysis. When the developing agent was dichloromethane:methanol = 1:1, the product and the substrate could be separated. The TLC plate results showed that there were multiple obvious product spots. The chiral analysis of the product was sent for HPLC detection, and it was found that the chiral ee value of the product catalyzed by the transaminase (SS-ATA) derived from Sciscionella sp. reached 95.40%, and the conversion rate reached 68.02%.

[0028] Table 1 Parameters of the ATA Cell Library Scanning Reaction System

[0029] Component (stock solution concentration) Volume of stock solution added in 100 μl PBS (pH 7.0, 0.1 M) 90 μl (±)-phenylethylamine 2.13 mg Substrate (100 g / L in DMSO) 10 μl PLP 0.0005 mg ATA enzyme 5 mg

[0030] Example 2 Expression of SS-ATA Enzyme

[0031] The SS-ATA bacterial strain was transferred into an LB test tube with Amp + resistance and cultured overnight at 37 °C. Then, the seed solution was transferred to a 2YT medium containing Amp + resistance at an inoculation amount of 1.5%. The bacteria were cultured at 37 °C. When the biomass OD 600 value reached about 0.8, IPTG induction was carried out, and the temperature was reduced to 25 °C. After 16 hours of expression, the bacterial cells were collected, and the cells were broken by ultrasound for electrophoresis analysis. The results are shown in Figure 1 , and the size of the protein was consistent with the expected value, and there was good soluble expression.

[0032] Optimization of Reaction Conditions in Example 3 - Selection of Isopropylamine Concentration

[0033] Optimize the dosage of isopropylamine in the conversion process. The ratio of isopropylamine to the substrate is 3:1, 5:1, and 7:1. Add isopropylamine and 9.4 mL of water to the reaction kettle in sequence. Then, adjust the pH value to about 9.0 with HCl, and then add 50 μL of coenzyme PLP (2.0 g / L), 0.3 g of substrate (30 g / L), and 0.5 g of transaminase. React with shaking at 30 °C. Monitor the reaction by TLC plate until the reaction ends. Take samples and send them for HPLC detection. Analyze the reaction results. The specific data are shown in Table 2. The screening results show that when the ratio of isopropylamine to water is 3:1, the conversion effect is the best, the conversion rate is 92.00%, and the ee value is 98.98%.

[0034] Table 2 Optimization Results at Different Isopropylamine Concentrations

[0035]

[0036] Optimization of Reaction Conditions in Example 4 - Selection of pH

[0037] Optimize the pH in the conversion process. The pH is set to 6.0 - 10.0. Based on the optimization results of Example 3, add 0.27 g of isopropylamine and 3.6 mL of water to the reaction kettle in sequence. Then, adjust the pH value with HCl, and then add 20 μL of coenzyme PLP (2.0 g / L), 0.2 g of substrate (50 g / L), and 0.2 g of transaminase. React with shaking at 30 °C. Monitor the reaction by TLC plate until the reaction ends. Take samples and send them for HPLC detection. Analyze the reaction results. The specific data are shown in Table 3. The screening results show that when the pH is 8.0, the conversion rate can reach 98.50%, and the ee value can reach 99.92%. The HPLC spectrum of substrate conversion is as shown in Figure 2 shown, and the chiral HPLC spectrum is as shown in Figure 3 shown.

[0038] Table 3 Optimization Results at Different pH Values

[0039]

Claims

1. A method for preparing the chiral intermediate compound I of zavilapam, characterized in that, This method uses compound II as a substrate and converts it into compound I under the catalysis of a transaminase, and the amino acid sequence of the transaminase is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, wherein The nucleotide sequence of the transaminase is shown in SEQ ID NO.

2.

3. The preparation method according to claim 1, characterized in that, The transaminase is derived from Sciscionella sp.

4. The preparation method according to claim 1, characterized in that, The transaminase participates in the catalytic reaction in the form of enzyme powder, enzyme clear liquid, homogenate, cells containing the enzyme, immobilized cells, immobilized enzyme, etc.

5. The preparation method according to claim 1, characterized in that, The transaminase expression strain is selected from Escherichia coli, yeast, Streptomyces or Bacillus subtilis.

6. The preparation method according to claim 1, characterized in that, The concentration of the substrate is 1 - 50 g / L.

7. The preparation method according to claim 1, wherein Isopropylamine needs to be added in the reaction, and the molar ratio of isopropylamine to compound II is 1:1 - 7:

1.

8. The preparation method according to claim 1, characterized in that, The reaction needs to control the pH to be 6.0 - 11.0.

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