Clopidogrel chiral intermediate and enzyme catalytic conversion method thereof

Abstract

The invention discloses a clopidogrel chiral intermediate and an enzyme catalytic conversion method thereof, and the enzyme catalytic conversion method comprises the following steps: modifying nano silicon dioxide particles by using hydrophilic silane and hydrophobic silane to obtain modified silicon dioxide nano particles; taking the obtained modified nano silicon dioxide particles as a stabilizer, dissolving (R, S)-o-chlorophenylglycine methyl ester in a solvent to serve as an oil phase, taking a buffer solution containing protease as a water phase, and emulsifying through a homogenizer to obtain a Pickering emulsion enzyme catalysis reaction system; and putting the obtained reaction system on a constant-temperature shaking table to react, after the reaction is finished, carrying out centrifugal demulsification and separation to obtain an oil phase and a water phase, and concentrating and crystallizing the oil phase to obtain (S)-2-chlorophenylglycine methyl ester. According to the invention, the direct conversion of the enantiomer is realized under mild conditions, the utilization rate of the raceme is improved, and the enantiomer excess value of the obtained S-type isomer is high.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a clopidogrel chiral intermediate and an enzyme-catalyzed conversion method thereof. Background Art

[0002] As an inhibitor of platelet aggregation, clopidogrel is mainly used for patients with recent myocardial infarction, recent ischemic stroke or confirmed peripheral arterial disease and acute coronary syndrome. It is also an irreplaceable basic drug in the treatment of acute coronary syndrome. The chiral raw material (S)-o-chlorophenylglycine methyl ester in the production of clopidogrel is generally first methylated by racemic o-chlorophenylglycine, and then split by L-(+) tartaric acid to obtain a single-configuration optically active isomer. Benzaldehyde is used as a catalyst to increase the reaction rate and promote the effective kinetic splitting. However, this method has problems such as time-consuming operation, high reaction temperature, and large amount of solvent.

[0003] As an efficient catalyst, biological enzymes have been well applied in the synthesis of chiral intermediates. In Chinese patent CN101864464A, immobilized penicillin G acylase (PGA) is used to selectively hydrolyze the racemate N-phenylacetyl-(R, S)-o-chlorophenylglycine to (S)-o-chlorophenylglycine, and then the (S)-o-chlorophenylglycine methyl ester is obtained by methyl esterification, while the (R)-N-phenylacetyl-o-chlorophenylglycine configuration remains unchanged, and after racemization, it can continue to be split with modified enzymes. This method shows that enzymes, as a green and environmentally friendly catalyst, provide a simple process and low cost method for the preparation of chiral clopidogrel intermediates, but the method requires acylation of the amino group first, which increases the reaction process. In Chinese patent CN104293875A, immobilized penicillin G acylase is used to directly react with racemic 2-chlorophenylglycine methyl ester to prepare (S)-2-chlorophenylglycine, thereby avoiding the protection and deprotection of reactive groups. However, this method demethylates o-chlorophenylglycine methyl ester, while the intermediate required for the synthesis of clopidogrel is an esterification product. There is a subsequent methylation step, which increases the operation links. In addition, the utilization rate of the hydrolysis product is not high and the yield is low. Summary of the invention

[0004] In view of the shortcomings of the existing methods, the present invention provides a clopidogrel chiral intermediate and an enzyme-catalyzed conversion method thereof.

[0005] To achieve the above-mentioned purpose, the present invention provides an enzyme-catalyzed conversion method for a chiral intermediate of clopidogrel, which uses racemic o-chlorophenylglycine methyl ester as a starting material and utilizes a Pickering emulsion interfacial enzyme-catalyzed reaction to prepare a single enantiomer of (S)-2-chlorophenylglycine methyl ester in a green and efficient manner, realizes direct conversion of enantiomers under mild conditions, improves the utilization rate of the racemate, and obtains an S-type isomer with a high enantiomeric excess.

[0006] A method for enzymatic conversion of a clopidogrel chiral intermediate comprises the following steps:

[0007] S1, modifying nano-silica particles using hydrophilic silane and hydrophobic silane to obtain modified silica nano-particles;

[0008] S2, using the modified nano-silica particles obtained in step S1 as a stabilizer, dissolving (R, S)-o-chlorophenylglycine methyl ester in a solvent as an oil phase, using a buffer solution containing a protease as an aqueous phase, and emulsifying by a homogenizer to obtain a Pickering emulsion enzyme catalytic reaction system;

[0009] S3. The reaction system obtained in step S2 is placed on a constant temperature shaker for reaction. After the reaction is completed, the emulsion is broken by centrifugation to separate the oil and water phases, and the oil phase is concentrated and crystallized to obtain (S)-2-chlorophenylglycine methyl ester.

[0010] Preferably, the hydrophilic silane in step S1 is 3-aminopropyltrimethoxysilane.

[0011] Preferably, the hydrophobic silane in step S1 is one or more of n-octyltrimethoxysilane, n-decyltrimethoxysilane and dodecyltrimethoxysilane.

[0012] Preferably, the molar ratio of the hydrophilic silane to the hydrophobic silane in step S1 is 1:1-4.

[0013] Preferably, in step S2, the mass ratio of (R,S)-o-chlorophenylglycine methyl ester:protease:modified nano-silica particles is 1:0.1-2:0.2-1.

[0014] Preferably, the solvent in step S2 is toluene or n-heptane, and the mass concentration of the (R,S)-o-chlorophenylglycine methyl ester in the oil phase is 0.05-0.2 g / mL.

[0015] Preferably, the buffer solution in step S2 is Tris-HCl, with a pH of 7.0-9.0, and the concentration of the protease in the buffer solution is 0.005-0.15 g / mL.

[0016] Preferably, the protease in step S2 is one or more of Bacillus licheniformis protease, Aspergillus oryzae protease, Aspergillus niger protease, Bacillus subtilis protease and Bacillus polymyxa protease.

[0017] Preferably, the reaction temperature in step S3 is 25-40° C., and the reaction time is 12-36 h.

[0018] A (S)-2-chlorophenylglycine methyl ester synthesized by the above conversion method.

[0019] The reaction equation of the enzymatic conversion method of the chiral intermediate of clopidogrel of the present invention is as follows:

[0020]

[0021] The positive beneficial effects of the present invention are:

[0022] 1. The Pickering emulsion enzyme catalytic reaction system of the present invention has the characteristics of large interface area and fast mass transfer rate, provides suitable conditions for the enzyme-catalyzed conversion of R-type o-chlorophenylglycine methyl ester, avoids cumbersome group protection and deprotection steps, and more importantly, avoids the formation of o-chlorophenylglycine, an ester hydrolysis product, and realizes direct conversion of enantiomers under mild conditions, thereby improving the utilization rate of the racemate, and the enantiomeric excess of the obtained S-type isomer is as high as 87.3%.

[0023] 2. The Pickering emulsion interfacial enzyme-catalyzed direct conversion method adopted in the present invention does not require the use of immobilized enzymes, greatly shortens the reaction process, uses free enzymes to improve reaction efficiency and reduce costs. After the reaction is completed, the separated enzymes and nanoparticles can be reused multiple times without affecting the conversion efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the results of liquid chromatography testing of the product of Example 1 of the present invention;

[0025] Figure 2 This is a graph showing the results of liquid chromatography testing of the product of Example 2 of the present invention;

[0026] Figure 3 This is a graph showing the results of liquid chromatography testing of the product of Example 3 of the present invention;

[0027] Figure 4 This is a graph showing the results of liquid chromatography testing of the product of Example 4 of the present invention;

[0028] Figure 5 This is a graph showing the results of liquid chromatography testing of the product of Example 5 of the present invention;

[0029] Figure 6 This is a graph showing the results of liquid chromatography testing of the product of Example 6 of the present invention;

[0030] Figure 7 This is a graph showing the results of liquid chromatography testing of the product of Example 7 of the present invention;

[0031] Figure 8 This is a graph showing the results of liquid chromatography testing of the product of Example 8 of the present invention;

[0032] Fig. 9 This is a graph showing the results of liquid chromatography testing of the product of Example 9 of the present invention;

[0033] Fig.10 This is a graph showing the results of liquid chromatography testing of the product of Example 10 of the present invention;

[0034] Fig.11 This is a graph showing the results of liquid chromatography testing of the product of Example 11 of the present invention;

[0035] Fig.12 This is a graph showing the liquid chromatography test results of the product of Example 12 of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with some specific embodiments.

[0037] Example 1

[0038] A method for enzymatic conversion of a clopidogrel chiral intermediate comprises the following steps:

[0039] 0.25g (R,S)-2-chlorophenylglycine methyl ester was dissolved in 5mL toluene, 0.025g Bacillus subtilis 1398 neutral protease (enzyme activity ≥ 50,000 U / g) was dissolved in 5mL Tris-HCl buffer solution with a pH value of 7.0, and then 0.1g 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane were added to the mixed solution of the above two modified SiO2 nanoparticles (diameter 30nm) in a molar ratio of 1:1. The mixture was emulsified by a homogenizer to prepare a Pickering emulsion. The system was placed in a constant temperature shaker at 30°C and 150rpm for 12h. After the reaction was completed, the organic layer was separated by centrifugation and concentrated to obtain the chiral intermediate of clopidogrel (S)-2-chlorophenylglycine methyl ester. After determination, see Figure 1 , the S-enantiomer excess value reached 65.5%. The protease in the aqueous phase does not need to be separated, and the enzyme-containing aqueous phase can be directly reused for the next experiment.

[0040] Example 2

[0041] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 1, except that: the amount of Bacillus subtilis 1398 neutral protease is 0.50 g, and the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:2, see Figure 2 , the S-enantiomeric excess ee% reached 71.3%

[0042] Example 3

[0043] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 2, except that the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:3, see Figure 3 , the S-enantiomeric excess ee% reached 75.2%.

[0044] Example 4

[0045] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 2, except that the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:4, see Figure 4 , the S-enantiomeric excess ee% reached 77.6%.

[0046] Example 5

[0047] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 1, except that: 0.025 g of Bacillus licheniformis 2709 alkaline protease (enzyme activity ≥ 2.4 U / g) is dissolved in 5 mL of Tris-HCl buffer solution with a pH value of 9.0, and the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:1, with a particle diameter of 100 nm and an amount of 0.25 g, see Figure 5 , the S-enantiomeric excess ee% reached 86.3%.

[0048] Example 6

[0049] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 5, except that the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:2, see Figure 6 , the S-enantiomeric excess ee% reached 87.3%.

[0050] Example 7

[0051] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 5, except that the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:3, see Figure 7 , the S-enantiomeric excess ee% reached 86.0%.

[0052] Example 8

[0053] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 5, except that the molar ratio of 3-aminopropyltrimethoxysilane to n-octyltrimethoxysilane is 1:4. Figure 8 , the S-enantiomeric excess ee% reached 82.4%.

[0054] Example 9

[0055] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 1, except that: 1 g of (R, S)-o-chlorophenylglycine methyl ester is dissolved in 5 mL of toluene, 0.75 g of Aspergillus niger 537 acid protease (enzyme activity ≥ 50,000 U / g) is dissolved in 5 mL of Tris-HCl buffer solution with a pH value of 9.0, and then 0.2 g of 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:1 modified nano-silica (particle diameter 50 nm) is added to the mixed solution of the above two, and the Pickering emulsion is prepared after emulsification by a homogenizer, and the system is placed in a constant temperature shaker at 150 rpm at 40°C for 36 hours, see Fig. 9 , the S-enantiomeric excess ee% reached 49.4%.

[0056] Example 10

[0057] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 9, except that the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:2, see Fig.10 , the S-enantiomeric excess ee% reached 50.2%.

[0058] Embodiment 11

[0059] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 9, except that the stabilizer is nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:3, see Fig.11 , the S-enantiomeric excess ee% reached 55.6%.

[0060] Example 12

[0061] The enzymatic conversion method of the chiral intermediate of clopidogrel in this example is basically the same as that in Example 9, except that the stabilizer is a nano-silica modified with 3-aminopropyltrimethoxysilane and n-octyltrimethoxysilane in a molar ratio of 1:4, see Fig.12 , the S-enantiomeric excess ee% reached 60.5%.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for enzymatic conversion of a chiral intermediate of clopidogrel, characterized in that: The steps include: S1, modifying nano-silica particles using hydrophilic silane and hydrophobic silane to obtain modified silica nano-particles; S2, using the modified nano-silica particles obtained in step S1 as a stabilizer, dissolving (R, S)-o-chlorophenylglycine methyl ester in a solvent as an oil phase, using a buffer solution containing a protease as an aqueous phase, and emulsifying by a homogenizer to obtain a Pickering emulsion enzyme catalytic reaction system; S3. The reaction system obtained in step S2 is placed on a constant temperature shaker for reaction. After the reaction is completed, the emulsion is broken by centrifugation to separate the oil and water phases, and the oil phase is concentrated and crystallized to obtain (S)-2-chlorophenylglycine methyl ester.

2. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The hydrophilic silane in step S1 is 3-aminopropyltrimethoxysilane.

3. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The hydrophobic silane in step S1 is one or more of n-octyltrimethoxysilane, n-decyltrimethoxysilane and dodecyltrimethoxysilane.

4. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The molar ratio of the hydrophilic silane to the hydrophobic silane in step S1 is 1:1-4.

5. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: In step S2, the mass ratio of (R,S)-o-chlorophenylglycine methyl ester:protease:modified nano-silica particles is 1:0.1-2:0.2-1.

6. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The solvent in step S2 is toluene or n-heptane, and the mass concentration of the (R, S)-o-chlorophenylglycine methyl ester in the oil phase is 0.05-0.2 g / mL.

7. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The buffer solution in step S2 is Tris-HCl, with a pH of 7.0-9.0, and the concentration of the protease in the buffer solution is 0.005-0.15 g / mL.

8. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The protease in step S2 is one or more of Bacillus licheniformis protease, Aspergillus oryzae protease, Aspergillus niger protease, Bacillus subtilis protease and Bacillus polymyxa protease.

9. The method for enzymatic conversion of clopidogrel chiral intermediate according to claim 1, characterized in that: The reaction temperature in step S3 is 25-40° C., and the reaction time is 12-36 hours.

10. (S)-2-chlorophenylglycine methyl ester synthesized by the conversion method according to any one of claims 1 to 9.

Images