Synthesis method of brivaracetam
The synthesis of (R)-4-propyl-pyrrolidone as an intermediate by enzymatic method has simplified the synthesis process of bovacetam, solved the problems of complex routes and high cost in the prior art, and achieved green and efficient industrial production.
Patent Information
- Application Number
- CN202411467327.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-02-18
AI Technical Summary
The existing synthesis route of bovacetam is complex and costly, and requires separation and purification of chiral intermediates, resulting in a decrease in yield.
(R)-4-propyl-pyrrolidone synthesized by the introduction of enzymatic method as an intermediate, 3-cyanocapronitrile hydrolysis was catalyzed by nitrile hydrolase to synthesize (R)-3-cyanocaproic acid and obtained by hydrogenation reduction (R)-3-aminomethylhexanoic acid followed by amidation and reaction separation to obtain bovacetam product.
It simplifies the process flow, reduces costs, is environmentally friendly, green and efficient, is suitable for industrial production, and improves the yield of bovacetam.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug synthesis, and in particular to a method for synthesizing brivaracetam. Background Art
[0002] As a new generation of anti-epileptic drugs, Brivaracetam has broad market prospects and currently has a variety of synthetic routes. For example, the invention patent with publication number WO2007065634 discloses a preparation route for Brivaracetam, which uses n-pentene as a starting material, obtains (R)-2-hydroxypentanol through asymmetric hydroxylation, reacts with thionyl chloride to obtain (4R)-4-propyl-ethylene sulfite, hydrates with ruthenium trichloride, and oxidizes with sodium periodate to obtain (4R)-4-propyl-ethylene sulfate, which then reacts with dimethyl malonate to obtain (S)-6,6-dimethyl-1-propyl-5,7-dioxospira1-octane-4,8-dione, which then reacts with (S)-2-aminobutyramide to obtain a mixture of a pair of positional isomers, which is then methylated and decarboxylated to obtain Brivaracetam. However, its disadvantages are that the reaction route is complicated and the cost is high. The synthesis process involves the separation and purification of chiral intermediates, which will greatly reduce the yield.
[0003] In other synthetic routes of brivaracetam, (R)-4-propyl-pyrrolidone is also a key chiral intermediate for the synthesis of brivaracetam, which can be prepared by ring-closing (R)-3-aminomethylhexanoic acid. (R)-3-aminomethylhexanoic acid can be synthesized by hydrolyzing 3-cyanohexanenitrile with nitrilase to (R)-3-cyanohexanoic acid, and then synthesized by hydrogenation reduction of (R)-3-cyanohexanoic acid. This enzymatic synthetic route is of great significance for the industrial green synthesis of brivaracetam, and can avoid the disadvantages of (R)-4-propyl-pyrrolidone in conventional preparation processes that chiral preparation, separation and purification are required, which can simplify the process and reduce costs. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a method for synthesizing brivaracetam, which can simplify the process, reduce costs, and is environmentally friendly, green and efficient, and suitable for industrialization by introducing (R)-4-propyl-pyrrolidone synthesized by an enzymatic method as an intermediate.
[0005] The purpose of the present invention is achieved through the following technical solutions: The present invention provides a method for synthesizing brivaracetam, comprising the following steps: (1) 3-cyanohexanenitrile is catalyzed by nitrilase to obtain (R)-3-cyanohexanoic acid, which is then catalytically hydrogenated to obtain (R)-3-aminomethylhexanoic acid, which is then amidated to obtain the compound represented by Formula I; (2) reacting the compound represented by formula I with methyl-2-bromobutyrate in an organic solvent in the presence of a base to obtain a compound represented by formula II; (3) subjecting the compound represented by formula II to aminolysis, and separating the resulting mixture by chromatography to obtain the brivaracetam product represented by formula III;
[0006] 3-Cyanocyanohexanenitrile is hydrolyzed by nitrilase to synthesize (R)-3-cyanohexanoic acid, and then (R)-3-cyanohexanoic acid is hydrogenated and reduced to synthesize (R)-3-aminomethylhexanoic acid. (R)-3-aminomethylhexanoic acid is amidated and ring-closed to prepare (R)-4-propyl-pyrrolidone, i.e., the compound shown in formula I. (R)-4-propyl-pyrrolidone and methyl-2-bromobutyrate are reacted in an organic solvent in the presence of a base to obtain the compound shown in formula II, which is then subjected to aminolysis, and the chiral isomers of the reaction mixture are separated by chromatography to obtain the buvacethamine product. The buvacethamine synthesis process of the present invention is simple, efficient, environmentally friendly, and suitable for industrialization.
[0007] Preferably, in step (1), the reaction conditions of the hydrogenation catalysis are: in the presence of a nickel catalyst and hydrogen, water is used as a solvent, the pressure is controlled at 2 to 3 MPa, and the reaction is carried out at room temperature for 8 to 12 hours.
[0008] Preferably, in step (1), the amidation is as follows: first, (R)-3-aminomethylhexanoic acid and dichlorothionyl are heated to 30-40° C. in dichloromethane to react to obtain a reactant; then, a portion of the reactant, triethylamine and N,N'-dicyclohexylcarbodiimide are stirred and mixed in tetrahydrofuran at room temperature, cooled to -20--10° C. and reacted for 30-40 min, and then another portion of the reactant is added dropwise and the reaction is continued for 60-80 min.
[0009] The thionyl chloride first reacts with the carboxyl group in (R)-3-aminomethylhexanoic acid to obtain an acyl halide, which then undergoes a ring-closing reaction with the amino group to generate an amide. The ring-closing reaction is promoted by controlling the reaction conditions and adding triethylamine and N,N'-dicyclohexylcarbodiimide to obtain a higher reaction yield.
[0010] Preferably, the molar ratio of the (R)-3-aminomethylhexanoic acid to dichlorothionyl is 1:2-4; the ratio of the added amounts of the (R)-3-aminomethylhexanoic acid to dichloromethane is 0.10-0.15 mol:100 mL; the mass percentage of the one part of the reactants to the other part of the reactants is 30-40%:60-70%, and the total percentage is 100%; the ratio of the added amounts of the reactants, triethylamine, N,N'-dicyclohexylcarbodiimide and tetrahydrofuran is 1 g:3-5 g:8-9 g:200 mL.
[0011] Preferably, in step (1), the nitrilase is an immobilized nitrilase, and the preparation method thereof comprises the following steps: S1: adding carboxyl-terminated PLGA (poly(lactic acid-co-glycolic acid)) to dichloromethane, and then adding a n-hexane solution containing hydroxyapatite to obtain a suspension; S2: adding macroporous resin to the suspension and then sonicating, and then adding polyvinyl alcohol aqueous solution and sonicating again; S3: heating to volatilize the organic solvent, filtering, adding the obtained product to a lysine solution, then adding a dicyclohexylcarbodiimide ethanol solution and stirring to react, filtering to obtain a carrier; S4: adding the carrier into a buffer solution and adding nitrilase for adsorption; then adding glutaraldehyde solution for immobilized enzyme reaction to obtain immobilized nitrilase.
[0012] The present invention first adsorbs carboxyl-terminated polylactic acid-glycolic acid copolymer and hydroxyapatite on the surface of a macroporous resin, wherein the macroporous resin is a macroporous weakly basic anion exchange resin, and electrostatic interaction occurs between free amine on the surface of the macroporous resin and the carboxyl group in the carboxyl-terminated polylactic acid-glycolic acid copolymer, and at the same time, the calcium ions in the hydroxyapatite coordinate with the carboxyl group and the amino group, so that a polylactic acid film containing hydroxyapatite is formed on the surface of the macroporous resin, and the introduction of polylactic acid with biological activity can also promote the adsorption effect on the enzyme. Afterwards, a polyvinyl alcohol aqueous solution is added to help the deposition of the carboxyl-terminated polylactic acid-glycolic acid copolymer on the surface of the resin particles, and a structure with a certain shape of protrusion is formed, and the load stability of the polylactic acid is improved by the interface effect. Then, the added n-hexane is a poor solvent for polylactic acid, and in the process of heating and volatilizing the organic solvent, a plurality of protrusion structures on the surface of the resin will form porous, thereby increasing the specific surface area for adsorption of the enzyme, and the hydroxyapatite exposed by the porous structure will have a good adsorption effect on the enzyme. The added lysine will dehydrate with the free amine in the macroporous resin under the catalysis of N, N-dicyclohexylcarbodiimide to generate an amide bond, and the lysine side chain will be grafted on the resin surface, so that the nitrilase can be better fixed by the cross-linking method, reducing the loss of enzyme activity. Therefore, the present invention can first adsorb the nitrilase on the carrier by the adsorption method through the prepared carrier, and compared with the resin adsorption on the conventional flat surface, the steric hindrance effect generated by the direct immobilization of the enzyme on the resin micropore wall can be reduced, and the spatial range of the immobilized nitrilase catalysis can be expanded, and then the nitrilase is cross-linked and fixed by glutaraldehyde, while the enzyme loading rate is increased, the structural stability of the enzyme fixed by the adsorption method is improved, and then the activity and stability of the obtained immobilized nitrilase are both high.
[0013] Preferably, in S1, the n-hexane solution containing hydroxyapatite is prepared by dispersing hydroxyapatite powder in n-hexane, the amount of hydroxyapatite added relative to the concentration of n-hexane is 0.05-0.08 g / mL; the particle size of the hydroxyapatite is 50-100 nm. The amount of carboxyl-terminated PLGA added relative to the concentration of the mixed volume of dichloromethane and n-hexane is 40-60 g / L; the mixed volume ratio of dichloromethane and n-hexane is 8-9:1.
[0014] The mixing volume ratio of dichloromethane and n-hexane, the concentration of polylactic acid, the concentration of hydroxyapatite in n-hexane, etc. will all affect the pore size and porosity of the porous structure, and the formed pore size is 10-25 μm.
[0015] Preferably, in S2, the macroporous resin is a macroporous weakly basic acrylic anion exchange resin; the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated PLGA is 10-12:1. The volume ratio of the suspension to the polyvinyl alcohol aqueous solution is 1:10; the concentration of the polyvinyl alcohol aqueous solution is 2-4 g / L. The ultrasonic time is 5-10 min; and the second ultrasonic time is 1-2 h.
[0016] Preferably, in S3, the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1g:3-6mL:0.4-0.5mL; the concentration of the lysine solution is 0.05-0.1g / mL; the concentration of the dicyclohexylcarbodiimide ethanol solution is 0.1-0.2g / mL. The heating and volatilization of the organic solvent is heating to 40-50°C and stirring for 3-5h, and the stirring speed is 1000-1500rpm; the stirring reaction is stirring at room temperature for 1-2h.
[0017] Preferably, in S4, the mass volume ratio of the carrier, buffer and glutaraldehyde solution is 1g:10mL:1-5mL; the mass concentration of glutaraldehyde in the glutaraldehyde solution is 10-15%. The amount of nitrilase used is: 16-20mg / g resin in terms of protein content; the temperature of the immobilized enzyme reaction is 30-40°C, and the reaction time is 1-3h.
[0018] Preferably, in step (1), the nitrilase catalyzed reaction is carried out at 30-35° C. and 600-800 r / min in a water bath for 22-26 hours.
[0019] Preferably, in step (2), the base is potassium hydride, sodium hydride, sodium methoxide, potassium methoxide or sodium isopropoxide; the organic solvent is tetrahydrofuran or toluene; the molar ratio of the compound represented by formula I to methyl-2-bromobutyric acid methyl ester is 1:1.1-1.4; the ratio of the added amount of the compound represented by formula I, the base and tetrahydrofuran is 1mmol:1.4-1.5mmol:4mL; and the reaction is carried out at -5 to 5°C for 30 to 50min.
[0020] Preferably, in step (3), the aminolysis is as follows: adding the compound represented by formula II to ammonia water at 0 to 5°C and stirring for reaction for 5 to 7 hours, the volume concentration of ammonia water is 50 to 60%, and the ratio of the added amount of the compound represented by formula II to ammonia water is 1 to 1.5 mmol: 2 mL.
[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) By introducing (R)-4-propyl-pyrrolidone synthesized by an enzymatic method as an intermediate, the process can be simplified, the cost can be reduced, and the process is environmentally friendly, green and efficient, and suitable for industrialization; (2) By modifying the macroporous resin, the specific surface area and adsorption active groups are increased, the steric hindrance effect of directly immobilizing the enzyme on the resin micropore wall is reduced, and the spatial range of immobilized nitrilase catalysis is expanded. While increasing the enzyme loading rate, the structural stability of the enzyme immobilized by adsorption is improved, resulting in higher enzyme activity and substrate conversion rate. DETAILED DESCRIPTION
[0022] The technical scheme of the present invention is described below with specific examples, but the protection scope of the present invention is not limited thereto. The content of the reaction products is detected by chromatography.
[0023] 1. Immobilized nitrilase S1. Dissolve the carboxyl-terminated PLGA (average molecular weight 50 kDa) in dichloromethane, add a n-hexane solution containing hydroxyapatite (average particle size 50-100 nm) and stir. The concentration of the carboxyl-terminated PLGA added relative to the mixed volume of dichloromethane and n-hexane is 40-60 g / L, and the mixed volume ratio of dichloromethane and n-hexane is 8-9:1 to obtain a suspension.
[0024] S2. After adding macroporous weakly basic anion exchange resin to the suspension, ultrasonicate for 5-10 minutes, the mass ratio of macroporous weakly basic anion exchange resin to carboxyl-terminated PLGA is 10-12:1; then add 2-4g / L polyvinyl alcohol aqueous solution, the volume ratio of the suspension to the polyvinyl alcohol aqueous solution is 1:10, and ultrasonicate again for 1-2h.
[0025] S3, heating the reactant obtained in step S2 to 40-50°C and stirring for 3-5h, with a stirring speed of 1000-1500rpm, volatilizing the organic solvent, and then filtering; adding the obtained product to a lysine solution with a concentration of 0.05-0.1g / mL, stirring for 1-2h at room temperature, and then adding a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1-0.2g / mL, stirring and reacting at room temperature for 1-2h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1g:3-6mL:0.4-0.5mL, and filtering after the reaction is completed to obtain the carrier.
[0026] S4, the carrier is added to PBS buffer, and then a pure enzyme solution containing 1 mg / mL nitrilase (commercially available) is added, the amount of nitrilase used is 16-20 mg / g resin in terms of protein content, and an adsorption reaction is carried out in a water bath shaker at 30-40° C. and 100-150 rpm for 1-2 h; then a PBS buffer containing 10-15% glutaraldehyde is added, and an immobilized enzyme reaction is carried out in a water bath shaker at 30-40° C. and 100-150 rpm for 1-2 h, followed by washing with deionized water 3-5 times, placing on filter paper and drying at room temperature to obtain immobilized nitrilase, which is stored in a 4° C. refrigerator for standby use.
[0027] In a specific embodiment of the present invention, in S1, the n-hexane solution containing hydroxyapatite is prepared by ultrasonically dispersing hydroxyapatite powder in n-hexane, and the concentration of hydroxyapatite added relative to the volume of n-hexane is 0.05-0.08 g / mL.
[0028] In a specific embodiment of the present invention, in S2, the macroporous weakly basic anion exchange resin is a macroporous weakly basic acrylic anion exchange resin D314 / D315, containing free amines and having a particle size of 0.80-1.25 mm.
[0029] In a specific embodiment of the present invention, in S4, the mass volume ratio of the carrier, the PBS buffer and the PBS buffer containing glutaraldehyde is 1 g:10 mL:1-5 mL, and the pH of the PBS buffer is 6-8.
[0030] 2. Synthesis of Brivaracetam (1) Immobilized nitrilase and 3-cyanohexanenitrile are added to PBS buffer in a ratio of 10 mL: 1 g: 0.3-0.4 g. The mixture is reacted at 30-35° C. and 600-800 r / min in a water bath for 22-26 h to prepare (R)-3-cyanohexane acid.
[0031] (2) (R)-3-cyanohexanoic acid is added to water to obtain a (R)-3-cyanohexanoic acid aqueous solution with a concentration of 200 g / L, a nickel catalyst is added to the (R)-3-cyanohexanoic acid aqueous solution, the mass ratio of the nickel catalyst to the (R)-3-cyanohexanoic acid is 1:1-2, the hydrogen pressure is controlled to be 2-3 MPa, and the reaction is carried out at room temperature for 8-12 hours to obtain (R)-3-aminomethylhexanoic acid.
[0032] (3) Amidation of (R)-3-aminomethylhexanoic acid is performed, and the tail gas is treated during the process. First, (R)-3-aminomethylhexanoic acid and dichlorothionyl are heated to 30-40°C in dichloromethane to react to obtain a reactant; then, a portion of the reactant, triethylamine and N,N'-dicyclohexylcarbodiimide are stirred and mixed in tetrahydrofuran at room temperature, cooled to -20--10°C and reacted for 30-40 minutes, and then another portion of the reactant is added dropwise and the reaction is continued for 60-80 minutes to obtain the compound shown in Formula I.
[0033] (4) reacting the compound represented by formula I and methyl-2-bromobutyric acid methyl ester in an organic solvent (tetrahydrofuran or toluene) in the presence of a base (potassium hydride, sodium hydride, sodium methoxide, potassium methoxide or sodium isopropoxide), wherein the molar ratio of the compound represented by formula I to methyl-2-bromobutyric acid methyl ester is 1:1.1-1.4, and the ratio of the amount of the compound represented by formula I, the base and tetrahydrofuran added is 1 mmol:1.4-1.5 mmol:4 mL, and reacting at -5-5°C for 30-50 min to obtain a compound represented by formula II; (5) Add the compound represented by formula II into 50-60% ammonia water at 0-5° C., stir and react for 5-7 hours, wherein the ratio of the compound represented by formula II to ammonia water is 1-1.5 mmol: 2 mL, and separate the reaction mixture by chromatography to obtain the brivaracetam product represented by formula III.
[0034] In a specific embodiment of the present invention, in step (1), the molar ratio of (R)-3-aminomethylhexanoic acid to dichlorothionyl is 1:2-4; the ratio of the added amount of (R)-3-aminomethylhexanoic acid to dichloromethane is 0.10-0.15 mol:100 mL; the mass percentage of one part of the reactants and the other part of the reactants is 30-40%:60-70%, and the total percentage is 100%; the added amount ratio of the reactants, triethylamine, N,N'-dicyclohexylcarbodiimide and tetrahydrofuran is 1 g:3-5 g:8-9 g:200 mL.
[0035] Example 1 (Preparation of immobilized nitrilase) The macroporous weakly basic anion exchange resin was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content, and an adsorption reaction was carried out for 2 hours at 30° C. and 130 rpm in a water bath shaker; then a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, the PBS buffer and the PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL, and an immobilized enzyme reaction was carried out for 1.5 hours at 30° C. and 130 rpm in a water bath shaker, and then the mixture was washed with deionized water 4 times, and dried on a filter paper at room temperature to obtain an immobilized nitrilase, which was stored in a refrigerator at 4° C. for later use.
[0036] Example 2 (Preparation of immobilized nitrilase) S1. Dissolve a carboxyl-terminated polylactic acid-hydroxyacetic acid copolymer (average molecular weight 50 kDa) in dichloromethane, add a n-hexane solution containing hydroxyapatite (average particle size 100 nm) and stir, the concentration of hydroxyapatite added relative to the volume of n-hexane is 0.06 g / mL, the concentration of carboxyl-terminated polylactic acid-hydroxyacetic acid copolymer added relative to the mixed volume of dichloromethane and n-hexane is 50 g / L, the mixed volume ratio of dichloromethane and n-hexane is 9:1, and a suspension is obtained.
[0037] S2. After adding macroporous weakly basic anion exchange resin to the suspension, ultrasonicate for 10 minutes, the mass ratio of macroporous weakly basic anion exchange resin to carboxyl-terminated polylactic acid-glycolic acid copolymer is 10:1; then add 3g / L polyvinyl alcohol aqueous solution, the volume ratio of suspension to polyvinyl alcohol aqueous solution is 1:10, and ultrasonicate again for 1h.
[0038] S3. The reactant obtained in step S2 is heated to 45°C and stirred for 4 hours at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 hour, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1 g / mL is added, and the reaction is stirred at room temperature for 1 hour, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.4 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0039] S4, the carrier was added to PBS buffer (100mM, pH = 7.4), and then a pure enzyme solution containing 1mg / mL nitrilase in terms of protein content was added, the amount of nitrilase used was 18mg / g resin in terms of protein content, and the adsorption reaction was carried out in a water bath shaker at 30 ° C, 130rpm for 2h; then a PBS buffer containing 10% glutaraldehyde (100mM, pH = 7.4) was added, the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1g: 10mL: 5mL, and the immobilized enzyme reaction was carried out in a water bath shaker at 30 ° C, 130rpm for 1.5h, and then washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a 4 ° C refrigerator for standby use.
[0040] Example 3 (Preparation of immobilized nitrilase) S1. Dissolve a carboxyl-terminated polylactic acid-hydroxyacetic acid copolymer (average molecular weight 50 kDa) in dichloromethane, add a n-hexane solution containing hydroxyapatite (average particle size 50 nm) and stir, the added amount of hydroxyapatite relative to the concentration of n-hexane is 0.08 g / mL, the added amount of the carboxyl-terminated polylactic acid-hydroxyacetic acid copolymer relative to the concentration of the mixed volume of dichloromethane and n-hexane is 45 g / L, the mixed volume ratio of dichloromethane and n-hexane is 9:1, and a suspension is obtained.
[0041] S2. After adding macroporous weakly basic anion exchange resin to the suspension, ultrasonicate for 10 minutes, the mass ratio of macroporous weakly basic anion exchange resin to carboxyl-terminated polylactic acid-glycolic acid copolymer is 12:1; then add 2 g / L polyvinyl alcohol aqueous solution, the volume ratio of the suspension to the polyvinyl alcohol aqueous solution is 1:10, and ultrasonicate again for 2 hours.
[0042] S3. The reactant obtained in step S2 is heated to 45°C and stirred for 4 hours at a stirring speed of 1500 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.1 g / mL, stirred at room temperature for 2 hours, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.2 g / mL is added, and the reaction is stirred at room temperature for 2 hours, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.5 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0043] S4, the carrier was added to PBS buffer (100mM, pH = 7.4), and then a pure enzyme solution containing 1mg / mL nitrilase in terms of protein content was added, the amount of nitrilase used was 18mg / g resin in terms of protein content, and the adsorption reaction was carried out in a water bath shaker at 30 ° C, 130rpm for 2h; then a PBS buffer containing 10% glutaraldehyde (100mM, pH = 7.4) was added, the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1g: 10mL: 5mL, and the immobilized enzyme reaction was carried out in a water bath shaker at 30 ° C, 130rpm for 1.5h, and then washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a 4 ° C refrigerator for standby use.
[0044] Example 4 (Synthesis of Buvaracetam) (1) In 10mL 100mM, pH=7.4 PBS buffer, add 1g immobilized nitrilase (Example 2), add 0.3g 3-cyanohexanenitrile, and react for 24h at 30°C and 700r / min in a water bath. Take a sample of 200μL, add 30μL 6M HCl to terminate the reaction and extract with ethyl acetate. Centrifuge the reaction solution at 8000rpm for 10min, take the supernatant and add ethyl acetate to extract at a volume ratio of 3:1, adjust the pH of the aqueous phase to 1-2 with hydrochloric acid solution, add ethyl acetate to extract at a volume ratio of 3:1, add NaOH aqueous solution with a pH of 12 to the organic phase for back extraction, take the aqueous phase to obtain (R)-3-cyanohexanoic acid aqueous solution, and add water to adjust the concentration to 200g / L.
[0045] (2) Add 25 g of nickel catalyst to 100 mL of (R)-3-cyanohexanoic acid aqueous solution, control the hydrogen pressure to 3 MPa, and react at room temperature for 10 h to obtain (R)-3-aminomethylhexanoic acid. The reaction solution is filtered, and the filtrate is centrifuged at 8000 rpm for 10 min. The supernatant is extracted by adding ethyl acetate at a volume ratio of 3:1. The aqueous phase is adjusted to pH 1-2 by hydrochloric acid solution, and then ethyl acetate is extracted by adding ethyl acetate at a volume ratio of 3:1. The organic phase is back-extracted by adding NaOH aqueous solution with a pH of 12. The aqueous phase is taken to obtain an aqueous solution of (R)-3-aminomethylhexanoic acid, and the (R)-3-aminomethylhexanoic acid is obtained by reduced pressure distillation.
[0046] (3) (R)-3-aminomethylhexanoic acid is amidated. 0.1 mol of (R)-3-aminomethylhexanoic acid is added to 100 mL of dichloromethane, and then 0.25 mol of dichlorothionyl is added dropwise. The mixture is heated to 35°C and reacted until the raw material disappears as detected by TLC. The reaction is stopped. The mixture is concentrated under reduced pressure to obtain 20.1 g of the reactant. 0.3 g of the reactant, 1.4 g of triethylamine and 2.6 g of N,N'-dicyclohexylcarbodiimide are stirred and mixed in 200 mL of tetrahydrofuran at room temperature, cooled to -20°C and reacted for 30 min, and then 0.7 g of the reactant is added dropwise and the reaction is continued for 80 min. After the reaction is completed, 3 times the volume of water is added and mixed, and the aqueous phase is extracted by distillation under reduced pressure to obtain the compound shown in Formula I.
[0047] (4) Under nitrogen atmosphere, 14.3 mmol of sodium methoxide was added to 40 mL of tetrahydrofuran, and then 10 mmol of the compound represented by formula I and 13.5 mmol of methyl-2-bromobutyrate were added, and the mixture was stirred evenly, and then stirred at -5°C for 45 min. After the reaction solution was warmed to room temperature, an equal volume of water was added, and the mixture was extracted after distillation under reduced pressure to obtain the compound represented by formula II.
[0048] (5) 12 mmol of the compound represented by formula II was added to 20 mL of 55% ammonia water at 0°C and stirred for reaction for 7 h. The white precipitate produced during the reaction was filtered out, washed with water and dried, and the reaction mixture was separated by chromatography to obtain the brivaracetam product represented by formula III.
[0049] Comparative Example 1 The difference from Example 2 is that in step S1 of preparing the immobilized nitrilase, no hydroxyapatite is added.
[0050] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and an n-hexane solution was added and stirred. The concentration of the carboxyl-terminated polylactic acid-glycolic acid copolymer added was 50 g / L relative to the mixed volume of dichloromethane and n-hexane, and the mixed volume ratio of dichloromethane and n-hexane was 9:1 to obtain a suspension.
[0051] Comparative Example 2 The difference from Example 2 is that in step S1 of preparing the immobilized nitrilase, the concentration of hydroxyapatite in n-hexane is too high.
[0052] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The concentration of the added hydroxyapatite relative to the volume of n-hexane was 0.12 g / mL, and the concentration of the added carboxyl-terminated polylactic acid-glycolic acid copolymer relative to the mixed volume of dichloromethane and n-hexane was 50 g / L. The mixed volume ratio of dichloromethane to n-hexane was 9:1, and a suspension was obtained.
[0053] Comparative Example 3 The difference from Example 2 is that in step S2 of preparing the immobilized nitrilase, no polyvinyl alcohol is added.
[0054] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1.
[0055] Comparative Example 4 The difference from Example 2 is that in step S3 of preparing the immobilized nitrilase, lysine is not added.
[0056] (3) The reactant obtained in step (2) was heated to 45° C. and stirred for 4 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered to obtain the carrier.
[0057] Comparative Example 5 The difference from Example 2 is that in step S4 of preparing the immobilized nitrilase, the concentration of glutaraldehyde is too high.
[0058] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content, and the adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h; then a PBS buffer (100 mM, pH = 7.4) containing 20% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL, and the immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0059] Comparative Example 6 The difference from Example 4 is that N,N'-dicyclohexylcarbodiimide is not added in step (3).
[0060] (3) (R)-3-aminomethylhexanoic acid is amidated. 0.1 mol of (R)-3-aminomethylhexanoic acid is added to 100 mL of dichloromethane, and then 0.25 mol of dichlorothionyl is added dropwise. The mixture is heated to 35°C and reacted until the raw material disappears as detected by TLC. The reaction is stopped. The mixture is concentrated under reduced pressure to obtain 20.1 g of the reactant. 0.3 g of the reactant and 1.4 g of triethylamine are stirred and mixed in 200 mL of tetrahydrofuran at room temperature, cooled to -20°C and reacted for 30 min, and then 0.7 g of the reactant is added dropwise and the reaction is continued for 80 min. After the reaction is completed, 3 times the volume of water is added and mixed, and the aqueous phase is extracted by distillation under reduced pressure to obtain the compound shown in Formula I.
[0061] Comparative Example 7 The difference from Example 4 is that the reactants in step (3) are not subjected to stepwise reaction.
[0062] (3) (R)-3-aminomethylhexanoic acid is amidated. 0.1 mol of (R)-3-aminomethylhexanoic acid is added to 100 mL of dichloromethane, and then 0.25 mol of dichlorothionyl is added dropwise. The mixture is heated to 35°C and reacted until the raw material disappears as detected by TLC. The reaction is stopped. The mixture is concentrated under reduced pressure to obtain 20.1 g of the reactant. 1 g of the reactant, 1.4 g of triethylamine and 2.6 g of N,N'-dicyclohexylcarbodiimide are stirred and mixed in 200 mL of tetrahydrofuran at room temperature, cooled to -20°C and reacted for 110 min. After the reaction is completed, 3 times the volume of water is added and stirred, and the aqueous phase is extracted by distillation under reduced pressure to obtain the compound shown in Formula I.
[0063] In 10mL 100mM, pH=7.4 PBS buffer, add 1g immobilized nitrilase (Example 1-3 and Comparative Example 1-5), add 0.3g 3-cyanohexanenitrile, and react for 24h under 30℃, 700r / min water bath conditions. Take 200μL of sample, add 30μL 6M HCl to terminate the reaction and extract with ethyl acetate. Take 200μL of the upper organic phase and dry it with anhydrous sodium sulfate, add 7.5μL methanol and 3.75μL diazomethane, and use gas chromatography to measure the conversion rate of substrate and the enantiomeric excess (ee) of product (R)-3-cyanohexanoic acid. According to the above steps, the immobilized nitrilase was reused 5 times in the synthesis of (R)-3-cyanohexanoic acid, and the conversion rate of substrate was measured by gas chromatography.
[0064] Activity assay reaction system: Add 1g immobilized nitrilase and 0.3g 3-cyanohexanenitrile to 10mL 100mM, pH=7.4 PBS buffer, and react for 15min at 30℃ and 700r / min in a water bath. Take 200μL of sample, add 30μL 6M HCl to terminate the reaction and extract with ethyl acetate. Take 200μL of the upper organic phase, dry it with anhydrous sodium sulfate, add 7.5μL methanol and 3.75μL diazomethane, detect the product concentration by liquid chromatography, and calculate the enzyme activity.
[0065] Pure enzyme activity determination reaction system: Take 1 mL of pure enzyme solution with a protein concentration of 1 mg / mL, add 0.03 g of 3-cyanohexanenitrile, and react for 15 minutes in a water bath at 30°C and 700 r / min. The post-treatment and detection methods are the same as above, and the free enzyme activity is calculated.
[0066] Protein adsorption rate = (total protein amount in the initial enzyme solution - total protein amount in the residual solution) / total protein amount in the initial enzyme solution.
[0067] Definition of enzyme activity: Under the conditions of PBS buffer (100 mM, pH = 7.4) at 30°C, the amount of enzyme required to generate 1 μmol (R)-3-cyanohexanoic acid per minute is one activity unit (1U).
[0068] Enzyme activity recovery rate = enzyme activity of immobilized enzyme / total free enzyme activity used for immobilization * 100%.
[0069] The yield of the compound represented by formula I = the mass of the compound represented by formula I actually obtained / the mass of the compound represented by formula I theoretically obtained * 100%.
[0070] The yield of brivaracetam = the mass of brivaracetam product / the total mass of the mixture before chromatography separation (including brivaracetam and its isomers) * 100%.
[0071] Table 1 Table 2 Yield of the compound represented by formula I / % Yield of Brivaracetam / % Example 4 86.48 52.96 Comparative Example 6 73.15 -- Comparative Example 7 82.93 -- As shown in Table 1, the comparison between Example 1 and Examples 2-3 shows that the present invention can ensure the firmness of the binding between the carrier and the enzyme and obtain an immobilized nitrilase with high activity by modifying the macroporous resin. The data of Comparative Examples 1-5 show that the component raw materials added to the immobilized nitrilase will affect the immobilization effect and structural stability of the enzyme, thereby causing a decrease in enzyme activity, substrate conversion rate, etc.
[0072] As shown in Table 2, the present invention can simplify the process, reduce costs, and is environmentally friendly, green and efficient, suitable for industrialization by introducing (R)-4-propyl-pyrrolidone synthesized by an enzymatic method as an intermediate, and can obtain a higher yield of Buvaracetam. The data of Comparative Examples 6-7 show that the control of the condition parameters in the amidation preparation process of the compound represented by Formula I has a greater impact on the yield, and obtaining a compound represented by Formula I with a higher yield is more conducive to improving the efficiency of the entire process and reducing costs.
[0073] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for synthesizing brivaracetam, characterized in that: The steps include: (1) 3-cyanohexanenitrile is catalyzed by nitrilase to obtain (R)-3-cyanohexanoic acid, which is then catalytically hydrogenated to obtain (R)-3-aminomethylhexanoic acid, which is then amidated to obtain the compound represented by Formula I; (2) reacting the compound represented by formula I with methyl-2-bromobutyrate in an organic solvent in the presence of a base to obtain a compound represented by formula II; (3) subjecting the compound represented by formula II to aminolysis, and separating the resulting mixture by chromatography to obtain the brivaracetam product represented by formula III; 2. According to the method for synthesizing brivaracetam according to claim 1, in step (1), the reaction conditions of the hydrogenation catalysis are: in the presence of a nickel catalyst and hydrogen, the pressure is controlled to be 2 to 3 MPa, and the reaction is carried out at room temperature for 8 to 12 hours.
3. According to the method for synthesizing brivaracetam according to claim 1 or 2, in step (1), the amidation is as follows: first, (R)-3-aminomethylhexanoic acid and dichlorothionyl are heated to 30-40° C. in dichloromethane to react to obtain a reactant; then, a portion of the reactant, triethylamine and N,N'-dicyclohexylcarbodiimide are stirred and mixed in tetrahydrofuran at room temperature, cooled to -20--10° C. and reacted for 30-40 min, and then another portion of the reactant is added dropwise and the reaction is continued for 60-80 min.
4. According to the synthesis method of brivaracetam according to claim 3, the molar ratio of (R)-3-aminomethylhexanoic acid to thionyl chloride is 1:2-4; the added amount ratio of (R)-3-aminomethylhexanoic acid to dichloromethane is 0.10-0.15 mol:100 mL.
5. According to the synthesis method of brivaracetam according to claim 3, the mass percentage of the one part of the reactants and the other part of the reactants is 30-40%:60-70%, and the total percentage is 100%; the ratio of the added amount of the reactants, triethylamine, N,N'-dicyclohexylcarbodiimide and tetrahydrofuran is 1g:3-5g:8-9g:200mL.
6. The method for synthesizing brivaracetam according to claim 1, wherein in step (1), the nitrilase is an immobilized nitrilase, and the preparation method thereof comprises the following steps: S1: adding carboxyl-terminated PLGA to dichloromethane, and then adding a hexane solution containing hydroxyapatite to obtain a suspension; S2: adding macroporous resin to the suspension and then sonicating, and then adding polyvinyl alcohol aqueous solution and sonicating again; S3: heating to volatilize the organic solvent, filtering, adding the obtained product to a lysine solution, then adding a dicyclohexylcarbodiimide ethanol solution and stirring to react, filtering to obtain a carrier; S4: adding the carrier into a buffer solution and adding nitrilase for adsorption; then adding glutaraldehyde solution for immobilized enzyme reaction to obtain immobilized nitrilase.
7. The method for synthesizing brivaracetam according to claim 6, wherein in S4, the amount of nitrilase used is: 16-20 mg / g resin in terms of protein content; the temperature of the immobilized enzyme reaction is 30-40° C., and the reaction time is 1-3 h.
8. The method for synthesizing brivaracetam according to claim 1, 6 or 7, wherein in step (1), the nitrilase-catalyzed reaction is carried out at 30-35°C and 600-800 r / min in a water bath for 22-26 hours.
9. The method for synthesizing brivaracetam according to claim 1, wherein in step (2), the base is potassium hydride, sodium hydride, sodium methoxide, potassium methoxide or sodium isopropoxide; the organic solvent is tetrahydrofuran or toluene; the molar ratio of the compound represented by formula I to methyl-2-bromobutyric acid methyl ester is 1:1.1-1.4; the ratio of the added amount of the compound represented by formula I, the base and tetrahydrofuran is 1 mmol:1.4-1.5 mmol:4 mL; and the reaction is carried out at -5-5°C for 30-50 min.
10. The method for synthesizing brivaracetam according to claim 1 or 9, wherein in step (3), the aminolysis is as follows: adding the compound represented by formula II to ammonia water at 0 to 5° C. and stirring for reaction for 5 to 7 hours, wherein the volume concentration of ammonia water is 50 to 60%, and the ratio of the amount of the compound represented by formula II to ammonia water is 1 to 1.5 mmol: 2 mL.
Citation Information
Patent Citations
3-carboxy- 2-oxo-1 -pyrrolidine derivatives and their uses
WO2007065634A1