A method for synthesizing L-2-aminobutyric acid, L-2-aminobutyric acid amide hydrochloride and application thereof

Abstract

The application provides a synthesis method and application of L-2-aminobutyric acid and L-2-aminobutyric acid amide hydrochloride, and belongs to the technical field of synthesis of chiral drug intermediates. The method for synthesizing L-2-aminobutyric acid by an enzyme method comprises the following steps: mixing raw materials, reacting, and separating to obtain L-2-aminobutyric acid, wherein the raw materials comprise crotonic acid, L-phenylalanine, a solvent and L-phenylalanine deaminase, and can further comprise an ammonium salt. The method has the advantages of cheap and easily available materials, high reaction selectivity, high atom utilization rate, high yield, good product quality and the like, and successfully solves the production problems of low material reaction selectivity, waste of isomer separation materials and high material cost in the traditional process.

Description

Technical Field

[0001] This invention belongs to the field of chiral drug intermediate synthesis technology, and relates to a method for synthesizing L-2-aminobutyric acid and L-2-aminobutyramide hydrochloride and their applications. Background Technology

[0002] Levetiracetam is currently the only drug used to treat focal and secondary generalized epilepsy. It possesses both anti-epileptic and anti-seizure properties, allowing for the simultaneous treatment and prevention of epilepsy. Levetiracetam has advantages such as good tolerability, few drug interactions, low incidence of adverse reactions, and a high treatment safety index. The chemical structure of levetiracetam is shown below:

[0003] .

[0004] L-2-aminobutyramide hydrochloride is a key intermediate in the development of the chiral antiepileptic drug levetiracetam. Its chemical structure is shown below:

[0005] .

[0006] L-2-aminobutyramide hydrochloride contains both amino and amide functional groups, making it an important intermediate in organic synthesis.

[0007] Existing technical records regarding synthetic routes for L-2-aminobutyramide hydrochloride include:

[0008] 1. 2-Bromobutyric acid / methyl 2-bromobutyrate route.

[0009] Chinese invention patent CN102020584A discloses a method for synthesizing L-2-aminobutyramide hydrochloride, an intermediate of the chiral drug levetiracetam. Starting with 2-bromobutyric acid, it first reacts with thionyl chloride to generate 2-bromobutyryl chloride, which then reacts with ammonia to obtain a racemic 2-aminobutyramide. This is resolved by L-tartaric acid and then converted to a salt to obtain the final product.

[0010] Chinese invention patent CN102898324A discloses a method for preparing (S)-2-aminobutyramide hydrochloride. Using methyl 2-bromobutyrate as the starting material, it reacts with ammonia in one step to obtain racemic 2-aminobutyramide, which is then resolved by L-tartaric acid and finally salted to obtain the final product. While the prior art reports high yields, short reaction steps, and simple operation, 2-bromobutyric acid / methyl 2-bromobutyrate is expensive, resulting in high raw material costs, and the production process generates a large amount of wastewater, which is detrimental to environmental protection.

[0011] 2. The Hein Route.

[0012] This method has the drawbacks of high temperature and pressure during hydantoin hydrolysis, which poses significant safety risks; in addition, the reaction releases a large amount of ammonia gas, and the desalination cost is also high.

[0013] 3. Propionaldehyde route.

[0014] Chinese invention patent CN101928229A discloses a process for producing L-2-aminobutyramide hydrochloride, an intermediate of levetiracetam. Using propionaldehyde as a raw material, a Streat reaction is carried out with an aqueous solution of ammonia, ammonium chloride, and sodium cyanide to obtain 2-aminobutyrone. This is then hydrolyzed under alkaline conditions and atmospheric pressure to obtain 2-aminobutyramide, which is finally resolved into salt to obtain L-2-aminobutyramide hydrochloride. This method is simple to operate, avoids high-temperature and high-pressure reactions, and yields a high-quality product. However, this method uses the highly toxic substance sodium cyanide, posing safety risks during the synthesis process. Furthermore, the reaction generates a large amount of wastewater containing ammonium chloride and excess sodium cyanide, resulting in high treatment costs.

[0015] 4. Transaminase pathway.

[0016] Chinese invention patent CN106834372A discloses a method for preparing (S)- or (R)-2-aminobutyramide via transaminase biocatalysis. Using 2-carbonylbutyramide as a raw material, and in the presence of an amino donor, transaminase is used as a biocatalyst to catalyze the conversion of the carbonyl group on 2-carbonylbutyramide to an amino group, forming (S)- or (R)-2-aminobutyramide. However, the raw material 2-carbonylbutyramide has poor stability, making it difficult to purchase or prepare, which is detrimental to industrial production.

[0017] 5. Ester hydrolysis route.

[0018] Chinese invention patent CN111057735A discloses a Bacillus amyloliquefaciens esterase in the resolution of N BOC DL α Applications of methyl gamma-aminobutyrate.

[0019] Chinese invention patent CN112195203A discloses an enzymatic method for synthesizing (S)-2-aminobutyramide, wherein the substrate (S) is synthesized in the presence of an amino donor under the action of a catalytic amount of lipase. 2 Methyl gamma-aminobutyrate undergoes catalytic ammonolysis to yield the corresponding product (S). 2 Aminobutyramide.

[0020] Chinese invention patents CN113816872A and CN115943137A both disclose (R / S) 2 (S) is obtained by resolving aminobutyrate as a raw material under the action of ester hydrolases. 2 Glycine butyrate, further ammonolyzed, yields (S). 2 Aminobutyramide.

[0021] However, methods such as ester hydrolysis generally require an isomer separation step, which makes it difficult to recover the separated isomers, resulting in serious material waste and low atom utilization. Summary of the Invention

[0022] In view of the above-mentioned problems existing in the prior art, the purpose of this invention is to provide a simple, economical and environmentally friendly enzymatic synthesis method and application of L-2-aminobutyric acid and L-2-aminobutyramide hydrochloride, so as to further improve the reaction selectivity and atom utilization rate, improve the yield and product quality, and at the same time solve the production problems of low material reaction selectivity, waste of isomerized materials and high material costs in traditional processes.

[0023] To achieve the above-mentioned objectives, the present invention provides a method for synthesizing L-2-aminobutyric acid, comprising the following steps:

[0024] The raw materials are mixed, reacted, and separated to obtain L-2-aminobutyric acid;

[0025] The raw materials include crotonic acid, L-phenylalanine, solvent, and L-phenylalanine ammonia-lyase.

[0026] Preferably, the solvent is selected from alcohol solvents or ether solvents.

[0027] More preferably, the alcohol solvent is selected from at least one of methanol, ethanol, propanol or butanol; the ether solvent is selected from at least one of diethyl ether, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran or dioxane.

[0028] More preferably, and as a specific embodiment of the present invention, the solvent is isopropanol.

[0029] Preferably, the raw material further includes an ammonium salt.

[0030] More preferably, the ammonium salt is selected from at least one of ammonium acetate, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium maleate, ammonium malate, ammonium citrate, ammonium tartrate, ammonium fumarate, ammonium bicarbonate, and ammonium carbonate.

[0031] Ammonium salts can serve as a supplement to the ammonia source of phenylalanine, and together they participate in the reaction of L-phenylalanine ammonia-lyase to synthesize L-2-aminobutyric acid.

[0032] Preferably, the ratio of crotonic acid, phenylalanine, ammonium salt and L-phenylalanine ammonia-lyase is 188:36-360:0-505:80-8000, in g:g:g:U.

[0033] In a further selection, the ratio of crotonic acid, phenylalanine, ammonium salt and L-phenylalanine ammonia-lyase was 188:120:240:800, in g:g:g:U.

[0034] Preferably, the reaction temperature is 10-60°C.

[0035] More preferably, and as a specific example of the present invention, the reaction temperature is 35°C.

[0036] On the other hand, the present invention provides a method for synthesizing L-2-aminobutyramide hydrochloride, comprising the method described in any one of the above-mentioned methods; the method comprises the following synthetic route:

[0037] .

[0038] Preferably, the method includes the following steps:

[0039] S1. Mix the raw materials, react them, and separate them to obtain L-2-aminobutyric acid;

[0040] S2. Mix L-2-aminobutyric acid obtained in step S1, ammonia water and ammonium chloride, pass liquid ammonia through, keep the reaction at a temperature, separate, and obtain L-2-aminobutyramide.

[0041] S3. Mix the L-2-aminobutyramide obtained in step S2 with ethanol, pass hydrogen chloride through, recrystallize, and separate to obtain L-2-aminobutyramide hydrochloride.

[0042] In step S1, the raw materials include crotonic acid, L-phenylalanine, solvent, and L-phenylalanine ammonia-lyase.

[0043] More preferably, in step S1, the raw material further includes an ammonium salt.

[0044] Preferably, in step S2, the weight concentration of ammonia monohydrate in the ammonia water is 15%-25%, more preferably 20%.

[0045] Preferably, in step S2, the ratio of L-2-aminobutyric acid, ammonia, ammonium chloride and liquid ammonia is 190-200g: 700-800g: 150-250g: 75-100g.

[0046] More preferably, and as a specific example of the present invention, the ratio of L-2-aminobutyric acid, ammonia, ammonium chloride and liquid ammonia is 193g:750g:200g:87.5g.

[0047] Preferably, in step S2, the temperature of the heat preservation reaction is 0-5℃, and the heat preservation reaction time is 10-14h.

[0048] More preferably, and as a specific example of the present invention, the heat preservation reaction time is 12 hours.

[0049] Preferably, in step S3, the ethanol is anhydrous ethanol and the hydrogen chloride is gaseous hydrogen chloride.

[0050] Preferably, in step S3, the recrystallization specifically involves heating and refluxing in anhydrous ethanol solvent, and then slowly cooling to 20-25°C.

[0051] In another aspect, the present invention provides the application of the above-described method for synthesizing L-2-aminobutyric acid or the above-described method for synthesizing L-2-aminobutyramide hydrochloride in the synthesis of pharmaceutical intermediates.

[0052] Preferably, the pharmaceutical intermediate is a levetiracetam intermediate.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] This invention provides a simple, economical, and environmentally friendly method for preparing L-2-aminobutyramide hydrochloride. The method utilizes phenylalanine ammonia-lyase and its corresponding amino donor, phenylalanine, to catalyze a bio-enzymatic reaction to obtain L-2-aminobutyric acid. Then, the amino group is modified, and salt is formed to obtain L-2-aminobutyramide hydrochloride. This method offers advantages such as inexpensive and readily available materials, high reaction selectivity, high atom utilization, high yield, and good product quality. It also successfully solves the production problems of low material reaction selectivity, waste of isomers during resolution, and high material costs associated with traditional processes. Detailed Implementation

[0055] Terminology and Declarations of this Invention:

[0056] 1. Articles “a,” “a kind,” and “the”: These include plural objects unless otherwise explicitly specified as a single (kind) object.

[0057] 2. Numerical Range: Unless otherwise expressly stated, all ranges or ratios disclosed herein shall be construed as including any and all subranges or subratios contained herein. For example, a stated range or ratio of 1 to 30 shall be considered to be included between the minimum value of 1 and the maximum value of 30, and includes any subranges or subratios, integers, decimals, or subranges or subratios consisting of integers or decimals, including endpoints.

[0058] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following description is merely an exemplary illustration of the scope of protection of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of protection of the present invention.

[0059] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0060] The sources of some reagents in the following examples and comparative examples are shown in Table 1 below:

[0061] Table 1

[0062]

[0063] In the following examples, the L-phenylalanine ammonia-lyase used was extracted from fresh plant tissue. The extraction method is as follows:

[0064] Take fresh potato tubers, peel them, wash them thoroughly with deionized water, and chop them. Add L-phenylalanine ammonia-lyase (PAL) extraction reagent and a small amount of quartz sand as abrasive. Grind the mixture in an ice bath until homogenized, filter through four layers of gauze, and centrifuge the filtrate at 10,000 rpm for 15 minutes at 4°C. The supernatant is the L-phenylalanine ammonia-lyase. Then, use a colorimetric assay kit to detect the enzyme activity units of the obtained L-phenylalanine ammonia-lyase (see the kit instructions for the detection method).

[0065] Example 1

[0066] A method for the enzymatic synthesis of L-2-aminobutyramide hydrochloride. The synthetic route is as follows:

[0067] .

[0068] The synthesis steps are as follows:

[0069] Step S1.

[0070] In a 5000mL four-necked flask, 188g of crotonic acid (approximately 2.18mol), 120g of L-phenylalanine (approximately 0.73mol), 240g of ammonium acetate (approximately 3.11mol), and 1200mL of isopropanol solvent were added. Then, 800U of the prepared L-phenylalanine ammonia-lyase (PAL) was slowly added with stirring. The reaction was carried out at 35℃ for 48 hours. HPLC analysis showed that the peak area percentage of the crotonic acid was less than 2%. After the reaction, diatomaceous earth was added and the mixture was filtered to recover the L-phenylalanine ammonia-lyase (PAL). The solvent was removed by vacuum distillation of the filtrate. Using dichloromethane / methanol (50:1~1:50) as eluent, the product was obtained by silica gel column chromatography, yielding 193g of L-2-aminobutyric acid (approximately 1.87mol). The purity of the L-2-aminobutyric acid product was determined to be 95.9%.

[0071] Yield of L-2-aminobutyric acid = number of moles of L-2-aminobutyric acid product ÷ number of moles of crotonic acid feedstock × 100%.

[0072] The yield of the product L-2-aminobutyric acid was calculated to be 85.71%.

[0073] Step S2.

[0074] The following raw materials were added to a 3000mL four-necked flask: L-2-aminobutyric acid obtained in step S1, 750g of 20% ammonia water, and 200g of ammonium chloride. The mixture was stirred at room temperature for 30 minutes until completely dissolved. The temperature was then lowered to below 0℃, and 87.5g of liquid ammonia was introduced first. The reaction was maintained at 0-5℃ for 12 hours. HPLC analysis showed that the peak area percentage of the L-2-aminobutyric acid was less than 1%. After the reaction was complete, 150g of dichloromethane was added for extraction three times, resulting in layer separation. The combined dichloromethane layers were collected and washed once with 50g of saturated brine, resulting in further layer separation. The dichloromethane layers were collected and concentrated to obtain 180g of L-2-aminobutyramide (approximately 1.76mol). The purity of the product L-2-aminobutyramide was determined to be 95.4%.

[0075] Yield of L-2-aminobutyramide = number of moles of L-2-aminobutyramide product ÷ number of moles of L-2-aminobutyric acid added × 100%.

[0076] The yield of the product L-2-aminobutyramide was calculated to be 94.16%.

[0077] Step S3.

[0078] In a 1000 mL four-necked flask, L-2-aminobutyramide obtained in step S2 was added, along with 540 g of anhydrous ethanol. The mixture was stirred at room temperature for 30 minutes, and the filtrate was collected. 75 g of hydrogen chloride gas was passed through the filtrate at room temperature (20-30°C), precipitating a large amount of white solid. The mixture was stirred for 1 hour, then heated to reflux for 1 hour, slowly cooled to 20-25°C, and stirred for 1 hour. The mixture was then filtered, washed with 100 g of anhydrous ethanol, and dried. 232 g (approximately 1.67 mol) of L-2-aminobutyramide hydrochloride was obtained. HPLC analysis showed the purity of the product L-2-aminobutyramide hydrochloride to be 99.7%, with an ee value of 99.9%.

[0079] Yield of L-2-aminobutyramide hydrochloride = number of moles of L-2-aminobutyramide hydrochloride ÷ number of moles of L-2-aminobutyramide added × 100%.

[0080] The yield of the product L-2-aminobutyramide hydrochloride was calculated to be 94.98%.

[0081] Example 2

[0082] Compared with Example 1, the difference is that in step S1, the amount of L-phenylalanine is changed to 36g, the amount of ammonium acetate is changed to 505g, and the amount of L-phenylalanine ammonia-lyase is changed to 80U. The rest of the synthesis steps are the same.

[0083] Example 3

[0084] Compared with Example 1, the difference is that in step S1, the amount of L-phenylalanine is changed to 480g, the amount of ammonium acetate is changed to 0g, and the amount of L-phenylalanine ammonia-lyase is changed to 8000U. The rest of the synthesis steps are the same.

[0085] Example 4

[0086] Compared with Example 1, the difference is that in step S1, the solvent is changed to an equal volume of methanol, while the rest of the synthesis steps are the same.

[0087] Example 5

[0088] Compared with Example 1, the difference is that in step S1, the solvent is changed to an equal volume of tert-butanol, while the rest of the synthesis steps are the same.

[0089] Example 6

[0090] Compared with Example 1, the difference is that in step S1, the solvent is changed to an equal volume of diethyl ether, while the rest of the synthesis steps are the same.

[0091] Example 7

[0092] Compared with Example 1, the difference is that in step S1, the solvent is changed to an equal volume of dioxane, while the rest of the synthesis steps are the same.

[0093] Example 8

[0094] Compared with Example 1, the difference is that in step S1, the reaction temperature is changed to 10°C, while the rest of the synthesis steps are the same.

[0095] Example 9

[0096] Compared with Example 1, the difference is that in step S1, the reaction temperature is changed to 60°C, while the rest of the synthesis steps are the same.

[0097] Comparative Example 1

[0098] Compared with Example 1, the difference is that in step S1, L-phenylalanine ammonia-lyase is replaced with alanine aminotransferase with the same enzyme activity, while the rest of the synthesis steps are the same.

[0099] The purity and yield of L-2-aminobutyric acid obtained in step S1 of Examples 1-9 and Comparative Example 1 were calculated and summarized in Table 2.

[0100] Table 2

[0101]

[0102] The above experimental results demonstrate that the system of L-phenylalanine ammonia-lyase with phenylalanine and ammonium salt can effectively induce chiral addition of amino groups to crotonic acid to obtain L-2-aminobutyric acid. On the one hand, this reaction exhibits high purity and yield, while in Comparative Example 1, the use of alanine aminotransferase did not result in the formation of L-2-aminobutyric acid. On the other hand, the addition of ammonium acetate significantly reduces the amount of phenylalanine, the organic amino donor, further lowering the material costs of the reaction.

[0103] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for synthesizing L-2-aminobutyric acid, characterized in that, Includes the following steps: The raw materials are mixed, reacted, and separated to obtain L-2-aminobutyric acid; The raw materials include crotonic acid, L-phenylalanine, solvent, and L-phenylalanine ammonia-lyase.

2. The method for synthesizing L-2-aminobutyric acid according to claim 1, characterized in that, The solvent is selected from alcohol solvents or ether solvents.

3. The method for synthesizing L-2-aminobutyric acid according to claim 1, characterized in that, The raw materials also include ammonium salts.

4. The method for synthesizing L-2-aminobutyric acid according to claim 2, characterized in that, The ammonium salt is selected from at least one of ammonium acetate, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium maleate, ammonium malate, ammonium citrate, ammonium tartrate, ammonium fumarate, ammonium bicarbonate, and ammonium carbonate.

5. The method for synthesizing L-2-aminobutyric acid according to claim 1 or claim 4, characterized in that, The proportions of the raw materials used are as follows: Crotonic acid: Phenylalanine: Ammonium salt: L-phenylalanine ammonia-lyase = 188:36-360:0-505:80-8000, in g:g:g:U.

6. The method for synthesizing L-2-aminobutyric acid according to claim 1, characterized in that, The reaction temperature is 10-60℃.

7. A method for synthesizing L-2-aminobutyramide hydrochloride, characterized in that, Includes the method for synthesizing L-2-aminobutyric acid according to any one of claims 1-6; The following synthetic routes are included:

8. The method for synthesizing L-2-aminobutyramide hydrochloride according to claim 7, characterized in that, Includes the following steps: S1. Mix the raw materials, react them, and separate them to obtain L-2-aminobutyric acid; S2. Mix L-2-aminobutyric acid obtained in step S1, ammonia water and ammonium chloride, pass liquid ammonia through, keep the reaction at a temperature, separate, and obtain L-2-aminobutyramide. S3. Mix the L-2-aminobutyramide obtained in step S2 with ethanol, pass hydrogen chloride through, recrystallize, and separate to obtain L-2-aminobutyramide hydrochloride. In step S1, the raw materials include crotonic acid, L-phenylalanine, solvent, and L-phenylalanine ammonia-lyase.

9. The method for synthesizing L-2-aminobutyramide hydrochloride according to claim 8, characterized in that, In step S1, the raw material also includes ammonium salt.

10. The use of the method for synthesizing L-2-aminobutyric acid according to any one of claims 1-4 or the method for synthesizing L-2-aminobutyramide hydrochloride according to any one of claims 5-9 in the synthesis of pharmaceutical intermediates.

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