WO2010019469A2

WO2010019469A2 - Preparation of (s)-2-aminobutyric acid

Info

Publication number: WO2010019469A2
Application number: PCT/US2009/053137
Authority: WO
Inventors: Michael Lloyd; Matthew Bycroft; Srinivas Gade
Original assignee: Dr. Reddy's Laboratories Ltd.; Dr. Reddy's Laboratories, Inc.
Priority date: 2008-08-11
Filing date: 2009-08-07
Publication date: 2010-02-18
Also published as: WO2010019469A3

Abstract

Processes for the preparation of (S)-2-aminobutyric acid, embodiments including selective hydrolysis of racemic N-protected-2-aminobutyric acid using an acylase enzyme, such as one derived from Thermococcus litorolis or Aspergillus melleus.

Description

PREPARATION OF (S)-2-AMINOBUTYRIC ACID

INTRODUCTION

Aspects of the present application relate to processes for the preparation of (S)-2-aminobutyric acid. Embodiments comprise the selective hydrolysis of a racemic N-protected-2-aminobutyric acid, using an acylase enzyme.

The amino acid (S)-2-aminobutyhc acid is an important building block in the synthesis of pharmaceutically active compounds such as levetiracetam, known to be useful for the treatment of epilepsy and other neurological disorders. Optically active amino acids can be made by a number of known methods.

For example, International Application Publication No. WO 2005/001107 and M. L. Youshko et al, in Tetrahedron Asymmetry, 15 (12), pp. 1933-1936 (2004), both describe the selective hydrolysis of alpha-amino acid esters and amides using acylase enzyme. Japanese Published Patent Application No. 2002-253294 describes the stereo-selective hydrolysis of racemic alpha-amino amides using microorganisms such as Rhodococcus, to give (S)-alpha-amino amides and (R)-alpha-amino acids.

Amino acid enantiomers may be prepared using various biotransformation methods. Organic Process Research and Development 2002, 6(4), 533-538 describes the synthesis of (S)-2-aminobutyhc acid via the use of microbial alpha- transaminases. Tetrahedron Letters 1987, 28(32), 3745-3746 describes the conversion of alpha-keto acids into L-alpha-amino acids using cloned E. coli Aspartate transaminase. Tetrahedron Asymmetry 1997, 8(3), 367-370 describes the protease catalyzed hydrolysis of amino acids and esters. Tetrahedron Asymmetry 1997, 8(19), 3197-3200 describes the L-acylase resolution of N- acetyl-DL-amino acids. Journal of the American Chemical Society 1989, 111 (16), 6354-6364 describes the enantioselective hydrolysis of N-acyl amino acids catalyzed by acylase I enzyme. Journal of Organic Chemistry 1987, 52, 2608- 2611 describes the acylase resolution of N-acetyl -2-aminobutyric acid.

Biochemical Society Transactions 2004, 32(2), 290-292 describes a method comprising the resolution of a racemic N-acylamino acid using L-aminoacylase. SUMMARY

Aspects of the present application relate to processes for the preparation of (S)-2-aminobutyric acid. Embodiments comprise the selective hydrolysis of a racemic N-protected-2-aminobutyric acid using an acylase enzyme. In aspects, a process comprises reacting a compound of Formula I, which is in racemic form,

Formula I wherein R is an alkyl or aryl group and M is hydrogen or a cation, with a L-or D- specific acylase enzyme to produce a single enantiomer of an amino acid of Formula II.

Formula Il

The compound of Formula Il may be further converted to the amino amide of Formula III.

Formula III

An embodiment relates to process for preparing a compound of Formula II, wherein M is hydrogen or a metal cation,

Formula Il comprising selectively hydrolyzing a compound of Formula I, wherein R is an alkyl or aryl group,

Formula I in the presence of an acylase enzyme.

A specific embodiment includes a process for preparing (S)-2-aminobutyhc acid, comprising selectively hydrolyzing racemic N-benzoyl-2-aminobutyhc acid using an acylase enzyme obtained from Thermococcus litorolis or Aspergillus melleus.

DETAILED DESCRIPTION

Aspects of the present application relate to processes for the preparation of (S)-2-aminobutyric acid. An embodiment comprises the selective hydrolysis of racemic N-protected-2-aminobutyric acid using an acylase enzyme.

An embodiment of a process comprises reacting a compound of Formula I, which is in racemic form,

Formula I wherein R is a CrC₅ alkyl or optionally a substituted aryl residue, and M is hydrogen or a cation, with a L-or D-specific acylase enzyme to produce a single enantiomer of an amino acid of Formula II.

Formula Il

An aryl residue in Formula I can be unsubstituted phenyl or phenyl substituted by one or more substituents such as methyl, hydroxymethyl, 2- hydroxyethyl, hydroxy, methoxy, ethoxy, amino, methylamino, ethylamino, t- butylamino, dimethylamino, diethylamino, carboxy, methoxycarbonyl, isopropoxycarbonyl, sec-butoxycarbonyl, t-butoxycarbonyl, fluorine, chlorine, bromine, acetyl or pivaloyl, nitro, oxo and/or cyano.

The compound of Formula Il may be further converted to an ester derivative, which may be further treated with ammonia to obtain a single enantiomer of amino amide of Formula III.

Formula III

In an embodiment, the acylase is L-specific. A useful L-specific acylase enzyme can be derived from Thermococcus litorolis. Another useful L-specific acylase enzyme can be derived from Aspergillus melleus. In embodiments, the present application provides the selective hydrolysis of racemic N-benzoyl-2-aminobutyric acid using L-acylase enzyme derived from Thermococcus litorolis or Aspergillus melleus.

The process may be schematically depicted as follows:

The racemic amino acid to be used as a starting material in the present application may contain R- and S-isomers in equal amounts, or one of the isomers may be in excess. The selective hydrolysis may be carried out by subjecting the racemic amino acid to contact with the L-acylase enzyme. The reaction may be carried out in an aqueous medium. The acylase enzyme may be used either in soluble form wherein the acylase is dissolved in water or in an immobilized form wherein acylase is rendered insoluble in water. Various methods for using acylase in the water insoluble form may be used. For example, acylase may be adsorbed onto an ion exchange resin.

The pH may be adjusted to be about neutral to basic, by the addition of a base. The base may be an alkali hydroxide such as sodium or potassium or lithium hydroxide. In embodiments, the pH is adjusted to be in the range of about 6.5 to about 9.5.

The reaction may be conducted under any suitable conditions, for example at ambient to an elevated temperature. In embodiments, the reaction mixture is heated to elevated temperatures. In embodiments, the reaction is conducted at temperatures about 60⁰C to about 85°C. The duration of hydrolysis may vary from about 1 hour to about 24 hours, or longer. In embodiments, the concentrations of racemic amino acid may be in the range of from about 100 to 500 grams per liter. The starting compound may be added in a single lot or in multiple portions.

The solution obtained from the reaction mixture comprises a mixture of (S)- 2-aminobutyric acid and (R)-N-benzoyl-2-aminobutyric acid, and these may be separated from each other by methods known in the art such as crystallization or extraction with a water immiscible solvent.

The reaction mixture may be acidified by the addition of a mineral acid, such as hydrochloric acid. In an embodiment, a process of the present application further comprises the step of separating (R)-N-benzoyl-2-aminobutyhc acid by extraction into a water immiscible solvent. Water immiscible solvents for this purpose include, but are not limited to: hydrocarbons such as hexane, heptane, cyclohexane, benzene, xylene, and toluene; esters such as ethyl acetate, isopropyl acetate, isobutyl acetate, and t-butyl acetate; and halogenated hydrocarbons such as dichloromethane and chloroform.

The (S)-2-aminobutyric acid may be converted into an amide derivative using conventional methods. In an embodiment, (S)-2-aminobutyric acid may be esterified and then converted into an amino amide through the following reactions:

NH₂ NH₂ NH₂

^{^}COOH ^^ COOMe ^^ CONH₂

(S)-2-aminobutyric acid may be treated with thionyl chloride and methanol and the reaction mixture may be stirred until completion of the reaction. The product may be isolated by following conventional procedures, for example, by evaporation of the solvent. Optionally, the reaction mixture may be filtered to remove a solid.

The above product may be treated with ammonia to obtain the amide derivative of the (S)-2-aminobutyric acid. The resulting amide may be converted to the drug compound levetiracetam using known methods. For example, (S)-2- aminobutyhc acid may be converted to (S)-2-aminobutyramide via an ester derivative. (S)-2-aminobutyramide may be converted to levetiracetam, for example by following the process disclosed in U.S. Patent No. 4,943,639.

The isolated (R)-N-benzoyl-2-aminobutyhc acid may be racemized and subjected to a selective hydrolysis method, as hereinbefore described.

The racemization of (R)-N-benzoyl-2-aminobutyric acid may be carried out by heating the optically active N-benzoyl-2-aminobutyhc acid, optionally in the presence of a high boiling solvent. The reaction may be carried out at temperatures of, for example, 40°C-200°C, in some embodiments at about 50⁰C- 150°C. The reaction may be carried out under atmospheric pressure; however, it is also possible to operate under elevated or reduced pressures.

In an embodiment, the racemization may be accomplished by heating a mixture of optically active N-benzoyl-2-aminobutyric acid and an alkali metal hydroxide, such as sodium hydroxide or potassium hydroxide. This reaction may be carried out in the presence or absence of a solvent. Useful solvents include water and organic solvents.

In another embodiment, (R)-N-benzoyl-2-aminobutyhc acid may also be converted to its racemic form by reacting with acetic anhydride. The compound of Formula I may be prepared by reacting the racemic amino acid of Formula IV,

Formula IV wherein M is hydrogen or a cation, with an acylating agent using conventional methods.

The acylating agent may be an acetyl halide or acetic anhydride, to provide the corresponding acetyl derivative, or a benzoyl halide, to provide a benzoyl derivative. In an embodiment, an acylating agent is benzoyl chloride.

The following examples will further describe certain specific aspects and embodiments, and are provided solely for purposes of illustration. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention and no limitation of the invention is implied.

EXAMPLE 1 : SYNTHESIS OF N-BENZOYL-2-AMINOBUTYRIC ACID.

A mixture of 2-aminobutyhc acid (200 g) and water (1 L) was stirred by means of an overhead stirrer. The mixture was cooled to below 10⁰C and the pH was adjusted to about 9 by drop-wise addition of 46-48% sodium hydroxide solution. Benzoyl chloride (270.5 mL) was added drop-wise to the mixture and the pH was maintained above 8.5 by further addition of 46-48% sodium hydroxide solution (250 ml_ added in total). After the completion of the addition, the mixture was allowed to warm to about 22-25°C and stirred for 20 hours. The mixture was acidified to pH about 2.5 with concentrated HCI (150 ml_) and extracted with ethyl acetate (2 L). The organic layer was dried over magnesium sulfate and evaporated under reduced pressure to yield N-benzoyl-2-aminobutyric acid as a white solid. Yield: 385 g.

EXAMPLE 2: SYNTHESIS OF N-BENZOYL-2-AMINOBUTYRIC ACID. A mixture of 2-amino butyric acid (50 g) and water (250 mL) was cooled to

5-10⁰C and the pH was adjusted to about 9 by drop-wise addition of 48% sodium hydroxide solution. Benzoyl chloride (81.8 g) was added drop-wise to the mixture and the pH was maintained above 8.5 by further addition of 48% sodium hydroxide solution (104 mL added in total). After completion of the addition, the mixture was allowed to warm to about 20-25⁰C and stirred for 20 hours. The mixture was acidified to pH about 2.5 with concentrated HCI (60 mL). The formed solid was collected by filtration and dried. Yield: 100.4 g.

EXAMPLE 3: L-ACYLASE BIO-RESOLUTION OF N-BENZOYL-2- AMINOBUTYRIC ACID.

A suspension of N-benzoyl-2-aminobutyric acid (364.5 g) in water (3.5 L) was placed into a 5 L vessel. The mixture was stirred by means of an overhead stirrer, heated to 65°C and the pH was adjusted to 8 by drop-wise addition of sodium hydroxide solution (100 mL). L-acylase enzyme solution (90 mL, 238500 units) was added to the mixture and stirring was continued for 19 hours at 65°C. The mixture was cooled to room temperature, acidified to pH 3 with concentrated hydrochloric acid and extracted with ethyl acetate (2*1.5 L). Concentration of the aqueous phase yielded (S)-2-aminobutyric acid hydrochloride. Yield: 23 g. EXAMPLE 4: L-ACYLASE BIO-RESOLUTION OF N-BENZOYL-2- AMINOBUTYRIC ACID.

A suspension of N-benzoyl-2-aminobutyric acid (50 g) in water (260 mL) was placed into a vessel and heated to 65°C. The pH was adjusted to 8 by drop- wise addition of sodium hydroxide solution (5M, 48 mL). L-acylase enzyme solution (1.9 mL) was added to the mixture and stirring was continued at 65°C for 19 hours. The mixture was cooled to room temperature, acidified to pH 3 with hydrochloric acid (6N, 38 mL) and the aqueous layer was washed with ethyl acetate (3*50 mL). Concentration of the aqueous phase yielded a solid, to which was added isopropyl alcohol (100 mL). The solvent was removed by evaporation under reduced pressure and the obtained solid was mixed with isopropyl alcohol (100 mL) and the slurry was stirred at 25-30⁰C for 2 hours. The solid was filtered, washed with isopropyl alcohol (10 mL) and dried. Yield: 25.4 g.

EXAMPLE 5: SYNTHESIS OF METHYL (S)-2-AMINOBUTYRATE HYDROCHLORIDE.

A suspension of (S)-2-aminobutyhc acid hydrochloride salt (90 g) in methanol (1 L) was cooled to below 5°C. Thionyl chloride (80 mL) was added drop-wise and, after the addition was completed, the mixture was allowed to warm to room temperature and was stirred for 18 hours. The insoluble material was removed by filtration and the filtrate was concentrated to dryness under reduced pressure to obtain methyl (S)-2-aminobutyrate hydrochloride as a white solid. Yield: 109 g.

EXAMPLE 6: SYNTHESIS OF (S)-2-AMINOBUTYRAMIDE HYDROCHLORIDE.

Into a 2 L vessel, methyl (S)-2-aminobutyrate hydrochloride (131 g) was placed. The vessel was cooled to 5°C and pre-cooled aqueous ammonia (35%, 1.4 L) was added. The mixture was stirred at 5°C for 18 hours. The mixture was concentrated under reduced pressure to obtain (S)-2-aminobutyramide hydrochloride as a white solid. Yield: 116 g. EXAMPLE 7: SYNTHESIS OF METHYL (S)-2-AMINOBUTYRATE HYDROCHLORIDE.

Into a 3-neck round bottom flask with a condenser attached, (S)-2- aminobutyhc acid (164 g) and methanol (500 mL) were placed. The mixture was stirred and HCI gas (67 g) was bubbled through the reaction mixture for 30 minutes. The mixture was heated to 55°C and stirred overnight. The mixture was allowed to cool to room temperature and inorganic salts were removed by filtration. The filter cake was washed with methanol. The solvent was removed from the filtrate under reduced pressure. The residue was dissolved in dichloromethane and remaining insoluble material was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a white solid. Yield: 103 g.

EXAMPLE 8: RACEMIZATION OF (R)-N-BENZOYL^-AMINOBUTYRIC ACID. Into a 1 L round bottom flask, (R)-N-benzoyl-2-aminobutyric acid (90 g) was placed and a mixture of sodium acetate (5.5 g) in ethyl acetate (500 mL) was added. The mixture was heated to 80⁰C and acetic anhydride (70 mL) was added drop-wise over 15 minutes. The mixture was stirred at 80⁰C for 4 hours. The mixture was allowed to cool to room temperature and aqueous NaOH (5M, 300 mL) was added. The mixture was vigorously stirred for 2 hours. The phases were separated and the aqueous phase was acidified to pH 3 by the addition of HCI. The aqueous phase was then extracted with ethyl acetate (2*250 mL). The combined organic extract was dried over magnesium sulfate and the solvent was removed under reduced pressure to obtain a white solid. Yield: 46 g.

EXAMPLE 9: RACEMIZATION OF (R)-N-BENZOYL-2-AMINOBUTYRIC ACID. Into a round bottom flask, sodium hydroxide (97 g) and water (125 mL) were stirred at room temperature to produce a clear solution. (R)-N-benzoyl-2- aminobutyric acid (25 g) was added and the mixture was heated to reflux for 12 hours. The mixture was cooled to 25-35°C, the pH was adjusted to 2 by the addition of hydrochloric acid (45 mL). The separated solid was collected by filtration, washed with water (50 mL) and dried. Yield: 18 g. EXAMPLE 10: RACEMIZATION OF (R)-N-BENZOYL^-AMINOBUTYRIC ACID.

Into a 1 L round bottom flask, (R)-N-benzoyl-2-aminobutyric acid (5.0 g) was placed and heated to 120⁰C for about 24 hours. The flask was cooled to 25- 35°C and the solid was collected. Yield: 5.0 g.

Claims

CLAIMS:

1. A process for preparing a compound of Formula II, wherein M is hydrogen or a metal cation,

Formula I in the presence of an acylase enzyme.

2. The process of claim 1 , wherein a compound of Formula I is N- benzoyl-2-aminobutyric acid.

3. The process of claim 1 , wherein an acylase enzyme is obtained from Thermococcus litorolis or Aspergillus melleus.

4. The process of claim 1 , wherein an acylase enzyme stereo- selectively hydrolyzes the compound of Formula I.

5. The process of claim 1 , wherein an acylase enzyme is L-specific.

6. The process of claim 1 , wherein hydrolyzing is conducted in a reaction medium comprising water.

7. The process of claim 1 , wherein hydrolyzing is conducted in the presence of a base.

8. The process of claim 1 , wherein hydrolyzing is conducted in the presence of an alkali metal hydroxide, alkali metal alkoxide, or alkali metal carbonate.

9. The process according to claim 1 , wherein hydrolyzing is conducted in a pH range of 7-10 and at temperatures of 20°C-70°C.

10. The process according to claim 1 , having the reaction scheme:

wherein M and R are as described in claim 1.

11. The process of claim 1 , wherein a compound of Formula Il is further converted to the amino amide of Formula III.

Formula III

12. The process of claim 11 , wherein the compound of Formula III is further converted to levetiracetam.

13. A process for preparing (S)-2-aminobutyhc acid, comprising selectively hydrolyzing racemic N-benzoyl-2-aminobutyric acid using an acylase enzyme obtained from Thermococcus litorolis or Aspergillus melleus.

14. The process of claim 13, wherein an acylase enzyme is L-specific.