This application claims priority to Indian patent application No. 1243/CHE/2009 filed on May 29, 2009, the contents of which are incorporated by reference in their entirety

Field of the invention

The present invention relates to an enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate, an intermediate in the preparation of R-Baclofen.

Background of the Invention

(R)- 4-amino-3- (4-chlorophenyl)-butanoic acid (R-Baclofen) represented by formula I, is a useful antispastic agent.

Formula I

Baclofen is used for treating spasm of skeletal muscles that cause muscle clonus, rigidity, and pain due to multiple sclerosis.

Chemically, baclofen is related to gamma-aminobutyric acid (GABA), a naturally-occurring neurotransmitter in the brain. Neurotransmitters are chemicals that nerves use to communicate with one another. GABA released by some nerves causes the activity of other nerves to decrease. It is believed that baclofen, acting like GABA, blocks the activity of nerves within the part of the brain that controls the contraction and relaxation of skeletal muscle.

Baclofen was first disclosed and claimed in US 3,471,548 and its European equivalent is GB 1017439.

WO 9722578 disclosed a process for the resolution of baclofen to (R) - Baclofen using S alpha methyl benzyl amine. US 5843765 disclosed biological production of substantially and enontiomerically pure (R) -Baclofen and its related compounds from racemic mixtures.

Chemoenzymic enantioselective synthesis of baclofen, disclosed in Canadian Journal of Chemistry (1994), 72(11), 2312-17, reported two different chemoenzymic enantioselective syntheses of baclofen In the first approach, the key step is the highly stereoselective enzymic hydrolysis of di-Me 3-(4-chlorophenyl)glutarate by chymotrypsin in an aq. medium. In the second approach, the key step is the enzyme-catalyzed esterification of 2-(4-chlorophenyl)-1,3-propanediol by acetic anhydride in the presence of a lipase in an org. medium.

U.S. Pat. No. 6410306 disclosed enantioselective enzymatic hydrolysis of substituted esters of glutaric acid. The enzymes used in the said enzymatic hydrolysis were selected from the class of lipases.

Accordingly, there remains a need for improved processes for preparing R-Baclofen that is convenient and cost efficient on a commercial scale. The process according to the present invention relates to an enzymatic process for the preparation of the (S)-monoester intermediate of Baclofen which in-turn is converted to R-Baclofen. The enzymatic process of the present invention is eco-friendly, cost effective and commercially viable.

Object and Summary of the Invention

The present invention relates to an enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate, an intermediate in the preparation of R-Baclofen.

In one aspect, the present invention relates to an enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate, wherein the enzyme is selected from lipase family.

In another aspect, the present invention relates to (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate having a purity of more than 99%.

Detail Description of the Invention

The present invention relates to an enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate.

In one embodiment, the present invention relates to an enzymatic process for the preparation of an intermediate of (R) -Baclofen wherein the enzyme is selected from Lipase family.

In another embodiment, the present invention relates to an enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate as summarized in scheme I

In another embodiment, the present invention relates to an enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate in enantiomeric excess comprising the steps of;

i) hydrolyzing 3-(4-chlorophenyl) glutarate with an enzyme in presence of a buffer, and ii) Isolating (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate

According to the present invention, ethyl 3-(4-chlorophenyl)glutarate (5 g) is dispersed in a buffer having pH of about 8 to 9 followed by addition of enzyme selected from lipases, maintaining the pH at about 8 using sodium hydroxide. On the completion of the reaction monitored by HPLC, the pH of the filtrate was adjusted to about 3.0 to 4.0 using cone, sulfuric acid followed by isolation of the precipitated products by filtration. The acid precipitant was dried to afford (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate. The (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate, prepared above is in-turn converted to R-baclofen following the procedures known in the art.

The enzyme used in the present invention is selected from lipases such as Porcine Pancreatic Lipase, CAL-A, lyophilized Candida lipolytica Lipase, Geotrichum Candidum Lipase, Pseudomonas aroginosa Lipase, Aspergillus niger Lipase, Pseudomonas cepacia Lipase, Pseudomonas fluorescens Lipase, Candida antartica, Rhizopus delemar Lipase, Rhizopus oryzae Lipase, Penicillium camembertii Lipase, Penicillium camembertii Lipase, Mucor javanicus Lipase, Penicillium roqueforti Lipase, Pseudomonas cepacia Lipase, CAL-B, lyophilized microbial, lyophilized Lipase, Thermomyces sp.

Lipase.Alcaligines sp., Chromobacterium viscosum Lipase.Candida utilis Lipase, Rhizopus niveus Lipase, Pseudomonas sp. Lipoprotein Lipase, Thermomuces lanuginosus Lipase, Rhizomucor miehei Lipase, Pseudomonas species Lipase, Wheat Germ Lipase, Rhizopus arrhizus Lipase, Pancreatic Lipase 250, Novozyme-435 and the like.

The present invention is carried out in presence of a buffer selected from sodium phosphate, potassium phosphate or any mixtures thereof.
The present invention is carried out at a pH from 8 to 9.

In another embodiment of the present invention relates to (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate having a purity more than 99 % ee.

The advantages of the present invention are a) the enzyme used in the present invention is cheap and can be reused thus making the invention cost effective, b) to avoid the use of chiral axillaries and lower temperatures, c) performing the reaction in the presence of a buffer at ambient temperature.

In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
Experimental

Example 1: Preparation of (S)-monoester of ethyl 3-(4-chlorophenyl)glutarate

Ethyl 3-(4-chlorophenyl)glutarate (5 g) was dispersed in 10 mM sodium phosphate buffer pH 8.0
(45 ml) in a 3-neck round bottom flask equipped with an electrode and pH delivery tube
connected to a pH stat. The mixture was stirred and adjusted the pH 8.0 at 30 to 40°C. The
reaction was initiated with the addition of 500 mg of CAL B (Novozyme-435) and the pH 8.0 was
maintained by automatic titration of 0.5M NaOH solution. The reaction was monitored by HPLC.
After complete conversion of the starting material, reaction was terminated by filtering off the enzyme. The pH of the filtrate was adjusted to 3.5 to 4.0 using 20 % H2S04 followed by isolation of the precipitated product by filtration and dried to afford (S)-monoester more than 90 % yield with 98 to 99 % ee.

Example 2:

Ethyl 3-(4-chlorophenyl)glutarate (50 g) was dispersed in 10 mM sodium phosphate buffer pH 8.0 (450 ml) in a 3-neck round bottom flask equipped with an electrode and pH delivery tube connected to a pH stat. The mixture was stirred and adjust the pH 8.0 at 38 to 40°C. The reaction was initiated with the addition of 5 g of CAL B (Novozyme-435) and the pH 8.0 was maintained by pH-Stat with 2M NaOH solution. The reaction was monitored by HPLC, after complete conversion of the starting material reaction was terminated by filtering off the enzyme. The pH of the filtrate was adjusted 3.5 to 4.0 using 20 % H2S04 followed by extraction with ethyl acetate. The organic layer was separated and dried over sodium sulphate and the solvent was evaporated on rota-evaporator to get the (S)-monoester 43 g (95 % yields) with 98 to 99 % ee.

Example 3:

Ethyl 3-(4-chlorophenyl)glutarate (50 g) was dispersed in 10 mM sodium phosphate buffer pH 8.0 (450 ml) in a 3-neck round bottom flask equipped with an electrode and pH delivery tube connected to a pH stat. The mixture was stirred and adjust the pH 8.0 at 35 to 40 OC. The reaction was initiated with the addition of 5 ml of liquid Candida antartica lipase B (CAL B L) and the pH 7.9-8 was maintained by pH-Stat with 2M NaOH solution. The reaction was monitored by HPLC, after complete conversion of the starting material reaction, the pH was adjusted 3.5 to 4.0 using 20 % H2S04 followed by extraction with ethyl acetate. The organic layer was separated and dried over sodium sulphate and the solvent was evaporated on rota- evaporator to get the (S)-monoester 40 g (89 % yields) with 97 to 98 % ee.

We Claim

1) Enzymatic process for the preparation of (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate in enantiomeric excess comprising the steps of;

i) hydrolyzing 3-(4-chlorophenyl) glutarate with an enzyme in presence of a buffer, and ii) Isolating (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate

2) The process according to claim 1, wherein the buffer is selecting from sodium phosphate buffer, potassium phosphate buffer, tris(hydroxymethyl)aminomethane or a mixture thereof.

3) The process according to claim 1, wherein the enzyme is selecting from Porcine Pancreatic Lipase, CAL-A, lyophilized Candida lipolytica Lipase, Geotrichum Candidum Lipase, Pseudomonas aroginosa Lipase, Aspergillus niger Lipase, Pseudomonas cepacia Lipase, Pseudomonas fluorescens Lipase, Candida rugosa Lipase, Rhizopus delemar Lipase, Rhizopus oryzae Lipase, Penicillium camembertii Lipase, Penicillium camembertii Lipase, Mucor javanicus Lipase, Penicillium roqueforti Lipase, Pseudomonas cepacia Lipase, CAL-B, Candida antartica lipase B (CAL B L),lyophilized microbial, lyophilized Lipase, Thermomyces sp. Lipase, Alcaligines sp., Chromobacterium viscosum Lipase, Candida utilis Lipase, Rhizopus niveus Lipase, Pseudomonas sp. Lipoprotein Lipase,Thermomuces lanuginosus Lipase, Rhizomucor miehei Lipase, Pseudomonas species Lipase, Wheat Germ Lipase,Rhizopus arrhizus Lipase, Pancreatic Lipase 250, Novozyme-435.

4) The process according to claim 3, wherein enzyme is selecting from CAL-A, CAL-B, Candida antartica lipase B (CAL B L) or Novozyme-435.

5) The process according to claim 1, wherein the hydrolysis is carried out at a temperature of 30 to 40°C.

6) The process according to claim 1, wherein hydrolysis is carried out at a pH from 8 to 9.

7) The process according to claim 6, wherein pH is maintained using metal hydroxides such as sodium hydroxide.

8) (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate having a purity more than about 99 % ee.

9) (S)-monoester of ethyl 3-(4-chlorophenyl) glutarate prepared according to claim 1 and further conversion to (R)- Baclofen.