This application claims priority to Indian patent application No 2719/CHE/2009 filed on,
November 9, 2009 the contents of which are incorporated by reference in their entirety.

Field of the Invention

The present invention relates to novel enzymatic process for the preparation of (S)-monoester intermediate of pregabalin. The present invention further relates to a process for the preparation of pregabalin using the (S)-monoester intermediate. The present invention also relates to one pot process for the preparation of monoamide intermediate of (S)-Pregabalin.

Background of the Invention

(S)-(+)-3-(aminomethyl)-5-methylhexanoic acid (S-Pregabalin) is a y-amino butyric acid, having the chemical structure as shown below,

LYRICA (pregabalin) has been found to activate GAD (L-glutamic acid decarboxylase). Pregabalin has a dose dependent protective effect on-seizure, and is a CNS-active compound. Pregabalin is useful in anticonvulsant therapy, due to its activation of GAD, promoting the production of GABA, one of the brain's major inhibitory neurotransmitters, which is released at 30 percent of the brains synapses. Pregabalin has analgesic, anticonvulsant, and anxiolytic activity.

Pregabalin has been prepared in various ways. Typically, a racemic mixture of 3-aminomethyl-5-methyl-hexanoic acid is synthesized and subsequently resolved into its R-and S-enantiomers. Such methods may employ an azide intermediate, a malonate intermediate, or Hofmann synthesis. See, respectively, U. S. Patent No. 5,563,175 to R. B. Silverman et al.; U. S. Patent Nos. 6,046,353; 5,840,956 and 5,637,767 to T. M. Grote et al.; and U. S. Patent Nos. 5,629,447 and 5,616,793 to B. K. Huckabee & D. M.

Sobieray, which are herein incorporated by reference in their entirety and for all purposes. In each of these methods, the racemate is reacted with a chiral acid (a resolving agent) to form a pair of diastereoisomeric salts, which are separated by known techniques, such as fractional crystallization and chromatography. These methods thus involve significant processing beyond the preparation of the racemate, which along with the resolving agent, adds to production costs. Moreover, the undesired R-enantiomer is frequently discarded since it cannot be efficiently recycled, thereby reducing the effective throughput of the process by 50%.

Pregabalin has also been synthesized directly using a chiral auxiliary,(4R,5S)-4- methyl-5-phenyl-2-oxazolidinone. In U.S. Patent Nos. 6,359,169; 6,028,214; 5,847,151; 5,710,304; 5,684,189; 5,608,090 and 5,599,973 all to R. B. Silverman et al, which are herein incorporated by reference in their entirety and for all purposes. Although these methods provide Pregabalin in high enantiomeric purity, they are less desirable for large-scale synthesis because they employ comparatively costly reagents (e.g. the chiral auxiliary) that are difficult to handle, as well as special cryogenic equipment to reach required operating temperatures, which can be as low as -78°C.

U.S. patent application 2003/0212290, discusses a method of making Pregabalin via asymmetric hydrogenation of a cyano-substituted olefin to produce a chiral cyano precursor of (S)-3-aminomethyl-5-methylhexanoic acid. The cyano precursor is subsequently reduced to give Pregabalin. The asymmetric hydrogenation employs a chiral catalyst that is comprised of a transition metal bound to a bisphosphine ligand, such as (R,R)- Me-DUPHOS. The method results in substantial enrichment of Pregabalin over (R)-3-(aminomethyl)-5-methylhexanoic acid.

The method discussed in U.S. Patent Application No. 2003/0212290, disclosed a commercially viable method for preparing Pregabalin, but further improvements would be desirable for various reasons. For example, bisphosphine ligands, including the proprietary ligand (R,R)-Me-DUPHOS, are often difficult to prepare because they possess two chiral centers, which adds to their cost. Furthermore, asymmetric hydrogenation requires the use of special equipment capable of handling H2, which adds to capital costs.

Therefore there is a need to develop an eco-friendly, safe and economically and industrially feasible process for large scale production of S-pregabalin eliminating the use of costly chiral ligands or auxiliaries, column chromatography. Present invention discloses an enzymatic process for desymmetrization of 3-Isobutyl-pentanedioic acid dialkyl ester using lipases to produce (S)-monoester intermediate of pregabalin which in turn is converted to S-pregabalin.

Object of the invention:

The main object of the present invention relates to enzymatic process for the preparation of (S)-monoester intermediate of pregabalin.
Another object of the present invention relates to further conversion of the (S)-monoester intermediate to pregabalin.

Another object of the present invention relates to one pot process for the preparation of monoamide intermediate of pregabalin.

Summary of the invention:

One aspect of the present invention relates to enzymatic process for the preparation of (S)-monoester intermediate of pregabalin comprising the steps of adding 3-isobutyl -pentanedioic acid dialkyl ester to buffer/cosolvent, adding enzyme and maintaining the pH with base, adjusting to acidic pH and isolating (S)-monoester intermediate.

Yet another aspect of the present invention relates to novel process for the preparation of pregabalin comprising the steps of adding 3-isobutyl-pentanedioic acid dialkyl ester to buffer/cosolvet, adding enzyme maintaining the pH with base, adjusting to acidic pH, isolating (S)-monoester intermediate, converting (S)-monoester intermediate into
monoamide, treating the monoamide with a mixture of bromine and sodium hydroxide to obtain Pregabalin.

Yet another aspect of the present invention relates to novel process for the preparation of monoamide intermediate comprising the steps of combining 3-isobutyl -pentanedioic acid dialkyl ester with suitable enzyme which stereoselctively hydrolyzing dialkyl ester compound and reacting with ammonia to obtain monoamide.

Detailed Description of the Invention:

The present invention relates to an enzymatic process for the preparation of (S)-monoester intermediate of pregabalin. The present invention further relates to process for the preparation of pregabalin using the (S)-monoester intermediate prepared by the enzymatic process. The present invention also provides one pot process for the preparation of monoamide intermediate of Pregabalin.

In one aspect, the present invention provides, a process for the preparation of (S)-monoester intermediate of pregabalin as illustrated in Scheme 1.

In one embodiment, the present invention provides, an enzymatic process for the
preparation of (S)-monoester intermediate of pregabalin comprising the steps of

i) adding 3-isobutyl -pentanedioic acid dialkyl ester (1) to buffer/cosolvent,
ii) adding enzyme in the presence a base,
iii) maintaining desired pH by adding base,
iv) adjusting the reaction mass pH to acidic, and
v) isolating (S) - monoester intermediate (2).

In another embodiment, the present invention provides, a process for the preparation of pregabalin using the (S)-monoester intermediate as illustrated in Scheme 2:

In yet another embodiment, the present invention provides, a chemo-enzymatic process for the preparation of pregabalin comprising the steps of:

i) adding 3-isobutyl -pentanedioic acid dialkyl ester(l) to buffer,
ii) adding enzyme in the presence of a base,
iii) adjusting the reaction mass pH to acidic,
iv) isolating (S) - monoester intermediate^),
v) converting (S) - monoester intermediate^) into monoamide(3),
vi) treating monoamide(3) with mixture of bromine and sodium hydroxide, and
vii) isolating (S)-Pregabalin.

According to the present invention, 3-isobutyl -pentanedioic acid dialkyl ester of formula (1) is added to buffer/cosolvent and added an enzyme selected from Upases and maintaining desired pH by adding base. The pH of the reaction mass is adjusted to acidic thus isolating (S) - monoester intermediate of formula (2). The (S) - monoester intermediate (2) is then converted into corresponding monoamide of formula (3) using aqueous ammonia followed by treatment with bromine and sodium hydroxide to afford S-pregabalin.

According to the present invention, the buffer used in the preparation process is selected from sodium phosphate buffer or potassium phosphate buffer (0.01 M to 1M), Tris-HCl buffer. The cosolvent selected from polar and nonpolar organic solvent such as methanol, ethanol, isopropanol, methylene chloride, ethylene chloride and the like.

According to the present invention, the enzyme used in the present invention is selected from Lipozymes, 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, 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,CAL-B (Novozyme-435), Candida antartica lipase (IMMCALB-T2-150) and the like.

According to the present invention, the enzyme is added at ambient temperature of about 20 to 45 °C by maintaining the pH of the reaction mixture at about 6 to 8.5.

According to the present invention, the base used in the preparation process is selected form sodium hydroxide, potassium hydroxide and the like.

According to the present invention, the pH adjustments are made by using acids selected from sulfuric acid or hydrochloric acid or their mixtures thereof.

In yet another embodiment, the present invention provides enantiomerically pure (S) -monoester having enantiomeric purity of about 80 -99 %.

In yet another embodiment, the present invention provides enantiomerically pure Pregabalin having enantiomeric purity of about 99 %.

In another aspect, the present invention provides a one pot chemo-enzymatic process for the preparation of monoamide (3)
comprising the steps of:

i) combining 3-isobutyl-pentanedioic acid dialkyl ester (1) with suitable enzyme
which stereoselctively hydrolyzing of compound of formula 1;

wherein R is substituted or unsubstituted C1-C6 alkyl

ii) reacting with ammonia; and

iii) isolating monoamide (3).

In one embodiment of the present invention, the (S) - monoester intermediate of formula (2) is not isolated during the preparation of monoamide (3).

In another embodiment of the present invention, the enzyme which stereoselctively hydrolyzing 3-isobutyl-pentanedioic acid dialkyl ester (1) is selected from Lipozymes, 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, 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,CAL-B (Novozyme-435), Candida antartica lipase (IMMCALB-T2-150) and the like.

In one more embodiment of the present invention, the enzymes are used in a combination with buffer. The buffer used in this process is selected from sodium phosphate buffer or potassium phosphate buffer or mixtures thereof.

In one more embodiment of the present invention, the reaction is carried out by adjusting the pH of the reaction mixture about 6.0 to 9.0, more preferably 6.5 to 8.0 and most preferably 7.0 to 8.0 by adding a base. The base used in this process is selected form sodium hydroxide, potassium hydroxide preferably sodium hydroxide.

In one more embodiment of the present invention, the reaction is done for about 0.5 to 50 hours, preferably 10-40 hours and more preferably 15-30 hours. The reaction mass is optionally extracted into water immiscible organic solvent and ammonia solution is added to obtain monoamide compound (3). The water immiscible organic solvent used is selected from the group consisting of methylethylketone, toluene, methyl tert-butyl ether, diisopropylether. Preferably methyl tert-butyl ether.

In yet another embodiment of the present invention, the monoamide compound (3) is further converted into (S)-Pregabalin by the conventional prior art methods.

In yet another embodiment, the present invention provides, enantiomerically pure Pregabalin having enantiomeric purity of about 99 %.

The following non-limiting examples illustrate specific embodiments of the present invention. They should not construe it as limiting the scope of present invention in anyway.

Examples: Example: 1
3-Isobutyl-pentanedioic acid dialkyl ester 1 (25 g) was added to 100 mM sodium phosphate buffer pH 7.9 (250 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 7.9 at 25 to 35°C. The reaction was initiated with the addition of 2.5 g of CAL B (Novozyme-435) and the pH 7.9 was maintained by automatic titration with 3M sodium hydroxide 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 4.2 using 20 % sulphuric acid / dil hydrochloric acid followed by extraction with ethyl acetate/methylene dichloride. The organic layer was separated and dried over sodium sulphate and the solvent was evaporated on rota evaporator to get the (S)-monoester 2 (21 g) around 95 % yield with 90 to 99 % ee.

Example: 2
(S)-Monoester 2 (10 g) was placed in autoclave and aqueous ammonia 30% (140 ml) was charged. The mixture was heated with stirring in autoclave for 3 days at 65 to 70 C. After that reaction mixture was cooled at room temperature and the water was distilled out completely. The residue was cooled up to 10 to 15 °C with stirring and the pH was adjusted up to pH 1-2 with HC1, the slid was precipitated out filtered and washed with water and dry to get monoamide 3 (6 g).

Example: 3
A suspension of monoamide 3 (10 g) in 50% sodium hydroxide solution (lOmL) at pH 7 to 8, a bromine in 50% sodium hydroxide solution was added with stirring and thereafter the reaction mixture was heated up to 75 to 80°C. After completion of the reaction it was cooled up to 45°C and quenched by the addition of 37% HCI solution. Again reaction mixture was heated up to 80 to 90°C for one hrs, cool the reaction mass up to 5 to 10°C and the solid was filtered and washed with chilled water and dry to get the (SyPregabalin (6 g) with 90 to >99% ee.

Example 4: Process for the preparation of R (-)-3-(Carbamoylmethyl)-5-methyl hexanoic acid
3-Isobutyl pentane dionic acid (200 g) was added to a mixture of Di potassium hydrogen phosphate (60.2 g), Potassium dihydrogen phosphate (7.2 g) and water. To this reaction mixture Lypozyme CAL B (20 ml) was added and pH was adjusted to 7.9 by the addition of 2M sodium hydroxide solution. To this Methyl tertiary butyl ether (1 Lit) was added and organic layer was separated and filtered throw hyflow. 10% Sodium chloride solution was added to the organic layer and organic layer was separated. Methyl tertiary butyl ether was distilled off under vacuum and the reaction mass was cooled. To this reaction mass Ammonia solution (1440 ml) was added at 0-5 °C. The temperature was raised to 25- 35 °C and maintained for 3-4 days. The reaction mass was cooled to 15-20 °C and pH was adjusted to 3 by adding CP HC1. The reaction mass was filtered and washed with chilled water. The obtained wet cake was dried under normal pressure to yield R(-)-3-(Carbamoylmethyl)-5-methyl hexanoic acid (90 g).

Example 5: Process for the preparation of (S)-Pregabalin
R(-)-3-(Carbamoylmethyl)-5-methyl hexanoic acid (100 g) was added to a mixture of sodium hydroxide (106.8 g) and water (700 ml). To this reaction mixture bromine (85.3 g) was slowly added at 5-10 °C and temperature was raised to 45-55 °C and stirred for 1 hour. The pH of the reaction mixture was adjusted to 6.8 by the addition of CP HC1 (135 ml) and maintained for 2-3 hours. The reaction temperature was cooled and obtained solid was filtered and washed with water. The obtained solid was recrystallized from mixture of water (90 ml) and isopropyl alcohol (210 ml) to yield crude (S)-Pregabalin (60
g).

Example 6: Purification of crude (S)-Pregabalin
To crude (S)-Pregabalin (100 g), a mixture of water (500 ml) and isopropyl alcohol (500 ml) was added and temperature was raised to 80-90 °C. To this reaction mixture PS-133 carbon (10 g) was added filtered through hyflow. The reaction mass was cooled to 5-10 °C and filtered, washed with isopropyl alcohol. The obtained residue was dried to yield pure (S)-Pregabalin (95 g).

We claim:

1. A one pot process for the preparation of monoamide (3)
comprising the steps of:

a) combining 3-isobutyl-pentanedioic acid dialkyl ester (1) with suitable
enzyme which stereoselctively hydrolyzing of compound of formula 1;
wherein R is substituted or unsubstituted C1-C6 alkyl

b) reacting with ammonia; and

c) isolating monoamide (3).

2. The process according to claim 1, the enzyme is selected from the group consisting of Lipozymes, 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, 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,CAL-B (Novozyme-435), Candida antartica lipase (IMMCALB-T2-150).

3. The process according to Claim 1, wherein enzymes are used in combination with buffer.

4. The process according to claim 3, wherein the buffer is selected from sodium phosphate buffer or potassium phosphate buffer or mixtures thereof.

5. The process according to claim 1, wherein step a, is carried at pH 6.5-9 by optionally adding base.

6. The process according to claim 5, wherein the base is selected from sodium hydroxide or potassium hydroxide.

7. The process according to claim 1, wherein hydrolysis of compound of formula 1 is carried out for about 0.5 to 50 hours.

8. The process according to claim 1, wherein after hydrolysis, the reaction mass is optionally extracted into water immiscible organic solvent selected from methylethylketone, toluene, methyl tert-butyl ether or diisopropylether.

9. The process according to claim 1, which further comprises conversion of monoamide (3) to (S)-pregabalin.

10. Enantiomerically pure (S)-Pregabalin having enantiomeric purity 99 % or more, obtained by the process according to Claim 9.