Title of Invention

"ENZYMATIC PROCESS FOR THE PREPARATION OF S-(+)-4(3-THIENYL)PHENYL-α-METHYLACETIC ACID"

Abstract

The present invention relates to an enzymatic process for preparing 5-(+)-4-(3-thienyl)phenyl-α-methylacetic acid, by enantioselective hydrolysis of the corresponding racemic ester using an extracellular lipase; particularly, the present invention relates to a process for the preparation of S-(+)-4-(3-thienyl)phenyl-α-methylacetic acid, of structural formula (3) in the accompanying diagram, by enantioselective hydrolysis of racemic ester of the acid (1), of structural formula (2), in the accompanying diagram, in a buffer medium or buffer containing a co-solvent if required in presence of commercially available lipases derived from the microorganisms of the genera Candida, Mucor, Rhizopus, Pseudomonas and Aspergillus.

Full Text

Field of the invention The present invention relates to an enzymatic process for the preparation of S-(+)-4-(3-thienyl)phenyl-a-methylacetic acid, by enantioselective hydrolysis of the corresponding racemic ester using an extracellular lipase. More particularly, the present invention relates to a process for the preparation of S-(+)-4-(3-thienyl)phenyl-α-methylacetic acid, of structural formula (3) in the accompanying diagram, by enantioselective hydrolysis of racemic ester of the acid (1), of structural formula (2), in the accompanying diagram, in a buffer medium or buffer containing a co-solvent if required in presence of commercially available lipases derived from the microorganisms of the genera Candida, Mucor, Rhizopus, Pseudomonas and Aspergillus. However, particularly preferred is the lipase from Candida rugosa. Background and prior art reference 4-(3-thienyl)phenyl-a-methylacetic acid is an intermediate for methyl 4-(3-thienyl)phenyl-α-methylacetate, an antiinflammatory agent approved by WHO under the name Atliprofen. This drug has been found to be effective and well tolerated both in rheumatoid arthritis and osteoarthritis. In addition, side effects found with other NSAIDs like haematological, renal, hepatic and coagulation abnormalities are absent and gastrointestinal problems are insignificant [Drugs of the Future, 1988, 13 (5), 411-2, www.phanrtabiz.com Nov 20, 2000]. A number of 2-arylpropionic acids are known as antiinflammatory agents and among the best known are ibuprofen, flurbiprofen, ketoprofen, and suprofen (all of which are substituted a-methylbenzeneacetic acids), and naproxen (a substituted a-methylnaphthaleneacetic acid). It is to be recognized that the a-methylarylacetic acid molecule is chiral at the a-carbon atom and, therefore, exists in two enantiomeric forms viz. the R and S forms. The S-enantiomers of these a-methylarylacetic acids possess greater antiinflammatory activity than the R-enantiomers [ Lombardino J.G.(ed.); Nonsteroidal Antiinflammatory Drugs, John Wiley & Sons, New York, 1985, 303]. S-(+)- naproxen for example, is having 28 times greater antiinflammatory activity than the R-enantiomer [HarrisonJ.T.; Lewis,B.; Nelson,0.; Rooks,W.; Roszkowski,A.; Tomolonis,A.; Fried,J.H., J Med. Chem., 1970, 13, 203]. The chemical synthesis [Marwah, A.K.; Sastry,C.V.R.; Shridhar,D.R., GB2181728 (1987)] of 4-(3-thienyl)phenyl-a-methylacetic acid leads to a racemic mixture of R and S enantiomers. Hence, there is a need to develop a resolution method in order to separate the individual enantiomers from the racemic mixture. The chemical resolution methods reported for other arylpropionic acids are cumbersome and expensive and usually involve selective stoichiometric crystallization of a diastereomeric salt by the use of an expensive amine such as cinchonidine [HarrisonJ.T.; Lewis,B.; Nelson.O.; Rooks,W.; Roszkowski,A.; Tomolonis,A.; FriedJ.H., J Med. Chem., 1970, 13, 203] or α-methylbenzylamine [Manimaran,T; Impastato,FJ,(Ethyl Corp.), US5015764 (1991)]. Alternatively, the racemic acids are also resolved by enantioselective hydrolysis of the corresponding ester by using extracellular lipases from microbial origin [Gu,Q.M.; Chen,C.S.; Sih C.J., Tetrahedron letters, 1763 (1986)]. Similarly, reference may be made to another process reported in prior art [BattistelJE.; Bianchi,D.;.Cesti,P.; Pina,C, Biotechnology and Bioengineering, 1991, 659] wherein a process for the enantioselective hydrolysis of racemic naproxen esters is described for obtaining the S-acid by using a lipase from Candida rugosa. Objects of the invention The main objective of the present invention is to provide a process for the preparation of 5-(+)-4-(3-thienyl)phenyl-α-methylacetic acid through kinetic resolution of the corresponding racemic esters. Yet another objective of the present invention is to provide a process for the preparation of S-(+)-4-(3-thienyl)phenyl-α-methylacetic acid using an extracellular lipase enzyme in a buffer medium. Summary of the Invention Accordingly, the present invention provides an enzymatic process for preparing S-(+)-4-(3-thienyl)phenyl-αa-methylacetic acid, by enantioselective hydrolysis of the corresponding racemic ester using an extracellular lipase. More particularly, the present invention relates to a process for the preparation of .V-( + )-4-(3-thienyl)phenyl-a-methylacclic acid, of siructural formula (3) in the accompanying diagram, by cnantioselcctive hydrolysis of racemic ester of the acid (1), of structural formula (2), in the accompanying diagram, in a buffer medium or buffer containing a co-solveni if required in presence of commercially available lipases derived from the microorganisms of the genera Candida, Mucor, Rhizopus, Pscudomonas and Aspergillus. However, particularly preferred is the lipase from Candida rugosa. Statement of invention Accordingly, the present invention provides an enzymatic process for the preparation of S-(+)-4-(3-thienyl)phenyl-α-methylacetic acid of through kinetic resolution of the corresponding racemic ester comprising steps of: a) adding a mixture of racemic ester of 4-(3-thienyl)phenyl-α-methylacetic acid to de-ionised water containing an organic co-solvent compound such as herein described, stirring and maintaining the mixture at a temperature in the range of 35 to 40°C and pH at 7.0 using an alkaline buffer; b) adding an extracellular lipase such as hereindescribed to the mixture of step (a) and maintaining the pH at 7 by adding an alkaline solution such as herein described followed by stirring for 24 to 120 hours; c) adjusting the pH of the mixture obtained from step (c) to 8-10 by adding an alkaline solution and saturating the mixture with sodium chloride solution; d) extracting the solution of step (c) with an organic solvent such as hereindescribed to remove unreacted ester of 4-(3-thienyl)phenyl-α-methylacetic acid and separating aqueous layer; e) acidifying the aqueous layer of step (d) to a pH 2.0 by adding a mineral acid such as herein described to obtain a solution; f) extracting the solution of step (e) with an organic solvent such as herein described; g) evaporating the organic solution of step (f) to obtain enantiomerically pure (S)-4-(3-thienyl)phenyl-a-methylacetic acid of formula (3) in 97-99% excess. ' In the above process, the ester is selected from a group consisting of cyclic or acyclic alkyl ester, haloalkyl ester or alkoxy ester. An another embodiment of the present invention provides the use of cyclic or acyclic ester containing carbon atoms 1 to 20. An embodiment of the present invention provides the use of haloalkyl ester selected from 2-chloromethyl, 2-bromomethyl, 2-chloroethyl or 2-bromoethyl ester. An embodiment of the present invention provides the use of alkoxy alkyl ester is selected from 2-methoxymethyl ester, 2-ethoxymethyl ester, 2-methoxyethyl ester or 2-ethoxyethyl ester. Yet another embodiment of the present invention provides the use of phosphate buffer of pH ranging from 6 to 8. Still yet another embodiment of the present invention, provides the use of extracellular lipase selected from genus Candida, Mucor, Rhizopus, Pseudomonas, or Aspergillus for enantioselective hydrolysis of the ester. Still yet another embodiment of the present invention provides the use of extracellular lipase Candida rugosa. An another embodiment of the present invention, the extracellular lipase used in the native form and carried fixed form. Still another embodiment of the present invention, provides use of optionally an organic co-solvent selected from dimethyl formamide, dimethyl sulfoxide or tetrahydrofuran. Yet another embodiment of the invention provides a process wherein the alkali solution used is selected from sodium hydroxide or potassium hydroxide. Still yet another embodiment of the present invention provides the removal of un-reacted ester of 4-(3-thienyl)phenyl-α-methylacetic acid by extraction with an organic solvent selected from a group consisting of dichloromethane, chloroform, diethyl ether, ethyl acetate, propyl acetate, butyl acetate, benzene or toluene. In yet another embodiment of the present invention, provides the use of mineral acid aqueous sulphuric acid or hydrochloric acid for acidifying the reaction mixture. Yet another embodiment of the present invention, provides isolation of enantiomerically pure S(+)-4-(3-thienyl)phenyl-α-methylacetic acid of formula (3) by extracting with an organic solvent selected from dichloromethane, chloroform, diethyl ether, ethyl acetate, propyl acetate, butyl acetate, benzene or toluene. The novelty of the present invention lies in the preparation of the S-(+)-4-(3-thienyl)phenyl-α-methylacetic acid with excellent selectivity. The S-acid (3) and R-ester (4) that were the products of the process of this invention were subsequently purified by chemical means typically by recrystallization from an organic solvent and / or column chromatography and characterization by physical and spectral methods. Brief description of the accompanying drawing: Figure 1 represents an acid of formula (1), its racemic ester of formula (2); S-(+) acid of formula (3) and R-(-) ester of formula (4), Where R is -CH3, -CH2CH2CH2CH3, - CH2 CH2C1, or - CH2 CH2OC2H5 The following examples are presented to illustrate the present invention and therefore should not be construed to limit the scope of the invention. Example 1 Synthesis of 4-(3-thienyl)phenyl-α-methyl-2-chIoroethyIacetate To a solution of 4-(3-thienyl)phenyl-α-methylacetic acid (lg, 0.0043mol) in 30 ml toluene was added 2-chloroethanol (0.57ml, 0.0086 mol) and 0.lg p-toluenesulphonic acid and the reaction mixture was refluxed for 9 hours with simultaneous removal of water. The reaction mixture was given washings with 1% Na2C03 solution (10ml x 2) so that pH is 7 and subsequently water washings (10ml x 2). After the evaporation of the solvent the compound was purified by column chromatography using toluene as an eluent. The product (2c)"was obtained as a solid compound in 80% yield with m.p. 42°C. IR(cm"1 KBr) = 1728, 1168, 781 1HNMR (300MHZ,Δ) = 1.5 (3H, d, J= 7.1), 3.6 (2H, t, J= 5.7), 3.7 (1H, q, J= 7.1), 4.3 (2H, m), 7.2-7.5 (7H, m). Example 2 Synthesis of 4-(3-thienyl)phenyl-α-methyl-2-ethoxyethylacetate To a solution of 4-(3-thienyl)phenyl-α-methylacetic acid (lg, 0.0043mol) in 30 ml toluene was added 2-ethoxyethanol (0.84ml, 0.0086 mol) and 0.lg p-toluenesulphonic acid and the reaction mixture was refluxed for 5 hours with simultaneous removal of water. The reaction mixture was given washings with 1% Na2C03 solution (10ml x 2) so that pH is 7 and subsequently water washings (10ml x 2). After the evaporation of the solvent the compound was purified by column chromatography using toluene as an eluent. The liquid compound (2d) was obtained in 91% yield. IR(cm"' KBr) = 2975, 1733, 1175, 1123, 782 1H NMR (300MHz,δ) =1.1 (3H, t, J= 7), 1.5 (3H, d, J= 7.1), 3.4 (2H, q, J= 7), 3.5 (2H, t, J= 6.3), 3.7 (1H, q, J= 7.1), 4.2 (2H, m) 7.2-7.5 (7H, m). Example 3 Synthesis of 4-(3-thienyl)phenyl-α-methylbutylacetate To a solution of 4-(3-thienyl)phenyl-α-methylacetic acid (lg, 0.0043mol) in 30 ml toluene was added n-butanol (0.81ml, 0.0086 mol) and 0.lg p-toluenesulphonic acid and the reaction mixture was refluxed for 10 hours with simultaneous removal of water. The reaction mixture was given washings with 1% Na2CO3 solution (10ml x 2) so that is pH is 7 and subsequently water washings (10ml x 2). After the evaporation of the solvent the compound was purified by column chromatography using toluene as an eluent. The liquid compound (2b) was obtained in 88% yield. lR(cm"' KBr) = 2959, 1730, 1166, 780 1H NMR (300MHz,δ) = 0.86 (3H, t, J= 7.3), 1.2 (2H, m), 1.5 (5H, m), 3.7 (1H, q, J= 7.1), 4.0 (2H, t, J= 6.6), 7.2-7.5 (7H, m). Example 4 Synthesis of (S)-(+)-4-(3-thienyl)phenyl-α-methylacetic acid A) 3.0g butyl ester (2b) was added to 90 ml deionised water taken in 250 ml beaker and stirred with an overhead mechanical stirrer maintaining the temperature 37°C of the solution. The pH of the reaction medium was set at 7.0 by connecting it to a pH-stat system and when the pH stabilises, 800 mg of the Candida rugosa lipase was added. The pH was maintained by automatic addition of 0.098N sodium hydroxide solution. After 84 hours the reaction was stopped. The volume of alkali consumed was 31.19ml (conversion of ester is 30%). pH of the reaction mixture was made 8-10 using 0.1N NaOH and the solution was saturated with sodium chloride. The reaction mixture was extracted with ethyl acetate (50ml x 3) to isolate the unreacted ester enriched as R-ester. The S-acid (0.7g) was isolated as white solid after acidifying the aqueous part to pH 2.0 with 0.1N hydrochloric acid and extracting with ethyl acetate (50ml x 3), having enantiomeric excess 97.86 % as determined by HPLC. Physical and spectroscopic data of (S)-4-(3-thienyl)phenyl-α-methylacetic acid: a. Melting point: 189°C b. [α]D : +48.48° at 589 nm [c=l, absolute ethanol, ee 97.86 %, HPLC, chiral AGP] c. IR (KBr): 3101, 2936, 1691 and 776 cm"1 d. 1H NMR (300MHz,5): 1.5 (3H, d, J = 7.1), 3.7 (1H, q, J = 7.1), 7.3-7.5 (7H, m) e. MS: [M]+232. HPLC analysis data: a. Column: Chiral - AGP (150 x 4 mm), ChromTech, Sweden. b. Mobile phase : i. Butyl ester: 20 mM Phosphate buffer (pH = 7.0) : acetonitrile (98 : 2) ii. Acid: 10 mM Phosphate buffer (pH =6.0) : acetonitrile (98 : 2) c. Retention time : R-acid : 36.05 and S- acid : 48.22 min ; S-ester (2b): 6.7 and R-estcr (2b): 8.5 min, respectively. The above procedure, as in example 4 (A), was repeated with the 2-chloroester (2c) of the r,acemic acid (1). After 24-hour reaction and usual workup lead to the isolation of unreacted R-ester with ee 70% and product S-acid with ee 71% (by HPLC, chiral AGP). Following the same procedure, as in example 4 (A), the experiment was repeated with n-butyl ester (2b) and a lipase from Aspergillus Niger (120u/mg, Amano Pharmaceutical, Japan). After 72-hour reaction, the enantiomeric excess of the product 5-acid was found to be 72% (by HPLC, chiral AGP). D) The same procedure, as in example 4 (C), was repeated with a lipase from Rhizopus oryzae (150u/mg, Amano Pharmaceutical, Japan). After 24 hour reaction the hydrolysed acid obtained, however, in this case was found to be with R-configuration having 54% ee (by HPLC, chiral AGP). We claim : 1. An enzymatic process for the preparation of S-(+)-4-{3-thienyl)phenyl-α-methylacetic acid, the said process comprising steps of : a) adding a mixture of racemic ester of 4- (3-thienyl)phenyl-α-methylacetic acid to de-ionised water containing an organic co-solvent compound such as herein described, stirring and maintaining the mixture at a temperature in the range of 35 to 40 °C and pH at 7.0 using an alkaline buffer; b) adding an extracellular lipase such as hereindescribed to the mixture of step (a) and maintaining the pH at 7 by adding an alkaline solution such as herein described followed by stirring for 24 to 120 hours; c) adjusting the pH of the mixture obtained from step (c) to 8-10 by adding an alkaline solution and saturating the mixture with sodium chloride solution; d) extracting the solution of step (c) with an organic solvent such as hereindescribed to remove unreacted ester of 4-(3-thienyl)phenyl-α-methylacetic acid and separating aqueous layer; e) acidifying the aqueous layer of step (d) to a pH 2.0 by adding a mineral acid such as herein described to obtain a solution; f) extracting the solution of step (e) with an organic solvent such as herein described; g) evaporating the organic solution of step (e} to obtain enantiomerically pure (S)-4-(3-thienyl)phenyl-a-methylacetic acid in 97-99% excess. 2. A process as claimed in claim 1 wherein in step (a) the organic co-solvent compound is selected from dimethyl formamide, dimethyl sulfoxide and tetrahydrofuran. 3. A process as claimed in claim 1 wherein the ester is selected from a cyclic or acyclic alkyl ester, haloalkyl ester or alkoxy ester. 4. A process as claimed in claim 3 wherein the cyclic or acyclic ester has 1 to 20 carbon atoms. 5. A process as claimed in claim 3 wherein the haloalkyl ester is selected from 2-chloromethyl, 2-bromomethyl, 2-chloroethyl or 2-bromoethyl ester. 6 . A process as claimed in claim 3 wherein the alkoxy alkyl ester is selected from 2-methoxymethyl ester, 2-ethoxymethyl ester, 2-methoxyethyl ester or 2-ethoxyethyl ester. 7. A process as claimed in claim 1 wherein the alkaline buffer is selected from a phosphate buffer having pH of 6 to 8 . 8. A process as claimed in claim 7 wherein the phosphate buffer is selected from a sodium dihydrogen ortho-phosphate and sodium phosphate dibasic buffer and potassium dihydrogen ortho-phosphate and potassium phosphate dibasic buffer. 9. A process as claimed in 1 wherein the extracellular lipase is obtained from organisms of the genus Candida, Mucor, Rhizopus, Pseudomonas, or Aspergillus. 10. A process as claimed in claim 9 wherein the extracellular lipase is obtained from Candida rugosa. 11. A process as claimed in claim in 1, wherein the extracellular lipase is in native or carrier fixed form. 12 . A process as claimed in claim 1 wherein the alkali solution is sodium hydroxide or potassium hydroxide having concentration 0.05-0.1 N. 13 . A process as claimed in claim 1 wherein the organic solvent is selected from the group consisting of dichloromethane, chloroform, diethyl ether, ethyl acetate, propyl acetate, butyl acetate, benzene and toluene. 14 . A process as claimed in claim 1 wherein the mineral acid is aqueous sulphuric acid or hydrochloric acid. 15. An enzymatic process for the preparation of S-(+)-4-(3-thienyl)phenyl-a-methylacetic acid substantially as herein described with reference to the accompanying examples.

Documents:

875-del-2002-abstract.pdf

875-del-2002-claims.pdf

875-del-2002-complete specification (granted.pdf

875-del-2002-correspondence-others.pdf

875-del-2002-description (complete).pdf

875-del-2002-drawings.pdf

875-del-2002-form-1.pdf

875-del-2002-form-13.pdf

875-del-2002-form-2.pdf

875-del-2002-form-26.pdf

875-del-2002-form-3.pdf

875-del-2002-form-5.pdf