ARYLALKANOIC ACID RESOLUTION

Abstract: Arylalkanoic acids are produced by biotransformation using suitable microorganisms and, in particular, (S)-ketoprofen in greater than 95 % purity from racemic ketoprofen ethyl ester.

Description: ARYLALKANOIC ACID RESOLUTION Field of the Invention This invention relates to arylalkanoic acid resolution. Background of the Invention Many pharmaceutically-active compounds produced by chemical synthesis are obtained and sold as mixtures of stereoiso ers. However, it is often the case that only one these stereoisomers is pharmaceutically active. The contaminating enantiomer often has very poor, if any, activity or, in some instances, has unwanted physiological side-effects and shows toxicity. Work by a number of researchers has shown that the anti-inflammatory activity of the 2-arylpropionic acids naproxen and ibuprofen is found with the (S) enantiomer. The same is true for ketoprofen, currently manufactured and sold as a racemate. Arylalkanoic acids may be resolved by biotransformation, according to the following reaction scheme: wherein R' is an alkyl group, Ar is an aromatic residue and, for example, R is an aliphatic residue of 1 to 4 carbon atoms. One particular object behind the present invention is to provide an economic route to the production of optically pure (S)-ketoprofen. Summary of the Invention The present invention provides, inter alia, a method for the resolution of (S -ketoprofen from a racemic mixture. This method involves biocatalytic hydrolysis of an ester of racemic ketoprofen, to yield a SUBSTITUTE SHEET biotransformation broth containing ketoprofen acid substantially enriched in one enantiomer and ketoprofen ester substantially enriched in the other enantiomer. Provided that the acid product of the biotransformation is sufficiently enriched with the desired enantiomer, further improvement of the optical purity can readily and economically be achieved using standard chemical procedures by forming a salt with a chiral amine of high optical purity, followed by crystallisation from solution. The residual ketoprofen ester from the biotransformation can readily be purified, chemically racemised and recycled for use in further biotransformations, to minimise raw material costs. Description of the Invention A preferred aspect of the present invention is based on the discovery of a biocatalyst suitable for use in the biotransformation described above: a microbe (ENZA-I3) has been identified that is extremely useful in carrying out the required resolution. This organism was originally isolated by screening samples from the sewage works for growth on ethanol as a sole source of carbon. Subsequent screening of the organism on plates containing ethyl ketoprofen showed that, following growth, there was extensive clearance of the non-water-soluble ethyl ketoprofen around ENZA-I3 colonies (indicative of potential ester hydrolysis) . Subsequent screening in liquid media showed ENZA-I3 to be considerably more active at hydrolysing ethyl ketoprofen than other isolates during the liquid screen. This strain has proved to have a number of characteristics advantageous for the resolution of (SJ- ketoprofen from the racemic ketoprofen ester, as follows: (a) It hydrolyses short-chain ketoprofen esters very rapidly. (b) It produces (S)-ketoprofen acid from racemic ethyl ketoprofen with very high enantioselectivity such that, at low conversions, greater than 95% purity (SJ- ketoprofen can be obtained. At conversions approaching 50% (40-50%) this reduces to 90% purity. This selectivity is obtained without the need to purify out contaminating activities (which can be expensive) or over-express the activity of interest by cloning. (c) By changing the substrate from ethyl to methyl ketoprofen it is possible to change the selectivity of the biocatalyst such that (R) -ketoprofen acid is preferentially accumulated instead of the (SJ enantiomer. Thus, by taking the biotransformation to greater than 50%, (SJ-ketoprofen can be produced as the methyl ester. (d) The organism grows rapidly at ambient temperatures with a doubling time of 1.5 to 2 hours, enabling biocatalyst to be easily and economically prepared. The isolated strain has been identified by Centralbureau Voor Schi melcultures (CBS) in the Netherlands as Trichosporon laibacchii (Windisch) which is also classified as Endomyces laibacchii. Several alternative strains of this species are publicly available from the CBS. These strains, for instance CBS 5791, 5790, 5381 and 2495, have been obtained and tested alongside isolate ENZA-I3. These tests show that some of these strains are almost as good as isolate ENZA-I3 at carrying out the biotransformation. In the CBS 1990 catalogue (32 nd edition) , these strains are classified as Trichosporon beiqelii. however they have subsequently been renamed by the CBS as Endomyces laibacchii. Strains of T. beiqelii that have been tested have been found to have similar selectivity to ENZA-I3, although they are not as active. ENZA-I3 has been deposited with the International Mycological Institute, Kew, UK on 20th August 1991, under the terms of Budapest Treaty, where it has been given the accession number 348917. Further characteristics of the deposited microbe ENZA- 13 are given below: Fermentative growth: Glucose -ve - SUBSTITUTE SHEET Aerobic growth: D-Glucose +ve D-Galactose -f-ve L-Sorbose +ve D-Glucosamine +ve D-Ribose +ve D-Xylose +ve L-Arabinose +ve D-Arabinose -ve L-Rhamnose +ve Sucrose +ve Maltose +ve αα-Trehalose +ve Methyl α-glucoside +ve Cellobiose +ve Salicin +ve Arbutin +ve Melibiose +ve Lactose +ve Raffinose +ve Melezitose +ve Inulin -ve Soluble starch +ve Glycerol +ve Meso-erythritol -ve Ribitol -ve Xylitol -ve L-Arabinitol +ve D-Glucitol +ve D-Mannitol -ve Galactitol +ve Myoinositol +ve Gluconolactone -ve D-Gluconate +ve D-Glucuronate +ve D-Galacturonate -ve Di-lactate +ve Succinate +ve Citrate +ve Methanol -ve Ethanol +ve Use of nitrogen source: Nitrate -ve on nitrate Nitrite -ve Ethylamine +ve L-lysine +ve Cadaverine +ve Creatine -ve Creatinine +ve Growth: +ve at 25°C, 30°C -ve at 35°C, 37°C Appearance: Colonies - cream, membranous Filaments - well-developed pseudohyphae/ septae hyphae arthrocandida Asci - none Teliospores/basidia - none The following Examples illustrate the invention. The following media were used in Examples 1 to 5 (in which (SJ- ketoprofen was produced) : *Ivanhoe Chemical Company, IL, USA. All media was adjusted to pH 6.5 with sodium hydroxide prior to autoclaving. All media were heat-sterilised at 121°C for between 20 and 40 minutes prior to inoculation. The glucose was sterilised separately from the rest of the medium as a 50% solution. The biotransformation mixture is as follows: SUBSTITUTE SHEET Sodium phosphate 100 M, pH 6.5 Yeast extract 10 g/1 Tween 80 5 g/1 Antifoam (XFO 371) 1 ml/1 Ketoprofen ethyl ester was added to the biotransformation mixture, to give a final concentration of 50 g/1 after the addition of the inoculum. The biotransformation medium was heat-sterilised at 121°C for between 20 to 40 minutes; the ketoprofen ethyl ester was heat-sterilised separately. The trace element solution used was as follows: CaCl 2 .2H 2 0 3.57 g/1 ZnO 2.0 g/1 0.85 g/1 4.8 g/1 2.0 g/1 5.4 g/1 0.3 g/1 CoCl 2 .6H 2 0 2.4 g/1 HC1 250 ml/1 Example l Cells (ENZA-I3) were inoculated onto YM (Difco) agar plates and incubated at 23°C for 2 days. A single colony was then transferred into 75 ml seed medium in a 500 ml shake flask. This was then grown aerobically at 23°C for 24 hours. The culture was then transferred into a 2.8 1 fermenter containing 1.5 1 of growth medium at 23°C. Growth continued for 10 hours with sufficient aeration and stirring to maintain aerobic conditions. 150 ml of this culture was then transferred to 1.35 1 biotransformation medium in a 2.8 1 vessel. During the biotransformation, an agitation of 1200 rpm and an aeration of 0.5 w were maintained. The temperature was controlled at 23°C. A sample taken 20 hours after the start showed a ketoprofen concentration of 7 g/1 with a purity of 98% (SJ -ketoprofen. A sample taken 73 hours after the start of the biotransformation showed a ketoprofen concentration of 23.3 g/1 with a purity of 94% (SJ -ketoprofen. Example 2 A similar methodology to that described in Example 1 was used, but on a larger scale. Cells were inoculated onto YM (Difco) agar plates and incubated for 2 days. Single colonies were then transferred onto each of four, 1 1 conical flasks containing 250 ml seed medium. After 1 day's growth, the contents of all flasks were transferred into a 15 1 fer enter containing 9 1 growth medium. After 10 hours' aerobic growth, 51 of cell broth was transferred into a 751 vessel containing 451 biotransformation medium to start the biotransformation. An air flow rate of 0.2 vvm and an agitation rate of 500 rpm were used to maintain aerobic conditions and ensure good mixing. Analysis of a sample taken 71 hours after the start of the biotransformation showed that 17 g/1 ketoprofen had been accumulated with a purity of 93% in favour of the (SJ enantiomer. Example 3 Cultures of various strains available from CBS and shown in the following Table were transferred from fresh YM agar plates into 250 ml flasks containing 25 ml of growth medium without glucose. After 24 hours' growth, 5.0 ml of each culture was transferred into baffled 250 ml flasks containing 20 ml of biotransformation media. Samples were taken after shaking at 23°C for 72 hours. The results are tabulated below: SHEET * Difficult to quantify accurately due to low concentrations, but selectivity for the (S) enantiomer (to a greater or lesser extent) observed for strains. Example 4 Effect of Temperature Studies show that at 23 and 26°C performance of the biocatalyst during the biotransformation is similar. At 20°C the rate of biotransformation is similar to that at 23°C, but the enantioselectivity of the biocatalyst drops such that in a standard biotransformation (Example 1) the purity of the acid product is only 88% (S) -ketoprofen after 70 hours rather than 93-94%. At 30°C, the rate of catalysis falls by about 40-50% and the enantioselectivity is also not good. Example 5 Effect of pH Activity is seen between pH 4.5 and 7.5 (and almost certainly higher though not tested) . The selectivity of the biocatalyst decreases significantly below pH 4.5. The optimal pH is considered to be between 6.5 and 7.5. Thus using the standard method (Example 1) with the pH controlled at 4.5, a sample at 66 hours had 8 g/1 (purity 91% (S) -ketoprofen) ; at pH 6.5 after 66 hours, the sample had 24 g/1 (purity 94% (S) -ketoprofen) ; at pH 7.5 after 66 hours, the sample had 23 g/1 (purity 80% (SJ -ketoprofen) . Example 6 Effect of Chain Length Cells were grown in a medium containing 25 g/1 yeast extract, 10 g/1 potassium dihydrogen phosphate, 0.5 g/1 magnesium sulphate heptahydrate, 2 g/1 ammonium sulphate and 1 ml/1 trace element solution. (CaCl 2 .2H 2 0 - 53 g/1, FeS0 4 .7H 2 0 - 2 g/1, MnS0 4 .H 2 0 - 100 mg/1, ZnS0 4 .7H 2 0 - 200 mg/1, CuS0 4 - 40 mg/1, CoCl 2 .6H 2 0 - 60 mg/1, NaMo0 4 - 40 mg/1, H 3 BO 3 - 30 mg/1), with the pH adjusted to 6.5 with sodium hydroxide. After overnight growth, the cultures were transferred at 20% into 250 ml baffled flasks containing 25 ml of the same medium, but with different ketoprofen esters added to 10 g/1 final concentration. The rate of biotransformation and the enantiomeric purity of the ketoprofen product was then measured. The results were: SUBSTITUTE SHEET

Claims: CLAIMS 1. A process for preparing predominantly one enantiomer of an optically-active arylalkanoic acid, from a mixture of enantiomers of an ester of the arylalkanoic acid, which comprises contacting the ester with a material characterised by the ability to produce (S)-ketoprofen in greater than 95% purity from racemic ketoprofen ethyl ester. 2. A process for preparing predominantly one enantiomer of an optically-active arylalkanoic acid, from a mixture of enantiomers of an ester of the arylalkanoic acid, which comprises contacting the ester with a material characterised by the ability to convert first and second esters of an arylalkanoic acid to the acid with selectivity for opposite enantiomers of the esters. 3. A process according to claim 2, wherein the first ester is ketoprofen ethyl ester and the material is enantioselective for the ester of (S)-ketoprofen. 4. A process according to claim 2 or claim 3, wherein the first or second ester is ketoprofen methyl ester and the material is enantioselective for the ester of (R)- ketoprofen. 5. A process according to any preceding claim, wherein the material is a microorganism of the genus Trichosporon or Endomyces♦ 6. A process according to any preceding claim, wherein the material has the characteristics of Trichosporon ENZA 1-3, IMI 348917. 7. A process according to any preceding claim, wherein the one enantiomer is (S)-ketoprofen. 8. A process according to any preceding claim, which is conducted at a pH of 4.5 to 7.5. 9. A process according to any any preceding claim, which is conducted at 20 to 30°C. 10. A process according to any preceding claim, wherein the ester is the methyl or ethyl ester.

Inventors: CARGANICO GERMANO, EVANS CHRISTOPHER THOMAS, STABLER PETER JOSEPH, WISDOM RICHARD ANTHONY, CARGANICO, GERMANO, EVANS, CHRISTOPHER, THOMAS, STABLER, PETER, JOSEPH, WISDOM, RICHARD, ANTHONY
Applicants: MENARINI LAB, LABORATORIOS MENARINI S.A.
Application year: 1992
Main classification: C12R1
IPC codes: C07D213, C07D401, C12P7, C12P17, C12P41, C12R1