AU663110B2 - Arylalkanoic acid resolution - Google Patents

Description

L Y OPI DATE 16/03/93 AOJP DATE 27/05/93 APPLN. ID 24772/92 lllll 111111 lllIIllllllllll 927 PCT NUMBER PCT/EP92/01896 111111111 1111111111 11111111111111111 AU9224772

PCT)

(51) International Patent Classification (11) International Publication Number: WO 93/04190 C12P 41/00, 7/40 (C12P 7/40 Al C12R 1:645) (43) International Publication Date: 4 March 1993 (04.03.93) (21) International Application Number: PCT/EP92/01896 (74) Agent: BIANCHETTI, Giuseppe; Studio Consulenza Brevettuale, Via Rossini, 8, 1-20122 Milano (IT).

(22) International Filing Date: 19 August 1992 (19.08.92) (81) Designated States: AU, BB, BG, BR, CA, CS, FI, HU, JP, Priority data: KP, KR, LK, MG, MN, MW, NO, PL, RO, RU, SD, 9118150.3 22 August 1991 (22.08.91) GB US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG).

(71) Applicant (for all designated States except US): LABORA- TORIOS MENARINI S.A. [ES/ES]; Alfonso XII, 587, E-08912 Badalona Published With international search report.

(72) Inventors; and Inventors/Applicants (for US only) EVANS, Christopher, Thomas (GB/GB]; Stable Cottage, Fowlmere Road, Heydon, Hertfordshire SG8 8PU McCAGUE, Raymond [GB/GB]; 5 Mansfield Close, Milton, Cambridgeshire CB4 6EE TAYLOR, Stephen, John, Clifford [GB/GB]; 52 Cambridge Road, Ely, Cambrigdeshire CB7 4MT CARGANICO, Germano [IT/ ES]; Alfonso XII, 587, E-08912 Badalona (ES).

(54)Title: ARYLALKANOIC ACID RESOLUTION (57) Abstract In a process for preparing predominantly one enantiomer of an optically-active 2-arylalkanoic acid by biotransformation, using an appropriate enzyme, of a mixture of enantiomers of an ester of the acid, the ester is derived from an alcohol of more than 4 carbon atoms, and the enzyme is in crude or only partially purified form. For example, S-ketoprofen can be thus prepared economically and in high ee, from racemic octyl ketoprofen.

-i- WO 93/04190 PCT/EP92/01896 1 ARYLALKANOIC ACID RESOLUTION Field of the Invention This invention relates to a process for resolving arylalkanoic acids, by a biotransformation according to the following reaction scheme: Ar enzyme Ar> COH Ar K CO R' Co 2 R' I R R. H R H 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.

Background of the Invention In the prior art, a preference has often been made for carrying out such biotransformations with a small alkyl residue for Thus, in EP-A-0196625, where 2-halopropionic acids are resolved by Candida cylindracea lipase (CCL) in a two-phase system, R' is defined as Ct-C 4 alkyl. Moreover, later work with this enzyme, as described by Wu et al, J. Am. Chem. Soc. (1990) 112, 1990-5, concentrates on using the methyl ester for resolution of arylpropionic acids. The enzyme is purified, by addition of sodium deoxycholate to the enzyme solution and subsequent precipitation -of the protein in 1:1 ethanol/diethyl ether.

This preference, for esters of ketoprofen at least, when using a crude preparation of CCL, is supported by the Table, below: enantiospecificity, as determined by the enantiomeric excess (ee) of the acid formed, declines with chain length from R'=methyl to R'=n-propyl.

Cambou and Klibanov, Appl. Biochem. Biotechnol. (1984) 9:255-60, report that CCL hydrolysis is selective for octyl 2-chloropropionate but not for the methyl ester. However, the failure of this methyl ester to exhibit SUBSTITUTE

SHEET

WO 93/04190 PCT/EP92/01896 2 enantiospecificity is considered to be due to its watersolubility, causing it not to form an interface.

Summary of the Invention As also shown in the Table, it has now surprisingly been found that when the esters are derived from increasingly longer chain alcohols, the ee subsequently increases to a higher figure than that obtainable with the methyl ester, of approximately 98% at and C 10 n-alkyl.

According to the present invention, the reaction illustrated above is conducted using crude (or partiallypurified) enzyme, and R' has more than 4, e.g. 5, 6 or 7 to to 20 C atoms. Subsequent work with purified enzyme showed that high ee was achieved whatever the chain length, but the purification adds to the overall cost.

Description of the Invention CCL has been identified as the most promising enzyme for enantiosspecific hydrolysis of racemic esters of ketoprofen. This enzyme gives the fastest rate and, unlike the majority of other active lipases, preferentially hydrolyses the S-ester, enabling simple purification of S-ketoprofen.

It appears that contaminating activities in crude CCL do not carry out the non-selective hydrolysis with esters of long-chain alcohols. Thus, there is advantage in using a long chain ester to enable the economy of using a crude or only partially pure enzyme. More particularly, the fact that, say, octyl ketoprofen can be used as a substrate with partially purified enzyme producing S-ketoprofen with ee of greater than 95% allows the development of a commercial process.

The following Examples illustrate the invention or are comparative.

Examples CCL is used as the biocatalyst to resolve acemic mixtures of various alkyl esters of ketoprofen; Sketoprofen is formed. The same reaction conditions are used, i.e. 10 ml of 0.1 M potassium phosphate, pH 7.5 plus i: t; SUBSTITUTE

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WO 93/04190 PCT/EP92/01896 3 2 ml cyclohexane, 100 mg alkyl ketoprofen, 50 mg enzyme, 30 0 C. Results are given in the Table.

The CCL is used in crude form, or after a threestep purification, comprising water extraction, filtration and column separation, as follows: The enzyme was resuspended at 50 mg/ml in deionised water. After stirring at room temperature for 30 minutes, the insoluble material was spun out. The supernatant was diafiltered against 10 volumes of McIlvaine's buffer mM K 2

HPO

4 10 mM citric acid, pH 3.7) at room temperature. Following diafiltration, the enzyme solution was concentrated to 1/5 of its original volume. The enzyme solution was bonded onto an AP-sephadex C-50 column ml enzyme solution: 20 ml of gel) equilibrated with Mcllvaine's buffer. The column was washed with McIlvaine's buffer 20 mM NaCl. After the first peak had eluted and the absorbance (at 280 nm) had returned to baseline, Mcllvaine's buffer 200 mM NaCl was used to elute a second protein peak.

The final step of purification yielded two activities. The protein eluted at a higher salt concentration was found to be highly enantiospecific.

The respective properties of the CCL biocatalysts were: Crude Purified Enzyme Enzyme Mass 50 g 2.1 g Powder Activity 150 U/mg 680 U/mg Yield (100%) 19% E (enantiospecifity ratio of enzyme)B 15 >200 A Assayed using olive oil as substrate, 37 0 C, pH B 100 ml 0.1 M ketoprofen: pH 6.5, 2 ml cyclohexane, 100 mg methyl ketoprofen, 30,000 U C'L, 30 0

C.

Table: Enantiomeric excess values(%) of S-ketoprofen by Candida cylindracea lipase hydrolysis of esters from various alcohols.

SUBSTITUTE SHEET WO 93/04190 WO 9304190PCT/EP92/01086 n-alcohols Using crude Using enzyme no. carbon enzyme purified on a SP atoms Sephadex gel column ee c E ee c E 1 75 0.57 39 99 0.48 >200 2 54 0.43 5 3 34 0.45 3 4 70 0.44 89 0.33 26 6 92 0.27 33 7 97 0.28 8 98 0.32 136 >99 0.23 >200 198 0.18 127 18 195 0.16 43 eC--fnVl SUBSTITUTE SHEET

Claims (4)

1. A process for preparing predominantly the S-enantiomer of ketoprofen by biotransformation, using crude preparations of Candida Cylindracea lipase, of a racemic ester of ketoprofen, characterised in that the ester is derived from an alcohol of formula R1OH, wherein R 1 is an alkyl group having from 4 to 20 carbon atoms with the proviso that R 1 is not n-hexyl.

2. A process according to claim 1, wherein the alcohol has from 8 to 10 carbon atoms.

3. Processes for the preparation of predominantly the S-enantiomer of ketoprofen substantially as hereinbefore described in any one of the Examples. I 4 DATED THIS 2nd DAY OF June 1995 LABORATORIS MENARINI S.A. By their Patent Attorneys KELVIN LORD AND COMPANY PERTH, WESTERN AUSTRALIA 4 0 i4 i- 7 I INTERNATIONAL SEARCH REPORT International Application No PCT/EP 92/01896 1 CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all)fi According to International Patent Classification (EPC) or to both National Classification and IPC .Int.Cl. 5 C12P41/O0; C12P7/40; //(C12P7/40,C12Rl:645)

11. FEELDS SEARCHED Minimum Documoentation SearcheiP Classification System Classification Symbols Int.C1. 5 C12P Docsumentation Searchsed other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched 3 Ell. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Document, It with indication, where appropriate, of the relevant passages' 12 Reevant to Claim NoJ t J X EP,A,0 407 033 (RHONE POULENC) 9 January 1991 see examples 18-24 see claims; examples 50-55 X EP,A,0 227 078 (WISCONSIN ALUMNI RESEARCH) 1 July 1987 see claims; example 29 A AGRICULTURAL AND BIOLOGICAL CHEMISTRY. 1-4 vol. 45, no. 6, June 1981, TOKYO JP pages 1389 1392 SHINOBU IRIUCHIJIMA ET AL. 'Asymmetric hydrolysis of (+/-)-alpha-substituted carboxylic acid esters with microorganisms.' see the whole document 0Special categories of cited documents 10 'T later document published after the international filing date document defining the general state of the art which is not or priority date and not in conflict with the application but consdere tobe o paltssar elevncecited to understand ihe principle or theory underlying the consdere tobe o paticuar elevnceinvention 'E earlier document but published 0n or after the international docusment of particular relevace; the claimed Jnventlon filing date I cannot be considered novel or cannot be considered to -V document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establith the publication date of another docusment of patclrrlvne he claimed invention citation or other special reasan (as specified) cno eonsidetrft nvolve aninventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in th 'e c c. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 04 DECEMBER 1992 4 Intesusatscnal Searching Authority Signature of Authorized Officer E1JROPEAN PATENT OFFICE DELANGHE L. L Pamn tCTISA/21O (seca se) (Jmuary t985) PCT/EP 92/01896 International Application No m. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM TIE SECOND SHEET) Category Citation of Doaument, with Indication, where appropriate, of the relevant passages Relevant to Claim No. A TETRAHEDRON LETTERS. 1,2 vol. 27, no. 16, 1986, OXFORD GB pages 1763 1766 QU-MING GU ET AL. 'A facile enzymatic resolution process for the preparation of (+)-S-2-(6-Methoxy-2-naphthyl)propionic acid (Naproxen)' see the whole document A CHEMICAL ABSTRACTS, vol. 101, no. 19, 1,2 1984, Columbus, Ohio, US; abstract no. 169019c, CAMBOU, BERNARD ET AL. 'Lipase-catalyzed production of optically active acids via asymmetric hydrolysis of esters.Effect of the alcohol moiety.' page 533 see abstract APPL.BIOCHEMBIOTECHNOL. vol. 9, nc. 3, 1984, ENG pages 255 260 A JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 1,2 vol. 107, 1985, GASTON, PA US pages 7072 7076 GERALD KIRCHNER ET AL. 'Resolution of racemic mixtures via lipase catalysis in organic solvents.' see the whole document P,X WO,A,9 113 163 (RHONE POULENC) 1,3-5 September 1991 see claims FII I ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION No. EP 9201896 SA 64266 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The memers; are as contained in the European Patent Office EDP file on The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. 04/12/92 Patent document Pblication Patent family Publication cited in search report dat member(s) date EP-A-0407033 09-01-91 US-A- 5108916 28-04-92 AU-A- 5824290 07-01-91 CA-A- 2057007 06-12-90 WO-A- 9015146 13-12-90 EP-A-0227078 0 1-07-87 AU-B- 599944 02-08-90 AU-A- 6676286 25-06-87 JP-A- 62190097 20-08-87 WO-A-9113163 05-09-91 None W For more details about this annex see Off~naj Journal of the European Pstept Office No. 12182