WO2016110708A1

WO2016110708A1 - Process

Info

Publication number: WO2016110708A1
Application number: PCT/GB2016/050035
Authority: WO
Inventors: Barrie RHODES; Jonathan Moseley; Andrew Wells
Original assignee: Aesica Pharmaceuticals Limited
Priority date: 2015-01-08
Filing date: 2016-01-08
Publication date: 2016-07-14
Also published as: GB201500277D0

Abstract

There is described a process for the preparation of a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst capable of stereo selective de-esterification of the ester; provided that the biocatalyst is not Aspergillus oryzae; Bacillus cereus C71; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2.

Description

PROCESS

Field of the Invention

The present invention relates to novel process for biotechnological preparation of flurbiprofen resolved in the form of optical S(+) and R(-) isomers.

More particularly, the invention provides a process for preparing a single enantiomer of flurbiprofen in high enantiomeric excess (ee).

Background of the Invention

2-(3-fluoro-4-phenyl-phenyl)propanoic acid (flurbiprofen) is generally commercially available as the racemate, i.e. (R,S)-(±)- 2-(3-fluoro-4-phenyl-phenyl)propanoic acid (R,S)-(±)-flurbiprofen), i.e. a mixture of enantiomers (R)-(-)-flurbiprofen and (S)-(+)- flurbiprofen. (R,S)-(±)-flurbiprofen is commercially available as a non-steroidal antiinflammatory drug (NSAID) which is therapeutically useful in the treatment or alleviation of, inter alia, inflammation and pain. However, in the racemate, (R,S)-(±)-flurbiprofen, it is the (S)-(+)-enantiomer, (S)-(+)-flurbiprofen, that is the therapeutically active species as an NSAID.

The (R)-(-)-enantiomer is generally inactive as an NSAID. However, there has been recent interest in (R)-(-)-flurbiprofen for the treatment of other disorders, such as Alzheimer's disease and cancer. Therefore, there remains a need for a method of preparing the optically pure (S)-(+) or (R)-(-) enantiomers of flurbiprofen, and salts thereof.

Attempts have been made at preparing the optically pure (S)-(+) or (R)-(-) enantiomers of flurbiprofen by the biocatalytic hydrolysis of esters of flurbiprofen.

Thus, for example, US Patent application No. 2003/170835 describes an esterase for the production of optically active aryl propionic acids, such as ibuprofen, ketoprofen and flurbiprofen. More specifically, US 2003/170835 describes an esterase derived from Pseudomonas sp. BHY-1 which unsymmetrically hydro lyses the racemic ester of a carboxylic acid to produce the corresponding optically pure carboxylic acid. Also, Pseudomonas sp. BHY-1 has a stereo selective hydrolase activity to convert racemic ester of aryl propionic acid to one-enantiomer aryl propionic acid. In addition, Lee et al, "Preparation of enantiomerically pure (^-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP", Journal of Molecular Catalysis B: Enzymatic 26 (2003) 149-156; describes the esterase PFl-K from Pseudomonas sp. KTCC 10122BP which exhibited a fairly high enantioselectivity towards the hydrolysis of racemic flurbiprofen ethyl ester to (S)-(+)-flurbiprofen.

However, none of the aforementioned methods have been successfully developed commercially.

Other prior art documents describe the enzymatic hydrolysis of a racemic flurbiprofen ester. A number of different biocatalysts have been unitised, including Aspergillus oryzae, Bacillus cereus C71, C. Candida Antarctica lipase B (Novozym ® 435), Candida cylindracea, Candida rugosa lipase, Carica papaya lipase, Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2. However, the known chemical methods for resolving the S-(+) and R-(-) isomers have generally been commercially unsatisfactory in that they need costly optically active reactants, such as, methylbenzylamine, and the like.

Thus, there has been a long felt need for a suitable commercial method that would allow the resolution of the S-(+) and R-(-) isomers of flurbiprofen in a simple and inexpensive way, and that, also would be efficient and commercially feasible.

Summary of the Invention

An object of the present invention is to provide a method of enzymatic asymmetric de- esterification using one or more suitable biocatalysts that are capable of asymmetrically de-esterifying an enantiomer of flurbiprofen ester, that is, a suitable biocatalyst capable of selectively de-esterifying the single enantiomer of a racemic or non-racemic mixture of flurbiprofen ester, in order to yield a substantially enantiomerically pure flurbiprofen acid comprising the selected enantiomer, whilst leaving the ester of the unselected enantiomer substantially unchanged.

We have now found a novel enzymatic method of preparing substantially optically pure (S)-(+) or (R)-(-) enantiomers of flurbiprofen by the biocatalytic hydrolysis of esters of flurbiprofen. The method of the invention may desirably include a step of racemisation of the unwanted flurbiprofen enantiomer, thus enhancing the efficiency of the preparation of the desired enantiomer.

Thus, according to a first aspect of the present invention there is provided a process for the preparation a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises subjecting a flurbiprofen ester, comprising a mixture (R)-(-)- flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst capable of stereo selective de-esterification of the ester; provided that the biocatalyst is not Aspergillus oryzae; Bacillus cereus C71 ; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2.

The flurbiprofen ester may be in racemic form or may comprise a non-racemic mixture of (R)-(-)-flurbiprofen and the (S)-(+)-flurbiprofen. In a preferred embodiment the process comprises the selective de-esterification of racemic flurbiprofen ester.

It will be understood by the person skilled in the art that the stereo selective de- esterification may selectively de-esterify S-(+)-flurbiprofen ester, e.g. to provide S-(+)- flurbiprofen, or a salt thereof; or the stereo selective de-esterification may selectively de- esterify R-(-)-flurbiprofen ester, e.g. to provide R-(-)-flurbiprofen, or a salt thereof, as desired. Thus, the process of the invention provides access to both enantiomers, i.e. S- (+)-flurbiprofen and R-(-)-flurbiprofen, in high ee; one as the acid the other as the ester that can chemically be hydrolysed to the acid after separation. Thus, the process of the invention may comprise the stereo selective de-esterification of S-(+)-flurbiprofen ester. Alternatively, the process of the invention may comprise the stereo selective de-esterification of R-(-)-flurbiprofen ester. The nature of the biocatalyst may vary, depending upon, inter alia, the chosen selective de-esterification, i.e. S-(+)-flurbiprofen ester or R-(-)-flurbiprofen ester; the nature of the ester; etc.

Thus, for example, for the selective de-esterification of S-(+)-flurbiprofen ester, the biocatalyst may preferably be selected from the group consisting one or more of Candida antarctica lipase A (IMMCALA (Chiralvision)); Candida antarctica lipase A (CLEA- 101-ST (Clea Technologies)); Candida antarctica lipase A (FE999 - Novozym 735 (Novozym)); Lipase 3.101 (Evocatal); Lipase 3.108 (Evocatal); Lipase evo-1.3.101.S (Evocatal); Lipase evo-1.3.108.S (Evocatal); Candida antarctica lipase A (NovoCor ADL (Chiralvision)); Pseudomonas fluorescens spray dried cells (PDNC40.17 (Biocatalysts Ltd.)); Pseudomonas fluorescens liquid (PDNC40.18 (Biocatalysts Ltd.)); and Candida rugosa lipase isoform PDNC40/2 (Biocatalysts Ltd.).

For the selective de-esterification of R-(-)-flurbiprofen ester, the biocatalyst may preferably be selected from the group consisting of one or more of Protease from Bacillus species (Savinase CLEA (Clea Technologies)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)); Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)); Aspergillus niger lipase (PDNC40/1 (Biocatalysts Ltd.)); Kluyveromyces lactis lipase (PDNC40/3 (Biocatalysts Ltd.)); Candida antarctica lipase B cross linked enzyme aggregate (CLEA-102-B4 (Clea Technologies)); Mucor javanicus lipase (PDNC40/4 (Biocatalysts Ltd.)); Lipase 3.137 (Evocatal); and Aspergillus niger lipase (PDNC40/5 (Biocatalysts Ltd.)). In a preferred embodiment of the present invention, for the selective de-esterification of S-(+)-flurbiprofen ester, the biocatalyst may preferably be selected from the group consisting of one or more of Candida antarctica lipase A (IMMCALA (Chiralvision)); Lipase 3.108 and Lipase 3.101. In a particularly preferred embodiment of the invention the process comprises the use of the biocatalyst Candida antarctica lipase A (IMMCALA (Chiralvision)), for the selective de-esterification of S-(+)-flurbiprofen ester.

In an alternative preferred embodiment of the present invention, for the selective de- esterification of R-(-)-flurbiprofen ester, the biocatalyst may preferably be selected from the group consisting one or more of Protease from Bacillus species (Savinase CLEA (Clea Technologies)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)); and Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)).

In a particularly preferred embodiment of the invention the process comprises the use of the biocatalyst Protease from Bacillus species (Savinase CLEA (Clea Technologies )); for the selective de-esterification of R-(-)-flurbiprofen ester. The amount of biocatalyst used in the process of the invention may vary depending, inter alia, upon the nature of the biocatalyst. The amount may vary with the choice of free enzyme or enzyme carrier is a supported enzyme is used. In the case of a supported enzyme, recovery and reuse or use in a packed bed reactor may be desirable.

Typical amounts used are illustrated in Tables I and II:

Table I

(S)

Typical

Conversion Selectivity

Enzyme Manufacturer concentration

(up to) (up to)

used

IMMCALA Chiral vision 50 95 lOmg/mL

CLEA-101-ST Clea Technologies 50 85 lOmg/mL

NovoCor ADL Chiral vision 50 85 lOmg/mL

PDNC40.18 Biocatalysts Ltd. 50 85 0.2mg/mL

PDNC40.17 Biocatalysts Ltd. 30 85 3mg/mL

Lipase 3.101 Evocatal 50 91 2mg/mL

Lipase 3.108 Evocatal 50 91 2mg/mL

FE999 - Novozym

Novozym 50 70 8mg/mL 735

Lipase evo-

Evocatal 30 85 4mg/mL 1.3.101.S

Lipase evo-

Evocatal 30 85 4mg/mL 1.3.108.S

PDNC40/2 Biocatalysts Ltd. 40 85 lOmg/mL Table II

(R )

Typical

Conversion Selectivity

Enzyme Manufacturer concentration

(up to) (up to)

used

Novozym 435 Novozymes 40 50 15mg/mL

Savinase CLEA Clea Technologies 11 90 30mg/mL

Alcalase CLEA Clea Technologies 30 70 30mg/mL

Mann-Associates

Lipase AE07 28 71 30mg/mL

(Cambridge)

Lipase 3.137 Evocatal 25 40 2mg/mL

CLEA-102-B4 Clea Technologies 40 45 5mg/mL

PDNC40/1 Biocatalysts Ltd. 35 65 lOmg/mL

PDNC40/3 Biocatalysts Ltd. 25 55 lOmg/mL

PDNC40/4 Biocatalysts Ltd. 30 45 lOmg/mL

PDNC40/5 Biocatalysts Ltd. 21 35 lOmg/mL

PDNC40/8 Biocatalysts Ltd. 25 85 lOmg/mL

The ester may vary, but may comprise, for example, a linear or branched CI -20 alkyl, C2- 20 alkenyl, CI -20 alkoxy, aryl, e.g. phenyl, phenoxy, heterocyclic; and substituted derivatives thereof. For the avoidance of doubt the term ester shall also include thioester unless specifically stated otherwise. In a preferred embodiment of the invention, the ester is a simple alkyl ester, e.g. alkyl CI -4, preferably methyl or ethyl.

In the process of the invention the biocatalyst may comprise a free enzyme or a suitable supported or immobilised version of the enzyme. As a biocatalyst, the enzyme may be used in a raw or purified form. Alternatively, the process may comprise the use of the appropriate enzyme producing microorganism in situ, e.g. in the culture broth of the microorganism, with the racemic or non-racemic flurbiprofen ester. In a further alternative the biocatalyst may comprise an enzyme in an immobilised form. Such immobilised form may comprise one or more of an enzyme physically adsorbed onto an inert carrier, occlusion of an enzyme in the lattices of a polymerised gel, cross-linking of the enzyme protein with a bifunctional reagent and covalent binding of the enzyme protein to a reactive insoluble support matrix.

The stereo selective de-esterification process according to the present invention may be conducted at temperatures in the range of from about 10°C to about 60°C, preferably in from about 20°C to about 50°C, more preferably at about 40°C.

The stereo selective de-esterification process will generally be carried out at a pH which provides optimum activity for the biocatalyst. Thus, the pH may vary depending, inter alia, upon the nature of the biocatalyst. The pH of the reaction mixture may also vary and may be from about 5 to about 9, preferably from about 6 to about 8, most preferably neutral pH.

The concentration of the starting racemic or non-racemic ester in the reaction mixture may vary from about 1% to about 90% w/w, preferably about 10 to about 80% w/w, preferably about 20 to about 70% w/w, preferably about 30 to about 60% w/w, preferably 45 to 55% w/w, e.g. about 50% w/w.

The stereo selective de-esterification process may be carried out in a variety of solvents, which may vary depending upon, inter alia, the nature of the ester, the biocatalyst, etc. Suitable solvents, include, but shall not be limited to alcohols, such as t-butanol or isopropyl alcohol; ethers such as cyclopentyl methyl ether (CPME) or methyl tert-butyl ether (MTBE); other suitable solvents include acetone, acetonitrile, dimethylsulfoxide (DMSO), dioxane, zso-octane, toluene and water; and mixtures thereof. Exemplary combinations include, but shall not be limited to, water & CPME, water & dioxane, water and DMSO, water & isooctane, water & MTBE, water & acetone, water & isopropyl alcohol and water & t-butanol. Specific combinations which may be mentioned include, for example, water & 10% CPME, water & 10% dioxane, water & 10% DMSO, water & 20% DMSO, water & 10% isooctane, water & 10% MTBE, water & 30% MTBE, MTBE & 10%) water, MTBE & 50% water, water & acetone, isopropyl alcohol & 10% water and t-butanol & 10% water. Preferred solvents include one or more of dimethylsulfoxide (DMSO), zso-octane, methyl tert-butyl ether (MTBE) and toluene; and mixtures thereof.

The duration of stereo selective de-esterification reaction may vary and may be from about 12 to about 120 hours, for example, from about 24 to about 96 hours, or from about 48 to about 72 hours.

At the end of the stereo selective de-esterification reaction, the reaction mixture may contain one stereo isomer of flurbiprofen acid and another stereo isomer of flurbiprofen ester. However, more aptly the reaction mixture will be enriched with one stereo isomer of flurbiprofen acid. For example, the reaction mixture may comprise S-(+)-flurbiprofen acid and R-(-)-flurbiprofen ester; or it may comprise R-(-)-flurbiprofen acid and S-(+)- flurbiprofen ester. The stereo isomer of flurbiprofen acid may desirably be isolated as the salt, for example by basifying the reaction mixture stereo isomer of flurbiprofen acid salt may be isolated by aqueous/ organic solvent extraction. The remaining stereo isomer of flurbiprofen ester may be isolated and the ester hydrolysed to yield the stereo isomer of flurbiprofen acid. Alternatively, stereo isomer of flurbiprofen ester may be subjected to a racemisation enabling it to be utilised as a starting material in the stereo selective de-esterification reaction of the invention.

The racemisation of the flurbiprofen ester may be carried out using conventional methods known to the person skilled in the art. However, in one aspect of the invention the racemisation may comprise an organocatalytic racemisation, for example, in the presence of water. Organocatalytic racemisation in the presence of water is especially suitable for combining the racemisation with biocatalyst de-esterification, e.g. enzymatic ester hydrolysis in water. In the organocatalytic racemisation of this aspect of the invention, the organocatalyst may desirably be an organic nitrogen-type Lewis base. Such Lewis bases include, but shall not be limited to l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and l,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD). The organic nitrogen-type Lewis base according to this aspect of the invention may comprise a free base or may comprise a suitable solid or polymer supported base; and mixtures thereof. The racemisation may be carried out in an organic solvent, such as an ether. Preferred ethers include, but shall not be limited to methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE). Preferably, the racemisation is carried out in an aqueous/ organic two-phase medium. Such a two phase medium will generally comprise an excess of the organic solvent. One example of a two phase medium is 5% v/v water and 95% v/v MTBE.

Thus according to this aspect of the invention there is provided a process for the preparation a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of: (i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of a biocatalyst capable of stereo selective de- esterification of the ester; provided that the biocatalyst is not Aspergillus oryzae; Bacillus cereus C71 ; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2; and

(ii) racemising the flurbiprofen ester.

It will be understood by the person skilled in the art the racemisation step (ii) may be repeated in order to increase the yield of the desired flurbiprofen enantiomer is achieved.

The two phase organic nitrogen-type Lewis base racemisation of flurbiprofen ester is novel per se. Therefore, according to a preferred aspect of the invention there is provided a process for the preparation a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of a biocatalyst capable of stereo selective de- esterification of the ester; and

(ii) carrying out a two phase organic nitrogen-type Lewis base racemisation of flurbiprofen ester. In a preferred aspect of the invention the stereo selective de-esterification of the ester comprises the use of a biocatalyst other than Aspergillus oryzae; Bacillus cereus C71 ; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens

ES-2.

The invention provides a process for the preparation of S-(+)-flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst selected from the group consisting one or more of Candida antarctica lipase A (IMMCALA (Chiralvision)); Candida antarctica lipase A (CLEA-101-ST (Clea Technologies)); Candida antarctica lipase A (FE999 - Novozym 735 (Novozym)); Lipase 3.101 (Evocatal); Lipase 3.108 (Evocatal); Lipase evo-1.3.101.S (Evocatal); Lipase evo-1.3.108.S (Evocatal); Candida antarctica lipase A (NovoCor ADL (Chiralvision)); Pseudomonas fluorescens spray dried cells (PDNC40.17 (Biocatalysts Ltd.)); Pseudomonas fluorescens liquid (PDNC40.18 (Biocatalysts Ltd.)); and Candida rugosa lipase isoform 1 DNC40/2 (Biocatalysts Ltd.); and

(ϋ) carrying out a two phase organic nitrogen-type Lewis base racemisation of flurbiprofen ester.

In a preferred embodiment of this aspect of the invention, for the selective de- esterification of S-(+)-flurbiprofen ester, the biocatalyst may preferably be selected from the group consisting of one or more of Candida antarctica lipase A (IMMCALA - (Chiralvision)); Lipase 3.108 and Lipase 3.101. Alternatively, the invention provides a process for the preparation of R-(-)-flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst selected from the group consisting one or m/ore of Protease from Bacillus species (Savinase CLEA (Chiralvision)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)); Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)); Aspergillus niger lipase (PDNC40/1 (Biocatalysts Ltd.)); Kluyveromyces lactis lipase (PDNC40/3 (Biocatalysts Ltd.)); Candida antarctica lipase B cross linked enzyme aggregate (CLEA-102-B4 (Clea Technologies)); Mucor javanicus lipase (PDNC40/4 (Biocatalysts Ltd.)); Lipase 3.137 (Evocatal); and Aspergillus niger lipase (PDNC40/5 (Biocatalysts Ltd.)); and

(ii) carrying out a two phase organic nitrogen-type Lewis base racemisation of flurbiprofen ester.

In a preferred embodiment of this aspect of the invention for the selective de-esterification of R-(-)-flurbiprofen ester, the biocatalyst may preferably be selected from the group consisting of one or more of Protease from Bacillus species (Savinase CLEA (Clea Technologies)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)); and Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)). According to this aspect of the invention the organic nitrogen-type Lewis base may be either l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and l,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD); or a similar base absorbed onto solids. The flurbiprofen enantiomer, that is S-(+)-flurbiprofen, or a salt thereof, or R-(-)- flurbiprofen, or a salt thereof, produced within a range of >95% enantiomeric excess (ee), preferably >96% ee, preferably >97% ee, more preferably >98% ee and most preferably >99% ee. Thus, according to this aspect of the invention there are provided an enantiomer of flurbiprofen prepared by dynamic kinetic racemic resolution. In one aspect the flurbiprofen is S-(+)-flurbiprofen, or a salt thereof. In another aspect of the invention the flurbiprofen is R-(-)-flurbiprofen, or a salt thereof. In a yet further aspect of the invention there is provided a pharmaceutical composition comprising flurbiprofen, or a salt thereof, prepared according to the process hereinbefore described.

As used herein, the term "salt" shall mean a "pharmaceutically acceptable salt" and refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which are not biologically or otherwise undesirable.

Salts are desirably pharmaceutically acceptable base addition salts, which can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminium, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropyl amine, and ethanolamine. The pharmaceutically acceptable salts of the present invention can be synthesised from a parent 2-(3-fluoro-4- phenyl-phenyl)propanoic acid, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the 2-(3-fluoro-4-phenyl-phenyl)propanoic acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Lists of additional suitable salts can be found, e.g., in "Remington's Pharmaceutical Sciences", 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley- VCH, Weinheim, Germany, 2002).

Other salts include ammonium or amino acid salts which are water soluble thereby being preferred. Complex salts with basic amino acids can be used directly and mixed salts with neutral or acidic amino acids are previously converted into the alkali metal, alkaline earth metal or ammonium salts. A preferred amino acid is lysine. Preferably, the flurbiprofen salt is prepared indirectly by adding the bases required for the salt formation. Amino acid salts may comprise an essential amino acid, such as, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and tyrosine; or a non-essential amino acid, such as, alanine, arginine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, asparagines and selenocysteine. Alternatively, the salt may comprise an amino sugar, such as meglumine. If a flurbiprofen enantiomer is isolated as an ester, the esters will generally be pharmaceutically acceptable esters. Such esters may be produced directly in the process of the present invention or may be produced by reacting the flurbiprofen acid with an appropriate alcohol. Esters of pharmaceutically acceptable alcohols can be formed with organic alcohols, such esters include, but shall not be limited to, e.g. acetate, acetoxyethyl ester (axetil), aspartate, benzoate, besylate, camsylate, cinnamate, citrate, edisylate, esylate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glucuronate, glycolate, hexafluorophosphate, hibenzate, isethionate, lactate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulphate, 2-napsylate, naphthylate, nicotinate, orotate, oxalate, palmitate, pamoate, propionate, pyruvate, saccharate, salicylate, stearate, succinate, tartrate, *- toluenesulfonate, tosylate, trifluoroacetate, and the like.

The invention will now be described by way of example only.

Example 1

Preparation of S(+) Flurbiprofen by Enzyme Catalysis Materials

Flurbiprofen C0₂Et (S) Flurbiprofen (R) Flurbiprofen

OEt

The enzyme IMMCALA is slurried in buffer pH 7. The racemic Flurbiprofen O-ethyl ester is dissolved in organic solvent and added to the reaction flask and stirred at 40°C for 48 h.

Apparatus

250 ml 3-neck round bottom flask, magnetic stirrer/heater with temperature control, 250 mL heating mantle, pH stat (or equivalent). Procedure

Preparation of S(+) Flurbiprofen (Enzyme Catalysed) 1. Charge the enzyme IMMCALA (2.0 g), 80 mL of deionised water and 10 mL of sodium phosphate 0.1 M buffer pH 7 to a 250 ml 3 -neck round bottom flask.

2. Commence stirring to disperse the enzyme. 3. Add 5.0 g of racemic Flurbiprofen O-ethyl ester dissolved in 10 mL of organic solvent.

4. Adjust the pH of the rapidly stirred reaction mixture to pH 7.0 (titrant 0.5M NaOH). 5. Heat the reaction vessel contents to an internal temperature of 40°C under pH control (set to 7.0) for up to 48 h (or until NaOH is no longer taken up).

6. After that time, turn the stirrer and the heating off and allow the reaction mixture to cool to room temperature.

Work Up

7. The reaction mixture is basified to pH 12-13 using 1 N NaOH added dropwise. 8. Filter the reaction mixture through a pad of damp Celite. The ester starting material may be recovered by rinsing the Celite with hexane.

9. Transfer the solution to a 500 mL separating funnel. 10. Extract the aqueous phase with isohexane (50 mL), allow to separate, then tap off the isohexane phase and keep. Also keep the combined aqueous layers.

11. Repeat step 10 a second time. 12. Repeat step 10 a third time.

13. Wash the combined isohexane extracts with a sodium hydroxide solution (20 mL).

Keep the combined aqueous layers. 14. Combine the isohexane phases and dry over magnesium sulphate for 15 mins before filtering. If this organic phase still contains some fibrous material, filter it through an isohexane damp pad of Celite.

15. Remove the isohexane by rotary evaporation to give an oil {ester starting material).

16. The combined aqueous layers are acidified using HCl 6 N dropwise to reach pH 2-3 (effervescence and the mixture turns white).

17. Transfer the solution to a 500 mL separating funnel. Extract the aqueous phase with MTBE (50 mL), allow to separate, then tap off the MTBE phase and keep.

Repeat step 18 a second time.

Repeat step 18 a third time. Wash the combined MTBE extracts with a saturated sodium chloride solution (20 mL). Add 2 g of magnesium sulphate to the combined MTBE extracts, stir with a spatula for 2 minutes and filter out. Transfer the organic MTBE layer to a flask. The aqueous layer may be discarded. Remove the MTBE by rotary evaporation to give an oil that solidifies upon standing to give a white solid.

Claims

1. A process for the preparation of a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst capable of stereo selective de-esterification of the ester; provided that the biocatalyst is not Aspergillus oryzae; Bacillus cereus C71 ; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2.

2. A process according to claim 1 wherein the flurbiprofen ester is in racemic form.

3. A process according to claim 1 wherein the flurbiprofen ester comprises a non- racemic mixture of (R)-(-)-flurbiprofen and the (S)-(+)-flurbiprofen.

4. A process according to any one of the preceding claims wherein the stereo selective de-esterification comprises de-esterification of S-(+)-flurbiprofen ester, to provide S-(+)-flurbiprofen, or a salt thereof.

5. A process according to any one of claims 1 to 3 wherein the stereo selective de- esterification comprises de-esterification of R-(-)-flurbiprofen ester, to provide R-(-)- flurbiprofen, or a salt thereof.

6. A process according to claim 4 wherein the biocatalyst is selected from the group consisting one or more of Candida antarctica lipase A (IMMCALA (Chiralvision)); Candida antarctica lipase A (CLEA-lOl-ST (Clea Technologies)); Candida antarctica lipase A (FE999 - Novozym 735 (Novozym)); Lipase 3.101 (Evocatal); Lipase 3.108 (Evocatal); Lipase evo-1.3.101.S (Evocatal); Lipase evo-1.3.108.S (Evocatal); Candida antarctica lipase A (NovoCor ADL (Chiralvision)); Pseudomonas fluorescens spray dried cells (PDNC40.17 (Biocatalysts Ltd.)); Pseudomonas fluorescens liquid (PDNC40.18 (Biocatalysts Ltd.)); and Candida rugosa lipase isoform 1 DNC40/2 (Biocatalysts Ltd.).

7. A process according to claim 6 wherein the biocatalyst is selected from the group consisting one or more of Candida antarctica lipase A (IMMCALA (Chiralvision)); Lipase 3.108 and Lipase 3.101.

8. A process according to claim 7 wherein the biocatalyst is Candida antarctica lipase A (IMMCALA (Chiralvision)), for the selective de-esterification of S-(+)- flurbiprofen ester.

9. A process according to claim 5 wherein the biocatalyst is selected from the group consisting one or more of Protease from Bacillus species (Savinase CLEA (Clea Technologies)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann- Associates Cambridge)); Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)); Aspergillus niger lipase (PDNC40/1 (Biocatalysts Ltd.)); Kluyveromyces lactis lipase (PDNC40/3 (Biocatalysts Ltd.)); Candida antarctica lipase B cross linked enzyme aggregate (CLEA-102-B4 (Clea Technologies)); Mucor javanicus lipase (PDNC40/4 (Biocatalysts Ltd.)); Lipase 3.137 (Evocatal); and Aspergillus niger lipase (PDNC40/5 (Biocatalysts Ltd.)).

10. A process according to claim 9 wherein the biocatalyst is selected from the group consisting one or more of Protease from Bacillus species (Savinase CLEA (Clea Technologies)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)) and Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)).

11. A process according to claim 10 wherein the biocatalyst is Protease from Bacillus species (Savinase CLEA (Clea Technologies)), for the selective de-esterification of R-(-)- flurbiprofen ester.

12. A process according to any one of the preceding claims wherein the ester comprises a linear or branched CI -20 alkyl, C2-20 alkenyl, CI -20 alkoxy, aryl, e.g. phenyl, phenoxy, heterocyclic; and substituted derivatives thereof.

13. A process according to any one of the preceding claims wherein the ester comprises a thioester.

14. A process according to any one of claims 1 to 12 wherein the ester is a simple alkyl ester, e.g. alkyl CI -4.

15. A process according to claim 14 wherein the ester is methyl or ethyl.

16. A process according to any one of the preceding claims wherein the biocatalyst comprises a free enzyme or a suitable supported or immobilised version of the enzyme.

17. A process according to any one of the preceding claims wherein the enzyme is used in purified form.

18. A process according to claim 16 wherein the enzyme is in an immobilised form.

19. A process according to claim 18 wherein the biocatalyst comprises Candida antarctica lipase A covalently bonded to a microporous resin (IMMCALA (Chiralvision)).

20. A process according to any one of claims 1 to 15 wherein the process comprises the use of an enzyme producing microorganism in situ.

21. A process according to any one of the preceding claims wherein the process is conducted at a temperature in the range of from about 10°C to about 60°C.

22. A process according to any one of the preceding claims wherein the process is conducted at a pH of from about 5 to about 9.

23. A process according to any one of the preceding claims wherein the concentration of the starting racemic or non-racemic ester in the reaction mixture is from about 1% to about 90% w/w.

24. A process according to any one of the preceding claims wherein the process is carried out in a solvent selected from one or more of an alcohol, such as t-butanol or isopropyl alcohol; an ether, such as cyclopentyl methyl ether (CPME) or methyl tert-butyl ether (MTBE); acetone, acetonitrile, dimethylsulfoxide (DMSO), dioxane, zso-octane, toluene and water; and mixtures thereof.

25. A process according to any one of the preceding claims wherein the solvent is selected from one or more of dimethylsulfoxide (DMSO), zso-octane, methyl tert-butyl ether (MTBE) and toluene; and mixtures thereof.

26. A process according to any one of the preceding claims which comprises isolating the stereo isomer of flurbiprofen acid as a salt.

27. A process according to any one of the preceding claims which comprises isolating the stereo isomer of flurbiprofen ester.

28. A process according to any one of claims 1 to 26 which comprises subjecting the stereo isomer of flurbiprofen ester to a racemisation.

29. A process according to claim 28 wherein the racemisation comprises an organocatalytic racemisation.

30. A process according to claim 29 wherein the organocatalyst is an organic nitrogen- type Lewis base.

31. A process according to claims 29 or 30 wherein the organocatalyst is 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) or l,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD).

32. A process according to any one of claims 29 to 31 wherein the organocatalyst is 1 , 8-diazabicyclo [5.4.0]undec-7-ene (DBU) .

33. A process according to any one of claims 29 to 32 wherein the organic nitrogen- type Lewis base comprises the free base.

34. A process according to any one of claims 29 to 32 wherein the organic nitrogen- type Lewis base comprises a suitable solid or polymer supported base.

35. A process according to any one of claims 29 to 34 wherein the racemisation is carried out in an organic solvent.

36. A process according to claim 35 wherein the racemisation is carried out in an organic ether solvent.

37. A process according to claim 36 wherein the organic ether solvent is methyl tert- butyl ether (MTBE) or ethyl tert-butyl ether (ETBE).

38. A process according to claims 36 or 37 wherein the racemisation is carried out in an aqueous/ organic two-phase medium.

39. A process according to claim 38 wherein the aqueous/ organic two-phase medium comprises water and MTBE.

40. A process for the preparation a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst capable of stereo selective de- esterification of the ester; provided that the biocatalyst is not Aspergillus oryzae; Bacillus cereus C71 ; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2; and

(ii) racemising the flurbiprofen ester.

41. A process according to claims 40 wherein the racemisation step (ii) is repeated.

42. A process for the preparation a stereospecific form of flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst capable of stereo selective de- esterification of the ester; and

43. A process according to claims 40 to 42 wherein the stereo selective de- esterification of the ester comprises the use of a biocatalyst other than Aspergillus oryzae ; Bacillus cereus C71 ; Candida Antarctica lipase B (Novozym ® 435); Candida cylindracea; Candida rugosa lipase; Carica papaya lipase; Pseudomonas sp. KCTC 10122BP and Serratia marcescens ES-2.

44. A process for the preparation of S-(+)-flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

45. A process according to claim 44 wherein the biocatalyst is selected from the group consisting of one or more of Candida antarctica lipase A (IMMCALA (Chiralvision)); Lipase 3.108 and Lipase 3.101.

46. A process for the preparation of R-(-)-flurbiprofen, or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(i) subjecting a flurbiprofen ester, comprising a mixture (R)-(-)-flurbiprofen and (S)-(+)-flurbiprofen, to the action of an biocatalyst selected from the group consisting one or more of Protease from Bacillus species (Savinase CLEA (Chiralvision)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)); Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)); Aspergillus niger lipase (PDNC40/1 (Biocatalysts Ltd.)); Kluyveromyces lactis lipase (PDNC40/3 (Biocatalysts Ltd.)); Candida antarctica lipase B cross linked enzyme aggregate (CLEA-102-B4 (Clea Technologies)); Mucor javanicus lipase (PDNC40/4 (Biocatalysts Ltd.)); Lipase 3.137 (Evocatal); and Aspergillus niger lipase (PDNC40/5 (Biocatalysts Ltd.)); and

47. A process according to claim 46 wherein the biocatalyst is selected from the group consisting of one or more of Protease from Bacillus species (Savinase CLEA (Clea Technologies)); Bacillus licheniformis lipase (PDNC40/8 (Biocatalysts Ltd.)); Pseudomonas stutzeri lipase (Lipase AE07 (Mann-Associates Cambridge)); and Protease from Bacillus licheniformis (Alcalase CLEA (Clea Technologies)).

48. A process according to any one of claims 44 to 47 wherein the organic nitrogen- type Lewis base may be either l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,5,7- triazabicyclo [4.4.0] dec-5 -ene (TBD) .

49. An enantiomer of flurbiprofen, or a salt thereof, prepared by a process according to any one of claims 1 to 48.

50. An enantiomer of flurbiprofen according to claim 49 wherein the enantiomer is S- (+)-flurbiprofen, or a salt thereof.

51. An enantiomer of flurbiprofen according to claim 49 wherein the enantiomer is R- (-)-flurbiprofen, or a salt thereof.

52. A pharmaceutical composition comprising an enantiomer of flurbiprofen, or a salt thereof, according to any one of claims 49 to 51.

53. A process, enantiomer or pharmaceutical composition as hereinbefore described with reference to the accompanying description.