WO2006006851A2

WO2006006851A2 - Process for the preparation of an enantiomerically enriched ester or the corresponding acid thereof

Info

Publication number: WO2006006851A2
Application number: PCT/NL2005/000490
Authority: WO
Inventors: Daniel Mink; Joannes Gerardus Theodorus Kierkels
Original assignee: Dsm Ip Assets B.V.
Priority date: 2004-07-08
Filing date: 2005-07-07
Publication date: 2006-01-19
Also published as: WO2006006851A3

Abstract

The invention relates to a process for the preparation of an enantiomerically enriched ester or corresponding acid thereof by enzymatic conversion of the corresponding mixture of enantiomers. The enantiomerically enriched ester/acid has a remote and quarternary chiral center. The process of the invention is particularly suitable for the preparation of enantiomerically enriched 5,6-dihydro-4-hydroxy-2-pyrones, which are important building docks for the synthesis of a number of pharmaceutically active compounds.

Description

PROCESS FOR THE PREPARATION OF AN ENANTIOMERICALLY ENRICHED

ESTER OR THE CORRESPONDING ACID THEREOF

The invention relates to a process for the preparation of an enantiomerically enriched compound of formula 1

wherein R^A stands for OH or for SH or wherein R^A stands for R¹⁵, wherein R¹⁵ stands for SR¹² or OR¹², wherein R¹² stands for an ester residue or wherein R^A stands for OR¹⁴ or SR¹⁴, wherein R¹⁴ stands for an ester residue which is not the same as R¹² and wherein R², R³ and R⁴ each independently stand for an optionally substituted (hetero)aryl, an optionally substituted alkyl, OR⁵, CO₂R⁶, C(O)R⁷, SR⁸, NR⁹R¹⁰,

OC(O)R¹¹ wherein R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ each independently stand for H, an optionally substituted alkyl or for an optionally substituted (hetero)aryl, wherein R², R³ and R⁴ are not the same and wherein R² and R³, R² and R⁴ or R³ and R⁴ may form a (hetero)cycloalkyl together with the carbon atom to which they are attached, provided that said (hetero)cycloalkyl does not have a plane of symmetry and wherein R²³ stands for

wherein R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ each independently stand for O, S or NR²², wherein R²² stands for an alkyl, aryl, aralkyl or alkaryl and wherein I, m, n, o, p, q, r, s, t, u, v, w, x and y each independently stand for 0 or 1 and wherein l + m + n + o + p + q + r + s + t + u + v + w + x + y = z, wherein z stands for 3, 4, 5, 6, or 7.

Enzyme catalyzed stereoselective conversions for compounds with remote chiral centers are known. With remote chiral center is meant that the chiral center is at least three bonds away from the reactive center. For example, in

Hydrolases in organic synthesis. Eds. Kazlauskas and Bornscheuer. Wiley-VCH, 1999, it is disclosed that some carboxylic acids with remote sterocenter can be enzymatically converted with some degree of stereoselectivity. Hughes et. al. (1990) J. Org. Chem. 55, 6252-6259 show the asymmetric hydrolysis of esters having remote prochiral centers. Bhalerao et. al. report on the lipase-catalyzed regio- and stereoselective hydrolysis of (ω-2)-acetoxy-ω-bromoalkenoates by a lipase from Candida cylindracea. Hedenstrόm et. al. (2002), Tetrahedron asymmetry, 13, 835-844 report, the highly stereoselective Candida rugosa lipase-catalyzed esterification of the 2- to 8-methyldecanoic acids, showing enantiomeric ratio's ranging from E = 2.8 - 68. Fadnavis et. al. (1997) Tetrahedron Asymmetry, 8, 337-339 show a lipase catalyzed stereoselective esterification of racemic α-lipoic, having a chiral center four carbon atoms away from the reactive center, which resulted in the formation of a product with a maximum enantiomeric excess of 23.8%.

Enzyme catalyzed stereoselective conversion of compounds having quaternary chiral centers are also known. For example Yee et. al. (1992) J. Org. Chem., 57, 3525-3527 describe the enzyme catalyzed stereoselective hydrolysis of tertiary α-substituted carboxylic acids esters. Sugai et. al. disclose the enzymatic preparation of enantiomerically enriched tertiary α-benzyloxy acid esters using a lipase derived from Candida rugosa, for which enzyme they report E-ratios between E = 10 - 52. Spero et. al. (1996) J. Org. Chem., 61 , 7398-7401 show stereoselective synthesis of the (S)-α.α-disubstituted phenethylamine from α,α-disubstituted amino acid esters using a lipase.

However, up till now, no example of a process wherein a compound having a remote and quaternary chiral center is stereoselective^ converted has been reported.

Therefore, it is the object of the invention to provide a process for the stereoselective conversion of a compound having a remote and quaternary chiral center.

This object is achieved by the present invention by reacting a stereoselective hydrolytic enzyme with a mixture of enantiomers of a compound of formula 1A, (1A)

wherein R¹ stands for OH, SH, SR¹² or OR¹², wherein R¹² and R²³, R², R³ and R⁴ are as defined above in the presence of water, H₂S, an alcohol of formula HOR¹⁴ or a thiol of formula HSR¹⁴, wherein R¹⁴ stands for an ester residue but is not the same as R¹² acting as a nucleophile and either collecting the remaining enantiomerically enriched compound of formula 1A

wherein R²³, R¹, R², R³ and R⁴ are as defined above, or collecting

- in case the nucleophile is the alcohol - the resulting enantiomerically enriched ester of formula 3

wherein R²³, R², R³, R⁴ and R¹⁴ are as defined above,

- in case the nucleophile is the thiol, wherein R¹⁴ stands for an ester residue but is not the same as R¹- the resulting enantiomerically enriched compound of formula 4

wherein R²³, R², R³ and R⁴ are as defined above,

- in case the nucleophile is water and R¹ stands for OR¹², SR¹² or SH - the resulting enantiomerically enriched acid of formula 2,

wherein R²³, R², R³ and R⁴ are as defined above,

- in case the nucleophile is H₂S and R¹ stands for OR¹², SR¹² or OH, the resulting enantiomerically enriched compound of formula 2A,

(2A) , wherein R²³, R², R³ and R⁴ are as defined above.

An additional advantage of the process of the present invention is that usually the resulting product and/or remaining compound has a relatively high enantiomeric excess (ee).

Chiral center has its conventional meaning in the art. For example, a C-atom having four different substituents is a chiral center. Quaternary center has its conventional meaning in the art. In the compound of formula 1A, the chiral center is the C-atom substituted with R², R³ and R⁴. This chiral center is also a quaternary center. Reactive center has its conventional meaning in the art. For example, in the compound of formula 1A, the reactive center is -C(O)R¹.

Preferably in the compounds of formula 1 , 1A, 2, 2A, 3, 4, one of R², R³ or R⁴ stands for OR¹³, wherein R¹³ stands for H, an optionally substituted alkyl or for an optionally substituted (hetero)aryl, preferably H; one of R², R³ or R⁴ stands for a C₁- Cβ-alkyl-, a Ce-Cnraryl-CrCn-alkyl- or a

rest group; and one of R², R³ or R⁴ stands for a Ci-C₈-alkyl- rest group.

The ester residue represented by R¹² or by R¹⁴ preferably stands for an alkyl group, for instance an alkyl group with 1-6 C-atoms or an aryl group, for instance an aryl group with 6-12 C-atoms, in particular a methyl, ethyl, propyl, isobutyl or tert. butyl group.

Preferably, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹³ each independently stand for H, an optionally substituted alkyl of 1-12 C-atoms, more preferably of 1-8 C- atoms (C-atoms of the substituents included) or for a (hetero)aryl of 2-10 C-atoms (C- atoms of the substituents included). Preferably in the heteroaryl, the heteroatom(s) is/are chosen from the group of N₁ O and S.

In the compounds of formula 1 , 1A, 2, 2A, 3, 4, R² and R³, R² and R⁴ or R³ and R⁴ may form a (hetero)cycloalkyl of preferably 3-6 C-atoms, provided that said (hetero)cycloalkyl does not have a plane of symmetry. R²³ may for example represent a C₃-C₇ alkyl. Preferably, z stands for

3 or 4. Preferably R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ stand for O.

In the compounds of formula 1 , 1A, 2, 2A, 3 and 4, the C-atom to which R², R³ and R⁴ are attached should be chiral.

Preferably R²² stands for an alkyl, aryl, aralkyl or alkaryl each of which is preferably not more than 10 C-atoms.

Examples of substituents include alkyl, (hetero)aryl, sulfonyl, alkoxycarbonyl, amidocarbonyl, nitrile, hydroxy, alkoxy, aryloxy, thioalkyl, mercapto, amino and fluorine.

In the framework of the invention with the term 'enantiomerically enriched' is meant 'having an enantiomeric excess (e.e.) of either the (R)- or (S) - enantiomer of a compound'. Preferably, the enantiomeric excess is > 80%, more preferably > 85%, even more preferably > 90%, in particular >95%, more in particular > 97%, even more in particular > 98%, most in particular > 99%.

With 'hydrolytic enzyme' is meant an enzyme with the ability to hydrolyze an ester group to form the corresponding acid group. Hydrolytic enzymes are known to react with esters to form first a so-called enzyme-acyl complex, after which the enzyme-acyl complex is attacked by a nucleophile to form the resulting product and the free enzyme. This resulting product may for example be a carboxylic acid in case H₂O iS the nucleophile or a (different) ester in case the nucleophile is an alcohol or a thiol. Which ester is formed depends on the alcohol or thiol used. For instance if an alcohol of formula HOR¹⁴, wherein R¹⁴ stands for an ester residue, but is not the same as R¹² is present in the process of the invention the resulting reaction product is the enantiomerically enriched ester of formula 3

wherein R^23t R², R³, R⁴ and R¹⁴ are as defined above. For instance, if a thiol of formula HSR¹⁴, wherein R¹⁴ stands for an ester residue, but is not the same as R¹², is present in the process of the present invention, the resulting product is the enantiomerically enriched compound of formula 4

wherein R²³, R², R³, R⁴ and R¹⁴ are as defined above. With 'stereoselectivity' of the hydrolytic enzyme is meant that the hydrolytic enzyme preferably catalyzes the conversion of one of the enantiomers of the compound of formula 1. In the compound of formula 1 , at least the C-atom substituted with R², R³ and R⁴ is chiral (since R², R³ and R⁴ are not the same) and the stereoselectivity of the hydrolytic enzyme should at least discriminate for this chiral center.

If a hydrolytic enzyme with a specific stereoselectivity is used, one enantiomer is preferably converted. This means that if a hydrolytic enzyme with an opposite stereoselectivity is used, the other enantiomer is preferably converted.

The enantiomer that is preferably converted strongly depends on the structure of the substrate and the stereoselectivity of the enzyme. Empirical methods that can predict the enantiomer that is preferably converted by the enzyme exist (Hydrolases in organic synthesis. Eds. Kazlauskas and Bomscheuer. Wiley-VCH, 1999). In addition, the person skilled in the art may also rely on experimental data. Thus, an enzymatic reaction will either yield the (S)-product or the (R)-product, leaving behind the remaining substrate with the opposite stereochemical configuration. It is known to the person skilled in the art that the stereochemical outcome of an asymmetric hydrolysis can be directed by choosing a hydrolase from a different class (Kurt Faber, Biotransformations in Organic Chemistry, a textbook, fourth complete revised and extended edition, Springer-Verlag Berlin Heidelberg, 2000, p. 97). Furthermore, it is common general knowledge that it it possible to obtain both (R)- and (S)-selective enzymes either by isolation of the enzymes from their natural environment or by genetic engineering of a known enzyme. An example of naturally occurring enzymes with opposite stereoselectivities are (R)-HNL of Prunus amygdalus (PaHNL, E.C. 4.1.2.10) and (S)-HNL from Hevea brasiliensis (HbHNl, E.C. 4.1.2.39) (Avi, M. et a/., 2004, Tetrahedron 60, pp 10411-10418. An example of a screening method which results in enzymes with opposite stereoselectivities is described by DeSantis G. et al., 2002. J. Am. Chem. Soc, vol 124, no 31 , p. 9025. Alternatively, one may mutate the enzyme, more in particular it's active site in order to obtain opposite stereoselectivity, such as for instance described for vanillyl-alcohol oxidase by Heuvel van den, R. H. H. et a/., 2000, vol. 97. no17, pp 9455-9460.

The stereoselectivity of an enzyme may be expressed in terms of E- ratio, the ratio of the specificity constants V_max /K_m of the two enantiomers as described in C-S. Chen, Y Fujimoto, G. Girdaukas, C. J. Sih., J. Am. Chem. Soc. 1982, 704, 7294-7299. Preferably, the hydrolytic enzyme has an E-ratio > 5, more preferably an E- ratio > 10, even more preferably an E-ratio > 50, most preferably an E-ratio > 100. The E-ratio of an enzyme may be enhanced using mutagenesis techniques known in the art, for example by using the gene site saturation mutagenesis (GSSM) method as described by DeSantis, G. et al., 2003, J. Am. Chem. Soc. VoI 125, no 38, p11477.

A stereoselective hydrolytic enzyme suitable for use in the present invention may for example be found in one of the general classes of hydrolytic enzymes, for instance in the group of esterases, lipases, proteases, peptidases or acylases, preferably in the group of esterases or lipases. The hydrolytic enzyme may be derived from both eukaryotic and prokaryotic cells, including but not limited to those from the following mammalian sources: porcine liver, porcine pancreas, for example commercially available porcine pancreatic lipase type Il (L-3126, Sigma); porcine kidney and bovine pancreas; those from the plant source wheat germ; those from the following mold genera: Absidia; Aspergillus; Fusarium; Gibberella; Mucor, Neurospora; Trichoderma; Rhizopus; Rhizomucor, for example Rhizomucor miehei; Thermomyces, for example Thermomyces lanugenousus; those from the following bacterial genera: Achromobacter, Alcaligenes; Bacillus; for example Bacillus licheniformis; Brevibacterium; Corynebacterium; Providencia; Pseudomonas, for example Pseudomonas fluorescens, Pseudomonas cepase or Pseucomonas alcaligenes;

Serratia; Rhodococcus, those from the following yeast genera: Candida, for example Candida rugose or Candida Antarctica; and those from the Actonomycete genus Nocardia.

Preferably, the stereoselective hydrolytic enzyme is found in the group of enzymes classified as carboxylic ester hydrolases (EC 3.1.1 ) or in the group of enzymes classified as peptidases, for example EC 3.4.1 , EC 3.4.11 , EC 3.4.21 , more preferably EC 3.4.21.62, EC 3.4.22 or EC 3.4.23.

A stereoselective hydrolytic enzyme may also be found in the group of commercially available hydrolytic enzymes. Examples of commercially available hydrolytic enzymes are: enzymes supplied by Fluka: Candida cylindracea lipase, lipase Hog pancreas, lipase Pseudomonas fluorescens, lipase Aspergillus oryzae, lipase Rhizopus niveus, lipase Rhizomucor miehei, lipase Candida antarctica, lipase Mucor javanicus, lipase Rhizopus arrhizus, lipase Penicillium roqueforti, lipase Candida lipolytica, lipoprotein lipase Pseudomonas sp., type B, lipoprotein lipase Pseudomonas cepacia, lipoprotein lipase Chromobacterium viscosum, esterase Bacillus stearothermophilus, esterase Bacillus thermoglucosidasius, esterase Mucor miehei, esterase hog liver; enzymes supplied by Altus: Candida rugosa lipase, lipase Mucor miehei, Candida antarctica B lipase, Candida antarctica A lipase, Chiro-CLEC-CR, Chiro-CLEC-CR (slurry), porcine liver esterase, penicillin acylase, subtilisin Carlsberg, Chiro-CLEC-BL (slurry), Chiro-CLEC-PC (slurry), Chiro-CLEC-EC (slurry), Aspergillus oryzae protease, PeptiCLEC-TR (slurry); enzymes supplied by Recombinant Biocatalysis: ESL-001-07, ESL001-01 , ESL-001-01 with stabilizer, ESL-001-02, ESL- 001-03, ESL-001-05; enzymes supplied by Boehringer-Mannheim: Chirazyme L4 (Pseudomonas sp.), Chirazyme L5 {Candida antarctica fraction A)₁ Chirazyme L1 (Burkholderia), Chirazyme L6 (porcine pancreas), Chirazyme L7, Chirazyme L8; enzymes supplied by DSM (formerly Gist-Brocades): Naproxen esterase, Lipomax, Genzyme, Lipoprotein lipase; enzymes supplied by Novo: Novozyme 868, Novozyme 435, immobilized Candida Antarctica lipase, Nagase enzyme, Lipase A-10FG (Rhizopus javanicus); enzymes supplied by Amano, Amano AYS, Amano PS, Amano PSD, Amano AKD11 , Amano AKD111.

The stereoselective hydrolytic enzyme may be used in any form. For example, the hydrolytic enzyme may be used - for example in the form of a dispersion, a solution or in immobilized form - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess the required stereoselective hydrolytic enzyme activity, or in a lysate of cells with such activity.

It will be clear to the average person skilled in the art that use can also be made of mutants of naturally occurring (wild type) enzymes with hydrolytic activity in the process according to the invention. Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild type enzymes using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild type enzyme and by effecting the expression of the thus modified DNA in a suitable (host) cell. Mutants of the stereoselective hydrolytic enzyme may have improved properties with respect to (stereo)selectivity and/or activity and/or stability and/or solvent resistance and/or pH prophile and/or temperature prophile. A stereoselective hydrolytic enzyme may for example be selected for the process of the invention by screening several enzymes or host cells expressing genes encoding enzymes for the presence of stereoselective hydrolytic activity. In general, the person skilled in the art is aware of how to screen for enzymes with a desired activity. Usually for selection of a suitable enzyme, conditions under which the substrate (in this case the compound of formula 1A) and the enzyme are brought into contact are chosen such that it is a good compromise between on the one hand the stability of the enzyme, the substrate and the reaction product and on the other hand the reaction velocity (which usually increases at higher temperatures). Screening for enzymes may be performed at any scale. For practical reasons, if large numbers of enzymes are screened, a reaction volume between 0.15ml and 10ml is used.

For example, it is possible to screen for enzymes suitable for the stereoselective hydrolysis of a compound of formula 1A, wherein R¹ does not stand for H (the 'hydrolysis screening') by monitoring the progress of the hydrolysis of the compound of formula 1A in an aqueous solution in the presence of an enzyme using an analytical method, for example TLC, HPLC or GC.

Examples of aqueous solutions are water and water with co-solvent, for example a water-miscible organic solvent or a water-immiscible solvent. Examples of water-miscible organic solvents include methanol, ethanol, aceton, dioxane, acetonitrile, tetrahydrofuran, dimethylsulfoxide and dimethylformamide. Examples of water-immiscible organic solvents include methyl-t-butyl ether, methyl-isobutyl ketone, toluene, hexane, xylene and iso-octane. The amount of co-solvent is in principle not critical and is usually chosen between 5 and 25 % v/v. In case the substrate is liquid it may be present in water as such. In case the substrate is solid, it may be advantageous that a co-solvent is also present. For example, it is also possible to screen for enzymes suitable for stereoselective conversion of a compound of formula 1 A into an ester of formula 3 or a compound of formula 4 (the '(trans)esterification screening') by monitoring the progress of the conversion of a compound of formula 1A in an organic solvent in the presence of an alcohol of formula HOR¹⁴ or a thiol of formula HSR¹⁴, wherein R¹⁴ is as defined above and in the presence of an enzyme using an analytical method, for example TLC, HPLC or GC.

Examples of organic solvents are, methanol, ethanol, aceton, dioxane, acetonitril, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, t-butanol, methyl-t-butyl ether, methyl-isobuyl ketone, toluene, hexane, xylene and iso-octane. Uusally, water-immiscible organic solvents are preferred. The solvent may also be the alcohol of formula HOR¹⁴ or the thiol of formula HSR¹⁴.

The person skilled in the art knows when to apply techniques for shifting the equilibrium of a (trans) esterification. Also, the person skilled in the art knows which techniques are suitable for shifting the equilibrium of a (trans)esterification. Examples of such equilibrium shifting techniques are, for example techniques that remove the ester residue represented by R¹² in formula 1A, for example the application of reduced pressure, pervaporation etc.

The enzyme/substrate ratio in the '(trans)esterification screening¹ or of the 'hydrolysis screening' is in principle not critical and may be chosen between 1/20 and 2/1. The amount of substrate used is in principle also not critical and may for example be between 5mM and 1.5 M.

The pH of the 'hydrolysis screening' is in principle not critical and may for example be chosen between 5 and 10, preferably between 6 and 8 and may be kept constant by using a buffered aqueous solution using a buffer concentration of for example between 1OmM and 50OmM. Alternatively, the pH of the screening reaction may be kept constant by using an automated pH-stat system. The temperature of the '(trans)esterification screening' or of the 'hydrolysis screening' is in principle not critical and may be chosen between 20 and 40⁰C. Alternatively, if an enzyme is sought, which should operate at high temperature, the temperature may be chosen a lot higher. The choice of the reaction conditions of the process of the invention depends on the choice of hydrolytic enzyme. Usually, the temperature of the process is chosen between 0 and 90°C, in particular between 10 and 40⁰C; usually the pH of the process is chosen between 4 and 10.

The choice of solvent depends on which nucleophile is used. For instance if the nucleophile is water or H₂S, the solvent may for example be water, an aqueous solvent, for example water with a water miscible organic solvent, for instance t-butanol, dioxane, methanol, ethanol, tetrahydrofuran, aceton or dimethylsulfoxide; or a two-phase system of water and a water immiscible solvent, for example toluene, hexane, heptane, methyl f-butyl ether, methyl iso-butyl ketone. If the nucleophile is an alcohol or a thiol, the solvent is preferably an organic solvent comprising at least 1 equivalent of HOR¹⁴ or HSR¹⁴, wherein R¹⁴ stands for an ester residue, but is not the same as R¹². Examples of organic solvents, which may comprise the alcohol or thiol are THF, CH₃CN, heptane, toluene, hexane, methyl-t-butyl-ether, methyl-iso-butyl ketone. Of course the organic solvent may also be the same as the alcohol or thiol used as the nucleophile.

With 'mixture of enantiomers' is meant a random mixture of (R) and (S)-enantiomers. Typically, a racemic mixture of the compound of formula 1A is used (i.e. when (R): (S) is 1 :1), but of course the process of the invention may also be performed, - for further enantiomeric enrichment -, on an already enantiomerically enriched mixture of enantiomers.

Collecting includes for example isolation by means of conventional methods, for example ultrafiltration, concentration, column chromatography, extraction or crystallization and further reaction of the obtained product (resulting enantiomerically enriched acid of formula 2, ester of formula 3, compound of formula 4 or remaining enantiomerically enriched ester of formula 1).

The resulting and/or remaining enantiomerically enriched compounds produced in the process of the invention can suitably be used as building blocks in the preparation of pharmaceuticals. For example the process of the invention is in particular very suitable for the preparation of enantiomerically enriched compounds of formula 5

wherein R³ respectively R⁴ stands for a CrCβ-alkyl-, a

or a C₃- C₈-cycloalkyl-CrC₄alkyl- rest group and wherein R⁴ respectively R³ stands for a Ci-C₈- alkyl- rest group and wherein R³ and R⁴ are not the same and wherein R¹² stands for a C₁-C₄ alkyl or benzylrest. These compounds are of particular interest, since these compounds may be cyclisized in the presence of a base to form the corresponding enantiomerically enriched compound of formula 6

wherein R³ and R⁴ are as defined above. Cyclization of the compound of formula 5 can be performed in a manner known per se, for instance by using a base as described in WO 02/068403 or in US 6,500,963 B2. Examples of bases suitable for the cyclization of the compound of formula 5 include: carbonate, OH, metalhydrides, for instance sodiumhydride; organometals, metalamides, for instance butyllithium; metaldialkyamides, for instance lithiumdiethylamide, lithiumdiisopropylamide and metalhexamethyldisilizanes, for instance lithiumhexamethyldisilizane. As metal cations may, for example be used lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, titan, silicium, tin- and lanthanoide, preferably lithium or sodium, more preferably lithium.

A process for the preparation of enantiomerically enriched compounds of formula 6 is disclosed in US 6,500,963. A disadvantage of said non- enzymatic process is that the process requires the use of an additional compound (a chiral amino alcohol), which either may need to be recycled in a non-qunatitative manner or which is lost.

The process of the invention is a commercially feasible enzymatic process. For instance, it only requires the use of catalytic amounts of stereoselective hydrolytic enzyme and the recovery of (expensive) chiral amino alcohol is not necessary.

Enantiomerically enriched 5,6-dihydro-4-hydroxy-2-pyrones, such as the compounds of formula 6 are important building blocks for the synthesis of a number of pharmaceutically active compounds, for instance for non-peptidic HIV protease inhibitors, more specifically in the potent and orally bioavailable HIV-protease inhibitor tipranavir (US 6,500,963 B2). Also described in US 6,500, 963 B2 is a process for the preparation of tiphnavir from enantiomerically enriched 5,6-dihydro-4-hydroxy-2- pyrones, in particular from enantiomerically enriched 5,6-dihydro-4-hydroxy-6- phenethyl-6-propyl-2H-pyran-2-on. The preparation of the racemic mixture of the compound of formula 5 is for instance described in WO 02/068403, hereby included by reference.

The invention will now be elucidated by way of the following example without however being limited thereto.

Examples Example 1

0.94 g (3.07 mmol) (R₁S)- δ-Hydroxy-S-oxo-δ-phenethyl-octanoic acid ethyl ester was added to 40 mL of a 50 mM potassium phosphate buffer, pH 8.0. The resulting emulsion was stirred vigorously for 10 minutes at 4O⁰C. The enzymatic conversion was started by adding an enzyme solution containing a protease derived from Bacillus licheniformis (Alcalase 2,5L DX from Novozymes). The pH of the reaction mixture was kept at pH 8.0 by adding 1 mol/L NaOH using a Metrohm pH stat, model 718 Stat Titrino.

Periodically, samples were taken an analyzed by means of chiral HPLC. In order to do so, about 0.5 mL of reaction mixture was acidified with 50 μL 37% HCI. To this 0.5 mL ethylacetate was added. The resulting mixture was stirred for 3 minutes after which the organic and aqueous layer were separated with an Eppendorf centrifuge. After separating the organic layer and evaporating the ethylacetate the samples were analyzed by means of chiral HPLC. The resulting product was (R)-5-hydroxy-3-oxo-5- phenethyl octanoic acid. The results are presented in table 1 below.

Table 1. Stereoselective enzymatic hydrolysis of (R₁S)- 5-Hydroxy-3-oxo-5-phenethyl- octanoic acid ethyl ester into (R)-5-hydroxy-3-oxo-5-phenethyl octanoic acid.

Example 2

The procedure as mentioned in example 1 was repeated, however, this time using 0.93 g (3.2 mmol) (R₁S)- 5-Hydroxy-3-oxo-5-phenethyl-octanoic acid methyl ester. The resulting product was (R)-5-hydroxy-3-oxo-5-phenethyl-octanoic acid. The results of this experiment are shown in the table 2 below. Table 2. Stereoselective enzymatic hydrolysis of (R₁S)- 5-Hydroxy-3-oxo-5-phenethyl- octanoic acid methyl ester into (R)-5-hydroxy-3-oxo-5-phenethyl-octanoic acid.

Example 3: Determination of activity and enantioselectivity of different hvdrolvtic enzymes.

Approximately 20-50 mg of the enzyme was suspended in 2 ml 200 mM Kpi buffer pH 7.0. The enzymatic reactions were started by addition of 100 μl of (R₁S)- 5-Hydroxy-3- oxo-5-phenethyl-octanoic acid ethyl ester. The reaction mixtures were incubated for 24 hours at room temperature on an orbital shaker at 350 rpm.

Samples were taken and analyzed by means of chiral HPLC. In order to do so, about 0.5 ml. of reaction mixture was acidified with 50 μl_ 37% HCI. To this 0.5 ml. ethylacetate was added. The resulting mixture was stirred for 3 minutes after which the organic and aqueous layer were separated with an Eppendorf centrifuge. After separating the organic layer and evaporating the ethylacetate the enantiomeric excess of the product (ee_P) and the remaining substrate (ee_s) were analyzed by means of chiral HPLC, using a Diacel Chiralpak AD column (250 mm x 4.6 mm I. D.). The HPLC was operated at 22°C at a flow rate of 1.2 mL/min using a mobile phase consisting of n-heptane/ethanol/trifluoroacetic acid (93/7/0.1 ). Detection of components was performed using a UV-detector at a wavelength of 254 nm.

The HPLC data were used to calculate the intrinsic enantioselectivity of the enzyme, referred to as the enantiomeric ratio (E-ratio). The E-ratio can be considered as an enzyme constant and describes the ability of the enzyme to distinguish between the two enantiomers of a racemic mixture, in an enzyme catalyzed resolution reaction. An enzyme may have a preference for either the (R)-enantiomer or the (S)-enantiomer. The E-ratio, commonly used in screening procedures, was first introduced by Chen et. a/. (J. Am. Chem. Soc. (1982), 104: 7294). In case of a non-selective reaction an E- ratio of 1 is obtained. The results are presented in the table below.

Claims

Process for the preparation of an enantiomerically enriched compound of formula 1

wherein R^A stands for OH or for SH or wherein R^A stands for R¹⁵, wherein R¹⁵ stands for SR¹² or OR¹², wherein R¹² stands for an ester residue or wherein R^A stands for OR¹⁴ or SR¹⁴, wherein R¹⁴ stands for an ester residue which is not the same as R¹² and wherein R², R³ and R⁴ each independently stand for an optionally substituted (hetero)aryl, an optionally substituted alkyl, OR⁵, CO₂R⁶, C(O)R⁷, SR⁸, NR⁹R¹⁰, OC(O)R¹¹ wherein R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ each independently stand for H, an optionally substituted alkyl or for an optionally substituted (hetero)aryl, wherein R², R³ and R⁴ are not the same and wherein R² and R³, R² and R⁴ or R³ and R⁴ may form a (hetero)cycloalkyl together with the carbon atom to which they are attached, provided that said (hetero)cycloalkyl does not have a plane of symmetry and wherein R²³ stands for

wherein R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ each independently stand for O, S or NR²², wherein R²² stands for an alkyl, aryl, aralkyl or alkaryl and wherein I, m, n, o, p, q, r, s, t, u, v, w, x and y each independently stand for 0 or 1 and wherein l + m + n + o + p + q + r + s + t + u + v + w + x + y = z, wherein z stands for 3, 4, 5, 6, or 7, comprising the steps of reacting a stereoselective hydrolytic enzyme with a mixture of enantiomers of a compound of formula 1A,

wherein R²³ is as defined above, wherein R¹ stands for OH, SH, SR¹² or OR¹², wherein R¹² and R², R³ and R⁴ are as defined above in the presence of water, H₂S, an alcohol of formula HOR¹⁴ or a thiol of formula HSR¹⁴, wherein R¹⁴ stands for an ester residue but is not the same as R¹² acting as a nucleophile and either collecting the remaining enantiomerically enriched compound of formula 1A

wherein R²³, R¹, R², R³ and R⁴ are as defined above, or collecting

wherein R²³, R², R³, R⁴ and R¹⁴ are as defined above, - in case the nucleophile is the thiol, wherein R¹⁴ stands for an ester residue but is not the same as R¹- the resulting enantiomerically enriched compound of formula 4

(4) wherein R²³, R², R³ and R⁴ are as defined above,

wherein R²³, R², R³ and R⁴ are as defined above,

, wherein R²³, R², R³ and R⁴ are as defined above. 2. Process according to claim 1 , wherein the resulting enantiomerically enriched acid of formula 2 is the acid represented by formula 7

wherein R³ respectively R⁴ stands for a CτC₈-alkyl-, a C₆-Cio-aryl-Ci-C₄-alkyl- or a C₃-C₈-cycloalkyl-CrC₄alkyl- rest group and wherein R⁴ respectively R³ stands for a Ci-C₈-alkyl- rest group and wherein R³ and R⁴ are not the same and wherein R¹² stands for a C₁-C₄ alkyl or benzylrest, is prepared from the corresponding ester of formula 1 , represented by the ester of formula 5

wherein R³, R⁴ and R¹² are as defined above. Process according to claim 1 , wherein the remaining enantiomerically enriched ester of formula 1 A is the enantiomerically enriched ester of formula 5

wherein R³ respectively R⁴ stands for a Ci-Cs-alkyl-, a

or a C₃-C₈-cycloalkyl-C₁-C₄alkyl- rest group and wherein R⁴ respectively R³ stands for a Ci-C₈-alkyl- rest group and wherein R³ and R⁴ are not the same and wherein R¹² stands for a C₁-C₄ alkyl or benzylrest is collected. Process according to claim 3, wherein the remaining enantiomerically enriched ester of formula 5 is the (R)-ester. Process according to claim 3 or claim 4, wherein the remaining enantiomerically enriched ester of formula 5, wherein R³, R⁴ and R¹² are defined as in claim 3, is cyclisized in the presence of a base in a manner known per se to form the corresponding enantiomerically enriched compound of formula 6

wherein R and R are defined as in claim 3. 6. Process according to claim 5, wherein the enantiomerically enriched compound of formula 6 is further converted into a non-peptidic HIV-protease inhibitor, preferably tiprinavir in a manner known per se.