WO2007091110A1 - Enzymatic resolution process for the preparation of cycli-c beta-amino acid and ester enatiomers - Google Patents

Abstract

The invention relates to a process for the preparation of cyclic β-amino acid and ester enantiomers of general formula (I) wherein R is hydrogen or optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and heteroaryl, wherein each ring may optionally be condensed; A is alkylene or alkenylene, and one or more carbon atoms of each groups may optionally be replaced with one or more heteroatoms; Rl and R2 are hydrogen or halogen, =(O)n, optionally halogenated alkyl, alkylidene, alkoxy, optionally protected hydroxy, optionally protected amino, mono- or dialkylamino and optionally substituted phenyl, or Rl and R2 taken together with one or any two atoms of the ring to which they are attached, form a saturated, unsaturated or aromatic fused homo- or heterocyclic ring; n is 0, 1 or 2; * denotes a chiral carbon atom; and the salts thereof, comprising the steps of hydrolysing a mixture of cyclic β-amino ester enantiomers with a stereoselective hydrolytic enzyme, and separating the obtained acid enantiomer and the unreacted ester enantiomer. The invention provides an enzymatic resolution procedure for the preparation of enantiomers of both the cis- and trans-cyclic β-amino acids and esters useful for the synthesis ' of biologically active agents.

Description

RESOLUTION PROCESS

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to an enzymatic resolution process for the preparation of cyclic β-amino acid and ester enantiomers and the salts thereof.

Until recently, investigations on the cyclic β-amino acid stereoisomers [Fϋlop, F.; Chem. Rev. 101, 2181 (2001); Fϋlδp, F.; Studies in Natural Product Chemistry Vol. 22, Atta-ur- -Rahman, Ed., Elsevier Science Publishers, pp. 273 (2000)] have been mainly considered as studies of academic interest. A decade ago, however, (lR,2S)-2-arninocyclopentane-l-carboxylic acid (cispentacin) proved to be an antifungal antibiotic. Cispentacin was isolated independently by two Japanese groups from Bacillus cereus [Konishi, M.; Nishio, M.; Saitoh, K.; Miyaki, T.; Oki, T.; Kawaguchi, H. J.; Antibiotics 42, 1749 (1989); Oki, T.; Hirano, M.; Tomatsu, K.; Numata, K.; Kamei, H. J.; Antibiotics 42, 1756 (1989)] and Streptomyces setonii [Iwamoto, T.; Tsujii, E.; Ezaki, M.; Fujie, A.; Hashimoto, S.; Okuhara, M.; Kohsaka, M.; Imanaka, H.; Kawabata, K.; Inamoto, Y.; Sakane, K. J.; Antibiotics 43, 1 (1990); Kawabata, K.; Inamoto, Y.; Sakane, K.; Iwamoto, T.; Hashimoto, S. J.; Antibiotics 43, 513 (1990)] and its strong antifungal (anti- -Candida albicans agent) properties were demonstrated.

Cyclic β-amino acids may also be used as building blocks for the preparation of modified (unnatural) analogues of biologically active peptides. The activity and/or the stability of a naturally occurring pharmacologically active peptide can be increased by insertion of a cyclic β-amino acid in place of an α-amino acid of the peptide. Cyclic β-amino acids may exist in cis and trans diastereomeric forms, and - due to the two stereocentres present in the molecule - there are altogether four enantiomers for one structure.

It has recently been established that oligopeptide chains built up from trans-2- -aminocyclopentane-1-carboxylic acid or trαw-2-aminocyclohexane-l-carboxylic acid can fold into a stable helical structure [Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.; Huang, X.; Barchi, J. J.; Gellman, S. H.; Nature, 387, 381 (1997); Hetenyi, A.; Mandity, I. M.; Martinek, T. A.; Tόth, G. K.; Fϋlδp, F.; J. Am. Chem. Soc. 127, 547 (2005)], while the oligomers built up from the cis isomer, e.g. (lR,2S)-2-aminocyclopentane-l- -carboxylic acid, present a strand conformation [Martinek, T. A.; Tόth, G. K.; Vass, E.; Hollόsi, M.; Fϋlδp, F.; Angew. Chem. Int. Ed. 41, 1718 (2002)].

Cyclic β-amino acids have also been successfully used in the synthesis of drags [e.g.

Hiratate, Akira; Tatsuzuki, Makoto; Busujima, Tsuyoshi; Preparation of Heterocyclic

Compounds Containing 2-Substituted Cycloalkane-Carboxylic Acid Derivative Moiety as Cysteine Protease Inhibitors. PCT Int. Appl (2005), 120 pp. CODEN: PIXXD2

WO 2005000793 Al 20050106 CAN 142:114079; Kanno, Hideyuki; Yoshino, Toshiharu;

Nagata, Tsutomu; Mochizuki, Akiyoshi; Preparation of Diamides and Their Use as Factor

Xa Inhibitors and Blood Coagulation Inhibitors for Oral Treatment of Thrombotic Diseases,

Jpn. KoM Tokkyo Koho (2004), 227 pp. CODEN: JKXXAF JP 2004210716 A2 20040729 CAN 141 :156930; Marcin, Lawrence R.; Higgins, Mendi A.; Preparation of Arylsύlfonyl-

-Amino-Cycloalkyl-Carboxamides as Inhibitors of β-Amyloid Production. U.S. Pat. Appl.

PuU. (2005), 17 pp. CODEN: USXXCO US 2005113442 Al 20050526 CAN 142:481832]. Various examples show that in the cases of compounds containing a chiral carbon atom, the desired biological effect usually resides in one of the enantiomers of the racemic mixture. Thus, there is a growing demand for enantiomerically pure biologically active products prepared from cyclic β-amino acids, e.g. in drug research it is extremely important to synthesize all of the possible enantiomers of an active chiral compound in order to study the pharmacological character of each enantiomer separately.

2. Description of the Prior Art

The methods used to date for the synthesis of cyclic β-amino acid enantiomers [Fulδp, F.; Chem. Rev. 101, 2181 (2001)] can be divided into two main groups. One group involves the stereospecific methods, e.g. catalytic asymmetric hydrogenation, or reduction of chiral enamine derivatives by using chiral catalysts or other chiral auxiliary reagents to ensure the selectivity of the reaction in order to obtain the desired enantiomer only. In these cases various difficulties can arise such as, e.g. in asymmetric hydrogenations, the syntheses often involve the use of rather expensive chiral catalysts, or the removal of the remaining traces of catalyst in the products may be problematic. The reduction of chiral enamines usually demands rather expensive chiral auxiliary reagents with complicated structures, which should be split off from the product, and this final step can lead to a significant loss in molecular weight. Such methods may create a source of pollution for the environment, thus they are not recommended for large-scale syntheses.

The other group of methods for producing enantiopure cyclic β— amino acids involves processes which use suitable precursors, e.g. a β-lactam; or which start from a racemate or a mixture of enantiomers of different ratio and result in the desired enantiomeric product. Effectively some cyclic β-amino acid enantiomers can be prepared from cyclic β-lactams via enzyme-catalyzed hydrolysis of the lactam ring [Forrό, E.; Fϋlop, F.; Org. Lett, 5, 1209 (2003)]. A disadvantage of this method is that the syntheses of starting racemic β-lactams are often cumbersome and proceed with low yields; furthermore, resulting from the structure of β-lactams, exclusively the enantiomers of the cis cyclic β-amino acids can be prepared in the case of small- or medium-sized rings.

One of the methods leading to the resolution of racemates goes through the preparation of diastereomeric salts, while an other applies enzyme-catalyzed kinetic resolution. In the case of earlier methods involving the preparation of diastereomeric salts in most cases only enantiomerically enriched products are obtained.

In order to use the enzyme-catalyzed kinetic resolution method for the preparation of cyclic β-amino acid ester enantiomers the enzyme-catalyzed esterification of the corresponding acids has been studied [Kanerva, L. T.; Csomόs, P.; Sundholm, O.; Bernath, G.; Fϋlδp, F.; Tetrahedron: Asymmetry 7, 1705 (1996)]. The authors disclosed that protecting groups are necessary to block the appropriate functional groups; otherwise for example in the course of the enzyme-catalyzed esterification of the racemic amino acid with an achiral alcohol, the danger of the formation of an intramolecular amide should be faced. A further disadvantage of this method is observed when the carboxylic function is protected by an ester (e.g. ethyl ester) group and an enzyme-catalyzed trans-esterification of a protected esterified carboxylic function is performed. In this case namely, the unreacted ester and the selectively produced ester enantiomer are present in the reaction mixture at the same time, and the similarity of their chemical properties renders their separation highly difficult. Such procedures can not be used for industrial-scale syntheses. To eliminate these difficulties, the same authors analysed the enzyme-catalyzed N-acylations of some cyclic β-amino esters in order to obtain the corresponding products in enantiopure form. One disadvantage of this method is that the separation of the products is time consuming: column chromatography is necessary. Furthermore an even greater disadvantage blocking the industrial application of this method is that the hydrolysis of the N-acylated amide enantiomer into the β-amino acid raises difficulties; this step is not even described in the article. These hydrolysis reactions are usually not too efficient, the yields are low and epimerization of the obtained enantiomers can occur.

One of the enzyme-catalyzed resolution methods the enzyme-catalyzed ester hydrolysis is disclosed in patent EP 1054996 Bl wherein this method is applied for the hydrolysis of racemic heterocyclic β-lactam ester compounds with a single chiral centre leading to enantiopure cyclic α-amino acids possessing an amino function as part of the cycle therefore there is no need to use protecting groups. Katayama et al. utilize the ester hydrolysis reaction [Katayama, S.; Ae, N.; Nagata, R.; Tetrahedron Asymmetry, 9, 4295, (1998)] to prepare β-amino acid enantiomers with a quinoline skeleton; compounds also with a single chiral centre and containing a secondary >NH as amino function forming part of the ring. A similar method is disclosed in patent EP 1367129 Bl for the preparation of the S-enantiomer of the acyclic 3-amino-3-phenylpropionic acid, which contains also only one chiral centre. International application WO 2005/085462 Al describes a stereoselective enzymatic method, performed in buffer solution in order to synthesize enantiopure acyclic β₂-amino acids (e.g. 3- -amino-2-benzylpropionic acid and ester) with a primary amino group and one chiral centre.

Lloyd et al. [Lloyd, R.C.; Lloyd, M.C.; Smith, M.E.B.; Holt, K.E.; Swift, J.P.; Keene, P.A.; Taylor, S.J.C.; McCague, R.; Tetrahedron 60, 717-728 (2004)] prepared N-Boc-protected cyclic γ-amino acid enantiomers via enzyme-catalyzed ester hydrolysis. They emphasize that in the case of compounds with more complex chirality and more accentuated rigidity (from conformational point of view), the elaboration of new enzymatic techniques might be necessary, since it is not always possible to use methods that worked well for α-amino acids for the preparation of enantiopure cyclic amino acids.

SUMMARY OF THE INVENTION

In order to avoid the above-mentioned disadvantages which might occur in the course of preparation of cyclic β-amino acid and ester enantomers (such as the need for protecting groups, or restriction to the formation of the cis enantiomers only), and in order to develop an adequate enzymatic process that is well scalable and can be applied at an industrial scale, we have studied the enzyme-catalyzed hydrolyses of racemic or enantiomerically enriched cyclic cis and trans β-amino esters, and have surprisingly found that all of the corresponding cyclic β-amino acid and ester enantiomers can be prepared in said simple way with good enantiomeric purity.

DETAILED DESCRIPTION OF THEINVENTION

The present invention is directed to the process of the preparation of cyclic β-amino acid and ester enantiomers

(i) of general formula (I) wherein

R is hydrogen atom or selected from the group consisting of alkyl alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and heteroaryl groups; which groups may optionally be mono- or polysubstituted; and wherein each ring independently may optionally be condensed with one or more homo- or heterocyclic rings, and one or more carbon atoms of the saturated and unsaturated rings may optionally be replaced with one or more heteroatoms selected from nitrogen, oxygen and sulfur atom; A is C_2-15 alkylene or C_2-15 alkenylene group containing one or more double bonds, and one or more carbon atoms of each groups may optionally be replaced with one or more heteroatoms selected from nitrogen, oxygen and sulfur atom; Rl and R2 each are hydrogen atom or independently selected from the group consisting of halogen, a group of general formula =(O)_n, optionally halogenated C_1-7 alkyl, C_1-7 alkylidene, Cj_-7 alkoxy, optionally protected hydroxy, optionally protected amino, mono- or di(C_1-7 alkyl)amino and phenyl optionally substituted with one or more halogen or

C_1-7 alkyl groups, or Rl and R2 taken together with one or any two atoms of the ring to which they are attached, form a saturated, unsaturated or aromatic fused homo- or heterocyclic ring of 3 to 15 members, wherein the heterocyclic ring may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur; n is 0, 1 or 2;

* denotes a chiral carbon atom; and the salts thereof. The invention provides an enzymatic resolution procedure for the preparation of both cis- and trans-cyc\\c β-amino acids and esters in enantiopure forms with high chemical and optical purities, in good chemical yields. The process of the present invention involves the enantioselective enzymatic hydrolysis of cyclic β-amino esters

(H) of general formula (II) wherein

R is as defined above, but different from hydrogen, and

A, Rl, R2 and n are as defined above; comprising carrying out enantioselective enzymatic hydrolysis on racemic or enantiomerically enriched mixtures of the cyclic β-amino esters, wherein only one of the cyclic β-amino ester enantiomers is selectively hydrolysed by the enzyme to give one enantiomer of the corresponding cyclic β-amino acid of general formula (I) wherein

R is hydrogen and A, Rl, R2, n and * are as defined above; and separating the obtained cyclic β-amino acid enantiomer and the unreacted enantiomer of the cyclic β-amino ester of general formula (I) wherein R is as defined above, but different from hydrogen and A, Rl, R2, n and * are as defined above; and optionally hydrolysing the unreacted ester enantiomer to give the corresponding amino acid enantiomer by a suitable hydrolysis method; and/or transforming the obtained amino acid enantiomer into the corresponding ester enantiomer by a suitable esterification method; and/or preparing enantiopure salts thereof.

The procedure of the invention, under optimized conditions, affords both enantiomers

(i.e. the one of the hydrolysed amino acid and the other of the unreacted amino ester) in high enantiomeric excess (>99%). The conversion is practically complete (starting from a racemate, a maximum conversion of 50% may be reached), and the products are obtained in good chemical yields (>45%). Further advantages of this method are the facts that the amino group need not necessarily be protected, and the products can be easily separated. The obtained amino acid enantiomer precipitates from organic solvents, whereas it is soluble in water, so that it can be easily washed off from the surface of the enzyme: it can be extracted into water. The unreacted ester enantiomer can be isolated as an ester or, without any preliminary purification, it can be submitted to acidic hydrolysis to form the corresponding β- -amino acid enantiomer. This procedure can be successfully used for the industrial-scale resolution of both cis- and nww-substituted cyclic β-amino acids. From an industrial aspect, another important advantage of this method is the fact that the racemic starting cis- and trans- -β-amino acid esters can easily be prepared on a large scale [e.g. Fϋlδp, F.; Chem. Rev. 101, 2181 (2001) or by any other esterification method known in the art e.g. Houben-Weyl; Methoden der organischen Chemie Vol. VIII (1952) and Vol. 20 (2003) Thieme Verlag, Germany], and the enzymes can be re-used without a significant loss in activity.

DEFINITIONS

As utilized herein, in the terms "protected amino" and "protected hydroxy" protecting groups mean groups used in organic chemistry, generally for the protection of a hydroxy or amino group (e.g. McOmie; Protecting Groups in Organic Chemistry, Plenum Press, New York, 1973, Greene and Wutts; Protecting Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons, New York, 1991).

R substituent stands for a hydrogen atom or is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and heteroaryl groups, which groups may optionally be mono- or polysubstituted and wherein each ring independently may optionally be condensed with one or more homo- or heterocyclic rings and one or more carbon atoms of the saturated and unsaturated rings may optionally be replaced with one or more heteroatoms selected from nitrogen, oxygen and sulfur atom. As utilized herein, the substituent R means, for example, a hydrogen atom or an optionally substituted group selected from the group consisting of C_1-1O alkyl, C_2-Io alkenyl, (C_2-I₀ alkenyl)-(C_1-10 alkyl), C_2-10 alkynyl, (C_2-10 alkynyl)-(Ci_-10 alkyl), C_3-15 cycloalkyl, (C_3-15 cycloalkyl)-(C_1-10 alkyl), C_3-I₅ cycloalkenyl, (C_3-15 cycloalkenyl)-(C_1-10 alkyl), C_3-15 cycloalkynyl, (C_3-I5 cycloalkynyl)-(Ci.]₀ alkyl),

alkyl), heterocyclyl-(C_1-10 alkyl), heteroaryl-(C_1-io alkyl), aryl, heteroaryl, saturated and unsaturated heterocyclyl groups; wherein alkyl, alkenyl and alkynyl chains may be straight or ramifying carbon chains; one or more heteroatoms of heterocyclyl rings and/or heteroaryl groups are chosen from the group of nitrogen, oxygen and sulfur; aryl is of 6 to 14 members and preferably of 6 to 10 members; heteroaryl is of 5 to 14 members and preferably of 5 to 10 members; saturated and unsaturated heterocyclyl groups are of 3 to 16 members and preferably 5 to 7 members; cycloalkyl., cycloalkenyl, cycloalkynyl, aryl, heteroaryl, saturated and unsaturated heterocyclyl rings independently may optionally be condensed with one or more saturated or unsaturated homo- or heterocyclic rings of 5 to 7 members, with aryl rings of 6 to 10 members or with heteroaryl rings of 5 to 10 members.

Preferably R stands for a hydrogen atom, Cj.io alkyl, C_2-7 alkenyl, (C_2-7 alkenylHC^ alkyl), C_2-7 alkynyl, (C_2-7 alkynyl)-(C_1-7 alkyl), C_3-10 cycloalkyl, (C_3-10 cycloalkyl)-(C_1-7 alkyl), C_3-10 cycloalkenyl, (C_3-10 cycloalkenyl)-(d.₇ alkyl), C_3-10 cycloalkynyl, (C_3-10 cycloalkynyl)- -(Cj_-7 alkyl), aryl-(C_1-7 alkyl), heterocyclyl-(C₁.₇ alkyl), heteroaryl-(C_1-7 alkyl), aryl, heteroaryl, saturated and unsaturated heterocyclyl group; wherein each group independently may optionally be substituted with one or more groups selected from halogen, halogeno(C_1-7 alkyl), nitro, amino, mono- or di(C_1-7 alkyl)amino, (C_1-7 alkyl)thio and C_1-7 alkoxy; and wherein aryl is phenyl or naphthyl; saturated and unsaturated heterocyclyl groups are of 5 to 7 members and contain one or more heteroatoms chosen from nitrogen, oxygen and sulfur; heteroaryl is of 5 to 10 members and contains one or more heteroatoms chosen from nitrogen, oxygen and sulfur; and cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclyl and/or heteroaryl rings may optionally be condensed with one or more saturated or unsaturated homo- or hererocyclic rings (containing one or more heteroatoms chosen from nitrogen, oxygen and sulfur) of 5 to 7 members and/or with aryl rings of 6 to 10 members. For example R may stand for hydrogen, optionally substituted C_1-10 alkyl e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, methoxymethyl, methoxyethyl, trifluoromethyl, trifluoroethyl, trichloroethyl, dimethylamino-ethyl, methylthioethyl; C_2-7 alkenyl e.g. allyl, butenyl, iso-butenyl; C_2-7 alkynyl e.g. propargyl; (C_3-10 cycloalkyl)-(C_1-7 alkyl) e.g. cyclohexylmethyl, cyclohexylethyl, cyclohexylbutyl, adamantylmethyl; optionally substituted or condensed aril e.g. phenyl, naphthyl, fluorenyl, (mono- or poly)chlorophenyl, (mono- or polynitro)-phenyl; optionally condensed aryl-(Ci_-7 alkyl) e.g. benzyl, fluorenylmethyl; saturated and unsaturated heterocyclyl and/or heteroaryl, heterocyclyl-(Ci.₇ alkyl), heteroaryl - -(C_1-7 alkyl), wherein heterocyclyl or heteroaryl are for example pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridinyl, piperidinyl, piperazinyl, pyrimidinyl, pyrazinyl, thiophenyl, furanyl, thiazolyl, oxazolyl, thiazinyl, morpholinyl, oxazinyl, indolyl, quinolinyl, tetrahydroquinolyl, pyranyl, dioxolanyl, benzothiophenyl, azepinyl, oxepinyl, thiepinyl and the like. More preferably R stands for a hydrogen atom, C_1-7 alkyl, e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, penthyl, 2-methylpenthyl, hexyl, heptyl; most preferably for a hydrogen atom, methyl or ethyl.

"A" stands for C_2-15 alkylene or C_2-15 alkenylene group containing one or more double bonds, and one or more carbon atoms of each groups may optionally be replaced with one or more heteroatoms selected from nitrogen, oxygen and sulfur atom. Preferably A is C_2-10 alkylene e.g. ethanediyl, propanediyl, butanediyl, pentanediyl or hexanediyl; or C_3-10 alkenylene containing one double bond e.g. ethenediyl, propenediyl, butene-1-diyl or butene- -2-diyl group.

Rl and R2 each stand for hydrogen atom or are independently selected from the group consisting of halogen, a group of general formula =(O)_a, optionally halogenated C_1-7 alkyl,

C_1-7 alkylidene, C_1-7 alkoxy, optionally protected hydroxy, optionally protected amino, mono- or di(C_1-7 alkyl)amino and phenyl optionally substituted with one or more halogen or C_1-7 alkyl groups, or Rl and R2 taken together with one or any two atoms of the ring to which they are attached, form a saturated, unsaturated or aromatic fused homo- or heterocyclic ring of 3 to 15 members, wherein the heterocyclic ring may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur. Preferably Rl and R2 stand for a hydrogen atom, C_1-7 alkyl,

C_1-7 alkylidene e.g. a methylidene; or a phenyl group; or preferably Rl and R2 taken together with any two atoms of the ring to which they are attached, form a saturated, unsaturated fused homo- or heterocyclic ring of 3 to 15 members, wherein the heterocyclic ring may contain oxygen as heteroatom e.g. norbornane, norbornene, oxanorbornane, oxanorbornene ring.

As utilized herein, the symbol n is 0, 1 or 2, preferable n is 0 or 1.

As utilized herein, the symbol * denotes a chiral carbon atom with R or S absolute configuration.

As utilized herein the term "enantiomerically enriched" means a mixture of enantiomers wherein the ratio of enantiomers [(R)/(S)] differs from 1.

Acidic and/or basic salts can be prepared from the enantiomers formed via the above enzymatic procedure applying any method known in the art. Preferred salts are the salts formed with inorganic acids.

The enzymes suitable for use in the process of invention belong to the family of hydrolytic enzymes, e.g. esterases, lipases, proteases, peptidases or acylases; preferred enzymes are esterases or Upases. The enzymes can be commercially available products e.g. as described in patent application WO 2005/085462 Al (the content of pages 4-6 thereof is considered to be built into this specification as reference) or can be supplied individually by specific firms, furthermore different genetically modified forms thereof can also be used. The enzyme may be in crude form or a purified extract or may be immobilized by different techniques.

In a preferred embodiment of the invention, suitable enzymes include lipases, especially CAL-B {Candida antarctica lipase B) preparations prepared by different immobilization techniques; more preferable enzymes are Lipolase, Novozym 435 and Chirazyme L-2.

The enzymatic procedure may be carried out in an organic solvent, in a mixture of organic solvents (including multiphase systems), in a mixture of aqueous and organic solvents, or in mono- or multiphase systems thereof, as well as in ionic liquids [e.g. l-butyl-3- -methylimidazolium hexafluorophosphate and the like with reference to Park, S.; Kazlauskas, R. J., Curr. Op. Biotechnol. 14, 432 (2003) built into this specification] or in compressed gases [e.g. propane, ethane, CO₂ and the like with reference to Almeida M.C. et al., Enz. Microb. Tech. 22, 494 (1998) built into this specification]. The organic solvent may be an apolar or a polar solvent or the mixture or a multiphase system thereof, hi a preferred embodiment of the invention halogenated hydrocarbon solvents, e.g. dichloromethane, dichloroethane, chlorobenzene and the like; ketone-type solvents e.g. acetone, 2-butanone, acetophenone and the like; alcohol-type solvents e.g. ethanol, propanol, isopropanol, butanol, pentanol, octanol and the like; hydrocarbons e.g. toluene, hexane, heptane and the like; ether- -type solvents e.g. diethyl ether, tetrahydrofuran, diisopropyl ether, tert-butyl-methyl ether, 1,2-dimethoxyethane and the like; or other polar solvents such as nitrile-type solvents e.g. acetonitrile and the like; amide-type solvents e.g. N,N-dimethylformamide, N,N-dimethyl- acetamide and the like; amine-type solvents e.g. N,N-diiso-propylethylamine and the like can be used as reaction medium.

As utilized herein the term "aqueous solvent" means water, an aqueous buffer medium, aqueous solutions of inorganic and/or organic cations and anions.

The temperature of the process of invention may be varied between wide limits, starting from the temperature where the enzyme activity begins to work and terminating at the temperature of the denaturation, but in all cases the optimum temperature for a given enzyme is determined by its optimum activity. That temperature or interval of temperatures is considered as optimum temperature where the enzymatic process exhibits maximum enantioselectivity and the reaction time needed to reach 50% conversion is the shortest. Preferable temperatures of the process of invention for CAL-B enzyme lie in the range from 60 to 70 ⁰C. The generalization and efficacy of the method of invention will be elucidated by way of the following examples without, however, being limited thereto.

Instruments and methods used in the analytical determination of the compounds synthesized:

¹H NMR: Bruker (400 MHz); Gas chromatograph: Varian 3900, Chrompack CP 9002; Optical rotations: Perkin-Elmer 341 polarimeter; Melting points (M.p.): Kofler apparatus.

Determination of enantiomeric excess (ee) for a mixture of (+) and (-) enantiomers [with composition given as the mol or mass fractions F(+) and F(-) (where F(+) + F(-) = 1)]: ee = | F(+) - F(-) |

(a) In order to determine the progress of the enzymatic reactions, the free NH₂ group of the racemic starting methyl, ethyl or isopropyl cis- and /rαm-2-aminocycloalkane- and cycloalkene-l-carboxylate was acylated [acetic anhydride, 4-dimethylamino-pyridine and pyridine (10:90 m/m)] into the corresponding racemic N-acylated derivatives (derivatization), and the enantiomers were then separated by using a gas chromatograph equipped with a chiral column (CP-Chirasil-Dex CB (1), L-VaI (2)] [A: 120 ⁰C for 5 min → 190 °C (rate of temperature rise 20 ⁰C min^"1, 140 kPa), B: 120 °C for 2 min → 190 °C (rate of temperature rise 10 ⁰C min^"1, 140 kPa), C: 120 °C for 15 min → 190 ⁰C (rate of temperature rise 20 °C min^"1, 140 kPa), D: 120 ⁰C for 2 min → 190 °C (rate of temperature rise 20 °C min^"1, 140 kPa)].

(b) The ee values for the β-amino acid enantiomers produced were determined with a gas chromatograph equipped with a chiral column (CP-Chirasil-Dex CB (1), L-VaI (2)] after double derivatization [in the first step, the COOH was esterifϊed with diazomethane; then, in the second step, the free NH₂ group was acylated with acetic anhydride in the presence of 4- -dimethylaminopyridine and pyridine (10:90 m/m)]. In the case of the /rα«-f-2-(aminocyclohex- -4-ene)-l-carboxylic acid enantiomers, the double derivatization was performed for the corresponding saturated compound (after a catalytic reduction), as described [(b)]. The retention times are given in Table A. Table A Retention times for the enantiomers

The enantioselectivity (E), which shows how many times faster one enantiomer is transformed into the product than its antipode, is calculated as a function of the enantiomeric excesses for the substrate (ees) and the product (eep):

(E)-{ln[(l-ee_s)/(l+ee_s/eep)]}/{ln[(l+ee_s)/(l+ee_s/eep)]} The absolute configurations were proved by comparing the [α] values with the literature data.

EXAMPLES Example 1 Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers and the corresponding salts, respectively

A) Lipolase (450 mg, 30 mg mL^"1; lipase B from Candida antarctica, Sigma- Aldrich) and water (9.0 μL, 0.50 mmol) are added to racemic ethyl cis-2- -aminocyclohexane-1-carboxylate (172 mg, 1.00 mmol) in diisopropyl ether (15 mL), and the mixture is shaken in an incubator shaker (Innova 4080) at 65 °C for 31 h. The reaction is stopped by filtering off the enzyme at 49% conversion (ee_s = 94%; ee_P = 97%; E = 234). The solvent is evaporated off, leaving the unreacted ethyl (lR,2S)-2-aminocyclohexane-l- -carboxylate as a yellowish oil, which is hydro lysed through reflux (4 h) with 18% aqueous HCl solution (w/w) into (lR,2S)-2-aminocyclohexane-l-carboxylic acid hydrochloride {83 mg, 46%; [OC]D = -8.4 (c = 0.5; H₂O); M.p. 229-231 °C (recrystallized from EtOH and Et₂O); ee = 99%}.

Or, the above-mentioned ethyl (lR,2S)-2-aminocyclohexane-l-carboxylate (50 mg) is transformed through reflux (1 h) with 24% aqueous HBr solution (w/w) into (lR,2S)-2-

-aminocyclohexane-1-carboxylic acid hydrobromide {61 mg, 93%; [OC]D = -6 (c = 0.25; H₂O); M.p. 223-225 °C (recrystallized from EtOH and Et₂O); ee = 99%} .

The filtered off enzyme is washed with distilled water (3x15 mL), and the water is evaporated off, yielding crystalline (lS,2R)-2-aminocyclohexane-l-carboxylic acid {62 mg, 43%; [α]D = +21 (c = 0.28; H₂O); M.p. 264-266 ⁰C (recrystallized from H₂O and (CH₃)₂CO); ee = 98%}. When (lS,2R)-2-aminocyclohexane-l-carboxylic acid (50 mg) is treated with 22% HCl/EtOH (5 niL), (lS,2R)-2-aminocyclohexane-l-carboxylic acid hydrochloride is obtained {55 mg,

88%; [α]D = +8.3 (c = 0.325 in H₂O); M.p. 230-233 ⁰C (recrystallized from EtOH and Et₂O)₅ ee = 99%}. ¹H NMR (400 MHz, D₂O) data for (1S.2R)- 2-aminocyclohexane-l-carboxylic acid: 1.46-2.02 (8H, m, 4xCH₂), 2.63-2.67 (IH, m, H-I), 3.45-3.49 (IH, m, H-2). ¹H NMR (400 MHz, D₂O) data for (1S.2R)- and (lR,2S)-2-aminocyclohexane-l- -carboxylic acid hydrochlorides are identical with the literature data [Forrό, E.; Fϋlop, F.; Org. Lett., 5, 1209 (2003)]. B) Preparative-scale synthesis of czs-2-aminocyclohexane-l-carboxylic acid and the corresponding salt enantiomers, respectively

Following the procedure described above [A)], the reaction of racemic ethyl cis-2-

-aminocyclohexane-1-carboxylate (6 g, 35.034 mmol) and water (315 μL, 17.514 mmol) in diisopropyl ether (180 mL) in the presence of Lipolase (5.4 g) at 65 ⁰C leads to 49% conversion in 69 h (ees = 93%; eep = 97%; E = 225). The reaction mixture is worked up according to the above-mentioned procedure [A)].

25

(lR,2S)-2-Aminocyclohexane-l-carboxylic acid hydrochloride {2.83 g, 45%; [α]o = -8.6 (c = 0.35; H₂O); M.p. 230-232 °C (recrystallized from EtOH and Et₂O); ee = 99%}.

(lS,2R)-2-Aminocyclohexane-l-carboxylic acid {2.356 g, 47%; [<X]D = +22 (c = 0.25 in H₂O); M.p. 261 -262 ⁰C (recrystallized from H₂O and (CH₃)₂CO); ee = 97%} .

(lS,2R)-2-Aminocyclohexane-l-carboxylic acid hydrochloride {[OC]D = +8.4 (c = 0.3 in H₂O); M.p. 230-234 °C (recrystallized from EtOH and Et₂O); ee = 99%}.

Example 2 Preparation of m-2-aminocyclohexane-l-carboxylic acid and ester enantiomers from different esters

A) The reaction of racemic methyl cw-2-aminocyclohexane-l-carboxylate

(7.8 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) in the presence of Lipolase (30 mg) at 65 °C is performed according to the procedure described in Example 1. The progress of the reaction is followed by taking samples from the reaction mixture at intervals and analysing them, after double derivatization [(see Qj)], by gas chromatography. The reaction reaches 50% conversion in 35 h (ees = 94%; eep = 94%; E = I 15).

B) The reaction of racemic isopropyl α.y-2-aminocyclohexane-l-carboxylate (9.2 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) in the presence of Lipolase (30 mg) at 65 ⁰C is performed according to the procedure described in Example 1. The progress of the reaction is followed by taking samples from the reaction mixture at intervals and analysing them, after double derivatization [(see (b)] by gas chromatography. The reaction reaches 43% conversion in 36 h (ee_s = 73%; ee_P = 97%; E = 144).

Examples 3-6

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers using different enzymes

Following the procedure described in Example 1, the reaction of racemic ethyl cis-2- -aminocyclohexane-1-carboxylate (8.6 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) at 65 °C is performed in the presence of different enzymes [including, for example, Chirazyme L-2 (lipase B from Candida antarctica; Roche), Novozym 435 (lipase B from Candida antarctica; Novo Nordisk), Chirazyme L-5 (lipase A from Candida antarctica; Novo Nordisk) or PPL (porcine pancreatic lipase), Table I].

Table 1 Preparation of cw-2-aminocyclohexane- 1 -carboxylic acid and ester enantiomers using different enzymes

Temperature

Example Lipase (30 mg (⁰C) Reaction Conversion ee_s ee_p E mL^"1) time (h) (%) (%)

3 Chirazyme L-2 65 26 48 89 96 147

4 Novozym 435 65 26 47 87 97 187

5 Chirazyme L-5^a 65 48 15 10 58 4

6 PPL 45 48 6 6 99 211 ^a20% (w/w) of lipase adsorbed on Celite in the presence of sucrose.

Examples 7-13 Preparation of c/s-2-aminocyclohexane-l-carboxylic acid and ester enantiomers in different solvents

Following the procedure described in Example 1, the reaction of racemic ethyl cis-2- -aminocyclohexane-1-carboxylate (8.6 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in the presence of Lipolase (30 mg) at 65 ⁰C is performed in different solvents (1 mL). The characteristics of the reactions after 28 h are presented in Table 2.

Table 2 Preparation of c/s-2-aminocyclohexane-l-carboxylic acid and ester enantiomers in different solvents Example Solvent (1 mL) Conversion ee_s ee o/

7 1,4-dioxane 19 23 99 249

8 (CH₃)₂CO 6 6 99 211

9 THF 12 14 99 228

10 Et₂O 45 80 98 245

11 tert-BuOMe 49 92 96 161

12 toluene 29 40 99 295

13 n-hexane 38 61 98 185

Examples 14-18

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers at different temperatures

Following the procedure described in Example 1, the reaction of racemic ethyl cis-2- -aminocyclohexane-l-carboxylate (8.6 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in the presence of Lipolase (30 mg) in diisopropyl ether (1 mL) is performed at different temperatures (Table 3). Table 3

Preparation of cz.s-2-aminocyclohexane-l-carboxylic acid and ester enantiomers at different temperatures

Example Temperature Reaction Conversion ee_s (%) ee_p (%) E

(⁰C) time (%)

(h)/(day)

14 25 20/5 11/47 12/87 99/97 223/187

15 45 20 21 26 99 256

16 60 19 36 55 99 346

17 70 19 36 55 98 172

18 80 19 29 40 98 146

Example 19

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers without the addition of water to the reaction mixture

Following the procedure described in Example 1, the reaction of racemic ethyl cis-2- -aminocyclohexane-1-carboxylate (8.6 mg, 0.05 mmol) in the presence of Lipolase (30 mg) in diisopropyl ether (1 mL) at 65 °C is performed without water added to the reaction mixture. The reaction reaches 43% conversion after 29 h (ees ~ 75%; eep = 98%; E = 224).

Examples 20-24

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers using different enzyme quantities

Following the procedure described in Example 1 , the reaction of racemic ethyl cis-2- -aminocyclohexane-1-carboxylate (8.6 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) at 65 °C is performed with different quantities of Lipolase. The results are presented in Table 4. Table 4

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers using different Lipolase quantities

Example Lipolase (mg ml/¹) Reaction Conversion ee_s ee_p E time (h) (%) (%) (%)

20 10 25 41 70 99 418

21 20 25 45 79 98 239

22 40 25 48 91 97 209

23 50 22 50 95 96 183

24 75 22 51 99 94 170

Examples 25-29

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers using different co-solvents

Following the procedure described in Example 1, the reaction of racemic ethyl cis-2- aminocyclohexane-1-carboxylate (8.6 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) in the presence of Lipolase (30 mg) at 65 ⁰C is performed in the presence of different co-solvents. The characteristics of the reactions after 27 h are presented in Table 5.

Table 5

Preparation of m-2-arninocyclohexane-l-carboxyric acid and ester enantiomers using different co-solvents

Example Co-solvent Conversion ee_s (%) ee_p (%) E

(0.025 mmol) (%)

25 triethylamine 47 86 98 276

26 ethanol 33 48 98 159

27 2-octanol 38 59 98 180

28 tert-amyl-alcohol 34 50 96 80

29 N,N-diisopropyl- 35 53 98 168

-ethylamine Examples 30-32

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers using regenerated enzyme

Following the procedure described in Example 1, the hydrolysis of racemic ethyl cis- -2-aminocyclohexane-l-carboxylate (8.6 mg, 0.05 mmol) is performed with water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) in the presence of regenerated Lipolase (30 mg Ix- -used, 2x-used and 3x-used, respectively) at 65 °C. The characteristics of the reactions after 24 h are presented in Table 6. After a preparative-scale hydrolysis, the enzyme is washed with water and left to dry at room temperature for a period of 3 days.

Table 6

Preparation of cw-2-aminocyclohexane-l-carboxylic acid and ester enantiomers with regenerated enzyme

Example Lipolase (30 mg mL^" ) Conversion (%) ee_s (%) ee_p (%) E

30 used Ix 43 74 98 244

31 used 2x 38 59 97 120

32 used 3x 32 46 97 103

Example 33 Preparation of m-2-aminocyclohexane-l-carboxylic acid and ester enantiomers with addition of the enzyme in portions

Following the procedure described in Example 1, the hydrolysis of racemic ethyl cis-

-2-aminocyclohexane-l-carboxylate (1.0 g, 5.839 mmol) is performed in diisopropyl ether (60 mL). The Lipolase (1.8 g) is added to the reaction mixture in 3 portions (0.6 g to start the reaction, 0.6 g after 24 h, and 0.6 g after 48 h). The reaction reaches 49% conversion after 58 h (ee_s = 94%; ee_P = 97%; E = 234).

Example 34

Preparation of cw-2-aminocyclopentane-l-carboxylic acid and ester enantiomers and the corresponding salts, respectively

Following the procedure described in Example 1, the hydrolysis of racemic ethyl cis- 2-aminocyclopentane-l-carboxylate (157 mg, 1.00 mmol) is performed in diisopropyl ether (15 mL) using Lipolase (450 mg) as catalyst and water (9.0 μL, 0.50 mmol) as nucleophile. The reaction mixture is worked up after 87 h, at 48% conversion (ees = 85%; eep = 93%; E = 74): (lR,2S)-2-Aminocyclopentane-l-carboxylic acid hydrochloride {69.4 mg, 42%; [OC]D = -5 (c = 0.21; H₂O); M.p. 164-166 °C (recrystallized from EtOH and Et₂O); ee = 94%}.

The resulting (lR,2S)-2-aminocyclopentane-l-carboxylic acid hydrochloride is converted by ion-exchange chromatography (Varion KSL) to (lR,2S)-2-aminocyclopentane-

-1-carboxylic acid {[<X]D = -9 (c = 0.22; H₂O); M.p. 207-210 °C (recrystallized from H₂O and (CHs)₂CO); ee = 98%}.

(lS,2R)-2-Aminocyclopentane-l-carboxylic acid {54.1 mg, 42%; [α]D⁵ = +8 (c = 0.225; H₂O); Mp. 218-229 ⁰C (recrystallized from H₂O and (CH₃)₂CO); ee = 96%}.

(lS,2R)-2-Aminocyclopentane-l-carboxylic acid (100 mg, 0.63 mmol) with SOCl₂ (0.09 mL, 1.22 mmol) in EtOH affords the corresponding ethyl (lS,2R)-2-

25 -aminocyclopentane-1-carboxylate hydrochloride {115 mg, 76%; [OC]D = +10 (c = 0.45; EtOH); M.p. 66-68 °C (recrystallized from EtOH and Et₂O); ee = 98%}.

(lS,2R)-2-Aminocyclopentane-l-carboxylic acid hydrochloride {[cφ = +5 (c = 0.22 in H₂O); M.p. 162-166 ⁰C (recrystallized from EtOH and Et₂O); ee = 96%}. ¹H NMR (400 MHz, D₂O) data for (lS,2R)-2-aminocyclopentane-l-carboxylic acid: 1.70- -2.12 (6H, m, 3xCH₂), 2.84-2.89 (IH, m, H-I), 3.72-3.73 (IH, m, H-2).

¹H NMR (400 MHz, D₂O) data for (1S,2R> and (lR,2S)-2-aminocyclopentane-l- -carboxylic acid hydrochlorides are identical with the literature data [Forrό, E.; Fϋlόp, F.; Org. Lett., 5, 1209 (2003)].

Example 35

Preparation of frγm.ϊ-2-aminocyclohexane-l-carboxylic acid and ester enantiomers

Following the procedure described in Example 1, the hydrolysis of racemic ethyl trans-2- -aminocyclohexane-1-carboxylate (1 g, 5.839 mmol) is performed with water (52.5 μL, 2.919 mmol) in diisopropyl ether (60 mL) in the presence of Lipolase (1.8 g, 30 mg mL^"1) at 65 ⁰C. The reaction mixture is worked up after 68 h, at 49% conversion (ees - 92%; eep = 97%; E = 217).

Hydrolysis of the unreacted ester enantiomer affords (lS,2S)-2-amino-cyclohexane-l- -carboxylic acid hydrochloride {482 mg, 46%; [α]D = +51 (c = 0.21 in H₂O); M.p. 198-201 ⁰C (recrystallized from EtOH and Et₂O); ee = 99%}.

Through the enzymatic hydrolysis (lR,2R)-2-aminocyclohexane-l -carboxylic acid

{404 mg, 48%; [<X]D ^' = -65 (c = 0.26; H₂O); M.p. 263-265 °C (recrystallized from H₂O and CH₃)₂CO); ee = 99%} is obtained.

(lR,2R)-2-Aminocyclohexane-l -carboxylic acid hydrochloride {[cφ = -51 (c = 0.21 in H₂O); M.p. 194-197 °C (recrystallized from EtOH and Et₂O); ee = 99%}. ¹H NMR (400 MHz, D₂O) data for (lR,2R)-2-aminocyclohexane-l-carboxylic acid: 1.29-2.11 (8H, m, 4xCH₂), 2.14-2.23 (IH, m, H-I), 3.20-3.27 (IH, m, H-2).

Example 36

Preparation of cw-2-aminocyclooctane-l -carboxylic acid and ester enantiomers

Following the procedure described in Example 1, the hydrolysis of racemic ethyl cis-

2-aminocyclooctane-l-carboxylate (0.5 g, 2.508 mmol) is performed in diisopropyl ether (30 niL) using Lipolase (0.9 g, 30 mg mL^"1) as catalyst and water (22.6 μL, 1.254 mmol) as nucleophile. The reaction mixture is worked up after 8 days, at 42% conversion (ees = 54%; eep = 74%; E = l l):

(lR,2S)-2-Aminocyclooctane-l-carboxylic acid hydrochloride {236 mg, 45%; [α]D = +6.8 (c = 0.39; H₂O); M.p. 204-207 ⁰C (recrystallized from EtOH and Et₂O); ee = 54%}. (lS,2R)-2-Aminocyclooctane-l-carboxylic acid {172 mg, 40%; [CC]D = -11 (c = 0.22; H₂O); M.p. 241-242 °C (recrystallized from H₂O and (CH₃)₂CO); ee = 77%}. ¹H NMR (400 MHz, D₂O) data for (lS,2R)-2-aminocyclooctane-l -carboxylic acid: 1.51-1.94 (12H, m, 6xCH₂), 2.78-2.81 (IH, m, H-I), 3.61-3.63 (IH, m, H-2).

Example 37

Preparation of cw-2-amino-3-cyclohexene-l -carboxylic acid and ester enantiomers

Following the procedure described in Example 1, the hydrolysis of racemic ethyl cis-2- -amino-3-cyclohexene-l-carboxylate (1 g, 5.909 mmol) is performed with water (53.2 μL, 2.954 mmol) in diisopropyl ether (60 mL) in the presence of Lipolase (1.8 g, 30 mg mL^"1) at 65 ⁰C. The reaction mixture is worked up after 66 h, at 49% conversion (ees = 93%; eep = 95%; E = 133): (lR,2S)-2-Amino-3-cyclohexene-l-carboxylic acid hydrochloride {472 mg, 45%; [α]D = +121 (c = 0.21 in H₂O); M.p. 204-210 ⁰C (recrystallized from EtOH and Et₂O); ee = 98%}.

(lS,2R)-2-Amino-3-cyclohexene-l-carboxylic acid {383 mg, 46%; [<X]D = -120 (c = 0.25; H₂O); M.p. 234-236 °C (recrystallized from H₂O and CH₃)₂CO); ee = 99%}.

25 (lS,2R)-2-Amino-3-cyclohexene-l-carboxylic acid hydrochloride {[CC]D = -120 (c = 0.24 in H₂O); M.p. 205-211 °C (recrystallized from EtOH and Et₂O); ee = 99%}. ¹H NMR (400 MHz, D₂O) data for (lS,2R)-2-amino-3-cyclohexene-l-carboxylic acid: 1.85- -2.18 (4H, m, 2xCH₂), 2.74-2.78 (IH, m, H-I), 3.97-3.99 (IH, m, H-2), 5.74- -6.15.(2H, m, CHCH). ¹H NMR (400 MHz, D₂O) data for (1S,2R)- and (lR,2S)-2-amino-3-cyclohexene-l- -carboxylic acid hydrochlorides are identical with the literature data [Forrό, E.; Fulδp, F.; Tetrahedron: Asymmetry, 15, 2875 (2004)].

Example 38 Preparation of c/^-2-amino-4-cyclohexene- 1 -carboxylic acid and ester enantiomers

Following the procedure described in Example 1, the hydrolysis of racemic ethyl cis-2- -amino-4-cyclohexene-l-carboxylate (1 g, 5.909 mmol) is performed with water (53.2 μL, 2.954 mmol) in diisopropyl ether (60 mL) in the presence of Lipolase (1.8 g, 30 mg mL^"1) at 65 ⁰C. The reaction mixture is worked up after 72 h, at 50% conversion (ees = 93%; eep = 94%; E = 110): (lR,2S)-2-Amino-4-cyclohexene-l -carboxylic acid hydrochloride {472 mg, 45%; [OC]D = -28 (c = 0.11 in H₂O); M.p. 205-210 °C (recrystallized from EtOH and Et₂O); ee = 98%}.

(lS,2R)-2-Amino-4-cyclohexene-l-carboxylic acid {383 mg, 46%; [<X]Ό = +34 (c = 0.26; H₂O); M.p. 234-239 °C (recrystallized from H₂O and CH₃)₂CO); ee = 98%}.

25

(lS,2R)-2-Amino-4-cyclohexene-l-carboxylic acid hydrochloride {[CC]D = +28 (c = 0.24 in H₂O); M.p. 205-209 ⁰C (recrystallized from EtOH and Et₂O); ee = 99%}.

¹H NMR (400 MHz, D₂O) data for (lS,2R)-2-amino-4-cyclohexene-l-carboxylic acid: 2.25-

-2.51 (4H, m, 2xCH₂), 2.75-2.77 (IH, m, H-I), 3.78-3.79 (IH, m, H-2), 5.64-

-5.84.(2H, m, CHCH).

¹H NMR (400 MHz, D₂O) data for (1S.2R)- and (lR,2S)-2-amino-4-cyclohexene-l- -carboxylic acid hydrochlorides are identical with the literature data [Forrό, E.; Fϋlδp, F.;

Tetrahedron: Asymmetry, 15, 2875 (2004)]. Example 39

Preparation of trα«s-2-amino-4-cyclohexene-l-carboxylic acid and ester enantiomers

Following the procedure described in Example 1, the hydrolysis of racemic ethyl trans-2- -amino-4-cyclohexene-l-carboxylate (1 g, 5.909 mmol) is performed with water (53.2 μL, 2.954 mmol) in diisopropyl ether (60 mL) in the presence of Lipolase (1.8 g, 30 mg mL^'1) at 65 ⁰C. The reaction mixture is worked up after 58 h, at 50% conversion (ees = 95%; eep = 96%; E = 183):

₂5

(lS,2S)-2-Amino-4-cyclohexene-l-carboxylic acid hydrochloride {461 mg, 44%; [α]D = +123 (c = 0.27 in H₂O); M.p. 182-184 ⁰C (recrystallized from EtOH and Et₂O); ee = 99%}. (lR,2R)-2-Amino-4-cyclohexene-l-carboxylic acid {375 mg, 45%; [«]D ^' = -152 (c = 0.29; H₂O); M.p. 268-270 ⁰C (recrystallized from H₂O and CH₃)₂CO); ee = 99%}.

(lR,2R)-2-Amino-4-cyclohexene-l-carboxylic acid hydrochloride {[CC]D = -121 (c = 0.25 in H₂O); M.p. 182-185 ⁰C (recrystallized from EtOH and Et₂O); ee = 99%}.

When ethyl-(lS,2S)-2-amino-4-cyclohexene-l-carboxylate (50 mg) is treated with 22% (w/w) HCl/EtOH (3 mL), ethyl-(lS,2S)-2-amino-4-cyclohexene-l-carboxylate hydrochloride was obtained {57.1 mg, 94%; [OC]D = +122 (c = 0.255 in EtOH); M.p. 134-135 °C (recrystallized from EtOH and Et₂O), ee = 99%}.

¹H NMR (400 MHz, D₂O) data for (lR,2R)-2-amino-4-cyclohexene-l-carboxylic acid: 1.17- -2.61 (5H, m, 2xCH₂ and H-I), 3.52-3.58 (IH, m, H-2), 5.64-5.8 l.(2H, m, CHCH). ¹H NMR (400 MHz, D₂O) δ (ppm) data for ethyl-(lS,2S)-2-arnino-4-cyclohexene-l- -carboxylate hydrochloride: 1.28 (3H, t, J = 7 Hz, CH₃), 2.19-2.58 (4H, m, 2xCH₂), 2.92-2.98 (IH, m, H-I), 3.75-3.81 (IH₅ m, H-2), 4.22-4.27 (2H, m, CH₂CH₃), 5.68-5.77 (2H, m, CHCH).

Claims

CLAIMSWhat is claimed is:

1. A process for the preparation of cyclic β-amino acid and ester enantiomers of general formula (I)

(i) wherein

R is hydrogen atom or selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cyclo-alkynyl, aryl and heteroaryl groups, which groups may optionally be mono- or polysubstituted and wherein each ring independently may optionally be condensed with one or more homo- or heterocyclic rings and one or more carbon atoms of the saturated and unsaturated rings may optionally be replaced with one or more heteroatoms selected from nitrogen, oxygen and sulfur atom; A is C_2-15 alkylene or C_2-15 alkenylene group containing one or more double bonds, and one or more carbon atoms of each groups may optionally be replaced with one or more heteroatoms selected from nitrogen, oxygen and sulfur atom;

Rl and R2 each are hydrogen atom or independently selected from the group consisting of halogen, a group of general formula =(O)_n, optionally halogenated C_1-7 alkyl, C_1-7 alkylidene, C_1-7 alkoxy, optionally protected hydroxy, optionally protected amino, mono- or di(Cμ₇ alkyl)amino and phenyl optionally substituted with one or more halogen or C_1-7 alkyl groups, or

Rl and R2 taken together with one or any two atoms of the ring to which they are attached, form a saturated, unsaturated or aromatic fused homo- or heterocyclic ring of 3 to 15 members, wherein the heterocyclic ring may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur; n is 0, 1 or 2; * denotes a chiral carbon atom; and the salts thereof, comprising the steps of hydrolysing a racemic or enantiomerically enriched mixture of cyclic β-amino esters of general formula (II)

(H) wherein R is as defined above, but different from hydrogen, and A, Rl, R2 and n are as defined above; with a stereoselective hydrolytic enzyme, wherein only one of the cyclic β-amino ester enantiomers is selectively hydrolysed by the enzyme to give one enantiomer of the corresponding cyclic β-amino acid of general formula (I) wherein R is hydrogen and A, Rl, R2, n and * are as defined above; and separating the obtained cyclic β-amino acid enantiomer and the unreacted enantiomer of the cyclic β-amino ester of general formula (I) wherein

R is as defined above, but different from hydrogen and A, Rl, R2, n and * are as defined above; and optionally hydrolysing the unreacted ester enantiomer to give the corresponding amino acid enantiomer; and/or transforming the obtained amino acid enantiomer into the corresponding ester enantiomer; and/or preparing enantiopure salts thereof.

2. The process according to claim 1 wherein the cyclic β-amino acid and ester enantiomers are of general formula (I) wherein R is a hydrogen atom, Cj_-10 alkyl, C_2-7 alkenyl, (C_2-7 alkenyl)-(C_1-7 alkyl), C_2-7 alkynyl, (C_2-7 alkynyl)-(C_1-7 alkyl), C_3-J0 cycloalkyl, (C_3-10 cycloalkyl)-(C_1-7 alkyl), C_3-10 cycloalkenyl, (C_3-10 cycloalkenyl)-(C[_-7 alkyl), C_3-10 cycloalkynyl, (C_3-I0 cycloalkynyl)- -(Ci_-7 alkyl), aryl-(Ci_₇ alkyl), heterocyclyl-(Ci_-7 alkyl), heteroaryl-(C_1-7 alkyl), aryl, heteroaryl, saturated and unsaturated heterocyclyl group; wherein each group independently may optionally be substituted with one or more groups selected from halogen, halogeno(Ci_-7 alkyl), nitro, amino, mono- or di(C_1-7 alkyl)amino, (C_1-V alkyl)thio and C_1-7 alkoxy; and wherein aryl is phenyl or naphthyl; saturated and unsaturated heterocyclyl groups are of 5 to 7 members and contain one or more heteroatoms chosen from nitrogen, oxygen and sulfur; heteroaryl is of 5 to 10 members and contains one or more heteroatoms chosen from nitrogen, oxygen and sulfur; and cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclyl and/or heteroaryl rings may optionally be condensed with one or more saturated or unsaturated homo- or hererocyclic rings (containing one or more heteroatoms chosen from nitrogen, oxygen and sulfur) of 5 to 7 members and/or with aryl rings of 6 to 10 members;

A is C_2-15 alkylene or C_2-15 alkenylene group containing one or more double bonds;

Rl and R2 each are hydrogen atom or independently selected from the group consisting of C_1-7 alkyl, C_1-7 alkylidene and phenyl, or

Rl and R2 taken together with any two atoms of the ring to which they are attached, form a saturated, unsaturated or aromatic fused homo- or heterocyclic ring of 3 to 15 members, wherein the heterocyclic ring may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur; n is 0, 1 or 2;

* denotes a chiral carbon atom; and the salts thereof; and the stereoselective hydrolytic enzyme is a lipase.

3. The process according to claim 1 or claim 2 wherein the cyclic β-amino acid and ester enantiomers are of general formula (I) wherein R is hydrogen or Cj_-7 alkyl;

A is C_2-10 alkylene or C_3-10 alkenylene group containing one double bond; Rl and R2 each are hydrogen, or

Rl and R2 taken together with any two atoms of the ring to which they are attached, form a saturated, unsaturated fused homo- or heterocyclic ring of 3 to 15 members, wherein the heterocyclic ring may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur; and the salts thereof.

4. The process according to any one of claims 1-3, wherein the cyclic β-amino acid and ester enantiomers and the salts thereof are

(lR,2S)-2-aminocyclohexane-l -carboxylic acid hydrochloride, ethyl (lR,2S)-2-aminocyclohexane-l -carboxylate, (lR,2S)-2-aminocyclohexane-l-carboxylic acid,

(lR,2S)-2-aminocyclohexane-l-carboxylic acid hydrobromide,

(1 S,2R)-2-aminocyclohexane-l -carboxylic acid,

(lS,2R)-2-aminocyclohexane-l-carboxylic acid hydrochloride, ethyl ( 1 S,2R)-2-aminocyclohexane- 1 -carboxylate, methyl (lR,2S)-2-aminocyclohexane-l -carboxylate, methyl (1 S,2R)-2-aminocyclohexane-l -carboxylate, isopropyl (lR,2S)-2-aminocyclohexane-l-carboxylate, isopropyl ( 1 S,2R)-2-aminocyclohexane- 1 -carboxylate,

(lR,2S)-2-aminocyclopentane-l -carboxylic acid hydrochloride, (1 R,2S)-2-aminocyclopentane- 1 -carboxylic acid,

(1 S,2R)-2-aminocyclopentane- 1 -carboxylic acid,

(lS,2R)-2-aminocyclopentane-l -carboxylic acid hydrochloride, ethyl (lR,2S)-2-aminocyclopentane- 1 -carboxylate, ethyl (IS ,2R)-2-aminocyclopentane- 1 -carboxylate, ethyl (1 S,2R)-2-aminocyclopentane-l -carboxylate hydrochloride,

(lS,2S)-2-aminocyclohexane-l -carboxylic acid hydrochloride,

( 1 R,2R)-2-aminocyclohexane- 1 -carboxylic acid,

(lR,2R)-2-aminocyclohexane-l -carboxylic acid hydrochloride, ethyl (1 S,2S)-2-aminocyclohexane-l -carboxylate, ethyl (lR,2R)-2-aminocyclohexane-l-carboxylate,

(lR,2S)-2-aminocyclooctane-l -carboxylic acid hydrochloride,

(lS,2R)-2-aminocyclooctane-l-carboxylic acid, ethyl (lR,2S)-2-aminocyclooctane- 1 -carboxylate, ethyl ( 1 S,2R)-2-aminocyclooctane- 1 -carboxylate, (lR,2S)-2-amino-3-cyclohexene-l-carboxylic acid hydrochloride,

( 1 R,2S)-2-amino-3 -cyclohexene- 1 -carboxylic acid,

(1 S,2R)-2-amino-3 -cyclohexene- 1 -carboxylic acid,

(1 S, 2R)-2-amino-3 -cyclohexene)- 1 -carboxylic acid hydrochloride, ethyl ( 1 R,2S)-2-amino-3 -cyclohexene- 1 -carboxylate, ethyl ( 1 S,2R)-2-amino-3 -cyclohexene- 1 -carboxylate,

(lR,2S)-2-amino-4-cyclohexene-l-carboxylic acid hydrochloride,

( 1 R,2S)-2-amino-4-cyclohexene- 1 -carboxylic acid, (1 S,2R)-2-amino-4-cyclohexene- 1 -carboxylic acid hydrochloride,

(lS,2R)-2-aniino-4-cyclohexene-l-carboxylic acid, ethyl (lR,2S)-2-amino-4-cyclohexene-l -carboxylate, ethyl (1 S,2R)-2-amino-4-cyclohexene-l -carboxylate,

(lS,2S)-2-amino-4-ciklohexene-l-carboxylic acid hydrochloride, (1 S,2S)-2-amino-4-cyclohexene- 1 -carboxylic acid,

(lR,2R)-2-amino-4-cyclohexene-l -carboxylic acid hydrochloride,

(lR,2R)-2-amino-4-cyclohexene-l -carboxylic acid, ethyl (lS,2S)-2-amino-4-cyclohexene-l-carboxylate hydrochloride, ethyl (lS,2S)-2-amino-4-cyclohexene-l -carboxylate and ethyl ( 1 R,2R)-2-amino-4-cyclohexene- 1 -carboxylate.

5. The process according to any one of claims 1-4, wherein the stereoselective hydrolytic enzyme is used in an organic solvent, a mixture of organic solvents, a mixture or multiphase system of an organic and aqueous solvent, an ionic liquid and a compressed gas.

6. The process according to claim 5 wherein the organic solvent is a hydrocarbon, a halogenated hydrocarbon, a ketone-, an alcohol-, an ether-, a nitrile-, an amide- or an amine-type solvent.

7. The process according to claim 5 or claim 6 wherein the organic solvent is hexane, toluene, acetone, dioxane, tetrahydrofuran, diethyl ether, diisopropyl ether or tert- -butylmethyl ether.

8. The process according to any one of claims 1-7, wherein the stereoselective hydrolytic enzyme is a CAL-B-type enzyme, used either in original or in regenerated form and at an optimum temperature ensuring optimum enzyme activity.

9. The process according to any one of claims 1-8, wherein the stereoselective hydrolytic enzyme is added in portions to the reaction mixture.