WO2009007759A1 - Resolution process - Google Patents

Resolution process Download PDF

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Publication number
WO2009007759A1
WO2009007759A1 PCT/HU2008/000076 HU2008000076W WO2009007759A1 WO 2009007759 A1 WO2009007759 A1 WO 2009007759A1 HU 2008000076 W HU2008000076 W HU 2008000076W WO 2009007759 A1 WO2009007759 A1 WO 2009007759A1
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optionally
enantiomer
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general formula
hydrogen atom
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PCT/HU2008/000076
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French (fr)
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Enikö FORRO
Ferenc FÜLÖP
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Szégédi Tudomanyegyetem
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • the invention relates to an enzymatic resolution process for the preparation of enantiopure cyclic cis ⁇ -amino acids and derivatives and the salts thereof.
  • One of the enantiomers of cyclic cis ⁇ -amino acids (lS,4R)-4-aminocyclopent-2- -ene-1-carboxylic acid is a key-intermediate used in one of the syntheses of purine and pyrimidine carbonucleosides with significant antiviral activity (e.g. abacavir, carbovir), since the (IS, 4R) absolute configuration of this intermediate is analogous with that of the two asymmetric centres in the cyclopentene ring of the side-chain — which is essential for the biological activity - of the natural nucleosides.
  • nucleosides can be obtained by known methods, for example by reduction [Daluge, S. M.; Martin, M.T.; Sickles, Barry R.; Livingston, D.A; Nucleosides, Nucleotides & Nucleic Acids 19, 297-327 (2000)] and subsequent coupling reaction [Crimmins, M.
  • the antipode ⁇ -amino acid enantiomer is also used as starting material for substances showing valuable biological activity [Levy, D.E.; Bao, M.; Cherbavaz, D.B.; Tomlinson, J.E.; Sedlock, D.M.; Homey, C.J.; Scarborough, R.M.; J. Med Chem. 46, 2177-2186 (2003)]. Further on the saturated analogue 3-amino-cyclopentane-carboxylic acid is a starting material applied in the search for biologically active compounds [Evans, C; McCague, R.; Roberts, S.M.; Sutherland, A.G.; J. Chem. Soc, Perkin Trans. 1, 656-657 (1991)].
  • N-acylation for example N-benzoylation
  • the resulted N-acyl derivative is transformed into a diastereomeric salt pair with (+)-cis-2-(benzyl-amino)-cyclohexane-l -methanol or (+)- ⁇ -methyl-benzylamine, then the diastereomeric salt pairs are separated and the (-) enantiomer of cis-4-(benzoyl-amino)- -cyclopent-2-ene-l-carboxylic acid is obtained by known steps.
  • This method leads to the pure enantiomers via a multi-step process starting from the racemic 2-azabicyclo[2.2.1]hept-5-ene- -3-one.
  • the enantiomer acids can be obtained after the non-enzymatic hydrolysis of the starting racemic 2- -azabicyclo[2.2.1]hept-5-ene-3-one via several steps (acylation, esterification, desacylation) and the resolution is carried out by selective cleavage of the ester groups linked to the cyclopentene ring with esterase or lipase enzymes.
  • EP 424064 Al describes a process for the resolution of the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one using the lactamase activity of ENZA-I (Rhodococcus equi NCIMB 41213) and ENZA-20 (Psedomonas solanacearum NCIMB 40249) strains, in which the (+) and (-) enantiomers of 2-azabicyclo[2.2.1]hept-5-ene-3- -one, as well as the (-) and (+) enantiomers of cis-4-aminocyclopent-2-ene-l-carboxylic acid, respectively - formed via the opening of the lactam ring - are obtained. Taylor et al.
  • the corresponding (1R,4S) enantiomer acid or esters thereof can be obtained from the unreacted (lS,4R)-azabicyclo[2.2.1]-hept-5-ene-3-one enantiomer separated after the enzymatic reaction using known methods, e.g. Forr ⁇ , E.; FUl ⁇ p, F.; Org. Lett. 5, 1209-1212 (2003) and Forr ⁇ , E; Arva, J.; F ⁇ l ⁇ p, F.; Tetrahedron: Asymmetry, 12, 643-649 (2001). In our further experiments the enzymatic reaction is extended to the saturated cyclopentane ring containing analogues and the protected derivatives as well.
  • lipase enzymes which are commercially available, stable, have high stereoselectivity and can be used on industrial scale. Furthermore lipase enzymes have not been used for the synthesis of enantiomers of cyclic cis ⁇ -amino acids starting from the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one or the derivatives thereof.
  • the process of our invention does not require the presence of an activating group, on the other hand instead of the (1S,4R) enantiomer the (1R,4S) enantiomer is hydrolyzed yielding the desired (lS,4R)-4- -aminocyclopent-2-ene-l-carboxylic acid enantiomer.
  • the invention is directed to a process for the preparation of enantiomers of cyclic cis ⁇ -amino acids and their derivatives of the general formula (I)
  • R 1 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;
  • R 2 is hydrogen atom or an amino protecting group; the dotted line in the ring is optionally an additional chemical bond; and * is a chiral carbon atom with defined configuration — and the salts thereof.
  • the process of the present invention involves the enantioselective enzymatic hydrolysis of lactams of general formula (II)
  • step al) or a2) wherein R 2 and the dotted line are as defined above and * is in step al) (1R,4S), while in step a2) (1S,4R) configuration — into the appropriate enantiomer of the general formula (I) — wherein R 1 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; R 2 and the dotted line are as defined above; and * is in step al) (1S,3R), while in step a2) (1R,4S) configuration —
  • the procedure of the invention affords both enantiomers (i.e. the one of the hydrolyzed ⁇ -amino acid and the other of the unreacted lactam) in high enantiomeric excess (>99%).
  • the conversion of the process is practically complete (starting from a racemic mixture, a maximum conversion of 50% for the individual enantiomers may be reached) and both products are obtained in good chemical yields (>48%). Further advantage of the process is that it is not necessary to use protecting or activating groups.
  • the non-protected enantiomer ⁇ -acid precipitates from organic solvents, whereas it is soluble in water, so it can be easily washed off and/or extracted from the surface of the enzyme with water, it can be extracted into water.
  • the non-hydrolyzed enantiomer of the lactam can easily be isolated from organic solvents.
  • the process of the invention can advantageously be used for the preparation of enantiopure cyclic cis ⁇ -amino acids on industrial scale.
  • the substituent R 1 means, for example, a hydrogen atom or an optionally substituted group selected from the group consisting of Ci-io alkyl, C 2- io alkenyl, (C 2- I 0 a!kenyl)-(Ci.io alkyl), C 2- I 0 alkynyl, (C 2- I 0 alkynylHd.io alkyl), C 3-15 cycloalkyl, (C 3- I 5 cycloalkyl)-(Ci.io alkyl), C 3- I 5 cycloalkenyl, (C 3- is cycloalkeny I)-(C MO alkyl), C 3- I 5 cycloalkynyl, (C 3 - I5 cycloalkynyl)-(Cj.io alkyl), aryl-(Ci-io alkyl), heterocyclyl- -(C 1-10 alkyl), heteroaryHd.io alkyl), aryl
  • R 1 stands for a hydrogen atom, C LIO alkyl, C 2-7 alkenyl, (C 2-7 alkenyl)- -(C 1-7 alkyl), C 2-7 alkynyl, (C 2-7 alkynyl)-(C,. 7 alkyl), C 3-10 cycloalkyl, (C 3-10 cycloalkyl)-(Ci -7 alkyl), C 3-10 cycloalkenyl, (C 3 .
  • 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
  • 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.
  • R may stand for hydrogen, optionally substituted Ci -I0 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- io cycloalkyl)-(Ci.
  • Ci -I0 alkyl e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, methoxymethyl, methoxyethyl, trifluoromethyl,
  • 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-(C 1-7 alkyl) e.g.
  • 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.
  • R 1 stands for a hydrogen atom, C 1-7 alkyl, e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, 2-methylpentyl, hexyl, heptyl; most preferably for a hydrogen atom, methyl or ethyl groups.
  • R substituent stands for hydrogen atom or an amino protecting group.
  • amino protecting group means groups which are commonly used for the protection of amino groups in organic chemistry, the introduction and cleavage thereof can be carried by known methods (e.g. McOmie; Protecting Groups in Organic Chemistry, Plenum Press, NY., 1973, Greene and Wutts; Protecting Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons, NY., 1991).
  • Preferred protecting groups are acyl or acyloxy groups.
  • enantiomerically enriched mixture stands for a mixture of enantiomers wherein the ratio of the enantiomers [(R/(S)] is different from one.
  • 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.
  • enantiomer acids obtained according to the process of the invention can be transformed by known methods into enantiomer esters [e.g. Csuk, R.; Dorr, P.; Tetrahedron: Asymmetry, 5, 269-276 (1994)].
  • lactams used in the process of the invention as starting materials are known compounds [Daluge, S.; Vince, R. J.; Org. Chem.; 43, 2311-20 (1978), Jagt, J. C; Van Leusen, A. M. J.; Org, Chem., 39, 564-566 (1974)].
  • the enzymes suitable for use in the process of invention are lipase enzymes, which belong to the family of hydrolytic enzymes. These enzymes are commercially available.
  • suitable enzymes include CAL-B (Candida Antarctica lipase B) preparations prepared by different immobilization techniques; more preferable enzymes are Lipolase, Novozym 435, Chyrazime L-2.
  • the process of the invention can 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 system thereof, as well as in ionic liquids [for example in 1-butyl- -3-methyl-imidazolium hexafluorophosphate and the like with reference to Park, S.; Kazlauskas, R. J.; Curr. Op. Biotechnol. 14, 432 (2003) considered to be incorporated into this specification] or in compressed gases [for example propane, ethane, carbon dioxide and the like with reference to M.C. Almeida et al., Enz. Microb. Tech.
  • the organic solvents may be an apolar or a polar solvent or the mixture or the multiphase system thereof.
  • halogenated hydrocarbon solvents such as dichloromethane, dichloroethane, chlorobenzene and the like; keton-type solvents, such as acetone, 2-butanone, acetophenone and the like; alcohol-type solvents, such as ethanol, propanol, isopropanol, butanol, pentanol, octanol and the like; hydrocarbons, such as toluene, hexane, heptane and the like; ether-type solvents, such as diethyl ether, tetrahydrofuran, diisopropyl ether, tert-butyl methyl ether, 1,2- -dimethoxyethane and the like; or other polar solvents, for example nitrile
  • aqueous solvent stands for water, an aqueous buffer medium, aqueous solutions containing inorganic and/or organic cations and anions.
  • the process of the invention can be carried out also in solid phase, without solvent addition, in the presence of an equivalent amount of water.
  • the temperature of the process of the 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 optimal 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 0 C.

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Abstract

The invention relates to a process for the preparation of enantiomers of cyclic cis γ-amino acids and their derivatives of formula (I) wherein R1 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 may optionally be condensed; R2 is hydrogen atom or an amino protecting group; the dotted line is optionally an additional chemical bond; * is a chiral carbon atom with defined configuration - and the salts thereof; comprising the steps of hydrolyzing a racemic or enantiomerically enriched mixture of an optionally protected lactam: 2-azabicyclo[2.2.2]heptane-3-one or the 5,6- -unsaturated derivative thereof with a lipase enzyme, wherein cyclic cis γ-amino acids with (1R,3S), or (1S,4R) absolute stereochemistry, respectively, can directly be obtained, and separating the obtained acid enantiomer and the unreacted lactam enantiomer; and optionally hydrolyzing the unreacted lactam enantiomer to give the corresponding amino acid enantiomers and derivatives thereof.

Description

RESOLUTION PROCESS
BACKGROUND OF THE INVENTION
1. Technical Field of the Invention
The invention relates to an enzymatic resolution process for the preparation of enantiopure cyclic cis γ-amino acids and derivatives and the salts thereof.
In the series of the cyclic cis γ-amino acids 4-aminocyclopent-2-ene-l-carboxylic acid, 3-aminocyclopentane-l-carboxylic acid and the enantiomers thereof are known compounds, and due to their GABA analogue structure they posses GABA receptor agonist, partial agonist and antagonist activity, in addition they can be used in the synthesis of peptides with definite non-natural secondary structures, furthermore they can be used for the design and synthesis of self-assembling peptide nanotubes [Allan, R. D.; Dickenson, H. W.; Fong, J.; Structure- -activity studies on the activity of a series of cyclopentane GABA analogues on GABAA receptors and GABA uptake; Eur. J. Pharmacol, 122, 339-348 (1986); Ordonez, M.; Cativiela, C; Stereoselective synthesis of γ-amino acids; Tetrahedron: Asymmetry 18, 3-99 (2007)].
(It is to be noted here that in this specification the following numbering and nomenclature are applied for the compounds: cis-4-aminocyclopent-2-ene-l-carboxylic acid and their (1S,4R) (-), (1R,4S) (+) enantiomers, as well as cis-3-amino-cyclopentane-l- -carboxylic acid, 2-azabicyclo[2.2.1]hept-5-en-3-one and 2-aza-bicyclo[2.2.1]heptane-3-one.)
One of the enantiomers of cyclic cis γ-amino acids (lS,4R)-4-aminocyclopent-2- -ene-1-carboxylic acid is a key-intermediate used in one of the syntheses of purine and pyrimidine carbonucleosides with significant antiviral activity (e.g. abacavir, carbovir), since the (IS, 4R) absolute configuration of this intermediate is analogous with that of the two asymmetric centres in the cyclopentene ring of the side-chain — which is essential for the biological activity - of the natural nucleosides. Consequently starting from said acid enantiopure nucleosides can be obtained by known methods, for example by reduction [Daluge, S. M.; Martin, M.T.; Sickles, Barry R.; Livingston, D.A; Nucleosides, Nucleotides & Nucleic Acids 19, 297-327 (2000)] and subsequent coupling reaction [Crimmins, M. T.; New developments in the enantioselective synthesis of cyclopentyl carbocyclic nucleosides; Tetrahedron 54, 9229-9272 (1998); Ferrero, M.; Gotor, V.; Biocatalytic selective modifications of conventional nucleosides, carbocyclic nucleosides, and C-nucleosides; Chem. Rev. 100, 4319-4347 (2000)].
On the other hand the antipode γ-amino acid enantiomer is also used as starting material for substances showing valuable biological activity [Levy, D.E.; Bao, M.; Cherbavaz, D.B.; Tomlinson, J.E.; Sedlock, D.M.; Homey, C.J.; Scarborough, R.M.; J. Med Chem. 46, 2177-2186 (2003)]. Further on the saturated analogue 3-amino-cyclopentane-carboxylic acid is a starting material applied in the search for biologically active compounds [Evans, C; McCague, R.; Roberts, S.M.; Sutherland, A.G.; J. Chem. Soc, Perkin Trans. 1, 656-657 (1991)].
2. Description of the Prior Art
Since the formation of the adequate absolute configuration of the asymmetric centres is essential for both, the synthesis of active ingredients of drugs and the search for biologically active compounds, several methods are developed for the resolution of cyclic y- -amino acids. For example according to the patent application EP 590685 Al the conventional method of diastereomeric salt pair formation is used for the resolution. In the course of the process after N-acylation (for example N-benzoylation) of the racemic cis-4-aminocyclopent- -2-ene-l-carboxylic acid, obtained from the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one by hydrolysis, the resulted N-acyl derivative is transformed into a diastereomeric salt pair with (+)-cis-2-(benzyl-amino)-cyclohexane-l -methanol or (+)-α-methyl-benzylamine, then the diastereomeric salt pairs are separated and the (-) enantiomer of cis-4-(benzoyl-amino)- -cyclopent-2-ene-l-carboxylic acid is obtained by known steps. This method leads to the pure enantiomers via a multi-step process starting from the racemic 2-azabicyclo[2.2.1]hept-5-ene- -3-one.
In addition to the conventional resolution methods enzymatic resolution processes are also described. For example R. Csuk et al. [Csuk, R.; Dorr, P.; Tetrahedron: Asymmetry 5, 269-276 (1994)] disclose the preparation of the adequate (lS,4R)-enantiomer of N-acyl- amino-carboxylic acid (enantiomeric excess: ee 49.7 %) and the (lR,4S)-enantiomer of N- -acyl-amino-carboxylic acid methyl ester (enantiomeric excess: ee >99 %) by enantioselective ester cleavage of the racemic cis-methyl-[4-(acyl-amino)-cyclopent-2-ene-l-carboxylate] - obtained from the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one by hydrolysis - with pig liver esterase enzyme. The enantiomers (lR,4S)-N-acyl-amino.-carboxylic acid and (1S,4R)-N- -acyl-amino-carboxylic acid ester are obtained by the above authors with high enan- tioselectivity by hydrolysis of N-acyl-amino esters having longer alkyl chain (n-butyl, n- -hexyl) - obtained by cross esterification of the above mentioned racemic N-acyl-amino- -carboxylic acid methyl esters - with lipase enzyme. According to these methods the enantiomer acids can be obtained after the non-enzymatic hydrolysis of the starting racemic 2- -azabicyclo[2.2.1]hept-5-ene-3-one via several steps (acylation, esterification, desacylation) and the resolution is carried out by selective cleavage of the ester groups linked to the cyclopentene ring with esterase or lipase enzymes.
The patent specification EP 424064 Al describes a process for the resolution of the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one using the lactamase activity of ENZA-I (Rhodococcus equi NCIMB 41213) and ENZA-20 (Psedomonas solanacearum NCIMB 40249) strains, in which the (+) and (-) enantiomers of 2-azabicyclo[2.2.1]hept-5-ene-3- -one, as well as the (-) and (+) enantiomers of cis-4-aminocyclopent-2-ene-l-carboxylic acid, respectively - formed via the opening of the lactam ring - are obtained. Taylor et al. [Taylor, S.J.C.; McCague, R.; Wisdom, R.; Lee, C; Dickson, K.; Ruecroft, G.; O'Brien, F.; Littlechild, J.; Bevan, J.; Roberts, S.M.; Evans, C.T.; Tetrahedron: Asymmetry 4, 1117-1128 (1993)] describe the use of the more selective and stable ENZA-25 and ENZA-22 strains with lactamase activity more suitable for the resolution of 2-azabicyclo[2.2. l]hept-5-ene-one.
SUMMARY OF THE INVENTION
In the course of our experiments aiming at the preparation of the enantiomers of the cyclic cis γ-amino acids and derivatives thereof it is surprisingly found that the (1S,4R)- -enantiomer of 4-aminocyclopent-2-ene-l-carboxylic acid can directly be obtained from the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one by stereoselective enzymatic hydrolysis using lipase enzyme. The corresponding (1R,4S) enantiomer acid or esters thereof can be obtained from the unreacted (lS,4R)-azabicyclo[2.2.1]-hept-5-ene-3-one enantiomer separated after the enzymatic reaction using known methods, e.g. Forrό, E.; FUlδp, F.; Org. Lett. 5, 1209-1212 (2003) and Forrό, E; Arva, J.; Fϋlδp, F.; Tetrahedron: Asymmetry, 12, 643-649 (2001). In our further experiments the enzymatic reaction is extended to the saturated cyclopentane ring containing analogues and the protected derivatives as well.
The advantage of our process is that instead of the above mentioned lactamase enzymes, which are not easily available, it uses lipase enzymes, which are commercially available, stable, have high stereoselectivity and can be used on industrial scale. Furthermore lipase enzymes have not been used for the synthesis of enantiomers of cyclic cis γ-amino acids starting from the racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one or the derivatives thereof. According to the process of our invention for example the (lS,4R)-4- -aminocyclopent-2-ene-l-carboxylic acid, which is used in the synthesis of Carbovir, can be obtained in one step, in high enantiomeric excess, on industrial scale.
The selective hydrolytic effect of lipase enzymes on the amide bond of the racemic 2- -azabicyclo[2.2.1]hept-5-ene-3-one or on the saturated analogue thereof found in our invention is all the more surprising because according to the international patent application WO 99/10519 Al acylase type enzymes, among others the pig pancreas lipase, can only be used for resolution of those racemic 2-azabicyclo[2.2.1]hept-5-ene-3-one derivatives, which contain an activating group on the nitrogen atom and the resolution results in the hydrolysis of (1S,4R) enantiomer beside the (1R,4S) enantiomer of 2-azabicyclo[2.2.1Jhept-5-ene-3-one containing the activating group. In contrast to this method the process of our invention does not require the presence of an activating group, on the other hand instead of the (1S,4R) enantiomer the (1R,4S) enantiomer is hydrolyzed yielding the desired (lS,4R)-4- -aminocyclopent-2-ene-l-carboxylic acid enantiomer.
DETAD-ED DESCRIPTION OF THE INVENTION
The invention is directed to a process for the preparation of enantiomers of cyclic cis γ-amino acids and their derivatives of the general formula (I)
Figure imgf000005_0001
(I)
— wherein
R1 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;
R2 is hydrogen atom or an amino protecting group; the dotted line in the ring is optionally an additional chemical bond; and * is a chiral carbon atom with defined configuration — and the salts thereof.
The process of the present invention involves the enantioselective enzymatic hydrolysis of lactams of general formula (II)
Figure imgf000006_0001
(ID wherein R2 and the dotted line are as defined above; comprising carrying out enantioselective lipase catalyzed enzymatic hydrolysis on racemic or enantiomerically enriched mixtures of the lactam, wherein al) only the (1S,4R) enantiomer of a lactam of the general formula (II) — wherein R2 is hydrogen atom or amino protective group and the dotted line is different from an additional bond — is selectively hydrolyzed to give the corresponding enantiomer of cyclic γ-amino acid of the general formula (I) — wherein the R1 is hydrogen atom, R2 is as defined above, the dotted line is different from an additional bond, and * is (1R,3S) configuration — and separating the obtained acid enantiomer and the unreacted lactam enantiomer; or a2) only the (1R,4S) enantiomer of a lactam of the general formula (II) — wherein R2 is hydrogen atom or amino protective group and the dotted line is an additional bond — is selectively hydrolyzed to give the corresponding enantiomer of cyclic γ-amino acid of the general formula (I) — wherein R1 is hydrogen atom, R2 is as defined above, the dotted line is an additional bond and * is (1S,4R) configuration — and separating the obtained acid enantiomer and the unreacted lactam enantiomer; and b) transforming the unreacted enantiomer lactam of the general formula (III)
Figure imgf000006_0002
(III) obtained in step al) or a2) — wherein R2 and the dotted line are as defined above and * is in step al) (1R,4S), while in step a2) (1S,4R) configuration — into the appropriate enantiomer of the general formula (I) — wherein R1 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; R2 and the dotted line are as defined above; and * is in step al) (1S,3R), while in step a2) (1R,4S) configuration — ; and optionally transforming the obtained enantiopure cyclic γ-amino acid of the general formula (I) — wherein R1 is hydrogen atom, R2, the dotted line and * are as described above — into an enantiomer ester of the general formula (I) — wherein R1 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; R2, * and the dotted line are as defined above — by a suitable esterification method; or transforming the obtained enantiopure compound of the general formula (I) — wherein the meaning of R1 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; R2, * and the dotted line are as defined above — into an enantiomer acid by any suitable known method; and/or cleaving the protective group of an obtained enantiopure compound of the general formula (I) — wherein R1 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; * and the dotted line are as defined above and R2 is an amino protecting group — by a suitable known method; and/or preparing enantiopure salts thereof.
The procedure of the invention, under optimized conditions, affords both enantiomers (i.e. the one of the hydrolyzed γ-amino acid and the other of the unreacted lactam) in high enantiomeric excess (>99%). The conversion of the process is practically complete (starting from a racemic mixture, a maximum conversion of 50% for the individual enantiomers may be reached) and both products are obtained in good chemical yields (>48%). Further advantage of the process is that it is not necessary to use protecting or activating groups.
Separation of products can be carried out by any of the usual methods. According to a preferable method of the invention the non-protected enantiomer γ-acid precipitates from organic solvents, whereas it is soluble in water, so it can be easily washed off and/or extracted from the surface of the enzyme with water, it can be extracted into water. The non-hydrolyzed enantiomer of the lactam can easily be isolated from organic solvents.
The process of the invention can advantageously be used for the preparation of enantiopure cyclic cis γ-amino acids on industrial scale.
DEFINITIONS
As utilized herein, the substituent R1 means, for example, a hydrogen atom or an optionally substituted group selected from the group consisting of Ci-io alkyl, C2- io alkenyl, (C2-I0 a!kenyl)-(Ci.io alkyl), C2-I0 alkynyl, (C2-I0 alkynylHd.io alkyl), C3-15 cycloalkyl, (C3-I5 cycloalkyl)-(Ci.io alkyl), C3-I5 cycloalkenyl, (C3-is cycloalkeny I)-(C MO alkyl), C3-I5 cycloalkynyl, (C3-I5 cycloalkynyl)-(Cj.io alkyl), aryl-(Ci-io alkyl), heterocyclyl- -(C1-10 alkyl), heteroaryHd.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 R1 stands for a hydrogen atom, CLIO alkyl, C2-7 alkenyl, (C2-7 alkenyl)- -(C1-7 alkyl), C2-7 alkynyl, (C2-7 alkynyl)-(C,.7 alkyl), C3-10 cycloalkyl, (C3-10 cycloalkyl)-(Ci-7 alkyl), C3-10 cycloalkenyl, (C3.10 cycloalkeny I)-(C J-7 alkyl), C3-1O cycloalkynyl, (C3-1O cycloalkynyl)-(C1-7 alkyl), aryl-(C1-7 alkyl), heterocyclyI-(Ci-7 alkyl), heteroaryl-(C1-7 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(C1-7 alkyl), nitro, amino, mono- or di(C1-7 alkyl)amino, (C1-7 alkyl)thio and C1.7 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 Ci-I0 alkyl e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, methoxymethyl, methoxyethyl, trifluoromethyl, trifluoroethyl, trichloroethyl, dimethylamino-ethyl, methylthioethyl; C2-7 alkenyl e.g. allyl, butenyl, iso-butenyl; C2-7 alkynyl e.g. propargyl; (C3-io cycloalkyl)-(Ci.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-(C1-7 alkyl) e.g. benzyl, fluorenylmethyl; saturated and unsaturated heterocyclyl and/or heteroaryl, heterocyclyl-(C1-7 alkyl), heteroaryl- -(C1-7 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 R1 stands for a hydrogen atom, C1-7 alkyl, e.g. methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, 2-methylpentyl, hexyl, heptyl; most preferably for a hydrogen atom, methyl or ethyl groups.
R substituent stands for hydrogen atom or an amino protecting group. As utilized herein in the term "amino protecting group" means groups which are commonly used for the protection of amino groups in organic chemistry, the introduction and cleavage thereof can be carried by known methods (e.g. McOmie; Protecting Groups in Organic Chemistry, Plenum Press, NY., 1973, Greene and Wutts; Protecting Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons, NY., 1991). Preferred protecting groups are acyl or acyloxy groups.
As utilized herein, the symbol * denotes a chiral carbon atom with R or S absolute configuration.
As utilized herein, the term "enantiomerically enriched mixture" stands for a mixture of enantiomers wherein the ratio of the enantiomers [(R/(S)] is different from one.
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 enantiomer acids obtained according to the process of the invention can be transformed by known methods into enantiomer esters [e.g. Csuk, R.; Dorr, P.; Tetrahedron: Asymmetry, 5, 269-276 (1994)].
The lactams used in the process of the invention as starting materials are known compounds [Daluge, S.; Vince, R. J.; Org. Chem.; 43, 2311-20 (1978), Jagt, J. C; Van Leusen, A. M. J.; Org, Chem., 39, 564-566 (1974)].
The enzymes suitable for use in the process of invention are lipase enzymes, which belong to the family of hydrolytic enzymes. These enzymes are commercially available. In a preferred embodiment of the invention suitable enzymes include CAL-B (Candida Antarctica lipase B) preparations prepared by different immobilization techniques; more preferable enzymes are Lipolase, Novozym 435, Chyrazime L-2.
The process of the invention can 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 system thereof, as well as in ionic liquids [for example in 1-butyl- -3-methyl-imidazolium hexafluorophosphate and the like with reference to Park, S.; Kazlauskas, R. J.; Curr. Op. Biotechnol. 14, 432 (2003) considered to be incorporated into this specification] or in compressed gases [for example propane, ethane, carbon dioxide and the like with reference to M.C. Almeida et al., Enz. Microb. Tech. 22, 494 (1998) considered to be incorporated into this specification]. The organic solvents may be an apolar or a polar solvent or the mixture or the multiphase system thereof. According to a preferred embodiment of the invention halogenated hydrocarbon solvents, such as dichloromethane, dichloroethane, chlorobenzene and the like; keton-type solvents, such as acetone, 2-butanone, acetophenone and the like; alcohol-type solvents, such as ethanol, propanol, isopropanol, butanol, pentanol, octanol and the like; hydrocarbons, such as toluene, hexane, heptane and the like; ether-type solvents, such as diethyl ether, tetrahydrofuran, diisopropyl ether, tert-butyl methyl ether, 1,2- -dimethoxyethane and the like; or other polar solvents, for example nitrile-type solvents, such as acetonitril and the like; amide-type solvents, such as N,N-dimethylformamide, N5N- -dimethylacetamide and the like; amine-type solvents, such as N,N-diisopropylethylamine and the like can be used as reaction medium.
As utilized herein the term "aqueous solvent" stands for water, an aqueous buffer medium, aqueous solutions containing inorganic and/or organic cations and anions.
The process of the invention can be carried out also in solid phase, without solvent addition, in the presence of an equivalent amount of water.
The temperature of the process of the 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 optimal 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) The ee values for the unreacted γ-lactam enantiomers were determined with a gas chromatograph equipped with a chiral column [CP-Chirasil-Dex CB (120 0C for 25 min → 160 0C, rate of temperature rise 10 0C min'1, 140 kPa)].
(b) The ee values for the γ-amino acid enantiomers produced were determined with a gas chromatograph equipped with a chiral column [CP-Chirasil-Dex CB (120 0C for 25 min — > 160 °C, rate of temperature rise 10 °C min"1, 140 kPa, after double derivatization: in the first step, the COOH was esterified 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].
(c) The ee values for the γ-amino ester enantiomers were determined with a gas chromatograph equipped with a chiral column [CP-Chirasil-Dex CB (120 °C for 25 min → 160 0C, rate of temperature rise 10 °C min'1, 140 kPa, after acylation of the free NH2 group with acetic anhydride in the presence of 4-dimethylaminopyridine and pyridine (10:90 m/m]. The retention times are given in Table A.
Table A Retention times for the enantiomers
Figure imgf000012_0001
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-ees)/(l+ees/eep)]}/{ln[(l+ees)/(l+ees/eeP)]}
The absolute configurations were proved by comparing the [α] values with the literature data.
EXAMPLES Example 1
Preparation of 4-aminocyclopent-2-en-l-carboxylic acid and ester enantiomers and the salts thereof
A) (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2-azabicyc- lo[2.2. l]hept-5-en-3-one
Lipolase (300 mg, 30 mg mL"1; lipase B from Candida antarctica, Sigma-Aldrich) and water (9.0 μL, 0.50 mmol) are added to racemic 2-azabicyclo[2.2.1]hept-5-en-3-one (109 mg, 1.00 mmol) in .diisopropyl ether (10 mL), and the mixture is shaken in an incubator shaker (Innova 4080) at 60 °C for 2 h. The reaction is stopped by filtering off the enzyme at 50% conversion (ees = 98%; eeP > 99%; E > 500).
The filtered off enzyme is washed with distilled water (3x15 mL), and the water is evaporated off, yielding crystalline (lS,4R)-4-aminocyclopent-2-en-l-carboxyh'c acid [55 mg,
43%; [α]D = -244 (c = 0.3; H2O); M.p. > 200 0C (recrystallized from H2O and (CH3)2CO); ee
> 99%].
1H NMR (400 MHz, D2O, 25 0C, TMS) data for (lS,4R)-4-aminocyclopent-2-en-l- -carboxylic acid: δ = 2.00-2.54 (m, 2H, CH2), 3.50-3.52 (m, IH, CHCOOH), 4.33-4.35 (m, IH, CHNH2), 5.94-6.28 (m, 2H, CHCH).
The organic solvent of the reaction mixture is evaporated off, leaving the unreacted
25
(lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one as a white crystalline product [48 mg, 44%; [CC]D = +545 (c = 0.25; CHCl^; M.p. 97-100 0C (recrystallized from iPr2O); ee = 98%]. 1H NMR (400 MHz, CDCl3, 25 °C, TMS) data for (lS,4R)-2-azabicyclo[2.2.1]hept-5-en- -3-one: δ = 2.23-2.42 (m, 2H, CH2), 3.23 (s, IH, CHCO), 4.34 (m, IH, CHNH), 5.54 (bs, IH, NH), 6.67-6.80 (m, 2H, CHCH). B) Gram-scale synthesis of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid, ethyl ester and hydrochloride salts thereof and (lS,4R)-2-azabicyclo[2.2.1]hept-5-en- -3 -one
Following the procedure described above [(A)], the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (1 g, 9.16 mmol) and water (82.4 μL, 4.58 mmol) in diisopropyl ether (50 mL) in the presence of Lipolase (1.5 g) at 60 0C leads to 50% conversion in 4 h (ees > 99%; eep > 99%; E > 1000). The reaction mixture is worked up according to the above-mentioned procedure [A)].
(lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid [559 mg, 48%; [OC]D = -243 (c = 0.34;
H2O); ee > 99%].
(lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one as a white crystalline product [462 mg, 46%;
[α]D = +543 (c = 0.3; CHCl3); M.p. 98-99 0C (recrystallized from iPr2O); ee > 99%].
When (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid (200 mg) is treated with 22% HCl/EtOH (w/w) (5 mL), (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid hydrochloride is obtained [223 mg, 87%; [CC]D = -1 10.9 (c = 0.55, H2O); M.p. 205-208 0C (recrystallized from EtOH and Et2O), ee > 99%].
(lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid (100 mg, 0.78 mmol) with SOCl2 (O.U mL, 1.44 mmol) in EtOH affords the corresponding ethyl (lS,4R)-4-
-aminocyclopent-2-en-l-carboxylate hydrochloride [116 mg, 78%; [CX]D = -107 (c = 0.4; EtOH); M.p. 170-171 0C (recrystallized from EtOH and Et2O); ee = 99%].
C) Synthesis of (lR,4S)-4-aminocycloρent-2-en-l-carboxylic acid and ethyl ester, and the corresponding hydrochloride and hydrobromide salts, respectively
The ring opening of the γ-lactam (lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one (200 mg) obtained in the enzymatic hydrolysis described in B) through reflux (2 h) with 18% aqueous HCl solution (w/w) results the (lR,4S)-4-aminocyclopent-2-en-l-
-carboxylic acid hydrochloride [268 mg, 90%; [α]" = +11 1 (c = 0.35; H2O); M.p. 208-209 0C (recrystallized from EtOH and Et2O); ee > 99%].
Or, the above-mentioned (lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one (100 mg) is transformed through reflux (1 h) with 24% aqueous HBr solution (w/w) into (lR,4S)-4- -aminocyclopent-2-en-l-carboxylic acid hydrobromide [161 mg, 84%; +108.3 (c = 0.15; H2O); ee > 98%]. The obtained (lR,4S)-4-aminocyclopent-2-en-l-carboxylic acid hydrochloride is converted by ion-exchange chromatography (Varion KSM) to (lR,4S)-4-
-aminocyclopent-2-en-l-carboxylic acid {[OC]D = +244 (c = 0.3; H2O); ee > 99%}; identification data found in literature for (lR,4S)-4-aminocyclopent-2-en-l-carboxylic acid
[α]D =+242 (c=2; H2O)[1], M.p.:>200 0C decomp. m.
The above-mentioned (lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one (100 mg) is transformed through reflux (1 h) with 22% HCl/EtOH solution (w/w) into the corresponding ethyl (lR,4S)-4-aminocyclopent-2-en-l-carboxylate hydrochloride [153 mg, 88%; [OC]D = +105 (c = 0.25; EtOH); M.p. 168-172 0C (recrystallized from EtOH and Et2O); ee = 98%]. 1H NMR (400 MHz, D2O, 25 0C, TMS) data δ (ppm) for (lR,4S)-4-aminocyclopent-2-en-l- -carboxylic acid are similar to those of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid. 1H NMR (400 MHz, D2O, 25 0C, TMS) data for (lR,4S)-4-aminocyclopent-2-en-l- -carboxylic acid hydrochloride are similar to those of (lS,4R)-4-aminocyclopent-2-en-l- -carboxylic acid hydrochloride: δ = 2.05-2.71 (m, 2H, CH2), 3.73 (m, IH, CHCOOH), 4.40 (m, IH, CHNH2), 5.97-6.24 (m, 2H, CHCH).
1H NMR (400 MHz, D2O, 25 0C, TMS) data for ethyl (lR,4S)-4-aminocyclopent-2-en-l- -carboxylate hydrochloride are similar to those of ethyl (lS,4R)-4-aminocyclopent-2-en- -1-carboxylate hydrochloride: δ = 1.22-1.26 (t, 3H, J = 7.12 Hz, CH3), 2.03-2.71 (m, 2H, CH2), 3.70-3.73 (m, IH5 CHCOOH), 4.15-4.20 (m, 2H, CH2CH3), 4.38 (m, IH, CHNH2), 5.94-6.23 (m, 2H, CHCH).
Example 2
Preparative-scale synthesis of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one on addition of the enzyme in portions
Following the procedure described in Example 1, the ring cleavage of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.0 g, 45.81 mmol) is performed in diisopropyl ether (100 mL). The Lipolase (2 g) is added to the reaction mixture in 5 portions (0.4 g to start the reaction, 0.4 g after 12 h, 0.4 g after 24 h, 0.4 g after 36 h and 0.4 g after 48 h). The reaction reaches 49% conversion after 56 h (ees = 94%; eeP > 99%; E > 500).
Examples 3-9 Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one using different enzymes
Following the procedure described in Example 1, the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.5 mg, 0.05 mmol) and water (0.91 μL, 0.05 mmol) in diisopropyl ether (1 mL) at 60 0C 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), PPL (porcine pancreatic lipase, Sigma-Aldrich), lipase PS (Pseudomonas cepacia, Amano Pharmaceuticals), lipase AK (Pseudomonas βuorescens, Amano Pharmaceuticals) and lipase AY (Candida rugosa, Fluka), Table I].
Table 1
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxyIic acid and (lS,4R)-2- -azabicyclo[2.2.l]hept-5-en-3-one using different enzymes
Example Lipase Temperature Reaction Conversion ees a eep b E
OO mg mL'1) CQ time (%) (%) (%)
3 Chirazyme L-2 60 140 min 41 68 > 99 > 200
4 Novozym 435 60 140 min 40 65 > 99 > 200
5 Chirazyme L-5C 60 140 min 15 17 > 99 > 200
6 Lipase PSC 45 64 h 10 11 > 99 > 200
7 Lipase AKC 45 64 h 14 16 > 99 > 200
8 PPL 45 64 h 20 25 > 99 > 200 aees enantiomeric excess for the lactam beep enantiomeric excess for the acid c20% (w/w) of lipase adsorbed on Celite in the presence of sucrose
Examples 9-15
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one in different solvents
Following the procedure described in Example 1, the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.5 mg, 0.05 mmol) and water (0.91 μL, 0.05 mmol) in the presence of Lipolase (30 mg) at 60 0C is performed in different solvents (1 mL). The characteristics of the reactions are presented in Table 2.
Table 2
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one in different solvents
Example Solvent Reaction Conversion ees eep E
(I mL) time (%) (%) (%)
9 1,4-dioxane 36 h 22 28 > 99 > 200
10 THF 36 h 7 7 > 99 > 200
11 Et2O 50 min 18 21 > 99 > 200
12 tert-BuOMe 50 min 35 54 > 99 > 200
13 toluene 36 h 33 49 > 99 > 200
14 n-hexane 50 min 34 51 > 99 > 200
15 CH2Cl2 36 h 20 24 > 99 > 200
Example 16
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one in water
Following the procedure described in Example 1, the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.5 mg, 0.05 mmol) in the presence of Lipolase (30 mg) at 60 °C is performed in water (1 mL). The reaction reaches 45% conversion after 19 h (ees = 82%; eep > 99%; E > 500).
Example 17
Preparative-scale synthesis of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one in water and on addition of the enzyme in portions
Following the procedure described in Example 1, the ring cleavage of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (1.0 g, 9.16 mmol) is performed in water (20 mL). The Lipolase (1.6 g) is added to the reaction mixture in 8 portions (0.2 g to start the reaction and 0.2 g after every 24 h). The reaction reaches 50% conversion after 8 days (ees > 99%; eep > 99%; E > 500).
Examples 18-22
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one at different temperatures
Following the procedure described in Example 1, the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.5 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in the presence of Lipolase (30 mg) in iPr2O at 60 0C is performed at different temperatures. (Table 3).
Table 3
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one at different temperatures
Example Temperature Reaction Conversion ees (%) eep (%) E
(0C) time (%)
18 30 17 h 43 74 > 99 > 200
19 45 50 min 11 12 > 99 > 200
20 60 50 min 26 34 > 99 > 200
21 70 50 min 29 40 > 99 > 200
22 80 50 min 48 86 > 99 > 200
Examples 23-28
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one without the addition of water or using different amounts of added water
Following the procedure described in Example 1, the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.5 mg, 0.05 mmol) in the presence of Lipolase (30 mg) at 60 0C is performed in diisopropyl ether (1 mL) using different amounts of added water. The results are presented after 140 min in Table 4. Table 4
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- azabicyclo[2.2.1]hept-5-en-3-one using different amounts of added water
Example H2O Conversion ees (%) eep E
(equiv.) (%) (%)
23 - 50 >99 >99 >200
24 0.5 50 >99 >99 >200
25 1 42 73 >99 >200
26 2 36 55 >99 >200
27 5 24 32 >99 >200
28 10 11 12 >99 >200
Examples 29-34
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one using different amounts of enzyme quantities
Following the procedure described in Example 1, the reaction of racemic 2- -azabicyclo[2.2.1]hept-5-en-3-one (5.5 mg, 0.05 mmol) and water (0.45 μL, 0.025 mmol) in diisopropyl ether (1 mL) at 600C is performed with different quantities of Lipolase. The results are presented after 50 min in Table 5.
Table 5
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.l]hept-5-en-3-one using different Lipolase quantities
Example Lipolase Conversion ees eep E
(mgmL"1) (%) (%) (%)
29 10 15 18 >99 >200
(50 after 20 h) (98) (>99) (> 200)
30 20 23 29 >99 >200
31 30 32 46 >99 >200
32 40 33 49 >99 >200
33 50 36 56 >99 >200
34 75 39 64 >99 >200 Examples 35-37
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one regenerated enzyme
Following the procedure described in Example 1, the ring cleavage of racemic 2- -azabicyclo[2,2.1]hept-5-en-3-one (5.5 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 that had already been used in 1, 2 or 3 cycles) at 60 0C. The characteristics of the reactions after 2 h are presented after 50 min 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 5 days.
Table 6 Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2-
-azabicyclo[2.2.1]hept-5-en-3-one with regenerated enzyme Example Lipolase (30 mg mL" ) Conversion (%) ees (%) eep (%) E
35 used once 28 39 > 99 > 200
36 twice used 25 33 > 99 > 200
37 3 times used 23 30 > 99 > 200
Example 38
Preparation of 3-aminocyclopentane-l-carboxylic acid enantiomers and the salts thereof in gram-scale
A) Preparation of (lR,3S)-3-ammocyclopentane-l-carboxylic acid, the salt thereof and (lR,4S)-2-azabicyclo[2.2.1]heptane-3-one
The hydrolysis of racemic 2-azabicyclo[2.2.1]heptane-3-one (1 g, 9.00 mmol) is performed in diisopropyl ether (50 mL) at 60 0C, using Lipolase (1.5 g) as catalyst and water (81 μL, 4.5 mmol) as nucleophile. The reaction mixture is worked up after 91 h, at 50% conversion (ees > 99%; eeP = 98%; E > 500):
(lR,3S)-3-aminocyclopentane-l-carboxylic acid [488 mg, 42%; [α]D5 = -10.6 (c = 0.35; H2O); M.p. > 200 0C with decomposition (recrystallized from H2O and Me2CO), ee = 98%]. 1H NMR (400 MHz, D2O, 25 0C, TMS) data for (lR,3S)-3-aminocyclopentane-l - -carboxylic acid: δ = 1.79-2.26 (m, 6H, 3xCH2), 2.79-2.84 (m, IH, CHCOOH), 4.85 (m, IH, CHNH2). (lR,4S)-2-azabicyclo[2.2.1]heptane-3-one [470 mg, 47%; [<X]D = +158 (c = 0.45; CHCl5); M.p. 78-81 0C (recrystallized from 1Pr2O); ee > 99%].
The 1H NMR (400 MHz, CDCl3, 25 °C, TMS) data for (1 R,4S)-2-azabicyclo [2.2.1 ]heptane-3- -one: δ = 1.38-1.94 (m, 6H, 3xCH2), 2.74 (s, IH, CHCO), 3.89 (m, IH, CHNH), 5.88 (bs, IH, NH). When (lR,3S)-3-aminocyclopentane-l-carboxylic acid (200 mg) was treated with 22% HCl/EtOH (4 mL), (lR,3S)-3-aminocyclopentane-l-carboxylic acid hydrochloride [225 mg,
88%; [α]D = -10.8 (c = 0.6; H2O); M.p. 177-180 °C (recrystallized from EtOH and Et2O), ee = 99%] was formed.
B) Preparation of (lS,3R)-3-aminocyclopentane-l-carboxylic acid hydrochloride Enantiomeric (lR,4S)-2-azabicyclo[2.2.1]heρtane-3-one (200 mg, 1.79 mmol), obtained by enzymatic hydrolysis described in A) is dissolved in 18% HCl (10 mL) and refluxed for 2 h.
White crystals of (lS,3R)-3-aminocyclopentane-l-carboxylic acid hydrochloride [241 mg,
81%; M.p. 175-177 0C]; [OC]D = +10.7 (c = 0.5; H2O); ee = 97%] are obtained. 1H NMR (400 MHz, D2O, 25 °C, TMS) δ (ppm) data for (1R.3S)- and for (1S,3R>3- -aminocyclopentane-1-carboxylic acid hydrochlorides are identical: δ = 1.77-2.40 (m, 6H, 3xCH2), 2.97-3.01 (m, IH, CHCOOH), 3.72-3.75 (m, IH, CHNH2).
Example 39
Preparation of N-Boc-4-aminocyclopent-2-en-l-carboxylic acid and N-Boc-2- -azabicyclo[2.2.1]hept-5-en-3-one and enantiomers in preparative-scale
A) Preparation of (lS,4R)-N-Boc-4-aminocyclopent-2-en-l-carboxylic acid, the hydrochloride salt thereof and (lS,4R)-N-Boc-2-azabicyclo[2.2.1]hept-5-en-3-one
The hydrolysis of racemic N-Boc-2-azabicyclo[2.2.1]hept-5-en-3-one (500 mg, 2.39 mmol is performed in diisopropyl ether (30 mL), at 30 0C, using Lipolase (0.9 g) as catalyst and water (21 μL, 1.18 mmol) as nucleophile. The reaction mixture is worked up after 18 h using column chromatography (eluant: EtOAc . hexane = 1 :3), at 50% conversion (ees = 97%; eeP = 96%; E > 200):
(lS,4R)-N-Boc-4-aminocycloρent-2-en-l-carboxylic acid [238 mg, 44%; [<X]D = -40.8 (c = 0.25; H2O); m.p. 130-132 °C (recrystallized from H2O and Me2CO); ee = 96%]. 1H NMR (400 MHz, DMSO-d6, 25 0C, TMS) data for (lS,4R)-N-Boc-4-aminocyclopent-2- -en-1-carboxylic acid: δ = 1.37-1.42 (overlapping the s of 9H, 3xCH3 and m of 2H, CH2), 2.04-2.06 (d, IH, J = 8,6 Hz, CHCO),.2.26-2.28 (d, IH, J = 8,6 Hz, CHNH), 6.72-6.97 (m, 2H, CHCH).
(lS,4R)-N-Boc-2-azabicyclo[2.2.1]hept-5-en-3-one [223 mg, 44%; [α]r>5 = +187 (c = 0.28 in CHCl3); m.p. 77-80 0C (recrystallized from iPr2O); ee = 96%].
The 1H NMR (400 MHz, DMSOd6, 25 °C, TMS) data for (lS,4R)-N-Boc-2- -azabicyclo[2.2.1]hept-5-en-3-one: δ = 1.37-1.42 (overlapping the s of 9H, 3xCH3 and m of 2H, CH2), 2.04-2.06 (d, IH, J = 8,6 Hz, CHCO), 2.26-2.28 (d, IH, J = 8,6 Hz, CHNH), 6.72- 6.97 (m, 2H, CHCH).
When (lS,4R)-N-Boc-4-aminocyclopent-2-en-l-carboxylic acid (100 mg) was treated with 22% HClTEtOH (4 mL), (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid hydrochloride [57 mg, 79%; [<x]o = -101.6 (c = 0.4 in H2O); m.p. 201-204 0C (recrystallized from EtOH and Et2O), ee = 95%] was formed.
B) Enantiomeric (lS,4R)-N-Boc-2-azabicyclo[2.2.1]hept-5-en-3-one (100 mg, 0.47 mmol), obtained by enzymatic hydrolysis described in A) is dissolved in 18% HCl (10 mL) and refluxed for 2 h, afforded white crystals of (lR,4S)-4-aminocyclopent-2-en-l-carboxylic acid hydrochloride [64 mg, 82%; m.p.202-205 0C; [α]σ = +100.7 (c = 0.3 in H2O); ee = 95%].
Example 40
Preparation of (lS,4R)-4-aminocyclopent-2-en-l-carboxylic acid and (lS,4R)-2- -azabicyclo[2.2.1]hept-5-en-3-one without solvent
Following the procedure described in Example 1 a mixture of thoroughly powdered racemic 2-azabicyck>[2.2.1]hept-5-en-3-one (55 mg, 0.5 mmol), Lipolase (150 mg) and water (4.5 μL, 0.25 mmol) is gently shaken at 60 0C. The reaction leads to 47 % conversion in 33 h (ees = 88%; eeP > 99%; E > 500).
References: [1] Taylor, S.J.C.; McCague, R.; Wisdom, R.; Lee, C; Dickson, K.; Ruecroft, D.; O'Brien, F.;
Littlechild, J.; Bevan, J.; Roberts, S.M.; Evans, C. T.; Tetrahedron: Asymmetry 4, 11 17-
1128 (1993) [2] Evans, C; McCague, R.; Roberts, S.M.; Sutherland, A.G.; J. Chem. Soc. Perkϊn Trans ],
656-657 (1991)

Claims

CLAIMSWhat is claimed is:
1. A process for the preparation of enantiomers of cyclic cis γ-amino acids and their derivatives of the general formula (I)
Figure imgf000023_0001
(I)
— wherein
R1 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;
R2 is hydrogen atom or an amino protecting group; the dotted line in the ring is optionally an additional chemical bond; and
* is a chiral carbon atom with defined configuration — and salts thereof comprising an enantioselective enzymatic hydrolysis carried out on racemic or enantiomerically enriched mixtures of lactam of general formula (II)
Figure imgf000023_0002
(H)
— wherein R2 and the dotted line are as defined above — hydrolyzing thereby selectively only one of the enantiomers of the lactam to give the corresponding enantiomer of cyclic cis γ-amino acid of general formula (I) — wherein R1 is hydrogen atom, R2, the dotted line and * are as defined above — and separating the obtained enantiomer of cyclic cis γ-amino acid and the enzimatically unreacted lactam enantiomer of general formula (III)
Figure imgf000023_0003
(III) — wherein R2, the dotted line and * are as defined above — characterized by that the enzymatic hydrolysis is carried out with a lipase enzyme wherein al) only the (IS, 4R) enantiomer of a lactam of the general formula (II) — wherein R2 is hydrogen atom or amino protective group and the dotted line is different from an additional bond — is selectively hydrolyzed to give the corresponding enantiomer of cyclic γ-amino acid of the general formula (I) — wherein the R1 is hydrogen atom, R2 is as defined above, the dotted line is different from an additional bond, and * is (1R,3S) configuration — and separating the obtained acid enantiomer and the unreacted lactam enantiomer; or a2) only the (1R,4S) enantiomer of a lactam of the general formula (II) — wherein R2 is hydrogen atom or amino protective group and the dotted line is an additional bond — is selectively hydrolyzed to give the corresponding enantiomer of cyclic γ-amino acid of the general formula (I) — wherein R1 is hydrogen atom, R2 is as defined above, the dotted line is an additional bond and * is (IS, 4R) configuration — and separating the obtained acid enantiomer and the unreacted lactam enantiomer; and b) transforming an unreacted enantiomer lactam of the general formula (III) obtained in step al) or a2) — wherein R2 and the dotted line are as defined above and * is in step al) (1R,4S), while in step a2) (IS, 4R) configuration — into the appropriate enantiomer of the general formula (I) — wherein R1 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; R2 and the dotted line are as defined above; and * is in step al) (1S,3R), while in step a2) (1R,4S) configuration — ; and optionally i) transforming an obtained enantiopure cyclic γ-amino acid of the general formula (I) — wherein R1 is hydrogen atom, R2, the dotted line and * are as described above — into an enantiomer ester of the general formula (I) — wherein R1 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; R2, * and the dotted line are as defined above — by a suitable esterification method; or ii) transforming an obtained enantiopure compound of the general formula (I) — wherein the meaning of R1 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; R2, * and the dotted line are as defined above — into an enantiomer acid by any suitable known method; and/or iii) cleaving a protective group of an obtained enantiopure compound of the general formula (I) — wherein R1 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;
* and the dotted line are as defined above and R2 is an amino protecting group — by a suitable known method; and/or iv) preparing enantiopure salts thereof.
2. The process according to claim 1 characterized in that CAL-B {Candida Antarctica lipase B) type enzymes are applied as lipase enzymes.
3. The process according to claim 2, characterized in that differently immobilized forms of CAL-B are applied.
4. The process according to claim 2 or claim 3, characterized in that Lipolase, Novozym 435, Chyrazime L-2 enzymes are applied.
5. The process according to any one of claims 1-4, characterized in that the enzyme catalyzed hydrolysis is carried out in an organic solvent, in a mixture of organic solvents, in a mixture of aqueous and organic solvents or in mono- or multiphase system thereof, in an ionic liquid, in a compressed gas or without solvents.
6. The process according to claim 5, characterized in that the organic solvent used is a hydrocarbon, a halogenated hydrocarbon, a keton-, an alcohol-, an ether-, a nitril-, an amide- or an amine-type solvent.
7. The process according to claim 6, characterized in that the organic solvent used is hexane, toluene, acetone, dioxane, tetrahydrofuran, diethyl ether, diisopropyl ether or tert- butyl methyl ether.
8. The process according to any one of claims 1-7, characterized in that CAL-B type enzymes are used either in original or in regenerated form and the enzyme catalyzed hydrolysis is carried out at an optimum temperature ensuring optimum en2yme activity.
9. The process according to any one of claims 1-8, characterized in that the enzyme is added in portions into the reaction mixture.
10. The process according to any one of claims 1-9, characterized in that the enantiomer lactam of the general formula (III) - wherein R2, the dotted line and * are as defined in claim 1 step b) - is isolated from the reaction mixture.
1 1. The process according to any one of claims 1-10 for the preparation of ( 1 S,4R)-4-aminocyclopent-2-ene- 1 -carboxylic acid, ethyl-[(l S,4R)-4-aminocyclopent-2-ene-l -carboxylate],
( 1 R,4S)-4-aminocyclopent-2-ene- 1 -carboxylic acid, ethyl-[( 1 R,4S)-4-aminocyclopent-2-ene- 1 -carboxylate] ,
(1 R,3 S)-3-aminocyclopentane- 1 -carboxylic acid,
(1 S,3R)-3-aminocyclopentane-l-carboxylic acid,
(lS,4R)-N-Boc-4-aminocyclopent-2-ene-l -carboxylic acid and the salts thereof characterized in that the appropriate starting materials are used.
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WO1999010519A1 (en) * 1997-08-22 1999-03-04 Glaxo Group Limited Process for preparing enantiomerically enriched n-derivatised lactams
WO2000003032A1 (en) * 1998-07-09 2000-01-20 Lonza Ag Method for producing (1r,4s)-2-azabicylco[2.2.1]hept-5-en-3-on derivatives
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EP0424064A1 (en) * 1989-10-16 1991-04-24 Chiroscience Limited Chiral azabicyloheptanone and a process for their preparation
WO1999010519A1 (en) * 1997-08-22 1999-03-04 Glaxo Group Limited Process for preparing enantiomerically enriched n-derivatised lactams
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