LV13635B - Enzymatic resolution of racemic 3-aryl-4-aminobutyric acids - Google Patents

Enzymatic resolution of racemic 3-aryl-4-aminobutyric acids Download PDF

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LV13635B
LV13635B LV060028A LV060028A LV13635B LV 13635 B LV13635 B LV 13635B LV 060028 A LV060028 A LV 060028A LV 060028 A LV060028 A LV 060028A LV 13635 B LV13635 B LV 13635B
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Grigorijs Veinbergs
Maksims Vorona
Antons Lebedevs
Aleksandrs Cernobrovijs
Ivars Kalvins
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Olainfarm As
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    • 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

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Abstract

A process for the enzymatic resolution of racemic 3-aryl-4-aminobutyric acid ester into their R- and S-enantiomers, wherein aryl is represented by phenyl group (fenibut) or p-chlorophenyl group (baclofen) and ester group by saturated or unsaturated alkyl containing from 2 to 8 carbon atoms. The process disclosed includes following steps: (1) selective cyclization of 3(S)-aryl-4-aminobutyric acid ester into 4(S)-aryl-2-pyrrolidinone using racemic 3-aryl-4-aminobutyric acid ester in water solution in the presence of alfa-chymotrypsin; (2) acidification of reaction mixture to pH<2.0 and separation of 4(S)-aryl-2-pyrrolidinone and 3(R)-aryl-4-aminobutyric acid ester by extraction; (3) isolation of 4(S)-aryl-2-pyrrolidinone from organic phase and 3(R)-aryl-4-aminobutyric acid ester from water phase and their conversion into respectively R- and S-isomers of 3-aryl-4-aminobutyric acid by acidic hydrolysis.

Description

Enzymatic resolution of racemic 3-aryl-4-aminobutyric acidEnzymatic resolution of racemic 3-aryl-4-aminobutyric acid

Background of the invention.Background of the invention.

The invention relates to the isolation of pure R- and 5-enantiomers from racemic 3-aryl-4aminobutyric acids by the means of selective enzymatic resolution. For example pure R- and Senantiomers of racemic 4-amino-3-phenylbutyric acid (fenibut) or 4-amino-3-pchlorophenylbutyric acid (baclofen) can be obtained by this method. It is known that only Renantiomer of fenibut is acting as mood enhancer and tranquilizer (Allan et al.. Tetrahedron, 1990, 46, No7, 2511-2524.). Similarly only Λ-enantiomer of baclofen is the bearer of antispastic activity (N. E. Bowery, Trends Pharm. Sci., 1982, 31, 411-413). Optical antipodes of these drugs: R-enantiomers of fenibut and baclofen are not only less active but even antagonistic to their R-antipodes and their presence in racemic mixture makes necessary to administer higher doses of drugs.The invention relates to the isolation of pure R- and 5-enantiomers from racemic 3-aryl-4-aminobutyric acids by means of selective enzymatic resolution. For example, pure R- and senantiomers of racemic 4-amino-3-phenylbutyric acid (phenibut) or 4-amino-3-pchlorophenylbutyric acid (baclofen) can be obtained by this method. It is known that only a renantiomer of phenibut is acting as a mood enhancer and tranquilizer (Allan et al., Tetrahedron, 46, 1990, No. 7, 2511-2524, 1990). Similarly only the β-enantiomer of baclofen is the bearer of antispastic activity (N.E. Bowery, Trends Pharm. Sci., 31, 411-413 (1982)). Optical antipodes of these drugs: The R-enantiomers of phenibut and baclofen are only less active but even antagonistic to their R-antipodes and their presence in the racemic mixture makes it necessary to administer higher doses of the drugs.

That is why the elaboration or effective process for the optical resolution of racemic 3-aryl-4aminobutyric acids opens way to the improvement of the target therapeutic activity of fenibut and baclofen.That is why the elaboration or effective process for the optical resolution of racemic 3-aryl-4aminobutyric acids opens the way to the improvement of the target therapeutic activity of fenibut and baclofen.

Description of the prior artDescription of the prior art

Several methods of racemic 3-aryl-4-aminobutyric acids resolution into R- and R-enantiomers are documented in literature. These are mainly chromatographic separations which include tedious steps of protection and subsequent deprotection of appropriate amino acid [I. Basova et al., SU 1432051 (1986). N. Langlois et. al. Tetrahedron, 1996, 52, No 48, 15117-15126. R. D. Allan et al.. Tetrahedron, 1990, 46, No7, 2511-2524. R. E. Zeile, Synthesis, 1991, 1023.]. Direct resolution of racemic 3-aryl-4-aminobutyric acid was achieved by using columns packed with expensive chiral stationary phase [C. Vaccher, J. Chromatogr. 1991, 542, 502-507]. Some methods are represented by cumbersome preferential crystallization of diastereoisomeric salts using optically active bases cinchonidine or L-(-)-a-methylbenzylamine as resolution aģent [M. Soborcinska et al., Pol. J. Chem., 1979, 53, 435-446. A. F. Wildervanck, et al., (US 6051734, 2000.].Several methods of racemic resolution of 3-aryl-4-aminobutyric acids into R and R enantiomers are documented in literature. These are mainly chromatographic separations which include tedious steps of protection and subsequent deprotection of the appropriate amino acid [I. Basova et al., SU 1432051 (1986). N. Langlois et. al. Tetrahedron, 1996, 52, No 48, 15117-15126. R. D. Allan et al., Tetrahedron, 46, No. 7, 1990, 2511-2524. R. E. Zeile, Synthesis, 1023, 1991]. The direct resolution of racemic 3-aryl-4-aminobutyric acid was achieved using columns packed with expensive chiral stationary phase [C. Vaccher, J. Chromatogr. 1991, 542, 502-507]. Some methods are represented by cumbersome preferential crystallization of diastereoisomeric salts using optically active bases of cinchonidine or L - (-) -? -Methylbenzylamine as a resolution agent [M. Soborcinska et al., Pol. J. Chem., 1979, 53, 435-446. Wildervanck, A. F., et al., U.S. Pat.

Optical resolution was achieved also by methods based on the combination of Chemical and enzymatic transformations [R. Chenevret, M. Desjardins, Can. J. Chem., 1994, 72, 2312-2317. R. V. Muralidhar, R. R. Chirumamilla et al., Med. Fac. Landbouww. Univ. Gent, 2001, 66, Nr 3a, 227-232]. R-Enantiomer of baclofen was also isolated from its racemic mixture by selective microbial degradation of its R-enantiomer therefore excluding the latter from utilization [W. Levadoux et al., D. US 5483765, 1998].Optical resolution was achieved also by methods based on a combination of chemical and enzymatic transformations [R. Chenevret, M. Desjardins, Can. J. Chem., 1994, 72, 2312-2317. Muralidhar R. V., Chirumamilla R. R., et al., Med. Fac. Landbouww. Univ. Gent., 2001, 66, No. 3a, 227-232]. The R-enantiomer of baclofen was also isolated from its racemic mixture by selective microbial degradation of its R-enantiomer therefore excluding the latter from utilization [W. Levadoux et al., D. US 5483765, 1998].

In general, ali mentioned methods are not convenient for large-scale production of pure enantiomeric drugs, because of ignoring technological and especially economical aspects of the availability and the cost of reaģents and materiāls.In general, the methods mentioned are not convenient for large-scale production of pure enantiomeric drugs because of the technological and especially economical aspects of availability and the cost of the reagent and material.

Therefore easy and effective optical resolution method of racemic 3-aryl-4-aminobutyric acids 5 into their R- and 5-enantiomers vvas the aim of the present invention.Therefore, an easy and effective optical resolution method of racemic 3-aryl-4-aminobutyric acids 5 into their R- and 5-enantiomers vvas the aim of the present invention.

Summary of the inventionSummary of the invention

Present invention provides a simple process for the cheap production of optically pure R- and 5enantiomers of 3-aryl-4-aminobutyric acid. After extensive investigations vve discovered, that R10 and 5-enantiomers of 3-aryl-4-aminobutyric acid can be produced from easily available racemic ester of 3-aryl-4-aminobutyric acid 2 by its treatment vvith enzymes selected from protease family vvhich catalyze the hydrolysis of ester group and the formation of peptide bond.The present invention provides a simple process for the cheap production of optically pure R- and 5enantiomers of 3-aryl-4-aminobutyric acid. After extensive investigations, the R10 and 5-enantiomers of the 3-aryl-4-aminobutyric acid can be prepared from the readily available racemic ester of the 3-aryl-4-aminobutyric acid 2 by its treatment by vvith enzymes selected from the protease family vvhich catalyze the hydrolysis of the ester group and the formation of the peptide bond.

Selective cyclization of 3-aryl-4-aminobutyrate 5-enantiomer in the presence of protease provided the formation of reaction mixture containing only tvvo products: 4(5)-aryl-215 pyrolidinone (35) and 3-aryl-4-aminobutyrate 2R in 7?-enantiomeric form. We found, that these compounds can be easily separated from each other by conventional extraction technique and then converted into target 3(R)-aryl-4-aminobutyric acid 17? and 3(5)-aryl-4-aminobutyric acid 15 by acidic hydrolysis (Scheme 1).Selective cyclization of the 3-aryl-4-aminobutyrate 5-enantiomer in the presence of the protease provided the reaction mixture containing only tvvo products: 4 (5) -aryl-215 pyrrolidinone (35) and 3-aryl-4-aminobutyrate 2R in 7? -enantiomeric form. We found that these compounds can be easily separated from each other by the conventional extraction technique and then converted to target 3 (R) -aryl-4-aminobutyric acid 17? and 3 (5) -aryl-4-aminobutyric acid 15 by acidic hydrolysis (Scheme 1).

Scheme 1.Scheme 1.

Detailed description of the inventionDetailed description of the invention

According to the invention, a process for the enzymatic resolution of the racemic mixture of formula 2 vvherein Ar is phenyl or/?-halo substituted phenyl, R is alkyl group and * mārks chiral carbon atom, is provided by the exposing of the racemic mixture of formula 2 to the catalyzing action of protease in its free or immobilized state in suitable aqueous or aqueous-organic cosolvent medium. These enzymes react selectively only vvith 5-enantiomer of 2 converting it into 4(5)-aryl-2-pyrrolidinone (35), but do not attach the R-enantiomer of 2. We found that obtainedAccording to the invention, the process for the enzymatic resolution of the racemic mixture is 2 vvherein Is phenyl or /? - halo substituted phenyl, R is an alkyl group, and * the chiral carbon atom provided by the exposing of the racemic mixture of formula 2 to the catalyzing action of the protease in its free or immobilized state in a suitable aqueous or aqueous-organic cosolvent medium. These enzymes react selectively only with the 5-enantiomer of 2 converting it into 4 (5) -aryl-2-pyrrolidinone (35), but do not attach the R-enantiomer of 2. We found that obtained

3.3.

enantiomeric products: 4(5)-aryl-2-pyrrolidinone (30) and 3(7?)-aryl-4-aminobutyric acid ester (2R), due to their different solubility in two-phase vvater/organic soivent system at low pH can be effectively separated by extraction and then converted into enantiomeric 17? and 15” 3-aryl-4aminobutyric acids respectively by acidic hydrolysis.enantiomeric products: 4 (5) -aryl-2-pyrrolidinone (30) and 3 (7R) -aryl-4-aminobutyric acid ester (2R), due to their different solubility in a two-phase water / organic solvent system at low pH can be effectively separated by extraction and then converted into enantiomeric 17? and 15 ”3-aryl-4aminobutyric acids respectively by acidic hydrolysis.

The preferred reaction conditions for the enzymatic resolution of racemic esters of 3-aryl-4aminobutyric acids 2 include:The preferred reaction conditions for the enzymatic resolution of racemic esters of 3-aryl-4aminobutyric acids 2 include:

1. application of a-chymotrypsin in vvater soluble or in vvater insoluble immobilized reusable state for the resolution of 3-aryl-4-aminobutyric acid esters;An application of? -Chymotrypsin in water soluble or in water insoluble immobilized reusable state for resolution of 3-aryl-4-aminobutyric acid esters;

2. the preferred substituents R in 2 vvhich are represented by saturated or unsaturated alkyl group containing from 2 to 8 carbon atoms;2. the preferred substituents R in 2 are represented by a saturated or unsaturated alkyl group containing from 2 to 8 carbon atoms;

3. the usage of aqueous or aqueous-organic co-solvent reaction medium vvith pH betvveen 6.0-7.0;3. use of aqueous or aqueous-organic co-solvent reaction medium vvith pH 6.0-7.0;

4. reaction temperature betvveen 20-40°C;4. reaction temperature betvveen 20-40 ° C;

5. the stirring or shaking of reaction mixture from 1 to 72 hours.5. stirring or shaking the reaction mixture from 1 to 72 hours.

Thus obtained mixture of optically active 4(5)-aryl-2-pyrrolidinone (35) and 3(7?)-aryl-4aminobutyric acid ester (27?) can be separated by extraction. Any of not mixing vvith vvater organic solvents (hydrocarbons such as hexane, benzene, toluene; halogenated hydrocarbons such as chloroform, methylene chloride; esters such as ethyl acetate; ketones, ethers) may be employed for this purpose. Reaction products 27? and 3,S can be isolated from vvater and organic phases by conventional procedures such as evaporation or crystallization, if necessary. We found that 3(7?)-aryl-4-aminobutyric acid esters 27? are readily soluble in vvater in acidic conditions and can be also extracted at neutral pH 7 and isolated from organic phase by evaporation or crystallization, if necessary.The resulting mixture of optically active 4 (5) -aryl-2-pyrrolidinone (35) and 3 (7) - aryl-4-aminobutyric acid ester (27) can be separated by extraction. Any of the mixing vvith vvater organic solvents (hydrocarbons such as hexane, benzene, toluene; halogenated hydrocarbons such as chloroform, methylene chloride; esters such as ethyl acetate; ketones, ethers) may be employed for this purpose. Reaction products 27? and 3, S can be isolated from water and organic phases by conventional procedures such as evaporation or crystallization, if necessary. We found that 3 (7?) - aryl-4-aminobutyric acid esters 27? are readily soluble in vvater in acidic conditions and can also be extracted at neutral pH 7 and isolated from organic phase by evaporation or crystallization, if necessary.

The preferred conditions for the isolation of substantially pure optically active 4(5)-aryl-225 pyrrolidinone (35) from reaction mixture include:Preferred conditions for the isolation of substantially pure optically active 4 (5) -aryl-225 pyrrolidinone (35) from reaction mixture include:

1. the acidification of reaction mixture till pH<2.0;1. the acidification of the reaction mixture to pH <2.0;

2. the extraction of 4(5)-aryl-2-pyrrolidinone (35) vvith organic soivent preferably vvith ethyl acetate, methylene chloride, toluene or benzene and the follovving evaporation of organic phase.2. the extraction of 4 (5) -aryl-2-pyrrolidinone (35) with organic solvent, preferably ethyl acetate, methylene chloride, toluene or benzene, and evaporation of the organic phase.

The preferred conditions for the isolation of substantially pure optically active 3(7?)-aryl-4aminobutyric acid ester (27?) from reaction mixture, after the elimination of 4(5)-aryl-2pyrrolidinone (35) by extraction can be performed by tvvo methods including:The preferred conditions for the isolation of substantially pure optically active 3 (7?) - aryl-4-aminobutyric acid ester (27?) From the reaction mixture, after the elimination of 4 (5) -aryl-2-pyrrolidinone (35) by extraction can be performed by tvvo methods including:

1. the evaporation of vvater phase;1. the evaporation of the vater phase;

2. the neutralization of vvater phase till pH 7.0, the extraction of 3(7?)-aryl-4-aminobutyric acid ester (27?) vvith organic solvent preferably vvith ethyl acetate, methylene chloride, toluene or benzene and the evaporation of organic phase.2. the neutralization of the water phase to pH 7.0, the extraction of 3 (7?) - aryl-4-aminobutyric acid ester (27?), An organic solvent preferably of ethyl acetate, methylene chloride, toluene or benzene and evaporation of the organic phase.

The follovving examples are illustrating but not restricting the present invention.The follovving examples are illustrating but not restricting the present invention.

General procedure for the synthesis and enzymatic resolution of racemic 3-aryl-4aminobutyric acid ester 2.General procedure for the synthesis and enzymatic resolution of racemic 3-aryl-4aminobutyric acid ester 2.

Protocol 1.Protocol 1.

A. Thionyl chloride (0.616 ml, 8.4 mM) vvas added to a cooled to -16°C mixture of 3-aryl-4aminobutyric acid 1 (5.5 mM) in 20 ml of appropriate alcohol. Obtained solution vvas boiled for 4 hours, and evaporated under reduced pressure yielding hydrochloric salt of 3aryl-4-aminobutyric acid ester 2 (98.0%-99.5% purity according to HPLC analysis).A. Thionyl chloride (0.616 mL, 8.4 mM) was added to a cooled to -16 ° C mixture of 3-aryl-4-aminobutyric acid 1 (5.5 mM) in 20 mL of the appropriate alcohol. Obtained solution was boiled for 4 hours and evaporated under reduced pressure to yield hydrochloric salt of 3aryl-4-aminobutyric acid ester 2 (98.0% -99.5% purity according to HPLC analysis).

B. Racemic 3-aryl-4-aminobutyric acid ester 2 25 mg vvas dissolved in 5 ml of 0.1 M phosphate buffer vvith pH 6.0-7.0 vvith/vvithout addition of 10% of organic co-solvent (dioxane, acetone etc.). Water soluble a-chymotrypsin’ 5 mg (or 25 mg of achymotrypsin immobilized on the suspension of SiCh (1 g) according to the procedure [K. Watanabe, G.P. Royer, Journal of Molecular Catalysis, 1983, 22, 145]) vvas added to the reaction mixture and the latter vvas stirred at 20~40°C for 72 hours. The reaction progress leading to the formation of 4(5)-aryl-2-pyrrolidinone (35) vvas monitored by HPLC.B. Racemic 3-Aryl-4-Aminobutyric Acid Ester 2 25 mg vvas dissolved in 5 ml of 0.1 M phosphate buffer vith pH 6.0-7.0 vith / vithout addition of 10% of organic co-solvent (dioxane, acetone etc.). Water soluble a-chymotrypsin '5 mg (or 25 mg of achymotrypsin immobilized on a suspension of SiCh (1 g) according to the procedure [K. Watanabe, GP Royer, Journal of Molecular Catalysis, 1983, 22, 145]) vvas added and the reaction mixture was stirred at 20 ~ 40 ° C for 72 hours. The reaction progresses to the formation of 4 (5) -aryl-2-pyrrolidinone (35) vvas monitored by HPLC.

C. After the end of enzymatic process2 the reaction mixture vvas acidified by the addition of 2N HCI to pH <2.0 and 4(5)-aryl-2-pyrrolidinone (35) vvas extracted by 3x20 ml of methylene chloride. Thus obtained organic and vvater phases vvere evaporated to dryness giving residues of 4(5)-aryl-2-pyrrolidinone (35) and 3(7?)-aryl-4-aminobutyric acid ester (27?) vvhich vvere converted in hydrochloric salts of 5- and 7?-enantiomers 15 and 17? of 3aryl-4-aminobutyric acid by their treatment in boiling 2N HCI for 20 hours and follovving evaporation of reaction mixture. Their optical purity vvas evaluated on the base of chiral HPLC analysis data3.C. After the end of enzymatic process 2 the reaction mixture was acidified by the addition of 2N HCl to pH <2.0 and 4 (5) -aryl-2-pyrrolidinone (35) was extracted by 3x20 ml of methylene chloride. The resulting organic and aqueous phases are evaporated to dryness to give residues of 4 (5) -aryl-2-pyrrolidinone (35) and 3 (7?) - aryl-4-aminobutyric acid ester (27?). of 5- and 7? -enantiomers 15 and 17? of 3aryl-4-aminobutyric acid by their treatment in boiling 2N HCl for 20 hours and follovving evaporation of the reaction mixture. Their optical purity was evaluated on the base of chiral HPLC analysis data 3 .

Protocol 2.Protocol 2.

The enzymatic resolution of 3-aryl-4-aminobutyric acid (1) on the stages A and B vvas realized according to Protocol 1.The enzymatic resolution of 3-aryl-4-aminobutyric acid (1) on steps A and B is realized according to Protocol 1.

C. After the end of enzymatic process 2 the reaction mixture vvas acidified by the addition of 2N HCI to pH <2.0 and 4(5)-aryl-2-pyrrolidinone 35 vvas extracted by 3x20 ml of ethyl acetate and evaporated to dryness giving residue of 4(5)-aryl-2-pyrrolidinone 35. The vvater phase vvas treated vvith 5N NH4OH to pH 7 and 3(7?)-aryl-4-aminobutyric acid ester (27?) vvas extracted by 3x20 ml of ethyl acetate. Thus obtained organic pahse vvasC. After the end of enzymatic process 2 the reaction mixture was acidified by the addition of 2N HCl to pH <2.0 and 4 (5) -aryl-2-pyrrolidinone 35 was extracted by 3x20 ml of ethyl acetate and evaporated to dryness giving residue of 4 (5) -aryl-2-pyrrolidinone 35. The water phase is treated vvith 5N NH4OH to pH 7 and 3 (7?) - aryl-4-aminobutyric acid ester (27?) vvas extracted by 3x20 ml of ethyl acetate . Thus obtained organic pahse vvas

LV 13635 5 evaporated to dryness giving residue of 3(7?)-aryl-4-aminobutyric acid ester (27?). Both enantiomeric products were converted in hydrochloric salts of 5- and 7?-enantiomers 15 and 17? of 3-aryl-4-aminobutyric acid by their treatment in boiling 2N HCI for 20 hours and following evaporation of reaction mixture. Their optical purity was evaiuated on the base of chiral HPLC analysis data 3.LV 13635 5 Evaporated to dryness affording a residue of 3 (7?) - aryl-4-aminobutyric acid ester (27?). Both enantiomeric products were converted in the hydrochloric salts of 5- and 7? -Enantiomers 15 and 17? of 3-aryl-4-aminobutyric acid by their treatment in boiling 2N HCl for 20 hours and the following evaporation of the reaction mixture. Their optical purity was evolved on the base of chiral HPLC analysis data 3 .

1a-chymotrypsin activity: 65-85 units/mg in the case of immobilized a-chymotrypsin the suspension of catalyst after the end of enzymatic conversion was filtered off, vvashed by distilled vvater and re-used repeatedly, if necessary. 1 a-chymotrypsin activity: 65-85 units / mg in case of immobilized a-chymotrypsin the suspension of the catalyst after the end of the enzymatic conversion was filtered off, vashed by distilled vvater and re-used repeatedly if necessary.

3 mobil phase - HC104 (pH 1.0); stationarv phase - CROWNPAK CR(+)5a 3 mobile phase - HC104 (pH 1.0); stationarv phase - CROWNPAK CR (+) 5a

The effectiveness of enzymatic resolution and the enantiomeric excess for 3-aryl-4-aminobutyric acids 7?- and 5-enantiomers obtained according the Protocol A and B are presented in the TableThe Effects of Enzymatic Resolution and Enantiomeric Excess on 3-Aryl-4-Aminobutyric Acids 7 - and 5-Enantiomers Obtained according to Protocol A and B

1.1.

Table 1Table 1

The conditions of enzymatic resolution process, yields and the enantiomeric excess for 3-aryl-4aminobutyric acid 7?- and 5-enantiomers (17? and 15)The conditions of the enzymatic resolution process, yields and enantiomeric excess for 3-aryl-4-aminobutyric acid 7? - and 5-enantiomers (17? And 15)

Example Nr (Protocol) Example No. (Protocol) R R Ar With Time (h) Time (h) t(°C) t (° C) Medium* Medium * Yield ** (%) Yield ** (%) Enantiomeric excess (%,ee) Enantiomeric excess (%, ee) 15 15th 17? 17? 1(1) 1 (1) Et No. Ph Ph 72 72 20-22 20-22 A A 66 66 92 92 64 64 2(1) 2 (1) z'-Pr z'-Pr Ph Ph 72 72 20-22 20-22 A A 54 54 76 76 53 fifty three 3(D 3 (D zz-Pr zz-Pr Ph Ph 72 72 20-22 20-22 A A 78 78 96 96 76 76 4(1) 4 (1) Allyl Allyl Ph Ph 24 24th 20-22 20-22 A A 68 68 74 74 67 67 5(2) 5 (2) Allyl Allyl Ph Ph 72 72 20-22 20-22 A A 86 86 96 96 84 84 6(1) 6 (1) ZZ-Bu ZZ-Bu Ph Ph 24 24th 20-22 20-22 A A 74 74 90 90 72 72 7(2) 7 (2) zz-Bu zz-Bu Ph Ph 72 72 20-22 20-22 A A 98 98 98 98 97 97 8(2) 8 (2) Octyl Octyl Ph Ph 72 72 20-22 20-22 A A 62 62 76 76 60 60 9(1) 9 (1) zz-Bu zz-Bu p-CIPh p-CIPh 72 72 20-22 20-22 B B 98 98 >98 > 98 >97 > 97 10(2) 10 (2) zz-Bu zz-Bu /z-CIPh / z-CIPh 72 72 20-22 20-22 C C 98 98 >98 > 98 >97 > 97

* A - phosphate buffer (pH 6.0-7.0); B - phosphate buffer/dioxane ratio 10:1; C - phosphate buffer/acetone ratio 10:2.* A - phosphate buffer (pH 6.0-7.0); B - phosphate buffer / dioxane ratio 10: 1; C - phosphate buffer / acetone ratio 10: 2.

** reaction yield vvas calculated on the basis of decrease in the size of 3-aryl-4-aminobutyric acid ester 2 peak on HPLC chart.** reaction yield vvas calculated on basis of reduction in size of 3-aryl-4-aminobutyric acid ester 2 peak on HPLC chart.

Claims (3)

1. Racemiskā 3-aril-4-aminosviestskābes estera (1) sadalīšana optiskajos izomēros, kas atšķiras ar to, ka racemāta sadalīšanu veic ar proteāžu palīdzību, kuras izvēlētas no grupas, kas sastāv no α-himotripsīna, papaīna vai subtilizīna:Claims 1. Catalytic resolution of the 3-aryl-4-amino-butyric acid ester (1) into optical isomers, characterized in that the racemate is cleaved by proteases selected from the group consisting of α-chymotrypsin, papain or subtilisin: ROOCROOC ROHROH ProteazeProtease ROOCROOC NH,NH, HOOCHOOC HOOCHOOC ArWith 2R ©2R © h3oh 3 o NH,NH, ArWith ArWith 17?17? HOOC Ύ NH2 ĀrHOOC Ύ NH 2 Ex Ar ir izvēlēts no grupas, kas sastāv no fenil- vai para-halogēnfenil- un R ir izvēlēts no grupas, kas sastāv no piesātinātas vai nepiesātinātas alkilgrupas ar 2-8 oglekļa atomiem, kura atšķiras ar to, ka sastāv no sekojošiem etapiem:Ar is selected from the group consisting of phenyl or para-halophenyl and R is selected from the group consisting of 2-8 carbon atoms, saturated or unsaturated, characterized in that it comprises the following steps: 10 (1) racemiskās 3-aril-4-aminosviestskābes skābes estera sastāvā esošā 3(6)-aril-4aminosviestskābes estera selektīvu ciklizāciju par 4(5)-aril-2-pirolidinonu (36);10 (1) selective cyclization of 3 (6) -aryl-4-amino-butyric acid ester in racemic 3-aryl-4-amino-butyric acid ester to 4 (5) -aryl-2-pyrrolidinone (36); (2) 4(S)-aril-2-pirolidinona (36) un 3(7?)-aril-4-aminosviestskābes estera (27?) atdalīšanu ar ekstrakciju pēc reakcijas maisījuma paskābināšanas līdz pH<2.0;(2) separating 4 (S) -aryl-2-pyrrolidinone (36) and 3 (7R) -aryl-4-amino-butyric acid ester (27R) by extraction after acidifying the reaction mixture to pH <2.0; (3) 4(6)-aril-2-pirolidinona (36) izdalīšanu no organiskās fāzes un 3(7?)-aril-415 aminosviestskābes estera (27?) izdalīšanu no ūdens fāzes ar ietvaicēšanu vai kristalizāciju.(3) separating 4 (6) -aryl-2-pyrrolidinone (36) from the organic phase and separating 3 (7?) - aryl-415 amino-butyric acid ester (27?) From the aqueous phase by evaporation or crystallization. 2. Process pēc p. 1., kas atšķiras ar to, ka proteāze ir a-himotripsīns.2. The process after p. 1. characterized in that the protease is α-chymotrypsin. 3. Process pēc p. 1., kas atšķiras ar to, ka α-himotripsīns tiek lietots tā ūdenī šķīstošā formā.3. The process after p. 1. characterized in that α-chymotrypsin is used in its water-soluble form. 4. Process pēc p. 1., kas atšķiras ar to, ka α-himotripsīns tiek lietots tā ūdenī nešķīstošā4. The process after p. 1. characterized in that α-chymotrypsin is used in its water-insoluble form 20 imobilizētā formā.20 in immobilized form. 5. Process pēc p. 1., kas atšķiras ar to, ka procesu pielieto racemiskās 3-fenil-4-aminosviestskābes esteru sadalīšanai (7?) un (6) enantiomēros.5. The process after p. 1. A process characterized in that the process is used to separate the esters of the racemic 3-phenyl-4-amino-butyric acid into the (7?) And (6) enantiomers. 6. Process pēc p. 1., kas atšķiras ar to, ka procesu pielieto racemiskās 3-p-hlorfenil-4aminosviestskabes esteru sadalīšanai (7?) un (6) enantiomēros.6. The process after p. 1. A process characterized in that the process is used for the resolution of the esters of the racemic 3-p-chlorophenyl-4-aminobutyric acid into its (7?) And (6) enantiomers. 25 7. Process pēc p. 1., kas atšķiras ar to, ka procesu pielieto racēmiskās 3-aril-4aminosviestskābes esteru, tajā skaitā zemāko un vidējo alkilesteru, kā ari nepiesātināto alkilesteru sadalīšanai enantiomēros.25 7. The process after p. The process of claim 1 wherein the process is for the resolution of the racemic 3-aryl-4-amino-butyric acid esters, including the lower and middle alkyl esters, as well as the unsaturated alkyl esters. 8. Process pēc ρ. 1., kas atšķiras ar to, ka procesu pielieto racēmisko 3-aril-4aminosviestskābju etilesteru sadalīšanai enantiomēros.8. Process after ρ. 1. A process wherein the ethyl esters of racemic 3-aryl-4-amino-butyric acids are resolved into enantiomers. 9. Process pēc ρ. 1., kas atšķiras ar to, ka procesu pielieto racēmisko 3-aril-4aminosviestskābju κ-propilesteru sadalīšanai enantiomēros.9. Process after ρ. 1. A process characterized in that the process is used to divide the κ-propyl esters of racemic 3-aryl-4-amino-butyric acids into enantiomers. 10. Process pēc ρ. 1., kas atšķiras ar to, ka procesu pielieto racēmisko 3-aril-4aminosviestskābju Zz<?-propilesteru sadalīšanai enantiomēros.10. Process after ρ. 1. A process characterized in that the process is used for the resolution of the Z 2 - propyl esters of the racemic 3-aryl-4-amino-butyric acids into enantiomers. 11. Process pēc ρ. 1., kas atšķiras ar to, ka procesu pielieto racēmisko 3-aril-4aminosviēstskābju «-butilesteru sadalīšanai enantiomēros.11. Process after ρ. 1. A process characterized in that the process is used for the resolution of the racemic 3-aryl-4-amino-butyric acid t-butyl esters into enantiomers. 12. Process pēc ρ. 1., kas atšķiras ar to, ka procesu pielieto racēmisko 3-aril-4aminosviestskābju alilesteru sadalīšanai enantiomēros.12. Process after ρ. 1. The process of claim 1 wherein the allyl esters of racemic 3-aryl-4-amino-butyric acids are resolved into enantiomers. 13. Process pēc ρ. 1., kas atšķiras ar to, ka procesu pielieto racēmisko 3-aril-4aminosviestskābju oktilesteru sadalīšanai enantiomēros.13. Process after ρ. 1. The process of claim 1 wherein the octyl esters of racemic 3-aryl-4-amino-butyric acids are resolved into enantiomers. 14. Process pēc ρ. 1, kas atšķiras ar to, ka etapā (1) pielietotais šķīdinātājs ir ūdens.14. The process after ρ. 1, characterized in that the solvent used in step (1) is water. 15. Process pēc ρ. 1, kas atšķiras ar to, ka etapā (1) pielietotais šķīdinātājs ir ūdens maisījums ar organisko šķīdinātāju, kas nejaucas vai vāji jaucas ar ūdeni un ir izvēlēts no grupas, kas sastāv no alifatiskajiem ogļūdeņražiem, aromātiskajiem ogļūdeņražiem, halogēnalkāniem, ēteriem, esteriem, ketoniem un to maisījumiem.15. Process after ρ. 1, characterized in that the solvent used in step (1) is a mixture of water and an organic solvent which is immiscible or slightly miscible with water and is selected from the group consisting of aliphatic hydrocarbons, aromatic hydrocarbons, haloalkanes, ethers, and mixtures thereof. 16. Process pēc ρ. 1., kas atšķiras ar to, ka etapu (1) veic pie 20° līdz 40°C.16. Process after ρ. The process of claim 1, wherein step (1) is carried out at 20 ° to 40 ° C. 17. Process pēc ρ. 1., kas atšķiras ar to, ka etapu (1) veic pie reakcijas vides pH no 6 līdz 7.17. Process after ρ. 1. characterized in that step (1) is carried out at a reaction medium pH of 6 to 7. 18. Process pēc ρ. 1 kas atšķiras ar to, ka etapā (3) 3(7?)-aril-4-aminosviestskābes esteri (27?) ekstrahē no ūdens fāzes pēc tās neitralizācijas līdz pH 7.18. Process after ρ. 1, characterized in that in step (3), the 3- (7?) - aryl-4-amino-butyric acid esters (27?) Is extracted from the aqueous phase after neutralization to pH 7. 19. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai lieto organisko šķīdinātāju, kas ir izvēlēts no grupas, kas sastāv no heksāna, benzola, toluola, hloroforma, metilēnhlorida, dihloretāna, etilacetāta, metilacetāta, dietilētera un meti 1 -/erc-butilētera.19. Process after ρ. 1, characterized in that in steps (2) and (3) an organic solvent selected from the group consisting of hexane, benzene, toluene, chloroform, methylene chloride, dichloroethane, ethyl acetate, methyl acetate, diethyl ether and methyl is used for the extraction. / erc-butyl ether. 20. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto heksānu..20. The process after ρ. 1, characterized in that hexane is used for extraction in steps (2) and (3). 21. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto benzolu.21. Process after ρ. 1, wherein benzene is used for extraction in steps (2) and (3). 22. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto toluolu.22. The process after ρ. 1, characterized in that toluene is used for extraction in steps (2) and (3). 23. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto hloroformu.23. The process after ρ. 1, characterized in that chloroform is used for extraction in steps (2) and (3). 24. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto metilēnhlorīdu.24. Process after ρ. 1, characterized in that in steps (2) and (3), methylene chloride is used for extraction. 25. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto dihloretānu.25. Process after ρ. 1, characterized in that dichloroethane is used for extraction in steps (2) and (3). 26. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto etilacetātu.26. The process after ρ. 1, characterized in that ethyl acetate is used for extraction in steps (2) and (3). 27. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto metilacetātu.27. Process after ρ. 1, wherein methyl acetate is used for extraction in steps (2) and (3). 28. Process pēc ρ. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto dietilēteri.28. Process after ρ. 1, characterized in that diethyl ether is used for extraction in steps (2) and (3). 29. Process pēc p. 1 kas atšķiras ar to, ka etapos (2) un (3) ekstrakcijai izmanto metil-tercbutilēteri.29. The process after p. 1, characterized in that in steps (2) and (3), methyl-tert-butyl ether is used for the extraction. 30. Process pēc p. 1. kas atšķiras ar to, ka etapā (3) iegūto 3(7?)-aril-4-aminosviestskābes esteri (2R) pārvērš R-enantiomērajā 3-aril-4-aminosviestskābē (17?) ar skābās hidrolizēs30. The process after p. 1. A process comprising the step of converting the 3 (7R) -aryl-4-amino-butyric acid ester (2R) obtained in Step (3) into the R-enantiomeric 3-aryl-4-amino-butyric acid (17R) by acid hydrolysis. 5 paņēmienu.5 techniques. 31. Process pēc p. 1., kas atšķiras ar to, ka etapā (3) iegūto 4(S)-aril-2-piroIidinonu (3S) pārvērš S-enantiomērajā 3-aril-4-aminosviestskābē (15) ar skābās hidrolizēs paņēmienu.31. The process after p. The process of claim 1, wherein the 4 (S) -aryl-2-pyrrolidinone (3S) obtained in step (3) is converted into the S-enantiomeric 3-aryl-4-amino-butyric acid (15) by acidic hydrolysis.
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