JPH0614797A - Production of optically active 2-arylpropionic acid and its ester - Google Patents

Abstract

PURPOSE:To efficiently produce the subject optically active 2-arylpropionic acid compound of high optical purity. CONSTITUTION:An enzyme derived from a microorganism belonging to Klebsielia is allowed to act on a racemic mixture of an optically active 2- arylpropionic acid ester or its salt so as to optically selectively hydrolyze it.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an optically active 2-arylpropionic acid and its antipodal ester.

[0002]

2. Description of the Related Art Conventionally, many compounds having a pharmacological action have been put to practical use as a mixture of stereoisomers.
However, in many cases the desired activity is present in only one stereoisomer, and in addition, the undesired stereoisomer is
It is often known to be toxic. Therefore, in order to provide an effective and safe drug, it is strongly desired to provide a stereochemically pure compound. Although 2-arylpropionic acid-based compounds are widely used as antiphlogistic and analgesic agents, most of them are supplied as racemic mixtures, and for the above reasons, optically pure compounds and optically active compounds for their preparation are used. The supply of raw materials was strongly desired. Conventionally, as a method for producing an optically active 2-arylpropionic acid or its ester, a method by asymmetric synthesis or a method of resolving a racemate from a diastereomeric salt is known, but in recent years, the stereochemistry of an enzyme has been known. Synthetic or resolution methods utilizing selective catalysis have been developed. As these methods, a corresponding optically active acid is obtained by optically and selectively hydrolyzing an ester of 2-arylpropionic acid with a microorganism-derived enzyme such as Bacillus, Pseudomonas, Arthrobacter, and Mucor. A method for obtaining the same (JP-A-63-45234) is known. However, since the enzyme used conventionally does not have sufficient optical selectivity and activity for the substrate, it cannot be said to be an advantageous method for practical and industrial application.

[0003]

DISCLOSURE OF THE INVENTION The present invention uses an enzyme having both high activity for 2-arylpropionic acid type compounds and high optical selectivity, and efficiently uses optically active 2-arylpropionic acid or The aim is to provide a practical and industrial process for obtaining the ester.

[0004]

MEANS FOR SOLVING THE PROBLEMS The present inventors have found that an enzyme derived from a microorganism belonging to the genus Klebsiella, particularly Klebsiella oxytoca, has a high hydrolysis reaction for a group of ester compounds of 2-arylpropionic acid type. Therefore, the present invention has been completed by discovering that it has high selectivity and high optical selectivity.

That is, the present invention has the general formula I

[0006]

[Chemical 3]

(In the formula, R ₁ represents a halogen, a linear or branched lower alkyl group, an amino group, a nitro group or a thienyl group having or not having a lower alkyl group as a substituent, and R ₂ is a quaternary group as a substituent. An optically active 2 represented by an amino group or a salt thereof, a lower alkoxy group, an oxysulfonyl group or a lower alkyl group having or not having a group selected from the group consisting of a salt and a halogen, and * represents an active center. -A general formula II characterized in that an enzyme derived from a microorganism belonging to the genus Klebsiella is allowed to act on a mixture of both enantiomers of an arylpropionic acid ester or a salt thereof to cause optical selective hydrolysis.

[0008]

[Chemical 4]

A process for producing an optically active 2-arylpropionic acid represented by the formula (wherein R ₁ has the same meaning as described above and * represents an active center) and its enantiomer ester.

The enzyme used for carrying out the method of the present invention may be any enzyme as long as it is an enzyme derived from a microorganism belonging to the genus Klebsiella and having the above-mentioned optical selective hydrolysis activity, but especially Klebsiella oxytoca (Klebsiella).
It is preferred to use a microorganism belonging to oxytoca), especially an ester-degrading enzyme produced by Klebsiella oxytoca SNSM-87 strain (Ministry of Industrial Science and Technology No. 12953). For details of the esterase and the method for producing the same, refer to the patent application (reference number 1009) entitled “Novel esterase A and method for producing the same” of the same applicant on the same date.
2029) As described in the specification. In carrying out the method of the present invention, the enzyme can be used in the form of being contained in the bacterial cells of the microorganism or a culture thereof, or as an enzyme collected from the culture.

The mixture of both enantiomers of the optically active 2-arylpropionic acid ester represented by the above general formula I or the salt thereof, which is a substrate for the enzymatic reaction in the present invention, can be easily synthesized by a known method. For example, a method in which thionyl halide is allowed to act on a racemic mixture of optically active 2-arylpropionic acid to form an acid halide, and then alcohol is allowed to act in the presence of a base catalyst to effect esterification, racemic acid and alcohol are concentrated in sulfuric acid, D.I. C. C. (1,3-dicyclohexylcarbodiimide) in the presence of a dehydration condensation agent, dehydration condensation in the presence of an amino alcohol such as choline and racemic acid in the presence of an acid catalyst, glycol ester of racemic acid in chlorosulfonic acid, etc. The method of obtaining an alkyl ester having a terminal sulfoxy group by acting the sulfonating agent can be exemplified.

In the reaction of the present invention, a mixture of both enantiomers of the 2-arylpropionic acid ester or a salt thereof is added to cells containing the enzyme or a culture thereof, or water or a buffer containing the enzyme collected from the culture. Add and stir. The pH of the reaction solution at this time is 3 to 11, preferably 6 to 10. The pH can be kept constant by appropriately dropping an alkali during the progress of the reaction to neutralize the generated acid. The reaction temperature is 10 ° C to 100 ° C, preferably 2
A temperature of 0 ° C to 90 ° C is suitable. After completion of the reaction, the produced optically active 2-arylpropionic acid and unreacted 2-arylpropionic acid ester are separated by a commonly used separation operation such as a solvent extraction method, a crystal precipitation method, or a column chromatography method. The separated optically active 2-arylpropionic acid can be made into a product having a higher chemical purity and a higher optical purity by subjecting it to ordinary purification steps such as recrystallization. Further, the unreacted optically active 2-arylpropionic acid ester thus separated is hydrolyzed by a conventional method to obtain optically active 2 which is an antipode of the above acid.
-Aryl propionic acid. Furthermore, the above ester and acid can be easily racemized by the action of a strong base or the like, and can be reused as a raw material for the method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples.

Reference Example 1 Preparation of Cell Culture Solution Soluble starch 1.0%, polypeptone 0.5%, yeast extract 0.5%, sodium chloride 0.5%, magnesium sulfate 0.05%, manganese chloride 0. Klebsiella oxytoca SNSM- in a 3 liter jar fermenter containing 2.5 liters of a medium consisting of 001%, calcium chloride 0.05%, potassium monophosphate 0.2%, ammonium sulfate 0.2%, pH 6.0. 87 strains
953), and aerated and agitated at 37 ° C for 120 hours (4
(00 rpm) culture was performed to obtain a culture solution.

Reference Example 2 Collection of bacterial cells The bacterial cells were collected from the bacterial cell culture solution obtained by the same operation as in Reference Example 1 by centrifugation to obtain 36 g of wet bacterial cells.

Reference Example 3 Enzyme Collection Klebsiella oxytoca was added to a 30 liter jar fermenter containing 20 liters of the same medium as in Reference Example 1.
Inoculated SNSM-87 strain (Microtechnology Research Institute, No. 12953),
After culturing with aeration and stirring (300 rpm) at 37 ° C. for 92 hours, the bacterial cells were collected from the culture solution by centrifugation, and 393 g of the wet bacterial cells obtained were 54 g of monopotassium phosphate and EDTA · 2N.
a 1.5 g, TritonX-100 2.8 ml, and lysozyme chloride 1.2 g were dispersed in 3.9 liters of a liquid,
Lyse by leaving at 7 ° C for 24 hours, remove insoluble matter by centrifugation, add 35 (w / v)% ammonium sulfate to the supernatant, dissolve, and leave at 4 ° C overnight, then salt out by centrifugation. The substances are collected and 5 mM Tris-HCl buffer (p
H8.0), the salted-out product was dissolved, and dialyzed against the same buffer solution overnight to desalt, followed by vacuum freeze-drying.
0.8 g was obtained.

Example 1 Production of (S) -2- (4-iodophenyl) propionic acid (hereinafter abbreviated as IPP) Methyl (R, S) -2- (4-iodophenyl) propionate 29.01 g ( 100 mmol), the above reference example 3
2.0 g of the enzyme prepared in 1. was added to 100 ml of 0.3M potassium phosphate buffer (pH 8). The reaction solution was kept at 35 ° C for 2
After shaking and stirring for 4 hours, the mixture was centrifuged and a mixture of the ester and the generated acid was collected from the reaction solution. To this, 20 ml of toluene, 15 (w / v)% sodium carbonate aqueous solution 2
0 ml was added, and the mixture was stirred and separated. The aqueous phase was acidified with hydrochloric acid (pH 2 to 5), and the precipitated crystals were collected by filtration and dried to obtain 11.6 g of optically active (S) -IPP (yield 43%). H
The optical purity by PLC was 99% or more. HPLC analysis conditions Column: OPTIPAC TA (manufactured by Waters) Mobile phase: Hexane / IPA = 97.5 / 2.5 Flow rate: 1 ml / min. Detection: UV, 235nm

Example 2 Production of (S) -2- (4-iodophenyl) propionic acid 0.6 g of the bacterial cell wet product obtained in Reference Example 2 was suspended in 10 ml of 0.3 M potassium phosphate buffer to prepare a substrate. As (R, S) -2
Ethyl-(4-iodophenyl) propionate was added to 0.3
1 g (1 mmol) was added, and the mixture was stirred at 35 ° C. for 24 hours (pH 8.0). After that, by the same operation as in Example 1, (S)-
0.1 g (36% yield) of IPP was obtained. The optical purity by HPLC was 99% or more.

Example 3 Production of (S) -2- (4-iodophenyl) propionic acid Instead of methyl (R, S) -2- (4-iodophenyl) propionate as a substrate, (R, S)- Methoxyethyl 2- (4-iodophenyl) propionate 33.4 g
(S) -IPP 13.8 g (yield 50%) was obtained in exactly the same manner as in Example 1 except that (100 mmol) was used.
The optical purity by HPLC was 99% or more. Further, the organic phase was dried to dryness to obtain 16.5 g (yield 49%) of 99% optical purity of methoxyethyl (R) -2- (4-iodophenyl) propionate in the form of oil.

Example 4 Production of (S) -2- (4-iodophenyl) propionic acid Instead of methyl (R, S) -2- (4-iodophenyl) propionate as a substrate, (R, S)- 23.9 g of 2-chloroethyl 2- (4-iodophenyl) propionate
(S) -IPP12.4g (45% of yield) was obtained completely like Example 1 except having used (100 mmol).
The optical purity by HPLC was 99% or more.

Example 5 Production of (S) -2- (4-iodophenyl) propionic acid N, N, N, trimethylammoniumethyl chloride of (R, S) -2- (4-iodophenyl) propionic acid chloride 39.8 g (100 mmol) of salt and 2.0 g of the enzyme prepared in Reference Example 3 were added to 100 ml of 0.3 M potassium phosphate buffer (pH 8). The reaction solution was shaken and stirred at 35 ° C. for 24 hours, acidified with concentrated hydrochloric acid (pH 2 to 5), and then stirred at 100 ° C.
ml water was added and extracted with ether (200 ml, 2
Times). The obtained ether phases were combined, washed with water and saturated saline, dried over magnesium sulfate, and then the ether was distilled off to obtain 8.0 g of optically active (S) -IPP (yield 29%). The optical purity by HPLC was 99% or more.

Example 6 (S) -2- {4- [2-
Production of (3-methyl) thienyl] phenyl} propionic acid (hereinafter abbreviated as TPP) Methyl (R, S) -2- {4- [2- (3-methyl) thienyl] phenyl} propionate 2.6 g ( 10 mm
6 g of the microbial cell wet product obtained in Reference Example 2 was added to 100 ml of 0.3 M potassium phosphate buffer (pH 8). 3 reaction liquids
After shaking and stirring at 5 ° C. for 24 hours, the mixture was centrifuged, and a mixture of the ester and the generated acid was collected from the reaction solution. To this, 20 ml of toluene and 20 ml of a 15 (w / v)% sodium carbonate aqueous solution were added, and the mixture was stirred and separated. The aqueous phase was acidified with hydrochloric acid (pH 2 to 5), and the precipitated crystals were collected by filtration and dried to obtain 1.0 g of optically active (S) -TPP (yield 41
%). The optical purity by HPLC was 99% or more. HPLC analysis conditions Column: Chiralcel OD (manufactured by Daicel Chemical Industries, Ltd.) Mobile phase: Hexane / IPA = 94/6 Flow rate: 1 ml / min. Detection: UV, 272nm

Example 7 Production of (S) -2- (4-isobutylphenyl) propionic acid (abbreviated as IBU hereinafter) 100 ml of 0.3M potassium phosphate buffer solution 100 ml of the cell culture medium obtained in Reference Example 1 And 0.23 g of ethyl (R, S) -2- (4-isobutylphenyl) propionate (1
mmol) was added (pH 8.0), and the mixture was shaken and stirred at 35 ° C. for 24 hours. The reaction solution was centrifuged, and a mixture of the ester and the generated acid was collected from the reaction solution. To this, 2 ml of toluene, 2 m of 15 (w / v)% sodium carbonate aqueous solution
1 was added, and the mixture was stirred and separated. Acidify the aqueous phase with hydrochloric acid (p
H2-5) and the precipitated crystals were collected by filtration and dried to obtain 41 mg of optically active (S) -IBU (yield 20%). HP
The optical purity by LC was 99% or more. HPLC analysis conditions Column: ULTRON EV-OVM (manufactured by Shinwa Kako Co., Ltd.) Mobile phase: 0.02M potassium phosphate buffer (pH 3) / acetonitrile = 95/5 Flow rate: 1 ml / min. Detection: UV, 220nm

Example 8 Production of (S) -2- (4-iodophenyl) propionic acid 100 ml of 0.3M potassium phosphate buffer and 100 ml of the cell culture solution obtained in Reference Example 1 and (R, S)- Add 0.44 g of potassium salt of sulfooxyethyl 2- (4-iodophenyl) propionate (1 mmol) (pH 8.0), 3
The mixture was shaken and stirred at 5 ° C for 24 hours. The reaction solution was acidified with concentrated hydrochloric acid (pH 2 to 5), 100 ml of water was added, and the mixture was extracted with ether (200 ml, twice). The obtained ether phases were combined, washed with water and saturated saline, dried over magnesium sulfate, and then the ether was distilled off to obtain an optically active (S)-
55 mg of IPP was obtained (20% yield). The optical purity by HPLC was 99% or more.

Example 9 Production of (S) -4-nitrophenyl-2-propionic acid (hereinafter abbreviated as NPP) 25.3 g of methoxyethyl (R, S) -4-nitrophenyl-2-propionate as a substrate Optically active (S) -N in the same manner as in Example 1 except that (100 mmol) was used.
3.9 g of PP was obtained (20% yield). The optical purity by HPLC was 99% or more. HPLC analysis conditions Column: OPTIPAC TA (manufactured by Waters) Mobile phase: Hexane / IPA = 97.5 / 2.5 Flow rate: 1 ml / min. Detection: UV, 272nm

Example 10 Preparation of (S) -2- (4-aminophenyl) propionic acid (hereinafter abbreviated as APP) 2-chloroethyl (R, S) -2- (4-aminophenyl) propionate 22. 8 g (100 mmol) and 2.0 g of the enzyme prepared in Reference Example 3 were added to 100 ml of 0.3 M potassium phosphate buffer (pH 8). 35 ° C of reaction solution
Then, the mixture was shaken and stirred for 50 hours and then centrifuged, and a mixture of the ester and the generated acid was collected from the reaction solution. The obtained mixture was separated by a chromatograph (column: silica gel, mobile phase: hexane / ethyl acetate), and the optical activity (S)-
3.3 g of APP was obtained (20% yield). The optical purity by HPLC was 99% or more. HPLC analysis conditions Column: ULTRON EV-OVM (manufactured by Shinwa Kako Co., Ltd.) Mobile phase: 0.02M potassium phosphate buffer (pH = 5) /
Acetonitrile = 90/10 Flow rate: 1 ml / min. Detection: UV, 255nm

[0027]

As described above, according to the present invention,
An optically active 2-arylpropionic acid compound having high optical purity can be efficiently obtained.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kazutoshi Toyota 2-3-2 Muroya, Nishi-ku, Kobe-shi, Hyogo Nagase & Co., Ltd. (72) Inventor Kensaku Uzura 2-chome, Muroya, Nishi-ku, Kobe, Hyogo No. 3 Nagase & Co., Ltd. (72) Inventor Toshinori Onishi 2-3 2-3 Muroya, Nishi-ku, Kobe-shi, Hyogo Nagase & Co., Ltd.

Claims (3)

[Claims] 1. The general formula I: (In the formula, R ₁ is a halogen, a linear or branched lower alkyl group,
An amino group, a nitro group or a thienyl group having or not having a lower alkyl group as a substituent, R ₂ is a quaternary amino group or a salt thereof, a lower alkoxy group,
The oxysulfonyl group represents a lower alkyl group having or not having a group selected from the group consisting of salts and halogens thereof, and * represents an active center), and the optically active 2-arylpropionic acid ester or salt thereof is represented by A general formula II characterized in that an enzyme derived from a microorganism belonging to the genus Klebsiella is allowed to act on a mixture of both enantiomers to hydrolyze optically selectively. (Wherein R ₁ has the same meaning as described above and * represents an active center), and a process for producing an optically active 2-arylpropionic acid and an antipodal ester thereof. 2. The method according to claim 1, wherein the enzyme is derived from a microorganism belonging to Klebsiella oxytoca. 3. The enzyme is Klebsiella oxytoca SNSM-
The method according to claim 1 or 2, wherein the enzyme is derived from strain 87 (Microtechnology Research Institute, Microbiology No. 12953).