CH664767A5 - METHOD FOR PRODUCING 2- (SUBSTITUTEDPHENYL) -PROPIONIC ACID BY MICROORGANISMS. - Google Patents

Description

DESCRIPTION The present invention relates to a process for the microbiological production of 2- (substituted-phen-15-nyl) -propionic acid from l- (substituted-phenyl) -l-phenylethane. The 2- (substituted phenyl) propionic acid has an anti-inflammatory effect and an analgesic activity in humans and animals. In particular, 2- (substituted-iertphenyl) propionic acid has been widely used as an excellent anti-inflammatory agent. To date, only chemical synthesis processes are known for their production. Japanese Patent Nos. 40-7491 (1965) and No. 47-18105 (1972) and Japanese Laid-Open No. 25- No. 50-4040 (1975) can be used as an example of such known chemical synthesis processes. These known methods generally comprise a large number of method steps, the execution of which is usually complicated and susceptible to faults; consequently, the known methods are not necessarily satisfactory. 30 The method disclosed in Japanese Patent Application Laid-Open No. 50-4040 (1975) includes, for example, the following process steps:

Isobutylbenzene +

II

A1C1,

he 0 CHj 130 ° C

i soBu- (C J> CH-CH2CH2COOH CH,

during 3 h

Cu (0C0CHj) 2 in CgHg

Pb (OCOCHj) 4

- ♦ isoBu

^ '<* H,

CH-CH = CH,

t-BuOH

¥ isoBu - <()> ÇH-C00H

- <o>

[2- (4-isobutylphenyl) propionic acid]

KMn04 CH j

It is the object of the present invention to provide a process for the preparation of 2- (substitutedphenyl) propionic acid which is not based on a chemical synthesis process in order to avoid the aforementioned complexity and the difficulties mentioned which are inherent in the known chemical synthesis processes are.

In the course of attempts to determine a method for producing 2- (substitutedphenyl) propionic acid using certain bacterial genera, it has been found that a specific bacterial genus that has recently been isolated from a soil in Iwaka City, Fukushima Prefecture, Japan, has been found Has the ability to oxidize l- (substitutedphenyl) -1-phenylethane in 2- (substitutedphenyl) propionic acid. This microbiological transformation

The novel process for the preparation of 2- (substitutedphenyl) propionic acid according to the invention was developed based on this method, which comprises fewer process steps than known chemical synthesis processes.

According to the present invention, the 60 process for the preparation of 2- (substitutedphenyl) propionic acid is distinguished by inoculating a microorganism into a nutrient medium which contains l- (substitutedphenyl) -l-phenylethane which is of the type of Pseudomonas belongs and has the ability to convert l- (substituted phenyl) -l-65 phenylethane to 2- (substituted phenyl) propionic acid by growing the microorganism on this medium and letting it produce 2- (substituted phenyl) propionic acid, and that the 2- (substituent)

3rd

664 767

iertphenyl) propionic acid collects and removes from the culture.

In the following the invention is described in detail.

The invention relates to a process for the preparation of 2- (substituted phenyl) propionic acid, in which bacteria which belong to the genus Pseudomonas and which have the ability to l- (substituted phenyl) -l-phenylethane 2- (sub-substituted phenyl) propionic acid to oxidize, inoculated into a nutrient medium containing l- (substitutedphenyl) -l-phenylethane. The substituent of 2- (substituted phenyl) propionic acid is the same as for l- (substituted phenyl) -l-phenyl-ethane. The inoculated bacteria are then grown, oxidizing l- (substitutedphenyl) -l-phenylethane to 2- (substituted-phenyl) propionic acid. Finally, the 2- (substitutedphenyl) propionic acid thus obtained is removed and collected.

The newly isolated bacteria used here, which are used in the present invention for converting l- (substituted-phenyl) -l-phenylethane into 2- (substituted-phenyl) -propionic acid, obviously belong to the genus Pseudomonas. This was recognized by examining their bacteriological properties, citing “Bergy's: Manual of Determinative Bacteriology, 8th Edition” and “The Prokaryotes”. After it was found that these bacteria differ from known bacteria of the genus Pseudomonas in that they use 1- (substitutedphenyl) -l-phenylethane, it was decided that these newly isolated bacteria should belong to a new genus called "Pseudomonas cepacia" has been. A strain of this bacterial genus “Pseudomonas cepacia” was obtained on May 25, 1984 under the number FERM-BP 534 from the Fermentation Research Institute, Agency of Industriai Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi, 1-chome, Yatabe-machi, Isubuka-gun, Ibaraki-ken 305, Japan.

The bacteriological properties of Pseudomonas cepacia are as follows:

(1) Morphological properties

Shape of a cell Size of a cell

Pleomorphism of a cell motility

Number of goats Formation of spores Fruit coloring Acid resistance rod-like

1.0 to 1.5 p.m width 1.5 to 2.5 (in length none positive greater than one no negative negative

Broth gelatin culture

Litmus milk culture

(1) Growth only on the surface of the medium

(2) Liquefaction of the gelatin: (-) (1) The appearance of the medium did not change, coagulation: (-) Liquefaction: (-)

(3) Effect of temperature on growth

Temperature (° C)

growth

4th

(")

15 10

(-)

30th

(+)

37

(+)

41

(")

20th

(4) Effect of pH on growth pH

growth

25 4.0

5.0

(-)

6.0

(+)

8.0

(+)

9.0

(-)

30 10.0

(-)

(5) Generation of acid and / or gas from carbohydrate

carbohydrate

Generation of acid

Generation of gas

L-arabinose

(+)

(\

D-xylose

(+)

(-)

D-glucose

(+)

(-)

D-mannose

(+)

(-)

D-fructose

(+)

(-)

D-galactose

(+)

(-)

Moltose

(+)

(-)

Sucrose

(+)

(-)

lactose

(+)

(-)

Trehalose

(+)

(-)

D-sorbitol

(+)

(* ")

D-mannitol

(+)

(-)

Inositol

(+)

(-)

Glycerol

(")

(-)

Strength

(")

(-)

(6) Other properties

(2) State of growth (color, gloss, appearance, etc. ^ Test criterion result

Experimental unit results

(Medium) growth in air (+

Growth under exclusion of air (-

Level (1) formation of pigments: (+) hydrolysis of starch (-

Broth agar culture pale yellow pigment eo reduction of a nitrate (-

(2) gloss: (+) the surface of denitrification (-

Colonies was smooth MR experiment (-

Inclined (1) formation of pigments: (+) VP test (-

Broth agar culture pale yellow pigment indole formation (-

(2) gloss: (+) the surface of it formation of hydrogen sulfide (-

Colonies were smoothly utilizing citric acid

Liquid (1) No growth in a culture according to Koser (-

Bouillon culture in a Christensen culture (-

664 767

4th

Utilization of a nitrate

(-)

Utilization of an ammonium salt

(+)

Formation of a pigment

(+) yellow pigment

Formation of urease

(-)

Formation of ÇMdÏS

(t)

Formation of catalase

(+)

O / F experiment after the oxidative

Hugh Leifson method

The bacterial genus Pseudomonas cepacia can be mentioned as a preferred example for use in the method according to the invention. On the other hand, however, all artificial or natural mutants of these bacteria as well as all other microorganisms belonging to the genus Pseudomonas and which produce 2- (substituted-phenyl) -propionic acid from l- (substituted-phenyl) -l-phenylethane can also be used in the context of the present invention Find.

Any carbon donor which is usually part of a nutrient medium can be used in the cultivation of the bacteria in the context of the present invention. However, in order to stop the bacteria from producing 2- (sub-substitutedphenyI) propionic acid in high yield, l- (substituted-phenyl) -l-phenylethane is advantageously used either as the only carbon donor or as part of the carbon donor. In this case, the l- (substitutedphenyl) -l-phenylethane is selected in accordance with the desired 2- (substitutedphenyl) propionic acid. In particular, the substituent or the substituent group of the l- (substitutedphenyl) -l-phenylethane should be the same as the substituent or the substituent group in the 2- (substitutedphenyl) propionic acid to be obtained.

The concentration of the l- (substitutedphenyl) -l-phenylethane used as the carbon donor is 0.01 to 0.5% by weight, this total amount being able to be added all at once or in portions.

A substance according to the following formula (I) can be given as l- (substitutedphenyl) -l-phenylethane:

in which R includes from the group

straight-chain alkyl groups with 1 to 4 carbon atoms,

Alkyl groups with branched chains and 3 or 4 carbon atoms,

- CF3-CH2CH2- and

- CF3-CHCH2-

I.

CHs is selected.

As starting materials, e.g. the following substances are suitable:

- 1 - (4-Methylphenyl) -1-phenylethane

- 1 - (4-ethylphenyl) -1-phenylethane

- 1 - (4-Propylphenyl) -1-phenylethane

- 1 - (4-isobutylphenyl) -1-phenylethane

- l- [4- (3,3,3-trifluoropropyl) phenyl] -l-phenylethane and

- 1 - [4- (2-Trifluoromethylpropyl) phenyl] -1-phenylethane nitrate, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate and polypeptone can be used.

In addition, as inorganic phosphorus and potassium donors in the nutrient medium, disodium hydrogen phosphate,

Raliumöilijdro ^ onpIiogDliflt, DïKaliumliydrogciiplioipIiat or similar use. Finally, magnesium sulfate, calcium chloride, iron chloride or the like are used as a small addition of metallic elements in the nutrient medium for the bacteria.

Other additives in the culture medium can be yeast extract, corn broth, meat extract and the like in order to promote good growth of the bacteria.

As a culture method for the bacteria, a culture with shaking, a culture with stirring, a forced-ventilation immersion culture or a combination thereof, e.g. a forced-ventilated, stirred diving culture or the like can be used.

The bacteria are preferably grown at a temperature of 25 to 35 ° C. and at a pH between 6 and 8 for a period of 3 to 35 days.

After the end of the cultivation, the 2- (substituted-phenyl) propionic acid-containing supernatant can be obtained by subjecting the grown material to filtration or centrifugation in order to remove the bacterial bodies therefrom. Finally, the purified acid is obtained by subjecting the supernatant to further processing which may include solvent extraction, dehydration, decolorization, fractionation and column chromatography, individually or in combination.

For example, one can proceed as follows: After the end of the cultivation, the grown material is subjected to centrifugation in order to obtain the supernatant fraction. If necessary, this is acidified by adding acid and the desired substance is washed with an organic solvent, e.g. Dichloromethane or the like, extracted. After the extract is dehydrated and condensed, the condensate is subjected to silica gel chromatography, the condensate being eluted and fractionated using a 7: 1 mixture of benzene and ethyl acetate as the eluent. The fraction thus obtained, which contains the desired substance, is condensed and the desired product is obtained in the form of crude crystals which can be purified by recrystallization if necessary.

Some exemplary embodiments of the method according to the invention are explained in more detail below.

example 1

In a conical flask with a capacity of 300 ml, a quantity of 100 ml of an aqueous, liquid nutrient medium with pH = 7.2 was filled with the following composition:

- 0.065 g

KzHPOt

- 0.026 g

KH2PO4

- 0.134 g

Na2HPC> 4- I2H2O

- 0.005 g

NH4CI

- 0.068 g

MgS04-7H20

- 0.083 g

CaCh

- 0.0008 g

FeCh-ôHîO

- 0.2 g

Polypeptone

- 0.3 g

Yeast extract

- 1 1

distilled water

- 0.05 g of l- [4- (3,3,3-trifluoropropyl) phenyl] -l-phenylethane

5

10th

15

20th

25th

30th

35

40

45

50

55

bO

65

As a nitrogen donor for growing the bacteria according to the present invention, without limitation, potassium can be made according to the teaching of Japanese Patent Application Laid-Open No. 58-29725 (1983).

5

664 767

After the flask was sterilized under pressure for 15 min at a temperature of 121 ° C., this solution was given Pseudomonas cepacia (strain no. KTB-03, under no. FERM-BP 534 from the Fermentation Research Institute, Agency of Industriai Science and Technology , Ministry of International Trade and Industry, 1-3 Higashi, 1-chome, Yatabe-machi, Isubuka-gun, Ibaraki-ken 305, Japan). The culture prepared in this way was kept at a temperature of 30 ° C. for 28 days, the flask being rotated and shaken regularly.

After removing the bacterial bodies from the cultivated material by centrifugation, the pH of the supernatant was reduced to below 2 by adding aqueous 6N hydrochloric acid, and the desired substance was extracted from the supernatant thus treated with the aid of 100 ml of chloroform. After this extract was dissolved in an aqueous 1N sodium hydroxide solution, this aqueous solution was acidified by further addition of hydrochloric acid, and this solution was subjected to extraction with chloroform again. By condensing the chloroform layer under reduced pressure

Example 2

The procedure in this example was the same as in Example 1, with the difference that l- [4- (2-trifluoromethylpropyl) phenyl] -l-phenylethane, prepared according to the teaching of Japanese Patent Application Laid-Open No. 58-29725 (1983) was used as the starting material instead of l- [4- (3,3,3, -Tri-

In order to completely remove the solvent, a slightly viscous, reddish-brown liquid was obtained.

This liquid was then subjected to silica gel chromatography using a mixture of benzene and ethyl acetate as an eluent so as to purify the liquid, and the resulting fraction was recrystallized from n-He-xan with the result that finally 27 mg of white crystals could be obtained. This corresponds to a yield of 61%, based on the starting material, 1 - [4- (3,3,3-trifluoropropyl) phenyl] -1-phenylethane.

The melting point of the crystals thus obtained was between 75 and 76.5 ° C., and the crystals could be identified as 2- [4- (3,3,3-trifluoropropyl) phenyl] propionic acid by examining the Nuclear magnetic resonance, the infrared absorption spectrum and the mass spectrum in comparison with an authentic pattern (cf. Japanese Patent Application Laid-Open No. 58-65237 [1983]).

The conversion of the starting material to the 2o substance obtained can be represented as follows:

fluoropropyl) phenyl] -l-phenylethane. 12 mg of purified white crystals were obtained. This corresponds to a yield of 27%, based on the starting material 1- [4- (2-trifluoromethylpropyl) phenyl] -1-phenylethane.

The conversion of the starting material to the substance obtained can be represented as follows:

3 ^> CHCH2- <S) -ÇH- {p)

CFj N 'CH3 N

Example 3

The procedure in this example was the same as in Example 1, with the difference that 0.1 g of l- (4-isobutylphenyl) -1-phenylethane instead of 0.05 g of 1- [4-3,3,3-trifluoropropyl) -phenyl] -l-phenylethane was used. 61 mg of purified white crystals were obtained. This corresponds to a yield of 70.5%, based on the starting material, l- (4-isobutylphenyl) -l-phenylethane.

The melting point of these crystals was between 75 and 77 ° C, and the nuclear magnetic resonance spectrum, the infrared absorption spectrum and the mass spectrum of these crystals were correspondingly the same as for an authentic 45 pattern of 2- (4-isobutylphenyl) propionic acid .

The conversion of the starting material to the substance obtained can be represented as follows:

3 ^ CKCH, -0) VcH- / 0> * ■ CHCH, - <("" "iVcH-COOH

Example 4

Example of breeding for a short period of time

50 ml of the same nutrient solution as in Example 1 and 0.01 g of l- (4-isobutylphenyl) -l-phenylethane were introduced into a conical flask with a capacity of 300 ml. After the nutrient medium had been sterilized under pressure for 15 min at 121 ° C., the same strain of Pseudomonas cepacia as in Example 1 was inoculated into the sterilized nutrient medium. The inoculum was kept at a temperature of 30 ° C for three days with the flask being rotated and shaken.

After the material thus grown was subjected to centrifugation to remove the bacterial bodies therefrom, the supernatant was removed by adding 6N salt.

55 acid brought to a pH below 2, and the desired substance was extracted from the thus treated supernatant with the aid of 50 ml of chloroform. After the chloroform layer had been condensed under reduced pressure, 10 ml of the condensate was measured exactly and subjected to a 60 high-speed liquid chromatography analysis. It was found that 4 mg of 2- (4-iso-butylphenyl) propionic acid had been formed. This corresponds to a yield of 46% with respect to the starting material, 1 - (4-isobutylphenyl) -1-phenylethane.

65

Example 5

In the same manner as in Example 1, 0.2 g of 1- (4-isopropylphenyl) -! - phenylethane was grown in the same culture

664 767 6

The strain of Pseudomonas cepacia according to Example 1 ver- The conversion of the starting material to the obtained one and converted into 2- (4-isopropylphenyl) propionic substance can be represented as follows:

changes. The amount of the product obtained was 49 mg,

which corresponds to a yield of 28.6%, based on the starting material, l- (4-isopropylphenyl) -l-phenylethane. 5

^ CH / OVçHX ^ O) ^ -— 3 ^ CH- / OVÇH-cooh ch3 ^ n— 'CH3N—' CH3 ^ 'CH3

G

Claims (4)

664 767 2nd PATENT CLAIMS 1. A process for the preparation of 2- (substituted phenyl) propionic acid, characterized in that inoculating in a nutrient medium which contains l- (substituted phenyl) -l-phenylethane, a microorganism which belongs to the type of Pseudomonas and which has the ability , l- (Substituted-iertphenyl) -l-phenylethane in 2- (SubstitutedphenyI) propionic acid to convert that the microorganism is grown on this medium and let it produce 2- (Substitutedphenyl) propionic acid and that so prepared 2- (substituted phenyl) propionic acid collects and removed from the culture. 2. The method according to claim 1, characterized in that the l- (substitutedphenyl) -l-phenylethane is a substance of the formula (I): in which R includes from the group straight-chain alkyl groups with 1 to 4 carbon atoms, Alkyl groups with branched chains and 3 or 4 carbon atoms, - CF3-CH2-CH2 and - CF3-CHCH2- I. CH3 is selected. 3. The method according to claim 1, characterized in that the microorganism is grown at a temperature of 25 to 35 ° C and at a pH of 6 to 8. 4. The method according to claim 1, characterized in that the concentration of l- (substitutedphenyl) -l-phenylethane in the nutrient medium is 0.01 to 0.5 wt .-%. (I) 10th