DE3523082A1 - METHOD FOR PRODUCING 2- (SUBSTITUYLPHENYL) -PROPIONIC ACID BY MICROORGANISMS - Google Patents

Description

description

The invention relates to a method for producing 2- (Substituylphenyl) propionic acid and in particular a process for the microbiological production of 2- (Substituylphenyl) propionic acid from 1- (substituylphenyl) -1-phenylethane.

2- (Substituylphenyl) propionic acid or 2 ~ (substituted? Phenyl) propionic acid has anti-inflammatory and analgesic activity in humans and animals, and in particular 2- (4-isobutylphenyl) propionic acid widely used as an excellent anti-inflammatory agent. For their preparation are only chemical synthesis processes known. These synthesis methods are described in, for example, Japanese Patent Publication Nos. 40-7491 (1965) and 47-18105 (1972), Japanese Patent Laid-Open 50-4040 (1975), etc., but the known methods generally involve many steps and their associated Actions are complicated and problematic; that is, the conventional methods are not necessarily satisfactory are.

For example, includes that described in Japanese Patent Laid-Open No. 50-4040 (1975) Procedure the following stages:

25 isobutylene +

AlCl.

CH ₃ . 130 ⁰ C 3 hours

isoBu- / OV ^CH ~ ^CH 7 ^CH 2 ^COOH

CH.

Cu (OCOCH ₃ ) ₂ in Pb (OCOCH ₃ ) ₄

t-BuOH

KMnO "

isoBu

(2- (4-1sobutylpheny1) propionic acid)

The object of the present invention is to provide a process for the preparation of 2- (substituylphenyl) propionic acid create, which is not based on a chemical synthesis process, to the complexity and the above mentioned To overcome difficulties of conventional chemical synthesis methods.

The task is solved by a method of production of 2- (Substituylphenyl) propionic acid, characterized by the stages of

Inoculating a microorganism belonging to the genus Pseudomonas and having an activity of converting 1- (substituylphenyl) -1-phenylethane to 2- (substituylphenyl) propionic acid into a culture medium containing 1- (substituyl) -1-phenylethane,

Culturing the microorganism therein, wherein the microorganism is used to produce 2- (substituylphenyl) propionic acid is caused, and isolation of the 2- (substituylphenyl) propionic acid so prepared from the so cultured material.

When investigating a process for the preparation of 2- (substituylphenyl) propionic acid using a special bacterial species was found that a soil in the city of IWAKA in the for the first time Prefecture FUKUSHIMA in Japan isolated bacterial species 1- (Substituylphenyl) -1-phenylethane to form 2- (Substituylphenyl) -propionic acid oxidized; based on this microbiological conversion, the inventive method for the production of 2- (Substituylphenyl) -propionic acid created, compared with the conventional chemical synthesis process involves fewer steps. 35

The present invention relates to a process for the production of 2- (substituylphenyl) propionic acid, characterized by the steps of inoculating bacteria belonging to the genus Pseudomonas and having an activity for converting 1- (substituylphenyl) ~ 1-phenylethane to 2- ( Substituylphenyl) propionic acid, the substituent of the latter being the same as the substituent of 1- (Substituylphenyl) -1-phenylethane, in a culture medium containing 1- (Substituylphenyl) -1-phenylethane, cultivation of the bacteria thus inoculated therein, the 1- (Substituylphenyl) -1-phenylethane is oxidized to 2- (Substituylphenyl) -propionic acid, and isolation of the 2- (Substituylphenyl) -propionic acid formed therein.

The bacteria isolated for the first time in the context of the invention and used according to the invention for the conversion of 1- (substituylphenyl) -1-phenylethane to 2- (substituylphenyl) propionic acid belong to the genus Pseudomonas , which is evident from an investigation of their bacteriological properties, with Bergy's manual of Determinative Bacteriology, 8th Edition and The Prokaryotes; Since the bacteria according to the invention are different from the known Pseudomonas species with regard to the use of 1- (substituylphenyl) -1-phenylethane, this bacterial species isolated for the first time is to be regarded as new; this new species has been given the name Pseudomonas Cepacia . A strain of Pseudomonas Cepacia has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, under the deposit number FERM-BP 534, with the deposit date of May 25, 1984.

Pseudomonas cepacia exhibits the following bacteriological characteristics:

(1) Morphological properties: shape of a cell: rod-shaped

Size of a cell: 1.0 to 1.5. Um width and

1.5 to 2.5 µm in length Pleomorphism of a cell: none Movement ability: positive Number of flagella: more than one

Spore formation: no Gram staining: negative Acid resistance: negative

(2) State of growth (color, luster, appearance, etc.)

5 test item (culture conditions) test results

Broth agar plate culture

Broth agar tilt culture

(1) Production of pigment: (+) pale yellow pigment

(2) Gloss: (+) the surface of the colonies was smooth.

(1) Production of pigment: (+) pale yellow pigment

(2) Gloss: (+) the surfaces of the colonies were smooth.

Liquid bouillon

(1) no growth

Broth-gelatin stab culture

(1) Growth only on the surface of the medium

(2) Liquefaction of the gelatin: (-)

Lack mus-Milch-Kul door

(1) The appearance of the medium did not change,

Coagulation: (-)
Liquefaction: (-)

(3) Effect of temperature on growth

Temperature (° C) growth

(4) Effect of pH on growth

pH growth

4.0 5.0 6.0 8.0

9.0

10.0

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

Carbohydrate Manufacture of acid Manufacture of gas
5

L-arabinose (+) (-)

D-xylose (+) (-)

D-glucose (+) (-)

D-mannose (+) (-)

D-fructose (+) (-)

D-galactose (+) (-)

Maltose (+) (-)

Sucrose (+) (-)

Lactose (+) (-)

Trehalose (+) (-)

D-sorbitol (+) (-)

D-mannitol (+) (-)

Inositol (+) (-)

Glycerine (-) (-)

Strength ( - ) ( - )

(6) Other properties

Test item test result 5

Growth under aerobic conditions (+)

Growth under anaerobic conditions (-)

Hydrolysis of starch (-)

Reduction of nitrate (-)

Denitration (-)

MR test (-)

VP test (-)

Formation of indole (-)

Formation of hydrogen sulfide (-)

5 Use of citric acid

in the culture medium according to Koser (-)

in the culture medium according to Christensen (-)

Use of nitrate (-)

Use of an ammonium salt (+)

Formation of pigment (+) (+) yellow pigment

Formation of urease (-)

Formation of oxidase (+)

Formation of catalase (+)

O / F test (Hugh-Leifson method) Oxidative

Although Pseudomonas Cepacia can be mentioned as an example of a bacterial species suitable according to the invention, any artificial or natural mutant thereof and all other microorganisms belonging to the genus Pseudomonas and producing 2- (substituylphenyl) propionic acid from 1- (substituylphenyl) -1-phenylethane , can be used in the present invention.

In the cultivation of the bacteria, any carbon source can be used in the present invention, which are usually is used as a nutritional component; but around the bacteria to produce 2- (Substituylphenyl) -propionic acid To cause high yield is 1- (Substituylphenyl) -1-phenylethane used as the sole carbon source or as a component of the carbon source. In In these cases, 1- (Substituylphenyl) -1-phenylethane is used selected according to the desired 2- (substituylphenyl) propionic acid. That is, the substituent or the Substituyl group in the 1- (substituylphenyl) -1-phenylethane used the same as the substituent or the substituyl group in the desired 2- (substituylphenyl) -propionic acid is.

The concentration of 1- (substituylphenyl) -1-phenylethane to be added as the carbon source is 0.01 to 0.5 wt%, and the total amount of the carbon source can be be added once or several times in proportional portions.

As the 1- (substituylphenyl) -1-phenylethane can be used by the formula I.

ORIGINAL INSPECTED

(D

reproduced compound are mentioned, where in the formula R is a radical group selected from the group consisting of straight-chain alkyl groups with 1 to 4 carbon atoms, branched alkyl groups with 3 to carbon atoms, CF -, - CH ₀ CH ₀ - and CF ₀ --CHCH ₀ - and

^{322 3} OH ²

The following compounds are exemplified as more preferred starting materials:

1- (4-methylphenyl) -1-phenylethane, 1- (4-Ethylpheny1) -1-phenylethane, 1 - (4-propylphenyl) -1-phenylethane, 1- (4-isobutylphenyl) -1-phenylethane, 1 - [4- (3,3,3-Trifluoropropyl) phenyl] -1 -phenylethane and 1 - [4- {2-trifluoromethylpropyl) phenyl] -1 -phenylethane.

According to the invention, potassium nitrate, ammonium nitrate, Ammonium sulfate, ammonium chloride, ammonium phosphate and polypeptone can be used without any limitation.

ORIGINAL INSPECTED

Furthermore, disodium hydrogen phosphate, KaJinm dihydrogen phosphate, dipotassium hydrogen phosphate and the like are used. Magnesium sulfate, Calcium chloride, ferric chloride and the like used as trace food metal elements for the bacteria.

Yeast extract, Corn steeple, meat extract and the like are used for beneficial growth of bacteria.

As a method of culturing the bacteria, shaken cultures, stirred cultures and inside can be used aerated cultures are called, and a method combining the above techniques can be used to Example, an internally aerated culture with stirring and the like can be used.

The cultivation is suitably carried out at a temperature of 25 to 35 C and a pH of 6 to 8 for 3 to Run for 35 days.

After cultivation is complete, it becomes 2- (substituylphenyl) propionic acid containing · supernatant obtained by subjecting the cultured material to a filtration or Subjects to centrifugation to remove the bacterial bodies. The purified acid can be obtained by subjecting the supernatant to a process that includes the steps of solvent extraction, dehydration, Decolorization, fractionation, column chromatography individually or in combination.

For example, after the cultivation is completed, the cultivated material is subjected to centrifugation in order to to obtain the supernatant fraction, and after acidifying the fraction by adding acid, if necessary the target substance is extracted with an organic solvent such as dichloromethane and the like. After Drying and evaporation of the extract, the residue is subjected to chromatography on silica gel, the Elute and fractionate residue using a 7: 1 mixture of benzene and ethyl acetate as the eluant will. The fraction thus obtained, which contains the target substance, is evaporated, with the desired product in raw crystals, which are purified by recrystallization, etc. as necessary.

The present invention is explained in more detail below with the aid of examples.

example 1

100 ml of an aqueous liquid culture medium (pH = 7.2) composed of 0.065 g of K ₂ HPO-, 0.026 g of KH ₂ PO ₄ , 0.134 g of Na ₂ HPO ₄ .12H ₂ O, 0.005 g NH ₄ Cl, 0.068 g MgSO ₄ .7H ₂ O, 0.083 g CaCl ₃ , 0.0008 g FeCl ₃ -OH ₂ O, 0.2 g polypeptone, 0.3 g yeast extract and 1 l of distilled water and 0.05 g of 1 - [4- (3,3,3-trifluoropropyl) phenyl] -1-phenylethane, which had been synthesized according to JP-OS 58-29725 (1983), introduced and after sterilizing the flask for 15 minutes At 121 ° C. under pressure, Pseudomonas Cepacia (strain No. KTB-03, deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan under the deposit number FERM-BP 534) was inoculated into the culture medium thus prepared and for 28 days cultivated at 30 ⁰ C with turning and shaking the flask.

After removing the bacterial bodies from the medium cultivated in this way by centrifugation, the pH of the The supernatant was adjusted to less than 2 by adding 6N aqueous hydrochloric acid and removed from the so treated The supernatant became the target substance with 100 ml of chloroform extracted. After dissolving the extract in an aqueous 1N hydroxide solution, the aqueous solution was repeated by Addition of hydrochloric acid acidified and the solution was subjected to one more extraction with chloroform subject. By evaporation of the chloroform phase at reduced pressure for the complete removal of the A slightly viscous liquid of reddish brown color was obtained.

The liquid thus obtained was subjected to silica gel chromatography using a mixture of benzene and ethyl acetate as an eluent to purify the liquid, and the fraction thus obtained was recrystallized from η-hexane, whereupon 27 mg of white crystals in a yield of 61% based on the Starting material 1 - [4- (3,3,3-Trifluorpropy 1) pheny I] -1-phenylethane were obtained.

The melting point of the crystals thus obtained was 75 to 76 _f 5 ° C, and the obtained crystals were identified as 2- 1 ⁴ ~ ( ³ ' ³ ' 3-Tr if luoropropyl) phenyl] propionic acid identified (see JP-OS 58-65237 (1983)).

The conversion of the starting material into the target substance is shown below:

CF ₃ CH ₂ CH ₂

CF ₃ CH ₂ CH ₂

Example 2

By cultivating the bacteria in the manner described in Example 1 with the difference that instead of 1- [4- (2-trifluoropropyl) phenyl] -1-phenylethane from Example 1 as starting material 1 - [4- (2-trifluoromethylpropyl) -phenyl] -1-phenylethane _f which had been synthesized according to JP-OS 58-29725 (1983) was used, 12 mg of purified white crystals were obtained in a yield of 27% based on the starting material H 4- (2- Triflormethylpropyl) -phenyl] -1-phenylethane.

The conversion of the starting material into the target substance is shown below.

³ - CHCH. -iOh CH- (O

CF

CH.

CF.

CHCH

ORlGiNAL INSPECTED

Example 3;

By cultivating the bacteria on the in Example 1 described way with the difference that instead of the 0.05 g of H4- (3,3,3-trifluoropropyl) phenyl] -1-phenylethane from Example 0.1 g of 1- (4-isobutylphenyl) -1-phenylethane was used, a 61 mg of purified white was obtained Crystals in a yield of 70.5% based on the starting material 1- (4-isobutylphenyl) -1-phenylethane.

The melting point of the crystals thus obtained was 75 bis 77 ° C and its NMR spectrum, infrared absorption spectrum and The mass spectrum was consistent with that of an authentic sample of 2- (4-isobutylphenyl) propionic acid.

The conversion of the starting material into the target compound is shown below.

CH.

CH.

■ CHCH

Example 4 Example of a short-term culture

After introducing 50 ml of the culture medium described in Example 1 and 0.01 g of 1- (4-isobutylphenyl) -1-phenylethane into a 300 ml Erlenmeyer flask and subjecting the medium to high pressure sterilization for 15 minutes at 121 ° C, the same strain of Pseudomonas Cepacia was inoculated into the culture medium sterilized in this way as in Example 1, and the inoculum was cultivated for 3 days at 30 ° C. while rotating and shaking the flask.

After the thus cultured material was centrifuged, whereby the bacterial bodies were removed, the supernatant became acidified to pH less than 2 by the addition of 6N hydrochloric acid and then the Target substance extracted with 50 ml of chloroform. After evaporation of the chloroform phase under reduced pressure accurately measured 10 ml of the residue by high speed liquid chromatography analysis subjected, and it was found that 4 mg of 2- (4-isobutylphenyl) propionic acid in a yield of 46% based on the starting material 1- (4-isobutylphenyl) -1-phenylethane had been formed.

Example 5

In the manner described in Example 1, 0.2 g of 1- (4-isopropylphenyl) -1-phenylethane was used in the culture of the same strain of Pseudomonas Cepacia as in Example 1 and converted to 2- (4-isopropylphenyl) propionic acid. The amount of the product was 49 mg, corresponding to a yield of 28.6% based on that

Starting material 1- (4-isopropylphenyl) -1-phenylethane.

The conversion of the starting material into the target compound

is shown below.

CE

CH

CH.

CH:

CH-COOH CH "

ORIGINAL INSPECTED

Claims (2)

Claims 1. A process for the preparation of 2- (Substituylphenyl) -pro-, pionic acid, characterized in that one ^J microorganism belonging to the genus Pseudonomas and ^J -an activity for the conversion of 1- (Substituylphenyl) -1-phe- . nylethane to 2- (Substituylphenyl) propionic acid, in a 0.01 to 0.5 wt.% 1- (Substituylphenyl) -1-phenylethane culture medium containing inoculated and the microorganisms thus inoculated at a temperature of 25 Cultivated up to 35 ° C and a pH value of 6 to 8. 2. The method according to claim 1, characterized in that there is one as 1- (substituylphenyl) -1-phenylethane Compound of formula (I) (D is used in which R is a radical group selected from the group consisting of straight-chain alkyl groups with 1 to 4 carbon atoms, branched alkyl groups with 3 to 4 carbon atoms, CF ₃ -CH ₂ CH ₂ - and CF ₃ -CHCH ₂ -. CH ₃