GB2160866A - Preparing 2-arylpropionic acids - Google Patents

Abstract

A 2-(substituted phenyl)propionic acid is prepared using a micro-organism belonging to the genus Pseudomonas, which can convert a 1-(substituted phenyl)-1-phenylethane. The starting material may be of the formula:- <IMAGE> wherein R is C1-4 alkyl, CF3CH2CH2 or <IMAGE>

Description

SPECIFICATION Preparing 2-aryipropionic acids The present invention relates to a process for the production of 2-(substituted phenyl) propionic acids.

2-(Substituted phenyl) propionic acids have anti-phlogistic and analgesic activity in humans and animals. 2-(4-lsobutylphenyl) propionic acid, for example, has been widely used as an anti-inflammatory agent. Many chemical syntheses are known, such as those disclosed in GB-A-0971700, GB-A-1091403 and GB-A-1459084.

The known processes comprise many steps, and are generally complicated and troublesome. For instance, JP-A-75004040 discloses reacting isobutylbenzene with 5-methyidihydro-2(3H)-furanone in the presence of AICI3 at 130 C for 3 hours, reacting the resultant 4-(4-isobutylphenyl) pentanoic acid with copper diacetate in benzene and lead tetraacetate, and reacting the thus-obtained 3-(4-isobutylphenyl)-1butene with t-butyl alcohol and KM nO4., to give 2-(4-isobutylphenyl)propionic acid.

According to the present invention, a process for the production of a 2-(substituted phenyl)propionic acid ("arylpropionic acid" herein) comprises culturing a medium containing a 1-(substituted phenyl)-1phenylethane ("diphenylethane" herein) and a suitable micro-organism belonging to the genus Pseudomonas.

A bacterial species newly isolated from soil in Iwaki City, Fukushima-Prefecture, Japan, oxidises a diphenylethane to an arylpropionic acid, e.g. when inoculated into a culture medium. The species having this characteristic has been found to belong to the genus Pseudomonas, as a result of examination of its bacteriological properties, while referring to Bergy's Manual of Determinative Bacteriology, 8th Ed. and The Prokaryotes. After finding that the bacterium differs from the known Pseudomonas species in its capability of utilising a diphenylethane, it has been decided that the newly-isolated bacterial species is novel; it has been named Pseudomonas cepacia. A strain (KTB-03) of Pseudomonas cepacia was deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, on 25th May 1984, and has the deposition number FERM-BP 534.

The bacteriological properties of Pseudomonas cepacia are as follows: (1) Morphological properties: Shape of cell: rod-like Size of cell: 1.0 to 1.5 wm in width and 1.5 to 2.5 zm in length Pleomorphism of cell: none Motility: positive Number of flagellae: more than one Spore-formation: none Gram-staining: negative Acid-resistance: negative (2) Growth state (colour, lustre, appearance) Test Culture Test Re sults Bouillon-agar plate (1) productivity of pigment: (+) pale yellow pigment (2) lustre:(+) the surfaces of colonies were smooth Bouillon-agar slant (1) productivity of pigment: (+) pale yellow pigment (2) lustre:(+) the surfaces of colonies were smooth Bouillon liquid (1) did not grow Bouillon-gelatin stab (1) grown only on the surface of the medium (2) liquefaction of gelatin:(-) Litmus-milk (1) the appearance of the medium did not change, coagulation:(-) liquefaction::(-) (3) Effect of temperature on growth Temperature (C) Growth 4 (-) 10 (-) 30 (+) 37 (+) 41 (-) (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 Production of acid Production of gas L-arabinose (+) (-) D-xylose (+) (-) D-glucose (+) (-) D-mannose (+) (-) D-fructose (+) (-) D-galactose (+) (-) Maltose (+) (-) Sucrose (+) Lactose ( + ) (-) Trehalose (+) (-) D-sorbitol (+) (-) D-mannitol (+) (-) Inositol (+) (-) Glycerol (-) (-) Starch (-) (-) (6) Other characteristics Growth under aerobic conditions (+) Growth under anaerobic conditions (-) Hydrolysis of starch (-) Reduction of nitrate (-) Denitrification (-) MR-test (-) VP-test (-) Formation of indole (-) Formation of hydrogen sulphide (-) Utilisation of citric acid in Koser's culture medium (-) in Christensen's culture medium (-) Utilisation of nitrate (-) Utilisation of ammonium salt (+) Formation of pigment (+) yellow pigment Formation of urease (-) Formation of oxidase (+) Formation of catalase (+) O/F test (Hugh-Leifson's Method) oxidative In general, any artificial or natural mutant, and all micro-organisms belonging to the genus Pseudomonas, which produce an arylpropionic acid from a diphenylethane, can be utilised in the present invention.

In the cultivation, any carbon source which is usually used as a nutrient component may be used, but in order to make the bacteria produce the arylpropionic acid in high yield, the diphenylethane is used as the sole carbon source or one component of the carbon source. The diphenylethane is selected corresponding to the desired arylpropionic acid.

The concentration of diphenylethane as the carbon source is preferably 0.01 to 0.5% by weight The total amount of the carbon source may be added all at once or in several portions.

Preferred diphenylethanes have the formula

wherein R is straight-chain C14 alkyl, branched-chain C34 alkyl, CF3-CH2-CH2-or CF3-CH (CH3)-CH2-. More preferred starting materials include 1 -(4-methyl ph enyl )-1 -phenylethane, 1 -(4-ethyl phenyl)-1 -phenylethane, 1 -(4-propylphenyl)-1 -phenylethane, 1 -(4-isobutylphenyl)-1 -phenylethane, 1-[4-(3,3,3-trifluoropro pyl)phenyl]-1 -phenylethane and 1 -[4-(2-trifluoromethylpropyl)phenyl]-1 -phenylethane.

Examples of the nitrogen source to be used in the process according to the present invention are potassium nitrate, ammonium nitrate, ammonium sulphate, ammonium chloride, ammonium phosphate and polypeptone.

In addition, examples of the inorganic phosphorus and potassium sources used in the culture medium are disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate. Magnesium sulphate, calcium chloride and ferric chloride are examples of minor nutrients providing metal elements for the bacteria. Yeast-extract, cornsteep liquor and/or meat-extract, for example, are used for good bacterial growth.

Examples of suitable techniques for conducting the invention are shaking, stirring and submerged aeration culture, or a combination of two or more may be used, e.g. submerged aeration culture under stirring. The cultivation is suitably carried out at a temperature of 25 to 35 C and a pH of 6 to 8, e.g. for 3 to 35 days.

After cultivation, the cultured material may be subjected to filtration or centrifugation in order to remove the bacterial material. A supernatant containing the arylpropionic acid is obtained. The purified acid can be obtained by subjecting the supernatant to conventional procedures such as solvent-extraction, dehydration, decoloration, fractionation and column-chromatography, alone or in any combination.

For instance, after the cultivation, the cultured material is subjected to centrifugation. After acidifying the supernatant by adding an acid, if necessary, the object substance is extracted with an organic solvent such as dichloromethane. After subjecting the extract to dehydration and condensation, the condensate is subjected to silica gel chromatography, thereby eluting and fractionating the condensate, using a 7:1 (v/v) mixture of benzene and ethyl acetate as the eluent. The thus-obtained fraction, containing the object substance, is condensed, thereby obtaining the object product as crude crystals which are purified according to necessity, e.g. by recrystallisation.

The following Examples illustrate the invention.

Example 1 A composition was prepared from 65 mg K2HPO4, 26 mg KH2PO4, 134 mg Na2HPO4.12H20, 5 mg NH4CI, 68 mg MgSO4.7H20, 83 mg CaCI2, 0.8 mg FeCl3.6H20, 200 mg polypeptone, 300 mg yeast extract, one litre distilled water and 50 mg 1-[4-(3,3,3- trifluoropropyl)phenyl]-1-phenylethane (see JP-A-83029725). 100 ml of this aqueous liquid culture medium (pH 7.2) were introduced into a 300 ml conical flask. After sterilising the flask for 15 min at 121 C under pressure, Pseudomonas cepacia FERM-BP 534 was inoculated into the culture medium which was then cultured for 28 days at 30 C, while rotating and shaking the flask.

After removing the bacterial material from the cultured material by centrifugation, the pH of the supernatant was adjusted to below 2 by the addition of 6N aqueous hydrochloric acid, and product was then extracted with 100 ml chloroform. The extract was dissolved in 1N aqueous sodium hydroxide, 6N aqueous hydrochloric acid was added, and the solution was subjected to extraction with chloroform. By condensing the chloroform layer under reduced pressure to remove the solvent completely, a slightly viscous liquid, reddish-brown in colour, was obtained.

The melting point of the crystals was 75-76.5 C. They were identified as 2-[4-(3,3,3-trifluoropropyl)phenyl]propionic acid from a comparison of the NMR, infra-red absorption and mass spectra with those of an authentic specimen (see JP-A-83065237).

Example 2 Following the procedure of Example 1, but using 1-[4-(2-trifluoromethylpropyl) phenyj-1 -phenylethane (see JP-A-83029725) instead of 1-[4-(3,3,3-trifluoropropyl)-phenyl]-1-phenylethane as the starting material, 12 mg purified white crystals were obtained in a yield of 17% with respect to the starting material; 2-[4 (2-trifluoromethylpropyl)phenyl]propionic acid is the object compound.

Example 3 Following the procedure of Example 1, but using 0.1 g 1-(4-isobutylphenyl)-1-phenylethane instead of 0.05 g 1-[4-(3,3,3-trifluoropropyl)phenyl]-1-phenylethane as the starting material, 61 mg purified white crystals were obtained in a yield of 70.5% with respect to the starting material.

The melting point of the crystals was 75-77 C, and the NMR, infra-red absorption and mass spectra were the same as those of an authentic specimen of 2-(4-isobutylphenyl) propionic acid.

Example 4 After introducing 50 ml of the same culture medium as in Example 1 and 0.01 g 1-(4-isobutylphenyl)-1phenylethane into a 300 ml conical flask, and subjecting the medium to high pressure sterilisation for 15 min at 121 C, the same strain of Pseudomonas cepacia as in Example 1 was inoculated into the sterilised culture medium, and the inoculum was cultured for 3 days at 30 C while rotating and shaking the flask.

After subjecting the thus-cultured material to centrifugation, thereby removing the bacterial material, the supernatant was acidified to a pH lower than 2 by adding 6N aqueous hydrochloric acid. Product was then extracted from the supernatant with 50 ml chloroform. After condensing the chloroform layer under reduced pressure, 10 ml of the condensate (measured off accurately) were subjected to high-speed liquid chromatographic analysis. It was found that 4 mg 2-(4-isobutylphenyl)propionic acid were formed, a yield of 46% with respect to the starting material.

This is an example of short period culture.

Example 5 Following the procedure of Example 1, 0.2 g 1-(4-isopropylphenyl)-1-phenylethane was utilised in the culture, and converted into 2-(4-isopropylphenyl)-propionic acid. The amount of the product was 49 mg, corresponding to a yield of 28.6% with respect to the starting material.

Claims (7)

1. A process for the production of a 2-(substituted phenyl)propionic acid, which comprises culturing a culture medium containing a 1-(substituted phenyl)-1-phenylethane and a micro-organism belonging to the genus Pseudomonas and having the characteristic ability to convert a 1-(substituted phenyl)-1-phenylethane to a 2-(substituted phenyl)propionic acid.

2. A process according to claim 1, wherein the 1-(substituted phenyl)-1-phenylethane has the formula

wherein R is straight-chain C1w, alkyl, branched-chain C2A alkyl, CF3-CH2-CH2-or CF3- -CH(CH3)-CH2-.

3. A process according to claim 1 or claim 2, which is carried out at a temperature of 25 to 35 C and a pH of 6 to 8.

4. A process according to any preceding claim, wherein the culture medium contains from 0.01 to 0.5% by weight of the 1-(substituted phenyl)-1-phenylethane.

5. A process according to claim 1, substantially as described in any of the Examples.

6. Pseudomonas cepacia, FERM-BP 534.

7. A bacterium of the genus Pseudomonas cepacia, having the characteristic defined in claim 1.