JPH05304973A - Production of carbostyril and/or 6-hydroxycarbostyril - Google Patents

Abstract

PURPOSE:To produce carbostyril and/or 6-hydroxycarbostyril from quinoline which is an inexpensive industrial raw material by utilizing the microbial enzymic reaction. CONSTITUTION:A microorganism, belonging to the genus Pseudomonas and having the ability to convert quinoline into carbostyril and/or 6- hydroxycarbostyril is cultured in a culture medium containing the quinoline as a main carbon source or the microorganism or its treated substance is brought into contact with the quinoline in an aqueous medium to extracellularly produce and accumulate the objective carbostyril and/or 6-hydroxycarbostyril. The produced and accumulated carbostyril or 6-hydroxycarbostyril or both are then collected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing carbostyril and / or 6-hydroxycarbostyril using an enzyme reaction of a microorganism. Carbostyril and / or 6-hydroxycarbostyril are compounds expected to be widely applied as synthetic intermediates of therapeutic agents effective for myocardial infarction and arrhythmia, which have a phosphodiesterase inhibitory action and / or a platelet aggregation inhibitory action.

[0002]

2. Description of the Related Art Conventionally, carbostyril and / or 6-
Hydroxycarbostyryl (a) is a method for producing carbostyril and / or 6-hydroxycarbostyril by alkali melting quinoline and / or 6-hydroxyquinoline with potassium hydroxide (J. Org. Chem., 36 , 3490 (19
71)), (b) A method for producing 6-hydroxycarbostyryl by N-oxidizing 6-hydroxyquinoline with peracid, rearrangement with acetic anhydride to give acetyloxycarbostyril, and then hydrolyzing it (J .Org.Chem., 33 , 1089
(1968), (c) p-anisidine was reacted with cinnamoyl chloride to obtain N-cinnamoyl-p-anisidine, and then this compound was cyclized by Friedel-Crafts reaction in the presence of Lewis acid to give 6-hydroxycarbostyryl. (Eur.J.Med.Chem.-Chim.The
r., 20 .121 (1985) ), a method for producing a carbostyril cyclized to (d) o-amino cinnamate (Ber.dt.Chem.Ges., 1
8,2395, (1885)) and the like are known.

However, in the methods (a) and (b), the danger of using peracid, the danger of melting the raw material with alkali, etc.
When the reaction is carried out on an industrial scale in addition to various adverse conditions, there is a great risk, and a decomposition reaction occurs, so that the target product can be obtained only in low purity and low concentration. Further, regarding the production of 6-hydroxycarbostyril, there is also a problem of difficulty in obtaining 6-hydroxyquinoline. (C), (d)
According to the method, raw materials are difficult to obtain, the reaction process is long, the cost is industrially large, and it is extremely economically disadvantageous. Regarding the action of microorganisms on quinoline, some reports have been made on the decomposition of quinoline from the viewpoint of preventing environmental pollution [Biol. Chem. Hopper-Seyler, 370, 1183 (1989)].
However, there is no report aiming at producing carbostyril and / or 6-hydroxycarbostyril using quinoline as a carbon source.

[0004]

An object of the present invention is to utilize a microbial enzyme reaction having a high selective specificity under mild temperature and normal pressure, from quinoline which is an inexpensive industrial raw material, with high selectivity. It is intended to provide a method for producing carbostyril and / or 6-hydroxy carbostyril.

[0005]

[Means for Solving the Problems] When the present inventors searched for a microorganism having the ability to oxidize quinoline from the natural world, it was recognized that it belongs to the genus Pseudomonas isolated from the soil of a tar chemical plant in Kitakyushu City, Fukuoka Prefecture. It was found that the strain has such an ability, and that culturing in a medium containing quinoline as a carbon source produces carbostyril and / or 6-hydroxycarbostyril in the culture, and completed the present invention. That is, the present invention cultivates a microorganism belonging to the genus Pseudomonas and having the ability to convert quinoline to carbostyril and / or 6-hydroxycarbostyril in a medium containing quinoline as a main carbon source, or The method comprises contacting with quinoline in an aqueous medium to produce and accumulate carbostyril and / or 6-hydroxycarbostyril outside the cell body, and then collecting the produced and accumulated carbostyril and / or 6-hydroxycarbostyril. The present invention relates to a method for producing carbostyril and / or 6-hydroxycarbostyril.

The microorganisms used in the present invention belong to the genus Pseudomonas and include carbostyril and / or 6-
Although any microorganism may be used as long as it has the ability to produce hydroxycarbostyril, a representative microorganism is Pseudomonas alcaligenes NSCC817 strain isolated from the soil by the present inventors. The NSCC817 strain has the following mycological properties. I. Morphological properties (1) Bacterial morphology: Bacillus (2) Size: 0.5-0.8 μm x 1.5-2.5 μm (3) Motility: Yes (4) Bengal hair: Extremely bent hair (5) Polymorphism: None ( 6) Spores: None (7) Gram stain: Negative II. Growth conditions in various media (1) Meat juice agar plate culture: Good growth, round, light yellowish brown (2) Meat juice agar slope culture: Good growth (3) Meat juice liquid culture: Good growth, cloudiness, (4) Meat juice stab culture : Grows on the surface layer, does not liquefy gelatin

III. Physiological properties (1) Reduction of nitrate: Positive (2) Denitrification reaction: Negative (3) Indole formation: Negative (4) Pigment formation: Negative (5) Catalase: Positive (6) Oxidase : Positive (7) DNase: Negative (8) Acylamidase: Positive (9) Lipase: Negative (10) Urease: Negative (11) Arginine dihydrolase: Negative (12) Lysine decarboxylase: Negative (13) Ornithine decarboxylase: Negative (14) β-Galactosidase: Negative (15) OF test: Does not decompose sugar (16) Growth at 41 ° C: Positive (17) Esculin hydrolysis: Negative (18) Growth in citrate medium: Negative (19) Range of growth: Temperature 25-41 ° C, pH 6
Grows at ~ 9. (20) Attitude toward oxygen: aerobic

(21) Carbon source assimilation D-glucose-D-fructose-D-xylose-D-galactose-D-mannitol-L-arabinose-D-mannose-L-rhamnose-maltose-lactose-sucrose-malon Acid-succinic acid + maleic acid + acetic acid + propionic acid + methanol-ethanol + testosterone-IV. Separation source Tar chemical factory soil in Kitakyushu City, Fukuoka Prefecture

Based on the above-mentioned mycological properties, Bergey's Manual of Systematic Bacteriologyvo
This strain was identified as Pseudomonas alcaligenes NSCC817 strain since it was identified by Lume 1) and it was in agreement with the mycological properties of Pseudomonas alcaligenes. This strain has been deposited in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 12586.

The production of carbostyril and / or 6-hydroxycarbostyril in the present invention may be carried out by culturing the above-mentioned microorganism in a quinoline-containing medium (hereinafter referred to as the first method). You may carry out by making it contact with quinoline in an aqueous medium (henceforth a 2nd method). First, the first method will be described. In culturing the present microorganism, an ordinary method for culturing a microorganism is used. As a carbon source, either a natural medium or a synthetic medium can be used as long as it is a medium containing quinoline, nitrogen source, inorganic salts, vitamins and other nutritional factors. The carbon source is preferably composed of only quininone, but in addition to this, a small amount of another assimilable carbon source, for example, an organic acid such as succinic acid, maleic acid, acetic acid or propionic acid can be appropriately used. The amount of the other carbon source used is preferably within the range that does not affect the productivity of the desired product, carbostyril and / or 6-hydroxycarbostyril, and is usually 5 to 100 parts by weight of quinoline.
It is preferably 50 parts by weight.

As the nitrogen source, ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, ammonium phosphate and other inorganic nitrogen sources, peptone, meat extract, corn steep liquor, yeast extract, casamino acid, soybean powder, etc., alone or in combination. Used.
It is also possible to utilize nitrogen in the quinoline molecule as a nitrogen source without adding a nitrogen source. As the inorganic salt, potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, iron sulfate, ferric chloride, zinc sulfate, copper sulfate, ammonium molybdate, boric acid, potassium iodide, sodium chloride and the like are appropriately selected. Can be added. Examples of nutritional factors necessary for growth include vitamins, amino acids, nucleic acids and salts thereof. Natural organic nutrients containing nutritional factors such as peptone, meat extract, corn steep liquor, yeast extract and casamino acid can also be added appropriately. A suitable medium for the production of carbostyril and / or 6-hydroxycarbostyril is prepared by mixing the above-mentioned medium components in appropriate amounts.

Optimally, the culturing is carried out by an aerobic culturing method, for example, a shaking culturing method or an aeration and stirring culturing method.
The liquid stationary culture method can be appropriately combined. The culture temperature is preferably 20 to 40 ° C, particularly 25 to 35 ° C, and the pH of the medium may be 6 to 9. The concentration of quinoline in the medium may be usually 0.03 to 0.3% (w / v), but preferably 0.
It is from 05 to 0.1% (w / v). Medium with quinoline as carbon source
When shake-cultured at about pH 7.0 at 30 ° C., carbostyril is produced and accumulated in the culture medium from about the first day of culture and from about the second day of culture. From this, it is considered that 6-hydroxycarbostyril is produced via carbostyril. When carbostyril is intended, the culture is terminated relatively early, and when 6-hydroxycarbostyril is intended, comparison is made. For a long period of time. The culturing is usually completed in 4 to 5 days, but is completed when the production amount of carbostyril and / or 6-hydroxycarbostyril reaches the maximum.

Next, the second method will be described. The second method is a method in which the microorganism or a treated product thereof is subjected to a catalytic reaction with quinoline in the presence of oxygen gas in an aqueous medium, for example, under the atmosphere or under aeration conditions, and growth of the microorganism may or may not occur. Examples of the processed products include immobilized cells and preparations of extracted enzymes from the cells.

In this reaction, the treated product such as the above-mentioned microorganism (which may be a culture solution of the above-mentioned microorganism or a concentrate thereof) or an immobilized bacterial cell of the above-mentioned microorganism, an enzyme extracted from the above-mentioned microorganism cell is treated in an aqueous medium. Quinoline is present in the suspended suspension to a pH of 6-9, 20-40 ° C, preferably 25-3.
By reacting at 5 ° C. usually with shaking or under aeration and stirring, carbostyril is carried out in 1 to 2 days, and 6 in 2 to 5 days.
-Hydroxycarbostyril can be mainly accumulated extracellularly. The concentration of quinoline in the reaction solution is 0.03 to 0.3% (w / v), especially 0.05 to 0.1% (w / v
v) is appropriate. The second method can also be carried out by continuously converting quinoline to a target substance by a bioreactor system using immobilized cells obtained by immobilizing the cells of the present invention by a conventional method.

In any of the first and second methods, the separation of carbostyril and / or 6-hydroxycarbostyril from the culture or reaction-terminated solution is usually carried out by separating the fermentatively produced organic compound from the culture solution. The method used may be used. For example, it may be carried out by a method usually used for separation from the culture broth after removal of bacterial cells. For example, the culture broth after removing the bacterial cells is extracted with a suitable organic solvent and concentrated under reduced pressure. Examples of the organic solvent that can be used during extraction include ethyl acetate, diethyl ether, acetone, butanol, and tetrahydrofuran. The concentrated solution is further subjected to TLC or the like to separate carbostyril and / or 6-hydroxycarbostyril. In addition, FD-MS, ¹
It can be performed by 1 H-NMR, IR, UV, melting point and the like.

[0016]

EXAMPLES Next, the method of the present invention will be specifically described by way of examples. Example 1 Seed culture was carried out by inoculating 10 ml of the medium shown in Table 1 containing 0.03 w / v% of quinoline in a thick test tube with Pseudomonas alcaligenes NSCC817 strain (Microtechnology Research Institute No. 12586) at 30 ° C. It was carried out by shaking culture for 2 days. For the main culture, a medium of the same composition containing 0.05 w / v% of quinoline was added to the culture medium.
It was carried out by dispensing 500 ml to an Erlenmeyer flask with L folds, inoculating 10 ml of the seed culture solution, and culturing at 30 ° C. for 24 hours with a rotary shaking culture machine at 150 rotations / min. The culture-finished solution was centrifuged at 8,000 rpm for 15 minutes to remove the cells,
When the supernatant was analyzed by HPLC, it was found that the supernatant contained 59 mg / L of carbostyril. The supernatant was extracted 3 times with 2 volumes of ethyl acetate. Combine the extracts and concentrate to dryness using a rotary evaporator. Dissolve the powder containing carbostyril in methanol and develop by TLC (toluene / ethyl acetate / acetic acid = 60/48/8) to obtain a spot corresponding to carbostyril. Was scraped off, extracted with ethyl acetate, and the extract was concentrated to dryness. FD-MS, ¹ H-NMR, IR,
It was confirmed to be carbostyril by UV and melting point. Finally, 12 mg of carbostyril was obtained per liter of culture supernatant.

Example 2 Seed culture was carried out by inoculating 10 ml of the medium shown in Table 1 containing 0.03 w / v% of quinoline in a large test tube with Pseudomonas alcaligenes NSCC817 strain (Microtechnology Research Institute No. 12586). Incubate at 30 ℃ for 24 hours, and further add 0.05w / quinoline
300 ml of a medium of the same composition containing v% was dispensed into a 2 L Erlenmeyer flask with folds, 20 ml of the test tube culture solution was inoculated, and 3
It was carried out by shaking culture at 0 ° C. for 24 hours in a rotary shaking culture machine. For the main culture, 2.7 L of quinoline 0.075 w / v% of the same composition was dispensed into a 5 L fermentor, 300 ml of seed culture was inoculated, 30 ° C., stirring speed 500 rpm, aeration rate
It was carried out by aeration and stirring culture at 0.5 VVM (L / L / min) for 85 hours.

The culture broth was centrifuged at 8,000 rpm for 15 minutes to remove the cells, and the supernatant was extracted three times with twice the volume of ethyl acetate. The extracts were combined, concentrated to dryness with a rotary evaporator, the powder containing 6-hydroxycarbostyril was dissolved in methanol, and the mixture was developed by TLC (toluene / ethyl acetate / acetic acid = 60/48/8) to give 6-hydroxy. A spot corresponding to carbostyril was scraped off, extracted with ethyl acetate, and the extract was concentrated to dryness. FD-MS, melting point, I
It was confirmed to be 6-hydroxycarbostyryl by R, UV and ¹ H-NMR. Finally 1 L of culture supernatant
2 mg of 6-hydroxycarbostyril were obtained each.

[0019]

[Table 1] Table 1. Medium composition _{─────────────────────── K 2 HPO 4 · 3H 2} O 0.79 g MgSO 4 · 7H 2 O 0.20 g FeSO 4 · 7H 2 O 0.02 g NaCl 1.00 g (NH ₄ ) ₂ SO ₄ 1.00 g H ₃ BO ₃ 0.50 mg CuSO ₄ / 5H ₂ O 0.04 mg KI 0.10 mg FeCl ₃ / 6H ₂ O 0.20 mg MnSO ₄ / 4H ₂ O 0.40 mg ZnSO ₄ / 7H _{2 O 0.40 mg (NH 4)} 6 Mo 7 O 24 · 4H 2 O 0.20 mg biotin 0.10 mg vitamin B ₁₂ 0.03 mg deionized water 1000 ml pH 7.2 ──────────────── ───────

[0020]

INDUSTRIAL APPLICABILITY According to the method of the present invention, carbostyril and / or 6-, which are useful as intermediates for the synthesis of pharmaceuticals, etc.
Hydroxycarbostyril can be produced from quinoline, which is an inexpensive industrial raw material, at room temperature and atmospheric pressure and with a high degree of purity with little by-products.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Pseudomonas and capable of converting quinoline to carbostyril and / or 6-hydroxycarbostyril is cultured in a medium containing quinoline as a main carbon source, or the microorganism or a treated product thereof is used. Contact with quinoline in an aqueous medium to produce and accumulate carbostyril and / or 6-hydroxycarbostyril outside the cell body, and then produce and accumulate carbostyril and / or
Alternatively, 6-hydroxycarbostyril is collected, and a method for producing carbostyril and / or 6-hydroxycarbostyril.