JPH08217760A - Novel antibiotic b-2607a - Google Patents

Abstract

PURPOSE: To obtain an antibiotic, B-4607A, which shows antibiotic effect against gram-positive and -negative bacteria in human and animals such as dogs and is useful as an antimicrobial against infections caused by a variety of bacterial. CONSTITUTION: The culture mixture of SANK 73794 strain in Vibrio separated from a hermit crab is inoculated to effect the solid culture, the shaking culture, or aeration agitation culture in a medium containing 1-10wt.%, based on the medium, of glucose or fructose as a carbon source, 0.1-6wt.% of soybean flour or wheat bran as a nitrogen source, preferably at 17-23 deg.C to obtain the compound given in the formula. In this culture, inorganic salts as nutrients are sodium or ammonium salts and silicone oil, vegetable oil or surfactants are used as a defoaming agent.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel compound B-4607A having an antibacterial activity, its production method and its producing strain.

[0002]

2. Description of the Related Art It is known that microorganisms belonging to the genus Vibrio separated from seawater produce, for example, bromodiphenyl ether having antibacterial activity (Experient).
ia 47 , 632 (1991)). However, bromodiphenyl ether is a bromobenzene compound. In addition, Maguneshijin that of marine bacteria Vibrio Gazogenesu (Vibrio gazogenes) to production (Magnesidin) are known (J.Antibiotics, 47,
257 (1994)). This compound is a triketone compound that chelate with magnesium.

A large number of derivatives having a phenazine skeleton as a mother nucleus, which B-4607A has, include synthetic products and natural products,
Examples of natural products having a phenazine-1-carboxylic acid structure include, for example, the antibiotics griseolutein A and B [J. Antibiotics Ser.A, 7 , 15 to 16 ( Streptomyces griseoluteus ) produced by Streptomyces griseoluteus. 1954); J. Antibio
tics Ser.A, 12 , 55 (1959); Chem.Pharm.Bull. 6 , 539〜543
(1958)], an antibiotic Saphenamycin produced by St. canarius [J. Antibiotics 35 , 141
2, (1982)] and Pseudomonas sp . ( Psendomonas sp .)
DC-86-M (J.An.
tibiotics 39 , 624, (1986)], and DOB-41 [J. Antibioti
cs, 41 , 589 (1988)] and the like are known. However, there is no compound having ester-linked glycine which is a characteristic of B-4607A.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have identified SANK 73794 belonging to the genus Vibrio isolated from hermit crabs.
Novel compound B-4607 having antibacterial activity from strain culture
The present invention has been completed by finding that A is produced.

[0005]

B-4607A of the present invention has the following structural formula and physicochemical properties.

[0006]

Embedded image

(1) Property of substance: orange-yellow powder (2) Molecular formula: C ₁₇ H ₁₅ N ₃ O ₅ (3) Molecular weight: 341 (measured by FAB-MS method) (4) High resolution mass spectrometry: C ₁₇ H ₁₆ N ₃ O ₅ [(M + H ⁺ ), FAB
-Measured by MS method] Actual value = 342.1060 Calculated value = 342.1090 (5) Elemental analysis (%): (Trifluoroacetate dihydrate (C
₁₇ H ₁₅ N ₃ O ₅・ CF ₃ COOH ・ 2H ₂ O)) Actual value = C: 44.47, H: 4.39, N: 8.54, F: 11.76 Calculated value = C: 44.97, H: 3.97, N: 8.28 , F: 11.23 (6) Infrared absorption spectrum: ν _max ^cm-1 potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below.

3306,2963,2647,1676,1603,1543,1460,144
0,1203,1135 (7) Ultraviolet absorption spectrum: λ _max ^nm (ε) The ultraviolet absorption spectrum measured in an aqueous solution is as shown below.

263 (25200), 350 (sh), 365 (3900) (8) High performance liquid chromatography: Separation column: Senshupack, ODS, H-2151, (column size φ6 × 150 mm) Senshu Kagaku Co., Ltd. Made.

Solvent: 25% CH ₃ CN / 0.2% trifluoroacetic acid solution Flow rate: 1.5 ml / min Wavelength: 220-400 nm (photodiode array detection) Retention time: 7.95 min (9) ¹ H-nuclear magnetic resonance spectrum: (Δ: ppm) Nuclear magnetic resonance spectrum (400MHz) measured using tetramethylsilane as an internal standard in deuterated dimethyl sulfoxide.
Is as shown below.

8.71 (1H, d, J = 8.0), 8.56 (1H, d, J = 9.2), 8.20
(1H, dd, J = 9.2, J = 8.0), 8.11 (1H, d, J = 8.0), 7.49 (1H, d, J =
8.0), 5.91 (2H, s), 4.16 (3H, s), 3.91 (2H, s) (10) ¹³ C-nuclear magnetic resonance spectrum: (δ: ppm) Dioxane (67.0 ppm The nuclear magnetic resonance spectrum (90 MHz) measured by using the above is shown below.

168.5 (s), 166.7 (s), 152.1 (s), 140.5 (s), 14
0.3 (s), 136.5 (d), 135.2 (s), 134.0 (d), 133.2 (d), 131.5
(d), 129.6 (s), 123.2 (s), 121.1 (s), 109.0 (d), 63.0 (t), 5
6.0 (q), 40.0 (t).

(11) Solubility: Soluble in water, methanol, dimethylsulfoxide, dimethylformamide, insoluble in ethyl acetate, chloroform, acetone, ethyl ether, hexane, etc. (12) Color reaction: Positive in ninhydrin reaction (13) thin layer chromatography: Rf value: 0.17 adsorbent: silica gel (Merck, Art.5715) developing solvent: CHCl ₃ -MeOH: (9 1) the present invention, the above SANK seventy-three thousand seven hundred and ninety-four strain producing B-4607A Hokkaido It is a marine bacterium isolated from hermit crabs collected on the coast of Abashiri City.

The mycological properties of the SANK 73794 strain, which is a B-4607A producing bacterium, are as follows.

1. Morphological properties When observed after culturing in a marine agar (manufactured by Difco) at 26 ° C. for 24 hours, the cells were 0.5 to 0.6 μm in diameter and 1.0 to 1.5 in length.
It has a rod shape of μm, has monopolar hairs, and moves. It does not sporulate and Gram stain is negative.

2. Growth state on marine agar (manufactured by Difco) Colonies cultured for 24 hours at 26 ° C. are somewhat brownish white, opaque and circular, flat, and full-edge. Does not form water soluble dyes.

3. Physiological properties (1) Growth in a medium containing no NaCl :-( 2) Growth in a medium containing NaCl: 0.5%: + (3) Growth in a medium containing NaCl: 10.0%: + (weak) (4) A medium was prepared by an OF (oxidative-fermentative) test [Hugh Leifson method] and artificial seawater. Glucose was degraded under aerobic and anaerobic conditions.

(5) Catalase: + (6) Oxidase: + (7) Reduction of nitrate: + (8) Hydrolysis of starch: + (9) Liquefaction of gelatin: + (10) Production of DNase: + (11 ) Lipase production:-(12) Growth temperature: Slightly grows at 7 ° C, 23 ° C-42 ° C.
Shows good growth. Grows slightly at 45 ° C, but 5
Does not grow at 0 ° C.

(13) Arginine dihydrolase :-( 14) Ornithine decarboxylase :-( 15) Lysine decarboxylase:-(16) Nutritional requirement :-( 17) Utilization of carbon compound: D-glucose + cellobiose- 3. D-mannose-lactose + L-arabinose-melibiose + D-mannitol + ethanol + rhamnose-mesoinositol-sucrose + L-serine +4. Chemotaxonomic properties (1) G + C (guanine + cytosine) content of DNA: 49.9
% (2) Quinone: Ubiquinone Q-8 SANK 73794 strain with the above-mentioned mycological properties is Vergiz
Manual of Systematic Bacteriology, 1
According to the classification of Volume (1984), it belongs to the genus Vibrio. However, the characteristics of SANK 73794 strain are
Manual of Systematic Bacteriology Volume 1 (1984) and the latest International Journal of Systematic Bacteriology (Int
It does not agree with the characteristics of the genus Vibrio described in the ernational Journal Systematic Bacteriology). Therefore, the present inventors have established the SANK 73794 strain as Vibrio.
rio) was identified as a new species.

This bacterium has been internationally deposited as a new species of the genus Vibrio in accordance with the Budapest Treaty, at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry (deposit number, (FERM
BP-4977): Original deposit date, January 25, 1995).

The SANK 73794 strain has been described above,
As is well known, the various properties of the fungi of the genus Vibrio are not constant, and are susceptible to mutations by natural or artificial operations (for example, ultraviolet irradiation, irradiation, chemical treatment, etc.). The 73794 strain is similar in this respect. The SANK 73794 strain referred to in the present invention includes all mutants thereof. Moreover, these mutants also include those obtained by genetic methods such as recombination, transduction, transformation and the like. That is, B- belonging to the genus Vibrio
The SANK 73794 strain, its mutants, and strains that are not clearly distinguished from them, which produce 4607A, are all included in the SANK 73794 strain.

In order to obtain the novel compound B-4607A according to the invention, the cultivation of these microorganisms is carried out in a medium such as that used for the production of other fermentation products. Such a medium contains a carbon source, a nitrogen source and an inorganic salt that can be assimilated by the microorganism.

Generally, as carbon sources, glucose, fructose, maltose, sucrose, mannitol,
Glycerol, dextrin, oats, rye, corn starch, potato, corn flour, soybean flour, cottonseed oil, molasses, citric acid, tartaric acid and the like can be used alone or in combination. Generally, the amount is variable at 1-10% by weight of the medium amount.

As the nitrogen source, a substance containing a protein is generally used in the fermentation process. Suitable nitrogen sources include soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate,
It is a nitrogen source for animal-based plants such as pharmamin, fish meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, and malt extract, plant-based extracts, and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate. The nitrogen sources are used alone or in combination in the range of 0.1-6% by weight of the medium amount.

The nutrient inorganic salts incorporated into the medium are usual salts capable of obtaining ions such as sodium, ammonium, calcium, potassium, magnesium, iron, phosphate, sulfate, chloride and carbonate. In addition, trace amounts of metals such as cobalt, manganese, and strontium, other bromides, fluorides,
Also included are salts for obtaining trace ions such as borates and silicates.

In liquid culture, defoaming agents such as silicone oil, vegetable oil, and surfactant are used.

Vibrio sp. SANK 73
The pH of the medium for culturing the 794 strain and producing B-4607A is 6.5.
It can be changed to -7.5.

The growth temperature of the bacterium is 7 ° C to 45 ° C, but the growth is good in the range of 17 ° C to 26 ° C.
17 ° C to 23 ° C is suitable for the production of 4607A.

B-4607A is obtained by aerobically culturing, but a commonly used aerobic culture method such as solid culture method, shaking culture method, aeration and stirring culture method is used.

For small-scale culture, from 17 ° C to 23 ° C
It is preferable to carry out shaking culture at ℃ for several days.

Culturing is initiated in a Erlenmeyer flask by 1-2 stages of seed development. The carbon medium and the nitrogen source can be used together in the medium at the seed developing stage. The seed flask is shaken in a constant temperature incubator, for example, at 20 ° C. for 1 to 3 days, or until it grows sufficiently. The grown seed is used to inoculate a second seed medium, or production medium. If an intermediate development step is used, it is grown in essentially the same way and partially used to inoculate the production medium. Inoculate flasks at constant temperature 1 to 3
Shake for days or until maximum production is reached, and at the end of incubation centrifuge or filter the contents of the flask.

In the case of large-scale culture, it is preferable to culture in a suitable tank equipped with a stirrer and an aeration device. According to this method, the nutrient medium can be prepared in the tank. The nutrient medium is heated to 125 ° C. to sterilize it, and after cooling, the sterilized medium is inoculated with the seed that has been previously grown. Culture is 17
It is carried out by aeration stirring at ℃ to 23 ℃. This method is suitable for obtaining large amounts of compounds.

B-4607A produced with the progress of culture
The change with time of the amount of can be measured using high performance liquid chromatography. Normally, the maximum production of B-4607A is reached after 19 to 96 hours of culture.

After the completion of the culture, B-4607A existing in the liquid portion of the culture medium and the cells is separated from the cells and other solid portions by a filtration operation or centrifugation using diatomaceous earth as a filter aid. It can be obtained by purifying B-4607A existing in the filtrate or supernatant and in the bacterial cells by utilizing its physicochemical properties. For example, B-4607A present in the filtrate or the supernatant is activated carbon or an adsorbing resin such as Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Co.), or DIAION HP-10. , HP-20, CHP-20, HP
-50 (manufactured by Mitsubishi Kasei) is used. B-4607A
The liquid containing B is passed through the layer of the adsorbent as described above to adsorb and remove impurities, or B-4607A is adsorbed and then eluted with methanol water, acetone water, n-butanol water, etc. Is obtained by In addition, B-4607A existing in the cells can be obtained by removing the organic solvent extracted with 50-90% water-containing acetone or water-containing methanol and then performing the same purification operation as that of the filtrate.

The B-4607A thus obtained was further subjected to adsorption column chromatography using a carrier such as silica gel, magnesium-silica gel florisil, Sephadex LH-20 (Pharmacia). It can be purified by partition column chromatography and high performance liquid chromatography using normal phase and reverse phase columns.

B-4607A can be separated and purified by repeatedly using the above-mentioned separation and purification means alone or in appropriate combination.

[0037]

The B-4607A of the present invention is a novel compound which has not been published in the literature and exhibits an antibacterial action against Gram-positive and Gram-negative bacteria in animals (eg, humans, dogs, cats, rabbits, etc.). , It is useful as an antibacterial agent against various bacterial infections.

When B-4607A of the present invention is used as a medicine, it can be administered in various forms according to a conventional method. The administration form is, for example, powder, granules, tablets, capsules, syrups, liquids, etc., orally or injectable (intravenous, intramuscular, subcutaneous), drip, suppository, ointment, ointment. It can be safely administered parenterally in the form of a drug. These various preparations are pharmaceutical preparations such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, solubilizers, suspension agents, coating agents, diluents, etc. It is possible to formulate with auxiliary agents known in the art. The dose varies depending on the disease to be treated, the route of administration, the number of administrations and the like, but for example, for adults, it is preferable to administer 20 mg to 2000 mg daily in one or several divided doses depending on the symptoms.

[0039]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto.

Example 1. B-4607A jar culture A) Culture Vibrio sp. SANK 737945 strain 23 on Marine Agar (Difco) Marine Agar Slant
Cultivation is carried out for 1 day at ℃, suspended in 3 ml of artificial seawater, and 0.1 ml of the suspension is aseptically inoculated into a 500 ml Erlenmeyer flask containing 100 ml of sterilized medium having the composition described below.
Culturing was carried out at 200 ° C. (rotating radius of 7 cm) on a rotary shaker for 24 hours. Fifteen ml of the bacterium was sterilized in two 30-liter jar fermenters containing 15 liters of aseptically sterilized medium having the same composition, and cultured at 23 ° C for 45 hours under aeration (7.5 liter / min) and stirring (100 rpm). did.

PSS-X medium Bactopeptone 10 g Sodium succinate 1 g (NH ₄ ) ₂ SO ₄ 1 g MgSO ₄ .7H ₂ O 1 g FeCl ₂ .nH ₂ O 2 mg MnSO ₄ .nH ₂ O 2 mg yeast extract 1 g artificial seawater 1 L pH 6 Adjust to pH 8) B) Isolate 30 liters of the obtained culture broth was adjusted to pH 3 with hydrochloric acid and separated into supernatant and cells by centrifugation. To the cells, 1 liter of acetone was added, stirred and filtered for extraction. Separated from the residue, the extract was added to the centrifugal supernatant and adsorbed on a column of Diaion HP-20 (4 liters), then washed with 10% acetone water,
Elution was performed with 50% acetone water. This was concentrated under reduced pressure, and finally freeze-dried to give an orange-yellow crude powder (56.6
g) was obtained.

Next, this crude powder was added to 0.2% containing 10% CH ₃ CN.
% TFA solution (500ml), Cosmo seal column
After adsorbing on 140 C ₁₈ -OPN (5 × 25 cm: manufactured by Nacalai Tesque, Inc.), 0.2% containing 20% to 25% CH ₃ CN.
Elution with a% TFA solution. Collect the active fractions,
Concentration and freeze-drying were performed to obtain an orange-yellow powder (420 mg) containing B-4607A.

To obtain a pure product, HPLC fractionation or column chromatography using LH-20 is most suitable.

Preparative HPLC is Senshupack ODS, H-4251
(φ10 × 250 mm) was used and developed with a 30% CH ₃ CN / TFA solution at 4 ml / min. The peaks from 14 minutes to 16 minutes were collected, concentrated and lyophilized, and isolated as a TFA salt of B-4607A.

The powder containing B-4607A was dissolved in methanol and developed with methanol using a LH-20 column (6.5 × 50 cm). The elution fractions of the activity peak were collected while monitoring by HPLC, concentrated, and finally dissolved in a small amount of water and lyophilized to obtain a pure product of B-4607A.

Test Example 1. Antibacterial activity of B-4607A The minimum inhibitory concentration (MIC) of B-4607A against general Gram positive and negative bacteria is normal agar medium (Eiken Chemical Co., Ltd.).
It was measured by the agar medium dilution method using

The results are shown in Table 1.

[0048]

[Table 1] ──────────────────────────────────── Minimum inhibitory concentration (MIC) ( μgm / ml) ──────────────────────────────────── Staphylococcus aureus 209P 0.4 Staphylococcus aureus 56R 0.8 Staphylococcus aureus 535 (MRSA) 0.8 Enterococcus faecalis 681 6.2 Escherichia coli NIHJ 0.4 Escherichia coli 609 0.4 Salmonella enteritidis 1.5 Klebsiella pneumoniae 806 6.2 Klebshera pneumera serrata serrata sera 157 1420 0.4 Morganella Morgany 1510 1.5 Pseudomonas Elginosa 1001 12.5 Pseudomonas Elginosa NO7 6.2 Pseudomonas El Ginosa 3719 12.5 ──────────────────────────────────── As is clear from Table 1, the B-4607A is It showed an excellent effect on general Gram positive and negative cells.

[0049]

From the above, the novel compound B-4607 of the present invention
A has an excellent antibacterial action and is useful as an antibacterial agent against various bacteria.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Hideyuki Shiozawa 1-25-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Hideji Takahashi 1-25-2 Hiromachi, Shinagawa-ku, Tokyo Sankyo stock company

Claims (4)

[Claims] 1. A B-4607A having the following structure: 2. A method for producing B-4607A, which comprises culturing a B-4607A-producing bacterium belonging to the genus Vibrio and collecting B-4607A from the culture. 3. The method according to claim 2, wherein the B-4607A-producing bacterium belonging to the genus Vibrio is a new strain of genus Vibrio (Vibrio sp.) SANK 73794 strain. 4. A Vibrio sp. SANK 73794 strain of the genus Vibrio having B-4607A producing ability.