JPH0578355A - B-1015 compound - Google Patents

Abstract

PURPOSE:To provide a new compound B-1015 having antibacterial activity and derived from the cultured product of Alcaligenes faecalis SANK 74291 strain separated from coronated turban shell (Lunnella coronata). CONSTITUTION:The B-1015 compound expressed by formula. The compound of formula can be produced by culturing a strain belonging to the genus Alcaligenes and capable of producing B-1015 compound [Alcaligenes faecalis SANK 74291 (FERM BP-3551)] and separating from the cultured product.

Description

Detailed Description of the Invention

[0001]

The present invention relates to B-101 having an antibacterial action.
5 It relates to a compound and a method for producing the same.

[0002]

2. Description of the Related Art Heretofore, compounds having a pyrazolopyridine skeleton have not been obtained from microorganisms. Slightly different bacteria from the genus Alcaligenes sp. (UC91252) and Streptomyces plicatu
s (UC8272)] is known to produce nitrosofungin (Nitrosofungin) having an antifungal effect [J. Antibiotics, 36, 1425-1430, (1983
Year)] is Alcaligene
s faecalis) has not been reported to produce compounds with antibacterial activity.

[0003]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have found that Alcaligenes faecalis SANK 7 isolated from Lunella coronata (GMELIN)
Novel compound B with antibacterial activity from 4291 strain culture
The present invention was completed by finding that a compound was produced.

[0004]

The B-1015 compound of the present invention has the following structural formula (I) and physicochemical properties. 1) Structural formula

[0005]

[Chemical 2]

2) Property of substance: acidic yellow powder 3) Molecular formula: C ₇ H ₈ N ₂ O ₅ S (high resolution negative
(Measured by FAB-MS) 4) Molecular weight: 232 (Measured by high resolution negative FAB-MS) 5) Elemental analysis value: (%) Calculated as C ₇ H ₈ N ₂ O ₅ S monohydrate monoammonium salt Value C 31.46, H 4.90, N 15.72, S
11.99 Measured value C 36.22, H 5.01, N 16.73, S
9.73 6) Infrared absorption spectrum: ν _max cm ^-1 Potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below. 3190, 1656, 1615, 1589, 1495, 1401, 1309, 1214, 11
15, 1047, 888, 827, 635 7) Ultraviolet absorption spectrum: λ _max nm (ε) The ultraviolet absorption spectrum measured in an aqueous solution is as shown below. Neutral water: 245 (4800), 345 (13400) Acidic water: 238 (4800), 320 (9300), 345 (Sh) Alkaline water: 260 (400), 350 (10200), 390 (9900)
, 420 (Sh) 8) Specific rotation: [α] _D ²⁵ = -10.1 ° (C 0.93, water) 9) High performance liquid chromatography: Separation column; Senshupack ODS H-2151 (column size φ 6 x 150 mm , Senshu Kagaku Co., Ltd.) Solvent: Water containing 16% acetonitrile-0.5% Pic A (manufactured by Waters) Flow rate: 1.5 ml / min Wavelength: 220-350 nm (detection by photodiode array) Retention time: 8.1 min 10) ¹ H-nuclear magnetic resonance spectrum: (δ: ppm) A nuclear magnetic resonance spectrum (270 MHz) measured by using tetramethylsilane as an external standard in heavy water is as shown below. 7.49 (H, d, J = 8.8 Hz), 6.92 (H, d, J = 2.9 Hz), 6.75 (H, dd, J
₁ = 8.8 Hz, J ₂ = 2.9 Hz), 4.82 (2H, s) 11) ¹³ C-nuclear magnetic resonance spectrum: (δ: ppm) Nuclei measured in deuterated methanol using tetramethylsilane as an internal standard Magnetic resonance spectrum (100 MHz)
Is as shown below. 162.0 (s), 144.0 (s), 140.4 (s), 119.4 (d), 115.9 (d),
115.3 (d), 59.5 (t) 12) Solubility: Soluble in water, methanol, dimethylsulfoxide and dimethylformamide, insoluble in ethyl acetate, acetone, chloroform and hexane. 13) Color reaction: positive for iodine.

The B-1015 compound of the present invention can be converted into a salt according to a conventional method. Examples of such salts include salts of alkali metals such as lithium, sodium and potassium; salts of alkaline earth metals such as calcium and barium; aluminum salts; salts of basic amino acids such as lysine and arginine; ammonia, Examples thereof include amine salts such as methylamine and dimethylamine. It is preferably a pharmacologically acceptable salt.

The B-1015 compound of the present invention has various isomers. In formula (I), these isomers and mixtures of these isomers are all represented by a single formula. Therefore, the present invention includes all of these isomers and a mixture of these isomers.

The above S for producing the B-1015 compound of the present invention
The ANK74291 strain was obtained from Lunella coronata (GMELIN), a mussel that was collected on the coast of Hachimanno, Ito City, Shizuoka Prefecture. ) 0.1 g, agar
Bacteria isolated using 15 g of artificial seawater (1000 ml, pH 7.4).

SANK 74291, a B-1015 compound producing bacterium
The mycological properties of the strain are as follows. 1. Morphological properties on ordinary agar medium (manufactured by Eiken Chemical Co., Ltd.) at 26 ° C, 24
The cells were 0.8 μm in diameter and
It is a bacillus with a diameter of 1.0-2.0 μm, has pericardial hair, and moves.
It does not sporulate and Gram stain is negative.

2. Growth on normal agar medium Colonies cultured at 26 ° C for 24 hours were light yellowish-brown white, round, flat, and full-edge, and the medium had a slightly pale olive color due to the formation of water-soluble pigment. Present. When the culture is continued, expansion of colonies due to movement on the surface of the medium is observed.

3. Physiological properties (1) Seawater requirement: Seawater is not required for growth. (2) Requirement of NaCl: No NaCl is required for growth. (3) OF (oxidative-fermentative)
Test [Hugh Leifson Method]: D
-Does not decompose glucose and D-xylose. (4) Catalase: + (5) Oxidase: + (6) Behavior toward oxygen: aerobic (7) Reduction of nitrate:-(8) Reduction of nitrite: + (9) Hydrolysis of starch:-(10 ) Urease hydrolysis :-( 11) Tween 80 degradation :-( 12) Gelatin liquefaction :-( 13) Growth temperature: It grows weakly at 5 ° C and 23-37.
It shows good growth at ℃. Grows weakly at 42 ℃, 50
Does not grow at ℃. (14) Utilization of carbon compounds: Iizuka and Komagata's medium (edited by Takeharu Hasegawa, “Classification and Identification of Microorganisms (below)”, 133-134)
The results were as follows when 0.2 g of yeast extract was added to the page). L-arabinose: -D-xylose: -D-glucose: -D-fructose: -D-mannose: -D-mannitol: -sodium acetate: + calcium gluconate:
-Glycine: + (15) Chemotaxonomic properties 1) G + C (guanine + cytosine) content of DNA: 56.2 mol% (HPLC method). 2) Quinone system: Ubiquinone Q-8.

The SANK 74291 strain having the above-mentioned mycological properties was analyzed by Bergey's Manual of Systematic Bacteriology.
eriology), Volume 1 (1984) and Akagawa
And Yamasato's report (International
International Journal of Systematic Bacteriolog
y), Vol. 39, pp. 462-466 (1989)). As a result, it was a Gram-negative aerobic bacillus with pericardium, G + C of DNA was 56.2 mol%, and quinone was ubiquinone Q-8.
faecalis SANK 74291) strain (depositor, Institute of Industrial Technology, Institute of Microbial Science and Technology: Deposit No., Microtech Lab. 3551
No., FERM BP-3551: Original deposit date, September 6, 1991).

To obtain the B-1015 compounds of the present invention, culturing of these microorganisms is carried out in a medium such as those used to produce other fermentation products. Such a medium contains a carbon source, a nitrogen source and an inorganic salt that can be assimilated by microorganisms.

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 varies with 1-10% by weight.

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 oil, cottonseed flour, casein hydrolyzate, pharmamine, fish meal, corn steep liquor,
Peptone, meat extract, yeast, yeast extract, malto extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like. The nitrogen sources are used alone or in combination within the range of 0.2-6% by weight of the medium amount.

The nutrient inorganic salts incorporated into the medium are ordinary salts capable of obtaining ions such as sodium, ammonium, calcium, phosphate, sulfate, chloride and carbonate. It also contains trace amounts of metals such as potassium, calcium, cobalt, manganese, iron and magnesium.

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

Alkaline Genes faecalis
The pH of the medium for culturing the nes faecalis) SANK 74291 strain and producing the B-1015 compound can be changed to 5.0-7.4.

The growth temperature of the bacterium is 4 ° C. to 42 ° C., but the range of 23 ° C. to 37 ° C. shows good growth.
20 ° C to 28 ° C is suitable for the production of the 1015 compound.

The B-1015 compound can be obtained by aerobically culturing, but a commonly used aerobic culturing method, for example, a solid culturing method,
The shaking culture method, aeration stirring culture method and the like are used.

For small-scale culture, from 22 ° C to 26 ° C
It is preferable to carry out shaking culture at ℃ for several days. Culture is initiated in an Erlenmeyer flask with 1-2 stages of seed development. The carbon medium and the nitrogen source can be used together in the medium of the seed developing stage. Seed flask in a constant temperature incubator 23
Shake at ℃ 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. Shake the inoculated flask at constant temperature for 1-2 days, and after 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 sterilized by heating to 125 ° C and, after cooling, the sterile medium is inoculated with the pre-grown seeds. Culture is 22 ℃
Aeration-stirring is performed at a temperature of up to 26 ° C. This method is suitable for obtaining large amounts of compounds.

The change with time in the amount of B-1015 compound produced with the progress of culture can be measured using high performance liquid chromatography. Usually 19 hours to 4
The maximum production of B-1015 compound is reached after 8 hours of culture.

After the completion of the culture, the B-1015 compound present in the liquid portion of the culture medium is separated from the bacterial cells and other solid portions by a filtration operation or centrifugation using diatomaceous earth as a filter aid, and the filtrate is obtained. Alternatively, it can be obtained by extracting and purifying the B-1015 compound present in the supernatant by utilizing its physicochemical properties. For example, B- present in the filtrate or supernatant
1015 compounds are, for example, activated carbon or adsorbent resins such as Amberlite XAD-2 and XAD-4 (produced by Rohm and Haas), Diaion HP-10 and HP.
-20, CHP-20, HP-50 (manufactured by Mitsubishi Kasei Co., Ltd.) and the like are used. The liquid containing the B-1015 compound is passed through the layer of the adsorbent as described above to adsorb and remove impurities, or
After adsorbing the B-1015 compound, it can be obtained by elution with methanol water, acetone water, n-butanol water or ammonia-added methanol, acetone water, n-butanol water and the like.

Further, it can be purified by being adsorbed and desorbed on an anion or cation exchanger by utilizing its behavior as an acidic substance. Examples of anion exchangers are DEAE-cellulose (Brown), DEAE-Sephadex, QAE-Sephadex (Pharmacia), Duolite A-
2 (made by Diamond Shamrock Chemical Co., Ltd.), Amberlite IRA-68 (made by Rohm and Haas Co., Ltd.),
Dowex 1 × 4, 21K, SBR-P (manufactured by Dow Chemical Co.), etc. can be used. As a cation exchanger, Amberlite IRC-50 (made by Rohm and Haas),
Dowex 50 W (manufactured by Dow Chemical Co.) can be used. In addition, by utilizing the properties of acidic substances as described above,
A method in which a quaternary ammonium salt such as dimethyl benzyl cetyl ammonium chloride is dissolved in a solvent immiscible with water, for example, dichloromethane, and the B-1015 compound is extracted as a quaternary ammonium salt from an aqueous solution is also used.

The B-1015 compound thus obtained is further subjected to adsorption column chromatography using a carrier such as silica gel, magnesium-silica gel florisil, Sephadex LH-20 (Pharmacia) and the like. It can be purified by conventional partition column chromatography and high performance liquid chromatography using normal phase and reverse phase columns.

The B-1015 compound can be separated and purified by repeating the above separation and purification means alone or in combination as appropriate.

[0029]

[Function] The B-1015 compound of the present invention is a novel compound which has not been published in the literature and is used in animals (eg, humans, dogs, cats, rabbits, etc.)
In the above, since it shows an antibacterial action against Gram positive and negative bacteria, it is useful as an antibacterial agent against various bacterial infections.

When the B-1015 compound of the present invention is used as a medicine, it is mixed with itself or an appropriate pharmaceutically acceptable carrier, excipient or diluent according to a conventional method, and powder, granules,
It can be safely administered orally or parenterally in the form of tablets, capsules, injections and the like. The dose varies depending on the disease to be treated, the route of administration and the number of administrations, 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.

[0031]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1. B-1015 Cultivation of compounds A) Culture Alkaline Genes faecalis SANK 74291 strain was prepared by using Marine Agar Slant (manufactured by Difco).
Incubate for 3 days at 22 ° C above, inoculate one platinum loop into a 500 ml Erlenmeyer flask containing 100 ml of sterilized medium of the composition described below, and incubate at 26 ° C with a rotary speed of 200 rpm (7 cm radius of rotation). It was cultivated for 24 hours on a shaker. Aseptically sterilize 300 ml of the same medium 15
30 liter jar fermenter including liter
The bacteria were cultivated in two units and cultured at 26 ° C for 23 hours under aeration (15 liters / minute) and stirring (100 rpm).

Medium composition Brain Heart Infusion (Difco) 37 g Artificial seawater (Jamarin S, Jamarin Laboratory) 800 ml Distilled water 200 ml .....・ ・ ・ ・ ・ ・ ・ No pH adjustment.

B) Isolation 32 liters of the obtained culture broth was removed by a Sharpless separator to remove solids, and the separated liquor (35 liters) was activated carbon (3 liters).
Liter) column. Water (6 liters) and 2
After washing with 0% acetone water (9 liters), elution was carried out with 50% acetone water (12 liters). Acetone under reduced pressure,
Distilled off with a rotary evaporator. When the concentrate was freeze-dried, 61.3 g of yellow powder was obtained. A 56 g portion of this powder was dissolved in 1.5 liters of 0.05 M phosphate buffer (pH 4.2) and equilibrated with the same concentration of phosphate buffer (pH 4.2) to obtain Sephadex DEAE A-25 (300 ml). Attached to the column. 0.1 with same concentration of phosphate buffer (pH 4.2)
Elution was performed sequentially with M saline solution (1.5 liters), 0.2 M saline solution (1.5 liters), and 0.25 M saline solution (1.5 liters), and Staphylococcus aureus 209P (Staphy
lococcus aurens 209P) was used as a test strain, and fractions showing antibacterial activity (0.25 M saline solution fraction) were collected and desalted using activated carbon. That is, activated carbon (100 ml) was packed in a column, and a 0.25 M saline fraction having antibacterial activity was added. After washing with water (1 liter), elution was carried out with 500 ml of acetone: 0.1 N-aqueous ammonia (1: 1 v / v). The eluate was concentrated with a rotary evaporator under reduced pressure, and the concentrate was finally freeze-dried to obtain 600 mg of a yellow crude powder.

Further, in order to obtain a pure B-1015 compound from this crude powder, it was applied to a DIAION CHP-20P column. That is, 600 mg of coarse powder is dissolved in a small amount (about 10 ml) of water,
It was applied to a Diaion CHP-20P (500 ml) column packed with water. Develop with water, fractionate into 15 ml fractions, collect fractions 33-45 showing antibacterial activity, concentrate on a rotary evaporator, and freeze-dry to obtain 200 mg of B-
1015 compound was obtained as a yellow powder.

Test Example 1. Antibacterial activity of B-1015 compound The minimum inhibitory concentration (MIC) of the B-1015 compound against general Gram positive and negative bacteria was measured by the agar plate dilution method using muller hinton broth (manufactured by Difco). The results are shown in Table 1.

[0037]

[Table 1]

[0038]

From the above, the B-1015 compound of the present invention exhibits an antibacterial action and is useful as an antibacterial agent against various bacteria.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] June 26, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction content]

2) Property of substance: acidic yellow powder 3) Molecular formula: C ₇ H ₈ N ₂ O ₅ S (high resolution negative
(Measured by FAB-MS) 4) Molecular weight: 232 (Measured by high resolution negative FAB-MS) 5) Elemental analysis value: (%) Calculated as C ₇ H ₈ N ₂ O ₅ S monohydrate monoammonium salt Value C 31.46, H 4.90, N 15.72, S
11.99 Measured value C 36.22, H 5.01, N 16.73, S
9.73 6) Infrared absorption spectrum: ν _max cm ^-1 Potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as shown below. 3190, 1656, 1615, 1589, 1495, 1401, 1309, 1214, 11
15, 1047, 888, 827, 635 7) Ultraviolet absorption spectrum: λ _max nm (ε) The ultraviolet absorption spectrum measured in an aqueous solution is as shown below. Neutral water: 245 (4800), 345 (13400) Acidic water: 238 (4800), 320 (9300), 345 (Sh) Alkaline water: 260 (400), 350 (10200), 390 (9900)
, 420 (Sh) 8) High Performance Liquid Chromatography: Separation column; Senshupack ODS H-2151 (column size φ 6 x 150 mm, Senshu Kagaku Co., Ltd.) Solvent: 16% acetonitrile-0.5% Pic A (Waters Inc.) Water content: 1.5 ml / min Wavelength: 220-350 nm (detection by photodiode array) Retention time: 8.1 min 9) ¹ H-nuclear magnetic resonance spectrum: (δ: ppm) In heavy water, as an external standard The nuclear magnetic resonance spectrum (270 MHz) measured using tetramethylsilane is as shown below. 7.49 (H, d, J = 8.8 Hz), 6.92 (H, d, J = 2.9 Hz), 6.75 (H, dd, J
₁ = 8.8 Hz, J ₂ = 2.9 Hz), 4.82 (2H, s) 10) ¹³ C-nuclear magnetic resonance spectrum: (δ: ppm) Nuclei measured in deuterated methanol using tetramethylsilane as an internal standard The magnetic resonance spectrum (100 MHz) is as shown below. 162.0 (s), 144.0 (s), 140.4 (s), 119.4 (d), 115.9 (d),
115.3 (d), 59.5 (t) 11) Solubility: Soluble in water, methanol, dimethylsulfoxide and dimethylformamide, insoluble in ethyl acetate, acetone, chloroform and hexane. 12) Color reaction: positive for iodine.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Shuji Takahashi 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Hideyuki Haruyama 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Kentaro Kodama 33 Miyukigaoka, Tsukuba-shi, Ibaraki Sankyo Co., Ltd.

Claims (3)

[Claims] 1. Formula (I): A compound having B-1015. 2. A method for producing a B-1015 compound, which comprises culturing a B-1015 compound-producing bacterium belonging to the genus Alcaligenes and collecting the B-1015 compound from the culture. 3. In [Claim 2], the B-1015 compound-producing bacterium belonging to the genus Alkaline Genes is Alkaline Genes faecalis SANK 74291 strain (Microtechnology Research Institute No. 3551).
No.) manufacturing method.