JPH0645593B2 - New substance CC12 - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel substance CC12 having antibacterial properties and a method for producing the same.

(Prior Art) Conventionally, as a tetramic acid derivative, for example, tirandamycin (Nirte, MJetal., J.Chem.Soc.P)
erkin TransI, Vol. 85, pp. 1057-1065 (1980)).

(Problems to be Solved by the Invention) The present inventors have found that a novel substance CC12 having antibacterial properties is produced from a culture of Streptomyces sp. SANK 60390 strain belonging to the genus Streptomyces isolated from soil. And completed the present invention.

(Means for Solving the Problems) The novel substance CC12 of the present invention has the following structural formula and physicochemical properties.

1) Structural formula 2) Appearance; colorless powder 3) Melting point; 161-166 ° C 4) Specific rotation: ▲ [α] ¹⁸ _D ▼ + 75.1 ° (c 1.0, in methanol) 5) Solubility: Water, methanol, ethanol Dissolved, slightly dissolved in acetone,
Insoluble in ethyl acetate, chloroform and hexane.

6) Thin layer chromatography: Rf value; 0.20 adsorbent; Silica gel 60 F254 (trade name, manufactured by Merck) Developing solvent: chloroform-methanol-acetic acid-water (8: 4: 1: 1) Rf value; 0.22 adsorbent Silica gel 60 F254 (trade name, manufactured by Merck) Developing solvent: Chloroform-methanol-29% ammonia water (3: 3: 1) 7) High resolution mass spectrum (m / z): 855.5483 (M + H) ⁺ 8) Element analysis: (%) Found C64.32, H8.37, N6.36, O20.00 calcd C66.02, H8.72, N6.55, O18.71 9 ) molecular _formula: C _{47 H} 74 _N 4 O ₁₀ 10) Ultraviolet absorption spectrum: λ _max nm (ε) in methanol 207 (19600), 245 (9900), 282 (10000) 0.01 Normal hydrochloric acid-in methanol 207 (18900), 250 (6600), 282 (9900) 0.01 208 (19900), 245 (9700), 281 (9600) in normal sodium hydroxide-methanol 11) Infrared absorption spectrum Le: Infrared absorption spectrum as measured in [nu _max cm ^-1 potassium bromide (KBr) tablet method is shown in Figure 1.

12) ¹ H-nuclear magnetic resonance spectrum: (δ: ppm) The nuclear magnetic resonance spectrum measured at 500 MHz in deuterated methanol is as shown in FIG.

13) ¹³ C-nuclear magnetic resonance spectrum: (δ: ppm) The nuclear magnetic resonance spectrum measured at 125 MHz in deuterated methanol is as shown in FIG.

12.0 (q), 12.0 (q), 16.5 (q), 17.1 (q), 17.9 (q), 23.4 (t), 23.4 (q), 24.1 (t), 29.6 (t), 29.9 (t), 32.2 (t), 33.6 (d), 36.7 (t), 37.4 (t), 37.9 (t), 37.9 (d), 39.2 (t), 41.7 (t), 43.7 (d), 44.0 (d), 45.0 (d), 48.2 (t), 50.7 (t), 54.5 (s), 57.6 (d), 70.2 (d), 71.1 (d), 73.7 (d), 75.5 (d), 77.6 (d), 78.3 (d), 83.7 (d), 103.0 (s), 120.2 (d), 123.0 (d), 124.0 (d), 129.8 (d), 134.3 (d), 134.3 (d), 135.0 (d), 139.7 (s), 140.4 (s), 141.1 (s), 155.8 (s), 181.0 (s), 192.3 (s), 204.0 (s), The novel substance CC12 of the present invention has various isomers. In the above formula, all of these isomers and mixtures of isomers are represented by a single formula. Therefore, the present invention includes all of these isomers and a mixture of these isomers.

The novel substance CC12 of the present invention can be converted into a salt according to a conventional method. Examples of such salts include alkali metals such as lithium, sodium and potassium; alkaline earth metals such as calcium and barium; aluminum; or basic amino acids such as lysine and arginine; and bases such as hydrochloric acid and sulfuric acid. , Inorganic acids such as phosphoric acid, hydrobromic acid, nitric acid; organic acids such as acetic acid, oxalic acid, maleic acid, malic acid, succinic acid, benzoic acid; . Among such salts, a pharmacologically acceptable salt is preferable.

The Streptomyces sp. SANK 60390 strain, which is a bacterium producing the novel substance CC12, was isolated from the soil collected in Ohata-cho, Shimokita-gun, Aomori Prefecture, and its mycological properties are as follows.

1. Morphological characteristics SANK 60390 strain is 28 ℃ at 7 ℃ on agar medium for strain identification.
It grows relatively well in 14 days of culture. The basal hyphae satisfactorily elongate and branch, exhibiting a light yellowish orange color, but no rupture or nocardia-like zigzag elongation is observed. The aerial hyphae are relatively well formed and have a brownish white, light brownish gray or olive gray color. 10 to 50 or 50 at the tip of the aerial mycelium
It forms more than one spore chain and exhibits a spiral shape. The surface of spores is smooth. The spore stalks are settled only on aerial hyphae, and no special organs such as sporangium, flagelliospores, sclerotium, and axle bifurcation are observed.

2. Properties on various culture media The properties after culturing on various culture media at 28 ° C for 14 days are as shown in the following table. The display of the color tone indicates the color chip number of the "Standard color table" for the Japan Color Research Institute.

3. Physiological Properties The physiological properties of the SANK 60390 strain observed for 2 to 21 days after culturing at 28 ° C are as shown in the following table.

Further, the carbon source assimilation ability of the SANK 60390 strain observed after culturing at 28 ° C. for 14 days using the Pridham-Gotrieve agar medium (ISP9) is shown in the following table.

The cell wall of the SANK 60390 strain is the method of B. Becker et al.
cker et al., Applied Microbiology, Volume 12, 421-423.
Page, 1984], LL-diaminopimelic acid and glycine were detected, confirming that the cell wall type is type I. In addition, the sugar component in the whole cell wall of the SANK 60390 strain was analyzed by the method of MP Lechevalier [MP Lechevalier, Journal of Laboratory & Clinic
al Medicine, 71, 834, 1968], no characteristic pattern was observed.

From the above, it was revealed that this strain belongs to the genus Streptomyces among actinomycetes, and therefore Streptomyces sp. (Streptomyces sp.) SANK 60390 (Microtechnological Research Institute No. 11523; FERM P-11523) Was named.

The SANK 60390 strain was identified by ISP [The International Streptomyces Project The Internat
ional Streptomyces Project) standard, Bergey's Manual of Systematic Bacteriolo
gy) Volume 4, The Actinomycetes
Volume 2 and recent literature on actinomycetes.

Although the CC12-producing bacterium has been described above, it is well known that various properties of actinomycetes are not constant and naturally and artificially change easily, and the strains usable in the present invention are: It includes all strains producing CC12 belonging to the genus Streptomyces.

The novel compound CC12 of the present invention is obtained by aerobically culturing a CC12-producing bacterium belonging to the genus Streptomyces in an appropriate medium,
It can be produced by collecting the target substance from the culture.

The medium may contain any nutrient source that can be utilized by CC12-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch and fats and oils can be used as a carbon source, soybean powder as a nitrogen source,
Organic substances such as cottonseed meal, dried yeast, yeast extract and corn steep liquor and inorganic substances such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be used. Also, if necessary, sodium chloride, potassium chloride, phosphate,
Inorganic salts such as heavy metal salts can be added. In order to suppress foaming during fermentation, a suitable antifoaming agent according to a conventional method,
For example, silicone oil can be added.

As the culture method, the aerobic liquid culture method is most suitable, as in the generally used antibiotic production method. A suitable culture temperature is 20-37 ° C, preferably 25-30 ° C.
With this method, the amount of CC12 produced reaches the maximum in 4 days of both shaking culture and aeration-agitation culture.

In this way, a culture with accumulated CC12 is obtained. In the culture, CC12 is partially present in the cells, but most of it is present in the culture filtrate.

Any purposeful method can be used to harvest CC12 from such cultures. One of the methods is based on the principle of extraction. Specifically, regarding CC12 in the culture filtrate, a method of extracting it with a water-immiscible solvent for CC12, such as butanol, or the intracellular For CC12, there is a method in which a bacterial cell assembly obtained by filtration, centrifugation, etc. is treated with methanol, ethanol, acetone or the like and collected. The culture itself may be subjected to the above extraction operation without separating the cells. Countercurrent partitioning with an appropriate solvent can also be included in the extraction category.

Another method for collecting CC12 from the culture is based on the principle of adsorption, which is already liquid.
12 content, for example, a culture filtrate or an extract obtained by carrying out the extraction operation as described above, a suitable adsorbent such as silica gel, activated carbon,
The target CC12 is adsorbed by using "Diaion HP20" (trade name, manufactured by Mitsubishi Kasei Co., Ltd.) and the like.
CC12 can be obtained by eluting with a suitable solvent. The CC12 crude product is obtained by concentrating and drying the CC12 solution thus obtained under reduced pressure.

In order to further purify the crude product of CC12 thus obtained, the extraction method and the adsorption method described above may be combined with a gel filtration method, high performance liquid chromatography, etc., as necessary, and performed a required number of times. For example, an adsorbent such as silica gel, column chromatography using a gel filtration agent such as "Sephadex LH-20" (trade name, manufactured by Pharmacia), "YMC pack" (trade name, manufactured by Yamamura Kagaku), etc. The high performance liquid chromatography and the countercurrent distribution method used can be appropriately combined and carried out. Specifically, for example, the CC12 rough standard is "Sephadex LH
Apply a “-20” column, elute the active fraction with methanol, and concentrate to dryness to obtain pure CC12.

The CC12 of the present invention is a novel substance whose pattern is unprinted, has an antibacterial activity against animals (eg, humans, dogs, cats, rabbits, etc.) and is useful as an antibacterial agent.

Therefore, the substance of the present invention can be used as an antibacterial agent or an infectious disease therapeutic agent.

The substance of the present invention as an antibacterial agent has a dosage form which is determined by any route of administration for the purpose of purpose and depending on the route of administration, such as powder,
It can be safely administered orally or parenterally in the form of granules, tablets, capsules, injections and the like. The drug is usually in a form diluted with a pharmaceutically acceptable carrier or diluent.

When the substance of the present invention is administered as a medicine, one of typical methods is to dissolve it in distilled water for injection or physiological saline and inject it. Specifically, there are methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into vein or artery, and local administration by injection.

The dose of the substance of the present invention is determined in consideration of the results of animal tests and various situations so that the total dose does not exceed a certain amount when administered continuously or intermittently. The specific dose varies depending on the administration method, the situation of the patient or the animal to be treated, such as age, weight, sex, susceptibility, diet, administration time, drugs used in combination, the patient or the degree of the disease, but for example, It is preferable to administer 0.01 to 5 g per day for an adult once or in several divided doses depending on the symptoms.

(Example) Next, the present invention will be described more specifically with reference to Examples and Test Examples.

Example 1. Production of new substance CC12 1) Culture The medium used was prepared by dissolving the components having the following composition in 1 L of water and adjusting the pH to 7.0.

Medium composition Glycerin 20 g Molases 10 g Casein 5 g Polypeptone 1 g Calcium carbonate 4 g Dispense and sterilize 100 ml each of the above medium into a 500 ml Erlenmeyer flask with a wart, Streptomyces sp. SANK 603
90 strains were inoculated and rotary culture was carried out at 200 rpm for 4 days at 27 ° C.

2) Isolation After culturing under the above conditions, 2 L of the culture broth was filtered and the filtrate was adsorbed on a Diaion HP20 (trade name, manufactured by Mitsubishi Kasei Co., Ltd.) column (3 cmφ × 25 cm). After washing the column with 50% methanol, elute with 500 ml of 50% acetone, and eluate 2
It was concentrated to 00 ml. This was extracted three times with 200 ml of butanol, concentrated, and then silica gel (trade name "Wakogel C20
0 ”, Wako Pure Chemical Industries, Ltd.) column (2 cmφ × 20c
m) and developed with chloroform-methanol-29% aqueous ammonia (5: 3: 1). The crude powder of CC12 obtained by concentrating the active fractions was dissolved in a small amount of methanol, and chromatographed with methanol in a Sephadex LH-20 column (Pharmacia) (2.5 cmφ × 50 cm). By concentrating and drying the active fraction, 28 mg of pure CC12 was obtained.

Test Example 1. Antibacterial spectrum The minimum inhibitory concentration of CC12 against general bacteria is normal agar medium (Eiken Chemical Co., Ltd.), and mold against Sabouraud agar medium (Eiken Chemical Co., Ltd.) by agar dilution method. It was measured.

The results are shown below.

(Effects of the Invention) As described above, the novel substance CC12 of the present invention exhibits an antibacterial action against bacteria, particularly Gram-positive bacteria, and is useful as an antibacterial agent.

[Brief description of drawings]

FIG. 1 shows the infrared absorption spectrum of CC12. FIG. 2 shows the ¹ H-nuclear magnetic resonance spectrum of the same substance. FIG. 3 shows the ¹³ C-nuclear magnetic resonance spectrum of the same substance.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1: 465) (C12N 1/20 C12R 1: 465)

Claims (3)

[Claims] 1. A novel substance CC12 represented by the following formula or a salt thereof. 2. A method for producing CC12, which comprises culturing a CC12-producing bacterium belonging to the genus Streptomyces and collecting CC12 from the culture. 3. The method according to claim 2. CC12 producing bacteria belonging to the genus Streptomyces in Streptomyces spp.
SANK 60390 strain (Ministry of Microbiology Research No. 11523; FERM P-11523)
Is a manufacturing method.