JPH0449277A - Novel substance cc12 - Google Patents

Abstract

NEW MATERIAL:The novel substance CC12 expressed by formula and its salt. USE:Antibacterial agent exhibiting antibacterial action against bacteria, especially Gram-positive bacteria. PREPARATION:A CC12-producing bacterial strain belonging to genus Streptomyces [preferably Streptomyces sp.SANK 60390 (FERM P-11523)] is cultured and the objective CC12 is separated from the culture product.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) 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, tyrandamycin (No1te, M
,J,etal,,J,C,hem,soc,Pe
rkin Trans I, vol. 85, 1057-1065
Page (1980)) is known.

(Problems to be Solved by the Invention) The present inventors have discovered Streptomyces sp. SANK 6039, which belongs to the genus Streptomyces and was isolated from soil.
A new substance with antibacterial properties CC'12 was developed from a culture of 0 strains.
The present invention was completed by discovering that this can be produced.

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

1) Structural formula 2) Appearance; Colorless powder 3) Melting point; 161-166°C 4) Specific optical rotation: [α]. +75. (c 1.0° in methanol) 5) Solubility: Soluble in water, methanol, ethanol, slightly soluble in acetone,
Insoluble in ethyl acetate, chloroform, and hexane.

6) Thin layer chromatography m: ■Rf value; 0.20 Adsorbent Nijiri Gel 60 F254 (trade name, manufactured by Merck & Co., Ltd.) Developing solvent: Chloroform-methanol-acetic acid-water (8:
4:1:1) ■Rf value: 0.22 Adsorbent Nijiri Kagel 60 F254 (trade name, manufactured by Merck & Co., Ltd.) Developing solvent: Chloroform-methanol-29% aqueous ammonia (3:3:1) 7) High-resolution mass spectrum (m/z):855.54
83 (M+H)" 8) Elemental analysis: (%) Actual value C64.32, H8.37, N 6.36.0
20.00 Calculated value C66.02, H8.72, N 6.55.0
18.71 9) Molecular formula: C47H74N 401°10) Ultraviolet absorption spectrum: λ,,, ax nm (E)■207 (19500), 245 (9900) in methanol,
282 (10000) ■ 0.01N hydrochloric acid in methanol 207 (18900), 250 (6600),
282 (9900) ■ 0.01 N sodium hydroxide in methanol 208 (19900), 245 (
9700), 281 (9600) 11) Infrared absorption spectrum. , xcm"-' Potassium bromide (KBr)
The infrared absorption spectrum measured by the tablet method is shown in FIG.

12)'H-nuclear magnetic resonance vector = (δ: pp
m) The nuclear magnetic resonance spectrum measured at 500 MHz in heavy methanol is shown in FIG.

13) 13C-nuclear magnetic resonance vector: (δ: p
pm) The nuclear magnetic resonance spectrum measured at 125 MHz in heavy methanol is shown in FIG.

12.0 (q), 12.0 (q), 16.5
((]), 17, i (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), 5
4.5 (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 (rJ), 134.3 (d), 13
4.3 (d), 135.0 (d), 139.7 (
s), 140.4 (s), 141.1 (s), 1
55.8 (s), igl, o (s), 192.3
(s), 204.0 (s), Novel substance of the present invention CC1
2 has various isomers. In the above formula, all these isomers and mixtures of isomers are represented by a single formula.

Therefore, the present invention includes all of these isomers and mixtures of these isomers.

The novel substance CC12 of the present invention can be converted into a salt according to a conventional method. Such salts include, for example, 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. , phosphoric acid, hydrobromic acid, nitric acid; acetic acid, oxalic acid, maleic acid,
Examples include salts with organic acids such as malic acid, succinic acid, and benzoic acid; Among such salts, pharmacologically acceptable salts are preferred.

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

1. Morphological characteristics The SANK 60390 strain was grown on agar medium for strain identification, 28
It grows relatively well when cultured at ℃ for 14 days. The basal hyphae are well elongated and branched, showing a light yellowish-orange color, but no fractures or Nocardia-like zigzag elongation are observed. Aerial mycelium is relatively well formed and exhibits brownish white, light brownish gray, or olive gray color. 1 at the tip of aerial hyphae
Forms chains of 0 to 50 or more than 50 spores and exhibits a spiral shape. The surface of the spore is smooth. Spore stalks are only attached to aerial hyphae, and special organs such as sporangia, flagellar spores, sclerotia, and axle branches are not 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. Color tones are indicated using the color chip numbers of the Japan Color Research New Edition ``Standard Color Table''.

Sucrose/Nitrate Agar G: HeM Gelcose/Asparagine Agar R: SP2G=8M: R: SP2G: Glycerin/Asparagine Agar (ISP 5) AM: R: Good, flat, pale yellowish orange. Not good, velvety, light brownish white 4-Kohashi (4-8-9) Good quality, flat, pale yellowish orange, slightly formed, white 4-Kohashi (4-8-8) Very good, flat, pale yellow stripes (4-8-9) Well formed, velvety, 4 yellow stripes (2-7-9) 4 yellow stripes (6-8-9) Starch/inorganic salts Agar (ISP 4) Tyrosine Agar (ISP 7) Nutrient Agar (DIFCO) G: Good, flat, 4 yellows M: Well formed, velvety, brownish white (1-6-6) R: 4 yellows (6-8-9) SP: Not produced G: Very good, flat, pale yellow stripes (4-8-9) 8M: Well formed, velvety, 4 yellow stripes (2-7 -9) R: 4 yellow stripes (6-8-9) SP: No production G: Good, flat, pale yellowish orange M2 Not so good, velvety, white R: 4 yellow stripes (4 -8-9) Yeast extract G varnish/malt extract agar (ISP 2) AM: Oatmeal agar (ISP 3) R: SP2G: 8M: R: SP: Water agar G: 8M: R: Very good , flat, 4 yellow stripes (4-8-9) abundantly formed, velvety, olive ash (1-6-10) yellow stripes (8-7-9) good production, flat, 4 yellow stripes Richly formed, velvety, brown-white (1-6-6) 4 yellow-colored stripes (4-7-10) Poorly formed, flat, pale yellowish-orange (2-9-9) Slightly formed , Light brownish gray (2-7-8) Light brownish gray (2-8-8) Potato extract G: Not very good, flat, Carrot extract agar Light yellowish orange (2-9-9) AM: Good,
Velvety, gray brown (4-5-6) R: 4 Yellow Laws (2-7-9) SP: No production G: Growth, AM: Aerial mycelium, R: Back side, SP: Soluble pigment 3, Physiological properties SANK observed for 2 to 21 days after culturing at 28°C
The physiological properties of strain 60390 are shown in the table below.

Deicing of starch Liquefaction of positive gelatin Reduction of positive nitrate
Coagulation of positive milk
Peptonization of positive milk
Positive Melanin-like pigment production (medium 1) * Negative
Growth temperature range (Medium 3)* 15-36℃
Salt tolerance 7%*: Medium 1
Tryptone Yeast Extract Broth (ISP 1) 2 Tyrosine Agar (ISP 7) 3 Yeast Extract Malt Extract Agar (ISP 2) Also, using Pridham-Gotlieb agar medium (ISP 9), 28°C, 14 SANK observed after one day of culture
The carbon source assimilation ability of the 60390 strain is shown in the table below.

D-glucose + D-fructose ±L-arabinose + L-rhamnose ±D-xylose Sucrose + inositol
Raffinose + D-Mannitol -
The cell wall of the control substrate-degrading SANK 60390 strain was prepared using the method of Becker et al. [B, Becker et al., Appl.
ied Microbiology.

12, pp. 421-423, 1984], LL-diaminopimelic acid and glycine were detected, and it was confirmed that the cell wall type was type II. In addition, the sugar components in the whole cell wall of SANK 60390 strain were analyzed using the M.P.
Lechevalier, Journal of
Laboratory & C11nical Medi
cine, Vol. 71, p. 834, 1968], no characteristic pattern was observed.

From the above, it was found that this bacterial strain belongs to the genus Streptomyces among actinomycetes, and therefore, it is classified as Streptomyces sp+.
ANK 60390 (Microtechnology Research Institute No. 11523;
FERM P11523).

The SANK 60390 strain was identified by ISP C Di.
The International Streptomyces Project
omyces Project) Standards, Bersey's
Bergey's Manual of
Systematic Bacteriology Volume 4, The Actinomycetes
ycetes) Volume 2 and recent literature on actinomycetes.

The bacteria that produce CC12 have been explained above, but it is well known that the properties of actinomycetes are not constant and can easily change naturally or artificially, and the strains that can be used in the present invention include: This includes all strains that produce CC12 belonging to the genus Streptomyces.

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

The medium can contain any nutrient source that can be utilized by the CC12-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, fats and oils can be used as carbon sources, and soybean flour, soybean flour and nitrogen can be used as nitrogen sources.
Organic materials such as cottonseed meal, dried yeast, yeast extract and corn staple liquor and inorganic materials such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. In addition, 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,
For example, silicone oil can also be added.

The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The culture temperature is suitably 20-37°C, preferably 25-30°C. In this method, the production amount of CC12 can be determined by shaking culture,
Both the aeration and agitation culture reached the maximum after 4 days of culture.

In this way an enriched culture of CC12 is obtained. In the culture, a portion of CC12 exists in the bacterial cells, but most of it exists in the culture filtrate.

Any convenient method can be used to harvest CC12 from such cultures. One method is based on the principle of extraction, and specifically, for CC12 in the culture filtrate, it can be extracted with a water-immiscible solvent for CC12, such as butanol, or Regarding CC12, there is a method of recovering bacterial cell aggregates obtained by filtration, centrifugation, etc., by treating them with methanol, ethanol, acetone, etc. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting CC12 from cultures is based on the principle of adsorption, in which CC12 is already in liquid form.
12-containing substances, such as culture filtrate or the extract obtained by performing the extraction operation as described above, using a suitable adsorbent such as silica gel, activated carbon, etc.
CC12 can be obtained by adsorbing the target CC12 using Diaion HP20J (trade name, manufactured by Mitsubishi Kaseikyoku Co., Ltd.) or the like, and then eluting it with an appropriate solvent. The CC12 solution thus obtained is concentrated to dryness under reduced pressure to obtain a crude sample of CC12.

In order to further purify the crude sample of CC12 obtained in this way, the extraction method and adsorption method described above may be combined with a gel filtration method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary.

For example, adsorbents such as silica gel, [Sephadex L
Column chromatography using gel filtration rules such as H-204 (trade name, manufactured by Pharmacia), high-performance liquid chromatography using rYMC pack j (trade name, manufactured by Yamamura Kagaku Co., Ltd.), and countercurrent distribution method as appropriate. Can be implemented in combination.

Specifically, for example, a crude sample of CC12 is placed in a Sephadex LH-2OJ column, the active fraction is eluted with methanol, and concentrated to dryness to obtain a pure product of CC12.

CC12 of the present invention is a novel substance that has not been described in any literature, has 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 a therapeutic agent for infectious diseases.

The substances of the invention as antibacterial agents can be administered by any convenient route of administration and in the form determined by the route of administration employed, 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 diluted form with a pharmaceutically acceptable carrier or diluent.

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

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

(Example) Next, the present invention will be explained in more detail by giving examples and test examples.

Example 1. Production of novel substance CC12 1) Culture The medium used was one in which components having the following composition were dissolved in IL water and adjusted to pH 7.0.

Medium composition Glycerin 20 g Molasses 10 g Casein 5 g Polypeptone 1 g Calcium carbonate 4 g 100 ml of the above medium was dispensed into sterilized Erlenmeyer flasks with warts, inoculated with Streptomyces sp. SANK 60390 strain, and heated to 27°C. Te4
Rotational culture was performed at 200 rpm for 1 day.

2) Isolation After culturing under the above conditions, 2 L of the culture solution was filtered, and the filtrate was adsorbed onto a Diaion HP20 (trade name, manufactured by Mitsubishi Kasei Corporation) column (3 cmφ x 25 cm). column 5
After washing with 0% methanol, 500 ml of 50% acetone
The eluate was concentrated to ZOOml. This 2
Extracted three times with 00 ml of butanol, concentrated, applied to a silica gel (trade name: Wako Gel C200J, manufactured by Wako Pure Chemical Industries, Ltd.) column (2 cm φ x 20 cm), and extracted with chloroform-methanol-29% aqueous ammonia (5: 3
:1). The crude powder of CC12 obtained by concentrating the active fraction was dissolved in a small amount of methanol and applied to a Sephadex LH-20 column (manufactured by Pharmacia) (
Chromatography was performed using methanol in a vacuum chamber (2,50 mφ x 50 cm). By concentrating the active fraction to dryness, 28 mg of pure CC12 was obtained.

Test example 1. Antibacterial Spectrum The minimum inhibitory concentration of CC12 against general bacteria was measured by the agar medium dilution method using ordinary agar medium (manufactured by Eiken Kagaku ■) and Sabouraud agar medium (manufactured by Eiken Kagaku ■) for fungi.

Staphylococcus 7 ureus 209P Staphyi D
Kotsukas 7 Uraeus 56R Staphyi ■Gotsugasu Aureus 535 (MR3A) EnteU] Tucas faecalis 681 Escherisi 7 Collie NIHJ Escherisi 7 Collie 609 Clef” Shera 21 Monier 806 7° Lotius 7゛elh゛1420 Pseudomonas ercinosa 1001 Cancellus fluhicans SC Cryptococcus netrumans
580637 Suberus fumicatus
10569 Trichophyton mentacus °゛■Phytes SC3,1 6,2 6,2 〉100 〉200 〉200 〉200 〉200 〉200 〉50 〉50 〉50 (Effect of the invention) In this way, the novel substance CC1 of the present invention -2 exhibits antibacterial activity against bacteria, particularly Durham-positive bacteria, and is useful as an antibacterial agent.

[Brief explanation of the drawing]

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

Claims (1)

[Claims] 1. A novel substance CC12 represented by the following formula and a salt thereof. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. A method for producing CC12, which is characterized by culturing CC12-producing bacteria belonging to the genus Streptomyces and collecting CC12 from the culture. 3. In claim 2, the CC12-producing bacterium belonging to the genus Streptomyces is Streptomyces sp.
60390 strain (FERMP No. 11523; FERMP
-11523).