JPS61152295A - Novel antibiotic ss46506a and its preparation - Google Patents

Abstract

NEW MATERIAL:An antibiotic SS46506A of the formula. It has following physical and chemical properties: elementary analyses (%): C 36.96, H 1.43, N 7.11, Cl 37.25; molecular weight: 384 (mass spectroscopy); molecular formula: C12H6N2 O4Cl4; melting point: 216-220 deg.C (decomp.); [alpha]<25>D=-37.2 deg. (c=1 in acetone); positive to iodine reaction, negative to FeCl3 and Dragendorff's reations; a weakly acidic substance; pale yellow needles; soluble in chloroform, methanol, ethyl acetate, ether, acetone, insoluble in water, n-hexane. USE:Antimicrobism against gram-positive bacteria, cutaneous fungi and rice blastcausative fungus. PREPARATION:A strain in Streptomyces such as Streptomyces Sp. S46506 (FERM P-8020) is cultured, preferably under aerobic conditions at 27 deg.C for 3-7 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic SS 46506 A and a method for producing the same.

[Conventional technology]

Conventionally, antibiotics produced by microorganisms, such as Streptomyces nou
-rsei) produced by Nystat
in) (E, L, Hanzen 3; 5ci
ence, 112 volumes.

2911 Sada, No. 423, 1950], Pseudomonas pyrroc.
Pyrronidoline (Pyrro
lnitrin) [Kei Arima et al.; Agricultural and Biological Chemistry, Vol. 28, p. 575, (
1964)], Actinosporangium, Bicuminophyllum SF-2080 (Actinosporan
gium vitamin-nophilum SF
-2080) produced by the pyrrolomycin group (Pyrr
olornycins) [Norio Ezaki et al.;
-90 (No. 199) is known.

[Problem that the invention seeks to solve]

An object of the present invention is to obtain a new antibiotic useful as a pharmaceutical, veterinary drug, agricultural chemical, or a material for conversion thereto.

[Failure to solve the problem]

The present inventors isolated numerous microorganisms from natural soil,
As a result of conducting various studies on the product, we found that a strain isolated from the soil in Kamagaya City, Chiba Prefecture produces SS 46506 A, a new antibiotic that has excellent antibacterial activity particularly against clam-positive bacteria, dermatophytes, and potato blast fungi. I found out that
The present invention has been developed.

That is, the present invention provides novel antibiotic SS 46506A
and a manufacturing method thereof.

Producing the antibiotic 5S46506A of the present invention8 46
Strain 506 has the following mycological properties.

1. Morphology Spore-forming hyphae are simply branched from aerial hyphae, and their tips are spiral-shaped. Axle branches are not allowed.

Conidia consist of a chain of 10 or more spores, the shape of the spore is oval to cylindrical, and the thickness is 0.5 to 0.8
X is 0.8 to 1.2 μm. The spore surface is smooth. Structures such as sporangia, flagellated spores, sclerotia, and division of basal hyphae are not observed.

2. Growth status on various media The growth status of strain 846506 on various media is shown in the following table. Observation was performed after culturing at 28°C for 14 days.

In addition, the description of the color is published by Nippon Shokuhaku Business Co., Ltd. f1981.
(2013) 1 Color Name Rate Dictionary' system name.

3. Physiological properties (1) Growth temperature range (yeast starch agar medium, 14-day culture) Optimum growth temperature 25-35℃ Growth temperature 16-41℃ (2 Liquefaction of gelatin Positive (3) Hydrolysis of starch Positive (4) Coagulation of skimmed milk Negative Peptonization of skimmed milk Positive (5) Production of melanin-like pigments Negative (6) Reduction of nitrates Positive (7) Decomposition of cellulose Negative 4, Carbon source utilization Strain 846506 is Pridham -Growth on Godelive agar medium was extremely poor, so a starch agar medium with starch removed was used as the basal medium.Growth was determined after 14 days of culture at 28°C.

Utilizes; L-arabinose, D-glucose, D-fructose, raffinose, D-mannite Slightly utilized; D-xylose, sucrose, L-rhamnose not utilized; Inositol 5 Diaminopimelic acid in all bacterial cells As a result of analyzing diaminopimelic acid in the body, LL-diaminopimelic acid was detected.

Strain 846506 hardly forms aerial hyphae on a normal actinomycete identification medium, but a few form on potato/carrot agar and plain agar.

The tip of the aerial hyphae forms a spiral (5 spirals). Some of them stick together and form a hemisphere. The latter is pseudosporangium, which is one of the characteristics of Actinosporangium bacteria.
ium), but no spherical pseudospore glue has been observed so far.

In addition, strain 846506 has the above-mentioned mycological properties and produces pyrrolomycin C, so it has been classified as Actenosporangium vitamina International Journal of Systematic Bacteriophys (Int. J. 5yst, Bac-
teriol, ) vol. 22, pp. 139-148, 19
1972], suggesting a close relationship. However, 5465
The 06 strain differs in that the spore-forming hyphae are spiral-shaped, yellow soluble pigment is observed, and characteristic pseudosporangia cannot be observed.

Based on the above characteristics, strain 846506 was determined to be a strain belonging to the genus Streptomyces.

The tips of the aerial hyphae are spiral-shaped, with ten or more spores chained together, and the spore surface is smooth. The color of the aerial mycelia is in the white color series, with no characteristic color on the underside and a yellow soluble pigment observed. Does not produce melanin-like pigments. Based on these properties, E, Ktster's search formula (I
nt, J, 5yst, Bact, eriol.

22, pp. 139-148, 1972), 846
I searched for the species to which the 506 strains were thought to belong, but nothing matched.

Streptomyces fragilis M 0303-' f F
8 (5treptornyces fragilis
MG 303-f F 8) Mosquito, hyloromycin C
Pyrroromycin B (Pyrro
lomycjn B) has been reported to be able to survive [Reunion et al.; The Journal of Antibiotics (J, Antibiotics), Vol. 37, 125
3-1256, 1984].

Therefore, this strain and Streptomyces fragilis (Int.
J, 5yst, Bacteriol, vol. 8, pp. 319-321, 1968), 84
Strain 6506 hardly forms aerial mycelium in a normal identification medium, forms a complete spiral, the color of the aerial mycelium is white color series, no red color is observed on the back side, D-mannit. The two differ in properties such as the use of , D-fructose, and raffinose.

As mentioned above, the present inventors could not find any species to which strain 846506 is thought to belong among the known species.
46506 strain Streptomyces sp. 846506
(Streptomyces sp, S 46506
) and has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under the deposit number 8020 (FERM P-802).
0).

The antibiotic SS 46506A of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic SS 46506 A producing strain, not only the above-mentioned 846506 strain but also its artificial mutants or natural mutants can be used as antibiotic SS 4650.
Any material capable of producing 6A can be used in the present invention.

The artificial mutant strain 846506 can be easily obtained by, for example, ultraviolet irradiation, cobalt-60 irradiation, chemical mutagenic agents, etc., as in the case of other actinomycetes.

Next, the cultivation of the bacterial strain in the production of the antibiotic SS 46506A will be explained. That is, for culturing the antibiotic s S 46506 A-producing strain belonging to the genus Streptomyces, a normal method for culturing actinomycetes is used.

As the nutrient medium, any synthetic medium, semi-synthetic medium, or natural medium can be used as long as it contains appropriate amounts of assimilable carbon sources, nitrogen sources, inorganic substances, etc. Examples of carbon sources include glucose, fructose, glycerin, mannitol, and starch.

Molasses etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. As nitrogen sources, inorganic or organic nitrogen compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, monosodium glutamate, etc., or natural products, such as soy flour, yeast extract, peptone, meat extract, dried yeast, cottonseed meal, etc. , proteose peptone, casamino acid, corn φ steep liquor, etc. may be used alone or in combination. As the inorganic substance, for example, calcium carbonate, sodium chloride, steel sulfate, manganese chloride, cobalt chloride, nickel chloride, etc. are used alone or in combination. In addition, organic and inorganic substances that aid the growth of the 846506 strain and promote the production of the antibiotic 5S46506A can be appropriately added to the medium.

As the culture method, methods commonly used for antibiotic production are employed, but liquid culture methods, particularly deep agitation culture methods, are most suitable. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 25 to 35°C, but it is generally preferable to culture at around 27°C. Antibiotic SS 46506
In the case of A, shaking culture or deep agitation culture, the production amount reaches its maximum after 3 to 7 days of culture. When the amount of antibiotic SS4.6506A accumulated in the culture solution reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

As shown in the examples below, SS 46506 A can be isolated from the culture solution by using various methods alone or in appropriate combinations, taking into account the physicochemical properties of the antibiotic. That is, since the antibiotic 5S46506 A normally exists in the culture F solution and the bacterial cells, the culture is separated into the bacterial cells and the F solution by centrifugation or filtration, and each of them is separated by a conventional separation method such as a solvent extraction method. , ion exchange resin method, gel filtration method, adsorption or distribution column chromatography method, dialysis method, precipitation method, etc. or by combining them as appropriate.
Separate and purify 46506A.

Examples of freeze-friendly separation and purification include the following methods. That is, the culture that has completed fermentation is centrifuged, and F
Separate into liquid and bacterial cells. Add water such as ethyl acetate and an organic solvent that does not mix freely with the F solution, stir and extract. An organic solvent that mixes freely with water, such as acetone or methanol, is added to the bacterial cells and stirred to extract the active ingredient.After the organic solvent is distilled off, the active ingredient is extracted with an organic solvent such as ethyl acetate. F
The liquid and the extract from the bacterial cells are combined, the solvent is distilled off, and the residue is subjected to silica gel column chromatography. The obtained active fractions are collected, the solvent is distilled off, and the fraction is secreted in a suitable solvent for crystallization to obtain pale yellow needle crystals of pure antibiotic SS 46506A.

S S 46506 A obtained as described above has the following physical and chemical properties.

<Physical and chemical properties> ■ Elemental analysis: Carbon 3696%, Hydrogen 1.43% Nitrogen 7
．． 11%, chlorine 37.25% ■ Molecular weight: 384 (mass spectrum) ■ Molecular formula:
CI2'H6N204Ct4■ Melting point: 216-22
0°C (decomposition) ■ Specific optical rotation: (a) D = -37,2
°C (C-1 acetone) ■ Ultraviolet absorption spectrum: 1st
Figure λMeOH(t): 220nm (sh), 276n'
m(6800)aX 285nm'(6950), 294nm(6500),
322nff+ (!5300) ■ Infrared absorption spectrum: Figure 2 ■ 'H-NMR spectrum: Figure 3 ■ 13C-NMR Nuvector (22,5MH2) deuterated chloroform: l [dimethyl sulfoxide-10:1].

δfppm): 147.7(s), 130.9(s
), 129.4(S), 129.3(d'), 126.
1S), 125.1(d).

123,7(s),,122.4-(S),116
．． 4(S), 105.4(s), 90.6(t), 6
8.6(d) [Phase] Color reaction: Iodine reaction is positive, ferric chloride reaction and Dragendorff reaction are negative.

■ Distinguish between basic, neutral, and acidic Weakly acidic substances 0 Shape and color of substance: Pale yellow needle-like crystals ■ Rf value in thin layer chromatography: Support, silica gel plate F2!14 (manufactured by Merck & Co.) Chloroform: Acetone (2o:x) 0.51n-hexane:ether ('3:1) 0.190 Solubility in solvents: chloroform, methanol, ethyl acetate,
Soluble in ether and acetone, insoluble in water and n-hexane.

■ Structural formula The above physical and chemical properties and structural formula were compared with those of known antibiotics, but no corresponding substances were found.
06 A is a new antibiotic.

[Effect]

The antibiotic SS 46506A of the present invention has the following biological properties.

■ Antibacterial action: Table 1 shows the minimum growth inhibitory concentration (MIC) of the antibiotic SS 465'06 A against various microorganisms.

Below is Table 1 in the margin [Effects of the invention] As is clear from Table 1, antibiotic 5S46506A
has extremely strong antibacterial activity against gram-positive bacteria, dermatophytes, and potato fungi. Therefore antibiotic SS 4650
6A is useful as a pharmaceutical, veterinary drug, agricultural chemical, or an intermediate raw material thereof.

〔Example〕

Next, an example will be given and explained.

Example Glucose 1%, Soluble Starch 3T, Polypeptone 80.5
%, soybean flour 1 t, dry yeast 1%, corn staple-
Prepare a liquid medium (pH 7.4) with a composition of 0.5% liquor and 0.2 parts calcium carbonate, and add 500 ml! , 100 me was dispensed into a Yosaka Lough flask and sterilized. This was inoculated with Streptomyces sp. 846506 strain, and 27
The cells were cultured with shaking at ℃ for 4 days to obtain a first seed bacterial solution. A medium with the same composition was inoculated with the first seed bacterial solution and cultured with shaking at 27°C for 2 days to obtain a second bacterial culture.

After sterilizing 16 tons of medium with the same composition and sterilizing it, inoculating it with 240 ml of the above-mentioned 22i1 day right solution, culturing at 27°C, rotating at 450 times/min, and aeration at 1.
The cells were cultured for 110 hours at 6 t/min.

After culturing, the culture solution is centrifuged and separated into P solution and bacterial cells.
10 tons of ethyl acetate was added to 13 tons of the obtained tear fluid and stirred to extract the active fraction. On the other hand, 3 tons of acetone was added to the bacterial cells and stirred to extract the active fraction twice.

The acetone extract was concentrated under reduced pressure to remove acetone, and then the active ingredients were extracted twice with 2 tons of ethyl acetate. The mixture was combined with the ethyl acetate extract of liquid P and concentrated under reduced pressure to obtain a brown oily substance. n-hexane:ether:acetone (9
Silica gel 6 filled with a mixture of : 3 : 0.5)
0 (Merck & Co., Ltd. Art.

9385) column (4CnTφx 35 cm) was covered with the brown powder obtained by dissolving the brown oily substance in acetone, adding a small amount of carbon dioxide and distilling off the solvent, and adding n-hexane:ether:acetone (9:3). :0.5), and the fractions extracted from the s S 46506A beach were collected and concentrated under reduced pressure to obtain a yellowish brown oil. Next, the yellow-brown oil was dissolved in acetone, a small amount of quartzite was added, the solvent was distilled off, and the yellow-brown powder was prefilled with a mixture of n-hexane: ethyl acetate: acetic acid (500:50:1'). silica gel 6
0 (Merck Art, 9385) column (4 cmφ
X, 35 cm), developed with n-hexane:ethyl acetate:acetic acid-1000:120:2, and sS4
The 6506 h elution fractions were collected and concentrated under reduced pressure to obtain a pale yellow powder. This was recrystallized with ether/n-hexane and 5S4
2.02 pale yellow needles of 6506A were obtained.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of the antibiotic SS 46506 A measured in a methanol solution. FIG. 2 shows the infrared absorption spectrum of the antibiotic 5S465o6A measured as a potassium bromide tablet. Figure 3 shows antibiotic SS 46506A(7)H-NM
This is an R spectrum measured in a deuterated acetone solution using TMS as a reference substance. that's all

Claims (1)

[Claims] 1. A novel antibiotic SS46506A represented by the following formula ▲ Numerical formulas, chemical formulas, tables, etc. are available. 2. SS46, a new antibiotic belonging to the genus Streptomyces
A method for producing a novel antibiotic SS46506A, which comprises culturing 506A-producing bacteria and collecting the novel antibiotic SS46506A from the culture. 3. The SS46506A producing bacterium is Streptomyces sp.
．． The manufacturing method according to claim 2, which is ¥S46,506).