JPS60126093A - Novel antibiotic substance ss7313b and its production - Google Patents

Abstract

PURPOSE:To provide a novel antibiotic substance SS7313B having specific elemental analysis value, EI-mass spectrum, melting point, specific rotation, UV- absorption spectrum, molecular formula, etc. CONSTITUTION:The novel antibiotic substance of the present invention produced by the microbial strain (FERM-P No.7367) is named as SS7313B. The culture of the strain capable of producing the antibiotic substance SS7313B can be carried out by the conventional cultivation method for actinomycetes. Usually, it is preferable to culture in a liquid medium under shaking or in a deep tank with aeration, under aerobic condition at 25-30 deg.C. The antibiotic substance SS7313B produced by the cultivation exists usually in the cell and in the culture liquid. Accordingly, the SS7313B is separated and purified by separating the cell from the liquid by filtration, etc., and separating the SS7313B from both fractions by conventional method. The SS7313B has a molecular formula of C7H8N2O4, and a plane structure of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic E187313B and a method for producing the same.

The present inventors isolated a large number of microorganisms in natural soil,
As a result of various research on the product, a strain isolated from the soil of Kitakyushu City, Fukuoka Prefecture was found to have antitumor effects.
The present invention was completed by discovering the production of a novel antibiotic 887313B that also has antibacterial activity against plant pathogenic bacteria.

That is, the present invention provides a novel antibiotic 887313B and a method for producing the same.

8731 producing the antibiotic 5s7313B' of the present invention
The three strains have the following mycological properties.

(1) Morphology Spore-forming hyphae are aerial hyphae with simple branching, and no axillary branching is observed. The tips of aerial hyphae are straight or wavy. No spore paste, flagellated spores, or sclerotia are observed. Also, no division of basal hyphae was observed. According to electron microscopy, the surface of the spores is smooth and thick, and the shape is cylindrical. The size of the spores is 0.5-0.7 x 0.7-0.9 μm,
Usually 50 or more are chained together.

(2) Growth conditions in various media (27°C.

(Culture for 14 days) The growth conditions of strain E17313 on various media are shown in the table below.

(3) Physiological properties ■ Growth temperature range (yeast extract/malt extract agar medium, 1
4-day culture) Temperature for growth: 8-37C Optimal temperature for growth: 27-30℃ ■ Liquefaction of gelatin Positive ■ Hydrolysis of starch Positive ■ Coagulation of skim milk Negative ■ Bezotonization of skim milk Positive ■ Production of melanin-like pigments Positive ■ Reduction of nitrate Positive ■Decomposition of cellulose Negative (4) Utilization of carbon source (Lidham-Godliep agar medium, cultured at 27°C for 14 days) ■D-glucose + ■L-arabinose − ■Sucrose − ■D-xylose + ■Inositol - ■D ~ Mannitol + ■D-Fructose + ■L-Rhamnose - 0 Raffinose - [Phase] Galactose Mo0 Salicin + (Note) + is used, - is not used.

Based on the above properties and the cell wall composition containing LL-cyamopimeline W1), it is clear that the E17313 strain attaches to Streptomyces. Furthermore, the mycological properties of the same strain were described in Waxman's "Thθactinomycetas" J Vol. 2 (1).
961), Scherling and Gottlieb's technical report "International Journal of Systematic Bacteriolozo
ays℃θmaticBacter1o1ogy)
J Vol. 18, pp. 69, 279 (1968), No. 1
Volume 9, p. 391 (1969), Vol. 22, p. 265 (1972) and "Percy's Manual 7J/. of Determinative Bacteriolozo (Berg.
ey's Manual of Detertoina
tiveaactariology) J 8th edition (19
1974) When searching for related species of 313 strains of El8F, 873 were found.
Strain 13 has yellow color series aerial mycelium, straight or wavy spore chains with more than 50 spores, smooth spore surface, and produces melanin-like pigments. Resotomycecaboulensis (Strsp.
tomycesQaVOuren day 18) and Streptomyces griseopruneus (streptomyces
S grismbrunous) and other rice plants are recognized.

Next, the results of a comparison between strain 87313 and the above two types of standards are shown in the following table.

As seen in the margin ependyma below, strain 87313 and Streptomyces griseoprunneus differ in the color tone of aerial hyphae, color tone of the underside, and soluble pigment. On the other hand, Streptomyces
Although the color tone and soluble pigments on the underside are slightly different from those of ``Kijiji urensis,'' other mycological properties match well between the two. Therefore, strain 87313 was identified as a strain belonging to Streptomyces giulensis.

In order to distinguish strain 8'7313 from known strains, the present inventors investigated Strezotomyces ΦI urensis B 7313.
(8toreptomyces cavourensi
e87313) and was given accession number 7357 (yicny
p-7357).

Antibiotic 887313a of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. The antibiotic 87313B producing strain is 87 above.
The 313 strain can be used for misdiagnosis 131, even if it is an artificial mutant or a natural mutant, as long as it has the ability to primarily contain the antibiotic 887313B. The artificial mutant strain 87313 mentioned above is similar to other actinomycetes, such as ultraviolet rays, cobalt-60 irradiation, chemical modification! A
It can be easily obtained by using an inducing agent or the like.

Next, the cultivation of the bacterial strain in the production of antibiotic 887313B will be explained. That is, the usual culture method for actinomycetes is used to culture the antibiotic 887313B-producing strain that grows on Strezotomyces almanica.

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.

As carbon sources, glucose, fructose, xylose, galactose, starch, glycerin, dextrin ζ, molasses, etc. are used singly 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 soybean extract, soybean flour, yeast extract, dried yeast, cottonseed meal, etc. Bezotapur, Zolotine, Soitone, etc. are used alone or in combination.

As the inorganic substance, for example, calcium carbonate, sodium chloride, copper sulfate, manganese chloride, zinc sulfate, etc. are used alone or in combination. In addition, a substance that helps the 87313 strain to develop and promotes the production of 887313B, or a general antifoaming agent such as silicone oil or Adecanol (trade name) can also be added to the medium as appropriate.

As the culture method, methods used in the production of general antibiotics are employed, but shaking culture using a liquid medium or deep aeration culture is usually preferred. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 25 to 30°C, but it is generally preferred to culture at around 27°C. antibiotic 13e
+7313B reaches its maximum production after 2 to 6 days of culture in both shaking culture and deep aeration culture. Antibiotic 5s7313B usually exists in bacterial cells and culture solution, so its isolation and purification can be carried out by separating the culture into bacterial cells and culture F solution by p-filtration or centrifugation, and then separating the bacterial cells and culture F solution. It is preferable to carry out an appropriate combination of conventional separation methods such as solvent extraction, precipitation, ion exchange resin, glucoplasty, adsorption or distribution column chromatography, and dialysis.

Examples of preferred separation and purification methods include the following methods. That is, first, the culture is subjected to centrifugation to obtain bacterial cells and culture F solution. The culture broth is extracted using an organic solvent, such as ethyl acetate. The bacterial cells are extracted with a hydrophilic solvent such as methyl alcohol or acetone, the extract is concentrated under reduced pressure, and distilled water is added to dissolve it to form an aqueous solution.
Extract with ethyl acetate etc. in the same way as the liquid, and combine with the ethyl acetate extract of J@Nitro P solution. The extract thus obtained was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography. The obtained active fractions are distilled off under reduced pressure, and the residue is subjected to Sephadex LJ (-20 column chromatography).The active fractions are collected and concentrated under reduced pressure, and then recrystallized with a suitable solvent such as ethyl ether to obtain antibiotic 8B7313.
Colorless columnar crystals of B are obtained. Antibiotic 8873138 obtained as described above has the following physicochemical and biological properties.

(1) Physical and chemical properties ■ Elemental analysis CHBL Experimental value (%) 45.70 4.35 15.11 Theoretical value ←) 45.66 4.38 15.21 ■ m Neemas spectrum (70θV) M m/z184 ■ Melting point 100℃ (decomposition) ■Specific rotation [α] = -14.3° (c = 0.37. Methyl alcohol) ■Ultraviolet absorption spectrum Figure 1 292 nm (840) ■Infrared absorption spectrum (KBr method) Figure 2: IH-NMR spectrum (90 MHz) Measured in deuterated acetone solution using TMBtl as a reference substance. Figure 3 δ (ppm): 3.40 (IH, dd', J = 4.0
．． 1.3H2), 3.70 (I H, d d # J
24-0-2. OH-z), 4.79 (IH.

br, d, J=+3.2Hz), 5.69 (I H
, d, J=8.2Hz), 6.43(IH, br,
), 8.00 (IH, br, ), 8.98 (IH,
br, ), 11.04 (IH, br-)■”O-NM
R spectrum (22.5 MHz) Measured in a dichloromethyl sulfoxide solution using TMEI as a reference substance.

δ (pPm): 189.8.170.4 e 170
．． 2-93.8, 65-9.

53.0, 52.4 ■ Solubility in solvents Soluble in water, methyl alcohol, ethyl alcohol, and acetone. Slightly soluble in chloroform and ethyl acetate. Slightly soluble in n-hexane.

[Phase] Color reaction 2. Positive with 4-zonitrophenylhydrazone test, yellow color with vanillin-sulfuric acid reagent.

■Distinction between basic, dispersive, and neutral Neutral (color and properties of substance Colorless columnar crystals (ethyl ether recrystallization) ) 0 Molecular Formula % Formula % (1Φ Structural Formula Based on the above physical and chemical measurements and separate research, the antibiotic 8S7313B of the present invention was determined to be a compound represented by the following planar structural formula.

This compound has two isomers, one with the following structure and its mirror image, and Honda Meisha includes both of them.

(2) Physiological Properties - Antitumor Effect The therapeutic effect of antibiotic 8E+7313B on Ehrlich carcinoma (Ehrlich) was tested in mice using the method described below. The results are shown in Figure 1. In addition, the antitumor activity in the table is the average survival days (0) of the untreated group.
The ratio of mean survival days (T) of the treatment group to that of the treatment group was expressed as a percentage.

〔experimental method〕

2×10a bt cells were transplanted into the peritoneal cavity of engineered OR mice (♀2 Nippon Talea) and removed for 24 hours.
s was intraperitoneally administered once a day for a total of 10 times.

Table 1 B) ■Antibacterial effect Antibiotic 887313B has antibacterial activity against plant pathogens. Table 2 shows the minimum W inhibiting concentration (Mlc) for various microorganisms.

[Test bacteria culture conditions]

For inoculum size 10' cells/wLt0 bacteria, Mueller-Hinton Agar (DifcO 1)
The cells were cultured for 18 to 20 hours at 37° C. in a 8-cell culture medium, and in the case of yeast and mold, they were cultured at 28° C. for 120 hours in a glucose-pezotone medium.

The properties described above were compared with those of known antibiotics similar to the compound of the present invention, but no corresponding substances were found (5S73i).
3B was determined to be a new antibiotic.

As is clear from these results, 1 antibiotic day B731
3B has shown a strong therapeutic effect on Ehrlich carcinoma ascites carcinoma in mice. It also has antibacterial activity against plant pathogens. In view of these antitumor and antibacterial activities, antibiotics ss7-313B are useful substances as antitumor antibiotics, and are also useful substances as agricultural chemicals.

Next, an example will be given and explained.

Example 1 Streptomyces cavourensis 87313 (Imitation Koken Bacterial Collection No. 7357), which produces 887313B, was mixed with glycerin 1.5-1 cottonseed meal 1.5% and sodium chloride 0.3%.
, L-7s/.

Inoculated into a liquid medium containing Ragin 0.2% (pH 7.0),
A seed culture solution is prepared by culturing with shaking at 7°C for 2 days. Next, 1.5% glycerin, 1% glucose, and 1.5% cottonseed meal.
%, sodium chloride 0.3%, L-Asuno Qyap
0.2% calcium carbonate, 0.3% calcium carbonate (pH 7.0)
The liquid medium was placed in a Zoya Fermenter with a volume of 16Ji301, and the seed culture solution 20ON was inoculated into this medium, and the aeration was continued.
161/min, stirring number 400r, pm, culture temperature 27℃
The cells were cultured for 4 days under these conditions. After the culture is completed, the culture solution is centrifuged, 1OA' of acetic acid IF-Af is added to the obtained P solution (13J), and extracted three times. On the other hand, methyl alcohol 51 was added to the bacterial cells, stirred, and treated with P. After concentrating the methyl alcohol of this extract under reduced pressure, 1j of water was added to the residual liquid, and this was extracted three times with 1j of ethyl acetate. This was combined with the extract from the filtrate, the solvent was concentrated under reduced pressure, and 500 rrrlf of chloroform was added to the resulting oily residue to remove insoluble matter, which was then subjected to silica gel column chromatography. The above chloroform 4° solution was passed through a 4 cm x 30 cm column (Merck Co., Ltd.) filled with chloroform in advance, and eluted with chloroform. After removing both eluted fractions, chloroform-methyl alcohol was added. Elution was performed with a mixed solution (96:4).

Of the eluted fractions, both fractions containing 5E17313B were collected and concentrated by IE, yielding 1.2% of the crude 887313B as a white powder.
f was obtained. Next, the white powder of crude E187313B was dissolved in a small amount of methyl alcohol, and this was added to Sephadex-LH2α, which had been filled with methyl alcohol in advance.
column (36 IL x 30 columns) and eluted with methylalpour. Both fractions containing E187313B were collected and concentrated under reduced pressure, and the residue was recrystallized from ethyl ether to obtain colorless columnar crystals of tf387313s.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic 87313B (in methyl alcohol solution). Figure 2 shows the infrared absorption spectrum of antibiotic SE+7313B (XSR tablet). Figure 3 shows the spectrum of antibiotic 81117313B. H-N MR spectrum (in deuterated acetone solution)'5c
show.

Claims (1)

[Claims] 1. Novel antibiotic 887 having the following physical and chemical properties
313B 07c Elementary analysis CHN Experimental value 66) 45.70 4.35 15.11 Theoretical value (→ 45.66 4.38 15.21 ■E Neemas spectrum (70eV) M” m/z 184 ■Melting point 100℃ (Decomposition) ■Specific rotation [α]-=-14.3° (0=0.37゜Methyl alcohol) ■Ultraviolet absorption spectrum Figure 1 292℃m (840) ■Infrared absorption spectrum (K-ho) No. Figure 2 ■1111-1I spectrum (90 MHz) TMS in deuterated acetone solution was added as a reference substance. Figure 3 δ (ppm): 3.40 (In, aa, I = 4.0
．． 1.3Hz), 3.70 (IH, ad, J=4.0
．． 2.0Llz), 4.79 (IH, br, d, J=8
．． 2Hz), 5.69 (IH. a, , T=8.2Hz), 6.43 (IH, br
,)8.00(IH,br,),59scxa,br,
), 11.04 (IH, bτ,) ■Is C-NMR spectrum (22,5MHz) 1i
The measurement was carried out using TM8 in 7 methyl sulfoxide solution as a reference substance. δ(PI)m): 189.8, 170.4, 1
70.2.93.8°65.9, 53.0, 52.
4. ■Solubility in solvents Soluble in water, methyl alcohol, ethyl alcohol, and acetone. Slightly soluble in chloroform and ethyl acetate. Slightly soluble in n-hexane. (Phi color reaction 2. Positive with 4-dinitrophenylhydrazone reagent, Pani') 7-Tit, shows yellow color with acid reagent. ■Distinction between basic, acidic, and neutral Neutral @ Color and properties of the substance Colorless columnar crystals (ethyl ether ester recrystallization) σHg) layer Chromadog, roughy Support: Silica Gluzolate Fzs4 (manufactured by Merck & Co.) ■Molecular formula % formula % 2, a novel antibiotic 8E17313BO3 according to claim 1, whose planar structural formula is represented by the following formula, a novel antibiotic 8E17313BO3 for Strezotomyces stripes. When antibiotic 8S7313B is isolated and collected, a new antibiotic 8137313B with all the characteristics
manufacturing method. 4. The production method according to claim 3, wherein the 887313B substance-producing bacterium is Streptomyces kagoulensis 557313m.