JPS61115081A - Novel antibiotic ss21020d and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic SS21010D shown by the formula. [Physical and chemical properties] Molecular weight and molecular formula: 748, C41H52N2O11 Mass spectrum (SIMS) (M+H)<+> m/z 749. Melting point: 145 deg.C(decomposition). Specific rotatory powder: [alpha]<20>D+390 deg.(C 0.1, MeOH). Solubility: soluble in chloroform, acetone, methanol, pyridine, or acidic water, insoluble in n-hexane. Basic, yellow ish red crystal. Color reaction: positive in Dragendorff's reagent, etc. USE:A drug. An antibacterial agent and an anti-cancer. PREPARATION:A mold such as Streptomyces sp. SS21020 strain [FERM P-7798] belonging to the genus Streptomyces, capable of producing antibiotic SS21010D, inoculated into a nutrient source-containing medium, cultivated aerobically and antibiotic SS21020D is collected from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic SS21020D and a method for producing the same.

[Conventional technology]

Conventionally, various antibiotics produced by microorganisms 1, such as Streptomyces pluricallescens (Streptmyces
Pluramyein A produced by ces pluricolororeacens
) (Maeda et al.: The Journal of Antibiotics Series A, Vol. 9, p. 75 (1956)), Neoplura V y A (Neoplura
mycin A) (Kondo et al.; The Journal of Antibiotics.

23 volumes, 354 members (1970); 3o also has 1143 pages (
1977)], Streptomyces griseus (St
Rubiflavin A produced by Reptomyaes griaeus
(H, Nadig et al.; Helhetica chimica acta, 6
Volume 3, page 2446 (1980): A, ABZa
108 et al.: Anti-microhyal agents and chemotherapeutics.

1964, page 68 (1965)] is known.

[Problem that the invention seeks to solve]

The purpose of the present invention is to obtain a new antibiotic useful as a pharmaceutical.

[Means for solving problems]

The present inventors isolated a large number of microorganisms from natural soil and conducted extensive research on their products. As a result, a strain isolated from soil in Adachi Ward, Tokyo was found to have antitumor activity and excellent Novel antibiotic SS21020 with antibacterial activity
They discovered that Dl can be produced and completed the present invention.

That is, the present invention provides a novel antibiotic SS21020D and a method for producing the same.

8210 producing the antibiotic SS21020D of the present invention
The 20 strains have the following mycological properties.

(1) Morphology Spore-forming hyphae are more simply branched than aerial hyphae, and no axillary branching is observed. The tip of the aerial hyphae is straight and rarely 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;
The shape is cylindrical. Spore size is 0.5-0.7
O, 7 to 1.0 μm, and usually 10 or more are chained together.

(2) Growth status on each culture medium (27°C, 14 days culture) The growth status of strain 821020 on various media is as shown in the following table. Colors were described using the "Color Name Dictionary" published by Nippon Shikiken Project ■, and indicated by their family names. Note that the numbers in parentheses in Table 1 indicate color standard numbers.

Extra details below (3) Physiological properties ■ Growth temperature range (yeast/malt agar medium, 14-day culture) Optimum growth temperature 27-30℃ Possible growth temperature 10-37℃ ■ Liquefaction of gelatin positive ■ Hydrolysis of starch positive ■ Coagulation of skim milk Negative Peptonization of skim milk Positive ■ Production of melanin-like pigments Negative ■ Reduction of nitrate Positive ■ Decomposition of cellulose Negative (4) Assimilation of carbon sources (Pridham-Godelive agar medium, cultured at 27°C for 14 days) L-arabinose, D-xylose, D-glucose,
D-fructose, L-rhamnose.

Utilizes D-mannitol, galactose, and salicin.

Do not use sucrose, inositol, or raffinose.

(5) Diaminopimelic acid in @ Diaminopimelic acid in all bacterial cells was analyzed and LL-diaminopimelic acid was detected.

Contains the above morphological characteristics and LL-diaminopimelic acid! D. It is clear that strain 821020 is a strain belonging to the genus Streptomyces.

The mycological properties of strain 821020 can be summarized as follows. The tips of aerial hyphae are straight and rarely wavy. Ten or more spores are chained together, and the spore surface is smooth. The color of the aerial mycelium is the yellow color series, and the color of the 71 sides ranges from yellow to brown.

The color of the back surface changes with pH on oatmeal agar medium. The soluble dye is generally faint yellow in color, but a slight amount of indicator uniform dye t- is visible on the nutrient agar medium. Does not produce melanin-like pigments.

Based on the above properties, Waxman's ``The actinomycetea''
J Vol. 2 (1961), Scherling and Gottlieb's ISP Report, International Journal of Systematic Bacteriology (Inter.
ational Journal of System
atic Bacterilog) rl Volume 18, 69
Page, 279 pages (1968), Volume 19, 391 pages (1968)
1969), Volume 22.

265 pages (1972) and “Birshi’s Manual op Determinative Bacteriology (B.
erg@y's Manual of Determi
From native Bacteriology) J 8th edition, and from related substance producing bacteria of SS21020D, 82
As a result of searching for 1020 species, the following four species were listed as closely related species.

Streptomyces pluricolorescens (8trep
tomyces pluricolorescensl
Streptomyces griseua Streptomyces chrysomallu
a) Streptomyces citreofluorescens (S
treptomyces citreofluoras
cens) and the 821020 strain, Streptomyces pluricolorescens differs in that it generally has shorter spore chains (and does not utilize L-arabinose and does not reduce nitrate). Streptomyces chrysomalus differs in that its chains are wavy, that it does not utilize L-arabinose and L-rhamnose, and that the color of the back side is different. Streptomyces citreofluorescens differs in that it does not recognize the color of the back side that changes due to the liquefaction of gelatin, it does not reduce nitrate, and it recognizes a strong yellow soluble pigment.Streptomyces citreofluorescens uses salicin. What I don't do,
They differ in that they do not show a color on the backside that changes with pH, and that they contain a strong yellow soluble pigment.

As mentioned above, the 821020 strain does not match any of the food sources, so the present inventors differentiated the 821020 strain from known strains by Streptomyces sp.
tomyces sp, ) 821020, and was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology, as the 77th
No. 98 (FERM P-7798).

The antibiotic SS21020D of the present invention is produced by, for example, inoculating the above-mentioned strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic SS21020D-producing strain, not only the above-mentioned 821020 strain, but also any artificial or natural mutant strain thereof can be used in the present invention as long as it has the ability to produce the antibiotic SS21020D. The above-mentioned artificial mutant strain 821020 can be easily obtained using, for example, ultraviolet rays, cobalt-60 irradiation, chemical mutagenic agents, etc., as in the case of other actinomycetes.

Next, culturing of bacterial strains in the production of antibiotic SS21020D will be explained. That is, a normal actinomycete culture method is used to culture the antibiotic SS21020D producing strain belonging to the genus Streptomyces.

As a nutrient medium, it is an assimilable carbon source and nitrogen source.

Any synthetic medium, semi-synthetic medium, or natural medium can be used as long as it contains an appropriate amount of inorganic substances.

As the carbon source, for example, glucose, fusictose, glycerin, mannitol, starch, molasses, etc. can be used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. Nitrogen sources include inorganic or organic nitrogen compounds 1 such as ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, monosodium glutamate, etc., or natural products 1 such as soy flour, yeast extract peptone, meat extract, dried yeast, cottonseed meal, protein Auspeptone.

Casamino acids, corn steep liquor, etc. are used alone or in combination. As an inorganic substance.

For example, calcium carbonate, sodium chloride, steel sulfate, manganese chloride, zinc chloride, etc. are used alone or in combination. In addition, it helps the growth of 821020 stocks.

A substance that promotes the production of 8S21020D or a general antifoaming agent such as silicone oil or Adekanol (trade name) can also be added to the medium as appropriate.

As the culture method, methods commonly used for the production of antibiotics 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 culturing is 25 to 30°C, but it is generally preferable to culture at around 27°C. The production amount of antibiotic SS21020D is 3 in both shaking culture and deep agitation culture.
Maximum reached after ~7 days of culture. Antibiotics in culture medium
When the accumulated amount of SS210200 reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

Isolation of SS21020D from the culture solution is carried out by using various methods alone or in appropriate combinations, taking into consideration the physicochemical properties of a single antibiotic, as shown in Examples below.

That is, since the antibiotic SS21020D normally exists in the culture solution and bacterial cells, the bacterial cells are separated from the culture by centrifugation or filtration, and the bacterial cells and culture F solution are separated by a conventional separation method 1, such as a solvent extraction method, Ion exchange resin method, gel filtration method, adsorption or distribution column chromatography method, dialysis method1. Antibiotic SS2102 can be prepared by precipitation method etc. alone or in appropriate combination.
0D t-Separate and purify.

Preferred examples of separation and purification include the first method. After fermentation, the culture is centrifuged.

Separate into P solution and bacterial cells. The bacterial cells are extracted with a solvent that dissolves the substance described below, such as chloroform or methanol, to obtain an extract, which is further concentrated under reduced pressure to remove the solvent, and an aqueous solution is obtained.

After treating the liquid from which the bacterial cells have been removed and the liquid obtained by treating the bacterial cells with a nonionic porous resin 1 such as HP-20 (manufactured by Mitsubishi Kasei) to adsorb the active ingredient, Elute using methanol, acetone, etc. The eluate was distilled off under reduced pressure, and to the residue was added form with a pI (8 to 9) and shaken to transfer the active ingredient to the chloroform layer.The aqueous layer was removed and the chloroform solution was distilled off under reduced pressure. death.

The residue is subjected to silica gel column chromatography. The obtained active fraction was distilled off under reduced pressure, and the residue was purified with Sephadex L.
Subjected to H-20 column chromatography. The active fractions are collected and concentrated under reduced pressure, and then subjected to silica gel column chromatography again.
Yellow-red crystals of 21020D are obtained.

SS21020D obtained as described above has the following physical and chemical properties.

Physical and chemical properties〉 ■Elemental analysis HN Experimental value (%) 66.15 6.78 4.02
Theoretical value (%) 65,76 7.00 &74
0 Molecular weight and molecular formula % Formula % ■ Ultraviolet absorption spectrum diagram 1 λ rabbit? H(・)・243(54700), 269(32,300)42
8 (10,3001) Infrared absorption spectrum Figure 2 ■' H-NMR spectrum (90 MHz) Figure 3 Measurements were made in deuterated chloroform solution using TMS as a reference substance.

■”C-NMR, X hect/l/ (215MHz
) Measured as TMSt reference material in deuterated chloroform solution.

δ(pptn): 18&3(s), 1812(s
), 179.0(s).

170.2 (s), 159.9 (sl, 155
．． 8 (a), 150.0 (a), 140.0 (
1), 13 & 6 (II), 137.2 (ml.

13L3 (d), 130.2 (d), 127.
4 (d), 126.1 (s), 12 & 8 (s)
, 12 & 8 (d), 119.0 (i).

11aO(s), 11α9(d), 77.3 (
d), 75.6 (1).

75.1 (d), 7t, 8 (d), 71.8
(d), 70.9 (d).

69.6 (d), 67.4 (d), 67.2
(d), 57.3 (s).

40.4 ((1), 40.4 ((t), 36.
8 ((11,36,8(ql.

3a6(t), 2&3(t), 241(ql, 2λ5(
(L).

18.9 ((11,17,6((11,1&5 ((
1), IL4 (q1■ Solubility Soluble in chloroform, acetone, methanol, pyridine, acidic water. Insoluble in n-hexane.

0 Basic, acidic, neutral distinction basic Color and properties of 0 substances yellow-red crystal O color reaction Positive for Dragendorff reagent.

0 Thin layer chromatography carrier Nijiri gel plate F7, (manufactured by Merck & Co., Ltd.) 1 person -
IS Indicator 0 Structural Formula As is clear from the above physical and chemical properties, the antibiotic SS21020D of the present invention has the above-mentioned known Pluramicne A (molecular weight near 72. Molecular formula: C41HstNt On )-
Neopurramycin A (molecular weight near 30, molecular formula: C1
, Horse N! 0. . ), Lubiflavin A (molecular weight near 30.
It is a new compound that is different from the molecular formula: C4 01°).

[Effect]

The antibiotic SS21020D of the present invention has the following biological properties.

■ Antibacterial action Table 1 shows the minimum inhibitory concentration (MIC) of the antibiotic SS21020D against various microorganisms.

Table 1: Antitumor Effect The therapeutic effect of antibiotic SS21020D on murine leukemia P-388 was tested by the following method.

The results are shown in Table 2. The survival effect in Table 1 is expressed as a percentage of the ratio of survival days (T) in the treated group to survival days (C) in the untreated group.

Experimental method: lXl0' P-388 cells were injected into CDF, mouse (Zan2
Transferred to the peritoneal cavity of a Japanese Charles River, and after 24 hours, once a day to SS21020D 1.

It was administered intraperitoneally 10 times in total.

Table 2 [Effects of the invention] In view of these antibacterial and antitumor effects, antibiotic SS2
1020D is useful as an antibacterial and anticancer agent.

〔Example〕

Next, an example will be given and explained.

Example A liquid medium having the composition of 0.5% crucose, glycerin λOfk, "jHfose peptone (manufactured by Difco) LO96, yeast extract O,a*, 0.5 S of sodium chloride, and 0.3 S of calcium carbonate was used. pH 7.0,
1211j was dispensed into a 500-Yosaka flask and sterilized. This was combined with Streptomyces sp. 821020 strain (
Inoculated with ``Feikoken Bacillus No. 7798''.

A seed culture solution was prepared by culturing with shaking at 27°C for 2 days.

Pour 16 J of liquid medium having the same medium composition into a 301-volume jar fermenter, and add 200 J of the above seed culture solution to it.
d, culture temperature 27℃, stirring number 459 r, p,
The cells were cultured for 5 days under conditions of 161 m/min and an air flow rate of 161/min. After culturing, centrifuge the culture solution.

The obtained filtrate (14 J) was used for 2 times! The active substance was adsorbed through a nonionic porous resin HP-20 (trade name, manufactured by Mitsubishi Kasei), washed with water, and eluted with methanol. The eluted methanol fraction was concentrated under reduced pressure, the residue was dissolved in a small amount of water, the pH of the aqueous solution was adjusted to pH 9.0, and the mixture was extracted with chloroform. The extracted chloroform solution was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography. Prepare chloroform, methanol, water, acetic acid (7:4) in advance.
A column of Kieselgel 60 (manufactured by Merck & Co., Ltd.) (direct & 4 cm: length 30 cm) packed with a mixture of 1:1)
The above residue was dissolved in a small amount of the same mixture, passed through a silica gel column, and eluted with the same mixture.

Of the eluted fractions, both fractions containing 5821020D were collected and concentrated under reduced pressure. The resulting residue was then dissolved in a small amount of methanol, passed through a Sephadex-LH200 column (diameter 2, length 50 m) filled with methanol in advance, and eluted with methanol. SS210
Both fractions containing 20D were collected and concentrated under reduced pressure, acetone gold was added to the residue, the precipitate was removed, the supernatant was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography. Chloroform-methanol-concentrated ammonia water (4:
A column of Kieselgel 60 (manufactured by Merck & Co., Ltd.) packed with a mixture of 1:0.1) (length 50 cm)
In step 1, the above residue was dissolved in a small amount of the same mixed solution, passed through a silica gel column, and eluted with the same mixed solution. 8
Fractions containing 821020D were collected and concentrated under reduced pressure, and the residue was dissolved in a small amount of 0.5M pH 9.0 buffer (sodium carbonate/boric acid) and extracted with chloroform. The extracted chloroform solution was concentrated under reduced pressure, and the residue was recrystallized from ethyl ether to obtain yellow-red crystal 35 of SS21020D.

[Brief Description of the Drawings] Figure 1 shows the ultraviolet absorption spectrum of the antibiotic 8821020D of the present invention, Figure 2 shows its infrared absorption spectrum, and Figure 3 shows the infrared absorption spectrum of the antibiotic 8821020D of the present invention.
The figure is a diagram showing the IH-NMR spectrum of the same. Written amendment to the above procedures (voluntary) May 31, 1985

Claims (1)

[Claims] 1. Antibiotic SS2102 having the following physicochemical properties
0D. (1) Elemental analysis C H N Experimental value (%) 66.15 6.78 4.02 Theoretical value (%) 65.76 7.00 3.74 (2) Molecular weight and molecular formula 748, C_4_1H_3_2N_2O_1_1 (3) Mass spectrum (SIMS) (M+H)^+m/z 749 (4) Melting point 145℃ (decomposition) (5) Specific optical rotation [a]^2^5_D+390° (C 0.1, MeOH
) (6) Ultraviolet absorption spectrum Figure 1 λ^M^e^O^H_m_a_x (■); 243 (54,700), 269 (32,300) 42
8(10,300) (7) Infrared absorption spectrum (Figure 2) (8)^1H-NMR spectrum (90 MHz) Figure 3 Measurement was carried out in a deuterated chloroform solution using TMS as a reference substance. (9)^1^3C-NMR spectrum (22.5MHz
) Measurement was carried out in a deuterated chloroform solution using TMS as a reference substance. δ (ppm): 188.3 (s), 183.2 (s),
179.0 (s), 170.2 (s), 159.9 (s
), 155.8 (s), 150.0 (s), 140.0
(s), 138.6 (s), 137.2 (s), 133
．． 3(d), 130.2(d), 127.4(d), 1
26.1(s), 125.8(s), 125.8(d)
, 119.0(s), 116.0(s), 110.9(
d), 77.3(d), 75.6(s), 75.1(d
), 71.8(d), 71.8(d), 70.9(d)
, 69.6(d), 67.4(d), 67.2(d),
57.3(s), 40.4(q), 40.4(q), 3
6.8(q), 36.8(q), 33.6(t), 28
．． 3(t), 24.1(q), 23.5(q), 18.
9(q), 17.6(q), 13.5(q), 12.4
(q) (10) Solubility Soluble in chloroform, acetone, methanol, pyridine, and acidic water. Insoluble in n-hexane. (11) Distinction between basic, acidic, and neutral Basic (12) Color and properties of substance Yellow-red crystal (13) Color reaction Positive for Dragendorff reagent. (14) Thin layer chromatography carrier: Silica gel plate F_2_5_4 (manufactured by Merck & Co., Ltd.) 2. The antibiotic according to claim 1, which has a structure represented by the following formula ▲ Numerical formula, chemical formula, table, etc. ▼ Material SS21020D. 3. Antibiotic SS2102 belonging to the genus Streptomyces
Cultivate 0D-producing bacteria and extract antibiotic SS21 from the culture.
Antibiotic SS21 characterized by collecting 020D
Manufacturing method of 020D. 4. The SS21020D substance producing bacterium is Streptomyces sp. S210
20 strain (KAIKOKEN BIYORI NO. 7798).