JPS6178395A - Novel antibiotic substance ss21020b and its preparation - Google Patents

Abstract

NEW MATERIAL:Antibiotic substance SS21020B having the following physical and chemical properties. Elemental analysis (%), C 66.78, H 7.27, N 3.45; molecular weight, 732, molecular formula, C41H52N2O10; melting point, 162 deg.C (decomposition); specific rotation, [alpha]D<25>=+289 deg. (C=0.1, MeOH); solubility, soluble in chloroform, ether acetone, methanol, pyridine and acidic water, insoluble in n-hexane; basic; yellowish red crystal; color reaction, positive to Dragendorff reagent, etc. USE:Antibacterial agent and antitumor agent. PREPARATION:A bacterial strain belonging to Streptomyces genus, e.g. Streptomyces sp S21020 (FERM P-7798) is cultured at about 27 deg.C for 3-7 days preferably by submerged culture under agitation.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic SS21020B and a method for producing the same.

[Conventional technology]

Conventionally, various antibiotics produced by microorganisms, such as Streptomyces burulicolorescens (Streptmyces
Pluramycin A produced by Ces pluricolorescens
) (Maeda et al.: The Journal of Antibiotics Series A, Volume 9, 75th (1956)
)], Neoplura Maishi A (Neoplura
mycin A) [Kondo et al.: The Journal of Antibiotics.

Volume 23, page 354 (19, 70); Volume 30, 1143
N (1977)], Streptomyces griseus (
Rubiflavin A produced by Streptomyces griseus
) (H, Nadig et al.; Helvetica Chimica Acta, Vol. 63, 24461 (1980); A, As
Zalos et al.
and chemotheraphy.

1964, page 68 (1965)] is known.

[Problem that the invention seeks to solve]

The object of the present invention is to obtain a novel antibiotic useful for medical use.

[Means for solving problems]

The present inventors isolated numerous microorganisms from natural soil,
As a result of various research on the product, a strain isolated from soil in Adachi Ward, Tokyo was discovered to be SS2102OB, a novel antibiotic with antitumor activity and excellent antibacterial activity.
'If' production was discovered and the present invention was completed.

That is, the present invention provides a novel antibiotic SS2102OB and a method for producing the same.

Producing the antibiotic SS2102OB of the present invention 821
The 020 strain has the following mycological properties.

(1) Morphology Spore-forming hyphae are more simply branched than aerial hyphae, and no axillary branching is observed. The tips of the aerial hyphae are straight and rarely wavy. No sporangia, 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 the shape is cylindrical. Spore size is 0.5~0.7 x 0
．． The diameter is 7 to 1.0 μm, and usually 10 or more are chained together.

(2) Growth status on various media (27°C, 14 days culture) S2] The growth status of the 020 strain on various media is as shown in the following table. Colors are described using the "Color Name Dictionary" published by Nippon Shikiken Project (Kiyuki), and indicated by their family names. The color standard numbers are shown in parentheses in the table.

Blank space 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 nitrates Positive ■ Decomposition of cellulose Negative (4) Assimilation of carbon sources (Pridham-Gotlieb 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 bacterial cells As a result of analyzing diaminopimelic acid in all bacterial cells, LL-diaminopimelic acid was detected.

From the above morphological characteristics and the presence of LL-diaminopimelic acid, it is clear that strain 821020 is a strain belonging to the genus Streptomyces.

The mycological properties of strain 821020 are 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 yellow color series, and the color of the underside is yellow to brown, and the pH level on oatmeal agar medium is
Recognize the color of the back side that changes. The soluble pigment is mostly faint yellow, but a slight amount of indicator uniform pigment is observed on the nutrient agar medium. Does not produce melanin-like pigments.

Based on the above properties, Waxman's book [The actinomycetes]
J Vol. 2, 1961), Scherling and Gottlieb's ISP Report, International Journal of Systematology (Inte.
rnational Journal of Syst
Ematic Bacteri-ology l No. 18
Vol. 69, 279 negative (1968), Vol. 19, 3
p. 91 (1969), Vol. 22, p. 265 (197
2 years) and ``No (- Seeds Manual of Terminal Bacteriology (Bergey's
Manual of Determi-native
13acteriology) J 8th edition, also 3
As a result of searching for the species of strain 821020 from related substance producing bacteria of 82102OB, the following four species were listed as closely related species.

Streftmyces pluricolorescens (Strep
tomyces pluricolorescens
l Streptomyces griseus 1 Streptomyces chrysomallu
s) Streptomyces citreofluorescens (
Streptomyces citreofluore
When comparing the above four species with the 821020 strain, Streptomyces pluricolorescens differs in that it generally has shorter spore chains, does not utilize L-arabinose, and does not reduce nitrate. Streptomyces griseus differs in that its spore chains are wavy, that it does not utilize L-arabinose and L-rhamnose, and that the color of its underside is different. Strephtomyces chrysomalus has wavy spore chains, does not utilize D-mannitol, and has a pH
It differs in that it does not recognize the color of the back side that changes due to heat, it does not recognize any liquefaction of gelatin, it does not reduce nitrates, and it recognizes a strong yellow soluble dye. Streptomyces citreofluorescens does not use salicin,
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 species, so in order to distinguish the 521020 strain from known strains, the present inventors used Streptomyces sp.
tomyces sp. l 521020 and was deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology as Fiber Science and Technology Research Institute No. 7798 (FERM P-7798).

The antibiotic SS210211B of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic SS21f12OB producing strain,
In addition to the 821020 strain mentioned above, any artificial mutant strain or natural variable strain thereof can be used in the present invention as long as it has the ability to produce the antibiotic 5821020B. 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 SS21020B will be explained. That is, a normal actinomycete culture method is used to culture the antibiotic SS21020B 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, fructose, glycerin, mannitol, 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. , groteose peptone, casamino acids, corn stave liquor, etc. may be used alone or in combination. As the inorganic substance, for example, calcium carbonate, sodium chloride, steel sulfate, manganese chloride, zinc chloride, etc. are used alone or in combination. Others 821020
Substances that aid the growth of the strain and promote production of SS21020B or common antifoaming agents 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 SS21020B 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 5821020B 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, SS21020B is isolated from the culture solution by using various methods alone or in appropriate combinations, taking into consideration the physicochemical properties of this antibiotic.

That is, since the antibiotic SS21020B is normally present in the culture lid liquid and the bacterial cells, the bacterial cells are separated from the culture by centrifugation or p-filtration, and the bacterial cells and culture filtrate are separated by conventional separation means such as a solvent. Antibiotic SS210 can be prepared by extraction method, ion exchange resin method, gel tear filtration method, adsorption or distribution column chromatography method, dialysis method, precipitation method, etc. alone or in appropriate combinations.
Separate and purify 2OB.

Examples of preferable separation and purification include the following methods.

After fermentation, the culture is centrifuged and separated into filtrate 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 such as HP-20 (manufactured by Mitsubishi Kasei) to adsorb the active ingredient, Elute using methanol, acetone, etc. This eluate was distilled off under reduced pressure, and the residue was
Add H8-9 water and chloroform and shake to transfer the active ingredient to the chloroform layer. The aqueous layer was removed, the chloroform solution was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography. The obtained active fraction was distilled off under reduced pressure, and the residue was subjected to Sephadex LH-20 column chromatography.

The active fractions are collected, concentrated under reduced pressure, and then recrystallized from a suitable solvent, such as ethyl ether, to obtain antibiotic 5821020B.
yellow-red crystals are obtained.

SS2102OB obtained as described above has the following physical and chemical properties.

Physical and chemical properties〉 ■Elemental analysis HN Experimental value (chi) 66.78 7.27 3.45
Theoretical value (忤) 67.19 7.15 3.82
■Molecular mushrooms and molecular formula% Formula% ■Ultraviolet absorption spectrum Figure 1 84eOH λmax (ε): 243 (48,3001,2
68 (29,3001°426 (9,300)) ■Infrared absorption spectrum (KBr method) Figure 2 ■'H-NMII spectrum (90B/I) (z )
Figure 3: Measurement was carried out using TMS as a reference substance in a deuterated chloroform solution.

■13C-NMR, ;<hek) k (22,5MH
z) Measured in deuterated chloroform solution using TMS as a reference substance.

δ(pl)m): 188.2 (s), 183
．． 2 (s), 179.2 (sl.

172.3 (sl, 159.9(s), 155.
9(a), 149.8(s).

140.3 (s), 138.5 (sl, 137
．． 3 (8), 133.2 (dl.

128.8 (dl, 126.0 (a), 126
．． 0 (s), 125.6 (di.

123.6(d), 119.3(s), 116.2
(s), 109.7(d).

77.4 (di, 75.2(d), 73.7 (
s), 72.0 (d), 71.1 (di, 69
．． 4 (dl, 67.5 (d), 67.5 Fd
l, 57.6 (sl.

40.4 (ql, 40.4 (ql, 38.5
(t), 36.9 (ql, 36.9(ql, 3
4.0 (t), 28.4 ft), 26.2
(q), 23.9 (Q).

18.9 (Q), 17.6 (ql, 12.9
(Q), 12.6 (ql■ Solubility Soluble in chloroform, ether, acetone, methanol, pyridine, acidic water. Insoluble in n-hexane.

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

0 Thin layer chromatography carrier Nijilica gel spray) Fz54 (manufactured by Merck & Co., Ltd.) ■ Structural formula As is clear from the above physical and chemical properties, the antibiotic SS2102OA of the present invention is the above-mentioned known pluramycin A (molecular needle = 772, molecular formula: C4 , HstN20u l,
Neoburramycin A (molecular weight near 30, molecular formula: C4
+Hy+NtO+o), rubiflavin A (molecular weight = 7
30, molecular formula: C41■l5oNto+o) is a new compound.

[Effect]

The antibiotic SS21020B of the present invention has the following biological properties.

■ Antibacterial activity The minimum inhibitory concentration (MICI) of the antibiotic SS21020B against various microorganisms is shown in Table 1.

Table 1: Antitumor Effect The therapeutic effect of the antibiotic SS2102QB on murine leukemia P-388 was tested by the following method.

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

Experimental method: 1X10' P-388 cells were injected into CDF, mouse (81
Transferred into the abdominal cavity of a Japanese Charles River ('1llL,
After 24 hours, SS21020B'kl was intraperitoneally administered once a day for a total of 10 times.

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

〔Example〕

Next, an example will be given and explained.

Example Glucose 0.5%, Glycerin 2.0%, Proteose Peptone (Difco) 1.0%, Yeast Extract 0
．． 3%, sodium chloride 0.5%, calcium carbonate 0.3% p) I7.0
The mixture was sterilized by dispensing 120 m/l into a 5QQm/Yosakaro flask. In this, Streptomyces sp. 8210
20 strains (Feikoken Bacteria No. 7798) were inoculated and subjected to shake culture at 27°C for 2 days to prepare a food culture solution.

A liquid medium 16A having the same medium composition was placed in a 30A jar fermenter, and 200% of the seed culture liquid was added to it.
+d inoculated, culture temperature 27°C, stirring number 45 Or,
pm and an air flow rate of 16.8/min for 5 days. After the completion of the culture, the culture solution was centrifuged and the obtained filtrate (1
4Al was passed through a 2J3 nonionic porous resin HP-20 (trade name, manufactured by Mitsubishi Kasei) to adsorb the active substance, and after washing with water, it was eluted with methanol. The eluted methanol fraction was concentrated under reduced pressure, the residue was dissolved in a small amount of water, and the p of the aqueous solution was
The solution was adjusted to pH 9.0 and extracted with chloroform. The extracted chloroform solution was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography. Kieselgel 60 (manufactured by Merck) (+M diameter 4
(The above residue was dissolved in the same mixture of 1 and 1 parts in a column of -rn=length 3 (l rrrH)), passed through a silica gel column, and eluted with the same mixture. Among the eluted fractions, fractions containing SS21020B were collected and concentrated under reduced pressure. Next, the obtained residue was dissolved in methanol from a small company, and this was added to a Sephadex-LH20 column (if'j diameter 2 (
'In: length 50 m) and eluted with methanol. The aliquoted fractions of 5821020H were collected and concentrated under reduced pressure, and the residue was dissolved in a subtotal of 0.5 molar pH 9.0 buffer (sodium carbonate/hydrochloride) and extracted with chloroform. The extracted chloroform solution was completely concentrated under reduced pressure, and the residue was crystallized with ethyl ether.
30 mg (c-obtained) of yellow-red crystals of 020B.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the antibiotic SS 2102 OB of the present invention, FIG. 2 shows its infrared absorption spectrum, and FIG. 3 shows its IH-NMR spectrum. Written amendment to the above procedure (voluntary) May 31, 1985 Item 1, Office I (Government Manabu Shiga, 1981-Q-Patent Application No. 202826, No. 2)
Title of the invention Novel antibiotic 8821020B and manufacturing method for 7 3 Supplementary name = IS (Relationship with '11) Applicant address 2-12-4 Nihonbashihama-cho, Chuo-ku, Tokyo Name Name Nisnis Pharmaceutical Co., Ltd. Representative Yasutomichi Nogata 4, Agent IFII liT, Fue 2100, Fue! Table of Contents 6, "Detailed Description of the Invention" column 7 of the specification to be amended, Contents of the amendment (1) Description (2) Same page, page 12, bottom line, rp-7798” should be changed to rp-7798J. (3) Same, page 20, line 2, r SS21020A J is corrected to r SS21020B J. (4) Same, page 20, bottom line, “minimum growth” is changed to “ ``Minimum growth.'' Correct the phrase ``Minimum growth.'' In silica gel column chromatography, first the two fractions containing SS21020B are eluted, then the fraction containing 5s21020A and D, and finally the SS2102 fraction.
Both fractions containing 0E are eluted. ”

Claims (1)

[Claims] 1. Antibiotic SS2102 having the following physicochemical properties
0B. (1) Elemental analysis C H N Experimental value (%) 66.78 7.27 3.45 Theoretical value (%) 67.19 7.15 3.82 (2) Molecular weight and molecular formula 732, C_4_1H_5_2N_2O_1_0 (3) Mass spectrum (SIMS) (M+H) m/Z 733 (4) Melting point 162℃ (decomposition) (5) Specific optical rotation [α] ^2^5_D+289° (C0.1, MeOH) (6) Ultraviolet absorption spectrum Figure 1 λ ^M^e^O^H_m_a_x(ε): 243(48
, 300), 268 (29,300), 426 (9,3
00) (7) Infrared absorption spectrum (KBr method) Fig. 2 (8)^1H-NMR spectrum (90 MHz) Fig. 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): 188.2 (s), 183.2 (s),
179.2 (s), 172.3 (s), 159.9 (s
), 155.9 (s), 149.8 (s), 140.3
(s), 138.5 (s), 137.3 (s), 133
．． 2(d), 128.8(d), 126.0(s), 1
26.0(s), 125.6(d), 123.6(d)
, 119.3(s), 116.2(s), 109.7(
d), 77.4(d), 75.2(d), 73.7(s
), 72.0(d), 71.1(d), 69.4(d)
, 67.5(d), 67.5(d), 57,6(s),
40.4(q), 40.4(q), 38.5(t), 3
6, 9(q), 36.9(q), 34.0(t), 28
．． 4(t), 26.2(q), 23.9(q), 18.
9(q), 17.6(q), 12.9(q), 12.6
(q) (10) Solubility Soluble in chloroform, ether, 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.) 2. The antibiotic according to claim 1, which has a structure represented by the following formula ▲ Numerical formula, chemical formula, table, etc. ▼ Material SS21020B. 3. Antibiotic SS2102 belonging to the genus Streptomyces
Cultivate 0B-producing bacteria and extract antibiotic SS21 from the culture.
Antibiotic SS21 characterized by collecting 020B
Manufacturing method of 020B. 4. The SS21020B substance producing bacterium is Streptomyces sp. S210
20 strain (KAIKOKEN BIYORI NO. 7798).