JPH0398591A - New antibiotic substance having antitumor activity and production thereof - Google Patents

Abstract

PURPOSE:To enable obtaining of a new antibiotic substance KO-3988C, etc., with high activity and reduced side effects by culturing a microorganism, belonging to the genus Streptomyces and capable of producing the aforementioned antibiotic substance in a culture medium. CONSTITUTION:Streptomyces sp. KO-3988 (FERM P-10369) strain having the following properties is obtained by separating thereof from soil in ATAMI City in SHIZUOKA Prefecture. Morphological properties; linear aerial mycelium and cylindrical spore size of 1.4X1.9mum. Growth conditions; well growing on glucose nitrate agar and producing a soluble coloring matter. Growth temperature range; 15-35 deg.C. Utilizing D-glucose, etc., without utilizing raffinose, etc. The resultant strain in then aerobically cultured in a culture medium containing glucose, etc., at 20-37 deg.C and neutral pH for 3-6 days to afford a culture, which is subsequently centrifuged and extracted to produce in antibiotic substance KO-3988C or KO-3988H, expressed by formula I (R is group expressed by formula II to VII) and having antitumor activity.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention provides a novel antibiotic KO-39 having antitumor activity.
88C to KO-3988H and a manufacturing method thereof.

(Issue 8 to be solved by the prior art and the invention) As is well known, antibiotics are substances produced by microorganisms and inhibit the growth of microorganisms and other cells. Among these antibiotics, certain antibiotics isolated from the culture filtrate of actinomycetes in soil, such as adriamycin, daunorpicin, mitomycin, and pleomycin, have so-called tumoricidal cell activity that inhibits the growth of cancer cells. It is known.

However, although it is simply called an anticancer drug, it depends on the tissue type of cancer,
Cancers that occur in the human body, including blood cancers and solid cancers, are extremely numerous and biologically diverse, and each cancer has a slightly different origin, drug sensitivity, and malignancy. Cancer treatment is not always easy.

Therefore, the current situation is that we have no choice but to rely on highly effective drugs, that is, drugs with strong cytotoxicity in a broad sense.

As described above, current anticancer drugs are still insufficient in terms of tumor selectivity and also have various problems in terms of side effects.

Under these circumstances, there is a strong desire for the emergence of newly regulated cancer drugs with relatively high tumor activity and reduced side effects, and the present inventors are working diligently to provide new antibiotics with antitumor activity. I did a lot of research.

(Means for Solving the Problems) The inventors of the present invention isolated bacterial strains from various soils and conducted intensive research on the metabolites produced by the strains, with the aim of searching for anticancer antibiotics as described above. As a result, the culture of bacterial strain KO-3988 isolated from freshly collected soil showed no antibacterial activity against Durum-positive bacteria, Durum-negative bacteria, and some fungi, but did not exhibit antibacterial activity against HeLa cells (human). A cell line derived from squamous epithelial tissue of the uterine cervix.
We have discovered that a new antibiotic has been produced that has the effect of suppressing the proliferation of eLa) cells, and have completed the present invention.

When we investigated the physicochemical properties of the new antibiotic, we found that there is no other substance that has the same physicochemical properties as the new antibiotic, so we named it antibiotic KO-398.
It was decided to call it 8C or KO-3988H.

These new antibiotics provided by this invention KO-
3988C to KO-3988H have the physical and chemical properties described below, and are antibiotics KO-3988C to KO-3988H belonging to the Streptomyces family.
It can be produced by culturing production bacteria in a medium, accumulating these antibiotics in the culture, and collecting antibiotics KO-3988C to KO-3988H from the culture.

This invention will be explained in detail below.

Antibiotic-producing bacteria The bacteria that produce antibiotics KO-3988C to KO-3988H belong to the genus Strebtomyces.

For example, strain KO-3988, which belongs to the genus Streptomyces and was isolated by the present inventors from the soil of Atami City, Shizuoka Prefecture,
This is an example of a strain most effectively used in the present invention.

The mycological properties of strain KO-3988 are as follows.

(I) Morphological properties Vegetative hyphae develop well on various agar media, and no division is observed. Aerial hyphae are moderately colonized on tyrosine agar and shucoulos nitrate agar, but poorly colonized on other media. The color ranges from white to brown. When observed under a microscope, aerial hyphae are linear and chains of 20 or more spores are observed. Spore size is 1.4XO. The spores have a cylindrical shape with a diameter of 9 μm and a smooth surface. Note that sclerotia, sporangia, and zoospores are not observed.

(If) Properties on various media The above-mentioned antibiotic-producing bacteria were cultured using various media, and the antibiotic-producing bacteria described above were cultured using various media.
g) and De Gottlieb (D. Gott I Ie
Method b) (International Journal of
Systematic Bacteriology Volume 16, 31
3, p. 3, 1966). The results are shown below for each culture condition used.

In addition, one color tone is determined as a standard color using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958), and its code is included in parentheses along with the color chart name. I wrote this. Moreover, unless otherwise specified, these results are the results of observation using one culture medium at 27° C. for 2 weeks.

(1) Sucrose/nitrate agar ■Growth Moderately grown Alabaster tint (13ba) ■Back side Alabaster tint (13ba)
■Aerial mycelia Moderate epiphytic white to alabaster tint (from a to 13 ha) ■Soluble pigments Not produced (2) Glucose ◆Asparagine agar (ISP) ■Growth Good growth Light tan (3 gc) ■Back side Light amber ( 3 1c) ■Aerial mycelium Slightly epiphytic white (a) ■Soluble pigment Not produced (3) Glycerol-asparagine agar (IsP) ■Growth Good growth Nude tan (4 gc) ■Back side Nude tan (4 gc) ■Aerial mycelium
Moderately epiphytic (5 cb) ■Soluble pigment Dusty Beach (5 ec) (4) Starch/Inorganic Salt Agar (ISP) ■Growth
Good growth Pale bink to nude tan (3 ca to 4 ge) ■Back side Veil pink to light spice brown (3 ca to 11 g) ■Aerial mycelium Medium epiphytic white to beige gray (a to 3 ih) ■Soluble pigment Peach tan (5 gc) (5) Tyrosine agar (ISP) ■Growth Good growth Mustard brown (2 ni) ■Back side Mustard tan (21 g) ■Aerial mycelium
Good epiphyte white to natural (a to 2 dC) ■Soluble pigment Covert brown (21i) (6) oatmeal agar (IsP) ■Growth Moderately grown bamboo (2 gc) ■Backside bamboo (2 gc) ■Ki mycelium
No epiphyte ■ No production of soluble pigment (7) Yeast extract/malt extract agar (ISP) ■ Growth
Poor growth ■ Back side ■ Aerial mycelium ■ Soluble pigment (8) nutrient agar (ISP) ■ Growth Moderate growth Bamboo ( 2 gc) ■ Back side Bamboo ( 2 gc) ■ Aerial mycelium
No epiphyte ■Soluble pigment Bamboo (2 gc) (9) Glycerol/calcium malate agar ■Growth Good growth Camel (3 1e) ■Back side Camel (3 ie) ■Aerial mycelia
■Soluble pigment Dusty Coral (6 gc) (
IO) Glucose/Beptone Agar ■Growth Good growth Corktan to Tile Red (41e to 5 ne) ■Back side Rust Brown (5 pg) ■Aerial mycelium No epiphyte ■Soluble pigment Tile Red (5 ne) (11
) Peptone Yeast Extract Agar (IP'S) ■ Growth
Medium growth natural (3 dc) ■Back side natural (3 dc) ■Aerial mycelium
No epiphyte ■Soluble pigment Spice brown (3 pn) (1
2) Glucose/Nitrate Agar ■Growth Good growth Light tan (3 gc) ■Back side Camel (3 ie) ■Aerial mycelium
Non-adhesive ■Soluble pigment Light Tan (3 gc) (III)
Physiological properties (1) Production of melanin pigment (a) Tyrosine agar (b) Peptone/yeast iron agar (c) Glucose/peptone ◆ Gelatin medium (21-23°C) (ii) Tributone/yeast solution Positive, positive, positive, positive (2) Tyrosinase reaction Positive (3) Hydrogen sulfide production Positive (4) Nitrate reduction Negative (5) Gelatin liquefaction (2)
1-23℃) Positive (glucose/peptone e-gelatin medium) (6) Starch hydrolysis Positive (
7) Coagulation of skim milk (27℃) Negative (8) Pebutonization of skim milk (27℃) Positive (9) Growth temperature range 15-35℃ Optimum temperature for growth
27°C (i0) Availability of carbon sources (Priedum-Gotlieb agar medium) - Utilize D-glucose, L-arabinose, D-xylose, D-mannitol, D-fructose, and i-inositol.

●Uses melibiose and L-rhamnose to some extent.

・Raffinose and sucrose are not used.

(11) Decomposition of cellulose Negative (IV
) Cell wall composition Diaminobimelic acid in the cell wall is of the LL type.

The mycological properties of this antibiotic-producing bacterium have been described in detail above, and can be summarized as follows.

Diaminobimelic acid in the cell wall of Honkan is of the LL type.

The morphology of aerial hyphae is linear and forms long spore chains.
The surface of the spore is smooth.

Regarding various cultural properties, vegetative mycelium exhibits a brown color tone, and aerial mycelium exhibits a white or brown color tone. Note that the soluble pigment produces a brown pigment.

From the above results, this bacterial strain belongs to the genus Strebtomyces, and according to Pridham and Tresner's classification (Burgess Manual of Determinative Bacteriology, 8th edition, pp. 748-829, 1974) ), it is thought to be a species belonging to the white or red series.

In addition, this bacterial strain is Strebtomyces SB K O
- 3 9 8 8 (Strepta+yces
sp. Ko-3988), which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology
No. 89).

Above, antibiotics KO-3988C or KO-3988
Although H-producing bacteria have been explained, the mycological properties of actinomycetes are generally extremely variable and not constant. Actinobacteria can be naturally or normally treated with ultraviolet irradiation, X-ray irradiation, or mutagenic agents (e.g., N-methyl-N
It is a well-known fact that mutations can be made by artificial mutation means using 0-N nitrosoguanidine, ethyl methanesulfonate, etc.). Such artificial mutant strains as well as natural mutants belong to the genus Streptomyces, and are not suitable for antibiotics KO-3988C or KO-3988H.
Any strain capable of producing can be used in the present invention.

Production of a novel compound In the present invention, first, bacteria producing antibiotics KO3988C to KO-3988H are cultured in a suitable medium.

In culturing this bacterium, a method for culturing actinomycetes is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, an inorganic salt is used.

As assimilable carbon sources, glucose, molasses, lees, dextrin, cellulose, corn glycerin, organic acids, and the like are used alone or in combination. Digestible nitrogen sources include organic nitrogen sources such as pebtone, meat extract, yeast extract, dried yeast, soy flour, corn stew liquor cottonseed meal, casein, soy protein digests, amino acids and urea, or nitrates and Inorganic nitrogen compounds such as ammonium salts are used alone or in combination. In addition, sodium salt as necessary,
Inorganic salts such as calcium, potassium, magnesium, and phosphate salts can be added. moreover,
If necessary, micronutrients, growth promoters, or precursors that promote the growth of this bacterium and the production of the new antibiotic compounds KO-3988C to KO-3988H may be appropriately added.

The culture is preferably carried out under aerobic conditions such as shaking culture or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. The pi{ of the medium is preferably around neutrality. Cultivation can be carried out at a temperature of 20 to 37°C, but usually 24 to 37°C.
It is best to maintain the temperature at 30°C, preferably around 27°C. In the case of liquid culture, the culture time is usually 3 to 6 days.
This antibiotic is produced and accumulated. Preferably, the culture is terminated when the amount accumulated during the culture reaches the maximum.

These culture compositions, medium liquid properties, culture temperature, stirring speed,
Culture conditions such as the amount of aeration and the like are adjusted or selected according to the type of idle plants used, external conditions, etc. so as to obtain preferable results.

If foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants are conveniently used.

The novel compounds, antibiotics KO-3988C to KO-3988H, are accumulated in the culture thus obtained. In particular, since antibiotics KO-3988C to KO-3988H are contained in the culture filtrate and cultured bacterial cells, the culture may be filtered by adding a filter aid (e.g., Celite and High Flow Super Cell) as necessary. It is advantageous to separate the culture filtrate and the bacterial cells by centrifugation or to collect the antibiotics KO-3988C to KO-3988H from a mixture of the culture filtrate and a concentrated methanol extract of the bacterial cells. .

Antibiotics KO-3988C to KO-3988H are
Insoluble in hexane and water, and many organic solvents such as alcoholic solvents (e.g. methanol and ethanol), chloroformic solvents (e.g. dichloromethane and chloroform), and ketonic solvents (e.g. acetone and methyl isobutyl ketone). In order to separate and purify the present antibiotic substance from the culture filtrate and cultured bacterial cells, it is preferable to use a purification method that utilizes these properties. Usually, the culture filtrate and the methanol extract of bacterial cells are concentrated separately, then the respective concentrates are mixed, and the antibiotic is extracted from this mixture using a non-hydrophilic solvent (e.g., ethyl acetate and chloroform). Transfer to the organic solvent layer. The organic solvent layer thus obtained is washed with a dilute aqueous ethylenediamine tetrahydrochloride solution to remove metal ions, etc., if necessary. After that, various dehydrating agents (for example, anhydrous sodium sulfate and bead gel) are added to dehydrate. The solvent in the dehydrated organic solvent layer is then removed under reduced pressure. In this concentration operation, antibiotics KO-3988G to KO-3
Although 988H is stable, it is usually preferable to conduct the heating at a temperature of 50° C. or lower. Add organic solvents such as hexane and petroleum ether to the residue to prepare antibiotic KO-3.
988C to KO-3988H can be precipitated. The obtained precipitate was washed several times with hexane, etc., and then filtered with suction or centrifuged to remove the antibiotic KO-3988C.
to obtain a crude product of KO-3988H.

To further purify the above crude product, antibiotic KO-
Many means can be used that utilize the difference in solubility between 3988C or KO-3988H and the congested substance, the difference in distribution between two immiscible liquids, or the difference in adsorption power to various adsorption carriers.

Among these, chromatography is particularly effective. Chromatography is effective for purifying this antibiotic.
Adsorption chromatography using adsorption resins such as silica gel, alumina, activated carbon cellulose, and hydroxyapatite HP-20, reverse phase partition chromatography using silanized silica gel and octadecylsilanized silica gel, and Cef7dex LH-20. and gel filtration chromatography based on molecular sieves using Toyovar, etc., and ion exchange chromatography using DEAE-cellulose, DEAE-Sephadex, DEAE Toyovar, etc.

Antibiotics KO-3988C to KO-3988H are
Separation and purification can be carried out by using the above-mentioned methods such as chromatography, electrophoresis, countercurrent distribution, ultrafiltration, and distillation alone, in combination in any order, or repeatedly. For example, the above crude product is dissolved in a small amount of chloroform, adsorbed on silica gel, and subjected to column chromatography using a chloroform-methanol mixed solution. The obtained active fraction was concentrated under reduced pressure, dissolved in a small amount of chloroform, and subjected to gel filtration chromatography based on molecular sieves using a chloroform-methanol mixed solution.
88C to KO-3988H can be separated and purified.

Below, antibiotics KO-3988C or KO-3988
The physicochemical and biological properties of H are described.

■． Physical and chemical properties (KO-3988C) (1) Structural formula Soluble in methane, chloroform, ethyl acetate, butyl acetate, and acetone (9) Distinction between basic, neutral, and acidic: Neutral substance (1O) - Color Reaction: Positive for H2S04 and iodine coloring (11) Appearance: Pale yellow needle-shaped crystals <KO-398
8D> (1) Structural formula: (2) (3) (4) (5) (6) (7) (8) ? Elementary composition: C2■H2605 Molecular weight: 370 Melting point = 213-215 Specific rotation = [α] D − -38° (C-0.55
, chloroform) Ultraviolet absorption spectrum: 221,267,298.4
08r+ll Infrared absorption spectrum: 32B0.1B55.161
51555.1390,1280.1195 cm-'
Solubility in solvents: Insoluble in hexane and water, methanol, ethanol, dichloro (2) (3) (4) (5) Elemental composition: C, 2H2.06 Molecular weight: 386 Melting point: 177-179°C Specific rotation: [α] o = -95° (C - 0.53
, chloroform) (6) Ultraviolet absorption spectrum: 221.287,29
2.407rv (7) Infrared absorption spectrum; 3350.18EiO
, 1B301575, 1440, 1275. l1[i0
Mark (8) Solubility in solvents: insoluble in hexane and water, soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone (9) Basic, neutral, acidic distinction: Neutral Substance (10) Color reaction: Positive for H2SO4 and iodine color development (l1) Appearance: pale yellow powder <KO-3988E) (1) Structural formula: (2) Elemental composition: C 22H 240 b (3) Molecular weight: 384 (4) Melting point = 184-186°C (5) Specific optical rotation = [α]2'- -79° (C 0.
2B, chloroform) (6) Ultraviolet absorption spectrum: 215.240 (sh
). 265.298.400nm (7) Infrared absorption spectrum: 3400.1665.
1B30.1570, 1440.1295.1200
am-' (8) Solubility in solvents: insoluble in hexane and water, soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone (9) Distinction between basic, neutral, and acidic: Neutral substance (10) Positive for color reaction diH2SO4 and iodine color development (1l) Appearance: Pale yellow powder (KO-3988F> (1) Structural formula: (9) Basic, neutral, acidic distinction: Neutral substance (lO) color reaction: Positive for H2, SO4, and iodine (11) Appearance: Pale yellow powder (KO-3988G> (1) Structural formula: (2) Elemental composition: C 22 H 260 b ( 3) Molecular weight: 386 (4) Melting point: 115-118°C (5) Specific rotation: [α] o = -13° (C-
0.35, chloroform) (6) Ultraviolet absorption spectrum: 218,286,29
4.400ni (7) Infrared absorption spectrum; 3400.1660.
1B351575.1440.1290.1195 c
m-' (8) Solubility in solvents: Insoluble in hexane and water, soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone (2) (3) (4) (5) ? Elementary composition 2C2■H240? Molecular weight: 400 Melting point: 121-124°C Specific optical rotation: [α] D” +12° (C-0.33
, chloroform) (6) Ultraviolet absorption spectrum: 219. 266,2
97,406nm (7) Infrared absorption spectrum: 3350,1860.
1B35, 1580.1400, 1285.1165
cm-' (8) Solubility in solvents: insoluble in hexane and water, soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone (9) Basic, neutral, and acidic levels. Neutral substance (10) Color reaction: Positive for H2SO4 and iodine color development (11) Appearance: Pale yellow powder <KO-3988H) (1) Structural formula: ? 2) Elemental composition: C2 ■ H 260 8 (3) Molecular weight =
418 (4) Melting point: 75-78°C (5) Specific rotation: [α] D- -52° (C10.
25, chloroform) (6) Ultraviolet absorption spectrum: 220.268,29
0,405nm (7) Infrared absorption spectrum: 3350.1660.
1B301575.1400, 1290.1185 c
m-' (8) Solubility in solvents: insoluble in hexane and water, soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone (9) Distinction between basic, neutral, and acidic: medium (10) Color reaction: Positive for H2S04 and iodine color development (1L) In physical and chemical properties beyond the appearance of a pale yellow powder, the molecular weight can be determined by field desorption (by elemental composition or high-resolution mass spectrum). FD) Measured by mass spectrometry. Moreover, the ultraviolet absorption spectrum was measured in methanol, and the infrared absorption spectrum was measured by the KBr disc method. Furthermore, nuclear magnetic resonance spectra were obtained using TM in deuterated chloroform.
This was done using the S standard.

The antibacterial spectrum of each antibiotic is shown below.

■． Biological properties (1) Antibacterial spectrum test of KO-3988C Bacillus MIC' Yu/wJ) Staphylococcus aureus FDA209P Staphylococcus aureus ATCC6538P Bacillus subtilis ATCC6633 Bacillus subtilis IFO3001 Micrococcus luteus ATC
C9431 Mycobacterium smegmatis ATC
C607 Eschlichia e Collie NIHJ Eschlichia Collie NIHJ JC-2 Crepziira punumoniae ATCC10031 Salmonella
Chibimium buloteus vulgaris IFO3167 Pseudomonas aeruginosa IFO3080 Candida Albicans Satsuriromyces cerevisiae ATCC9763 Crybutococcus neoforman Microsvorum dibcerum trichophyton Mentagrophytopenicillium Hercules IFO4674 Botrytis
Cine 1 Noscleotinia Cinerepilicularia Oryzemucor ● Racemosus IF04581>+000 )+000 )1000 >1000 >1000 >1000 >1000 >1000 >1000 )1000 >1000'>1000'>1000'>1000')1000'＞1000'＞1000' ) 1000' ＞1 000'＞1000'＞1000'＞1000' ゜Agard Dilution Note; ``MIC - Minimum Inhibitory Concentration; ゜Using Nutrient Agar (Agar is 1.0㎢): 1 using agar (2) Antibacterial spectrum of KO-3988D゜Agard dilution method; ``MIC-minimum inhibitory concentration; ゛using nutrient agar (agar is 1.0%); using 1 agar (4) KO -3988F antibacterial spectrum 5 Agar dilution method; bMIC - minimum inhibitory concentration; 'Nutritional agar used (agar is 1.0%); 6 Boteto agar used (3) KO-3988E antibacterial spectrum test bacterium Staphylococcus ●Aureus FDA209P Staphylococcus aureus ATCC6538P Bacillus ●Subtilus ATCC6633 Bacillus subtilis IFO3001 Micrococcus ●Luteus ATC
C9431 Mycobacterium Smegmatis ATC
C607 Eschlichia Collie NIHJ Eschlichia Collie NIHJ JC-2 Crepziira Punumoniae ATCC10031 Salmonella
Chibimium Proteus●Vulgaris IFO3167MICゝmue
/1) >1000e >1000' )1000c >1000''>1000'>tooo'>1000')1000'')1000'>tooo'>1000' 1 Agar dilution method;゛Nutritional agar used (agar is 1.0%); 4 Potato agar used (5) KO-3-988G antibacterial spectrum test bacteria MIC'''mu《/1) Bacillus subtilis ATCC6633 Bacillus saptillus IFO3001 Micrococcus luteus ATCC9431 Mycobacterium Smegmatis ATCC607 Escherichia coli NIHJ Escherichia coli NIHJ JC-2 Crepziira punumoniae ATCC10031 Salmonella e
Chibimium buloteus vulgaris IFO3167 >1000° >1000'>1000'''>I 000° >1000'>ioooe>1000c)1000'''>1000''' ゜Agardilution wide; 1 old C-1 smallest Growth inhibitory concentration: ゛Nutritional agar used (agar is 1.0%); 4 Potato agar used (6) KO-3988H antibacterial spectrum test bacteria MIC' k/m) Staphylococcus aureus FDA209P Staphylococcus aureus Aureus ATCC6538P Bacillus subtilis ATCC6633 Bacillus subtilis IFO3001 Micrococcus Luteus ATC
C9431 Mycobacterium smegmatis ATC
C607 Eschlichia Collie NIHJ Eschlichia Collie NIHJ JC-2 Klebziira Punumoniae ATCC10031 Salmonella
Chibimium Proteus 9 vulgaris IFO3167 Pseudomonas aeruginosa IF03080 Candida Albicans Saccharomyces cerevisiae ATCC9763 Crybutococcus neoforman Microsvorum Gypserum L. Lycophyton Mentagrophytoveninrium Herque IFO4674 Botrytis
Cinelescleotinia cinerebilicularia ● Oryzemucor ● Racemosus IF04581) 1000'>iooo'>1000'>1000'>1000'>1000'>1000' )10rot )1000'>1000'>1000'>1000'>1000')1000')+000'>1000'>1000')1000'>1000')1000')1000'>1000' 1 Agar dilution method; bMIC-minimum inhibitory concentration; ゛Use of nutrient agar ( Agar is 1.0%): As is clear from the results obtained using 4-pot agar, antibiotic KO-3988
It was found that C to KO-3988H did not exhibit antibacterial activity against Durum-positive bacteria, Durum-negative bacteria, and fungi.

■． Cell killing activity of antibiotics KO-3988C to KO3988H In an in vitro activity test against 81B melanoma cells, which are malignant melanomas, the minimum growth concentration (MIC) of antibiotics KO-3988C to KO-3988H was measured. The results are shown in Table 1.

Table 1 Antibiotics KO-3988C KO-3988D KO-3988E KO-3988F KO-39880 KO-3988H MIC (■/1) 0.78 1.6 0.78 0.78 0.78 0.78 The following is carried out The present invention will be explained in more detail by giving examples, but the present invention is not limited to these examples.

(Example) A. Culture of Strebtomyces KO-3988 Agar containing 1.0% glucose, 0.5% pebtone, 0.5% meat extract, 0.3% salt, and 1.2% agar Using a slant medium, Streptomyces SB KO −
3 9 8 8 (FERN-Pl0389) 27
The cells were cultured at ℃ for 6 days.

Separately, in a 500 ml Sakaro flask, a liquid culture (pH 7 .0
) 100 ml was prepared and sterilized.

One platinum loop of KO-3988 was inoculated into the ε-necked flask from the agar slant medium, and a reciprocal force seeker (amplitude 1
7 cms (120 reciprocations per minute) and shaking culture at 27°C for 72 hours to obtain seeds, and place them in a 3047 volume jar fermenter with 2.0% starch, 1.0% soybean flour, and 0.3% salt.
%, and medium 2 containing 0.3% calcium carbonate (
1 and sterilized, 200 ml of the seed mother was aseptically transplanted, and cultured with aeration with stirring to obtain a culture extract of about 20 ml.
I got 1. 40 (in a liter tank, 2.0% starch, 1.0% soybean flour, 0.3% salt, and 0 calcium carbonate)
．． After charging and sterilizing 200 g of a medium containing 3%, 20 Dl of the cultured seeds were aseptically transplanted and cultured with aeration with stirring to obtain about 200 g of cultured seeds.

Ethyl acetate was added to the culture solution obtained in step A above and thoroughly stirred to transfer and dissolve the antibiotic into the organic solvent. After standing still, the ethyl acetate layer containing the antibiotic was separated. Anhydrous sodium sulfate was added to this organic solvent (ethyl acetate layer) for dehydration. The dehydrated organic solvent is concentrated under reduced pressure to remove the solvent,
Approximately 300 g of an oily residue containing antibiotics KO-3988C to KO-3988H was obtained.

C, silica gel chromatography The oily residue obtained in step B above was transferred to a silica gel ram (inner diameter 80 mm, length 600 m+s) filled with silica gel (manufactured by Merck & Co., Ltd.).
) was adsorbed in advance using chloroform, and chromatography was performed while continuously changing the developing solution from chloroform to methanol. The obtained active fraction was concentrated under reduced pressure to obtain antibiotics KO-3988C to KO-
17 g of a crude product containing 3988H was obtained.

A column with an inner diameter of 30 mm and a length of 400 mm was used, and the column was filled with Sephadex LH-20. Using such a column, the crude product dissolved in a small amount of methanol was passed through the column. Using methanol as the developing solvent, antibiotics KO-3988C to KO-39
88H was sequentially eluted, and pure products of each antibiotic were obtained from each elution part. The results are shown in Table 2. Antibiotics KO
The physicochemical properties and biological properties of -3599C to KO-3599H are as described above.

Table 2 Antibiotics KO-3988C or KO obtained in C above
From the crude product containing -3988H, antibiotic KO-
Column chromatography was performed to obtain pure 3988G to KO-3988H. In addition, this column chromatography antibiotic KO-3988C KO-3988D KO-3988E KO-3988F KO-39880 KO-3988H Yield (g) 0.35 1.5 0.021 0.010 0.00511i 0.035 ( Effects of the Invention) According to the present invention, a novel antibiotic having antitumor activity and a method for producing the same are provided.

Claims (3)

[Claims] (1) Antibiotic KO- expressed by the following formula 3988C to KO-3988H. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (Here, R is ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼) (2) Cultivate a bacterium belonging to the genus Streptomyces and producing the antibiotic KO-3988C to KO-3988H according to claim 1 in a medium, accumulate the antibiotic in the culture, and collect the antibiotic from the culture. The method for producing an antibiotic according to claim 1, characterized in that the antibiotic is collected. (3) The bacteria belonging to the genus Streptomyces is Streptomyces KO-3988 (Feikoken Bacteria-P-10369).
) The manufacturing method according to claim 2.