JPH0555111B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to novel antitumor antibiotics and their production and harvesting. The structure of the antitumor antibiotic of the present invention has not yet been identified. However, in view of their unique physical, chemical and biological properties, applicants believe that BBM-1675 antibiotics are novel substances. European Patent Application No. 95154A1 describes Actinomadeura pulveraceus
6049 (ATCC 39100) is disclosed.
Although the structure of the WS6049 antibiotic has not yet been clarified, the characteristics that characterize the antibiotic indicate that WS6049-A and WS6049-B are structurally related to the BBM-1675 antibiotic of the present invention. It is shown. However, the spectral data
Both WS6049-A and WS6049-B are owned by the applicant.
This indicates that it is not identical to any of the BBM-1675 components. Furthermore, European Patent Application Publication No.
The producing bacteria described in No. 95154A1 are ISP medium No. 2 and 3.
and 4 in its aerial mycelial color, positive milk peptonation and positive utilization of D-fructose, D-mannite, trehalose and cellulose, Actinomadura verrucospora used in the present invention.
verrucosospora). According to the present invention, there is provided a novel anti-tumor antibiotic conjugate, herein referred to as BBM-1675, which complex can be applied to BBM- 1675—Producer strain, most preferably Actinomadeura verruchosospora strain H964—92
(ATCC 39334) or Actinomadeula verruchosospora strain A1327Y (ATCC 39638) or mutants thereof until a substantial amount of the BBM-1675 complex is produced by the strain in the medium, and the complex is Produced by optionally harvesting from the culture medium. In addition, according to the present invention, two main biologically active components BBM-1675 complex (BBM
-1675A ₁ and _A2 ) and four trace bioactive components BBM-1675 complex (BBM
-1675A ₃ , A ₄ , B ₁ and B ₂ ). These components can be separated and purified by conventional chromatography operations. BBM―
The 1675 complex and its bioactive components exhibit both antibacterial and antitumor activity. The present invention relates to a novel anti-tumor antibiotic conjugate, herein referred to as BBM-1675, and also to certain strains of Actinomadeula verruchosospora,
Most especially Actinomadeula verruchosospora strains
H964-92 and its mutant strain Actinomadeura
Concerning its production by fermentation of Verrucosospora strain A1327Y. The parent strains listed above are from Argentina, Misiones, and Pto Esperanza.
It was isolated from a soil sample collected in Esperanza. Biologically pure cultures of this fungus are kept at the American Type Culture Collection in Washington, DC.
Culture Collection) and ATCC
It was added to the permanent collection of microorganisms as 39334. Mutant strain A1327Y was then obtained by conventional nitrosoguanidine (NTG) treatment of strain H964-42 and has been deposited with the American Type Culture Collection as ATCC 39638. Many antibiotics - As in the case of production cultures, Actinomadeula verrucosospora strain H964-42
Alternatively, fermentation of strain A1327Y produces a mixture or complex of component substances. Two major bioactive ingredients, BBM-1675A ₁ and A ₂ and four minor bioactive ingredients, BBM-1675A ₃ , A ₄ , B ₁
and B ₂ is the BBM produced during the fermentation process.
Isolated from the 1675 complex. BBM-1675 and components BBM-1675A ₁ , A ₂ ,
A ₃ , A ₄ , B ₁ and B ₂ exhibit antibacterial activity against a broad spectrum of microorganisms, including especially Gram-positive bacteria. The BBM-1675 complex and its isolated bioactive components also exhibit phage-inducing properties in lysogenic bacteria. Two of the components, BBM-1675A ₁ and A ₂ , have been screened in vivo against various murine tumor lines and show inhibitory activity against L-1210 leukemia, B16 melanocytoma, and Lewis lung cancer. BBM-1675A ₃ and A ₄ are Mouse P-
It was found to be active against 388 leukemia.
Accordingly, the conjugate and its bioactive components can be used as antimicrobial agents or antitumor agents for inhibiting mammalian tumors. Microorganisms Streptomyces strain No. H964-92 was isolated from soil samples and can be prepared as a biologically pure culture for characterization by conventional methods. Strain H964-92 forms spore chains in aerial hyphae that exhibit a straight, curved, or hooked shape. The spores are spherical or oblong and have a ribbed surface. Aerial mycelium is poorly produced in most media. The color of the aerial mycelial mass is white, which later becomes pinkish or even blueish in some agar media. The basal hyphae are colorless or pale pink. Growth temperature is 15℃
In the range of ~43℃. Cell wall amino acid composition and total cell hydrolyzate sugar content indicate that strain H964-22 belongs to cell wall type _B. Menaquinone is MK-9 (H ₆ ). MK―
9 (H ₈ ). Main morphological along with chemical cell wall composition characteristics,
Based on cultural and physiological characteristics, strain H964
-92 can be classified as belonging to the genus Actinomadeula. The original strain H964-92 produced slightly normal growth and also carried poor aerial hyphae, but H964-42
A mutant strain showing good growth and improved aerial mycelium formation was obtained by treatment with NTG (nitrosoguanidine). The mutant strain (designated strain A1327Y) facilitated further taxonomic investigation and was identified as Actinomadeula verruchosospora. Methods The media and procedures used to study culture characteristics and carbohydrate availability were obtained from the International Streptomyces Project (Intl. J. Syst.
Bacteriol), Vol. 16, pp. 313-340, 1966]. SA Waksman (The Actinomyces)
Actinomycetes) Volume 2] and GM Lvedemann (GMLvedemann) [Intle J. Schist.
Bacteriol. Volume 21, pages 240-247, 1971]
was also used. Cell wall - amino acid composition and whole cell hydrolyzate sugar components were determined by Becker et al., Appl. Microbiol.
Microbiol, Vol. 13, pp. 236-213, 1965 and Lechevalier and Lechevalier, the Actinomyces, H. Prauser, Jena, Gustav Fisher
Verlag), pp. 393-405, 1970. Menaquinones are described by Collins et al., J. Zen. Microbiol (J.Gen.
100, pp. 221-231, 1977;
The menaquinone composition was also determined by Yamada et al., J. Zen. Apple.
Microbicl. (J.Gen.Appl.Microbicl.), No. 23
vol., pp. 331-335, 1977. Morphology Strain H964-92 forms basal and aerial hyphae.
The mycelium is long, branched and undivided into short threads. Aerial hyphae, spore chains are formed uniaxially or at the tips of the hyphae. Spiral branches of spore chains are also observed near the hyphal tips. These spore chains contain 2-10 spores/chain and are straight, bent or hooked. The spores have a warty surface and are circular to oval (0.5-0.6 x 0.6-1.4 μm) with rounded or pointed ends. After maturation, each spore is often separated by a blank pod. No motile spores, sporangia or sclerotia granules are seen on any of the media studied. Culture and Physiological Characteristics The growth of strain H964-92 is poor to fair on both synthetic and natural organic media. Aerial mycelium formation is generally poor, but oatmeal agar (ISP No.
3 medium), inorganic salt-starch agar (ISP No. 4 medium)
and common in Bennett's agar. Natural mutants lacking aerial hyphae occur frequently. The color of aerial mycelia is white, and later becomes pale pink on oatmeal agar, inorganic salt-starch agar, and glycerol-asparagine agar (ISP No. 5 medium). The color of the aerial mycelial mass changes to a bluish color when cultured for a long time (5 months) on oatmeal agar, glycerol-asparagine agar, and tyrosine agar. The color of the basal hyphae is determined by Tuapetsk agar, tyrosine agar, yeast extract-malt extract agar (ISP No. 2 medium), peptone-yeast extract-iron agar (ISP No. 6)
It is colorless to yellowish with medium) and Bennett's agar, and pink with glucose-asparagine agar and glycerol-asparagine agar. Melanoids and other diffusible pigments are not produced. Mutant strain No. obtained from this strain.
A1327Y mainly forms pale blue aerial mycelia and carries abundant aerial spore masses. Strain H964-92 at 15℃, 28℃, 37℃ and 43℃
However, it does not grow at 10℃ or 47℃.
This strain is sensitive to NaCl at 7% and 0.01%
and is resistant to lysozyme. The culture and physiological characteristics of the production strains are shown in Tables 1 and 2, respectively. Table 3 shows the availability of carbon sources.

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[Table] Not growing

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[Table] Cell wall composition and total cell sugar components The purified cell wall of strain H964-92 contains mesodiaminopimelic acid but lacks glycine. The whole cell hydrolyzate was madeulose (3-O-
methyl-D-galactose), glucose and ribose. Cell wall amino acids and total cell sugar components indicate that strain H964-92 is located in cell wall type _B. Two major components of menaquinone were identified as MK-9 (H ₆ ) and MK-(H ₈ ). Classification position of strain H964-92 Strain H964-92 has the following main characteristics.
(1) Aerial mycelia spore chains, short, straight, bent or hooked. (2) Spores: The surface of warts. (3) Aerial mycelium: pink or blueish in color. (4) Basal hyphae: pinkish in some medium. (5) Diffuse dye: None. (6) Mesophilic. (7) Cell wall type _B. (8) Menaquinone series: MK-9 (H ₆ ) and MK-9
( _H8 ). These main characteristics indicate that strain T964-92 belongs to the genus Actinomadeula. The earliest species of the genus Actinomadeula were isolated from mammals. Obtained from several bacterial strains or plant materials. However, many of the new species that have been proposed recently have been isolated from soil. Good fellow
et al., J.Gen.Microbiol,
According to numerous taxonomy and reviews of Actinomadeula and related actinobacteria by Vol. 112, pp. 95-111, 1979, most soil-derived Actinomadeula species are cluster No. It is classified as .7. J. Furment. Technol. (J.Ferment.Technol.), No.
49, pp. 904-912, 1971, Nomura and Obara reported five saprophytic species of the genus Actinomadeula,
Also, Preobrazhenskaya etc. [Actinomyces
Actinomycetes and Related Organisms, Vol.
12, pp. 30-38, 1977] presents keys for the identification and classification of Actinomadeula species. As a result of comparing descriptions of 30 species including microorganisms disclosed in patents, strain H964-92 was found to be the Actinomadura coerulea disclosed in the above-mentioned publication of Preobratschenskaya et al.
It also appears to be most similar to Actinomadeula verruchosospora described in the Nomura article cited above. Strain H964-92 was transformed into A. Verrucosospora strains
It was directly compared with KCCA 0147 and found to be closely related to A. verrucosospora in morphological, cultural and physiological characteristics. Therefore, the bacterial strain
H964-92 is classified as a new strain of Actinomadeula verruchosospora. For the production of BBM-1675, the present invention uses a special bacterial strain Actinomadeura verruchosospora strain.
Although H964-92 (ASCC 39334) and its mutant strain (referred to as strain A1327Y (ATCC 39638)) are described in detail, the present invention is limited to these microorganisms or microorganisms that have been fully disclosed with the culture characteristics described in the text. It should be understood that this is not the case.
It is specifically contemplated that the present invention encompasses strain H964-92 and all natural and artificial BBM-1675-producing variants. Antibiotic Production The BBM-1675 antibiotic of the present invention can be used in the production of Actinomadeula verruchosospora in a normal aqueous nutrient medium.
BBM-1675-producing strain preferably ATCC 39334
Alternatively, it can be produced by culturing a strain of Actinomadeula verruchosospora having the identifying characteristics of ATCC 39638 or a mutant strain thereof. Grow the microorganisms in a nutrient medium containing known nutrient sources for actinomycetes, i.e. assimilable carbon, nitrogen sources plus any inorganic salts and other known growth factors. Although deep aeration conditions are preferably used for large quantity antibiotic production, surface culture and bottles may also be used for limited quantity production. General procedures used for culturing other actinomycetes can be applied to the present invention. The nutrient medium contains a suitable assimilable carbon source such as glycerin, L(+)-arabinose, D-xylose, D-ribose, D-rhamnose, D-glucose, D-fructose, sucrose, cellbiose, soluble starch, D-mantol. or must contain isositol.
As a nitrogen source, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. may be used alone, or an organic nitrogen source such as peptone,
It can be used in combination with meat extract, yeast extract, corn steep liquor, soybean flour, cottonseed flour, etc. If necessary, nutritional inorganic salts providing sources of sodium, potassium, calcium, ammonium, phosphate, nitrate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron, etc. may also be added. . Production of BBM-1675 antibiotics can be carried out at any temperature that permits favorable growth of the producing bacteria, such as 15-45°C, and is preferably carried out at temperatures of about 27-32°C. Optimum production is usually obtained after a culture period of about 68-180 hours, depending on whether shake flask, stirrer or tank fermentation is used. When tank fermentation is to be carried out, it is desirable to produce a nutrient inoculum in the nutrient broth by inoculating the Pross medium with a slant or soil culture or a freeze-dried culture of the producer bacteria. After obtaining an active inoculum in this way, it is transferred aseptically to a fermentation tank medium. Antibiotic production can be monitored by a paper disc-agar diffusion assay using Staphylococcus aureus 209P as the test organism. Isolation and Purification Upon completion of fermentation, the BBM-1675 complex can be obtained from the broth by conventional isolation procedures such as solvent extraction. Thus, for example, the whole broth can be separated into a mycelial cake and a broth supernatant by filtration or centrifugation. Antibiotics in mycelium cake are
Suspend the cake in methanol to remove insoluble materials,
Then, it can be collected by concentrating the methanol extract. The active components in the broth supernatant can be collected by extraction with n-butanol. The n-butanol and methanol extracts are then combined and azeotropically evaporated to an aqueous solution where the majority of the antimicrobial activity is deposited as an oily solid. The solid is then dissolved in methanol and the solution is filtered. Concentrate the liquid and add to a mixture of ethyl acetate and water. The resulting organic extract contains crude BBM-1675 complex, which can be precipitated from solution by addition of an antisolvent such as n-hexane. The crude BBM-1675 complex contains two major bioactive components, BBM-1675A ₁ and A ₂ , and four minor active components, BBM-1675A ₃ , A ₄ , B ₁ and B ₂ ,
It is a mixture of several components including. These biologically active components can be separated and purified by conventional chromatography operations. In one operation, the crude BBM-1679 complex is first dissolved in methanol and purified by Sephadex LH-20 column chromatography using methanol as the eluent. This partially purified complex was then chromatographed on a silica gel column and eluted in a stepwise manner with increasing concentrations of methanol to remove BBM-
A mixture of 1675A ₁ , BBM-1675A ₂ , A ₃ and A ₄ and a mixture of BBM-1675B ₁ and B ₂ can be obtained. Component _A1 can be further purified by Sephadex LH-20 column chromatography using methanol as the eluent.
A mixture of A ₂ , A ₃ and A ₄ was prepared in a bonder pack using increasing concentrations of aqueous acetonitrile as the eluent.
C ₁₈ can be separated by chromatography at Waters Associates Lnc. blend
B ₁ and B ₂ can be separated by silica gel column chromatography using a mixture of chloroform and methanol as an eluent. Further details of suitable chromatographic separation procedures are provided in the Examples below. Physicochemical Properties of BBM-1675 Components The six bioactive components of the BBM-1675 complex can be distinguished from each other by two TLC systems as shown in the following table.

[Table] Separation of BBM-1675A ₂ , A ₃ , and A ₄ was difficult with a conventional phase TLC system, but could be achieved with reversed phase TLC. Individual BBM-1675 components exhibit similar solubility and color reactions to each other. For example, these components are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride. These components show positive reactions with ferric chloride, Ehrlich and Tollens reagents, but negative reactions with Sakaguchi, ninhydrin and Anthrone tests. The characteristic physicochemical properties of BBM-1675 components are shown in Table 5 below.

Table: The UV absorption maximum of the BBM-1675 component was observed at 253, 282 and 320 nm, which did not shift in acidic or alkaline solutions. BBM-1675A ₁ ,
The IR and PMR spectra of A ₂ , A ₃ and A ₄ are shown in Figures 1-4 and 5-8, respectively.
The 360MHz PMR of BBM-1675A ₁ contains two aromatics (7.50 and 8.59ppm) and one NH
(11.79ppm) along with proton, 1 acetyl (δ: 2.11ppm), 1 N-CH ₃ (2.52ppm), 4
OCH ₃ (3.42, 3.80, 3.88 and 3.98ppm) and one exomethylene (4.57 and
4.48ppm). The CMR spectrum of BBM-1675A ₁ is a triple intensity signal (δ: 56.0ppm, COH ₃ )
55 carbon signals including . BBM―
The molecular formula of 1675A ₁ is based on proton and ¹³ CNMR spectra, trace analysis and molecular weight measurements by HPLC and SIMS (secondary ion mass spectrometry).
It is inferred that C ₅₇ H ₇₂ N ₄ O ₃₂ . Structural study of BBM-1675A ₁ When treated with 0.5N HCl-CH ₃ OH at room temperature,
BBM-1675A ₁ loses its biological activity and produces lipophilic chromophoric substances (compounds) with several unidentified fragments. Compound () exhibits similar UV absorption to the parent antibiotic, which indicates that compound () has a BBM-
This suggests that it retains the chromophore structure of _1675A1 . Two other chromophore fragments related to compound () were obtained by alkaline hydrolysis of BBM-1675A ₁ . 0.05N KOH at 55℃ for 1 hour
Hydrolysis with CH ₃ OH produced compounds () with UV absorption maxima at 252, 284, 197 (Shoulder) and 322 nm. On the other hand, reaction with 1N KOH--CH ₃ OH yields an acidic chromophoric substance called compound (). The physicochemical properties of compounds (), () and () are summarized in Table 6 below.

Table Structural information for () and () was provided from the following spectral data and chemical transformations.
¹³ C and proton NMR in compound ()
4OCH ₃ , 1=CH ₂ , The presence of 2C=0 and 1>NH groups was indicated. The NMR spectrum of compound () is similar to that of (), and one of the four _OCH3 groups observed in formula ()
They differed only in the absence of one. Such differences, along with the acidic properties of compound (),
suggested that it was the methyl ester of compound (). Heating to reflux with 1N methanolic KOH quantitatively converted compound () to compound (), while treatment with diazomethane converted compound () to compound (). C ₂ H ₅ in OH
Treatment of compound () with NaBH ₄ yields a reduction product (compound (), M ⁺ : m/z 267), which replaces the -COOCH ₃ groups of compound ().
NMR showed a --CH ₂ OH group. Upon hydrogenation over palladium-plated charcoal, compound () produced a dihydro derivative (compound (), M ⁺ : m/z 297). The proton NMR spectrum of compound () showed a new doublet methyl signal and the absence of the exomethylene group present in compound (). Furthermore, one of the _three OCH groups has a high magnetic field (δ:
3.50ppm). These results are for the compound()
inside indicates the presence of a group, which upon hydrogenation forms the compound ()
In reduced to the base. Compound () at 80℃ for 3 hours at 1.5N
When heated with methanolic hydrogen chloride and chromatographed the hydrolyzate on a silica gel column, a weakly basic compound (compound (), M ⁺ :
m/z 211). IR spectrum and physicochemical properties indicated that compound () contained NH ₂ group. Compound () was identified as methyl 4,5-dimethoxy-anthranilate by comparative IR and NMR studies with the standard. Therefore, the structure of compound ()-() was determined as follows. When treated with 0.05N KOH―CH ₃ OH at 55℃,
Compound () is a novel chromogenic fragment (formula,
C ₁₅ H ₂₁ NO ₇ , M ⁺ : m/z 327) and the sugar (formula). The NMR spectrum of () is
One singlet C—CH ₃ and three downfield OCH ₃
In addition to _two upfield OCH groups and two aromatic protons commonly observed in compounds of the formula, and. Further hydrolysis with 1.5N methanolic hydrogen chloride yields the compound,
This one was identical to the one obtained earlier. After removal, the hydrolyzate was treated with 2,4-dinitrophenylhydrazine to precipitate a yellow solid,
This substance was identified as 2,4-dinitrophenylhydrazone of pyruvate. The compound is thus methyl 4,5-dimethoxy-N-(2',2-dimethoxypropionyl) anthranilate.
Although the compound did not show a molecular ion peak,
m/ in the mass spectrum consistent with the molecular formula C ₇ H ₁₄ O ₄
It showed an M-OCH ₃ peak at z 131.
The NMR spectrum showed a 2,6 dideoxyhydropyranose structure for the compound. This designation is supported by the ¹³ C-NMR of Eq.
In addition to the 15 carbon signal that can be specified in the compound
1C-CH ₃ , 1-CH ₃ , 30-CH', and 1 aromatic carbon were produced. The C ₃ -OH of the sugar was esterified with the carboxyl group of the sugar in the low magnetic field (δ:
The _C3 proton of the sugar appears at 5.39ppm, octet). The above results are summarized below. Compounds, and structural compounds of Compound Compound The molecular weight of the compound (457) corresponds to about 1/3 of the total molecules of BBM-1675A ₁ (presented formula
_C57H72N4O32 ; _calculated MW _{= 1324} ). BBM―
The partial structure of 1675A ₁ is thought to be as follows. After the filing _of U.S. _patent application Ser. It turns out that some of the characterization characteristics that were used were inaccurate. Following additional chromatographic purification procedures detailed in Examples 3 and 6 below, BBM
-1675A ₁ and A ₂ were isolated in more purified form and characterized in detail as shown below.
Also, the elemental analysis data for components A ₃ and A ₄ were corrected to indicate the presence of sulfur in these compounds, and HPLC retention times were calculated for these two components. The corrected physicochemical properties of BBM-1675 components are summarized below. BBM-1675A _1Chemical structural formula: Characteristics: White to pale yellow crystals; Melting point: 156-158° (decomposed) Elemental analysis:

[Table] λmax (nm) Absorption coefficient 320 12.4 280 sh (shoulder) 253 25.1 210 25.5 No significant change with acids or bases Light exposure: Solvent - CHCl ₃ [α] ²⁴゜_D = -207゜ (c = 0.0351) The second analysis showed the following light intensity. [α] ²⁷゜_D = -191゜ (c = 0.5, CTHCl ₃ ) Infrared absorption spectrum: See Figure 9 Main absorption bands (KBr) 985, 1015, 1070, 1110, 1150, 1210,
1250, 1308, 1380, 1405, 1446, 1520,
1592, 1608, 1668, 1715, 2920, 2960,
3360, 3440, cm ^-1 Mass spectrum: Instrument - VG - ZAB - 2F FAB - MS - Thioglycerol molecular mass range ion (m/z): 1249,
1357, 1463; by addition of NaCl (m/
z): 1271, 1379, 1485, 1597. FAB-MS-NB (MB: Matrix,
mw154); molecular mass range ion (m/
z): 1249, 1283, 1403, 1555; by addition of NaCl (m/z): 1249, 1271,
1303, 1425, 1483, 1577. FAB-MS-Glycerin-DMSO: Molecular mass range ion (m/z): 1215,
1247, 1279, 1293, 1325, 1353; NaCl
By the addition of (m/z): 1215, 1237,
1269, 1325, 1347, 1375. Instrument: Kratos MS-50 FAB-MS-thioglycerol: Molecular mass range ion (m/z): 1357, 1463 Molecular weight (according to the mass spectrum data above) Apparent MW=
1248 Nuclear magnetic resonance spectrum: Equipment - WM360 Brucker Solvent: CDCl ₃ ¹ NMR: 360MHz δ (ppm): 11.75
(1H, s); 8.55 (1H, s); 7.45 (1H,
s); 6.61 (1H, m); 6.23 (1H, brs);
6.17 (1H, brs); 5.93 (1H, d, J=
9.3); 5.82 (1H, d, J = 9.3); 5.7 (1H,
brs); 5.49 (1H, m); 5.45 (1H, d, J
= 2.3); 5.38 (1H, brs); 4.95 (1H, d,
j=10.2), 4.64 (2H, m); 4.54 (1H,
d, J=2.3); 4.2 (1H, s); 4.15−3.35
(26-28H) [4.10 (1H, m); 4.02 (1H,
brs); 3.95 (3H, s); 3.85 (3H, s);
3.79 (3H, s); 3.46 (1H, m); 3.40
(3H, s)]; 2.82-2.70 (3H, brm);
2.50 (3H, s), 2.47 (1H, m); 2.38-
2.11 (5H); 2.12 (1H, m; 2.11 (3H,
s); 160-1.05 (22H) [1.39 (3H, d,
J = 6.3); 6.31 (3H, d, J = 6.3); 1.29
(3H, d, J = 6.3); ^1.08 (6H)] See Figure 10 13C NMR: 90.3MHz See Figure 11 In a separate test, purified BBM-1675A ₁ of ¹³
The C NMR spectrum of was measured on a sample dissolved in CDCl ₃ (80 MHz). The main peaks are shown below. CMR of BBM−1675A ₁ (80MHz in CDCl ₃ )

[Table] BBM-1675A ₂ chemical structural formula: Characteristics: White crystals: Melting point 147-149℃ Elemental analysis: C: 52.71% H: 5.94% N: 3.94% S: 9.39%, O (difference): 28.01% Ultraviolet absorption spectrum: Equipment - Varian UV, Kyary 219 Solvent - Methanol Concentration - 0.02052g / λmax (nm) Absorption coefficient 320 12.2 282 16.3 252 26.2 214 25.8 No significant change with acids or bases Light intensity: [α] ²⁷ ° _D = -179.4 ° (c 0.5, CHCl ₃ ) Infrared Spectrum: See Figure 12 Equipment: Beckman
IR Model 4240 Main absorption bands (KBr): 950, 1015, 1070, 1100,
1155, 1213, 1250, 1313, 1375, 1405, 1450,
1520, 1595, 1610, 1685, 1735, 2940, 2980,
3440cm ^-1 . Mass spectrum: Instrument: VG-ZAB-2F FAB-MS-thioglycerol molecular mass range ion (m/z): 968,
1249, 1355, 1357, 1463, 1569; NaCl
(m/z): 990, 1271,
1379, 1485, 1593, FAB-MS-MB
(MB: Matrix, mw154); Molecular mass range ion (m/z): 1249, 1403,
1419, 1555, 1571, 1587; by addition of NaCl (m/z): 1249, 1271, 1425,
1441, 1457, 1483, 1577. FAB-MS-Glycerin-DMSO; Molecular mass range ion (m/z): 1215,
1231, 1247, 1263, 1279, 1293, 1309,
1325, 1326, 1341, 1353, 1369. Molecular weight (according to the above mass spectrum data): Apparent
MW-1248 Nuclear magnetic resonance spectrum: See Figure 13 Instrument: WM360 Brutzker Solvent: CDCl ₃ ¹ H NMR360MHz δ (ppm): 11.91
(1H, s); 8.62 (1H, s); 7.58 (1H,
s); 6.56 (1H, m); 6.22 (1H, s);
6.15 (1H, brs); 5.91 (1H, d, J=
9.6); 5.83 (1H, d, J = 9.6); 5.70
(1H, m); 5.45 (1H, d, J=2.2);
5.44 (1H, s); 5.34 (1H, brs); 4.95
(1H, d, J=10.2); 4.75 (1H, m);
4.65 (1H, d, J = 6.8); 4.54 (1H, d,
J=2.2); 4.47 (1H, m); 4.18 (1H,
s); 4.10 (1H, brs); 4.05-3.50 (20-
24H); [3.96 (3H, s); 3.87 (3H,
s); 3.77 (3H, s)]; 3.46 (1H, m);
3.39 (3H, s); 2.79 (1H, m); 2.73
(2H, m); 2.50 (3H, s); 2.50 (1H,
m); 2.38-2.22 (3H); 2.14 (1H, m);
2.10 (3H, s); 1.98 (2H, m); 1.65-
1.45 (6-8H); 1.38 (3H, d, J=
6.0); 1.34 (3H, d, J = 6.0); 1.22
(3H, d, J=6.8); 1.10 (6H). ¹³ C NMR 90.3MHz See Figure 14 ¹³ C NMR of BBM-1675A ₂ purified in separate tests
Spectra were measured on samples dissolved in CDCl ₃ (80MHz). The main peaks are shown below.

【table】

[Table] Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) data A for BBM-1675 components Study No. 1 - Summary

[Table] B Study No. Purification A ₁ and A ₂ Component TLC and HPLC TLC Chromatography Analtech GHLF Silica Gel Uniplates were used for all normal phase chromatography. One-dimensional plate with dimensions of 2.5cm x 10cm
Used for TLC. These were developed into a glass cylinder with dimensions of 6.4 cm (outer diameter) x 12 cm and containing 10 ml of eluate. A plate with dimensions of 7.5 cm x 10 cm was used for two-dimensional TLC. The sample was applied to the lower left corner, 1 cm from the edge. First, transfer the plate to a tank containing 50 ml of the first eluate (width 12.7 cm, depth
8.6 cm, height 13 cm). The plate was then air dried, rotated 90° counterclockwise, and developed in a second tank containing 50 ml of the second eluate. Watman analytical precoating C-18
Silica gel plates were used for all reversed phase chromatography. A plate measuring 2.5 cm x 7.6 cm was developed in a glass cylinder containing 10 ml of eluent. The normal phase plate was observed under 254nm UV light. The plate was inserted into a glass cylinder (outer diameter 6.4 cm x 12 cm) containing I ₂ crystals. The plate was then re-examined approximately 2 minutes later. reverse phase plate
Made visible under 254nm UV light only. The bands were detected by fluorescence and rapid cooling of the impregnating dye. Analytical HPLC The following components were used to construct the analytical HPLC system. Water Associates Model 600A Solvent Delivery System Pump; Varian Varichrome Model VUV-10UV/vis Detector, set to 254nm 0.10D;
Recordal Series 5000 Recorder; Waters Associates Model 660 Solvent Programmer; Waters Associates Model
U6K injector; Artek, μ-Pondapack C ₁₈ (10 μ) column (4.6 mm i.d. x 25 cm), Whattman Company Pel ODS (0.03-0.038 mm) guard column (4.6 mm i.d. x 5 cm). these members
316 stainless steel tubing (1.6mm outer diameter - 0.23
mm inner diameter). Eluate 2 for all analyses.
Pumped in at ml/min. The following members were used to construct the preparative HPLC intermediate pressure liquid chromatography system. Field Metering, Incorporated, Model RD-
SY2CSCFMI Love Pump; Field Metering, Inc., Model PD-60LF
FMI Pulse Dampener; Cardboard tube (8.65cm outside diameter)
15ml made up of polypropylene wrapping (3.0mm outside diameter x 1.5mm inside diameter) wrapped around
Sample loop; Glenco Series 3500 Universal LC column; Instrument Specialties Company Model UA-5 Absorbance/Fluorescence Monitor, Type 6
With optical unit; Instrumentation Specialties Company model
590 Flow Interrupter Valve; and Instrumentation Specialties Company Model 328 Fraction Collector. Polypropylene and Teflon tubing (3.0mm outer diameter x 1.5mm inner diameter) in the order in which these members are listed.
and Glenco Multifit connectors and valves. Glenco Series 3500 Universal LC
The column was slurry packed with the defined absorbent in the given solvent using standard techniques. The space between the settled bed and the top of the tube was filled with standard oxen sand. Pump the eluate at maximum speed, which does not exceed 60 psi back pressure (approximately 20 ml/min). Gradient Elution A Glenco gradient elution apparatus consisting of two chambers of equal diameter, height and volume connected in tandem with Teflon valves was used for all gradient elutions. One chamber served as a mixing chamber and the other as a static reservoir. The less polar solvent was initially kept in the mixing chamber. The more polar solvent was kept in the static chamber. Teflon coated magnetic stirring rod (1.0 x 3.7cm)
were placed in both chambers and powered by a Thomas Model 15 Magnematic Stirrer. The eluate was pumped into the intermediate pressure HPC system from the mixing chamber via a polypropylene tube (1.5 mm inner diameter x 3.0 mm outer diameter). As the eluate exited the mixing chamber, the solvent in the static reservoir was free to replace it, resulting in a linear eluate gradient. BBM-1675A ₁ and A ₂ TLC analysis BBM-1675A ₁ and A ₂ on regular phase plate
The observed Rf values for are summarized in Table 9 below. Rf was calculated by dividing the measured distance of the center of the zone from the sample application point by the measured distance of the solvent front from the sample application point.

[Table] 50% acetone in Ruskelisolve B 0.38 0.31
BBM with normal phase plates expanded in two directions
The observed Rf values for -1675A ₁ and A ₂ are summarized in Table 10 below. The position of the spot is expressed in Cartesian coordinates. The X coordinate is the Rf value of the second listed solvent system. The Y coordinate is the Rf value of the first listed solvent system.

Table 11 below summarizes the Rf values observed for BBM-1675A ₁ and A ₂ on C-18 reverse phase TLC plates developed with % acetone binary eluent.

TABLE The Rf values observed for BBM-1675A ₁ and A ₂ on C-18 reverse phase TLC plates developed with ternary eluent are summarized in Table 12 below.

【table】

[Table] Acetonitrile: Methanol:
_{0.1M NaH2PO4}
40%:30%:30% 0.00 0.00
HPLC analysis of BBM-1675A ₁ and A ₂ BBM-1675A ₁ and BBM-1675A ₂ in C-18
Single, binary and ternary eluents were assayed on a reverse phase silica gel column. The K' values observed for these compounds are summarized in Tables 13, 14 and 15 below. K′ was calculated using the following formula. K′=TR−T ₀ /T ₀ where TR is the measured retention time from the time of injection to the peak tip and T ₀ is the empty volume time.

[Table] Methanol 0.00 0.00
a = No compound eluted from the column.

[Table] le
a = No compound eluted from the column.

[Table] Biological properties of BBM-1675 components The antibacterial activity of BBM-1675 components was measured against various bacteria (Gram-positive, Gram-negative, and acid-resistant) and molds by serial double agar dilution method. Nutrient agar is used for Gram-positive and Gram-negative bacteria, and No. 1001 medium (3) is used for acid-resistant bacteria.
% glycerin _, 0.3% monosodium L-glutamate, 0.2% peptone, 0.31% _Na2HPO4 , 0.1%
_KHPO4 , 0.005% ammonium citrate, 0.001
% _MgSO4 and 1.5% agar) were used. For mold, Sabrod agar medium was used. As shown in Table 16, six BBM-1675 components (A ₁ , A ₂ , A ₃ ,
Each component (A ₄ , B ₁ , B ₂ ) exhibited a broad spectrum of antibacterial activity. BBM—1675A ₁ , A ₂ , A ₃ and
_A4 is particularly high against Gram-positive bacteria.

【table】

Table: A second antibacterial test was carried out on purified A ₁ and A ₂ (prepared in Example 3 below) and components A ₃ and A ₄ .

[Table] The prophage-inducing activity in the lysogenized bacterium E. coli W1709 (λ) was reported in Nature, Vol. 196, pp. 783-784, 1962, Lein et al. BBM-1675 components were measured according to the method. Plug counts were performed on agar plates containing test materials (T) and control plates (C). T/ with plaque count greater than 3.0
The C ratio was considered significant and the lysogenization-inducing activity (lLB activity) was expressed as the minimum inducing concentration of the test compound. As shown in Table 17, the BBM-1675 component showed strong lLB activity in lysogenized bacteria, suggesting that the component has antitumor activity.

[Table] The antitumor activity of BBM-1675A ₁ and A ₂ was determined in various mouse tumor lines. Lymphocytic leukemia P-
388, lymphoid leukemia L-1210, melanocytoma
B16 and Lewis lung cancer at a dose of 10 ⁶ each,
10 ⁵ , 5×10 ⁵ and 10 ⁶ cells/mouse were implanted intraperitoneally into male BDF ₁ mice. Mice were administered graded doses of test compounds intraperitoneally 24 hours after tumor implantation. Treatments were carried out once on day 1 only, on days 1, 4 and 7 (q3dx3), once a day for 9 days (qd1→9) or once a day for 11 days (qd1→11). Ingredient A ₃
and _A4 was tested on a q3d×3 schedule only for P-388 leukemia due to insufficient material supply. BBM-1675A ₁ , A ₂ , A ₃ and A ₄ were dissolved in 0.9% saline containing 10% dimethyl sulfoxide,
Chromomycin A ₃ (Toyomycin, Takeda Pharmaceutical Co., Ltd.) used as a reference compound was dissolved in 0.9% saline. The number of deaths or survivors of treated and untreated mice was recorded daily and the median survival time (MST)
was calculated for each of the test group (T) and control group (C). T/C equal to or greater than 125%
The values indicate that a significant antitumor effect was achieved.
The results are shown in Tables 18-23. BBM—1675A ₁ and A ₂
showed extremely strong antitumor activity against P-388 leukemia with a maximum T/C value of 160%. These components exhibit approximately 100-3000 times more activity than chromomycin A ₃ in terms of minimum effective doses. However, BBM-
1675A ₃ and A ₄ are components against P-388 leukemia.
The activity was lower than that of A ₁ or A ₂ (Table
19). BBM-1675A ₁ and A ₂ also showed activity against L-1210 leukemia (Table 12), B16 melanocytoma (Table 2) and Lewis lung carcinoma (Table 23).
The toxicity of BBM-1675A ₁ and A ₂ was determined in male ddY mice by intraperitoneal or intravenous administration. BBM
-1675A ₁ was approximately 10 times more toxic than BBM-1675A ₂ (Table 24). BBM—1675A ₁ and
The therapeutic index of _A2 was 4-8 and 8-20 times better than chromomycin _A2 in the P-388 leukemia line, respectively (Table 25). P-388 and L- inoculated intravenously at 5×10 ⁵ and 10 ⁴ cells/mouse, respectively.
A second experiment will be conducted by intravenously administering BBM-1675 components against 1210 leukemia. In these experiments, adriamycin was used as the reference drug, dissolved in 0.9% saline and 1.
Administered on days 4 and 7. The results are shown in Tables 26 and 27. Both components A ₁ and A ₂ were superior to Adriamycin in terms of maximum T/C value, minimum effective dose and range of activity.

【table】

【table】

【table】

【table】

【table】

【table】

【table】

【table】

【table】

【table】

[Table] * Minimum effective dose

【table】

【table】

Table: The antitumor activity of components BBM-1675A ₁ and A ₂ was also determined in a second study against P-388 leukemia, L-1210 leukemia, and B16 melanocytoma in mice. The results of these tests are presented in Tables 28, 29 and
Shown in 30. The methods used in these tests are
Cancer Chemther.Res 3rd
Vol., pp. 1-87 (3 parts), 1972.

【table】

【table】

【table】

【table】

【table】

【table】

Table: After further purification of BBM-1675A ₁ according to Example 4, samples of the purified compound were tested against L-1210 leukemia, P-388 leukemia, and B16 melanocytoma in mice. These results are shown below.

【table】

【table】

【table】

【table】

[Table] The above screening data is based on purified
BBM-1675A ₁ component has substantially the same antitumor activity as the previously screened less purified sample. This compound has exceptionally high efficacy. Because activity was shown against murine P-388 leukemia at a dose of 25 nonagrams/Kg on a schedule of 5 times per day. In trials against P-388 and L-1210 leukemia, BBM-
1675A ₁ is also effective when administered as a single injection once a day, three injections on each fourth day, or five times a day. Compounds against B16 melanocytoma ip
It is simultaneously effective when administered intravenously to animals bearing subcutaneous tumors, as it is when administered intraperitoneally to animals bearing tumor implants. This property of advantageous pharmacological delivery of drugs to tumors at distant sites is surprising for antitumor antibiotics. As mentioned above, BBM-1675 components have potent antimicrobial activity and are therefore useful in the therapeutic treatment of mammals and other animals for infectious diseases caused by such microorganisms. Additionally, this component can be used in other common antimicrobial applications, such as disinfection of medical and dental equipment. The induction of prophages in lysogenic bacteria and the activity shown against murine tumor lines indicate that the BBM-1675 components are also therapeutically useful in inhibiting the growth of mammalian tumors. Accordingly, the present invention provides antimicrobial or tumor suppressive doses of BBM-1675A ₁ , A ₂ , A ₃ , A ₄ , B ₁ or B ₂ or pharmaceutical compositions thereof effective in an animal host suffering from a bacterial infection or malignant tumor system. The present invention provides a method of therapeutically treating a host comprising administering an agent. According to another feature, the invention provides an effective antimicrobial or tumor inhibiting amount of BBM-1675A ₁ , A ₂ , in combination with an inert pharmaceutically acceptable carrier or diluent,
Pharmaceutical compositions comprising A ₃ , A ₄ , B ₁ or B ₂ are provided. These compositions can be prepared in any pharmaceutical form adapted for parenteral administration. Formulations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They can also be prepared in the form of sterile solid compositions that can be dissolved in sterile water, saline, or some other sterile injectable medium immediately before use. It will be appreciated that the actual preferred amount of BBM-1675 antibiotic used will vary depending on the particular ingredients, the particular formulation, the mode of application and the particular site, host and disease being treated. Many factors that modify the action of a drug are considered by those skilled in the art, such as age, weight, sex, diet, time of administration, route of administration, rate of excretion, host condition, drug combinations, response susceptibility, and severity of disease. . Administration can be continuous or periodic within the maximum tolerated dose. The optimal ratio for a given situation can be determined by one skilled in the art using routine dosage determination trials in view of the guidelines set forth above. As described above, the dosage of BBM-1675 anti-tumor antibiotic varies depending on the patient's age, condition, type of disease, and type of BBM-1675 anti-tumor antibiotic administered, but in general, the dosage is 0.00001 per day. ~10mg,
Preferably, a dose of 0.00001 to 1 mg can be administered to the patient, but this dose can be increased or decreased depending on the patient's symptoms, and the dosage can be adjusted to suit the patient's symptoms. The amount can be determined within a range that does not cause toxicity and can be higher, or the amount can be determined within a range that maintains the efficacy of the drug and can be lower than that. be able to. Further, when used in combination with other drugs, a smaller amount than the above dosage can be used. Further, the administration can be carried out orally or parenterally, for example, by subcutaneous injection, and it can also be administered to the skin as an external preparation. The following examples are presented for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 Fermentation of BBM-1675 Actinomadeura strain No. H964-92 was mixed with 1% malt extract, 0.4% glucose, 0.4% yeast extract,
They were grown and maintained on agar slants containing 0.05% CaCO ₃ and 1.6% agar. Using a well-grown agar slant, 3% soluble starch, 3% dry yeast, 0.3% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.05%
_MgSO4.7H2O , 0.2% _NaCl and 0.1% _CaCO3
and the pH was adjusted to 7.0 before sterilization. Rotate the nutrient medium on a shaker (250 rpm)
and incubated at 32°C for 72 hours, and 5 ml of the growth was incubated with 3
% Amashiyo molasses, 1% cornstarch, 1% fishmeal,
0.1% _CaCO3 and 0.005% _CuSO4.5H2O (before _{sterilization} )
The mixture was transferred to a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium (PH7.0). Fermentation was carried out on a rotary shaker for 6 days at 28°C. The antibiotic activity in the fermentation broth was determined using Staphylococcus spp.
Measured by paper disk agar diffusion using Staphylococcus aureus 209P. The antibiotic titer reached a maximum value of about 1 mcg/ml after 5 days of fermentation. Fermentation of BBM-1675 was carried out in a stirred jar fermentor. 500 ml of the inoculum growth prepared as described above was added to 20 ml of fermentation medium consisting of the same components as used in the shake flask fermentation.
Moved to Jiahua Mentor. Fermentation aeration speed
It was carried out at 32°C at 12/min and stirring 250 rpm. Under these conditions, antibiotic production reached a maximum value of about 0.9 mcg/ml after 68-76 hours of fermentation. Fermentation studies were also conducted in fermentation tanks. Seed culture at 30° _C for 4 days with 3% soluble starch, 3% dry yeast, 0.3% _K2HPO4 , 0.1 _{% KH2PO4} , 0.05%
Shaking was carried out in an Erlenmeyer flask containing nutrient medium consisting _{of MgSO4.7H2O} , 0.2% NaCl and 0.1% _CaCO3 . The seed culture was inoculated into a 200 seed tank containing 130 seed medium with the same composition as above, and the seed tank was stirred for 31 hours at 30° C. and 240 rpm. Using a second seed culture: 1% cornstarch, 3% Amashiyo molasses, 1% fishmeal, 0.005%
Fermentation medium containing _{SuSO4.5H2O and} 0.1% _CaCO3
3000 were inoculated. Production tank at 28℃ and 164rpm
It was operated at an air flow rate of 2000/min. As the fermentation progresses, the pH of the broth gradually increases and reaches 170-
It reached 7.7-7.8 after 180 hours, and at this time it was 1.7mcg/ml.
peak antibiotic activity was obtained. Example 2 Isolation and Purification of BBM-1675 Component The harvested fermentation broth (3000, PH7.8) was separated into mycelial cake and supernatant with the help of a Sharpless centrifuge. Suspend the mycelium cake in methanol 1600, and mix the mixture with 1
I stirred for hours. Insoluble material was removed and the methanolic extract was concentrated in vacuo to 43. The active ingredients contained in the broth supernatant were extracted with n-butanol.
Two extractions of 1000 portions were taken. The n-butanol and concentrated methanol extracts were combined and azeotropically evaporated to an aqueous solution (20) with occasional addition of water, where most of the antibiotic activity was deposited as an oily solid. This solid was digested in 30 methanol and the insoluble material was removed. The methanol extract was then concentrated in vacuo to a 10 solution to which 40 parts of ethyl acetate and 3 parts of water were added. After stirring for 30 minutes, the organic layer was separated, dried over sodium sulfate, and concentrated in vacuo. When the concentrate is added to n-hexane 20, the crude
A pale yellow solid (90.14 g, titer: 55 mcg/mg) of BBM-1675 complex was obtained. The complex was found by TLC to be a mixture of two major components, BBM-1675A ₁ and A ₂ , and several minor components. These were separated and purified by repeated chromatography performed in a cooled room to prevent deterioration. BBM-1675 complex (20g) was mixed with methanol (20g).
Sephadex (Sephadex)
It was packed into an LH-20 (φ5.5 x 85 cm) column. The column was developed with methanol and elution was monitored by bioassay using Staphylococcus aureus 209P. The active eluates were combined, concentrated in vacuo, and lyophilized to yield a semi-purified solid of BBM-1675 complex (4.19 g). The solid was chromatographed on silica gel chloroform (φ5.0×50 cm) cm ² using chloroform and increasing amounts (1→5% v/v) of methanol as eluent. The eluate was evaluated for antibacterial activity (against S. aureus) and TLC (SiO ₂ ; CHCl ₃ - CH ₃ OH=5:1v/v).
were pooled based on and concentrated in vacuo. Almost homogeneous BBM-1675A ₁ (yield after evaporation: 351
mg) was first eluted with 2% methanol (chloroform), then a mixture of BBM-1675A ₂ , A ₃ and A ₄ (507 mg) and then a mixture of BBM-1675B (210 mg).
mg) was eluted with 3% methanol in chloroform. BBM-1675A ₁ solid to Cephadex LH
-20 column (φ2.0×80cm) and developed with methanol. Concentration of the active fraction to dryness in vacuo and crystallization of the residual solid from methanol yielded colorless platelets of pure BBM-1675A ₁ (124
mg) (this material is the starting material for Example 3A) was obtained. Complex BBM - 1675A ₂ , A ₃ and A ₄
was separated by chromatography on a Bondapak C ₁₈ (Waters, φ3.0×50 cm) column. Elution was carried out with aqueous acetonitrile and the active eluent was subjected to TLC [Merck, silanized (C ₁₈ reversed phase silica gel):
_CH3CN - _H2O =75:25v/v). 20 trace components A ₄ (33 mg) and A ₃ (18 mg) in this order
% acetonitrile and then the other major component A ₂ (310 mg) (this material is the starting material for Example 3B) was eluted with 50% acetonitrile. Chromatography was performed on a silica gel column (φ3.0 x 40 cm) using chloroform and methanol using solids containing BBM-1675B ₁ and B ₂ as a developing solvent. The active fractions eluted with 4% methanol in chloroform were combined and evaporated to give pure BBM-1675B ₁ (7 mg). Another active fraction eluted at 5% methanol concentration and was evaporated to yield BBM-1675B ₂ (8 mg). Reference example 1 Further purification of BBM-1675A ₁ and A ₂ A Purification of BBM-1675A ₁ Glenco column, 2.67cm inner diameter x 75
cm. Baker bonded phase octadecyl (C ₁₈ ) silica gel (40μ particle size) in methanol was slurry packed. Column at intermediate pressure
Connect to HPLC system, and eluate 1.5
(41.6% acetonitrile - 2.6% methanol - 36.8
% 0.1M ammonium acetate). Partially purified BBM-1675A ₁ (100.5 mg) obtained according to the purification procedure of Example 2 was dissolved in 2 ml of acetonitrile and placed in the sample loop. The sample was pressed into the column. The column was eluted with the above eluent and a fraction of 87 ml was collected. eluent
Monitored at 240nm and 340nm. Pool fractions 55-71 and weigh in chloroform
Extracted twice with 1500 ml. After evaporating the chloroform to dryness, 89.8 mg of residue C was obtained. Glenco column, 1.5cm inner diameter x 20cm. WOelm silica gel (60-200μ particles) 12g
was slurry packed. Residue C was applied to the column in chloroform solution. The column was eluted with 500 ml linear steps of chloroform to 10% methanol in chloroform collecting 20-25 ml fractions. After analysis by silica gel TLC, fractions 6-9 were pooled and evaporated to dryness to yield residue D.
73mg was obtained. Form silica gel (63
-200μ particles) 12g was packed into slurry. Residue D was dissolved in approximately 2 ml of CHCl ₃ and applied to the column. The chloroform was replaced with 25 ml of Skellisolve B. The column was then eluted with 500 ml linear step Skellisolve B to 60% acetone in Skellisolve B collecting 28-25 ml fractions. Fractions 19-23 were pooled and evaporated to dryness to yield _65.6 mg of pure BBM-1675A1. This residue was analyzed using three TLC systems (5 in chloroform).
% methanol; 5% methanol in ether; and 50% acetone in Skellisolve B, silica gel)
It was found to be homogeneous by HPLC (C-18 silica gel - 41.5% acetonitrile: 21.5% methanol: 37.0%, 0.1M ammonium acetate). Gel permeation chromatography on purified BBM-1675A ₁ 2.5cm inner diameter x 45cm Pharmacia
The column was slurry packed with Sephatex LH-20 in methanol and adjusted to a 33.4 cm chromatography bed. Purified BBM-1675A ₁ (approx.
120 mg) was dissolved in 2 ml of methanol and transferred to a 2.5 ml sample container. The sample was applied to the column and elution was started with methanol at 1.75 ml/min to collect 10 ml fractions (Fac - 100 fraction collector). The eluate was monitored with an Isco UA-5 detector at 254 nm. BBM-1675A ₁ is 0.79-0.91 Ve/Vt
It was observed that elution occurred at (Ve = elution volume; Vt
= bet amount). B Purification of BBM-1675A ₂ A 2.65 cm id x 75 cm Glenco column was slurry packed with Baker bonded phase octadecyl (C ₁₈ ) silica gel (40μ particle size) in methanol. Connect the column to a medium pressure HPLC system and eluent
1.5 (50% acetonitrile-20% methanol-
Equilibrated with 30% 0.1M ammonium acetate).
Partially purified product obtained by the procedure of Example 2
BBM-1675A ₂ (76.9 mg) was dissolved in 2 ml of acetonitrile and placed in the sample loop. The sample was pressed into the column. Elute the column with the above elution at 87
ml fraction was collected. Eluent 254 and
Monitored at 340nm. Fractions 31-38 were pooled and extracted twice with 500 ml weighed portions of chloroform. When chloroform is evaporated to dryness, a homogeneous
65.8 mg of BBM-1675A ₂ was obtained. BBM-1675A ₂ is 2TLC system, 1 2-dTLC
It was homogeneous by analysis and HRLC. Reference Example 2 Suitable extraction method for BBM-1675A ₁ The raw fermentation liquid (6.8) obtained according to the general procedure of Example 1 was placed in a polypropylene container with a faucet at the bottom (top 12 cm diameter; bottom 10 cm diameter; height sa37
cm). An equal volume of chloroform was added. The mixture was stirred for 2 hours with a CRC-air driven stirrer at good speed. Add about 4 (1.3Kg) of Dicalite (superagent),
It was mixed in. The mixture was passed through a pad of Dicalite held in a No. 12 Buchner funnel. 19 liquid bottle with a vacuum outlet [Ace No.
5396-06]. Chloroform 2
I washed it with The liquid was transferred to a 20 minute funnel and the phases were allowed to separate. The lower layer (chloroform) was removed. A slurry pack of 91 g of Whelum silica gel (63-200 μ particles) was packed into a 2.5 cm inner diameter × 40 cm Glenco tube. The chloroform phase was pumped into the column using an FMI RPY-2CSD pump. The column was washed and rinsed with 600 ml of fresh chloroform.
The chloroform eluate was discarded. The column was then eluted with 600 ml of 10% methanol in chloroform.
The eluent was evaporated to dryness, yielding 547 mg of residue A. Residue A was dissolved in 50 ml of chloroform. Chloroform solution of Deicalite in one round bottom flask
Added to 20g. 200 ml of Skellisolve B was added to form a slurry. The solvent was removed on a rotary evaporator. Skelisolve residue B300
Slurry was made into ml. Transfer the slurry to an Ace flask chromatography tube (part no.
B5872-14) (41mm inner diameter x 45.7cm) was filled.
A glass fiber stopper was inserted into the neck of the stopcock between the stopcock and the column tube. A 1 cm layer of standard Ottawa sand was added over the glass fibers. The stopcock, fiberglass, and sand bed were aerated by passing a pressurized flow of Skelesolve B (5.7 psi) through them.
The slurry was added to the column to form a packed bed under pressure flow. Avoid letting the column dry out. After a stable column bed was obtained, a 2 cm layer of Otsutawa sand was added to the top of the bed. The bed was eluted with an additional 600-700 ml of Skellisolve B. The bed was eluted with 500 ml of toluene. Evaporation of the toluene eluent to dryness gave 93 mg of residue B. Reference example 1 is the partially purified BBM-1675A ₁ .
It can also be further purified according to the procedure described in . Example 3 BBM using mutant strain H964-92-A1327Y-
1675 Fermentation of complex Actinomadeula verrucosospora strain no.
Mutant strain A1327Y obtained by NTG treatment of H964-92
using 2% soluble starch, 1% glucose,
A nutrient medium containing 0.5% yeast extract, 0.5% NB-amine type A and 0.1% CaCO ₃ and adjusted to a PH of 7.0 before sterilization was inoculated. The nutrient medium was incubated at 32°C for 4 days on a rotary shaker (250 rpm). 5ml of growth
was transferred to a 500 ml Erlenmeyer flask. This flask contains 3% sweet molasses, 1% cornstarch, 1% fishmeal, 0.005%, CuSO ₄ 5H ₂ O, 0.05%
It contained 100 ml of fermentation medium consisting of % _MgSO4.7H2O and _0.1 % _CaCO3 , and the pH was adjusted to 7.0 before sterilization. Fermentation was carried out on a rotary shaker for 7 days at 28°C.
Antibiotic production reached a maximum value of approximately 1.5 mcg/ml. Example 4 Isolation and purification of BBM-1675 components The fermentation broth collected from Example 3 (3000, PH
7.6) was separated into mycelium cake and supernatant using a shear press centrifuge. The mycelial cake was stirred for 1 hour with 2000 ml of mezeethanol and the insoluble material was removed. The active components contained in the broth supernatant were extracted with n-butanol 1800. The methanol and n-butanol extracts were combined and azeotropically concentrated with occasional addition of water to form an aqueous solution (20), in which most of the antibiotic activity was deposited as an oily solid. The mixture was shaken three times with 20 portions of ethyl acetate to extract the active components. Pool the extracts and
The insoluble solution was removed by filtration and evaporated to 4 in vacuo. Add the concentrate to n-hexane while stirring.
When added to 30, a pale colored solid of crude BBM-1675 complex (81.7 g, titer: 59 mcg/mg) was obtained. The complex was analyzed by TLC and HPLC to reveal two main components,
It was found to be a mixture of BBM-1675A ₁ and A ₂ and several minor components. These components were separated and purified through a series of chromatographies carried out in a cold room to prevent deterioration. Crude BBM-1675 complex (20 g) was dissolved in methanol (20 ml), and Cephadex LH-
It was packed into 20 columns (φ5.5×85cm). The column was developed with methanol and elution was monitored by bioassay using Staphylococcus aureus 209P. The active eluates are pooled, concentrated in vacuo, and lyophilized to produce BBM-
1675 complex semi-purified solid (4.86 g, potency;
203mcg/mg) was obtained. The solid was then transferred to a silica gel column (φ3.0×
70 cm). Antibacterial activity against S. aureus and TLC of elution fluid
(SiO ₂ , CHCl ₃ -MeOH5: 1 v/v) and concentrated in vacuo. BBM―1675A ₁
(425 mg after evaporation, titer: 960 mcg/mg) was first eluted with 2% methanol in chloroform and then
BBM—Mixture of 1675A ₂ , A ₃ and A ₄ (730mg,
The BBM-1675B complex (200 mg, titer: 190 mcg/mg) was then eluted with 3% methanol in chloroform. The above medium A ₁ was mixed with 2% methanol in benzene on silica gel (column:
Chromatography was performed again using a φ2.2 x 44 cm).
Bioactive eluent was analyzed by HPLC [Lichrosorb RP-18: CH ₃ CN-MeOH-0.1M
CH ₂ COONH ₄ =5:2:3, v/v) and the fraction containing homogeneous BBM-1675A ₁ was evaporated to dryness in vacuo. When the residual solid is crystallized from methanol (10 ml), BBM-1675A ₁
(197 mg, titer: 1000 mcg/mg) was obtained as colorless prismatic crystals. BBM—Complex of 1675A ₂ , A ₃ and A ₄ (537
mg) Bondapack C ₁₈ (Waters, φ2.0×42
cm) and separated by column chromatography. Elution was carried out with aqueous acetonitrile and the bioactive eluent was subjected to TLC (Merck, silanization: CH ₃ CN-
H ₂ O=75:25, v/v). Trace ingredients
BBM-1675A ₄ (45 mg, titer: 410 mcs/mg) and A ₃ (19 mg, titer: 300 mcs/mg) were sequentially eluted with 20% acetonitrile, then the main components,
BBM-1675A ₂ (203 mg) was eluted with 50% acetonitrile. BBM-1675A ₂ fractions were crystallized from chloroform-n-hexane to deposit colorless rods (70 mg, titer: 290 mcg/mg).
The solid containing the BBM-1675 mixture was chromatographed on a silica gel column (φ3.0×40 cm) using chloroform and methanol as the developing solvent. The active fractions eluted with 4% methanol in chloroform were pooled and evaporated to give pure BBM-1675B ₁ (7 mg, titer: 180 mcg/
mg) was obtained. The other active fraction was eluted with 5% methanol concentration and was evaporated.
BBM-1675B ₂ (8 mg, titer: 140 mcg/mg) was obtained.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of partially purified BBM-1675A ₁ (KBr pellet). Figure 2 shows the infrared absorption spectrum of partially purified BBM- _1675A2 (KBr pellet). Figure 3 shows the infrared absorption spectrum of BBM-1675A ₃ (KBr pellet). Figure 4 shows the infrared absorption spectrum of BBM-1675A ₄ (KBr pellet). Fifth
The figure is partially purified in CDCl ₃ (60MHz)
This shows the proton magnetic resonance spectrum of BBM-1675A ₁ . Figure 6 shows the proton magnetic resonance spectrum of partially purified BBM-1675A ₂ in CDCl ₃ (60 Hz). Figure 7 shows the frequency of CDCl ₃ (60MHz)
This shows the proton magnetic resonance spectrum of BBM-1675A ₃ . Figure 8 shows BBM in CDCl ₃ (60MHz)
The proton magnetic resonance spectrum of 1675A ₄ is shown.
Figure 9 shows the infrared absorption spectrum of purified BBM-1675A ₁ (KBr pellet). Figure 10 shows CDCl ₃
Figure 3 shows the proton magnetic resonance spectrum of purified BBM-1675A ₁ at (360MHz). Figure 11 shows CDCl ₃
^13C magnetic resonance spectrum of purified BBM-1675A ₁ in (90MHz) is shown. Figure 12 shows purified BBM.
Infrared absorption spectrum of 1675A ₂ (KBr pellet)
shows. Figure 13 shows purification in CDCl ₃ (360MHz)
This shows the proton magnetic resonance spectrum of BBM-1675A ₂ . Figure 14 shows purification in CDCl ₂ (90.3MHz)
The ¹³ C magnetic resonance spectrum of BBM-1675A ₂ is shown.

Claims (1)

[Claims] 1 (a) Chloroform, ethyl acetate, acetone,
Soluble in ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (b) Positive reaction with ferric chloride, Ehrlich and Tollens reagents and Sakaguchi, ninhydrin and Negative reaction in the Anthrone test; (c) substantially the following infrared absorption spectrum (KBr)
Shows the main absorption bands of: 3420, 2940, 2920, 1720, 1690, 1610, 1600,
1520, 1455, 1410, 1375, 1315, 1255, 1155,
1100, 1075, 1020, and 990 cm ^-1 (d) Measurement of proton magnetic resonance spectrum [ ¹ H-NMR, 60 MHz; δppm] after dissolving in CDCl ₃ showed the following value; 11.80 (1 H, s ), 8.55 (1H, s), 7.45 (1H,
s), 6.50 (1H, m), 6.37 (1H, br), 6.18 (1H,
brs), 5.81 (2H, brs), 5.68 (1H, m), 5.5―
5.4 (3H, m), 5.05 (1H, brs), 4.88 (1H, m),
4.70 (1H, m), 4.54 (2H, m), 4.40 (1H,
brs), 4.2-3.6 (24-26H), 3.40 (3H, s),
3.35 (1H, m), 2.85-2.50 (3H, m), 2.37
(3H, s), 2.12 (3H, s), 1.60−1.05 (24−
26H). (e) It has a melting point in the range of about 125-127°C; (f) It has a light intensity [α] ²⁷ _D = -161° (c0.5, CHCl ₃ ); (g) 54.55% carbon, hydrogen 6.46%, nitrogen 3.73%, sulfur
(h) CHCl ₃ by silica gel thin layer chromatography;
- Rf value 0.72 in CH ₃ OH (5:1/v/v) solvent system
and an Rf value of 0.28 in a CN ₃ CH—H ₂ O (75:25 v/v) solvent system by reverse phase silica gel thin layer chromatography; (i) methanol, 0.01N HCl—CH ₃ OH and
When dissolved in 0.01N NaOH-CH ₃ OH, it exhibits the following ultraviolet absorption maximum: UV λmax nm (E1% 1cm): 253 (286) Methanol 282 (158) 320 (122) UV λmax nm (E1% 1cm): 253 (287) 0.01N HCl―CH ₃ OH 282(160) 320(126) UV λmax nm(E1% 1cm) :252(280) 0.01N HaOH―CH ₃ OH 283(162) 318(120) (j) C ₁₈ reverse phase silica gel column and CH ₃ CN—
CH ₃ OH―0.1M CH ₃ COONH ₄ (5:2:3v/
v) exhibits a high performance liquid chromatography retention time of 8.0 minutes in a solvent system; (k) is effective in inhibiting the growth of various bacteria and fungi; (l) induces prophage in lysogenic bacteria; and (m) BBM- _1675A , an antitumor antibiotic effective in inhibiting the growth of murine P-388 leukemia; (a) Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, and soluble in benzene and water. (b) Positive reactions with ferric chloride, Ehrlich and Tollens reagents and negative reactions with Sakaguchi, ninhydrin and Anthrone tests; (c) Virtually next infrared absorption spectrum (KBr)
Shows the main absorption bands; 3390, 2940, 2920, 1720, 1670, 1610, 1600,
1525, 1450, 1410, 1380, 1315, 1255, 1215,
1155, 1110, 1080, 1020, and 945 cm ^-1 (d) Measurement of proton magnetic resonance spectrum [ ¹ H-NMR, 60 MHz; δppm] after dissolving in CDCl ₃ showed the following value; 11.80 ( 1 H , br), 8.57 (1H, s), 7.52 (1H,
s), 6.82 (1H, m), 6.09 (1H, m), 5.82 (2H,
brs), 5.5-5.3 (4-5H, m), 5.02 (1H, m),
4.88 (1H, m), 4.65-4.40 (3H, m), 4.27
(1H, m), 4.15-3.55 (26-28H), 3.34 (3H,
s), 3.0-2.6 (2H, br), 2.48 (3H, s), 2.45
-2.15 (4H, m), 2.10 (3H, s), 1.91 (3H,
s), 1.5-1.15 (20H). (e) It has a melting point in the range of about 123-126°C; (f) It has a light intensity [α] ²⁷ _D -176° (c 0.5, CHCl ₃ ); (g) 54.65% carbon and 6.29% hydrogen. , nitrogen 3.51%, sulfur
It has an approximate elemental composition of 8.07% and 27.48% oxygen (difference); (h) _CHCl3 by silica gel thin layer chromatography.
-CH ₃ OH (5:1 v/v) solvent system showed an Rf value of 0.71, and reversed phase silica gel thin layer chromatography showed an Rf value of 0.71 in CH ₃ CN-H ₂ O (75:25 v/v) solvent system.
0.78; (i) methanol, 0.01N HCl—CH ₃ OH and
When dissolved in 0.01N NaOH-CH ₃ OH, it exhibits the following ultraviolet absorption maximum: UV λmax nm (E1% 1cm): 253 (257) Methanol 282 (153) 320 (117) UV λmax nm (E1% 1cm): 253 (258) 0.01N HCl―CH ₃ OH 282(155) 320(118) UV λmax nm(E1% 1cm) :252(266) 0.01N NaOH―CH ₃ OH 283(160) 318(118); (j) _C18 reverse phase silica gel column and _CH3CN—
CH ₃ OH―0.1M CH ₃ COONH ₄ (5:2:3v/
v) High performance liquid chromatography retention time
5.1 minutes; (k) effective in inhibiting the growth of various bacteria and fungi; (l) inducing prophage in lysogenic bacteria; and (m) inhibiting the growth of murine P-388 leukemia. BBM-1675A ₄ is an antineoplastic antibiotic that is effective in (a) soluble in chloroform, ethyl acetate, ethanol and methanol, slightly soluble in benzene and water, and soluble in n-hexane and carbon tetrachloride; insoluble; (b) positive reactions with ferric chloride, Ehrlich and Tollens reagents, and negative reactions with Sakaguchi, ninhydrin and Anthrone reagents; (c) an infrared absorption spectrum (KBr) that is substantially
Shows the main absorption bands; 3400, 2940, 2920, 1710, 1680, 1610, 1600,
1520, 1455, 1420, 1370, 1315, 1255, 1212,
1155, 1120, 1075, 1020, and 990 cm ^-1 (d) Measurement of proton magnetic resonance spectrum [ ¹ H-NMR, 80 Mmm ³ ; δppm] after dissolving in CDCl ₃ showed the following value; 11.80 [ 1H, s), 8.53 (1H, s), 7.45 (1H,
s), 7.00 (1H, brs), 6.90-6.40 (2H, m),
6.20 (1H, brs), 5.85 (2H, d), 5.62 (1H,
m), 5.5-5,3 (3H, m), 5.13 (1H, s),
5.1-4.9 (2H, m), 4.65 (1H, m), 4.55 (2H,
m), 4.18 (1H, s), 4.0-3.3 (26H), 2.75
(3H, s), 2.50 (3H, s), 2.4-1.8 (11H),
1.6―1.0 (18H). (e) It has a melting point in the range of about 159-161°C; (f) It has an optical density [α] ²⁷ _D -171° (c 0.5, CHCl ₃ ); (g) It has a melting point in the range of about 159-161°C; _CHCl3
-CH ₃ OH (5:1 v/v) solution system showed an Rf value of 0.63, and reversed phase silica gel thin layer chromatography showed an Rf value of 0.23 in CH ₃ CN-H ₂ O (75:25 v/v) solvent system. (h) Methanol, 0.01N HCl—CH ₃ OH and
When dissolved in 0.01N NaOH-CH ₃ OH, it exhibits the following ultraviolet absorption maximum: UV λmax nm (E1% 1cm): 253 (225) Methanol 282 (140) 320 (104) UV λmax nm (E1% 1cm: 253 ( ₍ i) _Various (j) antitumor antibiotic BBM-1675B ₁ that induces prophage in lysogenized bacteria, or (a) chloroform, ethyl acetate, acetone, ethanol and Soluble in methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (b) positive reaction with ferric chloride, Ehrlich and Tollens reagents, and Sakaguchi, Negative reaction in ninhydrin and anthrone tests; (c) substantially the following infrared absorption spectrum (KBr)
Shows the main absorption bands; 3400, 2940, 2920, 2840, 1720, 1660, 1610,
1530, 1450, 1380, 1300, 1255, 1200, 1150,
1120, 1070, 1010, and 980 cm ^-1 (d) have a melting point in the range of about 156-159°C; (e) have a light intensity [α] ²⁷ _D -122° (C 0.5, CHCl ₃ ); (f) CHCl ₃ by silica gel thin layer chromatography
- CH ₃ OH (5:1v/v) shows an Rf value of 0.60,
And by reverse phase silica gel thin layer chromatography, CH ₃ CN-H ₂ O (75:5v/v) showed an Rf value of 0.16; (g) methanol, 0.01N HCl-CH ₃ OH and
When dissolved in 0.01N NaOH-CH ₃ OH, it exhibits the following ultraviolet absorption maximum: UV λmax nm (E1% 1cm): 248 (212) Methanol 279 (141) 318 (103) UV λmax nm (E1% 1cm): 248 (210) 0.01N HCl―CH ₃ OH 279(140) 318(103) UV λmax nm(E1% 1cm) :248(233) 0.01N NaOH―CH ₃ OH 278(150) 318(110); (h) It is effective in inhibiting the growth of various bacteria and fungi; (i) Antitumor antibiotic BBM-1675B ₂ induces prophage in lysogenic bacteria. 2 BBM of Actinomadura verrucosospora
1675A ₃ , BBM-1675A ₄ , BBM-1675B ₁ or
BBM-1675B ₂ -Bacterial strains selected from production strains are grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources to produce a substantial amount of BBM-
1675A ₃ , BBM-1675A ₄ , BBM-1675B ₁ , or
BBM-1675A _{3 ,} BBM-1675A ₄ , BBM-
BBM-1675A ₃ , BBM characterized by collecting a substance selected from 1675B ₁ or BBM-1675B ₂
A method for producing an antitumor antibiotic selected from -1675A ₄ , BBM-1675B ₁ or BBM-1675B ₂ . 3 BBM of Actinomadeula verruchosospora
-1675A ₃ - The production strain was grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources.
Cultivate and then remove from the medium until a substantial amount of BBM-1675A ₃ is produced by the microorganism in the medium.
The method according to claim 2, characterized in that BBM-1675A ₃ is collected. 4 BBM-1675A ₃ - Identification characteristics of the producing bacteria Actinomadeura Verrucosospora strain H964-92
(ATCC39334), Actinomadeula verrucosospora strain A1327Y (ATCC 39638), or a mutant strain thereof. 5 BBM of Actinomadeula verruchosospora
-1675A ₄ - The production strain is grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources.
Cultivate and then remove from the medium until a substantial amount of BBM-1675A ₄ is produced by the microorganism in the medium.
The method according to claim 2, characterized in that BBM-1675A ₄ is collected. 6 BBM-1675-A ₄ - Identification characteristics of the producing bacteria Actinomadeura Verrucosospora strain H964-92
(ATCC 39334), Actinomadeula verrucosospora strain A1327Y (ATCC 39638), or a mutant strain thereof. 7 BBM of Actinomadeula verruchosospora
-1675B ₁ - Production strains were grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources.
Cultivate and then remove from the medium until a substantial amount of BBM-1675B ₁ is produced by the microorganism in the medium.
The method according to claim 2, characterized in that BBM-1675B ₁ is collected. 8 BBM-1675B ₁ - Identification characteristics of the producing bacteria Actinomadeura verrucospora strain H964-92 (ATCC
39334), Actinomadeura verruchosospora strains
The method according to claim 7, which is A1327Y (ATCC39638) or a mutant strain thereof. 9 BBM of Actinomadeula verruchosospora
-1675B ₂ - The production strain was grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources.
Cultivate and then remove from the medium until a substantial amount of BBM-1675B ₂ is produced by the microorganism in the medium.
The method according to claim 2, characterized in that BBM-1675B ₂ is collected. 10 BBM-1675B ₂ - Identification characteristics of the producing bacteria Actinomadeura Verrucosospora strain H964-92
(ATCC 39334), Actinomadeula verrucosospora strain A1327Y (ATCC 39638), or a mutant strain thereof. 11 BBM-1675A ₃ , BBM-1675A ₄ , BBM-
An antibacterial agent containing a substance selected from 1675B ₁ or BBM-1675B ₂ as an active ingredient. 12 BBM-1675A ₃ , BBM-1675A ₄ , BBM-
An antitumor agent containing a substance selected from 1675B ₁ or BBM-1675B ₂ as an active ingredient.