JPH0229079B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel anti-tumor antibiotic, namely Esperamicin, with the formula: Antitumor antibiotic BBM-1675A ₁ and formula: The present invention relates to an antitumor antibiotic BBM-1675A ₂ and its production and collection. The structures of some of the antitumor antibiotics related to the present invention have not yet been identified. but,
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 Actinomadura pulveraceus ( Actinomadura pulveraceus)
6049 (ATCC 39100) is disclosed.
Although the structure of the WS6049 antibiotic is not yet clear, the characterizing properties given to 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 WS6049A and WS6049B are BBM of the applicant.
- Indicates that it is not identical to any of the 1675 components. Furthermore, the producing bacteria described in European Patent Application No. 95154A1 showed positive milk peptonation and positive D-fructose, D-mannite, trehalose in the color of its aerial mycelia on ISP medium No. 2, 3 and 4. and Actinomadura verrucosospora used in the present invention in positive utilization of cellulose.
can be clearly distinguished from 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 A ₂ ) 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 antitumor antibiotic conjugate, herein referred to as BBM-1675, and also 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
ATCC39334 (added to the permanent collection of microorganisms as an international deposit under the Budapest Treaty). Mutant strain A1327Y was then obtained by conventional nitrosoguanidine (NTG) treatment of strain H964-42;
It has been deposited with the American Type Culture Collection as ATCC39638 (International Deposit under the Budapest Treaty). 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 its 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 types of ingredients, BBM-1675A ₁
and _A2 have been subjected to in vivo screening 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 were found to exhibit activity against murine P-388 leukemia. Therefore, the complex and its bioactive components can be used as antibacterial agents or antitumor agents to inhibit 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. Spores are spherical or oblong with a warty 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 Actinomodula 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]. S.A. Waksman [The Actinomycetes, Volume 2] and G.M.
GMLvedemann [Intle J.
Cyst 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. 236-243, 1965 and Lechevalier and Lechevalier, the Actinomycetes, H. Prauser, Jena, Gustav Fisher
Verlag), pp. 393-405, 1970. Menaquinones are described by Collins et al., J. Zen. Microbiol (J.Gen.
Mirobiol), Vol. 100, pp. 221-230, 1977, identified by mass spectrometry;
The menaquinone composition was also determined by Yamada et al., J. Zen. Apple.
Microbiol (J.Gen.Appl.Microbiol.), No. 23
vol., pp. 331-335, 1977. Morphology Strain H964-92 forms basal and aerial hyphae.
The basal hyphae are long, branched and undivided into short threads. In 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 yellow in color with medium) and Bennett's agar, and pink in glucose-asparagine agar and glycerol-asparagine agar. Melanoids and other diffusible pigments are not produced. Mutant strain No.A1327Y obtained from this strain
mainly forms pale blue aerial hyphae 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. [Table] [Table] [Table] Extract - Nitrate
Broth [Table] No growth [Table] Glycerol + + +
D(−)〓arabinose − − −
L(+)〓arabinose + + +
D〓Xylose + + +
D〓ribose + − −
[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 ). Taxonomic position of strain H964-92 Strain H964-92 has the following main characteristics.
(1) Aerial mycelial 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) Diffusing dye: None. (6) Mesophilic. (7) Cell wall type _B. (8) Menaquinone series:
MK-9 (H ₆ ) and MK-9 (H ₈ ). These main characteristics indicate that strain H964-92 belongs to the genus Actinomadeula. The earliest species of the genus Actinomadeula were isolated from mammals.
Some strains were also obtained from plant material. However, many of the new species that have been proposed recently have been isolated from soil. Good-fellow
et al., J. Zen. Microbiol (J.Gen.Microbiol),
According to numerous taxonomy and reviews of Actinomadeula and related actinomycetes 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 et al. [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 Preobratchenskaya 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. So, the strain
H964-92 is classified as a new strain of Actinomadeula verrucospora. 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 39368)) 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 associated with the present invention is preferably produced by a BBM-1675-producing strain of Actinomadeula verruchosospora in a conventional aqueous nutrient medium.
It can be produced by culturing a strain of Actinomadeula verruchosospora or a mutant strain thereof having the identifying characteristics of ATCC39334 or ATCC39638. The microorganisms are grown in a nutrient medium containing known nutrient sources for actinomycetes: 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- Must contain mannitol or inositol. Nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. alone, or organic nitrogen sources such as peptone, meat extract, fermented rice extract, corn steep, etc.
It can be used in combination with 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 broth medium with a slant or soil culture or a lyophilized 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 chloroform plus increasing concentrations of methanol to remove the 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 Inc. blend
B ₁ and B ₂ can be separated by silica gel column chromatography using a mixture of chloroform and methanol as the 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] [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. The 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
of OCH ₃ (3.42, 3.80, 3.88 and 3.98ppm) and one exomethylene (4.57 and
5.48ppm). The CMR spectrum of BBM-1675A ₁ is a triple intensity signal (δ: 56.0ppm, OCH 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 ()
This suggests that the chromophore structure of BBM-1675A ₁ is retained. Two other chromophore fragments related to compound () were obtained by alkaline hydrolysis of BBM-1675A ₁ . 0.05N at 55℃ for 1 hour
KOH―252, 284, 297 when hydrolyzed with CH ₃ OH
(shoulder) and a compound () having a UV absorption maximum at 322 nm were produced. On the other hand, 1N KOH―
Reaction with CH ₃ OH gives an acidic chromophoric substance designated as compound (). The physicochemical properties of compounds (), () and () are summarized in Table 6 below. Table Structural information for formulas () and () was provided from the following spectral data and chemical transformations. ¹³ C and proton NMR indicated the presence of 4OCH ₃ , 1=CH ₂ , 2C=0 and 1>NH groups in compound (). Compound()
The NMR spectrum of () was similar to that of (), differing only in the absence of one of the four _OCH3 groups observed in formula (). Such differences, together with the acidic nature of compound (), suggested that compound () was the methyl ester of compound (). Compound () was quantitatively converted to compound () by heating to reflux with 1N methanolic KOH, while compound () was converted to compound () by treatment with diazomethane. C ₂ H ₅ in OH
Treatment of compound () with NaBH ₄ yields a reduction product (compound (), M ⁺ : m/z267), which replaces the -COOCH ₃ groups of compound ().
NMR showed _-CH2OH 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 appeared in the compound () at a high magnetic field (δ: 3.50 ppm). These results are in the compound () indicates the presence of a group, which upon hydrogenation in the compound () reduced to the base. Compound () at 80℃ for 3 hours
When heated with 1.5N methanolic hydrogen chloride and subjected to silica gel column chromatography of the hydrolyzate, weakly basic compounds (compound (),
M ⁺ :m/z211) was generated. IR spectrum and physicochemical properties indicated that the compound () contained _NH2 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/z327) and sugar (formula)
was cleaved into The NMR spectrum of () is
One singlet C—CH ₃ and three downfield OCH ₃
In addition to two high-field _OCH3 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 M-OCH ₃ peak at z131. NMR
The spectrum showed a 2,6-dideoxyhydropyranose structure for the compound. This designation is supported by the ¹³ C-NMR of the equation, which is
In addition to the 15 carbon signals that can be assigned to a compound, 1C—
CH ₃ , 1-CH ₃ , 3O-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. The molecular weight of the compound (457) corresponds to approximately 1/3 of the total molecule of BBM-1675A ₁ (presented formula
C ₅₇ H ₇₂ N ₄ O ₃₂ : Calculated MW = 1324). BBM―
1675A ₁ is considered to be: After filing U.S. Patent Application No. 495,231, it was discovered that the components BBM-1675A ₁ and A ₂ (prepared according to Example 2 below) were not in fact completely pure and that no use was made to define such components. 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° (decomposition) Elemental analysis: [Table] Ultraviolet absorption spectrum: Equipment: Varian UV, Cary 219 Solvent: Methanol concentration: 0.01356g/λ _nax ( nm) Absorption coefficient 320 12.4 280 sh (shoulder) 253 25.1 210 25.5 No significant change with acids or bases Optical rotation: Solvent - CHCl ₃ [α] 24°D = -207° (c = 0.0351) In the second analysis It showed the following optical rotation. [α] 27°D = -191° (c = 0.5, CHCl 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; with addition of NaCl (m/z): 1271,
1379, 1485, 1597. FAB-MS-MB (MB: matrix, m.
w.154); 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 ions (m/z): 1215, 1247, 1279,
1293, 1325, 1353; by addition of NaCl (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 above mass spectrum data) 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);
2.38-2.22 (5H); 2.12 (1H, m); 2.11
(3H, s); 1.60-1.05 (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
¹³ C NMR spectra were measured on samples 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 structure: 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/ λ _nax (nm) Absorption coefficient 320 12.2 282 16.3 252 26.2 214 25.8 No significant change with acid or base Optical rotation: [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: Equipment VG-ZAB-2F FAB-MS-thioglycerol molecular mass range ion (m/z): 968, 1249,
1355, 1357, 1463, 1569; by addition of NaCl (m/z): 990, 1271, 1379, 1485,
1593, FAB-MS-MB (MB: matrix, mw154); molecular mass range ion (m/s): 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 (based on the mass spectrum data above): Apparent
MW=1248 Nuclear magnetic resonance spectrum: See Figure 13 Equipment: WM360 Brutzker Solvent: CDCl ₃ ¹ H NMR 360MHz δ (ppm): 1.91 (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 NMR90.3MHz See Figure 14 ¹³ C NMR of BBM-1675A ₂ purified in a separate test
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 for BBM-1675 components A Study No. 1 - Summary [Table] B Study No. 2 Purification A ₁ and A _Two- 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 in a glass cylinder with dimensions of 6.4 cm (outer diameter) x 12 cm and containing 10 ml of eluent. 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 first 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 quenching the fluorescence 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-Pondapak 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 RP—
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 connected with 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 Otsukwa 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. A 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 HPLC system from the mixing chamber via a polypropylene tube (1.5 mm inner diameter x 3.0 mm outer diameter). As the eluate exits the mixing chamber, the solvent in the static reservoir freely replaces this liquid;
This resulted 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] 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: α = No compound eluted from the column.
[Table] 25% methanol in water 1.25 1.25
a = No compound eluted from the column.
[Table] [Table] a=No elution. b = Not measured. Biological Properties of BBM-1675 Components The antibacterial activity of BBM-1675 components was determined on 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 types of 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 A ₄
A second antibacterial test was carried out on purified A ₁ and A ₂ (produced in Example 3 below) and components A ₃ and A ₄ . [Table] [Table] Prophage-inducing activity in the lysogenizing bacterium E. coli W1709 (λ), Nature, Vol. 196, pp. 783-784, 1962, Lein ), etc., the BBM-1675 component was measured. Plaque counts were performed on agar plates containing test materials (T) and control plates (C). A T/C ratio of plaque count greater than 3.0 was considered significant and lysogenization-inducing activity (lLB activity) was expressed as the minimum inducing concentration of the test compound. As shown in Table 17, BBM—
1675 component shows strong lLB activity in lysogenic bacteria,
This suggests that the component has antitumor activity. Table 17 Lysogenization-inducing activity antibiotic MIC of BBM-1675 components ^* (mcg/ml) BBM-1675A ₁ 0.0063 BBM-1675A ₂ 0.0125 BBM-1675A ₃ 0.05 BBM-1675A ₄ 0.10 BBM-1675B ₁ 0.10 BBM-1675B ₂ 0.20 *Minimum inducing concentration 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 dosed intraperitoneally with graded doses of the test compound 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). Components A ₃ and A ₄ were tested only on P-388 leukemia on a q3d x 3 schedule 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). A T/C value equal to or greater than 125% indicates that a significant anti-tumor effect has been achieved. The results are shown in Tables 18-23. BBM-1675A ₁ and A ₂ showed extremely potent antitumor activity against P-388 leukemia with a maximum T/C value of 160%. These ingredients are recommended for minimum effective doses of chromomycin.
Approximately 100-3000 times more active than _A3 . but,
BBM-1675A ₃ and A ₄ were less active against P-388 leukemia than components A ₁ or A ₂ (Table 19). BBM-1675A ₁ and A ₂ are also L-
It also showed activity against 1210 leukemia (Table 12), B16 melanocytoma (Table 22) and Lewis lung cancer (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 _A2 have therapeutic indexes of 4-8 and 8-20, respectively, than chromomycin _A3 in P-388 leukemia lines.
It was twice as good (Table 25). 5x each intravenously
A second experiment was performed by intravenous administration of the BBM-1675 component to P-388 and L-1210 leukemia inoculated with 10 ⁵ and 10 ⁴ cells/mouse. In these experiments, adriamycin
was used as a reference drug, dissolved in 0.9% saline and administered on days 1, 4 and 7. Table 26 results
and shown in 27. Maximum T/ for both components A ₁ and A ₂
It was superior to adriamycin in terms of C value, minimum effective dose and active range. [Table] Circles indicate significant antitumor activity.
[Table] [Table] Circles indicate significant antitumor activity.
[Table] [Table] Circles indicate significant antitumor activity.
[Table] Circles indicate significant antitumor activity.
[Table] [Table] Circles indicate significant antitumor activity.
[Table] [Table] Circles indicate significant antitumor activity.
[Table] [Table] *Minimum Effective Dose [Table] [Table] Circles indicate significant antitumor activity.
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 shown 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 6, a sample of the purified compound was added to mouse L-1210 leukemia, P -Tested against 388 leukemia and B16 melanocytoma. These results are shown below. [Table] [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. Reagent for P-388 and L-1210 leukemia, BBM-
1675A ₁ is also effective when administered as a single injection, once or three times a day, on each fourth day, or five times a day. Compounds against B16 melanocytoma ip
It is equally 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 activity 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 an effective antibacterial or tumor suppressive dose of BBM-1675A ₁ , A ₂ , A ₃ , A ₄ , B ₁ or
A method of therapeutically treating a host comprising administering B ₂ or a pharmaceutical composition thereof is provided. According to another feature, the present 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. Optimal application ratios 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 0.0001 to 1 mg can be administered to the patient, but this dose can be higher or lower 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 it can be higher, or the amount can be determined within a range where the drug is effective, and it can be lower. You can also. 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% _CaCO3 and 1.6% agar. Using a well-grown agar slant medium, add 3% soluble starch, 3% dry yeast,
0.3% _K2HPO4 , 0.1 _{% KH2PO4 ,} 0.05% _MgSO4・
Contains _7H2O , 0.2% NaCl and 0.1% _CaCO3 ,
It was inoculated into a nutrient medium whose pH was adjusted to 7.0 before sterilization.
The nutrient medium was incubated for 72 hours on a rotary shaker (250 rpm)32
℃, and 5 ml of the growth was mixed with 3% amashiyo molasses, 1% cornstarch, 1% fishmeal, 0.1%
_CaCO3 and 0.005% _CuSO4.5H2O (PH before sterilization ₎
7.0) into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium. Fermentation was carried out on a rotary shaker for 6 days at 28°C. Antibiotic activity in the fermentation broth was determined by paper disc agar diffusion using Staphylococcus aureus 209P as the test organism.
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 is reduced by 68–76
After fermentation for hours a maximum value of about 0.9mcg/ml was reached. 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 _{CuSO4.5H2O 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 a silica gel column (φ5.0×50 cm) using chloroform and increasing amounts (1→5% v/v) of methanol as eluent. The eluate was analyzed for antibacterial activity (against S. aureus) and TLC ( _SiO2 ; _CHCl3 - _CH3OH =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 (in chloroform), then a mixture of BBM-1675A ₂ , A ₃ and A ₄ (507 mg) and then a mixture of BBM-1675B (210 mg) with 3% methanol in chloroform. . BBM-1675A ₁ solid is safe indexed
It was applied to an LH-20 column (φ2.0 x 80 cm) and developed with methanol. The active fraction was concentrated to dryness in vacuo and the residual solid was crystallized from methanol to give colorless platelets (124 mg) of pure BBM-1679A ₁ (this material is the starting material for Example 3A). . The complexes BBM-1675A ₂ , A ₃ and A ₄ were separated by chromatography on a Bondapak C ₁₈ (Waters, φ3.0×50 cm) column. Elution is carried out with aqueous acetonitrile and the active eluent is 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). Example 3 Further purification of BBM-1675A ₁ and A ₂ A Purification of BBM-1675A ₁ Glenco column, 2.67 cm i.d. x 75
cm. Baker bonded phase octadecyl (C18) 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 - 21.6% 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 acetonitrile 2
ml 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. Glen column, 1.5cm inner diameter x 20cm. 12 g of Woelm silica gel (60-200 micron particles) 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, the fraction 6-
9 were pooled and evaporated to dryness, leaving a residue D 73
mg was obtained. A 1.5 cm ID x 20 cm Glenco column was slurry packed with 12 g of Whelum silica gel (63-200 micron particles) in Skellysolve B. Residue D was dissolved in approximately 2 ml of CHCl ₃ and applied to the column. Add chloroform to 25ml of Skerisolve B
Replaced with 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 and 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 Sephadex LH-20 in methanol and adjusted to a 33.4 cm chromatography bed. Purified BBM-1675A ₁ (approx. 120
mg) in 2 ml of methanol and transferred to a 2.5 ml sample container. The sample was applied to the column and methanol elution was started 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 was observed to elute at a Ve/Vt of 0.79-0.91 (Ve = elution volume; Vt = bed volume). B. Purification of BBM-1675A ₂ A 2.65 cm ID x 75 cm Glenco column was slurry packed with Baker bonded phase octadecyl (C18) 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 eluent above.
87ml 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, the homogeneity is
65.8 mg of BBM-1675A ₂ was obtained. BBM-1675A ₂ is 2TLC system, 1 2-dTLC
It was homogeneous by analysis and HPLC. Example 4 Extraction method suitable 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 equipped with a faucet at the bottom (top 12 cm diameter; bottom 10 cm diameter; height length 37cm)
Moved to. An equal volume of chloroform was added. The mixture was stirred for 2 hours with a CRC-air driven stirrer at good speed. Dicalite
Approximately 4 (1.3 kg) of super-aiding agent was added and mixed. The mixture was passed through a pad of Dicalite held in a No. 12 Buchner funnel. 19 liquid bottle with vacuum outlet [Ace No.5396-06]
collected in. The pine was washed with chloroform 2. 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 to yield 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 using 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 purged of air by passing a pressurized stream of Skellisolve 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. Example 3 This partially purified BBM-1675A ₁
It can also be further purified according to the procedure described in . Example 5 BBM using mutant strain H964-92-A1327Y-
1675 Fermentation of complex Actinomodula 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% Amashiyo 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 6 Isolation and purification of BBM-1675 components The fermentation broth collected from Example 5 (3000, PH
7.6) was separated into mycelium cake and supernatant using a shear press centrifuge. The mycelial cake was stirred with 2000 methanol for 1 hour and the insoluble materials were 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
Insoluble materials were removed by filtration and evaporated in vacuo.
And so. Add 30% of the concentrate to n-hexane while stirring.
When added to the solution, a pale yellow 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 (4.86g, potency:
203mcg/mg) was obtained. The solid was then transferred to a silica gel column (φ3.0×
70 cm). The eluent was S. Antibacterial activity and TLC against Aureus
( _SiO2 , _CHCl3 -MeOH=5:1 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 a mixture of BBM-1675A ₂ , A ₃ and A ₄ (730
mg, titer: 340 mcg/mg) then the BBM-1675B complex (200 mg, titer: 190 mcg/mg) was eluted with 3% methanol in chloroform. Above BBM
1675A ₁ was rechromatographed on silica gel (column: 2.2 x 44 cm) with 2% methanol in benzene. Bioactive eluent was analyzed by HPLC [Lichrosorb RP-18: CH ₃ CN-MeOH
−0.1M CH ₃ COONH ₄ =5:2:3, v/v)
and the fraction containing homogeneous BBM-1675A ₁ was evaporated to dryness in vacuo. Crystallizing the residual solid from methanol (10 ml) gives BBM
-1675A ₁ (197mg, titer: 1000mcg/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: _CH3CN-
H ₂ O=75:25, v/v). Trace ingredients
BBM-1675A ₄ (45 mg, titer: 410 mcg/mg) and A ₃ (19 mg, titer: 300 mcg/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 drawings]

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 MHz). 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 at (90.3MHz) 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 Formula: Antitumor antibiotic BBM-1675A ₁ or formula: Antitumor antibiotic BBM-1675A ₂ . 2 BBM of Actinomadura verrucosospora
1675A ₁ or BBM - 1675A ₂ - Selected production strains are grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources in substantial quantities.
until one selected from BBM-1675A ₁ or BBM-1675A ₂ is produced in the medium by the microorganism.
Culture, then BBM-1675A ₁ or BBM from the medium
-1675A A formula characterized by taking a compound selected from ₂ : Antitumor antibiotic BBM-1675A ₁ or formula: A method for producing a compound selected from antitumor antibiotic BBM- _1675A2 having 3 BBM of Actinomadeula verruchosospora
-1675A 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-1675A ₁ is produced by the microorganism in the medium.
The method according to claim 2, characterized in that BBM-1675A ₁ is collected. 4 BBM-1675A ₁ Identification of the producing bacteria Characteristics Actinomadeura verrucosospora strain H964-92 (ATCC
39334), Actinomadeura verruchosospora strains
The method according to claim 3, comprising A1327Y (ATCC 39638) or a mutant strain thereof. 5 BBM of Actinomadeula verruchosospora
-1675A ₂ - culturing the production strain in an aqueous nutrient medium containing an assimilable carbon source under deep aeration conditions until a substantial amount of BBM-1675A ₂ is produced by the microorganism in the medium; Next, from the medium, BBM
The method according to claim 2, characterized in that 1675A ₂ is collected. 6 BBM-1675A ₂ - Identification characteristics of the producing bacteria Actinomadeura Verrucosospora strain H964-92 (ATCC
39334), Actinomadeula percosospora strains
The method according to claim 5, which is A1327Y (ATCC 39638) or a mutant strain thereof. 7 Formula: Antitumor antibiotic BBM-1675A ₁ or formula: An antibacterial agent containing a compound selected from BBM-1675A ₂ as an active ingredient. 8 Formula: Antitumor antibiotic BBM-1675A ₁ or formula: An antitumor agent containing as an active ingredient a compound selected from BBM-1675A ₂ , an antitumor antibiotic having the following properties.