KR920001366B1 - Bbm-1675 new antitumor antibiotic complex - Google Patents

Abstract

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Description

Preparation method of new anticancer antibiotic complex BBM-1675

1 shows the infrared absorption spectrum of partially purified BBM-1675 A ₁ (KRr pellets).

2 shows the infrared absorption spectrum of partially purified BBM-1675 A ₂ (KBr pellet).

3 shows the infrared absorption spectrum of BBM-1675 A ₃ (KBr pellet).

4 shows the infrared absorption spectrum of BBM-1675 A ₄ (KBr pellet).

5 shows the proton magnetic resonance spectra of BBM-1675 A ₁ partially purified in CDCl ₃ (6 MHz).

6 shows the proton magnetic resonance spectra of BBM-1675 A ₂ partially purified in CDCl ₃ (60 MHz).

Figure 7 shows the proton magnetic resonance spectra of BBM-1675 A ₃ in CDCl ₃ (60 MHz).

8 shows the proton magnetic resonance spectrum of BBM-1675 A ₄ in CDCl ₃ (60 MHz).

9 shows the infrared absorption spectrum of the purified BBM-1675 A ₁ (KRr pellets).

10 shows the proton magnetic resonance spectra of BBM-1675 A ₁ purified in CDCl ₃ (360 MHz).

Figure 11 shows the ¹³ C magnetic resonance spectrum of BBM-1675 A ₁ purified in CDCl ₃ (90.3 MHz).

12 shows the infrared absorption spectrum of purified BBM-1675 A ₂ (KBr pellet).

Figure 13 shows the proton magnetic resonance spectra of purified BBM-1675 A ₂ in CDCl ₃ (360 MHz).

14 shows the ¹³ C magnetic resonance spectrum of BBM-1675 A ₂ purified in CDCl ₃ (90.3 MHz).

The present invention relates to new anticancer antibiotics and their production and recovery.

The anticancer antibiotic compound of the present invention has not yet been identified in terms of structure. However, due to its unique physical, chemical and biological properties, Applicants believe that BBM-1675 antibiotics are novel.

In European Patent Publication No. 95154 A ₁ , Actinomadura Fulveraceus S.P. nov. No. 6049 (Actinomadura pulveraceus sp.nov No. 6049) produces anti-cancer antibiotics named WS 6049-A and WS 6049-B. The fermentation of [ATCC 39100] is disclosed. Although the structure of the WS 6049 antibiotics is not yet known, the properties of these antibiotics suggest that WS 6049-A and WS 6049-B are structurally related to the BBM-1675 antibiotics of the present invention. However, the spectral data shows that neither of WS 6049 A nor WS 6049 B is identical to any of the BHM-1675 components of the present invention.

Furthermore, the product organisms disclosed in European Patent Application Publication No. 95154A 1 are characterized in terms of the color of the airborne mycelium in ISP mediums 2,3 and 4, in terms of positive milk peptonation properties, and also in D. In terms of the positive availability properties of fructose, D-mannitol, trehalose and cellulose, they are clearly distinguished from Actinomadura (verrucosospora) used in the present invention.

A new anticancer antibiotic complex, referred to herein as BBM-1675, is a material obtained according to the present invention, the complex comprising actinomaduran beru which produces BBM-1675 in an aqueous nutrient medium containing carbon and nitrogen sources assimilable under aerobic immersion conditions. Actinomadura (verrucosopora) strains, most preferably Actinomadura, verrucosopora strains H964-92 [ATCC 39334 (KFCC 10089)] or actinomadura berukosospora strains A 1327 Y [ATCC 39638 (KFCC 10089)] or mutants thereof are obtained by culturing until a substantial amount of said BBM-1675 complex is produced by said organism in said medium and said complex is selectively recovered from said medium. . The invention also provides two major biologically active components of the BBM-1675 complex, termed BBM-1675 A ₁ and A ₂ , and four less important of the complex, termed BBM-1675 A ₃ , A ₄ , B ₁ and B ₂ . Provide biologically active ingredients. These components can be separated and purified by conventional chromatography techniques. The BBM-1675 complex and its components with biological activity exhibit both antibacterial and anticancer activity.

The present invention relates to novel anticancer antibiotic complexes and specific actinomadurra berukosospora strains, more specifically actinomadura berukosospora strain H964-92 or actinomadura berukose, designated herein as BBM-1675. The preparation of the said preparation by fermentation of the mutant whose name is Pora strain A 1327 Y is related. The above-mentioned parent strain was isolated from soil specimens collected from Pto Esperanza, Misiones, Argentina. Biologically pure microbial cultures have been deposited with the American Type Culture Collection of Washington, D.C., and have been permanently deposited with the accession number of ATCC 39334 (KFCC 10089). Subsequently, mutant strain A 1327 Y was obtained by conventional nitrosoguanidine (NTG) treatment of strain H964-92 and has been deposited with the American Bacterial Conservation Association under accession number of ATCC 39638.

Fermentation of actinomadura berukosospora strain H964-92 or strain A 1327 Y, as in the case of cultures producing many antibiotics, results in mixtures or complexes of the component substances. Two major biological activities BBM-1675 A ₁ and A ₂ and four less important biological activities BBM-1675 A ₃ , A ₄ , B ₁ , and B ₂ produced during the fermentation process. Separated from the complex.

BBM-1675 and its components BBM-1675 A ₁ , A ₂ , A ₃ , A ₄ , B ₁ and B ₂ exhibit antimicrobial activity against a wide range of microorganisms, particularly gram positive bacteria. The BBM-1675 complex and components with biological activity isolated therefrom also have the activity of inducing propagation in risogenic bacteria. Two of these components, BBM-1675 A ₁ and A _2, have been used to detect in vivo the tumor systems of various mice and to detect L-1210 leukemia, p-388 leukemia, B ₁₆ hematoma, and Lewis lung cancer. It has been demonstrated to have an inhibitory effect on. BBM-1675 A ₃ and A ₄ show activity against mouse p-388 leukemia. Therefore, the complex of the present invention and its components having biological activity can be usefully used as an antimicrobial agent or an anticancer agent for inhibiting mammalian donation.

microbe

Actinomycete strain H964-92 was isolated from soil samples and prepared in a biologically pure culture state according to conventional methods for characterization purposes. Strain H964-92 forms a short spore chain on aerial mycelium, which shows a straight, serpentine or bent shape. These spores are round or oval and have a warty surface. Aerial mycelium is poorly formed on most media. The color of the airborne object is white, which in turn changes to pale pink, or also blue on some agar media. The substrate mycelium is colorless or pale pink. Growth temperature range is 15 ℃ to 43 ℃. It can be seen from the cell wall amino acid composition and the whole cell hydrolysis sugar component that strain H964-92 belongs to cell wall type III _B. Menakinone was identified as MK-9 (H ₆ ) .MK-9 (H ₈ ). Based on the main morphological, cultural and physiological properties along with the chemical properties of the cell wall composition, strain H964-92 is classified as belonging to the genus Actinomadura.

Native strain H964-92 produces airborne myceliums with poor growth and porosity, but variants with good growth properties and improved airborne mycelium properties are obtained by treating H964-92 with NTG (nitrosoguanidine). As the taxonomic studies accelerated from this variant, designated strain A 1327 Y, it was soon identified as actinomadura berukosospora.

Way

The media and processes used to investigate the culture and carbohydrate utilization characteristics are those recommended by the International Streptomyces project (Intl. J. Syst. Bacteriol. 16: 313-340, 1966). Published by S. Waxmann [The Actinomycetes Vol. 2] and G. M. Lebedman [Intl. J. Syst. Bacteriol. 21: 240-247, 1971]. Another medium was also used. Cell Wall Amino Acid Components and Whole Cell Hydrolyzate Sugars by Becker et al., Appl. Microbiol 13: 236-243, 1965, The Actinomycetes Ed. H. Prauser. Jena, Gustav by Lechevalier. Fischer Verlag), pp. 393-405, 1970. Menaquinone was confirmed by mass spectrum analysis according to the method of Collins et al. In J.Gen. Microbiol, 100: 221-230, 1977, and the component of menaquinone was found by J.Gen.Appl.Microbiol 23: 331- 335, based on the system published in 1977.

morphology

Strain H964-92 forms both substrate and aerial mycelium. The matrix mycelia are long, branched, and do not segment into short filaments. In airborne mycelium, short spore chains are formed morphologically at the end of mycelia. Branches of spore rings, such as spiral wounds, were also observed at the end of mycelia. These spore chains contain 2 to 10 spores in the chain and are straight, serpentine or curved. These spores have a wart-like surface and are round or oval (0.5-0.6 × 0.6-1.4 μm) with round or dotted ends. After maturation, each spore often separates with empty encapsulation. Motile spores, spore sacs or hard granules were not observed in any of the media investigated.

Culture and Physiological Properties

Growth regulation of strain H964-92 is not satisfactory in both chemical medium and natural organic medium. The formation of airborne mycelium is generally poor, but odorous agar [ISP NO.3 medium], inorganic salt-starch agar [ISP No. 4 medium] and Suitable in Bennett agar. Very often spontaneous variants lacking aerial mycelium are produced. The airborne mycelium is white in color and later turns to pale pink in oatmeal agar, inorganic salt-starch agar and glycerol-asparagine agar (ISP No. 5 medium). In addition, the color of the object changes to blue after a long incubation period (5 months) in oatmeal agar, glycerol-asparagine agar and tyrosine agar. The color of the substrate mycelium is Zapec agar, tyrosine agar, yeast extract-malt extract agar [ISP No. 2 medium], peptone-yeast extract-iron agar (ISP No. 6 medium (ISP) No. 6 medium)] and colorless or yellowish in Bennett agar, and pale pink in glucose-asparagine agar and glycerol-asparagine agar No melanoids and other diffuseable pigments are produced. Y is obtained from the original strain, mainly forming light blue airborne mycelium and having abundant airborne spore mass.

Strain H964-92 grows at 15 ° C, 28 ° C, 37 ° C and 43 ° C but not at 10 ° C or 47 ° C. It is also sensitive to 7% NaCl and resistant to 0.01% lysozyme. Culture and physiological characteristics of the resulting strains are shown in Tables 1 and 2, respectively. The utilization of the carbon source is shown in Table 3.

TABLE 1

TABLE 2

TABLE 3

Cell Wall Composition and Whole Cell Sugar

The cell wall of purified strain H964-92 contains mesodi-aminopimelic acid but lacks glycine. Whole cell hydrolysates show the presence of madurose (3-0-methyl-D-galactose), glucose and ribose. Cell wall amino acids and whole cell sugar components indicate that strain H964-92 belongs to cell wall type III _B. Two major constituents of menaquinone were identified as MK-9 (H ₆ ) and MK-9 (H ₈ ).

Taxonomic location of strain H964-92

Strain H964-92 has the following main characteristics.

(1) Air spore chains: short, straight, serpentine or curved (2) Spores: wart-like surfaces (3) Airborne mycelium: pink or blue (4) Substrate mycelium: light pink on some mycelium (5) Diffuse pigments: none (6) mesophilic (7) cell wall type III _B (8) menaquinone systems: MK-9 (H ₆ ) and MK-9 (H ₈ )

These major characteristics indicate that strain H964-92 belongs to the genus Actinomadura. It was isolated from mammals of early species of the genus Actinomadura. Some strains were also obtained from plants. However, most of the new species recently proposed have been separated from the soil.

According to Goodfellow et al. (J.Gen. Miirobiol, 112: 95-111 (1979)), most of the actinomadura species have been isolated from the soil based on Actinomyces associated with the Actinomymadura review. It is classified as group 7 among 14 groups. Strain H964-92 is mostly associated with species of population 7. Nonomura and Ohara reported five decaying species of the genus Actinomadura in J. Ferment.Technol. 49: 904-912 (1971), and Nonomura [J.Ferment.Technol. 52: 71-77 (1974)] and Actinomycetes and Related Organisms 12: 30-38 (1977) published a key statement on the identification and classification of actinomadura species. As a result of comparing 30 technical details including microorganisms published in each of the patents, strain H964-92 was actinomadura koerulia described in the reference foamed by Preobrazenskaya et al. And Actinomadura berukosospora published by Nonomura cited above.

Direct comparison of strain H964-92 with actinomadura berukosospora strain KCCA-0147 revealed that it was closely related to actinomadura berukosospora in morphological, culture and ecological characteristics. Therefore, strain H964-92 is classified as a novel actinomadura berukosospora strain.

In connection with the production of BBM-1675, the present invention relates to the specific strain actinomadura berukosospora strain H964-92 [ATCC 39334 (KFCC 10089)] and strain A 1327 Y [ATCC 39638 (KFCC 10090)] and mutants thereof. It will be described in detail using the strain, but it is to be noted that the present invention is not limited to the microorganisms described above or the microorganisms described by the culture characteristics disclosed herein. In particular, the present invention also encompasses strain H964-92 and all natural and artificial BBM-1675-producing mutants and mutants thereof.

Antibiotic production

The BBM-1675 antibiotic of the present invention is an actinomadura berukosospora strain producing BBM-1675, preferably an acti having the same properties as ATCC 39334 (KFCC 10089) or ATCC 39638 (KFCC 10090) or mutants thereof. Nomadura berukosospora strains can be prepared by culturing in conventional aqueous nutrient media. The organism is grown in a nutrient medium containing known actinomyces nutrients, ie carbon and nitrogen assimilable sources, and other known growth factors that are optional inorganic. Surface cultures and bottles may be used for the production of limited amounts of antibiotics, but initial soaking conditions are preferred for the mass production of antibiotics. Other general processes available for the cultivation of actinomyces can also be applied to the present invention.

Crude medium, moderately assimilable carbon sources such as glycerol, L (+)-rubber sugar, D-wood sugar, D-ribose, L-rhamnose, D-glucose, D-fructose, sucrose, cellobiose, soluble starch, D It should contain something like mannitol or inositol. As the nitrogen source, ones such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate and the like can be used alone or in combination with peptone, meat extract, yeast extract, corn steep liquor, soy flour, cottonseed flour and the like as an organic nitrogen source. Nutritive inorganic salts which supply sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron and the like may also be added as necessary.

The production of BBM-1675 antibiotics is effectively carried out at a certain temperature, such as 15-14 ° C., which is conducive to the satisfactory growth of the organisms producing them, and is conveniently carried out at temperatures of approximately 27-32 ° C. Typically, optimum production is achieved using shake flasks, agitated or tank fermentations and is obtained after a culturing period of about 68-180 hours. When the tank fermentation is performed, it is preferable to inoculate the broth medium by slope culture, soil culture, or lyophilized culture of the produced organism to produce an inoculum having growth power in the nutrient broth. After obtaining an inoculum with activity in this manner, it is transferred to the fermentation tank incubator aseptically. Antibiotic production can also be monitored using paper disk-agar diffusion analysis using Staphylococcus aureus 209P as a test organism.

Separation and Purification

Once fermentation is complete, the BBM-1675 complex can be recovered from the broth medium by conventional separation procedures, such as solvent extraction. Thus, for example, the entire broth medium can be separated into mycelium cake and broth supernatant by filtration or centrifugation. Antibiotics in the mycelium cake can be recovered by suspending the cake in methanol, filtering the insoluble material, and then concentrating the methanolic extract. Active antibiotics in the juice supernatant can be recovered by extraction with n-butanol. Subsequently, the aforementioned n-butanol and methanol extracts are combined and then azeotropically evaporated to form an aqueous solution, where most of the antibiotic is precipitated as an oily solid. The solid material is then dissolved in methanol, the solution is filtered and the filtrate is concentrated and added 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 reaction of antisolvent such as n-hexane.

The crude BBM-1675 complex is composed of two major biologic components, BBM-1675 A ₁ and A ₂ and four less important biological activities, BBM-1675 A ₃ , A ₄ , B ₁ and B ₂ It is a mixture of several components, including.

Components having these biological activities can be separated and purified by conventional chromatography techniques. In one procedure, the crude BBM-1675 complex is first dissolved in methanol and then purified by Sephadex LH-20 column chromatography using methanol as elution solvent. The partially purified complex is then chromatographed on a silica gel column and eluted in a concentration gradient step using chloroform and concentration gradient methanol, a mixture of BBM-1675 A ₁ and BBM-1675 A ₂ , A ₃ and A ₄ . And BBM-1675 B ₁ and B ₂ are obtained. In addition, the A ₁ component can be purified by Sephadex LH-20 column chromatography using methanol as the eluent. A mixture of A ₂ , A ₃ and A ₄ can be separated by chromatography on a Bondapak C ₁₈ column (Waters Associates, Inc.) using aqueous acetonitrile with increasing concentration (concentration-gradient) as eluent. The mixture of B ₁ and B ₂ components can be separated by silica gel column chromatography using a mixture of chloroform and methanol as eluent. Further details of the preferred chromatographic separation procedures are described in the Examples below.

Physical and Chemical Properties of BBM-1675 Constituents

The six biological and active ingredients of the BBM-1675 complex can be distinguished from each other by the two TLC systems as shown in the table below.

TABLE 4

Separation of BBM-1675 A ₂ , A ₃ and A ₄ is difficult by normal phase TLC systems but can be achieved by reverse phase TLC. Each BBM-1675 component shows similar solubility and color reactions. For example, they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, poorly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride. In addition, it shows a positive reaction with ferric chloride, erlich and tolene reagents, but negative reactions in Sakaguchi, ninhydrin and anthrone tests.

The unique physicochemical properties of the BRM-1675 components are shown in Table 5 below.

TABLE 5

UV absorption maxima of the BBM-1675 components were observed at 253, 282 and 320 nm, which did not migrate in acidic or alkaline solutions. The IR and PMR spectra of BBM-1675 A ₁ , A ₂ , A ₃ and A ₄ are shown in FIGS. 1-4 and 5-8, respectively, in the accompanying drawings. The 360 MHz PMR of the BBM-1675 A ₁ has one acetyl (δ: 2.11 ppm), one N-CH ₂ (2.52 ppm), with two aromatics (7.50 and 8.59 ppm) and one NH (11.79 ppm) proton Four OCH ₃ (3.42,3.80,3.88 and 3.98 ppm) and one exomethylene (4.57 and 5.48 ppm) groups were indicated. The NMR spectrum of BBM-1675 A ₁ showed 55 carbon signals including triple intensity signals (δ: 56.0 ppm, OCH ₃ ). The molecular formula of BBM-1675 A ₁ was inferred to be C ₅₇ H ₇₂ N ₄ O ₃₂ based on proton and ¹³ C NMR spectra, microanalysis and molecular weight determination by HPLC and SIMS (Secondary Ion Mass Spectroscopy).

Structural study of BBM-1675 A ₁

Treatment with 0.5N HCl-CH ₃ OH at room temperature, BBM-1675 A ₁ loses its biological activity and, together with several unidentified fragments, gives an affinity chromophore material (Compound 1). Compound 1 exhibits a similar UV absorption pattern to the parent antibiotic, suggesting to have a chromophore structure of BBM-1675 A ₁ . Two other chromophore fragments associated with Compound I were obtained by alkaline hydrolysis of BBM-1675 A ₁ , when hydrolyzed with 0.05N KOH-CH ₃ OH at 55 ° C. for 1 hour, UV at 252,284,297 (parallel) and 322 nm While providing compound II having an absorption maximum, the reaction in KOH-CH ₃ OH gives an acidic chromophore pointing to compound III. The physicochemical properties of compounds I, II and III are summarized in Table 6 below.

TABLE 6

Information on the structure of compounds II and III was provided from the following spectral data and chemical transformations.

, 2개의 ＞C=0 및 1개의 ＞NH기가 존재함을 가리켰다. 화합물 Ⅲ의 NMR 스펙트럼은 화합물 Ⅱ에서 관찰된 OCH₃기 4개중 1개가 없는 것만 다르고, 화합물 Ⅱ와 유사하였다. 화합물 Ⅲ의 산성 특성과 함께, 상술한 차이는 화합물 Ⅱ가 화합물 Ⅲ의 메틸 에스테르라는 것을 시사한다. 1N 메탄올성 KOH와 함께 환류하에 가열하면, 화합물 Ⅱ는 정량적으로 화합물 Ⅲ으로 전환되는 한편, 디아조메탄으로 처리하면 화합물 Ⅲ이 화합물 Ⅱ으로 전환되었다. 화합물 Ⅱ를 C₂H₅OH중에서 NaBH₄로 처리하면, 화합물 Ⅱ의 -COOCH₃기 위치에서 NMR결과 CH₂OH기를 나타내는 환원 생성물(화합물 Ⅳ, M⁺:m/z 267)이 수득되었다. 목탄상 팔라듐을 사용하여 수소화 처리하면, 화합물 Ⅱ는 이중 탈수소유도체(화합물 Ⅴ, M⁺:m/z 297)를 제공한다. 화합물 Ⅴ의 양성자 NMR 스펙트럼은 새로운 이중 메틸시그날과 화합물 Ⅱ에 존재하는 엑소메틸렌기의 결손을 보여주었다. 또한, OCH₃기중 하나는 화합물 Ⅴ중의 보다 높은 장(場)(δ:3.50ppm)에서 나타났다. 이들 결과는 화합물 Ⅱ내에서의

기의 존재를 가리키는데, 이것은 화합물 Ⅴ에서

기로 수소화처리 하여서 환원된다.C ₁₃ and proton NMR are four OCH ₃ , 1 = C ₂ , 7 in compound II.

, 2> C = 0 and 1> NH groups. The NMR spectrum of compound III was similar to compound II except that no one of the four OCH ₃ groups observed in compound II was absent. Along with the acidic properties of compound III, the above differences suggest that compound II is the methyl ester of compound III. Heating under reflux with 1N methanolic KOH resulted in quantitative conversion of compound II to compound III, while treatment with diazomethane converted compound III to compound II. Treatment of compound II with NaBH ₄ in C ₂ H ₅ OH yielded a reduced product (compound IV, M ⁺ : m / z 267) which exhibits a CH ₂ OH group at the -COOCH ₃ group position of compound II. When hydrotreated with palladium on charcoal, compound II provides a double dehydrogen derivative (compound V, M ⁺ : m / z 297). The proton NMR spectrum of compound V showed a new double methyl signal and a deficiency of the exomethylene group present in compound II. In addition, one of the OCH ₃ groups appeared at the higher field (δ: 3.50 ppm) in compound V. These results were obtained in Compound II.

Indicates the presence of a group, which in compound V

It is reduced by hydrogenation with a group.

Compound II was heated with 1.5 N methanolic hydrogen chloride at 80 ° C. for 3 hours, and the hydrolyzate was chromatographed on a silica gel column to give a weakly basic compound (Compound VI, M ⁺ : m / z 211). From the IR spectra and physicochemical properties it is inferred that compound VI contains NH ₂ groups. Compound VI was compared with the true sample by IR and NMR, and found to be methyl 4,5-dimethoxy-anthranilate. As a result, the structure of compound II-VI was determined as shown below.

When treated with 0.05N KOH-CH ₃ OH at 55 ° C., Compound I was split into new chromophore fragments (Compound VII, C ₁₅ H ₂₁ NO ₇ , M ⁺ : m / z 327) and sugar (Compound VII). NMR spectra of VIII show _three low long OCH ₃ and two aromatic protons commonly found in compounds II, V and VI plus one single C-CH ₃ and two high long OCH ₃ groups gave.

In addition, hydrolysis of X with 1.5 N methanolic hydrogen chloride produced Compound VI, which is consistent with that obtained previously from II. After removal of VI, the hydrolyzate was treated with 2,4-dinitrophenylhydrazine to precipitate a yellow solid, which was found to be the 2,4-dinitro phenylhydra zone of pyruvic acid. Therefore, compound VII is methyl 4,5-dimethoxy-N- (2 ', 2'-dimethoxy-propionyl) -anthranilate. Compound VII did not show a molecular ion peak but showed a M-OCH ₃ peak at m / z 131 in the mass spectrum according to the molecular formula of C ₇ H ₁₄ O ₄ . NMR spectra indicated the 2,6-dideoxyhexo-pyranose structure in compound VII. This problem was supported by the ¹³ C-NMR of compound I, which is one C-CH ₃ , one -CH ₂ , three 0-CH <and one in addition to the fifteen carbon signals that can be assigned to compound VII. Anomeric carbons. Sugar's C ₃ proton, which appears in the low field (δ: 5.39 ppm, octet), reflects that the sugar's C-OH is esterified by the carboxyl group of char. The results are summarized below.

The molecular weight (457) of compound I corresponds to approximately 1/3 of the total molecular weight of BBM-1675 A ₁ (inference equation C ₅₇ H ₇₂ N ₄ O ₃₂ ; calculated MW = 1234). The substructure of BBM-1675 A ₁ is inferred as follows.

After the US filing date of parent application Ser. No. 495,231, the definitions of these components and the fact that the components BBM-1675 A ₁ and A ₂ described above and produced according to Example 2 below are not actually completely pure. It has been found that the specific characteristics used are inaccurate. As described in more detail in Examples 3 and 6 below, according to further chromatographic purification techniques, BBM-1675 A ₁ and A ₂ are identified in more purified form and sufficiently realistically as described below. It became. In addition, elemental analysis data for components A ₃ and A ₄ were modified to indicate the presence of sulfur in these compounds, and HPLC retention periods were calculated for these two components. Summarized below are the modified physicochemical properties of the BBM-1675 components.

No significant changes in reaction with acids or bases.

The second analysis showed the following optical rotations.

Infrared absorption spectrum: see drawing 9

Main Absorption Band (KBr): 985,1015,1070,1110,1150,1210,1250,1308,1380,1405,1446,1520,1592,1608,1668,1715,2920,2960,3360,3440cm ^-1

Molecular mass range with addition reaction of ions (m, z): 1249, 1357, 1463: NaCl (m / z): 1271, 1379, 1485, 1597.

FAB-MS-MB (MB: Metrics, m.w. 154); Mass range ion (m / z): 1249, 1283, 1403, 1555; With addition reaction of NaCl (m / z): 1249,1271,1303,1425,1483,1577.

Fab-MS-glycerol-DMSO: molecular mass range ions (m / z): 1215,1247,1279,1293,1325,1353; With addition reaction of NaCl (m / z): 1215,1237,1247,1269,1325,1347,1375.

Instrument: Kratos MS-50

FAB-MS-thioglycerol: molecular mass

Range ion (m / z): 1357,1463.

Molecular weight apparent MW = 1248

(Based on mass spectral data above)

Nuclear Magnetic Resonance Spectrum: Instrument-WM 360 Brooker

Solvent: CDCl ₃

See drawing 10

¹³ C NMR: 90.3 MHz

See drawing 11

¹³ C NMR spectra of BBM-1675 A ₁ purified in the separation test were determined in samples dissolved in CDCl ₃ (80 MHz). The main peak is shown below.

Multiplicity-q = quartet; d = doublet; u = indeterminate t = triplet; s = single line

Elemental Analysis: C; 52.71 (%), H; 5.94%, N; 3.94%, S: 9.39%, O (series): 28.01%

Ultraviolet absorption spectrum: Instrument-Berian UV, carry 219

Solvent: Methanol

Concentration: 0.02052 g / l

No significant change in reaction with acids or bases.

Optical rotation:

Infrared spectrum: see figure 12

Instrument: Beckman IR Model 4240

Main Absorption Band (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; With addition reaction of NaCl (m / z): 990,1271,1379,1485,1593.

FAB-MS-MB (MB: Metrics, m.w. 154); Molecular mass range ion (m / z): 1249,1403,1419,1555,1571,1587; With addition reaction of NaCl (m / z): 1249,1271,1425,1441,1457,1483,1577.

FAB-MS-glycerol-DMSO; Molecular mass range ions (m / z): 1215,1231,1247,1263,1279,1293,1309,1325,1326,1341,1353,1369.

Molecular weight: apparent MW = 1248

(Based on the mass spectrum data described above)

Nuclear Magnetic Resonance Spectrum: See Figure 13

Gauge: WM 360 Brooker

Solvent: CDCl ₃

See Drawing 14

In a separate test, ¹³ C NMR spectra of purified BBM-1675 A ₂ were measured on samples dissolved in CDCl ₃ (80 MHz). The main peaks are:

TABLE 7

Data from thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) for BBM-1675 components

A. Study No. 1-Summary

TABLE 8

B. TLC and HPLC TLC Chromatography for Reserted No. 2-Refined A ₁ and A ₂ Components

Analtech GHLF silica gel uniplate was used in all normal phase chromatography. Plates measured 2.5 cm × 10 cm were used in one-dimensional TLC. They were 6.4 cm (o.d) x 12 cm and developed in a glass cylinder containing 10 ml eluent. Plates of 7.5 cm x 10 cm were used in two-dimensional TLC. Samples were applied to the lower left corner 1 cm away from the edge. The plate was initially developed in a tank containing 50 ml of the first eluent (12.7 cm wide, 8.6 cm deep and 13 cm high), then air dried, rotated 90 ° in the direction of the needle, and then the second tank containing the second 50 ml. Deployed within.

Whatman analysis Precoated C-18 silica gel plates were used in all reverse phase chromatography. A 2.5 cm by 7.6 cm size plate was developed in a glass cylinder containing 10 ml of eluent.

The normal phase plate was first irradiated with 254 nm UV and then inserted into a glass cylinder (I. 6.4 cm od per 12 cm) containing I ₂ crystals. The plate was then irradiated again after approximately 2 minutes. Reversed-phase plates were visually observed only at 254 nmUV light. Zones were detected by finding sites where the fluorescence of the injected dye was highly concentrated.

Analytical HPLC

The analytical HPLC system was assembled using the following components. Water-associated Model 6000A Solvent Delivery System Pump, Varian Varichrome Model VUV-10uv / vis Detection Set at 254 nm 0.10D, Fishing Vessel Record Serial Number 5000 Recorder, Water-Associated Model 660 Solvent Programmer , Al-Tech, μ-Bondac C ₁₈ (10μ) column (4.6nm id ×) with water-associated model U6K injector, Whatmansa Pell ODS (0.03-0.038mm) stabilizer column (4.6mm id × 5cm) These components are combined with 316 stainless steel tubing (1.6 mm od, -0.23 nm id). Eluent was pushed out at 2 ml / min for all assays.

Preparative HPLC

The medium pressure liquid chromatography system was assembled using the following components. Fluid Metering's Model RP-SY 2CSC FMI Lab Lamp, Fluid Metering's Model PD-60LF FMI Driven Brake, Polypropylene Tubing (3.0mm od × 1.5mm id) Assembled with 15ml Sample Ring Surrounding Cardboard Tube (8.65cm od) ), GlencoSeries 3500 Universal LC Column, Instrument Specialist's Model UA-5 Absorbance / Type 6 Optical Unit Fluorescence Detector, Instrument Specialist's Model 590 Flow Isolator Valve, and Instrument Specialist's Model 328 Fractional Collector. These components were valve-coupled in the order listed above with polypropylene and Teflon tubing (3.0 mm o.d. x 1.5 mm i.d.) and clenco multifit connectors.

The Glenco Series 3500 Universal LC column was slurry packed with the absorbent in the indicated solvent using standard techniques. The gap between the fixed bed and the tube cover was filled with standard typo and sand. Eluent was introduced at a maximum rate not exceeding 60 psi back pressure (approximately 20 ml / min).

Concentration gradient elution

A Glenco concentration gradient eluent consisting of two combustion chambers of equal diameter, height and volume connected in series with a Teflon valve was used in all concentration gradient elution processes. One combustion chamber is used as a mixed combustion chamber and the other is used as a static reservoir. Less polar solvents were initially observed in the mixed combustion chamber. More polar solvents were observed in the static reservoir. Teflon-coated magnetic stirs (1.0 × 3.7 cm) were placed in both combustion chambers and run using a Thomas model 15 magnet-matic stirrer. Eluent was introduced into the medium pressure system from the mixed combustion chamber through polypropylene tubing (1.5 mm ID x 3.0 mm OD). A linear gradient of the eluent was created by allowing the eluent to be freed from the mixed combustion chamber and freely substituted as a solvent in the static reservoir.

TLC analysis of BBM-1675A ₁ and A ₂

Summarized in Table 9 below are the Rf values observed for BBM-1675A ₁ and A ₂ on normal phase plates. Rf is calculated by dividing the measured distance from the sample application point to the center point of the sample development location by the measured distance of the solvent surface from the sample application point.

TABLE 9

Summarized in Table 10 below are the observed Rf values of BBM-1675A ₁ and A ₂ in normal phase plates developed in two dimensions. The location of these points 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 10

Summarized in Table 11 below is the observed Rf values of BBM-1675A ₁ and A ₂ in C-18 reversed phase TLC plates developed with binary eluent.

TABLE 11

Summarized in Table 12 below are the observed Rf values of BBM-1675A ₁ and A ₂ in C-18 reversed phase TLC plates developed with ternary eluent.

TABLE 12

HPLC Analysis of BBM-1675A ₁ and A ₂

BBM-1675A ₁ and BBM-1675A ₂ were analyzed on a C-18 reversed phase silica gel column using one-, two- and three-way eluents. Summarized in Tables 13, 14 and 15 below are the K 'values observed for these compounds. This K 'was calculated using the following formula.

TR is the holding period measured from the injection time to the peak, and TO is the void volume time.

TABLE 13

TABLE 14

TABLE 15

Biological Properties of BBM-1675 Components

The antimicrobial activity of the BBM-1675 components was measured using a series of 2-fold agar dilutions against various bacteria (gram positive, gram negative and acid resistant) and fungi. No. 1001 medium (3% glycerol, 0.3% sodium L-glutamate 0.2% peptone, 0.31% Na ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.005%) for gram-positive and gram-negative bacteria and acid-resistant organisms as a nutrient agar medium Ammonium citrate, 0.001% MgSO ₄ and 1.5% agar). Saburo agar medium was used for fungi. As shown in Table 16, each of the six BBM-1675 components (A ₁ , A ₂ , A ₃ , A ₄ , B ₁ , B ₂ ) showed a broad spectrum of antimicrobial activity. BBM-1675A ₁ , A ₂ , A ₃ and A ₄ showed a high level of activity against specialty Gram-positive bacteria.

TABLE 16

A second antibacterial test was carried out on purified A ₁ and A ₂ (as prepared in Example 3 below) and on components A ₃ and A ₄ . The data is summarized below.

BBM according to the method published by Line et al. In Nature 196: 783-784 (1962) regarding the propage-inducing activity in the Risogen bacterium E. coli W1709 (入). It was also measured for the -1675 component. Spot aggregation was performed on agar plates containing test substance (T) and control plate (C). Proportions indicating a spot aggregation of 3.0 or greater were considered important, and the Risogen Induced Activity (ILB activity) was expressed as the minimum induced concentration of the test compound. As shown in Table 17 below, since the BBM-1675 components exhibit strong ILB activity in Risogen, it is demonstrated that they can have anticancer activity.

TABLE 17

Anticancer activity of BBM-1675A ₁ and A ₂ was measured in various mouse tumor systems. Lymphocyte leukemia P-388, lymphatic leukemia L-1210, melanoma hematoma B 16 and Lewis lung cancer were inoculated in BDF ₁ male mice at inoculation levels of 10 ⁶ , 10 ⁵ , 5 × 10 ⁵ and 10 ⁶ cells per mouse, respectively. Transplanted into. Test compounds were administered intraperitoneally to mice 24 hours after tumor inoculation at graded doses. This treatment was carried out only once on the first day or once on the 1st, 4th and 7th days (q3d × 3), once every 9 days (qd1 × 9) or once every 11 days (qd1 → 11). Components A ₃ and A ₄ were only tested for P-388 leukemia by the q36 × 3 schedule due to the insufficient supply of material.

_{BBM-1675A 1, A 2,} A 3 and A ₄ 10% containing dimethyl sulfoxide was dissolved in 0.9% physiological saline solution, using a chromotherapy erythromycin A ₃ (Toyo last sour Takeda) as a control compound 0.9% It was dissolved in physiological saline. The mortality or survival of treated and untreated mice is recorded daily and the mean survival (MST) is calculated for each of the test (T) and control (C) groups. T / C values of 125% or higher indicate significant anticancer effects. The results are shown in Tables 18-23. BBM-1675A ₁ and A ₂ exhibited highly effective tumor therapeutic activity against p-388 leukemia with a maximum T / C value of 160%. They have approximately 100 to 3,000 times more activity than chromomycin A ₃ as the minimum effective dose. However, BBM-1675A ₃ and A ₄ are less active than component A ₁ or A ₂ against p-388 leukemia (Table 19). BBM-1675A ₁ and A ₂ also have activity against L-1210 leukemia (Table 21), B16 pigment cell tumor (Table 22) and Lewis lung cancer (Table 23). The toxicity of BBM-1675A ₁ and A ₂ was measured in ddy male mice by intraperitoneal or intravenous administration. As a result, BBM-1675A ₁ was approximately 10 times more toxic than BBM-1675A ₂ (Table 24). BBM-1675A ₁ and A ₂ is the therapeutic index of the p-388 leukemia based chromotherapy 4 to 8 times than the respective indices of erythromycin A ₃ and 8 to 20 times higher (Table 25). A second experiment was performed by intravenous administration of the BBM-1675 component in connection with p-388 and L-1210 leukemia, which were inoculated intravenously with 5 × 10 ⁵ and 10 ⁴ cells per mouse, respectively. In these experiments, adriamycin was used as a control reagent and the reagents were dissolved in 0.9% saline and administered on days 1,4 and 7. These 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 activity spectrum.

TABLE 18

Original figures indicate significant anticancer activity.

TABLE 19

Original figures indicate significant anticancer activity.

TABLE 20

Original figures indicate significant anticancer activity.

TABLE 21

Original figures indicate significant anticancer activity.

Table 22

Original figures indicate significant anticancer activity.

TABLE 23

Original figures indicate significant anticancer activity.

TABLE 24

TABLE 25

TABLE 26

TABLE 27

The anticancer activity of components BBM-1675 A ₁ and A ₂ was also measured by conducting a second test in connection with p-338 leukemia, L-1210 leukemia and B16 pigment cell tumor in mice. The results of these tests are shown in Tables 28, 29 and 30 below. Details of the methods used in these tests can be found in Cancer Chemother, Rep. 3: 1-87 (part 3), 1972.

TABLE 28

TABLE 29

TABLE 30

After further purification of BBM-1675 A ₁ according to Example 6, the purified compound samples were tested for mouse L-1210 leukemia, p-388 leukemia and B16 pigmentoma. The results of these tests are shown below.

Table 31

Table 32

Table 33

The data selected above indicates that the purified BBM-1675 A ₁ component has almost the same anticancer properties as the less purified samples that were conventionally selected. This compound has exceptionally high activity against mouse p-388 leukemia as it has been shown to be active at a dose of 25 nanograms / kg in a schedule of five times a day. In the trials for p-388 and L-1210 leukemia, BBM-1675A ₁ is effective for injection once a day, three injections every four days, or five injections once daily. For B16 pigmentoma, the compound had the same effect when administered intravenously to animals with subcutaneous tumors as well as when administered intravenously to animals with intraperitoneal tumor grafts. In other words, the successful pharmacological derivation of the drug for tumors in remote areas is unique among anticancer antibiotics.

As described above, since the BBM-1675 component has a strong antimicrobial activity, it is useful for treating mammals and other animals from infectious diseases caused by microorganisms. In addition, these components can be used for antibacterial agents such as disinfectants and dental parts and other common applications.

From the induction of propages in lysogen bacteria and the activity exhibited against the mouse tumor system, it can be seen that the BBM-1675 component is also therapeutically useful in inhibiting the growth of mammalian tumors.

Accordingly, the present invention provides a method for treating an animal host infected with a microorganism or a malignant tumor, the method comprising: BBM-1675 A ₁ , A ₂ , A ₃ , A ₄ , B ₁ or B ₂ or These pharmaceutical compositions are characterized by administration at effective antimicrobial or tumor-suppressive doses.

In a further aspect, the present invention provides a pharmaceutical composition comprising a tumor suppressor effective amount of BBM-1675 A ₁ , A ₂ , A ₃ , A ₄ , B ₁ or B ₂ together with a pharmaceutically acceptable inert carrier or diluent. . These compositions can be formulated in any pharmaceutical form suitable for parenteral administration.

Formulations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. It may also be prepared in the form of a sterile solid composition, which may be dissolved immediately before use in sterile water, saline or other sterile injectable media.

The amount of BBM-1675 antibiotic that is actually preferred depends on the particular ingredient, the particular composition formulated, the mode of application, and the particular site desired, the host, and the disease to be treated. Many factors that alter the action of a drug, such as age, weight, sex, diet, duration of administration, route of administration, excreta, host's condition, drug combination, response sensitivity and disease severity are those who are skilled in the art. Will be taken into account. Dosing can be continued or periodically within the maximum tolerated dosage range. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using routine dosage determination tests in accordance with the above guidelines.

The following examples are merely intended to illustrate the invention and are not intended to suggest the scope of the invention.

Example 1

Fermentation of BBM-1675

Actinomadura strain No. H 964-92 is grown and maintained in agar slope medium containing 1% malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% CaCO ₃ and 1.6% agar. 3% soluble starch, 3% dry yeast, 0.3% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ , 7H ₂ O, 0.2% NaCl and 0.1% CaCO using well grown agar slope culture Inoculate the vegetable medium containing ₃ and adjust the pH to 7.0 before sterilization. After incubating this vegetable medium at 32 ° C. for 72 hours on a rotary shaker (250 rpm), 5 ml of growth was added to 3% sugarcane molasses, 1% corn, starch, 1% fish food, 0.1% CaCO ₃ and 0.005% and Transfer to a 500 ml Erlenmeyer flask containing 100 ml fermentation medium consisting of CuSO ₄ H ₂ O (pre-sterilization pH 7.0). Fermentation is carried out at 28 ° C. for 6 days on a rotary shaker. Antibiotic activity in fermented broth medium is measured by agar diffusion method using Staphylococcus aureus 209P as the test organism. Antibiotic efficacy reaches a maximum of approximately 1 mcg / ml after 5 days of fermentation.

Fermentation of BBM-1675 is also carried out in a stirred bottle fermenter. 500 ml of the inoculated growth obtained is transferred to a 20 l bottle fermentor containing 10 l of fermentation medium consisting of the same components used in the shake flask presentation. Fermentation is carried out at 32 ° C. under aeration rate of 12 l / min and stirring at 250 rpm. Under these conditions, antibiotic production reaches a maximum of approximately 0.9 mcg / ml after 68-76 hours of fermentation.

Fermentation studies are also conducted in fermentation tanks. Inoculated cultures were prepared with a vegetable medium consisting of 3% soluble starch, 3% dry yeast, 0.3% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ .7H ₂ O, 0.2% NaCl and 0.1% CaCO ₃ . Shake for 4 days at 30 ° C. in the Erlenmeyer flask containing. The inoculation culture was inoculated into a 200 l inoculation tank containing 130 l inoculation medium having the composition as described above, and the inoculation tank was stirred at 30 ° C. at 240 rpm for 31 hours. A second inoculation culture is used to inoculate 3,000 l fermentation medium containing 1% corn starch, 3% sugarcane molasses, 1% fish food, 0.005% CuSO ₄ .5H ₂ O and 0.1% CaCO ₃ . The production tank is operated at 28 ° C. at 164 rpm with an aeration rate of 2,000 l / min. The pH of the broth medium gradually increased with fermentation, reaching 7.7-7.8 after 170-180 hours when peak antibiotic activity of 1.7 mcg / ml appeared.

Example 2

Isolation and Purification of BBM-1675 Components

The obtained fermented broth (3,000 L, pH 7.8) is separated into a mycelium cake and a supernatant using a Shapless centrifuge. The mycelium cape is suspended in 1,600 l of methanol and the mixture is stirred for 1 hour. Undissolved material is filtered off and the methanolic extract is concentrated to 43 l in vacuo. The active material contained in the juicy supernatant is recovered by extracting twice with n-butanol of 1,000 l each. The combined n-butanol extract and concentrated methane extract, followed by azeotropic evaporation of water with occasional addition of water yields an aqueous solution (20l) in which most of the antibiotic activator is precipitated as an oily solid.

The solid material is immersed in 30 l of methane and then the insoluble material is filtered off. The methanol extract is then concentrated in vacuo to give 10 l of solution, to which 40 l of ethyl acetate and 30 l of water are added. After stirring for 30 minutes, the organic layer was separated, dried over sodium sulfate, and evaporated to 4 l in vacuo. This concentrate was added to 20 l of n-hexane to give crude yellow BBM-1675 complex (90.14 g, activity: 55 mcg / mg) as a slightly yellow solid. The complex appears in TLC as a mixture of two important components, BBM-1675 A ₁ and A _2, and a few less important components. After separating these, the chromatography is repeated and purified, which is performed in a cooling chamber to prevent deterioration.

BBM-1675 complex (20 g) is dissolved in methanol (20 ml) and charged into a Sephadex LH-20 column (ψ5.5 × 85 cm). The column is developed with methanol and elution is monitored by biological potency assay with Staphylococcus aureus 209p. The active eluates are combined and concentrated in vacuo and then lyophilized to yield BBM-1675 complex (4.19 g), a semi-purified solid material. The solid is then chromatographed on a silica gel column (5.0 × 50 cm) using chloroform and increasing amount (1 → 5% v / v) methanol as eluent.

The eluate is combined based on antimicrobial activity (for S. aureus) and TLC (SiO ₂ ; CHCl ₃ —CH ₃ OH = 5: 1 v / v) and then concentrated in vacuo. The nearly homogeneous BBM-1675 A ₁ (output after evaporation: 351 mg) was first eluted with 2% methanol in chloroform and then eluted with a mixture (507 mg) of BBM-1675 A ₂ , A ₃ and A ₄ . BBM-1675 B mixture (210 mg) is eluted with 3% methanol in chloroform. The solid BBM-1675 A ₁ was applied to a Sephadex LH-20 (ø2.0 x 80 cm) column developed with methanol. The active fractions are concentrated and dried in vacuo. Crystallization of the residual solid material with methanol gives a colorless plate (124 mg) of pure BBM-1675 A ₁ (this material is the starting material of Example 3A). Complexes BBM-1675 A ₂ , A ₃ and A ₄ are separated by chromatography on a column of Vondapak C ₁₈ (water, ψ3.0 × 50 cm). After eluting with aqueous acetonitrile, the eluate with biological activity is tested by TLC (Merck, * Silanated: CH ₃ CN-H ₂ 0 = 75:25 v / v). Less important components A ₄ (33 mg) and A ₃ (18 mg) were sequentially eluted sequentially with 20% acetonitrile, followed by 50% of the other major component A ₂ (301 mg), which is the starting material of Example 3B. Eluted with acetonitrile.

The solid material containing BBM-1675 B ₁ and B ₂ is chromatographed on a silica gel column (ψ 3.0 × 40 cm) using developing solvents chloroform and methanol. The active fractions eluted with 4% methanol in chloroform are combined and then evaporated to give pure BBM-1675 B ₁ (7 mg). Another active fraction is eluted with 5% methane at a concentration and then evaporated to give BBM-1675 B ₂ (8m9).

^* C ₁₈ reversed phase silica gel

Example 3

Further purification of BBM-1675 A ₁ and A ₂

A. Purification of BBM-1675 A ₁

2.67 cm id × 75 cm glencocolums are slurry packed with Barker binding phase octadecyl (C18) silica gel (40 micron particle size) in methanol. The column is connected to a medium pressure HPLC system and then equilibrated with 1.5 l of eluent (41.6% acetonitrile-21.6% methanol-36.8% 0.1M ammonium acetate). Partially purified BBM-1675 A ₁ (100.5 mg) obtained by the purification process of Example 2 is dissolved in 2 ml of acetonitrile and then absorbed into the sample loop. Inject this sample into the column. This column is eluted with the eluent to collect 87 ml of oil. The eluent is monitored at 254 nm and 340 nm. Collect 55-71 fractions and extract twice with 1500 ml of chloroform. Evaporation and drying of this chloroform gave 89.8 mg of residue C.

The 1.5 cm i.d. × 20 cm glencocolums are slurry packed with 12 g of beryl silica gel (60-200 micron particles). Residue C is applied to the column in chloroform solution. The column is eluted with a 500 ml linear gradient of chloroform to 10% methanol in chloroform to collect 20-25 ml of oil. After analysis by TLC on silica gel, fractions 6-9 were combined, evaporated and dried to give 73 mg of residue D.

A 1.5 cm id × 20 cm Glenco column is slurry packed with 12 g of film silica gel (63-200 micron particles) in Skelesolve B. Residue D is dissolved in approximately 2 ml of CHCl ₃ and then applied to this column. Subsequently, chloroform is replaced with 25 ml of Skelsolve B, and then the column is eluted with a 500 ml linear gradient of 60% acetone in Skelesolve B to Skelesolve B to collect 28-25 ml of oil. Collecting fractions 19-23, evaporating and drying yields 65.6 mg pure BBM-1675 A ₁ .

This residue was prepared using three TLC systems (5% methanol in chloroform on silica gel, 5% methanol in ether and 50% acetone in Skelesolve B) and HPLC (C-18 silica gel-41.5% acetonitrile: 21.5% methanol: 37.0 % 0.1 M ammonium acetate).

Gel Permeation Chromatography with Purified BBM = 1675 A ₁

A 2.5 cm id x 45 cm Pharmacia column was slurry packed with Sephadex LH-20 in methanol and fitted to a 33.4 cm chromatographic bed. Purified BBM-1675 A ₁ (approximately 120 mg) is dissolved in 2 ml of methanol and then transferred to a 2.5 ml sample reservoir. This sample is applied to the column, and then eluting at 1.75 ml / min with methanol to collect 10 ml of oil (Pharmaciafrac-100 oil collector). The eluent is monitored at 254 nm with an Isco UA-5 detector. It is observed that BBM-1675 A ₁ elutes at Ve / Vt between 0.79 and 0.91 (Ve = elute volume, Vt = bed volume).

B. Purification of BBM-1675 A ₂

2.65 cm id × 75 cm glencolums are slurry packed with Barker binding phase octadecyl (C18) silica gel (40 micron particle size) in methanol. The column is coupled to a medium pressure HPLC system and then equilibrated with 1.5 l of eluent (50% acetonitrile-20% methanol-30% 0.1M ammonium acetate). Partially purified BBM-1675 A ₂ (76.9 mg) obtained by the procedure of Example 2 is dissolved in 2 ml of acetonitrile and then absorbed into the sample loop. Inject this sample into the column. The column is eluted with the eluent to collect 87 ml fractions. Eluent is monitored at 254 and 340 nm. Collect 31 to 38 fractions and extract twice with 500 ml of chloroform. Evaporation and drying of chloroform gave 65.8 mg of uniform BBM-1675 A ₂ .

BBM-1675 A ₂ is homogeneous in two TLC systems, one 2-d TLC analysis and HPLC.

Example 4

Preferred Extraction Process for BBM-1675 A ₁

The raw fermentation product (6.8 l) obtained according to the process of Example 1 was transferred to a polypropylene bagus (12 cm d, top: 10 cm d, bottom: 37 cm high) with a tap at the bottom, and the same volume of chloroform Add. The mixture is stirred for 2 hours at a significant rate using a CRC-airpulsed stirrer. Approximately 4 l (1.3 kg) of decalit (filter aid) is added and allowed to mix in it. This mixture is filtered over a decalite pad maintained in a No. 12 Buchner funnel. The filtrate is collected in a 19 l solution bottle equipped with a vacuum seal (Ace. No. 5396-06). The mat is washed with 2 l of chloroform. The filtrate is transferred to a 20 L separatory funnel and the phases allowed to separate. Remove the lower phase (chloroform).

2.5 i.d. × 40 cm Glenco tubes are slurry packed with 91 g of film silica gel (63-200 micron particle size). The chloroform phase is injected into the column using a FMI-2CSD pump. The column is washed with 600 ml of fresh chloroform. After removing the chloroform eluent, the column is eluted with 600 ml of 10% methanol in chloroform. Evaporation and drying of this eluent gave 547 mg of residue A.

Residue A is dissolved in 50 ml of chloroform. The chloroform solution is added to 20 g of decalite contained in a 1 l round bottom flask. Slurry is produced by adding approximately 200 ml of Skelesolve B. The solvent is transferred to a rotary evaporator. The residue is slurried in 300 ml Skelesolve B. This slurry is packed into an Ace flask chromatography tube (part number B5872-14) (41 mm id × 45.7 cm) according to the following procedure. Insert the glass wool plug into the spout of the Stop Cock between the stopper and the column tube. 1 cm layer of standard Ottawa sand is added onto the glass wool. The throttle, glass wool and sand bed are washed with air by passing a pressurized (5.7 psi) stream of Skeleton Solver B through them. The slurry is then added to the column and left to form a packed bed under pressurized flow. The column is not left to dry. After a stable column bed is obtained, 2 cm layer of Ottawa Sand is added to the top of the bed. Then elute the bed using an additional 600-70 ml of Skeleton Solve B. The bed is eluted with 500 ml of toluene. Evaporation and drying of the toluene eluate gave 93 mg of residue B. Then purify the partially purified BBM-1675 A ₁ again according to the procedure of Example 3.

Example 5

Fermentation of BBM-1675 Complex with Mutant H964-92-A 1327 Y

Containing 2% Soluble Starch, 1% Glucose, 0.5% Yeast Extract 0.5% NB-Amine Type A and 0.1% CaCO ₃ Using Mutant Strain A1327Y Obtained by NTG Treatment of Actinomadurra Berukosospora Strain H964-92 Inoculate the vegetable medium and adjust the pH before sterilization to 7.0. The vegetable medium was incubated on a rotary shaker (250 rpm) for 4 days at 32 ° C., then 5 ml of the grown product was 3% sugarcane molasses, 1% corn starch, 1% fish food, 0.005% CuSO ₄ · 5H ₂ O, 0.05 Transfer to a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium consisting of% MgSO ₄ 7H ₂ O and 0.1% CaCO ₃ and adjusted to pH 7.0 before sterilization.

Fermentation is carried out for 7 days at 28 ° C. on a rotary shaker. Antibiotic production reaches a maximum of approximately 1.5 mcg / ml.

Example 6

Isolation and Purification of BBM-1675 Components

The fermented broth (3,000 L, pH7.6) collected from Example 5 is separated into mycelium cake and supernatant using a Shapless centrifuge. The mycelium cake is stirred with 2,000 l of methanol for 1 hour and then the insoluble material is filtered off. The active material contained in the juicy supernatant is extracted with 1,800 l of n-butanol. When methanol and n-butanol extracts are combined, and sometimes azeotropically concentrated while adding water to the aqueous solution (20 l), most of the antibiotics are precipitated as an oily solid. The mixture is shaken three times with 20 l each of ethyl acetate to extract the actives. The extracts are combined, filtered to remove insoluble matters and then evaporated in vacuo to 4 l. This concentrate was added under stirring in 30 l of n-hexane to give a crude yellow BBM-1675 complex (81: 7 g, activity: 59 mcg / mg) as a pale yellow solid. This complex appears by TLC and HPLC to be a mixture of two major components BBM-1675 A ₁ and A ₂ with some less important components. These are separated and purified by a series of chromatography, which is carried out in a cooling chamber to prevent deterioration.

The crude BBM-1675 complex (20 g) is dissolved in methanol (20 ml) and then charged into a Sephadex LH-20 column (ø5.5 x 85 cm). The column is developed with methanol and the elution is monitored by biological efficacy test with Staphylococcus aureus 209p. The active eluate is collected, concentrated in vacuo and lyophilized to yield BBM-1675 complex (4.86 g, activity: 203 mcg / mg), which is a semi-pure solid. The solid material is then chromatographed on a silica gel column (ψ3.0 × 70 cm) using chloroform and increasing amount (1-5%) of methanol as the developing solvent. The eluate is collected based on antimicrobial activity against Staphylococcus aureus and TLC (SiO ₂ , CHCl ₃ -MeOH = 5: 1, v / v) and concentrated in vacuo. BBM-1675 A ₁ (425 mg after evaporation, Activity: 960 mcg / mg) was eluted first with 2% ethanol in chloroform and then a mixture of BBM-1675 A ₁ , A ₃ and A ₄ (732 mg, Activity: 340 mcg / mg ) Is eluted, and then the BBM-1675 B complex (200 mg, activity: 190 mcg / mg) is eluted with 3% methanol in chloroform. The BBM-1675 A ₁ is chromatographed again on silica gel (column: 2.2 × 44 cm) with 2% methanol in benzene. The eluate with biological activity was tested by HPLC (Richrosov RP-18: CH ₃ CN-MeOH-0.1MCH ₃ COONH ₄ = 5: 2: 3, v / v), and then homogeneous BBM-1675 A ₁ was prepared. The oil contained is evaporated and dried in vacuo. Crystallization of the current solid with methanol (10 ml) yielded BBM-1675 A ₁ (197 mg, activity: 1,000 mcg / mg) on a colorless prism.

The complex of BB3M-1675 A ₂ , A ₃ and A ₄ (537 mg) was isolated by column chromatography on Bonda Park C ₁₈ (water, ψ2.0 × 42 cm). Elution with aqueous acetonitrile was followed by elution with biological activity by TLC (Merck, Silaneized: CH ₃ CN-H ₂ O = 75: 25, v / v). Less important components BBM-1675 A ₄ (45 mg, activity: 410 mcg / mg) and A ₃ (19 mg, activity: 300 mcg / mg) were eluted successively with 20% acetonitrile, followed by main component BBM-1675 A ₂ ( 203 mg) was eluted with 50% acetonitrile. Crystallization of the BBM-1675 A ₂ fraction with chloroform-n-hexane precipitates a colorless rod-like substance (70 mg, active 290 mcg / mg). The solid material containing the BBM-1675B mixture is chromatographed on a silica gel column (ψ3.0 × 40 cm) with chloroform and methanol as developing solvents. Collecting and evaporating the active fraction eluted with 4% methanol in chloroform gave pure BBM-1675 B ₁ (7 mg, activity: 180 mcg / mg). Another active fraction was eluted at 5% methanol concentration and then doubled to give BBM-1675 B ₂ (8 mg, activity: 140 mcg / mg).

Claims (12)

Actinomadurra verrucosusfra strains producing BBM-1675 A ₁ were subjected to aerobic immersion under a pH of about 7.0 to 7.8 and a temperature of about 28 to 32 ° C. in an aqueous nutrient medium containing carbon and nitrogen assimilation. After incubation for about 2 weeks, the obtained BBM-1675 complex was eluted with 2% methanol in chloroform on a silica gel column and chromatographed to give BBM-1675 A ₁ , which was prepared using methanol on a Sephadex LH-20 column. A method for preparing an anticancer antibiotic BBM-1675 A ₁ having the following characteristics, characterized in that it is column chromatographed, the obtained active fraction is concentrated to dryness in vacuo, and the residue is crystallized with methanol. To pale yellow crystals; (b) soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, poorly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (c) positive reactions with ferric chloride, erlich and tolene reagents, and negative in Sakaguchi, nin hydrin and anthrone tests; (d) shows infrared absorption spectrum KBr as shown in FIG. 9; (e) when dissolved in CDCl ₃ , shows a proton magnetic resonance spectrum as shown in FIG. 10; (f) has a melting point in the range of about 156-158 ° C .; (g) optical rotation

Has; (h) As a result of mass spectrometry, the polymer had an apparent molecular weight of 1248. (i) 52.17% carbon, 6.15% hydrogen, 4.63% nitrogen. It has an elemental composition close to 9.09% of sulfur and 27.96% of oxygen (based on the series), and (j) shows a Rf value of 0.74 for the solvent system CHCl ₃ -CH ₃ OH (5: 1 v / v) in silica gel thin film chromatography. Reversed-phase silica gel thin layer chromatography showed Rf of 0.18 for the solvent system CH ₃ CN-H ₂ O (75:25 v / v), and (k) UV absorption when dissolved in methanol at a concentration of 0.013569 / l. Maximal value and absorption rate;

(l) retention time 13.3 min in high-performance liquid chromatography using C ₁₈ reversed-phase silica gel column and solvent system CH ₃ CN-CH ₃ OH-0.1M CH ₃ COONH ₄ (5: 2: 3: v / v); (m) is effective in inhibiting the growth of various bacteria and fungi; (n) has propagation induction in the ribogen bacteria; (o) Effective at inhibiting p-388 leukemia, L-1210 leukemia, B16 pigment cell tumor and Lewis lung cancer in mice. The microorganism producing the bacterium BBM-1675 A 1 according to claim 1, wherein the microorganism producing BBM-1675 A ₁ is actinomadura berukosospora strain H964-92 [ATCC 39334 (Korean Deposit No. KFCC-10089, Depositary Organization; 5. 24)] or actinomadura berukosphora strain A 1327 Y [ATCC 39638 (Korean Deposit No. KFCC-10090, Depositary Organization: Korean spawn association, date of deposit 1984. 5. 24)]. Actinomadurra Berukosospora strains producing BBM-1675 A ₂ were subjected to aerobic immersion under a pH of about 7.0 to 7.8 and a temperature of about 28 to 32 ° C. in an aqueous nutrient medium containing carbon and nitrogen assimilation. After incubation for about 2 weeks, the obtained BBM-1675 complex was chromatographed on silica gel column with 2% methanol in chloroform to elute BBM-1675 A ₁ and then BBM-1675 A ₂ , A ₃ and A ₄ were purified. Elution and column chromatography on Park C ₁₈ using 50% aqueous acetonitrile to give a BBM-1675 A ₂ fraction, which is crystallized with chloroform-n-hexane, characterized by A method for preparing the anticancer antibiotic BBM-1675 A ₂ : (a) has the appearance of white crystals; (b) soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, poorly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (c) positive reactions with ferric chloride, erlich and tolene reagents, and negative in Sakaguchi, nin hydrin and anthrone tests; (d) shows infrared absorption spectrum KBr as shown in FIG. 12; (e) when dissolved in CDCl ₃ , shows a proton magnetic resonance spectrum as shown in FIG. 13; (f) has a melting point in the range of about 147-149 ° C .; (g) optical rotation

-179.4 ° (c0.5, CHCl ₃ ); (h) measured by mass spectrometry, has an apparent molecular weight of 1248 and (i) an elemental composition close to 52.17% carbon, 5.94% hydrogen, 3.94% nitrogen, 9.39% sulfur, and 28.01% oxygen (by gradient) (j) Rf value of 0.71 for solvent system CHCl ₃ -CH ₃ OH (5: 1 v / v) in silica gel thin layer chromatography, solvent system CH ₃ CN-H ₃ O (75) in reversed phase silica gel thin layer chromatography. : 25 v / v) exhibits an Rf value of 0.21 and (k) an ultraviolet absorption maximum value and an absorption rate when dissolved in methanol at a concentration of 0.02052 g / l;

(l) retention time of 17.3 minutes in high performance liquid chromatography using a C ₁₈ reversed-phase silica gel column and a solvent system CH ₃ CN-CH ₃ OH-0.1M CH ₃ COONH ₄ (5: 2: 3: v / v); (m) is effective in inhibiting the growth of various bacteria and fungi; (n) has propagation induction in the ribogen bacteria; (o) It is effective in inhibiting the proliferation of p-388 leukemia, L-1210 leukemia, B16 pigment cell tumor and Lewis lung cancer in mice. 4. The microorganism producing the bacterium BBM-1675 A ₂ according to claim 3, wherein the microorganism producing actinomadura berukosospora strain H964-92 [ATCC 39334 (Korean Deposit No. KFCC-10089, Depositary Organization; Korean spawn association, Date of Deposit: 1984). 5. 24)] or actinomadura beruscophora strain A 1327 Y [ATCC 39638 (Korean Deposit No. KFCC-10090, Depositary Organization; Korean spawn association, Date of Deposit: May 24, 1984]). Actinomadurra verurosospora strains producing BBM-1675 A ₃ were subjected to aerobic immersion under a pH of about 7.0 to 7.8 and a temperature of about 28 to 32 ° C. in an aqueous nutrient medium containing carbon and nitrogen assimilation. After incubation for about 2 weeks, the obtained BBM-1675 complex was chromatographed on silica gel column with 2% methanol in chloroform to elute BBM-1675 A ₁ and then BBM-1675 A ₂ , A ₃ and A ₄ were purified. Elution and column chromatography on Park C ₁₈ using 20% aqueous acetonitrile to obtain a BBM-1675 A ₃ fraction yielding an anticancer antibiotic BBM-1675 A ₃ having the following characteristics: Method: (a) chloroform, ethyl acetate, acetone, ethane and soluble for methanol and methanol, poorly soluble for benzene and water and insoluble for n-hexane and carbon tetrachloride; (b) positive reactions with ferric chloride, erlich and tolene reagents, and negative in sakaguchi, nin hydrin and anthrone tests; (c) shows infrared absorption spectrum KBr as shown in FIG. 3; (d) when dissolved in CDCl ₃ , shows a proton magnetic resonance spectrum as shown in FIG. 7; (e) has a melting point in the range of about 125-127 ° C .; (f)

Has optical rotation; (g) having an elemental composition approximating 54.55% carbon, 6.46% hydrogen, 3.73% nitrogen, 7.49% sulfur and 27.77% oxygen (by series), and (h) solvent system CHCl ₃ -CH ₃ OH ( 5: 1 v / v) with an Rf value of 0.72 and reverse phase silica gel thin layer chromatography with an Rf value of 0.28 with respect to the solvent system CH ₃ CN-H ₂ O (75:25 v / v) (i) Methanol When dissolved in 0.01NHCl-CH ₃ OH and 0.01N NaOH-CH ₃ OH, the following ultraviolet absorption maximum values and absorption rates are exhibited; In methanol

(j) retention time 8.0 min in high performance liquid chromatography using a C ₁₈ reversed-phase silica gel column and a solvent system CH ₃ CN-CH ₃ OH-0.1M CH ₃ COONH ₄ (5: 2: 3: v / v); (k) is effective in inhibiting the growth of pseudobacteria and fungi; (1) has a propagation induction in the ribogen bacteria; (m) It is effective in inhibiting the proliferation of p-388 leukemia in mice. The microorganism producing the bacterium BBM-1675 A ₃ according to claim 5, wherein the microorganism producing actinomadura Berukosospora strain H964-92 [ATCC 39334 (Korean Deposit No. KFCC-10089, Depositary Organization; Korean spawn association, Date of Deposit: 1984). 5. 24)] or actinomadura beruscophora strain A 1327 Y [ATCC 39638 (Korean Deposit No. KFCC-10090, Depositary Organization; Korean spawn association, Date of Deposit: May 24, 1984]). Actinomadurra Berukosospora strains producing BBM-1675 A ₃ in aqueous nutrient medium containing carbon and nitrogen assimilation under aerobic immersion conditions at a pH of about 7.0 to 7.8 and a temperature of about 28 to 32 ℃ After incubating for about 2 weeks, the obtained BBM-1675 complex was eluted with 2% methanol in chloroform on a silica gel column to chromatograph the BBM-1675 A _1, and then BBM-1675 A ₂ , A ₃ and A ₄ were purified. Elution and column chromatography on 20 C aqueous acetonitrile on Park C ₁₈ yielded a BBM-1675 A ₄ fraction, to prepare an anticancer antibiotic BBM-1675 A ₄ having the following characteristics: Way; (a) chloroform, ethyl acetate, acetone, ethane and soluble in methanol, poorly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (b) positive reactions with ferric chloride, erlich and tolene reagents, and negative in sakaguchi, nin hydrin and anthrone tests; (c) shows an infrared absorption spectrometer KBr as shown in FIG. 4; (d) when dissolved in CDCl ₃ , shows a proton magnetic resonance spectrum as shown in FIG. 8; (e) has a melting point in the range of about 123-126 ° C .; (f)

Has optical rotation; (g) has an elemental composition approximating 54.65% carbon, 6.29% hydrogen, 3.51% nitrogen, 8.07% sulfur and 27.48% oxygen (by series), and (h) solvent system CHCl ₃ -CH ₃ OH ( 5: 1 v / v), Rf value 0.71, reverse phase silica gel thin layer chromatography, Rf value 0.78 for solvent system CH ₃ CN-H ₂ O (75:25 v / v), (i) methanol 0.01 When dissolved in N HCl-CH ₃ OH and 0.01 N NaOH-CH ₃ OH, the following ultraviolet absorption maximum is shown; In methanol

(j) retention time of 5.1 minutes in high performance liquid chromatography using a C ₁₈ reversed-phase silica gel column and a solvent system CH ₃ CN-CH ₃ OH-0.1M CH ₃ COONH ₄ (5: 2: 3: v / v); (k) is effective in inhibiting the growth of various bacteria and fungi; (l) has propagation induction in the ribogenateria; (m) It is effective in inhibiting the proliferation of p-388 leukemia in mice. The microorganism producing the bacterium BBM-1675 A ₄ according to claim 7, wherein the microorganism producing actinomydura berukosospora strain H964-92 [ATCC 39334 (Korean Deposit No. KFCC-10089, Depositary Organization; Korean spawn association, Date of Deposit: 1984). 5. 24)] or actinomadura beruscophora strain A 1327 Y [ATCC 39638 (Korean Deposit No. KFCC-10090, Depositary Organization; Korean spawn association, Date of Deposit: May 24, 1984]). The Akrinmadura berukosospora strain producing BBM-1675 B ₁ was subjected to aerobic immersion under a pH of about 7.0 to 7.8 and a temperature of about 28 to 32 ° C. in an aqueous nutrient medium containing carbon and nitrogen assimilation. After incubation for about 2 weeks, the obtained BBM-1675 complex was chromatographed with 3% methanol in chloroform on a silica gel column to give a mixture of BBM-1675 B ₁ and B ₂ , which was 4% in chloroform on a silica gel column. Chromatographing with methanol to obtain the BBM-1675 B ₁ fraction and evaporating it to produce an anticancer antibiotic BBM-1675 B ₂ having the following characteristics; (a) soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, poorly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (b) positive reactions with ferric chloride, erlich and tolene reagents, and negative in sakaguchi, nin hydrin and anthrone tests; (c) has a melting point in the range of about 159-161 ° C .; (d)

Has optical rotation; (e) Rf value of 0.63 for solvent system CHCl ₃ -CH ₃ OH (5: 1 v / v) in silica gel thin layer chromatography, and solvent system CH ₃ CN-H ₂ O (75:25) in reverse phase silica gel thin layer chromatography v / v) with an Rf value of 0.23 and (f) ultraviolet dissolution peaks when dissolved in methanol 0.01N HCl-CH ₃ OH and 0.01N NaOH-CH ₃ OH; In methanol

(g) is effective in inhibiting the growth of various bacteria and fungi; (h) It has propagation induction activity in Risogengeneria. The method of claim 9, wherein the microorganism producing BBM-1675 B ₁ is actinomadura Berukosospora strain H964-92 [ATCC 39334 (Korea Accession No. KFCC-10089, Depositary Organization; Korean spawn association, Date of Deposit: 1984). 5. 24)] or actinomadura beruscophora strain A 1327 Y [ATCC 39638 (Korean Deposit No. KFCC-10090, Depositary Organization; Korean spawn association, Date of Deposit: May 24, 1984]). Actinomadurra verrucosusfra strains producing BBM-1675 B ₂ were subjected to aerobic immersion under a pH of about 7.0 to 7.8 and a temperature of about 28 to 32 ° C. in an aqueous nutrient medium containing carbon and nitrogen assimilation. After incubating for about 2 weeks, the obtained BBM-1675 complex was chromatographed using 3% methanol in chloroform on a silica gel column to give a mixture of BBM-1675 B ₁ and B ₂ , which was 5% in chloroform on silica gel. Chromatographing with methanol to obtain the BBM-1675 B ₂ fraction and evaporating it to produce an anticancer antibiotic BBM-1675 B ₂ having the following characteristics; (a) soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, poorly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; (b) positive reactions with ferric chloride, erlich and tolene reagents, and negative in sakaguchi, nin hydrin and anthrone tests; (c) has a melting point within the range of about 156-159 ° C .; (d)

Has optical rotation; (e) Rf value of 0.60 for solvent system CHCl ₃ -CH ₃ OH (5: 1 v / v) in silica gel thin layer chromatography and solvent system CH ₃ CN-H ₂ O (75: 25 v / v), the Rf value is 0.16, and (f) the following ultraviolet absorption maximum and absorption are obtained when dissolved in methanol 0.01 N HCl-CH ₃ OH and 0.01 N NaOH-CH ₃ OH; In methanol

(l) effective in inhibiting the growth of various bacteria and fungi; (h) It has a propagation induction effect in the Risogenateria. The microorganism producing the bacterium BBM-1675 B ₂ according to claim 11, wherein the microorganism producing actinmadura berukosospora strain H964-92 [ATCC 39334 (Korean Deposit No. KFCC-10089, Depositary Organization; Korean spawn association, Date of Deposit: 1984). 5. 24)] or Actinomadura beruscophora strain A 1327 Y [ATCC 39638 (Korea Kit No. KFCC-10090, Depositary Organization; Korean spawn association, Date of Deposit: May 24, 1984. 5)].

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