JPS59232094A - Novel anti-tumor antibiotic substance complex bbm-1675 - Google Patents

Abstract

NEW MATERIAL:The antitumor antibiotic substance complex BBM-1675 composed of the main active components consisting of BBM-1675A1 and A2 and minor active components consisting of BBM-1675A3, A4, B1 and B2. The component 1675A1 has the following properties: Appearance, white or pale yellow crystal; apparent molecular weight (mass-spectrometry), 1,248; elemental compostion, C 52.17%, H 6.15%, S 9.09%, N 4.63% and O 27.96% (difference); melting point, 156-158 deg.C; specific rotation, [alpha]d<27>=-191 deg. (C 0.5, CHCl3); solubility, soluble in chloroform, acetone, methanol, etc., slightly soluble in water and benzene, and insoluble in n-hexane and CCl4. USE:An antibiotic substance having antitumor activity and antibacterial activity. PREPARATION:For example, the BBM-1675A1-producing strain of Actinomadura verrucosospora, e.g. H964-92 (ATCC 39334) is cultured in an aqueous nutrious medium by submerged technique under aeration, and the objective BBM-1675A1 is separated from the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel anti-tumor antibiotics and their production and harvesting.

The structure of the anti-inflammatory antibiotic of the present invention has not yet been identified. However, in view of their unique physical, chemical and biological properties, applicants believe that BBM-1675 antibiotics are novel substances.

European Patent Application No. 95154AI describes Actinomadura purperaceus CActinomadura
pulverace-ss) sp, Nov, 16604
WS6049 by fermentation of 9 (ATCC39100)
-A and the production of anti-inflammatory antibiotics designated WS6049-B are disclosed. Although the structure of WS6049 antibiotic is not yet clear, the characterizing properties given to the antibiotic indicate that WS6049-A
and WS6049-B have been shown to be structurally related to the BBM-1675 antibiotic of the present invention. However, the spectrum data is WS6049A and WS604
9B indicates that it is not identical to any of Applicant's BBM-1675 components. Furthermore, the producing bacteria described in European Patent Application No. 95154A1 showed the color of its aerial mycelia in ISP Baijitaka 2.3 and 4, positive milk peptonation and positive D-fructose, D-mannitol, In the positive use of trehalose oyohi cellulose, Actinomadeula verrucosus 5 (Ac t inomtx, duraveruc) used in the present invention.
OII08poτα).

According to the present invention, a novel anti-tumor antibiotic conjugate, herein referred to as BBM-1675, is provided. −
1675-Producer Strain, Most Preferred Actinomadeura Verrucospora Strain H964-'a2<ATCC39
334) or Actinomadeula verchuspora strain A1327Y (ATCC 39334-1675) produced by culturing until the complex is produced by the strain in the medium and optionally removing the complex from the medium. For example, two main bioactive components BEM-1675 complex (BEM 16
754 and A) and 4Ii of the BBM-1675 complex < BBM-1675A
3, AB, and B) are provided. These components can be separated and purified by conventional chromatography operations.

BBM-1675 complex and its bioactive components exhibit both antibacterial and antitumor activity.

The present invention relates to a novel anti-tumor antibiotic conjugate herein referred to as BBM-1675 and also relates to a novel anti-tumor antibiotic complex called BBM-1675 and also
Certain strains of Verchuspora, most particularly Actinomadeula Verrucospora strain H964-92 and its variant Actinomadeula Verrucospora strain A1321.
' Concerning its manufacture by fermentation. The parent strains mentioned above are Argentina, Misiones, Boudot C. Speranza (J't.

It was isolated from a soil sample collected at Espy de angα).

Biologically pure cultures of this bacterium are available in Washington, D.C.
The Aynerican Type Cultw
rtt Co11ection) and A.
It was added to the microbial permanent collection as TCC39334. Mutant strain A13271' was then transformed into strain H964.
-42 by conventional nitrosoguanidine (NTG) treatment and has been deposited in the American Type Culture Collection as ATCC 3963B25-.

As is the case with many antibiotic-producing cultures, fermentation of Actinomadeula verchuspora strain H964-42 or strain A1327Y produces hot metals or complexes of component materials. Two major bioactive components, BBM-
16754 and A2 and four trace bioactive components, B BM 1675 As, A4, B, and B,
was isolated from the BBM-1675 complex produced during the fermentation process.

BBM-1675 and its component EBM-1675A,
, A7, A3, A4, B1 and B exhibit antibacterial activity against a broad spectrum of microorganisms, including especially Gram-positive bacteria.

The BEM-1675 complex and its isolated bioactive components also exhibit phage-inducing properties in lysogenic bacteria. Two of the components, BBM 1675At and A, were subjected to in vivo screening against various mouse tumor systems and L-1210
Leukemia, B16 melanocytic cells and Lewis
Shows inhibitory activity against lung cancer. BBM 1675As
and A4 were found to exhibit activity against murine P-388 leukemia. Therefore, the conjugate and its biologically active components may be used as anti-carcinogenic agents or as anti-inflammatory agents for inhibiting mammalian tumors.
l11! Can be used as a tumor agent.

The microbial actinomycete strain 4H964-92 can be isolated from soil samples and prepared as a biologically pure culture for characterization by conventional methods. Strain H964-
92 forms on spore chains that exhibit a straight, curved or hooked shape in aerial hyphae. The spores are spherical or oblong and have a warty surface. Aerial mycelium is poorly produced in most media. The color of the aerial mycelial mass is white, which later acquires a pinkish hue, or even a bluish color on some agar plates. The basal hyphae are colorless or pale pink. Growth temperature is 15℃~
It is in the range of 43°C. Cell wall amino acid composition and total cell hydrolyzate sugar content indicate that strain H964-22 belongs to cell wall type mB. Menaki/y (me
naquinone) is A(K-9(He) ・MK
-9(H,).

Based on the main morphological, cultural and physiological characteristics as well as chemical cell wall composition characteristics, strain H964-92 can be classified as belonging to the genus Actinomadeula.

The original strain H964-92 produced slightly normal growth;
It also carried poor aerial mycelia, but the NT of H964-42
A mutant strain showing good growth and improved aerial mycelium formation was obtained by treatment with G to troguanidine). Mutant strain (
strain A1327Y) facilitated further taxonomic investigation and was identified as Actinomodula verruchospora.

Methods The media and procedures used to study culture characteristics and carbohydrate availability were developed by the International Streptomyces Project.
Strgptomy-ces Project
yst, Bacteriol), Volume 16, No. 313
-340 pages, 1966].

S, A, Waxman (S, Lupfaksmα匍 [The
The Actinomycetes
es) Volume 2] and G.

M, Revedemay (G, M, Lvadamann)
[Intro J.

Cyst bacteriol. 21, pp. 240-247, 1971] was also used. The cell wall-amino acid composition and whole cell hydrolyzate sugar components were determined by Pelaker (B
ecksr) etc., Able Microbiol<AI'P L
9Mi c deol) iol), vol. 29-13, p. 236-243, 1965 and LechgτaLier and Lechhevaria, di actinomycetes, H. Browser (H. Pra.
ustt de), Jena (Ja%α), Qsstav Fisher V
erlag), pp. 393-405, 1970. Menaquinone is Collins (Co)
llins) et al., J. Zen, Microbiol (J.

Gen, Mirobiol), Volume 100, No. 221
-230 pages, identified by mass spectrometry according to the procedure of 1977, and the menaquinone composition was determined by Yamada et al., J.
Zen, Apple, Micropiol (J, Gas, App
L, MicrobioL, ), Volume 23, No. 3
31-335, 1977.

Morphology Strain H964-92 forms basal and aerial hyphae. Group-30- The basal hyphae are long, branched and undivided into short systems.

In aerial hyphae, spore chains are formed uniaxially at the tip of the hyphae. Spiral-like branches of spore chains are also observed near the hyphal tip. These spore chains contain 2-10 spores/chain and are straight, bent or hooked. The spores cover the surface of the wart in a finite and circular to oval shape with rounded or sharp edges (0,5-0,6X O,6-1,4μ
m). After maturation, each spore is often separated by a blank pod. No motile spores, 5 spores or sclerotia granules are seen in any of the media studied.

Growth of strain H964-92 was poor to moderate on both synthetic and natural organic media. Aerial mycelium formation is generally poor1, but oatmeal agar (ISP/
%3 medium), inorganic salt-starch agar <ISP /164
medium) and Pennett's agar. Natural mutants lacking aerial hyphae occur frequently. The color of aerial mycelia is white, and later on oatmeal agar, inorganic salt-starch agar and glycerol-asparagine agar <ISP/
165 medium), it becomes pale pink. 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 colorless to yellowish on Czapek agar, tyrosine agar, yeast extract-malt extract agar (ISPI62 medium), peptone-yeast extract-iron agar (ISP scrap 6 medium), and Pennett agar, and glucose-asparagine. Agar and glycerol-
It is pink in color due to asparagine agar. Melanoids and other diffusible pigments are not produced. The mutant strain A6A1327V obtained from this strain forms pale blue aerial hyphae and carries abundant aerial spore masses.

Strain H964-92 was incubated at 15°C, 28°C, 37°C and 4
It grows at 3°C, but does not grow at 10°C or 47°C.

This strain is sensitive to NaCl at 7% and 0.01%
and is resistant to lysozyme.

33- -34- Glycerin-Asparagus G: Poor-Ordinary Gin Agar (I
SPAl, 5) R: Colorless-light yellowish pink (28
] A: Poor; light yellowish pink (28), light gray after 5 months (190) D: Inorganic salt-starch agar G: Rich Cl5P/164) R: Yellowish white (92)
) A: Rich; bright pink (4) Pinkish gray (10) D = Tyrosine-free agar G: Normal (ISP / l67) R: Yellowish white (9
2) A: Poor: light yellowish pink (28), very later (5 months) partially light grayish (190) D = non-nutrient agar G: poor - normal R: pale yellow (8)
9) A: None D: None 35- Normal Normal light yellowish pink (28) Light yellowish pink 1 color (28) -
Deep yellowish pink (27) Normal: White - Light pink Normal: White - Strong pink (
4) Color (2) None None Normal Normal Light yellowish pink (28) Light yellowish pink (28)
Normal: light bluish gray rich; light back color (185) (
190) None None Normal Normal Strong yellowish pink (26) Strong yellowish pink (26)
Jintong Two White - Light Pink Normal; White - Light Pink (4) (4) None No Poor Poor Colorless - Light Pink (7) Colorless - Light Pink (7
) None None Yeast-free extract-malt G: Rich kiss agar Cl5P, 462) R: Light yellow fi (89
) A: Poor: White (263) D = Oatmeal-free agar G: Normal Cl5P/163) R2 Colorless - pale pink (7) A: Poor; pinkish white (9) - light bluish gray (190) D : Bennett-free agar G: Abundance R: Yellow 90) A: Normal; White (263) Yellowish white (92) D: Peptone-free - Kada extract G: Normal - Iron agar <I; SP/166) E: Colorless A: No D=No □ 1 36- Rich Rich Strong yellowish pink (26) Strong yellowish pink (26)
Poor; white - pale pink Poor - white, pale pink (
7) Color (7) None Poor Poor pale yellowish pink (31) Light yellowish pink (31)
Very weak; clear flame chamber Very weak; clear pale blue (
184) Color (184) None
Mutyo Rich Strong yellowish pink (26) Strong yellowish pink (26)
Normal; pale yellowish pink, absent (31) and bluish white (189) absent, abundant, abundant, colorless, colorless, absent, absent Observed after culturing at 37°C for 3 weeks.

Abbreviation 20 = Growth, R = Reverse coloring, A = Aerial mycelium,
D = Diffuse dye 441 Colors and numbers in parentheses are
Andori, B, Sud; I5CC-NBS color name chart, illustrated with centroid colors. U.S., Dept. of Com., Cir., 553, Wash. 7D, C.,
November 1975 J <Kally, K, L, &D
,B.

JlLdd: l5CC-NBS coLor-na
me cha-rts 1l11Lstrated
with CentroidColors, U, S,
Dept., of the County, Cf., 553 Washi
ngton, D. C-+ Nov, 1975).

37- Table 2 Physiological Characteristics of Strain H964-92 Test Reaction Method and Medium Growth Temperature Range Maximum at 28°C-37°C Bennett agar growth, normal growth at 20°C and 43°C, gelatin growth at 10°C and 47°C Liquefaction Liquefaction Glucose
Peptone-gelatin medium Starch hydration Hydrolysis Starch agar decomposition In but-skimmed milk, Pep Difco (Di
-Reaction No tonification fco) Skim milk Melanin-like color No formation Tyrosine agar\cum. Formation Peptone-Yeast-Iron Agar and Tryptone-Fermented Extract Broth 38- Nitrate Reduction No reduction Czapek Glucose-Nitrate Pros and Glucose-Sukota Extract- Nitrate Broth Resistance NaC11 Grows below 5% Tryptone-Yeast 7% Does not grow on extract agar lysozyme resistant. 0.01% tryptone
Grows below yeast. Does not grow on extract agar 0.1% and grows at pH 5.0-9.5. Before tryptone-yeast, 4
．． 5 and extract agar 10.0 Table 3 Glycerol + 10.0
D(-)-arabinose --- L(±)-arabinose + 10
Ten - xylose + ten
10-ribose 10--L
-Rhamnose Shishi Juri-
Glucose - Galactose - - D-Fructose
Shishi Juri-mannose
−−− LH-Sorbose − −− Sucrose lactose −−− Cerbiose + Melibiose − −−Trehalose
Shi Shi Shi Raffinose
− −D(+)−fructose −−
− Soluble starch cellulose 10 dulcitol −−− Neusitol + −−D-mannitol 10−sorbitol − −− Salicin −−− Observation after culturing at 28°C for 3 weeks Basal medium: Bridam-Gottlieb
-Gottlieb) The purified cell wall of strain H964-92 in mineral medium contains mendiaminopimelic acid but lacks glycine. Whole cell hydrolysates show the presence of madulose (3-O-methyl-D-galactose), glucose and ribose.

Cell wall amino acids and total cell sugar components are from strain H964-9.
2 indicates that it is located in cell wall type IIIB. The two main components of menaquinone are MK-9 (#a)
and MK-(Hs).

Taxonomic position of strain H964-92 Strain H964-92 has the following key characteristics. (1
) Aerial mycelial spore chains, short, straight, bent or hooked. (2
) Spore 241- Surface of warts. (3) Aerial mycelium: pink or bluish color. (4) Basal hyphae: pinkish in some medium. (5) Diffusing dye: None. (6) Mesophilic. (7) Cell wall type IIIB.

(8) Menaquinone series: MK-9 (H6) and MK
-9<昂).

These main characteristics indicate that strain H964-92 belongs to the genus Actinomadeura. 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-Gen, Mic-robiol, 1st
12, pp. 95-111, 1979, that most soil-derived Actinomadeula species are grouped into clusters (Clwst) of the 14 described strain groups.
er)/167. J. Ferment;
Te42-Knorr, (J, Ferment, Techn.
), Vol. 49, No. 1, pp. 904-912, 19
In 1971, Nomura and Obara reported five saprophytic species of the genus Actinomadeula! Preobratchenskaya (Pr.
eobrazhenskaya) etc. [Actinomadeulus and Related Organisms (ACl
i −%mycgtesα%d Retated O
rganisms), Vol. 12, pp. 30-38, 1
[977] published a key for the identification and classification of Actinomadeula species. As a result of comparing descriptions of 30 species including microorganisms disclosed in patents, strain 11964-9 was found.
2 is Actinoma-d coerulea (Actinoma-d.
ura coeruLea) and most similar to Actinomadeura verchuspora described in the Nomura article cited above.

Strain H964-92 ± Perchospora strain KCCA-
0147 and was found to be closely related to A. Verruchospora in morphological, cultural and physiological characteristics. Therefore, strain H964-92 is classified as a new strain of Actinomadeura verchuspora.

For the production of BBM-1675, the present invention uses the special strain Actinomadeura belconspora 113 strain H964-
92<ASCC39334) and its mutant strain (referred to as strain A1327Y (ATCC39638)), but the present invention is not limited to these microorganisms or to microorganisms whose culture characteristics are fully disclosed in the text. You should understand that there is no such thing. The present invention is directed to strain H9.
64-92 and all natural and human BM-167
It is specifically contemplated to include 5-producing mutant strains.

Antibiotic Production The EBM-1675 antibiotic of the present invention is produced by producing BEM-1 of Actinomadeula verruchospora in a conventional aqueous nutrient medium.
675-Producer Strain Preferably, it can be produced by culturing a strain of Actinomadeula verrucospora or a mutant strain thereof having the identifying characteristics of ATCC 39334 or ATCC 39B3B. 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,
Must contain D-man 45-nitol or inositol. Nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. alone or in combination with organic nitrogen sources such as peptone, meat extract, fermented rice extract, corn steep liquor, soybean flour, cottonseed flour, etc. can be used. 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 the BEM-1675 antibiotic may be carried out at any temperature that allows favorable growth of the producing bacteria, such as 15-45°C, and is preferably carried out at temperatures of about 27-32°C.

Typically, optimal production is around 68-18 cm depending on whether shake flasks, shakers or tank fermentations are used.
Obtained after one incubation period at 0:46. When a full 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 producing bacteria. After obtaining an active inoculum in this way, it is transferred aseptically to a fermentation tank medium. For antibiotic production, Staphylococcus aureus was used as a test bacterium.
paper disc using eus) 209 P
It can be monitored by an agar diffusion assay.

Upon completion of the 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 by filtration or centrifugation into the mycelial shell and the broth supernatant. Antibiotics in the mycelial cake can be collected by suspending the cake in methanol, filtering out insoluble materials, and concentrating the methanol extract. The active components in the broth supernatant can be collected by extraction with n-butanol. Next, the above n-butanol and methanol extracts are combined and azeotropically evaporated to form an aqueous solution in which most of the antibacterial activity is deposited as an oily solid. The solids are then dissolved in methanol and the solution is filtered. Concentrate the filtrate 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 anti-solvent such as n-hexane.

The crude BBM-1675 complex contains two major bioactive components,
It is a mixture of several ingredients, including BBM 1675 A and A, and four minor active ingredients, BBM 1675As, Aa, B+ and B2. These biologically active components can be separated and purified by conventional chromatography operations. In one operation, coarse BB
The M-1679 complex was first dissolved in methanol and Sephadex LH using methanol as the elution solvent.
Purify by -20 column chromatography. This partially purified complex was then chromatographed on a silica gel column, eluting in a stepwise manner with chloroform plus increasing concentrations of methanol to increase BBM-1.
675AI, BBM-1675A2, a mixture of A3 and A4 and BBM-1675B.

and B2 can be obtained. A, the component is Sephadex LH- using methanol as the eluent.
Further purification can be achieved by 20 column chromatography. The mixture of A2, As and A4 can be separated by chromatography on Bondapak C88 (Waters Associates Inc.) using increasing concentrations of aqueous acetonitrile as the eluent. . Mixture B1, Bt, can be separated by silica gel column chromatography using a mixture of chloroform and methanol as the eluent. Further details of a suitable romatographic separation procedure are provided in the Examples below.

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.

50- Table 4 Rf value BBM 1675 As 0.74
0.18 BBM 1675 At O
,710,21EBM-1675As O,7
20,28EBM 1675 Aa 0.7
1 0.78EBM-167571? ,
0.63 0.23EBM 16
75 Bt 0.60 0-16 cost CI8 Reversed phase silica gel B BM 1675 Separation of At, A, and A4 was difficult with normal phase TLC system Force ζ Reverse phase TLC
I was able to achieve 1.

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,
It is also insoluble in n-hexane and carbon tetrachloride. These components give positive reactions with ferric chloride, Ehrlich and Tollens reagents, but negative reactions with the Itame, Ninhydrin and Anthrone tests.

The characteristic physicochemical properties of BBM-1675 components are shown in Table 5 below.

Table 5 Melting point (decomposition): 156-158°C 147-149°C
125-12'7 [α) (cO-5, CHCl5): 191°-1
794°-1 analysis Experimental value (%) C: 51
．． 52 53.81 55°H: 5.816.
316°N: 4.023.823°14°3 (z 282(195) 282(172) 282(]32
0 (143) 32G (128) 320
(Coin O,0INHC1lCHsOH: 253(
323) 253 (276) 253 (2 282 (19)
2) 282 (167) 282 (ko32
0 (144) 320 (128) 320
(CoinO,0LNNaOH-CHsOH:252(3
25) 252(289) 252(/:283(17)
2) 283(171) 283(]318(1363
318 (122) 318 (: Molecular weight (estimated value): 1.300 1,1
00 1.1 (tGa l HPLC+ Finep
akGEL-101 53- 531-s A4 BI B
t, 27℃ 123-126℃ 159-161℃ 1
56-159℃61° -176° -171°
-12280053,67 526,35 573,45 9136,53! 86) 253 (257) 253 (225)
248 (212) 158) '282 (153
3282 (140) 279 (141) L22)
320 (117) 320 (104) 318
(103)287) 253(2583253(2)
25) 248 (210) 601 282
(155) 282 (140) 279 (
140) 126) 320 (118) 320
(105) 318 (103) 280) 25
2 (266) 252 (236) 24B (23
3) 1623 283 (160) 282 (141
) 278 (150) 120) 318 (11
8) 318(105) 318(,110)1
0 1.400 The maximum UV absorption of BBM-1675 component is 253.282
and 3205 m, which did not shift in acidic or alkaline solutions. BBM 1675
The IR and PMR spectra of /L, A1, A3 and A4 are shown in Figures 1-4 and 5-8, respectively. B
The 360MHz PMB of BM 1675A is 2(l
1 acetyl (δ: 2.11 p), 1 N- H
s (2-52p%), 4 0CRs (3-42,
3-80, 3,88 and 3.98 ppm) and 1
exomethylene (4,57 and 5.48 pμ)
showed that. The CMR spectrum of BBM-1675A is a triple intensity signal (δ: 56.0p%'L, 0CHs
) showed 55 carbon signals including .

The molecular formula of BBM-1675AI is based on proton and cNMR spectra, trace analysis and HP
Based on molecular weight measurements by LC and SIMS (secondary ion mass spectrometry), C37H? It is inferred that it is 2N403t.

Structural study of EBM-1675A, when treated with 0-5 N HCI CHsOE at room temperature, BBM-1675A loses its biological activity and becomes a lipophilic chromophoric substance (compound I) with several unidentified fragments.
) is generated.

Compound (1) showed similar UV absorption to the parent biomaterial, suggesting that compound (1) retains the chromophore structure of BBM-1675A. Two other chromophore fragments related to compound (1) are BBM-167.
Obtained by alkaline hydrolysis of 5A. 55℃ for 1 hour
When hydrolyzed with 0.05NKOH-CH30H, 25
A compound (I[) having a UV absorption maximum at 2.284.297 (shoulder) and 322 nm was produced. on the other hand,
Reaction with INKOHCEsOH yields an acidic chromophoric substance designated as the compound Saliva. Compound (I), (II)
The physicochemical properties of and (1) are summarized in Table 6 below.

56 Table 6 Compound ■ Melting point: 82-83℃
9 (ff) (e O,2CHCl5)
'100' molecular formula
: CnHsINOt.

318 (6,300) MS ~: 457 (A
f+) 25 41 81 64 LC (Xylene y-”MEK-CffsOH=5:5:lv/
v): Rf O,5B' ”ME
K = methyl ethyl ketone 57- Compound ■ Compound ■ 133° 253-255℃ 00°C84PayyN□a C15HIllNOa2
52 (26,600) 248 (26,900) 28
3 (11,200) 295 (14,400) 295
(,M+) 281(M”3280
263 263 236 251 222 248 218 0.66 0.13 Structural information for formulas (II) and (2) was provided from the following spectral data and chemical transformations. 13C
And proton NMR shows that 40C &, 1
”C#t,? (:'-12.C=O and 1>NH
indicated the existence of a group. The NME spectrum of compound (2) was similar to that of (11), differing only in the absence of one of the four OCBs groups observed in formula (1).

The acidic nature of compound (III) suggested that the compound #(...) was a methyl ester of compound (t). Compound (1) was quantitatively converted to compound (III) by heating with W reflux with 1N methanolic KOH, while compound (1) was converted to compound '1m (1) by treatment with diazomethane. c, HsOH
Treatment of compound (1) with medium NaB&gt; yielded a reduction product (compound (IV), Af+: m/z 267), which replaced the -COOCHs group of compound 121(1) with -C1i by NMR. , showed a DH group. When water is added with palladium-coated charcoal, compound (U) is converted into a dihydro derivative (compound (V
), A/+.

m/z 297). The proton NMR spectrum of compound (7) showed a new doublet methyl signal and the absence of the exomethylene group present in compound (n). Furthermore, one of the oCH5 groups appeared in compound (9) at a high magnetic field (δ: L50 ppm). These results indicate that -C=CH in compound (II).

Indicating the presence of a CH3 group, which was reduced to a -CHCHs 0CH8 group in hydrogenation compound (9). Compound (n) at 80°C for 8 hours at 1.5
Heating with N methanolic hydrogen chloride and chromatography of the hydrolyzate on a silica gel column yielded a weakly basic compound (compound (Vl) 1.M+: m/z
211) was generated. The IR spectrum and physicochemical properties are that the compound (VT) is N1
-12 groups. Compound (■)
was identified as methyl 4,5-dimethoxy-anthranilate by comparison with standard JR and HA (R studies). Therefore, the structure of compound (II)-(VT) was determined as follows. Ta.

Upon treatment with 0.05 N KOR-CHsOH at 55°C, compound (I) forms a novel colored fragment (formula ■, C
15H21NO? , M+:yyb/z827) and the sugar (formula ■). The NMR spectrum in (■) consists of one singlet c-CH3 and three low-field OCH3
In addition, two high-field 0CR3 groups and two aromatic protons commonly observed in compounds of formulas ■, ■, and ■ were shown. Further hydrolysis of (1) with 1.5N methanolic hydrogen chloride yielded compound (2), which was identical to the previously obtained (2). After removing (1), the water decomposition product (2) was treated with 2.4-dinitrophenylhydrazine to precipitate a yellow solid, which was identified as 2,4-dinitrophenylhydrazone of pyruvic acid. So, the compound ■
is methyl 4,5-dimethoxy-N-(2', 2'-dimethoxypropionyl) anthranilate. Compound (1) did not show a molecular ion peak, but showed an M-OCII3 peak at yrL/g181 with a mass vector of 62-mass consistent with the molecular formula 〇JI404. The NMR spectrum is 2 for compound ■.
,6-siboxyhydropyranose structure. This designation is supported by the "C-NMR of formula I, which NMR
In addition to the 15 carbon signal that can be assigned to compound ■, I C
-CH3,1-CH, ,8O-CH', and 1 aromatic carbon were produced. The C3 proton of the sugar shown in the low magnetic field (δ: 5.89% recommended octet) indicates that the C3-OH of the sugar has been esterified with the carboxyl group of ■. The above results are summarized below.

Structures of compounds ■, ■ and ■ Compound ■ CIL3 0CHs63- Compound ■ Compound ■ The molecular weight (457) of compound ■ is E BM 1675
Corresponds to approximately × of all molecules of At (presentation formula C, sir, 7v
40. ; calculation MW64- = 1824). The partial structure of BBM-1675A1 is thought to be as follows.

υ After filing U.S. Patent Application Box 495,281, the above component BBM-16? 5 A, and A2 (prepared according to Example 2 below) were in fact not found to be completely pure and that some of the characterization characteristics used to define such components were inaccurate. . Following additional chromatographic purification procedures detailed in Examples 8 and 6 below, BEM-1675AI and A were isolated in highly purified form and characterized in detail as shown below.

We also corrected the elemental analysis data for components A and A4 to show the presence of sulfur in these compounds, and
PLC retention times were calculated for these two components. The corrected physicochemical properties of the BEM-1675 components are summarized below.

Melting point 156-158' (decomposed) Original purple analysis: Analysis 1 Analysis 2 Average C: 51.60% C: 52.74% C: 5
2.17%H", 6.31%H: 5.99
H', 6.15#: 5.81% N: 8,
94% N: 4.68% S: 8.47% S
: 9.71% S: 9.09%O (difference): 2
8.11% O (difference): 27.62% O (difference):
27.96% concentration - 0.01856. V//- λrrLtLx (nrn) Absorption coefficient 1820
12.4 280 aA (shoulder) 258
25.1 210 25.5 Optical rotation without significant change with acids or bases: Solvent - CHCl.

[α] = -207° (c = 0.0851) The second analysis showed the following optical rotation.

[α]. =-1916 (c=0.5, CHC2s) Infrared absorption band (KBr) 985.1015.1070.1110.1150.1
210.1250.1808.1880.1405.1
446.1520.1592.1608.1668.1
715.2920.2960.8860.8440゜c
rn-' Mass quictor: Apparatus-VG-ZAB-2FFAB-M
S-thioglycerol molecular mass range ion (door/g): 1249.1857.
1468: <m7g by addition of NaCl): 1271
．． 1879.1485.1597゜FAB-MS-MB
CMB knee r trix, m0w.

154): Molecular mass range ion (WL/g): 1249
．． 1288.1408.1555: 1 m/g by addition of NaCt): t249.1271.1808.142
5.1488.1577゜FAB-MS-Glycerin-
DMSO: Molecular mass range ion (rn/z):
1215.1247.1279.1298.1825.
1858; with addition of NaCt (m/g):
1215.1287.1269.1825.1847
．． 1375° Apparatus: Kratos MS-50 FAB-MS-thioglycerol bimolecular mass range ion (m7g): 1857.146848- Molecular weight (according to the mass data above) Apparent &W=12
48 Nuclear Magnetic Resonance Spectrum: Apparatus - WM860 Brwcker Solvent: CDCts "NMR: 860MHz δ<9'l)m):
11.75 <III.

8); 8.55 (IH, 8); 7.45 (I
Hl #); 6.61 (IH, ylL); 6.28
(IH, brs); 6.17 (IH, brs):
5.98 (iH,d, J=9.8);5. g2C
xH, d, J=9. s):5,7(iH,bra);5
．． 49 (IH, yrL); 5.45 (iH, d,
J=2.8); 5.88 (IH, bra);
4.95 CIH, d-, 7=10.2); 46
4 (2H.

yx); 4.54 (IH, d, J=2.8)
:4.2 (IH, g):4.15-8.85(26-
28H) (4,10 (IH, m); 4.02 (IH,
brs); 8.95 (8H, s); 8.85 (8H
, a), 8.7949- (8#, s): 8.46 (IH, m): 8-40 (
8II, s) ): 2.82-2.70 C8H
, bτm); 2.50 (8H,s), 2.47 (
IH, nz); 2,88-2.22 (5H);
2.12 (IH, nL); 2.11 (8H, s
): 1.60-1.05 (22H) (1,89(
8H, d, J=6.8): 6.81 <8H, d, J-
6,8); 1.29 (8H,d, J=6.8
), 1.08 (6H)) See Figure 10 CNMR: 90.8 MHz See Figure 11 Separate tests showed that purified BBM-1675A, c
NMR spectra were measured on samples dissolved in CDC1, (80 MHz). The main peaks are shown below.

CMR of HEM-1675AI (CDC1, medium 80Af
Hg)-F〇- B BM 1675 At Characteristics: White crystals: Melting point 147-149℃ Elemental analysis:
C: 52.71 cm H: 5.94% N: 8.94% S: 9.89% O (difference): 28.01% Ultraviolet absorber: Equipment - Varian UV, Carry 2
19 Solvent-methanol concentration: 0.02052 g/1 820 12.2 282 16.8 252 26.2 214 25.8 1 No significant change with acid or base Infrared spectrum: See Figure 12 Equipment: Beckman Icman, ) J
R model 4240 Main absorption band (MB de): 950.1015.1070
．． 1100.1155.1213.1250.1813
．． 1375.1405.1450.1520.1595
．． 1610.1685.1735.2940.2980
．． 8440cm-”.

Mass squictor: Equipment: VG-ZAB-2FFAB-M
S-thioglycerol molecular mass range ion (m/z): 968.1249.1
855.1857.1468.1569: by addition of NaCl (m7g): 990.1271.1379.1
485.1598, FAB-MS-MB (MB knee 1 trix, m, w, 154): molecular mass range ion (yyr/s) = 1249.1403.1419.1
555.1571.1587: by addition of NaCt (
rrL/z):1249.1271.1425.1'4
41.1457.1488.15T'l.

FAB-MS-Glycerin-DMSO; Molecular mass range ion (m7g): 1215.1231.1247.12
68.1279.1298.1809.1825.18
26.1341.1858.1869゜Molecular weight (according to the above mass scuttle data): Apparent A#=1
248 Nuclear Magnetic Resonance Spectrum: See Figure 18 Apparatus: WM860 Pruzker Solvent: CDCLm "II NMR860Mfiz δ(pT)m):
11.9.1 (IH.

a); 8-62 (IHy8); 7.58 (iH-,')
; 6.56 (IH, yyb); 6.22 (iH, a);
6-15.

(IH, brs); 5.91 (iH, d,, 7
-9.6); 5.88 <IH, d, J-9,6): 5
．． 70 (IH.

−7 to − m); 5.45 (IH, d, J=2.2); 5.44
(IH, s), 5.34 (IH, brs); 49
5 (LH, d, J = 10-2): 4.75 (IH, r
n): 4.65 <IH, d, J=6.8); 4.54
<111°d, J=2.2);, 4.47
(IH, m); 4.18 (IH, s): 4.10
(IH, brs'), 4.05-8.50 (2072
,4ff); (8,96(,811,8):8.8
7 (8H, a); 8.77 (8H, s)'); 8, . 4
6 (IH, yrL); 8.89 C8H, s):
2.79 (IH, m); 2.78 (2H, yx); 2
．． 50 (8II, s); 2.50 (IH, m); 2
．． 88-2.22 (8H); 2.14 (IH, m)
; 2.10 (8H.

s); 1.9g (2H,m); 1.65-
1-45 (6-8H); 1.88 (8H, d
, J=6.0); 1.84 (8H,d,,7=6.
0); 1-22 (8H, d,, r75- = 6.8); 1.10 (6H).

"CNMR90,8MHz See Figure 14 ISCNMR of BBM-1675A purified in separate tests
Spectra were measured on samples dissolved in cDcts (80 AfHz). The main peaks are shown below.

-76= BBM 1675J4 13.7' 16.
9 17.5 19.834.1 35.
1 39.3 47.657.6 62.
4 64.5 64.971.9 73,
6 75.8 76.183.3 86.
2 8B, 4 90.499.6, 103.8
107.1 112.4144.1 154.2 1
54.5 160.9 - Soichi 539 - 22.3 22.7 23.4 33.15
2.6 55.7 56.0 56.16
5.9 68.3 69,2 69.777
．． 1 77.7 78.1 78.397.
2 98.3 99.1 99.5123.
2 124.8 129.9 137.3167.9
192.2 -78- BN-16'15A1 (), 74 0.1
8 1&8EBM-1675A2 α7
1 α21 17.8BBM-16
75As 072 α28
&OBBM-16'15A4 0.71
α78 &IBm-1675B1
0.68 α28-BBM-1
675Bz O-60α16-band C
s Reverse phase silica gel PLC 79- TLC chromatography About all normal phase chromatography Analtech/GHL
F. Silica Gel Uniplate (Analtech
GHLFSilica Gttl Uniplates
)It was used. A plate with dimensions of 2.56 In x 10 m was used for one-dimensional TLC. These are 6.4m (outer diameter)
It was developed in a glass cylinder with dimensions of x 12 crn and containing 10 d of eluent. 7 for 2D TLC.
A plate with dimensions of 5a x 10cm was used. The sample was applied to the lower left corner, 1 cIn from the edge. First, transfer the plate to a tank (width: 12 mm) containing 50 ml of the first eluate.
．． 76 In, depth 8.6 saws, height 18 cm) was developed without a housing. The plate was then air-dried LA90 @ rotated counterclockwise and developed in a second tank containing a second eluate 50d.

Whatman Analytical Precoat C-
18 silica gel plates were used for all reverse phase chromatography. 2.580-σX7.6 cIn plate with eluent 10ya/
It was expanded in a glass cylinder containing a.

The normal phase plate was first observed under 254 nm UV light.

Plate I, glass cylinder containing crystal (outer diameter 6
．． 4 cm x 12 cIn). The plate was then re-examined approximately 2 minutes later. 254 zr reversed phase plate
n Made visible under UV light only. The bands were detected by quenching the fluorescence of the impregnating dye.

The following components were used to construct the analytical HPLC system.

Water Associates Model 600A Solvent
Delivery system Bonf: Varian Parichrome model VUV-10uv/vis daytector, 254n
m0.10. Set to Fisher Recordal Series 5000 Recorder: Waters Associates Model 660 Solvent Programmer: Injector to Waters Associates Model U6; Alchik, μm Bondapak C1g (10μ) Color A (4
, 6111111 internal diameter x 25 m), Whatman Company IL ODS (0,08-0,088) guard column (46 internal diameter x 5 m). These members are fitted with 816 stainless steel tubing (1,6mm outer diameter - 0,28v+
m inner diameter). The eluate was pumped 2 ml 11 minutes into all analyses.

The following members were used to construct the intermediate pressure liquid chromatography system. Fluid Metering, Inconsolidated, Model RP-SY2C8CFMI Lab Pump; Fluid Metering, Inconsolidated,
Model PD-60LF FMI Pulse Dumbener;
15 m sample roof consisting of polypropylene tubing (8.0 mm OD xi, 5 mm ID) wrapped around a cardboard tube (OD 8.65 cm); Grezz Series 3500 Universal LC column: Instrument・Shiartize Company Model UA
-5 Absorption/fluorescence monitor, type 6 optical unit key;
0 Flow Interrupter Valve: and Instrumentation S4Cialtize Company Model 328 Fraction Collector. Polypropylene and Teflon tubing (8
, 0 fin outer diameter x 1.5 mm inner diameter) and connected with Gretz Multifit connectors and valves.

A Gretzz Series 3500 Universal LC column was slurried back 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 Okwa sand. The eluate was pumped in at maximum speed at 60 psi back pressure (approximately 201 nl!).
/minute).

Gradient Elution A Grezz 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. - the chamber acts as a mixing chamber;
Others also worked as static reservoirs. The less polar solvent was initially kept in the mixing chamber. The more polar solvent was kept in a static chamber. Teflon-coated magnetic stirring rod (1, OX
8.7c1n) into the compartment and was driven by a Thomas Model 15 Magnematic agitator. The eluate was prepared using polypropylene tubing (1.5 mm inner diameter
8. 0 mm outside diameter) from the mixing chamber to the intermediate pressure HPLC system. As the eluate exits the mixing chamber, the solvent in the static reservoir freely interchanges with this fluid;
This resulted in a linear eluate gradient.

-8← BEM-1675A and At TLC analysis The observed Rf values for BBM-1675A and A2 on regular phase plates are summarized in Table 9 below.

Rf was calculated by dividing the measured distance of the center of the zone from the point of sample application by the measured distance of the solvent front from the point of sample application.

Table 9 Rf 4% methanol in chloroform 0°8B
0.80 dimethyl ether 5% methanol
0.89 - 50% Acetone in Schellisolp B 0.88 G, The observed Rf values for BBM-16751A and A in the normal phase plate developed in the 8 direction 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 10 Chloroform 94% methanol to diethyl ether 5% methanol (0,84,0,88)
(0,28,0,28) 50 thiacetone in chloroform 94% methanol solution in Skellisolve B. R observed for A
The f values are summarized in Table 11 below.

Table 11 The Rf values observed for EBM-1675AI and A on C-18 reverse phase TLC plates developed with ternary eluents are summarized in Table 12 below.

-82-88- BBM-1675A and HPLC analysis of A2 BBM
-1675A1 and B BM 1675 Ax C
-18 reverse phase silica gel columns using single, binary and ternary eluents. The values observed for these compounds are given in Tables 18.14 and 15 below. K' was calculated using the following formula.

TR-T.

K'=□O where TR is the measured retention time from the time of injection to the peak tip and To is the empty volume time.

Table 18 shows 'acetonitrile α
G Tetrahydrofuran α
α methanol o, o o
o, o.

α = No compound eluted from the column.

89 - In Table 14', 25% water α in acetonitrile
α 25% methanol in water 1.25
1-25α = No compound eluted from the column.

Table 15 Acetonitrile: Methanolone water 40%: 80%: 30% α
α-acetonitrile: methanol 0.11 MIId)
Ac4α%: 801%: 30%
1-7 8.05α% :200% = 3
0% 8.8 6.54&8% :2&
8% 2 3&8% 6.1b42.5%:
22.5%: 85.0% 7.8
b41.5% =215% :87.0%
9.7b90-α = did not elute.

b=No measurements were taken.

Biological Properties of BBM-1675 Component The antibacterial activity of BEM-1675 component was determined by serial double agar dilution method against various bacteria (Gram-positive, Durham-negative and rF
Ir acidic) and mold were measured. For Gram-positive and Durham-negative bacteria, use nutrient agar;
A6t 001 medium (3% glycerin,
0.3% sodium L-glutamate, 0.2% peptone, 0.81% Ha, HPO4, 0.1% KHPO, 0.005% ammonium citrate, 0.001% Mg
5O, and 1.5% agar) were used. For mold, Sabrod agar medium was used. As shown in Table 16, six ICBM-1675 components (A1, A2, A3, Mu, B
+ -Bt) showed a broad spectrum of antibacterial activity. BBM-1675A1, At, and A4 are especially highly active against Gram-positive bacteria! ! ! A, and A2 (produced in Example 8 below)
A second antibacterial test was conducted on components A3 and A4.

Lysogenic bacteria E, coli (E-coli) Wl 709 (
λ) prophage-inducing activity was measured using Nature (Nat
ure), Volume 196, Pages 788-784, 1962
BBM-16 according to the method of Lein et al.
Measurements were made for 75 components. Plaque counts were performed on agar plates containing test materials (G) and control plates (G). Tl with a larger plaque count than 8.0
The C ratio was considered significant and the lysogenization-inducing activity (t
LB activity) was expressed as the minimum attractable concentration of the test compound. As shown in Table 17, the BBM-1675 component showed strong tLB activity in lysogenized bacteria, thereby suggesting that the component has antitumor activity.

Table 17 BBM-1675A1 0.0068B
BM 1675A2 0.0125B
BM-1675A3 0.05BBM-1
675A40.10 BBM-1675B, 0.10BB
M-1675B, 0.20 US Minimum Inducible Concentration The antitumor activity of BBM-1675A1 and A2 was determined in various mouse tumor lines. lymphocytic leukemia P-888,
IJ lymphoid leukemia L-1210, melanocytoma B16
and Lewis lung cancer at an inoculum dose of 106.10'%5, respectively.
X10' and 106 cells/mouse were implanted intraperitoneally into male BDF1 mice. Mice were given graded doses of test compounds intraperitoneally 24 hours after tumor implantation. Treatment was done once only on the first white of the eyes, 1.4 and 7Cq8dX8), 9
Once a day for days (7dl+9) or once a day for 11 days (
qd1→11). Components A3 and A4 are q8d only for P-888 leukemia due to insufficient supply of materials.
The test was conducted on an X8 schedule.

BBM 1675A+, 4, A3 and Mt were dissolved in 0.9% saline containing 10% dimethyl sulfoxide,
and chromomycin Cch used as reference compound.
romomycin) A3 [Toyo
rnycin, Takeda Pharmaceutical Co., Ltd.] was dissolved in 0.9% saline. The number of deaths or survivals of treated and untreated mice was recorded daily, and the median survival time (NET) was determined in the test group (7').
and the control group (C).

A TIC value equal to or greater than 125 cm indicates that a significant anti-tumor effect has been achieved. The results are shown in Table 18-
It is shown in 28-9 two. BBM-1675AI and A showed extremely potent antitumor activity against P-888 leukemia with a maximum T/C4 of 160 chi. These ingredients have a minimum effective dose of about 10-aooo times the total activity of Chromo, Goisin A3 and more.

However, BBM-1675A3 and A4 are p-ass
Component A or A also had low activity against leukemia (Table 19). B BM 1675 A1 and A, also L-1210 leukemia (Table 12), B16
It also showed activity against melanocytoma (Table 22) 8 and Lewis lung carcinoma (Table 28). BBM-1675AI and A,
Toxicity was determined in male ddY mice by intraperitoneal or intravenous administration. BBM-1675A, is BEM 16
It was about 10 times more toxic than 75 At (Table 24). The therapeutic index of EEM-1675A and A, respectively, was 4-8 and 8-20 times better than chromomycin A in the P-888 leukemia lineage (Table 25). 5X10' and 1 intravenously, respectively.
P-888 and L-12 inoculated with 04 cells/mouse
A second experiment was conducted using intravenous administration of BBM-1675 components against 10 leukemias. In these experiments, adriamycin (αdriamycin) was used as the reference drug, dissolved in 0.9% saline and administered on days 1.4 and 7. The results are shown in Tables 26 and 27. Ingredients A8 and A! Both were superior to adriamycin in terms of maximum T/C value, minimum effective dose, and range of activity.

-105- Table 24 BBM-1675A, 0,019 0.010
0.00046BBM-1675A2 0.18
α10 0.0072 Yen-11- 313M-1675A+ 6B >46 2
15 5BHM-16T5At 180 72
2 24 24CnronornycinAs
B B None 9j) 28 28 US
Minimum Effective Dose 106-550-Component B The antitumor activity of BM 1675 A1 and A2 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 30 below.

The methods used in these trials were
3, pp. 1-87 (3 parts), 1972.

qd1→9 12.8 TOX6
．． 4 6.0 3.2 13.0 1.6 14.5 0.8 16.5 0.4 16.0 0.2 15.0 0.1 13.0 0.05 12.0 0.025 12 ．． OBBM-16754d, 1 256
TOXDM SO→sal ine
12 g 12.564
27.0 32 26.0 16 16.0 8 15.5 4 15.0 2 12.0 1 12.0 0.5 10.0 d, 1, 5 & 9 128 T
OX64 TOX 32 TOX 16 24.5 8 17.5 4 15.0 2 15.0 1 12.5 0.5 12.0 111- TOX TOX 1/675
-1.5 4/6163 -1
．． 2 6/6181 -1.6
6/6206 -2.3 6/6
200 -0.9 6/6188
-0.8 515163 -0
．． 4 6/6150 +0.1 6
/6 150 -0.7 6/6TOX
TOX O/6156-3.
5 4/6338 -1.9
6/6325 -2.0 6/62
00 -1.8 6/6194
-1.9 6/618g -0
,76/6 150 -0.5 6/6150
0 6/6125 +0.2
6/6 TOX TOX O/6TOX
TOX O/6TOX
-1,32/ 6 306 -1.3 515219
-1.1 6/6188 0
6/6188 +0.1 6/
6 156 -0.4 6/6150
-0.4 6/6BBM-1675A,
qdl-490,2511,0()JI SQ→
sal inn 64
TOX32 6.0 16 8.0 8 15.5 4 17.0 2 15.0 1 14.0 0.5 14.0 0.25 12.0 0.125 12.0 Comparison 5aline
8.0 Tumor liquid species: Intraperitoneal transplantation 106 ascites cancer cells Host: CDF♀ mouse Toxicity: d, survival at 5 Ius<4/6 evaluation 2 MST = median survival time effect: %T/ C=<MET treatment/MST comparison control)
X100 boundary: %T/C≧125 was considered significant antitumor activity.

-"NSC38270=olibomycin (olivom
ycin) A112- 553- 138 -0.4 6/6TOX
TOX 1/675 -2
．． 9 4/6100 -1.9
6/6194 -1.3 6/6
213 -1.8 6/6188
-1.1 6/6175 -0.
5 6/6175 -0.6
6/6150 -0.1 6/61
50 +0.1 6/6 - +0.5 10/10 -113- qd1→9 12.8 TOX6.
4 8. ．． 0 3.2 8.0 1.6 9.0 0.8 8.5 0.4 8.0 0.2 8.0 0.1 7.0 0.05 7.0 0.025 6. OBBM-1675A, d, 1 2
56 TOXl 28' 7.0 64 7.5 32 8.0 16 7.0 89.5 48.5 28.0 1, 8.0 0.5 8.0 114- 554- TOX -2,43/6 114 -1.6 6/6114
-1.7 6/6129 -
2.1 6/6121 -1.6
6/6114 -1.0 6/
6114 -0.5 5/6100
+0.3 6/6100 +0
．． 3 6/686 -0.6
6/6TOX TOX O/610
0 -1.8 5/6107 -
1.3 4/6114 -2.2
5/6100 -2.3 6/
6136 -1.4 6/6121
-1.1 6/6114
=0.8 6/6114 0
6/6114 -0.1 6/
6 After further purification of BBM 1675 AI according to Example 6, a sample of the purified compound was injected into mouse L-1210
Tested against leukemia, P-888 leukemia and B16 melanocytoma.

These results are shown below.

116-555- The above screening data is based on purified BBM-16
The 75A+ component has substantially the same antitumor activity as the previously screened unpurified sample. This compound has exceptionally high efficacy. That is because activity was shown against murine P-888 leukemia at a dose of 25 nonagrams/Jcy on a 5 times daily schedule. In trials against P-888 and L-1210 leukemia, BBM-1675A, as a single injection,
It is also effective to administer the drug every 4 days or 5 times per day for multiple injections.

For B16 melanocytic cells, the compounds are effective when administered intravenously to animals bearing subcutaneous tumors, as when administered intraperitoneally to animals bearing 'LF tumor transplants. Such properties, such as advantageous pharmacological delivery of drugs to tumors at distant sites, are surprising for antitumor antibiotics.

As mentioned above, the BBM-1675 component has potent antimicrobial activity and is 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 disinfecting medical and dental bags.

The induction of prophages in lysogenic bacteria and the activity shown against murine tumor lines indicate that the BEM-1675 components are also therapeutically useful in inhibiting the growth of mammalian tumors.

Accordingly, the present invention provides effective antimicrobial or tumor suppressive doses of BBM-
1675A, , A, , A3, AB, or B, or a pharmaceutical composition thereof.

According to another feature, the invention provides an effective antimicrobial or tumor inhibiting amount of BBM 1675 A+,4,A8, in combination with an inert pharmaceutically acceptable carrier or diluent.
The present invention provides pharmaceutical compositions including A4, B, and Frozen B. 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 composition, the mode of application and the particular site, host and disease being treated. Many factors that alter the action of a drug are considered by those skilled in the art, including age, weight, sex, diet, time of administration, route of administration, rate of excretion, host condition, drug combinations, response susceptibility, and disease. That's about it. 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.

The following examples are presented for illustrative purposes only and are not intended to limit the scope of the invention.

Actinomadeura strain 16H964-92 was mixed with 1% malt extract, 0.4% glucose, 0.4% yeast extract, and 0.4% malt extract.
The cells were grown and maintained on agar slants containing 0.05% CaCOs and 1.6% agar. Using a well-grown agar slant, 8% soluble starch, 8% dry yeast, 0.8% 2HPO, 0,1% mio<, 126-0.05% MgSO4, 7&Q10, 2% NaCL and 0 7) H containing 1% CaCO3 and T'H before sterilization
The nutrient medium was incubated at 82°C for 72 hours on a rotary shaker (250 rprn), and 5 ml of the growth was mixed with 8% sweet potato molasses, 1% cornstarch, 1 chi fishmeal, 0.1% CaCO3 and 0.1% CaCO3.
005% Cu5Ch ・5H20 (pH 7 before sterilization,
500++ containing 100 mA' of fermentation medium consisting of 0)
+A' Transferred to Erlenmeyer flask. Fermentation was carried out on a rotary shaker for 6 days at 28°C. Staphylococcus aureus (Staphylococcus αureas) 2
Measured by paper disk agar diffusion using 09P. Antibiotic titer is approximately 1 rncg/m after 5 days of fermentation
The maximum value of l was reached.

Fermentation of rBBAI-1675 was also carried out in an agitated jar fermentor. 500 m of inoculum growth prepared as described above was transferred to a 201 jar fermenter containing 10 t of fermentation medium consisting of the same components as used in the shake flask fermentation. Fermentation was carried out at 82° C. with an aeration rate of 12 t/min and agitation of 250 rpm. Under these conditions, antibiotic production reached a maximum value of about 0.9 mcV after 68-76 hours of fermentation.

Fermentation studies were also conducted in fermentation tanks. Seed culture at 30°C for 4 days with 3% soluble starch, 3% dry yeast, 0.3
%KJP O4,0,1%KIItPO,,0,05%
MQSO, 7 Ht 0.

It was shaken in an Erlenmeyer flask containing a nutrient medium consisting of 0.2% NaCL and 0.1% CaCO. The seed culture was added to 180 t of seed medium having the same composition as above.
The seed tank was inoculated with 200 liters of p.c., and the seed tank was stirred for 81 hours at 80° C. and 240 r7yrn.

A second seed culture was used to prepare 1 T corn starch, 3 T cane molasses, 1% fishmeal, 0.005% Cw SO+ ・51
Fermentation medium a containing 1t O and 0.1% caco3,
It was inoculated to o o o z. Production tank at 28℃ 16
It was operated at an air flow rate of 2,00011 minutes at 4 rpm. As the fermentation progressed, the pH of the prosthesis gradually increased and reached 7.7-7.8 after 170-180 hours, when a peak antibiotic activity of 1.7 mcjJ/ml was obtained.

The harvested fermentation process (8,0004 pH 7-8) was separated into mycelial cake and supernatant with the help of a Sharpless centrifuge. The mycelium cake was suspended in 1.600 t of methanol and the mixture was stirred for 1 hour.

The insoluble material was evaporated and the methanolic extract was concentrated in vacuo to 481 ml. The active components contained in the Pross supernatant were extracted twice with partial amounts of Loubutanol 1000. Le 129- The monobutanol extract and the concentrated methanol extract were combined, and water was added at any time for azeotropic evaporation to form an aqueous solution (20
t), where most of the antibiotic activity was deposited as an oily solid. This solid was digested in 80 ml of methanol and the insoluble material was removed. The methanol extract was then concentrated in vacuo to form a solution of 10, to which were added 401 tons of ethyl acetate and 80 tons of water. After stirring for 80 minutes, the organic layer was separated, dried over sodium sulfate, and dried in vacuo for 4 hours.
It was concentrated to t. When the concentrate was added to 20 tons of luhexane, a pale yellow solid (90.14 g) of crude BEM-1675 complex was obtained.
, titer: 55mcvη) was obtained. The complex is TLC
It was found to be a mixture of two major components, BBM-1675A and A, and several minor components. These were separated and purified by repeated chromatography performed in a cooled room to prevent deterioration.

130-BBM-1675 complex (20g) was dissolved in methanol (20g).
ml) and Sephadex (5ephatex).
) It was packed into a LH-20 (φ5.5×85 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 separated from a silica gel column (φ5.0x50cr) using chloroform and increasing amounts (1→5% V/V) of methanol as eluent.
Chromatography was performed with n).

The eluate was evaluated for antibacterial activity (against S. aureus) and T.L.
C(Si02; CHCLB-CR20II=5
: 1 v/v) and concentrated in vacuo. is mostly homogeneous BM-' 1
675,4. (Yield 2351 after evaporation was first eluted with 2% methanol (in chloroform) and then BBM-1
Mixture of 675At, As and A4 (507mg)
The WCBBM-1675B mixture (210 mg) was then eluted with 3% methanol in chloroform. BBM 1
The 675A+ solid was applied to a Sephadex LH-20 column (φ2.O×806rn) and developed with methanol. The active fraction is concentrated to dryness in vacuo and the remaining solid is crystallized from methanol to yield pure BM-
Colorless platelets (124 cm) of 1679A (this material is the starting material for Example 3A) were obtained.

Composite BBM-1675A, A3 and No. 4 were bonded to Bondapak C, 6 (Waters).
%ters), φB, 0x50): 1) by chromatography on a column of Elution was carried out with aqueous acetonitrile and the active eluent was analyzed by TLC [Merck
k), Silanization (Cts reverse phase silica gel): CH*
CN HtO=75:25 v/v).

Minor components A4 (88 η) and As (xsmv) were eluted in this order with 20% acetonitrile, followed by the other major component At (810 η) (this material was used in Example 8B).
) was eluted with 50% acetonitrile.

The solid containing BBM-1675B and B2 was subjected to chromatography on a silica gel column (φB, Ox 40 cm ) using chloroform and methanol as a developing solvent. Kuro Ho 71. The active fractions eluted with 4% methanol in A were combined and evaporated to give pure BM 1615B+ (7 Ing). Another active fraction was eluted at 5% methanol concentration and was evaporated to yield HEM-1675B.

(8 Da) was obtained.

Glenco column, 2.67 C11 + id x75 cIn.

=IS3- was slurry packed with Baker bonded phase octadecyl (Cl8) silica gel (40μ particle size) in methanol. The column was connected to a medium pressure HPLC system and equilibrated with eluent 1.51 (41.6% acetonitrile-21.6% methanol-36.8% 0.1M ammonium acetate).

Partially purified BBM 16754 (100,5 rv) obtained according to the purification procedure of Example 2 was dissolved in 2 ml of acetonitrile and placed in the sample loop.

The sample was pressed into the column. The column was eluted with the above eluent and a fraction of 87 m/m was collected. eluent at 240
nrn and 840 nm. 7 lactations 55-71 were pooled and extracted twice with chloroform weighing 1500 m/l. When the chloroform was evaporated to dryness, a residue C89.8 was obtained.

Gretz column, 1.5 cm i.d. x 20 cIn, silica gel (60-20
0μ particles) 12g was super-13←rally punctured. 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 for 20 min.
-25 mJ fraction. After analysis by silica gel TLC, fractions 6-9 were pooled and evaporated to dryness to give a residue D7B.

12 g of Whelum silica gel (68-200 micron particles) in Shell'/5olve B was slurried back into a 1.5 crn id x 20 c1n Glenco column. The residue was dissolved in approximately 2 d of ClICl3 and applied to the column. Chloroform in Skerisolve B 25! Replaced with /. The column was then eluted with 500 g linear steps of Skellisolve B to 60 thiacetone in Skellisol B to 2B-25! Collected 1 yon of /. Fractions 19-28 are pooled and evaporated to pure EM 1675 At.
65.6# was obtained.

This residue was analyzed using three TLC systems (5% methanol in chloroform: 5% methanol in ether;
3950% acetone, silica gel) and HPLCCC
-18 silica gel -41.5% acetonitrile: 2t5
% methanol 17.0% 0. It was homogeneous in terms of I Mw ammonium acetate).

Two knees 2.5 cm inner diameter x 45 cIn Pharmacia (Phar
rnacia) column in methanol.
The H-20 was slurried back and adjusted to an 88.4 cm chromatography bed. Purified BEM-1675
, 41 (approximately 120 mg) was dissolved in methanol 2-,
．． 5d sample container. Apply the sample to the column and elute with methanol at 1-75 mA! /min starting with 1
0! [Pharmacia Fac-100 Fraction Collector]. The eluate was monitored with an Isco UA-5 detector at 254 nm.

BBM-1675A1 has a Ve/V of 0.79-0.91
<Ve4 output amount observed to elute at t: Vt
= bed amount).

B, B Purification of BM-1675Ax A 2° 65 crn id x 756 rn Glenco column was slurried back with a synthetic-car bonded phase octadecyl (C18) silica gel (40 g particle size) in methanol.

The column was connected to a medium pressure HPLC system and the eluent L5
t(501 Acetonitrile - 20% Methanol - 80%
Equilibration was performed with 0.1M ammonium acetate). Partially purified BB$167 obtained by the procedure of Example 2
5. A2 (76.9 mF) in 1 liter of acetonitrile and 2 r
and placed in the sample loop. The sample was pressed into the column. The column was eluted with the above eluent and an 87d fraction was collected. , Seiki liquid 254 and 840
I watched it on nyn. 7 lactations 81-88 were pooled and extracted twice with 500 ml aliquots of chloroform. When chloroform is evaporated to dryness, homogeneous REM 1675 is obtained.
A265.8m9 was obtained.

B BM 1675 A2 is 2TLC system, 1) No 2-
It was homogeneous by dTIJc analysis and HPLC.

Example 4 Extraction method suitable for BBM-1675A Raw fermentation liquid (6,8
t) Polypropylene basin with faucet at the bottom (top 12 cIn diameter: bottom 10 cIn diameter; height 87 cm)
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. Approximately 4z (9 to 1,8) of Dicalite (filter aid) was added and mixed in. The mixture was filtered through a De13B-Iqualite pad held in a 412 Buchner funnel. Liquid bottle 19 with a vacuum outlet for liquid F [Ace/I65896-0
6:). The mat was washed with 2 tons of chloroform. The filtrate was transferred to a 201 separatory funnel and the phases were separated. The lower layer (chloroform) was removed.

91 g of Whelum silica gel (68-200 micron particles) was slurried back into a 2.5 crn internal diameter x 40 crn Glenco tube.

The chloroform phase was pumped into the column using a Fur RPY-2'C8D pump. The column was washed and rinsed with 600ml of fresh chloroform. Discard the chloroform eluate *. The column was then eluted with chloroform 910% methanol 600 WLt. This eluent was evaporated to dryness to yield a residue A347119.

■' Residue A was dissolved in chloroform 501. Add 1 chloroform solution to Dicalide 20I in a round bottom 7 flask! added to.

200ml of Skellisolve B was added to form a slurry.

The solvent was removed on a rotary evaporator. The residue was made into a slurry in Skarinlup B800ynl. Transfer the slurry to a Cease flask chromatography tube (part size B5872-14) (41 mm inner diameter x 45 mm) using the following procedure.
7cm). A fiberglass stopper was inserted into the neck of the stopcock between the stopcock and the column tube. A 1 cIn layer of standard Ottawa sand was added over the glass fibers. Stopcocks, glass fibers and sand beds were passed through them with a pressurized flow of Skelisorp B (5,7
psi).

The slurry was added to the column to form a packed bed under pressure flow. I made sure not to let the column dry out. After a stable column bed was obtained, a 2 cm layer of Ottawa sand was added to the top of the bed. Addition of bed skelesolve B 6
00-700 ml. 500 toluene for bed
It eluted with m1. The toluene eluent was evaporated to dryness to obtain a residue B98tn9. This partially purified EEM-1675 stock can also be further purified according to the procedure of Example 3.

Example 5 Actinomodula verrucospora strain/16H964
Using mutant strain 11B27Y obtained by -92ONTG treatment, 2% soluble starch, 1% glucose, 0.5% yeast extract, 0.5'JbNB-amine type A and 0
．． It was inoculated into a nutrient medium containing 1% CaCO5 and adjusted to pH 7.0 before sterilization. Place the nutrient medium on a rotary shaker (25
0 rpm) for 4 days at 82°C. 5ml of growth
was transferred to a 500 ml Erlenmeyer flask. This flask contains 8 grams of cane molasses, 1% cornstarch, 1
% fishmeal, 0.005% CuSO4・5#tQ, 0.05
%141−, MriS()4・7#tQ and 0.1%C
It contained a fermentation medium Zoom/ consisting of aCO3, 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 Seiryu reached a maximum value of approximately 1.5 mcvrnl.

Example 6' Isolation and Purification of RBM-1675 Components The fermentation broth (8,000 ts pH 7,6) collected from Example 5 was separated into mycelium cake and supernatant using a Sharpless centrifuge. The mycelium cake was stirred with 2,000 t of methanol for 1 hour and the insoluble materials were removed. □The active content contained in the broth supernatant was converted into 1' rubbutanol,
Extracted at 8'o t. methanol and n-
The butanol extracts were combined and azeotropically concentrated by adding water at any time to form an aqueous solution (-201), in which most of the antibiotic active ingredient was deposited as an oily solid. The mixture was shaken three times with 20 ml each of ethyl acetate to extract the active 142-component. The extracts were pooled, filtered to remove insoluble materials, and evaporated in vacuo to 4t. When the concentrate is added to 80t of n-hexane while stirring, crude BBM-1 is obtained.
675 complex pale yellow solid (81,7,9, potency: 5
9 rnc9/η) was obtained. The complex showed 92 main components, BBM-1675A,
It was found to be a mixture of A2 and several trace components. These components were separated and purified by a series of chromatographies carried out in a cold room to prevent deterioration.

Crude BBM-1675 complex (20,!ii') was dissolved in methanol (20mj) and Sephadex LH
-20 column (φ5.5 x 85 cm). The column was developed with methanol and elution was monitored by bioassay using Staphylococcus aureus 209P. The active eluates were pooled, concentrated in vacuo, and lyophilized to yield a semi-purified solid of BBM-1675 complex (4.86 g, titer: 208 mc9/r
ψ) was obtained. The solid was then chromatographed on a silica gel column (φ8.OX 70 cm) using chloroform and increasing amounts (1-5%) of methanol as developing solvents. The eluent was evaluated for antibacterial activity against S. aureus and TLC (Sift, CHCl, 3-M
eOH=5:1 v/v) and concentrated in vacuo. BBM-1675A, (425 after evaporation
#I9, titer: 960rILC,! lil/li) was first eluted with chloroform 92% methanol and then BBM
-Mixture of 1675A2, A3 and A4 (780 mg
, titer: 840 mcVzng) then BBM-16
75B complex (200rn9, titer: 190 rrL
tJ/m9) was eluted with 8 timethanol in chloroform. The above BBM-1675A1 was mixed with 2% methanol in benzene on silica gel (column: φ2.2x44cIn).
Chromatography was performed again. Bioactive eluent H
PLCC Liehrosorb BP
-18: CHsCN AfgOHO, IM CHsC
The homogeneous fractions containing BM-1675A1 were evaporated to dryness in vacuo. When the residual solid is crystallized from methanol (10 m/), BBM-1675, 4, (197
mg, titer: 1,000 m, cg/In9) was obtained as colorless prismatic crystals.

B B M 1675 At1As and A4 complex (587■) was added to Bondapak C08 (Waters,
φ2. It was separated by column chromatography using Ox42crrI). Elution was carried out with aqueous acetonitrile and the bioactive eluent was analyzed by TLC (Merck, silanization: CH,
CM-Hto==r 5 : 25, V′ν). Trace component E EM-1675A4 (45■, titer: 410vMjfl/1n9) and ABC19m9
: titer: 800 rncg/'nl) was sequentially eluted with 20 thiacetonitrile, and then the major component, 111-BBM-1675A, (208■), was eluted with 50% acetonitrile. When the E BM 1675 At fraction was crystallized from chloroform-n-hexane, colorless rod-like crystals (70 Ing, titer: 290 mcg/m
9) was deposited. The solid containing the BBM-1675 mixture was subjected to chromatography on a silica gel column (φB, □ x 40 an) using chloroform and methanol as a developing solvent. The active fractions eluted with 4-thimethanol in chloroform were pooled and evaporated to yield pure BM 1675Bt (7#117,
A titer of 180 mcfl/my) was obtained. The other active fraction was eluted with 5-timethanol concentration, which was evaporated to BBM-1675J3t (8rn9,
A titer of 140 mc9/71Q) was obtained.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of partially purified BBM 16754 (KBr-''e fret. -:L46- Figure 2 shows the infrared absorption spectrum of partially purified BBM 1675At (KBr Heret) Figure 3 shows the infrared absorption spectrum of BBM-1675A (
KBr-E! ). Figure 4 shows the infrared absorption spectrum of BBM-1675A4 (
KBr islet). Figure 5 shows the proton magnetic resonance spectrum of partially purified BBM-1675A in CDC1CDC13 (60 Mll z). Figure 6 shows the partially purified BBM-1675A in CDCAs (60 Mll z). Figure 7 shows the proton magnetic resonance spectrum of BBM- in CI) Cts (60 MHz).
1675A. This shows the rumored proton magnetic resonance spectrum. Figure 8 shows CDC1CDC13 (RBM-167 in 60)
5A. This shows the proton magnetic resonance spectrum of . FIG. 9 shows the infrared absorption spectrum (KBrdlet) of ABBM-1675AI. Figure 40 shows the proton magnetic resonance spectrum of purified BBM-1675A in CDCAs (860 MHz). Figure 11 shows the purified BBM-1675A, (013C magnetic resonance spectrum) in CDC1, (90,8MP1g). Figure 12 shows the infrared absorption spectrum (KBrBr fret) of purified BBM 1675At. Figure 13 shows the infrared absorption spectrum of purified BBM 1675At. L in CD Cts (860Mllz)
Figure 14 shows the proton magnetic resonance spectrum of BBM-1675A manufactured by N.
The I3c magnetic resonance spectrum of M-1675A is shown. 12/, f,, Jyonu'10 Temple 9 dezu 4-27 Continued from page 1 0 Inventor Hiroaki Okuma 3-19 Inaridai, Itabashi-ku, Tokyo 0 Inventor Yoguchi Hiroshi Kugayama, Suginami-ku, Tokyo 2-5-23 Procedural amendment (method) % formula % 1. Indication of the case 1982 Patent Application No. 96758 2. Name of the invention Novel anti-1111i antibiotic complex BBM-1675
3. Relationship with the case of the person making the amendment Manufacturer's name: Pristol-Myers Laboratory Co., Ltd. Akasaka Taisei Building (Telephone: 582-7161) Handwritten note 1 Specification 6 attached to the application, Contents of the amendment Procedural amendment 1i ( Method) % Formula % 1. Indication of the case 1982 Patent Translation No. 96758 2. Name of the invention Novel anti-abrasive and anti-abrasive substance complex BBM-16753/Relationship with the Sodemasa meeting Patent applicant Name: Drawing 6 attached to the Bristol-Myers Laboratory Co., Ltd. Contents of the amendments Attachment 1: dπ→)V However, the amendments to the contents are not included.

Claims (1)

[Scope of Claims] L in a substantially purified form, (a) having a white to pale yellow crystal appearance; (b)
Soluble in p-roloform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in hexane and carbon tetrachloride; and showed a positive reaction to Tollens' reagent and a negative reaction to the cross-grain, ninhydrin and Anthrone tests: (d) Infrared absorption spectrum substantially as shown in FIG.
KBr); (e) When dissolved in CDCll5, substantially
The proton magnetic resonance spectrum shown in the figure shows: (a) has a melting point in the range of about 156-158°C; (σ)
Optical rotation [α), = -191° (cO, 5, CHCl,
); (h) has an apparent molecular weight of 1248 as measured by mass spectrometry; (j) 52.17% carbon, 6.15% hydrogen;
4.63% nitrogen, 9.09% sulfur and 27.96% oxygen
% (difference) with approximate elemental composition: 0) Silica gel thin layer chromatography, with CHCl5
-CH,OH (5: 1 t'/, ) solvent system showed an Rf value of 0.74, and reverse phase silica gel thin layer chromatography showed an Rf value of 0.18 vf- in the CH3CMHtO (75: 25 Vv) solvent system. : (&) When dissolved in methanol at a concentration of 0.013569/l, it exhibits the following ultraviolet absorption maximum and absorption coefficient:
320 12.4 280 Shoulder 253 25.1 210 25.5 No significant change with addition of acid or base: (1) CU reverse phase silica gel column and solvent system CH3CN-CH5O
H0, IM CHsCOONHk (5: 2: 3V
v) exhibits a high performance liquid chromatography retention time of 13.3 minutes; (?L) is effective in inhibiting the growth of various bacteria and fungi; (?L) induces prophages in lysogenized bacteria; and 1
(O) Mouse P388 leukemia, L-1210 leukemia, B
BBM-1675A+, an antitumor antibiotic that is effective in inhibiting the growth of 16 melanocytoma and Lewis lung cancer. 2 in substantially purified form, ((L) white crystalline in appearance; (b) soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water; and Insoluble in n-hexane and carbon tetrachloride; (6) salt (positive reaction to IJ diiron, Ehrlich and Tollens reagents and negative reaction to the Itame, Ninhydrin and Anthrone tests; The infrared absorption spectrum (K
Br); (e) When dissolved in CDCIs, it exhibits a proton magnetic resonance spectrum substantially as shown in FIG.
Optical rotation [α)” = -179,4° (c O,5, C
lIC1jB): (h) Apparent molecular weight of 1248 as measured by mass spectrometry
(j) 52.71% carbon, 5.94% hydrogen
, nitrogen 3.94%, sulfur 9.39% and oxygen 28.0
It has an approximate elemental composition of 1% (difference): 4 (j) CHC by silica gel thin layer chromatography
IB-CIIsOH (5: 1 t'/- Rf in dissolution system
Reversed-phase silica gel thin layer chromatography showed an Rf value of 0.21 in CHsCN HtO (75: 25%) solvent system; (k) in methanol at a concentration of 0.020529/13; When dissolved, it exhibits the following ultraviolet absorption maximum and absorption coefficient: 5-λ(n) absorption coefficient 320 12.2282
16.3282
26゜2214 25.8 No significant change upon addition of acid or base 1; (1) CI8
Reversed phase silica gel column and CHsCN-CH30HO
, I M CHsCOONH+ (5: 2: , 3
Vg) Shows a high performance liquid chromatography retention time of 17.3 minutes in a solvent system; (e) Effective in inhibiting the growth of various bacteria and fungi; &L) Induces prophages in lysogenic bacteria; and (6) the antitumor antibiotic BBM-1675At, which is effective in inhibiting the growth of murine P388 leukemia, L-1210 leukemia, B16 melanocytoma, and Lewis lung carcinoma.
. a(α) Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride: (b) Chloride 1 positive reaction with ferric, Ehrlich and Tovans reagents and 1 negative reaction with the cross-cut, ninhydrin and anthrone tests; (C) Infrared absorption spectrum substantially as shown in Figure 3 <KB
(d) When dissolved in CDCIs, it exhibits the proton magnetic resonance spectrum shown in Figure 7: (g) has a melting point in the range of about 125-127°C; ω
Optical rotation [α] = -161° (c O-5, CHCl5
); (a) 54.55% carbon, 6.46% hydrogen, 3% nitrogen;
．． 73%, sulfur 7.49% and oxygen 27.77% (difference):
-CH3OH (5: IVv) solvent system with Ef value of 0.7
1, and then reversed-phase silica gel thin layer chromatography showed an Rf value of 0.28 in a CH3CN H20 (75: 25 v/v) solvent system; (gradient, methanol, O-01NHCII CHsOH
and when dissolved in 0.01 N NaOH-CH8OH, it shows the following ultraviolet absorption maximum, 320 (122) 320 (126) 31B (120) (j) C18 reversed phase silica gel column and CHsCN
CHsOH-0,I MCHRCOONII4 (5:
2:3 v/v) solvent system with high performance liquid chromatography retention time of 8.0 minutes; (&) Effective in inhibiting the growth of various bacteria and fungi: (1) In lysogenic bacteria and (c) an antitumor antibiotic B BM-1675As that is effective in inhibiting the growth of murine P-388 leukemia. 4(α) Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride: (b) Second chloride Showing positive reactions with iron, Ehrlich and Tollens reagents and negative reactions with cross-cut, ninhydrin and Anthrone tests; 9- (c) Infrared absorption spectrum α shown in actual value in Figure 4.
Br); Cd) When dissolved in CDCts, exhibits a proton magnetic resonance shear substantially as shown in Figure 8; (g) has a melting point in the range of about 123-126°C;
Optical rotation [α) fi7-176° (co, 5, CHCl
5); ((7) 54.65% carbon, 6.29% hydrogen, 3.51% nitrogen, 8.07% sulfur and 27.48% oxygen (
(difference); (h) CHC by silica gel thin layer chromatography;
l, -CH5OH (5: 1 v/v) solvent system showed an R1 value of 0.71 and reversed phase silica gel thin layer chromatography showed CHsCN HtO' (75: 25v/v)
Shows an Rf value of 0.78 in the solvent system; 10- (j) methanol, 0. OL# HCl-CH,O
When dissolved in H and 0.01N NaOH-CHBOH, it shows the following ultraviolet absorption extremes, 320 (117) 320 (118,) 318 (118); Ci) c, s reverse phase silica gel column and CH3C
N-C11, OHO, I Af Cll5COONHt
(5: 2: 3υ/υ) shows a high performance liquid chromatography retention time of 5.1 minutes: (k) Effective in inhibiting the growth of various bacteria and fungi.
(1) Induce prophages in lytic bacteria; and (-
BBM-1675A, an antitumor antibiotic that is effective in inhibiting the growth of murine P-388 leukemia. 5. (α) Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride: (b) No. chloride (c) has a melting point in the range of about 159-161°C;
Force Optical rotation [α) D 171 @ (60,5, CHC
Ls) Contains e: (g) CHC by silica gel thin layer chromatography
Rf! in l3-CH,OH (5: 1 v/v) solution system!
0.63 and reversed-phase silica gel thin layer chromatography showed CH3CN-H,0 (75:25 v/v
) shows an Rf value of 0.23 in a solvent system; shows the following ultraviolet absorption maximum when dissolved in methanol, 0.01 N HCt-CHsOH and QOIN NaOHCHsOH, 320 (104) 320 (105) 318 (105); (g) Various BBM-1675Bl is an antitumor antibiotic that induces prophages in lysogenic bacteria. 15-6, (α) chloroform, ethyl acetate, acetone, Soluble in ethanol and methanol, slightly soluble in benzene and water, and insoluble in roohexane and carbon tetrachloride; (6) Shows a positive reaction with ferric chloride, Ehrlich's and Tollen's reagents, and showed negative reactions in the cross-grain, ninhydrin and anthrone tests; (6) It has a melting point in the range of about 156-159°C: (
Optical rotation [α 7-122° (60,5, CHClm
>: (C) Silica gel thin layer chromatography (:H
Rf@0 with Cl-5-CHsOH (5:1 v/v)
．． 60 and Rf with CH3CN&O (75:25 vlυ) by reverse phase silica gel thin layer chromatography.
It shows a value of 0.16; methanol, 0. OINH
When dissolved in Cl-ClI5OH and 0.01-1←NNaOH-CH,Off, it shows the following ultraviolet absorption coefficient (7, 31B(103) 318(103) 318(110): (g) Various bacteria and fungi (A) BBM-1675B, an antitumor antibiotic that induces pro7age in lytic bacteria. Cultivate under deep aeration conditions in an aqueous nutrient medium containing carbon and nitrogen sources until a substantial amount of BBM-1675A is produced by the microorganism into the medium, and then extract BBM-1675A from the medium.
A method for producing an antitumor antibiotic BBM-1675A, which comprises collecting 1675At. 8.BBM 1675/it producing bacteria identified Characteristics Actinomadeura verchuspora strain H964-92<A
8. The method according to claim 7, comprising the strain A1327Y (ATCC39334), Actinomadeura verchuspora strain A1327Y (ATCC39638), or a mutant strain thereof. 9. EBM-16 of Actinomadeura belconsubora
754 = production strain produced under deep aeration conditions in an aqueous nutrient medium containing an assimilable carbon source in a substantial amount of B BM 1
675 Antineoplastic antibiotic BBM-1675, characterized in that BBM-1675A is collected from the medium after the culture until 675 Ax is produced by the microorganism in the medium.
Production method of At. I) Identification of BBM-1675,42-producing bacteria Features Actinomadeura verchuspora strain H964-92CA
10. The method according to claim 9, which is the strain A1327Y<ATCC39634), Actinomadeura verconspora strain A1327Y<ATCC39638), or a mutant strain thereof. Actinomadeura belconsubora BBM-167
5A3-Producer strains were grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources with substantial amounts of EB.
An anti-tumor antibiotic BEM characterized by culturing until M-1675A is produced by the microorganism in the medium, and then collecting EBM-1675A from the medium.
Production method of 1675As. L BBM-16'rsA, - Identification of the producing bacterium Characteristics Actinomadeura verchuspora strain H964-92CA
17. The method according to claim 11, wherein 17- TCC39334), Actinomadeura verchuspora strain A1327Y<ATCC3963B) or a mutant strain thereof. U, Actinomadeura verrucospora BBM-16
The 75 luyl-producing strain was grown under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources to produce a substantial amount of BB.
M'-1675A4 is produced by the microorganism in the medium, and then from the medium BBM-1675A,
An antitumor antibiotic BBM- characterized in that it collects
1675,44 production method. 14. Identification characteristics of BBM-1675-4-producing bacteria
14. The method according to claim 13, wherein the strain is Actinomadeura verchuspora strain A1327Y (ATCC39638) or a mutant strain thereof. 15. DI of Actinomadeura verchuspora) M-
cultivating the 1675B1-producing strain in an aqueous nutrient medium containing assimilable carbon and nitrogen sources under deep aeration conditions until a substantial amount of HEM-1675B1 is produced by the microorganism in the medium; Next, from the medium, BBM-1675B
Antitumor antibiotic B characterized by collecting 1 B
Production method of M 1675 Bt. 16. BBM-1675B, - Identification characteristics of the producing bacteria Actinomadeura belconspora strain H964-92CA
16. The method according to claim 15, which is an Actinomadeura verrucospora strain A1327Y (ATCC39334) or a mutant strain thereof. 17. BBM-1 of Actinomadeura verchuspora
675B, - the producing strain is cultivated under deep aeration conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources until a substantial amount of BBM-1675B is produced by the microorganism in the medium. , then BBM-1675B from the medium
2. A method for producing antitumor antibiotic BBM 1675Bt, which is characterized by collecting BBM 1675Bt. 1B, EBM 1675 Bt-producing bacteria identified Characteristics Actinomadeura verrucospora strain H964-92
(ATCC39334), Actinomadeura verchuspora strain Al327YCATCC39638) or a mutant strain thereof. B, BM 1675A1, BBM-16 effective antibacterial dose for animal hosts suffering from infections caused by 196 microorganisms.
75A2, B BAI-1675A3, BBM-167
54, BBM-1675BI or BBM-1675B
2. A method of therapeutically treating a host, comprising administering a. 20. Tumor-suppressing doses of BBM-1675AI, BBM-1675A, effective in animal hosts suffering from malignant tumors.
BBM-1675A3, BBM-16754, BBM-
1675B1''t or BBM-1675B2. 1675A,,BB
M-1675 bird, BBM-1675A BBM1675
A pharmaceutical composition containing B1 or BBM-1675B. 22. Effective tumor-inhibiting amounts of E B Af 1675 At , B B M in combination with a pharmaceutical carrier or diluent.
1675 A2, B B Af 1675A,,
BEM-1675A or BBM-1675B, or a pharmaceutical composition containing BBM-1675B. 23. A biologically pure culture of the microorganism Actinomadeura berchospora strain H964-92CATCC39334) capable of producing the antibiotic BBM-1675 in harvestable quantities by cultivation in an aqueous nutrient medium containing assimilable carbon and nitrogen sources. . 24. A biologically pure culture of the microorganism Actinomadeura verchuspora strain A2l-1327YCATCC39638) capable of producing the antibiotic BBM-1675 in harvestable quantities by cultivation in an aqueous nutrient medium containing assimilable carbon and nitrogen sources. .