CH663155A5 - Antitumour antibiotics - Google Patents

Abstract

The antitumour antibiotics BBM-1675 A1, A2, A3, A4, B1 and B2 are described. These compounds can be isolated from an antibiotic complex in which the first two occur as major components and the other four occur as subsidiary components. The complex is obtained by fermentation of certain strains of Actinomadura verrucosospora. The compounds can be used as active substances with antimicrobial action and antitumour action in pharmaceutical compositions.

Description

       

  
 



   The present invention relates to new antitu



  Morantibiotic compounds and their production and recovery. 



   The antitumor antibiotic compounds of the present inventions have not yet been structurally identified.  However, given their unique physical, chemical, and biological properties, BBM-1675 antibiotics are believed to be new substances. 



   EP-A1-95154 discloses the fermentation of Actinomadura pulveraceus sp.  nov  No.  6049 (ATCC 39100) to produce anti-tumor antibiotics designated WS 6049-A and WS 6049-B.  The structures of the WS 6049 antibiotics have not yet been elucidated, however, based on the characterization properties reported for WS 6049-A and WS 6049-B, it can be concluded that they are structurally related to the BBM-1675 antibiotics of the present invention could be. 

  However, the spectral data show that neither WS 6049 A nor WS 6049-B are identical to any of the BBM
1675 components.  Furthermore, the producing organism described in EP-A1-95154 can be clearly distinguished from the Actinomadura verrucosospora of the present invention, namely in the color of its aerial mycelium on ISP medium numbers.  2, 3 and 4, in its positive milk peptonization and in its positive use on D-fructose, D-mannitol, trephalose and cellulose. 



   The present invention provides novel anti-tumor antibiotic compounds, designated BBM-1675 Al, A2, A3, A4, B1 and B2 (in their entirety) in this specification, which are made by culturing a BBM-1675-producing strain of Actinomadura verrucosospora , in particular by Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638) or a mutant thereof in an aqueous nutrient medium which contains assimilable sources of carbon and nitrogen, under submergenic aerobic conditions.  The compounds BBM-1675 comprise two main bioactive components, namely BBM-1675 Al and A2, and the four bioactive secondary components BBM-1675 A3, A4, B1 and B2.  The components can be separated and purified by conventional chromatographic methods. 

  The bioactive components mentioned, also referred to in their entirety as BBM-1675 complex, all have antimicrobial and antitumor activity. 



   Description of the drawings
Fig.  1 shows the infrared absorption spectrum of partially purified BBM-1675 A, (KBr tablet)
Fig.  2 shows the infrared absorption spectrum of partially purified BBM-1675 A2 (KBr tablet)
Fig.  3 shows the infrared absorption spectrum of BBM-1675 A3 (KBr tablet)
Fig.  4 shows the infrared absorption spectrum of BBM-1675 A4 (KBr tablet)
Fig.  5 shows the magnetic proton resonance spectrum of partially purified BBM-1675 A1 in CDCl3 (60 MHz)
Fig.  6 shows the magnetic proton resonance spectrum of partially purified BBM-1675 A2 in CDC13 (60 MHz)
Fig.  7 shows the magnetic proton resonance spectrum of BBM-1675 A3 in CDCl3 (60 MHz)
Fig. 

   8 shows the magnetic proton resonance spectrum of BBM-1675 A4 in CDCl3 (60 MHz)
Fig.  9 shows the infrared absorption spectrum of purified BBM-1675 A1 (KBr tablet)
Fig.  10 shows the magnetic proton resonance spectrum of BBM-1675 Al in CDCl3 (360 MHz)
Fig.  11 shows the 13C magnetic resonance spectrum of purified BBM-1675 Al in CDCl (90. 3 MHz)
Fig.  12 shows the infrared absorption spectrum of cleaned BBM-1675 A2 (KBr-Tahlette)
Fig.  13 shows the magnetic proton resonance spectrum of purified BBM-1675 A2 in CDCl3 (360 MHz)
Fig. 

   14 shows the magnetic '3C resonance spectrum of purified BBM-1675 A2 in CDCl3 (90. 3 MHz). 



   The present invention relates to the new antitumor antibiotics as defined in claims 1-6 and their production by fermentation with certain strains of Actinomadura verrucosospora, as defined in claims 7-18.     As microorganisms such.  B.  Actinomadura verrucosospora strain H964-92 or mutants thereof in question e.g.  B.  Actinomadura verrucosospora strain A1327Y.  The parent strain above was isolated from a soil sample found at Pto Esperanza, Misiones, Argentina.  A biologically pure culture of the organism was  March 1983 at the American Type Culture Collection.    



  12301 Parklawn Drive, Rockville MD 20852 United States.  deposited and incorporated into the permanent collection of microorganisms as ATCC 39334.  The mutant strain A1327Y was later obtained by conventional nitrosoguanidine (NTG) treatment of the strain H964-92 and was  March 1984 deposited with the American Type Culture Collection as ATCC 39638. 



   As in the case of many cultures producing antibiotics, the fermentation Actinomadura verrucosospora strain H964-92 or strain A1327Y gives a mixture or a complex of component substances.    



  Two main bioactive components, BBM-1675 A, and A2.    



  and four bioactive minor components, BBM-1675 A3, A4, B1 and B2 were isolated from the BBM-1675 complex generated by the fermentation process. 



     BBM-1675 and its components BBM-1675 A1.     A2, A3, A4, B1 and B2 have antimicrobial activity against a wide range of microorganisms.  including gram positive bacteria.  The BBM-1675 complex and the bioactive components separated from it also have phag inducing properties in lysogenic bacteria.  Two of the components, BBM-1675 Al and A2, were subjected to in vivo screening against various mouse tumor systems and showed inhibitory activity against L-121 0 leukemia, P-388 leukemia, B16 melanoma and Lewis lung carcinoma.  BBM-1675 A3 and A4 have been shown to be active against mouse P-388 leukemia. 



  The complex and its bioactive components can thus be used as antimicrobial agents or as anti-tumor agents for the inhibition of tumors in warm-blooded animals. 



   The microorganism
Actinomycetes strain no.  H964-92 was isolated from a soil sample and prepared by conventional methods as a biologically pure culture for its characterization. 

 

  The strain H964-92 forms short spore chains on the aerial mycelium.  which have straight, flexible or curved shapes.  The spores are spherical or oval in shape and have a warty surface.  Aerial mycelium is weak in most media.  The color of the aerial mass is white and later shows a pink shade or further changes to a bluish color in certain agar media.  The color of the substrate mycelium is colorless or slightly pink.  The growth temperature ranges from 15 - 43 C. 

  The cell wall amino column. Implementation and the entire cell hydrolyzable sugar components.  which shows.  that the strain H9 (. 4-9 'belongs to cell wall type IIIB.  The menaquinone was identified as MK-9 (H6) MK-9 (H8).    



   Due to the main morphological cultivation and physiological characteristics in connection with the chemical cell wall composition characteristics, the strain H964-92 can be classified as belonging to the genus Actinomadura. 



   Although the original strain H964-92 showed only modest growth and carried only sparse aerial mycelia, the NTG (nitrosoguanidine) treatment of H964-92 improved the formation of aerial mycelium.  The variant, designated strain A1327Y, facilitated further taxonomic investigations and was subsequently identified as Actinomadura verrucosospora. 



   Methods
The media and procedures used for cultural characteristics testing and carbohydrate utilization were those developed by the International Streptomyces Project (Intl.     J.  Syst.    Bacteriol.  16: 313-340, 1966) were recommended.  Additional media created by S. A.  Waksman (The Actinomycetes, vol.  2) and G. . M.  Lvedemann (times.     J.  Syst.  Bacteriol.  21: 240-247, 1971) were also used.  The amino acid composition of the cell wall and the total cell hydrolyzate sugar components were determined according to the methods described by Becker et al.  in Appl.  Microbiol.  , 13: 236-243, 1965 and  Lechevalier and Lechevalier in The Actinomycetes, Ed.  H. 

  Prauser, Jena, Gustav Fischer Verlag, S.  393-405, 1970.  The menaquinones were identified by mass spectral analysis, according to the methods of Collins et al.  in J.  Gene.  Microbiol.  , 100: 221-230, 1977, and the menaquinone composition was shown based on the system described by Yamada et al.  in J.  Gene.  Appl.  Microbiol.  , 23: 331-335, 1977. 



   morphology
The H964-92 strain forms both substrates and aerial mycelia.  The substrate mycelium is long, branched and not fragmented into short fibers.  In the aerial mycelium, the short spore chains are monopodial or at the hyphal end.  Vertebral branches of spore chains are also observed near the hyphal end.  These spore chains contain 2-10 spores in a chain and are straightly movable or curved.  The spores have a warty surface and are spherical or elliptical (0.5-0.6 x 0.6-1.4 µm) in shapes with rounded or pointed ends.  After ripening, the spores are often separated from the empty shells.  Immobile spores, sporangia or hardened granules could not be noticed in any of the media tested. 



   Cultural and physiological characteristics
The growth of strain H-964-92 is weak to modest both in chemically defined media and in natural organic media.  The formation of the aerial mycelium is generally weak, but is modest in oats, flour, agar (ISP no.  3-medium) inorganic salt starch agar (ISP No.  4-medium) and Bennett's agar. 



  Spontaneous variants, which lack the aerial mycelium, occur in high frequency.  The color of the aerial mycelium is white, which later changes to a faint pink in oatmeal agar, inorganic salt starch agar and glycerin-asparagine agar (ISP no.  5-medium). 



  The color of the aerial mass changes to a bluish color after a long incubation (5 months) in oatmeal agar, glycerin-asparagine agar and tyrosine agar.  The color of the substrate mycelium is colorless to yellowish in Czapek agar, tyrosine agar, yeast extract-malt extract agar (ISP no.  2-medium), peptone yeast extract iron agar (ISP No.  6-medium) and is of a pink color in glucose-asparagine agar and glycerin-asparagine agar.  Melanoid and other diffusible pigments are not produced.  A variant no.  A1327Y, which was obtained from the original strain, predominantly forms a pale blue aerial mycelium and carries a large amount of aerial spore mass. 



   The strain H-964-92 grows at 15 CC, 28 "C, 37" C and 43 "C, but not at 10" C or at 47 "C.     It is sensitive to NaCl in 7% and resistant to lysozyme in 0.01%. 



   The cultural and physiological characteristics of the producing strain are shown in Tables 1 and  2 shown.  The recycling of the carbon sources is shown in Table 3. 



   Table 1
Cultural characteristics of strain H 964-92 (original strain ATCC 39334 and variant A 1327 Y) strain no.  H 964-92 original trunk variant no.  A 1 327Y Actinomadura verrucosospora (ATCC 39334) KCC A-0147 Trypton yeast extract agar G: abundant, flaky, modest, flaky, modest, flaky, (ISP no.    1) sedimented and not sedimented and not sedimented and not colored colored colored sucrose nitrate agar G: modestly low low (Czapek's Agar) R: colorless colorless colorless
A: scanty; light gray (264); none or scanty; reddish- none or scanty; faint to faint pink (7) white blue (185)
D: none none none glucose-asparagine agar G: modestly low low
R: white (263) to deep yellow-colorless colorless light-pink (27)
A:

   none or very sparse none or very sparse none or very sparse white faint pink (7) white
D: none none none
Table 1 (continued)
Cultural characteristics of strain H 964-92 (original strain ATCC 39334 and variant A 1327 Y)
Strain no.  H 964-92 original trunk variant no. 

  A 1 327Y Actinomadura verrucosospora (ATCC 39334) KCC A-0147 Glycerin-Asparagine Agar G: low to modestly modestly modest (ISP no.  5) R: colorless to slightly yellowish-light yellowish pink (28) slightly yellowish pink (28) to pink (28) deep yellowish pink (27)
A: low; slightly yellowish-modest; white to slightly modest; white to strong pink (28), after 5 mona-pink (4) pink (2) ten slightly bluish-gray (190)
D: none none none inorganic salt starch- G: quite modestly modest agar (ISP no.  4) R: yellowish white (92) slightly yellowish pink (28) slightly yellowish pink (28)
A: abundant; slightly pink (4) modest; slightly bluish; faint blue (185) to reddish gray (10) gray (190)
D: none none none tyrosine agar (ISP No.  7) G: modestly modestly modest
R:

   yellowish-white (92) strong yellowish-pink (26) strong yellowish-pink (26)
A: low; slightly yellowish-modest; white to modest: white to pink (28), much later (5 light pink (4) light pink (4)
Months) partly slightly bluish-gray (190)
D: none none none nutrient agar G: low to modestly low low
R: pale yellow (89) colorless to pale pink (7) colorless to pale pink (7)
A: none none none
D: none none none yeast extract-malt extract G: abundant abundant agar (ISP no.     2) R: pale yellow (89) strongly yellowish pink (26) strongly yellowish pink (26)
A: low; white (263) low; white to pale low; white to pale yellow (7) yellow (7)
D: none none none oatmeal agar G: modestly low low
R: colorless to pale pink (7) pale yellowish pink (31) pale yellowish pink (31)
A:

   low; reddish-white (9) very sparse; light pale very scanty; light pale to slightly bluish-gray blue (184) blue (184) (190)
D: none none none Bennett's Agar G: abundant abundant
R: greyish-yellow (90) strongly yellowish-pink (26) strongly yellowish-pink (26)
A: modest; white (263) modest; pale yellowish- none to yellowish-white (92) pink (31) and bluish-white (189)
D: none none none peptone yeast extract iron G: modestly abundant abundant agar (ISP no.  6) R: colorless colorless colorless
A: none none none
D: none none none * Observed after incubation at 37 C for 3 weeks. 

 

   ** Abbreviations: G = growth; R = reverse color; A = aerial mycelium; D = diffusible pigment. 



  *** Color and numbers in brackets correspond to the color standard in Kelly, K.  L.   & DB.    Judd: ISCC-NBS color-name charts illustrated with Centroid Colors.  U.  S.  Dept.  of Com.  Cir.  553, Washington, D. C. , Nov , 1975. 



   Table 2
Test reaction method and medium
Temperature range for maximum growth at Bennett's agar
Growth 28 37 "C; modest at 20 and 43" C; no growth at 10 and 47 C
Table 7 (continued) Test reaction method and medium gelatin liquefaction liquefaction glucose-peptone-gelatin medium starch hydrolysis hydrolysis starch agar plate reaction in skimmed no coagulation and fully-difco skimmed milk constant peptonization formation of the melanoid- not produced tyrosine agar, Peptone yeast iron agar pigments and trypton yeast extract broth nitrate reduction not reduced Czapek's glucose nitrate broth and glucose yeast extract nitrate broth resistance to NaCI growth at 5% or we tryptone yeast extract agar niger ;

   no growth at
7% lysozyme resistant.  Growth in trypton yeast extract agar
0.01% or less; no
Growth at 0.1%. 



  pH growth at pH tryptone yeast extract agar 5.0-9.5; no growth at pH 4.5 and 10.0. 



   Table 3
Use of carbon sources
Strain no.  H964-92 Actinomadura original variant verrucosospora
Strain no.  A1327Y KCC A-0147 Glycerin + + + D (-) - arabinose L (+) - arabinose + + + D-xylose + + + D-ribose + - - L-rhamnose + + + D-glucose + + + D- Galactose D-Fructose + + + D-Mannose -) -Sorbose Sucrose + + + Lactose Cellobiose + + + Melibiose Trehalose + + + Raffinose D (+) -Melezitose Soluble Starch + + + Cellulose + + + Dulcitol Inositol + - D- Mannitol + + + D-sorbitol salicin observer after incubation at 28 C for 3 weeks basic medium:

  Pridham-Gottlieb inorganic medium cell wall composition and total cell sugar components
The purified cell wall of strain H964-92 contains mesodiaminopimelic acid, but no glycine.  The entire cell hydrolyzate shows the presence of madurose (3-0 methyl-D-galactose), glucose and ribose.  The cell wall amino acid and the entire cell sugar components show that the strain H964-92 can be classified into cell wall type IIIB.  Two main components of the menaquinone have been identified as MK-9 (H6) and MK-9 (H8). 



   Taxonomic position of strain H964-92
The H964-92 strain has the following main characteristics: (1) Aerial spore chains: Short, straight, flexible or curved in shape.  (2) Spores: warty surface.  (3) Aerial mycelium: reddish or bluish color. 



  (4) Substrate mycelium: reddish in some media.  (5) Diffusible pigment: none.  (6) Mesophile.  (7) Cell wall type IIIB.  (8) Menaquinone system: MK - 9 (Hs) and MK- 9 (Hs).    



   These main characteristics show that the H964-92 strain belongs to the Actinomadura genus.  Early species of the genus Actinomadura were isolated from warm-blooded animals.  Some strains have also been obtained from plant materials.  However, many of the new species recently described have been isolated from the soil.  According to the numerical taxonomy and overview of the Actinomadura and the related Actinomycetes by Goodfellow et al.  in J.  Gene.  Microbiol.  , 112: 95-111 (1979), in particular Actinomadura species that originate from the soil are classified in group no.  7 of the 14 groups described.  Strain no.  H-964-92 is most similar to Group 7 species.  Nonomura and Ohara in J.  Ferment. 



  Technol.  , 49: 904-912 (1971) described five saprophytic species of the genera Actinomadura and Nonomura (J. 



  Ferment.  Technol.  52: 71-77 (1974) and Preobrazhenskaya et al.  (Actinomycetes and Related Organisms 12: 30 38 (1977)) published the keys for the identification and classification of the Actinomadura species.  As a result of comparison with the descriptions of the 30 species including organisms disclosed in patents, strain H964-92 appears most similar to Actinomadura coerulea according to the Preobrazhenskaya publication mentioned above and Actinomadura verrucosospora according to the Nonomura publication cited above. 



   The strain no.  H964-92 was compared directly to A. 



  verrucosospora strain KCC A-0147 and proved to be the A.  verrucosospora in the morphological, cultural and physiological characteristics as closely related.  Thus the strain H964-92 was classified as a new strain of Actinomadura verrucosospora. 



   It is understood that for the production of BBM-1675 the present invention, despite being described in detail with reference to the particular strain Actinomadura verrucosospora strain H964-92 (ATCC 39334) and its mutants strain A1327Y (ATCC 39638), is not applicable these microorganisms or to microorganisms, the characteristics of which are fully disclosed herein.  It is expressly intended that the invention encompass strains H964-92 and all natural and artificial BBM-1675 producing variants and mutants thereof. 



   The BBM-1675 antibiotics of the present invention can be prepared by culturing a BBM1675-producing strain of Actinomadura verrucosospora, preferably a strain of Actinomadura verrucosospora, with the identification characteristics of ATCC 39334 or ATCC 39638 or a mutant thereof, in a conventional aqueous nutrient medium.  The organism is grown in a nutrient medium containing known actinomycetes nutrient sources, e.g.  B.  Assimilable sources of carbon and nitrogen and optionally inorganic salts and other known growth factors. 



  Submerged aerobic conditions are preferred for the production of large quantities of the antibiotic, but can also be used for the production of limited quantities of surface cultures and bottles.  The general methods used for the cultivation of other Actinomycetes are also applicable in the present invention.  The nutrient medium should be suitable assimilable carbon sources such as glycerin, L - (+) - arabinose, D-xylose, D-ribose, L rhamnose, D-glucose, D-fructose, sucrose, cellobiose, soluble starch, D-mannitol or inositol. 

  As nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc.  can be used either alone or in combination with organic nitrogen sources such as peptones, meat extract, yeast extract, corn water, soybean powder, cottonseed flour, etc. 



  If necessary, inorganic nutrient salts can also be used as sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron and the like.  be used. 



   The production of the antibiotics BBM-1675 can be carried out at any temperature which allows a satisfactory growth of the producing organism, e.g.  B. 



     15-45 C, and is usually carried out at a temperature around 27 - 32 C  Usually the optimal production is obtained after incubation times of 68 - 180 h, depending on whether a shaking flask, a stirring vessel or tank fermentation is used.  When tank fermentation is carried out, it is desirable to prepare a vegetative inoculum in a nutrient broth by inoculating the broth culture with a slant or bottom culture of the producing organism.  After an active inoculum is obtained in this way, it is transferred aseptically into the fermentation tank medium.  Antibiotic production can be followed by a paper disc agar diffusion test using Staphylococcus aureus 209P as the test organism. 



   Isolation and cleaning
When fermentation is complete, the BBM1675 complex can be obtained from the broth by conventional isolation methods, such as liquid extraction.  For example, the entire broth can be separated into a mycelial cake and the broth supernatant by filtration or centrifugation.  The antibiotic in the mycelial cake can be obtained by slurrying the cake in methanol, filtering off the insoluble material and concentrating the methanolic extract.  The activity in the broth supernatant can be obtained by extraction with n-butanol. 

  The above-mentioned n-butanol and methanol extracts can then be combined and evaporated azeotropically, an aqueous solution being obtained which excretes most of the antibiotic activity as an oily solid.  The solid can then be dissolved in methanol and the solution filtered.  The filtrate is concentrated and added to a mixture of ethyl acetate and water.  The organic extract obtained contains the crude BBM-1675 complex, which can be precipitated from the solution, by adding an anti-solvent, such as n-hexane. 



   The raw BBM-1675 complex is a mixture of different components, including the two main bioactive components BBM-1675 A and A2 and the four bioactive secondary components BBM-1675 A3, A4, B1 and B2.  These bioactive components can be separated and purified by conventional chromatographic methods.  In one method, the crude BBM-1675 complex is dissolved in methanol and then purified by Sephadex LH-20 column chromatography using methanol as the eluent.  

  This partially purified complex can then be chromatographed on a silica gel column and then gradually eluted using chloroform and an increasing concentration of methanol to give BBM-1675 Al, a mixture of BBM-1675 A2, A3 and A4 and a mixture of Obtain BBM-1675 B1 and B2.  The A1 components can be further purified by Sepha dex LH-20 column chromatography using methanol as the eluent.  The mixtures of A2, A3 and A4 can be purified by chromatography on a Bondapak C18 column (Waters Associates, Inc. ) using increasing concentrations of aqueous acetonitrile as eluent. 

  The mixture of components B1 and B2 can be separated by silica gel column chromatography using a mixture of chloroform and methanol as the eluent.  Further details of the preferred chromatographic separation methods are provided by the following examples. 



  Physicochemical properties of the BBM-1675 components
The six bioactive components of the BBM-1675 complex can be distinguished from one another by two thin layer chromatography (TLC) systems, as shown in Table 4 below. 



   Table 4
TLC of BBM-1675 components
RF values components 5102 * silanized CHCL3-CH30H (S: I v / v) CH3CN-H20 (75: 25v / v) BBM-1675A1 0.74 0.18 BBM-1675A2 0.71 0.21 BBM-1675 A3 0.72 0.28 BBM-1675 A4 0.71 0.78 BBM-1675 B1 0.63 0.23 BBM-1675B2 0.60 0.16 * Cls reverse phase silica gel
The separation of BBM-1675 A2, A3 and A4 was difficult by ordinary phase TLC systems, but could be done by reverse phase TLC. 



   The individual BBM-1675 components show solubility and color reactions that are similar to each other.  Z. B. 



      they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride.  They give positive reactions with iron chloride, Ehrlich and Tollen's reagent, but negative reactions in the Sakaguchi, Ninhydrin and Anthron tests. 



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



   Table 5
Physico-chemical properties of the BBM-1675 components
BBM-1675 Al A2 A2 A4 B1 B2 Melting point (dec. ): 156-158 "147-149 125-127 123-126 159-161 156-159 [aai; 7 (c 0.5 CHCl3): -191 -179.4" -161 -176 -171 -122 Anal.  found (%) C: 51.52 53.81 55.00 53.67
H: 5.81 6.31 6.52 6.35
N: 4.02 3.82 3.57 3.45
0: 38.65 36.06 34.91 36.53 by difference UV #max nm (E 1cm):

   253 (325) 253 (281) 253 (286) 253 (257) 253 (225) 248 (212)
282 (195) 282 (172) 282 (158) 282 (153) 282 (140) 279 (141)
320 (143) 320 (128) 320 (122) 320 (117) 320 (104) 318 (103) in 0.01N Hc1-CH3OH: 253 (323) 253 (276) 253 (287) 253 (258) 253 ( 225) 248 (210)
282 (192) 282 (167) 282 (160) 282 (155) 282 (140) 279 (140)
320 (144) 320 (128) 320 (126) 320 (118) 320 (105) 318 (103) in 0.01N NaOH-CH3OH 252 (325) 252 (289) 252 (280) 252 (266) 252 (236 ) 248 (233)
283 (172) 283 (171) 283 (162) 283 (160) 282 (141) 278 (150)
318 (136) 318 (122) 318 (120) 318 (118) 318 (105) 318 (110) Molecular weight (approximate value):

   1,300 1,100 1,100 1,400 (Gel HPLC = high pressure liquid chromatography, Finepak, GEL-lol)
The UV absorption maxima of the BBM-1675 components were observed at 253, 282 and 320 nm; they did not shift either in an acidified or in an alkaline solution.  The IR and PMR spectra (magnetic proton resonance) of BBM-1675 A1, A2, A3 and A4 are shown in Fig.  1 - 4 or  in Fig.  5-8.  The 360 MHz PMR of BBM-1675 A1 have an acetyl (6: 2.11 ppm), an N - CH3 (2.52 ppm), four OCH3 (3.42, 3.80, 3.88 and 3, 98 ppm) and an exomethylene (4.57 and 5.48 ppm), along with two aromatic protons (7.50 and 8.59 ppm) and an NH proton (11.79 ppm. 

  The CMR spectrum) of BBM-1675 Al showed 55 carbon signals including a signal with triple intensity (o: 56.0 ppm, OCH3).  The molecular formula of BBM-1675 A, is derived as C57H72N4032, based on proton and 13C NMR spectra, microanalyses and molecular weight determinations by HPLC and SIMS (secondary ion mass spectrometry). 



   Structural studies of BBM-1675 Al
When treated with 0.5N HCI-CHIOH at room temperature, BBM-1675 A loses its bioactivity and results in a lipophilic chromophore substance (compound I) together with various unidentified fragments.  Compound I shows UV absorption which is similar to that of the corresponding antibiotic, which allows the conclusion that compound I contains the chromophore structure of BBM-1675 A1. 

  Two further chromophoric fragments, which are related to compound I, are obtained by alkaline hydrolysis of BBM-1675 A1: hydrolysis with 0.05N KOH - CH30H at 55 C for 1 h gives compound II with the UV absorption maxima at 252, 284, 297 (shoulder) and 322 nm, while the reaction in IN KOH-CH3OH gives an acidic chromophoric substance, which is designated compound III.  The physicochemical properties of compounds I, II and III are summarized in Table 6 below. 



   Table 6
Compound I Compound II Compound III F.  .    82-83 "C 133" C 253-255 iC [ai39 (c 0.2 CHCl3) -100 "0 0 molecular formula: C2lH3lNOlo C14H1, NO6 Cl3HlsNO6 UV Smai nm (±): 244 (21,850) 252 (26,600) 248 ( 26,900)
276 (9,400) 283 (11,200) 295 (14,400)
318 (6,300) 297 (sh8,800) 310 (13,500)
322 (11.700) MS m / z 457 (M +) 295 (M +) 281 (M +)
425 280 263
341 263 236
281 251 222
264 248 218 TLC (xylene- * MEK-CH3OH =. 

  Rf0.58 0.66 0.13 5: 5: 1 v / v) * MEK = methyl ethyl ketone
The structural information about the compounds II and III resulted from the following spectral data and the chemical transformation.  13C and proton NMR indicated the presence of four OCH3, one = CH2, seven
I -C =, branch C = 0 and a> NH group in compound II.  The NMR spectrum of compound III was similar to that of II, only differing in the absence of one of the four OCH3 groups observed with the compound of formula II.  This difference, together with the acidic nature of compound III, suggests that II is a methyl ester of compound III. 

  When heated under reflux in 1N methanol KOH, Compound II is converted quantitatively to Compound III while Compound III was converted to Compound II when treated with diazomethane.  The compound of the formula II with NaBH4 in C2H5OH gave a reduction product (compound IV, M +: mtz 267) which, as the NMR showed, had a - CH2OH group instead of the COOCH3 group of the compound II.  After hydrogenation with palladium on carbon, compound II gave a dihydro derivative (compound V, M +: mlz 297).  The proton NMR spectrum of compound V showed a new doublet methyl signal and the absence of the exomethyl group present in compound II. 

  Furthermore, one of the OCH3 groups in compound V appeared in a higher region (8: 3.50 ppm). 

 

  This result shows the presence of one
EMI10. 1
 Group in compound II, which by hydrogenation to the group
EMI10. 2nd
   was reduced in compound V.  Compound II was heated with 1.5N methanolic hydrogen chloride at 80 C for 3 h and the hydrolyzate was chromatographed on a silica gel column to give a weakly basic compound (Compound VI, M +: m! Z 911).    



  The IR spectrum and the physicochemical properties showed that the compound VI contained an NH2 group.  Compound VI was identified as methyl 4,5-dimethoxy-anthramilate by comparing the IR and NMR studies with an authentic sample.  As a result, the structures of Compounds 11 - VI were determined as shown below.   



   Structures of Compounds II, III, IV, V and VI Compound II Compound III
EMI11. 1


 <tb> <SEP> CB2
 <tb> CH30 <SEP> H- <SEP> y / 2 <SEP> CH
 <tb> <SEP> S <SEP> \ 0CH, <SEP> 3 <SEP> co-¸03
 <tb> cE3 <SEP> OCH3 <SEP> C <SEP> \ COOH
 <tb> 3 <SEP> u <SEP> OOE
 <tb> <SEP> \ <SEP> after
 <tb> <SEP> connection <SEP> IV
 <tb> <SEP> CE
 <tb> <SEP> 2Pd / c <SEP> CH3 <SEP> -c0-j2
 <tb> <SEP> + / MeOH <SEP> CH3 <SEP> o · <SEP> 20B <SEP> O3
 <tb> <SEP> connection <SEP> V
 <tb> <SEP> connection <SEP> VI
 <tb> <SEP> 30,
 <tb> <SEP> CH <SEP>> <SEP> NE2
 <tb> <SEP> L
 <tb> <SEP> CH3 <SEP> OOCH3 <SEP> CH3 <SEP> 0 <SEP> COOCH3
 <tb>
After treatment with 0.05N KOH - CH30H at 55 "C, the compound I was split into a new, chromophore fragment (compound VII, Cl5H2lNO7, M +: m / z 327) and a sugar (compound VIII).

  The NMR spectrum of VII showed a singlet C - C113 and two OCH3 groups in the high area in addition to three OCH3 groups in the low area and two aromatic protons, which were usually observed for the compounds II, V and VI. Further hydrolysis of VII with 1.5N methanolic hydrogen chloride gave compound VI, which was identical to that obtained from II. After removal of VI, the hydrolyzate was treated with 2,4-dinitrophenylhydrazine to precipitate a yellow solid which was identified as 2,4-dinitrophenylhydrazone of pyruvic acid. Thus compound VII is methyl 4,5-dimethoxy-N- (2 ', 2'-dimethoxypropionyl) anthranilate.

  The compound of formula VIII showed no molecular ion peak, but showed the M-OCH3 peak at m / z 131 in the mass spectrum, in agreement with the molecular formula C7HI404. The NMR spectrum revealed the 2,6-dideoxyhexopyranose structure for compound VIII. This assignment was confirmed by the 3C NMR spectrum of compound I, which gave an increase to one C - CH3, one - CHp, three 0 - CH <and an anomeric carbon atom, in addition to 15 carbon signals that could be attributed to compound VII. The C3 proton of the sugar, which appeared in a low region (8: 5.39 ppm, octet), showed that the C3 - OH of the sugar was esterified with the carboxy group of VII.

  The above results are summarized below.



   Structure of compounds I, VII and VIII compound 1
EMI12.1
 Compound VII Compound VI
EMI12.2


 <tb> <SEP> OCH
 <tb> 3 <SEP> 2 <SEP> OCH <SEP> NH-CO-C11-CH33 <SEP> CH30 <SEP> C <SEP> 2
 <tb> CH3O <SEP> OCH3 <SEP> + <SEP> CH30 <SEP> CH3OM1 <SEP> 3
 <tb> <SEP> 3 <SEP> 11 <SEP> COOCH
 <tb> <SEP> connection <SEP> VIII
 <tb> <SEP> OH
 <tb> CH3 <SEP> $ <SEP> OH
 <tb> <SEP> CH3 <SEP> CH3-CO-COOH
 <tb> <SEP> 3
 <tb>
The molecular weight of compound I (457) is responsible for about a third of the entire BBM-1675 A1 molecule (proposed formula C57H72N4032; calculated molecular weight = 1324).

  The substructure of BBM1675 A1 is considered as follows:
EMI12.3

After the registration date of the original application with the serial no. 495,231 in the United States, it was discovered that the components BBM-1675 Al and A2 described above and prepared in accordance with Example 2 were in fact not completely pure and that certain characterization properties used to define these compounds were inaccurate. According to additional chromatographic purification procedures, as fully described in Examples 3 and 6 below, BBM-1675 A1 and A2 were isolated in purer form and fully characterized as described below.

  Elemental analysis data for compounds A3 and A4 were also revised to show the presence of sulfur in these compounds and HPLC retention times were calculated for these two compounds. The revised physicochemical properties of the BBM-1675 components are summarized below.



     BBM-1675 A,
Description: white to pale yellow crystals;
F. 156-158 "C (dec.) Elemental Analysis Analysis 1 Analysis 2 Mean C: 51.60% C: 52.74% C: 52.17% H: 6.31% H: 5.99% H: 6 , 15 N: 5.31% N: 3.94% N: 4.63% S: 8.47% S: 9.71% S: 9.09% O (by Diffe- O (by Diffe- O ( by difference): 28.31% margin): 27.62% margin):

   27.96% ultraviolet absorption spectrum: Instrument-Varian UV,
Cary 219
Solvent methanol
Concentration 0.01356 g / l ha, (nm) absorption coefficient 320 12.4 280 sh (shoulder) 253 25.1 210 25.5
No significant change with acid or base Optical rotation: solvent - CHCl3 [a] D24 = -207 (C = 0.0351)
A second analysis showed the following optical rotation: [a] D27 = -191 "(C = 0.5, CHC13).



  Infrared absorption spectrum: See Fig. 9
Main absorption bands (KBr):
985, 1015, 1070, 1110, 1150, 1210, 1250,
1308, 1380, 1405, 1446, 1520, 1592, 1608,
1668, 1715, 2920, 2960, 3360, 3440, cm- 'mass spectra: instrument - VG-ZAB-2F
FAB-MS thioglycerin
Molecular mass range ions (m / z): 1249, 1357, 1463; with addition of NaCl (m / z): 1271.1379.1485.1597.



   FAB-MS-MB (MB: Matrix, M.G. 154); Molecular mass range ions (m / z): 1249, 1283, 1403, 1555; with addition of NaC1 (m / z): 1249, 1271, 1303, 1425, 1483, 1577.



   FAB-MS-Glycerin-DMSO: Molecular mass range ion (m / z): 1215, 1247, 1279, 1293, 1325.1353; with addition of NaCl (m / z): 1215, 1237, 1247, 1269, 1325, 1347,
1375.



   Instrument: Kratos MS-50
FAB-MS thioglycerin; Molecular mass range
Ions (m / z): 1357, 1463.



  Molecular weight (based on the mass spectral data described above):
Apparent MG = 1248 nuclear magnetic resonance spectra:
Instrument - WM360 Brucker solvent: CDC13
1NMR: 360 MHz 8 (ppm): 11.75 (1H, s); 8.55 (1H, s); 7.45 (1H, s); 6.61 (1H, m); 6.23 (1H, brs); 6.17 (1H, brs); 5.93 (1H, d, J = 9.3); 5.82 (1H, d, J = 9.3); 5.7 (1H, brs); 5.49 (1H, m); 5.45 (1H, d, J = 2.3); 5.38 (1H, brs); 4.95 (1H, d,
J = 10.2); 4.64 (2H, m); 4.54 (1H, d, J = 2.3); 4.2 (1H, s); 4.15-3.35 (26-28H) [4.10 (1H, m); 4.02 (1H, brs); 3.95 (3H, s); 3.85 (3H, s), 3.79 (3H, s); 3.46 (1H, m); 3.40 (3H, s)]; 2.82-2.70 (311, brm);

   2.50 (3H, s), 2.47 (1H, m);
2.38 - 2.22 (5H); 2.12 (1H, m); 2.11 (3H, s); 1.60-1.05 (22H) [1.39 (3H, d, J = 6.3); 6.31 (3H, d, J = 6.3) - 1.29 (3H, d, J = 6.33, 1.08 (6H) j
See Fig. 10 13CNMR: 90.3MHz
See Fig. 11
In a separate test, the 3C NMR spectrum of purified BBM-1675 Al was determined, for an example dissolved in CDCl3 (80 MHz). The main peaks are given below.



   CMR of BBM-1675 A1 (80 MHz in CDCl3)
Chemical shift in ppm (multiplicity *) BBM-1675 Al 13.7 (q) 16.6 (q) 17.5 (q) 19.8 (q) 22.2 (q) 22.6 (q)
23.4 (q) 29.0 (t) 34.0 (t) 35.1 (t) 39.5 (t) 47.2 (d)
52.5 (q) 55.6 (u) 56.0 (q) 57.1 (d) 62.4 (t) 64.5 (d)
67.7 (d) 68.2 (d) 68.8 (t) 69.2 (d) 69.6 (t) 70.2 (d)
71.9 (d) 76.0 (d) 76.6 (d) 77.1 (u) 77.3 (d) 83.4 (s)
86.6 (d) 88.4 (s) 90.5 (t) 97.2 (d) 98.3 (s) 99.0 (d)
99.6 (d) 103.8 (d) 107.6 (s) 112.5 (d) 123.1 (d) 124.9 (d) 13û.1 (d) 131.5 (s) 134.9 (s) 136.7 (s) 144.0 (s) 147.2 (s)
153.8 (s) 154.4 (s) 155.0 (s) 160.7 (s) 166.4 (s) 191.8 (s) * Multiplicity - q = quartet; d = doublet; u = uncertain; t = triplet; s = single.



   BBM-1675 A2 Description:
White crystals; F. 147-149 C Elemental analysis: C: 52.71%
H: 5.94%
N: 3.94%
S: 9.39%
O (by difference): 28.01% ultraviolet absorption spectra: Instrument - Varian UV, Cary 219 solvent: methanol concentration: 0.02052 g / l Bm (nm) absorption coefficient 320 12.2 282 16.3 252 26.2 214 25.8
No significant change with acid or base.



  Optical rotation [a] D27 = - 179.4 (c 0.5, CHC13) Infrared spectrum:
See Fig. 12
Instrument: Beckman IR Model 4240
Main absorption bands (KBr): 950, 1015, 1070, 1100,
1155, 1213, 1250, 1313, 1375, 1405, 1450, 1520, 1595,
1610, 1685, 1735, 2940, 2980, 3440, cml.

 

  Mass spectra:
Instrument: VG-ZAB-2F
FAB-MA thioglycerin
Molecular mass range ions (m / z): 968, 1249, 1355,
1357, 1463, 1569; with addition of NaCl (m / z): 990, 1271,
1379, 1485, 1593 FAB-MS-MB (MB: Matrix, M.G. 154);
Molecular mass range ions (m / z): 1249, 1403, 1419,
1555, 1571, 1587; with addition of NaCl (m / z): 1249,
1271, 1425, 1441, 1457, 1483, 1577.



   FAB-MS glycerin DMSO; Molecular mass range ion (mlz): 1215, 1231, 1247, 1263, 1279, 1293, 1309, 1325, 1326, 1341, 1353, 1369.



  Molecular weight: (based on the mass data described above):
Apparent MG = 1248 nuclear magnetic resonance spectra:
See Fig. 13
Instrument: WM 360 Brucker
Solvent: CDCl3 1 H NMR 360 MHz 8 (ppm): 11.91 (1H, s); 8.62 (1H, s);
7.58 (1H, s); 6.56 (1H, m); 6.22 (1H, s); 6.15 (1H, brs); 5.91 (1H, d, J = 9.6); 5.83 (1H, d, J = 9.6); 5.70 (1H, m); 5.45 (1H, d, J = 2.2); 5.44 (1H, s), 5.34 (1H, brs); 4.95 (1H, d, J = 10.2); 4.75 (1H, m); 4.65 (1H, d, J = 6.8); 4.54 (1H, d, J = 2.2); 4.47 (1H, m); 4.18 (1H, s); 4.10 (1H, brs),
4.05 - 3.50 (20 - 24H); [3.96 (3H, s); 3.87 (3H, s);

   3.77 (3H, s)]; 3.46 (1H, m); 3.39 (3H, s); 2.79 (111m); 2.73 (2H, m); 2.50 (3H, s); 2.50 (1H, m); 2.38-2.22 (3H): 2. 14 (1H, m); 2.10 (3H, s); 1.98 (2H, m); 1.65 - 1.45 (6-8H);
1.38 (3H, d, J = 6.0); 1.34 (3H, d, J = 6.0); 1.22 (3H, d, J = 6.8); 1.10 (6H).



   Ú C NMR 90.3 M11z
See Fig. 14
In a separate test, the 3C NMR spectrum of purified BBM-1675 An was determined, for a sample dissolved in CDCl3 (80 MHz). The main peaks are given below.



  BBM-1675 A2 13.7 16.9 17.5 19.8 22.3 22.7 23.4 33.1
34.1 35.1 39.3 47.6 52.6 55.7 56.0 56.1
57.6 62.4 64.5 64.9 65.9 68.3 69.2 69.7
71.9 73.6 75.8 76.1 77.1 77.7 78.1 78.3
83.3 86.2 88.4 90.4 97.2 98.3 99.1 99.5
99.6 103.8 107.1 112.4 123.2 124.8 129.9 137.3
144.1 154.2 154.5 160.9 167.9 192.2
Table 7
Physico-chemical properties of BBM-1675 A3, A4, B1, B2
A3 A4 B1 B2 Melting point (dec.) 125-127 C 123-126 C 159-161 C 156159 C [α] D27 (c0.5, CHCl3) -161 C -176 C -171 C -122 C Anal. found (%):

  C: 54.55 54.65
H: 6.46 6.29
N: 3.73 3.51
S: 7.49 8.07 UV max nm (E1'cm) 253 (286) 253 (257) 253 (225) 248 (212) in MeOH 282 (158) 282 (153) 282 (140) 279 (141)
320 (122) 320 (117) 320 (104) 318 (103) in 0.01N HC1-MeOH: 253 (287) 253 (258) 253 (225) 248 (210)
282 (160) 282 (155) 282 (140) 279 (140)
320 (126) 320 (118) 320 (105) 318 (103) in 0.01N NaOH-MeOH: 252 (280) 252 (266) 252 (236) 248 (233)
283 (162) 283 (160) 283 (141) 278 (150)
318 (120) 318 (118) 318 (105) 318 (110)
Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) data from BBM 1675 components A.

  Study No. 1 - Summary Table 8 TLC and HPLC of BBM-1675 components
TLC (Rf) HPLC (retention time in min)
SiO2 * Silanized Lichrosorb RP-18 CHCI3MeOH CH3CN-H20 CH3CN-MeOH-0.1M CH3COONH4 (5: 1 v / v) (75:25 v / v) (5: 2: 3 v; v) BBM-1675A1 0. 74 0.18 13.3 BBM-1675A2 0.71 0.21 17.3 BBM-1675A3 0.72 0.28 8.0 BBM-1675A4 0.71 0.78 5.1 BBM-1675B1 0.63 0 , 23 BBM-1675B2 0.60 0.16 * C, 8 reverse phase silica gel
B. Study No. 2 - TLC and HPLC for purified Al and A2 components
TLC chromatography
Analtech GHLF silica gel uniplates were used as plates for normal phase chromatography. 2.5 cm x 10 cm plates were used for the one-dimensional TLC.

  These were developed in glass cylinders measuring 6.4 cm (outer diameter) x 12 cm and containing 10 cm of eluent. For the two-dimensional TLC plates with the dimensions 7.5 cm x 10 cm were used. The samples were applied to the lower left corner 1 cm from the edge. The plates were first developed in a tank (12.7 cm wide, 8.6 cm deep, 13 cm high) containing 50 ml of the first eluent. The plates were then air dried 90 rotated counterclockwise and developed in a second tank with 50 ml of the second eluent.



   Whatman's analytical precoated Css silica gel plates were used for reverse phase chromatography. The 2.5 cm x 7.6 cm plates were developed in glass cylinders with 10 ml of eluent.



   The normal phase plates were viewed under ultraviolet light with a wavelength of 254 nm. The plates were then placed in a glass cylinder (6.4 cm outer diameter x 12 cm) containing I2 crystals. After approximately 2 minutes, the plates were then checked again. The reverse phase plates were viewed only with UV light with a wavelength of 254 nm. The zones were determined by examining the quenching of the fluorescence of an impregnated dye.



   Analytical HPLC
The following components were used to build an analytical HPLC system: Waters Associates Model 6000A solvent delivery system pump; Varian Varichrom model VUV-10 uv / vis detector for 254 nm 0.1 OD; Fisher Recordal Series 5000 writer; Waters Associates Model 660 Solvent Programmer; Waters Associates Model U6K Injector; Alltech, tt- Bondapack C15 (101l) column (4.6 mm inner diameter x 25 cm) with a Whatmann Co. Pell ODS (0.03 - 0.038 mm) protective column (4.6 mm inner diameter x 5 cm). The components were connected with 316 stainless steel tubes (1.6 mm outer diameter - 0.23 mm inner diameter). The eluent was conveyed at 2 ml / min for all analyzes.



   Preparative HPLC
The following components were used to build a medium pressure liquid chromatography system: Fluid Metering, Inc. Model RP-SY 2CSC FMI laboratory pump; Fluid Metering, Inc. Model PD-60LF FMI Pulse Humidifier; a 15 ml sample loop, made of polypropylene tube (3.0 mm outer diameter x
1.5 mm inner diameter) by wrapping around a cardboard tube (8.65 cm outer diameter); Glenco 3500 Series Universal LC Columns; Instrument Specialties Co. Model UA-5 Absorption / Fluorescence Monitor with Type 6 Optical Unit; Instrumentation Specialties Co. Model 590 Flow Cut-Off Valve; and an Instrumentation Specialties Co. Model 328 fraction collector.



  The components were connected with polypropylene and Teflon tubing (3.0 mm outer diameter x 1.5 inner diameter) and Glenco Multifit connectors and valves in the order shown.



   The Glenco Series 3500 Universal LC columns were packed by slurrying the defined adsorbent in the specified solvent using standard techniques. The empty space between the bed placed in the pipe end was filled with standard Ottawa sand. The eluent was conveyed at a maximum so that a pressure of 4.14 bar was not exceeded (approximately 20 ml / min).



   Gradient elution
A Glenco gradient elution apparatus, consisting of two chambers of the same diameter, height and volume, connected in tandem with a Teflon valve, was used for all gradient elutions. One chamber served as a mixing chamber and the other as a static reservoir. Less polar solvents were originally added to the mixing chamber. The more polar solvent was added to the static chamber. Teflon-coated magnetic stirring bars (1.0 x 3.7 cm) were placed in both chambers and driven with a Thomas Model 15 Magne-Matic magnetic stirrer.



  The eluent was conveyed from the mixing chamber in a medium pressure HPLC system via polypropylene tubing (1.5 mm inner diameter x 3.0 mm outer diameter). When the eluent was removed from the mixing chamber, the eluent from the static reservoir was allowed to replace it, creating a linear elution gradient.



   TLC analysis of BBM-1675 Al and A2
Table 9 below shows the observed Rf values for BBM-1675 A1 and A2 on normal phase plates. The Rf value is calculated by differentiating the measured distance from the center of the zone to the point of the sample application by the measured distance from the solvent front to the point of the sample application.

 

   Table 9
RF system / connection BBM-1675 Al BBM-1675 A2
4% methanol in chloroform 0.33 0.30
5% methanol in diethyl ether 0.39 50% acetone in Skellysolve B 0.38 0.31
Table 10 below summarizes the observed Rf values of BBM-1675 Al and A2 on normal phase plates developed in two dimensions.



  The position of the spots is expressed in Cartesian coordinates. The X coordinate is the Rf value of the second solvent system listed. The Y coordinate is the Rf value of the first solvent system listed.



   Table 10
RF system / connection BBM-1675 A, BBM-1675 A2
4% methanol in chloroform against
5% methanol in diethyl ether (0.34, 0.33) (0.28, 0.23)
4% methanol in chloroform against
50% acetone in Skelly solve B (0.33, 0.29)
Table 11 below shows the observed Rf values for BBM-1675 Al and A2 on C-18 reverse phase TLC plates developed in binary eluents.



   Table 11
RF system / compound BBM-1675 Al BBM-1675 A2 25% 0.5 M NaCl in acetonitrile 0.18 0.21 25% water in acetonitrile 0.00 0.00
Table 12 below summarizes the observed Rf values of BBM-1675 A and A2 on C-18 reverse phase TLC plates developed with ternary elution media.



   Table 12
RF system / connection BBM-1675A1 BBM-1675A2 acetonitrile: methanol: 0.5M NaCI 80%: 10% 10% 1.00 1.00 60%: 10%: 30% 0.57 0.50 40% 30%: 30% 0.32 0.22 30%. 50%: 20% 0.44 0.33 50%: 30%: 20% 0.62 0.54 40%: 40%. 20% 0.60 0.49 50%: 20%: 20% 0.42 0.34 60%: 20% 20% 0.74 0.69 acetonitrile methanol water 40%: 30%: 30% 0.00 0.00 acetonitrile.

  Methanol: 0.1M NH4OAc 40%: 30%: 30% 0.32 0.22 acetonitrile: Methanol: 0.1M NaH2PO4 40%: 30% 30% 0.00 0.00
HPLC analysis of BBM-1675 A1 and A2 BBM-1675 Al and BBM-1675 A2 were tested using a simple, a binary and a ternary eluent on C-18 reverse phase silica gel columns.



  The observed K 'values for these compounds are summarized in Tables 13, 14 and 15 below. The K 'value was calculated using the following formula:
K '= TR-To
To where TR is the retention time measured from the time of the injection to the peak and To is the time of the empty volume.



   Table 13
K 'system / compound BBM-1675 Al BBM-1675 A5 acetonitrile a a tetrahydrofuran a a methanol 0.00 0.00 a = compound was not eluted from the column.



   Table 14 System / compound BBM-1675 Al BBM-1675 A2 25% water in acetonitrile a a 25% methanol in water 1.25 1.25 a = compound was not eluted from the column.



   Table 15
Ternary eluent system / compound BBM-1675A1 BBM-1675A2 acetonitrile methanol water 40%: 30% 30% aa acetonitrile: methanol 0.1M NH, OAc 40%: 30% 30% 1.7 3.0 50% 20% 30% 3.8 6.5 43.3% 23.3 % 33.3% 6.1 b 42.5% 22.5% 35.0% 7.8 b 41.5% 21.5% 37.0% 9.7 ba = was not eluted b = not determined
Biological properties of the BBM-1675 components
The antimicrobial activity of the BBM-1 675 components was determined for a large number of bacteria (gram-positive, gram-negative and acid-resistant) and fungi using the serial double agar dilution method.

  The agar culture medium was used for gram-positive and gram-negative bacteria and medium No. 1001 (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na2HPO4, 0, 1% KH2PO4, 0.005% ammonium citrate, 0.001% MgSO4 and 1.5% agar) was used for acid-resistant organisms. Sabouraud agar medium was used for fungi. As shown in Table 16, each of the BBM-1675 components (A1, A2, A3, A4, B1 and B2) showed a wide range of antimicrobial activity. BBM1675 Al, A2, A3 and A4 showed a particularly high activity against gram-positive bacteria.



   Table 16
Antimicrobial activity of BBM-1675 components strain MIC in, µg / ml BBM-1675 At A2 A3 A4 B, B2 S. aureus 209 P <0.0008 0.0063 0.0063 0.0125 0.012 0.0063 S. aureus Smith <0.0008 0.0031 0.0063 0.0125 0.012 0.012 B. subtilis PCI 129 <0.0008 0.05 0.0125 0.0125 0.05 0.05 M. luteus 1001 0.0016 0.0063 0.0125 0.0125 0.1 0.1 M. navus <0.0008 0.0016 0.0063 0.0125 0.025 0.025 Mycobacterium 607 0.05 0.1 NT NT 0.05 0.025 E. coli NIHJ 0.1 0.8 1.6 3.1 0.8 3, 1 K. pneumoniae D11 0.4 0.8 1.6 3.1 0.8 0.8 P. aeruginosa D15 0.8 1.6 1.6 3.1 3.1 3.1 C. albicans IAM 4888 0.4 0.4 1.6 6.3 3.1 1.6 C.

   neoformans 1.6 3.1 1.6 6.3 6.3 12.5
A second antimicrobial test was carried out with purified Al and A2 (prepared according to Example 3 below) and with components A3 and A4.



  The data are summarized below.



   MIC by ADT (, µg / ml) strain BBM-1675 Al A2 A3 A4 S. aureus 209 P <0.0008 0.0063 0.0063 0.012 S. aureus Smith <0.0008 0.0031 0.0063 0.012 B. subtilis PCI 219 <0.0008 0.05 0.012 0.025 M. luteus 1001 0.0016 0.0063 0.012 0.05 M. flavus <0.0008 0.0016 0.0063 0.012 Mycobacterium 607 0.05 0.1 0.16 0.16 E. coli NIHJ 0.1 0.8 1.6 3.1 K. pneumoniae D11 0.4 0, 8 1.6 3.1 P.aeruginosaDl5 0.8 1.6 3.1 3.1 B. fragilis A20928 0.2 1.6 0.2 0.4 C. difficile A21675 0.4 0.8 0, 05 0.4 C. perfringens A9635 0.05 0.8 0.4 0.4 C. albicans IAM 4888 0.4 0.4 1.6 6.3 C. neoformans 1.6 3.1 1.6 6 , 3rd
The activity of the Prophag induction in the lysogenic bacterium E. coli W1709 (X) was determined for the BBM-1675 components according to the method of Lein et al in Nature 196:

  : 783-784 (1962). The plaque number was carried out on agar plates comprising the test material (T) and the control plate (C). A T / C ratio on the plaque number greater than 3.0 was considered significant and the lysogenic induction activity (ILB activity) was expressed as the minimum inducible concentration of the test compound. As shown in Table 17.



  components BBM-1675 showed strong ILB activity in lysogenic bacteria, indicating that they may have anti-tumor activity.



   Table 17
Lysogenic induction activity of
BBM-1675 component antibiotic MlC * (Ilg / ml) BBM-1675 A5 0.0063 BBM-1675 A2 0.0125 BBM-1675 A3 0.05 BBM-1675A4 0.10 BBM-1675 B1 0.10 BBM-1675 B2 0.20 * minimum inducible concentration
The anti-tumor activity of BBM-1675 Al and A2 was determined in different mouse tumor systems. P-388 lymphocytic leukemia, L1210 lymphoid leukemia, B16 melanotic melanoma and Lewis limb carcinoma were implanted intraperitoneally in male BDF1 mice, with an inoculum size of 106, 105, 5 x 105 and 106 cells per mouse. 24 hours after tumor inoculation, graduated doses of test compounds were administered intraperitoneally to the mice.



  Treatments were performed once on the first day only, on days 1, 4 and 7 (q3d x 3), once a day for 9 days (qd 1 <9) or 11 days (qd 1 -t 11). Components A3 and A4 were only tested against P-388 leukemia according to the q3d x 3 pattern, due to the scarcity of material.



     BBM-1675 A1, A2, A3 and A4 were dissolved in 0.9% saline containing 10% dimethyl sulfoxide and Chromomycin A3 (Toyomycin, Takeda) used as the reference compound was dissolved in 0.9% saline. Death or survival of the treated and untreated mice was recorded daily and the mean survival (MST) was calculated for each of the test (T) and control (C) groups. A T / C value that was equal to or greater than 125% that a meaningful anti-tumor effect was achieved. The results are shown in Tables 18-23. BBM-1675 A, and A2 showed a potent anti-tumor activity against P-388 leukemia with a maximum T / C of 160%.

  They are approximately 100-3000 times more active than Chromomycin A3 in terms of the minimum effective dose. However, BBM-1675 A3 and A4 were less active than Al or A2 component against P-388 leukemia (Table 19). BBM-1675 Al and A2 were also active against L-1210 leukemia (Table 21), B16 melanoma (Table 22) and Lewis lung carcinoma (Table 23). The toxicity of BBM-1675 A1 and A2 was determined in male ddY mice by intraperitoneal or intravenous administration; BBM-1675 A was about 10 times more toxic than BBM-1675 A2 (Table 24). The therapeutic indices of BBM-1675 Al and A2 were 4-8 and 8-20 times better than those of chromomycin A2 in the P-388 leukemia system (Table 25).

  Second experiments were carried out by intravenous administration of BBM-1675 components against P-388 and L1210 leukemias, which were inoculated intravenously in an amount of 5 × 105 and 104 cells per mouse. In these experiments, adriamycin was used as the reference agent, which was dissolved in 0.9% saline and administered on days 1, 4 and 7. The results are shown in Tables 26 and 27. Both components Al and A2 were superior to adriamycin in terms of maximum T / C, minimum effective dose and range of activity.



   Table 18
Effect of BBM-1675 Components on P-388 Leukemia (Day 1 Treatment)
Dose, ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 A1 0.03 19.0 152 -2 , 6 5/5 0/5
0.01 19.0 - 1.0 5/5 0/5
0.003 18.0 144 1.0 5/5 0/5
0.001 20.0 160 - 1.4 - 1.4 5/5 0/5
0.0003 16.0 i) 0.0 5/5 0/5
0.0001 15.0 120 -0.2 5/5 0/5
0.00003 14.0 112 -0.2 5/5 0/5 BBM-1675 A2 0.3 6.0 48 -3.8 3/5 0/5
0.1 20.0 8 - 1.8 5/5 0/5
0.03 18.0 144 -1.4 5/5 0/5
0.01 17.0 136 -0.6 5/5 0/5
0.003 17.0 (2) -0.4 5/5 Q / 5
0.001 16.0

     i) 0.0 5/5 0/5
0.0003 15.0 120 0.0 5/5 0/5
0.0001 14.0 112 0.0 5/5 0/5 chromomycin 1 19.0 152 -0.2 4/5 0/5 A3
0.3 17.0 (2) + +0.6 5/5 0/5
0.1 16.0 128 +0.8 5/5 0/5
0.03 14.0 112 0.0 5/5 0/5
0.01 14.0 112 -0.2 5/5 0/5 porters - 12.5 - +0.1 10/10 0/10 * day 1, i.p.



  The circle indicates significant anti-tumor activity.



   Table 19
Effect of BBM-1675 Components on P-388 Leukemia (Day 1, 4, and 7 Treatment)
Dose, ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 Al 0.03 7.0 56 -2 , 8 5/5 0/5
0.01 19.0 152 -1.0 5/5 0/5
0.003 19.0) -0.6 5/5 0/5
0.001 16.0 (i) -0.6 5/5 0/5 0.0003 17.0 136 - 0.4 5/5 0/5
0.0001 16.0 128 -0.2 5/5 0/5
0.00003 14.0 112 +0.4 5/5 0/5 BBM-1675 A2 0.3 tox. - - 2/5 0/5
0.1 11.0 88 -1.0 5/5 0/5
0.03 18.0 -1.2 5/5 0/5
0.01 18.0 144 -0.4 5/5 0/5
0.003 18.0 144 - 0.4 5/5 0/5
0.001 17.0

     -0.4 5/5 0/5
0.0003 16.0 128 -0.4 5/5 0/5
0.0001 16.0 128 -0.2 5/5 0/5
0.00003 15.0 120 -0.4 5/5 0/5 BBM-1675 A3 0.01 17.5 140 +0.2 4/4 0/4
0.001 15.0 120 +0.6 4/4 0/4
0.0001 13.5 108 +0.6 4/4 0/4 BBM-1675 A4 0.01 16.5 í) +0.2 4/4 0/4
0.001 14.0 112 +0.4 4/4 0/4
0.0001 12.5 100 +0.6 4/4 0/4 chromomycin 0.3 18.0 144 +0.6 5/5 1/5 A3
0.1 18.0) + +0.6 5/5 0/5
0.03 17.0 136 - -0.2 5/5 0/5
0.01 14.0 112 0.0 5/5 0/5 carriers - 12.5 - +0.4 10/10 0/10 * days 1.4 and 7, i.p.



  The circles indicate significant anti-tumor activity.



   Table 20
Effect of BBM-1675 components on P-388 leukemia (gd 1-> 9 treatment)
Dose, ip MST T / C Mean weight survivors on (mgikg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 Al 0.01 7.0 56 -1.8 5/5 0/5
0.003 13.0 104 -1.0 5/5 015
0.001 19.0 152 - 1.2 5/5 0/5
0.0003 19.0) -0.8 5/5 0/5
0.0001 18.0 144 0.0 5/5 0/5
0.00003 16.0) +0.2 5/5 0/5 0.00001 16.0) -0.2 5/5 0/5 BBM-1675 A2 0.1 6.0 48 -2.2 4 / 5 0/5
0.03 13.0 104 - 1.4 5/5 0/5
0.01 18.0 -1.0 - 1.0 5/5 0/5
0.003 18.0 í) - -0.6 5/5 0/5
0.001 18.0)

   -0.8 5/5 0/5
0.0003 17.0 í) - -0.4 5/5 0/5
0.0001 16.0) -0.4 5/5 0/5
0.00003 15.0 120 -0.6 5/5 0/5
0.00001 15.0 120 +0.4 5/5 0/5 chromomycin 0.3 9.0 72 -2.0 5/5 0/5 A3
0.1 18.0 144 +0.4 5/5 0/5
0.03 18.0 144 0.0 5/5 0/5
0.01 15.0 120 -0.2 5/5 0/5
0.003 13.0 104 -0.2 5/5 0/5 carriers - 12.5 - +0.4 10/10 0/10 * qd 1-> 9, i.p.



  The circle shows significant anti-tumor activity.



   Table 21
Effect of BBM-1675 components on L-1210 leukemia
Dose, ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 Al 0.003 14.5 -1.7 6 / 6 0/6
0.001 12.0 (i) -0.5 6/6 1/6
0.0003 12.0) +0.3 6/6 0/6
0.0001 11.0 116 +1.0 6/6 0/6 BBM-1675A2 0.03 10.5 111 -1.5 6/6 0/6
0.01 13.5 142 -1.2 6/6 0/6
0.003 13.0) -0.2 6/6 0/6
0.001 11.0 116 +1.3 6/6 0/6
0.0003 10.5 111 +1.0 6/6 0/6 chromomycin 0.3 8.5 89 - 1.2 6/6 0/6 A3
0.1 11.5 121 +1.2 6/6 0/6
0.03 11.0 116 +1.2 6/6 0/6
0.01 11.0 116 +1.3 6/6 0/6
0.01 11.0 116 +1.3 6/6 0/6
0.003 10.0 105 + 1.5 6/6 0/6 carriers <RTI

    ID = 22.11> - 9.5 - +1.4 12/12 0/12 * qd 19, i.p.



  The circle shows significant anti-tumor activity.



   Table 22
Effect of BBM-1675 components on B16 melanoma
Dose, ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 A1 0.003 10.0 61 -0.7 6/6 0/6
0.001 31.5 191 0.0 6/6 0/6
0.0003 40.5 8 +0.3 6/6 0/6
0.0001 27.0 (i) +0.8 6/6 0/6 0.00003 22.0 133 +1.8 6/6 0/6
0.00001 18.0 109 +2.2 6/6 0/6 BBM-1675 A2 0.03 11.0 67 -0.8 6/6 0/6
0.01 26.5 161 +0.3 6/6 0/6
Table 22 (continued)
Effect of BBM-1673 components on B16 melanoma
Dose,

   ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45
BBM-1675 A2 0.003 29.5) + +0.2 6/6 0/6
0.001 26.0) +0.8 6/6 0/6
0.0003 22.0 133 +0.2 6/6 0/6
0.0001 18.0 109 +0.2 6/6 0/6
0.00003 17.0 103 + 1.7 6/6 0/6 chromomycin 0.1 25.5 153 +2.3 6/6 0/6 A3
0.03 23.0) +2.2 6/6 0/6
0.01 21.0) +2.3 6/6 0/6
0.003 18.0 109 +2.2 6/6 0/6 carriers - 16.5 +2.1 12/12 0/12 * qd 1-> 9, i.p.

 

  The circle shows significant anti-tumor activity.



   Table 23
Effect of BBM-1675 components on lung carcinoma
Dose, ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 A1 0.003 10.0 91 - 1.7 5/6 0/6
0.001 31.5 i) -0.7 6/6 1/6
0.0003 21.5 i) -0.7 6/6 0/6
0.0001 21.0 + 1.0 6/6 0/6
0.00003 13.0 118 + 1.0 6/6 0/6
0.00001 11.5 105 +1.0 6/6 0/6 BBM-1675 A2 0.03 10.0 91 - 1.8 6/6 0/6 0.01 25.5) - 1.7 - 1.7 6/6 0/6
0.003 28.5 (i) 0.0 6/6 1/6
0.001 17.0 6) -0.3 6/6 0/6
0.0003 15.0 +1.2 6/6 0/6
0.0001 10.5 95 +0.5 6/6 0/6
0.00003 11.0 100 +0.8 6/6 0/6
Table 23 (continued)
Effect of BBM-1675 components on lung carcinoma
Dose,

   ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 Chromomycin 0.1 21.5 195 +1.2 6/6 1/6 3
0.03 17.0 +1.7 3/3 0/5
0.01 17.0 +1.5 6/6 0/6
0.003 11.5 105 + 1.7 6/6 0/6 carrier - 11.0 - +0.8 12/12 1/12 * qd -> i.p.



  The circle shows significant anti-tumor activity.



   Table 24
Toxicity of BBM-1675 components LD50 (mg / kg / d) single dose multiple dose i.p. I.V. I.p.



     BBM-1675 A1 0.019 0.010 0.00046 BBM-1675 A2 0.18 0.10 0.0072
Chromomycin A3 0.81 0.41 0.23
Table 25
Therapeutic indices LD50 / MED *
P-388 simple qd 1-> 9 L1210 B16 LL
BBM-1675 A, 63> 46 2 15 5
BBM-1675 A2 180 72 2 24 24
Chromomycin A3 8 8 inactive 23 23 * minimum effective dose
Table 26
Effect of BBM-1675 components on intravenously implanted P-388 leukemia
Dose,

   ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (%) change on d 5 (g) d 5 d 45 BBM-1675 Al 0.01 9.5 106 - 1.7 6/6 0/6
0.003 14.0) - -0.3 6/6 0/6
0.001 11.5 8 +0.3 6/6 0/6
0.0003 9.0 100 +0.3 6/6 0/6 BBM-1675 A2 0.1 7.0 78 -3.7 6/6 0/6
0.03 15.0 167-1.0 6/6 0/6
0.01 12.0) -0.5 6/6 0/6
0.003 9.0 100 + 1.0 6/6 0/6
Table 26 (continued)
Effect of BBM-1675 components on intravenously implanted P-388 leukemia
Dose, ip MST T / C mean weight survivors on (mg / kg / d *) (d) () change on d 5 (g) d 5 d 45 adriamycin 30 tox.

   - 0/6 0 (6
10 9.0 100 -1.5 6/6 0/6
3 12.0 6) + +0.7 6/6 0/6
1 9.0 100 +1.7 6/6 0/6 carrier - 9.0 + + 1.7 12/12 0/12 * Tagel, 4und7i.v.



  The circle shows significant anti-tumor activity.



   Table 27
Effect of BBM-1675 components on intravenously implanted L-1210 leukemia
Dose, ip MST T / C Mean weight survivors on (mg / kg / d *) (d) (% 3 change on d 5 (g) d 5 d 45 BBM-i675 Al 0.008 9.5 119 -2.0 4/6 0/6
0.004 14.0 8 -0.2 6/6 0/6 0.002 13.0 163 +0.2 6/6 0/6
0.001 9.5 119 +0.8 6/6 0/6
0.0005 9.0 113 +0.8 6/6 0/6 BBM-1675 A2 0.063 11.0) 1.8 -1.8 6/6 0/6
0.032 14.0 6) +0.2 6/6 0/6
0.016 10.5 +0.8 6/6 0/6
0.008 8.0 100 + 1.2 6/6 0/6
0.004 8.0 100 +0.8 6/6 0/6 Adriamycin I6 tox.

   2/6 0/6
8 12.0 150 +0.2 6/6 0/6
4 9.0 113 +1.5 6/6 0/6
2 8.0 100 +1.7 6/6 0/6 porters - 8.0 - + +1.4 12/12 0/12 * days 1.4 and 7, i.v.



  The circle indicates significant anti-tumor activity.



   The antitumor activity of components BBM-1675 A and A2 was also determined by a second test against P388 leukemia, L-1210 leukemia and B16 melanoma in mice. The results of these tests are shown in Tables 28, 29 and 30 below. Details of the methods used in these tests are in Cancer Chemother. Rep., 3: 1 -87 (para. 3), 1972.



   Table 28
Effect of BBM-1675 Al and A2 on P-388 leukemia material treatment schedule dose, IP MST effect AWC, gm survivors ltg / kg / d days MST,% T / C d S on d 5 (30) * NSC 38270 qdl- > 9 400 13.0 163 -0.6 6/6
200 11.0 138 -0.9 6/6 BBM-1675A1 d.l 51.2 20.0 250 -2.1 4/6 DMSO saline 25.6 18.0 225 - 1.8 6/6
12.8 16.5 206 -1.1 6/6
6.4 13.0 163 +0.1 6/6
3.2 12.0 150 -0.3 6/6
1.6 11.0 138 -0.3 6/6
0.8 10.5 131 0 6/6
0.4 10.0 125 +0.4 6/6
0.2 10.0 125 +0.3 6/6
0.1 10.0 125 0 6/6 d.1,

   5 + 9 25.6 8.0 100 - 1.8 6/6
12.8 13.5 169 -1.5 6/6
6.4 16.5 206 -0.8 6/6
3.2 16.0 200 -0.8 6/6
1.6 15.5 194 +0.3 6/6
0.8 12.5 156 +0.3 6/6
0.4 12.0 150 -0.1 6/6
0.2 11.5 144 + 0.2 6/6
0.1 12.0 150 +0.8 6/6
0.05 10.0 125 +0.8 6/6 qdl o9 12.8 TOX TOX TOX 1/6
6.4 6.0 75 - 1.5 4/6
3.2 13.0 163 -1.2 6/6
1.6 14.5 181 - 1.6 6/6
0.8 16.5 206 -2.3 6/6
0.4 16.0 200 -0.9 6/6
0.2 15.0 188 -0.8 5/5
0.1 13.0 163 -0.4 6/6
0.05 12.0 150 +0.1 6/6
0.025 12.0 150 -0.7 6/6 BBM-1675A2 d.1 256 TOX TOX TOX 0/6 DMSO saline 128 12.5 156 -3.5 4/6
64 27.0 338 - 1.9 6/6
32 26.0 325 - 2.0 6/6
16 16.0 200 -1.8 6/6
8 15.5 194 -1.9 6/6
4 15.0 188 -0.7 6/6
2 12.0 150 -0.5 6/6
1 12.0 150 0 6/6
0.5 10.0 125 +0.2 6/6 d.1.5 + 9 128 TOX TOX TOX 0/6
64 TOX TOX TOX 0/6
32 TOX TOX <RTI

    ID = 26.9> - 1.3 2/6
16 24.5 306 - 1.3 5/5
8 17.5 219 -1.1 6/6
4 15.0 188 0 6/6
2 15.0 188 +0.1 6/6
1 12.5 156 -0.4 6/6
0.5 12.0 150 -0.4 6/6 BBM- 1 675A2 DMSO saline qdlo9 0.25 11.0 138 -0.4 6/6
64 TOX TOX TOX 1/6
32 6.0 75 - 2.9 4/6
16 8.0 100 -1.9 6/6
8 15.5 194 - 1.3 6S6
4 17.0 213 -1.8 6/6
2 15.0 188 --1.1 6/6
Table 28 (continued)
Effect of BBM-1675 Al and A2 on P-388 leukemia material treatment schedule dose, IP MST effect AWC, gm survivors lay / kg / d days MST,

   % T / C d 5 am d 5 (30) BBM- 1 675A2 1 14.0 175 -0.5 6/6 DMSO saline qdle9 0.5 14.0 175 -0.6 6/6
0.25 12.0 150 -0.1 6/6
0.125 12.0 150 +0.1 6/6 control saline 8.0 - +0.5 10/10 solution tumor inoculum: 106 ascites cells i.p. implanted host: CDFl Q mice TOX: <4/6 live mice on day 5 Evaluation:

  MST = mean survival time Effect:% T / C = (MST treated / MST control) x 100 criterion:% T / C 2 125 was considered to be a significant anti-tumor activity * NSC 38270 = Olivomycin A
Table 29
Effect of BBM-1675 A1 and A2 on L-1210 leukemia material treatment plan dose, IP MST effect AWC, gm survivors Fg / kg / inj. Days MST,% T / C d 5 on d 5 (30) BBM-1675A1 d.l 51.2 12.0 171 -1.1 5/6
25.6 7.0 100 -2.3 6/6
12.8 9.0 129 - 1.1 5/6
6.4 9.5 136 -0.5 6/6
3.2 6.0 86 -1.7 6/6
1.6 7.0 100 -0.8 6/6
0.8 8.0 114 -0.4 6/6
0.4 7.0 100 +0.3 6/6
0.2 7.0 100 - 0.5 5/6
0.1 7.0 100 +0.8 5/6 d.l,

   5 + 9 25.6 TOX TOX TOX 1/6
12.8 9.0 129 -1.8 6/6
6.4 9.0 129 -0.8 6/6
3.2 8.0 114 -1.9 6/6
1.6 8.5 121 0 6/6
0.8 8.0 114 -0.4 6/6
0.4 7.5 107 -1.3 6/6
0.2 8.0 114 0 6/6
0.1 8.0 114 +0.4 5/6
0.05 7.0 100 +0.3 6/6 qd 1e9 12.8 TOX TOX -2.4 3/6
6.4 8.0 114 - 1.6 6/6
3.2 8.0 114 - 1.7 6/6
1.6 9.0 129 -2.1 6/6
0.8 8.5 121 - 1.6 6/6
0.4 8.0 114 - 1.0 6/6
0.2 8.0 114 -0.5 5/6
0.1 7.0 100 +0.3 6/6
0.05 7.0 100 +0.3 6/6
0.025 6.0 86 -0.6 6/6 BBM-1675A2 d.1 256 TOX TOX TOX 0/6
128 7.0 100 - 1.8 5/6
64 7.5 107 - 1.3 4/6
32 8.0 114 - 2.2 5/6
16 7.0 100 -2.3 6/6
8 9.5 136 -1.4 6/6
4 8.5 121 -1.1 6/6
2 8.0 114 -0.8 6/6
1 8.0 114 0 6/6
0.5 8.0 114 -0.1 6/6
Table 30
Effect of BBM-1675 Al and A2 on B16 melanoma material dose, IP MST effect AWC,

   gm survivors llg (m) or. Days MST,% T / C d5 on d 10 (61) mg / kg / inj.



  BBM-1675A1 3.2M TOX TOX -1.8 2/10
1.6 16.0 64 -1.8 10/10
0.8 34.5 168 - 1.8 10/10
0.4 56.5 226 -0.9 10/10 (2) b
0.2 47.0 188 -0.7 10/10
0.1 37.0 148 -0.4 10/10
BBM-1675A2 16M 13.0 52 -2.1 10/10
8 29.5 118 -2.0 10/10
4 43.5 174 -1.1 10/10
2 50.5 202 -2.1 10/10 (3) b
1 0.5 140 -1.0 10/10
0.5 38.0 152 -1.1 10/10
Control saline 25.0 - -0.1 10/10 solution a only one without tumor; MST d.m.o. = 55.0 (220%) b two without tumor; MST d.m.o. = 46.0 (184%)
Tumor inoculum: 0.5 ml of 10% porridge, i.p.



   Host: BDFI 9 mice
Treatment: qd 1 <9
TOX: <7/10 live mice on day 10
Evaluation: MST = mean survival time
Effect:% T / C = (MST treated / MST control) x 100
Criterion:% T / C 2 125 was considered a significant anti-tumor effect.



   After further cleaning of BBM-1675 Al tested according to 35 mice. The results of these tests are next
Example 6, samples of the purified compound were presented opposite.



     L-1210 leukemia, P-3 88 leukemia and B16 melanoma in
Table 31
Effect of Purified BBM-1675 Al on P-388 Leukemia (Day 1 Treatment)
Compound dose, route, schedule MST, d T / C (%) mean weight survivor (mg / kg dose) change on d 5 on d 5 BBM-1675A1 0.1024 i.p., qd> <1 1 TOX TOX 0/6
0.0512 17.5 159 -1.8 4/6
0.0256 16.5 150 -2.6 6/6
0.0128 17.5 159 -1.4 6/6
0.0064 15.5 141 -2.2 6/6
0.0032 15.5 141 -2.5 6/6
0.0016 16.5 150 -1.0 6/6
0.0008 15.0 136 -1.2 6/6
0.0004 15.0 136 -2.0 6/6
0.0256 i.p., q4d x 3;

  TOX TOX -1.5 1/6
0.0128 10.0 91 -2.5 5/6
0.0064 17.5 159 -1.9 6/6
0.0032 17.0 155 -0.8 6/6
0.0016 17.0 155 -2.0 6/6
0.0008 15.0 136 -1.7 6/6
0.0004 15.0 136 -0.4 6/6
0.0002 13.0 118 -0.8 6/6
0.0001 13.0 118 -1.3 6/6
0.00005 13.5 123 -1.0 6/6
Table 31 (continued)
Effect of purified BBM-1675 A1 on P-388 leukemia (treatment on day 1) compound dose, route, plan MST, d T / C (%) mean weight survivor (mg / kg dose) change on d 5 on d 5
0.0128 i.p., qd x 5 TOX TOX 0/6
0.0064 TOX TOX - 3.6 3/6
0.0032 17.5 155 -2.2 5/6
0.0016 14.5 132 -2.0 6/6
0.0008 15.5 141 -2.2 6/6
0.0004 16.0 145 -2.8 6/6
0.0002 17.0 155 - 1.3 6/6
0.0001 14.0 127 -1.6 5/6
0.00005 15.0 136 -1.6 6/6
0.000025 15.0 136 - 1.0 6/6 control (carrier3 1 x 106 i.p., q4d x 3; 11.0 100 -0.7 9/9 host: CDF1 female mice implantation height and location:

   1 x 106 cells, i.p.



   Table 32
Effect of purified BBM-1675A1 on L-1210 leukemia (treatment on day 1) compound dose, route, plan MST, d T / C (%) mean weight survivor (mg / kg dose) change on d 5 on d 5 BBM-1675A1 0.1024 ip, qd x 1 TOX TOX 1/6
0.0512 TOX TOX -2.0 0/6
0.0256 8.0 114 -2.9 4/6
0.0128 11.0 157 -2.0 6/6
0.0064 11.0 157 -1.9 6/6
0.0032 10.0 143 -2.0 6/6
0.0016 10.0 143 -2.6 5/6
0.0008 8.0 114 -0.4 6/6
0.0256 i.p., q4d x 3 TOX TOX -2.3 2/6
0.0128 10.5 150 -1.7 6/6
0.0064 11.0 157 -1.8 6/6
0.0032 11.0 157 -1.4 6/6
0.0016 10.5 150 - 1.9 6/6
0.0008 9.0 129 -0.6 6/6
0.0004 8.5 121 -0.7 6/6 0.00102 8.0 114 -0.5 6/6
0.0128 i.p.,

   qd x 5 TOX TOX -2.8 2/6
0.0064 7.0 100 - 1.8 5/6
0.0032 11.5 164 -1.0 6/6
0.0016 11.0 157 -1.5 6/6
0.0008 10.0 143 -1.6 5/6
0.0004 8.5 121 -0.4 6/6
0.0002 8.5 121 0.1 6/6
0.0001 8.5 121 0.0 6/6 control (carrier) 1 x 106 i.p., qd x 5 7.0 100 0.1 10/10 host: CDFI female mice implantation height and site: 1 x 106 cells, i.p.



   Table 33
Effect on purified BBM-1675 Al based on B16 melanoma (treatment on day 1) compound dose, route, plan MST, d T / C (%) mean weight survivor (mg / kg dose) change on d 5 on d 5 * BBM-1675A1 0.0064 ip, q4d x 3 16.5 110 -3.8 8/10
0.0032 22.5 150 -3.0 10/10
0.0016 25.0 167 -1.8 10/10
0.0008 22.0 147 -2.3 10/10
0.0004 24.0 160 -1.8 9/10
0.0016 i.p., qd x 9 27.0 180 -3.7 10/10
0.0008 27.0 180 -2.9 10/10
0.0004 26.0 173 -2.3 10/10
0.0002 24.5 163 -2.4 10/10
0.0001 25.0 170 -2.3 10/10 control (carrier) 0.5 ML i.p., qd x 9 15.0 100 -0.3 10/10 ** BBM-1675A1 0.0064 i.v.,

   q4d x 3 15.0 86 -4.7 10/10
0.0032 32.5 186 -2.1 10/10
0.0016 26.0 149 -1.4 10/10
0.0008 24.0 137 -0.4 10/10
0.0004 24.5 140 -0.0 10/10
0.0064 i.p., q4d x 3 18.0 103 -2.7 10/10
0.0032 23.0 131 -1.4 10/10
0.0016 24.0 137 -0.7 10/10
0.0008 25.0 146 -0.8 10/10
0.0004 21.5 123 -1.4 10/10 control (carrier) 0.2 ML iv, q4d x 3 17.5 100 -0.4 10/10 host: BDFI female mice * implantation height and location: 0, 5 ml 10% porridge, ip



  ** Implantation height and location: fragment, s.c.



   The above screening data indicate that the purified BBM-1675 Al component has essentially the same anti-tumor activities as the less purified sample that was previously screened. The compound has an exceptionally high potency, since activity was demonstrated at a dose of 25 nanograms / kg in the 5-day plan against P-388 leukemia in mice. For tests against P-388 and L-1210 leukemia, BBM-1675 Al is effectively administered as a single injection on day 1, three injections every fourth day, or 5 times a day. The compound was also effective against B16 melanoma when administered to animals having subcutaneous tumors and also intraperitoneally to animals administered i.p. Had tumor implants.

  This property of successful pharmacological delivery of a drug to a tumor at a distant site is unusual among antitumor antibiotics.



   As shown above, the BBM-1675 components have potent antimicrobial activity and are thus useful in the therapeutic treatment of warm-blooded animals and other animals against infectious diseases caused by such microorganisms. In addition, the compounds can also be used for conventional applications of antimicrobial agents, such as the disinfection of medical and dental equipment.



   Induction of prophage in lysogenic bacteria and activity against mouse tumor systems shows that the BBM-1675 components are also therapeutically useful for inhibiting the growth of warm-blooded animal tumors.



   It thus becomes a method for the therapeutic treatment of host animals. which are affected by a microbial infection or by a malignant tumor, the method, the administration of an effective antimicrobial or tumor-inhibiting dose of BBM-1675 A1, A2, A3, A4, B1 or B2 or a pharmaceutical composition thereof to the called host animal includes.



   The present invention provides a pharmaceutical composition which contains an effective antimicrobial or anti-tumor amount of BBM-1675 Al, A2, A3, A4 B1 or B2 in combination with an inert pharmaceutically acceptable carrier or diluent. These compositions can be presented in any pharmaceutical form. which is convenient for parenteral administration.

 

   Preparations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They can also be in the form of sterile. solid compositions are prepared which can be dissolved with sterile water, physiological saline or some other sterile injectable media immediately before use.



   The currently preferred amounts of BBM-1675 antibiotics can vary according to the particular components.



  the special formulations. the type of application and the special location. the host and the disease being treated are varied. There have to be many factors that can change the effects of the drug.



  be considered by the skilled person. for example, age, body weight, gender, diet, time of administration, route of administration, amount of excretion, condition of the host, drug combinations, sensitive reactions and seriousness of the condition. The administration can be carried out continuously or periodically within the maximum tolerable dose. The person skilled in the art can find out the optimum extent of the application for specified framework conditions using conventional dosage determination tests in view of the above-mentioned guidelines.



   The following examples serve to illustrate the present invention.



   example 1
Fermentation from BBM-1675
Actinomadura strain No. H964-92 was grown on an agar medium containing 1% malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% CaCO3 and 1.6% agar and kept alive. A well overgrown agar medium was used to inoculate the vegetative medium, which was 3% starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgS04-7H20, 0.2% NaC 1 and contained 0.1% CaCO3 with the pH adjusted to 7.0 prior to sterilization.

  The vegetative culture was incubated at 32 ° C. for 72 h in a rotary shaker (250 revolutions / min) and 5 ml of the culture were transferred into a 500 ml Erlenmeyer flask which contained 100 ml fermentation medium, this fermentation medium consisting of 3% molasses, 1% Corn starch, 1% fish meal, 0.1% CaCO3 and 0.005% CuSO4 5H20 (pH value before sterilization 7.0) The fermentation was carried out at 28 TLC for 8 days in a rotary shaker and the antibiotic activity in the fermentation broth was checked determined by paper disc agar diffusion using Staphylococcus aureus 209P as a test organism.

  The antibiotic effect reached a maximum of about 1 yg / ml after 5 d fermentation.



   The fermentation of BBM-1675 was also carried out in a stirred vessel fermenter. 500 ml of the inoculum, which was grown as mentioned above, was placed in a 20 1 fermenter containing 10 1 fermentation medium, which consisted of the same components as in the shake bottle fermentation. The fermentation was carried out at 32 ° C., with a degree of aeration of 12 l / min and stirring at 250 revolutions / min. Under these conditions, the antibiotic production reached a maximum of about 0.91 lg / ml after 68-76 h fermentation.



   Fermentation studies in fermentation tanks were also carried out. A seed culture was carried out for 4 d at 30 C in Erlenmeyer flasks containing a vegetative medium consisting of 3% soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4 7H2O, 0 , 2% Nach and 0.1% CaCO3 contained. The inoculum was inoculated in a 200 1 inoculation tank containing 1301 inoculation medium with the same composition as described above, and the inoculation tank was stirred at 240 revolutions / min at 30 ° C. for 31 hours.

  The second implant culture was used to inoculate 3000 liters of fermentation medium containing 1% corn starch, 3% molasses, 1% fish meal, 0.005% CuSO4-5H2O and 0.1% CaCO3. The production tank was in operation at 28 C, at 164 revolutions / min with a ventilation rate of 2000 l / min. The pH of the broth gradually increased as the fermentation progressed, reaching 7.7-7.8 after 170-180 h, producing a peak antibiotic activity of 1.7 yg / ml.



   Example 2
Isolation and cleaning of the BBM-1675 components
The harvested fermentation broth (3000 l, pH 7.8) was separated into a mycelium cake and a supernatant using a Sharpless centrifuge. The mycelium cake was suspended in 1600 liters of methanol and the mixture was stirred for 1 hour. The insoluble parts were filtered off and the methanolic extract was concentrated to 43 l in vacuo. The activity contained in the broth supernatant was obtained by extraction with two 1000 l parts of n-butanol. The n butanol extracts and the concentrated methanol extract were combined and evaporated azeotropically with the occasional addition of water to an aqueous solution (20 l), in which the antibiotic activity was largely reflected as an oily solid.

  The solid was dissolved in 30 liters of methanol and the insoluble parts were removed by filtration. The methanol extract was then concentrated in vacuo to a solution to which 40 liters of ethyl acetate and 30 liters of water were added. After stirring for 30 min, the organic layer was separated, dried over sodium sulfate and concentrated to 41 in vacuo. The addition of the concentrate in 20 1 n-hexane gave a pale yellow solid of the crude BBM-1675 complex (90.14 g, activity: 55 g / mg). Thin layer chromatography showed that the complex was a mixture of two major components BBM-1675 A1 and A2 and various minor components. They were separated and purified by repeated chromatography procedures which were carried out in a cold room in order to avoid decomposition.



   The BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and applied to a Sephadex LH-20 column (5.5 x 85 cm). The column was developed with methanol and the elution was followed by bioassay using Staphylococcus aureus 209P. The active eluates were combined, concentrated in vacuo and lyophilized to give a semi-pure solid BBM-1675 complex (4.19 g). The solid was then chromatographed on a silica gel column (5.0 x 50 cm) using chloroform and an increasing proportion (1st <5% v / v) of methanol as eluent.



   The eluates were combined on the basis of antibacterial activity (against S. aureus) and thin layer chromatography (SiO2; CHCI3 - CH3OH = 5: 1 v / v) and concentrated in vacuo. Almost homogeneous BBM-1675 A (yield after evaporation 351 mg) was first eluted with 2% methanol in chloroform and then a mixture of BBM1675 A2, A3 and A4 (507 mg) followed by the BBM-1675 B mixture (210 mg) with 3% methanol in chloroform. The solid BBM-1675 A1 was placed on a Sephadex LH20 column (2.0 x 80 cm) and developed with methanol. The active fractions were evaporated to dryness in vacuo and the remaining solid was recrystallized from methanol to give colorless platelets of pure BBM-1675 A1 (124 mg). This material is the starting material for Example 3A.

  The complex BBM-1675 A2, A3 and A4 was separated by chromatography on a column of Bondapak Cls (Waters 3.0 x 50 cm). Elution was carried out in aqueous acetonitrile and the bioactive eluates were tested by thin layer chromatography (Merck, * Silanized: CH3CN - H2O = 75:25 v / v). The secondary components Al (33 mg) and A3 (18 mg) were washed out successively with acetonitrile, a further main component being A2 * Cla reversed-phase silica gel.



  (301 mg) with 50% acetonitrile followed (this material was the starting material for Example 3B).



   The solid containing BBM-1675 B1 and B2 was chromatographed on a silica gel column (3.0 x 40 cm) with chloroform and methanol as the developing solvent. The active fractions eluted with 4% methanol in chloroform were pooled and evaporated to give the pure BBM-1675 B1 (7 mg). Another active fraction was eluted with 5% methanol to give evaporation BBM-1675 B2 (8 mg).



   Example 3
Further cleaning of BBM-1675 A1 and A2
A. Purification of BBM-1675 Al
A Glenco column measuring 2.67 cm internal diameter x 75 cm was packed in methanol by slurrying with a Baker phase-bonded (C 18) silica gel (40 tam particle size). The column was connected to a medium pressure HPLC system and equilibrated with 1.5 l eluent (41.6% acetonitrile - 21.6% methanol - 36.8% 0.1M ammonium acetate). The partially purified BBM-1675 Al (100.5 mg), which was obtained according to the cleaning method according to Example 2, was dissolved in 2 ml of acetonitrile and taken up in a sample loop. The sample was pumped onto the column. The column was eluted with the above eluent, collecting 87 ml fractions.

  Elution was monitored at 254 nm and 340 nm. Fractions 55-71 were pooled and extracted twice with 1500 ml aliquots of chloroform. The chloroform was evaporated to dryness to give 89.8 mg of residue C.



   A Glenco column with the dimensions 1.5 cm inner diameter x 20 cm was packed with 12 g Woelm silica gel (60-200 m particles) while being slurried. The residue C was applied to the column in chloroform solution. The column was eluted with 500 ml of a linear gradient from chloroform to 10% methanol in chloroform, collecting 10-25 ml fractions. After analysis by TLC on silica gel, fractions 6-9 were combined and evaporated to dryness to give 73 mg of residue D.



   A Glenco column with a mass of 1.5 cm inner diameter x 20 cm was packed in Skellysolve B with a slurry containing 12 g of Woelm silica gel (63-200 m particles). The residue D was dissolved in about 2 ml of CHC13 and applied to the column. The chloroform was displaced with 25 ml of Skellysolve B. The column was then eluted with 500 ml of a linear gradient from Skellysolve B to 60% acetone in Skellysolve B, collecting 28-25 ml fractions. Fractions 19-23 were pooled, evaporated to dryness to give 65.6 mg of pure BBM-1675 A.



   This residue was homogeneous in three thin layer chromatography systems (5% methanol in chloroform; 5% methanol in ether; and 50% acetone in Skellysolve B on silica gel) and on high pressure liquid chromatography (C18 silica gel - 41.5% acetonitrile: 21.5% methanol: 37 , 0% 0.1 M ammonium acetate).



  Gel permeation chromatography with purified BBM
1675 A1
A Pharmacia column measuring 2.5 cm internal diameter x 45 cm was packed in a slurry with Sephadex LH-20 in methanol and placed on a 33.4 cm chromatography bed. The purified BBM-1675 A, (approximately 120 mg) was dissolved in 2 ml of methanol and transferred to a 2.5 ml sample reservoir. The sample was applied to the column and the elution started with 1.75 ml with methanol, collecting 10 ml fractions (Pharmacia Frac-100 fraction collector). Elution was monitored at 254 nm using an Isco UA-5 detector.

  It was determined that BBM-1675 A was eluted at Ve / Vt of 0.79-0.91 (Ve = elution volume; Vt = bed volume).



   B. Cleaning BBM-1675 A
A Glenco column with a mass of 2.65 cm inner diameter x 7.5 cm was packed in methanol with a slurry of Baker's phase-bound octadecyl (C 1 8) silica gel (40 ptm particle size). The column was connected to a medium pressure HPLC system and equilibrated with 1.5 l of the eluent (50% acetonitrile - 20% methanol 30% 0.1M ammonium acetate). The partially purified BBM-1675 A2 (76.9 mg), which was obtained by the method according to Example 2, was dissolved in 2 ml of acetonitrile and taken up in a sample loop. The sample was pumped onto the column. The column was eluted with the above eluent, collecting 87 ml fractions.

  Elution was monitored at 254 and 340 nm. Fractions 31-38 were pooled and extracted twice with 500 ml aliquots of chloroform. The chloroform was evaporated to dryness to give 65.8 mg of homogeneous BBM-1675 A2.



   BBM-1675 A2 was homogeneous in 2 TLC systems, a 2d TLC analysis and in HPLC.



   Example 4
Preferred extraction method for BBM-1675 A,
Crude fermentation product (6.8 l) obtained according to the procedure of Example 1 was transferred to a polypropylene tank (12 cm in diameter, top; 10 cm in diameter, bottom; 37 cm in height) equipped with a tap at the bottom. An equal volume of chloroform was added. The mixture was stirred vigorously with a CRC air powered stirrer for 2 hours. Approximately 4 liters (1.3 kg) of dicalite (filter aid) was added, the whole being mixed. The mixture was filtered on a Dicalite cushion which was held in a No. 12 Buchner funnel. The filtrate was collected in a 19 1 solution bottle, which was provided with a vacuum vent (Ace. No. 5396-06).

  The mat was washed with 2 liters of chloroform. The filtrate was transferred to a 20 l separatory funnel and the phases were separated. The lower phase (chloroform) was removed.



   A Glenco tube with the dimensions 2.5 cm inner diameter x 40 cm was packed with 91 g of Woelm silica gel (63-200 lm particles) under slurry.

 

  The top chloroform phase was pumped through the column using an FMI RPY-2CSD pump. The column was washed with 60 ml of fresh chloroform. The chloroform eluate was discarded. The column was then eluted with 600 ml of 10% methanol in chloroform. This eluate was evaporated to dryness to give 547 mg of residue A.



   The residue A was dissolved in 50 ml of chloroform.



  The chloroform solution was added to 20 g of dicalite in an 11 round bottom flask. A slurry was created by adding approximately 200 ml of Skellysolve B. The solvents were removed on a rotary evaporator. The residue was slurried in 300 ml Skellysolve B. The slurry was packed into an Ace bottle chromatography tube (Part No. B5872-14) (41 mm inner diameter x 45.7 cm) by the following procedure. A glass wool plug was inserted into the neck of the tap between the tap and the column tube.



  A 1 cm layer of standard Ottawa sand was placed over the glass wool. The tap, glass wool and sand bed were cleaned of air by pressing Skellysolve B through under pressure (0.4 bar). The slurry was then placed on the column, forming a packed bed under pressure flow. The column was prevented from running dry. After a stable column bed was obtained, a 2 cm layer of Ottawa sand was placed on top of the bed. The bed was then eluted with an additional 600-700 ml of Skellysolve B. The bed was eluted with 500 ml of toluene. The toluene eluate was evaporated to dryness to give 93 mg of residue B. This partially cleaned BBM-1675 A1 can be further cleaned according to the procedure of Example 3.



   Example 5
Fermentation of the BBM-1675 complex below
Use of the variant H964-92-A1327Y
The variant strain A1327Y, which was obtained by NTG treatment of Actinomadura verrucosospora strain No. H964-92, was used to convert the vegetative medium, which contains 0.2% soluble starch, 1% glucose, 0.5% yeast extract, 0.5% NB-amine type A and contained 0.1% CaCO3, the pH being adjusted to 7.0 before sterilization.

  The vegetative culture was incubated at 32 ° C. for 4 d on a rotary shaker (250 revolutions / min) and 5 ml of the culture were transferred to a 500 ml Erlenmeyer flask which contained 100 ml of a fermentation medium which consisted of 3% molasses, 1 % Corn starch, 1% fish meal, 0.005% CuSO4 '5H2O, 0.05% MgSO4-7H2O and 0.1% CaCO3, the pH being adjusted to 7.0 before sterilization.



   The fermentation was carried out on the rotary shaker at 28 ° C. for 7 days. The antibiotic production reached a maximum of approximately 1.511 g / ml.



   Example 6
Isolation and cleaning of the BBM-1675 components
The purified fermentation broth from Example 5 (30001, pH 7.6) was separated into the mycelium cake and supernatant using a Sharpless centrifuge. The mycelium cake was stirred with 2000 l of methanol for 1 h and the insoluble materials were separated off by filtration. The activity contained in the broth supernatant was extracted therefrom with 1800 l of n-butanol. The methanol and n-butanol extracts were combined and concentrated azeotropically, by occasionally adding water to the aqueous solution (20 l), which excreted most of the antibiotic activity as an oily solid. The mixture was shaken three times with 20 l of ethyl acetate in order to extract the activity.

  The extracts were combined, filtered to remove the insoluble matter and evaporated to 41 in vacuo. The addition of the concentrate in 301 n-hexane with stirring gave the crude BBM-1675 complex (81.7 g, activity: 59, µg / mg) as a pale yellow solid. As shown by TLC and HPLC, the complex was a mixture of two main components, BBM1675 A1 and A2 and various secondary components. They were separated and purified by a series of chromatographies which were carried out in a cold room to avoid the decomposition.



   The crude BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and applied to a Sephadex LH-20 (5.5 x 85 cm). The column was developed with methanol and elution was followed by bioassay using Staphylococcus aureus 209P. The active eluates were combined, concentrated in vacuo and lyophilized to give the semi-pure BBM-1675 complex as a solid (4.86 g activity: 203 pg / mg).



  The solid was then chromatographed on a silica gel column (3.0 x 70 cm) using chloroform and an increasing amount (15%) of methanol as the developing solvent. The eluates were combined, based on antibacterial activity against S. aureus and TLC (SiO2, CHC 13 - MeOH = 5: 1, v / v) and concentrated in vacuo. BBM-1675 A1 (425 mg after evaporation, activity: 960 Clg / mg) was first eluted with 2% methanol in chloroform and then a mixture of BBM-1675 A2, A3 and A4 (732 mg, activity 340 Fg / mg) was followed by the BBM-1675 B complex (200 mg, activity 190 g / mg) with 3% methanol in chloroform.

  The above-mentioned BBM-1675 Al was re-chromatographed on silica gel (column: 2.2 × 44 cm) with 2% methanol in benzene. The bioactive eluates were checked by HPLC (Lichrosorb RP-18: CH3CN-MeOH-0, lMCH3COONH4 = 5: 2: 3, v / v) and the fractions containing homogeneous BBM-1675 Al were evaporated to dryness in vacuo. The remaining solid was recrystallized from methanol (10 ml) to give colorless prisms of BBM-1675 A (197 mg, activity: 1000, lg / mg).



   The complex of BBM-1675 A2, A3 and A4 (537 mg) was separated by column chromatography on Bondapak C1s (Waters, 2.0 x 42 cm). Elution was carried out with aqueous acetonitrile and the bioactive eluates were checked by TLC (Merck, silanized: CH3CN - H2O = 75:25, v / v). The secondary components BBM-1675 A4 (45 mg, activity 410, 1 g / mg) and A3 (19 mg, activity: 300, wg / mg) were washed out successively with 20% acetonitrile, followed by a main component, BBM-1675 A2 (203 mg) with 50% acetonitrile. The BBM-1675 A2 fraction was extracted from chloroform-n-hexane, whereby colorless rods were excreted (70 mg, activity: 290 pg / mg).

  The solid, which contained the BBM-1675 B mixture, was chromatographed on a silica gel column (3.0 × 40 cm) using chloroform and methanol as the developing solvent. The active fractions, which were extracted with 4% methanol in chloroform, were combined and evaporated to give pure BBM-1675 Bl (7 mg, activity: 180 pg / mg). Another active fraction was eluted with a concentration of 5% methanol, which after evaporation gave BBM-1675 B2 (8 mg, activity: 140 pg / mg).


    

Claims (22)

 PATENT CLAIMS 1. Antitumor antibiotic BBM-1675 A1, which appears in essentially pure form a) as white to pale yellow crystals; b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, is slightly soluble in benzene and water, and is insoluble in n-hexane and carbon tetrachloride; c) a positive reaction with ferric chloride, Ehrlich and Tollen's reagent and a negative reaction in sakaguchi, ninhydrin and anthrone tests; d) essentially has such an infrared absorption spectrum (KBr) as shown in FIG. 9; e) dissolved in CDCl3 has an essentially nuclear magnetic resonance spectrum as shown in FIG. 10; f) has a melting point in the range of 156-158 "C;  g) has an optical rotation of [a] D27 = - 191 "(c 0.5, CHC13); h) has an apparent molecular weight of 1248, determined by mass spectrography; i) an approximate elementary composition of 52.17% carbon, 6.15% hydrogen, 4.63% nitrogen, 9.09% sulfur and 27.96% oxygen, the last value being determined by difference; j) with silica gel thin-layer chromatography, an Rf value of 0.74 with has the solvent system CHCl3 - CH3OH (5: 1 v / v), and in reverse phase silica gel thin layer chromatography has an Rf value of 0.18 with the solvent system CH3CN - H20 (75:25 v / v);  k) dissolved in methanol at a concentration of 0.01356 gil the following ultraviolet absorption maxima and absorption coefficients have X = (nm) absorption coefficients 320 12.4 280 shoulder 253 25.1 210 25.5, with no significant changes occurring when acid or base is added ; 1) has a retention time of 13.3 min with high performance liquid chromatography with a C18 reverse phase silica gel column and the solvent system CH3CN - CH3OH - 0, IM - CH3COONH4 (5: 2: 3 via); m) is effective in inhibiting the growth of various bacteria and fungi; n) prophage induced in lysogen bacteria; and o) is effective in inhibiting the growth of P388 leukemia, L-1210 leukemia, B16 melanoma and Lewis lung carcinoma in mice 2nd Antitumor antibiotic BBM-1675 A2, which appears in essentially pure form a) as white crystals; b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, is slightly soluble in benzene and water and is insoluble in n-hexane and carbon tetrachloride; c) a positive reaction with ferric chloride, Ehrlich and Tollen's reagent and a negative reaction in Sakaguchi, Ninhydrin and Anthron tests; d) essentially has such an infrared absorption spectrum (KBr) as shown in FIG. 12; e) resolved in CDC13 essentially gives a nuclear magnetic resonance spectrum, as shown in FIG. 13; f) has a melting point in the range of 147-149 "C; g) has an optical rotation of [a] D27 = - 179.4 (c 0.5, CHCl3);  h) has an apparent molecular weight of 1248 as determined by mass spectrography; i) has an approximate elemental composition of 52.71% carbon, 5.94% hydrogen, 3.94% nitrogen, 9.39% sulfur and 28.01% oxygen, the last value being determined by difference; j) has an Rf value of 0.71 for silica gel thin layer chromatography with the solvent system CHCl3 - CH3OH (5: 1 vlv) and for reverse phase silica gel thin layer chromatography has an Rf value of 0.21 with the solution system CH3CN-H2O ( 75:25 v / v);  k) dissolved in methanol at a concentration of 0.02052 g / l following ultraviolet absorption maxima and absorption coefficients has Bmax (nm) absorption coefficients 320 12.2 282 16.3 282 26.2 214 25.8 with no significant changes in acids or base addition; 1) a retention time of 17.3 min in high performance liquid chromatography with a Cl8 reverse phase silica gel column and the solution system CH3CN - CH3OH - 0.1 M CH3COONH4 (5: 2: 3 v / v).  m) is effective in inhibiting the growth of various bacteria and fungi; n) prophage induced in lysogenic bacteria; and o) is effective in inhibiting P-388 leukemia, L-121 0 leukemia, B 16 melanoma and Lewis lung carcinoma in mice.  3. Antitumor antibiotic BBM-1675 A3, which a) is soluble in chloroform, ethyl acetate, acetone, ethanol or methanol, is slightly soluble in benzene and water, and is insoluble in n-hexane and carbon tetrachloride; b) have a positive reaction with iron chloride, Ehrlich's and Tollen's reagent and a negative reaction in Sakaguchi, Ninhydrin and Anthron tests; c) essentially has an infrared absorption spectrum (KBr) as shown in FIG. 3; d) dissolved in CDCI3 essentially has a magnetic proton resonance spectrum, as shown in FIG. 7; e) has a melting point in the range of 125-127 C; f) has an optical rotation of [a] D27 = - 1610 (c 0.5, CHC13);  g) has an approximate elemental composition of 54.55% carbon, 6.46% hydrogen, 3.73% nitrogen, 7.49% sulfur and 27.77% oxygen, the latter being determined by difference; h) has an Rf value of 0.72 for silica gel thin layer chromatography with the solvent system CHCl3 - CH3OH (5: 1 v / v) and an Rf value of 0.28 for reverse phase silica gel thin layer chromatography with the solvent system CH3CN - H2O (75 : 25 v / v); i) has the following ultraviolet absorption maxima when dissolved in methanol, 0.01N HCl - CH30H and 0.01N NaOH - CH30H UV #max nm (E1 cm): 253 (286) in methanol 282 (158) 320 (122) UV Xx nm (E, cm):  253 (287) in 0.01N HCl-CH3OH 282 (160) 320 (126) UV¸inx nm (E: 252 (280) in 0.01N NaOH-CH3OH 283 (162) 318 (120); j) has a retention time of 8.0 min with high-performance liquid chromatography on a Clg-reversed-phase silica gel column and the solvent system ClI3CN-CH3OH-0, lM CH3COONH4 (5: 2: 3 v / v); k) is effective in inhibiting the growth of various bacteria and fungi; 1) prophage induced in lysogenic bacteria; and m) is effective in inhibiting the growth of P388 leukemia in mice.  4. Antitumor antibiotic BBM-1675 A4 which a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, is slightly soluble in benzene and water and is insoluble in n-hexane and carbon tetrachloride; b) a positive reaction with ferric chloride, Ehrlich and Tollen's reagent and a negative reaction in Sakaguchi, Ninhydrin and Anthron tests; c) results essentially in an infrared absorption spectrum (KBr), as shown in Fig. 4; d) dissolved in CDCl3 essentially gives a magnetic proton resonance spectrum, as shown in FIG. 8; e) has a melting point in the range of 123-126 C; f) has an optical rotation of [a] D27 = - 176 (c 0.5 CHC13);  g) has an approximate elemental composition of 54.65% carbon, 6.29% hydrogen, 3.51% nitrogen, 8.07% sulfur and 27.48% oxygen, the latter value being determined by difference; h) has an Rf value of 0.71 in silica gel thin layer chromatography in the solvent system CHCl3 - CH3OH (5: 1 v / v) and an Rf value of 0.78 in reverse phase silica gel thin layer chromatography with the solvent system CH3CN - H2O (75:25 v / v); i) has the following ultraviolet absorption maxima when dissolved in methanol, 0.01N HCl-CH3OH and 0.01N NaOH-CH3OH UV UV #max nm (E 1cm) (E, cm): 253 (257) in methanol 282 (153) 320 (117) UV h ,,,, nm (E '%:  253 (258) in 0.01N HCl-CH3OH 282 (155) 320 (118) UV nm (E11rn): 252 (266) in 0.01N NaOH-CH3OH 283 (160) 318 (118); j) has a retention time of 5.1 min on high-performance liquid chromatography with a Cl8 reversed-phase silica gel column and the solvent system CH3CN-CH3OH-0.1M CH3COONH4 (5: 2: 3 v v); k) is effective in inhibiting the growth of various bacteria and fungi; 1) Prophage induced in lysogenic bacteria: and m) is effective in inhibiting P-388 leukemia in mice.  5. Anti-tumor antibiotic BBM-1675 B1. which a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, and is slightly soluble in benzene and water, and is insoluble in n-hexane and carbon tetrachloride; b) a positive reaction with ferric chloride, Ehrlich and Tollen's reagent and a negative reaction in sakaguchi, ninhydrin and anthrone tests; c) has a melting point in the range of 159-161 C; d) has an optical rotation of [a1n23 - 171 (c 0.5, CHCI3);  e) has an Rf value of 0.63 for silica gel thin layer chromatography in the CHC 13 - CH30H solvent system (5: 1 v / v) and an Rf value of 0.23 for reverse phase silica gel thin layer chromatography with the CH3CN - H2O solvent system ( 75:25 v / v); f) has the following ultraviolet absorption maxima when dissolved in methanol, 0, OlN HCl-CH3OH and 0.0 IN NaOH - CH3OH UV #max nm (E 1cm)): 253 (925) in methanol 282 (140) 320 (104) UV Ämax nm (E, cm):  253 (225) in0, OlN HCl-CH3OH 282 (140) 320 (105) UV kma \ nm (E1cm): 252 (236) in 0.01N NaOH-CH3OH 283 (141) 318 (105); g) is effective in inhibiting the growth of various bacteria and fungi; and h) prophage induced in lysogenic bacteria.  6. Anti-tumor antibiotic BBM-1675 B2. which a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, is slightly soluble in benzene and water and is insoluble in n-hexane and carbon tetrachloride; b) a positive reaction with ferric chloride, Ehrlich and Tollen's reagent and a negative reaction in Sakaguchi-.   Ninhydrin and anthrone tests result; c) has a melting point in the range of 156-159 ° C; d) has an optical rotation of [α] D27 = = 122 (c 0.5 CHC13); e) has an Rf value of 0.60 for silica gel thin layer chromatography in the solvent system CHC13 - CH30H (5: 1 v / v) and an Rf value of 0.16 for reverse phase silica gel thin layer chromatography in the solvent system CH3CN-H2O (75 v? v) having; f) has the following ultraviolet absorption maxima. when dissolved in methanol. 0.0lN HCl-CH3OH and 0.01N NaOH-CH3OH   UV Xma ,. nm (El cm): 248 (212) in methanol 279 (141) 318 (103) UV Äma ,, nm (EC,)): 248 (210) in 0, OlN HCl-CH3OH 279 (140) 318 (103) UV times nm (El cm): 248 (233) in 0, OlN NaOH-CH3OH 278 (150) 318 (110); g) is effective against the growth of various bacteria and fungi; and h) prophage induced in lysogenic bacteria.  7. A process for the preparation of the anti-tumor antibiotic BBM-1675 Al according to claim 1, characterized in that a BBM-1675 Al-producing strain of Actinomadura verrucosospora is cultivated in an aqueous nutrient medium which has assimilable sources of carbon and nitrogen under submergenic aerobic conditions until a substantial proportion of BBM-1675 A1 has been produced by the microorganism mentioned in the culture medium mentioned and BBM1675 Al is then obtained from the culture medium.  8. The method according to claim 7, characterized in that the BBM-1675 A, -producing organism has the identification characteristics of Actinomadura verrucosospora strain H964-92 ATCC 39334, or of Actinomadura verrucosospora strain A1327Y, ATCC 39638.  9. A process for the preparation of the anti-tumor antibiotic BBM-1675 A2 according to claim 2, characterized in that a BBM-1675 A2-producing strain of Actinomadura verrucosospora is cultivated in an aqueous nutrient medium which has assimilable sources of carbon and nitrogen under submergenic aerobic conditions until a substantial proportion of BBM-1675 A2 has been produced by the organism in the culture medium mentioned and then BBM-1675 A2 is obtained from the culture medium.  10. The method according to claim 9, characterized in that the BBM-1675 A2-producing organism has the identification characteristics of Actinomadura verrucosospora strain H964-92, ATCC 39334, or Actinomadura verrucosospora strain A1327Y, ATCC 39638.  11. The method for producing the anti-tumor antibiotic BBM-1675 A3 according to claim 3, characterized in that a BBM-1675 A3-producing strain of Actinomadura verrucosospora is cultivated in an aqueous culture medium which has assimilable sources of carbon and nitrogen under submergenic aerobic conditions until a substantial proportion of BBM-1675 A3 has been produced by the organism in the culture medium mentioned and then BBM-1675 A3 is obtained from the culture medium.  12. The method according to claim 11, characterized in that the BBM-1675 A3-producing organism has the identification characteristics of Actinomadura verrucosospora strain H964-92, ATCC 39334 or Actinomadura verrucosospora strain A1327Y, ATCC 39638.  13. A method for producing the anti-tumor antibiotic BBM-1675 A4 according to claim 4, characterized. that a BBM-1675 A4-producing strain of Actinomadura verrucosospora is cultivated in an aqueous nutrient medium which contains assimilable sources of carbon and nitrogen under submergenic aerobic conditions until a substantial proportion of BBM-1675 A4 is cultivated in the said culture medium by the organism mentioned has been obtained and then BBM-1675 A4 is obtained from the culture medium.  14. The method according to claim 13, characterized in that the BBM-1675 A4-producing organism has the identification characteristics of Actinomadura verrucosospora strain H964-92, ATCC 39334 or Actinomadura verrucosospora strain A1327Y, ATCC 39638.  15. The method for producing the anti-tumor antibiotic BBM-1675 Bl according to claim 5, characterized in that a BBM-1675 B1 -producing strain of Actinomadura verrucosospora is cultivated in an aqueous nutrient medium which has assimilable sources of carbon and nitrogen under submergenic aerobic conditions until a substantial proportion of BBM-1675 B1 has been produced by the organism in the culture medium mentioned and then BBM-1675 B1 is obtained from the culture medium.  16. The method according to claim 15, characterized in that the BBM-1675 Bl-producing organism has the identification characteristics of Actinomadura verrucosospora strain H964-92, ATCC 39334 or Actinomadura verrucosospora strain A1327Y, ATCC 39638.  17. A method for producing the anti-tumor antibiotic BBM-1675 B2 according to claim 6, characterized in that a BBM-1675 B2-producing strain of Actinomadura verrucosospora is cultivated in an aqueous nutrient medium which has assimilable sources of carbon and nitrogen under submergenic aerobic conditions until a substantial proportion of BBM-1675 B2 has been cultivated in the culture medium mentioned by the organism mentioned and then BBM-1675 B2 is obtained from the culture medium.  18. The method according to claim 17, characterized in that the BBM-1675 B2-producing organism has the identification characteristics of Actinomadura verrucosospora strain H964-92, ATCC 39334 or Actinomadura verrucosospora strain A1327Y, ATCC 39638.  19. Pharmaceutical composition containing an effective antimicrobial content of BBM-1675 A according to claim 1, BBM-1675 A2 according to claim 2, BBM-1675 A3 according to claim 3, BBM-1675 A4 according to claim 4, BBM-1675 Bl according to claim 5 or BBM1675 B2 according to claim 6 in combination with a pharmaceutical carrier or diluent.  20. Pharmaceutical composition containing an effective tumor-inhibiting amount of BBM-1675 Al according to claim 1, BBM-1675 A2 according to claim 2, BBM1675 A3 according to claim 3, BBM-1675 A4 according to claim 4, BBM-1675 B1 according to claim 5 or BBM -1675 B2 according to claim 6 in combination with a pharmaceutical carrier or diluent.    21. Biologically pure culture of the microorganism Actinomadura verrucosospora strain H964-92, ATCC 39334, which is capable of producing the antibiotic BBM-1675 in detectable quantity after cultivation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen as a means for carrying out the method according to one of claims 8, 10, 12, 14, 16 or 18.  22. Biologically pure culture of the microorganism Actinomadura verrucosospora strain A1327Y, ATCC 39638, which is capable of producing the antibiotic BBM-1675 in a detectable amount after cultivation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen as a means of Implementation of the method according to one of claims 8, 10, 12, 14, 16 or 18.