CY1465A

CY1465A - Bbm-1675,a new antitumor antibiotic complex

Info

Publication number: CY1465A
Application number: CY1465A
Authority: CY
Original assignee: Bristol Myers Co
Priority date: 1983-05-16
Filing date: 1989-07-21
Publication date: 1989-07-21
Also published as: CA1241282A; JPS59232094A; ZA843591B; ATA160984A; JPH0229079B2; JPH02128693A; KR920001366B1; FI841906L; DK163127C; YU43846B; SE8402619L; IT8420928A0; NL8401572A; SE8402619D0; YU84984A; IT1175498B; DK239784D0; FI81114B; GB2141425A; IL71824A

Description

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GB 2 141 425 A

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SPECIFICATION

BBM-1675, a new antitumor antibiotic complex

5 Background of the invention

1. Field of the invention

This invention relates to new antitumor antibiotic substances and to their production and recovery.

2. Description of the prior art

10 The antitumor antibiotic compounds of the present invention have not yet been identified in terms of structure. In view of their unique physical, chemical and biological properties, however, applicants believe that the BBM-1675 antibiotics are novel substances.

European Patent Publication No. 95154A1 discloses fermentation of Actinomadura puiveraceus sp. nov. No. 6049 (ATCC 39100) to produce antitumor antibiotics designated WS 6049-Aand WS 6049-B. The 15 structures of the WS 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that WS 6049-A and WS 6049-B may be related in structure to the BBM-1675 antibiotics of the present invention. Spectral data show, however, that neither WS 6049A nor WS 6049B is identical to any of applicants'BBM-1675 components. Moreover, the producing organism described in European Patent Application Publication No. 95154A1 may be clearly differentiated from Actinomadura 20 verrucosospora employed in the present invention in the color of its aerial mycelium on ISP Medium Nos. 2, 3 and 4, in its positive milk peptonization and in its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.

Summary of the invention

25 There is provided by the present invention a new antitumor antibiotic complex designated herein as BBM-1675, said complex being produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora most preferably Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a 30 substantial amount of said BBM-1675 complex is produced by said organism in said culture medium, and optionally recovering the complex from the culture medium. Also provided by the present invention are two major bioactive components of BBM-1675 complex designated as BBM 1675A! and A2 and four minor bioactive components of said complex designated BBM-1675A3, A4, Bt and B2. The components may be separated and purified by conventional chromatographic procedures. The BBM-1675 complex and its 35 bioactive components exhibit both antimicrobial and antitumor activity.

Description of the drawings Figure 1 shows the infrared absorption spectrum of partially purified BBM-1675 A, (KBr pellet).

Figure 2 shows the infrared absorption spectrum of partially purified BBM-1675 A2 (KBr pellet). 40 Figure 3 shows the infrared absorption spectrum of BpM-1675 A3 (KBr pellet).

Figure 4 shows the infrared absorption spectrum of BBM-1675 A4 (KBr pellet).

Figure 5 shows the proton magnetic resonance spectrum of partially purified BBM-1675 A1 in CDCI3 (60 MHz).

Figure 6 shows the proton magnetic resonance spectrum of partially purified BBM-1675 A2 in CDCI3 (60 45 MHz).

Figure 7 shows the proton magnetic resonance spectrum of BBM-1675 A3in CDCI3 (60 MHz).

Figured shows the proton magnetic resonance spectrum of BBM-1675 A4 in CDCi3 (60 MHz).

F/gwreS shows the infrared absorption spectrum of purified BBM-1675 A-i (KBr pellet).

Figure 10 shows the proton magnetic resonance spectrum of purified BBM-1675 A-i in CDCI3 (360 MHz). 50 Figure 77showsthe 13C magnetic resonance spectrum of purified BBM-1675 Ai in CDCI3 (90.3 MHz).

Figure 12 shows the infrared absorption spectrum of purified BBM-1675 A2 (KBr pellet).

Figure 13 shows the proton magnetic resonance spectrum of purified BBM-1675 A2 in CDCI3 (360 MHz). Figure 74shows the 13C magnetic resonance spectrum of purified BBM-1675 A2 in CDCI3 (90.3 MHz).

55 Detailed description

This invention relates to a novel antitumor antibiotic complex designated herein as BBM-1675 and to its preparation by fermentation of certain strains of Actinomadura verrucosospora, most particularly Actinomadura verrucosopora strain H964-92and a mutant thereof designated Actinomadura verrucosospora strain A1327Y. The above-mentioned parent strain was isolated from a soil sample collected at Pto 60 Esperanza, Misiones, Argentina. A biologically pure culture of the organism has been deposited (28th March, 1983) with the American Type Culture Collection, Washington, D.C. and added to its permanent collection of microorganisms as ATCC 39334. Subsequently, the mutant strain A1327Y was obtained by conventional nitrosoguanidine (NTG) treatment of strain H964-92 and was deposited with the American Type Culture Collection as ATCC 39638 on 21 st March, 1984.

65 As in the case of many antibiotic-producing cultures, fermentation of Actinomadura verrucosospora strain

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H964-92 or strain A1327Y results in the production of a mixture or complex of component substances. Two major bioactive components, BBM-1675 A! and A2, and four minor bioactive components, BBM-1675 A3, A4, B-, and B2, have been separated from the BBM-1675 complex produced during the fermentation process.

BBM-1675 and its components BBM-1675 A1r A2, A3, A4, B-i and B2 exhibit antimicrobial activity against a 5 broad spectrum of microorganisms including especially gram-positive bacteria. The BBM-1675 complex and separated bioactive components thereof also exhibit phage inducing properties in lysogenic bacteria. Two of the components, BBM-1675 A, and A2, have been submitted to in vivo screening against various mouse tumor systems and demonstrate inhibitory activity against L-1210 leukemia, P-388 leukemia, B16 melanoma and Lewis lung carcinoma, BBM-1675 A3 and A4 have been shown to exhibit activity against mouse P-388 10 leukemia. The complex and its bioactive components, therefore, may be used as antimicrobial agents or as antitumor agents for inhibiting mammalian tumors.

The microorganism

The actinomycete Strain No. H964-92 was isolated from a soil sample and prepared by conventional 15 procedures as a biologically pure culture for characterization. Strain H964-92 forms on the aerial mycelium short spore-chains which show straight, flexuous or hooked shapes. The spores are spherical or oval-shaped and have a warty surface. Aerial mycelium is poorly formed on most media. The aerial mass color is white which later turns to a pinkish shade, or further changes to a bluish color in some agar media. The color of substrate mycelium is colorless or pale pink. The growth temperature ranges from 15°Cto 43°C. The cell-wall 20 amino acid composition and whole cell hydrolyzate sugar components showthat strain H964-92 belongs to cell wall Type IIIB. The menaquinone was identified as MK-9(H6)'MK-9(Ha).

Based on the major morphological, cultural and physiological characteristics along with the chemical cell-wall composition characteristics, strain H964-92 can be classified as belonging to the genus Actinomadura.

25 Although the original strain H964-92 gave only moderate growth and bore scant aerial mycelia, a variant showing good growth and improved aerial mycelium formation was obtained by NTC (nitrosoguanidine) treatment of H964-92. The variant, designated strain A1327Y, facilitated further taxonomical investigation and was subsequently identified as Actinomadura verrucosospora.

30 Methods

The media and procedures used for examining cultural characteristics and carbohydrate utilization were those recommended by the International Streptomyces Project (Intl. J. Syst. Bacterioi. 16:313-340,1966). Additional media described by S. A. Waksman (The Actinomycetes, Vol. 2) and G. M. Lvedemann [Int J.

Syst. Bacterioi. 21: 240-247,1971) were also used. The cell wall-amino acid composition and whole cell 35 hydrolyzate sugar components were analyzed according to the methods described by Becker, et al. in Appf. Microbiol. 13: 236-243,1965 and by Lechevalierand Lechevalierin The Actinomycetes, Ed. H. Prauser, Jena, Gustav Fischer Verlag, pp. 393-405,1970, respectively. The menaquinone was identified by mass spectral analysis according to the procedure of Collins et al. in»/. Gen. Microbiol. 100: 221-230,1977, and the menaquinone composition was represented based on the system described by Yamada et al. in J. Gen. Appl. 40 Microbiol. 23: 331-335,1977.

Morphology

Strain H964-2 forms both substrate and aerial mycelia. The substrate mycelium is long, branched and not fragmented into short filaments. In the aerial mycelium, short spore-chains are formed monopodially or at 45 the hyphal tip. Whorl-like branches of spore-chain are also observed nearby the hyphal tip. These spore-chains contain 2 to 10 spores in a chain and are straight, flexuous or hooked in shape. The spores have a warty surface and are spherical to elliptical (0.5-0.6 x 0.6-1.4 (jum) in shape with rounded or pointed ends. After maturation each spore is often separated with empty sheath. Motile spores, sporangia or sclerotic granules are not seen in any media examined.

50

Cultural and physiological characteristics

Growth of strain H964-92 is poor to moderate in both chemically defined media and natural organic media. Formation of aerial mycelium is generally poor but is moderate in oat meal agar (ISP No. 3 medium), inorganic salts-starch agar (ISP No. 4 medium) and Bennett's agar. Spontaneous variants which lack aerial 55 mycelium occur at high frequency. The color of aerial mycelium is white which latertumsto pale pink in oat meal agar, inorganic salts-starch agar and glycerol-asparagine agar (ISP No. 5 medium). The aerial mass color further changes to a bluish color after long incubation (5 months) in oat meai agar, glycerol-asparagine agar and tyrosine agar. The color of substrate mycelium is colorless to yellowish in Czapek's agar, tyrosine agar, yeast extract-malt extract agar (ISP No. 2 medium), peptone-yeast extract-iron agar (ISP No. 6 medium) 60 and Bennett's agar, and is a pinkish color in glucose-asparagine agar and glycerol-asparagine agar.

Melanoid and other diffusible pigments are not produced. A variant No. A1327Y, which was obtained from the original strain, forms predominantly pale blue aerial mycelium and bears abundant aerial spore mass.

Strain H964-92 grows at 15°C, 28°C, 37°C and 43°C, but not at 10°C or at47°C. It is sensitive to NaCI at 7%, and resistant to lysozyme at 0.01%.

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

TABLE 1

Cultural Characteristics' of Strain H964-92 (original strain ATCC 39334 and variant AI327Y)

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Strain No. H964-92

Original Strain (ATCC 39334)

Variant No. A1327Y

Actinomadura verrucosospora KCC A-0147

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Tryptone-yeast extract 15 agar (ISP No. 1)

Sucrose-nitrate agar (Czapek's agar)

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G: abundant, floccose, moderate, floccose, sedimented and not sedimented and not pigmented pigmented poor colorless no or scant; pinkish white (9)

none

G: moderate R: colorless A: scant; light gray (264), to pale pink (7)

D:none moderate, floccose, sedimented and not pigmented poor colorless no or scant; pale blue (185)

none

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20

25 Glucose-asparagine agar G: moderate

R: white (263) to deep yellowish pink (277) A: no or very scant; pale pink (7) 30 D:none

Glycerol-asparagine agar G: poorto moderate

(ISP No. 5)

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Inorganic salts-starch agar (ISP No. 4)

50 Tyrosine agar [ISP No. 7)

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poor colorless no or very scant-

white none moderate light yellowish pink (28)

moderate; white to light pink (4)

R: colorless to light yellowish pink (28)

A: poor; light yellowish pink (28), after 5 months light bluish gray (190)

D:none

G:abundant

R: yellowish white (92)

A: abundant; light pink moderate; light bluish (4) to pinkish gray gray (190)

(10)

D:none none

G: moderate moderate

R: yellowish white (92) strong yellowish pink (26)

none moderate light yellowish pink (28)

A: poor; light yellowish pink (28), very later (5 months) partially light bluish gray (190)

D:none moderate; white to light pink (4)

none poor colorless no or very scant;

white none moderate light yellowish pink (28 to deep yellowish pink (27) moderate; white to strong pink (2)

none moderate light yellowish pink (28)

abundant; pale blue 1185)

none moderate strong yellowish pink (26)

moderate; white to light pink (4)

none

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60 Nutrient agar

G: poorto moderate R: pale yellow (89)

A:none D:none poor colorless to pale pink (7)

none none poor colorless to pale pink (7)

none none

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TABLE 1 - cont'd

Strain No. H964-92

Actinomadura

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Original Strain

verrucosospora

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(ATCC 39334)

Variant No. A 7327Y

KCCA-0147

Yeast extract-malt

G: abundant abundant abundant

extract agar

R: pale yellow (89)

strong yellowish strong yellowish

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{ISP No. 2)

pink (26)

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A: poor; white (263)

poor; white to pale poor; white to pale

pink (7)

D:none none none

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Oat meal agar

G: moderate poor poor

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(ISP No. 3)

R: colorless to pale pale yellowish pale yellowish

pink (7)

pink (31)

A: poor; pinkish very scant; vivid very scant; vivid

white (9) to light pale blue (184)

pale blue (184)

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bluish gray (190)

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D:none none none

Bennett's agar

G:abundant abundant abundant

R: grayish yellow (90)

strong yellowish strong yellowish

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pink (26)

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A: moderate; white (263) moderate; pale yellow-

none

to yellowish white ish pink (31) and

(92)

bluish white (189)

D:none none none

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Peptone-yeast extract-

G: moderate abundant abundant

iron agar (ISP No. 6)

R: colorless colorless colorless

A:none none none

D:none none none

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* Observed after incubation at 37°C for 3 weeks.

** Abbreviation: G = Growth; R = Reverse color; A = Aerial mycelium; D = Diffusible pigment *** Color and number in parenthesis follow the color standard in "Kelly, K. L. & D. B. Judd; ISCC-NBS

color-name charts illustrated with Centroid Colors. U.S. Dept. of Comm. Cir. 553, Washington, D.C., Nov., 1975". 40

KCC = Kaken Culture Collection of Kaken Chemical Company

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Range of temperature for growth

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Gelatin liquefaction

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Reactions in skimmed milk

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Formation of melanoid pigment

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Resistance to NaCI

Lysozyme

PH

TABLE 2

Physiological Characteristics

Response

Maximal growth at 28°C to 37°C.

Moderate at 20°C and 43°C. No growth at 10°C and 47°C.

Liquefied

Hydrolyzed

Not coagulated and completely peptonized

Not produced Not reduced

Growth at 5% or less. No growth at 7%.

Resistant. Growth at 0.01% or less. No growth at 0.1%.

Growth in 5.0 to 9.5. No growth at 4.5 and 10.0.

of Strain H964-92

Method and Medium Bennett's agar

Glucose-peptone-gelatin medium Starch agar plate Difco skimmed milk

Tyrosin agar, peptone-yeast-iron agar and tryptone-yeast extract broth.

Czapek's glucose-nitrate broth and glucose-yeast extract-nitrate broth

Tryptone-yeast extract agar

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TABLE 3

Utilization of Carbon Sources

5 Strain No. H964-92 Actinomadura

Original Variant verrucosospora strain No. A1327Y KCC A-0147

Glycerol

+

D(-)-Arabinose

-

L(+)-Arabinose

+

D-Xylose

+

D-Ribose

+

—

-

L-Rhamnose

+

D-Glucose

+

D-Galactose

-

D-Fructose

+

D-Mannose

-

L(-)-Sorbose

-

Sucrose

+

Lactose

-

Cellobiose

+

Melibiose

-

Trehalose

+

Raffinose

-

D(+)-Melezitose

-

Soluble starch

+

Cellulose

+

Dulcitol

-

Inositol

+

-

D-Mannitol

+

D-Sorbitol

-

Salicin

—

-

35 Observed after incubation at 28°C for 3 weeks

Basal medium: Pridham-Gottlieb inorganic medium

Cell-wall composition and whole cell sugar components Purified cell-wall of strain H964-92 contains mesodiaminopimelic acid but lacks glycine. The whole cell 40 hydrolyzate shows the presence of madurose (3-O-methyl-D-galactose), glucose and ribose. The cell-wall amino acid and whole cell sugar components indicate that strain H964-92 is placed in cell-wall Type Mb-Two major components of menaquinone were identified as MK-9(H6) and MK-9(H8).

Taxonomic position of strain H964-92 45 Strain H964-92 has the following major characteristics: (1) Aerial spore-chains: short, straight, flexuous or hooked in shape. (2) Spores: warty surface. {3) Aerial mycelium: pinkish or bluish color. (4) Substrate mycelium: pinkish in some media. (5) Diffusible pigment: none. (6) Mesophile. (7) Cell-wall Type lllB. (8) Menaquinone system: MK-9(H6)and MK-9(H8).

These major characteristics indicate that strain H964-92 is placed in the genus Actinomadura. Early species 50 of the genus Actinomadura were isolated from mammals. Some strains were also obtained from plant materials. However, many of the new species proposed recently were isolated from soil. According to the numerical taxonomy and review of the Actinomadura and related actinomycetes by Goodfellowet al. in J. Gen. Microbiol. 112:96-111 (1979), most Actinomadura species of soil origin are classified intoCluster No. 7 among the 14 clusters described. Strain No. H964-92 is most related to the species of Cluster?. Nonomura 55 and Ohara in J. Ferment Techno!. 49: 904-912 (1971) reported five saprophytic species of the genus Actinomadura and Nonomura [J. Ferment. Techno!. 52:71-77 (1974)] and Preobrazhenskaya etal. [Actinomycetes and Related Organisms 12: 30-38 (1977)] published the keys for identification and classification of the Actinomadura species. As a result of comparison with the descriptions of 30 species including organisms disclosed in patents, strain H964-92 appears most similar to Actinomadura coerulea gO described in the Preobrazhenskaya et al. reference above and to Actinomadura verrucosospora described in the Nonomura references cited above.

Strain No. H964-92 was directly compared with A. verrucosospora strain KCC A-0147 and was found to be closely related to A. verrucosospora in the morphological, cultural and physiological characteristics. Thus, strain H964-92 is classified as a new strain of Actinomadura verrucosospora.

65 It is to be understood that for the production of BBM-1675, the present invention, though described in

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detail with reference to the particular strain Actinomadura verrucosospora strain H964-92 (ATCC 39334) and the mutant strain thereof designated strain A1327Y (ATCC 39638), is not limited to these microorganisms or to microorganisms fuliy described by the cultural characteristics disclosed herein. It is specifically intended that the invention embrace strain H964-92 and all natural and artificial BBM-1675-producting variants and 5 mutants thereof.

Antibiotic production

The BBM-1675 antibiotics of the present invention may be prepared by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora, preferably a strain of Actinomadura verrucosospora having the 10 identifying 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 nutritional sources for actinomycetes, i.e. assimilable sources of carbon and nitrogen plus optional inorganic salts and other known growth factors. Submerged aerobic conditions are preferably employed for the production of large quantities of antibiotics, although for production of limited amounts, surface cultures and bottles may also 15 be used. The general procedures used for the cultivation of other actinomycetes are applicable to the present invention.

The nutrient medium should contain an appropriate assimilable carbon source such as glycerol, 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 20 nitrate, sodium nitrate, etc. may be used either alone or in combination with organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep liquor, soybean powder, cotton seed flour, etc. There may also be added, if necessary, nutrient inorganic salts to provide sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobait, iron, and the like.

25 Production of the BBM-1675 antibiotics can be effected at any temperature conducive to satisfactory growth of the producing organism, e.g. 15-45°C, and is conveniently carried out at a temperature of around 27-32°C. Ordinarily, optimum production is obtained after incubation periods of from about 68-180 hours, depending on whether shake-flask, stir-jar or tank fermentation is employed. When tank fermentation is to be carried out, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth 30 culture with a slant or soil culture or a lyophilized culture of the producing organism. After obtaining an active inoculum in this manner, it is transferred aseptically to the fermentation tank medium. Antibiotic production may be monitored by the paper disc-agar diffusion assay using Staphylococcus aureus 209P as the test organism.

35 Isolation and purification

When fermentation is complete, the BBM-1675 complex may be obtained from the broth by conventional isolation procedures, e.g. solvent extraction. Thus, for example, the whole broth may be separated by filtration or centrifugation into mycelial cake and broth supernatant. Antiobiotic in the mycelial cake may be recovered by suspending the cake in methanol, filtering off insoluble materials and concentrating the 40 methanolic extract. Activity in the broth supernatant may be recovered by extraction with n-butanol. The above-mentioned n-butanol and methanol extracts may then be combined and evaporated azeotropically to an aqueous solution which deposits most of the antibiotic activity as an oily solid. The solid may then be dissolved in methanol and the solution filtered. Filtrate is concentrated and added to a mixture of ethyl acetate and water. The resulting organic extract contains the crude BBM-1675 complex which may be 45 precipitated from solution by addition of an antisolvent such as n-hexane.

The crude BBM-1675 complex is a mixture of several components including two major bioactive components, BBM-1675 A-i and A2, and four minor bioactive components, BBM-1675 A3, A4, B, and B2. These bioactive components may be separated and purified by conventional chromatographic procedures. In one procedure the crude BBM-1675 complex is first dissolved in methanol and purified by Sephadex LH-20 50 column chromatography using methanol as the eluting solvent. This partially purified complex may then be chromatographed on a silica gel column and eluted in a stepwise manner using chloroform plus an increasing concentration of methanol to provide BBM-1675 A1, a mixture of BBM-1675 A2, A3 and A4 and a mixture of BBM-1675 B, and B2. The A-i component may be further purified by Sephadex LH-20 column chromatography using methanol as the eluting solvent. The mixture of A2, A3 and A4 may be separated by 55 chromatography on a column of Bondapak C18 (Waters Associates, Inc.) using increasing concentrations of aqueous acetonitrile as the eluant. The mixture of B, and B2 components may be separated by silica gel column chromatography using a mixture of chloroform and methanol as the eluting solvent. Further details of the preferred chromatographic separation procedures are provided in the examples which follow.

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Physico-chemical properties of BBM-1675 components

The six bioactive components of BBM-1675 complex are distinguishable from each other by two TLC systems as shown in the following Table.

TABLE 4

TLC of BBM-1675 Components Rf Values

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Component

Si02

CHCI3-CH30H(5:1 v/v)

* SHanized CH3CN-H20(75:25 v/v)

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BBM-1675 Ai BBM-1675 A2 15 BBM-1675 A3

BBM-1675 A4 BBM-1675 B-, BBM-1675 B2

20 * C-ia reverse phase silica gel

0.74 0.71 0.72 0.71 0.63 0.60

0.18 0.21 0.28 0.78 0.23 0.16

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25

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Separation of BBM-1675 A2, A3 and A4 was difficult by ordinary phase TLC systems but could be achieved by a reverse phase TLC.

The individual BBM-1675 components show solubility and color reactions similar to each other. For example, 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 ferric chloride, Ehrlich and Tollen's reagents but negative responses in Sakaguchi, ninhydrin and anthrone tests.

Characteristic physico-chemical properties of BBM-1675 components are shown in Table 5 below.

TABLE 5

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Physico-Chemical Properties of BBM-1675 Components

BBM-1675A-,

A3

a4

Bj B2

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Melting point (dec)

156 ~ 158°C 147 ~ 149°C 125 ~ 127°C 123 ~ 126°C 159

~ 161°C 156- 159°C

(a)" (c 0.5, CHCI3)

-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

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N

4.02

3.82

3.57

3.45

(by difference)

O

38.65

36.06

34.91

36.53

(UVXmax rim(Ei cm) in CH3OH

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50

in O.OINHCI-CH3OH

in 0.01 N NaOH-CH3OH

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253(325) 282(195) 320(143)

253 (281) 282(172) 320(128)

253 (286) 282(158) 320(122)

253 (257) 282(153) 320(117)

253 (225) 282(140) 320(104)

248 (212) 279 (141) 318(103)

253 (323) 282(192) 320(144)

253 (276) 282(167) 320(128)

253 (287) 282(160) 320(126)

253 (258) 282 (155) 320 (118)

253 (225) 282 (140) 320 (105)

248(210) 279(140) 318(103)

252 (325) 283(172) 318(136)

252 (289) 283(171) 318(122)

252(280) 283(162) 318(120)

252 (266) 283(160) 318(118)

252 (236) 282(141) 318(105)

248 (233) 278(150) 318(110)

1,300

1,100

1/100

1,400

60

65

Mo. wt.

(approximate value)

{Gel HPLC, Finepak (GEL-101)

The UV absorption maxima of BBM-1675 components were observed at 253,282 and 320 nm, which did not shift in acidic or alkaline solution. The IR and PMR spectra of BBM-1675 A-i, A2, A3 and A4 are shown in Figures 1-4 and Figures 5-8 respectively. The 360 MHz PMR of BBM-1675 A-i indicated one acetyl (8:2.11 ppm), one N-CH3(2.52 ppm), four OCH3(3.42,3.80,3.88 and 3.98 ppm) and one exomethylene (4.57 and 5.48 ppm) groups, along with two aromatic (7.50 and 8.59 ppm) and one NH (11.79 ppm) protons. The CMR

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spectrum of BBM-1675A-I exhibited 55 carbon signals including a triple intensity signal (8:56.0 ppm, OCH3). The molecular formula of BBM-1675A-I is deduced to be C57H72N4O32 based on proton and 13C NMR spectra, micronalysis and molecular weight determination by HPLC and SIMS (secondary ion mass spectrometry).

Structural study of BBM-1675A1 5

Upon treatment with 0.5N HCI-CH3OH at room temperature, BBM-1675A! loses its bioactivity and affords a lipophilic chromophore substance (compound I) along with several unidentified fragments. Compound I shows UV absorption similar to that of parent antibiotic suggesting that compound I retains the chromophoric structure of BBM-1675Ai. Two other chromophoric fragments related to compound ! are obtained by alkaline hydrolysis of BBM-1675Ai: hydrolysis with 0.05N KOH-CH3OH at55°Cforone hour -jq yields compound II having UV absorption maxima at 252,284,297 (shoulder) and 322 nm, while the reaction in 1N KOH-CH3OH affords an acidic chromophore substance designated compound III. Physico-chemical properties of compounds I, II and III are summarized in Table 6 below.

15

TABLE 6

Properties of Compounds /, // and III

15

Compound /

Compound II

Compound III

20

M.p.

82 ~ 83°C

133°

253 ~ 255°C

20

[oc]D9(C 0.2 CHCI3) :

-100°

0

25 Molecularformula

C2IH3-|NOio

C14H17NO6

C13H15NO6

25

. CH3OH

UVX nm (e) : max

30

244(21,850) 276 (9,400) 318 (6,300) 318 (6,300)

252 (26,600) 283 (11,200) 297 (sh8,800) 322(11,700)

248 (26,900) 295(14,400) 310(13,500)

30

MS m/z :

35

457 (M+)

425

341

281

264

295 (M+)

280

263

251

248

281 (M+)

263

236

222

218

35

TLC

(Xylene-*MEK-CH3OH=5:5:1 v/v) :

40

Rf 0.58

0.66

0.13

40

MEK = methyl ethyl ketone

45

50

Structural information about compounds II and III was provided from the following spectral data and chemical transformation. The 13C and proton NMR indicated the presence of four OCH3, one =CH2, seven -6=,two >C=0 and one >NH groups in compound II. The NMR spectra of compound III was similar to those of II, differing only in the absence of one of the four OCH3 groups observed for compound II. This difference, together with the acidic nature of compound 111, suggested that compound II is a methyl ester of compound III. When heated under reflux with 1N methanolic KOH, compound II was quantitatively converted to compound 111, while compound III was converted to compound II by treatment with diazomethane.

Treatment of compound II with NaBH4in C2H50H gave a reduction product (compound IV, M+:m/z 267) which showed a —CH2OH group in the NMR in place of the —COOCH3 group of compound II. Upon hydrogenation over palladium on charcoal, compound II afforded a dihydroderivative (compound V, M+:m/z 297). The proton NMR spectrum of compound V exhibited a new doublet methyl signal and the absence of

45

50

BNSDOCID: <GB 2141425A_

10

GB 2 141 425 A

10

the exomethylene group present in compound II. Furthermore, one of the OCH3 groups appeared at higher field (5: 3.50 ppm) in compound V. These results indicated the presence of a

—c=ch2

5 i 5

och3

group in compound II which was reduced by hydrogenation to 10 -CH-CH3 10

i och3

group in compound V. Compound II was heated with 1.5N methanolic hydrogen chloride at80°Cfor3 hours 15 and the hydrolyzate chromatographed on a silica gel column to afford a weakly basic compound (compound 15 VI, M+: m/z211).The IR spectrum and physico-chemical properties indicated that compound VI contained an NHZ group. Compound VI was identified as methyl 4,5-dimethoxy-anthranilate by comparative IR and NMR studies with an authentic sample. Consequently, the structures of compounds II-VI were determined as shown below.

20 20

Structures of compounds II, III, IV, V and V!

Compound II

Compound III

25

30

25

30

35

40

45

50

H+/MeOH

M/

Comoound VI

35

40

45

50

55 Upon treatment with 0.05N KOH-CH3OH at 55°C, compound i was split into a new chromophoric fragment 55 (compound VII, C-15H21NO7, M+ : ml? 327) and a sugar (compound VIII). The NMR spectrum of VII exhibited one singiet C-CH3 and two high-field 0CH3 groups in addition to three low-field 0CH3 and two aromatic protons commonly observed for compounds II, V and VI. Further hydrolysis of VII with 1.5N methanolic hydrogen chloride gave compound VI which was identical with that previously obtained from II. After 60 removal of VI, the hydrolyzate was treated with 2,4-dinitrophenylhydrazine to precipitate yellow solid which 60 was identified as 2,4-dinitrophenylhydrazone of pyruvic acid. Thus compound VII is methyl, 4,5-dimethoxy-N-(2',2'-dimethoxypropionyl)-anthranilate. Compound VIII did not show the molecular ion peak but did show the M-OCH3 peak at m/z 131 in the mass spectrum in agreement with the molecular formula of C7H1404. The NMR spectrum indicated 2,6-dideoxyhexopyranose structure for compound VIII. The assignment was 65 supported by the13C-NMR of compound I which gave rise to one C-CH3, one -CH2, three 0-CH< and one 65

BNSDOCID: <GB 2141425A_J_>

11

GB 2 1 41 425 A

11

anomeric carbon in addition to 15 carbon signals assignable to compound Vil. The C3 proton of the sugar appeared at low-field (5: 5.39 ppm, octet) revealing the C3-OH of the sugar was esterified by thecarboxyl group of VIII. The above results are summarized below:

5 Structures of compound I, VII and VIII

Compound I

Compound VII

Compound VI

oat3

ifl

15

v cs p-

CB,

I 3 OCH3

CH,Ovri*£rvVvJJH-CO-i-CH

I L '

at 3 o-^^/^-cooch3

Compound VIII .OH

SB

aj,Y*V

■NHj COOCH,

CH,

L—CHj-CO-COOH

20

The molecular weight of compound I (457) accounts for about one-third of the entire molecule of BBM-1675A! (proposed formula CS7H72N4032; calc'd MW = 1324). The partial structure of BBM-1675 A1 is considered to be as follows:

10

15

20

25

30

35

0

1

25

30

35

Subsequentto the U.S. filing date of parent application Serial No. 495,231, it was discovered that components BBM-1675 A-i and A2 described above and produced according to Example 2 below were in fact 40 not completely pure and that certain of the characterizing properties used to define such components were inaccurate. Following additional chromatographic purification procedures as described more fully in Examples 3 and 6 below, BBM-1675 An and A2 were isolated in more purified form and fully characterized as described below. Also, the elemental analysis data for components A3 and A4 was revised to show the presence of sulfur in these compounds and HPLC retention times were calculated for these two components.

40

BNSDOCID: <GB 2141425A_I_>

12

GB 2 141 425 A

12

Summarized below are the revised physico-chemical properties of the BBM-1675 components. BBM-1675A j

5 Description:

Elemental analysis:

10

15

Analysis 1

C: 51.60% H: 6.31% N: 5.31% S: 8.47%

white to pale yellow crystals; mp 156-158° (dec.)

Analysis 2

C: 52.74% H: 5.99 N: 3.94% S: 9.71%

Average

C: 52.17 H: 6.15 N: 4.63% S: 9.09%

O (by difference):28.31% O (by difference): 27.62% 0 (by difference):27.96% Ultraviolet absorption spectrum: Instrument-Varian UV, Cary 219

20

25

absorptivities

12.4

sh (shoulder) 25.1

25.5

30

Optical rotation:

35

40

Solvent - methanol Concentration = 0.01356 g/l

320 280 253 210

No significant change with acid or base

Solvent - CHCI3 [a]g4°= —207° (C=0.0351)

A second analysis showed the following optical rotation: [a]§7°=-191°(C=0.5, CHCI3).

Infrared absorption spectrum: See Figure 9

45 Mass spectra:

50

55

60

Major absorption bands (KBr):

985,1015,1070,1110,1150,1210, 1250,1308,1380,1405,1446,1520,1592,1608, 1668,1715,2920, 2960,3360,3440, cm-1

Instrument - VG-ZAB-2F FAB-MS-thioglycerol

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

FAB-MS-MB (MB:matrix, m.w. 154); Molecular mass range ions (m/z): 1249, 1283,1403,1555; with the addition of IMaCI (m/z): 1249,1271,1303,1425,1483, 1577.

FAB-MS-glycerol-DMSO: Molecular mass range ions (m/z): 1215,1247,1279, 1293,1325,1353; with addition of NaCI (m/z): 1215,1237,1247,1269,1325, 1347,1375.

Instrument: Kratos MS-50

FAB-MS-thiogfycerol; Molecular mass range ions (m/z): 1357,1463.

Molecular weight (based on above-described mass spectral data)-:

apparent MW = 1248

10

15

20

25

30

35

40

45

50

55

60

BNSDOCID: <GB 2141425A_I_>

13

GB 2 141 425 A 13

Nuclear Magnetic Resonance Spectra: Instrument-WM360 Brucker Solven: CDCI3

1NMR: 360 MHzS{ppm): 11.75 (1H,s); 8.55 (1H,s); 7.45 (1H, s); 6.61 (1H, m); 6.23 (1H, brs); 6.17 (1H, 5 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, 5

brs); 4.95 (1Hr d, J = 10.2); 4.64 (2H, m); 4.54 (1H, d, J=2.3); 4.2 (1H, s); 4.15-3.35 (26-28H) [4.10 (1H, m); 4.02 (1H, brs); 3.95 {3H, s); 3.85 (3H, s), 3.79 (3H, s); 3.46 (1H, m); 3.40 (3H( s)]; 2.82-2.70 (3H, brm); 2.50 (3H, s), 2.47 (1H, m); 2.38-2.22 (5H); 2.12 (1H, m); 2.11 (3H, s); 1.60-1.05 (22H) [1.39 (3H, d, J=6.3); 6.31 <3H,d,J=6.3); 1.29 (3H, d,J=6.3), 1.08 <6H)]

10 10

See Figure 10 13C NMR: 90.3 MHz

15 See Figure 11 15

In a separate test the 13C NMR spectrum of purified BBM-1675 A1 was determined for a sample dissolved in CDCI3 (80 MHz). Major peaks are indicated below.

20 CMR of BBM-1675A, (80 MHz in CDCL3) 20

Chemical shift in ppm (Multiplicity*)

BBM-1 675AT

25 23.4(q) 29.0(t) 34.0(t) 35.1 (t) 39.5(t) 47.2(d) 25

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)

89.5(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)

130.1(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)

30 99.6(d) 103.8(d) 107.6(s) 112.5(d) 123.1(d) 124.9(d) 30

*Multiplicity - q = quartet; d = doublet; u = uncertain 35 t = triplet; s = singlet 35

BBM-1675A2

Description: white crystals; mp 147-149°C

40 40

Elemental analysis: C: 52.71%

H: 5.94%

N: 3.94%

S: 9.39%

45 0(by difference): 28.01%' 45

Ultraviolet absorption spectra: Instrument-Varian UV. Cary 219

Solvent: methanol

50 50

Concentration: 0.02052 g/l

Xmax (nm) absorptivities

55 320 12.2 55

282 16.3

252 26.2

214 25.8

60 No significant change with acid or base. 60

BNSDOCID: <GB 2141425A_J_>

14

GB 2 141 425 A

14

Optical rotation: Infrared spectra:

Mass spectra:

10

15

Molecular weight: 20 (based on above-described mass spectral data)

[a]g7°=-179.4° (c 0.5, CHCI3)

See Figure 12

Instrument: Beckman IR Model 4240

Major absorption bands (KBr): 950,1015,1070,1100,1155,1213,1250,1313, 1375,14G5,1450,1520,1595,1610,1685,1735,2940,2980,3440, cm-1.

Instrument: VG-ZAB-2F FAB-MS-thioglycerol

Molecular mass range ions (m/z): 968,1249,1355,1357,1463,1569; with addition of NaCI (m/z): 990,1271,1379,1485,1593

FAB-MS-MB (MB: matrix, m.w. 154); Molecular mass range ions (m/z): 1249, 1403,1419,1555,1571,1587; with the addition of NaCI (m/z): 1249,1271,1425, 1441,1457,1483,1577.

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

apparent MW = 1248

10

15

20

Nuclear Magnetic: Resonance Spectra

25

30

35

See Figure 13

Instrument: WM 360 Bruckner Solvent: CDCI3

1H NMR 360 MHz 5(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)j 3.46 (1H, m); 3.39 {3H, s); 2.79 (1H, m); 2.73 (2H, m); 2.50 (3H, s); 2.50 (1H, m); 2.38-2.22 (3H); 2.14 (1H, m); 2.10 (3H,s); 1.98 (2H, m); 1.65-1.45 (6-8H); 1.38{3H, d, J=6.0); 1.34 (3H, d, J=6.0); 1.22 (3H, d, J=6.8); 1.10 (6H).

13C NMR 90.3 MHz

26

30

35

40

See Figure 14

In a separate test the 13C NMR spectrum of purified BBM-1675A2 was determined for a sample dissolved in CDCI3 (80 MHz). Major peaks are indicated below.

40

BBM-1675A2

13.7

16.9

17.5

19.8

22.3

22.7

23.4

33.1

45

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

50

144.1

154.2

154.5

160.9

167.9

192.2

45

50

: <GB 2141425A l_>

15

GB 2 141 425 A 15

TABLE 7

Physico-chemical Properties of BBM-1675 A3, A4, Bu B2

5

A3

A4

B,

Bz

Melting point (dec):

125 — 127°C

123- 126°C

159- 161°C

156-159

[a]o7 (c 0.5, CHCI3):

-161°C

-176°C

-171°C

-122°

10

Anal. Found (%):

C:

54.55

54.65

H:

6.46

6.29

N:

3.73

3.51

S:

7.49

8.07

15

UV Xmax nm (E]°£m):

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)

20

in 0.01 N HCl-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 0.01 N NaOH-MeOH:

252 (280)

252 (266)

252 (236)

248 (233)

25

283(162) 318(120)

283(160) 318(118)

283(141) 318(105)

278(150) 318(110)

10

15

20

25

Thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) data on BBM-1675 30 components

A. Study No. 1 - summary

30

35

40

TABLE 8

TLC and HPLC of BBM-1675 components

TLC (Ftf) HPLC (retention time,

in minutes

Si02 *SHanized Lichrosorb RP-18

CHCh-MeOH CH3CN-H20 CH3CN-MeOH-0.1M CH3C00NH4 (5:1 v/v) 75:25 v/v) (5:2:3 v/v)

35

40

BBM-1675A, 0.74

45 BBM-1675A2 0.71

BBM-1675A3 0.72

BBM-1675A4 0.71

BBM-1675B-] 0.63

BBM-1675B2 0.60

50

* C18 reverse phase silica gel

0.18

13.3

0.21

17.3

0.28

8.0

0.78

5.1

0.23

-

0.16

-

B. Study No. 2 - TLC and HPLC for purified A1 and A2 components TLC Chromatography

55 Analtech GHLF Silica Gel Uniplates were used for all normal phase chromatography. Plates measuring 2.5 55 cm x 10 cm were used for one-dimensional TLC. These were developed in glass cylinders measuring 6.4 cm (o.d.) by 12 cm and containing 10 ml of eluant. Plates measuring 7.5 cm x 10 cm were used for two dimensional TLC. The Sample was applied to the lower left hand corner 1 cm from the edges. The plate was developed first in a tank (12.7 cm wide, 8.6 cm deep, 13 cm high) containing 50 ml of the first eluant. The 60 plate was then air dried, rotated 90° counterclockwise, and developed in a second tank containing 50 ml of 60 the second eluant.

Whatman analytical precoated C-18 silica ge! plates were used for all reverse phase chromatography.

Plates measuring 2.5 cm x 7.6 cm were developed in glass cylinders measuring 10 ml of eluant.

Normal phase plates were viewed under 254 nv uv light first. The plates were then inserted into a glass 65 cylinder (6.4 cm o.d. by 12 cm) containing l2 crystals. The plates were then reexamined after approximately 2 65

BNSDOCID: <GB.

2141425A_I_>

16 GB 2 141 425 A

16

10

minutes. Reversed phase plates were visualized under 254 nm uv light only. Zones were detected by looking for quenching of the fluorescence of an impregnated dye.

Analytical HPLC

The following components were used to construct an analytical HPLC system: Waters Associates Model 6000A Solvent Delivery System pump; Varian Varichrom Model VUV-10uv/vis Detector set at 254 nm 0.1 OD; Fisher Recordal Series 5000 Recorder; Waters Associates Model 660 Solvent Programmer; Waters Associates Mode! U6K injector; Alltech, p.-BondapakCi8(10(i) column (4.6 mm i.d. x 25 cm) with a Whatman Co. Pell ODS (0.03-0.038 mm) guard column (4.6 mm i.d. x 5 cm). The components were connected with 316 stainless steel tubing (1.6 mm o.d. - 0.23 mm i.d.). Eluant was pumped at 2 ml/minforall analysts.

10

15

20

25

30

35

40

Preparative HPLC

The following components were used to construct a medium pressure liquid chromatography system: Fluid Metering, Inc. Model RP-SY 2CSC FMI Lab Pump; Fluid Metering, Inc. Model PD-60LF FMI Pulse Dampener; a 15 ml sample loop constructed of polypropylene tubing (3.0 mm o.d. x 1.5 mm i.d.) wrapped around a cardboard tube (8.65 cm o.d.); Glenco Series 3500 Universal LC columns; Instrument Specialties Co. Model U A-5 Absorbance/Fluoresence Monitor with a Type 6 optical unit; Instrumentation Specialities Co. Model 590 Flow Interrupter Valve; and an Instrumentation Specialties Co. Model 328 Fraction Collector. The components were connected with polypropylene and Teflon tubing (3.0 mm o.d. x 1.5 mm i.d.) and Glenco multifit connectors and valves in the order listed.

The Glenco series 3500 Universal LC Columns were slurry packed with the defined adsorbent in the designated solvent using standard techniques. The void between the settled bed and tube top was filled with standard Ottawa sand. Eluant was pumped at a maximum rate which would not exceed 60 psi back pressure (approximately 20 ml/min).

Gradient elution

A Glenco gradient elution apparatus consisting of two chambers of equal diameter, height and volume connected in tandem with a Teflon valve was used for all gradient elutions. One chamber served as a mixing chamber and one as a static reservoir. The less polar solvent was initially held in the mixing chamber. The more polar solvent was held in the static chamber. Teflon coated magnetic stirring bars (1.0 x 3.7 cm) were placed in both chambers and driven by Thomas Model 15 Magne-matic stirrers. Eluant was pumped from the mixing chamber to the medium pressure HPLC system through polypropylene tubing (1.5 mm ID x 3.0 mm OD). As eluant was removed from the mixing chamber, the solvent in the static reservoir was allowed to freely replace it, thus creating a linear gradient of eluant.

TLC analysis of BBM-1675A-, and As

Summarized in Table 9 below are the observed Rf values for BBM-1675Ai and A2 on normal phase plates. Rf is calculated by dividing the measured distance of the center of a zone from the point of sample application by the measured distance of the solvent front from the point of sample application.

TABLE 9

15

20

25

30

35

40

45

System/Compound

4% methanol in chloroform 5% methanol in diethyl ether 50% acetone in Skellysolve B

Rf

BBM-1675Ai BBM-1675A2 0.30 0.31

0.33 0.39 0.38

45

50 Summarized in Tabie 10 below are the observed Rf values of BBM-1675Ai and A2 on normal phase plates developed in two dimensions. The position of the spots are expressed in Cartesian coordinates. The X coordinate is the Rf values of the second listed solvent system. The Y coordinate is the Rf values of the first listed solvent system.

50

55

TABLE 10

55

Rf

60

System/Compound

4% methanol in chloroform vs 5% methanol in diethyl ether

4% methanol in chloroform vs 50% acetone in Skellysolve B

BBM-167SA, (0.34, 0.33) (0.33, 0.29)

BBM-1675AZ (0.28, 0.23)

60

BNSDOCID: <GB 2141425A_J_>

17

GB 2 141 425 A

17

Summarized in Table 11 belowarethe observed Rf values for BBM-1675Ai and A2on C-18 reversed phase TLC plates developed in binary eluants.

TABLE 11

Rf

10

15

20

25

30

35

SystemI Compound

25% 0.5 M NaCI in acetonitrile 25% water in acetonitrile

BBM-1675A,

0.18 0.00

BBM-1675A2

0.21 0.00

Summarized in Table 12 belowarethe observed Rf values of BBM-1675A! and A2on C-18 reversed phase TLC plates developed with ternary eluants.

TABLE 12

SystemlCompound

Rf

BBM-1675A, BBM-1675A,

Acetonitrile

Methanol

0.5 NaCL

80%

10%

1.00

60%

10%

30%

0.57

0.50

40%

30%

0.32

0.22

30%

50%

20%

0.44

0.33

50%

30%

20%

0.62

0.54

40%

20%

0.60

0.49

50%

20%

0.42

0.34

60%

20%

0.74

0.69

Acetonitrile

Methanol water

40%

30%

0.00

Acetonitrile

Methanol

0.1MNH4OAc

40%

30%

0.32

0.22

Acetonitrile

Methanol

0.1 M NaH2P04

40%

30%

0.00

10

15

20

25

30

35

HPLC analysis of BBM-1675A j andA2 40 BBM-1675A! and BBM-1675A2 were assayed using single, binary, and ternary eluants on C-18 reversed phase silica gel columns. Summarized in Tables 13,14 and 15 below are the observed K' values for these compounds. The K' was calculated using the following formula:

40

45

K' =

TR-To To

45

where TR is the retension time measured from time of injection to peak apex and To is the void volume time. 50 TABLE 13 50

55

SystemlCompound

Acetonitrile

Tetrahydrofuran

Methanol

K•

BBM- 167SA, BBM- 1675A2

a a

0.00

a a o.oo

55

a = compound did not elute from column.

18

GB 2 141 425 A

18

TABLE 14

SystemlCompound

25% water in acetonitrile 25% methanol in water

K'

BBM-1675A, a

1.25

BBM-1675A 2 a

1.25

10

a = compound did not elute from column.

TABLE 15

10

Ternary Eluants

15

20

25

SystemlCompound iC

BBM- 1675A, BBM- 1675A,

Acetonitrile

: Methanol water

40%

: 30%

30%

a a

Acetonitrile

: Methanol o.imnh4oac

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

b

15

20

25

30

35

40

a = did not elute b = not determined

Biological properties of BBM-1675 components

Antimicrobial activity of the BBM-1675 components was determined for a variety of bacteria {gram-positive, gram-negative and acid-fast) and fungi by the serial two-fold agar dilution method. Nutrient agar medium was used for gram-positive and gram-negative bacteria and No. 1001 medium (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% NaaHPCU, 0.1% KH2P04,0.005% ammonium citrate, 0.001% MgS04 and 1.5% agar) for acid-fast organisms. Sabouraud agar medium was used for fungi. As shown in Table 16, each of the six BBM-1675 components (A1( A2, A3, A4, B1(B2) showed a broad spectrum of antimicrobial activity. BBM-1675 A1r A2, A3 and A4 in particular were highly active against gram-positive bacteria.

TABLE 16

30

35

40

45

50

55

Antimicrobial Activity of BBM-1675 Components

MIC in mcgtmi

Strain

BBM-1675A,

As

^3

a4

By

B2

S. aureus 209P

<0.0008

0.0063

0.0125

0.012

0.0063

S. aureus Smith

<0.0008

0.0031

0.0063

0.0125

0.012

B. subtilis PCI 219

<0.0008

0.05

0.0125

0.05

M. luteus 1001

0.0016

0.0063

0.0125

0.1

M. f/avus

<0.0008

0.0016

0.0063

0.0125

0.025

Mycobacterium 607

0.05

0.1

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

P. aeruginosa D15

0.8

1.6

3.1

C. albicans IAM 4888

0.4

1.6

6.3

3.1

1.6

C. neoformans

1.6

3.1

1.6

6.3

12.5

45

50

55

BNSDOCID: <GB 2141425A_L>

19

GB 2 141 425 A

19

A second antimicrobial test was carried out on purified A-i and A2 (as prepared in Ex. 3 below) and on components A3 and A4. Data is summarized below.

MIC by ADT (mcglml)

5

Strain

BBM-1675A,

A2

A3

A4

S. aureus 209P

<0.0008

0.0063

0.012

S. aureus Smith

<0.0008

0.0031

0.0063

0.012

B. subtiiis PCI 219

<0.0008

0.05

0.012

0.025

10

M. luteus 1001

0.0016

0.0063

0.012

0.05

M. fiavus

<0.0008

0.0016

0.0063

0.012

Mycobacterium 607

0.05

0.1

0.16

E. co/f'NIHJ

0.1

0.8

1.6

3.1

K. pneumoniae D11

0.4

0.8

1.6

3.1

15

P. aeruginosa D15

0.8

1.6

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

C. albicans 1AM 4888

0.4

1.6

6.3

20

C. neoformans

1.6

3.1

1.6

6.3

The activity of prophage induction in lysogenic bacterium E. coli W1709 (K) was determined for BBM-1675 components according to the method of Lein et al. in Nature 196: 783-784 (1962). The plaque count was made on agar plates containing test material (T) and control plate (C). AT/C ratio of the plaque counts of 25 greater than 3.0 was considered significant and the lysogenic induction activity (ILB activity) was expressed 25 as the minimum inducible concentration of the test compound. As shown in Table 17, BBM-1675 components showed strong ILB activity in the lysogenic bacteria, thus suggesting that they may possess antitumor activity.

30 TABLE 17 30

Lysogenic induction Activity of BBM-1675 Components

Antibiotic MIC*(mcgimi)

35 BBM-1675 At 0.0063 35

BBM-1675 A2 0.0125

BBM-1675 A3 0.05

BBM-1675 A4 0.10

BBM-1675 B, 0.10

40 BBM-1675 B2 0.20 40

* minimum inducible concentration

The antitumor activity of BBM-1675 At and A2 was determined in various mouse tumor systems. 45 Lymphocytic leukemia P-388, lymphoid leukemia L-1210, melanotic melanoma B16and Lewis lung 45

carcinoma were implanted intraperitoneally into male BDF, mice at an inoculum size of 10s, 105,5 x 105and 106 cells per mouse, respectively. Graded doses of test compounds were administered to the mice intraperitoneally 24 hours after the tumor inoculation. The treatments were given once on the first day only, on day 1,4 and 7 (q3d x 3), once daily for 9 days (qd 1—>9) or 11 days (qd 1—>11). Components A3 and A4 were 50 tested only against P-388 leukemia by a q3d x 3 schedule due to short supply of material. 50

BBM-1675 A-i, A2, A3 and A4 were dissolved in 0.9% saline containing 10% dimethyl sulfoxide, and chromomycin A3 (Toyomycin, Takeda) employed as a reference compound was dissolved in 0.9% saline.

Death or survival of the treated and non-treated mice was recorded daily, and the median survival time (MST) was calculated for each of the test (T) and control (C) groups. AT/C value equal to or greater than 55 125% indicates that a significant antitumor effect was achieved. The results are shown in Tables 18 through 55 23. BBM-1675 At and A2 showed extremely potent antitumor activity against P-388 leukemia with a maximum T/C value of 160%. They are approximately 100 to 3000 times more active than chromomycin A3 in terms of minimum effective dose. BBM-1675 A3 and A4, however, were less active than component A1 or A2 against P-388 leukemia (Table 19). BBM-1675 At and A2 were also active against L-1210 leukemia (Table 21), 60 B16 melanoma (Table 22) and Lewis lung carcinoma (Table 23). The toxicity of BBM-1675 At and A2 was 60

determined in male ddY mice by intraperitoneal or intravenous administration; BBM-1675 A-i was about 10 times more toxic than BBM-1675 A2 (Table 24). The therapeutic indices of BBM-1675 Ai and A2 were 4 to 8 and 8 to 20 times better than those of chromomycin A3, respectively, in the P-388 leukemia system (Table 25). Second experiments were carried out by intravenous administration of BBM-1675 components against 65 P-388 and L-1210 leukemias, which were inoculated intravenously at 5 x 10s and 104 cells per mouse, 65

BNSDOCID: <GB 2141425A_J_>

20 GB 2 141 425 A

20

respectively. In these experiments, adriamycin was used as a 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 A-i and A2 were superior to adriamycin in terms of maximum T/C value, minimum effective dose and activity range.

TABLE 18

10

Effect of BBM-1675 Components on P-388 Leukemia (Day 1 treatment)

Average wt

Dose, ip

MST

TIC

change on

Survivors on

(mgikgiday*)

(days)

(%)

day 5 (g)

day 5

day 45

15

BBM-1675 A-i

0.03

19.0

(lib

-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

(165)

-1.4

5/5

0/5

0.0003

16.0

Cjjif)

0.0

5/5

0/5

20

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

(16^)

-1.8

5/5

0/5

25

0.03

18.0

(331)

-1.4

5/5

0/5

0.01

17.0

dUD

-0.6

5/5

0/5

0.003

17.0

ffff)

-0.4

5/5

0/5

0.001

16.0

(T2Sp

0.0

5/5

0/5

0.0003

15.0

120

0.0

5/5

0/5

30

0.0001

14.0

112

0.0

5/5

0/5

Chromomycin A3

1

19.0

(352*)

-0.2

4/5

0/5

0.3

17.0

(W)

+0.6

5/5

0/5

0.1

16.0

(?2|)

+0.8

5/5

0/5

35

0.03

14.0

112

0.0

5/5

0/5

0.01

14.0

112

-0.2

5/5

0/5

Vehicle

_

12.5

-

+0.1

10/10

0/10

10

15

20

25

30

35

40

* day 1,i.p.

Circle indicates a significant antitumor activity.

40

BNSDOCID: <GB 2141425A_L>

21

GB 2 141 425 A

21

TABLE 19

Effect of BBM-1675 Components on P-388 Leukemia

c

(Days

1, 4 and 7 treatment)

D

Average wt.

5

Dose, i.p

MST

TIC

change on

Survivors on

mgikgiday*)

(days)

(%)

day 5(g)

day 5

day 45

10

BBM-1675 A,

0.03.

7.0

56

-2.8

5/5

0/5

10

0.01

19.0

(|52)

-1.0

5/5

0/5

0.003

19.0

q52)

-0.6

5/5

0/5

0.001

16.0

-0.6

5/5

0/5

0.0003

17.0

d3g)

-0.4

5/5

0/5

15

0.0001

16.0

(Tib

-0.2

5/5

0/5

15

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

20

0.03

18.0

-1.2

5/5

0/5

20

0.01

18.0

Q44)

-0.4

5/5

0/5

0.003

18.0

Q®

-0.4

5/5

0/5

0.001

17.0

(136)

-0.4

5/5

0/5

0.0003

16.0

(TjEb

-0.4

5/5

0/5

25

0.0001

16.0

Q28)

-0.2

5/5

0/5

25

0.00003

15.0

120

-0.4

5/5

0/5

BBM-1675 A3

0.01

17.5

+0.2

4/4

0/4

0.001

15.0

120

+0.6

4/4

0/4

30

0.0001

13.5

108

+0.6

4/4

0/4

30

BBM-1675 A4

0.01

16.5

(511)

+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

35

Chromomycin A3

0.3

18.0

(344)

+0.6

5/5

1/5

0.1

18.0

U44)

+0.6

5/5

0/5

0.03

17.0

036)

-0.2

5/5

0/5

0.01

14.0

112

0.0

5/5

0/5

40

Vehicle

-

12.5

-

+0.4

10/10

0/10

* days 1,4 and 7, i.p.

Circle indicates a significant antitumor activity.

BNSDOCID: <GB 2141425A_L>

22 GB 2 141 425 A

22

TABLE 20

BBM-1675 At

10

15

Effect of BBM-1675 Components on P-388 Leukemia (qd 1->9 treatment)

Average wt. TiC change on (%) day 5(g)

Dose, ip (mgikgiday*)

0.01

0.003

0.001

0.0003

0.0001

0.00003

0.00001

MST (days)

7.0 13.0 19.0 19.0 18.0 16.0 16.0

-1.8 -1.0 -1.2 -0.8 0.0 +0.2 -0.2

Survivors on day 5 day 45

5/5 5/5 5/5 5/5 5/5 5/5 5/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5

10

15

20

25

30

35

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

(144)

-1.0

5/5

0/5

0.003

18.0

(34$)

-0.6

5/5

0/5

0.001

18.0

(344)

-0.8

5/5

0/5

0.0003

17.0

(Til)

-0.4

5/5

0/5

0.0001

16.0

dH>

-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 A3 0.3

9.0

72

-2.0

5/5

0/5

0.1

18.0

(144)

+0.4

5/5

0/5

0.03

18.0

m

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

Vehicle

12.5

-

+0.4

10/10

0/10

* qd 1-»9, i.p.

Circle indicates a significant antitumor activity.

20

25

30

35

TABLE 21

40

45

50

55

60

Effect of BBM-1675 Components on L-1210 Leukemia

BBM-1675 A,

BBM-1675 A2

Chromomycin A3

Vehicle

Average wt.

Dose

MST

TIC

change on

Survivors on

(mgikgiday*)

days

(%)

days (g)

day 5

day 45

0.003

14.5

(153)

-1.7

6/6

0/6

0.001

12.0

(f26)

-0.5

6/6

1/6

0.0003

12.0

di§>

+0.3

6/6

0/6

0.0001

11.0

116

+ 1.0

6/6

0/6

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

Off)

-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

0.3

8.5

89

-1.2

6/6

0/6

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

0.003

10.0

105

+1.5

6/6

0/6

9.5

+1.4

12/12

0/12

40

45

50

55

60

* qd 1—»0, i.p.

65 Circle indicates a significant antitumor activity.

65

BNSDOCID: <GB 2141425A_I_>

23

GB 2 141 425 A

23

TABLE 22

Effect of BBM-1675 Components on B16 Melanoma

Average wt.

10

15

20

25

Dose

MST

TIC

change on

Survivors on

<mgikgiday*)

(days)

(%)

day 5(g)

day 5

day 45

BBM-1675 A,

0.003

10.0

61

-0.7

6/6

0/6

0.001

31.5

(391)

0.0

6/6

0/6

0.0003

40.5

(24§)

+0.3

6/6

0/6

0.0001

27.0

Q6J)

+0.8

6/6

0/6

0.00003

22.0

OH)

+ 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

(361)

+0.3

6/6

0/6

0.003

29.5

079)

+0.2

6/6

0/6

0.001

26.0

QH)

+0.8

6/6

0/6

0.0003

22.0

(HD

+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 A3

0.1

25.5

(355)

+2.3

6/6

0/6

0.03

23.0

(J39)

+2.2

6/6

0/6

0.01

21.0

U27)

+2.3

6/6

0/6

0.003

18.0

109

+2.2

6/6

0/6

Vehicle

16.5

+2.1

12/12

0/12

30

* qd 1-*9, i.p.

Circle indicates a significant antitumour activity.

35

TABLE 23

Effect of BBM-1675 Components on Lewis Lung Carcinoma

Average wt.

Dose

MST

TIC

change on

40

(mgikgiday*)

(days)

(%)

day 5(g)

BBM-1675 At 0.003

10.0

91

-1.7

0.001

31.5

(286)

-0.7

0.0003

21.5

U95)

-0.7

45

0.0001

21.0

dsn)

+ 1.0

0.00003

13.0

118

+ 1.0

0.00001

11.5

105

+1.0

Survivors on

BBM-1675 A2 0.03

10.0

91

-1.8

50

0.01

25.5

@!>

-1.7

0.003

28.5

(25§)

0.0

0.001

17.0

-0.3

0.0003

15.0

Ci3eD

+ 1.2

0.0001

10.5

95

+0.5

55

0.00003

11.0

100

+0.8

60

Chromomycin A3

Vehicle

0.1

21.5

(195)

+ 1.2

0.03

17.0

(T5§)

+ 1.7

0.01

17.0

Q5l5

+ 1.5

0.003

11.5

105

+ 1.7

11.0

+0.8

day 5

day 45

5/6

0/6

6/6

1/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

1/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

1/6

5/5

0/5

6/6

0/6

6/6

0/6

12/12

1/12

* qd 1->11, i.p.

65 Circle indicates a significant antitumor activity.

10

15

20

25

30

35

40

45

50

55

60

65

BNSDOCID: <GB 2141425A_L>

24 GB 2 141 425 A

TABLE 24

Toxicity of BBM-1675 Components

5 LD50 (mgikgiday)

Multiple dose

Single dose

(qd 1->9)

i.p i.v.

i.p.

BBM-1675 An

0.019

0.010

0.00046

BBM-1675 A2

0.18

0.10

0.0072

Chromomycin A3

0.81

0.41

0.23

15

TABLE 25 Therapeutic Indices

20

Single

P-388 qd 1-*9

LDsolMED* L1210

BW

LL

BBM-1675 At BBM-1765A2 Chromomycin A3

63 180 8

>46 72 8

2 2

inactive

15 24 23

5 24 23

25

* mininum effective dose

TABLE 26

30 Effect of BBM-1675 Components on Intravenously Implanted

P-388 Leukemia

Average wt.

Dose

MST

TIC

change on

Survivors on

35

(mgikgiday*)

(days)

(%)

day 5(g)

day5

day 45

BBM-1675 At

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

(!H>

+0.3

6/6

0/6

40

0.0003

9.5

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

-1.0

6/6

0/6

0.01

12.0

QH)

-0.5

6/6

0/6

45

0.003

9.0

100

+1.0

6/6

0/6

(

Adriamycin

30

tox.

-

0/6

10

9.0

100

-1.5

6/6

0/6

3

12.0

+0.7

6/6

0/6

50

1

9.0

100

+1.7

6/6

0/6

Vehicle

_

9.0

-

+1.7

12/12

0/12

* days 1,4 and 7, i.v.

55 Circle indicates a significant antitumor activity.

24

5

10

15

20

25

30

35

40

45

50

55

BNSDOCID: <GB 2141425A_I_>

25

GB 2 141 425 A 25

TABLE 27

Effect on BBM-1675 Components on Intravenously Implanted L-1210 Leukemia

10

15

20

25

BBM1675 A-,

BBM-1675 Az

Adriamycin

Vehicle

Average wt.

Dose

MST

TIC

change on

(mgikgiday*)

(days)

(%)

day 5(g)

•0.008

9.5

119

-2.0

0.004

14.0

(17Bl?

-0.2

0.002

13.0

(iii)

+0.2

0.001

9.5

119

+0.8

0.0005

9.0

113

+0.8

0.63

11.0

638)

-1.8

0.032

14.0

(T7S

+0.2

0.16

10.5

(Hd

+0.8

0.008

8.0

100

+ 1.2

0.004

8.0

100

+0.8

Survivors on

16 8 4 2

tox.

-

12.0

+0.2

9.0

113

+ 1.5

8.0

100

+ 1.7

8.0

_

+ 1.4

day 5

day 45

4/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

2/6

0/6

6/6

0/6

6/6

0/6

6/6

0/6

12/12

0/12

10

15

20

25

* days 1,4 and 7, i.v.

30 Circle indicates a significant antitumor activity.

30

Antitumor activity of components BBM-1675 At and A2 was also determined by a second test against P-388 leukemia, L-1210 leukemia and B16 melanoma in mice. Results of these tests are shown below in Tables 28, 29 and 30. Details of the methods used in these tests have been described in Cancer Chemother. Rep. 3:1-87 35 (Part 3), 1972. 35

BNSDOCID: <GB 2141425A__L>

26

GB 2 141 425 A

26

TABLE 28

Effect of BBM-1675 A1 and A2 on P-388 Leukemia

10

15

20

25

30

35

40

45

50

55

60

65

Material * NSC 38270

BBM-1675A-, DMSO^saline

Treatment Dose, IP Schedule qd 1-»9

d.1

d.1,5&9

qd1-»9

BBM-1675 Az DMSO-»saline d.1

d.1,5&9

MST

Effect MST

AWC, gm

Survivors mgikgiday

Days

% 77 C

d.5

d.5 (30)

400

13.0

163

-0.6

6/6

200

11.0

138

-0.9

6/6

10

51.2

20.0

250

-2.1

4/6

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

15

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

20

0.1

10.0

125

0

6/6

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

25

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

30

0.1

12.0

150

+0.8

6/6

0.05

10.0

125

+0.8

6/6

12.8

TOX

1/6

6.4

6.0

75

-1.5

4/6

35

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

40

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

45

256

TOX

0/6

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

50

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

55

128

TOX

0/6

64

TOX

0/6

32

TOX

-1.3

2/6

16

24.5

306

-1.3

5/5

60

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

65

BNSDOCID: <GB 2141425A_L>

27

GB 2 141 425 A

27

TABLE 28 - cont'd

BBM-1675 A2 DMSO-»saline

10

15

20

25

qd1->9

Control

Tumor inoculum

Host

Tox

Evaluation

Effect

Criteria

* NSC 38270 = olivomycin A

0.25

11.0

138

-0.4

6/6

64

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

6/6

4

17.0

213

-1.8

6/6

2

15.0

188

-1.1

6/6

1

14.0

175

-0.5

6/6

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

Saline

8.0

»

+0.5

10/10

106 ascites cells implanted i.p.

CDFt 9 mice.

<4/6 mice alive on d.5

MST = median survival time.

%T/C = (MST treated/MST control) x 100.

%T/C > 125 considered significant antitumor activity.

10

15

20

25

30

BNSDOCID: <GB 2141425A_I_>

28 GB 2 141 425 A

28

TABLE 29

Effect on BBM-1675 A, and Az on L-1210 Leukemia

Material

Treatment Schedule

BBM-1675 A, d.1

10

15

20

25

30

35

40

45

50

d.1,5&9

qd 1—»9

BBM-1675 A2 d.1

Effect

AWC,

5

Dose, IP

MST

gm

Survivors on

p.glkglinj

Days

% TIC

d.5

d.5 (30)

51.2

12.0

171

-1.1

5/6

25.6

7.0

100

-2.3

6/6

10

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

15

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

25.6

TOX

1/6

20

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

25

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

30

12.8

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

35

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

0.5

7.0

100

+0.3

6/6

0.025

6.0

86

-0.6

6/6

40

256

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

45

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

50

0.5

8.0

114

-0.1

6/6

BNSDOCID: <GB 2141425A_L>

29

GB 2 141 425 A 29

TABLE 30

Effect of BBM-1675 A, and Az on B16 Melanoma

10

15

20

Effect

AWC

Dose, IP

MST

gm

Survivors

Materia!

Iig (m) or mglkg!inj

Days

% TIC

d.5

d. 10 (61)

BBM-1675 A-i

3.2M

TOX

-1.8

2/10

1.6

16.0

168

-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-1675 A2

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

10

15

20

25

3 Only one without tumor; MST d.m.o. = 55.0 (220%), b Two without tumor; MST d.m.o. = 46.0 (184%)

25

30

35

Tumor inoculum Host

Treatment Tox

Evaluation

Effect

Criteria

0.5 ml of a 10% brei, ip BDFt $ mice.

qd 1—>9

s7/10 mice alive on d.10

MST = median survival time

% T/C = (MST treated/MST control) x 100.

% T/C >125 considered significant antitumor activity.

After further purification of BBM-1675A! according to Example 6, samples of the purified compound were tested against L-1210 leukemia, P-388 leukemia and B16 melanoma in mice. Results of these tests are shown below.

30

35

BNSDOCID: <GB 2141425A_I_>

30

GB 2 141 425 A

30

TABLE 31

Effect of Purified BBM-1 67SAi on P388 Leukemia

10

15

20

25

30

35

40

Compound BBM-1675A,

Control (vehicle)

Host: CDF1 female mice 45 Implant level and site: 1x106 cells, i.p.

(Day 1 Treatment)

Average wt.

5

Dose

MST

TIC

change on

Survivors on fmg/kgidose)

Route, schedule

Days

(%)

day5

day 5

0.1024

i.p., qd x 1

TOX

0/6

10

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

15

0.0016

16.5

150

-1.0

6/6

0.0008

15.0

136

-1.2

6/6

0.0004

15.0

136

-2.6

6/6

0.0256

i.p.r q4d x 3;

TOX

-1.5

1/6

20

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

25

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

30

0.0128

i.p., qd x 5;

TOX

0/6

0.0064

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

35

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

40

1 x 106

i.p., q4d x 3;

11.0

100

-0.7

9/9

45

BNSDOCID: <GB 2141425A_

31

GB 2 141 425 A

31

TABLE 32

Effect of Purified BBM-1675A} on L-1210 Luekemia (Day 1 Treatment)

5

Average wt.

Dose MST TIC change on Survivors on

Compound (mg/kgldose) Route, schedule Days (%) day 5 day 5

10

BBM-1675A, 0.1024

i.p., qd x 1

TOX

1/6

10

0.0512

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

15

0.0032

10.0

143

-2.0

6/6

15

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

-2.3

2/6

20

0.0128

10.5

150

-1.7

6/6

20

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

25

0.0004

8.5

121

-0.7

6/6

25

0.0002

8.0

114

-0.5

6/6

0.0128

i.p., qd x 5

TOX

-2.8

2/6

0.0064

7.0

100

-1.8

5/6

30

0.0032

11.5

164

-1.0

6/6

30

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

35

0.0001

8.5

121

0.0

6/6

35

Control

(vehicle)

1 X 106

i.p., qd x 5

7.0

100

0.1

10/10

40

Host: CDF1 female mice

40

Implant level and site: 1 x 106 cells, i.p.

BNSDOCID: <GB 2141425A_I_>

32 GB 2141 425 A 32

TABLE 33

Effect of Purified BBM-1675A1 on B1S Melanoma (Day 1 Treatment)

Compound 10 *BBM-1675An

15

20

Average wt

Dose

MST

TIC

change on

Survh

(mglkgidose)

Route, schedule

Days

(%)

day 5

0.0064

i.p., q4d x 3

16.5

110

-3.8

mo

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

25.5

170

-2.3

10/10

10

15

20

Control

25

30

35

(vehicle)

0.5 ML

i.p., qd x 9

15.0

100

-0.3

10/10

**BM-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

25.5

146

-0.8

10/10

0.0004

21.5

123

-1.4

10/10

Control

(vehicle)

0.2 ML

i.v., q4d x 3

17.5

100

-0.4

10/10

25

30

35

40 Host: BDF1 female mice

*lmp!ant level and site: 0.5ML 10% BREl, i.p. **lmplant level and site: Fragment, s.c.

40

The above screening data indicates that the purified BBM-1675Ai component has substantially the same 45 antitumor properties as the less purified sample screened previously. The compound has exceptionally high 45 potency since activity has been demonstrated at a dose of 25 nanograms/kg on a daily times 5 schedule against P-388 leukemia in mice. On tests against P-388 and L-1210 leukemias, BBM-1675A-) is effective whether given as a single injection, day 1, every fourth day for 3 injections, or daily times 5. Against B16 melanoma the compound was equally effective given intravenously to animals bearing subcutaneous 50 tumors as when it was given intraperitoneally to animals bearing ip tumor implants. This property of 50

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

As shown above the BBM-1675 components possess potent antimicrobial activity and are thus useful in the therapeutic treatment of mammals and other animals for infectious diseases caused by such 55 microorganisms. Additionally the components may be utilized for other conventional applications of 55

antimicrobial agents such as disinfecting medical and dental equipment.

The induction of prophage in lysogenic bacteria and the activity shown against mouse tumor systems indicate that the BBM-1675 components are also therapeutically useful in inhibiting the growth of mammalian tumors.

60 The present invention, therefore, provides a method for therapeutically treating an animal host affected by 60 a microbial infection or by a malignant tumor which comprises administering to said host an effective antimicrobial ortumor-inhibiting dose of BBM-1675 Ai, A2, A3,A4, Bi or B2, or a pharmaceutical composition thereof.

In another aspect, the present invention provides a pharmaceutical composition which comprises an 65 effective antimicrobial ortumor-inhibiting amount of BBM-1675 Ai, A2, A3, A<, B-, or B2in combination with 65

BNSDOCID: <GB 2141425A_I_>

33

GB 2 141 425 A

33

an inert pharmaceutical acceptable carrier or diluent. These compositions may be made up in any pharmaceutical form appropriate for parenteral administration.

Preparations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be manufactured in the form of sterile 5 solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.

It will be appreciated that the actual preferred amounts of the BBM-1675 antibiotics used will vary according to the particular component, the particular composition formulated, the mode of application and the particular situs, host and disease being treated. Many factors that modify the action of the drug will be 10 taken into account by those skilled in the art, for example, age, body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease. Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines.

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

Example 1

Fermentation of BBM-1675

20 Actinomadura strain No. H964-92 was grown and maintained on an agar slant containing 1 % malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% CaC03 and 1.6% agar. A well-grown agar slant was used to inoculate vegetative medium containing 3% soluble starch, 3% dry yeast, 0.3% K2HPO4,0.1% KH2P04,0.05% MgS04-7H20,0.2% NaCI and 0.1% CaC03, the pH being adjusted to pH 7.0 before sterilization. The vegetative culture was incubated at 32°Cfor 72 hours on a rotary shaker (250 rpm) and 5 ml of the growth was 25 transferred into a 500-ml Erlenmeyer flask which contained 100 ml of fermentation medium composed of 3% cane molasses, 1% corn starch, 1 % fish meal, 0.1% CaC03and 0.005% CuS04-5H20 (pH 7.0 before sterilization). The fermentation was carried out at 28°C for six days on the rotary shaker. The antibiotic activity in the fermentation broth was determined by the paper-discagar diffusion using Staphylococcus aureus 209P as the test organism. The antibiotic potency reached a maximum of about 1 mcg/ml after five 30 days fermentation.

Fermentation of BBM-1675 was also performed in stir-jar fermenters. Five hundred mililiters of inoculum growth as prepared above was transferred into 20-liter jarfermenters containing 10 liters of fermentation medium which consisted of the same ingredients as used in the shake flask fermentation. The fermentation was carried out at 32°C with an aeration rate of 12 liters/minute and agitation at 250 rpm. Under these 35 conditions, the antibiotic production reached a maximum of about 0.9 mcg/ml after 68-76 hours of fermentation.

Fermentation studies were also carried out in fermentation tanks. A seed culture was shaken forfour days at30°C in Erlenmeyer flasks containing vegetative medium consisting of 3% soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgS04-7H20,0.2% NaCI and 0.1% CaC03. The seed culture was 40 inoculated to a 200-liter seed tank containing 130 liters of seed medium having the same composition as above, and the seed tank was stirred at 240 rpm at30°Cfor31 hours. The second seed culture was used to inoculate 3,000 liters of fermentation medium containing 1% corn starch, 3% cane molasses, 1 % fish meal, 0.005% CuS04-5H20 and 0.1% CaC03. The production tank was operated at 28°C at 164 rpm with an aeration rate of 2,000 liters/minute. The broth pH gradually rose with the progress of fermentation and reached 7.7-7.8 45 after 170-180 hours, when a peak antibiotic activity of 1.7 mcg/ml was produced.

Example 2

Isolation and purification of BBM-1675 components

The harvested fermentation broth (3,000 liters, pH 7.8) was separated to mycelial cake and supernate with 50 the aid of a Sharpless centrifuge. The mycelial cake was suspended in 1,600 liters of methanol and the mixture stirred for one hour. The insoluble materials were filtered off and the methanolic extract was concentrated in vacuo to 43 liters. The activity contained in the broth supernate was recovered therefrom by extraction with two 1,000-liter portions of n-butanol. The n-butanol extracts and concentrated methanol extracts were combined and evaporated azeotropically by occasional additions of water to an aqueous 55 solution (20 liters) which deposited most the antibiotic activity as an oily solid. The solid was digested in 30 liters of methanol and the insolubles were removed by filtration. The methanol extract was then concentrated in vacuo to a 10-liter solution, to which was added 40 liters of ethyl acetate and 30 liters of water. After being stirred for 30 minutes, the organic layer was separated, dried over sodium sulfate and evaporated in vacuo to 4 liters. Addition of the concentrate into 20 liters of n-hexane afforded a pale yellow 60 solid of crude BBM-1675 complex (90.14 g, potency: 55 mcg/mg). The complex was shown in TLC to be a mixture of two major components, BBM-1675 A, and A2/ and several minor ones. They were separated and purified by repeated chromatographies which were performed in a cooled room to prevent deterioration.

The BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and charged on a column of Sephadex LH-20 (0 5.5 x 85 cm). The column was developed with methanol and the elution monitored by bioassay 65 using Staphylococcus aureus 209P. The active eluates were combined, concentrated in vacuo and

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lyophilized to give a semi-pure solid of BBM-1675 complex (4.19 g). The solid was then chromatographed on a column of silica gel (0 5.0 x 50 cm) using chloroform plus an interesting amount (1-»5% v/v) of methanol as eiuants.

Eluates were pooled on the basis of antibacterial activity (vs. S. aureus) and TLC (Si02; CHCI3-CH3OH = 5:1 5 v/v and concentrated in vacuo. Nearly homogeneous BBM-1675 At (yield after evaporation: 351 mg) was eiuted first with 2% methanol in chloroform and then a mixture of BBM-1675 A2,A3 and A4 (507 mg) followed by BBM-1675 B mixture (210 mg) with 3% methanol in chloroform. The solid of BBM-1675 At was applied on a column of Sephadex LH-20 (0 2.0 x 80 cm) which was developed with methanol. The active fractions were concentrated in vacuo to dryness and the residual solid was crystallized from methanol to afford colorless 10 .plates of pure BBM-1675 A1 (124 mg) (this material is starting material for Example 3A.). The compled

BBM-1675 A2,A3 and A4 was separated by chromatography on a column of BondapakC18 (Waters, 03.0 x 50 cm). Elution was carried out with aqueous acetonitrile and the bioactive eluates were examined by TLC (Merck, *Silanized: CH3CN-H20 = 75:25 v/v). The minor components A4 (33 mg) and A3 (18 mg) were eiuted successively in that order with 20% acetonitrile followed by another major component A2 (301 mg) (this 15 material is starting material for Example 3B) with 50% acetonitrile).

The solid containing BBM-1675 Bi and B2 was chromatographed on a column of silica gel (03.0 x 40 cm) with chloroform and methanol as the developing solvent. The active fractions eiuted with 4% methanol in chloroform were combined and evaporated to afford pure BBM-1675 B-i (7 mg). Another active fraction was eiuted at 5% methanol concentration, which upon evaporation afforded BBM-1675 B2 (8mg). 20 *Cis reverse phase silica gel

Example 3

Further purification of BBM-1675A r and A?

A. Purification of BBM-1675A,

25 A 2.67 cm i.d. x 75 cm Glenco column was slurry packed with Baker bonded phase octadecy! (C18) silica gel (40 micron particle size) in methanol. The column was connected into a medium pressure HPLC system and equilibrated with 1.51 of eluant (41.6% acetonitrile -21.6% methanol - 36.8% 0.1 M ammonium acetate). Partially purified BBM-1 675At (100.5 mg) obtained according to the purification procedure of Example 2 was dissolved in 2 ml of acetonitrile and drawn into the sample loop. The sample was pumped onto the column. 30 The column was eiuted with the above eluant collecting 87 ml fractions. The eluant was monitored at 254 nm and 340 nm. Fractions 55 through 71 were pooled and extracted twice with 1500 ml aliquots of chloroform. The chloroform was evaporated to dryness to yield 89.8 mg of residue C.

A 1.5 cm i.d. x 20 cm Glenco column was slurry packed with 12 g of Woelm silica gel (60-200 micron particles). Residue C was applied to the column in a chloroform solution. The column was eiuted with a 500 35 ml linear gradient of chloroform to 10% methanol in chloroform collecting 20-25 ml fractions. After analysis by TLC on silica gel, fractions 6-9 were pooled and evaporated to dryness to yield 73 mg of residue D.

A 1.5 cm i.d. x 20 cm Glenco column was slurry packed with 12 g Woelm silica gel (63-200 micron particles) in Skellysolve B. Residue D was dissolved in approximately 2 ml of CHCI3 and applied to the column. The chloroform was displaced with 25 ml of Skellysolve B. The column was then eiuted with a 500 40 ml linear gradient of Skellysolve B to 60% acetone in Skellysolve B collecting 28-25 ml fractions. Fractions 19-23 were pooled and evaporated to dryness to yield 65.6 mg of pure BBM-1675Ai-

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

45

Gel permeation chromatography with purified BBM-1675A ?

A 2.5 cm i.d. x 45 cm Pharmacia column was slurry packed with Sephadex LH-20 in methanol and adjusted to a 33.4 cm chromatography bed. Purified BBM-1675AT (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 elution 50 commenced at 1.75 ml/min with methanol collecting 10 ml fractions [Pharmacia Frac-100 fraction collector]. The eluant was monitored at 254 nm with an Isco UA-5 detector. BBM-1675An was observed to elute at Ve/Vt of 0.79 to 0.91 (Ve= elution volume; Vt= bed volume).

B. Purification of BBM-1675A2

55 A 2.65 cm i.d. x 75 cm Glenco column was slurry packed with Baker bonded phase octadecyl (C18) silica gel (40 micron particle size) in methanol. The column was connected into the medium pressure HPLC system and equilibrated with 1.51 of eluant (50% acetonitrile - 20% methanol -30% 0.1 <M ammonium acetate). Partially purified BBM-1675A2 (76.9 mg) as obtained by the procedure of Example 2 was dissolved in 2 ml of acetonitrile and drawn into the sample loop. The sample was pumped onto the column. The column was 60 eiuted with the above eluant collecting 87 ml fractions. The eluant was monitored at 254 and 340 nm.

Fractions 31 through 38 were pooled and extracted twice with 500 ml aliquots of chloroform. The chloroform was evaporated to dryness to yield 65.8 mg of homogeneous BBM-1675A2.

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

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Example 4

Prefered extraction process for BBM-1675A /

Raw fermentation (6.8 I) obtained according to the general procedure of Example 1 was transferred to a polypropylene bucket (12 cm d,top; 10 cm d, bottom; 37 cm high) equipped with a faucet at the bottom. An 5 equal volume of chloroform was added. The mixture was stirred at a good rate with a CRC-air driven stirrer for 2 hours. Approximately 41 (1.3 kg) of Dicalite (filter aid) was added and allowed to mix in. The mixture was filtered on a Dicalite pad which was held in a No. 12 Buchnerfunnel. The filtrate was collected in a 191 solution bottle equipped with a vacuum take-off (Ace. No. 5396-06). The mat was washed with 2 liters of chloroform. The filtrate was transferred to a 201 separatory funnel and the phases allowed to separate. The 10 lower phase (chloroform) was removed.

A 2.5 cm i.d. x 40 cm Glenco tube was slurry packed with 91 g of Woelm silica gel (63-200 micron particles). Using an FMI RPY-2CSD pump, the above chloroform phase was pumped through the column. The column was rinsed with 600 ml of fresh chloroform. The chloroform eluant was discarded. The column was then eiuted with 600 ml of 10% methanol in chloroform. This eluant was evaporated to dryness to yield 15 547 mg of residue A.

Residue A was dissolved into 50 ml of chloroform. The chloroform solution was added to 20 g of Dicalite in a 11 round bottom flask. A slurry was created by adding approximately 200 ml of Skellysolve B. The solvents were moved in a rotary evaporator. The residue was slurried in 300 ml of Skellysolve B. The slurry was packed into an Ace fiask chromatography tube (Part No. B5872-14) (41 mm id x 45.7 cm) by the following 20 procedure. A glass wool plug was inserted into the throat of the stop cock between the cock and the column tube. A1 cm layer of standard Ottawa sand was added above the glass wool. The stopcock, glass wool and sand bed were purged of air by passing a pressurized (5.7 psi) flow of Skellysolve B through them. The slurry was then added to the column and allowed to form a packed bed under pressurized flow. The column was never allowed to go dry. After a stable column bed was obtained, a 2 cm layer of Ottawa sand was added 25 onto the top of the bed. The bed was then eiuted with an additional 600-700 ml of Skellysolve B. The bed was eiuted with 500 ml of toluene. The toluene eluant was evaporated to dryness to yield 93 mg of residue B. This partially purified BBM-1675Ai may then be further purified according to the procedure of Example 3.

Example 5

30 Fermentation of BBM-1675 complex using variant H964-92-A1327Y

A variant strain A1327Y, which was obtained by NTG treatment of Actinomadura verrucosospora strain No. H964-92, was used to inoculate vegetative medium containing 2% soluble starch, 1% glucose, 0.5% yeast extract, 0.5% NB-amine type A and 0.1% CaC03, the pH being adjusted to 7.0 before sterilization. The vegetative culture was incubated at 32°Cforfourdayson a rotary shaker (250 rpm) and 5 ml of the growth 35 was transferred into a 500 ml Erlenmeyer flask which contained 100 ml of fermentation medium composed of 3% cane molasses, 1% corn starch, 1%fish meal, 0.005% CuS04*5H20,0.05% MgS04-7H20 and 0.1% CaC03, the pH being adjusted to 7.0 before sterilization.

The fermentation was carried out at 28°Cfor7 days on the rotary shaker. The antibiotic production reached a maximum of ca. 1.5 mcg/ml.

40

Example 6

Isolation and purification of BBM-1675 components

The harvested fermentation broth from Ex. 5 (3,000 L, pH 7.6) was separated to mycelial cake and supernate by using a Sharpless centrifuge. The mycelial cake was stirred with 2,000 L of methanol for one 45 hour and the insoluble materials were removed by filtration. The activity contained in the broth supernate was extracted therefrom with 1,800 L of n-butanol. The methanol and n-butanol extracts were combined and concentrated azeotropically by occasional additions of water to an aqueous solution (20 L) which deposited most of the antibiotic activity as an oily solid. The mixture was shaken three times with 20 L each of ethyl acetate to extract the activity. The extracts were pooled, filtered to remove the insolubles and evaporated in 50 vacuo to 4 L. Addition of the concentrate into 30 L of n-hexane under stirring afforded pale yellow solid of crude BBM-1675 complex (81.7 g, potency: 59 mcg/mg).The complex was shown by TLC and HPLC to be a mixture of two major components, BBM-1675 A-i and A2 and several minor ones. They were separated and purified by a series of chromatographies which were carried out in a cold room to prevent deterioration. The crude BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and charged on a column of 55 Sephadex LH-20 (0 5.5 x 85 cm). The column was developed with methanol and the elution monitored by bioassay using Staphylococcus aureus 209P. The active eluates were pooled, concentrated in vacuo and lyophilized to give a semi-pure solid of BBM-1675 complex (4.86 g, potency: 203 mcg/mg). The solid was then chromatographed on a column of silica gel (0 3.0 x 70 cm) using chloroform and an increasing amount (1-5%) of methanol as developing solvents. The eluates were pooled on the basis of antibacterial activity 60 against S. aureusand TLC (Si02, CHCI3-MeOH = 5:1, v/v) and concentrated in vacuo. BBM-1675 A-i (425 mg after evaporation, potency: 960 mcg/mg) was eiuted first with 2% methanol in chloroform and then a mixture of BBM-1675 A2, A3, and A4 (732 mg, potency: 340 mcg/mg) followed by BBM-1675 B complex (200 mg, potency : 190 mcg/mg) with 3% methanol in chloroform. The above BBM-1675 A, was rechromatographed on silica gel (column: 0 2.2 x 44 cm) with 2% methanol in benzene. The bioactive eluates were examined by 65 HPLC(LichrosorbRP-18: CH3CN-MeOH-0.1MCH3COONH4 = 5:2:3, v/v) and the fractions containing

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homogeneous BBM-1675 A, evaporated in vacuo to dryness. The residual solid was crystallized from methanol (10 ml) to give colorless prisms of BBM-1675 At (197 mg, potency: 1,000 mcg/mg).

The complex of BBM-1675 A2, A3 andA; (537 mg) was separated by column chromatography on Bondapak C18 (Waters, 0 2.0 x 42 cm). Elution was carried out with aqueous acetonitrile and the bioactive eluates were 5 examined by TLC (Merck, silanized: CH3CN-H20 = 75:25, v/v). The minor components BBM-1675 A4 (45 mg, potency: 410 mcg/mg) and A3 (19 mg, potency: 300 mcg/mg) were eiuted successively with 20% acetonitrile followed by a major component, BBM-1675 A2 (203 mg) with 50% acetonitrile. The BBM-1675 Az fraction was crystallized from chloroforrri-n-hexane to deposit colorless rods (70 mg, potency: 290 mcg/mg). The solid containing BBM-1675 B mixture was chromatographed on a column of silica gel (0 3.0 x 40 cm) with 10 chloroform and methanol as developing solvent. The active fractions eiuted with 4% methanol in chloroform were pooled and evaporated to afford pure BBM-1675 B-i (7 mg, potency: 180 mcg/mg). Another active fraction was eiuted at 5% methanol concentration, which upon evaporation afforded BBM-1675 B2 (8 mg, potency: 140 mcg/mg).

Claims

15 CLAIMS

1. The antitumor antibiotic BBM-1675 A-i which in substantially purified form:

(a) appears as white to pale yellow crystals;

(b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and 20 water, and insoluble in n-hexane and carbon tetrachloride;

(c) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(d) exhibits an infrared absorption spectrum (KBr) substantially as shown in Figure 9;

(e) when dissolved in CDCl3 exhibits a proton magnetic resonance spectrum substantially as shown in 25 Figure 10;

(f) has a melting point in the range of about 156-158DC;

(g) has an optical rotation of [<x]l7 = -191°(c0.5, CHCf3);

(h) has an apparent molecular weight of 1248 as determined by mass spectroscopy;

(i) has an approximate elemental composition of 52.17% carbon, 6.15% hydrogen, 4.63% nitrogen, 9.09% 30 sulfur and 27.96% (by difference) oxygen;

(j) exhibits in silica gel thin layer chromatography an Rf value of 0.74 with the solvent system CHCI3-CH3OH {5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.18 with the solvent system CH3CN-H20 (75:25 v/v);

(k) when dissolved in methanol at a concentration of 0.01356 g/l exhibits the following ultraviolet 35 absorption maxima and absorptivities:

\max (nm) absorptivities

320 12.4

40 280 shoulder

253 25.1

210 25.5

with no significant change upon addition of acid or base;

45 (I) exhibits a high performance liquid chromatography retention time of 13.3 minutes with a C-|8 reversed • phase silica gel column and the solvent system CH3CIM-CH3OH-0.1M-CH3COONH4 (5:2:3 v/v);

(m) is effective in inhibiting the growth of various bacteria and fungi;

(n) induces prophage in lysogenic bacteria; and

(0) is effective in inhibiting the growth of P-388 leukemia, L-1210 leukemia, B16 melanoma and Lewis lung

50 carcinoma in mice.

2. The antitumor antibiotic BBM-1675 A2 which in substantially purified form:

(a) appears as white crystals;

(b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

55 (c) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

(d) exhibits an infrared absorption spectrum (KBr) substantially as shown in Figure 12;

(e) when dissolved in CDCI3 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 13;

60 (f) has a melting point in the range of about 147-149°C;

(g) has an optical rotation of [a]o7 = — 179.4° {c 0.5, CHCI3);

(1) has an approximate elemental composition of 52.71% carbon, 5.94% hydrogen, 3.94% nitrogen, 9.39% sulfur and 28.01% (by difference) oxygen;

65 (j) exhibits in silica gel thin layer chromatography an Rf value of 0.71 with the solvent system CHCI3-CH3OH

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(5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.21 with the solvent system CH3CN-H20 (75:25 v/v);

(k) when dissolved in methanol at a concentration of 0.02052 g/l exhibits the following ultraviolet absorption maxima and absorptivities:

5 5

kmsx (nm) absorptivities

320 12.2

282 16.3

10 282 26.2 10

214 25.8

with no significant change upon addition of acid or base;

(I) exhibits a high performance liquid chromatography retention time of 17.3 minutes with a C-|8 reversed

15 phase silica gel column and the solvent system CH3CN-CH3OH-O.IM CH3COONH4 (5:2:3 v/v); 15

(m) is effective in inhibiting the growth of various bacteria and fungi;

(n) induces prophage in lysogenic bacteria; and

(o) is effective in inhibiting the growth of P-388 leukemia, L-1210 leukemia, B16 melanoma and Lewis lung carcinoma in mice.

20 3. The antitumor antibiotic BBM-1675 A3 which: 20

(a) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, slightly soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride;

(b) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

25 (c) exhibits an infrared absorption spectrum (KBr) substantially as shown in Figure 3; 25

(d) when dissolved in CDCI3 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 7;

(e) has a melting point in the range of about 125-127°C;

(f) has an optical rotation of [ajp7 = - 161° (c0.5, CHCI3);

30 (g) has an approximate elemental composition of 54.55% carbon, 6.46% hydrogen, 3.73% nitrogen, 7.49% 30 sulfur and 27.77% (by difference) oxygen;

(h) exhibitsin silica gel thin layer chromatography an Rf value of 0.72 with the solvent system CHCI3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.28 with the solvent system CH3CN-H20 (75:25 v/v);

35 (I) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01 N HCI-CH3OH 35 and 0.01 IM NaOH-CH3OH

UVXmaxnm(E^J : 253(286)

in methanol 282(158)

40 320(122) 40

UV\maxnm(Ej*m) : 253(287)

in 0.01 N HCI-CH3OH 282 (160)

320(126)

UV \max nm (E,°£m) : 252(280)

in 0.01 N NaOH-CH3OH 283(162)

01Q M on\

320(126)

45 45

318(120);

50 (j) exhibits a high performance liquid chromatography retention time of 8.0 minutes with a Ci8 reversed 50 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;

(I) induces prophage in lysogenic bacteria; and

(m) is effective in inhibiting the growth of P-388 leukemia in mice.

55 4. The antitumor antibiotic BBM-1675 A4 which: 55

60 (c) exhibits an infrared absorption spectrum (KBr) substantially as shown in Figure 4; 60

(d) when dissolved in CDCI3 exhibits a proton magnetic resonance spectrum substantially as shown in Figure 8;

(e) has a melting point in the range of about 123-126°C;

(f) has an optical rotation of [a]" - 176° (c 0.5, CHCI3);

65 (g) has an approximate elemental composition of 54.65% carbon, 6.29% hydrogen, 3.51% nitrogen, 8.07% 65

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sulfur and 27.48% (by difference) oxygen;

(h) exhibits in silica gel thin layer chromatography an Rf value of 0.71 with the solvent system CHCI3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.78 with the solvent system CH3CN-H20 (75:25 v/v);

5 (i) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01 N HCI-CH30H 5 and 0.01 NNaOH-CH3OH

UVWnm(E^) : 253(257)

in methanol 282(153)

10 320(117) 10

UVKmaxnm(El^J : 253(258)

in 0.01 N HCI-CH3OH 282(155)

320(118)

15 15

UV Xmax nm (E5°cm) •' 252(266)

in 0.01 N Na0H-CH30H 283(160)

318(118);

20 (j) exhibits a high performance liquid chromatography retention time of 5.1 minutes with a C18 reversed 20 phase silica gel column and the solvent system CH3CN-CH3OH-O.IM CH3COONH4 (5:2:3 v/v);

(k) is effective in inhibiting the growth of various bacteria and fungi;

(I) induces prophage in lysogenic bacteria; and

(m) is effective in inhibiting the growth of P-388 leukemia in mice.

25 5. The antitumor antibiotic BBM-1675 Bt which: 25

(b) gives a positive reaction with ferricchloride, Ehrlich and Tollen's reagents and a negative reaction in Sakaguchi, ninhydrin and anthrone tests;

30 (c) has a melting point in the range of about 159-161°C; 30

(d) has an optical rotation of [a]o7 — 171° (c 0.5, CHCI3);

(e) exhibits in silica gel thin layer chromatography an Rf value of 0.63 with the solvent system CHCI3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin iayer chromatography an Rf value of 0.23 with the solvent system CH3CN-H20 (75:25 v/v);

35 (f) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01 N HCI-CH3OH 35 and 0.01 N NaOH-CH3OH

UVUnmlE^) : 253(225)

in methanol 282(140)

40 320(104) 40

UV X.max nm (Ei°om) : 253(225)

in 0.01 N HCI-CH3OH 282 (140)

320(105)

45 45

UVXmB)Cnm(E?*m) : 252(236)

inO.OINNaOH-CI-kOH 283(141)

318(105);

50 (g) is effective in inhibiting the growth of various bacteria and fungi; and 50

(h) induces prophage in lysogenic bacteria.

6. The antitumor antibiotic BBM-1675 B2 which:

55 (b) gives a positive reaction with ferric chloride, Ehrlich and Tollen's reagents and a negative reaction in 55 Sakaguchi, ninhydrin and anthrone tests;

(c) has a melting point in the range of about 156-159°C;

(d) has an optical rotation of [alo7 - 122° (c 0.5, CHCI3);

(e) exhibits in silica gel thin layer chromatography an Rf value of 0.60 with the solvent system

60 CHCI3-CH3OH (5:1 v/v) and exhibits in reverse phase silica gel thin layer chromatograpy an Rf value of 0.16 60 with the solvent system CH3CN-H20 (75:25 v/v);

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(f) exhibits the following ultraviolet absorption maxima when dissolved in methanol, 0.01 N HCI-CH3OH and 0.01 NNaOH-CH3OH

UV Xmax nm (E]°c m) : 248(212)

5 in methanol 279(141) 5

318(103)

UVXma!< nm (E,°cm) : 248(210)

in 0.01 N HCI-CH3OH 279(140)

10 318(103) 10

UVXmaxnrn(E{*J : 248(233)

in 0.01 N NaOH-CH3OH 278(150)

318(110);

15 15

(g) is effective in inhibiting the growth of various bacteria and fungi; and

(h) induces prophage in lysogenic bacteria.

7. The process for the production of the antitumor antibiotic BBM-1675 At which comprises cultivating a BBM-1675 AT-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing

20 assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount 20 of BBM-1675 At is produced by said organism in said culture medium and then recovering BBM-1675 At from the culture medium.

8. The process according to Claim 7 wherein the BBM-1675 AT-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosos-

25 pora strain A1327Y (ATCC 39638), or a mutant thereof. 25

9. The process for the production of the antitumor antibiotic BBM-1675 A2 which comprises cultivating a BBM-1675 A2-producing strain of Actinomadura verrucosospora in an aqeuous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 A2 is produced by said organism in said culture medium and then recovering BBM-1675 A2

30 from the culture medium. 30

10. The process according to Claim 9 wherein the BBM-1675 A2-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

11. The process for the production of the antitumor antibiotic BBM-1675 A3 which comprises cultivating a

35 BBM-1675 A3-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing 35 assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 A3 is produced by said organism in said culture medium and then recovering BBM-1675 A3 from the culture medium.

12. The process according to Claim 11 wherein the BBM-1675 A3-producing organism has the identifying

40 characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosos- 40 pora strain A1327Y (ATCC 39638), or a mutant thereof.

13. The process for the production of the antitumor antibiotic BBM-1675 A4 which comprises cultivating a BBM-1675 A4-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount

45 of BBM-1675 A4 is produced by said organism in said culture medium and then recovering BBM-1675 A4 45 from the culture medium.

14. The process according to Claim 13 wherein the BBM-1675 A4-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

50 15. The process for the production of the antitumor antibiotic BBM-1675 Bt which comprises cultivating a 50 BBM-1675 BT-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of BBM-1675 Bt is produced by said organism in said culture medium and then recovering BBM-1675 Bt from the culture medium.

55 16. The process according to Claim 15 wherein the BBM-1675 BT-producing organism has the identifying 55 characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof.

17. The process for the production of the antitumor antibiotic BBM-1675 82 which comprises cultivating a BBM-1675 B2-producing strain of Actinomadura verrucosospora in an aqueous nutrient medium containing

60 assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount 60 of BBM-1675 B2 is produced by said organism in said culture medium and then recovering BBM-1675 B2 from the culture medium.

18. The process according to Claim 17 wherein the BBM-1675 B2-producing organism has the identifying characteristics of Actinomadura verrucosospora strain H964-92 (ATCC 39334), Actinomadura verrucosos-

65 pora strain A1327Y (ATCC 39638), or a mutant thereof. 65

40

GB 2 141 425 A

40

19. A method for therapeutically treating an animal host affected by a microbial infection which comprises administering to said host an effective antimicrobial dose of BBM-1675 Au BBM-1675 A2,

BBM-1675 A3, BBM-1675 A„, BBM-1675 Bi or BBM-1675 B2.

20. A method for therapeutically treating an animal host affected by a malignant tumor, which comprises

5 administering to said host an effective tumor-inhibiting dose of BBM-1675 An, BBM-1675 A2, BBM-1675 A3, 5 BBM-1675 A4, BBM-1675 Bt or BBM-1675 B2.

21. A pharmaceutical composition comprising an effective antimicrobial amount of BBM-1675 A1r BBM-1675 A2, BBM-1675 A3, BBM-1675 A4, BB-1675 Bt or BBM-1675 B2 in combination with a pharmaceutical carrier or diluent.

10 22. A pharmaceutical composition comprising an effective tumor-inhibiting amount of BBM-1675 At, 10 BBM-1675 A2, BBM-1675 A3, BBM-1675 A4, BBM-1675 Bt or BBM-1675 B2 in combination with a pharmaceutical carrier or diluent.

23. A biologically pure culture of the microorganism Actinomadura verrucosospora strain H964-92 (ATCC 39334), said culture being capable of producing the antibiotic BBM-1675 in a recoverable quantity

15 upon cultivation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen. 15

24. A biologically pure culture of the microorganism Actinomadura verrucosospora strain A1327Y (ATCC 39638), said culture being capable of producing the antibiotic BBM-1675 in a recoverable quantity upon cultivation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen.

25. Chemical compound BBM-1675 Ai obtained by cultivating Actinomadura verrucosospora strain

20 H964-92 (ATCC 39334) orA1327Y (ATCC 39638). 20

26. Chemical compound BBM-1675 A2 obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) or A1327Y (ATCC 39638).

27. Chemical compound BBM-1675 A3 obtained by cultivating Actinomadura verrucosospora strain H-964-92 (ATCC 39334) or A1327Y (ATCC 39638).

25 28. Chemical compound BBM-1675A4obtainedbycultivating/4cft/7o/?7S£/t/ra verrucososporastrain 25

H964-92 (ATCC 39334) or A1327Y (ATCC 39638).

29. Chemical compound BBM-1675 B, obtained by cultivating Actinomadura verrucosospora strain H964-92 (ATCC 39334) or A1327Y (ATCC 39638).

30. Chemical compound BBM-1675 B2 obtained by cultivating Actinomadura verrucosospora strain

30 H964-92 (ATCC 39334) orA1327Y (ATCC 39638). 30

31. Chemical compound BBM-1675 At-

32. Chemical compound BBM-1675 A2.

33. Chemical compound BBM-1675 A3.

34. Chemical compound BBM-1675 A4.

35 35. Chemical compound BBM-1675 Bt- 35

36. Chemical compound BBM-1675 B2.

Printed in the UK for HMSO, D8818935, 10/84, 710Z Published by "Hie Patent Office, 25 Southampton Buildings, London, WC2A 1AY, from which copies may be obtained.

BNSDOCID: <GB 2141425A_I__>