DK163127B - ANTIBIOTIC ANTITUM TREATMENTS AND ANTIBIOTIC AGENTS, PROCEDURES FOR PREPARING THEM AND PHARMACEUTICAL PREPARATIONS CONTAINING THESE - Google Patents

Description

in

DK 163127 B

The present invention relates to novel antibiotic antitumor active compounds, antibiotic compounds and their preparation and recovery, and pharmaceutical compositions containing the compounds.

The antibiotic antitumor compounds of the present invention have not yet been identified for structure.

However, due to their unique physical, chemical and biological properties, the BBM-1575 antibiotics in question are believed to be novel compounds.

EP-A-95154A1 discloses the fermentation of Actinomadura pulveraceus sp. November No. 6049 (ATCC 39100) for the preparation of antibiotic antitumor agents called WS 6049-A and WS 6049-B. The structures of these WS 6049 antibiotics have not yet been elucidated, but the characterizing properties listed for these antibiotics suggest that WS 6049-A and WS 6049-B may be structurally related to BBM-1675 antibiotics of the present invention. . However, spectral data shows that neither WS 6049A nor WS 6049B are identical to any of the BBM-1675 components. This is evident from the C spectra, for example BBM-1675 Aj and -Ag have 7 methyl groups between 13 and 23 ppm, 20 while WS 6049A has 6 methyl groups and -B has 5 methyl groups in the same 13 region. Furthermore, detailed analyzes of H spectra and C-spec three show that Aj and Ag have a -N-CH (CHg) g side chain not present in WS 6049A and -B. Furthermore, WS 6049A and -B have a molecular weight between 1100 and 1200, while BBM-Aj and -Ag both have a molecular weight of 1248.

In addition, the producing organism described in European Patent No. 95154A1 can be distinguished from the Actinomadura verrucosospora used in the present invention for the color of its air mycelium on ISP mediums 2, 3 and 4, on the latter's positive milk peptoneization and on its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.

30 BBM-1675 A14 and -Bj_2 each exhibit a broad antimicrobial activity spectrum (see Table 16) and differ effectively from WS 6049A and -B in that they have documented antimicrobial activity against M.luteus 1001, M. flavus , Mycobacterium 607, K. pneumoniae Dll, and C. neoformans.

35 WS 6049A and -B have only documented antitumor activity against P-388 leukemia. In addition, BBM-1675 Ajg has unexpected antitumor activity against L-1210 leukemia, B16 melanoma and Lewis lung carcinoma as shown in Tables 21-23. Against B16 melanoma, BBM-1675 Aj was unexpectedly equally effective,

DK 163127 B

2 whether administered intravenously to animals with subcutaneous tumors or intraperitoneally to animals with ip-implanted tumor tissue.

In summary, it can be found that BBM-1675 and -Bjg have a documented wider antimicrobial activity spectrum than WS 6049A 5 and -B, and that BBM-1675 Aj ^ has documented wider antitumor activity than US 6049A and -B.

The present invention provides a new antibiotic antitumor active complex called BBM-1675 which is produced by growing a BBM-1675 producing strain of Actinomadura verrucosospora, most preferably Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura strain (ATCC 39638) or a mutant thereof, in an aqueous nutrient medium containing assimilable carbon and nitrogen sources under submerse aerobic conditions, until a significant amount of the BBM-1675 complex has been formed by the organism in the culture medium and if desired, the complex is recovered from the culture medium .

The present invention also provides two major bioactive components of the BBM-1675 complex called BBM-1675 Ax and A2 and four bioactive bi components of the complex called BBM-1675 A3, A4, Bx and Bz. The components can be separated and purified by ordinary chromatographic methods. The BBM-1675 complex and its bioactive components exhibit both antimicrobial and antitumor activity.

The antibiotic anti-tumor agent BBM-1675 A1 according to the invention is characterized by the characterizing part of claim 1. The antibiotic anti-tumor agent BBM-1675 Ag according to the invention is characterized by the characterizing part of claim 2. The antibiotic anti-tumor agent BBM-1675 µ according to the invention is characterized by the characterizing part of claim 3. The antibiotic anti-tumor agent BBM-1675 A A according to the invention is characterized by the characterizing part of claim 4. The antibiotic agent BBM-1675 Bj according to the invention is peculiar to the characterizing part of claim 5.

The antibiotic agent BBM-1675 Bg according to the invention is characterized by the characterizing part of claim 6.

Referring to the drawing, Figure 1 shows the infrared absorption spectrum of partially purified 35 BBM-1675 Ax (KBr pill), Figure 2 the infrared absorption spectrum of partially purified BBM-1675 A2 (KBr pill), Figure 3 the infrared absorption spectrum of BBM-1675 A3

DK 163127 B

Figure 3 (KBr pill), Figure 4 the infrared absorption spectrum of BBM-1675 A4 (KBr pill), Figure 5 the proton magnetic resonance spectrum of partially purified 5 BBM-1675 Ax in CDC13 (60 MHz), Figure 6 the proton magnetic resonance spectrum of partially purified BBM -1675 Az in CDC13 (60 MHz), Figure 7 the proton magnetic resonance spectrum of BBM-1675 A3 in CDC13 (60 MHz), Figure 10 the proton magnetic resonance spectrum of BBM-1675 A4 in CDC13 (60 MHz), Figure 9 the infrared absorption spectrum of purified BBM-1675 Ax (KBr pill), Figure 10 shows the proton magnetic resonance spectrum of purified 15 BBM-1675 A2 in CDC13 (360 MHz).

Figure 11 shows the 13C magnetic resonance spectrum of purified BBM-1675 A! in CDC13 (90.3 MHz).

Figure 12 is the infrared absorption spectrum of purified BBM-1675 A2 (KBr pill); Figure 13 is the proton magnetic resonance spectrum of purified BBM-1675 Az in CDC13 (360 MHz).

Figure 14 shows the 13C magnetic resonance spectrum of purified BBM-1675 A2 in CDC13 (90.3 MHz).

The invention relates to a novel antibiotic antitumor complex 25 called BBM-1675 and its preparation by fermentation of certain strains of Actinomadura verrucosospora, in particular Actinomadura verrucosospora strain H964-92 and a mutant thereof designated Actinomadura verrucosospora strain A132. The above parent strain was isolated from a soil sample collected at Pto Esperanza, Misiones, Argentina. A biologically pure culture of the organism has been deposited with the American Type Culture Collection, Washington, D.C. and added to its permanent collection of microorganisms such as ATCC 39334. Later, the mutant strain A1327Y was obtained by ordinary nitrosoguanidine (NTG) treatment of strain H964-92, and it was deposited with the American Type Culture Collection as ATCC 39638.

As is the case with many other antibiotic producing cultures, fermentation of Actinomadura verrucosospora strain H964-92 or strain A1327Y results in the preparation of a mixture or complex of drug components. Two major bioactive components, BBM-1675

DK 163127 B

4

Ax and A2, and four bioactive bi components, BBM-1675 A3, A4, Bx and B2, have been separated from the BBM-1675 complex prepared during the fermentation process.

Microorganism 5 Actinomycete strain No. H964-92 was soldered from a soil sample and prepared by conventional methods for a biologically pure culture for characterization. Strain H964-92 forms on the aerial mycelium short spore chains showing straight, curved or curved shapes. The grooves are spherical or oval shaped and have a warty surface. Air mycelium 10 is only formed to a small extent on most media. The color of the aerial mycelium is white, which later changes to a pinkish hue or further changes to a bluish color on some agar media. The substrate mycelium is colorless or slightly pink. The growth temperature ranges from 15 to 43 ° C. The amino acid composition of the cell wall and the sugar components of the whole cell hydrolyzate show that strain H964-92 belongs to cell wall type IIIg. The menaquinone was identified as ΜΚ-9 (Η6) · ΜΚ-9 (Η8).

Based on essential morphological, cultural and physiological characteristics, as well as the chemical composition characteristics of the cell wall, strain H964-92 can be classified as belonging to the genus Acti-20 nomadura.

Although the original strain H964-92 produced only moderate growth and carried sparse air mycelium, a variant was shown that showed good growth and improved air mycelium formation by NTG (nitrosoguanidine) treatment of H964-92. The variant, called strain A1327Y, facilitated further taxonomic investigation and was subsequently identified as Actinomadura verrucoso-spora.

methods

The media and methods used to study the culture characteristics and carbohydrate utilization were those recommended by "The International Streptomyces Project" (Intl.J.Syst.Bacteriol. 16, 313-340, 1966). Additional media described by S.A. Waksman ("The Actinomyce- tes", Vol. 2) and G.M. Lvedemann (Intl. J. Syst.Bacteriol. 21, 240-247, 1971) was also used. The amino acid composition of the cell wall and the sugar components of the whole cell hydrolyzate were analyzed according to methods described by Becker, et al., Respectively, in Appl. Microbiol. 13, 236-243, 1965 and by Lechevalier and Lechevalier in "The Actinomycetes",

Oath. H. Prauser, Jena, Gustav Fischer Verlag, pp. 393-405, 1970. Menaqui-

DK 163127 B

The nitrate was identified by mass spectrometric analysis according to the methods of Collins, et al. in J.Gen.Microbiol. 100, 221-230, 1977, and the menaquinone composition was designated, based on a system described by Yamada, et al., In J.Gen.Appl.Microbiol. 23, 331-335, 1977.

5 Morphology

Strain H964-92 forms both substrate and aerial mycelium. The substrate mycelium is long, branched and not fragmented in short strands. In the aerial mycelium, short spore chains are formed monopodially or at the hyphal tip. Whorl-like spore chains are also observed near the hyphal tip. These spore chains contain 2 to 10 spores in a chain and are straight, curved or curved in shape. The grooves have a warty surface and are spherical to elliptical (0.5-0.6 x 0.6-1.4 μαα) of shape with rounded or pointed ends. After ripening, each spore is often separated with an empty sheath. Moving spores, sporangia or skierotic granules are not seen in some of the investigated media.

Cultural and physiological characteristics Growth of strain H964-92 is low to moderate in both chemically defined media and natural organic media. Air mycelium formation is generally poor, but is moderate in oatmeal sagar (ISP # 3 medium), 20 inorganic salt-starch agar (ISP # 4 medium), and Bennett's agar.

Spontaneous variants lacking aerial mycelium occur with great frequency. The color of the aerial mycelium is white, which later turns to pale pink in oatmeal agar, inorganic salt-starch agar and glycerol-asparagine gin (ISP # 5 medium). The color of the aerial mycelium is further changed to a bluish color after long incubation (5 months) on oatmeal agar, glycerol asparagine agar and tyrosine agar. The substrate mycelium is colorless to yellowish in Czapek's agar, tyrosine agar, yeast extract malt extract agar (ISP # 2 medium), peptone yeast extract iron agar (ISP # 6 medium) and Bennett's agar, and is pinkish in glucose asparagine agar and glycerol aspirate. 30 raginagar. Melanoid and other diffusible pigments are not produced.

A variant No. A1327Y obtained from the original strain forms predominantly weak blue air mycelium and carries ample air trace mass.

Strain H964-92 grows at 15, 28, 37 and 43 ° C, but not at 10 or 47 ° C. It is sensitive to 7% NaCl and resistant to 0.01% lysozyme.

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

DK 163127B

6

Table 1

Cultural characteristics of strain H964-92 (native strain ATCC 39334 and variant A1327Y) 05 strain no., H964-92_ Actinomadura

Original strain Variant verrucosospora (ATCC 39334) No. Al327 Y KCC A-0147

Trypton / G: abundant, fluffy, moderate, fluffy, moderate, fluffy, yeast sedimented and sedimented and sedimented and staked agar non pigmented non pigmented non pigmented

10 (ISP

number 1)

Saccha— G: moderately low, low rose / ni- R: colorless, colorless, colorless, tratagar A: squat, pale nothing or squat or knee- (Czapeks (264), for weak leg, pinkish leg, pale blue <15 agar) pink (7) white (9) (185) D: nothing nothing nothing

Glucose / G: moderately low low asparagine- R: white (263) for colorless colorless agar dark yellow pink (27) A: nothing or very nothing or very nothing or very 20 squat, weak squat, white squat, white pink (7) D : nothing nothing nothing

Glycerol / G: rings for moderate to moderate moderate asparagine- R: colorless to light-light-yellow-light-yellow-light-yellow-pink-pink (28) red (28) to 25 OSP (28) dark-yellow No. 5) pink (27) A: low-light-yellow moderate, white to moderate, white pink (28), light pink (4) to bright light ether 5 months bright red (2) cheap gray (190) D: Nothing nothing nothing 1 35

Inorganic G: abundant moderately moderate salts / R: yellowish white (92) pale yellowish pink pale yellowish pink starch- (28) (28) agar A: abundant, light pale-moderate, pale blue-abundant, weak (ISP red (4) to light - corpse gray (190) blue (185) no. 4) reddish gray (10) D: nothing nothing nothing

DK 163127 B

7

Tyrosine- G: moderate moderate moderate agar R: yellowish white (92) strong yellowish strong yellowish (ISP pink (26) pink (26) no. 7) A; rings, pale yellowish moderate, white to moderate, white to pink (28), light pink (4) light pink (4) much later (5 05 months) partly bright bluish gray (190) D: nothing nothing nothing G: rings to moderate low rings agar R: pale yellow (89) colorless to pale colorless to pale pink (7) pink (7) .jO A: nothing nothing nothing D: nothing nothing nothing

Gaerek- G: abundant abundant abundant stretched / R: pale yellow (89) vigorous yellow vigorous yellow malty pink (26) pink (26) stretch agar A: rings, white rings, white to rings, white to (ISP (263) weak pink (7) faint pink (7) 15 # 2) D: nothing nothing nothing

Oatmeal G: moderately low Poor agar R: colorless to slightly yellowish slightly yellowish (ISP pale pink (7) pink (31) pink (31) no. 3) A: small, very pinkish, very squishy, white (9) for light-lively faint blue lively faint blue 20 bluish gray (190) (184) (184) D: nothing nothing nothing

Bennetts G: ample ample ample agar R: greyish yellow (90) strong yellowish strong yellowish pink (26) pink (26) A: moderate, white moderate, faint nothing (263) to yellowish yellowish pink 25 white (92) (31) and bluish white (189) D: nothing nothing nothing

Pepton / G: moderately abundant yeast- R: colorless colorless colorless stack / iron- A: nothing nothing nothing 30 agar (ISP D: nothing nothing no. 6) * Observed after incubation for 3 weeks at 37 ° C.

** Abbreviations: G = growth, R = backing color, A = air mycelium, D = diffusible pigment.

35 *** Color and numbers in parentheses follow the color standard of "Kelly, K.L.

& D.B. Judd, ISCC-NBS color labeling chart illustrated with Centroid Colors, U.S.Dept, of Comm. Cir. 553, Washington, D.C., November 1975 ".

DK 163127B

8

Table 2

Physiological characteristics of strain H964-92

Method and Method 05 Test_ Result_Dium__

Temperature range Maximum growth at Bennett's agar for growth 28 to 37 ° C. Moderate at 20 and 43 ° C. None at 10 and 47 ° C.

Melting of gela- Melted Glucose / peptone / gelatinous medium

Starch Hydrolysis Hydrolyzed Starch Agar Plate

Reactions in No Coagulation and "Difco" Skim Milk Skim Milk Complete Peptonization 15

Formation of Mala Negative Tyrosine Agar, Peptone / Noid Pigment Yeast / Iron Agar and Tryptone / Yeast Extract Substrate

Nitrate Reduction Negative Czapek's giucose / nitrate substrate and glucose / yeast extract kt / n itrate substrate

Resilience- Growth at 5% or Trypton / yeast extraction to NaCI less. No growth agar at 7%

Lysozyme Resistant. Growth at 0.01% or agar less. No growth at 0.1% pH Growth in 5.0 to 9.5. Tryptone / yeast extract

No growth at 4.5 agar and 10.0 1 35

DK 163127 B

9

Table 3

Exploitation of carbon sources

Strain # H964-92_ Actinomadura 05 Original Variant verrucosospora strain # A1327Y KCC A-0147

Glycerol + + + D (-) - Arabinose - L (+) - Arabinose + + + 10 D-Xylose + + + D-Ribose + L-Rhamnose + + + D-Glucose + + + D-Galactose - 15 D- Fructose + + + D-Mannose - - L (-) - Sorbose -

Sucrose + + +

Lactose - 20 Cellobiose +++

Melibiosis -

Trehalose +++

Raffinose - D (+) - Melezitosis - 25 Soluble Starch +++

Cellulose +++

Dulcitol -

Inositol + D-Mannito! + + + 30 D-Sorbitol -

Salicin -

Observed after incubation at 28 ° C for 3 weeks.

Basic medium: "Pridham-Gottlieb" inorganic medium.

35

DK 163127B

10

Cell wall composition and whole cell sugar components

Purified cell wall from strain H964-92 contains mesodiamino-pimelic acid but lacks glycine. The whole cell hydrolyzate shows the presence of madurose (3-O-methyl-D-galactose), glucose and ribose.

The cell wall amino acid and the whole cell sugar components suggest that strain H964-92 belongs to cell wall type ΙΙΙβ. Menaquinone's two major components were identified as MK-9 (H6) and MK-9 (H8).

10 Taxonomic location of strain H964-92

Strain H964-92 has the following main characteristics: 1) air trace chains: short, straight, curved or curved in shape, 2) spores: warty surface, 3) aerial mycelium: pink or bluish color, 4) substrate mycelium: pink in some media, 5) diffusible pigment: nothing, 6) mesophilic, 7 cell wall type Hig, and 8) menaquinone system: MK-9 (H6) and MK-9 (H8).

These main characteristics suggest that strain H964-92 belongs to the genus Actinomadura. Early species of the genus Actinomadura are isolated from mammals. Some strains were also obtained from plant materials. However, many of the newly proposed new species in soles are from soil. In accordance with the numerical taxonomy and overview of Actinomadura and related actinomycetes by Goodfellow, et al., In J.Gen.Microbiol. 112, 95-111 (1979), most of the 25 Actinomadura species of soil origin in Group 7 are classified among the 14 described groups. Strain No. H964-92 is most akin to the species in Group 7. Nonomura and Ohara reported in J.Ferment.

Technol. 49, 904-912 (1971) five saprophytic species of the genus Actinomadura, and in J.Ferment.Technol. 52, 71-77 (1974) (Nonomura) 30 and in "Actinomycetes and Related Organisms" 12, 30-38 (1977) (Preobrazhenskaya et al.), The keys for identifying and classifying Actinomadura species were published. As a result of comparison with the description of 30 species, including patent organisms, strain H964-92 appears to be most similar to Actinomadura 35 coerulea described in the above article by Preobrazhenskaya, et al., And Actinomadura verrucosospora disclosed. in the above article by Nonomura.

Strain # H964-92 was directly compared to A. verrucoso-

DK 163127B

11 spore strain KCC A-0147 and was found to be closely related to A verrucosospora in terms of morphological, cultural and physiological characteristics. Strain H964-92 was thus classified as a new strain of Actinomadura verrucosospora.

It is to be understood that the present invention is not limited to the specific Actinomadura verrucosospora, strain H964-92 (ATCC 39334), or the mutant strain thereof called strain A1327Y (ATCC 39638) or corresponding microorganisms with respect to BBM-1675 to the cultivation characteristics shown herein, although the invention is described in detail with reference to these microorganisms. It is particularly intended that the invention should encompass the use of strain H964-92 and all natural and artificial BBM-1675 producing variants and mutants thereof having substantially the same properties.

15 Antibiotic Preparation

The method of producing the antibiotic BBM-1675 Aj, -Ag, -Aj, -A ^, -Bj or -Bg is peculiar to the cultivation of Actinomadura verrucosospora H964-92 (ATCC 39334) or its mutant Actinomadura verrucosospora A1327Y (ATCC 39638) or another mutant thereof having substantially the same properties in an aqueous nutrient medium containing assimilable carbon and nitrogen sources under submerse aerobic conditions, until a significant amount of BBM-1675 Al, -Ag, -A , -Bj or -Bg have been produced by the organism in the culture medium, and then BBM-1675 extracts Aj, -Ag, -A ^, -A ^, -Bj or -Bg from the culture medium. The common methods used for the cultivation of other actinomycetes are useful in 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 nitrate, sodium nitrate, etc. can be used either alone or in combination with organic nitrogen sources such as peptone, meat extract, yeast extract, corn mold liquid, soybean powder, cotton seed meal, etc. to provide

DK 163127 B

12 sources of sodium / potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium manganese, cobalt, iron and the like.

Preparation of BBM-1675 antibiotics can be carried out at any temperature leading to a satisfactory growth of the producing organism, for example 15 to 45 °, and conveniently carried out at a temperature of about 27 to 32 ° C. Generally, optimum production is obtained after incubation periods pi from ca. 68 to 180 hours, depending on whether shake bottle, tube separator or tank fermentation is used. When tank fermentation is to be performed, it is desirable to prepare a vegetative inoculum in a nutrient substrate by inoculating the substrate with a scree or soil culture or a lyophilized culture of the producing organism. Having thus obtained an active inoculum, it is aseptically transferred to the fermentation tank medium. Antibiotic preparation can be followed by the paper disc agar diffusion test using Staphylococcus aureus 209P as the test organism.

Isolation and Purification When the fermentation is complete, the BBM-1675 complex can be obtained from the substrate by ordinary isolation methods, for example, solvent extraction. Thus, for example, the entire substrate can be separated by filtration or centrifugation into a mycelial cake and a substrate supernatant. Antibiotics in the mycelial cake 25 can be recovered by suspending the cake in methanol, filtering out the insoluble materials and concentrating the methanolic extract.

Activity in the substrate supernatant can be recovered by extraction with an n-butanol. The aforementioned n-butanol and methanol extracts can then be combined and evaporated azeotropically to an aqueous solution in which most of the antibiotic activity precipitates as an oily solid. The solid can then be dissolved in methanol and the solution filtered. The filtrate is concentrated and added to a mixture of ethyl acetate and water. The resulting organic extract contains the ri BBM-1675 complex which can be precipitated from the solution by the addition of an anti-solvent such as n-hexane.

The crude BBM-1675 complex is a mixture of several components comprising two major bioactive components, BBM-1675 and A2, and

DK 163127 B

13 four bioactive bee components, BBM-1675 A3, A4, Bx and B2. These bioactive components can be separated and purified by ordinary chromatographic methods. In one method, the crude BBM-1675 complex is first dissolved in methanol and purified by "Sephadex 05 LH-20" column chromatography using methanol as the eluent solvent. This partially purified complex can then be chromatographed on a silica gel column and eluted stepwise using chloroform plus an increasing concentration of methanol to provide BBM-1675 Ax, a mixture of BBM-1675 A2, 10 A3 and A4 and a mixture of BBM-1675 Bi and B2. That the component can be further purified by "Sephadex LH-20" column chromatography using methanol as the eluent solvent. The mixture of A2, A3 and A4 can be separated by chromatography on a column of "Bondapak C18" (Waters Associates, Inc.) using increasing concentrations of aqueous acetonitrile as the eluent. The mixture of the Bx and B2 components can be separated by silica gel column chromatography using a mixture of chloroform and methanol as the eluent solvent. Further details of the preferred chromatographic separation methods are provided in the following examples.

Physico-chemical properties of the BBM-1675 components

The six bioactive components of the BBM-1675 complex can be distinguished from each other by two TLC systems as shown in the following table. 1 35

DK 163127 B

14

Table 4 TLC of BBM-1675 components

Rf values__ 05 Si02 * Silanized

CHCl3-CH3OH CH3CN-H2O

Component (5: 1 vol / vol) (75:25 vol / vol) BBM-1675 Ai 0.74 0.18 BBM-1675 A2 0.71 0.21 BBM-1675 A3 0.72 0.28 BBM 1675 A4 0.71 0.78 BBM-1675 Bx 0.63 0.23 BBM-1675 B2 0.60 0.16 15 * C18 reverse phase silica gel.

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

20 The individual BBM-1675 components show similar solubility and color reactions. For example, they are soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride. They give positive reactions with ferric chloride, Ehrlich and 25 Tolien's reagents, but negative reaction with Sakaguchi, ninhydrin and anthron tests.

Characteristic physicochemical properties of BBM-1675 components are shown in Table 5 below. 1 35

DK 163127 B

15

U

island

Qv / λαα s ~ \ s ~ ss->.

[n No «O O CO CO O O

i- r ^ O r-'S'O CO LO r- CVJr-r- CNJr-T— CVlT-r- £ Q Sw 'j m $ 00 05 00 00 05 00 CO 00 00 «iffir- f ~. r- »tNr» i f *. r- CQ | ϊ— l NN (0 NN (0 NNC0 u 0 / * “S / - \ Λ / '" • s cd mo ^ r in g in g r- in v- CM M- O no CO <ί o Ν rr CVIr -r- N rrj 0 v-'wv-x / v_ / 0f, fZ MNO CONO NNC0 coli? 88 $ 88 $ 88 $ «υ y and r- \ rN / ^ -. / ^ s / ~ s / ^ s / -sr-sx-v fli CM f "CO f" · 00 in 00 CO o 00 2 V; irtiflr tfllflr (D (Or 5 CVIr-r- N rr N rr S ior * m in co £ CO {S CO cn in oo cm o co cm o cm co co o 0 * cm r- CO CO co CD in OD CM U5 00 CM U5 00 r- -ί

Jg <r- I in CO CM CM CO CM CM CO CM CM CO r- m Γ "· r j CD ^ r ~ is. Rcrvr.

• λι CO00CM N O CD OCMO

S i- OOlDCM CO CD CM 00 CD CM

CO Nrr Nrr Nrr m> 0 ONNr

Qj r- O ΙΛ in 05 o „, m ω -ivv co CM o (O CM o cm co od about c- ¢ {¾ ~ UD CD CO O · UD 00 CM LD 00 CM lp Mr r- _ O <| r- i in CO CM CM CO CM CM CO CM CM CO r- 0) n i_

m a) M

H -Q ^ _ (0 rv r-N r \ rN rv r-v »rNM ΜΙΌβ ΙΛ r-0 CONN MDN 00 In Ν C« · Nrr Nrr Nrr β)} *. r- r- CM CD w w w ^

$ f *. S CO °° v °° O (O CM O CO CM O CM CO CO O

Cl '. V- CO CO CO CD in 00 CM in 00 CM in 00 r- r- Φ <| r · I in co CM CM CO CM CM CO CM CM CO r- X.

in 'i ** 1, u

in OD / V / V / * V / "i /" V / "V

1 inmcococMrfmcMco

i COr- CMOD ^ i CMODrr CM Γ> · CO

a r— COr-r-COrrCOrr

· - i j o CM r * CM LO

> 1 CO 05 CO CM O CO CM O CM CO 00 O

u_ CO Si r_ fin ^ too in oo cm in oo cm in oo r- co QQ f. 7 Jn $ CM CM CO CM CM CO CM CM CO r-

U X Z O

i «^ x +; u CO a λ C _i a Ο ΰ e E ΐ% ϊη! Γ {I, !ί! ° «* g15 -g U ® ^ ω I V 0-537 r I S! v ±: ¾ c ° “I X X z 3 U C ID 3 *> T3 ^

Λ CD * "C in" 7 “7 C · CD

ω w O ^ O jj; iE> jUQ.

^ «Q ~ Ό ^ X ° ° fl)> C

E ^ J 2> υ ° ° S

(/) U ^ S »/ -J I— · - · · - -

DK 163127B

16 UV absorption maxima for the BBM-1675 components were observed at 253, 282 and 320 nm, which did not change in acidic or basic solution. The IR and PMR spectra of BBM-1675 Ax, A3 / A3 and A4 are shown in Figures 1 to 4 and Figures 5 to 8. respectively. The 360 MHz 05 PMR spectrum of BBM-1675 Aj suggests one acetyl group (6: 2, 11 ppm), one N-CH3 group (2.52 ppm), four OCH3 groups (3.42, 3.80, 3.88 and 3.98 ppm) and one exomethylene group (4.57 and 5.48 ppm) as well as two aromatic protons (7.50 and 8.59 ppm) and one NH proton (11.79 ppm). The CMR spectrum for BBM-1675A! exhibited 55 10 carbon signals including a signal of triple intensity (δ: 56.0 ppm, OCH3). The molecular formula for BBM-1675A! was concluded to be Η57Η72Ν4032 based on proton and C13 NMR spectra, microanalysis and molecular weight determination by HPLC and SIMS (secondary ion mass spectrometry).

15

Structural Study of BBM-1675 Ai

When treated with 0.5N HCl-CH 3 OH at room temperature, BBM-1675Aj loses its bioactivity and yields a lipophilic chromophore substance (compound I) along with several unidentified fragments. Compound (I) shows UV absorption similar to that of the parent antibiotic, suggesting that Compound (I) retains the chromophore structure of BBM-1675Al. Two other chromophore fragments coated with compound (I) fiss by basic hydrolysis of BBM-1675Al: hydrolysis with 0.05N KOH-CH3OH at 55 ° C for 1 hour yield compound (II) having 25 UV absorption maxima at 252. 284, 297 (shoulder) and 322 nm, while the reaction in 1N KOH-CH3OH gives an acidic chromophore substance called compound (111). Physicochemical properties of compounds (I), (II) and (III) are summarized in Table 6 below. 1 35

DK 163127 B

17

Table 6

Properties of compounds (I), (II) and (III)

Conn. (I) Ex. (II) Forb. (Ill)

Melting point 82 ~ 83 ° C 133 ° C 253 ~ 255 ° C

[or] 39 (c 0.2 CHC | 3) -100 ° 0 0

Molecular C 21 H 31 NO 10 C 14 H 17 NO 6 C 13 H 15 NO 6 .... CH 3 OH, 244 (21,850) 252 (26,600) 248 (26,900) in UV Λ max nm ^ ε; 276 (9,400) 283 (11,200) 295 (14,400) '318 (6,300) 297 (sh8,800) 310 (13,500) 322 (11,700) MS m / z 457 (M +) 295 (M +) 281 (M +) 425 280 263 341 263 236 281 251 222 15 264 248 218

TLC

(Xylene-1MEK-CH 3 OH = 5: 5: 1 v / v) Rf 0.58 0.66 0.13 MEK = methyl ethyl ketone.

20

Structural information on compounds (II) and (III) was provided from the following spectral data and chemical conversions.

13C and proton NMR indicated the presence of four OCH3 groups, group, seven -C = groups, two> C-O groups and one 25> NH group in compound (II). The NMR spectra of compound (III) were similar to spectra of compound (II), differing only in the absence of one of the four OCH3 groups observed in compound (II). This difference along with the acidic nature of compound (III) indicated 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 (III), while compound (III) was converted to compound (1i) by treatment with diazomethane. Treatment of compound (II) with NaBH4 in C 2 H 5 OH gave a.

reduction product (compound (IV), M: m / z 267), which showed a 35 -CH 2 OH group by NMR instead of compound (11) s -C00CH 3 group. Upon hydrogenation over palladium-pi-charcoal, compound (II) gave a dihydro derivative (compound (V), M +: m / z 297).

DK 163127B

18

The proton NMR spectrum of compound (V) exhibited a novel doubled methyl signal and lacked the exomethylene group present in compound (II). In addition, one of the OCH3 groups at higher field (δ: 3.50 ppm) appeared in compound (V). These results suggest the-05 site of a -C = CH2 in * OCH3 group in Compound (II) which is reduced by hydrogenation to -CH-CH3 in the OCH3 group in Compound (V). Compound (II) was heated with 1.5N methanolic hydrogen chloride at 80 ° C for 3 hours and the hydrolyzate chromatographed on a silica gel column to give a weak basic compound (compound (VI), M +: m / z 211). The IR spectrum and physicochemical properties suggested that compound (VI) contains an NH 2 group. Compound (VI) was identified as methyl 4,5-dimethoxy-anthranilate by comparative IR and NMR studies with a real sample. Accordingly, the structures of compounds (II) to (VI) were determined as shown below.

25 1 35

DK 163127 B

19 structures for compounds (II), (III), (IV), (v \ --- (V |)

Compound (II)

Compound (III)

05 λ. CH

3 | nOCH, —_-_ A J \ JH-C0-C

II 3 I Xoch3 CH30 '^ A'C00CH3 α3 \ ΧΜ

ίο NV

Compound (IV) isl * CH-15 \ Pd / C εΗ3 ° ν ^ \ ΝΗ-οο-ς ^ + \ Π 0CH3

H / MeOH \ I I

- CH 2 OH - CH 2 OH

20 \ Φ \ \ J Compound (V)

Compound (VI) CH 2

NH-CO-CH

3 Ίγ in 2 i \ I I I and 3 CH3 <T <i ^ X ^ OOCS3 CBsCTS ^ 'cooch3

When treated with 0.05N COH-CH 3 OH at 55 ° C, compound (J) was partitioned into a new chromophoric fragment (Compound (VII), C 15 H 21 NO 7, M +: m / z 327) and a sugar (Compound VIII). The NMR spectrum of (VII) showed one single C-CH3 group and two high-field OCH3 groups in addition to the three low-field OCH3 groups and two aromatic protons commonly observed in compounds (II), 35 (V) and (VI ). Further hydrolysis of (VII) with 1.5N methanolic hydrogen chloride gave compound (VI) identical to the one obtained from (II). After removal of (VI),

DK 163127 B

The 2-dinitrophenylhydrazine was precipitated with a yellow solid which was identified as 2,4-dinitrophenylhydrazone by pyruvic acid. Compound (VII) is thus methyl 4,5-dimethoxy-N- (2 ', 2, -dimethoxypropionyl) anthranilate. Compound (VIII) did not show any molecular ion peak but showed the M-OCH3 peak at m / z 131 in the mass spectrum in accordance with molecular formula C7H1404.

The NMR spectrum suggested 2,6-dideoxyhexopyranose structure for compound (VIII). The assignment was supported by 1SC NMR of compound (I), which resulted in one C-CH3 carbon atom, one -CH2 carbon atom, three O-CH <carbon atoms and one anomeric carbon atom in addition to the 15 compound (VII). The C3 proton in the sugar occurs at low field (6: 5.39 ppm, octet), revealing that the C3-OH group in the sugar was esterified with the carboxyl group in (VII). The above results are summarized below.

Structures for compounds (I), (VII) and (VIII)

Compound (I) Compound (VII) Compound (VI) <? CH3 OCH, ^ OHVCH3OH H + v CH3 ° '^ 7T'NH2

\. \\ 0033 -: - * 3 I 0CH3 -> I II

<= 3 ° ^ / C ° \ CH3O> <^ / ^ C00CH3 CH30 ^^ / ^ COOCH3

0 OH

/ \ Compound (VIII) \ / 3 ·

3 - CH, O- (1 'V ° H

3 - CH -, - CO-COOH 1 35

The molecular weight of compound (I) (457) counts approx. one-third of the entire molecule BBM-1675A! (suggested formula 057Η72Ν4032, calculated molecular weight = 1324). The substructure of BBM-1675 Ax is believed to be as follows:

DK 163127 B

21 CH, 0-l <^ SsV · NH-CO-C * CH 3 | I 2 I 0CH3 ca3o '^ / S: ov 05 S_-0H' Γ5- · 10

Following the filing date of Tribal Application No. 495,231 in U.S.A. it was discovered that components BBM-1675 Ax and A2 / described above and prepared in accordance with Example 2 below were in fact not completely pure and that some of the characterizing properties used to define such components were imprecise. Following the additional chromatographic purification procedures described in more detail in Examples 3 and 6 below, BBM-1675 Ax and A2 were isolated in a more purified form and completely characterized as described below. Elemental analysis data for components A3 and A4 were also revised to show the presence of sulfur in these compounds and the HPLC retention times were calculated for these two components. The revised physicochemical properties of the BBM-1675 components are summarized below.

25

BBM-1675 AND DESCRIPTION

White to slightly yellow crystals, mp 156 to 158 ° C (dec).

30 35

DK 163127 B

22

ELEMENT ANALYSIS

Analysis 1 Analysis 2 Average C: 51.60% C: 52.74% C: 52.17% 05 H: 6.31% H: 5.99% H: 6.15% N: 5.31% N: 3.94% N: 4.63% S: 8.47% S: 9.71% S: 9.09% O (by difference): O (by difference): O (by difference): 28.31% 27.62% 27.96% 10 ULTRAVIOLET ABSORPTION SPECTRUM Instrument: "Varian UV", "Cary 219"

Solvent: methanol

Concentration: 0.01356 g / L 15 ^ max ^ nm ^ Absorptivities 320 12.4 280 sh (shoulder) 253 25.1 20 210 25.5

No significant change with acid or base.

OPTICAL ROTATION Solvent: CHCl3 [a] 24 °; -207 ° (C = 0.0351)

Another analysis showed the following optical rotation: [α] D = 7 °: -191 ° (C = 0.05, CHCl 3).

INFRARED ABSORPTION SPECTRUM: see Figure 9 Main absorption bands (KBr): 985, 1015, 1070, 1110, 1150, 1210, 1250, 1308, 1380, 1405, 1446, 1520, 1592, 1608, 1668, 1715, 2920, 35 2960, 3360, 3440 cm -1.

DK 163127 B

23 MASS SPECTS Instrument: "VG-ZAB-2F" FAB-MS-thioglycerol, molecular mass ions (m / z): 1249, 1357, 05 1463; upon the addition of NaCl (m / z): 1271, 1379, 1485, 1597.

FAB-MS-MB (MB: matrix, molecular weight 154), molecular mass ions (m / z): 1249, 1283, 1403, 1555; upon the addition of NaCl (m / z): 1249, 1271, 1303, 1425, 1483, 1577.

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

Instrument: "Kratos MS-50" FAB-MS-thioglycerol, molecular mass ions (m / z): 1357, 1463.

MOLECULE WEIGHT (based on the above mass spectral data)

Apparent molecular weight: 1248.

20 NUCLEAR MAGNETIC RESONANCE SPECTS Instrument: "WM360 Brucker"

Solvent: CDCl 3 δ NMR: 360 MHz δ (ppm): 11.75 (1H, s); 8.55 (1H, s); 7.45 (1H, s); 6.61 (1H, m); 6.23 (1H, brs); 6.17 (1H, brs); 5.93 (1H, d, J = 9.3); 5.82 (1H, d, J = 9.3); 5.7 (1H, brs); 5.49 (1H, m); 5.45 (1H, d, J = 2.3); 5.38 (1H, brs); 4.95 (1H, d, J = 10.2); 4.64 (2H, m); 4.54 (1H, d, J = 2.3); 4.2 (1H, s); 4.15-3.35 (26-28H) [4.10 (1H, m); 4.02 (1H, brs); 3.95 (3H, s); 3.85 (3H, s); 3.79 (3H, s); 3.46 (1H, m); 3.40 (3H, s)]; 2.82-2.70 (3H, brm); 2.50 (3H, s); 2.47 (1H, m); 2.38-2.22 (5H); 2.12 (1H, m); 2.11 (3H, s); 1.60-1.05 (22H) [1.39 (3H, d, J = 6.3); 1.31 (3H, d, J = 6.3); 1.29 (3H, d, J = 6.3); 1.08 (6H)]. 1

See Figure 10

DK 163127 B

24 C NMR: 90.3 MHz

See Figure 11 05 In a separate test, the 13C NMR spectrum of purified BBM-1675 Ax was determined for a sample dissolved in CDCl3 (80 MHz). The main peaks are listed below.

CMR for BBM-1675Aj (80MHz in CDCI <0 10 Chemical shifts in ppm (multiplicity *) 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 (h) 56.0 (q) 57.1 (d) 62.4 (t) 64.5 (d) 67.7 (d) 68.2 (d) 68.8 (t) 69.2 (d) 69 , 6 (t) 70.2 (d) 71.9 (d) 76.0 (d) 76.6 (d) 77.1 (u) 77.3 (d) 83.4 (s) 86.6 (d) ) 88.4 (s) 90.5 (t) 97.2 (d) 98.3 (s) 99.0 (d) 99.6 (d) 103.8 (d) 107.6 (s) 112.5 (d) 123.1 (d) 124.9 (d) 130.1 (d) 131.5 (s) 134.9 (s) 136.7 (s) 144.0 (s) 147.2 (s) ) 153.4 (s) 154.4 (s) 155.0 (s) 160.7 (s) 166.4 (s) 191.8 (s) * Multiplicity: q = quartet, d = duplicate, u = uvis, t = triplet s = singlet.

25 BBM-1675 Α »

DESCRIPTION

White crystals, mp 147 to 149 ° C. 1 2 3 4 5 6

ELEMENT ANALYSIS

2 C: 52.71% 3 H: 5.94% 4 N: 3.94% 5 S: 9.39% 6 O (by difference): 28.01%

DK 163127 B

25 ULTRAVIOLET ABSORPTION SPECTS Instrument: "Varian UV", "Cary 219"

Solvent: methanol

Concentration: 0.02052 g / l 05 µmax Absorptivities 320 12.2 282 16.3 252 26.2 10 214 25.8

No significant change with acid or base.

OPTICAL ROTATION

[Α] 25 °: -179.4 ° (C = 0.5, CHCl 3).

INFRARED ABSORPTION SPECTRUM: see Figure 12 Instrument: "Beckman IR Model 4240" 20 Main absorption band (KBr): 950, 1015, 1070, 1100, 1155, 1213, 1250, 1313, 1375, 1405, 1450, 1520, 1595, 1610, 1685, 1735, 2940, 2980, 3440 cm "1.

Mass spectra

Instrument: "VG-ZAB-2F11 FAB-MS-thioglycerol, molecular mass ions (m / z): 968, 1249, 1355, 1357, 1463, 1569; upon the addition of NaCl (m / z): 990, 1271, 1379, 1485, 1593.

FAB-MS-MB (MB: matrix, molecular weight 154), molecular mass ions (m / z): 1249, 1403, 1419, 1555, 1571, 1587; upon the addition of NaCl (m / z): 1249, 1271, 1425, 1441, 1457, 1483, 1577.

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

MOLECULAR WEIGHT (based on the above mass spectral data)

Apparent molecular weight: 1248.

35

DK 163127B

26 Nuclear Magnetic Resonance Spectra: see Figure 13 Instrument: "WIVI360 Brucker"

Solvent: CDCl3 05 1 NMR: 360 MHz 6 (ppm): 11.91 (1H, s); 8.62 (1H, s); 7.58 (1H, s); 6.56 (1H, m); 6.22 (1H, s); 6.15 (1H, brs); 5.91 (1H, d, J = 9.6); 5.83 (1H, d, J = 9.6); 5.70 (1H, m); 5.45 (1H, d, J = 2.2); 5.44 (1H, s); 5.34 (1H, brs); 4.95 (1H, d, J = 10.2); 4.75 (1H, m); 4.65 (1H, d, J = 6.8); 4.54 (1H, d, J = 2.2); 4.47 (1H, m); 4.18 (1H, s); 4.10 (1H, brs); 4.05-3.50 (20-24H) (3.96 (3H, s): 3.87 (3H, s); 3.77 (3H, s)]; 3.46 (1H, m); 3, 39 (3H, s); 2.79 (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, s); d, J = 6.0); 1.34 (3H, d, J = 6.0); 1.22 (3H, d, J = 6.8); 1.10 (6H).

13 C NMR 90.3 MHz

See Figure 14.

In a separate test, the 13 C NMR spectrum of purified BBM-1675 Ag was determined for a sample dissolved in CDCl 3 (80 MHz). The main peaks are listed below. 1 2 3 4 5 6 35 13.7 16.9 17.5 19.8 22.3 22.7 23.4 33.1 2 34.1 35.1 39.3 47.6 52.6 55.7 56.0 56.1 3 57.6 62.4 64.5 64.9 65.9 68.3 69.2 69.7 4 71.9 73.6 75.8 76.1 77.1 77.7 78.1 78.3 5 83.3 86.2 88.4 90.4 97.2 98.3 99.1 99.5 6 99.6 103.8 107.1 112.4 123.2 124.8 129.9 137.3 144.1 154.2 154.5 160.9 167.9 192.2

DK 163127 B

27 u

ISLAND

/ "" S / »" \ / ~ S / "S / -S

tn CM r- CO © O CO co oo li Γ- O r- ^ O CO in r- CM t— ΐ— CM t— t- CM ^ c— j O WN-y'w '' vy '' - ' CD CM CO O) 00 00 CD OO 00 00 00 C * - in .— ^ Γ> · r · 'f I' · r · N γ-

ΕΟ] r I CM CM CO CM CM CO CM CM CO

u 0 _ y — vy — iz-s co in o <t m o in m t- m r: οι o ojrro co tt o CM t— t— CM t— t— CM t— r—

J Q VW V-ice ^ W

O) £ CO CM O 00 CM O CM 00 00 'S) L in oo cm in co cm moor- CQ t— 7 CMCMC0 CMCMC0 CMCMC0 0 «

OQ

01 0

CO

1 ou <f Tf. ηΛΛ ΛΛΛ / -Τ / -v n! f * co r- · oo in co co o oo «. r- mint- min »- ep cd t- t? CMt— t— (\ | r- t * (M f r · «Υ i 0 rn oo r · Is« · —'-- y ^ -y

a CO f2 «O CO CM O CO CM O CM CO OO

LP ti KJ '«t CD CO 00 UO CO CM m 00 CM moor-

You <™ 7 in CM CM CO CM CM CO CM CM CO

r— Σ ^ CQ 1 "» ϋ 03 S S- (o <2 * "ι_ υ ® £, —v y-N / -» ytn> y ~ \ / —t / - ·>

-Q k. <D 00 CM NOifl OCMO

® i! COmCMOOCDCMOOCDCM

IC CMt — r- CMt-t— CMt — r V) ί 0 LO CD CO 0> ww, -_ yy C - in r>. td

ro in ω v \ ^ V CO CM O CO CM O cm CO OO

σι «n r tt cd co m oo cm m oo cm m oo r-

φ1 <| r- i LO CM CM CO CM CM CO CM CM CO

0>

-X

W

E

Φ X.

- * ϋϊζΰϊ in «u.

*? o / —s x 2 Ϊ e * 1 s 2 1 5 s v 2? 3. ? ^ Σ i

+ J LO I I -L O

.se o U- Uro C X I 2 3 0; ro g- w S Si 2 2

B *> * o 5 Z

S «Ω„ ® v v s ~ c> 2 O ° W 3 <3 -

DK 163127 B

28

Comprehensive chromatography (TLC) and high performance liquid chromatography (HPLC) data for BBM-1675 components A. Study # 1 - Overview 05

Table 8 TLC and HPLC of BBM-1675 components HPLC (retention TLC (Rf) - time in minutes "Lichrosorb RP-18"

SiO2 * S in I and in serified CH3CN-MeOH- CHCl3-IVeOH CH3CN-H2O 0.1M CH3C00NH4 Component (5: 1 v / v) (75:25 v / v) (5: 2: 3 v / v) ) 15 BBM-1675 At 0.74 0.18 13.3 BBM-1675 A2 0.71 0.21 17.3 BBM-1675 A3 0.72 0.28 8.0 BBM-1675 A4 0.71 0, 78 5.1 BBM-1675 Bi 0.63 0.23 20 BBM-1675 B2 0.60 0.16 * C18 reverse phase silica gel.

B. Study # 2 - TLC and HPLC of purified Ai and Α 25 Components TLC chromatography "Analtech GHLF Silica Gel Uniplates" was used for all normal phase chromatography. Plates measuring 2.5 x 10 cm were used for one-dimensional TLC. These were prepared in glass cylinders measuring 6.4 cm (outer diameter) x 12 cm and containing 10 ml of eluent. Plates measuring 7.5 x 10 cm were used for two-dimensional TLC. The sample was pivoted on the lower left corner 1 cm from the edges. The plate was first developed in a tank (12.7 cm wide, 8.6 cm deep, 13 cm high) containing 35 ml of the first eluant. The plate was then air dried, turned 90 ° counterclockwise and evaporated in a second tank containing 50 ml of the second eluent.

DK 163127 B

29 l of Whatman analytical plates, which had been pre-coated with 018 silica gel, were used for all reverse phase chromatography.

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

05 Normal phase plates were first examined under 254 nm UV light.

The plates were then inserted into a glass cylinder (6.4 cm (outer diameter) x 12 cm) which contained 12 crystals. The plates were re-examined after approx. 2 minutes. Reversal phase plates were examined only under 254 nm UV light. Zones were detected by looking for suppression of the fluorescence of an im-10 dye.

Analytical HPLC

The following components were used to prepare an HPLO analytical system: a "Waters Associates Model 6000A 15 Solvent Delivery System" pump, a "Varian UV / VIS detector"

Varichrom Model VUV-10 "set at 254 nm 0.1 OD, a" Fisher Recordal Series 5000 "recording apparatus, a" Waters Associates Model 660 "solvent mixer, a" Waters Associates Model U6K "injector, a column of type 20 "Alltech, μ-Bodapak C18" (10μ) (4.6 mm (inner diameter) x 25 cm) with a "Whatman Co. Pell ODS "(0.03 to 0.038 mm) safety column (4.6 mm (inner diameter) x 5 cm). The components were cursed with" 316 "stainless steel pipes (1.6 mm (outer diameter) - 0.23 mm (inner diameter)) Eluent was pumped at 2 ml / minute in all 25 assays.

Preparative HPLC

The following components were used to prepare a medium pressure preparative HPLC system: a "Fluid 30 Metering, Inc. Model RP-SY 2CSC FMI" laboratory pump, a "Fluid Metering, Inc. Model PD-60LF FMI" pulse steam, a 15 ml sample loop made of polypropylene tube (3.0 mm (outer diameter) x 1.5 mm (inner diameter)) wrapped around a cardboard tube (8.65 cm (outer diameter)), columns of type "Glenco Series 3500 Universal LC ", absorbance / fluorescence monitor of the" Instrument Specialties Co. Model UA-5 "type with a" Type 6 "optical device, a flow cutoff valve of the" Instrumentation Specialties Co. Model 590 "type, and a

DK 163127 B

30 collector of type "Instrumentation Specialties Co. Model 328". The components were blended with polypropylene and teflon tubes (3/0 mm (outer diameter) x 1.5 mm (inner diameter)) and "Glenco" multifit connections and valves in the order provided.

The 05 "Glenco Series 3500 Universal LC" columns are slurry packed with the indicated absorbent in the prescribed solvent using standard techniques. The void between the sunken bed and the top of the pipe was filled with standard Ottawa sand. The eluent was pumped at a maximum rate which did not exceed a back pressure of 414 kPa (60 psi) (about 20 ml / minute).

Gradient

A "Glenco" gradient elution apparatus, consisting of two chambers of equal diameters, heights and volumes, tandem connected to a 15 Teflon valve, was used in all gradient elutions. One chamber served as a mixing chamber and one as a static reservoir. The less polar solvent was held to start in the mixing chamber. The more polar solvent was kept in the static chamber. Teflon-coated magnetic stirring pins (1.0 x 3.7 cm) were placed in both chambers and 20 were driven by stirrers of the "Thomas Model 15 Magnetic Matic" type.

Eluent was pumped from the mixing chamber to the medium pressure HPLC system through polypropylene tubes (1.5 mm (inner diameter) x 3.0 mm (outer diameter)). As the eluent was removed from the mixing chamber, the solvent in the static chamber was allowed to freely replace it, thus creating a linear elution gradient.

TLC analysis of ΒΒΜ-1675ΑΊ and A? Table 9 below summarizes the observed Rf values for 30 BBM-1675A! and Ag p in normal phase plates. Rf was calculated by dividing the measured distance from the center of a zone to the sample setting point by the measured distance from the solvent front to the sample setting point.

35

DK 163127 B

31

Table 9

Rf_

System / connection_ BBM-l675Ai BBM-1675A? 05 4% Methanol In Chloroform 0.33 0.30 5% Methanol in Diethyl Ether 0.39 50% Acetone in Skellysolve B 0.38 0.31 Table 10 below summarizes the Rf values observed for 10 BBM-1675AJ and A2 on normal phase plates developed in two dimensions.

The positions of the spots are expressed in right-angled coordinates. The x-coordinate is the Rf value for the second listed solvent system. The y-coordinate is the Rf value of the first solvent system mentioned.

15

Table 10

Rf_

System / Compound BBM-1675A1 BBM-1675A2 4% methanol in chloroform versus 5% methanol in diethyl ether (0.34, 0.33) (0.28, 0.23) 4% methanol in chloroform versus 50% acetone in " Divide Light Solve B "(0.33, 0.29) 25 Table 11 below summarizes the observed Rf values for BBM-1675A1 and A2 on C-18 reverse phase TLC plates induced in binary eluent systems.

Table 11

Rf_

System / compound_ BBM-1675Ai BBM-1675A2 25% 0.5M NaCl in acetonitrile 0.18 0.21 35 25% water in acetonitrile 0.00 0.00 In Table 12 below, the observed Rf values for

DK 163127 B

32 BBM-1675AJ and A2 on C-18 reverse phase TLC plates induced in ternary eluent systems.

Table 12 05

Rf_

System / connection_ BBM-1675A1 BBM-1675A «»

Acetonitrile: Methanol: 0.5M NaCl

80%: 10%: 10% 1.00 1.00 10 60%: 10%: 30% 0.57 0.50 40%: 30%: 30% 0.32 0.22 30%: 50%: 20 % 0.44 0.33 50%: 30%: 20% 0.62 0.54 40%: 40%: 20% 0.60 0.49 15 50%: 20%: 20% 0.42 0.34 60%: 20%: 20% 0.74 0.69

Acetonitrile: Methanol: Water 40%: 30%: 30% 0.00 0.00

Acetonitrile: Methanol: 0.1M NH4 OAc 40 40%: 30%: 30% 0.32 0.22

Acetonitrile: Methanol: 0.1M NaH 2 PO 4 40%: 30%: 30% 0.00 0.00 HPLC analyzes of BBM-1675Ai and A <, 25 BBM-1675A! and BBM-1675A2 were analyzed using single, binary, and ternary eluents on C-18 reverse phase silica gel columns. Tables 13, 14 and 15 below summarize the observed K 'values for these compounds. K 'was calculated using the following formula: 30 K «= TR ~ To To where TR is the retention time spleen from injection time to peak, and To is the void volume time.

35

DK 163127B

33

Table 13 K |

System / compound_ BBM-1675Ai BBM-1675A «> 05 Acetonitrile a a

Tetrahydrofuran and a

Methanol 0.00 0.00 a: the compound was not eluted from column 10

Table 14 JV_

System / connection_ BBM-1675At BBM-1675A? 25 25% water in acetonitrile a 25% methanol in water 1.25 1.25 a: the compound was not eluted from the column 20 Table 15

Ternary eluents K / _

System / compound_ BBM-1675Ai BBM-1675Ag 25 Acetonitrile: Methanol: Water 40%: 30%: 30% a a

Acetonitrile: Methanol: 0.1M NH 4 OAc 40%: 30%: 30% 1.7 3.0 50%: 20%: 30% 3.8 6.5 30 43.3%: 23.3%: 33.3 % 6.1 b 42.5%: 22.5%: 35.0% 7.8 b 41.5%: 21.5%: 37.0% 9.7 ba: not eluted 35 b: not determined 34

DK 163127B

Biological Properties of the BBM-1675 Components The antimicrobial activity of the BBM-1675 components was determined against a variety of bacteria (gram-positive, gram-negative and acid-fast) and fungi by the two-fold agar series dilution method.

05 Nutrient agar medium was used for gram-positive and gram-negative bacteria and medium # 1001 (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na2HPO4, 0.1% KH2PO4, 0.005% ammonium citrate, 0.001% MgSO 4 and 1.5% agar) for acid-resistant organisms. Sabouraud agar medium was used for fungi. As shown in Table 16, each of the 10 six BBM-1675 components (A4, A2, A3, A4, Bx, B2) showed a broad antimicrobial activity spectrum. BBM-1675 Al7 A2, A3 and A4 were particularly active against gram-positive bacteria.

Table 16 15 Antimicrobial Activity for BBM-1675 Components MIC in pg / ml ___ BBM

Tribe_ 1675Ai A9 Aa Ad Bj_ B? 20 S.aureus 209P <0.0008 0.0063 0.0063 0.0125 0.012 0.0063 S.aureus Smith <0.0008 0.0031 0.0063 0.0125 0.012 0.012 B. subtilis PCI 219 <0.0008 0.05 0.0125 0.0125 0.05 0.05 M.luteus 1001 0.0016 0.0063 0.0125 0.0125 0.1 0.1 M.flavus <0.0008 0.0016 0.0063 0.0125 0.025 0.025 25 Mycobacterium 607 0.05 0.1 NT NT 0.05 0.025 E.coli NIHJ 0.1 0.8 1.6 3.1 0.8 3.1 K. pneumoniae D11 0.4 0 , 8 1.6 3.1 0.8 0.8 P. aeruginosa D15 0.8 1.6 1.6 3.1 3.1 3.1 C. albicans I AM 4888 0.4 0.4 1, 6 6.3 3.1 1.6 30 C. neoformans 1.6 3.1 1.6 6.3 6.3 12.5

Another antimicrobial test was performed on purified Αχ and A2 (as prepared in Example 3 below) and on components A3 and A4.

Data is summarized below.

35

DK 163127 B

35 MIC at APT (ua / mn_

Strain_ BBIVM675Ai A9 Aa Aa S.aureus 209P <0.0008 0.0063 0.0063 0.012 S.aureus Smith <0.0008 0.0031 0.0063 0.012 05 B.subtilis PCI 219 <0.0008 0.05 0.012 0.025 M.luteus 1001 0.0016 0.0063 0.012 0.05 M.flavus <0.0008 0.0016 0.0063 0.012

Mycobacterium 607 0.05 0.1 0.16 0.16 E.coli NIHJ 0.1 0.8 1.6 3.1 10 K. pneumoniae D11 0.4 0.8 1.6 3.1 P.aeruginosa D15 0.8 1.6 3.1 3.1 B. fragilis A20928 0.2 1.6 0.2 0.4 C. difficile A21675 0.4 0.8 0.05 0.4 C. perfringens A9635 0 05 0.8 0.4 0.4 15 C. albicans IAM 4888 0.4 0.4 1.6 6.3 C. neoformans 1.6 3.1 1.6 6.3

The prophage induction activity on the lysogenic bacterium E.coli W1709 (λ) was determined for the BBM-1675 components according to the method of Lein, et al., In Nature 196, 783-784 (1962). The plate count was performed on agar plates containing test material (T) and control plate (C). A T / C ratio in plaque counts of greater than 3.0 was considered significant and the lysogenic induction activity (ILB activity) was expressed as the minimum inducible concentration of the test compound. As shown in Table 17, the BBM-1675 components showed strong ILB activity on lysogenic bacteria, suggesting that they may possess antitumor activity. 1 35

DK 163127B

36

Table 17

Lysogenic induction activity for the BBM-1675 components

Antibiotic MIC * (µg / ml) 05 BBM-1675 Aj. 0.0063 BBM-1675 A2 0.0125 BBM-1675 A3 0705 BBM-1675 A4 0710 BBM-1675 Bi 0710 BBM-1675 B2 0720 * Minimum Inducible Concentration

The anti-tumor activity of BBM-1675 Ax and A2 was determined in different mouse tumor systems. Lymphocyte leukemia P-388, lymphoid leukemia L-1210, melanotic melanoma B16, and Lewis lung carcinoma were implanted intraperitoneally in male BDFi mice with inoculum size p in 1067 10s7 5 x 105 and 106 cells per cell, respectively. mouse. Graduated doses of the test compounds were administered intraperitoneally to the mouse 24 hours after tumor inoculation. The treatments were given once on the first day7 on days 1, 4 and 7 (q3d x 3) 7 once daily for 9 days (qd 1 9) or 11 days (qd 1 11). Components A3 and A4 were tested for lack of material only against P-388 leu chemistry with a q3d x 3 scheme.

BBM-1675 Al7 A2 / A3 and A4 were dissolved in 079% saline containing 10% dimethylsulfoxide7 and chromomycin A3 (Toyomycin,

Takeda 7, used as a reference compound, was dissolved in 0.9% saline. The number of dead or survivors among the treated and untreated mice was recorded daily, and the median survival time (MST) was calculated for each of the test (T) and control (C) groups. A T / C value equal to or greater than 125% suggests that a significant antitumor effect was achieved. The results are shown in Tables 18 to 23. BBM-1675 A1 and A2 showed extremely potent antitumor activity against P-388 leukemia with a maximum T / C value of 160%.

35 They are approx. 100 to 3000 times stronger than chromomycin A3 expressed in minimal effective dose. However, BBM-1675 A3 and A4 were less active than components Ax or A2 against P-388 leukemia (Table 37

DK 163127B

19). BBM-1675 Αχ and A2 were also active against L-1210 leukemia (Table 21), B16 meiosis (Table 22), and Lewis lung carcinoma (Table 23). The toxicity of BBM-ieySAj and A2 was determined on male-ddY mice by intraperitoneal or intravenous administration; BBM-1675 Αχ was approx.

05 10 times more toxic than BBM-1675 A2 (Table 24). The therapeutic index of BBM-1675 Αχ and A2 was 4 to 8 and 8 to 20 times better than chromomycin A3's in the P-388 leukemia system, respectively (Table 25). Other experiments were performed by intravenous administration of the BBM-1675 components to P-388 and L-1210 leukemia, which were inoculated intravenously at 5 x 10 5 and 104 cells, respectively.

mouse. 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 Aj and A2 were better than adriamycin expressed at maximum T / C-value, minimum 15 effective dose and range of activity.

20 25 1 35

Table 18 The effect of BBM-1675 components on P-388 Leukemia (Day 1 treatment) 38

DK 163127 B

05 Average Weight- Survivor Dose, ip change of (mg / kg / MST T / C on day -— day *) (days) (%) 5 (g) Day 5 Day 45 BBM-1675 Ax 0.03 19.0 6¾) -2.6 5/5 0/5 0.01 19.0 Q5§> -1.0 5/5 0/5 0.003 18.0 i® -1.0 5/5 0/5 0.001 20.0 <3 | S> -1/4 5/5 0/5 0.0003 16.0 Q2§) 0.0 5/5 0/5 0.0001 15.0 120 -0.2 5 / 5 0/5 0.00003 14.0 112 -0.2 5/5 0/5 BBM-1675 A2 0.3 6.0 48 -3.8 3/5 0/5 15 0.1 20.0 < 5 £ fl> -1/8 5/5 0/5 0.03 18.0 <®> -1.4 5/5 0/5 0.01 17.0 d |> -0.6 5/5 0/5 0.003 17.0 OM) -0.4 5/5 0/5 0.001 16.0 0.0 5/5 0/5 0.0003 15.0 120 0.0 5/5 0/5 0, 0001 14.0 112 0.0 5/5 0/5 20> ___

Chromomycin A3 (19.0 Cl52) -0.2 4/5 0/5 0.3 17.0 +0.6 5/5 0/5 0.1 16.0 +0/8 5/5 0/5 0.03 14.0 112 0.0 5/5 0/5 0.01 14.0 112 -0.2 5/5 0/5

Vehicle - 12.5 - + 0.1 10/10 0/10 25 * Day 1, ip

Circle indicates significant anti-tumor activity 1 35

Table 19 The effect of BBM-1675 components on P-388 Leukemia (treatment on days 1, 4 and 7)

DK 163127 B

39 05 Average Weight- Survivor Dose, ip change of (mg / kg / MST T / C on day - day *) (days) (%) 5 (g) Day 5 Day 45 BBM-1675 Ax 0.03 7.0 56 -2.8 5/5 0/5 0.01 19.0 -1.0 5/5 0/5 10 0.003 19.0 <g |) -0.6 5/5 0/5 0.001 16.0 shout -0.6 5/5 0/5 0.0003 17.0 -0.4 5/5 0/5 0.0001 16.0 & §> -0.2 5/5 0/5 0 , 00003 14.0 112 +0.4 5/5 0/5 BBM-1675 A2 0.3 tox. - - 2/5 0/5 15 0.1 11.0 88 -1.0 5/5 0/5 0.03 18.0 -1.4 5/5 0/5 0.01 18.0 ® - 0.4 5/5 0/5 0.003 18.0 OM) -0.4 5/5 0/5 0.001 17.0 @ -0.4 5/5 0/5 0.0003 16.0 02® -0 , 4 5/5 0/5 0.0001 16.0 Qjg) -0.2 5/5 0/5 20 0.00003 15.0 120 -0.4 5/5 0/5 BBM-1675 As 0, 01 17.5 @) +0/2 4/4 0/4 0.001 15.0 120 +0.6 4/4 0/4 0.0001 13.5 108 +0.6 4/4 0/4 BBM- 1675 A4 0.01 16.5 +0.2 4/4 0/4 0.001 14.0 112 +0.4 4/4 0/4 25 0.0001 12.5 100 +0.6 4/4 0 / 4

Chromomycin A3 0.3 18.0 (1444) -0.6 5/5 1/5 0.1 18.0 ®> +0.6 5/5 0/5 0.03 17.0 Q3§) -0 , 2 5/5 0/5 0.01 14.0 112 0.0 5/5 0/5

Vehicle - 12.5 - +0.4 10/10 0/10 wv * days 1, 4 and 7, ip

Circle indicates a significant antitumor activity 1

Table 20 The effect of BBM-1675 components on P-388 Leukemia (qd 1-> 9 treatment) 40

DK 163127 B

05 Average Weight- Survivor Dose, ip change pi (mg / kg / NIST T / C by day - day *) (days) (%) 5 (g) Day 5 Day 45 BBM-1675 Ai 0.01 7 , 0 56 -1.8 5/5 0/5 1Q 0.003 13.0 104 -1.0 5/5 0/5 0.001 19.0 d5b -1.2 5/5 0/5 0.0003 19.0 052) -0.8 5/5 0/5 0.0001 18.0 q® ο, ο 5/5 0/5 0.00003 16.0 fi2S) +0.2 5/5 0/5 0.00001 16.0 Q2§ -0.2 5/5 0/5 BBM-1675 A2 0.1 6.0 48 -2.2 4/5 0/5 15 0.03 13.0 104 -1.4 5 / 5 0/5 0.001 18.0 &> -1.0 5/5 0/5 0.003 18.0 ® -0.6 5/5 0/5 0.001 18.0 GM) -0.8 5/5 0 / 5 0.0003 17.0 Π36) -0.4 5/5 0/5 0.0001 16.0 ® -0.4 5/5 0/5 0.00003 15.0 120 -0.6 5 / 5 0/5 20 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 +0.4 5/5 0/5 0.03 18.0 QH> 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 25 Vehicle - 12.5 - +0.4 10/10 0 / 10 * qd 1 + 9, ip

Circle indicates a significant anti-tumor activity 35

Table 21 The effect of BBM-1675 components on L-1210 Leukemia 41

DK 163127 B

Average Weight- Survivor 05 Dose, change pi (mg / kg / MST T / C by day - day *) (days) (%) 5 (g) Day 5 Day 45 BBM-1675 Aj 0.003 14.5 - 1.7 6/6 0/6 0.001 12.0 -0.5 6/6 1/6 0.0003 12.0 QaD +0.3 6/6 0/6 Ί0 0.0001 11.0 116 +1 , 0 6/6 0/6 BBM-1675 A2 0.03 10.5 111 -1.5 6/6 0/6 0.01 13.5 ® -1.2 6/6 0/6 0.003 13.0 Q3p -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 15 Chromomycin As 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

Vehicle - 9.5 - +1.4 12/12 0/12 20 * qd 1 + 9, ip

Circle indicates a significant antitumor activity 25 1 35

Table 22 BBM-1675 components effect on B16 melanoma

DK 163127B

42

Avg. Weight- Survivor Dose, change of (mg / kg / MST T / C in day - day *) (days) (%) 5 (g) Day 5 Day 45 BBM-1675 Ax 0.003 10.5 61 -0.7 6 / 6 0/6 0.001 31.5 09¾ 0.0 6/6 0/6 0.0003 40.5 (245) +0.3 6/6 0/6 10 0.0001 27.0 +0.8 6 / 6 0/6 0.00003 22.0 OID +1.8 6/6 0/6 0.00001 18.0 109 + 2.2 6/6 0/6 BBM-1675 A2 0.03 11.5 67 -0.8 6/6 0/6 0.01 26.5 +0.3 6/6 0/6 0.003 29.5 g2 |> +0.2 6/6 0/6 -c 0.001 26.0 QS & +0.8 6/6 0/6 0.0003 22.0 +0.2 6/6 0/6 0.0001 18.0 109 +0.2 6/5 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 03 © +2.1 6/6 0/6 0.01 21.0 Qg) +2, 3 6/6 0/6 20 0.003 18.0 109 + 2.2 6/6 0/6

Vehicle - 16.5 - +2.1 12/12 0/12 * qd 1 + 9, ip 25 Circle indicates significant anti-tumor activity 1 35

Table 23 Effect of BBM-1675 components on Lewis lung carcinoma

DK 163127B

43

Average Weight- Survivor 05 Dose, change of (mg / kg / NIST T / C by day - day *) (days) (%) 5 (g) Day 5 Day 45 BBM-1675 Ax 0.003 10.0 JJ1 -1.7 5/6 0/6 0.001 31.5 (Ziff) -0.7 6/6 1/6 0.0003 21.5 5l |> -0.7 6/6 0/6 Ί0 0.0001 21.0 ®) +1.0 6/6 0/6 0.00003 13.0 118 +1.0 6/6 0/6 0.00001 11.5 105 +1.0 6/6 0/6 BBM -1675 A2 0.03 10.0 91 -1.8 6/6 0/6 0.01 25.5 (jffib -1.7 6/6 0/6 0.003 28.5 G5® 0.0 6/6 1/6 0.001 17.0 -0.3 6/6 0/6 15 0.0003 15.0 Q§g) +1.2 6/6 0/6 0.0001 10.5 95 +0.5 6 / 6 0/6 0.00003 11.0 100 +0.8 6/6 0/6

Chromomycin A3 0.1 21.5 (pKp +1.2 6/6 1/6 0.03 17.0 qf> +1.7 5/5 0/5 0.01 17.0 +1.5 6 / 6 0/6 20 0.003 11.5 105 +1.7 6/6 0/6

Vehicle - 11.0 - +0.8 12/12 1/12 * qd 1 + 11, ip 25 Circle indicates significant anti-tumor activity 1 35

DK 163127 B

44

Table 24 BBM-1675 component toxicity LD50 (mg / kg / day) ^ Single dose Multiple dose (qd 1- »9) ip 'iv ip BBM-1675 Ax 0.019 0.010 0.00046 BBM-1675 A2 0.18 0.10 0.0072 10

Chromomycin A3 0.81 0.41 0.23 15

Table 25

Therapeutic Index LDS0 / MED * 20 - P-388

Single qd 1 * 9 L1210 B16 LL

BBM-1675 Aj 63> 46 2 15 5 25 BBM-1675 A2 180 72 2 24 24

Chromomycin A3 8 8 inactive 23 23 * minimum effective dose 30 35 45

DK 163127B

Table 26 BBM-1675 Components Effect on Intravenously Implanted P-388 Leukemia 05 Average Weight- Survivor Dose, Change in (mg / kg / MST T / C on Day - Day *) (Days) (%) 5 (g ) Day 5 Day 45 BBM-1675 At 0.01 9.5 106 -1.7 6/6 0/6 .Λ 0.003 14.0 ¢ 1¾ -0.3 6/6 0/6 10 0.001 11.5 Q2jp +0.3 6/6 0/6 0.0003 9.0 100 +0.3 6/6 0/6 'BBM-1675 A2 0.1 7.0 73 -3.7 6/6 0/6 0 , 03 15.0 And) -1.0 6/6 0/6 0.01 12.0 qH) -0.5 6/6 0/6 0.003 9.0 100 +1.0 6/6 0/6 15

Adriamycin 30 tox. - - 0/6 0/6 10 9.0 100 -1.5 6/6 0/6 3 12.0 (JH) +0.7 6/6 0/6 1 9.0 100 +1.7 6 / 6 0/6

Vehicle - 9.0 - +1.7 12/12 0/12 20 * days 1, 4 and 7, iv

Circle indicates significant anti-tumor activity 25 1 35 46

DK 163127B

Table 27 Effect of BBM-1675 Components on Intravenously Implanted L-1210 Leukemia 05 Average Weight- Survivor Dose, change pi (mg / kg / MST T / C on day - day *) (days) (%) 5 (g) ) Day 5 Day 45 BBM-1675 Ax 0.008 9.5 119 -2.0 4/6 0/6 in 0.004 14.0 ¢ 7¾ -0.2 6/6 0/6 υ 0.002 13.0 <5ϋ) + 0.2 6/6 0/6 0.001 9.5 119 +0.8 6/6 0/6 0.0005 9.0 113 +0.8 6/6 0/6 BBM-1675 A2 0.063 11.0 ( JH) "1'8 6/6 0/6 0.032 14.0 U7 £) +0.2 6/6 0/6 0.016 10.5 QU) +0.8 6/6 0/6 0.008 8.0 100 +1.2 6/6 0/6 0.004 8.0 100 +0.8 6/6 0/6

Adriamycin 16 tox. - - 2/6 0/6 8 12.0 050) +0.2 6/6 0/6 4 9.0 113 +1.5 6/6 0/6 2 8.0 100 +1.7 6 / 6. 0/6 20 Vehicle - 8.0 - +1.4 12/12 0/12 * days 1, 4 and 7, iv

Circle indicates a significant antitumor activity 25 1 35

DK 163127 B

47

Antitumor activity of components BBM-1675 A1 and A2 was also determined by another 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 05 of these tests have been described in Cancer Chemother. Rep. 3, 1-87 (Part 3), 1972.

Table 28 BBM-1675 Ai and AoS effect on P-388 leukemia 10

Active Avg. Over-

Treatment- Dose, ning weight-living ip, pg / MST MST dr, g per day

Material schedule kg / day days% T / C day 5 5 (30) * NSC 38270 qd1- »9 400 13.0 163 -0.6 6/6 15 200 11.0 138 -0.9 6/6 BBM- 1675AJ day 1 51.2 20.0 250 -2.1 4/6 DMSO 25.6 18.0 225 -1.8 6/6 saline 12.8 16.5 206 -1.1 6/6 6.4 13, 0 163 + 0.1 6/6 3.2 12.0 150 -0.3 6/6 pn 1.6 11.0 138 -0.3 6/6 0.8 10.5 131 0 6/6 0.4 10.0 125 +0.4 6/6 0.2 10.0 125 +0.3 6/6 0.1 10.0 125 0 6/6 Day 1, 25.6 8.0 100 -1.8 6/6 5 and 9 12.8 13.5 169 -1.5 6/6 25 6.4 16.5 206 -0.8 6/6 3.2 16.0 200 -0.8 6/6 1.6 15, 5 194 +0.3 6/6 0.8 12.5 156 +0.3 6/6 0.4 12.0 150 -0.1 6/6 0.2 11.5 144 +0.2 6 / 6 0.1 12.0 150 +0.8 6/6 30 0.05 10.0 125 +0.8 6/6 qd119 12.8 tox. tox. tox. 1/6 6.4 6.0 75 -1.5 4/6 3.2 13.0 163 -1.2 6/6 1.6 14.5 181 -1.6 6/6 0.8 16.5 206 -2.3 6/6 0.4 16.0 200 -0.9 6/6 35 0.2 15.0 188 -0.8 5/5 0.1 13.0 163 -0.4 6/6 0.05 12 , 0 150 + 0.1 6/6 0.025 12.0 150 -0.7 6/6

DK 163127 B

48

Table 28 (continued) BBM-1675A2 day 1 256 tox. tox. tox. 0/6 DNISO + 128 12.5 156 -3.5 4/6 Saline 64 27.0 338 -1.9 6/6 32 26.0 325 -2.0 6/6 05 16 16.0 200 -1, 8 6/6 8 15.5 194 -1.9 6/6 4 15.0 188 -0.7 6/6 2 12.0 150 -0.5 6/6 1 12.0 150 0 6/6 0 , 5 10.0 125 +0.2 6/6 -jQ day 1, 128 tox. tox. tox. 0/6 5 and 9 64 tox. tox. tox. 0/6 32 tox tox -1,3 2/6 16 24,5 306 -1,3 5/5 8 17,5 219 -1,1 6/6 4 15,0 188 0 6/6 2 15,0 188 + 0.1 6/6 1 12.5 156 -0.4 6/6 15 0.5 12.0 150 -0.4 6/6 qd1 + 9 0.25 11.0 138 -0.4 6 / 6 54 tox. tox. tox. 1/6 32 6.0 75 -2.9 4/6 16 8.0 100 -1.9 6/6 8 15.5 194 -1.3 6/6 20 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

Control saline 8.0 - +0.5 10/10 25

Tumor inoculum: 106 ascite cells implanted ip 30 Host: CDFi $ mice

Toxicity: <4/6 mice alive on day 5

Assessment: NIST = median survival time

Impact:% T / C = (NIST treated / NIST control) x 100

Criterion:% T / C έ 125 is considered significant antitumor activity 1

NSC 38270 = olivomycin A

Table 29

DK 163127 B

49 BBM-1675 Ai and AoS effect p! L-1210 leukemia

Active Avg. Over-

Treatment- Dose, ning weight-bearing 05 kling ip, pg / MST MST dring, g per day

Material schedule kg / inj. days% T / C day 5 5 (30) BBM-1675A day 1 51.2 12.0 171 -1.1 5/6 25.6 7.0 100 -2.3 6/6 12.8 9.0 129 -1.1 5/6 6.4 9.5 136 -0.5 6/6 10 3.2 6.0 86 -1.7 6/6 1.6 7.0 100 -0.8 6/6 0.8 8.0 114 -0.4 6/6 0.4 7.0 100 +0.3 6/6 0.2 7.0 100 -0.5 5/6 0.1 7.0 100 +0.8 5/6 day 1, 25.6 tox. tox. tox. 1/6 15 5 and 9 12.8 9.0 129 -1.8 6/6 6.4 9.0 129 -0.8 6/6 3.2 8.0 114 -1.9 6/6 1.6 8.5 121 0 6/6 0.8 8.0 114 -0.4 6/6 0.4 7.5 107 -1.3 6/6 0.2 8.0 114 0 6/6 20 0.1 8.0 114 +0.4 5/6 0.05 7.0 100 +0.3 6/6 qd1 + 9 12.8 tox. tox. -2.4 3/6 6.4 8.0 114 -1.6 6/6 3.2 8.0 114 -1.7 6/6 1.6 9.0 129 -2.1 6/6 0.8 8.5 121 -1.6 6/6 25 0.4 8.0 114 -1.0 6/6 0.2 8.0 114 -0.5 5/6 0.1 7.0 100 +0.3 6/6 0.05 7.0 100 +0.3 6/6 0.025 6.0 86 -0.6 6/6 BBM-1675A2 day 1 256 tox. tox. tox. 0/6 30 128 7.0 100 -1.8 5/6 64 7.5 107 -1.3 4/6 32 8.0 114 -2.2 5/6 16 7.0 100 -2.3 6 / 6 8 9.5 136 -1.4 6/6 4 8.5 121 -1.1 6/6 2 8.0 114 -0.8 6/6 1 8.0 114 0 6/6 35 0, 5 8.0 114 -0.1 6/6

DK 163127 B

50

Table 30 BBM-1675 Ai and Å9's effect on | B16 melanoma

Through 05 Effects.

Dose, Opening Weight-Over-M9 (wi) or MST MST Dr, Apply

Material mg / kg / check. days% T / C day 5 day 5 (30) BBM-1675Aj. 3.2M tox. tox. -1.8 2/10 1.6 16.0 64 -1.8 10/10 0.8 34.5 168 -1.8 10/10 10 0.4 56.5 226 -0.9 10 / I0 (2) b 0.2 47.0 188 -0.7 10/10 0.1 37.0 148 -0.4 10/10 BBM-1675A2 16M 13.0 52 -2.1 10/10 8 29, 5 118 -2.0 10/10 4 43.5 174 -1.1 10/10 15 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 20 25.

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

Tumor inoculum: 0.5 ml of a 10% knit, ip Host: BDFi? mouse

Treatment: qd 1 * 9

Toxicity: <7/10 mice alive on day 10 35 Assessment: MST = median survival time

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

Criterion:% T / C ^ 125 is considered as significant antitumor activity

DK 163127 B

51

After further purification of BBM-1675AJ in accordance with Example 6, samples of the purified compound were tested for L-1210 leukemia, P-388 leukemia and B16 melanoma in mice. Results of these tests are shown below.

05

Table 31

Purified BBM-1675AiS Effect on P-388 Leukemia (Treatment on Day 1)

Dose, average weight- Over- (mg / kg / Road, MST T / C change alive

Compound dose) schedule (days) (%) in day 5 of day 5 BBM-1675 Aj 0.1024 ip, tox. tox. 0/6 0.0512 qdxl 17.5 159 -1.8 4/6 0.0256 16.5 150 -2.6 6/6 15 0.0128 17.5 159 -1.4 6/6 0.0064 15.5 141 -2.2 6/6 0.0032 15.5 141 -2.5 6/6 0.0016 16.5 150 -1.0 6/6 0.0008 15.0 136 -1.2 6/6 0.0004 15.0 136 -2.0 6/6 20 0.0256 ip, tox. tox. -1.5 1/6 0.0128 q4dx3 10.0 91 -2.5 5/6 0.0064 17.5 159 -1.9 6/6 0.0032 17.0 155 -0.8 6/6 0.0016 17.0 155 -2.0 6/6 0.0008 15.0 136 -1.7 6/6 0.0004 15.0 136 -0.4 6/6 „0.0002 13.0 118 -0.8 6/6 25 0.0001 13.0 118 -1.3 6/6 0.00005 13.5 123 -1.0 6/6 0.0128 ip, tox. tox. 0/6 0.0064 qdx5 tox. tox. -3.6 3/6 0.0032 17.5 155 -2.2 5/6 0.0016 14.5 132 -2.0 6/6 30 0.0008 15.5 141 -2.2 6/6 0.0004 16.0 145 -2.8 6/6 0.0002 17.0 155 -1.3 6/6 0.0001 14.0 127 -1.6 5/6 0.00005 15.0 136 - 1.6 6/6 0.000025 15.0 136 -1.0 6/6

Control 1 x 106 ip, 11.0 100 -0.7 9/9 (vehicle) q4dx3 Host: CDFj female mice

Implant level and site: 1 x 10 6 cells, ip

DK 163127 B

52

Table 32

Purified BBM-1675ATS effect on L-1210 leukemia (treatment on day 1)

Dose, average weight- Over- (mg / kg / Road, MST T / C change alive

Compound dose) schedule (days) (%) at day 5 at day 5 BBM-1675 0.1024 ip, tox. tox. 1/6 0.0512 qdx1 tox. tox. -2.0 0/6 0.0256 8.0 114 -2.9 4/6 0.0128 11.0 157 -2.0 6/6 0.0064 11.0 157 -1.9 6/6 0 , 0032 10.0 143 -2.0 6/6 0.0016 10.0 143 -2.6 5/6 0.0008 8.0 114 -0.4 6/6 0.0256 ip, tox. tox. -2.3 2/6 15 0.0128 q4dx3 10.5 150 -1.7 6/6 0.0064 11.0 157 -1.8 6/6 0.0032 11.0 157 -1.4 6 / 6 0.0016 10.5 150 -1.9 6/6 0.0008 9.0 129 -0.6 6/6 0.0004 8.5 121 -0.7 6/6 0.0002 8.0 114 -0.5 6/6 20 0.0128 ip, tox. tox. -2.8 2/6 0.0064 qdx5 7.0 100 -1.8 5/6 0.0032 11.5 164 -1.0 6/6 0.0016 11.0 157 -1.5 6/6 0.0008 10.0 143 -1.6 5/6 0.0004 8.5 121 -0.4 6/6 0.0002 8.5 121 0.1 6/6 25 0.0001 8.5 121 0 , 0 6/6

Control 1x10® ip, 7.0 100 0.1 10/10 (vehicle) qdx5 30 Host: CDFj female mice

Implant level and location: 1 x 106 cells, ip 35

DK 163127 B

53

Table 33

Purified BBM-1675ATS effect on B16 melanoma (treatment on day 1) 05

Dose, average weight - Over- (mg / kg / Road, MST T / C change live Compound dose) schedule (days) (%) on day 5 on day 5 * BBM-1675 Ax 0.0064 ip, 16.5 110 -3.8 8/10 0.0032 q4dx3 22.5 150 -3.0 10/10 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 ip, 27.0 180 -3.7 10/10 0.0008 qd * 9 27.0 180 -2.9 10/10 0 , 0004 26.0 173 -2.3 10/10 15 0.0002 24.5 163 -2.4 10/10 0.0001 25.5 170 -2.3 10/10

Control 0.5 ML ip, 15.0 100 -0.3 10/10 (vehicle) qdx9 ** BBM-1675 Ax 0.0064 iv, 15.0 86 -4.7 10/10 20 0.0032 q4dx3 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 ip, 18.0 103 -2.7 10/10 0.0032 q4dx3 23.0 131 -1.4 10/10 25 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 0.2 ML iv, 17.5 100 -0.4 10/10 (vehicle) q4dx3 30 1 Host: BDF! female mouse * implant level and site: 0.5ML 10% BREED, ip ** implant level and site: Fragment, s.c.

DK 163127 B

54

The above screening data suggests that the purified BBM-1675Ax component has essentially the same anti-tumor properties as the less purified previously screened sample. The compound has exceptional high potency as activity has been demonstrated at a 05 dose of 25 ng / kg when administered 5 times daily to mice against P-388 leukemia. In tests against P-388 and L-1210 leukemia, BBM-1675Αχ is effective, whether given as a single injection on day 1, 3 injections every 4 days or 5 times daily. Against B16 melanoma, the compound was just as effective when given intravenously to animals with subcutaneous tumors as it was given intraperitoneally to animals with ip-implanted tumor tissue. This property of successful pharmacological delivery of a drug to a tumor at another site is uncommon among antibiotic antitumor agents.

As shown above, the BBM-1675 components possess a high antimicrobial activity and are thus useful in the therapeutic treatment of infectious diseases caused by such microorganisms in mammals and other animals. Furthermore, the components can be utilized for other conventional applications for antimicrobial agents such as disinfection of medical and dental equipment.

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

In another aspect, the present invention provides a pharmaceutical composition containing an effective antimicrobial amount of BBM-1675 Aj, A2, A3, A4, Bx or B2 combined with a pharmaceutically acceptable carrier or diluent. In yet another aspect, the present invention provides a pharmaceutical composition which contains an effective tumor inhibiting amount of BBM-1675 Ax, A2, A3, or A4 in combination with a pharmaceutically acceptable carrier or diluent. These compositions can be prepared in any pharmaceutical form suitable for parenteral administration.

Compositions according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions.

DK 163127 B

55, suspensions or emulsions. They can also be prepared in the form of sterile solid preparations which can be dissolved in sterile water, physiological saline or some other sterile injectable media immediately before use.

It will be appreciated that the actual preferred amounts of BBM-1675 antibiotics used will vary according to the particular component, the particular formulation, the mode of application and the particular site, host and disease being treated. Many factors that modify the effect of the drug will be considered by those skilled in the art, such as age, body weight, gender, diet, time of administration and route, excretion rate, host condition, drug combinations, response sensitivities, and severity of the disease. Administration may be done continuously or periodically within the maximum tolerated dose. The optimum amount of use for a given set of conditions can be determined by those skilled in the art by using ordinary dose determination tests in light of the above guidelines.

The invention is further illustrated by the following examples. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Example 1 2

Fermentation of BBM-1675 3

Actinomadura strain No. H964-92 was grown and stored on 4 slop agar containing 1% malt extract, 0.4% glucose, 0.4% yeast 5 stretched, 0.05% CaCO 3 and 1.6% agar. A well-grown oblique agar was used to inoculate a vegetative medium containing 3% soluble 7 starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% 8

Mg504 * 7H2O, 0.2% NaCl and 0.1% CaCO3, pH adjusted to pH

9 7.0 before sterilization. The vegetative culture was incubated at 32 ° C for 10 72 hours on a rotary shaker (250 rpm) and 5 ml of growth was transferred to a 500 ml Erlenmeyer flask containing 100 ml of solid 12 curing medium composed of 3% sugar cane molasses. , 1% corn starch, 13 1% fishmeal, 0.1% CaCO3 and 0.005% CuSO4'5H2O (pH 7.0 before sterilization). The fermentation was carried out at 28 ° C for 6 days on a rotary shaker. The antibiotic activity in the fermentation substrate was determined by paper disc agar diffusion using Staphylococcus aureus 209P as the test organism. The antibiotic strength reached a maximum of approx. 1 pg / ml after 5 days of fermentation.

DK 163127 B

56

Fermentation of BBM-1675 was also carried out in cabbage fermenters of tubes. 500 ml of inoculum liquid was prepared as above and transferred to 20 L dish fermenters containing 10 L fermentation medium which consisted of the same ingredients used in the shake-05 bottle fermentation. The fermentation was carried out at 32 ° C with an aeration rate of 12 l / min and stirring at 250 rpm. Under these conditions, antibiotic production reached a maximum of approx.

0.9 pg / ml after 68 to 76 hours of fermentation.

Fermentation studies were also performed in fermentation tanks. A seed culture was shaken for 4 days at 30 ° C in an Erlenmeyer flask containing a vegetative medium consisting of 3% soluble starch, 3% dry yeast, 0.3% K2HPO4, 0.1% KH2PO4, 0.05 % MgSO 4'7H20, 0.2%

NaCl and 0.1% CaCO3. The seed culture was inoculated into a 200 L seed pod containing 130 L seed medium of the same composition as above, and the seed tank was stirred at 240 rpm at 30 ° C for 31 hours. The second seed culture was used to inoculate 3000 L of fermentation medium containing 1% corn starch, 3% sugarcane molasses, 1% fishmeal, 0.005% CuSO4 * 5H2O and 0.1% CaCO3. The production tank was operated at 28 ° C and 164 rpm at an aeration rate of 2000 l / min. The pH of the substrate gradually increased with the progress of the fermentation, reaching 7.7 to 7.8 after 170 to 180 hours, when a peak antibiotic activity of 1.7 pg / ml was produced.

Example 2 Isolation and purification of BBM-1675 components

The harvested fermentation substrate (3000 L, pH 7.8) was separated into mycelial cake and supernatant by a "Sharpless" centrifuge. The mycelial cake was suspended in 1600 L of methanol and the mixture was stirred for 1 hour. The insoluble materials were filtered off and the 30 extracts were concentrated in vacuo to 43 I. The activity contained in the substrate supernatant was then extracted by extraction with two 1000 I portions of n-butanol.The n-butanol extracts and the concentrated methanol extract were combined and evaporated azeotropically, with the occasional addition of water to a aqueous solution 35 (20 L), whereby most of the antibiotic activity precipitated as an oily solid, the solid was dissolved in 30 L of methanol and the insoluble was removed by filtration.

DK 163127B

57 was centered in vacuo to a 10 L solution to which was added 40 L of ethyl acetate and 30 L of water. After 30 minutes of stirring, the organic layer was separated, dried over sodium sulfate and evaporated in vacuo to 4 I. Addition of the concentrate to 20 L of n-hexane gave a pale yellow yellow solid BBM-1675 complex (90.14 g, potency). : 55 pg / mg). The complex was shown by TLC to be a mixture of two major components, BBM-1675 Ax and A2, and several bi components.

They were separated and purified by repeated chromatographs performed in a cooled room to prevent deterioration.

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

The eluates were collected on the basis of antibacterial activity (against 20 S. aureus) and TLC (510 O; CHCl 3 -CH 3 OH = 5: 1 v / v) and concentrated in vacuo. First, almost homogeneous BBM-1675 At (yield after evaporation: 351 mg) was eluted 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 part of BBM-1675 Aj was applied to a column of "Sephadex LH-20" (φ 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 give colorless plates of pure BBM-1675 Ax (124 mg) (this material is the starting material of Example 3A). Complex BBM-1675 A2, A3 and A4 were separated by chromatography on a column of "Bondapak C18" (Waters, φ 3.0 x 50 cm). Elution was performed with aqueous acetonitrile, and the bioactive eluates were assayed by TLC (Merck, 11: CH 3 CN-H 2 O - 75:25 v / v). The bicomponents A4 (33 mg) and A3 35 C18 reverse phase silica gel.

DK 163127 B

58 (18 mg) was successfully eluted in this order with 20% acetonitrile followed by another major component A2 (301 mg) (this material is starting material in Example 3B) with 50% acetonitrile.

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

Example 3

Further purification of BBM-1675 Ai and A9 15 A. Purification of BBM-1675A,

A 2.67 cm (inner diameter) x 75 cm "Glenco" column slurry was packed with "Baker" bonded phase octadecyl (C18) silica gel (40 µm particle drying) in methanol. The column was pre-mixed with a medium pressure HPLC system and equilibrated with 1.5 L of eluent (41.6% acetonitrile - 21.6% methanol - 36.8% 0.1M ammonium acetate). Partially purified BBM-1675AX (100.5 mg) obtained according to the purification procedure of Example 2 was dissolved in 2 ml of acetonitrile and withdrawn into the test loop. The sample was pumped onto the column. The column was eluted with the above eluent, collecting 87 ml of fractions. The eluent was monitored at 254 nm and 340 nm. Fractions 55 to 71 were pooled and extracted twice with 1500 ml portions of chloroform. The chloroform was evaporated to dryness to give 89.8 mg of residue C.

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

A 1.5 cm (inner diameter) x 20 cm "Glenco" column was slurried with 12 g of "Woelm" silica gel (63 to 200 µm particles) in

DK 163127 B

59 "Dissolving Solve B". The residue D was dissolved in ca. 2 ml of CHCl 3 and applied to the column. The chloroform was replaced with 25 ml "Skellysolve B". The column was then eluted with a 500 ml linear gradient of "Skelllysolve B" to 60% acetone in "Sklyslysolve B", collecting 20 to 05 25 ml fractions. Fractions 19 to 23 were collected and evaporated to dryness to give 65.6 mg of pure BBM-1675Aj.

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

Gel chromatography with purified BBM-1675AT

A 2.5 cm (inner diameter) x 45 cm "Pharmacia" column was slurried with "Sephadex LH-20" in methanol and adjusted to a 33.4 cm chromatographic bed. Purified BBM-1675AX (about 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 began at 1.75 ml methanol / minute with methanol, collecting 10 ml fractions ["Pharmacia Frac-100" fraction collector]. The eluent was monitored at 254 nm with an "Isco UA-5" detector. BBM-1675AX is observed to elute at Ve / Vt of 0.79 to 0.91 (Ve = elution volume,

Vt = bearing volume).

B. Purification of BBM-1675Ag 25 A 2.65 cm (inner diameter) x 75 cm "Glenco" column was slurried with "Baker" bonded phase octadecyl (C18) silica gel (40 µm particle size) in methanol. The column was pre-mixed with a medium pressure HPLC system and equilibrated with 1.5 L of eluent (50% acetonitrile - 20% methanol - 30% 0.1M 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 withdrawn into the test loop. The sample was pumped onto the column. The column was eluted with the above eluent, collecting 87 ml of fractions. The eluent was monitored at 254 nm and 340 nm. Fractions 31 to 35 38 were pooled and extracted twice with 500 ml portions of chloroform. The chloroform was evaporated to dryness to give 65.8 mg of homogeneous BBM-1675A2.

DK 163127 B

60 BBM-1675A2 was homogeneous in two TLC systems, one two-dimensional TLC analysis and HPLC.

Example 4 05 Preferred extraction process for BBM-1675Ai

Rifermentation (6.8 L) obtained in accordance with the general methods of Example 1 was transferred to a polypropylene container (12 cm in diameter at the top, 10 cm in diameter at the bottom, 37 cm high) equipped with a tap at the bottom. An equal volume of chloroform was added. The mixture was stirred at good speed with a CRC air-driven stirrer for 2 hours. Ca. 4 l (1.3 kg) of "Dicalite" (filter aid added) and mixed. The mixture was filtered on a "Dicalite" pad held in a No. 12 BCichner funnel. The filtrate was collected in a 19 L solution flask equipped with a vacuum outlet 15 ("Ace" No. 5396-06). The cake was washed with 2 L of chloroform. The filtrate was transferred to a 20 I separating funnel and allowed to separate. The lower phase (chloroform) was removed.

A 2.5 cm (inner diameter) x 40 cm "Glenco" tube slurry was packed with 91 g of "Woelm" silica gel (63 to 200 µm 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 eluted with 600 ml of 10% methanol in chloroform. This eluent was evaporated to dryness to give 547 mg of residue 25 A.

The residue A was dissolved in 50 ml of chloroform. The chloroform solution was added to 20 g of "Dicalite" in a 1 L round bottom flask. A slurry was created by adding approx. 200 ml "Skyllysolve B". The solvents were removed on a rotary evaporator. The residue was slurried in 300 ml "Skellysolve B". The slurry was packed in an "Ace" flask chromatography tube (part no. B5872-14) (41 mm (inner diameter) x 45.7 cm) by the following procedure. A glass wool plug is inserted into the stopcock's neck between the tap and the column tube. A 1 cm layer of standard Ottawa sand was added to the top of the glass wool. The stopcock, glass wool 35 and sand bed were cleaned of air by pushing a flow of "Skellysolve B" through them at 39.33 kPa (5.7 psi). The slurry was then added to the column and allowed to form a packed bed underneath

DK 163127 B

61 pressure flow. The column was never allowed to run out. After obtaining a stable column bed, a 2 cm layer of Ottawa sand was added to the top of the bed. The bed was then eluted with an additional 600 to 700 ml "Skellysolve B". The bed was eluted with 500 ml of toluene. The toluene eluent was evaporated to dryness to give 93 mg of residue B. This partially purified BBM-1675Ai can then be further purified according to the procedure of Example 3.

Example 5

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

A variant strain A1327Y obtained by NTG treatment of Actinomadura verrucosospora strain # H964-92 was used to inoculate a vegetative medium containing 2% soluble starch, 1% glucose, 0.5% yeast extract, 5% NB-amine type A and 0.1%

CaCO3, adjusting the pH to 7.0 before sterilization. The vegetative culture was incubated at 32 ° C for 4 days on a rotary shaker (250 rpm) and transferred 5 ml of the growth to a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium composed of 3% sugar cane molasses, 1% corn starch, 1 % fishmeal, 0.005%

CuSO4 * 5H2O, 0.05% MgSO4 * 7H2O, and 0.1% CaCO3, adjusting the pH to 7.0 prior to sterilization.

The fermentation was carried out at 28 ° C for 7 days on a rotary shaker. Antibiotic production reached a maximum of 1.5 pg / ml.

25

Example 6

Insulation and cleaning of BBM-1675 components

The harvested fermentation substrate of Example 5 (3000 L, pH 7.6) was separated into mycelial cake and supernatant by means of a "Sharp-30 less" centrifuge. The mycelial cake was stirred in 2000 L of methanol for 1 hour and the insoluble materials were removed by filtration. The activity contained in the substrate supernatant was then extracted with 1800 L of n-butanol. The methanol and n-butanol extracts were collected and concentrated azeotropically, with occasional addition of water, to an aqueous solution (20 L) which precipitated most of the antibiotic activity as an oily solid. The mixture was shaken 3 times with 20 L of ethyl acetate each time to extract the activity. ex-

DK 163127 B

The 62 extracts were collected, filtered to remove insoluble material and evaporated in vacuo to 4 I. Addition of the concentrate to 30 I n-hexane with stirring afforded a pale yellow solid, rit BBM-1675 complex (81.7 g, strength: 59 pg / mg). The complex was shown by 05 of TLC and HPLC to be a mixture of two major components, BBM-1675 Ax and A2 / and several bi components. They were separated and purified by a series of chromatographs performed in a cold room to prevent deterioration.

Crude BBM-1675 complex (20 g) was dissolved in methanol (20 ml) and a column of "Sephadex LH 20" (φ 5.5 x 85 cm) was piped. The column was developed with methanol and elution was overcome by bioassay using Staphylococcus aureus 209P. The active eluates were pooled, concentrated in vacuo and lyzed to give a semi-pure solid, BBM-1675 complex (4.86 g strength: 15 203 pg / mg). The solid was then chromatographed on a silica gel column (φ 3.0 x 70 cm) using chloroform and an increasing amount (1 to 5%) of methanol as developing solvents. The eluates were collected on the basis of antibacterial activity against S. aureus and TLC (SiO 2; CHCl 3 -MeOH = 5: 1 v / v) and concentrated in vacuo. BBM-1675 Ax (425 mg after evaporation, potency: 960 pg / mg) was first eluted with 2% methanol in chloroform and then a mixture of BBM-1675 A2, A3 and A4 (732 mg, potency: 340 pg / mg) followed of BBM-1675 B complex (200 mg, potency: 190 pg / mg) with 3% methanol in chloroform. The above BBM-1675 Ax was re-chromatographed on silica gel (column: φ 2.2 x 44 cm) with 2% methanol in benzene. The bioactive eluates were examined by HPLC ("Lichrosorb RP-18": CH3 CN-MeOH-0.1M CH3COONH4 = 5: 2: 3 v / v) and the fractions containing homogeneous BBM-1675 Αχ were evaporated in vacuo to dryness. The residue (solid) was crystallized from methanol (10 ml) to give colorless prisms of BBM-1675 Αχ (197 mg, potency: 1000 pg / mg).

The complex of BBM-1675 A2, A3 and A4 (537 mg) was separated by column chromatography on "Bondapak C13" (Waters, φ 2.0 x 42 cm).

Elution was performed with aqueous acetonitrile, and the bioactive eluates were assayed by TLC (Merck, silanized: CH 3 CN-H 2 O = 75:25 v / v). In components A4 (45 mg, potency: 410 pg / mg) and A3 (19 mg, potency: 300 pg / mg) were successfully eluted with 20% acetonitrile

DK 163127 B

63 followed by a major component, BBM-1675 A2 (203 mg), with 50% acetonitrile. The BBM-1675 A2 fraction was crystallized from chloroform-n-hexane to precipitate colorless rods (70 mg, potency: 290 pg / mg). The solid containing the BBM-1675 B mixture was chromatographed on a silica gel column (φ 3.0 x 40 cm) with chloroform and methanol as developing solvents. The active fractions eluted with 4% methanol in chloroform were pooled and evaporated to give pure BBM-1675 Bj (7 mg, potency: 180 pg / mg). Another active fraction eluted with 5% methanol in chloroform at 10 evaporation gave BBM-1675 B2 (8 mg, strength 140 pg / mg).

15 20 25 1 35

Claims (10)

1. Antibiotic Anti-Tumor Agent BBM-1675 Ax CHARACTERIZED BY AT 5 in its substantially purified form: a) occurs as white to slightly yellow crystals, b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride; c) gives positive reaction with ferric chloride, Ehrlich and Tollen reagents and negative reaction with the Sakaguchi, ninhydrin and anthron tests; d) exhibits an infrared absorption spectrum (KBr) substantially as shown. in Figs. 9, 15 e) when dissolved in CDC13, a proton magnetic resonance spectrum exhibits substantially as shown in Fig. 10; f) has a melting point in the range of from ca. 156 to 158 ° C, 27 g) exhibit an optical rotation of [a] D = -19Γ (c 0.5, CHCl 3), h) has an apparent molecular weight of 1248 determined by mass spectrometry, i) has an approximate elemental composition of 52, 17% carbon, 6.15% hydrogen, 4.63% nitrogen, 9.09% sulfur and 27.96% (calculated as difference) oxygen, j) by thin layer chromatography on silica gel shows an Rf value of 25 0.74 with the solvent system CHC13-CH30H (5: 1 v / v) and by reverse phase thin layer chromatography on silica gel exhibit an Rf value of 0.18 with the solvent system CH3 CN-H 2 O (75:25 v / v), when dissolved in methanol at a concentration of 0.01356 g / l, the following ultraviolet absorption maxima and absorptivities exhibit: 30 µm (nm) Absorptivities 320 12.4 280 shoulder 253 25.1 35 210 25.5 without any significant change by the addition of acid or base DK 163127 B l) exhibits a retention time by HPLC on a C18 reverse phase silica gel column of 13.3 minutes with solution (5: 2: 3 v / v), m) is effective in inhibiting the growth of various bacteria and fungi, n) induces prophage in lysogenic bacteria, and o) is effective in inhibiting of the growth of P-388 leukemia, L-1210 leukemia, B16 melanoma, and Lewis lung carcinoma in mice. 2. Antibiotic antitumor agent BBM-1675 A2 CHARACTERIZED in that in substantially purified form: a) occurs as white crystals, b) is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water and insoluble in n-hexane and 15 carbon tetrachloride, c) give positive reaction with ferric chloride, Ehrlich and Tollen reagents and negative reaction with the Sakaguchi, ninhydrin and anthron tests, d) exhibit an infrared absorption spectrum (KBr) substantially 20 as shown in Figure 12 e) when dissolved in CDC13, exhibits a proton magnetic resonance spectrum substantially as shown in Fig. 13; f) has a melting point in the range of from ca. 147 to 149 ° C, 27 g) exhibit an optical rotation of [cr]] Q = -179.4 * (c 0.5, H) has an apparent molecular weight of 1248 as determined by mass spectrometry, i) has an approximate elemental composition of 52.71% carbon, 5.94% hydrogen, 3.94% nitrogen, 9.39% sulfur and 28.01 % (calculated as 30 difference) oxygen; j) for thin layer chromatography on silica gel exhibits an Rf value of 0.71 with the solvent system CHC13-CH30H (5: 1 v / v) and on reverse phase thin layer chromatography on silica gel exhibits an Rf value of 0.21 with the solvent system CH3 CN-H 2 O (75:25 v / v), 35 k) when dissolved in methanol at a concentration of 0.02052 g / l, exhibits the following ultraviolet absorption maxima and absorptivities: DK 163127B * max ( nm) Absorptivities 320 12.2 282 16.3 252 26.2 5 214 25.8 without any significant change by the addition of acid or base, 1. exhibit a retention time by HPLC on a C18 reverse phase silica gel column of 17.3 minutes with solvent system CH3CN-CH30H-0.1M CH3C00NH4 (5: 2: 3 v / v), 10 m) is effective vt to inhibit 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. 15 3. Antibiotic Antitumor Agent BBM-1675 A3 CHARACTERIZED IN: (a) it is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water, and insoluble in n-hexane and 20 carbon tetrachloride; with ferric chloride, Ehrlich and Tollen reagents and negative reaction in the Sakaguchi, ninhydrin and anthron tests, c) exhibit an infrared absorption spectrum (KBr) substantially as shown in Figure 3, d) when dissolved in CDC13, exhibit a proton magnetic Resonance spectrum substantially as shown in Figure 7, e) has a melting point in the range of from ca. 125 to 127 ° C, 27 f) exhibit an optical rotation of [α] ρ = - 161 ° (c 0.5, (G) has an approximate elemental composition of 54.55% carbon, 6.46% hydrogen, 3.73% nitrogen, 7.49% sulfur and 27.77% (calculated as difference) oxygen, h) by thin layer chromatography on silica gel, an Rf value of 35 0.72 with the solvent system exhibits CHC13-CH3 OH (5: 1 v / v) and in reverse phase thin layer chromatography on silica gel exhibits an Rf value of 0.28 with the solvent system CH3CN-H20 (75 : 25 vol / vol), i) when dissolved in methanol, 0.01N HCl-CH3OH and 0.01N DK 163127 B NaOH-CH3OH, exhibit the following ultraviolet absorption maxima: "λ max ™ * <Εί"> 253 l286 » in methanol 282 (158) 5 320 (122) Uv λ max ™ <Elcm> 253 <287) in 0.01N HCl-CH 2 OH 282 (160) 320 (126) 10 λ max ™> <E} «> 252 < 280 'in 0.01N NaOH-CH 3 OH 283 (162) 318 (120) 15 j) exhibits a retention time by HPLC on a C18 reverse phase silica gel column of 8.0 minutes by the solvent system CH3 CN-CH30H-0.1M CH3COONH4 (5: 2 : 3 vol / vol), k) is effective for inhibition (1) induces prophage in lysogenic bacteria; (m) is effective in inhibiting the growth of P-388 leukemia in mice; and (n) can be produced by culturing the organism Actinomadura verrucosospora H964-92 (ATCC 39334). . 4. Antibiotic antitumor agent BBM-1675 A4 CHARACTERIZED IN: (a) it is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; with ferric chloride, Ehrlich and Tollen reagents and negative reaction in the Sakaguchi, ninhydrin and anthron tests; c) exhibit an infrared absorption spectrum (KBr) substantially as shown in Figures 4, 35 resonance spectrum substantially as shown in Figure 8, e) has a melting point in the range of about 123 to 126 ° C, 27 f) exhibit an optical rotation of [aJD -176 ° (c 0.5, CHCl3), DK 163127B g) has an approximate elemental composition of 54.65% carbon, 6.29% hydrogen, 3 , 51% nitrogen, 8.07% sulfur, and 27.48% (calculated as difference) oxygen, h) by thin layer chromatography on silica gel exhibits an Rf value of 0.71 with the solvent system CHCl3-CH3OH (5: 1 vol and in reverse phase thin layer chromatography on silica gel exhibit an Rf value of 0.78 with the solvent system CH3 CN-H 2 O (75:25 v / v), i) when dissolved in methanol, Ο, ΟΙΝ HCl-CH 0.01N NaOH-CH 3 OH, exhibits the following ultraviolet absorption maxima: 10 UV λ max ™ <m) 253 <257> in methanol 282 (153) 320 (117) UV λ max ™ <E & 253 <258> in 0.01N HCl-CH30H 282 (155) 320 (118) UV λ max ™ <cm @ 252 <266> 20 in 0.01N NaOH-CH30H 283 ( 160) 318 (118) j) exhibit a retention time by HPLC on a C18 reverse phase silica gel column of 5.1 minutes with the solvent system CH3 CN (5: 2: 3 v / v), k) is effective in inhibiting the growth of various bacteria and fungi, l) inducing prophage in lysogenic bacteria, m) is effective in inhibiting the growth of P 388 leukemia in mice, and 30 n) can be produced by culturing the organism Actinomadura verrucosospora H964-92 (ATCC 39334). 5. Antibiotic BBM-1675 Bx CHARACTERIZED IN: (a) it is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water, and insoluble in n-hexane and carbon tetrachloride; reaction with ferric chloride, Ehrlich and Tollens reagents and negative reaction with the Sakaguchi, ninhydrin and DK 163127 B anthron tests; c) has a melting point in the range of approx. 159 to 16Γ0, 27 d) exhibit an optical rotation of [α] ρ -17Γ (c 0.5, CHCl13), e) by thin layer chromatography on silica gel exhibit an Rf value of 0.63 with the solvent system CHCC13-CH30OH ( 5: 1 v / v) and in reverse phase thin layer chromatography on silica gel exhibit an Rf value of 0.23 with the solvent system CH3 CN-H 2 O (75:25 v / v), f) when dissolved in methanol, Ο, ΟΙΝ HCl -CH30H and 0.01N NaOH-CH30H, exhibit the following ultraviolet absorption maxima: 10 "λ max ™ (Elcn,)" 3 (225) in methanol 282 (140) 320 (104) UV λ max nm (E m) 253 (225) in 0.01N HCl-CH 3 OH 282 (140) 320 (105) UV λ max nm (E m) 252 (236) 20 in i, ΟΙΝ NaOH-CH 3 OH 283 (141) 318 (105) g) is effective in inhibiting the growth of various bacteria and fungi, 25 h) induces prophage in lysogenic bacteria, and i) can be produced by culturing the organism Actinomadura verrucosospora H964-92 (ATCC 39334). 6. Antibiotic BBM-1675 B2 CHARACTERISTICS IN: a) it is soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, heavily soluble in benzene and water and insoluble in n-hexane and carbon tetrachloride, b) gives a positive reaction with ferric chloride, Ehrlich and Tollen reagents and negative reaction in the Sakaguchi, ninhydrin and anthron test, c) has a melting point in the range of about 156 to 159eC, 27 d) exhibit an optical rotation of [<*] q = -122 * (c 0.5, CHCl3), DK 163127 B e) by thin layer chromatography on silica gel exhibit an Rf value of 0.60 with the solvent system CHC13-CH30H (5: 1 v / v) and by reverse phase thin layer chromatography on silica gel exhibits an Rf value of 0.16 with the solvent system CH3CN-H2O (75:25 v / v), when dissolved in methanol , Ο, ΟΙΝ HC1-CH30H and 0.01N NaOH-CH30H, exhibit the following ultraviolet absorption maxima: UV λ max ™ <Ekm> 248 (212> in methanol 279 (141) 10 318 (103) UV λ max »<Elcm> 248 (210> in 0.01N HCl-CH3OH 279 (140) 318 (103) 15 w λ max ™ <E1 «> 248 <233> in Ο, ΟΙΝ Na0H-CH3 OH 278 (150) 318 (110) 20 g) is (h) induces prophage in lysogenic bacteria; and (i) can be produced by culturing the organism Actinomadura verrucosospora H964-92 (ATCC 39334). 25 7. Process for Preparing the Antibiotic BBM-1675 A1} BBM-1675 A2, BBM-1675 A3, BBM-1675 A4, BBM-1675 Bx, or BBM-1675 B2 FEATURED BY Cultivating Actinomadura verrucosospora H964-92 ( ATCC 39334) or its mutant Actinomadura verrucosporas A 1327Y (ATCC 39638) or another mutant thereof having substantially the same properties in an aqueous nutrient medium containing assimilable carbon and nitrogen sources, under substantial aerobic conditions, until a considerable amount of of BBM-1675 Alf BBM-1675 Az, BBM-1675 A3, BBM-1675 A4, BBM-1675 B3 or BBM-1675 B2 have been produced by the organism in the culture medium and then extract BBM-1675 Ax, BBM-1675 A2 , BBM-1675 A3, BBM-1675 A4, BBM-1675 Bx or BBM-1675 B2 from the culture medium. DK 163127 B 8. A pharmaceutical composition characterized in that it contains an effective antimicrobial amount of BBM-1675 Alt BBM-1675 A2, BBM-1675 A3, BBM-1675 A4, BBM-1675 Bj or BBM-1675 B2 in combination with a pharmaceutically acceptable carrier or diluent. 5 9. Pharmaceutical Composition CHARACTERISTICS OF THE INVENTION It contains an effective tumor inhibiting amount of BBM-1675 Ax, BBM-1675 Az, BBM-1675 A3 or BBM-1675 A4 in combination with a pharmaceutically acceptable carrier or diluent. 10 15 20