IE59204B1 - Antibiotics - Google Patents

Abstract

There is provided new antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, said substances being produced by selective chemical hydrolysis of the bioactive components BBM-1675A1 (esperamicin A1) or BBM-1675A2 (esperamicin A2) produced from Actinomadura verrucosospora. BBM 1675C and BBM 1675D have molecular weights of approx 855 and 695 respectively.

Description

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

The antitumor compounds of the present invention have not yet been identified in terms of structure. In view of their unique physical, chemical and biological properties’, however, applicant believes that the BBM-1675C and 3314-=-16750 antibiotics are novel substances.

Patent Specification No.., A^OcM-discloses fermentation of I r Actinomadura verrucosospora strain H9S4--92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638) to produce a new antitumor antibiotic complex designated as BBM-1675. Two major bioactive components of the BBM-1675 complex described therein were designated as BBM-1675A^ and ΒΒΜ-ΙδϊΞΑ^θ The structures of the BBM-1675A^ and BBM-1675A^ antibiotics, also known as esperamicin A^ and esperamicin A^, respectively, have not yet been elucidated, but both components exhibit excellent antimicrobial and antitumor activity.

United States Patent No. 4,530,835, issued July 23, 1985 to Bunge et al., discloses fermentation of an unidentified Actinomycete isolate WP-444 (ATCC 39363) to produce antitumor antibiotics designated CL-1577A and CL-X577B. The structures of the CL-1577 antibiotics have not yet been elucidated, but the characterising properties given for the antibiotics indicate that CL-1577A and CL-1577B are similar in structure to the BBM-1675 antibiotics, and especially BBM-1675Aj and A_ mentioned above in Tatent Specification No.

There is disclosed by R. H. Bunge et al., in J. Antibiotics# 37(12) , 1566-1571 (1984) the fermentation of Actinomadura sp. (ATCC 39363) to produce a bioactive complex from which two major components, PD 114,759 and PD 115,028# were * 5 isolated. In J. Chem. Soc. Chem. Commun,# 919=920 (1985)# J. H.

Wilton et al. described the partial structural elucidation of the antibiotics PD 114,759 and PD 115,028. The production, isolation and characterization of the PD 114,759 and PD 115,028 antibiotics appear to be identical to the above-mentioned CL--15 77A and CL-1577B antibiotics# respectively.

European Patent Application No. 95,154# published November 30, 1983, discloses fermentation of Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100) to produce antitumor antibiotics designated WS 6049-A and WS 6049-B. The structures of the WS 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that WS 6049-A and WS 6049-B are related in structure to the BBM-1675 antibiotics of Patent Specification Ho. and to the CL-1577 antibiotics of United States Patent No. 4,530,835. Spectral data show# however# that neither WS 6049-A nor WS 6049-3 is identical to any of the BBM=1675 components. Moreover# the producing organism described in European Patent Application No. 95,154 may be clearly differentiated from Actinomadura verrucosospora employed in Patent Specification No. in the color of its aerial mycelium on ISP Medium Nos. 2# 3 and 4, in its positive milk peptonization and in its positive utilization of D ·fructose# D-mannitol# trehalose and cellulose.

There is provided by the present invention new antitumor ,30 antibiotic substances designated herein as BBM-1675C and BBM-1675D, also known as 3MY-27305 and BMY-27307, respectively, said substances being produced by selective chemical hydrolysis of the bioactive components BBM=1675A^ (esperamicin A^) or BBM-1675A2 (esperamicin Aj), which are themselves produced by cultivating a ί BBM-1675"producing strain of Actinomadura verrucosospora. The bioactive substances B3M-1675C and BBM-1675D may be separated and purified by conventional chromatographic procedures, and both substances exhibit excellent antimicrobial and antitumor activity. * According to the invention there is provided the antitumor 1 antibiotic BBM-1675C which in substantially pure form ; (a) appears as an amorphous solid; (b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform; (c) exhibits in· silica gel thin layer chromatography an value of 0.28 with the solvent system toluene;acetone (3:2, v/v) ; (d) has an apparent molecular weight of 85 5 as determined by high resolution FAB mass spectroscopy; (e) has an ultraviolet absorption spectrxim in methanol solution substantially as shown in FIG. 1 exhibiting ultraviolet absorption maxima and afasorptivities at 210 nm (a ~ 21,770), 274 nm (a = 9,340) and 313 nm (shoulder) (a ~ 4,190) with no significant change upon addition of acid or base; (f) has an infrared absorption spectrum (KBr, film) substantially as shown ia FIG. 3 exhibiting principal absorption peaks at 540, 740, 955, 990, 1017, 1065, 1080, 1118, 1150, 1250, 1305, 1325, 1340, 1370, 1385, 1440, 1690, 1705, 1735, 2900, 2920, .2930, 2970, and 3450 reciprocal centimeters; (g) has a low resolution mass spectrum substantially as shown m FIG. 5 exhibiting a molecular ion [Mt-H.] ' of 856; (h) has a 360 MHs proton magnetic resonance spectrum in GDCl^ substantially as shown in FIG. 7 exhibiting signals at 6.54 (IH, dd, J«7.7, 7.0); 6.21 (IH, hrs); 5.87 (IH, d, J~9.6); 5.78 (IH, dd, J^9.6, 1.5); 5.66 (IH, hrd, J=2.9); 4.94 (IH, dd, J=10.3, 1.8); 4.61 (IH, d, J=7.7); 4.25 (IH, s); 4.09 (IH, q, J=2„S); 3.97 (IH, t, J=9.6); 3.92-3.53 (10H); 3.45 (IH, dt, J=10.3, 4.0)? 3.37 (3H, s); 2.77 (IE, m); 2.69 (IH, dt, J=9.9, 5.2); 2.49 (IH, de, J=10.3, 2.6); 2.48 (3H, s); 2.30 (2H, m) ; 2.13 (IH, m); 2.09 (3H, s) ; 1.50 (2H, m) ,- 1.37 (3H, d, 1.32 (3H, d, ύ·^6.3); and'1.08 (SH) parts per million downfield from tetramethylsilane? (i) has a 90.6 MBs carbon-13 magnetic resonance spectrum in CDClg substantially as shown in FIG. 9 exhibiting signals at 13.7, 17.5, 19.8, 22. 3, 22.7, 23.5, 34.2, 35.2, 39.5, 47.7, 52.7, .55..8, 56.1, 57.7, 62.4, 64.7, 67..4, 69.3, 69.8,.71.9, 76.1, 77.1, 77.7, 79.7, 83.2, 88.4, 97.3, 99.7, 123.4, 124.6 , 130.1, and 193.1 parts per million downfield hen te frame thyIs ilane.

There is also provided the antitumor antibiotic BSM-1675D which in substantially pure forms (a) appears as an amorphous solid; (b) is soluble in methanol, ethanol, acetone and tetrahydrofuran, and slightly soluble in chloroform; (•c) exhibits in silica gel thin-layer chromatography an R^ value of 0..,22 with the solvent system chloroform:methanol (5:0.5, v/v) and exhibits in reverse phase silica gel thin layer chromatography an.R^ value of 0.37 with the solvent system methanol:water (70:30, v/v); (d) has an apparent molecular weight of 695 as determined by high resolution FAB mass spectroscopy? (-) has an ultraviolet absorption spectrum in methanol solution substantially as shown in FIG. 2 exhibiting ultraviolet absorption maxima and · ab’sorptivities at 2X4 nm (a ~ 27,000), 4 nm (a ξ 12,800), and 325 nm (a ~ 5,400) with no significant change upon addition of acid or base; (f) has an infrared absorption spectrum (KBr, £ila) substantially as shown in FIG. 4 exhibiting principal absorption peaks at 735# 755# 910# 960, 1000, 1020# 1085, 1150, 1195, 1250# 1310, 1335# 1355# 1385,.1445# 1510, 1585, 1720# 1735, 2880, 2930, 2950, and 3400 reciprocal centimeters; (g) has a low resolution mass spectrum substantially as shown in FIG. 5 exhibiting a molecular ion [M+H]* of 655; (h) has a 350 MHz, proton magnetic resonance spectrum in CD Cl 3 t 10% CB^OD substantially as shown in FIG. 8 exhibiting signals at .43 (IK# dd# «>4.4, 10.3); 6.13 (IH# s) ; 5.81 (IK# d# >8.8); 5.70 (IH, d, J=8.8),° 5.48 (IH# 6 brs); 4.48 (IH, d# >8.1); 4.02 (IH, d, >2.0); 3.95-3.80 (solvent background); 3.77 (IH# t# >9.0); 3.70-3.40. (11H# brm) ; 3.35 (IH# ra); 3.28 (3H# s) j 3.22 (3H, brs); 2.66-2.55 (2H# ra); 2.38 (3H# s) ; 2.23-2.12 (2H# ra)#· 1.42· (IH# brdt); 1.22 (3H# d# >5-9); 0.94 (3H# d# >5.6); and 0.87 (3H, d, >5.9) carts parmillion downfield from tetramethylsilane; (i) has a 90.6 MHt carbon-13 magnetic resonance spectrum in CDCl^ + 10% CDjQD substantially as shown in FIG. 10 (FIG. 10A + 10B) exhibiting signals at 17.5# 21.6# 22.2# 23.0# 33.4,-39.21 46.4# 52.3, 55.8# '62.1# 57.8# 69.8# 70.X# 71.3# 75.8# 77.1# 78.1# 82.4, 83.3# 88.2# 97.4, 99.6# 122.6# 124.8# 130.1# 130.8# 134.3# 148.7# and 2.8 parts per million downfield from tetramethylsilane.

DESCRIPTION OF THE DRAWINGS FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

FIG.

F IG. 15 FIG.

FIG. 1 shows the ultraviolet ahsorpt ion spectrum of BBM-1675C. 2 shows the ultraviolet absorpt ion spectrum of BBM-1675D. 3 shows (KBr i, the infrared absorption film). spectrum of BBM-1675C 4 shows (KBr, the infrared absorption film). spectrum of BBM-1675D 5 shows the relative abundance : BBM-1675C. mass spectrum of 6 shows the relative abundance : mass spectrum of FIG. 103 BBM-1675D. shows the proton magnetic resonance spectrum of BBM-1675C in CDC1_ (360 MHz). shows the proton magnetic resonance spectrum of BBM-1675D in CDCl3 4- 10% CD3OD (360 MHz). shows the C magnetic resonance spectrum of BBM-1675C in CDC13 (90.6 MHz). 3 10A shows the C magnetic resonance spectrum (110-200 ppm) of BBM-1675D in CDCl3 + 10% CD3OD (90.6 MHz). shows the C magnetic resonance spectrum (0110 ppm) of B3M-1675D in CDC13 + 10% CD3OD (90.6 MHz).

FIG. 11A shows the proton magnetic resonance spectrum of compound 3A (α-anomer) in CDClg (360 MHz).

FIG. 11B shows the proton magnetic resonance spectrum of compound 3B (β-anomer) in CDClg (360 MHz).

This invention relates to two novel antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY--27307, respectively, said substances being produced by selective chemical hydrolysis of the bioactive components 3BM-1675A^ (esperamicin Aq) or BBM=1675Ag (esperamicin A^), which are themselves produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora, most preferably Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura ver ru co so spo r a strain A1327Y (ATCC 39638), or a mutant thereof. In another aspect, the present invention provides a process for producing the BBM-1675C substance by selective hydrolysis of the bioactive components ΒΕΜ-ΙοϊδΆ^ or BBM~1675Ag. In a further aspect, the present invention provides a process for the preparation of BBM-1675D by selective hydrolysis of the BBM-1675C substance or, more preferably, from the bioactive components 3BM-16 75A^ or B3M-3.6VSA^. The isolation and purification of BBM-1675C and BBM-1675D from the reaction mixture may be accomplished by conventional chromatographic procedures.

The bioactive substances BBM-1675C and 3BM-1675D exhibit antimicrobial activity against a broad spectrum of microorganisms and have also been shown to exhibit inhibitory activity against various mouse tumor systems, such as B-388 leukemia and B16 melanoma. The newly described substances of the present invention, therefore, may he used as antimicrobial agents or as antitumor agents for inhibiting mammalian tumors.

During the course of degradation studies to elucidate the structure of the antitumor antibiotics BBM-1675A^. (esperamicin A^) and BBM-1675A2 (esperamicin A2), a mixture of components were produced which lead to the isolation and identi~ fication of two inactive fragments, compounds of the Formulae 1 and 2, respectively. However, it was surprisingly found that the chemical degradation lead to the stepwise liberation of two bioactive fragments BBM-1675C and BBM-1S75D. Sven more surprising, it was found that the two different antibiotics BBM-1675A^ and A? produced the same bioactive fragments as illustrated in Scheme 1. Still more surprising, the smaller molecular weight fragments BBM-1675C and D (having approximately 70S and 55% of the molecular weight of the parent antibiotics BBM-1675Aj and A2, respectively) were found to be more effective than BBM-16 75A2 and comparable to ΒΒΜ-ΙβϊδΑ^ as antitumor and antimicrobial agents.

Scheme 1 BBM-1675D (esperamicin A2) The BBM--1575C and BBM-1675D substances may be prepared by selective chemical hydrolysis of the antibiotic ΒΒΜ··1ο75Α^ as outlined in Scheme 2.

Scheme 2 $ BBM-1575A. ~--u- „ > BBM-1675C 4(m.w. 1248) (m.w. 855) Formula 1: Compound of m.w. 425 which is amixture of e and β anomers eP/ch3oh BBM-1575D (m.w. 695) Formula 3: Compound of m.w. 192 which is a mixture of c and β anomers The starting BBM-16 75A1 compound is prepared according to the procedure ' described in - Patent Specification No. The purified BBM-1675A^ component is hydrolysed with a mineral or organic acid such as hydrogen chloride# sulfuric acid# p-toluenesulfonic acid# benzenesulfonic acid or the like# in an organic or mixed aqueous-organic inert solvent at a temperature of about 0°C to the refluxing temperature of the solvent until a substantial amount of the desired BBM-1675C or BBM-1675D is produced.

Preferably# the hydrolysis is carried out in C^-Cg alcohol solvents# and most preferably# the alcoholysis is carried out in methanol. The temperature of the reaction is not critical# but it is preferred to conduct the reaction at about ambient temperature to 60°C# and most preferably from about 40° to 60°C.

The selective hydrolysis of ΒΒΜ-ΙβΙδΑ^ proceeds in a stepwise manner with the initial production of the BBM-1675C antibiotic and the inactive fragment of Formula 1. Subsequent or continued treatment under hydrolysing conditions leads to the liberation of a mixture of a and β anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM-1675D. It should be appreciated by those skilled in the art that altering the reaction conditions such as time# temperature and concentration of acid will produce varying relative amounts of the antibiotics BBM-1675C and D. Thus# it is desirable to monitor the progress of reaction by thin layer chromatography as described in the examples herein.

When it is desired to prepare only the BBM-1675D antibiotic# the selective hydrolysis is preferably carried out with an organic acid such as p-toluenesulf onic acid as described herein to yield a quantitative amount of BBM-1675D.

The BBM-1675C and BBM-X6 75D substances may also be prepared by selective chemical hydrolysis of the antibiotic ΒΒΜ-ΙδΙδΑ^ as outlined in Scheme 3.

Scheme 3 JS) BBM-16 75A2—QH > BBM-16 75C 4 (m=v„ 1248) 3 (m,w. 855) Formula 2: Compound of m.w. 425 which is a mixture of a and 8 ancmers CH-OH Ψ BBM-1675D (m.w. 695) Formula 3 s Compound of m.w. 192 whidi is a mixture of a and β ananers The starting BBM-ISVSA^ compound is prepared according to the procedure described in Patent Specification , No™ . The selective hydrolysis of purified BBM-X675A2 likewise proceeds in a stepwise manner with the initial production of the SBM-1675C antibiotic and the inactive fragment of Formula 2. Continued treatment under hydrolyzing conditions leads to the liberation of a mixture of a and β anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM- 16750,, The reaction conditions utilized for the selective chemical hydrolysis of BBM-1675&2 are substantially' the same as those utilized for the hydrolysis of 3BM=1675A^ described above» In a manner similar to the production of BBM-1675D from 33M»1675A^, when it is preferred to produce only the 3BM-1S75D antibiotic, the hydrolysis of 3BM-1675A2 is carried out until substantially all of BBM-1S75A2 and BBM-1675C is converted to BBM -16 75D,, Most preferably, the hydrolysis is carried out with an organic acid such as p-toluenesulfonic acid.

The discovery, as described herein, that the same BBM-1S75C and D antibiotics are produced from two different antibiotics BBM=1675A1 and BBM-1675A2 with the concurrent loss of two inactive fragments of Formulas 1 and 2, respectively, and the thiosugar of Formula 3, provides an additional advantage for the present invention. Accordingly, in a further aspect of the present invention, there is provided a process for the selective hydrolysis of a mixture of BBM"-16 75A^ and A9 to produce BBM-1675C and D as illustrated in Scheme 4.

Scheme 4 BBM-1S75A1 BBM-1675A£ >BBM-16 75C BBM-1675D ί < This advantage becomes apparent when one considers that the relative amounts of BBM-1675A^ and produced in the fermentation process is subject to variability. The production of BBM-1675C and D is therefore independent of the relative amounts of ΒΒΜ-1675ΑΊ and A_ utilized as starting material in the present invention.

As described herein, the hydrolysis of the BBM-16 75Ap A2 and C antibiotics results in the release of an inactive thiosugar fragment. The said thiosugar was isolated to provide further information into the chemical structure of the BBM-1675C antibiotic and hence, for the BBM-1675A^ and A2 antibiotics. The compound of Formula 3 was identified as a mixture of a and β ancmers of a thiosugar which has the structure illustrated in Schemes 2 and 3. Further characterisation was made possible when the products of the alcoholysis, the a and β anomers, were separated. The proton magnetic resonance spectra {360 MHz) of the compound 3A (α-anomer) and compound 3B (β-anomer) are shown in FIGS. 11A and 11B, respectively. From an analysis of the spectral data, the thiosugar methyl glycosides of Formula 3 were tentatively assigned the relative stereochemistry of the formula At the present time, the absolute stereochemistry, i.e. D or L, has not yet been determined. Accordingly, based on the present interpretation of the spectral data, it is concluded that the thiosugar of Formula 3 (less the CHg group from the anomeric methoxy which is incorporated during the methanolysis) is a component in the structure of the antibiotic BBM-1675C and furthermore, is a component in the structure of the starting BBM-1675Aj and A^ antibiotics.

Physico-chemical Properties of BBM-1675C Description; amorphous solid Ultraviolet absorption spectrum Instrument Solvent Concentration See FIG. 1 Hewlett-Packard 8458 methanol 0.0155 g/l -mas (nm) absorptivities 210 274 21#770 9#340 4#190 313 sh (shoulder) No significant change is observed with acid or base.

Infrared absorption spectrum : See FIG. 3 Instrument s Nicolet 5DX FT'-IR Major absorption bands (KBr# film): 540# 740# 955# 990# 1017# 1065# 1080# 1118, 1150# 1250, 1305# 1325# 1340# 1370# 1385# 1440# 1690# 1705, 1735# 2900# 2920# 2930# 2970, 3450 cm Mass spectrum Instrument Method See FIG. 5 Finigan 4500 TSQ fast atom bombardment (FAB) ionization Matrix m/z Molecular Ion Relative Abundance glycerol 856 100% glycerol NaCl 878 [M+Na]* 100% dithiothreitol;dithioerythrito 1 856 [Μ+Η)+ 100% (3:1) (w; w) Instrument : Kratos MS-50 High resolution FAB (m/z) : [M-rH]^ = 856.3362 Molecular weight; apparent MW = 855 (based on above-described mass spectral data) Elemental composition: ^36®δ1^3°14S3 (based on above-described high resolution data) Proton Magnetic Resonance Spectrum: See FIG. 7 Instrument; WM 360 Bruker Solvent : CDC1-. 3 H NMR 360 MHz δ (ppm): 6.54 (IH, dd, 3=7,7, 7.0); 6.21 (IH, brs); 5.87 (IH, d, J=9.6); 5.78 (IH, dd, J=9.6, 1.5); 5.66 (IH, brd, J=2.9); 4.94 (IH, dd, J=10.3, 1.8); 4.61 (IH, d, J=7.7); 4.25 (IH, s); 4.09 (IH, q, J=2.6); 3.97 (IH, t, J=9. 6); 3 .92-3.53 (10H), 3. 45 (IH , dt, J— 10.3 , 4.0) 9 3.37 (3H, s) ; 2.77 (IH, m); 2.69 (IH, dt. J=9.S ), 5. 2} ΐ 2.49 (IH, dd, J=10„3, 2.6); 2.48 (3H, s); 2.30 (2H, rn) ; 2.13 I IH, m) ; 2.09 (3H, s); 1.50 (2H, m) ; 1.37 (3H, a, J=5. 9); 1 .32 (3H, d, J=6.3) ; 1.08 (6H) .

See FIG. 9 .

WM 360 Bruker C Magnetic Resonance Spectrum: Instrument; Solvent : CDCl3 13c NMR 90 .6 MHz δ (ppm) : 13.7 , 17.5, 19.8, 22.3, 22.7, 23.5, 34.2, 35.2 47.7, 52.7, 55.8, 56.1, 57.7, 62.4, 64.7, 67.4 69.8, 71.9, 76.1, 77.1, 77.7, 79.7, 83.2, 88.4 99.7, 123.4, 124.6 , 130.1, 193 .1. 39.5, 69.3, 97.3, Physico-chemical Properties of BBM-16 75D Description; amorphous solid Ultraviolet absorption spectrum; See FIG. 2 Instrument Solvent Concentration λ (nm) -max-5-214 274 325 Hewlett-Packard 8458 methanol 0.01 g/l absorptivities 27,000 12,800 ,400 No significant change is observed with acid or base, Infrared absorption spectrum; See FIG. 4 Instrument : Nicolet 5DX FT-IR Major absorption bands (KBr, film); 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385, 1720, 1735, 2880, 2930, 2960, 3400 cm* 1445, 1510, 1685, 1 Mass spectrum Instrument Method Matrix Molecular ion (m/z) Relative abundance See FIG. 6 Finigan 4500 TSQ fast atom bombardment (FAB) ionizat ion thioglycerol [M-4-H]+ = 695 100% Instrument High resolution FAB (m/z, Kratos MS-50 [iYH-H]* = 596.2794 Molecular weight; apparent MW = 695 (based on above-described mass spectral data) Elemental composition: C29H49N3°i2S2 (based on above-described high resolution data) 4- 4.

Correlation of [MtH] and [(M-s-H)+2]‘ relative abundances to their calculated values confirms the elemental composition derived from high resolution-FAB measurements.

Proton magnetic resonance spectrum: See FIG. 8 Instrument : WM 360 Bruker Solvent : CDClg + 10% CDgOD "H NMR 360 MHz δ (ppm) ; 6.43 (IH, dd, J=4.4, 10 ,, 3); 5,,13 (IH, s); 5.81 (IH, d, J=8.8); 5.70 (IH, d, J=8.8); 5.48 (IH, 6 brs); 4.48 (IH, d, J=8„,l); 4.02 (IH, d, J=2.0); 3.95-3.80 (solvent background); 3.77 (IH, t, J=9.0); 3.70-3.40 (11H, brm); 3.35 (IH, m); 3.28 (3H, s) ; 3.22 (3H, brs); 2.66-2.55 (2H, m); 2.38 (3H, s) ; 2.23-2.12 (2H, m); 1.42 (IH, brdt); 1.22 (3H, d, J=5.9); 0.94 (3H, d, J=6.6); 0.87 (3H, d, J=5.9) .

C Magnetic resonance spectrums See FIG. 10A and 103 Instrument : WM 360 Bruker Solvent : CDC1, + 10% CD^OD 3 3 A C NMR 90.6 MHz δ (ppm): 17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 6 2.1, 67.8, 6 9.8, 70.1, 71.3, 75.8, 77.1, 78.1, 82.4, 83.3, 88.2 , 97.4, 99.6, 122.6, 124.8, 130. 1, 130 .8, 134.3 , 148. 7, 192. 8.

Biological Properties of BBM-16 75 Substances Antimicrobial activity of the BBM-1675 substances was determined for a variety of gram-positive and gram-negative microorganisms. Table I below provides data in the form of results of an antimicrobial screening procedure involving the parent BBM--1675A^ component and the BBM-1675C and BBM-1675D substances of the present invention. In the screening procedure, each test compound at a uniform concentration of 10 yg/ml of solution impregnated on a paper strip was placed on the growth culture, and the measure of antibiotic activity is the resulting zone of inhibition from the paper strip. As shown in Table I, the BBM-1675C and D substances showed a broad spectrum of antimicrobial activity which were at least as effective as the BBM-1675A^ component; and in particular, the BBM-1675C and D substances were more effective as inhibitors of gram-negative organisms. sa TABLE I ANTIMICROBIAL ACTIVITY OF BBM-1675 SUBSTANCES Zone of Inhibition , mm Test Microorganism BBM- -1675A1 BBM-1675C BBM-16 5 Escherichia coli AS 19 22 52 51 Escherichia coli K 12 13 . 36 35 Escherichia coli P 1373 12 34 33 Escherichia coli R Azaserine 14 35 34 Escherichia coli R Netropsin 11 32 ' 32 10 Escherichia coli R Mitomycin C 12 35 34 Escherichia coli R Bleomycin 16 38 36 Escherichia coli R Daunomycin 19 45 44 Escherichia coli R Neomycin 24 53 52 Escherichia coli R Sibiromycin 14 32 30 15 Escherichia coli R Hedaxnycin 14 30 25 Escherichia coli R Aclacinomycin 15 41 40 Bacillus subtilis ATCC 6633 34 43 41 Klebsiella pneumoniae 17 35 35 Staphylococcus 209 P •32 47 44 20 Staphylococcus R Actinoleukin 33 35 33 Staphylococcus R Streptonigrin 37 50 48 Staphylococcus faecalis P1377 30 39 3-8 Streptococcus aureus Smith P 36 47 45 Staphylococcus aureus Smith R 40 55 53 25 Actinomycin D Staphylococcus aureus Smith R 17 32 31 Aureolic acid Acinetobacter 16 33 32 Micrococcus luteus 35 57 55 30 Saccharomyces cerevisiae petite 22 42 43 R = resistant to named antibiotic 1 Activity Against P-388 Leukemia Tables II and III contain the results of laboratory tests with CDF1 mice implanted intraperitoneally with a tumor β inoculum of 10 ascites cells of P-388 leukemia and treated with 5 various doses of BBM-1675A^, C or D. The substances were administered by intraperitoneal injection. Groups of six mice were used for each dosage amount, and they were treated with a single dose of the substance on the day after inoculation. A group of ten saline treated control mice was included in each series of experiments. The 3BM-1675A^ treated group in Table III was included as a direct comparison. A 30-day protocol' was employed with the mean survival time in days being determined for each group of mice and the number of survivors at the end of the 5-day period being noted. The mice were weighed before treatment and again on day four. The change in weight was taken as a measure of drug toxicity. Mice weighing 20 grams each were employed, and a loss in weight of up to approximately 2 grams was not considered excessive. The vehicle treated control animals usually died within nine days. The results were determined in terms of a % T/C which is the ratio of the mean survival time of the treated group to the mean survival time of the vehicle treated control group times 100. An effect in terms of % T/C equal to or greater than 125 indicates that a significant antitumor effect was achieved. The screening results in Table II show the initially unexpected level of antitumor activity of the BBM-1675C substance. In Table III, the results of a direct comparison of BBM-1675A1 (esperamiein Αχ) and the B3M-1675C and 33M-1675D substances are reported. The data suggest that B3M-16 75C is about comparable to BBM-1675A^ in potency and antitumor effectiveness and that it is not schedule dependent, while BBM-1675D is only slightly less effective.

Additionally, it is reported in the present invention that the same substances BBM-1675C and BBM-1675D can also be obtained from the 3BM-1675A2 (esperamicin A^) component. In comparison of the data reported herein for BBM-1675C and 3BM--16 75D and the data reported in Patent Specification No. /for the BBM-1675A2 component, it is surprisingly found that the substances BBM-1675C and D are more effective as antitumor agents than the parent BBM-1675A2 component from which they were derived.

TABLE II EFFECT OF BBM-1675C ON P-388 LEUKEMIA (Day 1 Treatment) Compound Dose, IP mg/kg/inj . MST days Effect MST % T/C AWC gm Day 4 Survivors Day 5 BBM-1675C 3.2 TOX TOX .. 0/6 0.8 TOX TOX = 0/6 0.2 TOX TOX = 0/6 0.05 TOX TOX -1.8 1/6 15 0.0125 11.0 • 122 -2.5 5/6 0.00 3125 13.5 150 -2.5 6/6 Vehicle - 9.0 100 0.4 10/10 Tumor inocul urn: 10^ ascit es cells implanted « JP β Host: CDF^ male mice Evaluation: MST = median survival time Effect: % T/C = (MST treated/MST control) x 100 Criteria: % T/C _> 12 5 considered significant antitumor activity AWC: average weight change (treated-control) in grams (on day 4) TABLE III EFFECT OF BBM-16 75 SUBSTANCES ON P -388 LEUKEMIA Effect AWC Compound Treatment Schedule Dose, IP mg/kg/inj. MST days MST % T/C gm Day 4 Survivors Dav 5 BBM- lSTSAg^ d. 1 0.0512 TOX . TOX 0/6 0.0256 TOX TOX - 0/6 0.0128 TOX TOX -1.8 3/6 0.0064 15.5 172 -0.3 6/6 0.0032 15.0 167 -0.6 6/ 6 0.0016 15.5 172 0.6 o /o 0.0008 12.5 139 0.3 o/ o 0.0004 12.0 133 1.4 6/6 0.0002 11.0 122 0.8 6/ 6 0.0001 11.5 128 1.4 6/6 BBM-16 75 C d. 1 0.0256 ΊΟΧ TOX — 0/6 0.0128 TOX TOX -0.8 3/6 0.0064 11.5 128 -0.3 6/ 6 0.00 32 14.5 161 -0.1 6/6 0.0016 10.5 117 0.0 6/ 6 0.0008 12.0 133 0.3 676 0.0004 11.5 128 0.8 6/6 0.0002 11.0 122 1.4 6/6 0.0001 11.0 122 0.8 6/ 6 0.00005 10.5 117 1.3 6/6 TABLE III (cont.) Compound Treatment Schedule Dose, IP mg/kg/inj. MST days MST % T/C gm Day 4 Survivors Day 5 BBM-1675D d. 1 0.0256 9.0 100 0.1 6/6 0.0128 11.5 128 0.3 6/6 0.0064 12.5 139 0.3 6/6 0.0032 12.0. 133 0.1 6/6 0.0016 11.5 128 0.8 6/6 0.0008 10.0 111 0.2 6/ 6 0.0004 10.0 111 0.5 6/6 0.0002 9.5 106 1.7 6/6 0.0001 9.5 106 1.7 6/6 0.00005 9.0 100 2.0 6/6 BBM-1675C d. 1^5 0.0032 16.0 178 -1.3 6/6 0.0016 13.5 150 -1.0 6/ 6 0.0008 13.5 150 -0.3 6/6 0.0004 12.0 133 -0.4 6/6 0.0002 12.0 133 -0.4 6/6 0.0001 11.0 122 -0.4 5/6 0.00005 11.0 122 0. 9 6/6 0.000025 8.5 94 2.2 6/ 6 0.0000125 8.0 89 2.4 6/6 0.00000625 8.0 89 2.4 6/ 6 Vehicle 9.0 100 2.4 10/10 Tumor inoculum; 10 ascites cells implanted ι.ρ.

Host; CDF^ female mice Evaluation; MST = median survival time Effect; % T/C = (MST treated/MST control) x 100 Criteria; % T/C 125 considered significant antitumor activity AWC; average weight change (treated-control) in grams (on day 4) Activity Against BI6 Melanoma Table IV contains results of antitumor tests using the B16 melanoma grown in mice. BDF^ mice were employed and inoculated subcutaneously with the tumor implant. A 60-day protocol was used. Groups of ten mice were used for each dosage amount tested, and the mean survival time for each group was determined. Control animals inoculated in the same way as the test animals and treated with the injection vehicle and no drug exhibited a mean survival time of 22.5 days. For each dosage level, the test animals were treated with the test compound on days 1, 5 and 9 by intraperitoneal injection. An effect in terms of %' T/C equal to or greater than 125 indicates that a significant antitumor effect was achieved. The results in Table IV show that in a direct comparison BBM-1675C was also effective in treatment of mice bearing BI 6 melanoma and was about comparable to BBM-1675A^ in potency.

TABLE IV EFFECT OF BBM-1675 SUBSTANCES ON BI6 MELANOMA (Day 1, 5 and 9 Treatments) Compound Dose, IP mg/kg/inj. MST days Effect MST % T/C AWC gm Day 12 Survivors Day 10 BBM-1675A1 0.0032 37.5 167 0.3 10/10 0.0016 37.5 167 0.3 ' 10/10 0.0008 38.5 171 1.4 10/10 0.0004 37.0 164 1.8 10/10 0.0002 34.5 153 2.0 10/10 0.0001 32.0 142 1.9 10/10 BBM-1675C 0.0008 31.5 140 0.6 10/10 0.0004 37.0 164 1.2 10/10 0.0002 31.0 138 0.6 10/10 0.0001 31.5 140 1.0 10/10 0.00005 27.5 122 0.8 10/10 0.000025 25.0 111 0.5 10/10 Vehicle - 22.5 100 0.3 10710 Tumor inoculum: 0.5 ml of a 10% brei, IP Host; BDF female mice Evaluation . MST = median survival time Effects % T/C = (MST treat ed/MST control) κ 100 Criteria: % T/C 2 125 cons idered significant antitumo r activity AWCs aver age weight change (treat ed-control) in grams (on day 12) As indicated by the antimicrobial and mouse tumor data provided above, BBM-1S75C and BBM-1675D are thus useful as antibiotics in the therapeutic treatment of mammals and other animals for infectious diseases and also as antitumor agents for therapeutically inhibiting the growth of mammalian tumors.

The present invention, therefore, provides a method for therapeutically treating an animal host affected by a microbial infection or by a malignant tumor which· comprises administering to said host an effective antimicrobial or tumor-inhibiting dose of BBM-1675C or BBM-1675D, or a pharmaceutical composition thereof.

The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumorinhibiting amount of BBM-1675C or BBM-1675D in combination with an inert pharmaceutically acceptable carrier or diluent. Such compositions may also contain other active antimicrobial or antitumor agents and may be made up in any pharmaceutical form appropriate for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs and preparations for parenteral administration such as sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.

For use as an antimicrobial agent, the BBM-1675C or BBM-1675D, or a pharmaceutical composition thereof is administered so that the concentration of active ingredient is greater than the minimum inhibitory concentration for the particular organism being treated. For use as an antitumor agent, optimal dosages and regimens of BBM-1675C or BBM-1675D for a given mammalian host can be readily ascertained by those skilled in the art. It will, of course, be appreciated that the actual dose of S8 BBM-1675C or BBM-1675D used will vary according to the particular composition formulated, the mode of application and the particular situs, host and disease being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease. Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines'.

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

Chemical Preparation and Isolation of BBM-1675C and BBM-1675D Example 1 A sample of 3BM-1675AJ (50· mg) was dissolved in 2.5 ml of methanol and treated with 2.5 ml of 0.1 molar solution of hydrogen chloride in methanol,. The reaction was allowed to proceed at a temperature of about 50°C, and the disappearance of the starting material (approximately 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography (TLC) on silica gel plates (Analtech, 250 micron, GF) with toluene;acetone (3:2, v/v) as the eluting solvent. After the starting material has been consumed, the reaction mixture was neutralized with a saturated solution of WaHCO^ in methanol, then evaporated under reduced pressure to yield a dry residue containing the bioactive fragments. The BBM-1675C substance was isolated from the residue by flash column chromatography on a 2 cm i.d. x 10 cm column packed with Woelm silica gel (32-63 micron particle size). The column was eluted with toluene;acetone (3;2, v/v) collecting 3 ml fractions. Each fraction was analyzed by TLC [silica gel with toluene;acetone (3;2, v/v) as eluent], and the TLC spots were visualized with a UV 254 nm light source and a ceric sulfate spray (1% ceric sulfate and 2,5% molybolic acid in 10% sulfuric acid). Fractions 6-12 (R- value for BBM-1675C is 0.28) were pooled and evaporated to dryness to yield 12 mg (35%) of substantially pure BBM-1675C.

The physico-chemical properties of BBM-1675C appear in the specification and the ultraviolet, infrared, mass, 3H NMR and 13 C NMR spectra of the compound appear as Figures 1, 3, 5, 7 and 9, respectively.

Example 2 When the reaction time of the procedure in Example 1 is extended, the amount of 3BM-1675C decreases, and two new products denoted as compound 3^ (R^ = 0.65) and B3M-1675D (R_ remains at baseline) [TLC: silica, toluene:acetone (3:2, v/v)] appear and become more prominent with time.

Compound BBM-1675D which usually accompanies the production of BBM-1675C was isolated from the chromatographic column described in Example 1 by eluting the column with chloroform:methanol (5:1, v/v). The appropriate fractions were pooled and evaporated to dryness to yield 18 mg of substantially pure BBM-1675D from the reaction described in Example 1.

The BBM-1675D substance exhibits one major spot at R^ = 0.37 in reverse phase TLC (Whatman*MKC^gF, 200 micron) using 3 0% water in methanol as the eluent and R, = 0.22 in normal phase silica gel TLC using chloroform:methanol (5:0.5, v/v) as the eluent.

Example 3 Substantial improvement in the yield of BBM-1675D can be achieved by using d-toluene sulfonic acid in place of hydrogen chloride in the chemical hydrolysis of 3BM-1675A2 or BBM-1675A^ *Trade Mark as illustrated by the procedures of Examples 3 and 5, respectively.

A sample of BBM-1675A2 (15.2 mg) was hydrolyzed with 0.03 molar solution of p—toluene sulfonic acid in methanol (1 ml) at a temperature of about 63°C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30°C. The BBM-1675D substance was isolated from the dry residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size). The column was eluted with chloroform:methanol (5:0.5, v/v), and the collected fractions were analyzed by TLC [silica gel with chloroform: me thanol (5:0.5, v/v) as eluent]. The applied chromatography conditions permitted the separation of the mixture of inactive compounds 2 and _3 (7 mg) from the bioactive BBM-1675D substance which has an R^ value of 0.22. The appropriate fractions were pooled and evaporated to dryness to yield 8 mg of substantially pure BBM-1675D in near quantitative yield.

The physico-chemical properties of BBM-1675D appear in 1 the specification and the ultraviolet, infrared, mass, H NMR and 13 C NMR spectra of the compound appear as Figures 2, 4, 6, 8 and combined 10A and 10B, respectively.

Example 4 A sample of BBM-1675A2 (40 mg) was treated with 5 ml of an 0.5 molar solution of hydrogen chloride in methanol at about 50°C for about 2 hours according to the general procedure and isolation method described in Example 1. After neutralization with NaHCOg and evaporation to dryness, the BBM-1675C substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) using toluene:acetone (3:2, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 8.4 mg of substantially pure BBM-1675C which is identical to the product isolated in Example 1.

The chromatographic column of above was then eluted with chloroform;methanol (5/0.25, v/v) and the fractions collected were pooled and evaporated to dryness to yield BBM-1675D. The BBM-1675D substance was further purified by an additional flash chromatography column with silica gel utilizing chloroform/methanol (5;0.5, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 6.3 mg of substantially pure BBM-1675D which is identical to the product isolated in Example 3.

Example 5 A sample of BBM-1675A^ (49.3 mg) was hydrolyzed with 0.037 M solution of p-toluenesulfonic acid in methanol (1.5 ml) at a temperature of about 60°C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 30°C to give a residue which contains BBM-1675D and the inactive compounds _1 and J3. The BBM-16 75D bioactive substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) utilizing chloroform/methanol (5/0.25, v/v) as the eluent. The appropriate fractions were combined· and evaporated to dryness to yield 27 mg of substantially pure BBM-1675D which is identical to the product isolated in Example 3.

Example 6 A sample of BBM-1675C (5.1 mg) was hydrolyzed with 0.5 molar solution of hydrogen chloride in methanol (1 ml) at about 40-50°C overnight. After neutralization with NaHCO^ and evaporation to dryness, the BBM-1675D bioactive substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) utilizing chloroform/methanol (5/0.25, v/v) as the eluent. The appropriate fractions yielded substantially pure 3BM-1675D which is identical to the product isolated in Example 3.

Example 7 When the general procedure of Examples 1 and 2 are repeated, except that the starting material 3BM-1675A^ is replaced by an equimolar amount of a mixture containing BBM-16 75A^ and BBM-1675A2, there is thereby produced the BBM-1675C and BBM-1675D substances.

Example 8 When the general procedure of Example 5 is repeated, except that the starting material BBM-1675A^ is replaced by an equimolar amount of a mixture containing BBM-1675A^ and BBM"1675A2, there is thereby produced the BBM-1675D substance.

Claims (22)

CLAIM'S s

1. The antitumor antibiotic 3BM-1675C which in substantially pure forms (a) appears as an amorphous solid; (b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform; (c) exhibits in silica gel thin layer chromatography an R_ value of 0.28 with the solvent system toluenes acetone (3s2, v/v); (d) has an apparent molecular weight of 855 as determined by high resolution FAB mass spectroscopy; (e) has an ultraviolet absorption spectrum in methanol solution substantially as shown in FIG. 1 exhibiting ultraviolet absorption maxima and absorptivities at 210 nm (a = 21,770), 274 nm (a = 9,340) and 313 nm (shoulder) (a = 4,190) with no significant change upon addition of acid or base; (f) has an infrared absorption spectrum (KBr, film) substantially as shown in FIG. 3 exhibiting principal absorption peaks at 540, 740, 955, 990, 1017, 1065, 1080, 1118, 1150, 1250, 1305, 1325, 1340, 1370, 1385, 1440, 1690, 1705, 1735, 2900, 2920, 2930, 2970, and 3450 reciprocal centimeters; (g) has a low resolution mass spectrum substantially as shown in FIG. 5 exhibiting a molecular ion of 856; (h) has a 360 MHz proton magnetic resonance spectrum in CDCl^ substantially as shown in FIG. 7 exhibiting signals at 6.54 (IH, dd, 3=7.7, 7.0); 6.21 (IH, brs); 5.87 (IH, d, J=9»6); 5.78 (IH, dd, J=9.6, 1.5); 5.66 (IH, brd, J=2.9); 4.94 (IH, dd, J=10.3, 1.8); 4.61 (IH, d, J=7.7); 4.25 (IH, s); 4.09 (IH, q, J=2.6); 3.97 (IH, t, J=9.6); 3.92-3.53 (10H); 3.45 (IH, dt, J=10.3, 4.0); 3.37 (3H, s); 2.77 (IH, m); 2.69 (IH, dt, 3=3.3, 5.2); 2.49 (IH, dd, J=10.3, 2.6); 2.48 (3H, s); 2.30 (2H, m) ; 2.13 (IH, m) ; 2.09 (3H, s); 1.50 (2H, xn) ; 1.37 (3H, d, J=5.9); 1.32 (3H, d, J=6.3); and 1.08 (6H) parts per million downfield from tetramethylsilane; (i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDClg substantially as shown in FIG. 9 exhibiting signals at 13.7, 17.5, 19.8, 22.3, 22.7, 23.5, 34.2, 35.2, 39.5, 47.7, 52.7, 55.8, 56.1, 57.7, 62.4, 64.7, 67.4, 69.3, 69.8, 71.9, 76.1, 77.1, 77.7, 79.7, 83.2, 88.4, 97.3, 99.7, 123.4, 124.6, 130.1, and 193.1 parts per m illion downfield from tetramethylsilane.

2. The antitumor antibiotic 3BM-1675D which in substantially pure form: (a) appears as an amorphous solid;' (b) is soluble in methanol, ethanol, acetone and tetrahydrofuran, and slightly soluble in chloroform; (c) exhibits in silica gel thin-layer chromatography an R^ value of 0.22 with the solvent system chloroformsmethanol (5:0.5, v/v) and exhibits in reverse phase silica gel thia layer chromatography an R^ value of 0.37 with the solvent system methanol:water (70:30, v/v); (d) has an apparent molecular weight of 695 as determined by high resolution FAB mass spectroscopy; (e) has an ultraviolet absorption spectrum in methanol solution substantially as shown in ?IG. 2 exhibiting ultraviolet absorption maxima and absorptivities at 214 nm (a = 27,000), 274 ran (a = 12,800), and 325 nm (a = 5,400) with no significant change upon addition of acid or bases; (f) has an infrared absorption spectrum (KBr, film) substantially as shown in FIG. 4 exhibiting principal 5 absorption peaks at 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385,.1445, 1510, 1685, 1720, 1735, 2880, 2930, 2960, and 3400 reciprocal centimeters? 10 (g) has a low resolution mass spectrum substantially as shown in FIG. 6 exhibiting a molecular ion [M+H] of 696? (h) has a 360 MHz proton magnetic resonance spectrum in CDCl3 + 10% CD^OD substantially as shown in FIG. 8 exhibiting signals at 15 6.43 (IH, dd, >4.4, 10.3); 6.13 (IH, s); 5.81 (IH, d, >8.8); 5.70 (IH, d, J=8.8); 5.48 (IH, 6 brs); 4.48 (IH, d, >8.1); 4.02 (IH, d, J=2.0); 3.95-3.80 (solvent background); 3.77 (IH, t, >9.0); 3.70-3.40 (UH, brm); 3.35 (IH, m) ;

3. 28 (3H, s); 3.22 (3H, brs); 2.66-2.55 (2H, m); 2.38 (3H, 20 s); 2.23-2.12 (2H, m) ; 1.42 (IH, fordt) ; 1.22 (3H, d, >5.9); 0.94 (3H, d, >6.6); and 0.87 (3H, d, >5.9) parts permillion downfield from tetramethylsilane; (i) has a 90.6 MHz carbon!3 magnetic resonance spectrum in CDCl, + 10% CD-OD substantially as shown in FIG. 10 3 3 25 (FIG. 10A + 10B) exhibiting signals at 17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 62.1, 67.8, 69.8, 70.1, 71.3, 75.8, 77.1, 78.1, 82.4, 83.3, 88.2, 97.4, 99.6, 122.6, 124.8, 130.1, 130.8, 134.3, 148.7, and 192.8 parts per million downfield from tetramethylsilane. 303. The process for the production of the antitumor antibiotic 3BM-1675C, which comprises hydrolyzing BBM-1675A^ or BBM-1675A 2 with a mineral or organic acid until a substantial amount of BBM-1675C is produced and then recovering BBM-1675C from the reaction medium.

4. The process for the production of the antitumor antibiotic SBM-1675D, which comprises hydrolysing ΒΒΜ-1675Α Ί or BBM-1675with a mineral, or organic acid until an amount of 3BM1675D is produced ’drich is at least sufficient far recovery and then recovering SBA1675D from the reaction medium.

5. The process for the production of the antitumor antibiotic BBH-167SD, which comprises hydrolysing 8BM-1675C with a mineral or organic acid until an anoint of BBH-1675D is produced which is at least sufficient for recovery and then recovering BBM-1S75D from tiis reaction medium.

6. The process for the production of the antitumor antibiotic B3M-1675C, which comprises hydrolyzing a mixture of SBM-1675A^ axid BBM-1675A^ with a mineral or organic acid until an amount of BBM-1675C is produced which is at least sufficient for recovery and then recovering BBM-1675C iron the reaction medium.

7. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing a mixture of 3ΒΜ-1675Α ί and BBM-1675Awith a mineral or organic acid until an amount of BBM-1675D is produced which is at least sufficient for recovery and then recovering BBM-1675D from the reaction medium.

8. The antitumor antibiotic BBM-1675C according to claim 1, substantially as hereinbefore specifically described with particular reference to the examples,,,

9. The antitumor antibiotic BBM-1679D according to claim 2, substantially as hereinbefore specifically described with particular reference to the examples.

10. A process for the production of BBM-1675C according to claim 1, substantially as hereinbefore specifically described with particular reference to the examples. *=> ώ. Ila ' A process for the production of BBM-1675D according to claim 2, substantially as hereinbefore specifically described with particular reference to the examples.

11.

12. A pharmaceutical composition comprising an effective antimi5 crobial amomt of BSM-1S75C or BBM 1675D in combination with a pharmaceutical carrier or diluent.

13. A pharmaceutical composition comprising an effective tumorinhibiting amount of BBM-1675C or BBM-1675D in combination with a pharmaceutical carrier or diluent. 10

14. A pharmaceutical compound as defined in claim 1 for use in a method of treatment of the human or animal body.

15. A pharmaceutical compound as defined in claim 2 for use in a method of treatment of the human or animal body.

16. A compound according to claims 1 or 2 for use as an antitumor 15 ·, antibiotic.

17. A compound according to either of claims 1 or 2 substantially as hereinbefore specifically described in each of the examples for the use hereinbefore specifically described.

18. A pharmaceutical composition according to either of, claims 12 20 or 13 substantially as hereinbefore specifically described in examples.

19. A compound when produced by a process as claimed in any one of claims 3 to 7 P 10 or 11.

20. The use of a substance according to either of claims 1 or 2 for the manufacture of a medicament which is ant .icumor antibiotic. 25

21. Use of BBM-1675C of BBM-1675D for treating an animal host affected by a microbial infection.

22. Use of BBM-1675C or BBM-1675D for treating an animal host affected by a malignant tumor sensitive to BBM-1675C or BBM-1675D. F. R. KELLY & C0. P AGENTS FOR THE APPLICANTS mSKJIrifSESS· SQDIBR ΟΟΪΦΑΝΪ 13 Sheets Sheet 1 s BRISTOIrfyQTERS SQUIBB COMPANY . 13 Sheets Sheet 2 _59204 A85M3SW€£ F. K. KW & C®