IE862280L - Bbm-1675c and d 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. [GB2179649A]

Description

92U4 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 aud biological properties', however f applicant believes that the BBM-1675C and BBM-1675D antibiotics are novel substances.

Patent Specification No*, k?00^|»discloses fermentation of Actinomadura verrucosospora strain H964-92 (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 BBM-1675A2» The structures of the BBM~1675A^ and BBM-16 7 5A, antibioticse also known as esperamicin A^ and esperamicin. respectively, have not yet been elucidated, but both components exhibit excellent antimicrobial and antitumor activity.

United States Patent No. 4,530f>835 ? issued July 23, 1985 to Bungs et al. t, discloses fermentation of an unidentified Actinomycete isolate WP-444 (ATCC 39363) to produce antitumor antibiotics designated CL-1577A and CL-1577B. The structures of the CL-1577 antibiotics have not yet been elucidatedbut 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-1675A^ and A^ mentioned above in Patent Specification No. 3 There is disclosed by R. H. Bunge et al., in J. Antibiotics, J37(12), 1566-1571 (1984) the fermentation of Actinomadura sp. (ATCC 39 363) 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 10 CL-1577B antibiotics, respectively.

European Patent Application No. 95,154, published November 30, 1983, discloses fermentation of Actinomadura pulveraceus sp. nov. Mo. 6049 (ATCC 39100) to produce antitumor antibiotics designated WS 6049-A and WS 6049-B. The structures 15 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-16 75 antibiotics of Patent Specification Ho. and to the CL-1577 antibiotics of United States Patent No. 20 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. Moreovert the producing organism described in European Patent Application No. 95,154 may be clearly differentiated from Actinomadura verrucosospora employed in Patent 25 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-fructoset, 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 BMY-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 , which are themselves produced by cultivating a 4 BBM-16 75-producing strain of Actinomadura verrucosospora. The bioactive substances BBM-IS75C and BBM-1675D may be separated and purified by conventional chromatographic procedures, and both substances exhibit excellent antimicrobial and antitumor activity. " 5 According to the invention there is provided the antitumor 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? 10 (c) exhibits in' silica gel thin layer chromatography an Rf value of 0.28 with the solvent system toluene;acetone (3s 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 spectrum in methanol solution 15 substantially as shown in FIG. 1 exhibiting ultraviolet absorption maxima and afasorptivities at 210 ma (a - 21 e, 770), 2 74 nm (a - 9,340) and 313 nia (shoulder) (a = 4,190) with no significant change upon addition of acid or base? (f) has an infrared absorption spectrum (KBr, tils) 20 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, IS 90, 1705, 1735, 2900, 2920, .2930, 25 2970, and 34S0 reciprocal centimeters; (g) has a low resolution mass spectrum subs ally as shown in FIG. 5 exhibiting a molecular ion [M-rHj ' of 05'6; (h) has a 360 MHz proton magnetic resonance spectrum in substantially as shown in FIG. 7 exhibiting signals CDCl^ at 6.54 (1H„ ddr J-7.7, 7.0); 6.21 (1H, brs); 5.87 (IB, d, J~9.6); 5.78 (1H, dd, J^9.6, 1.5); 5.66 (1H, brd, J=2.9); 4,94 (IE e dd, J—1Q.3, 1.8); 4.61 (1HC ds J=7„7); 4.25 (1H, s); 4.09 (1H, q, J^2.6); 3.97 (1H, tf J»9.6); 3.92-3.S3 5 (10H); 3.45 (IB, dt, J-10.3, 4.0); 3.37 (3Hf s); 2.77 (IE, m); 2.69 (1H, dt, J=9» 9, 5.2); 2.49 (1H, dd, J»10.3, 2.6); 2.48 (3H, s) ; 2*30 (2H, m) ; 2.13 (1H, in);.2.09 (3H, s) ; 1.50 (2H, m) ; 1.37 (3H, d, J^S.9); 1.32 (3H, d, J=6.3); and' 1.08 (6H) parts per million down field from tetramethylsilane? 10 (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, 19.0, 22. 3, 22.7, 23.5, 34.2, 35.2, 39.5, 47.7, . 5 2.7, .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, 15 124.6, 130. 1, and 193.1 parts per million downfield frosa te tr aiaethy Is ilane.

There is also provided the antitumor antibiotic BBM-1675D which in substantially pure form? (a) appears as an amorphous solid; 20 (b) is soluble in methanol, ethanol, acetone and tetrahydrofuraa, and slightly soluble in chloroform; (■c) exhibits in silica gel thin-layer chromatography an Rg value of 0.22 with the solvent system chloroform:methanol (5:0.5, v/v) and exhibits in reverse phase silica gel thin layer 25 chromatography an.R~ value of 0.37 with the solvent system methane Is water (70s30e> v/v)? ,(d) has an apparent molecular "height of 695 as determined by ■ high resolution FAB saass spectroscopy? 30 has an ultraviolet absorption spectrum in methanol solution substantially as shown in FIG. 2 exhibiting ultraviolet absorption maxima and * ab'sorptivities at 214 rm (a = 27,000), © 27 4 nm (a ~ 12,800) , and 325 nm (a = 5, 400) with no significant change upon addition of acid or base; (£) has an infrared absorption spectrum (K3r, fiia) substantially as shown in FIG. 4 exhibiting principal 5 absorption peaks at 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 12S0, 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]"1" of 656; (h) has a 360 MHz, proton magnetic resonance spectrum in CDCl^ * 10% CD^QD substantially as shown in FIG. 8 exhibiting signals at 15 6. 43 (IK, &&t, J=4.4, 10.3),- 6.13 (1H, s) ; 5.81 (IE, d, J=8.8); 5.70 (1H, d, J=8.8)j 5.48 (1H, 6 brs); 4 = 48 (1H, d, J=8.1); 4.02 (1H, d, J-2.Q); 3.95-3.80 (solvent background); 3.77 (1H, t, J=9. 0); 3.70-3-40. (11H, btm) ; 3*35 (1H, 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, n) ? 1.42- (1H, brdt) ; 1.22 (38, 'Je5.9); 0.94 (3H, d, J=6.6) ; and 0.87 (3H, d, J=5-9) carts per-million do^mfield from tetrasnathylsilanej (i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCl^ + 10% CD^OD siibstantially as shown in FIG. 10 25 (FIG. 1QA 4- 10B) exhibiting signals at 17.5, 21.6,- 22.2, 23.0, 33.4,- 39.2", 46.4, 52.3, 35.8, '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 19 2.8 parts par million downfield from tetraraethylsilane. ■ 7 DESCRIPTION OF THE DRAWINGS FIG. 1 shows the ultraviolet absorption spectrum of BBM-16 75C.

FIG. 2 shows the ultraviolet absorption spectrum of BBM=1675D,a FIG. 3 shows the infrared absorption spectrum of BBM-16 75C 5 (KBri, film).

FIG. 4 shows the infrared absorption spectrum of BBM-1675D (KBr? film).

FIG. 5 shows the relative abundance mass spectrum of BBM-1675C. 10 FIG. 6 shows the relative abundance mass spectrum of BBM-1675D.

FIG. 7 shows the proton magnetic resonance spectrum of BBM-1S75C in CDC1_ (360 MHz). 3 FIG. 8 shows the proton magnetic resonance spectrum of 15 BBM-1S75D in CDCl- + 10% CD.OD (360 MHz). 3 3 13 FIG. 9 shows the C magnetic resonance spectrum of BBM-1675C in CDC13 (90.6 MHz). 1 3 FIG. 10A shows the C magnetic resonance spectrum (110-200 ppm) of BBM—1675D in CDC13 + 10% CD30D (90.6 MHz}. 20 FIG. 103 shows the C magnetic resonance spectrum (0-110 ppm) of BBM-1675D in CDC13 + 10% CD^D (90.6 MHz).

FIG. 11A shows the proton magnetic resonance spectrum of compound 3A (os-anomer) in CDCl^ (360 MHz)„ FIG. 11B shows the proton magnetic resonance spectrum of compound 3B (B-anomer) in CDCl^ (360 MHz). 5 This invention relates to t^JO novel antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, also known as BMY-2730 5 and BMY-27307, respectively, said substances being produced by selective chemical hydrolysis of the bioactive components BBM-1675 A^ (esperamicin A1) or BBM-1675A^ (esperamicin 10 wkich are themselves produced by cultivating a BBM-1675-producing strain of Actinomadura verrucososporaf most preferably Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof. In another aspect, the present invention 15 provides a process for producing the BBM-1675C substance by selective hydrolysis of the bioactive components BBM~1675Ai or BBM-1675A2. 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 20 the bioactive components BBM~1675A^ or BBM-1675A2* 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-=16 75C and BBM-1675D 25 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 P-388 leukemia and B16 melanoma. The newly described substances of the 9 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-16 75A^. 5 (esperamicin A^) and BBM-1675A^ (esperamicin A2)? a mixture of components were produced which lead to the isolation and identification 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 10 bioactive fragments BBM-1675C and BBM-1S75D. Even more surprising, it was found that the two different antibiotics BBM-1675Aj 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 15 70% and 55% of the molecular weight of the parent antibiotics BBM~1675Aj and A2, respectively) were found to be more effective than BBM-1675A2 and comparable to BBM-1675A^ as antitumor and antimicrobial agents. 20 Scheme 1 BBM-16 75 A (esperamicin A^) BBM-1675A (esperamicin A2) BBM-1675C BBM-1675D The BBM-1675C and BBM-1675D substances may be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A^ as outlined in Scheme 2. 4 1 The starting BBM-16 75A1 compound is prepared according to the procedure ' described in - Patent Specification No* F-i The purified BBM-1675A^ component is hydrolysed with a mineral or organic acid 5 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* 10 Preferablye the hydrolysis is carried out in alcohol solvents, and most preferably„ the alcoholysis is carried out in methanol. The temperature of the reaction is not criticalP but it is preferred to conduct the reaction at about ambient temperature to 60°C, and most preferably from about 40° to 60°C„ 15 The selective hydrolysis of BBM-1675Ai 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 0 anomers of the thiosugar of 20 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 25 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-toluenesulfonic acid as described 30 herein to yield a quantitative amount of BBM-1675D.

The BBM-1675C and BBM-1675D substances may also be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A^ as outlined in Scheme 3. 1 2 Scheme 3 BBM-16 75A. d£) la BBM-16 75C v2 CH3OH ' (m„w. 1248) (m„w„ 855) O'-" ^ 2 och3 Fontaila 2 s Compound of m.w, 425 which is a mixture of a and 6 ancroers I^/CH-OH 3 CH- V 1S75D (m.w. 695) "\3 0 CH3S "/ OH Formula 3 s Compound of m„w„ 192 whicli is a mixture of a and 6 ananers 1 3 The starting BBM-16 75A2 compound is prepared according to the procedure described in Patent Specification < No„ /£ooj3'+~ - The selective hydrolysis of purified BBM-1675A2 likewise proceeds in a stepwise 5 manner with the initial production of the BBM-1675C antibiotic and the inactive fragment of Formula 2. Continued treatment under hydrolyzing conditions leads to the liberation of a mixture of a and $ anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM-1675D. 10 The reaction conditions utilized for the selective chemical hydrolysis of BBM~1675A2 are substantially' the same as those utilized for the hydrolysis of BBM-1675A^ described above* In a manner similar to the production of BBM-1675D from B3M~1675A1» when it is preferred to produce only the BBM-1675D 15 antibiotic, the hydrolysis of BBM-1675A^ is carried out until substantially all of BBM-1675A^ and BBM-1675C is converted to BBM-1675D. Most preferably, the hydrolysis is carried out with an organic acid such as p-toluenesulfonic acid, The discovery, as described herein,, that the same 20 BBM-1675C and D antibiotics are produced from two different antibiotics BBM-1675A^ 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 fo^r the present invention. Accordingly, in a further aspect of the 25 present invention, there is provided a process for the selective hydrolysis of a mixture of BBM-16 75 A ^ and to produce BBM-1675C and D as illustrated in Scheme 4.

Scheme 4 BBM-16 75A1 + BBM-1675A£ ^"BBM-16 75C BBM-16 75D 14 This advantage becomes apparent when one considers that the relative amounts of BBM-1675 A^ and A2 produced in the fermentation process is subject to variability. The production of BBM-1675C and D is therefore independent of the relative amounts 5 of BBM-1675A., and A2 utilized as starting material in the present invention- As described herein, the hydrolysis of the BBM-1675A^, Aj 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 A^ antibiotics. The compound of Formula 3 was identified as a mixture of a and j3 anomers of a thiosugar which has the structure illustrated in Schemes 2 and 3. Further characterization was made possible when the products of the ale oho lysis £, the a and 6 anomers , were separated. The proton magnetic resonance spectra {360 MHz) of the compound 3A (a-anomer) and compound 3B (f5-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 OH At the present time, the absolute stereochemistry, i.e. D or Lj, has not yet been determined. Accordingly, based on the present interpretation of the spectral data, it is concluded that 25 the thiosugar of Formula 3 (less the CH^ 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-1675A^ and A^ antibiotics. 10 15 20 5 10 15 20 . 25 I S Physico-chemical Properties of BBM-1675C Descriptions amorphous solid Ultraviolet absorption spectrums Instrument : Solvent : Concentration : See FIG. 1 Hewlett-Packard 84 58 methanol 0»0155 g/1 X_ (nm) -max—5—™ absorptivities 210 274 313 sh (shoulder) 21,77 0 9,340 4,190 Mo significant change is observed with acid or base* Infrared absorption spectrum : See FIG„ 3 Instrument : Micolet 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"1.

Mass spectrum Instrument Method See FIG. 5 Finigan 4500 TSQ fast atom bombardment (FAB) ionization Matrix m/z Molecular Ion Relative Abundance glycerol 856 glycerol + NaCl 87 8 dithiothreitol:dithioerythrito 1 85 6 (3 s1) (w;w) [M-i-H] ' [M-i-Ma] [M+H]4" 100% 100% 100% 1 s Instrument s Kratos MS-50 High resolution FAB (m/z) : [M-rH]+ = 856.3362 Molecular weight; apparent MW = 855 (based on above-described mass spectral data) 5 Elemental composition; C^gHg (based on above-described high resolution data) Proton Magnetic Resonance Spectrum? See FIG. 7 Instruments WM 360 Bruker Solvent ; CDC!0 1 10 aH NMR 360 MHz 6 (ppm)s 6.54 (1Hf dd, J=7.7, 7.0); 6.21 (1H, brs); 5.87 (1H, df J=9.6) ; 5.78 (1H, dd, J=9.6, 1.5); 5.66 (1H, brd, J=2.9) ; 4.94 {1H, dd, J=10.3,? 1.8); 4.61 (1H, d, J=7.7); 4.25 (1H, s); 4.09 (1H, q, J=2.6); 3.97 (1H, t, 15 J=9.6); 3.92=3.53 (10H), 3.45 (lfl, dt, J=10„3, 4.0); 3.37 (3H, s); 2.77 (1H, m); 2.69 (1H, dt, J=9.9, 5.2); 2.49 (1H, dd, J=10- 3, 2.6); 2.48 (3H, s) ; 2.30 (2H, m) ; 2.13 (1H, m); 2.09 (3H, s); 1.50 (2H, m); 1.37 (3H, d, J=5.9); 1.32 (3H, d, J=6.3); 1.08 (6H). 13 20 C Magnetic Resonance Spectrum; See FIG. 9 Instrument: WM 360 Bruker Solvent : CDCl_ 13 NMR 90.6 MHz 6 (ppm); 13.7, 17.5, 19.8, 22.3, 22.7, 23.5, 34.2, 35.2, 39.5, 25 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, 193.1. 17 Physico-chemical Properties of BBM-1675D Descriptions amorphous solid Ultraviolet absorption spectrum; Instrument : Solvent : Concentration ; See FIG„ 2 Hewlett-Packard 84 58 methanol 0.01 g/1 A (nm) -max-5 - absorptivities 10 214 274 325 27,000 12,800 5,400 No significant change is observed with acid or base, 15 Infrared absorption spectrum; See FIG. 4 Instrument : Nicolet 5DX FT-IR Major absorption bands (KBr, film)s 7 35, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385, 1445, 1510, 1685, 1720, 1735, 2880, 2930, 2960, 3400 cm"1.

Mass spectrum 20 Instrument Method Matrix Molecular ion (m/z) Relative abundance See FIG. 6 Finigan 4500 TSQ fast atom bombardment (FAB) ionizat ion thioglycerol [M+H]+ = 695 100% 25 Instrument High resolution FAB (m/z) Kratos MS-50 [M+H] = 696.2 794 Molecular weight; apparent MW = 695 (based on above-described mass spectral data) & 8 I Elemental compositions C29H49N3°i2S2 (based on above-described high resolution data) -j. j Correlation of [M4-H] 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 : CDCl^ + 10% CD^OD ~H NMR 360 MHz 6 (ppm) i 6.43 (1Ht dd, J=4.4, 10.3); 6.13 (1H, s)5.81 (1H, d, J=8.8); 5.70 (1H, d, J=8.8); 5.48 (1H, 6 brs); 4.48 (1H, d, J=8,., 1) ; 4.02 (1H, d, J=2„ 0) ; 3.95-3.80 (solven background); 3.77 (1H, t, J=9.0); 3.70-3.40 (11H, brm) 3.35 (1H, 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 (1H, brdt) 1.22 (3H, d, J=5.9); 0.94 (3H, d, J=6.6),« 0.87 (3H, d, J= 5. 9) . 1 3 C Magnetic resonance spectrums See FIG. 10A and 103 Instrument : WM 3S0 Bruker Solvent : CDC1, + 10% CD-,00 13 3 3 x C NMR 90.6 MHz 6 (ppm)s 17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52. 3, 55.8, 62. If. 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, 192.8.

I 9 Biological Properties of BBM-1675 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 5 results of an antimicrobial screening procedure involving the parent BBM-1675A^ component and the BBM-1675C and BBM-16 75D substances of the present invention. In the screening procedure,, each test compound at a uniform concentration of 10 Mg/ml of solution impregnated on a paper strip was placed on the growth 10 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 15 substances were more effective as inhibitors of gram-negative organisms. 10 15 ga TABLE I ANTIMICROBIAL ACTIVITY OF BBM-1675 SUBSTANCES Zone of Inhibition, mm BBM-1675Ax BBM-16 75C BBM-16 75D Test Microorganism Escheri chia coli AS 19 Escherichia coli K 12 Escheri chia coli P 1373 Bscherichia coli R Azaserine Escherichia coli R Netropsin Escherichia coli R Mitomycin C Escherichia coli R Bleomycin Escherichia coli R Daunomycin Escherichia coli R Neomycin Escherichia coli R Sibiromycin Escherichia coli R Hedamycin Escherichia coli R Aclacinomycin Bacillus subtilis ATCC 6633 Klebsiella pneumoniae 22 52 51 13 - 36 35 12 34 33 14 35 34 11 32 ' 32 12 35 34 16 38 36 19 45 44 24 53 52 14 32 30 14 30 25 15 41 40 34 43 41 17 35 35 Staphylococcus 209 P -32 47 44 2o 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 2 2 42 43 R = resistant to named antibiotic Activity Against P-388 Leukemia Tables II and III contain the results of laboratory tests with CDF., mice implanted intra per itoneally with a tumor 6 inoculum of 10 ascites cells of P-388 leukemia and treated with various doses of BBM-16 75A^ 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-1S75treated 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-1675A^ (esperamicin A^) and the BBM-1675C and BBM-1675D substances are reported,., The data suggest that BBM-1675C 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 BBM-1675A2 (esperamicin A2> components In comparison of the data reported herein for BBM-1675C and BBM-16 75D and the data reported in Patent 3 2 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.

CABLE II 20 EFFECT OF BBM-1675C ON P-388 LEUKEMIA (Day 1 Treatment) • Effect AWC Dose, IP MST MST gm Survivors 10 Compound mq/kg/ini. days % T/C Day 4 Day 5 BBM-1675C 3.2 TOX TOX 0/6 0.8 TOX TOX 0/6 (N i8 o 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 inoculum; 10 ascites cells implanted i„p.

Hosts CDF^ male mice Evaluations 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) n$ TABLE III EFFECT OF BBM-1675 SUBSTANCES ON P-388 LEUKEMIA Effect AWC Treatment Dose, IP MST MST gm Survi^ 5 Ccmpound Schedule mg/kg/inj. days % T/C Day 4 Day 5 BBM-16 75 A1 d. 1 0.0512 TOX .

TOX 0/6 0.0256 TOX TOX - 0/6 0.0128 TOX TOX -1.8 3/6 0.00 64 15.5 172 -0.3 6/6 10 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 15 0.0001 11.5 128 1.4 6/6 BBM-16 75 C d. 1 0.0256 TOX TOX — 0/6 0.0128 TOX TOX -0.8 3/6 0.0064 11.5 128 -0 o 3 6/ 6 0.00 32 14.5 161 -0.1 6/6 20 0.0016 10.5 117 0.0 6/ 6 0. 0008 12.0 133 0.3 6*/6 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 25 0.00005 10.5 117 1.3 6/6 2 4 TABLE III (cont.) Treatment 10 15 20 Compound BBM-1675D BBM-1675C d. 1- Vehicle Dose, IP mg/kg/ini.

MST days MST % T/C gm Day 4 Survi\ Day 5 0.0256 9.0 100 0.1 6/6 0.0128 11.5 128 0.3 6/6 0.00 64 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 0.00 32 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 9.0 100 2.4 10/11 25 30 Tumor inoculums 10° ascites cells implanted i.p.

Hosts CDF^ female mice Evaluations MST = median survival time Effects % T/C = (MST treated/MST control) x 100 Criteria? % T/C _> 125 considered significant antitumor activity AWCs average weight change (treated-control) in grams (on day 4) Activity Against B16 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 5 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 10 animals were treated with the test compound on days lt, 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 15 bearing B16 melanoma and was about comparable to BBM-1675A^ in potency.

TABLE IV EFFECT OF BBM-1675 SUBSTANCES ON B16 MELANOMA (Day 1, 5 and 9 Treatments) Ccsn pound Dose, IP mq/kg/inj MST davs Effect MST % T/C AWC gm Day 12 Survivors Day 10 BBM-1675A. 10 0.0032 0»0016 0.0008 0.0004 0.0002 0.0001 37.5 37.5 38.5 37.0 34.5 32-0 167 167 171 164 153 142 0.3 0.3 1.4 1. 8 2.0 1.9 10/10 10/10 10/10 10/10 10/10 10/10 BBM-16 75C 15 0.0008 0.0004 0.0002 0.0001 0.00005 0.000025 31.5 37.0 31.0 31.5 27.5 25. 0 140 164 138 140 122 111 0.6 1.2 0.6 1.0 0.8 0.5 10/10 10/10 10/10 10/10 10/10 10/10 Vehicle 22.5 100 0.3 10710 20 Tumor inoculums 0.5 ml of a 10% brei, IP Hosts female mice Evaluations MST = median survival time Effects % T/C = (MST treated/MST control) x 100 Criteria: % T/C _> 12 5 considered significant antitumor activity 25 AWC: average weight change {treated-control) in grams (on day 12) %1 As indicated by the antimicrobial and mouse tumor data provided above, BBM-1675C 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-1S75D,, or a pharmaceutical composition thereof* The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of BBM-1675C or BBM-1675D in combination with an inert pharmaceuticaily 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 waterf 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 5 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 10 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 15 invention.

Chemical Preparation and Isolation of BBM-1675C and BBM-1675D Example 1 A sample of BBM-16 751^ (50- mg) was dissolved in 2.5 ml of methanol and treated with 2,, 5 ml of 0.1 molar solution of 20 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 (AnaltechP 250 microns GF) with toluene;acetone 25 {3%2i 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 30 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 (3s2, v/v) as eluent], and the TLC spots were % 3 visualized with a UV 254 ran light source and a eerie sulfate spray (1% eerie 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 substan-5 tially pure BBM-1S75C.

The physico-chemical properties of BBM-16 75C appear in the specification and the ultraviolet, infrared, mass, H NMR and 1 3 C NMR spectra of the compound appear as Figures 1, 3* 5, 7 and 9, respectively. 1 0 Example 2 When the reaction time of the procedure in Example 1 is extended, the amount of BBM-1675C decreases, and two new products denoted as compound J3 (R^ = 0»65) and BBM-1675D (R- remains at baseline) [TLC? silica,, toluene?acetone (3?2, v/v)] appear and 15 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 (5sle v/v). The appropriate fractions were 20 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 = 0.37 in reverse phase TLC (Whatman*MKC^gF, 200 micron) using 3 0% water in methanol as the eluent and Rt = 0.22 in normal phase 25 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 p-toluenesulfonic acid in place of hydrogen 30 chloride in the chemical hydrolysis of BBM-1675A2 or BBM-1675A^ ^Trade Mark 3 0 as illustrated by the procedures of Examples 3 and 5, re spectively.

A sample of BBM-1675A2 (15,2 mg) was hvdrolyzed with 0.03 molar solution of p—toluenesulfonic acid in methanol (1 ml) 5 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 10 Woelm silica gel (32-63 micron particle size). The column was eluted with chloroform?methanol (5s0.5,. v/v) , and the collected fractions were analysed by TLC [silica gel with chloroformsmethanol (5;0.5, v/v) as eluent]. The applied chromatography conditions permitted the separation of the mixture 15 of inactive compounds 2 and 3^ (7 mg) from the bioactive BBM-16 75D substance which has an value of 0.22. The appropriate fractions were pooled and evaporated to dryness to yield 8 mg of substantially pure 3BM-1675D in near quantitative yield* The physico-chemical properties of BBM-1675D appear in 1 20 the specification and the ultraviolet, infrared* mass, H NMR and 13 C NMR spectra of the compound appear as Figures 2S 4, 6, 8 and combined 10A and 10B, respectively.

Example 4 A sample of BBM-1675A^ (40 mg) was treated with 5 ml of 25 an 0.5 molar solution of hydrogen chloride in methanol at about 5 0°C for about 2 hours according to the general procedure and isolation method described in Example 1. After neutralization with WaHCO^ and evaporation to dryness, the BBH-1675C substance was isolated from the residue by flash column chromatography on a 30 column packed with Woelm silica gel (32-63 micron particle size) using toluene;acetone (3s2, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 8.4 mg of substantially pure 3BM-16 75C which is identical to the product isolated in Example 1. 31 The chromatographic column of above was then eluted with chloroform^methanol (5s0.25, v/v) and the fractions collected were pooled and evaporated to dryness to yield BBM-16 75D. The BBM-1675D substance was further purified by an additional flash 5 chromatography column with silica gel utilizing chloroformsmethanol (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. 10 Example 5 A sample of BBM-1675A^ (49.3 mg) was hydrolyzed with 0.03 7 M solution of p-toluenesulfonic acid in methanol (1.5 ml) oh* * at a temperature of about 50°C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 15 3 0°C to give a residue which contains BBM-1675D and the inactive compounds JL 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 20 appropriate fractions were combined- and evaporated to dryness to yield 27 mg of substantially pure BBM-1575D 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 25 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) utiliz-30 ing chloroformsmethanol (5s0.25, v/v) as the eluent. The appropriate fractions yielded substantially pure BBM-1675D which is identical to the product isolated in Example 3.

«Rp 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 75A1 and BBM-1675A2f there is thereby produced the BBM-1675C and 5 BBM-167 5D 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=16 75A^ and 10 BBM~1675A2, there is thereby produced the BBM-1675D substance. 33

Claims (9)

1. CLAIMS s 1» The antitumor antibiotic BBM-1675C which in substan tially pure form; (a) appears as an amorphous solid? (b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofurar>. 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 = 21t>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, fi3ia) 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 [M+H]* of 856? (h) has a 360 MHz proton magnetic resonance spectrum in CDCl^ substantially as shown in FIG. 7 exhibiting signals at 3 41 6.54 (1H, dd, 3=1.1» 1.0)'. 6.21 (1H, brs)? 5,87 (1H, d, J=9.6); 5.78 (1H, dd, J=9.6, 1.5); 5.66 (1H, brd, J=2.9); 4.94 (1H, dd, J=10.3, 1.8); 4.61 (1H, d, J=7.7); 4.25 (1H, s); 4.09 (1H, q, J=2.6); 3,97 (1H, t{ J=9.6); 3.92-3.53 5 (1OH)j 3.45 (1H, dt, J=10.3, 4.0); 3.37 (3H, s); 2.77 (1H, m) ; 2.69 (1H, dt, J=9.9, 5.2); 2.49 (1H, dd, J=10.3, 2.6); 2.48 (3H, s); 2.30 (2H, m) ; 2.13 (1H, m) j 2.09 (3H, s) ; 1„50 (2H, m) ; 1.37 (3H, d, J=5.9); 1.32 (3H, d, J=6.3); and 1.08 (6H) parts per million downfield from tetramethylsilane; 10 (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 e 35.2, 39.5, 47.7, 52.7, 55.8, 56.1, 57.7, 62.4, 64.1, 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, 15 124.6, 130.1, and 193.1 parts per million downfield from tetramethylsilane.

2. The antitumor antibiotic BBM-1675D which in substan- • tially pure form? (a) appears as an amorphous solid;" 20 (b) is soluble in methanol, ethanol, acetone and tetrahydrofuran, and slightly soluble in chloroform; (c) exhibits in silica gel thin-layer chromatography an value of 0.22 with the solvent system chloroformsmethanol (5?0.5, v/v) and exhibits in reverse phase silica gel thin layer 25 chromatography an value of 0.37 with the solvent system methanol?water (70s 30, v/v); (d) has an apparent molecular weight of 695 as determined by high resolution FAB mass spectroscopy; (e) 30 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), 35 274 ran (a = 12,800), and 325 nm (a = 5,400) with no significant change upon addition of acid or base? (f) has an infrared absorption spectrum (KBrfilm) 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 CDCI^ + 10% CD^OD substantially as shown in FIG. 8 exhibiting signals at 15 6-43 (1H, dd, J=4.4, 10.3)? 6.13 (1H, s)? 5-81 (1H, d, J=8.8)? 5.70 (1H, d, J=8.8)? 5.48 (1H, 6 brs)? 4-48 (1H, d, J=8.1)? 4.02 (1H, d, J=2.0); 3.95-3.80 (solvent background)? 3-77 (1H, t, J-9 ,0); 3.70-3.40 (11H, brm)? 3.35 (1H, 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 (1H, brdt)y 1.22 (3H, d, J=5.9)? 0.94 (3H, d, J=6.6)? and 0.87 (3H, d, J=5 9) parts per-million downfield from tetramethylsilane? (i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDC1- + 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.lt* 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. 30

3. The process for the production of the antitumor anti biotic BBM-1675C, which comprises hydrolyzing BBM-1675A^ or BBM-1675^2 with a mineral or organic acid until a substantial 38 amount of BBM-1S75C is produced and then recovering BBM-1675C from the reaction medium.

4. ■ The process for the production of the antitumor anti biotic BBM-1675D, which comprises hydrolyzing BBM-1675A., or 5 BBM-1675Aj with a mineral or organic: acid until an amount of BEM-1675D is produced which is at least sufficient for recovery and then recovering 38M-1675D frcsn the reaction medium. S„ The process for the production of the antitumor anti biotic BBM-167SD, which comprises hydrolyzing BBM-1S75C with a 10 mineral or organic acid until an snount of BBH--1675D is produced which is at least sufficient for recovery and then recovering BBM-1675D from the reaction medium* 6. The process for the production of the antitumor antibiotic BBM-1675C, which comprises hydrolyzing a mixture of 15 BBM-1675and 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 fran the reaction medium, 7. The process for the production of the antitumor antibiotic BBM-16 75D, which comprises hydrolyzing a mixture of 20 BBM-16 75An and BBM-16"SA^ with a mineral or organic acid until an amount of BBM-1675D is produced which is at least sufficient fat recovery and then recovering BBM-1675D from the reaction medium. 8. The antitumor antibiotic BBM-1675C according to claim 1 e substantially as hereinbefore specifically described with particular reference 25 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 f. 30 substantially as hereinbefore specifically described with particular reference to the examples„ 3? lie A process for the production of BBM-1675D according to claim 2, substantially as hereinbefore specifically described with particular reference to the examples.. 12. A pharmaceutical composition comprising an effective antimi-5 crobial anoiast of BBM-1575C or BBM 1675D in combination with a pharmaceutical carrier or diluent. 13. A pharmaceutical composition comprising an effective tumor-inhibiting 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. 1

5. A pharmaceutical compound as defined in claim 2 for use in a method of treatment of the hunan or animal body. 1

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

7. 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. 1

8. A pharmaceutical composition according to either of. claims 12 20 or 13 substantially as hereinbefore specifically described in exanples. 1

9. A compound whan produced by a process as claimed in any one of claims 3 to 7, 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 antitumor antibiotic. 25 21. Use of BffiM 675C of BBM-1675D for treating an animal host affected by a microbial infection. 22. Use of BBM-1S75C or BBM-1675D for treating an animal host affected by a malignant tumor sensitive to BBM-1675C or BBM-1675D, F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.