CA1307256C - Bbm-1675c and d antitumor antibiotics - Google Patents
Bbm-1675c and d antitumor antibioticsInfo
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- CA1307256C CA1307256C CA000516111A CA516111A CA1307256C CA 1307256 C CA1307256 C CA 1307256C CA 000516111 A CA000516111 A CA 000516111A CA 516111 A CA516111 A CA 516111A CA 1307256 C CA1307256 C CA 1307256C
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Abstract
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). The new antitumor antibiotics exhibit both antimicrobial activity and antitumor activity.
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). The new antitumor antibiotics exhibit both antimicrobial activity and antitumor activity.
Description
~3~P7~
BACKGROUND OF THE INVENTION
l. Field of the Invention This invention relates to new antitumor antibiotic substances and to their production and isolation.
BACKGROUND OF THE INVENTION
l. Field of the Invention This invention relates to new antitumor antibiotic substances and to their production and isolation.
2. Disclosure Statement 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 BBM-1675D antibiotics are novel substances.
United Kingdom Patent Application No. 2,141,425, published December l9, 1984, discloses fermentation of Actinomadura verrucosospora strain ~964-92 (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-167SA2. The structures of the BBM-1675Al and BBM-167SA2 antibiotics, also known as esperamicin Al and esperamicin A2, 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-1577B. The structures of the CL-1577 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that CL-1577A and CL-1577B are similar in structure to the BB~-1675 antibiotics, and especially BBM-1675A
and A2 mentioned above in United Kingdom Patent Application No.
2,141,425.
:3 3~7~5i6 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 isolated. In J. hem. Soc. Chem. Commun., 919-920 (19~5), J. H.
Wilton et al. described the partial structural elucidation of the antibiotics PD 114,759 and PD 115,02~. The production, isolation and characterization of the PD 114,759 and PD 115,028 antibiotics appear to be identical to the above-mentioned CL-1577A and CL-1577B antibiotics, respectivelyO
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 United Kingdom Patent Application No. 2,141,425 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-B 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 United Kingdom Patent Application No. 2,141,425 in the color of its aerial mycelium on ISP Medium Nos. 2, 3 and 4, in its positive milk peptonization and in its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.
SUMMARY OF THE INVENTION
There is provided by the present invention new antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY-27307, respectively, said sub-stances being produced by selective chemical hydrolysis of the bioactive components BBM-1675Al (esperamicin Al) or BBM-1675A2 (esperamicin A2), which are themselves produced by cultivating a ~L3~ ii6 BBM-1675-producing strain of Actinomadura verrucosospora. The bioactive substances BBM-1675C and BBM-1675D may be separated and purified by conventional chromatographic procedures, and both substances exhibit excellent antimicrobial and antitumor activity.
DESCRIPTION OF THE DRAWINGS
FIG . 1 shows the ultraviolet absorption spectrum of BBM-1675C.
FIG. 2 shows the ultraviolet absorption spectrum of BBM-1675D.
IG. 3 shows the infrared absorption spectrum of BBM-1675C
(KBr, film).
IG. 4 shows the infrared absorption spectrum of BBM-1675D
(KBr, film~.
IG. 5 shows the relative abundance mass spectrum of BBM-1675C.
IG. 6 shows the relative abundance mass spectrum of BBM-1675D.
IG. 7 shows the proton magnetic resonance spectrum of BBM-1675C in CDC13 (360 MHz~.
IG. 8 shows the proton magnetic resonance spectrum of BBM-1675D in CDC13 + 10% CD30D t360 MHz).
IG. 9 shows the C magnetic resonance spectrum of BBM-1675C
in CDC13 (90.6 MHz).
FIG. lOA shows th~ 13C magnetic resonance spectrum ~110-200 ppm) of BBM-1675D in CDC13 + 10% CD30D ~90.6 MHz).
FIG. lOB shows the 13C magnetic resonance spectrum (0-110 ppm) of BBM-1675D in CDC13 + 10% CD30D t90.6 MHz~.
~3~
f TG. llA shows the proton magnetic resonance spectrum of compound 3A (~-anomer) in CDC13 (360 MHz).
IG. llB shows the proton magnetic resonance spectrum of compound 3B (~-anomer) in CDC13 (360 MHz).
DETAILED DESCRIPTION OF THE INVENTION
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 BBM-1675A1 (esperamicin A1~ or BBM-1675A2 (esperamicin A2), which are themselves produced by cultivating a BBM-1675-produclng strain of Actinomadura verrucosospora, most preferably Actinomadura verrucososp_ a strain H964-92 (ATCC
39334) or Actinomadura verrucosospora 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 BBM-1675Al 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 the bioactive components BBM-1675A1 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-1675C and BRM-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 P-388 leukemia and B16 melanoma. The newly described substances of the ~ ~7~
present invention, therefore, may be 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-1675Al (esperamicin A1) 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 Formulas 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-1675D. Even more surprising, it was found that the two different ant-ibiotics BBM-1675A1 and A2 produced the same bioactive fragments as illustrated in Scheme 1. Still more surprising, the smaller molecular weight fragments BBM-1675C and D (having approximately 7096 and 55g6 of the molecular weight of the parent antibiotics BBM-1675Al and A2, respectively) were found to be more effective than BBM-1675A~ and comparable to BBM-1675A1 as antitumor and antimicrobial agents.
Scheme (esperamicin A~
\
BBM-1575C ~BBM-1675D
/
(esperamicin A2) The BBM-1675C and BBM-1675D substances may be prepared by selective chemical hydrolysis of the antibiotic B~M-1675A1 as outlined in Scheme 2.
Scheme 2 CH~--OCH3 HO o ~ C~
675 1 CH30H ~ ~
~m.w. 1248) (m.w. 855) CH30 H
' ' ~ CH2 Compound 1 (m.w. 425) (A mixture of ~ and anomers) ~/CH30H
rO\
BBM-1675D C 3 ~ ~ oc}~3 (m.w. 695) OH
Compound 3 (m.w. 192) (A mixture of ~ and anomers) ~3~
The starting BBM-1675A1 compound is prepared according to the procedure described in United ~Cingdom Patent Application No. 2,141,425, published December 19, 1984. The purified BBM-1675Al component is hydrolyzed with a mineral or organic acid such as hydrogen chloride, sulfuric acid, E~-toluenesulfonic acid;
benzenesulfonic acid or the like, in an organic or mixed aqueous-organic inert solvent at a temperature of about 0C 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 Cl-C6 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 60C, and most preferably from about 40 to 60~C.
The selective hydrolysis of BBM-1675Al 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 hydrolyzing conditions leads to the liberation of a mixture of Cl and ~ anomers of the thiosugar of Formula 3 and the producticn of the antibiotic BBM-1675D. It should be appxeciated by those skilled in the art that altering the reaction conditions such as time, temperature and concentra-tion of acid will produce varying relative amounts of the antibiotics BBM-1675C and D. Thus, it is desirable to moni:tor 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 ~-toluenesulfonic acid as described 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-1675A2 as outlined in Scheme 3.
~30~
j g Scheme 3 CH ~ O ~ OCH3 2 3 0 C H 3 0x~
(m.w. 1248) (m.w. 855) CH30 NH
o~, C H2 Compound 2 (m.w. 4253 (A mixture of a and anomers3 H(~9/ CH 30H
~ / ~0 BBM- 16 75D + 3S ~ ~ OCH3 (m.w. 695) y OH
Compound 3 (m.w. 192) (A mixture of ~ and R
anomers) The skarting BBM-1675A2 compound is prepared according to the procedure described in United Kingdom Patent Application No. 2,141,425, published December 19, 1984. The selective hydrolysis of purified BBM-1675A2 likewise proceeds in a stepwise 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 produc-tion of the antibiotic BBM-1675D.
The reaction conditions utilized for the selective chemical hydrolysis of BBM-1675A2 are substantially the same as those utilized for the hydrolysis of BBM-1675A1 described above.
In a manner similar to the production of BBM-1675D from BBM-1675Al, when it is preferred to produce only the BBM-1675D
anti~iotic, the hydrolysis of BBM-1675A2 is carried out until substantially all of BBM-1675A2 and BBM-1675C is converted to BBM-1675D. Most pre~erably, the hydrolysis is carried out with an organic acid such as ~-toluenesulfonic acid.
The discovery, as described herein, that the same BBM-1675C and D antibiotics are produced from two different antibiotics BBM-1675Al 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~ 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-1675Al and A2 to produce BBM-1675C
and D as illustrated in Scheme 4.
Scheme 4 BBM-1675Al ~ BBM-1675A2 -~ BBM-1675C ~ BBM-1675D
This advanta~e becomes apparent when one considers that the relative amounts of BBM-1675A1 and A2 produced in the fermenta-tion process is subject to variability. The production of BBM-1675C and D is therefore independent of the relative amounts of BBM-1675Al and A2 utilized as starting material in the present invention.
As described herein, the hydrolysis of the BBM-1675A1, 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-1675A1 and A2 antibiotics. The compound of Formula 3 was identified as a mixture of ~ and ~
anomers of a thiosugar which has the structure illustrated in Schemes 2 and 3. Further characterization was made possible when the products of the alcoholysis, the and ~ anomers, were separated. The proton magnetic resonance spectra (360 MHz) of the compound 3A (~-anomer) and compound 3B (~-anomer) are shown in F~GS. llA and llB, 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
CH3S~OCH3 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 CH3 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-1675Al and A2 antibiotics.
( Ph sico-chemical Pro erties of BBM-1675C
Y _ _ P __ Description: amorphous solid Ultraviolet absorption spectrum: See FIG. 1 Instrument : Hewlett-Packard 8458 Solvent : methanol Concentration : 0.0155 g/l ax (nm) absorptivities 210 21,770 274 9,340 313 sh (shoulder) 4,190 No significant change is observed with acid or base.
Infrared absorption spectrum : See FIG. 3 Instrument : 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 1, Mass spèctrum : See FIG. 5 Instrument : Finigan 4500 TSQ
Method : fast atom bombardment tFAB) ionization Molecular Relative Matrix m/z Ion Abundance glycerol 856 [M-~H] 100%
glycerol + NaCl 878 [M+Na] 100%
dithiothreitol:dithioerythritol 856 [M+H] 100 (3:1) (w:w) ~ 3~
i Instrurnent : Kratos MS-50 High resolution FAB (m/z)- [M+H] = 856.3362 Molecular weight: apparent MW = 855 (based on above-described mass spectral data) Elemental composition: C36H61N3014S3 (based on above-described high resolution data) Proton Magnetic Resonance Spectrum: See FIG. 7 Instrument: WM 360 Bruker Solvent : CDC13 lH NMR 360 MHz ~ (ppm):
6.54 (lH, dd, J=7.7, 7.0); 6.21 (lH, brs); 5.87 (lH, d, J=9.6); 5.78 (lH, dd, J=9.6, 1.5); 5.66 (lH, brd, J=2.9); 4094 (lH, dd, J=10.3, 1.8); 4.61 (lH, d, J=7.7~; 4.25 (lH, s); 4.09 (lH, q, J=2.6); 3.97 (lH, t, J=9.6); 3.92-3.53 (lOH~, 3.45 ~lH, dt, J=10.3, 4.0);
United Kingdom Patent Application No. 2,141,425, published December l9, 1984, discloses fermentation of Actinomadura verrucosospora strain ~964-92 (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-167SA2. The structures of the BBM-1675Al and BBM-167SA2 antibiotics, also known as esperamicin Al and esperamicin A2, 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-1577B. The structures of the CL-1577 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that CL-1577A and CL-1577B are similar in structure to the BB~-1675 antibiotics, and especially BBM-1675A
and A2 mentioned above in United Kingdom Patent Application No.
2,141,425.
:3 3~7~5i6 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 isolated. In J. hem. Soc. Chem. Commun., 919-920 (19~5), J. H.
Wilton et al. described the partial structural elucidation of the antibiotics PD 114,759 and PD 115,02~. The production, isolation and characterization of the PD 114,759 and PD 115,028 antibiotics appear to be identical to the above-mentioned CL-1577A and CL-1577B antibiotics, respectivelyO
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 United Kingdom Patent Application No. 2,141,425 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-B 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 United Kingdom Patent Application No. 2,141,425 in the color of its aerial mycelium on ISP Medium Nos. 2, 3 and 4, in its positive milk peptonization and in its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.
SUMMARY OF THE INVENTION
There is provided by the present invention new antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY-27307, respectively, said sub-stances being produced by selective chemical hydrolysis of the bioactive components BBM-1675Al (esperamicin Al) or BBM-1675A2 (esperamicin A2), which are themselves produced by cultivating a ~L3~ ii6 BBM-1675-producing strain of Actinomadura verrucosospora. The bioactive substances BBM-1675C and BBM-1675D may be separated and purified by conventional chromatographic procedures, and both substances exhibit excellent antimicrobial and antitumor activity.
DESCRIPTION OF THE DRAWINGS
FIG . 1 shows the ultraviolet absorption spectrum of BBM-1675C.
FIG. 2 shows the ultraviolet absorption spectrum of BBM-1675D.
IG. 3 shows the infrared absorption spectrum of BBM-1675C
(KBr, film).
IG. 4 shows the infrared absorption spectrum of BBM-1675D
(KBr, film~.
IG. 5 shows the relative abundance mass spectrum of BBM-1675C.
IG. 6 shows the relative abundance mass spectrum of BBM-1675D.
IG. 7 shows the proton magnetic resonance spectrum of BBM-1675C in CDC13 (360 MHz~.
IG. 8 shows the proton magnetic resonance spectrum of BBM-1675D in CDC13 + 10% CD30D t360 MHz).
IG. 9 shows the C magnetic resonance spectrum of BBM-1675C
in CDC13 (90.6 MHz).
FIG. lOA shows th~ 13C magnetic resonance spectrum ~110-200 ppm) of BBM-1675D in CDC13 + 10% CD30D ~90.6 MHz).
FIG. lOB shows the 13C magnetic resonance spectrum (0-110 ppm) of BBM-1675D in CDC13 + 10% CD30D t90.6 MHz~.
~3~
f TG. llA shows the proton magnetic resonance spectrum of compound 3A (~-anomer) in CDC13 (360 MHz).
IG. llB shows the proton magnetic resonance spectrum of compound 3B (~-anomer) in CDC13 (360 MHz).
DETAILED DESCRIPTION OF THE INVENTION
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 BBM-1675A1 (esperamicin A1~ or BBM-1675A2 (esperamicin A2), which are themselves produced by cultivating a BBM-1675-produclng strain of Actinomadura verrucosospora, most preferably Actinomadura verrucososp_ a strain H964-92 (ATCC
39334) or Actinomadura verrucosospora 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 BBM-1675Al 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 the bioactive components BBM-1675A1 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-1675C and BRM-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 P-388 leukemia and B16 melanoma. The newly described substances of the ~ ~7~
present invention, therefore, may be 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-1675Al (esperamicin A1) 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 Formulas 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-1675D. Even more surprising, it was found that the two different ant-ibiotics BBM-1675A1 and A2 produced the same bioactive fragments as illustrated in Scheme 1. Still more surprising, the smaller molecular weight fragments BBM-1675C and D (having approximately 7096 and 55g6 of the molecular weight of the parent antibiotics BBM-1675Al and A2, respectively) were found to be more effective than BBM-1675A~ and comparable to BBM-1675A1 as antitumor and antimicrobial agents.
Scheme (esperamicin A~
\
BBM-1575C ~BBM-1675D
/
(esperamicin A2) The BBM-1675C and BBM-1675D substances may be prepared by selective chemical hydrolysis of the antibiotic B~M-1675A1 as outlined in Scheme 2.
Scheme 2 CH~--OCH3 HO o ~ C~
675 1 CH30H ~ ~
~m.w. 1248) (m.w. 855) CH30 H
' ' ~ CH2 Compound 1 (m.w. 425) (A mixture of ~ and anomers) ~/CH30H
rO\
BBM-1675D C 3 ~ ~ oc}~3 (m.w. 695) OH
Compound 3 (m.w. 192) (A mixture of ~ and anomers) ~3~
The starting BBM-1675A1 compound is prepared according to the procedure described in United ~Cingdom Patent Application No. 2,141,425, published December 19, 1984. The purified BBM-1675Al component is hydrolyzed with a mineral or organic acid such as hydrogen chloride, sulfuric acid, E~-toluenesulfonic acid;
benzenesulfonic acid or the like, in an organic or mixed aqueous-organic inert solvent at a temperature of about 0C 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 Cl-C6 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 60C, and most preferably from about 40 to 60~C.
The selective hydrolysis of BBM-1675Al 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 hydrolyzing conditions leads to the liberation of a mixture of Cl and ~ anomers of the thiosugar of Formula 3 and the producticn of the antibiotic BBM-1675D. It should be appxeciated by those skilled in the art that altering the reaction conditions such as time, temperature and concentra-tion of acid will produce varying relative amounts of the antibiotics BBM-1675C and D. Thus, it is desirable to moni:tor 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 ~-toluenesulfonic acid as described 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-1675A2 as outlined in Scheme 3.
~30~
j g Scheme 3 CH ~ O ~ OCH3 2 3 0 C H 3 0x~
(m.w. 1248) (m.w. 855) CH30 NH
o~, C H2 Compound 2 (m.w. 4253 (A mixture of a and anomers3 H(~9/ CH 30H
~ / ~0 BBM- 16 75D + 3S ~ ~ OCH3 (m.w. 695) y OH
Compound 3 (m.w. 192) (A mixture of ~ and R
anomers) The skarting BBM-1675A2 compound is prepared according to the procedure described in United Kingdom Patent Application No. 2,141,425, published December 19, 1984. The selective hydrolysis of purified BBM-1675A2 likewise proceeds in a stepwise 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 produc-tion of the antibiotic BBM-1675D.
The reaction conditions utilized for the selective chemical hydrolysis of BBM-1675A2 are substantially the same as those utilized for the hydrolysis of BBM-1675A1 described above.
In a manner similar to the production of BBM-1675D from BBM-1675Al, when it is preferred to produce only the BBM-1675D
anti~iotic, the hydrolysis of BBM-1675A2 is carried out until substantially all of BBM-1675A2 and BBM-1675C is converted to BBM-1675D. Most pre~erably, the hydrolysis is carried out with an organic acid such as ~-toluenesulfonic acid.
The discovery, as described herein, that the same BBM-1675C and D antibiotics are produced from two different antibiotics BBM-1675Al 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~ 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-1675Al and A2 to produce BBM-1675C
and D as illustrated in Scheme 4.
Scheme 4 BBM-1675Al ~ BBM-1675A2 -~ BBM-1675C ~ BBM-1675D
This advanta~e becomes apparent when one considers that the relative amounts of BBM-1675A1 and A2 produced in the fermenta-tion process is subject to variability. The production of BBM-1675C and D is therefore independent of the relative amounts of BBM-1675Al and A2 utilized as starting material in the present invention.
As described herein, the hydrolysis of the BBM-1675A1, 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-1675A1 and A2 antibiotics. The compound of Formula 3 was identified as a mixture of ~ and ~
anomers of a thiosugar which has the structure illustrated in Schemes 2 and 3. Further characterization was made possible when the products of the alcoholysis, the and ~ anomers, were separated. The proton magnetic resonance spectra (360 MHz) of the compound 3A (~-anomer) and compound 3B (~-anomer) are shown in F~GS. llA and llB, 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
CH3S~OCH3 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 CH3 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-1675Al and A2 antibiotics.
( Ph sico-chemical Pro erties of BBM-1675C
Y _ _ P __ Description: amorphous solid Ultraviolet absorption spectrum: See FIG. 1 Instrument : Hewlett-Packard 8458 Solvent : methanol Concentration : 0.0155 g/l ax (nm) absorptivities 210 21,770 274 9,340 313 sh (shoulder) 4,190 No significant change is observed with acid or base.
Infrared absorption spectrum : See FIG. 3 Instrument : 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 1, Mass spèctrum : See FIG. 5 Instrument : Finigan 4500 TSQ
Method : fast atom bombardment tFAB) ionization Molecular Relative Matrix m/z Ion Abundance glycerol 856 [M-~H] 100%
glycerol + NaCl 878 [M+Na] 100%
dithiothreitol:dithioerythritol 856 [M+H] 100 (3:1) (w:w) ~ 3~
i Instrurnent : Kratos MS-50 High resolution FAB (m/z)- [M+H] = 856.3362 Molecular weight: apparent MW = 855 (based on above-described mass spectral data) Elemental composition: C36H61N3014S3 (based on above-described high resolution data) Proton Magnetic Resonance Spectrum: See FIG. 7 Instrument: WM 360 Bruker Solvent : CDC13 lH NMR 360 MHz ~ (ppm):
6.54 (lH, dd, J=7.7, 7.0); 6.21 (lH, brs); 5.87 (lH, d, J=9.6); 5.78 (lH, dd, J=9.6, 1.5); 5.66 (lH, brd, J=2.9); 4094 (lH, dd, J=10.3, 1.8); 4.61 (lH, d, J=7.7~; 4.25 (lH, s); 4.09 (lH, q, J=2.6); 3.97 (lH, t, J=9.6); 3.92-3.53 (lOH~, 3.45 ~lH, dt, J=10.3, 4.0);
3.37 (3H, s); 2.77 (lH, m); 2.69 (lH, dt, J=9.9, 5.2);
- 2.49 (lH, dd, J=10.3, 2.6); 2.48 (3H, s); 2.30 (2H, m);
2.13 (lH, 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).
C Magnetic Resonance Spectrum: See FIG. 9 Instrument: WM 360 Bruker Solvent : CDC13 13C NMR 90.6 MHz ~ (ppm):
13.7, 17.5, 19.8, 22.3, 22.7, 23.5, 3~.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, 193.1.
~3~
, Physico-chemical Properties of BBM~1675D
Description: amorphous solid Ultraviolet absorption spectrum: See FIG. 2 Instrument : Hewlett-Packard 8958 Solvent : methanol Concentration : 0.01 g/l (nm) absorptivities 214 27,000 274 12,800 325 5,400 No significant change is observed with acid or base.
Infrared absorption spectrum: See FIG. 4 Instrument : Nicolet 5DX ~T-IR
Major absorption bands (KBr, film):
735, 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 : See FIG. 6 Instrument : Finigan 4500 TSQ
Method : fast atom bombardment ~FAB) ionization Matrix : thioglycerol Molecular ion (m/z): [M+H] = 696 Relative abundance : 100%
Instrument : Kratos MS-50 High resolution FAB tm/z): [M+H] = 696.2794 Molecular weight: apparent MW - 695 (based on above-described mass spectral data) : ( Elemental composition: C29H49N3O12S2 ~based on above-described high resolution data) Correlation of ¦M+H] and [(M+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 : CDCl3 + 10% CD30D
H NMR 360 MHz ~ (ppm)o 6.43 ~lH, dd, J=4.4, 10.3); 6.13 (lH, s); 5.81 (lH, d, J=8.8); 5.70 ~lH, d, J=8.8); 5.48 (lH, 6 brs); 4.48 (lH, d, J=8.1); 4.02 (lH, d, J=2.0); 3.95-3.80 (solvent background); 3.77 (lH, t, J-9.0); 3.70-3.40 (llH, brm);
3.35 (lH, 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 (lH, brdt); 1.22 (3H, d, J=5.9); 0.94 (3H, d, J=6.6); 0.87 (3H, d, J=5O9).
3C Magnetic resonance spectrum: See FIG. lOA and lOB
Instrument : WM 360 Bruker Soivent : CDC13 ~ 10% CD30D
13C NMR 90.6 MHz ~ (ppm):
17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 62.1, 67.8, 6908, 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.
~ ~50 3256 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 1 below provides data in the form of results of an antimicLobial screening procedure involving the parent BBM-1675A1 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 ~g/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 1, the BBM-1675C and D substances showed a broad spectrum of antimicrobial activity which were at least as effective as the BBM-1675Al component; and in particular, the BBM-1675C and D
substances were more effective as inhibitors of gram-negative organisms.
~3(~7%56 j TABLE_I
ANTIMICROBIAL ACTIVITY OF BBM-1675 SUBST~CES
Zone of Inhibition, mm Test Microorganism BBM-1675AlBBM-1675CBBM-1675D
Escherichia coli AS 19 22 52 51 Escherichi.a coli K 12 13 . 36 35 Escherichia coli P 1373 12 34 33 Escherichia c_ R Azaserine 14 35 34 Escherichia coli R Netropsin 11 32 32 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 Escherichia coli R Hedamycin 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 Staphylococcus R Actinoleukin 33 35 33 Staphylococcus R Streptonigrin 37 50 48 Staphylococcus faecalis P1377 30 39 38 Streptococcus aureus Smith P 36 47 45 -Staphylococcus aureus Smith R 40 55 53 Actinomycin D
Staphylococcus aureus Smith R 17 32 31 Aureolic acid Acinetobacter 16 33 32 Micrococcus luteus 35 57 55 Saccharomyces cerevisiae petite 22 42 43 R = resistant to named antibiotic ~3 I
Activity Against P-388 Leukemia Tables II and III contain the results of laboratory tests with CDF~ mice implanted intraperitoneally with a tumor inoculum of 10 ascites cells of P-388 leukemia and treated with various doses of BBM-1675Al, C or D. The substances were admin-istered 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 BBM~1675Al 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 con-sidered excessiveO 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 gro~p times 100. An effect in terms of % T/C equal to or greater than 125 indicates that a significant antitumor ef~ect 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-1675Al (esperamicin Al) and the BBM-1675C and BBM-1675D
substances are reported. The data suggest that BBM-1675C is about comparable to BBM-1675Al 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) component. In comparison of the data reported herein for BBM-1675C and BBM-1675D and the data reported in published ~.K. Patent ~3~
Application No. 2,141,925 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-1675A;~ compo-nent from which they were derived.
TABLE II
(Day 1 Treatment~
Effect AWC
Dose, IP MST MST gm Survivors Cc~Tpound mg/kg/inj. ~ % T/C Day 4 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 0.0125 11.0 122 -2.5 5/6 0.003125 13.5 150 -2.5 6/6 Vehicle - 9.0 100 0.4 10/10 Tumor inoculum: 10 ascites cells implanted i.p.
Host: CDFl male 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) TABLE III
-EFFECT OF BBM-1675 SUBSTANCES ON P-388 LEI~KE:MIA
Effect AWC
Treatment Dose, IP MST MST gm Survivors Compound Schedule mg/kg/in;.days % T/C Day 4 Day 5 BBM-1675Al 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 6/5 0.0016 15.5 172 0.6 6/6 0.0008 12.5 139 0.3 6/6 0.00~4 12.0 133 1.~ 6/6 0.0002 11.0 122 0.8 6/6 0.0001 11.5 128 1.~ 6/6 BBM-1675C d. 1 0.0256 TOX TOX - 0/6 0.0128 TOX TOX--0. 8 3 /6 0.0064 11.5 128 -0.3 6/6 0.0032 14.~i 161 ~0.1 6/6 0.0016 10.5 117 0.0 6/6 0.0008 1200 133 0.3 6/6 0.000~ 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 ~L3~
( TABLE III (cont.) Treatment Dose, IP MST MST gm Survivors Compound Schedule mg/kq/inj. ~y~ % T/C Day 4 Day 5 _ BBM-1675D d. 1 0.02569.0 100O.l 6/6 0.012811.5 1280.3 6/6 0.006412.5 1390.3 6/6 0.003212Ø 1330.1 6/6 0.001611.5 1280.8 6/6 0.0008lO.0 1110.2 6/6 0~0004lOoO 1110.5 6/6 0.00029.5 1061.7 6/6 0.00019.5 1061.7 6/6 0.000059.0 1002.0 6/6 BBM-1675C d. 1~5 0.003216.0 178-1.3 6/6 0.001613.5 150-1.0 6/6 0.000813.5 150-0.3 6/6 0.000412.0 133-0.4 6/6 0.000212.0 133~0~4 6/6 0.000111.0 122-0.4 5/6 0.0000511.0 1~20.9 6/6 0.0000258.5 94 2.2 6/6 0.0000125 8.0 89 2.4 616 0.00000625 8.0 89 2.4 6/6 Vehicle - 9.0 1002.4 10/10 -Tumor inoculum: 106 ascites cells implanted i.p.
Host: CDF1 female mice Evaluatio.n: 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 (t.reated-control) in grams (on day 4) Activity A~ainst B16 Melanoma Table IV contains results of antitumor tests using the Bl6 melanoma grown in mice. BDF1 mice were employed and inoc-ulated 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, S and 9 by intraperitoneal injection. An effect in terms of % TIC 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 B16 melanoma and was about comparable to BBM-1675Al in potency.
~3~
TABL_ IV
(Day 1, 5 and 9 Treatments) Effect AWC
Dose, IP MST MST gm Survivors Compound mg/kg/inj. ~ ~6 TJc Day 12 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 0O0004 37.0 1~4 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 1096 brei, IP
Host: BDF1 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 12) ~3~ 56 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-1675D, or a pharmaceutical composition thereof.
The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of BBM-16 75C or BBM-16 75D 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 adminis-tration 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 solu~ions, 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 inject-able medium immediately before use.
For use as an antimicrobial agent, the BBM-1675C or BBM-1675D, or a pharmaceutical composition thereof is adminis-tered 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 mam~alian host can be readily ascertained by those skilled in the art. It will, of course, be appreciated that the actual dose of ( 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 a~e, 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 Pre aration and Isolation of BBM-1675C and BBM-1675D
p Example l A sample of BBM-1675Al (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 50C, and the disappearance of the starting material (approximately 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography ITLC) 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 NaHCO3 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 elu~ed 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 - trade mark ~3~
visualized wi-th a UV ~54 nm light source and a ceric sulfate spray (1% ceric sulfate and 2.596 molybolic acid in 10% sulfuric acid). Fractions 6-12 (Rf value for BBM-1675C is 0.283 were pooled and evaporated to dryness to yield 12 mg (35%) of substan-tially pure BBM-1675C.
The physico-chemical properties of BBM-1675C appear in the specification and the ultraviolet, infrared, mass, lH NMR and 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 BBM-1675C decreases, and two new products denoted as compound 3 (Rf = 0.65) and BBM-1675D (Rf 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 Rf = 0.37 in reverse phase TLC (Whatman MKC18F, 200 micron) using 30% water in methanol as the eluent and Rf = 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 P-toluenesulfonic acid in place of hydrogen chloride in the chemical hydrolysis of BBM-1675A2 or BBM-1675A
trade mark . ~ ` ;,r~ . ~
!
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 E~-toluenesulfonic acid in methanol (1 ml) at a temperature of about 63C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30C. 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 (5:0.5l v/v), and the collected fractions were analyzed by TLC [silica gel with chloroform:methanol (5:0.5, v/v) as eluent]. The applied chromatography conditions permitted the separation cf the mixture of inactiv~ compounds 2 and 3 (7 mg) from the bioactive BBM-1675D
substance which has an Rf value of 0.22. The appropriate fractions were pooled and evaporated to dryness to yield 8 mg of substantially pure BB~1-1675D in near quantitative yield.
The physico-chemical properties of ~3BM-1675D appear in the specification and the ultraviolet, infrared, mass, 1H NMR and 13C NMR spectra o~ the compound appear as Figures Z, 4, 6, 8 and combined lOA and lOB, respectively.
Example 4 A sample of BBM-1675A2 (40 mg) was treated with 5 ml of an O.S molar solution of hydrogen chloride in methanol at about 50C for about 2 hours according to the general procedure and isolation method described in Example 1. After neutralization with NaHCO3 and evaporation to dryness, the BBM-1675C substance was isolated from the residue by flash column chromatograph~ Oll 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 ~.9 mg of substantially pure B~M-1675C which is identical to the product isolated in Example 1.
trade mark ~3~ t The chromatographic column of above was then eluted with chloroform:methanol (5:0.25, v/v) and the fractions collect-ed 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 u-tilizing 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-1675Al (49.3 mg) was hydrolyzed with 0.037 M solution of ~-toluenesulfonic acid in methanol (1.5 ml) at a temperature of about 60C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 30C to give a residue which contains BBM-1675D and the inactive compounds 1 and 3. 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 si7e) 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 ~xample 3.
Example 6 A sample of BBM-1675C (S.1 mg) was hydrolyzed with 0.5 molar solution of hydrogen chloride in methanol (1 ml) at about 40-50C overnight. Afte~r neutralization with NaHCO3 and evapo-ration to dryness, the BBM-1675D bioactive substance was isolated from the residue by flash column chrGmatography on a column *
packed with Woelm silica gel (32-63 micron particle size~ utiliz-ing chloroform:methanol (5:0.25, v/v) as the eluent. The appro-priate fractions yielded substantially pure BBM-1675D which is identical to the product isolated in Example 3.
trade mark ~3~)~7~e-6 Example 7 When the general procedure of Examples 1 and Z are repeated, except that the starting material BBM-1675Al is re-placed by an equimolar amount of a mixture containing BBM-1675A
and BBM-1675A2, there is thereby produced the BBM-1675C and BBM-1675D substances.
~ e 8 When the general procedure of Example 5 is repeated, except that the starting material BBM-1675A1 is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, there is thereby produced the BBM-1675D substance.
- 2.49 (lH, dd, J=10.3, 2.6); 2.48 (3H, s); 2.30 (2H, m);
2.13 (lH, 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).
C Magnetic Resonance Spectrum: See FIG. 9 Instrument: WM 360 Bruker Solvent : CDC13 13C NMR 90.6 MHz ~ (ppm):
13.7, 17.5, 19.8, 22.3, 22.7, 23.5, 3~.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, 193.1.
~3~
, Physico-chemical Properties of BBM~1675D
Description: amorphous solid Ultraviolet absorption spectrum: See FIG. 2 Instrument : Hewlett-Packard 8958 Solvent : methanol Concentration : 0.01 g/l (nm) absorptivities 214 27,000 274 12,800 325 5,400 No significant change is observed with acid or base.
Infrared absorption spectrum: See FIG. 4 Instrument : Nicolet 5DX ~T-IR
Major absorption bands (KBr, film):
735, 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 : See FIG. 6 Instrument : Finigan 4500 TSQ
Method : fast atom bombardment ~FAB) ionization Matrix : thioglycerol Molecular ion (m/z): [M+H] = 696 Relative abundance : 100%
Instrument : Kratos MS-50 High resolution FAB tm/z): [M+H] = 696.2794 Molecular weight: apparent MW - 695 (based on above-described mass spectral data) : ( Elemental composition: C29H49N3O12S2 ~based on above-described high resolution data) Correlation of ¦M+H] and [(M+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 : CDCl3 + 10% CD30D
H NMR 360 MHz ~ (ppm)o 6.43 ~lH, dd, J=4.4, 10.3); 6.13 (lH, s); 5.81 (lH, d, J=8.8); 5.70 ~lH, d, J=8.8); 5.48 (lH, 6 brs); 4.48 (lH, d, J=8.1); 4.02 (lH, d, J=2.0); 3.95-3.80 (solvent background); 3.77 (lH, t, J-9.0); 3.70-3.40 (llH, brm);
3.35 (lH, 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 (lH, brdt); 1.22 (3H, d, J=5.9); 0.94 (3H, d, J=6.6); 0.87 (3H, d, J=5O9).
3C Magnetic resonance spectrum: See FIG. lOA and lOB
Instrument : WM 360 Bruker Soivent : CDC13 ~ 10% CD30D
13C NMR 90.6 MHz ~ (ppm):
17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 62.1, 67.8, 6908, 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.
~ ~50 3256 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 1 below provides data in the form of results of an antimicLobial screening procedure involving the parent BBM-1675A1 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 ~g/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 1, the BBM-1675C and D substances showed a broad spectrum of antimicrobial activity which were at least as effective as the BBM-1675Al component; and in particular, the BBM-1675C and D
substances were more effective as inhibitors of gram-negative organisms.
~3(~7%56 j TABLE_I
ANTIMICROBIAL ACTIVITY OF BBM-1675 SUBST~CES
Zone of Inhibition, mm Test Microorganism BBM-1675AlBBM-1675CBBM-1675D
Escherichia coli AS 19 22 52 51 Escherichi.a coli K 12 13 . 36 35 Escherichia coli P 1373 12 34 33 Escherichia c_ R Azaserine 14 35 34 Escherichia coli R Netropsin 11 32 32 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 Escherichia coli R Hedamycin 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 Staphylococcus R Actinoleukin 33 35 33 Staphylococcus R Streptonigrin 37 50 48 Staphylococcus faecalis P1377 30 39 38 Streptococcus aureus Smith P 36 47 45 -Staphylococcus aureus Smith R 40 55 53 Actinomycin D
Staphylococcus aureus Smith R 17 32 31 Aureolic acid Acinetobacter 16 33 32 Micrococcus luteus 35 57 55 Saccharomyces cerevisiae petite 22 42 43 R = resistant to named antibiotic ~3 I
Activity Against P-388 Leukemia Tables II and III contain the results of laboratory tests with CDF~ mice implanted intraperitoneally with a tumor inoculum of 10 ascites cells of P-388 leukemia and treated with various doses of BBM-1675Al, C or D. The substances were admin-istered 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 BBM~1675Al 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 con-sidered excessiveO 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 gro~p times 100. An effect in terms of % T/C equal to or greater than 125 indicates that a significant antitumor ef~ect 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-1675Al (esperamicin Al) and the BBM-1675C and BBM-1675D
substances are reported. The data suggest that BBM-1675C is about comparable to BBM-1675Al 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) component. In comparison of the data reported herein for BBM-1675C and BBM-1675D and the data reported in published ~.K. Patent ~3~
Application No. 2,141,925 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-1675A;~ compo-nent from which they were derived.
TABLE II
(Day 1 Treatment~
Effect AWC
Dose, IP MST MST gm Survivors Cc~Tpound mg/kg/inj. ~ % T/C Day 4 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 0.0125 11.0 122 -2.5 5/6 0.003125 13.5 150 -2.5 6/6 Vehicle - 9.0 100 0.4 10/10 Tumor inoculum: 10 ascites cells implanted i.p.
Host: CDFl male 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) TABLE III
-EFFECT OF BBM-1675 SUBSTANCES ON P-388 LEI~KE:MIA
Effect AWC
Treatment Dose, IP MST MST gm Survivors Compound Schedule mg/kg/in;.days % T/C Day 4 Day 5 BBM-1675Al 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 6/5 0.0016 15.5 172 0.6 6/6 0.0008 12.5 139 0.3 6/6 0.00~4 12.0 133 1.~ 6/6 0.0002 11.0 122 0.8 6/6 0.0001 11.5 128 1.~ 6/6 BBM-1675C d. 1 0.0256 TOX TOX - 0/6 0.0128 TOX TOX--0. 8 3 /6 0.0064 11.5 128 -0.3 6/6 0.0032 14.~i 161 ~0.1 6/6 0.0016 10.5 117 0.0 6/6 0.0008 1200 133 0.3 6/6 0.000~ 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 ~L3~
( TABLE III (cont.) Treatment Dose, IP MST MST gm Survivors Compound Schedule mg/kq/inj. ~y~ % T/C Day 4 Day 5 _ BBM-1675D d. 1 0.02569.0 100O.l 6/6 0.012811.5 1280.3 6/6 0.006412.5 1390.3 6/6 0.003212Ø 1330.1 6/6 0.001611.5 1280.8 6/6 0.0008lO.0 1110.2 6/6 0~0004lOoO 1110.5 6/6 0.00029.5 1061.7 6/6 0.00019.5 1061.7 6/6 0.000059.0 1002.0 6/6 BBM-1675C d. 1~5 0.003216.0 178-1.3 6/6 0.001613.5 150-1.0 6/6 0.000813.5 150-0.3 6/6 0.000412.0 133-0.4 6/6 0.000212.0 133~0~4 6/6 0.000111.0 122-0.4 5/6 0.0000511.0 1~20.9 6/6 0.0000258.5 94 2.2 6/6 0.0000125 8.0 89 2.4 616 0.00000625 8.0 89 2.4 6/6 Vehicle - 9.0 1002.4 10/10 -Tumor inoculum: 106 ascites cells implanted i.p.
Host: CDF1 female mice Evaluatio.n: 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 (t.reated-control) in grams (on day 4) Activity A~ainst B16 Melanoma Table IV contains results of antitumor tests using the Bl6 melanoma grown in mice. BDF1 mice were employed and inoc-ulated 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, S and 9 by intraperitoneal injection. An effect in terms of % TIC 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 B16 melanoma and was about comparable to BBM-1675Al in potency.
~3~
TABL_ IV
(Day 1, 5 and 9 Treatments) Effect AWC
Dose, IP MST MST gm Survivors Compound mg/kg/inj. ~ ~6 TJc Day 12 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 0O0004 37.0 1~4 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 1096 brei, IP
Host: BDF1 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 12) ~3~ 56 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-1675D, or a pharmaceutical composition thereof.
The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of BBM-16 75C or BBM-16 75D 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 adminis-tration 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 solu~ions, 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 inject-able medium immediately before use.
For use as an antimicrobial agent, the BBM-1675C or BBM-1675D, or a pharmaceutical composition thereof is adminis-tered 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 mam~alian host can be readily ascertained by those skilled in the art. It will, of course, be appreciated that the actual dose of ( 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 a~e, 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 Pre aration and Isolation of BBM-1675C and BBM-1675D
p Example l A sample of BBM-1675Al (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 50C, and the disappearance of the starting material (approximately 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography ITLC) 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 NaHCO3 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 elu~ed 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 - trade mark ~3~
visualized wi-th a UV ~54 nm light source and a ceric sulfate spray (1% ceric sulfate and 2.596 molybolic acid in 10% sulfuric acid). Fractions 6-12 (Rf value for BBM-1675C is 0.283 were pooled and evaporated to dryness to yield 12 mg (35%) of substan-tially pure BBM-1675C.
The physico-chemical properties of BBM-1675C appear in the specification and the ultraviolet, infrared, mass, lH NMR and 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 BBM-1675C decreases, and two new products denoted as compound 3 (Rf = 0.65) and BBM-1675D (Rf 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 Rf = 0.37 in reverse phase TLC (Whatman MKC18F, 200 micron) using 30% water in methanol as the eluent and Rf = 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 P-toluenesulfonic acid in place of hydrogen chloride in the chemical hydrolysis of BBM-1675A2 or BBM-1675A
trade mark . ~ ` ;,r~ . ~
!
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 E~-toluenesulfonic acid in methanol (1 ml) at a temperature of about 63C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30C. 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 (5:0.5l v/v), and the collected fractions were analyzed by TLC [silica gel with chloroform:methanol (5:0.5, v/v) as eluent]. The applied chromatography conditions permitted the separation cf the mixture of inactiv~ compounds 2 and 3 (7 mg) from the bioactive BBM-1675D
substance which has an Rf value of 0.22. The appropriate fractions were pooled and evaporated to dryness to yield 8 mg of substantially pure BB~1-1675D in near quantitative yield.
The physico-chemical properties of ~3BM-1675D appear in the specification and the ultraviolet, infrared, mass, 1H NMR and 13C NMR spectra o~ the compound appear as Figures Z, 4, 6, 8 and combined lOA and lOB, respectively.
Example 4 A sample of BBM-1675A2 (40 mg) was treated with 5 ml of an O.S molar solution of hydrogen chloride in methanol at about 50C for about 2 hours according to the general procedure and isolation method described in Example 1. After neutralization with NaHCO3 and evaporation to dryness, the BBM-1675C substance was isolated from the residue by flash column chromatograph~ Oll 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 ~.9 mg of substantially pure B~M-1675C which is identical to the product isolated in Example 1.
trade mark ~3~ t The chromatographic column of above was then eluted with chloroform:methanol (5:0.25, v/v) and the fractions collect-ed 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 u-tilizing 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-1675Al (49.3 mg) was hydrolyzed with 0.037 M solution of ~-toluenesulfonic acid in methanol (1.5 ml) at a temperature of about 60C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 30C to give a residue which contains BBM-1675D and the inactive compounds 1 and 3. 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 si7e) 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 ~xample 3.
Example 6 A sample of BBM-1675C (S.1 mg) was hydrolyzed with 0.5 molar solution of hydrogen chloride in methanol (1 ml) at about 40-50C overnight. Afte~r neutralization with NaHCO3 and evapo-ration to dryness, the BBM-1675D bioactive substance was isolated from the residue by flash column chrGmatography on a column *
packed with Woelm silica gel (32-63 micron particle size~ utiliz-ing chloroform:methanol (5:0.25, v/v) as the eluent. The appro-priate fractions yielded substantially pure BBM-1675D which is identical to the product isolated in Example 3.
trade mark ~3~)~7~e-6 Example 7 When the general procedure of Examples 1 and Z are repeated, except that the starting material BBM-1675Al is re-placed by an equimolar amount of a mixture containing BBM-1675A
and BBM-1675A2, there is thereby produced the BBM-1675C and BBM-1675D substances.
~ e 8 When the general procedure of Example 5 is repeated, except that the starting material BBM-1675A1 is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, there is thereby produced the BBM-1675D substance.
Claims (7)
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, tetrahydrofuran and chloroform;
(c) exhibits in silica gel thin layer chromatography an Rf value of 0.28 with the solvent system toluene:acetone (3:2, 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 follows:
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 follows:
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 follows:
exhibiting a molecular ion [M+H]+ of 856;
(h) has a 360 MHz proton magnetic resonance spectrum in CDCl3 substantially as follows:
exhibiting signals at 6.54 (1H, dd, J-7.7, 7.0); 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 (10H), 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);
(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 Rf value of 0.28 with the solvent system toluene:acetone (3:2, 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 follows:
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 follows:
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 follows:
exhibiting a molecular ion [M+H]+ of 856;
(h) has a 360 MHz proton magnetic resonance spectrum in CDCl3 substantially as follows:
exhibiting signals at 6.54 (1H, dd, J-7.7, 7.0); 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 (10H), 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);
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 2tetramethylsilane;
(i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCl3 substantially as follows:
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 from tetramethylsilane.
2. The antitumor antibiotic BBM-1675D which in substan-tially 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 Rf 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 Rf 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 follows:
exhibiting ultraviolet absorption maxima and absorptivities at 214 nm (a = 27,000), 274 nm (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 (KBr, film) substantially as follows:
exhibiting principal 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;
(g) has a low resolution mass spectrum substantially as follows:
exhibiting a molecular ion [M+H]+ of 696;
(h) has a 360 MH proton magnetic resonance spectrum in CDCl3 +
10% CD3OD substantially as follows:
exhibiting signals at 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 Isolvent background);
(i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCl3 substantially as follows:
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 from tetramethylsilane.
2. The antitumor antibiotic BBM-1675D which in substan-tially 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 Rf 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 Rf 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 follows:
exhibiting ultraviolet absorption maxima and absorptivities at 214 nm (a = 27,000), 274 nm (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 (KBr, film) substantially as follows:
exhibiting principal 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;
(g) has a low resolution mass spectrum substantially as follows:
exhibiting a molecular ion [M+H]+ of 696;
(h) has a 360 MH proton magnetic resonance spectrum in CDCl3 +
10% CD3OD substantially as follows:
exhibiting signals at 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 Isolvent 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); 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 CDCl3 + 10% CD3OD substantially as follows:
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.
3. The process for the production of the antitumor anti-biotic BBM-1675C, which comprises hydrolyzing BBM-1675A1 or BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675C is produced and then recovering BBM-1675C
from the reaction medium.
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); 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 CDCl3 + 10% CD3OD substantially as follows:
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.
3. The process for the production of the antitumor anti-biotic BBM-1675C, which comprises hydrolyzing BBM-1675A1 or BBM-1675A2 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 BBM-1675D, which comprises hydrolyzing BBM-1675A1 or BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675D is produced and then recovering BBM-1675D
from the reaction medium.
from the reaction medium.
5. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing BBM-1675C with a mineral or organic acid until a substantial amount of BBM-1675D
is produced and then recovering BBM-1675D from the reaction medium.
is produced 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 BBM-1675A1 and BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675C is produced and then recovering BBM-1675C from the reaction medium.
7. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing a mixture of BBM-1675A1 and BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675D is produced and then recovering BBM-1675D from the reation medium.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US77033585A | 1985-08-27 | 1985-08-27 | |
| US770,335 | 1985-08-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1307256C true CA1307256C (en) | 1992-09-08 |
Family
ID=25088206
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000516111A Expired - Fee Related CA1307256C (en) | 1985-08-27 | 1986-08-15 | Bbm-1675c and d antitumor antibiotics |
Country Status (26)
| Country | Link |
|---|---|
| JP (2) | JPH0733393B2 (en) |
| KR (1) | KR920010226B1 (en) |
| AT (1) | AT392971B (en) |
| AU (1) | AU604464B2 (en) |
| BE (1) | BE905332A (en) |
| CA (1) | CA1307256C (en) |
| CH (1) | CH668598A5 (en) |
| CY (1) | CY1676A (en) |
| DE (1) | DE3629052C2 (en) |
| DK (1) | DK170671B1 (en) |
| ES (1) | ES2002728A6 (en) |
| FI (1) | FI83422C (en) |
| FR (1) | FR2586686B1 (en) |
| GB (1) | GB2179649A (en) |
| GR (1) | GR862160B (en) |
| HK (1) | HK793A (en) |
| HU (1) | HU197915B (en) |
| IE (1) | IE59204B1 (en) |
| IL (1) | IL79519A0 (en) |
| IT (1) | IT1229176B (en) |
| LU (1) | LU86562A1 (en) |
| NL (1) | NL8602165A (en) |
| PT (1) | PT83261B (en) |
| SE (2) | SE469632B (en) |
| SG (1) | SG109692G (en) |
| ZA (1) | ZA865796B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2177518T3 (en) * | 1987-01-30 | 2002-12-16 | American Cyanamid Co | DIHIDRO DERIVATIVES OF ANTIBIOTICS LL-E33288. |
| US4916065A (en) * | 1988-06-10 | 1990-04-10 | Bristol-Myers Company | BU-3420T Antitumor antibiotic |
| US5028536A (en) * | 1989-03-15 | 1991-07-02 | Bristol-Myers Squibb Company | Antitumor antibiotic BMY-41339 |
| US5086045A (en) * | 1989-03-15 | 1992-02-04 | Bristol-Myers Squibb Company | Antitumor antibiotic |
| CA2027601A1 (en) * | 1989-11-06 | 1991-05-07 | Koko Sugawara | Antitumor antibiotic bu-3983t |
| CA2039789A1 (en) * | 1990-04-27 | 1991-10-28 | Samuel J. Danishefsky | Calicheamicinone, derivatives and analogs thereof and methods of making the same |
| US5116845A (en) * | 1990-05-04 | 1992-05-26 | Bristol-Myers Company | BU-3420T antitumor antibiotic |
| US5264586A (en) * | 1991-07-17 | 1993-11-23 | The Scripps Research Institute | Analogs of calicheamicin gamma1I, method of making and using the same |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3148023A1 (en) * | 1981-12-04 | 1983-06-09 | Rudolf Dipl.-Ing. 8901 Oberottmarshausen Fischer | Heating boiler for hot flue gases |
| US4578271A (en) * | 1982-05-24 | 1986-03-25 | Fujisawa Pharmaceutical Co., Ltd. | Biologically active WS 6049 substances, a process for the production thereof and their pharmaceutical compositions |
| NZ208013A (en) * | 1983-05-16 | 1987-07-31 | Bristol Myers Co | Antitumour antibiotic bbm-1675 and production by cultivating actinomadura verrucosospora |
| JPS606194A (en) * | 1983-06-23 | 1985-01-12 | Meiji Seika Kaisha Ltd | Novel antibiotic substance sf-2288 and its preparation |
| US4530835A (en) * | 1983-07-08 | 1985-07-23 | Warner-Lambert Company | CL-1577 Antibiotic compounds and their production |
-
1986
- 1986-07-25 IL IL79519A patent/IL79519A0/en not_active IP Right Cessation
- 1986-08-01 ZA ZA865796A patent/ZA865796B/en unknown
- 1986-08-15 CA CA000516111A patent/CA1307256C/en not_active Expired - Fee Related
- 1986-08-19 GB GB08620118A patent/GB2179649A/en active Granted
- 1986-08-20 GR GR862160A patent/GR862160B/en unknown
- 1986-08-22 AU AU61751/86A patent/AU604464B2/en not_active Ceased
- 1986-08-22 FI FI863405A patent/FI83422C/en not_active IP Right Cessation
- 1986-08-26 LU LU86562A patent/LU86562A1/en unknown
- 1986-08-26 BE BE0/217084A patent/BE905332A/en not_active IP Right Cessation
- 1986-08-26 FR FR868612085A patent/FR2586686B1/en not_active Expired - Fee Related
- 1986-08-26 IE IE228086A patent/IE59204B1/en not_active IP Right Cessation
- 1986-08-26 DK DK406086A patent/DK170671B1/en not_active IP Right Cessation
- 1986-08-26 CH CH3417/86A patent/CH668598A5/en not_active IP Right Cessation
- 1986-08-26 IT IT8621527A patent/IT1229176B/en active
- 1986-08-26 SE SE8603597A patent/SE469632B/en not_active IP Right Cessation
- 1986-08-26 ES ES8601355A patent/ES2002728A6/en not_active Expired
- 1986-08-26 KR KR1019860007092A patent/KR920010226B1/en not_active IP Right Cessation
- 1986-08-26 NL NL8602165A patent/NL8602165A/en not_active Application Discontinuation
- 1986-08-27 PT PT83261A patent/PT83261B/en unknown
- 1986-08-27 DE DE3629052A patent/DE3629052C2/en not_active Expired - Fee Related
- 1986-08-27 AT AT2317/86A patent/AT392971B/en not_active IP Right Cessation
- 1986-08-27 JP JP61201199A patent/JPH0733393B2/en not_active Expired - Lifetime
- 1986-08-27 HU HU863709A patent/HU197915B/en not_active IP Right Cessation
-
1992
- 1992-02-13 SE SE9200428A patent/SE9200428L/en not_active Application Discontinuation
- 1992-10-16 SG SG1096/92A patent/SG109692G/en unknown
-
1993
- 1993-01-07 HK HK7/93A patent/HK793A/en not_active IP Right Cessation
- 1993-10-10 CY CY1676A patent/CY1676A/en unknown
-
1994
- 1994-08-19 JP JP6195156A patent/JPH07233186A/en active Pending
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