GB2179649A - Antibiotics - Google Patents
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- GB2179649A GB2179649A GB08620118A GB8620118A GB2179649A GB 2179649 A GB2179649 A GB 2179649A GB 08620118 A GB08620118 A GB 08620118A GB 8620118 A GB8620118 A GB 8620118A GB 2179649 A GB2179649 A GB 2179649A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
There is provided new antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, said substances being produced by selective chemical hydrolysis of the bioactive components BBM-1675A1 (esperamicin A1) or BBM-1675A2 (esperamicin A2) produced from Actinomadura verrucosospora. BBM 1675C and BBM 1675D have molecular weights of approx 855 and 695 respectively.
Description
SPECIFICATION
Antibiotics
This invention relates to new antitumor antibiotic substances and to their production and isolation.
Th 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-1 6750 and
BBM-1675D antibiotics are novel substances.
United Kingdom Patent Application No. 2,141,425, published December 19, 1984, discloses fermentation of
Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain Al 327Y (ATCC 39638) to produce a new antitumor antibiotic complex designated as BBM-1 675. Two major bioactive components of the BBM-1675 complex described therein were designated as BBM-1675A, and BBM-1675A2.
The structures of the BBM-1 675A1 and BBM-1 6752 antibiotics, also known as esperamicin A, 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-I 577B are similar in structure to the BBM-1675 antibiotics, and especially BBM-1675A, and A2 mentioned above in United Kingdom Patent
Application No. 2,141,425.
There is disclosed by R.H. Bunge et a/., in J. Antibiotics, 37 (12), 1566-1571(1984) the 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. InJ. Chem. Soc. Chem. Commun., 919-920 (1985), J. H. Wilton etna!.
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-1577A and CL-1 577B antibiotics, respectively.
European Patent Application No. 95,154, published November 30, 1983, discloses fermentation of
Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100) to produce antitumor antibiotics designated WS 6049-A and WS 6049-B. The structures of the WS 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that WS 6049-A and WS 6049-B are related in structure to the BBM-1675 antibiotics of 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.
There is provided by the present invention new autitumor 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 A2), which are themselves produced by cultivating a BBM-1675-producing strain of
Actinomadura verrucosospora. The bioactive substances BBM-1 6750 and BBM-1 675D may be separated and purified by conventional chromatographic procedures, and both substances exhibit excellent antimicrobial and antitumor activity.
Description of the drawings
Figure 1 shows the ultraviolet absorption spectrum of BBM-1675C.
Figure 2 shows the ultraviolet absorption spectrum of BBM-1675D.
FigureS shows the infrared absorption spectrum of BBM-1 6750 (KBr, film).
Figure 4 shows the infrared absorption spectrum of BBM-1675D (KBr, film).
Figure 5 shows the relative abundance mass spectrum of BBM-1675C.
Figure 6 shows the relative abundance mass spectrum of BBM-1675D.
Figure 7 shows the proton magnetic resonance spectrum of BBM-1 675C in CDCI3 (360 MHz).
Figure 8 shows the proton magnetic resonance spectrum of BBM-1 675D in CDCI3 + 10% CD3OD (360 MHz).
Figure 9 shows the '3C magnetic resonance spectrum of BBM-1675C in CDCI3 (90.6 MHz).
Figure lOA shows the 13C magnetic resonance spectrum (110-200 ppm) of BBM-1675D in CDCI3 + 10% CD30D (90.6MHz).
Figure lOB shows the 13C magnetic resonance spectrum (0-110 ppm) of BBM-1 675D in CDCI3 + 10% CD30D (90.6MHz).
Figure liA shows the proton magnetic resonance spectrum of compound 3A (z-anomer) in CDCI3 (360 MHz).
Figure 11B shows the proton magnetic resonance spectrum of compound 3B (p-anomer) in CDCI3 (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-1 675A1 (esperamicin A,) or BBM-1 675A2 (esperamicin A2), which are themselves produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora, most preferably Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain Al 327Y (ATCC 39638), or a mutant thereof.In another aspect, the present invention provides a process for producing the BBM-1 675C substance by selective hydrolysis of the bioactive components BBM-1675A, or BBM-1675A2. In a further aspect, the present invention provides a process for the preparation of BBM-1 675D by selective hydrolysis of the BBM-1 675C substance or, more preferably, from the bioactive components BBM-16752 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-l 675C and BBM-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 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-1675A (esperamicin A,) and BBM-1675A2 (esperamicin A2), a mixture of components were produced which lead to the isolation and identification of two inactive fragments, compounds of the Formulas 1 and 2, respectively.
However, it was surprisingly found that the chemical degradation lead to the stepwise liberation of two bioactive fragments BBM-1675C and BBM-1675D. Even more surprising, it was found that the two different antibiotics BBM-1675A 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 70% and 55% of the molecular weight of the parent antibiotics BBM-1675A, and A2, respectively) were found to be more effective than BBM-l 675A2 and comparable to BBM-1 675A1 as antitumor and antimicrobial agents.
Scheme 1
BBM- 16 75A1 (esperamicin ; BBM-1675C \ BBM- 16 75D BBM- 16 75A2 / / w BBM-1675A2 MM (esperamicin A2) The BBM-1 675C and BBM-1675D substances may be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A, as outlined in Scheme 2.
Scheme 2
CH3 OCH3 HO o HO 0 CH BBM-1675A1 Eot BBM-1675C + 39 (m.w. 1248) (m.w. 855) CH3OH OCH3 Compound 1 (m.w. 425) (A mixture of a and ss anomers) )/CH30H J BBM-1675D + CH3sCH3o OCH3 (m.w. 695) OH Compound 3 (m.w. 192)
(A mixture of a and B
anomers)
The starting BBM-1 675A, compound is prepared according to the procedure described in United Kingdom
Patent Application No. 2,141,425, published December 19, 1984. The purified BBM-1675A component is hydrolyzed with a mineral or organic acid such as hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid or the like, in an organic or mixed aqueous-organic inert solvent at a temperature of about 0 C to the refluxing temperature of the solvent until a substantial amount of the desired BBM-1675C or
BBM-1 675D is produced. Preferably, the hydrolysis is carried out in C,-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 60"C, and most preferably from about 40 to 60"C.
The selective hydrolysis of BBM-1 675A, proceeds in a stepwise manner with the initial production of the BBM-1 675C antibiotic and the inactive fragment of Formula 1. Subsequent or continued treatment under hydrolyzing conditions leads to the liberation of a mixture of ac and ss anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM-1675D. It should be appreciated by those skilled in the art that altering the reaction conditions such as time, temperature and concentration of acid will produce varying relative amounts of the antibiotics BBM-1 675C and D. Thus, it is desirable to monitor the progress of reaction by thin layer chromatography as described in the examples herein.
When it is desired to prepare only the BBM-1 675D antibiotic, the selective hydrolysis is preferably carried out with an organic acid such as p-toluenesulfonic acid as described herein to yield a quantitative amount of
BBM-1675D.
The BBM-1 675C and BBM-1 675D substances may also be prepared by selective chemical hydrolysis of the antibiotic BBM-1 675A2 as outlined in Scheme 3.
Scheme 3
cMMocH 15 SOCH3 OH Zip BBM-1675C C 30H CH3O 0 BBM-1675A2 855) CH3ONH (mew. 1248) CH 25 0 C H2 OCH3 30 Compound 2 (m.w. 425) (A mixture of a and B anomers) 35 H)/CH30H 40 45 50 CH3 55 BBM-1675D + CH3S z SOCH3 (m.w. 695) OH Compound 3 (m.w. 192) (A mixture of a and ss anomers) The starting 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-1 675A2 likewise proceeds in a stepwise manner with the initial production of the BBM-1 675C antibiotic and the inactive fragment of Formula 2. Continued treatment under hydrolyzing conditions leads to the liberation of a mixture of and 8 anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM-1 675D.
The reaction conditions utilized for the selective chemical hydrolysis of BBM-1 675A2 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 BBM-1675A,, when it is preferred to produce only the BBM-1675D antibiotic, the hydrolysis of BBM-1675A2 is carried out until substantially all of BBM-1 675A2 and BBM-1 675C is converted to
BBM-1 675D. Most preferably, the hydrolysis is carried out with an organic acid such as p-toluenesulfonic acid.
The discovery, as described herein, that the same BBM-1675C and D antibiotics are produced from two different antibiotics BBM-1 675A, and BBM-1675A2 with the concurrent loss of two inactive fragments of
Formulas 1 and 2, respectively, and the thiosugar of Formula 3, provides an additional advantage for the present invention. Accordingly, in a further aspect of the present invention, there is provided a process for the selective hydrolysis of a mixture of BBM-1 675Aa and A2 to produce BBM-1 675C and D as illustrated in Scheme 4.
Scheme 4
This advantage becomes apparent when one considers that the relative amounts of BBM-1675A, and A2 produced in the fermentation process is subject to variability. The production of BBM-1 675C and D is therefore independent of the relative amounts of BBM-1675Ar andA2 utilized as starting material in the present invention.
As described herein, the hydrolysis of the BBM-1 675A1, 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-1 675C antibiotic and hence, for the BBM-1675A, and A2 antibiotics. The compound of
Formula 3 was identified as a mixture of a and ss 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 cl and ss anomers, were separated.The proton magnetic resonance spectra (360 MHz) of the compound 3A (cr-anomer) and compound 3B (13-anomer) are shown in Figures 1 1A and 11 B, respectively. From an analysis of the spectral data, the thiosugar methyl glycosides of Formula 3 were tentatively assigned the relative stereochemistry of the formula
At the present time, the absolute stereochemistry, i.e. D or L, has not yet been determined. Accordingly, based on the present interpretation of the spectral data, it is concluded that the thiosugar of Formula 3 (less the 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-1675A' and A2 antibiotics.
Physico-chemical Properties of BBM- 7675C Description: amorphous solid
Ultraviolet absorption spectrum: See Figure 1
Instrument: Hewlett-Packard 8458
Solvent: methanol
Concentration: 0.0155 g/l #max(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 Figure 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-.
Mass spectrum: See FigureS Instrument: Finigan 4500 TSQ Method: fast atom bombardment (FAB) ionization
Molecular Relative
Matrix m/z lon Abundance glycerol 856 [M+H]+ 100% glycerol + NaCI 876 [M + Na] + 100% dithiothreitol:dithioerythritol 856 [M+H+ 100%
(3:1) (w:w)
Instrument: Kratos MS-50
High resolution FAB (m/z): [M+H]+ = 856.3362
Molecular weight: apparent MW = 855 (based on above-described high resolution data)
Elemental composition: C3sH6,N3014S3 (based on above-described high resolution data)
Proton Magnetic Resonance Spectrum: See Figure 7
Instrument:WM 360 Bruker
Solvent: CDCI3 'H NMR 360 MHz 6 (ppm): 6.54 (1 H,dd,J =7.7,7.0);6.21 (1 H, brs); 5.87 (1 H, d,J=9.6); 5.78 (1 H,dd,J =9.6,1.5); 5.66 (1 H, 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 (1 H, dt J = 10.3, 4.0); 3.37 (3H, s); 2.77 (1 H, m); 2.69 (1 H, dt, J = 9.9, 5.2); 2.49 (1 H,
dd, J = 10.3,2.6); 2.48 (3H, s); 2.30 (2H, m); 2.13 (1 H, 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).
13C Magnetic Resonance Spectrum: See Figure 9
Instrument: WM 360 Bruker
Solvent: CDCI3 13C NMR 90.6 MHz 5 (ppm): 13.7, 17.5, 19.8, 22.3,22.7,23.5,34.2, 36.2,39.6,47.7, 62.7, 66.8, 66.1,67.7, 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.
Physico-chemical Properties of BBM- 1 675D Description: amorphous solid
Ultraviolet absorption spectrum: See Figure 2
Instrument: Hewlett-Packard 8458
Solvent: methanol
Concentration: 0.01 g/l #max(n (nm) absorptivities
214 27,000
274 12,800 325 5,400
No significant change is observed with acid or base.
infrared absorption spectrum: See Figure 4
Instrument: Nicolet 5DX FT-IR
Major absorption bands (KBr, film):
735, 755, 910, 960, 1000, 1020. 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385, 1445, 1510, 1685, 1720, 1735, 2880, 2930, 2960, 3400 cm-.
Mass spectrum: See Figure 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 (m/z): [M+H]+ = 696.2794
Molecular weight: apparent MW = 695 (based on above-described high resolution data)
Elemental composition: C29H49N3012S2
(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 Figure 8
Instrument: WM 360 Bruker
Solvent: CDCI3 + 10% CD3OD 1H NMR 360 MHz 5 (ppm): 6.43(1H,dd,J=4.4,10.3);6.13(1H,s);5.81 6.81(1 H,d,J=8.8); 5.70(1 H,d,J=8.8); 6.48(1 H,6 brs);4A8 (1 H, d, J =8.1); 4.02 (1 H, d, J =2.0); 3.95-3.80 (solvent background); 3.77 (1 H, t, J =9.0); 3.70-3.40 (11 H,
brm); 3.35 (1 H, 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 (1 H, brdt); 1.22 (3H, d, J = 5.9); 0.94 (3H, d, J = 6.6); 0.87 (3H, d, J = 5.9).
13C Magnetic resonance spectrum: See Figures 1 OA and 1 0B
Instrument: WM 360 Bruker
Solvent: CDCIs + 10%CD3OD 13C NMR 90.6 MHz 5 (ppm): 17.6, 21.6,22.2,23.0, 33A, 39.2, 46A, 62.3, 66.8, 62.1, 67.8, 69.8,70.1 ,71.3,75.8, 77.1,78.1 , 82A, 83.3, 5 88.2,97.4,99.6, 122.6, 124.8, 130.1, 130.8, 134.3, 148.7, 192.8.
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 results of an antimicrobial screening
procedure involving the parent BBM-1 675A1 component and the BBM -1675C and BBM -1 675D 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 I, the BBM-1675C
and D substances showed a broad spectrum of antimicrobial activity which were at least as effective as the
BBM-1675A1 component; and in particular, the BBM-1 676C and D substances were more effective as
inhibitors of gram-negative organisms.
TABLE I
Antimicrobial activity of BBM- 1675 substances
Zone of inhibition, mm
Test Microorganism BBM- 1675A, BBM-1675C BBM- 1675D Escherichia coliAS 19 22 52 51
Escherichia coli K 12 13 36 35 Fscherici7ia coli P 1373 12 34 33 EscherichiacoliRAzaserine 14 35 34
Escherichia coli R Netropsin 11 32 32
Escherichia coll 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
Zone of inhibition, mm
Test Microorganism BBM- 1675A, BBM- 1675C BBM- 1675D
Escherichia coli R Aclacinomycin 15 41 40 Bacillus subtllis 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 P1 377 30 39 38
Streptococcus aureus Smith P 36 47 45 Staphylococcus aureus Smith R 40 55 53 Actinomycin D Stapf7ylococcus aureus Smith R 17 32 31 Aureolic acid
Acinetobacter 16 33 32 Micrococcus luteus 35 57 55 Saccharomycescerevisiae petite 22 42 43
R = resistant to named antibiotic
Activity against P-388 Leukemia
Tables II and lil contain the results of laboratory tests with CDF1 mice implanted intraperitoneally with a tumor inoculum of 106 ascites cells of P-388 leukemia and treated with various doses of BBM-1675A, C or D. The substances were administered by intraperitoneal injection. Groups of six mice were used for each dosage amount, and they were treated with a single dose of the substance on the day after inoculation. A group of ten seline treated control mice was included in each series of experiments. The BBM-1675A1 treated group in Table Ill 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-1 675C substance. In Table lil, the results of a direct comparison of BBM-1 675A1 (esporamicinA1) and the BBM-1 675C and BBM-1 675D substances are reported. The data suggest that BBM-1 675C is about comparable to BBM-1 675A1 in potency and antitumor effectiveness and that it is not schedule dependent, while BBM-1 675D is only slightly less effective.
Additionally, it is reported in the present invention that the same substances BBM-1 675C 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-1 675D and the data reported in published U.K. Patent Application No.
2,141,425 for the BBM-1675A2 component, it is surprisingly found that the substances BBM-1 675C and D are mera effective as antitumor agents than the parent BBM-1675A2 component from which they were derived.
TABLE II
Effect of BBM- 1675C on P-388 Leukemia
(Day 1 Treatment)
Effect AWC
Dose, lP MST MST gm Survivors Compound mg/kg/inj. days % TIC Day 4 Day5 BEM-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: 106 ascites cells implanted i.p.
Host: CD F, male mice
Evaluation: MST = median survival time
Effect 96 TIC = (MST treated/MST control) x 100
Criteria: % TIC > 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 Leukemia
Effect AWC
Treatment Dose, IP MST MST gm Survivors
Compound Schedule mg/kg/inj. days % TIC Day4 Day5
BBM-1675A1 d. 1 0.0512 TOX TOX - 0/6
0.0256 TOX TOX - 0/6
0.0128 TOX TOX - 1.8 3/6
0.0064 15.5 172 - 0.3 6/6
0.0032 15.0 167 -0.6 6/6
0.0016 15.5 172 0.6 6/6
0.0008 12.5 139 0.3 6/6
0.0004 12.0 133 1.4 6/6
0.0002 11.0 122 0.8 6/6
0.0001 11.5 128 1.4 6/6
BBM-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 14.5 161 -0.1 6/6
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
0.00005 10.5 117 1.3 6/6 BBM-1675D d. 1 0.0256 9.0 100 0.1 6/6
0.0128 11.5 128 0.3 6/6
0.0064 12.5 139 0.3 6/6
0.0032 12.0 133 0.1 6/6
0.0016 11.5 128 0.8 6/6
0.0008 10.0 111 0.2 6/6
0.0004 10.0 111 0.5 6/6
0.0002 9.5 106 1.7 6/6
0.0001 9.5 106 1.7 6/6
0.00005 9.0 100 2.0 6/6 SBM-1676C d.1o5 0.0032 16.0 178 -1.3 6/6
0.0016 13.5 150 -1.0 6/6
0.0008 13.5 150 -0.3 6/6
0.0004 12.0 133 -0.4 6/6
0.0002 12.0 133 -0.4 6/6
0.0001 11.0 122 -0.4 5/6 0.00005 11.0 122 0.9 6/6
0.000025 8.5 94 2.2 6/6 0.0000125 8.0 89 2.4 6/6
0.00000625 8.0 89 2.4 6/6
Vehicle - 9.0 100 2.4 10/10
Tumor inoculum: 106 ascites cells implanted i.p.
Host: CDF1 female mice
Evaluation: MST = median survival time
Effect: % T/C = (MST treated/MST control) x 100
Criteria: % T/C 2 125 considered significant antitumor activity
AWC: average weight change (treated-control) in grams (on day 4)
Activity against B16 Melanoma Table IV contains results of antitumor tests using the B1 6 melanoma grown in mice. BDF1 mice were employed and inoculated subcutaneously with the tumor implant. A 60-day protocol was used. Groups of ten mice were used for each dosage amount tested, and the mean survival time for each group was determined.
Control animals inoculated in the same way as the test animals and treated with the injection vehicle and no drug exhibited a mean survival time of 22.5 days. For each dosage level, the test animals were treated with the test compound on days 1, 5 and 9 by intraperitoneal injection. An effect in terms of % T/C equal to or greater than 125 indicates that a significant antitumor effect was achieved. The results in Table IV show that in a direct comparison BBM-1675C was also effective in treatment of mice bearing B16 melanoma and was about comparable to BBM-1675A1 in potency.
TABLE IV
Effect of BBM- 1675 substances on B16 melanoma
(Day 1, 5 and 9 Treatments)
Effect AWC
Dose, IP MST MST gm Survivors
Compound mg/kg/inj. days % T/C 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-1i675C 0.0008 31.5 140 0.6 10/10
0.0004 37.0 164 1.2 10/10
0.0002 31.0 138 0.6 10/10
0.0001 31.5 140 1.0 10/10
0.00005 27.5 122 0.8 10/10
0.000025 25.0 111 0.5 10/10
Vehicle - 22.5 100 0.3 10/10
Tumor inoculum: 0.6 my of a 10% brei, IP
Host: BDF1 female mice
Evaluation:MST = median survival time
Effect: % ,rc = (MST treated/MST control) x 100
Criteria: % T/C 2 126 considered significant antitumor activity
AWC: average weight change (treated-control) in grams (on day 12)
As indicated by the antimicrobial and mouse tumor data provided above, BBM-1 675C and BBM-1 675D 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-1676D, our a pharmaceutical composition thereof.
The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of BBM-1 675C or BBM-1 675D in combination with an inert pharmaceutically acceptable carrier or diluent Such compositions may also contain other active antimicrobial or antitumor agents and may be made up in any pharmaceutical form appropriate for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs and preparations for parenterai administration such as sterile aqueous or non-aqueous solutions, suspensions or emuisions.They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.
For use as an antimicrobial agent, the BBM-1 675C or BBM-1 675D, 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-1 676D 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 BBM-1 675C or BBM-1 675D used will vary according to the particular composition formulated, the mode of application and the particular situs, host and disease being treated.Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease. Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines.
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
Chemical Preparation and Isolation of BBM- 1675C and BBM-1675D
Example 1
A sample of BBM-1675A, (50 mg) was dissolved in 2.5 ml of methanol and treated with 2.5 ml of 0.1 molar solution of hydrogen chloride in methanol. The reaction was allowed to proceed at a temperature of about 50"C, and the disappearance of the starting material (approximately 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography (TLC) on silica gel plates (Analtech, 250 micron, GF) with toluene:acetone (3:2, v/v) as the eluting solvent. After the starting material has been consumed, the reaction mixture was neutralized with a saturated solution of 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 10cm column packed with Woelm silica gel (32-63 micron particle size). The column was eluted with toluene:acetone (3:2, v/v) collecting 3 ml fractions. Each fraction was analyzed by TLC
(silica gel with toluene:acetone (3:2, v/v) as eluent), and the TLC spots were visualized with a UV 254 nm light source and a ceric sulfate spray (1% ceric sulfate and 2.5% molybolic acid in 10% sulfuric acid). Fractions 6-12 (R, value for BBM-1 675C is 0.28) were pooled and evaporated to dryness to yield 12 mg (35%) of substantially
pure BBM-1675C.
The physico-chemical properties of BBM-1 676C appear in the specification and the ultraviolet, infrared, mass, 'H NMR and 13C 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-1 676C 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-1 676C 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 BBNí-1675D substance exhibits one major spot at Rf = 0.37 in reverse phase TLC (Whatman MKC18F, 20C micron) using 30% water in methanol as the eluent and Rf = 0.22 in normal phase silica gel TLC using chloroform:methanol (5:0.S, 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-1 675A2 or BBM-1675Aa as illustrated by the procedures of Examples 3 and 5, respectively.
A sample of BBM-1 676A2 (15.2 mg) was hydrolyzed with 0.03 molar solution of p-toluenesulfonic acid in methanol (1 ml) at a temperature of about 63"C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30 C. The BBM-1 676D substance was isolated from the dry residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size). The column was eluted with chloroform:methanol (5:0.5, v/v), and the collected fractions were analyzed by TLC silica gel with chloroform:methanol (6:0.5, v/v) as eluent].The applied chromatography conditions permitted the separation of the mixture of inactive compounds 2 and3 (7 mg) from the bioactive BBM-1 676D 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 BBM-1675D in near quantitative yield.
The physico-chemical properties of BBM-1675D appear in the specification and the ultraviolet, infrared, mass, 1H NMR and 13C NMR spectra of the compound appear as Figures 2,4, 6,8 and combined 10A and lOB, respectively.
Example 4
A sample of BBM-1 676A2 (40 mg) was treated with 5 ml of an 0.5 molar solution of hydrogen chloride in methanol at about 50"C for about 2 hours according to the general procedure and isolation method described in
Example 1. After neutralization with NaHCO3 and evaporation to dryness, the BBM -1 675C substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) using toluene:acetone (3:2, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 8.4 mg of substantially pure BBM-1675C which is identical to the product isolated in Example 1.
The chromatographic column of above was then eluted with chloroform:methanol (5:0.25, v/v) and the fractions collected were pooled and evaporated to dryness to yield BBM-1675D. The BBM-1675D substance was further purified by an additional flash chromatography column with silica gel utilizing chloroform:methanol (5:0.5, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 6.3 mg of substantially pure BBM-1676D which is identical to the product isolated in Example 3.
Example 5
A sample of BBM-1675A1 (49.3 mg) was hydrolyzed with 0.037 M solution of p-toluenesulfonic acid in methanol (1.5 ml) at a temperature of about 60"C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 30"C to give a residue which contains BBM-1 676D 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 size) utilizing chloroform:methanol (5:0.25, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 27mg of substantially pure BBM-1 676D which is identical to the product isolated in Example 3.
Example 6
A sample of BBM-1 676C (5.1 mg) was hydrolyzed with 0.5 molar solution of hydrogen chloride in methanol (1 ml) at about 40-50"C overnight. After neutralization with NaHCO3 and evaporation to dryness, the
BBM-1675D bioactive substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) utilizing chloroform:methanol (5:0.25, v/v) as the eluent. The appropriate fractions yielded substantially pure BBM-1 676D which is identical to the product isolated in Example 3.
Example 7
When the general procedure of Examples 1 and 2 are repeated, except that the starting material BBM-1 676A1 is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, there is thereby produced the BBM-1676C and BBM-1 676D subtances.
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-1675Ar and BBM-1675A2, there is thereby produced the BBM-1675D substance.
Claims (20)
1. The antitumor antibiotic BBM-1 676C which in substantially pure form:
(a) appears as an amorphous solid;
(b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform;
(c) exhibits in silica gel thin layer chromatography an Rf value of 0.28 with the solvent system
tcluene: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 shown in Figure 1
exhibiting ultraviolet absorption maxima and absorptivities at 210 nm (a = 21,770), 274 nm (a = 9,340)
and 313 nm (shoulder) (a = 4,190) with no significant change upon addition of acid or base;
(f) has an infrared absorption spectrum (KBr, film) substantially as shown in Figure 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 Figure 5 exhibiting a molecular ion [M+H]+ of 856;
(h) has a 360 MHz proton magnetic resonance spectrum in CDCI3 substantially as shown in Figure 7
exhibiting signals at
6.54 (1 H, dd, J=7.7, 7.0); 6.21 H, brs); 5.87 (1 H, d, J = 9.6);5.78 (1 H, dd, J =9.6, 1 .5); 5.66 (1 H, brd, J = 2.9);
4.94 (1 H, dd, J= 10.3, 1.8); 4.61(1 H, d, 7.7); 4.25 (1 H,
s); 4.09 (1 H, q, J = 2.6); 3.97 (1 H, t, J = 9.6); 3.92-3.53 (1 OH), 3.45 (1 H, dt, J = 10.3, 4.0); 3.37 (3H, s); 2.77 (1 H,
m); 2.69 (1 H, dt, J = 9.9, 5.2); 2.49 (1 H, dd, J = 10.3, 2.6);
2.48 (3H, s); 2.30 (2H, m);
2.13 (1 H, 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 tetramethylsilane;
(i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCI3 substantially as shown in Figure 9
exhibiting signals at 13.7, 17.5, 19.8, 22.3, 22.7, 23.5, 34.2, 35.2, 39.5, 47.7,
52.7, 55.8, 56.1, 57.7, 62.4, 64.7, 67.4, 69.3, 69.8, 71.9,
76.1, 77.1, 77.7, 79.7, 83.2, 88.4, 97.3, 99.7, 123.4,
124.6, 130.1, and 193.1 parts per million downfield from
tetramethylsilane.
2. The antitumor antibiotic BBM-1675D which in substantially pure form: (a) appears as an amorphous solid; (b) is soluble in methanol, ethanol, acetone and tetrahydrofuran, and slightly soluble in chloroform; (c) exhibits in silica gel thin-iayer 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 shown in Figure 2 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 shown in Figure 4 exhibiting principal absorption peaks at 735,755, 910, 960,1000,1020,1085,1150,1195,
1250,1310,1336,1366, 1386,1446,1610,1685, 1720, 1735, 2880, 2930, 2960, and 3400 reciprocal
centimeters;;
(g) has a low resolution mass spectrum substantially as shown in Figure 6 exhibiting a molecular ion [M + H] + of 696;
(h) has a 360 MHz proton magnetic resonance spectrum in CDCI3 + 10% CD3OD substantially as shown in
Figure 8 exhibiting signals at 6.43 (1 H, dd, J = 4.4, 1 0.3); 6.1 3 (1 H, s); 5.81 (1 H, d,
J=8.8); 5.70 (1 H, d, J=8.8); 5.48 (1 H, 6 brs); 4.48 (1 H, d,
J = 8.1); 4.02 (1 H, d, J = 2.0); 3.95-3.80 (solvent background);
3.77 (1 H, t, J = 9.0); 3.70-3.40 (11 H, brm); 3.35 (1 H, 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 (1 H, brdt);
1.22 (3H, d, J =6.9); 0.94 (3H, d, J = 6.6); and 0.87 (3H, d, J = 5.9) parts per
million downfield from tetramethylsilane;
(i) has a 90.6MHz carbon-13 magnetic resonance spectrum in CDCI3 + 10% CD3OD substantially as shown in
Figure 10 (Figure 1 OA + 1 OB) 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 antibiotic BBM-1675C, which comprises hydrolyzing
BBM-1675A, 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-1675A, or BBM-1 675A2 with a mineral or organic acid until a substantial amount of BBM-1675D is produced and then recovering BBM-1675D from the reaction medium.
5. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing BBM-1 676C with a mineral or organic acid until a substantial amount of BBM-1 676D 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-1675A, and BBM-1 675A2 with a mineral or organic acid until a substantial amount of
BBM-1675C is produced and then recovering BBM-1 676C from the reaction medium.
7. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing a mixture of BBM-1675A, 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 reaction medium.
8. The antitumor antibiotic BBM-1675C, substantially as hereinbefore specifically described with particular reference to the examples.
9. The antitumor antibiotic BBM-1679D, substantially as hereinbefore specifically described with particular reference to the examples.
10. A process for the production of BBM-1675C, substantially as hereinbefore specifically described with particular reference to the examples.
11. A process for the production of BBM-1675D, substantially as hereinbefore specifically described with particular reference to the examples.
12. A pharmaceutical composition comprising an effective antimicrobial amount of BBM-1675C or
BBM-1675D in combination with a pharmaceutical carrier or diluent.
13. A pharmaceutical compostion comprising an effective tumor-inhibitng amount of BBM-1 676C or BBM-1676D in combination with a pharmaceutical carrier or diluent.
14. A pharmaceutical compound as defined in claim 1 for use in a method of treatment of the human or animal body.
15. A pharmaceutical compound as defined in claim 2 for use in a method of treatment of the human or animal body.
16. A compound according to claims 1 or 2 for use as an antitumor antibiotic.
17. A compound according to either of claims 1 or 2 substantially as hereinbefore specifically described in each of the examples for the use hereinbefore specifically described.
18. A pharmaceutical composition according to either of claims 12 or 13 substantially as hereinbefore specifically described in examples.
19. A compound when produced by a process as claimed in any one of claims 3 to 7, 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.
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Cited By (8)
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EP0276485A2 (en) * | 1987-01-30 | 1988-08-03 | American Cyanamid Company | Dihydro derivatives of LL-E33288 antibiotics |
US4916065A (en) * | 1988-06-10 | 1990-04-10 | Bristol-Myers Company | BU-3420T Antitumor antibiotic |
EP0431323A1 (en) * | 1989-11-06 | 1991-06-12 | Bristol-Myers Squibb Company | Antitumor antibiotic BU-3983T |
US5028536A (en) * | 1989-03-15 | 1991-07-02 | Bristol-Myers Squibb Company | Antitumor antibiotic BMY-41339 |
EP0454494A2 (en) * | 1990-04-27 | 1991-10-30 | Yale University | Calicheamicinone, derivatives and analogs thereof and methods of making the same |
US5086045A (en) * | 1989-03-15 | 1992-02-04 | Bristol-Myers Squibb Company | Antitumor antibiotic |
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 |
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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 IE IE228086A patent/IE59204B1/en not_active IP Right Cessation
- 1986-08-26 BE BE0/217084A patent/BE905332A/en not_active IP Right Cessation
- 1986-08-26 NL NL8602165A patent/NL8602165A/en not_active Application Discontinuation
- 1986-08-26 ES ES8601355A patent/ES2002728A6/en not_active Expired
- 1986-08-26 LU LU86562A patent/LU86562A1/en unknown
- 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 DK DK406086A patent/DK170671B1/en not_active IP Right Cessation
- 1986-08-26 FR FR868612085A patent/FR2586686B1/en not_active Expired - Fee Related
- 1986-08-26 KR KR1019860007092A patent/KR920010226B1/en not_active IP Right Cessation
- 1986-08-26 CH CH3417/86A patent/CH668598A5/en not_active IP Right Cessation
- 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 PT PT83261A patent/PT83261B/en unknown
- 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
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1994
- 1994-08-19 JP JP6195156A patent/JPH07233186A/en active Pending
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0276485A2 (en) * | 1987-01-30 | 1988-08-03 | American Cyanamid Company | Dihydro derivatives of LL-E33288 antibiotics |
EP0276485A3 (en) * | 1987-01-30 | 1990-09-12 | American Cyanamid Company | Dihydro derivatives of ll-e33288 antibiotics |
EP1215212A2 (en) * | 1987-01-30 | 2002-06-19 | American Cyanamid Company | Dihydro derivatives of LL-E33288 antibiotics |
EP1215212A3 (en) * | 1987-01-30 | 2003-05-21 | Wyeth Holdings Corporation | Dihydro derivatives of LL-E33288 antibiotics |
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 |
EP0431323A1 (en) * | 1989-11-06 | 1991-06-12 | Bristol-Myers Squibb Company | Antitumor antibiotic BU-3983T |
EP0454494A2 (en) * | 1990-04-27 | 1991-10-30 | Yale University | Calicheamicinone, derivatives and analogs thereof and methods of making the same |
EP0454494A3 (en) * | 1990-04-27 | 1993-05-26 | Yale University | 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 |
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Legal Events
Date | Code | Title | Description |
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PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19960819 |