US 4,038,384 AGranted

Antibiotic A-28086 and process for production thereof

AssigneeEli Lilly and Company

InventorsDavid H. Berg, Robert L. Hamill, Marvin M. Hoehn

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Abstract

Antibiotic A-28086 complex, comprising microbiologically active, structurally related factors A, B, and D, produced by submerged aerobic fermentation of Streptomyces aureofaciens NRRL 5758. Individual factors A, B, and D are separated and isolated by chromatography. The A-28086 antibiotics are antibacterial, antifungal, antiviral, anti-PPLO, anticoccidial, insecticidal and acaricidal agents and also increase feed-utilization efficiency in ruminants.

Claims11

Dependent claims

Antibiotic A-28086 factor A which is a white crystalline compound when crystallized from acetone-water;

which is soluble in lower alcohols, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene;
but which is only slightly soluble in hexane;
and is insoluble in water;
which melts at about 98.degree.-100.degree. C., resolidifies, and remelts at about 195.degree.-200.degree. C. and which has:
a. a molecular weight of 764, as determined by mass spectrometry;
b. an approximate elemental composition of 66.69 percent carbon, 9.85 percent hydrogen, and 23.10 percent oxygen;
c. an empirical formula of C.sub.43 H.sub.72 O.sub.11, as determined by mass spectrometry;
d. a specific rotation of -54.degree. (c=0.2, methanol) when determined at 25.degree. C.;
e. an infrared absorption spectrum in chloroform with the following distinguishable absorption maxima:
2.85, 3.34, 5.83, 6.82, 7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (strong), 9.55 (strong), 9.60, 9.85, 10.15, 10.45, and 10.70 (weak) microns;
f. in its ultraviolet spectrum in ethanol only end absorption below 220 m.mu.;
g. a nuclear magnetic resonance spectrum in deuterochloroform with the following characteristics:
.delta.6.01, 4.21, 4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3.55, 2.83, 2.76, 2.74, 2.68, 2.66, 2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05, 1.96, 1.90, 1.85, 1.70, 1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18, 0.95, 0.93, 0.90, 0.88, 0,85, 0.77, 0.75, 0.73, 0.68, and 0.66 ppm;
h. a titratable group with a pK.sub.a value of 7.9 in 80% aqueous dimethylformamide;
i. a characteristic X-ray powder diffraction pattern (Cu.sup.+.sup.+ radiation, 1.5405 .lambda., nickel filter) having the following interplanar spacings in angstroms (d):
______________________________________ Relative d Intensity ______________________________________ 12.00 100 10.10 50 9.25 90 8.00 40 7.50 15 6.92 90 6.40 40 5.98 05 5.68 15 5.20 40 4.98 40 4.62 40 4.21 20 3.48 10 ______________________________________
j. an R.sub.f value of 0.24 on thin-layer chromatography over silica gel in a benzene-ethyl acetate (3:2) solvent system, using Bacillus subtilis ATCC 6633 as the detection organism;
k. the following R.sub.f values in the paper-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as detection organism:
______________________________________ R.sub.f Value Solvent System ______________________________________ 0.11 Water saturated with methyl iso- butyl ketone (MIKB) 0.41 Water saturated with MIBK plus 2% p-toluenesulfonic acid and 1% piperidine 0.54 Water:methanol:acetone (12:3:1)- adjusted to pH 10.5 with NH.sub.4 OH and then lowered to pH 7.5 with H.sub.3 PO.sub.4 0.48 1% MIBK, 0.5% NH.sub.4 OH in water 0.15 17.4 g K.sub.2 HPO.sub.4, 30 ml ethanol per liter of water 0.24 Benzene saturated with water 0.24 Water 0.75 water:MIBK:ethyl acetate (98:1:1) ______________________________________
l. an acid function capable of forming salts and ester derivatives;
and
m. at least one hydroxyl group capable of esterification;
an acyl-ester derivative selected from the group consisting of
1. A-28086 factor A acetyl ester derivative which is a white crystalline compound (from acetone-water);
which is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene;
is only slightly soluble in hexane;
and is insoluble in water;
which melts at about 100.degree.-103.degree. C.;
and which has:
a'. a molecular weight of about 807;
b'. an elemental composition of 67.67 percent carbon, 8.71 percent hydrogen, and 23.13 percent oxygen;
c'. an empirical formula of C.sub.45 H.sub.74 O.sub.12 ;
d'. an infrared spectrum in chloroform with the following observable absorption maxima:
2.85, 3.36, 3.38 (strong), 5.80, 6.83, 7.25, 7.52 (strong), 7.60 (weak), 7.80 (strong), 8.45 (strong), 8.80 (strong), 8.95 (strong), 9.10 (strong), 9.20, 9.63, 9.80 (strong), 10.12 (weak), 10.25 (weak), and 10.50 microns;
e'. an ultraviolet spectrum showing only end absorption;
f'. a nuclear magnetic resonance spectrum in which the carbinyl resonance is shifted downfield to approximately 5.3 ppm;
g'. a titratable group with a pK.sub.a value of 8.5 in 80% aqueous dimethylformamide;
h'. an acid function capable of forming salts and ester derivatives;
2.

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A-28086 factor A butyryl ester derivative which is a white crystalline compound (from acetone-water);

which is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene;
is slightly soluble in hexane;
and is insoluble in water;
which melts about 96.degree.-98.degree. C.;
and which has:
a'". a molecular weight of about 835;
b'". an approximate elemental composition of 67.60 percent carbon, 9.41 percent hydrogen, and 22.99 percent oxygen;
c'". an empirical formula of C.sub.47 H.sub.78 O.sub.12 ;
d'". an infrared spectrum in chloroform with the following observable absorption maxima:
2.89, 3.40, 3.45, 3.51, 5.85, 5.92 (strong), 5.97 (strong), 6.90, 7.30, 7.84 (weak), 8.55, 8.85 (weak), 9.01 (strong), 9.26, 9.76, 9.95, 10.31, and 10.64 microns;
e'". a nuclear magnetic resonance spectrum in which the carbinyl resonance is shifted downfield to approximately 5.3 ppm;
f'". an acid function capable of forming salts and ester derivatives;
4.

10 more elements

Dependencies are inferred from the claim text.

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Abstract

Antibiotic A-28086 factor A which is a white crystalline compound when crystallized from acetone-water;

which is soluble in lower alcohols, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene;
but which is only slightly soluble in hexane;
and is insoluble in water;
which melts at about 98.degree.-100.degree. C., resolidifies, and remelts at about 195.degree.-200.degree. C. and which has:
a. a molecular weight of 764, as determined by mass spectrometry;
b. an approximate elemental composition of 66.69 percent carbon, 9.85 percent hydrogen, and 23.10 percent oxygen;
c. an empirical formula of C.sub.43 H.sub.72 O.sub.11, as determined by mass spectrometry;
d. a specific rotation of -54.degree. (c=0.2, methanol) when determined at 25.degree. C.;
e. an infrared absorption spectrum in chloroform with the following distinguishable absorption maxima:
2.85, 3.34, 5.83, 6.82, 7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (strong), 9.55 (strong), 9.60, 9.85, 10.15, 10.45, and 10.70 (weak) microns;
f. in its ultraviolet spectrum in ethanol only end absorption below 220 m.mu.;
g. a nuclear magnetic resonance spectrum in deuterochloroform with the following characteristics:
.delta.6.01, 4.21, 4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3.55, 2.83, 2.76, 2.74, 2.68, 2.66, 2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05, 1.96, 1.90, 1.85, 1.70, 1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18, 0.95, 0.93, 0.90, 0.88, 0,85, 0.77, 0.75, 0.73, 0.68, and 0.66 ppm;
h. a titratable group with a pK.sub.a value of 7.9 in 80% aqueous dimethylformamide;
i. a characteristic X-ray powder diffraction pattern (Cu.sup.+.sup.+ radiation, 1.5405 .lambda., nickel filter) having the following interplanar spacings in angstroms (d):
______________________________________ Relative d Intensity ______________________________________ 12.00 100 10.10 50 9.25 90 8.00 40 7.50 15 6.92 90 6.40 40 5.98 05 5.68 15 5.20 40 4.98 40 4.62 40 4.21 20 3.48 10 ______________________________________
j. an R.sub.f value of 0.24 on thin-layer chromatography over silica gel in a benzene-ethyl acetate (3:2) solvent system, using Bacillus subtilis ATCC 6633 as the detection organism;
k. the following R.sub.f values in the paper-chromatographic systems indicated below, using Bacillus subtilis ATCC 6633 as detection organism:
______________________________________ R.sub.f Value Solvent System ______________________________________ 0.11 Water saturated with methyl iso- butyl ketone (MIKB) 0.41 Water saturated with MIBK plus 2% p-toluenesulfonic acid and 1% piperidine 0.54 Water:methanol:acetone (12:3:1)- adjusted to pH 10.5 with NH.sub.4 OH and then lowered to pH 7.5 with H.sub.3 PO.sub.4 0.48 1% MIBK, 0.5% NH.sub.4 OH in water 0.15 17.4 g K.sub.2 HPO.sub.4, 30 ml ethanol per liter of water 0.24 Benzene saturated with water 0.24 Water 0.75 water:MIBK:ethyl acetate (98:1:1) ______________________________________
l. an acid function capable of forming salts and ester derivatives;
and
m. at least one hydroxyl group capable of esterification;
an acyl-ester derivative selected from the group consisting of
1. A-28086 factor A acetyl ester derivative which is a white crystalline compound (from acetone-water);
which is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene;
is only slightly soluble in hexane;
and is insoluble in water;
which melts at about 100.degree.-103.degree. C.;
and which has:
a'. a molecular weight of about 807;
b'. an elemental composition of 67.67 percent carbon, 8.71 percent hydrogen, and 23.13 percent oxygen;
c'. an empirical formula of C.sub.45 H.sub.74 O.sub.12 ;
d'. an infrared spectrum in chloroform with the following observable absorption maxima:
2.85, 3.36, 3.38 (strong), 5.80, 6.83, 7.25, 7.52 (strong), 7.60 (weak), 7.80 (strong), 8.45 (strong), 8.80 (strong), 8.95 (strong), 9.10 (strong), 9.20, 9.63, 9.80 (strong), 10.12 (weak), 10.25 (weak), and 10.50 microns;
e'. an ultraviolet spectrum showing only end absorption;
f'. a nuclear magnetic resonance spectrum in which the carbinyl resonance is shifted downfield to approximately 5.3 ppm;
g'. a titratable group with a pK.sub.a value of 8.5 in 80% aqueous dimethylformamide;
h'. an acid function capable of forming salts and ester derivatives;
2.

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