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US 4,122,168 AGranted
Antibiotic A-35512 and process for production thereof
AssigneeEli Lilly and Company
InventorsKarl H. Michel, Calvin E. Higgens
Life of this patent
Estimating the term…
Abstract
Antibiotic A-35512 complex, comprising microbiologically active, related factors A, B, C, E and H, is produced by submerged aerobic fermentation of Streptomyces candidus NRRL 8156. The individual A-35512 factors are separated and isolated by chromatography. The A-35512 antibiotics are antibacterial and growth-promoting agents and increase feed-utilization efficiency in animals. In addition, A-35512 factor B is useful in the treatment of dental caries and acne.
Claims9
Antibiotic A-35512 factor A which is a white, amorphous, basic compound, having an approximate elemental composition of 54.29 percent carbon, 5.19 percent hydrogen, 5.58 percent nitrogen, 33.76 percent oxygen, and 1.69 percent chlorine;
- and which in its dihydrochloride form is a white amorphous compound which has these characteristics:
- (a) an approximate elemental composition of 51.03 percent carbon, 5.10 percent hydrogen, 4.75 percent nitrogen, 34.20 percent oxygen, and 4.80 percent chlorine;
- (b) an infrared absorption spectrum in KBr pellet with significant absorption maxima at the following frequencies (cm.sup.-1) :
- 3405 (strong), 3300 (shoulder), 2950 (weak), 1750 (weak), 1670 (strong), 1625 (shoulder), 1602 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1405 (weak), 1345 (shoulder), 1312 (medium), 1225 (medium), 1180 (weak), 1130 (weak), 1080 (strong), and 1020 (shoulder);
- (c) ultraviolet absorption spectra, in acidic and neutral methanol, with an absorption maximum at 282 nm (.epsilon. 11,700), and, in basic methanol, an absorption maximum at 292 nm (.epsilon. 14,000), and with end absorption at 225 nm;
- (d) a molecular weight of about 2106, as determined by titration;
- (e) four titratable groups in 66% aqueous dimethylformamide with pK.sub.a values of approxiamtely 7.35, 9.09, 10.49, and 12.44;
- (f) the following specific rotations:
- [.alpha]sub.D.sup.25 -100 (c 1, H.sub.2 O)
- [.alpha]sub.365.sup.25 -400 (c 1, H.sub.2 O)
- (g) is soluble in water, partially soluble in methanol and ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane;
- (h) an amino-acid analysis indicating the presence of at least five amino-acid residues, one of which is glycine;
- (i) an R.sub.f value of approxiamtely 0.21 on paper chromatography in a 1-butanol:pyridine:acidic acid:water (15:10:3:12) solvent system, using Sarcina lutea as the detection organism;
- (j) a .sup.13 C nuclear-magnetic-resonance spectrum in D.sub.2 O with the following characteristics:
- ______________________________________ No. PPM Height (%) ______________________________________ 3 175.3 0.8 4 173.0 2.1 5 172.1 2.0 6 171.4 1.5 7 170.9 2.7 8 170.5 2.3 9 169.5 1.6 10 159.0 1.3 11 157.9 2.3 12 156.2 2.6 13 155.6 2.3 14 155.3 2.4 15 154.4 1.1 16 136.3 1.4 17 136.0 1.0 18 135.1 1.4 19 133.5 1.2 20 129.6 1.6 21 129.1 1.7 22 128.7 1.8 23 127.5 1.0 24 126.0 1.4 25 124.3 2.8 26 122.1 1.6 27 109.9 1.3 28 107.4 1.6 29 101.7 0.9 30 77.6 3.8 31 76.3 4.6 32 75.5 2.6 33 74.8 2.5 34 74.5 2.4 35 73.4 3.7 36 72.8 6.0 37 72.0 4.4 38 70.9 7.0 39 69.6 2.8 40 67.4 90.9* 41 65.4 1.7 42 61.7 3.1 43 56.7 1.7 44 55.5 1.3 45 54.7 0.8 46 24.6 0.9 47 19.1 1.7 48 17.9 2.0 49 17.2 1.9 50 16.7 3.2 51 16.2 3.0 ______________________________________
- (k) is stable for 72 hours in aqueous solutions having a pH of from about 3 to about 10;
- or a pharmaceutically-acceptable acid-addition salt of A-35512 factor A.
14 more elements
Antibiotic A-35512 factor B which is a white amorphous, basic compound having:
- (a) an approximate elemental composition of 53.97 percent carbon, 4.75 percent hydrogen, 5.25 percent nitrogen, 34.29 percent oxygen, and 1.59 percent chlorine;
- (b) ultraviolet absorption spectra, in acidic and neutral methanol, with an absorption maximum at 282 nm (.epsilon. 15000) and, in basic methanol, an absorption maximum at 292 nm (.epsilon. 16000);
- (c) a molecular weight of about 2143, as determined by titration;
- (d) four titratable groups in 66% aqueous diemthylformamide with pK.sub.a values of approximately 7.15, 8.81, 10.20, and 12.00;
- (e) the following specific rotations:
- [.alpha]sub.D.sup.25 -123.degree. (c 1, H.sub.2 O)
- [.alpha]sub.36.sup. 25 -446.degree. (c 1, H.sub.2 O);
- (f) an empirical formula in the range of
- C.sub.97-99 H.sub.101-105 N.sub.8-9 O.sub.46-48 C1;
- and which in its dihydrochloride form is a white, hydroscopic, crystalline compound (from 50 percent aqueous methanol) which has these characteristics:
- (a') an approximate elemental composition of 52.57 percent carbon, 4.80 percent hydrogen, 5.66 percent nitrogen, 32.86 percent oxygen, and 4.51 percent chlorine;
- (b') an infrared absorption spectrum in KBr pellet with significant absorption maxima at the following frequencies (cm.sup.-1 ):
- 3420 (strong), 3300 (shoulder), 2950 (weak), 1752 (weak), 1675 (strong), 1630 (shoulder), 1605 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1410 (weak), 1345 (shoulder), 1312 (medium), 1225 (medium), 1180 (weak), 1135 (weak), 1080 (strong), and 1020 (weak);
- (c') ultraviolet absorption spectra in acidic and neutral methanol, with an absorption maximum at 282 nm (.epsilon. 12,000) and, in basic methanol, an absorption maximum at 292 nm (.epsilon. 14,000), and with end absorption at 225 nm;
- (d') a molecular weight of about 2027, as determined by titration;
- (e') four titratable groups in 66% dimethylformamide with pK.sub.a values of approximately 7.15, 8.87, 10.30, and 12.10;
- (f') the following specific rotations:
- [.alpha]sub.D.sup.25 -128.degree. (c 1, H.sub.2 O)
- [.alpha]sub.36.sup. 25 -475.degree. (c 1, H.sub.2 O);
- (g') is soluble in water, partially soluble in methanol and ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane;
- (h') an amino-acid analysis indicating the presence of at least five amino-acid residues, one of which is glycine;
- (i') an R.sub.f value of approximately 0.34 on paper chromatography in a 1-butanol:pyridine:acetic acid:water (15:10:3:12) solvent system, using Sarcina lutea as the detection organism;
- (j') a .sup.13 C nuclear-magnetic-resonance spectrum in D.sub.2 O with the following characteristics:
- ______________________________________ No. PPM Height (%) ______________________________________ 2 173.0 4.1 3 171.9 3.7 4 171.6 3.3 5 171.0 5.8 6 170.8 5.0 7 169.6 3.6 8 159.0 4.1 9 157.9 4.4 10 157.5 3.7 11 156.6 4.8 12 155.6 6.1 13 155.3 4.2 14 154.9 3.3 15 154.3 4.2 16 151.7 3.3 17 144.3 3.1 18 136.7 3.5 19 136.1 4.9 20 135.4 4.0 21 135.2 4.4 22 133.6 4.2 23 133.3 4.1 24 129.8 1.7 25 129.3 3.0 26 128.8 2.6 27 127.6 1.5 28 126.1 3.9 29 124.2 5.6 30 122.4 1.4 31 122.0 4.4 32 120.7 3.3 33 116.5 2.7 34 109.5 0.8 35 108.2 1.1 36 107.7 2.7 37 104.5 1.7 38 101.8 2.9 39 100.9 1.6 40 98.3 1.0 41 76.9 1.2 42 76.1 1.8 43 74.1 2.0 44 73.5 2.7 45 72.7 2.4 46 72.3 4.0 47 71.0 7.1 48 70.3 2.5 49 69.7 2.5 51 64.6 1.2 52 62.0 1.5 53 58.0 1.3 54 56.8 1.7 55 55.4 3.9 56 54.3 2.5 57 24.5 2.0 58 17.9 3.0 59 17.2 2.0 60 16.3 2.5 ______________________________________
- (k') is stable for as much as 72 hours in aqueous solutions having a pH of from about 3 to about 10;
- (l') an X-ray powder diffraction pattern (Cu.sup.++ radiation, 1.5405 .lambda., nickel filter) having the following interplanar spacings in angstroms (d):
- ______________________________________ Relative d Intensity ______________________________________ 17.15 100 12.90 80 10.85 70 9.25 70 8.87 60 8.22 50 7.86 50 6.93 40 6.20 40 5.62 40 5.04 05 4.02 02 3.54 02 ______________________________________
- (m') contains the following sugars:
- glucose, fucose, mannose, rhamnose, and 3-amino-2,3,6-trideoxy-3-C-methyl-L-xylo-hexopyranose;
- (n') has at least one hydroxyl group capable of esterification;
- or a pharmaceutically acceptable acid-addition salt of A-35512 factor B.
28 more elements
Antibiotic A-35512 factor C which is a white, amorphous, basic compound having:
- (a) an approximate elemental composition of 53.93 percent carbon, 5.15 percent hydrogen, 5.80 percent nitrogen, 32.35 percent oxygen, and 1.90 percent chlorine;
- (b) an empirical formula of approximately C.sub.84 H.sub.99 N.sub.8 O.sub.38 Cl, based on elemental composition;
- (c) a molecular weight of approximately 1862;
- and which is its dihydrochloride form is a white amorphous compound which has these characteristics:
- (a') an approximate elemental composition of 51.76 percent carbon, 5.07 percent hydrogen, 5.61 percent nitrogen, 30.29 percent oxygen, and 4.88 percent chlorine;
- (b') an infrared absorption spectrum in KBr pellet with significant absorption maxima at the following frequencies (cm.sup.-1):
- 3370 (strong), 3280 (shoulder), 3040 (shoulder), 2980 (shoulder), 2920 (weak), 1740 (weak), 1658 (strong), 1620 (weak), 1589 (medium), 1503 (strong), 1460 (weak), 1428 (medium), 1385 (weak), 1330 (weak), 1295 (medium), 1210 (strong), 1162 (medium), 1120 (weak), 1060 (strong), and 1005 (medium);
- (c') ultraviolet absorption spectra with an absorption maximum, in acidic and neutral methanol, at 282 nm (.epsilon. 14,600) and, in basic methanol, at 292 nm (.epsilon. 16,400);
- (d') a molecular weight of about 1982, as determined by titration;
- (e') three titratable groups in 66% dimethylformamide with pk.sub.a values of approximately 7.30, 8.92, and 10.99;
- (f') the following specific rotations:
- [.alpha]sup.2.sub. D -161.degree. (c 1.05, H.sub.2 O)
- [.alpha]sup.25.sub.365 -614.degree. (c 1.05, H.sub.2 O);
- (g') is soluble in water, dimethyl sulfoxide, and aqueous dimethylformamide, partially soluble in methanol and ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile, and dioxane;
- (h') an amino-acid analysis indicating the presence of five amino-acid residues, one of which appears to be glycine;
- (i') an R.sub.f value of approximately 0.46 on paper chromatography in a l-butanol:pyridine:acetic acid:water (15:10:3:12) solvent system, using Sarcina lutea as the detection organism;
- (j') a .sup.13 C nuclear-magnetic-resonance spectrum in D.sub.2 O with the following characteristics:
- ______________________________________ No. PPM Height (%) ______________________________________ 1 172.9 2.5 2 172.2 2.0 3 171.5 2.2 4 171.0 3.9 5 169.6 2.0 6 158.6 1.8 7 157.8 3.0 8 156.5 2.1 9 156.1 2.8 10 155.6 4.2 11 154.6 3.9 12 151.1 1.5 13 143.3 1.4 14 136.0 3.2 15 135.4 2.7 16 133.2 3.7 17 128.7 2.3 18 126.5 3.1 19 124.6 2.1 20 129.3 2.7 21 121.5 2.2 22 120.1 1.2 23 118.0 1.7 24 116.5 1.2 25 107.8 2.7 26 104.5 1.9 27 101.7 1.9 28 94.5 1.0 29 75.9 3.0 30 74.3 2.0 31 73.4 2.3 32 72.1 3.5 33 70.9 4.3 34 68.7 2.9 36 64.3 1.3 37 62.2 1.6 38 56.1 1.4 39 55.2 3.5 40 54.2 2.1 41 24.3 1.9 42 17.9 2.2 43 17.1 2.0 44 16.2 2.0 ______________________________________
- (k') is stable in aqueous solutions having a pH of from about 3 to about 10, for as long as 146 hours;
- (l') an empirical formula in the range of C.sub.83-85 H.sub.97-101 N.sub.8 O.sub.37-39 Cl.sub.3 and an approximate empirical formula of C.sub.84 H.sub.97 N.sub.8 O.sub.38 Cl.multidot.2HCl, based on elemental composition;
- ora pharmaceutically-acceptable acid-addition salt of A-35512 factor C.
18 more elements
Antibiotic A-35512 factor E which is a white, amorphous, basic compound having an approximate elemental composition of 54.84 percent carbon, 4.73 percent hydrogen, 5.26 percent nitrogen, 32.67 percent oxygen, and 1.72 percent chlorine;
- and which in its hydrochloride form is a white amorphous compound which has these characteristics:
- (a) an approximate elemental composition of 52.67 percent carbon, 4.59 percent hydrogen;
- 5.55 percent nitrogen, 33.51 percent oxygen, and 3.62 percent chlorine;
- (b) an infrared absorption spectrum in KBr pellet with significant absorption maxima at the following frequencies (cm.sup.-1):
- 3360 (strong), 3220 (shoulder), 2900 (weak), 1725 (weak), 1650 (strong), 1580 (medium), 1498 (strong), 1450 (weak), 1419 (weak), 1295 (medium), 1205 (medium), 1172 (medium), 1110 (weak), 1060 (strong), and 1000 (weak);
- (c) ultraviolet absorption spectra with the following absorption maxima:
- in neutral methanol, 270 nm (sh) and 359 nm (.epsilon. 16,216);
- in acidic methanol, 286 nm (.epsilon. 18,018) and 310 nm (sh);
- in basic methanol, 270 nm (sh), 300 nm (.epsilon. 16,216), and 354 nm (.epsilon. 17,568);
- (d) a molecular weight of about 2018, as determined by titration;
- (e) three titratable groups in 66% aqueous dimethylformamide with pK.sub.a values of approximately 6.30, 9.09, and 11.62;
- (f) the following specific rotation:
- [.alpha]sup.2.sub. D -108.3 (c 1, H.sub.2 O);
- (g) is soluble in water, partially soluble in methanol and ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane;
- (h) an amino-acid analysis indicating the presence of six amino-acid residues;
- (i) an R.sub.f value of approximately 0.64 on paper chromatography in a l-butanol:pyridine:acetic acid:water (15:10:3:12) solvent system, using Sarcina lutea as the detection organism;
- or a pharmaceutically-acceptable acid-addition salt of A-35512 factor E.
14 more elements
Antibiotic A-35512 factor H which is a white, amorphous, basic compound having:
- (a) an approximate elemental composition of 53.76 percent carbon, 5.32 percent hydrogen, 5.53 percent nitrogen, 33.48 percent oxygen, and 1.59 percent chlorine;
- (b) an empirical formula in the range of
- C.sub.85-87 H.sub.103-107 N.sub.8 O.sub.38-40 Cl;
- (c) a molecular weight of approximately 1908, based on elemental composition;
- and which in its hydrochloride form is a white amorphous compound which has these characteristics:
- (a') an approximate elemental composition of 53.10 percent carbon, 5.37 percent hydrogen, 5.35 percent nitroen, 30.12 percent oxygen, and 3.78 percent chlorine;
- (b') an infrared absorption spectrum in KBr pellet with significant absorption maxima at the following frequencies (cm.sup.-1):
- 3410 (strong), 3240 (shoulder), 2940 (weak), 1670 (strong), 1630 (shoulder), 1605 (strong), 1520 (strong), 1470 (weak), 1442 (weak), 1400 (weak), 1345 (shoulder), 1310 (mediium), 1225 (medium), 1180 (weak), 1135 (weak), 1080 (strong), and 1020 (shoulder).
- (c') ultraviolet absorption spectra which show, in acidic and neutral methanol, and absorption maximum at 282 nm (.epsilon. 12,500) and, in basic methanol, an absorption maximum at 292 nm (.epsilon. 14,000), and end absorption at 225 nm;
- (d') a molecular weight of approximately 1660, as determined by titration;
- (e') five titratable groups in 66% aqueous dimethylformamide with pK.sub.a values of approximately 5.0, 7.46, 9.80, 11.43 and 13.02;
- (f') the following specific rotation:
- [.alpha]sup.25.sub.D -123.5.degree. (c 1, H.sub.2 O);
- (g') is soluble in water, partially soluble in methanol and ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane;
- (h') an amino-acid analysis indicating the presence of five amino-acid groups, one of which is glycine;
- (i') approximate R.sub.f values in the following paper-chromatographic systems, using Sarcina lutea as the detection organism:
- ______________________________________ Solvent System R.sub.f Value ______________________________________ 1-butanol:pyridine:
- acetic acid:water (15:10:3:12) 0.15 CH.sub.3 OH:0.1N HCl (3:1) 0.47 1-propanol:NH.sub.4 OH:H.sub.2 O (6:3:1) 0.11 ______________________________________
- (j') a .sup.13 C nuclear-magnetic-resonance spectrum in D.sub.2 O with the following characteristics:
- ______________________________________ No. PPM Height (%) ______________________________________ 2 177.2 2.7 3 171.6 5.2 4 170.9 5.8 5 169.6 4.7 6 158.9 3.1 7 157.6 4.3 8 156.6 3.8 9 155.6 4.1 10 155.4 3.8 11 154.3 2.4 12 151.3 1.6 13 137.7 2.0 14 136.7 2.2 15 136.0 4.0 16 135.3 1.9 17 133.5 5.0 18 129.4 3.7 19 127.3 1.3 20 126.1 3.2 21 124.2 6.9 22 122.6 4.1 23 107.6 2.7 24 101.8 1.8 25 76.2 2.8 26 73.5 4.4 27 72.3 7.4 28 71.0 12.2 29 69.7 4.6 31 61.6 3.5 32 56.8 1.8 33 55.4 2.8 34 55.0 1.5 35 24.5 2.6 36 17.9 4.6 37 17.2 2.6 38 16.3 3.6 ______________________________________
- (k') is stable for as much as 72 hours in aqueous solutions having a pH of from about 3 to about 10;
- or a pharmaceutically-acceptable acid-addition salt of A-35512 factor H.
19 more elements
Antibiotic A-35512 complex produced by cultivating Streptomyces candidus having the same identifying characteristics of NRRL 8156 in a culture medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced.
The method of producing antibiotic A-35512 complex which comprises cultivating Streptomyces candidus having the same identifying characteristics of NRRL 8156 in a culture medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced.
Dependencies are inferred from the claim text.