Antibiotic A/16686 and process for preparation thereof

A process for producing an antibiotic substance selected from the group consisting of antibiotic A/16686 free bases and its physiologically-acceptable acid addition salts characterized, in the form of its hydrochloride, by

• (A) being a white crystalline material which melts at 224.degree.-226.degree. C.;
• (B) being very soluble in water and dimethylformamide;
• soluble in methanol, ethanol, propanol, and butanol;
• but insoluble in ethyl ether, petroleum ether, and benzene;
• (C) an approximate elemental composition of 51.73 percent carbon, 6.34 percent hydrogen, 9.96 percent nitrogen, 5.84 percent chlorine (total content), 4.74 percent chlorine ions, and 1 percent residue;
• (D) an infrared absorption spectrum in nujol with the following observable absorption maxima (see FIG. 1 of the accompanying drawings):
• 3290-3070, 2930 and 2860 (nujol), 1765, 1630, 1510, 1455, 1375 (nujol), 1230, 1175, 1130, 1065, 1030, 1015, 980, 840, and 820 cm.sup.-1 ;
• (E) an ultraviolet absorption spectrum with the following absorption maxima (see FIG. 2 of the accompanying drawings)
• (a) in methanol:
• ______________________________________ 232 nm (E .sub.1cm.sup.1% = 178) 265 nm (E .sub.1cm.sup.1% = 107) ______________________________________
• (b) in methanol containing 0.1 N HCl:
• ______________________________________ 231 nm (E .sub.1cm.sup.1% = 167) 270 nm (E .sub.1cm.sup.1% = 96) ______________________________________
• (c) in methanol containing 0.1 N NaOH:
• ______________________________________ 250 nm (E .sub.1cm.sup.1% = 232) 265 nm (shoulder) ______________________________________
• (d) in methanol containing pH 7.38 buffer:
• ______________________________________ 231 nm (E .sub.1cm.sup.1% = 167) 270 nm (E .sub.1cm.sup.1% = 96) ______________________________________
• (F) a specific rotation, [.alpha]sub.D.sup.24, of +49.7.degree. (c=0.43% in DMF)
• (G) the following characteristic reactions:
• ______________________________________ ninhydrin (3% ethanolic solution) negative ninhydrin (3% ethanolic solution + sodium acetate) positive 1% FeCl.sub.3 - 1% K.sub.3 Fe(CN).sub.6 positive (green) Molish positive Fehling negative Biuret positive Anthrone positive Millon negative H.sub.2 SO.sub.4 conc. negative KMnO.sub.4 positive ______________________________________
• (H) the following R.sub.f values in paper-chromatography on Whatman Nr. 1 paper using S.aureus ATCC 6538 as a detection organism:
• ______________________________________ Elution systems R.sub.f values ______________________________________ (1) n-butanol saturated with Sorensen 0.00 buffer pH 6.0 (2) n-butanol saturated with water 0.00 containing 2% of p-toluenesulfonic acid (3) n-butanol saturated with water con- 0.00 taining 2% ammonium hydroxide (4) Sorensen buffer, pH 6.0, saturated 0.05 with n-butanol (5) n-butanol:methanol:water 4:1:2 0.43 (6) ethyl acetate saturated with water 0.00 (7) n-butanol:acetic acid:water 2:1:1:
• 0.52 (8) n-butanol:pyridine:water 4:3:7 0.87 ______________________________________
• (I) The following R.sub.f values in various thin-layer-chromatographic systems indicated below:
• ______________________________________ Elution system (v/v/v) R.sub.f values ______________________________________ (1) n-propanol:n-butanol:N ammonium 0.15 hydroxide 2:3:4 (upper phase) (2) n-butanol:acetic acid:water 4:2:5 0.61 (3) n-butanol:ethanol:0.1 N hydrochloric 0.61 acid (4) chloroform:ethanol:10% acetic acid 0.00 4:7:2 (5) n-butanol:acetic acid:water 4:1:5 0.17 (6) methanol:10% aqueous ammonium 0.52 acetate:10% ammonium hydroxide 10:9:1 (7) n-butanol:pyridine:water:acetic acid 0.45 6:4:3:1 (8) methanol:10% aqueous ammonium acetate:10% 0.11 ammonium hydroxide 10:9:1 (9) 0.25 M aqueous NaH.sub.2 PO.sub.4 :acetonitrile 1:1 0.68 (10) methanol:10% aqueous ammonium acetate 1:1 0.65 (11) n-butanol:acetic acid:water 4:2:5 0.77 1 to 7 on Silicagel 60 Fe.sub.254 plates;
• 8,9 on Silicagel 60 F.sub.254 silanised and 10,11 on cellulose F plates ______________________________________
• (J) an amino-acid analysis, after acid hydrolysis in 6 N hydrochloric acid at 110.degree. C. for 6 hours, which indicates the presence of at least the following recognized amino acids:
• ornithine, aspartic acid, threonine, glycine, alanine, leucine, phenylalanine, p-hydroxy-phenylglycine, and hydroxy, chloro-substituted phenyl-glycine;
• which comprises cultivating the strain Actinoplanes sp. ATCC 33076 in a culture medium containing assimilable sources of carbon, assimilable sources of nitrogen and inorganic salts under submerged aerobic conditions until a substantial amount of antibiotic A/16686 is produced, and recovering said antibiotic activity by conventional procedures.

A process for producing an antibiotic substance selected from the group consisting of antibiotic A/16686 free base, and its non-toxic pharmaceutically acceptable acid addition salts characterized, in the form of its hydrochloride, by

• (A) being a white crystalline material which melts at 224.degree.-226.degree. C.;
• (B) being very soluble in water and dimethylformamide;
• soluble in methanol, ethanol, propanol, and butanol;
• but insoluble in ethyl ether, petroleum ether, and benzene;
• (C) an approximate elemental composition of 51.73 percent carbon, 6.34 percent hydrogen, 9.96 percent nitrogen, 5.84 percent chlorine (total content), 4.74 percent chlorine ions, and 1 percent residue;
• (D) an infrared absorption spectrum in nujol with the following observable absorption maxima (see FIG. 1 of the accompanying drawings):
• 3290-3070, 2930 and 2860 (nujol), 1765, 1630, 1510, 1455, 1375 and (nujol), 1230, 1175, 1130, 1065, 1030, 1015, 980, 840, and 820 cm.sup.1 ;
• (E) an ultraviolet absorption spectrum with the following absorption maxima (see FIG. 2 of the accompanying drawings):
• (a) in methanol:
• ______________________________________ 232 nm (E .sub.1cm.sup.1% = 178) 265 nm (E .sub.1cm.sup.1% = 107) ______________________________________
• (b) in methanol containing 0.1 N HCl:
• ______________________________________ 231 nm (E .sub.1cm.sup.1% = 167) 270 nm (E .sub.1cm.sup.1% = 96) ______________________________________
• (c) in methanol containing 0.1 N NaOH:
• ______________________________________ 250 nm (E .sub.1cm.sup.1% = 232) 265 nm (shoulder) ______________________________________
• (d) in methanol containing pH 7.38 buffer:
• ______________________________________ 231 nm (E .sub.1cm.sup.1% = 167) 270 nm (E .sub.1cm.sup.1% = 96) ______________________________________
• (F) a specific rotation [.alpha]sub.D.sup.24 of +49.7.degree. (c=0.43% in DMF)
• (G) the following characteristic reactions:
• ______________________________________ ninhydrin (3% ethanolic solution) negative ninhydrin (3% ethanolic solution + sodium acetate) positive 1% FeCl.sub.3 - 1% K.sub.3 Fe(CN).sub.6 positive (green) Molish positive Fehling negative Biuret positive Anthrone positive Millon negative H.sub.2 SO.sub.4 conc. negative KMnO.sub.4 positive ______________________________________
• (H) the following R.sub.f values in paper-chromatography on Whatman Nr. 1 paper, using S.aureus ATCC 6538 as a detection organism:
• ______________________________________ Elution system R.sub.f values ______________________________________ (1) n-butanol saturated with Sorensen 0.00 buffer pH 6.0 (2) n-butanol saturated with water 0.00 containing 2% of p-toluenesulfonic acid (3) n-butanol saturated with water containing 2% ammonium hydroxide (4) Sorensen buffer, pH 6.0, saturated 0.05 with n-butanol (5) n-butanol:methanol:water 4:1:2 0.43 (6) ethyl acetate saturated with water 0.00 (7) n-butanol:acetic acid:water 2:1:1 0.52 (8) n-butanol:pyridine:water 4:3:7 0.87 ______________________________________
• (I) The following R.sub.f values in the silica gel thin-layer-chromatographic systems indicated below:
• ______________________________________ Elution system (v/v/v) R.sub.f values ______________________________________ (1) n-propanol:n-butanol:N ammonium 0.15 hydroxide 2:3:4 (upper phase) (2) n-butanol:acetic acid:water 4:2:5 0.61 (3) n-butanol:ethanol:0.1 N hydrochloric 0.61 acid (4) chloroform:ethanol:10% acetic acid 0.00 4:7:2 (5) n-butanol:acetic acid:water 4:1:5 0.17 (6) methanol:10% aqueous ammonium 0.52 acetate:10% ammonium hydroxide 10:9:1 (7) n-butanol:pyridine:water:acetic acid 0.45 6:4:3:1 (8) methanol:10% aqueous ammonium acetate:10% 0.11 ammonium hydroxide 10:9:1 (9) 0.25 M aqueous NaH.sub.2 PO.sub.4 :acetonitrile 1:1 0.68 (10) methanol:10% aqueous ammonium acetate 1:1 0.65 (11) n-butanol:acetic acid:water 4:2:5 0.77 1 to 7 on Silicagel 60 Fe.sub.254 plates;
• 8,9 on Silicagel 60 F.sub.254 silanised and 10,11 on cellulose F plates ______________________________________
• (J) an amino-acid analysis, after acid hydrolysis in 6 N hydrochloric acid at 110.degree. C. for 6 hours, which indicates the presence at least of the following recognized amino acids:
• ornithine, aspartic acid, threonine, glycine, analine, leucine, phenylalanine, p-hydroxy-phenylglycine, and hydroxy, chloro-substituted phenyl-glycine;
• which comprises cultivating the strain Actinoplanes sp. ATCC 33076 in a culture medium containing assimilable soures of carbon, assimilable sources of nitrogen and inorganic salts under submerged aerobic conditions until a substantial amount of antibiotic activity is produced, recovering said antibiotic activity by separate extraction of broth and mycelium with an organic solvent selected from lower alkanols or chloroform, treating the antibiotic substance recovered from the extracting solvents with a mixture chloroform:ethanol:water 4:7:2, pouring the oily product which separates into water, separating the solidified product and further purifying it by silica gel column chromatography eluting with a mixture acetonitrile:
• 0.01 N HCl 1:1 whereby antibiotic A/16686 hydrochloride is recovered which is desalted by chromatography on a cross-linked dextran gel column;
• said process being further characterized in that, antibiotic A/16686 hydrochloride may be transformed into the corresponding free base or into another physiologically-acceptable acid addition salt by common procedures.