GB708686A - Antibiotic d-13 and its production - Google Patents

Abstract

<PICT:0708686/IV (b)/1> <PICT:0708686/IV (b)/2> An antibiotic D-13 is produced by cultivating a strain of Streptomyces vinaceusdrappus, which is described, or a mutant thereof, in an aqueous, nutrient-containing, carbohydrate solution under aerobic conditions at 20-30 DEG C., and preferably 24-28 DEG C., for 2 to 5 days, and is isolated from the filtered fermentation broth. Specified carbohydrate sources are glycerol, dextrin, starch and sugars, e.g. glucose; organic nitrogen sources are soybean meal, rolled oats, corn steep liquor, peptone and beef extract; growth substances are yeast, distillers solubles, and fermentation solubles; mineral salts are ammonium nitrate, potassium nitrate, sodium chloride, potassium chloride, calcium carbonate and sodium dihydrogen phosphate. Anti-foaming agents may be present. The antibiotic is isolated by (a) adsorption or (b) solvent extraction. Adsorption may be on activated carbon or cationic ion-exchange resins at the pH of the filtered broth, i.e. pH 7-8 and room temperatures. The adsorbent is eluted with aqueous water-miscible alcohols or ketones at pH 2.0, e.g. 10 per cent aqueous acetone, the pH of the eluate adjusted to 7-8 and a crude product obtained by freeze drying. Solvent extraction is by water-immiscible, neutral, alcohols, ketones, halogenated hydrocarbons or esters, e.g. butanols, pentanols, diethylketone, methylene chloride, ethyl acetate and amyl acetate, at a pH of 7 or higher. The extract is (a) concentrated, extracted with dilute aqueous acid, and the antibiotic precipitated by adjusting the pH to 7-9 with alkali; (b) distilled to remove the solvent azeotropically and the residual aqueous solution freeze dried, or seeded with crystals of D-13. The crude antibiotic is purified by (a) dissolving in water at pH 7.5-9.0, extracting with a water-immiscible solvent, e.g. butanol, treating the extract with dilute acid and adjusting the pH of the acid solution to 7-9; or (b) countercurrent distribution using the systems water-methylene chloride, or water-butanol. Antibiotic D-13 is effective in inhibiting the growth of mycobacteria; it is basic and forms salts with acids; it is sparingly soluble in water and methanol in the anhydrous form m. pt. 243-244 DEG C., more soluble in the hydrate form m. pt. 160-170 DEG C., <FORM:0708686/IV (b)/1> = 143 (alcohol); contains C = 56.91 per cent, H = 6.97 per cent, N = 13.51 per cent and O = 22.61 per cent. Ultra-violet absorption spectrum in 50 per cent aqueous ethanol maximum at 306 millimicrons E 1%/1 cm. = 512; in 50-50 ethanol-N/10 N NaOH, maxima at 272 millimicrons E 1%/1 cm. = 283, and 325 millimicrons maximum at 304 millimicrons E 1%/1 cm. = 451. Infra-red absorption spectrum of mineral oil suspension of anhydrous D-13 exhibits absorption at the following frequencies 3405, 3230, 1684, 1654, 1614, 1567, 1522, 1491, 1252, 1176, 1104, 1072, 1046, 1019, 855, 790, 758, 692. The anhydrous form of D-13 is obtained by crystallizing the crystalline form of D-13 from an anhydrous solvent such as methanol. Hydrochloride is produced by dissolving the antibiotic in hydrochloric acid and (a) freeze drying or (b) adding acetone, or a mixture of methanol and acetone. The sulphate and acetate may be prepared in a similar manner.