Patent GB-866600-A (GB)
Title: New antibiotic e73, compositions thereof and process for preparation of antibiotic e73, cycloheximide, and fungicidin
Filed: 1958-03-06
Abstract: <PICT:0866600/IV (b)/1> <PICT:0866600/IV (b)/2> A new antibiotic E73 is co-produced with the antibiotics cycloheximide and fungicidin by cultivating Streptomyces Albutus ATCC 2757 or a compound E73 producing a mutant thereof, in an aqueous nutrient medium containing a source of carbon, nitrogen and inorganic salts under submerged aerobic conditions.  A pH of 7,0, a temperature of 26 to 30 DEG C. and a duration of 1 to 5 days is employed.  A suitable medium contains glucose, soybean meal, potassium hydrogen phosphate, sodium chloride, distillers solubles and calcium carbonate.  The fermentation broth is filtered and the fungicidin recovered by solvent extraction of the mycelium by known methods, the E73 and cycloheximide being obtained by the solvent extraction of the clarified broth with a water-immiscible solvent at pH 2 to 8. Fungicidin is recovered by extracting the mycelium with n-butanol, concentrating, allowing crude fungicidin to precipitate, treating the precipitate with water-ethyl acetite-t-butanol (5 : 4.1.) and recovering the fungicidin from the aqueous layer.  A mixture of E73 and cycloheximide is obtained by extracting the clarified broth with a water-immiscible solvent such as chloroform, ethyl acetate or methyl isobutyl ketone at pH 2 to 8 and preferably pH7.0, concentrating and pouring into petroleum ether.  The crude is purified by chromatography on alumina using a solution of the crude in ethyl acetate and developing with ethyl acetate containing 2% methanol.  The cycloheximide E73 fraction is further purified by partition chromatography on silica gel using an aqueous methanol isopropyl ether system (1 : 5), Compound E73 is active against yeasts, fungi and protozoa and inhibits the growth of tumours; it melts at 140-141/C. and contains 59,9% carbon, 7,5% hydrogen and 4,4% nitrogen; [a ]25D=8,8 (1% methonal); molecular weight 287; soluble in water, alkanols, ether, chloroform, methylene chloride and acetone; hydrolyses at pH above 8,0 with liberation of ammonia (cycloheximide gives dimethylcyclohexanone): maxima in infra red (chloroform solution) at 3559, 2950, 1727, 1709, 1391, 1149, 1131, 1116, 1041, 1025, 946, 878 and 826 cm-1; forms a monoacetate and p-nitrobenzoate of melting points 170-171 DEG C. and 167-168 DEG C. respectively by treating a pyridine solution of E73 with acetic anhydride or p-nitrobenzoyl chloride.  Esters of E73 are produced with carboxylic acids in general e.g. propionate, butyrate, valerate, succinate, acrylate, maleate, palmitate, stearate, benzoate, toluate and naphthoate. E73 may be administered to man and animals (see Group VI).ALSO:Antibiotic E73 (see Group IV (b)), which is effective against fungi and tumours, is used orally and parenterally in admixture with a carrier.  It may be put up in aqueous or other injectable solutions, e.g. propylene glycol or as a suspension in peanut oil or sorbitol solution. It may be added to bacterial fermentations which are conducted at or near a neutral pH in aqueous media in order to prevent yeast contamination and the production of unwanted by-products.  Such fermentations are used in the production of ascorbic acid, gluconic acid, a -ketoglutaric acid, steroids, lysine and acetic acid.