Patent GB-846933-A (GB)
Title: New antifungal substance, pimaricin, and method of producing same
IPC: C12P19/62
Filed: 1957-07-17
Abstract: An antifungal antibiotic pimaricinis produced by aerobically fermenting a pimaricin-producing strain of Streptomyces gilvosporeus especially S. gilvosporeus ATCC 13326 in an aqueous nutrient medium containing a source of <PICT:0846933/IV(b)/1> <PICT:0846933/IV(b)/2> carbon, nitrogen and inorganic salts.  Specified carbon sources are starch, hydrolysed starch ; sugars such as lactose, maltose, dextrose, sucrose and molasses ; alcohols such as glycerol and mannitol ; organic acids such as citric acid and acetic acid. Nitrogen sources are proteins such as casein, zein and lactalbumin ; protein hydrolysates such as proteoses, peptones and peptides ; corn steep liquor, soybean meal, gluten, cotton-seed meal, fish meal, meat extracts, stick liquor, liver cake, yeast extracts, distillers solubles, amino acids, urea, ammonium and nitrates. Other additions are (a) mineral salts, e.g. sodium, potassium, calcium, magnesium, chlorides, sulphates and phosphates (b) trace elements, e.g. boron, cobalt, iron, copper, zinc, manganese, chromium and molybdenum.  The pH is preferably 6 to 8, the temperature of cultivation 17 to 42 DEG C. and preferably 26 to 30 DEG C. and the duration 48 to 120 hours. The antibiotic is isolated from the broth by (a) adsorption or (b) extraction with a water-miscible polar organic solvent such as methanol, butanol, acid acetone, ethanol, propanol, dimethylformamide, pyridine, methyl ethyl ketone and ethyl acetate. The broth is adjusted to pH 7,0 and filtered with a filter aid. Magnesol (a synthetic hydrated magnesium silicate), Fuller's earth or activated charcoal is added to the filtrate, and the antibiotic eluted therefrom with a water-miscible polar organic solvent especially methanol, which is then followed by 70% aqueous methanol. The eluates are pooled, concentrated and cooled to 3-5 DEG C.  The crude so formed is further purified by dissolving in glacial acetic acid, filtering and adjusting to pH 4,0 with aqueous ammonia. Alternatively, a solution in the minimum quantity of glacial acetic acid is poured into water.  The sulphate is prepared by dissolving the antibiotic in glacial acetic acid, filtering and adding sulphuric acid.  The picrate is prepared by adding picric acid to an ethanol solution of the antibiotic.  The addition of potassium hydroxide to a methanol solution of the antibiotic leads to the production of the monopotassium salt.  The N-acetyl derivative is prepared by reacting pimaricin with acetic anhydride in methanol.  The anti-fungal antibiotic is a white crystalline substance with amphoteric properties forming salts with acids and with alkali metals; it contains 58% carbon, 7,5% hydrogen, 2% nitrogen and 32,5% oxygen ; free base has [a ]d25 of 248 (0,5% in dimethylformamide); N-acetyl derivative has Nx of 1,522, Ny of 1,543 \sB 0,002 and has a positive optic sign ; ultraviolet absorption spectrum has the characteristics shown in Fig. 2 and the infra-red absorption spectrum is given in Fig. 1; it is soluble in benzene, dioxane, aqueous dioxane, pyridine, piperidine, methanol, ethanol but is insoluble in ether and petroleum ether.  It is distinguished from the antibiotics rimocidin and chromin (J. Antibiotics 6(5) 247-250, 1953) by paper strip chromatography using as solvent system the upper phase of a mixture of triethylamine, formamide and water 100 : 30 : 100.