Patent GB-815381-A (GB)
Title: Antibiotic designated nucleocidin and preparation thereof
Filed: 1956-04-18
Abstract: A new antibiotic Nucleocidin, effective against trypanosomes, amoebae and gram negative and gram positive bacteria is produced by aerobically fermenting an aqueous nutrient medium containing a source of carbon, nitrogen <PICT:0815381/IV (b)/1> <PICT:0815381/IV (b)/2> and nutrient salts with a nucleocidin-producing strain of Streptomyces calvus nov. spec.  The initial pH of the medium is 6.0 to 8.0, the temperature of fermentation 17-42 DEG  C. and preferably 26-30 DEG  C. and the duration of fermentation 48 hours. Specified carbon sources are starch, sugars such as lactose, sucrose, molasses; alcohols such as glycerol and mannitol and organic acids such as citric and acetic acid.  Nitrogen sources are proteins, e.g. casein, processes, peptones, peptides, casein hydrolysates, corn steep liquor, soya bean meal, gluten, cottonseed meal, fish meal, meat extracts, yeast extracts, amino acids, urea, ammonium and nitrate salts and others.  Nutrient salts are sodium, potassium calcium and magnesium, chloride, sulphate and phosphate and the trace metals boron, cobalt, iron, copper, zinc, manganese, chromium and molybdenum.  Nucleocidin is recovered by (a) adsorption or (b) solvent extraction or a combination of (a) and (b).  Adsorption on activated carbon is preferred using a pH of 6.5 to 8.0 and eluting with a polar solvent especially aqueous acetone.  Other eluants are aqueous methanol of pH 2 to 3 to 9.0; ethanol, propanol, butanol, beta methoxyethanol, beta ethoxy ethanol, dilute acids such as hydrochloric and acetic, pyridine and aqueous phenol.  The filtered fermentation liquor may be stirred with the activated carbon and filtered, the cake being stirred with the eluant to extract the antibiotic.  Alternatively, the fermentation liquor is passed down a column of the activated carbon, washed with water and then eluted. Nucleocidin can be absorbed on cationic exchange resins.  The eluate may be concentrated or lyophilized and the crude antibiotic used as such for the treatment of animals, or further purified.  Further purification may be by activated carbon chromatography, a solution of the antibiotic in e.g. 50 per cent aqueous acetone being passed down the column of carbon, which is developed with the same solvent to remove impurities, and then with a solvent containing a higher proportion of the acetone to remove the nucleocidin.  The antibiotic may be isolated from a clarified fermentation broth, or purified from an aqueous solution, if desired after saturating with ammonium sulphate, by extraction with n-butanol.  Crystals are produced by adjusting the pH of an aqueous acid solution of the antibiotic to pH 4.0. Examples relate to the preparation of the hydrochloride, sulphate, picrate and helianthate salts of the antibiotic. Nucleocidin is weakly basic, contains carbon 34.03 per cent, hydrogen 4.05 per cent, nitrogen 21.60 per cent, sulphur 8.30 per cent and oxygen 32.02 per cent, possibly C11H15-16N6O8S; [a ]24.5 = -33.3 (acid aqueous ethanol); soluble in water especially at pH 9.25, methanol and acetone, sparingly soluble in n-butanol, ethyl acetate, benzene and ether; it is stable in water at pH 3, pH 7 and pH 9 at room temperatures; the ultraviolet absorption at pH 7.0 (Fig. 2) shows a maximum absorption peak at 256 millimicrons; the infrared absorption spectrum (Fig. 1) shows absorption bands at the following frequencies expressed in microns: 2.87, 2.99, 3.13, 5.96, 6.18, 6.29, 6.58, 6.75, 7.00, 7.24, 7.36, 7.45, 7.71, 7.99, 8.24, 8.46, 8.76, 8.96, 9.26, 9.56, 9.83, 9.95, 10.20, 10.54, 11.15, 11.87, 12.55, 12.71, 13.16, 13.63 and 14.00.