Patent GB-823115-A (GB)
Title: A new antibiotic actinobolin and salts thereof
Filed: 1957-07-29
Abstract: A new antibiotic actinobolin is produced by growing Streptomyces griseoviridus var. atrofaciens, especially NRRL2563, in an aqueous nutrient medium containing a source of carbon and nitrogen under aerobic and preferably submerged aerobic conditions.  Suitable carbon sources are glucose, d-mannose, d-galactose, corn syrup, starch, malt liquor, molasses and glycerol in proportions of 0.5 to 5% by weight. Nitrogen sources are extract of hog stomach, <PICT:0823115/IV (a)/1> <PICT:0823115/IV (a)/2> amino acids, peptones, hydrolysed and unhydrolysed proteins, fish meal, soybean meal, corn steep liquor, meat extracts, peanut meal, inorganic nitrates, urea and ammonium salts in proportions not exceeding 6%.  Mineral salts may also be present in proportions of 0.1 to 1%, examples are sodium chloride, sodium bicarbonate, calcium carbonate and sodium acetate.  The pH of the medium is 6 to 8.5, the temperature of cultivation 20 DEG  to 35 DEG  C. and preferably 23 DEG  to 30 DEG  C., the duration of cultivation 3 to 6 days. Actinobolin is isolated from the clarified broth by adsorption on e.g. activated carbon at pH3 to 6 followed by elution with e.g. 20 to 80% aqueous acetone.  Further purification is by (a) adsorption at pH 5.5 to 6.5 and elution from an aluminium silicate cation exchange adsorbent such as Decalso and then from activated carbon, (b) adsorption and elution from a cation exchange resin such as "Amberlite IRC-50" (Registered Trade Mark), (c) treatment with a chelating agent, and/or (d) fractional precipitation with various solvents.  In the case of (c) an aqueous solution of a chelating agent such as ethylene-diamine-tetra-acetic acid is added to the purified concentrate until the resulting solution is free of colour, the solution is concentrated, extracted with acetone and the solvent removed.  The residue is treated with a waterimmiscible solvent such as ethyl acetate and the precipitate which forms is recrystallized from ethanol.  Actinobolin sulphate is obtained by adding sulphuric acid to actinobolin acetate in water to pH 1.2, treating with "Amberlite IR-45" to pH 3.5 filtering off the resin and freeze drying the filtrate.  By percolating an aqueous solution of actinobolin sulphate through "Amberlite IR-45" (a weak anion exchange resin in OH form), actinoblin free base is recovered from the effluent.  An actinobolin alumina complex is obtained by treating an ethanol solution of actinobolin acetate with aluminium chloride. Actinobolin is a broad spectrum antibiotic particularly effective against streptococci and may be used medicinally (see Group VI) and for removing iron from aqueous biological and medicinal products, e.g. tetraethylammonium chloride (see Group VI).  It is a stable, white substance, very soluble in water and in alcohols such as methanol; [a ]28D + 59 (0.5 phosphate buffer); contains 50.22% carbon, 6.88% hydrogen, 9.17% nitrogen and 33.73% oxygen (by difference); gives deep red with ferric chloride, reduces copper solutions, gives positive ninhydrin test; ultraviolet absorption in phosphate buffer at pH 7.0 shows maximum at 263 mm , in 0.1N NCl shows max. at 262 mm ; infra-red absorption shows maxima at 2.90, 3.20, 3.30, 3.36, 6.04, 6.27, 6.85, 7.09, 7.86, 8.10, 8.39, 8.77, 8.95, 9.28, 9.45, 9.93, 11.42, 11.74 and 13.08 microns.  It is amphoteric forming salts with acids, e.g. acetic and sulphuric.  The Rf value of actinobolin acetate in n-butanol (10) acetic acid (1), water (4) is 0.12 to 0.19.  Actinobolin and acid salts thereof form complexes with aluminium, iron, copper and cobalt.ALSO:Actinobolin (Group IV (b)), a broad spectrum antibiotic particularly effective against streptococcal infections, and its salts with acids, e.g. sulphate and acetate is put up in dosage form.  Aqueous solution of tetraethyl ammonium chloride are purified from trace amounts of iron by treatment with actinobolin or a salt thereof and adsorption of the resultant iron complex on activated carbon.