Patent GB-935327-A (GB)
Title: The antibiotic baí¬8509 and its components a, b and c and pharmaceutical compositions containing the same
Filed: 1959-09-24
Abstract: A new antibiotic designated BA-8509 and its components A. B and C are produced by cultivating an antibiotic BA-8509 producing strain of Streptomyces ambofaciens especially S. ambofaciens A.T.C.C. 13256 under submerged aerobic conditions in an aqueous nutrient medium containing a source of carbohydrate, organic nitrogen and inorganic salts preferably at 26 to 30 DEG C. and a pH of 7,0 for 24 to 96 hours.  A specified aqueous nutrient medium contains "Cerelose" (Registered Trade Mark), soya bean meal, distiller's solubles, sodium chloride, dipotassium phosphate and calcium carbonate.  In order to isolate the antibiotic, the broth is adjusted to a pH of 7,0 to 7,5 concentrated under reduced pressure below 40 DEG C. and poured into a large volume of methanol to precipitate the solid material containing the antibiotic.  The solid product is diluted with water, passed through a column of activated <PICT:0935327/IV(a)/1> <PICT:0935327/IV(a)/2> <PICT:0935327/IV(a)/3> charcoal, washed with a 0,1% phosphate buffer (pH 7,0-7,5) and eluted with a solvent consisting of acetone, pyridine, phosphate buffer (10%) and water. The crude material is separated into its components A, B, C and D by passage through a column of a quaternary anion exchange resin in its acetate form and elution with aqueous buffer phosphate.  The component D is the known compound 6-diazo-5oxo-L.norleucine.  The components A and C are purified by partition chromotography on silica gel. and elution with a solvent system of phosphate buffer (2,5%), n-butanol and iso-propanol followed by shaking with petroleum ether and water, adjusting the aqueous layer to pH 7,0, freeing from organic solvents and freeze drying.  The pale yellow product is freed from phosphate by extraction with a (1:9) mixture of methanol and methylene chloride, evaporation, dissolution in water and freeze drying.  The component B is purified by counter current distribution using a phenol, 0,1% phosphate buffer system and then ion exchange on the acetate form of "Dowex" (Registered Trade Mark) which is eluted with 0,25% phosphate buffer and purified by counter current distribution in a system consisting of tertiary butyl alcohol, tertiary amyl alcohol and water.  The antibiotic components A, B and C are readily soluble in water and have an indefinite melting point. <TABS:0935327/IV(a)/1> Oxidation of BA-8509 A with periodic acid gives formaldehyde and a product which on acid hydrolysis gives glutamic acid.  BA-8509 A has been shown to be N-acetyl-6-diazo-5-oxo-norleucine.  The components A, B and C are amphoteric forming salts with alkali and alkaline earth metals either by neutralisation of the metal hydroxide or by passing through an ion exchange resin in a cycle corresponding to the salt desired.  They decompose in acid solution giving nitrogen similarly to aliphatic diazo compounds. The antibiotic components are particularly active against malignant tumours and used in therapeutic compositions (see Group VI).  Specification 764,198 is referred to.ALSO:A therapeutic composition for oral and parenteral administration to humans and animals comprises the antibiotics BA-8509A, BA-8509B or BA-8509C or alkali or alkaline earth metal salts thereof (see Group IV (b)) and a pharmaceutical carrier. Specified carriers are water, aqueous ethand, syrup, isotonic saline, glycose, starch, lactose, calcium phosphate, an animal feedstuff or mixtures thereof.  The active ingredient is present in an amount which is at least 0.0001% of the final composition and in antitumour usage may comprise 10 to 90% of the composition. In antitumour use the composition may contain additional carcinostats, e.g. nitrogen mustard type carcinostats, 6-mercaptopurine, 8-azaguanine, urethane, 6-diazo-5-oxo-L-norleucine, azaserine, triethylenemelamine, mitomycin C, triethylenephosphoramide, 1,4-dimethylsulphonyloxybutane, the carcinostatic folic acid analogs or ethylcarbamate.  Specification 764,198 is referred to.