Patent GB-830874-A (GB)
Title: A new antibiotic mitomycin c and its production by fermentation
Filed: 1958-04-08
Abstract: <PICT:0830874/IV (b)/1> <PICT:0830874/IV (b)/2> A new antibiotic, mitomycin C, active against pathogenic bacteria, is produced by growing Streptomyces calspitosus NRRL 2564 in a liquid nutrient medium under aerobic and preferably submerged conditions.  The pH is 7.0, the temperature of cultivation is 26 DEG  to 32 DEG  C. and the duration 2 to 6 days.  The medium contains a source of (a) nitrogen such as peptone, meat extract, corn steep liquor, soy bean meal, yeast, urea, ammonium sulphate and ammonium nitrate; (b) carbon, e.g. glucose, starch, glycerol, soybean oil, maltose and dextrin; and (c) inorganic salts such as sodium chloride, potassium chloride, calcium carbonate, potassium phosphate, ferrous sulphate, magnesium sulphate and zinc sulphate.  Mitomycin C is recovered from the clarified broth by (a) adsorption, (b) solvent extraction. Adsorption is by means of carbon at pH 6 to 9, followed by elution with a solvent such as acetone, cyclohexanone, methyl isobutyl ketone, butanol, chloroform or a mixture thereof.  Acetone is the preferred solvent and the carbon is washed with methanol to remove water.  Extraction is at pH 6 to 9 by means of a water-immiscible solvent such as an alcohol, e.g. butanol, an ester, e.g. butyl acetate, a ketone, e.g. methyl isobutyl ketone and cyclohexanone, and chloroform.  Extraction may be increased by the simultaneous utilization of salting-out agents such as sodium chloride and ammonium sulphate.  Further purification is obtained by (A) chromatography on alumina and (B) countercurrent distribution. For (A), a solution of the crude antibiotic in chloroform is adsorbed on alumina and developed with chloroform containing increasing quantities of methanol up to 6%, the eluate containing 3 to 6% methanol being collected and the antibiotic recovered therefrom.  Ethanol or butanol may be used in place of methanol. Alternatively, after developing with methanol containing 0.5% methanol, the purple alumina layer is removed and the antibiotic recovered by elution with methanol per se.  For (B), the fixed phase is chloroform and the moving phase an aqueous phosphate buffer of pH 6.0 containing 10% sodium chloride.  Mitomycin C occurs as purple coloured needle-shaped crystals which do not melt even at 360 DEG C.; it contains 53.84% carbon, 5.14% hydrogen and 15.49% nitrogen; it is soluble in water, methanol, ethanol and chloroform; slightly soluble in carbon tetrachloride, benzene and ethyl ether and insoluble in petroleum ether; stable in aqueous solution at pH 5 to 11 even on heating, less stable in organic solvent solution; peaks are observed in the ultra-violet absorption spectrum (Fig. 1) at <FORM:0830874/IV (b)/1> 742; infra-red absorption spectrum peaks are observed at the following frequencies (cm.-1) 2930, 2857, 1700, 1640, 1596, 1545, 1448, 1370, 1333, 1230, 1170, 1155, 1125, 1060, 1025, 1002, 969, 952, 919, 895, 854, 824, 780 and 701.  It is active against many pathogenic micro-organisms including staphylococci and pneumoniae and is used medicinally (see Group VI).ALSO:A therapeutic composition comprises the antibiotic mitomycin C (see Group IV (b)) and a carrier which may be an ointment base, a solvent or an inert powder.  The antibiotic may also be put up in tablet and capsule form.