Patent GB-846130-A (GB)
Title: New antibiotic and derivatives and salts thereof, preparations containing the same and process for the manufacture of these substances
IPC: G01N33/487
Filed: 1957-03-19
Abstract: <PICT:0846130/IV(b)/1> <PICT:0846130/IV(b)/2> New antibiotics cinerubin A, cinerubin B, or a mixture thereof, are produced by the aerobic, preferably submerged, cultivation of streptomyces cinereoruber borbaz et al var. fructofermentu Nov. spec., or a mutant thereof, in an aqueous nutrient medium containing an inorganic salt, a nitrogenous compound and a carbohydrate.  Specified inorganic salts are chlorides, nitrates, carbonates or sulphates of the alkali metals, alkaline earth metals, magnesium, iron, zinc or manganese. Specified nitrogenous compounds, carbohydrates and growth promoters are amino acids and mixtures thereof, peptides and proteins and their hydrolysates such as peptone or tryptone, meat extracts, water-soluble fractions of cereal grains such as maize and wheat, distillers residues from alcohol manufacture and yeast, soya-bean and cottonseed meals, glucose, saccharose, lactose and starch. Fermentation is for 36 to 120 hours at 20 to 40 DEG C.  The nature of the medium determines the antibiotic produced.  Thus cinerubin A is chiefly formed by fermentation in a medium containing mannite and soya flour; cinerubin B from a medium containing glycerin, soya flour, sodium chloride, calcium carbonate and sodium nitrate; and a mixture of cinerubin A and B from a medium containing mannite, distillers solubles, sodium chloride and sodium nitrate. Cinerubin is isolated from the clarified broth by (a) extraction (b) adsorption. Residual cinerubin adsorbed on the mycelium is recovered by washing with an organic solvent at least partially miscible with water e.g. alcohols such as methanol, ketone such as acetone or with organic or inorganic acids such as acetic, hydrochloric or sulphuric.  The washings are added to the clarified broth.  Alternatively, the whole broth may be extracted. Extraction is at a pH above 7 by organic solvents immiscible with water e.g. esters of fatty acids such as ethyl acetate, hydrocarbons such as benzene, chlorinated hydrocarbons e.g. chloroform, ketones such as methyl propyl ketone, esters such as diethyl ether and glycol ether.  The extract is then treated with aqueous acid at below pH 5,0 e.g. acetic, hydrochloric, sulphuric or citrate or phosphate buffers.  The aqueous extract is adjusted to pH 7 and is then extracted with solvent.  The processes may be repeated. Initially or as a further step in purification, recovery may be by adsorption on active carbon or earths followed by elution with a solvent at least partially soluble in water e.g. acetone.  Further purification may be by (a) countercurrent distribution between an aqueous acid or an alcoholic aqueous solution and a solvent immiscible with water e.g. aqueous methanol-carbon tetrachloride, (b) chromatography and (c) crystallisation from organic solvents e.g. acetone, methanol and ethanol. (a) and (b) above may be used for separating cinerubin A from cinerubin B.  Cinerubin A is a red substance, melting point 155 to 157 DEG C, contains C=61,12%, H=7,03%, N=1,91% and OCH3=4,08%; ultra violet absorption spectrum:-234 mm  (log E=2,28), 258 mm  (log E=2,36), 292 mm  (log E=1,92), 475 mm  (log E=2,05), 495 mm  (log E=2,11) and 530 mm  (log E=1,94); infra-red spectrum bands are visible at the following wave lentghs: 2,9, 3,4, 3,5, 5,77, 6,06, 6,21, 6,81, 7,02, 7,06, 7,21, 7,55, 7,68, 7,88, 8,14, 8,56, 8,88, 9,05, 9,56, 9,84, 10,37, 10,78, 11,19, 11,31, 11,79, 12,32, 12,71, 13,10, 13,49, 13,75 and 14,21 microns.  On hydrolysis with dilute mineral acids cinerubin A produces two reducing sugars and the antibiotic cinerubinone.  This melts at 217-220 DEG C, contains C=63,70%, H=5,38%, (C)CH3=3,42%, OCH3 = 7,27% and active hydrogen = 1,26%; ultraviolet spectrum bands are visible at 234 mm  (log E=3,01), 258 mm  (log E=2,68), 294 mm (log E= 2,25), 470 mm  (log E=2,30), 495 mm  (log E=2,34), 515 mm (log E=2,28) and 530 mm  (log E=2,20).  Infra-red absorption spectrum bands are visible at the following wave lengths: 2,87, 3,00, 3,25, 3,43, 3,52, 5,78, 6,06, 6,24, 6,88, 7,07, 7,55, 7,69, 8,13, 8,30, 8,54, 8,84, 9,06, 9,34, 9,56, 9,75, 9,98, 10,07, 10,28, 10,47, 11,01, 11,59, 11,87, 12,02, 12,72, 13,31, 13,88, 14,10 and 14,49 microns. Cinerubin B forms orange-coloured crystals melts 180-181 DEG C., contains C=59,76%, N= 6,50%, O=30,44%, N=1,78%, OCH3=4,54% and CCH3=6,38%; ultraviolet absorption spectrum shows bands at 235 mm (log E=2,77), 258 mm  (log E=2,46), 291 mm  (log E=2,03), 297 mm , 394 mm , 415 mm , 471 mm , 488 mm  (log E=2,24), 497 mm  (log E=2,27), 519 mm  (log E=2,16) and 533 mm  (log E=2,10); infra red absorption bands are visible at the following wave lengths: 2,83, 3,38, 3,58, 5,71, 6,16, 6,78, 7,02, 7,47, 7,60, 8,06, 8,48, 8,75, 9,41, 9,72, 9,90, 10,18, 10,62, 11,62, 12,15, 12,43 and 12,85 microns. On hydrolysis with dilute mineral acids cinerubin B produces cinerubinone.  Cinerubin A and cinerubin B form acid and neutral salts with inorganic and organic acids such as hydrochloric, sulphuric, acetic, propionic, valeric, palmitic, oleic, succinic, citric, mandelic, glutamic and pantothenic acids.  The cinerubin A and B are distinguished from rhodomycin A and B by their different behaviour in paper chromatography.  Cinerubin A has RF value of 0,55 in system benzene formamide, whereas cinerubin B remains at the original drop point and cinerubinone has a value of 0,8.ALSO:New antibiotics cinerubin A or cinerubin B produced by culturing streptomyces cinereoruber corboz et al var. fructofermentans or a mutant thereof (see Group IV(b)) active against a wide range of microorganisms including influenza virus and against tumors, their salts, esters, and ethers, the acid hydrolysis product cinerubinone, or mixtures thereof, are formulated into pharmaceutical compositions in admixture with an organic or inorganic carrier suitable for enteral parenteral or local administration.  The antibiotics may be used in the form of their acid or neutral salts with inorganic or organic acids. Specified are the hydrochloride, sulphate, acetate, pantothenate, propionate, valerate, palmitate, oleate, succinate, citrate, mandelate and glutamate.  Suitable forms are ampoules, tablets, dragres, salves, creams, powders, suppositories, solutions, suspensions and emulsions.  Specified diluents are gelatine, lactose, starch, magnesium stearate, talc, vegetable oils, lauryl alcohols, gums, polyalkylene glycols, petroleum jelly, and cholesterol. Preserving, wetting or emulsifying and stabilising agents may be present together with other drugs.