Patent GB-795570-A (GB)
Title: Thiostrepton, its salts and method of manufacture
Filed: 1956-02-29
Abstract: A mixture of antibiotics containing one which has been designated thiostrepton, is produced by cultivating Streptomyces sp. 3705 (Wakeman Collection), or a mutant thereof, in an aqueous nutrient medium comprising an assimilable source of carbon nitrogen and inorganic salts, under aerobic and especially submerged aerobic conditions.  The pH of the medium is 6 to 8, the temperature of cultivation 20 DEG  to 35 DEG  C. and preferably 25 DEG  to 27 DEG C. and the duration of cultivation 72 to 168 hours.  Specified carbon sources are starch, dextrin and sugars such as maltose, lactose and glucose, glycerol insulin and fats.  Specified nitrogen sources are asparagine, soybean meal, ammonium sulphate, sodium nitrate and sodium nitrite.  Specified minerals are sodium chloride, sodium sulphate, calcium carbonate and cobalt chloride. Thiostrepton is isolated from the mycelial cake obtained by centrifuging or filtering the fermentation broth at a neutral pH by extraction with an organic solvent (1) such as chloroform, dioxane, a N,N-di-(lower alkyl) lower alkanoic acid amide, e.g. N,N-dimethyl formamide and N,N-dimethyl acetamide, and benzyl alcohol.  In one example, the mycelial cake is slurried with chloroform and filtered.  The residue is given a further extraction with chloroform, the extracts are concentrated and crude thiostrepton (A) precipitated by the addition of hexane. This is purified by dissolving in dioxane, adding carbon, filtering and precipitating the thiostrepton by the addition of water.  The mother liquor (B) contains a second antibiotic. Further purification is obtained by dissolving in dioxane and adding aqueous methanol.  Apart from thiostrepton, two other antibiotics are produced and are found predominantly in the clarified broth.  Small quantities may be <PICT:0795570/IV (b)/1> recovered from the crude (A) and mother liquor (B) above. Thiostrepton is active against grampositive bacteria; it is a weak base forming (a) salts with inorganic and organic acids, e.g. hydrochloridic and sulphuric and (b) complexes with alkaline earth metal salts such as calcium chloride and magnesium chloride; both (a) and (b) are hydrolysed by water; it melts with decomposition at 246 DEG  to 256 DEG  C.; it contains C = 51.75 per cent, H = 5.30 per cent; S = 9.22 per cent; N = 15.84 per cent; and O = 17.89 per cent; the equivalent weight is 380; it is soluble in the above solvents (1), insoluble in water, ether, acetone, benzene, hexane, ethyl acetate, amyl acetate, dibutyl ether, glycol, diethylene glycol, glycerol, corn oil, sesame oil and ethyl oleate; ultraviolet absorption shoulders of thiostrepton in methanol are at 240, 280 and 305 mu; infra red absorption peaks are observed at 3289, 1733, 1658, 1631, 1580, 1534, 1511, 1486, 1420, 1376, 1366, 1348, 1309, 1282, 1269, 1244, 1203, 1166, 1152, 1138, 1116, 1094, 1060, 1033, 1020, 984, 971, 951, 935, 921, 893, 956, 808, 780, 769, 760, 740, 730.  Thiostreptin shows the same antibiotic spectrum as penicillin and is used medicinally (see Group VI).ALSO:A new antibiotic thiostrepton produced by fermentation using Streptomyces sp. 3705 (Waksman Collection (see Group IV (b)) admixed with an inert pharmaceutically acceptable diluent may be administered (a) orally, in a two-piece capsule; or (b) intravenously, by mixing, immediately prior to injection, a solution in N,N-dimethyl acetamide with water to give a final composition containing about 10 per cent N,N-dimethyl acetamide and 90 per cent water.