Patent GB-795482-A (GB)
Title: Amphotericins, their salts and method of manufacture
IPC: C07H17/08
Filed: 1955-12-21
Abstract: <PICT:0795482/IV (b)/1> <PICT:0795482/IV (b)/2> Amphotericin, an antifungal antibiotic having two components amphotericin A and amphotericin B, is produced by cultivating Streptomyces sp. 3694 (Wakeman Collection-Rutgers Institute of Microbiology), or a mutant thereof, in an aqueous nutrient medium comprising an assimilable source of carbon and nitrogen, under submerged aerobic conditions. The temperature is 23 DEG  to 30 DEG  C. and preferably 25 DEG  C. and the duration 24 to 150 hours.  Specified carbon sources are starch, dextrin, maltose, glucose and mannitol which are particularly effective; lactose, sucrose, inositol and glycerol being less effective.  Specified nitrogen sources are asparagine, casein hydrolysate, soybean meal, beef extract and yeast extract.  Mineral salts such as sodium chloride, cobalt chloride and calcium carbonate may be present.  Amphotericin is isolated from the fermentation broth by employing three methods. (A) The mycelium separated from the broth is extracted at pH 2 to 3 by a solvent especially an alkanol such as isopropanol, n-propanol or n-butanol.  (B) The whole broth is alkanized at pH 11 to 12 by a base such as sodium hydroxide, filtered with filter aid, filtrate adjusted to pH 7.0 so that amphotericin precipitates.  (C) The whole broth is adjusted to pH 2 to 3 or 10 to 11 and then extracted with a solvent, e.g. an alkanol, filtered, filtrate adjusted to pH 7.0 and solvent removed at below 35 DEG  C. Amphotericin is separated into its components by (1) slurrying crude in an alcohol at pH and filtering, the residue is crude amphotericin B and the filtrate when neutralized gives crude amphotericin A.  (2) Slurrying crude amphotericin with a dialkyl alkanoic acid amide, e.g. dimethyl formamide, dimethyl acetamide or diethyl formamide.  The residue is crude amphotericin B and crude amphotericin A is recovered from the filtrate by adding aqueous alcohol or aqueous ketone. Amphotericin A and B may be further purified by treatment with solvents.  Amphotericin is amphoteric forming salts with both bases and acids.  Alkali metal and alkaline earth metal salts are produced by reacting amphotericin with an alkali metal or alkaline earth metal hydroxide.  Ammonium or amine salts are produced by reacting amphotericin with ammonium hydroxide or an organic nitrogen base. Alkaline earth metal salts e.g. calcium chloride or magnesium chloride form complexes with amphotericin.  Acid salts of amphotericin are formed with mineral acids such as hydrochloric, sulphuric or phosphoric and with organic acids such as acetic, citric or tartaric.  Soluble in glacial acetic acid and dimethyl formamide; less soluble in methanol, aqueous isopropanol and wet butanol, but more so as acid and alkaline pH.  Insoluble in water as neutral or acid pH soluble as alkaline pH.  Insoluble in ether, acetone ethyl acetate, chloroform and hexane.  Amphotericin A has the analysis C = 59.28 per cent, H = 8.43 per cent, N = 1.81 per cent and 0 = 30.48 per cent; [a ]2D = 136 DEG  (dimethyl formamide) = 28 DEG  (in methanol); it is crystalline, and has an absorption spectrum measured in methanolic HCl with bands at the following wavelengths: 228, 291, 304 and 318 millimicrons. Amphotericin B is crystalline; contains C = 56.70 per cent, H = 7.72 per cent, N = 1.87 per cent and O = 33.71 per cent; [a]2D DEG r. = 238 DEG  (dimethylformamide) and - 52.2 DEG (methanol), and has absorption spectrum measured in methanolic HCl with bands at the following wavelengths: 345, 362, 382 and 405 millimicrons.  Amphotericin is used medicinally (see Group VI).ALSO:The antibiotic amphotericin (see Group IV (b)), as the mixture or in the form of its components amphotericin A or amphotericin B, is incorporated in pharmaceutical tablets, alone or with the further addition of broad spectrum antibiotics such as oxytetracycline, chlortetracycline or tetracycline.