Patent GB-758726-A (GB)
Title: New antibiotic and process for its preparation
Inventor: NINET LEON
IPC: C07D/
Filed: 1953-09-07
Abstract: A new antibiotic, spiramycin (sequamycin), is produced by the aerobic cultivation of Streptomyces ambofaciens NRRL 2420, or a mutant thereof, in a medium containing assimilable sources of carbon, nitrogen and mineral salts until substantial antibiotic activity is produced in the medium.  Specified carbon sources are vegetable and animal oils, glycerol, starches, sugars such as sucrose, glucose, maltose and dextrose and sugar alcohols; nitrogen sources are amino acids, casein, fish meal, soybean meal, meat extracts, liver cake, distillers solubles, yeast extracts, corn steep liquor; mineral salts are calcium carbonate, sodium chloride, magnesium sulphate and potassium dihydrogen phosphor together with the trace metals. Initially the pH of the medium is adjusted to 6 to 7; the temperature of fermentation is 24 DEG  to 28 DEG  C. and preferably 26 DEG  C. and duration 3 to 4 days. Cultivation is preferably submerged. Spiramycin is isolated by solvent extraction at pH 8 to 11 and preferably at pH 9 with a water-immiscible solvent.  Specified are alcohols, e.g. butanol; alphatic ketones, e.g. methyl ethyl ketone and methyl isobutyl ketone; halogenated aliphatic hydrocarbons, e.g. chloroform, aromatic hydrocarbons, e.g. benzene and esters such as ethyl acetate and amyl acetate.  The extract either with or without concentration is extracted with water of pH below 4 and preferably pH 2 to 3.  After <PICT:0758726/IV(a)/1> <PICT:0758726/IV(a)/2> <PICT:0758726/IV(a)/3> concentrating the aqueous extract is treated with benzene or chloroform, concentrated, dehydrated pH adjusted to 5 with sulphuric acid and Spiramycin sulphate precipitated by the addition of ether.  Spiramycin base is an amorphous white powder forming acid salts with acids, e.g. sulphuric and hydrochloric; it is soluble in chloroform, chlorinated solvents, alcohols, hexane, benzene, ketone, ethyl acetate and amyl acetate; the sulphate is soluble in water and methanol, ethanol and butanol; the base contains 60 to 61 per cent C, 8.9 to 9.4 per cent H, 25.5 to 27 per cent O and 3.3 to 3.6 per cent N; molecular weight (400)n to (450)n where n is 2; gives a violet pink colouration with conc. strong mineral acids, e.g. sulphuric or hydrochloric; [a ]D20 = -70 to -75 DEG  (C = 2 per cent ethanol); its ultra violet absorption spectrum in alcoholic solution (E1%1 cm. = 346) shows a peak at 231 mm . The antibiotic congocidin is also present in the fermentation broth.  Spiramycin may be separated into three components Spiramycin I, II and III by countercurrent distribution using cyclohexane and an aqueous solution of Spiramycin containing 1 per cent disodium phosphate.  Spiramycin I is basic and contains carbon 60.3 per cent, hydrogen 8.7 per cent, oxygen 28.5 per cent and nitrogen 3.2 per cent; molecular weight 800; melting point 134-137 DEG  C.; displays in ethyl alcohol solution a maximum absorption at 232 mm .; neutral equivalent of 463; dissociation constant pKb of 7.7; [a ]D20 in methanol (C = 1 per cent) of -96 DEG , in ethanol (C = 1 per cent) of 91 DEG C. and in chloroform (C = 1 per cent) of -57; and in solid form exhibits characteristic absorption in the infra-red at the following frequencies expressed in reciprocal centimetres: 3470, 2970, 2940, 1736, 1455, 1378, 1317, 1275, 1237, 1160, 1122, 1090, 1052, 1015, 993, 905, 865, 840, 810 and 782.  Spiramycin II is basic and contains carbon 61.6 per cent, hydrogen 8.5 per cent, oxygen 26.8 per cent and nitrogen 3.1 per cent; in ethyl alcohol solution it displays maximum absorption at 232 mm ; molecular weight 800; neutral equivalent 464; dissociation constant of pKb 7.6; melting point of 130-133 DEG  C.; optical rotation [a ]D20 in methanol -86 DEG , in ethanol of -80 DEG , and in chloroform of -55 DEG ; and which in solid form exhibits characteristic absorption in the infra-red 3460, 2970, 2940, 1740, 1457, 1372, 1300, 1275, 1232, 1160, 1122, 1085, 1052, 1015, 993, 940, 905, 860, 840, 810, 782 and 685.  Spiramycin III is basic and contains carbon 61 per cent, hydrogen 8.5 per cent, oxygen 26.7 per cent, nitrogen 3.0 per cent; in ethyl alcohol solution shows maximum absorption at 232 mm ; molecular weight of 900; neutral equivalent 473; dissociation constant pKb of 7.6; melting point of 128-131 DEG C.; [a ]D20 in methanol of -83 DEG , in ethanol of -79 DEG  and in chloroform of -50 DEG  C.; and in solid form exhibits absorption in infra-red at the following frequencies expressed in reciprocal centimetres: 3470, 2970, 2940, 1740, 1460, 1380, 1370, 1300, 1280, 1240, 1185, 1162, 1122, 1085, 1052, 1015, 995, 906, 865, 842, 810, 782, 695 and 685.