Patent GB-1349179-A (GB)
Title: Antibiotic
IPC: A61K35/74
Filed: 1971-10-11
Abstract: 1349179 Antibiotic WS-4545 and its esters FUJISAWA PHARMACEUTICAL CO Ltd 11 Oct 1971 [12 Oct 1970] 47214/71 Heading C2A The new antibiotic WS-4545 and esters thereof, having the general Formula (I) wherein R 1 , R 2  and R 3  may be the same or different and each is a hydrogen atom or an acyl radical, are prepared by aerobically cultivating Streptomyces sapporonensis (ATCC 21532) or an antibiotic WS-4545-producing mutant thereof, in an aqueous nutrient medium to give antibiotic WS-4545 having the aforesaid formula in which R 1 , R 2  and R 3  are hydrogen atoms, and isolating the said antibiotic and if desired, acylating it to give the mono-, di- and/or tri-ester. The antibiotic has the chemical name 1- [(1,2,3 - trihydroxy - 2 - methyl) - propyl] - 5- methylene - 6 - hydroxy - 7,9 - dioxo - 2 - oxa- 8,10 - diazabicyclo[4,2,2]decane. It may be acylated by reacting with 1, 2 or 3 equivalents of an appropriate carboxylic acid or its acylating derivative, preferably in the presence of a base (e.g. pyridine) and if necessary, a condensing agent. When the R groups are to be different, a mono- or di-ester is first prepared and the remaining -OH groups or group then esterified with a different carboxylic acid or its acylating derivative. The acylation may produce a mixture of the mono-, di- and tri-esters, in which case they may be separated from each other by column chromatography. The acyl aroups may be linear, branched or cyclic alkyl-, aryl, aralkyl-, heterocyclic- or benzene-fusedheterocyclic-carbonyl groups, and may be substituted by halogen, alkoxy, amino, acylamino, acyloxy, nitro or hydroxy groups and the aliphatic chains may be interrupted by hetero atoms (e.g. O or S). Antibiotic WS-4545 is isolated by filtering the fermentation broth, and treating the filtrate, with an adsorbent solid (e.g. activated charcoal); or by adding a water-miscible organic solvent to the filtrate to precipitate impurities and then evaporating the supernatant to obtain the antibiotic; or by extracting the filtrate with a water-immiscible solvent for the antibiotic. If an adsorbent is used, the antibiotic is subsequently extracted therefrom into a suitable solvent. The solvent solution is then evaporated to small volume, and treated with a non-solvent liquid to precipitate the WS-4545, which is purified by adsorption and elution from a chromatographic column and re-crystallized. The antibiotic is a white crystalline solid, the crystals being either rhombic or monoclinic according to the crystallization procedure employed. The rhombic form melts at 187 to 189 C. (decomp.) and has an I.R. absorption spectrum as shown in Fig. 2, with peaks at 3400, 3300, 3160, 2900, 2840, 1703, 1670, 1450, 1390, 1370, 1295, 1260, 1220, 1195, 1140, 1120, 1080, 1030, 970, 960, 935, 895, 870, 820, 785, 725 and 680 cm.<SP>-1</SP>. The monoclinic crystals melt at 188 to 191 C. (decomp.) and have an I.R. spectrum as shown in Fig. 3, with peaks at 3500, 3400, 3270, 2900, 2840, 1685, 1640, 1455, 1417, 1375,1330, 1300, 1260, 1245, 1210, 1160, 1135, 1125, 1105, 1085, 1050, 1010, 995, 980, 940, 920, 900, 885, 870, 840, 805, 795, 760, 725, 690 and 675 cm.<SP>-1</SP>. The crystal forms are interconverted by dissolving the one form in a hot solvent and cooling to cause crystallization in the presence of crystals of the other form. The following characteristics are common to both forms of antibiotic WS-4545: soluble in water, methanol, ethanol and pyridine, slightly soluble in acetone and ethyl acetate, insoluble in ether, chloroform, benzene and n-hexane;   molecular weight 302; specific rotation [α]<SP>#23</SP> D = 63À5 (C=1% in MeOH); elementary analysis C=47À44%, H=6À16%, N=9À36%, O(by difference)=37À04%; no specific ultra-violet absorption; empirical formula C 12 H 18 N 2 O 7 ; positive to Fehling's and Molisch's tests, decolorizes KMnO 4  solution, faint positive reaction with ferric chloride, negative to ninhydrin, Tollen's and Ehrlich's tests. Pharmaceutical compositions having antibiotic activity against gram-negative microorganisms, comprise antibiotic WS-4545 and/or an ester thereof, of Formula (I) above, together with a solid, semi-solid or liquid pharmaceutical carrier or diluent.