Reactions of the nitroso analogue of chloramphenicol with reduced glutathione

Abstract

Nitroso-chloramphenicol (NOCAP) was synthetized by reduction of chloramphenicol (CAP) with zinc dust in a modification of the procedure published by Corbett and Chipko. The radioactive derivative was similarly prepared from [dichloroacetamido-1- 14C]CAP. NOCAP rapidly reacted with GSH with the formation of hydroxylamino-chloramphenicol (NHOHCAP), d -(−)-threo-1-(p- hydroxylaminophenyl)-2- dichloroacetamido-1,3- propanediol and glutathione disulfide (GSSG). In addition, a hydrophilic sulfinamide was formed (GSONHCAP), d -(−)-threo-1-(p- glutathionesulfinamidophenyl)-2- dichloroacetamido-1,3 - propanediol . Free amino-chloramphenicol (NH 2CAP), d -(−)-threo-1-(p- aminophenyl)-2- dichloroacetamido-1,3- propanediol , was not detected. The proportion of NHOHCAP formed increased with increasing GSH concn, at the expense of GSONHCAP. Analysis by stopped-flow spectroscopy revealed formation of a labile adduct in the reaction of NOCAP with GSH ( k = 5500 M −1 sec −1 at 37°, pH 7.4). This reaction was reversible because nearly all NOCAP could be extracted with ether from the labile intermediate. The equilibrium adduct/NOCAP was dependent on GSH concn ( K = 4500 M −1 at 37°, pH 7.4). The labile intermediate either isomerized to the sulfinamide, GSONHCAP (favoured by decreasing pH at constant GSH), or it was thiolytically cleaved by another GSH molecule to NHOHCAP and GSSG (favoured by increasing GSH at constant pH). At acid pH, GSONHCAP readily hydrolyzed to NH 2CAP and glutathionesulfinic acid. Thus, NOCAP reacts with thiols similar to nitrosobenzene. A scheme is presented for the proposed reaction mechanism. It is concluded that most of the NOCAP, if formed in the intestine or liver, will be rapidly disposed by reactions with GSH. Hence, toxic concns at the sensitive target, i.e. the bone marrow, may usually be prevented.