H-1-NMR SPECTROSCOPIC STUDIES ON THE REACTIONS OF HALOALKYLAMINES WITH BICARBONATE IONS - FORMATION OF N-CARBAMATES AND 2-OXAZOLIDONES IN CELL-CULTURE MEDIA AND BLOOD-PLASMA

H-1 NMR spectroscopic methods have been applied to compare the in vitr o reactivity of the renal papillary nephrotoxin 2-bromoethanamine (BEA ) with those of selected halide-substituted nephrotoxic analogues, 2-c hloroethanamine (CEA), 2-fluoroethanamine (FEA), and 1-phenyl-2-iodoet hanamine (PIEA). The primary 1H NMR-detectable transformation during a 24 h incubation of confluent Madin Darby canine kidney (MDCK) cells w ith BEA, CEA, and FEA (at concentrations up to the IC50 determined by neutral red uptake) was the appearance in cell culture media of 2-oxaz olidone (OX). Additional novel signals assigned as FEA carbamate (N-ca rbamoyl-2-fluoroethanamine) were observed in media collected following incubation of cells with FEA. We propose that N-carbamate intermediat es are formed from the spontaneous reaction of these haloalkylamines w ith HCO3--buffered growth media and that OX is formed from the carbama te via elimination of the hydrogen halide. Further H-1 NMR experiments , conducted for up to 8 h at 25 degrees C on 5 mM solutions of BEA, CE A, and FEA in (H2O)-H-2 containing a 20-fold excess of HCO3- at pH 7.6 , demonstrated a time-dependent decrease in the concentration of the f ree haloalkylamines accompanied by the production of N-carbamate inter mediates and OX. Under these pseudo-first-order reaction conditions, t he formation of OX from BEA was complete within approximately 6 h. In similar reaction conditions OX formation from CEA (24 h after initiati on) had reached 54% of its final equilibrium concentration. Equivalent experiments demonstrated that PIEA was almost completely converted to 4-phenyl-2-oxazolidinone (PHOX) within 2 h. These observations reveal the strong disposition of this series of haloalkylamines toward react ion with HCO3- and indicate that the compounds in this family may exis t only transiently as free amines in vivo, where there will virtually always be excess HCO3-. The physiological relevance of the in vitro fi ndings is further indicated by the MMR-detectable conversion of BEA to OX and also an alkylating aziridine (AZ) moiety in rat plasma contain ing BEA, The ability to form carbamoylated species and OX (or PHOX) ma y mediate the toxicity of this series of haloalkylamines and hence is potentially of considerable significance.