1H 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.
1H NMR spectroscopic methods have been applied to compare the in vitro reactivity of the renal papillary nephrotoxin 2-bromoethanamine (BEA) with those of selected halide-substituted nephrotoxic analogues, 2-chloroethanamine (CEA), 2-fluoroethanamine (FEA), and 1-phenyl-2-iodoethanamine (PIEA). The primary 1H NMR-detectable transformation during a 24 h incubation of confluent Madin Darby canine kidney (MDCK) cells with BEA, CEA, and FEA (at concentrations up to the IC50 determined by neutral red uptake) was the appearance in cell culture media of 2-oxazolidone (OX). Additional novel signals assigned as FEA carbamate (N-carbamoyl-2-fluoroethanamine) were observed in media collected following incubation of cells with FEA. We propose that N-carbamate intermediates are formed from the spontaneous reaction of these haloalkylamines with HCO(3-)-buffered growth media and that OX is formed from the carbamate via elimination of the hydrogen halide. Further 1H NMR experiments, conducted for up to 8 h at 25 degrees C on 5 mM solutions of BEA, CEA, and FEA in 2H2O containing a 20-fold excess of HCO3- at pH 7.6, demonstrated a time-dependent decrease in the concentration of the free haloalkylamines accompanied by the production of N-carbamate intermediates and OX. Under these pseudo-first-order reaction conditions, the formation of OX from BEA was complete within approximately 6 h. In similar reaction conditions OX formation from CEA (24 h after initiation) 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 reaction with HCO3- and indicate that the compounds in this family may exist only transiently as free amines in vivo, where there will virtually always be excess HCO3-. The physiological relevance of the in vitro findings is further indicated by the NMR-detectable conversion of BEA to OX and also an alkylating aziridine (AZ) moiety in rat plasma containing BEA. The ability to form carbamoylated species and OX (or PHOX) may mediate the toxicity of this series of haloalkylamines and hence is potentially of considerable significance.
Anthony
M L
ML
Department of Chemistry, Birkbeck College, University of London, UK. maria@chem.bbk.ac.uk.
Holmes
E
E
McDowell
P C
PC
Gray
T J
TJ
Blackmore
M
M
Nicholson
J K
JK
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002219:Carbamates / Q000378:metabolism* / Q000633:toxicity; D002460:Cell Line; D003470:Culture Media; D004285:Dogs; D005021:Ethylamines / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010080:Oxazoles / Q000378:metabolism* / Q000633:toxicity; D051381:Rats; D011916:Rats, Inbred F344
D001639:Bicarbonates; D002219:Carbamates; D003470:Culture Media; D005021:Ethylamines; D010080:Oxazoles; C004504:2-bromoethylamine