Nucleoside and DNA Adducts from N-Nitrosotolazoline

Abstract

The reaction of N-nitrosotolazoline, the nitrosation product of a representative imidazoline receptor drug tolazoline, with DNA, deoxyguanosine (dG), or deoxyadenosine (dA) produces adducts containing the 2-phenylacetamidoethyl group. The synthesis and characterization of 2-phenylacetamidoethyl-guanine derivatives (O6-dG, O6-Gua, N2-Gua, and 7-Gua) and 2-phenylacetamidoethyladenine derivatives (1-Ade, 3-Ade, 7-Ade, and N6-Ade) are described. In addition to the use of an established UV spectral method for confirming the structure of the alkyl adenines, a new 13C NMR method for determining the N-alkylation site is presented. In combination with the synthesized standards, HPLC MS/MS methods were used to determine the nature and the quantity of adducts produced. N-Nitrosotolazoline reacted with dG to give 7-(2-phenylacetamidoethyl)deoxyguanosine (major), O6-(2-phenylacetamidoethyl)deoxyguanosine, and 5'-O-phenyacetyldeoxyguanosine. The reaction of N-nitrosotolazoline with dA produced the 1-, 3-, 7-, N6, and 5-O'-2-phenylacetamidoethyl adenine and dA derivatives as well as several phenylacetyl adducts. Reaction of N-nitrosotolazoline with DNA in vitro resulted in the detection of 2-phenylacetamidoethyl adducts (adduct, relative %): 7-Gua, 60%; 3-Ade, 30%; O6-Gua, 8%; and 7-Ade, 2%. Comparison of these data with appropriate literature data, as well as our work on the mechanism of N-nitrosotolazoline hydrolytic decomposition, is consistent with the adducts being produced from a 2-phenylacetamidoethyldiazonium intermediate. The results show that N-nitrosotolazoline, and presumably other N-nitrosoimidazolines, if produced by endogenous nitrosation pathways, are capable of alkylating DNA without additional metabolic transformation and are probable carcinogens.