5H-Pyridophenoxazin-5-one (PPH), a new anticancer
iminoquinone, is able to inhibit a large number of lymphoblastoid and solid
tumor-derived cells at submicromolar concentrations. Molecular modeling calculations indicated that this compound might intercalate into the
DNA double strand. This was also supported by nuclear magnetic resonance studies. Since
free radicals arising from anticancer quinonic drugs have been proposed to be key species responsible for DNA cleavage, we have aimed to intercept and identify
free radicals from PPH generated under bioreductive conditions. The first and second monoelectronic reduction potentials of PPH were measured by means of cyclic voltammetry: the reduction potential of PPH is compatible with its reduction by compounds such as
NADH, and suggested that reduction of PPH may play a role in its cytotoxicity. The radical
anion PPH(*)(-) was detected by means of electron paramagnetic resonance spectroscopy, and its identification was supported by DFT calculations. EPR experiments in the presence of spin traps 5,5-dimethylpyrroline N-
oxide and 5-(diethoxyphosphoryl)-5-methylpyrroline N-
oxide suggested the occurrence of an electron transfer between the radical
anion of the
drug and
oxygen resulting in the formation of the
superoxide anion (O(2)(*)(-)). The enthalpy of the reaction of PPH(*)(-) with O(2) was determined both in the gas phase and in
solution at the B3LYP/6-31+G level using the isodensity PCM method, and the overall process in
dimethyl sulfoxide was predicted to be slightly exothermic. We propose that the monoelectronic reduction of PPH in the proximity of
DNA may eventually lead to radicals that could cause considerable damage to
DNA, thus accounting for the high cytotoxic activity of the
drug. Indeed, a comet assay (alkaline single-cell electrophoresis) showed that PPH causes
free radical-induced DNA damage.