Radical Chemistry and Cytotoxicity of Bioreductive 3‑Substituted Quinoxaline Di‑<i>N</i>‑Oxides

The radical chemistry and cytotoxicity of a series of quinoxaline di-<i>N</i>-oxide (QDO) compounds has been investigated to explore the mechanism of action of this class of bioreductive drugs. A series of water-soluble 3-trifluoromethyl (<b>4</b>–<b>10</b>), 3-phenyl (<b>11</b>–<b>19</b>), and 3-methyl (<b>20</b>-<b>21</b>) substituted QDO compounds were designed to span a range of electron affinities consistent with bioreduction. The stoichiometry of loss of QDOs by steady-state radiolysis of anaerobic aqueous formate buffer indicated that one-electron reduction of QDOs generates radicals able to initiate chain reactions by oxidation of formate. The 3-trifluoromethyl analogues exhibited long chain reactions consistent with the release of the HO^•, as identified in EPR spin trapping experiments. Several carbon-centered radical intermediates, produced by anaerobic incubation of the QDO compounds with N-terminal truncated cytochrome P450 reductase (POR), were characterized using <i>N</i>-<i>tert</i>-butyl-α-phenylnitrone (PBN) and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-<i>N</i>-oxide (DEPMPO) spin traps and were observed by EPR. Experimental data were well simulated for the production of strongly oxidizing radicals, capable of H atom abstraction from methyl groups. The kinetics of formation and decay of the radicals produced following one-electron reduction of the parent compounds, both in oxic and anoxic solutions, were determined using pulse radiolysis. Back oxidation of the initially formed radical anions by molecular oxygen did not compete effectively with the breakdown of the radical anions to form oxidizing radicals. The QDO compounds displayed low hypoxic selectivity when tested against oxic and hypoxic cancer cell lines <i>in vitro</i>. The results from this study form a kinetic description and explanation of the low hypoxia-selective cytotoxicity of QDOs against cancer cells compared to the related benzotriazine 1,4-dioxide (BTO) class of compounds