A Strategy for Selective O 6 ‐ Alkylguanine‐DNA Alkyltransferase Depletion Under Hypoxic Conditions

Cellular resistance to chemotherapeutics that alkylate the O ‐6 position of guanine residues in DNA correlates with their O 6 ‐alkylguanine‐DNA alkyltransferase activity. In normal cells high [ O 6 ‐alkylguanine‐DNA alkyltransferase] is beneficial, sparing the host from toxicity, whereas in tumor cells high [ O 6 ‐alkylguanine‐DNA alkyltransferase] prevents chemotherapeutic response. Therefore, it is necessary to selectively inactivate O 6 ‐alkylguanine‐DNA alkyltransferase in tumors. The oxygen‐deficient compartment unique to solid tumors is conducive to reduction, and could be utilized to provide this selectivity. Therefore, we synthesized 2‐nitro‐6‐benzyloxypurine, an analog of O 6 ‐benzylguanine in which the essential 2‐amino group is replaced by a nitro moiety, and 2‐nitro‐6‐benzyloxypurine is >2000‐fold weaker than O 6 ‐benzylguanine as an O 6 ‐alkylguanine‐DNA alkyltransferase inhibitor. We demonstrate oxygen concentration sensitive net reduction of 2‐nitro‐6‐benzyloxypurine by cytochrome P450 reductase, xanthine oxidase, and EMT6, DU145, and HL‐60 cells to yield O 6 ‐benzylguanine. We show that 2‐nitro‐6‐benzyloxypurine treatment depletes O 6 ‐alkylguanine‐DNA alkyltransferase in intact cells under oxygen‐deficient conditions and selectively sensitizes cells to laromustine (an agent that chloroethylates the O ‐6 position of guanine) under oxygen‐deficient but not normoxic conditions. 2‐Nitro‐6‐benzyloxypurine represents a proof of concept lead compound; however, its facile reduction (E 1/2 – 177 mV versus Ag/AgCl) may result in excessive oxidative stress and/or the generation of O 6 ‐alkylguanine‐DNA alkyltransferase inhibitors in normoxic regions in vivo .