(A) DNA alkylation assay employing linearized pBR322 DNA and the pyridone derivatives 17a or 17b or the unsaturated imines 15a or 15b. Conditions: Linearized pBR322 DNA (20 μM in base pairs (bps)), 15a (100, 10, 1, 0.5, or 0.1 μM), 15b (500, 100, 10, or 1 μM), 17a (500 or 100 μM), or 17b (500 or 100 μM), 37 °C, 15 h. Cisplatin (CP; 100 μM) and methylmethanesulfonate (MMS; 500, 100, or 10 μM) were used as positive controls for DNA cross-linking and alkylation, respectively. DNA was visualized using SybrGold. B. DNA alkylation assay employing linearized pBR322 DNA and the unsaturated imine 15a. Conditions: Linearized pBR322 DNA (20 μM in base pairs), 15a (100, 10, 1, 0.1, 0.05, 0.01, or 0.005 μM), 37 °C, 15 h.