DNA-targeted 1,2,4-benzotriazine 1,4-dioxides as hypoxia-selective analogues of Tirapazamine

Tirapazamine (TPZ) is a bioreductive hypoxia-selective cytotoxin currently in Phase II/III clinical trial in combination with radiotherapy or cisplatin-based chemotherapy. In order to develop TPZ analogues with improved solubility/potency and therapeutic indices, we prepared a series of compounds where a DNA-targeting chromophore is attached to the 3-position via a flexible linker. We measured binding of these compounds to calf thymus DNA, using an equilibrium dialysis method with HPLC analysis. The binding constants, KDNA, ranged from 100 M-1 to 7 x 105 M-1. DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore. The efficacy of the compounds in killing aerobic and hypoxic mouse SCCVII tumour cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C10) and the hypoxic toxicity relative to TPZ (RHT) was determined, together with the differential between the hypoxic and aerobic cytotoxicity, for each compound (HCR). There was a strong positive correlation between KDNA and RHT (spanning ca 70-fold range; r = 0.966) and no correlation between KDNA and HCR. The strongest binder in the series was 56 times more potent than TPZ under hypoxia and had an HCR of 188 (ca 258 for TPZ). These data support the hypothesis that, by targeting TPZ to DNA, higher levels of the toxic TPZ radical are formed in the vicinity of the DNA by reductive enzymes in the cell nucleus under hypoxia, leading to more DNA damage and increased cytotoxicity. This work, combined with other pharmacological studies, is expected to identify DNA-targeted TPZ analogues that have increased anti-tumour efficacy