Abstract
Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure-activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation. Single-electron metabolism was achieved by exposing the human carcinoma cell line T47D to each agent under hypoxic conditions, whilst concerted two-electron metabolism was assessed by stably expressing the cDNA for human NQO1 in a cloned cell line of T47D. The C-3 and C-5 positions of the indolequinone nucleus were modified to manipulate reactivity of the reduction products and the four prodrugs were identified as NQO1 substrates of varying specificity. Two of the four prodrugs, in which both C-3 and C-5 groups remained functional, proved to be NQO1-directed cytotoxins with selectivity ratios of 60- to 80-fold in the T47D (WT) versus the NQO1 overexpressing T47D cells. They also retained selectivity as hypoxic cytotoxins with oxic/hypoxic ratios of 20- to 22-fold. Replacement of the C-3 hydroxymethyl leaving group with an aldehyde group ablated all selectivity in air and hypoxia in both cell lines. Addition of a 2-methyl group on the C-5 aziridinyl group to introduce steric hinderance reduced but did not abolish NQO1-dependent metabolism. However, it enhanced single-electron metabolism-dependent DNA cross-linking in a manner that was independent of cytotoxicity. These data demonstrate that subtle structure-activity relationship exists for different cellular reductases and under certain circumstances distinct forms of DNA damage can arise, the cytotoxic consequences of which can vary. This study identifies a candidate indolequinone analogue for further development as a dual hypoxia and NQO1-directed prodrug