| Abstract
| - A series of indolequinones including derivatives of EO9 bearing various functional groups andrelated indole-2-carboxamides have been studied with a view to identifying molecular featureswhich confer substrate specificity for purified human NAD(P)H:quinone oxidoreductase (DT-diaphorase), bioreductive activation to DNA-damaging species, and selectivity for DT-diaphorase-rich cells in vitro. A broad spectrum of substrate specificity exists, but minor changesto the indolequinone nucleus have a significant effect upon substrate specificity. Modificationsat the 2-position are favorable in terms of substrate specificity as these positions are locatedat the binding site entrance as determined by molecular modeling studies. In contrast,substitutions at the (indol-3-yl)methyl position with bulky leaving groups or a group containinga chlorine atom result in compounds which are poor substrates, some of which inactivate DT-diaphorase. Modeling studies demonstrate that these groups sit close to the mechanisticallyimportant amino acids Tyr 156 and His 162 possibly resulting in either alkylation within theactive site or disruption of charge-relay mechanisms. An aziridinyl group at the 5-position isessential for potency and selectivity to DT-diaphorase-rich cells under aerobic conditions. Themost efficient substrates induced qualitatively greater single-strand DNA breaks in cell-freeassays via a redox mechanism involving the production of hydrogen peroxide (catalaseinhibitable). This damage is unlikely to form a major part of their mechanism of action in cellssince potency does not correlate with extent of DNA damage. In terms of hypoxia selectivity,modifications at the 3-position generate compounds which are poor substrates for DT-diaphorasebut have high hypoxic cytotoxicity ratios.
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