The design, synthesis, mechanisms of activation and biological activity of indolequinone phosphoramidate prodrugs

A series of 2- and 3-substituted indolequinone phosphoramidate prodrugs was synthesized and the biological activity evaluated. These prodrugs were designed to undergo enzymatic activation by DT-diaphorase (DTD) via quinone reduction and expulsion of the phosphoramidate substituent. The liberated phosphoramidate anion is capable of cross-linking DNA and is hypothesized to be the active species for this series of compounds. The interaction of these compounds with human DTD was investigated. All of the 2-substituted indolequinones are excellent substrates for DTD; while indolequinones substituted at the 3-position with bulky leaving groups are mechanism-based inactivators of DTD. Both series of analogs were demonstrated using 31P NMR to undergo rapid activation following two-electron reduction thus establishing a role for the activation of these prodrugs by two-electron reductases (i.e. DTD). To explore the selectivity of two-electron reductive activation, alternative mechanisms of activation were explored. Reductive elimination following one-electron (radiolytic) reduction of the phosphoramidate from the 3-position is sufficient to support one-electron reduction as a potential mechanism of activation in vivo. However, reductive elimination from the 2-position occurred approximately 150-fold slower and is unlikely to represent a physiologically significant activation pathway. The one- and two-electron reduction potentials for the 2- and 3-regioisomers (cyclic voltammetry) are identical, suggesting that differences in reduction potential cannot account for the different rates of activation following one-electron reduction. Activation of the indolequinone phosphoramidates with sulfur nucleophiles dimethyldithiocarbamate and glutathione was evaluated using 31P NMR. Expulsion of phosphoramidate anion from the 3-position occurred rapidly, supporting a role for this pathway in vivo, while expulsion from the 2-position is slow and unlikely to be physiologically relevant. In general, indolequinones substituted at the 2- and 3-positions with phosphoramidates capable of cross-linking DNA were potent cytotoxins and broad inhibitors of tumor cell growth in vitro. Evaluation of these compounds using the COMET assay supports the idea that the cytotoxicity of both series of compounds is due primarily to interstrand DNA cross-linking, a finding consistent with the hypothesized mechanism of action. The results from the above experiments support drug delivery from the 2-position as the more selective and superior prodrug strategy.