Laromustine

Abstract: A series of 4-nitrobenzyloxycarbonyl prodrug derivatives of O(6)-benzylguanine (O(6)-BG), conceived as prodrugs of O(6)-BG, an inhibitor of the resistance protein O(6)-alkylguanine-DNA alkyltransferase (AGT), were synthesized and evaluated for their ability to undergo bioreductive activation by reductase enzymes under oxygen deficiency. Three agents of this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1) and its monomethyl (2) and gem-dimethyl analogues (3), were tested for activation by reductase enzyme systems under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O(6)-BG following reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency, providing evidence for the preferential release of the AGT inhibitor O(6)-BG under hypoxia.

Abstract: Cloretazine {1,2-bis(methylsulfonyl)-l-[(2-chloroethyl)-2-(methylamino)carbonyl]hydrazine; VNP40101M; 101M} is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy against transplanted murine and human tumor models and has shown activity in clinical trials against relapsed or refractory acute myeloid leukemia. Base catalyzed activation of this prodrug generates two different reactive intermediates: chloroethylating species that covalently interact with DNA at the O 6 -position of guanine residues that progress to a G-C interstrand cross-link, and a carbamoylating agent, methyl isocyanate. Previous findings from this laboratory have provided initial evidence that methyl isocyanate can contribute to the efficacy of Clore-tazine by enhancing the cytotoxicity of the generated chloroethylating species. This action may be due in part to inhibition of the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT); however, activity in cells devoid of AGT indicates that other actions are involved in the synergistic cytotoxicity. Herein we demonstrate that O 6 -benzylguanine can also produce synergistic cell kill with the alkylating component of Cloretazine but differs from methyl isocyanate in that the enhancement occurs in AGT-containing cells, but not in cells devoid of AGT. Methyl isocyanate generated by the decomposition of 1,2-bis(methylsulfonyl)-1-[methylaminocarbonyl]hydrazine also acts to enhance the activity of a variety of DNA cross-linking agents, while only producing additive cytotoxicity with methylating agents. Flow cytometric studies using annexin as a marker for apoptosis indicate that in Chinese hamster ovary cells and in human leukemia cells Cloretazine-induced apoptosis is primarily caused by the generated methyl isocyanate. Comet assays designed to detect DNA cross-links in intact cells indicate that the chloroethylating species generated by the activation of Cloretazine produce DNA cross-links, with the co-generated methyl isocyanate increasing the degree of cross-linking produced by the reactive chloroethylating species. These findings provide further evidence that the methyl isocyanate produced by the activation of Cloretazine can be a major contributor to the cytotoxicity produced by this antineoplastic agent.

Abstract: To investigate the interaction of the electrophilic species generated by the decomposition of the antineoplastic prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M) on the ability of O6-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O6-chloroethylguanine and/or N1,O6-ethanoguanine DNA lesions. The contributions of inhibitory electrophilic species generated from VNP40101M towards AGT was assessed using analogues that selectively generated either the chloroethylating or the carbamoylating components of VNP40101M. The activity of AGT was determined from the inhibition of crosslink formation from O6-chloroethylguanine and/or N1,O6-ethanoguanine lesions. The half-lives of sulfonylhydrazine derivatives and isocyanates were measured using an acidification assay which gives a change in absorbance proportional to the release or consumption of small quantities of protons. Both of the reactive components produced by VNP40101M directly inactivated cloned human AGT; the carbamoylating moiety (IC50 about 13 μM) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC50 about 100 μM). These inhibitory actions were moderated by the addition of naked T5 bacteriophage DNA. Thus, AGT bound to DNA was markedly more resistant than free AGT to these electrophilic species. DNA also blocked the spontaneous loss of AGT activity which occurred upon incubation of this protein under mild conditions. The reaction of AGT with the methyl isocyanate generated from the decomposition of VNP40101M increased the net number of crosslinks generated by VNP40101M compared to a sulfonylhydrazine prodrug that formed the equivalent alkylating species in the absence of the cogeneration of methyl isocyanate. These actions may be of significance to the antineoplastic activity of VNP40101M.