Influence of Mustard Group Structure on Pathways of in Vitro Metabolism of Anticancer N-(2-Hydroxyethyl)-3,5-dinitrobenzamide 2-Mustard Prodrugs □ S Nuala A. Helsby, Michael A. Goldthorpe, Magdalene H. Y. Tang, Graham J. Atwell, Eileen M. Smith, William R. Wilson, and Malcolm D. Tingle Department of Molecular Medicine and Pathology (N.A.H.), Department of Pharmacology (M.A.G., M.H.Y.T., M.D.T.), and Auckland Cancer Society Research Centre (N.A.H., G.J.A, E.M.S., W.R.W.), Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand Received September 5, 2007; accepted November 9, 2007 ABSTRACT: The dinitrobenzamide mustards are a class of bioreductive nitro- aromatic anticancer prodrugs, of which a phosphorylated analog (PR-104) is currently in clinical development. They are bioactivated by tumor reductases to form DNA cross-linking cytotoxins. How- ever, their biotransformation in normal tissues has not been exam- ined. Here we report the aerobic in vitro metabolism of three N-(2 hydroxyethyl)-3,5-dinitrobenzamide 2-mustards and the cor- responding nonmustard analog in human, mouse, rat, and dog hepatic S9 preparations. These compounds have a range of mus- tard structures (–N(CH 2 CH 2 X) 2 where X  H, Cl, Br, or OSO 2 Me). Four metabolic routes were identified: reduction of either nitro group, N-dealkylation of the mustard, plus O-acetylation, and O-glucuronidation of the hydroxyethyl side chain. Reduction of the nitro group ortho to the mustard resulted in intramolecular alkyla- tion and is considered to be an inactivation pathway, whereas reduction of the nitro group para to the mustard generated poten- tial DNA cross-linking cytotoxins. N-Dealkylation inactivated the mustard moiety but may result in the formation of toxic acetalde- hyde derivatives. Increasing the size of the nitrogen mustard leav- ing group abrogated the ortho-nitroreduction and N-dealkylation routes and thereby improved overall metabolic stability but had little effect on aerobic para-nitroreduction. All four compounds underwent O-glucuronidation of the hydroxyethyl side chain and further studies to elucidate the relative importance of this pathway in vivo are in progress. The dinitrobenzamide mustards (DNBMs) are bioreductive antican- cer prodrugs designed to be activated by nitroreduction, either by endogenous one-electron reductases in hypoxic regions of tumors or by exogenous oxygen-independent two-electron reductases expressed in tumors using gene therapy. Enzymatic nitroreduction converts an electron-withdrawing nitro group ( p +0.78) to a hydroxylamine ( p -0.32) or amine ( p -0.66) metabolite; this “electronic switch” can be used to activate the latent nitrogen mustard moiety (Denny and Wilson, 1986; Palmer et al., 1990; Siim et al., 1997) The 2,4-dinitro- benzamide-5-mustard class (2,4-DNBM) was investigated first and shown to provide cytotoxicity and DNA cross-linking selectively under hypoxic conditions (Palmer et al., 1992). The 2,4-DNBMs were subsequently shown to be substrates for the oxygen-insensitive ni- troreductase nfsB from Escherichia coli (Anlezark et al., 1995; Wilson et al., 2002; Atwell et al., 2007), which is of interest for prodrug activation in the context of gene- or virus-directed enzyme prodrug therapy. We have shown that SN 23862 and other 2,4-DNBMs pro- vide efficient bystander effects (killing of adjoining cells) when activated by either endogenous one-electron reductases or the nfsB nitroreductase through the diffusion of an activated metabolite in which the nitro group para to the mustard has been reduced to the corresponding amine (Wilson et al., 2002; Helsby et al., 2004; Wilson et al., 2007). In addition to its metabolic activation in tumor cells, the 2,4-DNBM SN 23862 is extensively metabolized in tumor-bearing mice, with nitroreduction of either the 4- or 2-nitro group as well as sequential oxidative N-dealkylation of the mustard moiety (Kestell et al., 2000). The latter route yields the nontoxic 2,4-dinitro-5 amine, but the dechloroethylation reactions also generate chloroacetaldehyde, which is a reactive cytotoxin (Sood and O’Brien, 1993). Formation of Financial support for this project was provided by the Auckland Medical Research Foundation and the Health Research Council of New Zealand. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.018739. □ S The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. ABBREVIATIONS: DNBM, dinitrobenzamide mustard; 2,4-DNBM, 2,4-dinitrobenzamide 5-mustard; SN 23862, bischloroethyl-2,4-dinitrobenz- amide; 3,5-DNBM, 3,5-dinitrobenzamide 2-mustard; PR-104, 2-((2-bromoethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethyl meth- anesulfonate phosphate ester; CB 1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide; SN 27858, 2-((2-bromoethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}- 4,6-dinitroanilino)ethyl methanesulfonate, PR-104A, 1; SN 29546, 2-((2-chloroethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate, 2; SN 27686, 2-[bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide, 3; SN 29893, 2-(diethylamino)-N-(2- hydroxyethyl)-3,5-dinitrobenzamide, 4; LC, liquid chromatography; MS, mass spectrometry; HPLC, high-performance liquid chromatography; UDPGA, UDP-glucuronic acid; Rt, retention time; amu, atomic mass units. 0090-9556/08/3602-353–360$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 18739/3298125 DMD 36:353–360, 2008 Printed in U.S.A. 353