Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy Michael P. Hay, * William R. Wilson and William A. Denny Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand Received 27 February 2005; revised 30 March 2005; accepted 30 March 2005 Available online 25 April 2005 Abstract—A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3–NTR neo , WiDr/WiDr–NTR neo ), Chinese hamster (V79/V79–NTR puro ) and murine (EMT6/EMT6–NTR puro ) cell line pairs, and were com- pared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN 29427. The low solubility of the prodrugs (from 3 to 39 lM) precluded the determination of IC 50 values against the parent cell lines in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24 h period, although release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold). Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel (1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold) but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs. Ó 2005 Elsevier Ltd. All rights reserved. 1. Introduction The development of gene-directed enzyme prodrug ther- apy (GDEPT) 1–5 using an oxygen-insensitive nitrore- ductase from Escherichia coli B (the nfsB gene product NTR) has been the subject of considerable study 6 since the first description of NTR. 7–10 Two main classes of nitroaromatic substrate have been identified as prodrugs activated by NTR: 2,4-dinitrobenzamides and 4-nitro- benzylcarbamates. The first-identified NTR prodrug, the 2,4-dinitrobenzamide, CB 1954 (1) 7,11–13 has ad- vanced to clinical trial 14–16 and structure–activity rela- tionships (SAR) have been determined for related aziridines 17 and nitrogen mustards. 18–21 The bromomus- tard 2 (SN 29427) shows increased in vivo activity and a larger bystander effect compared to CB 1954 in preclin- ical models. 22 A second class of prodrugs under investi- gation is based on the 4-nitrobenzylcarbamate moiety (3), which undergoes reduction to the hydroxylamine and subsequent fragmentation to release a cytotoxic amine. 23–25 Masking the amine function as a carbamate provides significant deactivation of the cytotoxin by either electronic effects, where electronic release from the amine is required for activation of the cytotoxin [r p(NH 2 ) = À0.66, r p(OCONH) = À0.17], or by steric effects limiting binding to the site of action. A variety of cyto- toxic agents bearing a critical amine group have been examined as their 4-nitrobenzylcarbamate prodrugs for NTR including aniline mustard, 26 mitomycin, 26 enediy- nes, 27,28 seco-cyclopropylindoline derivatives, 29 pyrrolo- benzodiazepines, 30 anthracyclines 26 and tallimustine analogues. 31 Related nitrobenzyl phosphorodiamidate 32 0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2005.03.055 Keywords: Prodrug; Doxorubicin; Nitroreductase. * Corresponding author. Tel.: +64 9 3737599x86598; fax: +64 9 3737502; e-mail: m.hay@auckland.ac.nz Bioorganic & Medicinal Chemistry 13 (2005) 4043–4055