Abstract 738: Design and synthesis of novel N10-protected pyrrolobenzodiazepine (PBD) prodrugs for use in nitroreductase-mediated GDEPT therapies

Abstract

Abstract The over-expression of telomerase in cancer cells has been previously exploited for gene therapy strategies. One approach involves the use of a plasmid containing a telomerase promoter to control the expression of an exogenous nitroreductase enzyme capable of activating bioreductively-sensitive prodrugs. CB1954 is the most commonly studied prodrug for use in bioreductive GDEPT approaches, although it has a number of drawbacks including relatively low potency, inherent toxicities and a lack of patent protection. Therefore, we have designed some novel bioreductive prodrugs based on the sequence-selective DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumour agents. The PBDs interact covalently with DNA through formation of a covalent aminal bond between their electrophilic N10-C11 position and the nucleophilic C2-NH2 of guanine bases. The prodrug design concept involves the introduction of a bulky bioreductively-sensitive protecting group at the N10-position which effectively blocks interaction with DNA thus reducing potency. However, release of the N10-protecting group under bioreductive conditions restores the ability to interact with DNA along with the original biological activity. As proof-of-principle, we installed a p-nitrobenzylcarbamate group at the N10-position of a PBD. We found that upon reduction to the N10-(p-aminobenzylcarbamate), this grouping self-immolated to afford the biologically-active parent PBD, p-aminobenzyl alcohol and carbon dioxide. Control molecules including non-reducible N10-benzyl- and N10-SEM-protected analogues incapable of self-immolation were also synthesized. Along with the parent N10-unsubstituted PBD, these molecules were all evaluated in matched in vitro panels of human A2780 (ovarian), A549 (lung), C33a (cervical) and 5637 (bladder) tumour cells, one panel (“NTR+”) being transfected with plasmids containing the Nitroreductase (NTR) gene under the control of a CMV promoter, a surrogate for the telomerase promoter. The NTR+ panel was found to be more sensitive to the prodrug than the NTR- panel, with a rank order of sensitisation of 18.4 > 8.1 > 2.6 and 1.5 for the A2780, A549, C33a and 5637 cell lines, respectively. Crucially, the prodrug was significantly less cytotoxic in all cell lines (e.g., IC50 = 0.29 and 0.015μ in NTR- and NTR+ A2780 cells, respectively) compared to the parent non-N10-substituted PBD (e.g., IC50 = 0.000151and 0.00028µM in NTR- and NTR+ A2780 cells, respectively). The N10-benzyl and SEM control molecules were significantly less cytotoxic in both NTR+ and NTR- A2780 cells, with IC50 values ranging from 1.9 to 3.3μM. In preliminary in vivo experiments, the N10-(p-nitrobenzylcarbamate) prodrug was evaluated in a nude mouse human tumour xenograft model implanted with A2780 CMV-NTR cells. A clear tumour response to the prodrug was observed at non-toxic doses. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 738.