Abstract 3203: Targeting tumor hypoxia with prodrug conjugates of potent small-molecule inhibitors of tubulin polymerization

Abstract

Abstract Regions of pronounced hypoxia in malignant tumors represent an opportunity for selectivity in drug activation, and hence targeting and delivery. In this study, a series of bioreductively activatable prodrug conjugates (BAPCs) of small-molecule inhibitors of tubulin polymerization were synthesized and evaluated in biochemical and biological studies. The BAPCs are designed to be biologically inert until selectively cleaved under hypoxic conditions, by reductase enzymes found in the tumor, to release potent effector anticancer agents. BAPCs were evaluated in the following assays: (1) cytotoxicity using the regular sulforhodamine B assay (normoxic conditions) to confirm their reduced cytotoxicity in cancer cells in culture compared to the effector agents; (2) cleavage under anoxic conditions by the reductase enzyme, NADPH cytochrome P450 oxidoreductase (POR), that is implicated in the bioreductive cleavage of compounds with nitrothiophene and nitroimidazole triggers; and (3) differential cytotoxicity under hypoxic versus normoxic conditions in cancer cell lines with the established bioreductive compound tirapazamine (TPZ) as the control. The POR assay was modified by the addition of protocatechuate 3,4-dioxygenase to ensure anoxic conditions, and Triton X-100 to facilitate solubilization of the BAPCs. A series of unsubstituted, methyl, and gem-dimethyl nitrothiophene- and nitroimidazole-triggered prodrug conjugates (BAPCs) of two experimental anticancer agents (KGP03 and KGP18) were synthesized. KGP03 and KGP18 were inspired by the natural product combretastatin A-4 (CA4). A unique attribute of the selected anticancer agents is that both bind tubulin at the colchicine site, inhibit tubulin polymerization into microtubules, and thus function as pronounced cytotoxic, anti-proliferative agents in human cancer cell lines and as vascular disrupting agents (VDAs). The assays were validated with a series of nitrothiophene analogues of CA4 (Thomson et al., Mol. Cancer Ther. 2006 5:2886). In our evaluation of this series, the gem-dimethyl nitrothiophene analogue of CA4 was efficiently cleaved by POR and gave an average differential hypoxia cytotoxicity ratio (HCR: GI50 values of normoxic/hypoxic conditions) of 41 in the human A549 lung carcinoma cell line. The corresponding prodrug of combretastatin A-1 had an HCR = 26. The monomethyl nitroimidazole BAPCs of KGP03 and KGP18 produced positive HCRs in our initial assays. In preliminary studies, the CA4 BAPC demonstrated antivascular activity in an orthotopic syngeneic breast tumor mouse model (4T1/BALB/c). In the POR assay, the gem-dimethyl nitrothiophene analogue of KGP03 was completely cleaved in 24 h. Biological evaluation of these BAPCs indicates the occurrence of selective, hypoxic activation resulting in release of the potent inhibitors of tubulin polymerization (CA4, CA1, KGP03, KGP18). Citation Format: Kevin G. Pinney, Mary Lynn Trawick, Ralph P. Mason, Li Liu, David J. Chaplin, Blake A. Winn, Laxman Devkota, Tracy E. Strecker, Jeni Gerberich, Alex Winters, Yifan Wang, Matthew T. MacDonough. Targeting tumor hypoxia with prodrug conjugates of potent small-molecule inhibitors of tubulin polymerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3203. doi:10.1158/1538-7445.AM2017-3203