Benjamin J. Buckley, Ashraf Aboelela, Elahe Minaei, Longguang X. Jiang, Zhihong Xu, Umar Ali, Karen Fildes, Chen-Yi Cheung, Simon M. Cook, Darren C. Johnson, Daniel A. Bachovchin, Gregory M. Cook, Minoti Apte, Mingdong Huang, Marie Ranson, Michael J. Kelso

Metastasis is the cause of death in the majority (~90%) of malignant cancers. The oral potassium-sparing diuretic amiloride and its 5-substituted derivative 5-N,N-(hexamethylene)amiloride (HMA) reportedly show robust anti-tumor/metastasis effects in multiple in vitro and animal models. These effects are likely due, at least in part, to inhibition of the urokinase plasminogen activator (uPA), a key protease determinant of cell invasiveness and metastasis. This study reports the discovery of 6-substituted HMA analogs that show nanomolar potency against uPA, high selectivity over related trypsin-like serine proteases and minimal inhibitory effects against epithelial sodium channels (ENaC), the diuretic and anti-kaliuretic target of amiloride. Reductions in lung metastases were demonstrated for two analogs in a late-stage experimental mouse metastasis model and one analog completely inhibited formation of liver metastases in an orthotopic xenograft mouse model of pancreatic cancer. The results support further evaluation of 6-substituted HMA derivatives as uPA-targeting anticancer drugs.