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The urokinase plasminogen activator (uPA) plays a critical role in tumor cell invasion and migration and is a promising antimetastasis target. 6-Substituted analogues of 5- N,N -(hexamethylene)amiloride (HMA) are potent and selective uPA inhibitors that lack the diuretic and antikaliuretic properties of the parent drug amiloride. However, the compounds display pronounced selectivity for human over mouse uPA, thus confounding interpretation of data from human xenograft mouse models of cancer. Here, computational and experimental findings reveal that residue 99 is a key contributor to the observed species selectivity, whereby enthalpically unfavorable expulsion of a water molecule by the 5- N,N -hexamethylene ring occurs when residue 99 is Tyr (as in mouse uPA). Analogue 7 lacking the 5- N,N -hexamethylene ring maintained similar water networks when bound to human and mouse uPA and displayed reduced selectivity, thus supporting this conclusion. The study will guide further optimization of dual-potent human/mouse uPA inhibitors from the amiloride class as antimetastasis drugs.

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 antitumor/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 antikaliuretic 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.

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site.

Ab initio molecular electrostatic potential (MEP) patterns are used to determine the electrostatic requirements for the formation of a stable blocking complex between amiloride analogs and the epithelial sodium channel of Rana ridibunda. MEP maps calculated in the 3-21G(*) and STO-3G basis sets for amiloride and analogs with pyrazine ring modifications are used to interpret differences in the microscopic rate constants for analog-channel binding determined by Li et al. MEP maps of the protonated analogs are correlated to differences in the value of kon, the microscopic association constant. Those analogs with kon values similar to amiloride are found to have a MEP maximum that is localized over the side chain, as well as strong, distinguishing minima in the MEP pattern off the carbonyl oxygen and positions 3, 4, and 5 of the pyrazine ring. MEP maps of a model-encounter complex (protonated analog and formic acid anion) are correlated to differences in koff, the microscopic dissociation constant. The major conclusions of this work are that (1) a stable blocking complex is formed with analogs which have a deep, localized minimum off the 6 position of the pyrazine ring, (2) the stability of the blocking complex is directly related to the depth of that minimum, (3) substitution at position 5 affects not only the depth but also the location and size of the minimum off position 6, and (4) steric factors may influence the optimal binding of the 6-position ligand to the ion channel. The MEP analysis also suggests that the distance between the proton donors of the chelating guanidinium moiety and the deep, localized minimum off position 6 of the pyrazine ring may define an important spatial requirement for all those analogs which form a stable blocking complex with the channel.