Degree Name

Doctor of Philosophy


School of Chemistry, Faculty of Science


The altered protein expression and activity that promotes changes in cancer cells can provide drug targets for tumour-selective chemotherapy. Typically these treatments are devised based on biomarkers that have been identified as indicators of tumour grade and patient prognosis. Acquisition of enhanced proteolytic capability and invasive potential is widely recognised as the final breach of normal cellular behaviour and one which promotes tumour metastasis, and one of the key mediators is the urokinase plasminogen activation system. The system incorporates the serine protease urokinase plasminogen activator (uPA) and its cognate receptor uPAR, the upregulation of which results in the enhancement of tumour proteolytic and migratory capability. Both uPA and uPAR can be targeted with peptides and small molecules to effect a decrease in the metastatic ability of tumour cells. Another key protein in tumour development is the sodium hydrogen exchanger (NHE). NHEs are integral membrane proteins that exchange one intracellular proton for one extracellular sodium ion. Nine mammalian NHE isoforms (NHE1-NHE9) are known and several have been shown to be upregulated and/or activated in tumours, regulating intracellular pH and cell volume as well as establishing the well characterised low extracellular pH of solid tumours. Conveniently, clinically-used and orally administered potassium-sparing diuretic amiloride has been shown to inhibit both the proteolytic activity of uPA and the ion exchange of NHE1, providing an excellent lead for anticancer drug development. Section 1 explores structure-activity relationships of amiloride analogues as inhibitors of uPA. Initially, analogues substituted at the 5-position were targeted for synthesis from precursor 13, an acylguanidine derivative of the commercially available methyl ester 14. Other analogues targeted were 6-substituted derivatives which were to be accessed from 6-iodoester 57. Alternative acylguanidines, synthesised through guanidinylation of methyl ester precursors, were investigated to characterise the binding contribution of the acylguanidine unit of amiloride and to possibly identify new acylguanidine cores. Other analogues in which the acylguanidine was substituted with isosteric groups, or where exocyclic amino groups were substituted for hydrogen, were synthesised in order to establish the importance of these substituents for inhibitor binding. Overall, analogues with substituents at the 5-position were generally as potent or only slightly less potent than amiloride. Further analogues substituted at the 6-position with bulkier groups (i.e. 15, 58, 63 and 64) showed improved potency. Indeed 15 and 64 were the most potent uPA inhibitors identified in this study (6-fold increase in potency relative to amiloride). It was found that simple arylacylguanidines are not useful as uPA inhibitors and should not be pursued as alternative uPA inhibitor scaffolds, while analogues lacking either the 3- or 5-amino substituent were found to be inactive, demonstrating the importance of these groups. Compounds 11 and 12 were of particular interest in the study as they are reported as potent inhibitors of NHE-1 (11 Ki = 30 nM, 12 Ki = 13 nM). The two analogues produced 2-fold improvements in potency relative to amiloride against uPA. Compounds 15, 27 and 64 were the most potent inhibitors identified, suggesting that analogues containing double modifications at the 5- and 6-positions should be investigated further. Section 2 investigates two highly potent non-peptidic antagonists 83 and 84 (IC50 83 = 0.8 nM, IC50 84 = 33 nM) of the uPA:uPAR interaction that represent useful pharmacological tools for studying the plasminogen activation system and the effects that small molecule uPAR antagonists can have as antitumour/antimetastasis agents. Antagonists 83 and 84 were prepared by new solution phase and mixed solid/solution phase syntheses, respectively. The activities of 83 and 84 were assessed in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employ HMW-uPA as the competing ligand. Compounds 83 and 84 were found to be poor antagonists of the uPA:uPAR interaction in physiologically relevant cellular systems and are therefore not useful as small molecule probes for pharmacological investigations of the plasminogen activation system. The work highlights the importance of using HMW-uPA as the competing ligand in competition experiments aimed at measuring the potency of small molecule uPAR antagonists as potential pharmacological tools or anticancer drug leads.



Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.