Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator

4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups.This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.Multiple proteases, including matrix metalloproteases (MMP-2, MMP-9 and MMP-14), cysteine proteases (cathepsin B and cathepsin L), aspartyl protease (cathepsin D) and serine proteases (plasmin, matriptase and urokinase) participate in cancer cell growth, metastasis and angiogenesis [1-4]. High expression of proteases often correlates with a poor prognosis [5,6]. Urokinase (uPA) plays an especially important role in extracellular proteolysis that contributes to cancer cell metastasis. Many cancer cells secrete pro-uPA and its receptor uPAR; binding of pro-uPA to uPAR leads to its activation, with subsequent generation of plasmin by the uPA-catalyzed hydrolysis of extracellular plasminogen [7,8]. The increased production of plasmin leads to degradation of extracellular matrix both by plasmin itself and by other proteases that are activated by plasmin. The surface location of bound uPA provides directionality to the degradation of matrix, thereby assisting the directional migration of cancer cells.