Affinity-Enhanced Luminescent Re(I)- and Ru(II)-Based Inhibitors of the Cysteine Protease Cathepsin L

Two new Re­(I)- and Ru­(II)-based inhibitors were synthesized with the formulas [Re­(phen)­(CO)₃(<b>1</b>)]­(OTf) (<b>7</b>; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru­(bpy)₂(<b>2</b>)]­(Cl)₂ (<b>8</b>; bpy = 2,2′-bipyridine), where <b>1</b> and <b>2</b> are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds <b>7</b> and <b>8</b> were characterized using various spectroscopic techniques and elemental analysis; <b>7</b> and <b>8</b> both show exceptionally long excited state lifetimes. Re­(I)-based complex <b>7</b> inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor <b>1</b> with a second-order rate constant of 211000 ± 42000 M^–1 s^–1. Irreversible ligation of <b>7</b> to papain, a model of CTSL, was analyzed with mass spectroscopy, and the major peak shown at 24283 au corresponds to that of papain-<b>1</b>-Re­(CO)₃(phen). Compound <b>7</b> was well tolerated by DU-145 prostate cancer cells, with toxicity evident only at high concentrations. Treatment of DU-145 cells with <b>7</b> followed by imaging via confocal microscopy showed substantial intracellular fluorescence that can be blocked by the known CTSL inhibitor CLIK-148, consistent with the ability of <b>7</b> to label CTSL in living cells. Overall this study reveals that a Re­(I) complex can be attached to an enzyme inhibitor and enhance potency and selectivity for a medicinally important target, while at the same time allowing new avenues for tracking and quantification due to long excited state lifetimes and non-native element composition