Dual action agents containing a cysteine protease inhibitor and Rubased photosensitizer for photodynamic therapy (PDT) were designed, synthesized, and validated in 2D culture and 3D functional imaging assays of triple-negative human breast cancer (TNBC). These combination agents deliver and release Rubased PDT agents to tumor cells and cause cancer cell death upon irradiation with visible light, while at the same time inactivating cathespin B (CTSB), a cysteine protease strongly associated with invasive and metastatic behavior. In total five Rubased complexes were synthesized with the formula [Ru(bpy)2(l)](O2CCF3)2 (3), where bpy = 2,2′-bipyridine and 1 = a bipyridine-based epoxysuccinyl inhibitor; [Ru(tpy)(NN)(2)](PF6)2, where tpy = terpiridine, 2 = a pyridine-based epoxysuccinyl inhibitor and NN = 2,2′-bipyridine (4); 6,6′-dimethyl-2,2′-bipyridine (5); benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (6); and 3,6-dimethylbenzo[i]-dipyrido[3,2-a:2′,3′ -c]phenazine (7). Compound 3 contains a [Ru(bpy)3]2+ fluorophore and was designed to track the subcellular localization of the conjugates, whereas compounds 4–7 were designed to undergo either photoactivated ligand dissociation and/or singlet oxygen generation. Photochemical studies confirmed that complexes 5 and 7 undergo photoactivated ligand dissociation, whereas 6 and 7 generate singlet oxygen. Inhibitors 1–7 all potently and irreversibly inhibit CTSB. Compounds 4–7 were evaluated against MDA-MB-231 TNBC and MCF-10A breast epithelial cells in 2D and 3D culture for effects on proteolysis and cell viability under dark and light conditions. Collectively, these data reveal that 4–7 potently inhibit dye-quenched (DQ) collagen degradation, whereas only compound 7 causes efficient cell death under light conditions, consistent with its ability to release a Ru(II)-based photosensitizer and to also generate 1O2.

A series of Ru(ii) complexes bearing the tridentate 2,6-di(quinolin-2-yl)pyridine (dqpy) ligand was designed to undergo photoinduced ligand dissociation with red/near-IR light.

We report the synthesis and photochemical and biological characterization of the first selective and potent metal-based inhibitors of cytochrome P450 3A4 (CYP3A4), the major human drug metabolizing enzyme. Five Ru­(II)-based derivatives were prepared from two analogs of the CYP3A4 inhibitor ritonavir, 4 and 6: [Ru­(tpy)­(L)­(6)]­Cl2 (tpy = 2,2′:6′,2″-terpyridine) with L = 6,6′-dimethyl-2,2′-bipyridine (Me2bpy; 8), dimethylbenzo­[i]­dipyrido­[3,2-a:2′,3′-c]­phenazine (Me2dppn; 10) and 3,6-dimethyl-10,15-diphenylbenzo­[i]­dipyrido­[3,2-a:2′,3′-c]­phenazine (Me2Ph2dppn; 11), [Ru­(tpy)­(Me2bpy)­(4)]­Cl2 (7) and [Ru­(tpy)­(Me2dppn)­(4)]­Cl2 (9). Photochemical release of 4 or 6 from 7–11 was demonstrated, and the spectrophotometric evaluation of 7 showed that it behaves similarly to free 4 (type II heme ligation) after irradiation with visible light but not in the dark. Unexpectedly, the intact Ru­(II) complexes 7 and 8 were found to inhibit CYP3A4 potently and specifically through direct binding to the active site without heme ligation. Caged inhibitors 9–11 showed dual action properties by combining photoactivated dissociation of 4 or 6 with efficient 1O2 production. In prostate adenocarcinoma DU-145 cells, compound 9 had the best synergistic effect with vinblastine, the anticancer drug primarily metabolized by CYP3A4 in vivo. Thus, our study establishes a new paradigm in CYP inhibition using metalated complexes and suggests possible utilization of photoactive CYP3A4 inhibitory compounds in clinical applications, such as enhancement of therapeutic efficacy of anticancer drugs.

A new complex, [Ru(tpy)(dppn)(Cbz-Leu-NHCH2CN)]2+ (1, tpy = 2,2':6',2''-terpyridine, dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) was synthesized and its photochemical properties were investigated. This complex undergoes photorelease of the Cbz-Leu-NHCH2CN ligand, a known cathepsin K inhibitor, with a quantum yield, Φ450, of 0.0012(4) in water (λirr = 450 nm). In addition, 1 sensitizes the production of singlet oxygen upon visible light irradiation with quantum yield, ΦΔ, of 0.64(3) in CH3OH. The photophysical properties of 1 were compared with those of [Ru(tpy)(bpy)(Cbz-Leu-NHCH2CN)]2+ (2, bpy = 2,2'-bipyridine), [Ru(tpy)(dppn)(CH3CN)]2+ (3), and [Ru(tpy)(bpy)(CH3CN)]2+ (4) to evaluate the effect of the release of the Cbz-Leu-NHCH2CN inhibitor relative to the CH3CN ligand, as well as the role of dppn as the bidentate ligand for 1O2 production instead of bpy. Nanosecond transient absorption spectroscopy confirms the formation of the long-lived dppn-centered 3ππ* state in 1 and 3 with a maximum at ∼540 nm and τ ∼20 μs in deaerated acetonitrile. Complexes 1 and 3 are able to cause photoinduced damage to DNA (λirr ≥ 395 nm), whereas 2 and 4 do not photocleave DNA under similar experimental conditions. These results suggest that 1 is a promising agent for dual activity, both releasing a drug and producing singlet oxygen, and is poised to exhibit enhanced biological activity in phototochemotherapy upon irradiation with visible light.