Ru(II) Complexes with Absorption in the Photodynamic Therapy Window: 1O2 Sensitization, DNA Binding, and Plasmid DNA Photocleavage.

Two Ru(II) complexes, [Ru(pydppn)(bim)(py)]2+ [2; pydppn = 3-(pyrid-2'-yl)-4,5,9,16-tetraaza-dibenzo[a,c]naphthacene; bim = 2,2'-bisimidazole; py = pyridine] and [Ru(pydppn)(Me4bim)(py)]2+ [3; Me4bim = 2,2'-bis(4,5-dimethylimidazole)], were synthesized and characterized, and their photophysical properties, DNA binding, and photocleavage were evaluated and compared to [Ru(pydppn)(bpy)(py)]2+ (1; bpy = 2,2'-bipyridine). Complexes 2 and 3 exhibit broad 1MLCT (metal-to-ligand charge transfer) transitions with maxima at ∼470 nm and shoulders at ∼525 and ∼600 nm that extend to ∼800 nm. These bands are red-shifted relative to those of 1, attributed to the π-donating ability of the bim and Me4bim ligands. A strong signal at 550 nm is observed in the transient absorption spectra of 1-3, previously assigned as arising from a pydppn-centered 3ππ* state, with lifetimes of ∼19 µs for 1 and 2 and ∼270 ns for 3. A number of methods were used to characterize the mode of binding of 1-3 to DNA, including absorption titrations, thermal denaturation, relative viscosity changes, and circular dichroism, all of which point to the intercalation of the pydpppn ligand between the nucleobases. The photocleavage of plasmid pUC19 DNA was observed upon the irradiation of 1-3 with visible and red light, attributed to the sensitized generation of 1O2 by the complexes. These findings indicate that the bim ligand, together with pydppn, serves to shift the absorption of Ru(II) complexes to the photodynamic therapy window, 600-900 nm, and also extend the excited state lifetimes for the efficient production of cytotoxic singlet oxygen.

Differences in Photophysical Properties and Photochemistry of Ru(II)-Terpyridine Complexes of CH3CN and Pyridine.

A series of 22 Ru(II) complexes of the type [Ru(tpy)(L)(L')]n+, where tpy is the tridentate ligand 2,2';6,2″-terpyridine, L represents bidentate ligands with varying electron-donating ability, and L' is acetonitrile (1a-11a) or pyridine (1b-11b), were investigated. The dissociation of acetonitrile occurs from the 3MLCT state in 1a-11a, such that it does not require the population of a 3LF state. Electrochemistry and spectroscopic data demonstrate that the ground states of these series do not differ significantly. Franck-Condon line-shape analysis of the 77 K emission data shows no significant differences between the emitting 3MLCT states in both series. Arrhenius analysis of the temperature dependence of 3MLCT lifetimes shows that the energy barrier (Ea) to thermally populating a 3LF state from a lower energy 3MLCT state is significantly higher in the pyridine than in the CH3CN series, consistent with the photostability of complexes 1b-11b, which do not undergo pyridine photodissociation under our experimental conditions. Importantly, these results demonstrate that ligand photodissociation of pyridine in 1b-11b does not take place directly from the 3MLCT state, as is the case for 1a-11a. These findings have potential impact on the rational design of complexes for a number of applications, including photochemotherapy, dye-sensitized solar cells, and photocatalysis.

Acetonitrile Ligand Photosubstitution in Ru(II) Complexes Directly from the 3MLCT State.

The excited states of the series [Ru(tpy)(L)(CH3CN)]n+ (1-11) (n = 1, 2) containing bidentate ligands L with varying electron-donating ability were characterized through Arrhenius analysis of the temperature dependence of their excited-state lifetimes. Complexes 1-11 undergo photoinduced CH3CN dissociation upon 450 nm irradiation with ligand exchange quantum yields that increase with the energy barrier to populating a dissociative triplet ligand field (3LF) state from the lowest-energy triplet metal-to-ligand charge transfer (3MLCT) excited state. Combined with DFT calculations, the data indicate that ligand photodissociation in 1-11 occurs directly from the 3MLCT state instead of a 3LF state. This finding is in contrast to the generally accepted mechanism for ligand photodissociation in Ru(II) complexes and indicates that alternative pathways for photoinduced ligand dissociation are available. These results can widely impact design principles for applications that require ligand photodissociation, such as photochemotherapy and photocatalysis, as well as for those where photosubstitution is undesirable, such as solar energy conversion.

Ir(III)-Based Agents for Monitoring the Cytochrome P450 3A4 Active Site Occupancy.

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

Photocytotoxicity and photoinduced phosphine ligand exchange in a Ru(ii) polypyridyl complex.

Two new tris-heteroleptic Ru(ii) complexes with triphenylphosphine (PPh3) coordination, cis-[Ru(phen)2(PPh3)(CH3CN)]2+ (1a, phen = 1,10-phenanthroline) and cis-[Ru(biq)(phen)(PPh3)(CH3CN)]2+ (2a, biq = 2,2'-biquinoline), were synthesized and characterized for photochemotherapeutic applications. Upon absorption of visible light, 1a exchanges a CH3CN ligand for a solvent water molecule. Surprisingly, the steady-state irradiation of 2a followed by electronic absorption and NMR spectroscopies reveals the photosubstitution of the PPh3 ligand. Phosphine photoinduced ligand exchange with visible light from a Ru(ii) polypyridyl complex has not previously been reported, and calculations reveal that it results from a trans-type influence in the excited state. Complexes 1a and 2a are not toxic against the triple negative breast cancer cell line MDA-MB-231 in the dark, but upon irradiation with blue light, the activity of both complexes increases by factors of >4.2 and 5.8, respectively. Experiments with PPh3 alone show that the phototoxicity observed for 2a does not arise from the released phosphine ligand, indicating the role of the photochemically generated ruthenium aqua complex on the biological activity. These complexes represent a new design motif for the selective release of PPh3 and CH3CN for use in photochemotherapy.

Unlocking the Potential of Ru(II) Dual-action Compounds with the Power of the Heavy-atom Effect.

We report the synthesis, photochemical and biological characterization of two new Ru(II) photoactivated complexes based on [Ru(tpy)(Me2 bpy)(L)]2+ (tpy = 2,2':6',2''-terpyridine, Me2 bpy = 6,6'-dimethyl-2,2'-bipyridine), where L = pyridyl-BODIPY (pyBOD). Two pyBOD ligands were prepared bearing flanking hydrogen or iodine atoms. Ru(II)-bound BODIPY dyes show a red-shift of absorption maxima relative to the free dyes and undergo photodissociation of BODIPY ligands with green light irradiation. Addition of iodine into the BODIPY ligand facilitates intersystem crossing, which leads to efficient singlet oxygen production in the free dye, but also enhances quantum yield of release of the BODIPY ligand from Ru(II). This represents the first report of a strategy to enhance photodissociation quantum yields through the heavy-atom effect in Ru(II) complexes. Furthermore, Ru(II)-bound BODIPY dyes display fluorescence turn-on once released, with a lead analog showing nanomolar EC50 values against triple negative breast cancer cells, >100-fold phototherapeutic indexes under green light irradiation, and higher selectivity toward cancer cells as compared to normal cells than the corresponding free BODIPY photosensitizer. Conventional Ru(II) photoactivated complexes require nonbiorthogonal blue light for activation and rarely show submicromolar potency to achieve cell death. Our study represents an avenue for the improved photochemistry and potency of future Ru(II) complexes.

Photosensitive Ru(II) Complexes as Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4.

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.

Dual-Action Ru(II) Complexes with Bulky π-Expansive Ligands: Phototoxicity without DNA Intercalation.

We report the synthesis and photochemical and biological characterization of Ru(II) complexes containing π-expansive ligands derived from dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine (Me2dppn) adorned with flanking aryl substituents. Late-stage Suzuki couplings produced Me2dppn ligands substituted at the 10 and 15 positions with phenyl (5), 2,4-dimethylphenyl (6), and 2,4-dimethoxyphenyl (7) groups. Complexes of the general formula [Ru(tpy)(L)(py)](PF6)2 (8-10), where L = 4-7, were characterized and shown to have dual photochemotherapeutic (PCT) and photodynamic therapy (PDT) behavior. Quantum yields for photodissociation of monodentate pyridines from 8-10 were about 3 times higher than that of parent complex [Ru(tpy)(Me2dppn)(py)](PF6)2 (1), whereas quantum yields for singlet oxygen (1O2) production were ∼10% lower than that of 1. Transient absorption spectroscopy indicates that 8-10 possess long excited state lifetimes (τ = 46-50 µs), consistent with efficient 1O2 production through population and subsequent decay of ligand-centered 3ππ* excited states. Complexes 8-10 displayed greater lipophilicity relative to 1 and association to DNA but do not intercalate between the duplex base pairs. Complexes 1 and 8-10 showed photoactivated toxicity in breast and prostate cancer cell lines with phototherapeutic indexes, PIs, as high as >56, where the majority of cell death was achieved 4 h after treatment with Ru(II) complexes and light. Flow cytometric data and rescue experiments were consistent with necrotic cell death mediated by the production of reactive oxygen species, especially 1O2. Collectively, this study confirms that DNA intercalation by Ru(II) complexes with π-expansive ligands is not required to achieve photoactivated cell death.