Singlet Oxygen Formation vs Photodissociation for Light-Responsive Protic Ruthenium Anticancer Compounds: The Oxygenated Substituent Determines Which Pathway Dominates

Abstract

Ruthenium complexes bearing protic diimine ligands are cytotoxic to certain cancer cells upon irradiation with blue light. Previously reported complexes of the type [(N,N)2Ru­(6,6′-dhbp)]­Cl2 with 6,6′-dhbp = 6,6′-dihydroxybipyridine and N,N = 2,2′-bipyridine (bipy) (1 A ), 1,10-phenanthroline (phen) (2 A ), and 2,3-dihydro-[1,4]­dioxino­[2,3-f]­[1,10]­phenanthroline (dop) (3 A ) show EC50 values as low as 4 μM (for 3 A ) vs breast cancer cells upon blue light irradiation (Inorg. Chem.20177519). Herein, subscript A denotes the acidic form of the complex bearing OH groups, and B denotes the basic form bearing O– groups. This photocytotoxicity was originally attributed to photodissociation, but recent results suggest that singlet oxygen formation is a more plausible cause of photocytotoxicity. In particular, bulky methoxy substituents enhance photodissociation but these complexes are nontoxic (Dalton Trans201815685). Cellular studies are presented herein that show the formation of reactive oxygen species (ROS) and apoptosis indicators upon treatment of cells with complex 3 A and blue light. Singlet oxygen sensor green (SOSG) shows the formation of 1O2 in cell culture for cells treated with 3 A and blue light. At physiological pH, complexes 1 A -3 A are deprotonated to form 1 B -3 B in situ. Quantum yields for 1O2 (ϕΔ) are 0.87 and 0.48 for 2 B and 3 B , respectively, and these are an order of magnitude higher than the quantum yields for 2 A and 3 A . The values for ϕΔ show an increase with 6,6′-dhbp derived substituents as follows: OMe < OH < O–. TD-DFT studies show that the presence of a low lying triplet metal-centered (3MC) state favors photodissociation and disfavors 1O2 formation for 2 A and 3 A (OH groups). However, upon deprotonation (O– groups), the 3MLCT state is accessible and can readily lead to 1O2 formation, but the dissociative 3MC state is energetically inaccessible. The changes to the energy of the 3MLCT state upon deprotonation have been confirmed by steady state luminescence experiments on 1 A -3 A and their basic analogs, 1 B -3 B . This energy landscape favors 1O2 formation for 2 B and 3 B and leads to enhanced toxicity for these complexes under physiological conditions. The ability to convert readily from OH to O– groups allowed us to investigate an electronic change that is not accompanied by steric changes in this fundamental study.

Light‐responsive and Protic Ruthenium Compounds Bearing Bathophenanthroline and Dihydroxybipyridine Ligands Achieve Nanomolar Toxicity towards Breast Cancer Cells†

“…In our past work, we have demonstrated that protonation and deprotonation events (with 3 – 5 ) determine the lipophilic vs . hydrophilic properties (5), the UV‐Vis spectral features (6,7) and whether photodissociation or singlet oxygen formation is the dominant pathway for these ruthenium complexes (8). Many Ru(II) diimine complexes exhibit light‐driven toxicity by producing singlet oxygen in cells (9,10).…”

“…This can be explained by a lower energy 3 MLCT state for the basic forms which has been demonstrated computationally and experimentally. This 3 MLCT state can interact with 3 O 2 in solution to generate toxic 1 O 2 (8). While all three of our previously reported compounds ( 3 – 5 ) can generate singlet oxygen, complex 5 is the most photocytotoxic with EC 50 values in the presence of blue light of ~4 μM ( vs .…”

“…Our combined evidence points to the light‐driven toxicity for 5 (mostly 5 B at physiological pH) being due to singlet oxygen formation. While some photodissociative ligand loss does occur, the products of such ligand loss do not appear to be toxic, with these specific co‐ligands (8, 19, 20).…”

The Development of Ru(II)-Based Photoactivated Chemotherapy Agents

“…Considering the inherent acidity surrounding cancer cells, Papish and co-workers reported a series of pH-activated PACT agents, which can be activated by light- and pH-triggered ligand dissociation [ 118 , 119 , 120 ]. At a low pH value (pH = 5), complexes 57 – 61 existed in their acidic form, and the quantum yields for photodissociation were higher than in deprotonated form.…”

“…Thus, they investigated how synthetic changes to ligands and ligand protonation states can influence the quantum yields for 1 O 2 and photodissociation. Cytotoxicity studies showed that 1 O 2 formation is a more plausible cause of photocytotoxicity [ 120 ].…”