Structure–activity insights into benzimidazole-based Ir( <scp>iii</scp> ) cyclometallated complexes for cancer therapy

A family of cyclometallated Ir(III) complexes incorporating benzimidazole-based C^N ligands and ancillary diimines with distinct functionalities, 2-(pyridin-2-yl)-1<i>H</i>-benzo[<i>d</i>]imidazole (L1), 4-(1<i>H</i>-benzo[<i>d</i>]imidazol-2-yl)thiazole (L2), di(pyridin-2-yl)amine (L3), [2,2'-bipyridine]-4,4'-dicarboxylic acid (L4), and dibutyl[2,2'-bipyridine]-4,4'-dicarboxylate (L5), has been synthesised. All complexes display oxygen-sensitive emission in the 477-695 nm range; however, singlet oxygen (¹O₂) generation, detected by fluorescence spectroscopy, was only observed for complexes 1, 4, and 5, which exhibit non-structured emission bands associated with ³MLCT and ³LLCT transitions. In contrast, complexes 2 and 3 show structured emission profiles consistent with predominant ³LC character. The complexes exhibit distinct antitumour activity in A549 cells depending on the nature of the ancillary ligand. Complex 3, bearing an exocyclic amine donor, displayed efficient cellular uptake and significant dark cytotoxicity. Complexes 1 and 2, featuring endocyclic benzimidazole donors, showed lower cellular accumulation and no activity in the dark. A similar trend was observed for complex 4, while complex 5, its esterified analogue, showed remarkable chemotherapeutic activity and high cell uptake. The different p<i>K</i>_a values of 2, 3 and 4 further correlate with their internalization capacity. Upon light irradiation, complexes 2, 3, and 5 exhibited enhanced antiproliferative effects and induced apoptotic cell death, probably through reactive oxygen species (ROS) generation as indicated by flow cytometry. Nevertheless, only complex 2 retained phototherapeutic potential due to its low dark toxicity. Mechanistic studies revealed that complex 2 is capable of oxidising NADH under irradiation and generating H₂O₂. Overall, these results reveal clear structure-activity relationships and highlight complex 2 as a promising candidate for photodynamic therapy (PDT), while complexes 3 and 5 act predominantly as chemotherapeutic-like agents.