Microwave-assisted synthesis and photodynamic activity of tris-heteroleptic Ru(II) complexes with asymmetric polypyridyl ligands Received 1 April 2022, Accepted 15 July 2022, Available online 20 July 2022, Version of Record 26 July 2022.
1. Introduction
Photodynamic therapy (PDT) has emerged as a promising alternative cancer treatment or complement to chemotherapy, radiotherapy, or surgery. The success of PDT is attributed to its excellent spatial and temporal selectivity, negligible side effects, and non-invasiveness [1]. PDT is based on the excitation of generally nontoxic photosensitizers (PS) in the dark with visible or near-infrared light to alter the viability of cancer cells by, among others, generating high levels of reactive oxygen species (ROS) [2]. The production of ROS occurs because of energy or electron or hydrogen atom transfer from the triplet excited state of the PSs to dioxygen molecule. Therefore, compounds with long-lived triplet excited states are desirable for PDT.
Ruthenium(II) polypyridyl complexes are drawing particular attention because of their rich photophysical and outstanding biological properties. In addition to PDT, Ru complexes are used in photochemotherapy [3], [4], [5], [6], [7], imaging [8], [9], [10], [11], or solar energy conversion [12], [13], among others. The most successful Ru(II) compound TLD-1433 has recently entered phase II clinical trials as a photosensitizer for PDT against bladder cancer [14], [15]. Ruthenium(II) polypyridyl complexes possess a long list of advantages, which makes them a promising alternative for currently used therapeutics. Researchers often mention intense luminescence, large Stokes shift, high water solubility, and good photostability of Ru(II) polypyridyl complexes [16]. However, the longest-wavelength absorption band (usually a metal-to-ligand-charge-transfer MLCT transition) of most Ru(II) complexes is located in the blue region of the visible spectrum (<500 nm), which limits their phototherapeutic applications [17], [18]. Therefore, exploring new Ru(II) compounds with absorption spectra closer to the 'therapeutic window' of 600–850 nm is highly desirable for PDT. To take advantage of the structure of Ru compounds and explore each ligand in their structure, tris-heteroleptic Ru(II) compounds need to be synthesized. The selection of the solvent to introduce the first ligand into this heteroleptic structure is crucial in synthesis [19]. Carrying out the reaction in protic solvents or solvents with high boiling points results in obtaining a mixture of homoleptic compounds [19]. The introduction of the second ligand is also demanding; classical synthesis often leads to low yields and very long reactions; therefore, studies on tris-heteroleptic Ru(II) complexes are relatively rare. To improve this process, we propose the use of microwave synthesis at this stage. The appropriate choice of the ligands determines not only the photophysical and physicochemical properties of the Ru(II) complexes (lipophilicity, stability, reactivity of the excited states) but also completely controls the biological properties of the compounds, such as cellular uptake and localization, accumulation route or cytotoxicity [20].
Currently, one of the main drawbacks of PDT is the dependence of its efficiency on the oxygen level in the treated tissues [21]. Since most of the explored PDT systems operate through the energy transfer process (type II sensitization mechanism) and rely on singlet oxygen generation, the use of such systems in hypoxic tumors is inherently limited. Therefore, the focus of newly developed compounds is shifting towards the development of photosensitizers, the photosensitization activity of which will be based on both energy and electron/hydrogen atom transfer processes [22], [23].
This work focuses on the synthesis of three new tris-heteroleptic Ru(II) polypyridyl complexes. The 4,7-diphenyl-1,10-phenanthroline (dip) and 2,2-bipyridine (bpy) ligands are chosen to ensure the appropriate biological properties of the complexes, while 2,3-bis(2-pyridyl)quinoxaline (dpq) and its derivatives were applied to shift the MLCT transition of the compounds towards longer wavelengths. The modification of the synthetic protocol with the use of microwave irradiation allowed us to reduce the reaction times and achieve high yields of the desired complexes. The complexes were synthesized as a mixture of isomers and separated with the use of preparative chromatography. The Ru(II) complexes were characterized with the use of DFT calculations. Biological evaluation was performed only for the prevalent isomer of each synthesized Ru complex. Stereoisomers of Ru(II) polypyridyl complexes may interact differently with chiral biomacromolecules such as DNA or proteins. However, several studies have shown that most often only modest differences (if any) in biological activities between stereoisomers can be observed [20]. The potential of the compounds as cyto- and photocytotoxic agents was evaluated against the highly aggressive breast cancer cell line MDA-MB-231. Based on our previous work [24], we speculate that even a subtle modification of the structure in one of the ligands may lead to a different excited state behavior and photobiological activity of a compound. Our goal was to determine the most photoactive complex in vitro and investigate the basis of its photodynamic activity. Furthermore, the influence of irradiation on the antimetastatic activity of Ru(II) polypyridyl complexes was explored.
The presented studies showed that while the synthesized Ru(II) complexes differed in cytotoxic activity, irradiation with visible light greatly enhanced their antiproliferative properties. The phototoxicity of the compounds was caused by the generation of 1O2 and H2O2, which proved that the complexes can generate ROS via type I and II sensitization mechanisms. An additional benefit came from the enhanced antimetastatic activity of the Ru complexes upon visible light irradiation. This could provide the basis for exploring a potential combination of PDT and antimetastatic therapy using Ru(II) polypyridyl complexes.