Impact of the central atom and halido ligand on the structure, antiproliferative activity and selectivity of half-sandwich Ru(ii) and Ir(iii) complexes with a 1,3,4-thiadiazole-based ligand

Dalton Transactions PAPER Cite this: Dalton Trans., 2023, 52, 12717 Received 2nd June 2023, Accepted 10th August 2023 DOI: 10.1039/d3dt01696j rsc.li/dalton Impact of the central atom and halido ligand on the structure, antiproliferative activity and selectivity of half-sandwich Ru(II) and Ir(III) complexes with a 1,3,4-thiadiazole-based ligand† Radka Křikavová, * a Michaela Romanovová, b Zuzana Jendželovská, b Martin Majerník, b LukášMasaryk, a Pavel Zoufalý, a David Milde, c Jan Moncol, d Radovan Herchel, a Rastislav Jendželovský b and Ivan Nemec a,e Half-sandwich complexes [Ru(η 6 -pcym)(L1)X]PF 6 (1,3) and [Ir(η 5 -Cp*)(L1)X]PF 6 (2,4) featuring a thiadiazole-based ligand L1 (2-(furan-2-yl)-5-(pyridin-2-yl)-1,3,4-thiadiazole) were synthesized and characterized by varied analytical methods, including single-crystal X-ray diffraction (X = Cl or I, pcym = p-cymene, Cp* = pentamethylcyclopentadienyl). The structures of the molecules were analysed and interpreted using computational methods such as Density Functional Theory (DFT) and Quantum Theory of Atoms in Molecules (QT-AIM). A 1 H NMR spectroscopy study showed that complexes 1–3exhibited hydrolytic stability while 4underwent partial iodido/chlorido ligand exchange in phosphate-buffered saline. Moreover, 1–4demonstrated the ability to oxidize NADH (reduced nicotinamide adenine dinucleotide) to NAD + with Ir(III) complexes 2and 4displaying higher catalytic activity compared to their Ru(II) analogues. None of the complexes interacted with reduced glutathione (GSH). Additionally, 1–4exhibited greater lipophilicity than cisplatin. In vitro biological analyses were performed in healthy cell lines (CCD-18Co colon and CCD-1072Sk foreskin fibroblasts) as well as in cisplatin-sensitive (A2780) and -resistant (A2780cis) ovarian cancer cell lines. The results indicated that Ir(III) complexes 2and 4had no effect on human fibroblasts, demonstrating their selectivity. In contrast, complexes 1and 4exhibited moderate inhibitory effects on the metabolic and proliferation activities of the cancer cells tested (selectivity index SI > 3.4 for 4and 2.6 for cisplatin; SI = IC 50 (A2780)/IC 50 (CCD-18Co)), including the cisplatin-resistant cancer cell line. Based on these findings, it is possible to emphasize that mainly complex 4could represent a further step in the development of selective and highly effective anticancer agents, particularly against resistant tumour types. Introduction Conventional platinum-based anticancer drugs represent one of the most widely used groups of chemotherapeutics which have been used in clinical practice for more than 40 years. 1,2 However, due to their large number of side effects and low efficacy against some types of tumours, one of the main goals of medicinal chemists is to develop new agents with higher antiproliferative activity, lower general toxicity and the ability to kill cancer cells resistant towards the biological action of conventional drugs. 3–5 Currently investigated promising groups of potential nonplatinum drugs include ruthenium complexes (BOLD-100 6,7 and TLD1433 8,9 ), which have already entered clinical trials. Indeed, BOLD-100 is presently the most clinically advanced ruthenium-based agent, which has already received Orphan Drug Designations (ODDs) from the FDA in both gastric and †Electronic supplementary information (ESI) available: NMR, ESI+ mass spectra, 1 H NMR stability and interaction studies, crystallographic data, crystal structures and non-covalent interactions, and cellular experimental (metabolic activity, viability, MMP) results. CCDC 2266585–2266588. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d3dt01696j a Department of Inorganic Chemistry, Faculty of Science, Palacký University Olomouc, 17. listopadu 12, CZ-771 46 Olomouc, Czech Republic. E-mail: radka.krikavo[email protected] b Department of Cellular Biology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Šrobárova 2, 041 54 Košice, Slovakia c Department of Analytical Chemistry, Faculty of Science, Palacký University Olomouc, 17. listopadu 12, CZ-771 46 Olomouc, Czech Republic d Department of Inorganic Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Bratislava SK-81237, Slovakia e Central European Institute of Technology, Brno University of Technology, Purkyňova 123, 61200 Brno, Czech Republic This journal is © The Royal Society of Chemistry 2023 Dalton Trans.,2023,52, 12717–12732 | 12717 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue pancreatic cancers. 10 Based on the success of such agents, it seems rational to further investigate potentially effective anticancer complexes of not only ruthenium but also other noble metals, such as iridium which tends to form complexes with similar structural features. 11–13 One of the most interesting structural types of bioactive Ru and Ir complexes is undoubtedly represented by half-sandwich complexes of the general formula [M(η 6 /η 5 -arene/arenyl)(L)(X)] 0/n+ , which have been widely studied for their high cytotoxicity, acceptable selectivity and different mechanisms of action compared to platinumbased anticancer drugs. 14 The coordination sphere of biologically relevant half-sandwich coordination compounds is typically composed of three structural elements: (i) a η 5/6 -arene/yl ligand which stabilizes the oxidation state of the metal cation and can facilitate transport through a cell membrane; (ii) a monodentate ligand, often (but not necessarily) a leaving group (X, typically a halido ligand but also others) 15 which readily dissociates to allow coordination of the metal atom by the target biomolecules (iii) and an auxiliary ligand L which can regulate the reactivity of the complex molecule to various biomolecules (DNA, enzymes) and even play a key role in the interactions with them through hydrogen bonds or intercalation. Furthermore, the overall charge and counterion identity are other factors which could affect solubility, cell uptake, intracellular metabolism and generally, the fate of the complexes in the biological environment. 16–18 The main aim of this study was to prepare new Ru(II) and Ir(III) half-sandwich complexes with the general formulas [Ru(η 6 -pcym)(L1)X]PF 6 and [Ir(η 5 -Cp*)(L1)X]PF 6 , where pcym is 1-methyl-4-(propan-2-yl)benzene (para-cymene), Cp* is pentamethylcyclopentadienyl, X = Cl − or I − and L1 is a bidentate N-donor ligand derived from thiadiazole, 2-(furan-2-yl)-5- (pyridin-2-yl)-1,3,4-thiadiazole (Fig. 1). Compounds involving thiadiazole rings as scaffolds have been of great interest as core structures of antitumor agents due to their high reactivity and the presence of a toxophoric N–C–S moiety. 19 Diverse modifications of the thiadiazole rings in various positions have led to a variety of novel compounds with a wide spectrum of pharmacological activities, such as antifungal, 20 antibacterial, 21 antiviral, 22 anti-inflammatory, 23 analgesic, 24 antihelmintic 25 and of particular interest is the impressive anticancer/antitumor activity. 26–29 Several patents have been registered since 2008 concerning new thiadiazole ring-containing derivatives useful for the development of new anticancer drug molecules. 30 To the best of our knowledge, only two works have reported on the biological investigation of Ru(II) half-sandwich complexes involving thiadiazole-based ligands. The studies focused on Ru(II) complexes with a carbonic anhydrase inhibitor acetazolamide, which were found to be inactive in vitro on all tested cell lines, 31 however showed to be very potent inhibitors of tumour-associated carbonic anhydrase isoforms. 32 The thiadiazole derivative in this work contains two heterocyclic substituents, i.e. the pyridine and furan rings. Pyridine is present to enable a bidentate N,N-coordination mode. This motif has been frequently used in half-sandwich ruthenium/iridium complexes as an N-donor part of bidentate ligands, such as bipyridine, azopyridine, 2-phenylpyridine, picolinate, or as a terminal monodentate ligand as a part of structure–activity studies, particularly, in the pioneering works of Sadler et al. 14,33–35 The furanyl moiety brings another heterocyclic functionality and as the non-coordinating one, it could enable a varied array of non-covalent contacts due to its aromatic ring and heteroatom. Additionally, the furan ring is also an interesting moiety from the medicinal point of view, as multiple clinically approved pharmaceuticals, e.g. with antimicrobial, antiviral, anti-inflammatory, anti-ageing, and anticancer properties, contain this heterocycle in their structures. 36 Furthermore, another objective of this study was to investigate changes in the biological and chemical properties (e.g. solution stability, antiproliferative activity and cytotoxicity) of the prepared complexes upon replacement of the chlorido by the iodido ligand. In previous works on various half-sandwich complexes, 33,37,38 it was shown that a relevant difference in biological properties can be achieved by such structural variation. This work thus reports on half-sandwich complexes [M(η 6 /η 5 -arene/yl)(L1)X]PF 6 in which the influence of two variables, i.e. M = Ru/Ir, X = Cl/I, on structural properties, solution stability and antiproliferative activity was investigated. Results Synthesis and basic characterization The thiadiazole-based compound L1 (2-(furan-2-yl)-5-(pyridin2-yl)-1,3,4-thiadiazole) used in this work was prepared by a two-step synthetic procedure (Scheme 1), which was inspired by previously published protocols. 39,40 First, picolinic acid and 2-furoic hydrazide were converted via a 1,1′-carbonyldiimidazole coupling reagent into N-2furanyl-N‘-picolinoylhydrazine(I) in dichloromethane at room temperature. Second, Lawesson’s reagent was employed in the cyclization reaction of I under reflux and a nitrogen atmosphere in chloroform to yield L1 (Scheme 1). Compounds 1–4were prepared by the reaction between the corresponding dimeric complex precursors [M(μ-Cl)(η 6 /η 5 - arene/yl)Cl] 2 and L1, followed by the addition of the stabilizing PF 6− counter anions (NH 4 PF 6 ). During the preparation of 3 Fig. 1 Structural formulas of [Ru(η 6 -pcym)(L1)X]PF 6 (left) and [Ir(η 5 -Cp*) (L1)X]PF 6 (right) complexes, where X = Cl − (1,2)orI − (3,4). Paper Dalton Transactions 12718 |Dalton Trans.,2023,52, 12717–12732 This journal is © The Royal Society of Chemistry 2023 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online and 4, the chloride anions were precipitated by the addition of silver triflate, and then were substituted by iodide anions (KI) and again, microcrystalline products were isolated after the addition of PF 6− counter anions. Compounds 1–4were slightly soluble in water but were well soluble in organic media, such as N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol, ethanol or acetone. The purity and structure of L1 and resulting complexes 1–4were studied and confirmed by elemental analysis, mass spectrometry, FTIR, and NMR spectroscopy and crystal structures were determined by single crystal X-ray diffraction. The ESI+ mass spectra contained peaks with m/zvalues and isotopic distributions attributable to the ions (e.g. {[Ru(pcym) (L1)]-H} + , {[Ir(Cp*)(L1)]-H} + , [Ru(pcym)(L1)X] + , [Ir(Cp*)(L1)X] + ) agreeing well with the proposed formulas of the complex cations in 1–4(Fig. S1–S4†). The identity and purity of the ligand and complexes were investigated using high resolution 1 H and 13 C NMR spectroscopy (see the ESI, Fig. S5–S12†). The 1 H signals in L1 were all significantly shifted downfield upon coordination with the metal atoms (see ESI, Fig. S10†). The most significant change was observed for the signal assigned to the C13H hydrogen, adjacent to the coordination site, i.e. pyridine nitrogen, where the coordination shift, Δδ=δ complex −δ ligand , equalled 0.91 (1), 0.36 (2) 0.86 (3) and 0.38 ppm (4). Large shifts were also calculated for the hydrogen C16H with Δδranging between 0.39 and 0.49 ppm. In the carbon NMR spectra, which also showed significant shifting of most of the signals with respect to the spectrum of L1, the highest Δδs were observed for the signal of C16, i.e. 6.3–7.0 ppm downfield. Then the chemical shift of C13 also changed markedly, yet more so for Ru(II) complexes 1 and 3(∼6 ppm) than for Ir(III) complexes 2and 4(∼3 ppm). Similarly, significant upfield shifts of ca. 4.5 ppm, and 2.5 ppm in the spectra of Ru(II) and Ir(III) complexes, respectively, were observed for the signals corresponding to the quaternary carbon C5, which lies in the vicinity of nitrogen N4, i.e. the coordination site on the 1,3,4-thiadiazole ring. Crystal structures Single crystals were obtained for all the herein reported coordination compounds 1–4and their crystal structures were determined using single-crystal X-ray diffraction analysis (Table S1†). The coordination compounds consist of the complex cations and PF 6− anions. All the complex cations contain the bidentate L1 ligand, η 6 -pcym (1and 3)orη 5 -Cp* (2 and 4) ligands and monodentate halido ligands X (X = Cl − in 1 and 2,I − in 3and 4). The overall coordination geometry can be described best as three-legged piano stool pseudooctahedral. The longest metal–ligand (M–L) bond lengths were observed for bonds with the halides (in Å, 2.394(2) in 1, 2.3857(11) in 2, 2.6986(4) in 3and 2.6445(9) in 4), while the M–N bonds were significantly shorter (2.05–2.12 Å, Fig. 2). The distances between the centroids of the arene/yl ligands and metal atoms are shorter in the Ru complex cations (1.69 in 1and 3vs. 1.78 Å in 2and 4). The non-covalent interactions in the crystal structures of 1–4are mostly of weak nature, mainly the C–H⋯π,C–H⋯S, C–H⋯F, C–H⋯Cl (1and 2), C–H⋯I(3and 4) hydrogen bonds. Significant non-covalent interactions are summarized in ESI (Fig. S13–S16†) and some of the selected interactions are discussed in greater detail in the Discussion paragraph (vide infra). Lipophilicity studies Cytotoxicity and the ability of drugs to enter cells often correlate with their lipophilicity (hydrophobicity). Thus, one of the Scheme 1 Preparation of 2-(furan-2-yl)-5-(pyridin-2-yl)-1,3,4-thiadiazole (L1)via a two-step reaction procedure: (a) 2-furoic hydrazide, dichloromethane, room temperature and (b) Lawesson’s reagent, chloroform, reflux, overnight, nitrogen atmosphere, given with the atom numbering scheme. PA = pyridine-2-carboxylic acid and I = N‘-(furan-2-carbonyl)pyridine-2-carbohydrazide. Fig. 2 A perspective view illustrating the molecular structures of the complex cations [Ru(η 6 -pcym)(L1)Cl] + (A, complex 1), [Ir(η 5 -Cp*)(L1)Cl] + (B, 2), [Ru(η 6 -pcym)(L1)I] + (C, 3), and [Ir(η 5 -Cp*)(L1)I] + (D, 4). Hydrogen atoms have been omitted for clarity. The colour code used is as follows: light grey (carbon), green (chlorine), violet (iodine), dark blue (iridium), turquoise (ruthenium), light blue (nitrogen), red (oxygen), yellow (sulphur). Selected bond lengths (in Å): 1(A), d(Ru1–N1) = 2.052(10), d(Ru1–N3) = 2.116(10), d(Ru1–Cl1) = 2.394(2); 2(B), d(Ir1–N1) = 2.063 (4), d(Ir1–N3) = 2.114(4), d(Ir1–Cl1) = 2.3857(11); 3(C), d(Ru–N1) = 2.063 (3), d(Ru1–N3) = 2.120(3), d(Ru1–I1) = 2.6986(4); and 4(D), d(Ir1–N1) = 2.054(8), d(Ir1–N3) = 2.093(8), d(Ir1–I1) = 2.6445(9). Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2023 Dalton Trans.,2023,52, 12717–12732 | 12719 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online possible explanations for different cytotoxicity of 1–4(see below) may be based on their different lipophilicity, which is related to their ability to enter cancer cells. Lipophilicity can be determined by the octanol/water partition coefficient (log P), which was calculated for all studied compounds: log P =−0.96 ± 0.03 (for 1), −0.25 ± 0.01 (for 2), −0.03 ± 0.02 (for 3) and −0.07 ± 0.01 (for 4). The obtained results showed that the iodido compounds (3and 4) were more lipophilic than the chlorido ones (1and 2). Compounds 1–4were more lipophilic than cisplatin (CDDP, −2.21 ± 0.1). 41 Solution stability Solution stability of 1–4was investigated by 1 H NMR spectroscopy in water containing solvent mixtures of 150 µL of MeOD-d 4 and 350 µL of D 2 O (SM1) and in 150 µL of MeOD-d 4 and 350 µL of D 2 O with the addition of PBS (SM2, pH = 7.4, PBS stands for phosphate-buffered saline, concentration of the chloride anions in SM2 was 98 mM) (Fig. S17–S24†). In the solvent mixtures SM1 and SM2, 1–3showed overall hydrolytic stability because their spectra did not change over time. No new signals emerged up to 48 h. To exclude immediate hydrolysis, the obtained spectra were compared to those of dehalogenated complexes 1 h and 2 h , which unambiguously proved no occurrence of hydrolysis. The obtained signals corresponded neither to the signals of 1 h and 2 h , nor to those of ligand L1 measured under the same conditions. Analogically, in SM1 compound 4was hydrolytically stable. In contrast, though, new signals appeared in the spectrum of 4dissolved in SM2. The chemical shifts of the new set of signals agreed perfectly with the signals of the chlorido analogue, i.e. complex 2(Fig. 3). Many signals were overlapping, resulting in broad unresolved multiplets, however, discrete new signals appeared for C15H at 8.33 ppm (at 8.27 ppm for the iodido complex and 8.34 ppm for 2) and for C14H at 7.91 ppm (at 7.82 ppm for 4and 7.92 ppm for 2) as well as for Cp* hydrogens at 1.77 ppm (at 1.87 ppm for 4and 1.77 ppm for 2). Therefore, in the presence of chloride anions in solution, complex 4undergoes gradual iodido/chlorido ligand exchange. The conversion rate of the ligand exchange for 4in SM2 was 40% after standing at room temperature for 48 h. Interaction with GSH Reduced glutathione (GSH) is a tripeptide, which often coordinates to metal centres of complex molecules and is involved in the detoxification of many anticancer metallodrugs. 42,43 Importantly, it plays a vital role in the redox balance in the cell, therefore any interference with the equilibrium between GSH and its oxidized form GSSG (GSSG = glutathione disulphide) can result in pathological changes in cellular metabolism. 42,44 Possible interactions with GSH of 1–4were studied in a solvent mixture (SM3) of 150 µL of MeOD-d 4 and 350 µL of D 2 O with PBS (pH = 7.4) with 5 molar equivalents of GSH. The structural features of complexes 1–3remained unaltered because the positions of signals in their spectra did not change over time. In contrast, new signals appeared in the spectrum of 4, as it exhibited the same changes as described above in SM2 (i.e. the iodido/chlorido ligand exchange). The presence of GSH did not affect the conversion rate of the ligand exchange significantly, which was ca. 35% after 48 h. In addition, as far as any covalent interactions and/or GSHrelated ligand exchange reactions are concerned, the results showed that none of these occurred in the interaction system, as evidenced by the unaltered aliphatic part of the 1 H NMR spectra over 48 h. Similarly, complexes 1–4were catalytically inactive in the GSH-to-GSSG oxidation reaction, since negligible (1) or no GSH transformation was evidenced in the NMR study (Fig. S25–S32†). Interestingly, with respect to the spectrum of GSH alone in the same medium, the position of two signals of GSH between 3.5 and 4.0 ppm in the spectra of all the interaction mixtures was shifted already at 0 h. These two shifted signals belong to the protons attached to carbons directly neighbouring with the two terminal COOH groups of GSH. Since no covalent interactions and/or GSH-related ligand exchange reactions were detected in the spectra, it could be suggested that the shift of the signals is related to the immediate formation of a different array of non-covalent interactions of the COOH groups of GSH with the complexes present in the mixtures. Analogical shifts of the same GSH protons were observed by Y. Q. Hao et al. 45 Interaction with NADH The NADH/NAD + system is indispensable for cellular metabolism, as it plays multiple crucial roles especially in many enzymatic events in cells. The ability of ruthenium and iridium complexes to oxidize NADH to form NAD + has been reported in several studies. 46–48 Disordering of the NADH to NAD + ratio can lead to the disruption of various metabolic events eventually resulting in cell death. 49 In a mixture (SM4) Fig. 3 1 H NMR stability study of complex 4in SM2 (30% MeOD-d 4 /70% D 2 O with PBS), as observed at different time points (0 h or 48 h). The grey area shows the 1 H NMR spectrum of 4after 48 h confirming the occurrence of I − →Cl − ligand exchange with light blue colour denoting the signals of the original iodido complex and with yellow for the chlorido analogue. Other signals are not coloured due to overlap. For comparative purposes, the 1 H spectrum of chlorido complex 2in the same solvent mixture is shown (top). Paper Dalton Transactions 12720 |Dalton Trans.,2023,52, 12717–12732 This journal is © The Royal Society of Chemistry 2023 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online of 150 µL of MeOD-d 4 and 350 µL of D 2 O with PBS (pH = 7.4) and 5 molar equivalents of NADH, complexes 1–3remained intact and the positions of their signals in the spectra did not change over time up to 48 h (Fig. S33–S35†). On the other hand, similar to the above presented results, due to the presence of chloride anions in the solution, the stability of 4was lower and again, new signals corresponding to chlorido complex 2resulting from the ligand exchange were observed in the obtained spectra. Notably, the presence of NADH in the interaction mixture somewhat affected the iodido/chlorido exchange, whose conversion rate was 47% after 48 h (Fig. 4). In the spectra of all compounds new signals confirming the oxidation of NADH to NAD + were observed. Interestingly, ruthenium complexes 1and 3exhibited significantly lower ability to oxidize NADH (i.e. 14 and 10% NADH oxidation after 48 h, respectively) than their iridium congeners 2and 4(28% and 32%). In other words, the catalytic efficiency of 1–4 towards the NADH oxidation can be expressed as being ca. 0.7, 1.4, 0.5 and 1.6 molar equiv. per mol of a complex after 48 h, respectively. We did not detect the characteristic hydrido signal in the high-field region of the 1 H NMR spectra. Previously reported half-sandwich Ru(II) and Ir(III) complexes (with pyridine derived ligands) were also observed as potent oxidants of NADH. 47,50,51 Determination of IC 50 values and effects of tested complexes on metabolic activity in healthy CCD-18Co and CCD-1072Sk human fibroblasts To determine the IC 50 (half maximal inhibitory concentration) values of tested complexes in the CCD-18Co colon and CCD-1072Sk foreskin fibroblasts we used the well-established MTT assay. The MTT assay was performed 24 and 48 h after the exposure of the cells to tested complexes. As the reference drug, we used CDDP, a well-known chemotherapeutic agent, which is generally used in the treatment of ovarian cancer. The estimated IC 50 values derived from mean metabolic activity are shown in Table 1. Based on the obtained results, iridium complexes 2and 4which had the weakest activity against fibroblasts were chosen for subsequent experiments realized on ovarian cancer cells. Complexes 1and 3showed an inhibitory effect against healthy cells; nevertheless, complex 1 was also involved in further studies on the cancer cell lines for comparative purposes. Determination of IC 50 values and effect of tested complexes on the proliferation of A2780 and A2780cis ovarian carcinoma cells Complexes 1,2and 4were chosen for a study of a potential effect on proliferation and induction of cell death in CDDPsensitive A2780 and CDDP-resistant A2780cis ovarian carcinoma cells. At first, we determined the IC 50 values of selected complexes by the MTT assay. The MTT assay was performed 24 and 48 h after exposure of the cells to tested complexes. We used CDDP as the reference drug and the estimated IC 50 values derived from mean metabolic activity are shown in Table 2. The lowest IC 50 values were obtained for 1and 4. The IC 50 values for 2were not defined; therefore, this compound was excluded from subsequent analyses. Determination of IC 50 values in healthy and cancer cell lines allowed the calculation of the selectivity index, SI = (IC 50 (CCD-18Co)/IC 50 (A2780), which was more favourable for complexes 1and 4with SI > 5.8 and SI > 3.4, respectively, as compared to 2.6 for CDDP. On the other hand, with the second used healthy cell line, i.e. SI 2 = (IC 50 (CCD-1072Sk)/ IC 50 (A2780), complex 1was found non-selective with SI 2 = 0.4, in contrast with >3.4 (4) and 6.2 (CDDP). Additionally, differences in IC 50 values against sensitive and resistant cell lines enabled the calculation of the resistance factor RF, defined as RF = IC 50 (A2780cis)/IC 50 (A2780), which equals 1.4 (1), 1.6 (4) and 2.8 (CDDP). The impact of complexes 1and 4on cell proliferation was assessed by evaluation of metabolic activity (Fig. S36†), cell cycle distribution (Table 3) and total cell number (Fig. 5). Tested complexes showed a timeand dose-dependent inhibitory effect on the metabolic activity of both cancer cell lines. However, a stronger effect of tested complexes was observed in CDDP-sensitive A2780 cancer cells (Table 2, Fig. S36A and C†). Fig. 4 1 H NMR investigation of representative compound 1in SM4 (30% MeOD-d 4 /70% D 2 O with PBS+ 5 molar equivalents of NADH), as observed at different time points (0 h or 48 h). Asterisks denote the signals corresponding to NAD + originating from the oxidation of NADH in the mixture with 1(blue areas). For comparison purposes, the 1 H spectra of NADH and NAD + are shown (top). Table 1 The IC 50 values (µM) of tested complexes in CCD-18Co and CCD-1072Sk fibroblasts CCD-18Co CCD-1072Sk 24 h 48 h 24 h 48 h 1 4.31 ± 1.83 >50 1.92 ± 1.02 3.29 ± 0.88 2 >50 >50 >50 >50 3 3.18 ± 1.33 >50 2.18 ± 1.27 2.63 ± 0.12 4 >50 >50 >50 >50 CDDP >50 11.03 ± 0.62 >50 26.39 ± 8.22 Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2023 Dalton Trans.,2023,52, 12717–12732 | 12721 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Analyses of the total cell number and cell cycle distribution were performed 48 h after incubation of the cells with 5 and 25 µM of complexes 1and 4, and CDDP. Analogously to the results from the metabolic activity studies, we observed a dosedependent decrease in the total cell number in both cancer cell lines. The effect of tested complexes was weaker in CDDPresistant A2780cis cells in comparison with CDDP-sensitive A2780 cancer cells (Fig. 5). Flow cytometric analysis of cell cycle distribution revealed that the observed antiproliferative effect of tested complexes against CDDP-resistant cells was accompanied by increased accumulation of cells in the G1 phase of the cell cycle. These changes were attended by the reduction of cell population in the S phase of the cell cycle. In the case of complex 1, decreased percentage of cells in the G2/M phase of the cell cycle was also observed. After the exposure of CDDP-sensitive A2780 cancer cells to tested complexes we did not observe significant changes in the cell cycle distribution (Table 3). Effect of complexes 1 and 4 on the induction of cell death in A2780 and A2780cis ovarian carcinoma cells To determine whether the antiproliferative effect of complexes 1and 4was associated with the onset of cell death, we analysed cell viability and mitochondrial membrane depolarization. The analyses were performed 48 h after incubation of the cells with 5 and 25 µM of complexes 1and 4, and CDDP. However, we did not observe any effect on the viability or mitochondrial membrane potential of used cancer cells after exposure to tested complexes. Significant changes in viability and mitochondrial membrane depolarization were achieved only after the treatment of both cancer cell lines with CDDP (Fig. S37†). Discussion Computational evaluation of structures It is well established that the structural and electronic properties of molecules determine their biological and pharmacological properties. 52 Furthermore, any structural modification leads to the formation of a different array of non-covalent interactions. This may be of crucial importance in the biological environment, as varied non-covalent interactions of a suitable drug with its target molecule result in functional modification of the biologically relevant molecules with different consequences for cellular metabolism. 53 Therefore, we took a closer look at structures and energetically available structural modifications of 1–4. The crystal structures were determined by single crystal X-ray analysis of all the reported compounds, which enabled mutual comparison between organic compound L1/complexes, Table 2 The IC 50 values (µM) of selected complexes in A2780 and A2780cis ovarian carcinoma cell lines A2780 A2780cis 24 h 48 h 24 h 48 h 1 14.90 ± 5.21 8.69 ± 1.75 16.11 ± 5.79 12.48 ± 4.83 2 >25 >25 >25 >25 4 >25 14.70 ± 6.72 >25 23.36 ± 1.03 CDDP 20.35 ± 1.39 4.27 ± 0.70 >25 11.96 ± 2.71 Fig. 5 The effect of 1,4and CDDP on the total cell number of A2780 and A2780cis ovarian carcinoma cell lines. The total cell number was analysed 48 h after treatment of cells with selected complexes. The experimental groups were compared with the untreated control (*p< 0.05, ** p< 0.01, *** p< 0.001). Table 3 The effect of 1,4and CDDP on cell cycle distribution of A2780 and A2780cis ovarian carcinoma cell lines. Changes in the cell cycle distribution [%] were analysed 48 h after treatment of cells with the selected complexes. The experimental groups were compared with the untreated control (* p< 0.05, ** p< 0.01, *** p< 0.001) A2780 A2780cis G0/G1 S G2/M G0/G1 S G2/M Control 64.60 ± 4.27 23.34 ± 2.33 12.06 ± 2.09 58.10 ± 1.07 28.69 ± 1.30 13.21 ± 0.24 DMSO (0.25%) 64.33 ± 5.36 24.14 ± 2.81 11.53 ± 2.99 59.01 ± 0.70 27.94 ± 0.49 13.04 ± 0.51 1 (5 µM) 65.88 ± 6.10 23.50 ± 3.39 10.63 ± 2.77 58.59 ± 1.65 28.25 ± 1.14 13.16 ± 0.51 1 (25 µM) 71.42 ± 5.56 18.40 ± 3.74 10.18 ± 2.06 63.66 ± 0.96** 25.50 ± 0.84* 10.84 ± 0.73* 4 (5 µM) 62.41 ± 6.01 26.53 ± 3.25 11.07 ± 2.96 58.02 ± 0.56 29.23 ± 0.25 12.76 ± 0.50 4 (25 µM) 72.61 ± 5.51 19.50 ± 2.94 7.90 ± 2.61 63.51 ± 1.22** 24.89 ± 0.47* 11.60 ± 0.83 CDDP (5 µM) 9.49 ± 1.02*** 85.21 ± 7.22*** 5.30 ± 7.20 35.77 ± 3.22* 30.63 ± 0.31 33.60 ± 3.07*** CDDP (25 µM) 66.23 ± 6.05 27.39 ± 1.99 6.38 ± 5.27 24.49 ± 15.82*** 58.28 ± 17.79** 17.23 ± 2.14 Paper Dalton Transactions 12722 |Dalton Trans.,2023,52, 12717–12732 This journal is © The Royal Society of Chemistry 2023 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Ru/Ir complexes and chlorido/iodido complexes. After analysing the crystal structures of compounds 1–4, the most striking difference was found in the conformation of ligand L1 in Ru/Ir complexes, which is evidenced by the orientation of the furanyl rings. In Ir(III) compounds 2and 4, the furanyl rings are oriented towards the metal centre and the thiadiazole nitrogen atoms (further abbreviated as the Zconformation), whereas in Ru(II) compounds 1and 3, they are oriented away from the metal centre (Econformation, see Fig. 2). The difference in the orientation could potentially be attributed to the different aryl ligands present in compounds 1–4(η 6 -pcym in 1 and 3,η 5 -Cp* in 2and 4). However, it is apparent that the aryl ligands do not induce any steric hindrance. In fact, some of the methyl groups on the aryl ligands participate in weak intramolecular C–H⋯N hydrogen bonding with the thiadiazole nitrogen atom, stabilizing the structure of the molecule by one (1and 3) or by a bifurcated pair (2and 4) of hydrogen bonds. The strengths of the non-covalent interactions were evaluated employing the quantum theory of atoms in molecules (QT-AIM) by means of interaction energy (E int ), and wave functions were calculated by DFT theory using the ORCA 4.2.1 program. 54 The calculations were performed as single-point convergence on the appropriate fragments of the crystal structure at B3LYP and ZORA-def2-TZVP levels of theory (special basis sets: old-ZORA-TZVP for Ru and I, SARC-ZORA-TZVP for Ir). 55 All QT-AIM calculations were performed using the Multiwfn package. 56 It was revealed that E int of these contacts is comparable for all the intramolecular C–H⋯N hydrogen bonds (1.5–2.3 kcal mol −1 ) with the weaker second interactions in the bifurcated pairs (kcal mol −1 , 0.86 in 2and 0.72 in 4). Importantly, the presence of the PF 6− anion induces significant non-covalent interactions in 1–4. Apart from the weak C–H⋯F interactions observed in all compounds, each PF 6− anion forms either one (3) or two (1,2, and 4)F⋯S interactions with the thiadiazole sulphur atom from the L1 ligand (Fig. 6A and the ESI, Fig. S13†). Although the F⋯S distances in these contacts are relatively long, they are mostly shorter than the sum of the van der Waals radii (∑R vdw (F,S) = 3.27 Å, see ESI Fig. S13†): d(F⋯S, in Å) = 1, 3.246(7), 3.422(6); 2, 3.024(3), 3.156(4); 3, 3.212(4); and 4, 3.025(7), 3.156(8). The interaction energies for these contacts range from 0.7 to 1.9 kcal mol −1 . The calculations performed using the non-covalent interaction (NCI) method 57 confirm that these interactions are indeed weakly attractive (see ESI Fig. S13†). Interestingly, in the molecules adopting the Zconformation (2, and 4) the hydrogen atom in the fifth position of the furanyl ring forms a weak C–H⋯F interaction with the PF 6− anion (in Å): 2,d(C19⋯F5) = 3.434(6), 4,d(C19⋯F3) = 3.459(8). Another significant distinction between the crystal packing of the Eand Zcompounds is the presence of the centrosymmetric R 22 (6) 58 synthon, which is formed by hydrogen bonding between neighbouring furanyl moieties through C–H⋯O interactions (Fig. 6B and see ESI Fig. S14†). These interactions are relatively weak with long donor⋯acceptor distances (in Å, 3.406(7) in 2, 3.580(18) in 4) Fig. 6 (A) Perspective view illustrating the non-covalent interactions (black dashed lines) between the PF 6− anion and the complex cation in the crystal structure of 4. (B) Perspective view highlighting the interactions between the furanyl rings and iodido ligands. Hydrogen atoms, except for those involved in non-covalent interactions, have been omitted for clarity. (C) Graphical comparison displaying the relative electronic energies for the ground states of E/Z-isomers of L1 and 1–4, along with the transition states (TS #1 and TS #2 ) derived from DFT calculations. The colour code used is as follows: light grey (carbon), green (chlorine), violet (iodine), dark blue (iridium), turquoise (ruthenium), light blue (nitrogen), red (oxygen), yellow (sulphur). Selected lengths of non-covalent interactions (in Å): (A), d(C7⋯N2) = 3.422(8), d(C14⋯F5) = 3.660(6), d(C19⋯F3) = 3.459(8), d(F3⋯S1) = 3.156(8), d(F5⋯S1) = 3.025(7) and (B) d(C21⋯O1) = 3.580(18), d(I1⋯O1) = 3.633(8). Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2023 Dalton Trans.,2023,52, 12717–12732 | 12723 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online resulting in low E int (1.10 kcal mol −1 in 2and 0.50 kcal mol −1 in 4). Notably, the formation of this synthon is also supported by interactions with the halido ligands. In 2, a pair of Cl⋯O contacts are formed with d(Cl⋯O) = 3.670(4) Å, while I⋯O contacts in 4are slightly shorter with a distance of d(I⋯O) = 3.633 (8) Å. However, calculations using the electron localization function (ELF) 59 indicate that these interactions are not reliant on the formation of a σ-hole on the halogen atom (see ESI Fig. S15†). 60 Consequently, it is unsurprising that these interactions are only weakly attractive, as evident from the NCI plots (see ESI Fig. S15†), and exhibit relatively low calculated E int (0.40 kcal mol −1 in 2and 0.86 kcal mol −1 in 4). As the orientation of the furanyl ring is different in the crystal structures of 1–4, there are E(dihedral angle S–C–C–O close to 0°) and Z(dihedral angle S–C–C–O close to 180°) isomers of L1 observed in these complexes, we decided to investigate this phenomenon also by theoretical methods employing density functional theory (DFT). Herein, ORCA 5.0 software was utilized, 61 and r 2 SCAN functional 62 was applied to all calculations together with the atom-pairwise dispersion correction (D4). 63 The geometry optimization was carried out in water with the C-PCM implicit solvation model 64,65 and again, Ahlrichs def2-TZVP basis set was used for all atoms, with ECP for Ru, Ir and I. 66 The relative energies were investigated for both Eand Z-isomer for L1 and complexes 1–4.Itis evident that the lowest electronic energy is found for the E-isomers of all compounds, and the Z-isomers have energy higher by 2–3 kJ mol −1 (Fig. 6c and see ESI Fig. S16†). The selected bond distances of optimized molecular geometries are listed in Table S2†and show nice agreement with the X-ray data, confirming good performance of the selected theoretical method. The data are also supplemented by Mayer bond orders. Generally, there is a very small variation in Ru–N and Ir–N bond distances and Mayer bond orders within 1–4. However, Mayer bond orders are significantly lower for Ir–X(X = Cl and I) bonds in 2(0.774) and 4(0.668) compared to Ru–X bonds in 1(0.937) and 3(0.934), respectively. In particular, the Ir–I bond in 4is evidently much weaker, which is in accordance with the lower solution stability of this complex to halogen-ligand substitution (see the Solution stability section). Moreover, in all cases, the transition states (TS #1 and TS #2 ) were identified and confirmed by the presence of one imaginary frequency. The activation energy (energy barrier) for the E–Zisomeric reaction was found close to 34–35 kJ mol −1 for 1–4and slightly lower for the compound L1 itself, close to 29–30 kJ mol −1 , which means that the kinetics of such reaction is slowed down by the coordination of L1. In summary, the structural and computational investigations demonstrate that the orientation of the furanyl rings in 1–4is dominantly governed by the collective influence of weak non-covalent interactions within the crystal structure, rather than intramolecular interactions. Biological studies As demonstrated previously, the structure–activity modifications of this type of Ru(II) and Ir(III) with the general formula [M(η 6 /η 5 -arene/yl)(L)X] 0/n+ can be achieved by the choice of both a suitable bidentate ligand L and a monodentate ligand X. 18,34,67 The original idea behind such a design was that an arene/yl ligand should control lipophilicity and stabilize the oxidation state of the metal, a chelating ligand was present for ensuring additional stability and a monodentate ligand, X, was initially included as a site for activation, expectedly by aquation. 33,68,69 Hydrolysis of half-sandwich complexes was suggested to be an important activation step leading to the formation of covalent bonds with biomolecular targets. 70 However, it has been proven that, intriguingly, it is not only the identity of X, but also the specific combination of monodentate X and chelating ligands that influences the hydrolysis rate significantly. At the same time, evidence has shown that for this type of organometallic complexes, hydrolysis does not necessarily have to be the activation step. 33,71 Therefore, we thoroughly investigated the solution stability of 1–4towards hydrolysis and interactions with selected biomolecules. It was confirmed that 1–4were stable in water (D 2 O with the addition of MeOD for solubility) for a period of 48 h. This is important information because rapid hydrolysis of halido (X) half-sandwich [M(η 6 /η 5 -arene/yl)(L)(X)] + complex cations to their aqua species [M(η 6 /η 5 -arene/yl)(L)(H 2 O)] 2+ can result in strong binding to biomolecules, which may lead to deactivation and decrease in their cytotoxicity. 33,71 Analogically, after the addition of PBS buffer (in a MeOD/ D 2 O solution mixture) solution stability was also observed, but in this case only for 1–3. Interestingly, complex 4underwent partial I − to Cl − ligand exchange, due to the presence of an excess of the chloride anions originating from PBS in solution. Such ligand exchange has been observed for similar half sandwich complexes previously. 71,72 It is important to note that the concentration of the chloride anions in the studied solutions (98 mM) was very close to that in the extracellular environment (110 mM), but significantly higher than that in the intracellular (4 mM) environment of mammalian cells. 73 It is also of importance that the extent of the ligand substitution was 40% after 48 h thus indicating slow reaction kinetics. Similarly to previous reports on analogous half-sandwich complexes, 49,51 1–4catalysed the conversion of NADH to NAD + , suggesting that the mechanism of action is possibly related to changes in the redox homeostasis in the cell. These types of catalytically active complexes change the equilibrium related to the important NADH/NAD + redox couple in cells. Such a change may have significant consequences for cellular metabolism, such as a substantial increase in ROS levels or interference with the lactate dehydrogenase-catalysed lactate– pyruvate conversions. 34,35 Herein, the Ir complexes (2and 4) were significantly more efficient oxidizers than Ru complexes (1and 3). NADH oxidation occurred with the efficiency of ca. 0.7, 1.4, 0.5 and 1.6 molar equiv. per mol of a complex after 48 h for 1–4, respectively. Similarly, as in the solution stability study performed in the absence of NADH, complexes 1–3 remained stable and did not undergo any structural changes in the mixture containing NADH (in MeOD/D 2 O with PBS). On Paper Dalton Transactions 12724 |Dalton Trans.,2023,52, 12717–12732 This journal is © The Royal Society of Chemistry 2023 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online the other hand, the chloride anion presence again induced the I − to Cl − ligand exchange (47% after 48 h) for 4also in the presence of NADH. Furthermore, we also investigated the interactions of 1–4 with GSH and it was found that 1–4did not react with GSH, no oxidation of GSH or formation of GSH adducts with complex molecules was observed whatsoever. Again, 1–3were stable in this solution mixture but 4underwent partial I − to Cl − ligand exchange to a very similar extent as in other experiments (35%). The investigation of the effect of 1–4on metabolic activity of healthy fibroblasts CCD-18Co and CCD-1072Sk (Table 1) revealed that after 48 h significant inhibitory activity was shown by Ru complexes only towards the CCD-1072Sk cell line and both 1and 3were significantly more active than CDDP. In contrast, Ir complexes 2and 4showed no activity (IC 50 > 50 µM), which made them interesting for the following investigations. In the case of studies in cancer cell lines A2780 and A2780cis (Table 2), only 1and 4exhibited an inhibitory effect on metabolic activity, which was further comparable to that of CDDP in the case of 1. Unfortunately, the results showed that complex 1was more active in healthy cells than in cancer cells. Therefore, taking into consideration the presented results, complex 4might be understood as the most suitable candidate for further studies. Although the activity of 4in ovarian carcinoma cells was weaker than that of 1and CDDP, it showed the best activity profile, as it was clearly selective towards cancer over healthy cells with the selectivity index (SI = (IC 50 (CCD-18Co)/IC 50 (A2780)) > 3.4, as compared to 2.6 for CDDP. Differential selectivity of an antiproliferative agent toward cancer cells compared to healthy cells is clearly an important factor as it increases the probability of tumourspecific cytotoxicity, which is related to decreased side-effects during treatment. All in all, based on the results for complexes 1–4, it is evident that the change of the metal from Ru to Ir resulted in lower toxicity to normal cells while halide switch from Cl to I led to higher activity in cancer cells. Higher cytotoxicity of iodido with respect to chlorido Ru, Ir, Rh or Os half sandwich complexes has been previously reported in a few studies. 33,37,38 However, this study reports on the situation, when the difference in the determined IC 50 values between 2 and 4is significant, and the IC 50 value for 2could not be determined up to the highest tested concentration, proving that the halide switch indeed turned on the desired activity. Additionally, although the tested complexes share partial cross-resistance with CDDP, complex 4exhibited somewhat better ability to overcome resistance in A2780cis cells than CDDP, as the resistance factor, defined as RF = IC 50 (A2780cis)/ IC 50 (A2780), dropped from 2.8 for CDDP to 1.6 for 4. Lastly, in spite of the notably higher effect on metabolic activity of 1, both 1and 4seem to influence the cell cycle in a very similar manner, (Table 3) leading to the accumulation of cells in the G1 phase of the cell cycle and reduction of their number in the S and G2/M phases, which is in agreement with the previous reports. 33 Because of the fact that the analysed complexes affected the metabolic activity status and cell cycle but did not influence the viability of the cells, it is possible to conclude that the effect of both 1and 4is cytostatic not cytotoxic. These findings agree with previously reported results. Sačková et al. 74 showed that despite the decreased metabolic activity status analysed by the MTT assay, the cell viability could remain unchanged. Subsequently, cell death analyses realized by Mikešová et al. 75 and Babinčák et al. 76 also proved and enriched these findings. Thus, the decrease in the metabolic activity of the cells does not necessarily result from the cytotoxic action of tested compounds. In relation to our analyses, cell death induction is not the substantial cause of lower total cell number observed 48 h after application of 1and 4. More specifically, this phenomenon seems to be preferentially associated with the antiproliferative effect of the analysed complexes. Therefore, it can be highlighted that the results of our study again confirmed that the MTT assay is a very powerful tool to assess primarily cell metabolic activity. Unfortunately, this is often overlooked in published studies that interpret the results in relation to secondary processes or states of cells, such as viability and consequently drug-induced cytotoxicity. 77 Thus, in order to draw well-founded conclusions, it should be emphasized that careful and rational interpretation of the data in combination with different types of cell-based assays is necessary. In summary, it was found that the presented complexes did not require hydrolysis as an activation step. The results of lipophilicity studies (Ir complexes are more lipophilic than Ru analogues) and NADH oxidation efficiency investigation (Ir complexes are more efficient oxidants) do not correlate with the general findings of the antiproliferative activity study, in which Ru complex 1was labelled as the most potent one. However, looking at antiproliferative activity profile more closely, complex 1has to be excluded from further studies, since it was shown to negatively influence metabolic activity more in healthy than in cancer cells. Therefore, overall best results were found for Ir-iodido complex 4with possibly the most promising therapeutic index. Complex 4was more lipophilic than its Ru analogue and was the most efficient catalyst of the NADH-to-NAD + oxidation reaction, which could indicate that the mechanism of action is likely related to the disruption of cellular redox balance. These results highlight the importance of studies performed not only in cancer but also in normal cells to properly evaluate the pharmacological potential of anticancer drug candidates. Nevertheless, it should be pointed out that the results for complex 4represent a stepping stone for further studies, in which various structural modifications will be necessary in order to increase the stability of the iodido complex in the physiological environment with high chloride concentrations, as it was found for complex 4that partial I − -to-Cl − ligand exchange occurred in such an environment. It might be speculated that this reaction could contribute to a general decrease in anticancer activity of 4, which thereby partially transforms to complex 2(i.e. the chlorido counterpart of 4) which was evaluated as inactive up to the highest concentration level. Dalton Transactions Paper This journal is © The Royal Society of Chemistry 2023 Dalton Trans.,2023,52, 12717–12732 | 12725 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online C. Sanchez-Cano, F. Ponte, E. Sicilia and P. J. Sadler, Chem. Sci., 2020, 11, 5466–5480. 73 L. Costanzo, Physiology, Elsevier, Inc, 6th edn, 2018, ISBN: 978-0-323-47881-6. 74 V. Sačková, P. Fedoročko, B. Szilárdiová, J. Mikešand J. Kleban, Phytochem. Phytobiol., 2006, 82, 1285–1291. 75 L. Mikešová, J. Mikeš,J.Kovaľ, K. Gyuraszová, L.’Čulka, J. Vargová, B. Valeková and P. Fedoročko, Photodiagn. Photodyn. Ther., 2013, 10, 470–483. 76 M. Babinčák, R. Jendželovský, J. Košuth, M. Majerník, J. Vargová, K. Mikulášek, Z. Zdráhal and P. Fedoročko, Cancers, 2021, 13, 1646. 77 M. Ghasemi, T. Turnbull, S. Sebastian and I. Kempson, Int. J. Mol. Sci., 2021, 22, 12827. 78 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Adv., 2015, 71,3–8. 79 L. J. Bourhis, O. V. Dolomanov, R. Gildea, J. A. K. Howard and H. Puschmann, Acta Crystallogr., Sect. A: Found. Adv., 2015, 71,59–75. 80 O. Dolomanov, L. J. Bourhis, R. Gildea, J. A. Howard and H. Puschmann, J. Appl. Crystallogr., 2009, 42, 339–341. 81 J. Koziskova, F. Hahn, J. Richter and J. Kožíšek, Acta Chim. Slovaca, 2016, 9, 136–140. 82 CrysAlisPro 1.171.40.82a, Rigaku Oxford Diffraction, 2020. 83 C. F. Macrae, I. Sovago, S. J. Cottrell, P. T. A. Galek, P. McCabe, E. Pidcock, M. Platings, G. P. Shields, J. S. Stevens, M. Towler and P. A. Wood, J. Appl. Crystallogr., 2020, 53, 226–235. 84 P. Štarha, Z. Trávníček, H. Crlíková, J. Vančo, J. Kašpárková and Z. Dvořák, Organometallics, 2018, 37, 2749– 2759. 85 J. Kleban, J. Mikeš, B. Szilardiová, J. Kovaľ,V.Sačková, P. Solár, V. Horváth, J. Hofmanová, A. Kozubík and P. Fedoročko, Photochem. Photobiol., 2007, 83, 1174– 1185. 86 J. Mikeš,J.Kovaľ, R. Jendželovský, V. Sačková, I. Uhrinová, M. Kello, L. Kuliková and P. Fedoročko, Photochem. Photobiol. Sci., 2009, 8, 1558–1567. Paper Dalton Transactions 12732 |Dalton Trans.,2023,52, 12717–12732 This journal is © The Royal Society of Chemistry 2023 Open Access Article. Published on 11 August 2023. Downloaded on 2/19/2024 1:17:03 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online