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We report the synthesis and characterization of novel pentamethylcyclopentadienyl (Cp*) iridium(III) complexes [(Cp*)Ir(4-methyl-4'-carboxy-2,2'-bipyridine)Cl]PF6 (Ir-I), the product (Ir-II) from amide coupling of Ir-I to dibenzocyclooctyne-amine, and its conjugate (Ir-CP) with the cyclic nona-peptide c(CRWYDENAC). The familiar three-legged 'piano-stool' configuration for complex Ir-I was confirmed by its single crystal X-ray structure. Significantly, copper-free click strategy has been developed for site-specific conjugation of the parent complex Ir-I to the tumour targeting nona-cyclic peptide. The approach consisted of two steps: (i) the carboxylic acid group of the bipyridine ligand in complex Ir-I was first attached to an amine functionalized dibenzocyclooctyne group via amide formation to generate complex Ir-II; and (ii) the alkyne bond of dibenzocyclooctyne in complex Ir-II underwent a subsequent strain-promoted copper-free cycloaddition with the azide group of the modified peptide. Interestingly, while complex Ir-I was inactive towards A2780 human ovarian cancer cells, complex Ir-II exhibited moderate cytotoxic activity. Targeted complexes such as Ir-CP offer scope for enhanced activity and selectivity of this class of anticancer complexes. Show less

A series of ruthenium(ii) complexes with N-heterocyclic carbene ligands were successfully synthesized by transmetalation reactions between silver(i) N-heterocyclic carbene complexes and [RuCl₂(p-cymene)]₂ in dichloromethane under Ar conditions. All new compounds were characterized by spectroscopic and analytical methods. These ruthenium(ii)-NHC complexes were found to be efficient precatalysts for the transfer hydrogenation of ketones by using 2-propanol as the hydrogen source in the presence of KOH as a co-catalyst. The antibacterial activity of ruthenium N-heterocyclic carbene complexes 3a-f was measured by disc diffusion method against Gram positive and Gram-negative bacteria. Compounds 3d exhibited potential antibacterial activity against five bacterial species among the six used as indicator cells. The product 3e inhibits the growth of all the six tested microorganisms. Moreover, the antioxidant activity determination of these complexes 3a-f, using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as reagent, showed that compounds 3b and 3d possess DPPH and ABTS antiradical activities. From a concentration of 1 mg ml^-1, these two complexes presented a similar scavenging activity to that of the two used controls gallic acid (GA) and butylated hydroxytoluene (BHT). From a concentration of 10 mg ml^-1, the percentage inhibition of complexes 3b and 3d was respectively 70% and 90%. In addition, these two Ru-NHC complexes exhibited antifungal activity against Candida albicans. Investigation of the anti-acetylcholinesterase activity of the studied complexes showed that compounds 3a, 3b, 3d and 3e exhibited good activity at 100 μg ml^-1 and product 3d is the most active. In a cytotoxicity study the complexes 3 were evaluated against two human cancer cell lines MDA-MB-231 and MCF-7. Both 3d and 3e complexes were found to be active against the tested cell lines showing comparable activity with examples in the literature. Show less

The rational design of anticancer agents that acts in specific biological targets is one of the most effective strategies for developing chemotherapeutic agents. Aiming at obtaining new ruthenium (II) compounds with good cytotoxicity against tumor cells, a series of new complexes of general formula [RuCl(PPh₃)(Hdpa)(NN)]Cl [PPh₃ = triphenylphosphine, N-N = 2,2'-dipyridylamine (Hdpa) (1), 1,2-diaminoethane (en) (2), 2,2'-bipyridine (bipy) (3), 5,5'-dimethyl-2,2'-bipyridine (dmbipy) (4), 1,10-phenanthroline (phen) (5) and 4,7-diphenyl-1,10-phenanthroline (dphphen) (6)] were synthesized. The complexes were characterized by elemental analysis and spectroscopic techniques (IR, UV/Visible, and 1D and 2D NMR) and three of their X-ray structures were determined: [RuCl(PPh₃)(Hdpa)₂]Cl, [RuCl(PPh₃)(Hdpa)(en)]Cl and [RuCl(PPh₃)(Hdpa)(dmbipy)]Cl. All the complexes are more cytotoxic against the cancer cell line than against the non-tumor cell line, highlighting complexes 1 and 5, which have an index selectivity of 18 and 15, respectively. The binding constants of compounds 1-6 with human serum albumin (HSA) were determined by tryptophan fluorescence quenching, indicating moderate to strong interactions. The binding mode of the complexes to calf thymus (CT) DNA was explored by several techniques, which reveal that only the dphphen compound 6 causes distortions in the secondary and tertiary structures of DNA. The studies demonstrated that the nature of the NN co-ligand and the presence of the PPh₃ and Hdpa ligands are features that can influence the binding affinity of the complexes by the biomolecules and in the cytotoxic activity of the complexes. Overall, the complexes with diimine co-ligand are much more cytotoxic than compound 2 with the aliphatic diamine. Show less

We report the synthesis, characterisation and cytotoxicity of six cyclometalated rhodium(III) complexes [Cp^XRh(C^N)Z]^0/+, in which Cp^X = Cp*, Cp^ph, or Cp^biph, C^N = benzo[h]quinoline, and Z = chloride or pyridine. Three x-ray crystal structures showing the expected "piano-stool" configurations have been determined. The chlorido complexes hydrolysed faster in aqueous solution, also reacted preferentially with 9-ethyl guanine or glutathione compared to their pyridine analogues. The 1-biphenyl-2,3,4,5,-tetramethylcyclopentadienyl complex [Cp^biphRh(benzo[h]quinoline)Cl] (3a) was the most efficient catalyst in coenzyme reduced nicotinamide adenine dinucleotide (NADH) oxidation to NAD⁺ and induced an elevated level of reactive oxygen species (ROS) in A549 human lung cancer cells. The pyridine complex [Cp^biphRh(benzo[h]quinoline)py]⁺ (3b) was the most potent against A549 lung and A2780 ovarian cancer cell lines, being 5-fold more active than cisplatin towards A549 cells, and acted as a ROS scavenger. This work highlights a ligand-controlled strategy to modulate the reactivity and cytotoxicity of cyclometalated rhodium anticancer complexes. Show less

We report on chemistry and cytotoxic studies of four new ruthenium (II) complexes containing uracil derivatives. All compounds are neutral, presenting the formula [Ru(PPh₃)₂(2TU)₂] (1), [Ru(PPh₃)₂(6m2TU)₂] (2), [Ru(dppb)(2TU)₂] (3) and [Ru(dppb)(6m2TU)₂] (4), where PPh₃ = triphenylphosphine; dppb = 1,4-bis(diphenylphosphino)butane, 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil. They were characterized using NMR, UV-vis and IR spectroscopies, microanalytical analysis and mass spectrometry. Furthermore, the crystal structures of 1-4 were determined by single-crystal X-ray diffraction. The coordination of 2-thiouracil derivatives with ruthenium increases regions able to carry out hydrogen bonds with the biological targets, such as DNA. We evaluated the interaction of the complexes with DNA by UV/Vis spectrophotometric titration, and as a result, the values of DNA-binding constants are in the range of 0.8-1.8 × 10⁴ M^-1. Moreover, the interaction of the complexes with BSA was investigated. In vitro, activities against B16-F10 (mouse melanoma), HepG2 (human hepatocellular carcinoma), HL-60 (human promyelocytic leukemia) and K562 (human chronic myelocytic leukemia) and non-tumor cells: PBMC (human peripheral blood mononuclear cells activated with concanavalin A - human lymphoblast) were carried out. Cytotoxicity assays revealed that complexes (2) and (4) present biological activity against tumor cells comparable with oxaliplatin, the reference platinum drug, revealing that they are promising molecules for developing new antitumor compounds. Show less

Ruthenium is attracting considerable interest as the basis for new compounds to treat diseases, and studies have shown that complexes with different structures have significant antineoplastic and antimetastatic potential against several types of tumors, including tumors resistant to cisplatin drugs. We examined the cytotoxic, genotoxic, and pro-apoptotic activities of six ruthenium complexes containing amino acid with general formulation [Ru(AA)(bipy)(dppb)]PF₆, where AA = amino acid (alanine, glycine, leucine, lysine, methionine, or tryptophan); bipy = 2,2´-bipyridine; and dppb = [1,4-bis(diphenylphosphine)butane], against A549 (lung carcinoma) and K562 (chronic myelogenous leukemia) cancer cells. The results show that the ruthenium complexes tested were able to induce cytotoxicity in A549 and K562 cancer cells. Complex 1 containing alanine inhibited the cell viability of A549 and K562 tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and the induction of DNA damage and cell cycle arrest. Complex 1 was able to induce caspase-mediated apoptosis in K562 cells through the mitochondrial dysfunction, the upregulation of apoptotic genes, and the downregulation of Bcl2 anti-apoptotic gene. Besides being cytotoxic to K562 and A549 cells, ruthenium complex containing alanine shows low cytotoxicity and genotoxicity against non-tumor cells. These results suggest that the ruthenium (II) complex is a potential safe and efficient antineoplastic candidate for leukemia treatment. Show less

This study describes a series of newly synthesized phosphine/diimine ruthenium complexes containing the lawsone as bioligand with enhanced cytotoxicity against different cancer cells, and apoptosis induction in prostatic cancer cells DU-145. The complexes [Ru(law)(N-N)₂]PF₆ where N-N is 2,2'-bipyridine (1) or 1,10-phenanthroline (2) and [Ru(law)(dppm)(N-N)]PF₆, where dppm means bis(diphenylphosphino)methane, N-N is 2,2'-bipyridine (3) or 1,10-phenanthroline (4), and law is lawsone, were synthesized and fully characterized by elemental analysis, molar conductivity, NMR, UV-vis, IR spectroscopies and cyclic voltammetry. The interaction of the complexes (1-4) with DNA was evaluated by circular dichroism, gel electrophoresis, and fluorescence, and the complexes presented interactions by the minor grooves DNA. The phosphinic series of complexes exhibited a remarkably broad spectrum of anticancer activity with approximately 34-fold higher than cisplatin and 5-fold higher than doxorubicin, inhibiting the growth of 3D tumor spheroids and the ability to retain the colony survival of DU-145 cells. Also, the complex (4) inhibits DU-145 cell adhesion and migration potential indicating antimetastatic properties. The mechanism of its anticancer activity was found to be related to increased reactive oxygen species (ROS) generation, increased the BAX/BCL-2 ratio and subsequent apoptosis induction. Overall, these findings suggested that the complex (4) could be a promising candidate for further evaluation as a chemotherapeutic agent in the prostate cancer treatment. Show less

A series of nanomaterials based on mesoporous silica have been synthesised and functionalised with a photoactive polypyridyl ruthenium(ii) complex, namely [Ru(bipy)2-dppz-7-hydroxymethyl][PF6]2 (bipy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), by various methods. The functionalisation reactions were based on the covalent binding to different ligands attached to the pores of the mesoporous nanoparticles and a simple physisorption using polyamino-functionalised mesoporous silica nanoparticles. The resulting nanostructured systems have been characterised by XRD, XRF, BET, SEM and TEM, observing the incorporation of the metallodrug onto the nanostructured silica in a different way depending on the synthetic method used in the loading reactions. In our studies, we have also observed that functionalisation with the metallodrug causes changes in the structural and textural features of the materials. The phototherapeutic activity of the ruthenium-functionalised materials in HeLa cervical cancer cells has been tested and the preliminary results are presented herein. Show less

The use of Photodynamic Therapy (PDT) for the treatment of several kinds of cancer as well as bacterial, fungal or viral infections has received increasing attention during the last decade. However, the currently clinically approved photosensitizers (PSs) have several drawbacks, including photobleaching, slow clearance from the organism and poor water solubility. To overcome these shortcomings, many efforts have been made in the development of new types of PSs, such as Ru(II) polypyridyl complexes. Nevertheless, most studied Ru(II) polypyridyl complexes have a low absorbance in the spectral therapeutic window. In this work, we show that, by carefully selecting substituents on the polypyridyl complex, it is possible to prepare a complex absorbing at a much higher wavelength. Specifically, we report on the synthesis as well as in-depth experimental and theoretical characterisation of a Ru(II) polypyridyl complex (complex 3) combining a shift in absorbance towards the spectral therapeutic window with a high ¹O₂ production. To overcome the absence or poor selectivity of most approved PSs into targeted cells/bacteria, they can be linked to targeting moieties. In this line, compound 3 was designed with reactive aldehyde groups, which can be used as a highly versatile synthetic precursor for further conjugation. As a proof of concept, 3 was reacted with benzylamine and the stability of the resulting conjugate 4 was investigated in DMSO, PBS and cell media. 4 showed an impressive ability to act as a PDT PS with no measurable dark cytotoxicity and photocytotoxicity in the low micromolar range against cancerous HeLa cells from 450 nm up to 540 nm. Show less

Previous studies on the neutral and cationic half-sandwich iridium(iii) and ruthenium(ii) complexes showed that the charge and the substitution pattern of the bidentate ligands, as well as the nature of the accompanying counteranion have a significant effect on their biological activities. In this contribution, a series of zwitterionic and cationic half-sandwich iridium(iii) and ruthenium(ii) complexes containing sulfonate groups have been prepared and characterized. The different locations of counteranions between these two kinds of complexes exert great influence on the cytotoxic activity towards cancer cells. The various possible mechanism of actions (MoAs) of the complexes were determined by flow cytometry. This work has shown for the first time the different biological activities between zwitterionic and cationic half-sandwich complexes. Show less

Among all molecules developed for anticancer therapies, photodynamic therapeutic agents have a unique profile. Their maximal activity is specifically triggered in tumors by light, and toxicity of even systemically delivered drug is prevented in nonilluminated parts of the body. Photosensitizers exert their therapeutic effect by producing reactive oxygen species via a light-activated reaction with molecular oxygen. Consequently, the lowering of pO₂ deep in solid tumors limits their treatment and makes essential the design of oxygen-independent sensitizers. In this perspective, we have recently developed Ir(III)-based molecules able to oxidize biomolecules by type I processes under oxygen-free conditions. We examine here their phototoxicity in relevant biological models. We show that drugs, which are mitochondria-accumulated, induce upon light irradiation a dramatic decrease of the cell viability, even under low oxygen conditions. Finally, assays on 3D tumor spheroids highlight the importance of the light-activation step and the oxygen consumption rate on the drug activity. Show less

This paper describes the synthesis of new 6-aminoflavone (6AFl (3)) and 6-aminochromone (6AC (4)) complexes with Cu(ii) and Ru(ii) ions ([Cu(6AC)₂Cl₂] (3a), [Cu(6AFl)₂Cl₂] (4a), [Ru(p-cymene)(6AC)Cl₂] (4b)) and comparison of their properties with the previously described 7-aminoflavone (7AFl (1)) and 7-amino-2-methylchromone (7A2MC (2)) analogues. The cytotoxic effect of all these complexes against two human leukaemia cell lines (HL-60 and NALM-6), melanoma WM-115 cells and COLO205 cells, is determined. The cytotoxicity of copper(ii) complexes, especially [Cu(6AFl)₂Cl₂] (3a) was higher than ruthenium(ii) complexes with the same ligands. Their cytotoxic potency was also stronger in comparison to the referential agents like cisplatin. The pro-oxidative properties were determined for the most active complexes and their ability to generate ROS (reactive oxygen species)/RNS (reactive nitrogen species) in cancer cells was confirmed. The type of ligand and the chemical structure of the tested complexes had an influence on the level of ROS/RNS generated in cancer cells. The redox properties of the copper complex compounds were evaluated by cyclic voltammetry, and compared with the data for Ru(ii) complexes. The reduction and oxidation processes of Ru(iii)/Ru(ii) and Cu(ii)/Cu(i) were described as quasi-reversible. Show less

Half-sandwich ruthenium(ii) complexes [(η⁶-p-cymene)Ru(C^N)-(X)]^0/+ (X = Cl, py or 4-NMe₂-py) containing a cyclometalated 2-ppy or 1-ppz with a non-coordinated CHO group as a handle for further functionalization have been synthesized to achieve selective cytotoxicity to cancer cells, the more potent compounds acting as proteosynthesis inhibitors; this is a new mode of action for half-sandwich metal complexes. Show less

Three iridium(III) complexes ([Ir(Hppy)₂(L)](PF₆) (Hppy = 2-phenylpyridine, L = 5-nitrophenanthroline, NP), 1; 5-nitro-6-amino-phenanthroline (NAP), 2; and 5,6-diamino-phenanthroline (DAP) 3 were synthesized and characterized. The cytotoxicities of Ir(III) complexes 1-3 against cancer cell lines SGC-7901, A549, HeLa, Eca-109, HepG2, BEL-7402, and normal NIH 3T3 cells were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) method. The results showed that the three iridium(III) complexes had moderate in vitro anti-tumor activity toward SGC-7901 cells with IC₅₀ values of 3.6 ± 0.1 µM for 1, 14.1 ± 0.5 µM for 2, and 11.1 ± 1.3 µM for 3. Further studies showed that 1-3 induce cell apoptosis/death through DNA damage, cell cycle arrest at the S or G0/G1 phase, ROS elevation, increased levels of Ca²⁺, high mitochondrial membrane depolarization, and cellular ATP depletion. Transwell and Colony-Forming assays revealed that complexes 1-3 can also effectively inhibit the metastasis and proliferation of tumor cells. These results demonstrate that 1-3 induce apoptosis in SGC-7901 cells through ROS-mediated mitochondrial damage and DNA damage pathways, as well as by inhibiting cell invasion, thereby exerting anti-tumor cell proliferation activity in vitro. Show less

The rational design by the introduction of fluorine into a compound has achieved success in the development of organic anticancer drugs. However, the fluorine effect in metal-based anticancer complexes has rarely been reported. In this contribution, we report the synthesis, characterization, chemical reactivity, and biological activity of a series of half-sandwich zwitterionic iridium(III) complexes containing different substituents in the η⁵-Cp^R ring. The molecular structures for complexes Ir1-Ir4 and Ir7 were determined by single-crystal X-ray crystallography techniques. Notably, the asymmetrically substituted fluoro complexes Ir4 and Ir6 in solution show two conformational isomers. These complexes have sufficient stability, exhibit fluorescence emission, and show potent catalytic activity in converting NADH to NAD⁺. The effect of the substituents in the η⁵-Cp^R ring for these zwitterionic complexes on their anticancer activity was systematically investigated. Surprisingly, the presence of fluorinated substituents gives rise to a significant increase in the anticancer activity. The lipophilicity and cellular uptake levels of these complexes appeared to be the primary factors for their cytotoxicity in this system. A microscopic mechanism study showed that the typical complex Ir4 entered A549 cancer cells through an energy-dependent pathway and was mainly located in lysosomes. Furthermore, an increase in ROS level, apoptosis induction, and cell-cycle perturbation together contribute to the anticancer potency of these zwitterionic complexes. Show less

Benzoylthiourea derivatives feature several donor atoms capable of coordinating to metal centers. We report here a series of Ru(η⁶ -p-cymene) complexes employing benzoylthiourea derivatives as ligands. Such ligands often coordinate to metal centers through their S and O donor atoms. We isolated complexes where the ligands were mono- or bidentately coordinated to Ru involving the S donor atom and surprisingly in bidentate coordination mode a deprotonated thiourea nitrogen resulting in a 4-membered ring structure around the metal center. DFT calculations were used to explain the differences in coordination behavior. These were complemented by stability studies and biological investigations of the compounds as anticancer agents. Several of the synthesized derivatives exhibited significant cell growth inhibitory activity, with the complexes featuring bidentate ligands being more potent than their monodentate counterparts. This can be explained by the higher stability of the former under the conditions employed in cell culture assays. Show less

Fine-tuning of the properties of a recently reported 1,3-indandione-based organoruthenium complex is attempted to optimize the stability under physiological conditions. Previous work has shown its capacity of inhibiting topoisomerase IIα; however, fast aquation leads to undesired reactions and ligand cleavage in the blood stream before the tumor tissue is reached. Exchange of the chlorido ligand for six different N-donor ligands resulted in new analogs that were stable at pH 7.4 and 8.5. Only a lowered pH level, as encountered in the extracellular space of the tumor tissue, was capable of aquating the complexes. The 50% inhibitory concentration (IC₅₀) values in three human cancer cell lines differed only slightly, and their dependence on the utilized leaving group was smaller than what would be expected from their differences in cellular accumulation, but in accordance with the very minor variation revealed in measurements of the complexes' lipophilicity. Show less

In this study, five ruthenium arene complexes with fluorene-bearing N,N-(1) and N,O-(2) donor Schiff base ligands were synthesized and fully characterized. Cationic ruthenium complexes 3[X], ([Ru(η⁶-C₆H₆)(Cl)(fluorene-N[double bond, length as m-dash]CH-pyridine)][X] (where X = BF₄, PF₆, BPh₄), were obtained by reacting ligand 1 with [Ru(η⁶-C₆H₆)Cl₂]₂ in the presence of NH₄X salts, whereas neutral complex 4, Ru(η⁶-C₆H₆)(Cl)(fluorene-N[double bond, length as m-dash]CH-naphtholate), was isolated by reacting ligand 2 with the same precursor. It was possible to obtain a cationic version of the latter, 5[BF₄], by reacting 4 with AgBF₄ in the presence of pyridine. All compounds were fully characterized by NMR and HR-ESI-MS whereas some of them were also analyzed by single crystal X-ray analysis. Their in vitro antiproliferative activity was also assessed in human breast cancer cell lines, notably MCF-7 and T47D. Complex 4 and its cationic counterpart 5[BF₄] were found to be the most cytotoxic compounds of the series (IC₅₀ = 6.2-16.2 μM) and displayed higher antiproliferative activities than cisplatin in both cell lines. It was found that 5[BF₄] undergoes a ligand exchange reaction and readily converts to 4 in the presence of 0.1 M NaCl, explaining the similarity in their observed cytotoxicities. Whereas 3[BF₄] and 3[PF₆] were found inactive at the tested concentrations, 3[BPh₄] displayed a considerable cytotoxicity (IC₅₀ = 16.7-27.8 μM). Notably, 3[BPh₄], 4 (and 5[BF₄]) were active against T47D, a cisplatin resistant cell line. Interestingly, 4 (16.4 μM) was found to be less cytotoxic than 3[BPh₄] and cisplatin (6.6 and 7.9 μM, respectively) in breast healthy cells (MCF-12A). However, in comparison to 4 and cisplatin (at 10 μM), a lower in vivo toxicity was observed for complex 3[BPh₄] on the development of zebrafish (Danio rerio) embryos. Show less

Multidrug resistance mediated by the overexpression of ABC transporters is a major challenge in cancer chemotherapy. Here, we report the synthesis of an organoruthenium complex, RuF, that was designed to surmount multidrug resistance by combining ABCG2 inhibition and cancer cell cytotoxicity, yielding synergistic efficacy. Show less

An organoruthenium(II) complex with pyrithione (2-mercaptopyridine N-oxide) 1 a has previously been identified by our group as a compound with promising anticancer potential without cytotoxicity towards non-cancerous cells. To expand the rather limited research on compounds of this type, an array of novel chlorido and 1,3,5-triaza-7-phosphaadamantane (pta) organoruthenium(II) complexes with methyl-substituted pyrithiones has been prepared. After thorough investigation of the aqueous stability of these complexes, their modes of action have been elucidated at the cellular level. Minor structural alterations in the ruthenium-pyrithionato compounds resulted in fine-tuning of their cytotoxicities. The best performing compounds, 1 b and 2 b, with a chlorido or pta ligand bound to ruthenium, respectively, and a methyl group at the 3-position of the pyrithione scaffold, have been further investigated. Both compounds trigger early apoptosis, induce the generation of reactive oxygen species and G1 arrest in A549 cancer cells, and show no strong interaction with DNA. However, only 1 b also inhibits thioredoxin reductase. Wound healing assays and mitochondrial function evaluation have revealed differences between these two compounds at the cellular level. Show less

Three new ruthenium(II)-arene complexes with pyrido[2',3':5,6]pyrazino[2,3-f][1, 10]phenanthroline (ppf) of general formula: C1 ([(ƞ⁶-benzene)Ru(ppf)Cl]PF₆, C2 ([(ƞ⁶-toluene)Ru(ppf)Cl]PF₆) and C3 ([(ƞ⁶-p-cymene)Ru(ppf)Cl]PF₆) have been synthesized. The structures of complexes were determined by elemental analysis, IR, ESI-MS, as well as with ¹H and ¹³C NMR spectroscopy. Cytotoxic activity has been evaluated in three different human neoplastic cell lines (A549, A375, LS 174T) and in one human non-tumor cell line (MRC-5), by the MTT assay. Complexes C1-C3 showed IC₅₀ values in the micromolar range below 100 µM. Complex C3, carrying ƞ⁶-p-cymene as the arene ligand, exhibited cytoselective activity toward human malignant melanoma A375 cells (IC₅₀ = 15.8 ± 2.7 µM), and has been selected for further analyses of its biological effects. Drug-accumulation study performed in the A375 cells disclosed that C3 possess lower ability of entering the cells compared to cisplatin and distributes approximately equally in the cytosol and membrane/organelle fraction of cells. Investigations in the 3D model of A375 cells, disclosed different effects of the complex C3 and cisplatin on growth of multicellular tumor spheroids (MCTSs). While the size of cisplatin-treated MCTSs decreased with time, MCTSs treated with C3 continued to growth. Differences in structural organization and biological activity of this type of ruthenium(II)-arene complexes versus cisplatin in A375 malignant melanoma cells pointed out their different modes of action, and necessity for further biological studies and optimizations for potential applications. Show less

The monocationic chloro complexes containing chelating N∩N ligands: [(η⁶-p-cymene)Ru(L1-4)Cl]⁺ (1-4), where L1 = 4-methyl-1,10-phenantroline, L2 = dipyrido[3,2-a:2',3'-c]phenazine, L3 = 11-chloro-dipyrido[3,2-a:2',3'-c]phenazine, L4 = 11-nitro-dipyrido[3,2-a:2',3'-c]phenazine; p-cymene = 1-methyl-4-isopropylbenzene) have been prepared and characterized as the hexafluorophosphate salts. The biological activity of 1-4 has been investigated in selected 2D monolayer cell cultures (A549, PANC-1, MDA-MB-231, MRC-5). All investigated ruthenium complexes showed similar or even better cytotoxicity to cisplatin. However, there was no significant reduction in growth of PANC-1 cells in a 3D cell culture of multicellular tumor spheroids (MCTS) after treatment with 2-4, while the cisplatin treatment induced retardation in MCTS growth. Flow cytometry analysis of the cell cycle of PANC-1 cells shows that 3 caused changes of cell cycle phase distribution characterized by slight accumulation of cells in the G2-M phase. Absence of the Sub-G1 phase in the cell cycle of the treated cells indicated that there was no fragmentation of DNA for the analyzed time intervals (48 and 72 h treatment). Fluorescent microscopy, after acridine orange/ethidium bromide staining, revealed that the investigated ruthenium complexes induced some characteristics of apoptotic morphology (shrinking and condensation of chromatin) with notably preserved integrity of the plasma membrane. Investigation of cellular uptake and DNA - fraction accumulation performed by inductively coupled plasma mass spectrometry in PANC-1 cells with equimolar concentrations (5 μM) of 2-4 and cisplatin showed more efficient cellular uptake and DNA - fraction accumulation of complex 3 compared to complexes 2 and 4. Show less