👤 Szatmári I

The effect of polypyridyl Ru(II) complexes on the ability of cancer cells to migrate and invade, two features important in the formation of metastases, is evaluated. In vitro studies are carried out on breast cancer cell lines, MDA-MB-231 and MCF-7, as well as melanoma cell lines A2058 and A375. Three Ru(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and as a third ligand 2,2'-bipyridine (bpy), or its derivative with either 4-[3-(2-nitro-1H-imidazol-1-yl)propyl] (bpy-NitroIm), or 5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moiety attached are examined. The low sub-toxic doses of the studied compounds greatly affected the cancer cells by inhibiting cell detachment, migration, invasion, transmigration, and re-adhesion, as well as increasing cell elasticity. The molecular studies revealed that the Ru(II) polypyridyl complexes impact the activity of the selected integrins and upregulate the expression of focal adhesion components such as vinculin and paxillin, leading to an increased number of focal adhesion contacts. Show less

We have synthesized a series of novel substituted sulfonyl ethylenediamine (en) Ru^II arene complexes 1-8 of [(η⁶-arene)Ru(R¹-SO₂-EnBz)X], where the arene is benzene, HO(CH₂)₂O-phenyl or biphenyl (biph), X = Cl or I, and R¹ is phenyl, 4-Me-phenyl, 4-NO₂-phenyl or dansyl. The 'piano-stool' structure of complex 3, [(η⁶-biph)Ru(4-Me-phenyl-SO₂-EnBz)I], was confirmed by X-ray crystallography. The values of their aqua adducts were determined to be high (9.1 to 9.7). Complexes 1-8 have antiproliferative activity against human A2780 ovarian, and A549 lung cancer cells with IC₅₀ values ranging from 4.1 to >50 μM, although, remarkably, complex 7 [(η⁶-biph)Ru(phenyl-SO₂-EnBz)Cl] was inactive towards A2780 cells, but as potent as the clinical drug cisplatin towards A549 cells. All these complexes also showed catalytic activity in transfer hydrogenation (TH) of NAD⁺ to NADH with sodium formate as hydride donor, with TOFs in the range of 2.5-9.7 h^-1. The complexes reacted rapidly with the thiols glutathione (GSH) and N-acetyl-L-cysteine (NAC), forming dinuclear bridged complexes [(η⁶-biph)₂Ru₂(GS)₃]^2- or [(η⁶-biph)₂Ru₂(NAC-H)₃]^2-, with the liberation of the diamine ligand which was detected by LC-MS. In addition, the switching on of fluorescence for complex 8 in aqueous solution confirmed release of the chelated DsEnBz ligand in reactions with these thiols. Reactions with GSH hampered the catalytic TH of NAD⁺ to NADH due to the decomposition of the complexes. Co-administration to cells of complex 2 [(η⁶-biph)Ru(4-Me-phenyl-SO₂-EnBz)Cl] with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, partially restored the anticancer activity towards A2780 ovarian cancer cells. Complex 2 caused a concentration-dependent G1 phase cell cycle arrest, and induced a significant level of reactive oxygen species (ROS) in A2780 human ovarian cancer cells. The amount of induced ROS decreased with increase in GSH concentration, perhaps due to the formation of the dinuclear Ru-SG complex. Show less

Despite their outstanding properties as potential photosensitizers for photodynamic therapy (PDT), Ir(III) biscyclometalated complexes need both further developments to overcome remaining limitations and in-depth investigations into their mechanisms of action to reach clinic application in the treatment of cancer. This work describes the synthesis of a family of Ir(III) complexes of general formula [Ir(C^N)₂(N^N')]Cl (N^N' = thiabendazole-based ligands; C^N = ppy (2-phenylpyridinate) (Series A), or dfppy (2-(2,4-difluorophenyl)pyridinate) (Series B)) and their evaluation as potential PDT agents. These complexes are partially soluble in water and exhibit cytotoxic activity in the absence of light irradiation versus several cancer cell lines. Furthermore, the cytotoxic activity of derivatives of Series A is enhanced upon irradiation, particularly for complexes [1a]Cl and [3a]Cl, which show phototoxicity indexes (PI) above 20. Endocytosis was established as the uptake mechanism for [1a]Cl and [3a]Cl in prostate cancer cells by flow cytometry. These derivatives mainly accumulate in the mitochondria as shown by colocalization confocal microscopy experiments. Presumably, [1a]Cl and [3a]Cl induce death on cancer cells under irradiation through apoptosis triggered by a multimodal mechanism of action, which likely involves damage over mitochondrial DNA and mitochondrial membrane depolarization. Both processes seem to be the result of photocatalytic oxidation processes. Show less

Title: Rhenium(I)-tricarbonyl complexes with methimazole and its selenium analogue: Syntheses, characterization and cell toxicity. Abstract: This study explores the effect of a thione/selone ligand on the cell toxicity (in vitro) and light activity of diimine Re(CO)3+ complexes. Six rhenium(I) complexes with general formula fac-[Re(CO)3(N,N')X]+ were prepared, where X = 2-mercapto-1-methylimidazole (methimazole; MMI), and 1-methylimidazole-2-selone (MSeI); N,N' = 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen) and 2,9-dimethyl-1,10-phenanthroline (dmphen). Their triflate salts were characterized using single-crystal X-ray diffraction, 1H, 13C and 2D NMR, UV-vis and vibrational spectroscopy. Their cytotoxic properties were tested, showing significant cytotoxicity (IC50 = 8.0-55 μM) towards the human breast cancer cell line MDA-MB-231. The half-inhibitory concentration (IC50) for fac-[Re(CO)3(dmphen)(MMI)]+, the most toxic complex in this series (8.0 ± 0.2 μM), was comparable to that of the corresponding aqua complex fac-[Re(CO)3(dmphen)(H2O)]+ with IC50 = 6.0 ± 0.1 μM. The fac-[Re(CO)3(bpy)(MMI/MSeI)]+ complexes were somewhat less toxic towards the human embryonic kidney cell line HEK-293 T after 48 h of exposure. The stability of the complexes upon irradiation was monitored using UV-vis spectroscopy, with no CO released when exposed to UV-A light (λ = 365 nm). Show less

Platinum complexes are used in chemotherapy, primarily as antineoplastic agents. In this study, we assessed the cytotoxic and cytostatic properties of a set of osmium(II), ruthenium(II), iridium(III) and rhodium(III) half-sandwich-type complexes with bidentate monosaccharide ligands. We identified 5 compounds with moderate to negligible acute cytotoxicity but with potent long-term cytostatic activity. These structure-activity relationship studies revealed that: (1) osmium(II) p-cymene complexes were active in all models, while rhodium(III) and iridium(III) Cp* complexes proved largely inactive; (2) the biological effect was influenced by the nature of the central azole ring of the ligands-1,2,3-triazole was the most effective, followed by 1,3,4-oxadiazole, while the isomeric 1,2,4-oxadiazole abolished the cytostatic activity; (3) we found a correlation between the hydrophobic character of the complexes and their cytostatic activity: compounds with O-benzoyl protective groups on the carbohydrate moiety were active, compared to O-deprotected ones. The best compound, an osmium(II) complex, had an IC₅₀ value of 0.70 µM. Furthermore, the steepness of the inhibitory curve of the active complexes suggested cooperative binding; cooperative molecules were better inhibitors than non-cooperative ones. The cytostatic activity of the active complexes was abolished by a lipid-soluble antioxidant, vitamin E, suggesting that oxidative stress plays a major role in the biological activity of the complexes. The complexes were active on ovarian cancer, pancreatic adenocarcinoma, osteosarcoma and Hodgkin's lymphoma cells, but were inactive on primary, non-transformed human fibroblasts, indicating their applicability as potential anticancer agents. Show less

Iron-sulfur (FeS) proteins are ancient and fundamental to life, being involved in electron transfer and CO2 fixation. FeS clusters have structures similar to the unit-cell of FeS minerals such as greigite, found in hydrothermal systems linked with the origin of life. However, the prebiotic pathway from mineral surfaces to biological clusters is unknown. Here we show that FeS clusters form spontaneously through interactions of inorganic Fe2+/Fe3+ and S2- with micromolar concentrations of the amino acid cysteine in water at alkaline pH. Bicarbonate ions stabilize the clusters and even promote cluster formation alone at concentrations >10 mM, probably through salting-out effects. We demonstrate robust, concentration-dependent formation of [4Fe4S], [2Fe2S] and mononuclear iron clusters using UV-Vis spectroscopy, 57Fe-Mössbauer spectroscopy and 1H-NMR. Cyclic voltammetry shows that the clusters are redox-active. Our findings reveal that the structures responsible for biological electron transfer and CO2 reduction could have formed spontaneously from monomers at the origin of life. Show less

Morpholine motif is an important pharmacophore and, depending on the molecular design, may localize in cellular acidic vesicles. To understand the importance of the presence of pendant morpholine in a metal complex, six bidentate N,O-donor ligands with or without a pendant morpholine unit and their corresponding ruthenium(II) p-cymene complexes (1-6) are synthesized, purified, and structurally characterized by various analytical methods including X-ray diffraction. Complexes 2-4 crystallized in the P2₁/c space group, whereas 5 and 6 crystallized in the P1̅ space group. The solution stability studies using ¹H NMR support instantaneous hydrolysis of the native complexes to form monoaquated species in a solution of 3:7 (v/v) dimethyl sulfoxide-d₆ and 20 mM phosphate buffer (pH* 7.4, containing 4 mM NaCl). The monoaquated complexes are stable for at least up to 24 h. The complexes display excellent in vitro antiproliferative activity (IC₅₀ ca. 1-14 μM) in various cancer cell lines, viz., MDA-MB-231, MiaPaCa2, and Hep-G2. The presence of the pendant morpholine does not improve the dose efficacy, but rather, with 2-[[(2,6-dimethylphenyl)imino]methyl]phenol (HL1) and its pendant morpholine analogue (HL3) giving complexes 1 and 3, respectively, the antiproliferative activity was poorer with 3. MDA-MB-231 cells treated with the complexes show that the acidic vesicles remain acidic, but the population of acidic vesicles increases or decreases with time of exposure, as observed from the dispersed red puncta, depending on the complex used. The presence of the 2,6-disubstituted aniline and the naphthyl group seems to improve the antiproliferative dose. The complex treated MDA-MB-231 cells show that cathepsin D, which is otherwise present in the cytosolic lysosomes, translocates to the nucleus as a result of exposure to the complexes. Irrespective of the presence of a morpholine motif, the complexes do not activate caspase-3 to induce apoptosis and seem to favor the necrotic pathway of cell killing. Show less

Four bipyridine-type ligands variably derivatized with two bioactive groups (taken from ethacrynic acid, flurbiprofen, biotin, and benzylpenicillin) were prepared via sequential esterification steps from commercial 2,2'-bipyridine-4,4'-dicarboxylic acid and subsequently coordinated to ruthenium(II) p-cymene and iridium(III) pentamethylcyclopentadienyl scaffolds. The resulting complexes were isolated as nitrate salts in high yields and fully characterized by analytical and spectroscopic methods. NMR and MS studies in aqueous solution and in cell culture medium highlighted a substantial stability of ligand coordination and a slow release of the bioactive fragments in the latter case. The complexes were assessed for their antiproliferative activity on four cancer cell lines, showing cytotoxicity to the low micromolar level (equipotent with cisplatin). Additional biological experiments revealed a multimodal mechanism of action of the investigated compounds, involving DNA metalation and enzyme inhibition. Synergic effects provided by specific combinations of metal and bioactive fragments were identified, pointing toward an optimal ethacrynic acid/flurbiprofen combination for both Ru(II) and Ir(III) complexes. Show less

In this work, the various biological activities of eight organoruthenium(II) complexes were evaluated to reveal correlations with their stability and reactivity in aqueous media. Complexes with general formula [Ru(η⁶-p-cymene)(X,Y)(Z)] were prepared, where (X,Y) represents either an O,O-ligand (β-diketone), N,O-ligand (8-hydroxyquinoline) or O,S-pyrithione-type ligands (pyrithione = 1-hydroxypyridine-2(1H)-thione) with Cl^- or 1,3,5-triaza-7-phosphaadamantane (PTA) as a co-ligand (Z). The tested complexes inhibit the chlamydial growth on HeLa cells, and one of the complexes inhibits the growth of the human herpes simplex virus-2. The chlorido complexes with N,O- and O,S-ligands displayed strong antibacterial activity on Gram-positive strains including the resistant S. aureus (MRSA) and were cytotoxic in adenocarcinoma cell lines. Effect of the structural variation on the biological properties and solution stability was clearly revealed. The decreased bioactivity of the β-diketone complexes can be related to their lower stability in solution. In contrast, the O,S-pyrithione-type complexes are highly stable in solution and the complexation prevents the oxidation of the O,S-ligands. Comparing the binding of PTA and the chlorido co-ligands, it can be concluded that PTA is generally more strongly coordinated to ruthenium, which at the same time decreased the reactivity of complexes with human serum albumin or 1-methylimidazole as well as diminished their bioactivity. Show less

Ruthenium complexes are developed as substitutes for platinum complexes to be used in the chemotherapy of hematological and gynecological malignancies, such as ovarian cancer. We synthesized and screened 14 ruthenium half-sandwich complexes with bidentate monosaccharide ligands in ovarian cancer cell models. Four complexes were cytostatic, but not cytotoxic on A2780 and ID8 cells. The IC₅₀ values were in the low micromolar range (the best being 0.87 µM) and were similar to or lower than those of the clinically available platinum complexes. The active complexes were cytostatic in cell models of glioblastoma, breast cancer, and pancreatic adenocarcinoma, while they were not cytostatic on non-transformed human skin fibroblasts. The bioactive ruthenium complexes showed cooperative binding to yet unidentified cellular target(s), and their activity was dependent on reactive oxygen species production. Large hydrophobic protective groups on the hydroxyl groups of the sugar moiety were needed for biological activity. The cytostatic activity of the ruthenium complexes was dependent on reactive species production. Rucaparib, a PARP inhibitor, potentiated the effects of ruthenium complexes. Show less

Platinum-based complexes are one of the most successful chemotherapeutic agents having a significant ground in cancer chemotherapy despite their side effects. During the past few decades, Ru(II) complexes have been emerging as efficient alternatives owing to their promising activities against platinum-resistant cancer. The pathway of action, lipophilicity, and cytotoxicity of a Pt or Ru complex may be tuned by varying the attached ligands, the coordination mode, and the leaving group. In this work, we report a family of Pt(II) and Ru(II) complexes (1-5) of three N,O and N,N donor-based trimethoxyanilines containing Schiff bases with the general formula [Pt^II(L)(DMSO)Cl], [Ru^II(L)(p-cymene)Cl], [Ru^II(L)(p-cymene)Cl]⁺, and [Pt^II(L)Cl₂]. All of the complexes are characterized by different analytical techniques. ¹H NMR and electrospray ionization mass spectrometry (ESI-MS) data suggest that the N,O-coordinated Pt(II) complexes undergo slower aquation compared to the Ru(II) analogues. The change of the coordination mode to N,N causes the Ru complexes to be more inert to aquation. The N,O-coordinating complexes show superiority over N,N-coordinating complexes by displaying excellent in vitro antiproliferative activity against different aggressive cancer cells, viz., triple-negative human metastatic breast adenocarcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. In vitro cytotoxicity studies suggest that Pt(II) complexes are more effective than their corresponding Ru(II) analogues, and the most cytotoxic complex 3 is 10-15 times more toxic than the clinical drugs cisplatin and oxaliplatin against MDA-MB-231 cells. Cellular studies show that all of the N,O-coordinated complexes (1-3) initiate disruption of the microtubule network in MDA-MB-231 cells in a dose-dependent manner within 6 h of incubation and finally lead to the arrest of the cell cycle in the G2/M phase and render apoptotic cell death. The disruption of the microtubule network affects the agility of the cytoskeleton rendering inhibition of tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a key step in angiogenesis. Complexes 1 and 2 inhibit VEGFR2 phosphorylation in a dose-dependent fashion. Among the Pt(II) and Ru(II) complexes, the former displays higher cytotoxicity, a stronger effect on the cytoskeleton, better VEGFR2 inhibition, and strong interaction with the model nucleobase 9-ethylguanine (9-EtG). Show less

Sulfonamides have a broad range of therapeutic applications, which include the inhibition of various isoforms of carbonic anhydrases (CAs). Among the various CA isoforms, CA IX is overexpressed in tumors and regulates the pH of the tumor microenvironment. Herein we present five new ruthenium(II) p-cymene complexes (1-5) of Schiff base ligands (L1-L4) of 4-(2-aminoethyl)benzenesulfonamide by varying the aldehyde to enhance the selective cytotoxicity toward cancer cells. All of the complexes are stable to aquation for the observed period of 24 h except 1, which aquated within 1 h, but the monoaquated species is stable for 24 h. The two imidazole derivatives, 1 and 2, are cytotoxic to the cancer cells MDA-MB-231 and MIA PaCa-2 but not to the noncancerous cells CHO and MDCK. The enhanced toxicity in hypoxia against MDA-MB-231 may be due to the greater expression of CA IX in hypoxia, as per the immunofluorescence data. The most cytotoxic complexes, 1 and 2, are lipophilic, whereas 3-5 show high hydrophilicity and are not cytotoxic up to 200 μM. Complexes 1 and 2 also show a higher cellular accumulation in MDA-MB-231 than the nontoxic yet solution-stable complex 5. The cytotoxic complexes bind with the model nucleobase 9-ethylguanine but have slow reactivity toward cellular tripeptide glutathione. Both 1 and 2 induce apoptosis by depolarizing the mitochondrial membrane potential and arrest the cell cycle in the SubG1 phase. Show less

Solution chemical properties of two novel 8-hydroxyquinoline-D-proline and homo-proline hybrids were investigated along with their complex formation with [Rh(η⁵-C₅Me₅)(H₂O)₃]²⁺ and [Ru(η⁶-p-cymene)(H₂O)₃]²⁺ ions by pH-potentiometry, UV-visible spectrophotometry and ¹H NMR spectroscopy. Due to the zwitterionic structure of the ligands, they possess excellent water solubility as well as their complexes. The complexes exhibit high solution stability in a wide pH range; no significant dissociation occurs at physiological pH. The hybrids and their Rh(η⁵-C₅Me₅) complexes displayed enhanced cytotoxicity in human colon adenocarcinoma cell lines and exhibited multidrug resistance selectivity. In addition, the Rh(η⁵-C₅Me₅) complexes showed increased selectivity to the chemosensitive cancer cells over the normal cells; meanwhile, the Ru(η⁶-p-cymene) complexes were inactive, most likely due to arene loss. Interaction of the complexes with human serum albumin (HSA) and calf-thymus DNA (ct-DNA) was investigated by capillary electrophoresis, fluorometry and circular dichroism. The complexes are able to bind strongly to HSA and ct-DNA, but DNA cleavage was not observed. Changing the five-membered proline ring to the six-membered homoproline resulted in increased lipophilicity and cytotoxicity of the Rh(η⁵-C₅Me₅) complexes while changing the configuration (L vs. D) rather has an impact on HSA or ct-DNA binding. Show less

Drug resistance to existing anticancer agents is a growing clinical concern, with many first line treatments showing poor efficacy in treatment plans of some cancers. Resistance to platinum agents, such as cisplatin, is particularly prevalent in the treatment of ovarian cancer, one of the most common cancers amongst women in the developing world. Therefore, there is an urgent need to develop next generation of anticancer agents which can overcome resistance to existing therapies. We report a new series of organoruthenium(II) complexes bearing structurally modified pyrithione ligands with extended aromatic scaffold, which overcome platinum and adriamycin resistance in human ovarian cancer cells. The mechanism of action of such complexes appears to be unique from that of cisplatin, involving G₁ cell cycle arrest without generation of cellular ROS, as is typically associated with similar ruthenium complexes. The complexes inhibit the enzyme thioredoxin reductase (TrxR) in a model system and reduce cell motility towards wound healing. Importantly, this work highlights further development in our understanding of the multi-targeting mechanism of action exhibited by transition metal complexes. Show less

Photodynamic therapy (PDT) is a cancer treatment still bearing enormous prospects of improvement. Within the toolbox of PDT, developing photosensitizers (PSs) that can specifically reach tumor cells and promote the generation of high concentration of reactive oxygen species (ROS) is a constant research goal. Mitochondria is known as a highly appealing target for PSs, thus being able to assess the biodistribution of the PSs prior to its light activation would be crucial for therapeutic maximization. Bifunctional Ir(III) complexes of the type [Ir(C^N)₂(N^N-R)]⁺, where N^C is either phenylpyridine (ppy) or benzoquinoline (bzq), N^N is 2,2'-dipyridylamine (dpa) and R either anthracene (1 and 3) or acridine (2 and 4), have been developed as novel trackable PSs agents. Activation of the tracking or therapeutic function could be achieved specifically by irradiating the complex with a different light wavelength (405 nm vs. 470 nm respectively). Only complex 4 ([Ir(bzq)₂(dpa-acr)]⁺) clearly showed dual emissive pattern, acridine based emission between 407-450 nm vs. Ir(III) based emission between 521 and 547 nm. The sensitivity of A549 lung cancer cells to 4 evidenced the importance of involving the metal center within the activation process of the PS, reaching values of photosensitivity over 110 times higher than in dark conditions. Moreover, complex 4 promoted apoptotic cell death and possibly the paraptotic pathway, as well as higher ROS generation under irradiation than in dark conditions. Complexes 2-4 accumulated in the mitochondria but species 2 and 4 also localizes in other subcellular organelles. Show less

Ruthenium-based complexes currently attract great attention as they hold promise to replace platinum-based drugs as a first line cancer treatment. Whereas ruthenium arene complexes are some of the most studied species for their potential anticancer properties, other types of ruthenium complexes have been overlooked for this purpose. Here, we report the synthesis and characterization of Ru(II) cyclopentadienyl (Cp), Ru(II) cyclooctadienyl (COD) and Ru(III) complexes bearing anastrozole or letrozole ligands, third-generation aromatase inhibitors currently used for the treatment of estrogen receptor positive (ER +) breast cancer. Among these complexes, Ru(II)Cp 2 was the only one that displayed a high stability in DMSO and in cell culture media and consequently, the only complex for which the in vitro and in vivo biological activities were investigated. Unlike anastrozole alone, complex 2 was considerably cytotoxic in vitro (IC₅₀ values < 1 μM) in human ER + breast cancer (T47D and MCF7), triple negative breast cancer (TNBC) (MBA-MB-231), and in adrenocortical carcinoma (H295R) cells. Theoretical (docking simulation) and experimental (aromatase catalytic activity) studies suggested that an interaction between 2 and the aromatase enzyme was not likely to occur and that the bulkiness of the PPh₃ ligands could be an important factor preventing the complex to reach the active site of the enzyme. Exposure of zebrafish embryos to complex 2 at concentrations around its in vitro cytotoxicity IC₅₀ value (0.1-1 μM) did not lead to noticeable signs of toxicity over 96 h, making it a suitable candidate for further in vivo investigations. This study confirms the potential of Ru(II)Cp complexes for breast cancer therapy, more specifically against TNBCs that are usually not responsive to currently used chemotherapeutic agents. Show less

Glioblastomas (GBs) are highly aggressive and malignant brain tumors, which are highly resistant to conventional multimodal treatments, leading to their abysmal prognosis. Herein, we designed two organometallic half-sandwich Ru(ii)-η6-p-cymene complexes containing Schiff bases derived from 3-aminoquinoline and 2-hydroxy-benzaldehyde (L1) and 2-hydroxy-naphthaldehyde (L2), namely [Ru(η6-p-cymene)(L1)Cl] (1) and [Ru(η6-p-cymene)(L2)Cl] (2), respectively, and studied their activity on GB cells. Both complexes were structurally characterized using single-crystal X-ray diffraction, which exhibited their half-sandwich three-legged piano-stool geometry. Furthermore, we studied their physicochemical behavior, solution speciation, aquation kinetics, and photo-substitution reactions using various spectroscopic methods. The complexes exhibited a moderate binding affinity with calf-thymus (CT)-DNA (Kb ∼ 105 M-1). The complexes effectively interacted with human serum albumin (HSA) (K ∼ 105 M-1) with preferential tryptophan binding, as determined via synchronous fluorescence studies. The in vitro studies showed their significant antiproliferative activity against an aggressive human GB cell line, LN-229 (IC50 = 22.8 μM), with moderate selectivity relative to normal mouse fibroblast L929 cells. Notably, [Ru(η6-p-cymene)(L1)Cl] (1) exhibited a higher selectivity index (S.I.) than [Ru(η6-p-cymene)(L2)Cl] (2) and cisplatin. We evaluated the clonogenic potential of the GB cells using a colony formation assay in the presence of complex 1. Excitingly, it showed ∼75% inhibition of the clonogenic potential of GB cells at the IC50 concentration. Complex 1 also effectively lowered the migratory potential of the GB cells, as assessed by the wound healing assay. The studied compound led to the apoptosis of GB cells, as evidenced by nuclear condensation, blebbing, and enhanced caspase 3/7 activity, and thus has anticipated utility in the treatment of GBs using photochemotherapy. Show less

The use of photodynamic therapy (PDT) against cancer has received increasing attention over recent years. However, the application of the currently approved photosensitizers (PSs) is limited by their poor aqueous solubility, aggregation, photobleaching and slow clearance from the body. To overcome these limitations, there is a need for the development of new classes of PSs with ruthenium(II) polypyridine complexes currently gaining momentum. However, these compounds generally lack significant absorption in the biological spectral window, limiting their application to treat deep-seated or large tumors. To overcome this drawback, ruthenium(II) polypyridine complexes designed in silico with (E,E')-4,4'-bisstyryl-2,2'-bipyridine ligands show impressive 1- and 2-Photon absorption up to a magnitude higher than the ones published so far. While nontoxic in the dark, these compounds are phototoxic in various 2D monolayer cells, 3D multicellular tumor spheroids and are able to eradicate a multiresistant tumor inside a mouse model upon clinically relevant 1-Photon and 2-Photon excitation. Show less

Due to the great potential expressed by an anticancer drug candidate previously reported by our group, namely, Ru-sq ([Ru(DIP)₂(sq)](PF₆) (DIP, 4,7-diphenyl-1,10-phenanthroline; sq, semiquinonate ligand), we describe in this work a structure-activity relationship (SAR) study that involves a broader range of derivatives resulting from the coordination of different catecholate-type dioxo ligands to the same Ru(DIP)₂ core. In more detail, we chose catechols carrying either an electron-donating group (EDG) or an electron-withdrawing group (EWG) and investigated the physicochemical and biological properties of their complexes. Several pieces of experimental evidences demonstrated that the coordination of catechols bearing EDGs led to deep-red positively charged complexes 1-4 in which the preferred oxidation state of the dioxo ligand is the uninegatively charged semiquinonate. Complexes 5 and 6, on the other hand, are blue/violet neutral complexes, which carry an EWG-substituted dinegatively charged catecholate ligand. The biological investigation of complexes 1-6 led to the conclusion that the difference in their physicochemical properties has a strong impact on their biological activity. Thus, complexes 1-4 expressed much higher cytotoxicities than complexes 5 and 6. Complex 1 constitutes the most promising compound in the series and was selected for a more in depth biological investigation. Apart from its remarkably high cytotoxicity (IC₅₀ = 0.07-0.7 μM in different cancerous cell lines), complex 1 was taken up by HeLa cells very efficiently by a passive transportation mechanism. Moreover, its moderate accumulation in several cellular compartments (i.e., nucleus, lysosomes, mitochondria, and cytoplasm) is extremely advantageous in the search for a potential drug with multiple modes of action. Further DNA metalation and metabolic studies pointed to the direct interaction of complex 1 with DNA and to the severe impairment of the mitochondrial function. Multiple targets, together with its outstanding cytotoxicity, make complex 1 a valuable candidate in the field of chemotherapy research. It is noteworthy that a preliminary biodistribution study on healthy mice demonstrated the suitability of complex 1 for further in vivo studies. Show less

The utilization of photodynamic therapy (PDT) for the treatment of various types of cancer has gained increasing attention over the last decades. Despite the clinical success of approved photosensitizers (PSs), their application is sometimes limited due to poor water solubility, aggregation, photodegradation, and slow clearance from the body. To overcome these drawbacks, research efforts are devoted toward the development of metal complexes and especially Ru(II) polypyridine complexes based on their attractive photophysical and biological properties. Despite the recent research developments, the vast majority of complexes utilize blue or UV-A light to obtain a PDT effect, limiting the penetration depth inside tissues and, therefore, the possibility to treat deep-seated or large tumors. To circumvent these drawbacks, we present the first example of a DFT guided search for efficient PDT PSs with a substantial spectral red shift toward the biological spectral window. Thanks to this design, we have unveiled a Ru(II) polypyridine complex that causes phototoxicity in the very low micromolar to nanomolar range at clinically relevant 595 nm, in monolayer cells as well as in 3D multicellular tumor spheroids. Show less

The well-documented cytotoxic activity of coordinatively saturated and substitutionally inert polypyridyl Ru(ii) complexes substantiates their high potency as antiproliferative agents against primary tumors. However, the primary cause of cancer morbidity and mortality responsible for about 90% of cancer deaths is the occurrence of metastasis. Therefore, scientists have to concentrate their efforts on designing compounds affecting not only the primary tumor, but also efficiently inhibiting metastasis. Herein, we report two families of Ru(ii) polypyridyl complexes bearing 2,2'-bipyridine substituted by a semicarbazone 2-formylopyridine moiety as one of the ligands and 4,4'-di-tert-butyl-2,2'-dipyridyl or 4,7-diphenyl-1,10-phenanthroline as auxiliary ligands. These complexes strengthen cells' adherent properties and inhibit the activity of metalloproteinases (MMPs) in vitro, which is relevant in anti-metastatic treatment. The in vitro studies were performed on human lung adenocarcinoma (A549) and human pancreatic cancer (PANC-1) cells, which have a well-documented invasive potential. The induced alteration of the tumor cells' adhesion properties correlated with the high cytotoxic effect exerted by the complexes and their excellent cellular uptake. It was also proved that both complexes directly inhibit M-MP2 and M-MP9 enzyme activities, which are essential for the development of tumor metastasis. The results of this study indicate that the biological properties of polypyridyl Ru(ii) complexes extend beyond the standard cytotoxic activity and represent an important step towards designing new anti-metastatic agents. Show less

Three new ruthenium(II)-arene complexes, [Ru(η⁶-p-cymene)(L¹)Cl₂] (C1) where L¹ is N-((4 methoxyphenyl)carbamothioyl)benzamide; [Ru(η⁶-p-cymene)(L²)Cl₂] (C2) where L² is 4-(3-benzoylthioureido)benzoic acid and [Ru(η⁶-p-cymene)(L³)Cl₂] (C3) where L³ is methyl 4-(3- benzoylthioureido)benzoate have been synthetized, characterized and evaluated for their antimicrobial and anticancer activity. Characterization was performed using ¹H and ¹³C NMR, IR spectroscopy, mass spectrometry, electrical conductivity measurements and X-Ray diffraction analysis. X-Ray diffraction analysis of C1 showed typical expected "piano-stool" geometry with ruthenium coordinated to ligand via nitrogen and sulfur atoms of benzoylthiourea derivatives. Interesting, in herein described complex, upon coordination the four-membered ring was formed, instead of six-membered chelate common for this type of ligands. Cytotoxic activity was determined in human cervix adenocarcinoma (HeLa) cell line and IC₅₀ values ranged from 29.68 to 52.36 μM and the complexes were more active than related ligands (except in case of C2 where it is found that IC₅₀ value is close to IC₅₀ value of related ligand). Complex [Ru(η⁶-p-cymene)(L¹)Cl₂] (C1) expressed the highest cytotoxic activity with IC₅₀ value of 29.7 μM. Complexes and ligands were tested against nine Gram-positive and Gram-negative bacteria and one yeast- Candida albicans. Clinical Candida spp. strains from microbiological laboratories were included in testing processes as well. Minimum inhibitory concentrations values ranged from 62.5 μg/ml for complexes against Candida albicans to over 1000 μg/ml for several bacterial species. Show less

Herein the design and synthesis of a new 8-hydroxyquinoline derivative, (S)-5-chloro-7-((proline-1-yl)methyl)8-hydroxyquinoline (HQCl-Pro), with good water solubility and multidrug resistance reversal activity are reported. In this work the proton dissociation processes of HQCl-Pro and its complex formation with [Rh(η⁵-C₅Me₅)(H₂O)₃]²⁺, [Ru(η⁶-p-cymene)(H₂O)₃]²⁺ and [Ru(η⁶-toluene)(H₂O)₃]²⁺ were investigated by the combined use of pH-potentiometry, UV-visible spectrometry and ¹H NMR spectroscopy. Our results revealed the prominent solution stability of the complexes in all cases. The lipophilicity of the complexes increased with the chloride ion concentration, and the complexes showed moderate log D values (-0.8 to +0.4) at pH 7.4 at all tested Cl^- concentrations. The formation of mixed hydroxido complexes from the aqua complexes was characterized by relatively high pK_a values (8.45-9.62 in chloride-free medium). Complexation processes are much slower with the Ru(η⁶-arene) triaqua cations than with [Rh(η⁵-C₅Me₅)(H₂O)₃]²⁺. Both the pK_a values and H₂O/Cl^- exchange constants of the Ru-complexes are lower by 0.5-1.0 orders of magnitude than those of the Rh analogue. Arene loss (p-cymene and toluene) and oxidation were found in the case of Ru-complexes when an excess of HQCl-Pro and aromatic (N,N) bidentate ligands was added. The cytotoxicity and antiproliferative effect of HQCl-Pro and its complexes were assayed in vitro. In contrast to the structurally familiar 8-hydroxyquinoline, HQCl-Pro and its Rh(η⁵-C₅Me₅) complex were somewhat more effective against drug resistant Colo 320 adenocarcinoma human cells compared to the drug sensitive Colo 205 cells. The Ru- and Rh-complexes showed a similar metal uptake level after 4 h, while a longer incubation time resulted in higher cellular Rh concentration. Show less

The organoiridium complex Ir[(C,N)₂(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (¹O₂) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of ¹O₂ generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action. Show less

Twenty-four novel organometallic osmium(II) phenylazopyridine (AZPY) complexes have been synthesised and characterised; [Os(η⁶-arene)(5-RO-AZPY)X]Y, where arene = p-cym or bip, AZPY is functionalized with an alkoxyl (O-R, R = Me, Et, ⁿPr, ⁱPr, ⁿBu) or glycolic (O-{CH₂CH₂O}_nR*, n = 1-4, R* = H, Me, or Et) substituent on the pyridyl ring para to the azo-bond, X is a monodentate halido ligand (Cl, Br or I), and Y is a counter-anion (PF₆^-, CF₃SO₃^- or IO₃^-). X-ray crystal structures of two complexes confirmed their 'half-sandwich' structures. Aqueous solubility depended on X, the AZPY substituents, arene, and Y. Iodido complexes are highly stable in water (X = I ⋙ Br > Cl), and exhibit the highest antiproliferative activity against A2780 (ovarian), MCF-7 (breast), SUNE1 (nasopharyngeal), and OE19 (oesophageal) cancer cells, some attaining nanomolar potency and good cancer-cell selectivity. Their activity and distinctive mechanism of action is discussed in relation to hydrophobicity (RP-HPLC capacity factor and Log P_o/w), cellular accumulation, electrochemical reduction (activation of azo bond), cell cycle analysis, apoptosis and induction of reactive oxygen species (ROS). Two complexes show ca. 4× higher activity than cisplatin in the National Cancer Institute (NCI) 60-cell line five-dose screen. The COMPARE algorithm of their datasets reveals a strong correlation with one another, as well as anticancer agents olivomycin, phyllanthoside, bouvardin and gamitrinib, but only a weak correlation with cisplatin, indicative of a different mechanism of action. Show less

(p-Cymene)-ruthenium bioconjugates ML (1) and ML₂ (2), bearing phosphane ligands substituted with chiral or non-chiral amino acid esters, L, were synthetized and characterized by instrumental methods (NMR, CD, MS) and DFT calculations (using the wB97xD functional). Cytotoxic activity of complexes 1 and 2 was investigated by using human cervical carcinoma cell line (HeLa) and MTT assay. Four (2_pG, 2_pA, 2_mG and 2_mA) out of ten synthesized ruthenium complexes showed significant toxicity, with IC₅₀ values of 5-30 μM. Evaluation of the potential biomolecular targets of bioconjugates 2 by UV-Vis, fluorescence and CD spectroscopy revealed no measurable interaction with DNA, but micromolar affinity for proteins. The cytotoxicity of bioconjugates 2 is in correlation with their BSA binding constants, i. e. bioconjugates with lower IC₅₀ values show higher binding affinities towards BSA. Compound 2_mG with value of IC₅₀ 16 μM was selected for further biological characterization. The higher level of toxicity towards tumor compared to normal cell lines indicates its selective activity, important characteristic for potential medical use. It was detected 2_mG caused increase of cells in the S phase of cell cycle and consequential decrease of cells in G0/G1 phase. Additionally, 2_mG caused dose- and time-dependent increase of SubG0/G1 cell population, suggesting its ability to induce programmed cell death. Further investigation determined autophagy as the mode of cell death. The role of GSH in HeLa cells response to investigated organometallic ruthenium complexes was confirmed using specific regulators of GSH synthesis, buthionine sulfoximine and N-acetyl-cysteine. Pre-treatment of cells with ethacrynic acid and probenecid emphasized the role of GSH in detoxification of 2_mG compound. The amount of total ruthenium accumulation in the cell did not correlate with toxicity of 2_pG, 2_pA, 2_mG and 2_mA, suggesting structure dependent differences in either cell uptake or kinetics of ruthenium complexes detoxification. We speculate that ruthenium complexes bind protein-based biomolecules further triggering cell death. Based on the gained knowledge, the synthesis and development of more tumor-specific ruthenium-based complexes as potential anticancer drugs can be expected. 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

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

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 purpose of this work was to screen the antitumor actions of two metal organoruthenium-8-hydroxyquinolinato (Ru-hq) complexes to find a potential novel agent for bone, lung and breast chemotherapies. We showed that ruthenium compounds (1 and 2) impaired the cell viability of human bone (MG-63), lung (A549) and breast (MCF7) cancer cells with greater selectivity and specificity than cisplatin. Besides, complexes 1 and 2 decreased proliferation, migration and invasion on cell monolayers at lower concentrations (2.5-10 μM). In addition, both compounds induced genotoxicity revealed by the micronucleus test, which led to G2/M cell cycle arrest and induced the tumor cells to undergo apoptosis. On the other hand, in multicellular 3D models (multicellular spheroids; MCS), 1 and 2 overcame CDDP presenting lower IC50 values only in MCS of lung origin. Moreover, 1 outperformed 2 in MCS of bone and breast origin. Finally, our findings revealed that both compounds inhibited the cell invasion of multicellular spheroids, showing that complex 1 exhibited the most important antimetastatic action. Taken together, these results indicate that compound 1 is an interesting candidate to be tested on in vivo models as a novel strategy for anticancer therapy. Show less