📚 BiometalDB

Combinational use of drugs has been a common strategy in cancer treatment because of synergistic advantages in reducing dose and toxicity, minimizing or delaying drug resistance. To improve the efficacy of chemotherapy, various potential combinations have been investigated. Ruthenium complex is considered a potential alternative of the platinum-based drugs due to its significant efficacy and safety. Previously, we reported that ruthenium(II) complex (Δ-Ru1) has great anticancer potential and minor toxicity toward normal tissues. However, the therapeutic efficacy and mechanism of action of ruthenium(II) complex combined with other anticancer drugs is still unknown. Here, we investigated the combinational effect of Δ-Ru1 and doxorubicin in different cancer cells. The data assessed by Chou-Talalay method showed significant synergism in MCF-7 cells. Furthermore, the results in antiproliferation efficacy indicated that the combination showed strong cytotoxicity and increasing apoptosis of MCF-7 cells in 2D and 3D multicellular tumor spheroids (MCTSs). Significant inhibition of MCF-7 cells accompanied with increased ROS generation was observed. Furthermore, the expression of PI3K/AKT was significantly down-regulated, while the expression of PTEN was strongly up-regulated in cells treated with combination of Δ-Ru1 and doxorubicin. The expression of NF-κB and XIAP decreased while the expression of P53 increased and associated with apoptosis. These findings suggest that the combination of ruthenium complex and doxorubicin has a significant synergistic effect by down-regulating the PI3K/AKT signaling pathway in MCF-7 cells. This study may trigger more research in ruthenium complex and combination therapy that will be able to provide opportunities for developing better therapeutics for cancer treatment. Show less

There is an urgent need for more effective, less toxic cancer therapy agents. Motivated by this need, we synthesized a small panel of N-heterocyclic carbene (NHC)-coordinated ruthenium(II) arene complexes Ru1-Ru6 with the formula [Ru(p-cymene)(L)Cl]PF₆ (L = NHC ligand with varying substituents). Cell-based in vitro studies revealed that despite the structural similarity, Ru1-Ru6 exhibited distinct cytotoxic activities against cancer cells. In particular, Ru4 and Ru6, which bear n-octyl and pentamethylbenzyl motifs, respectively, were the most active at inducing apoptosis. In human ovarian A2780 cancer cells, Ru4 and Ru6 showed the highest cytotoxicities with IC₅₀ values of 2.74 ± 0.15 μM and 1.98 ± 0.10 μM, respectively, and they were approximately 2-fold more potent than cisplatin (IC₅₀ = 5.55 ± 0.37 μM). In addition to the cell killing capacity, inhibition of cell migration was validated by using these two optimized complexes. Mechanistic studies revealed that Ru4 and Ru6 complexes induced apoptosis in a caspase-dependent manner, primarily through intracellular reactive oxygen species (ROS) overproduction and cell cycle arrest at G1 phase. Furthermore, in a preclinical metastatic model of A2780 tumor xenograft, administration of Ru4 and Ru6 (20 μmol/kg) resulted in a marked inhibition of tumor progression and metastasis. Finally, a substantially alleviated systemic toxicity was observed for both complexes in comparison with cisplatin in animals. Overall, this study greatly increases our understanding of NHC-coordinated Ru(II) arene metallodrugs, aiding further investigation of their therapeutic potential in the treatment of metastatic cancers. Show less

The curative effect of sorafenib in hepatocellular carcinoma (HCC) is limited and sorafenib resistance remains a major obstacle for HCC. To overcome this obstacle, a new photoactive sorafenib-Ru(II) complex Ru-Sora has been designed. Upon irradiation (λ = 465 nm), Ru-Sora rapidly releases sorafenib and generates reactive oxygen species, which can oxidize intracellular substances such as GSH. Cellular experiments show that irradiated Ru-Sora is highly cytotoxic toward Hep-G2 cells, including sorafenib-resistant Hep-G2-SR cells. Compared to sorafenib, Ru-Sora has a significant photoactivated chemotherapeutic effect against Hep-G2-SR cancer cells and 3D Hep-G2 multicellular tumor spheroids. Furthermore, Ru-Sora inducing apoptosis and ferroptosis is proved by GSH depletion, GPX4 downregulation, and lipid peroxide accumulation. Metabolomics results suggest that Ru-Sora exerts photocytotoxicity by disrupting the purine metabolism, which is expected to inhibit tumor development. This study provides a promising strategy for enhancing chemotherapy and combating drug-resistant HCC disease. Show less

A novel three components approach for the synthesis of bioactive Ru-arene pyridinylmethylene complexes has been developed using pyridine carboxaldehyde, amino pyridine and dichloro (p-cymene) ruthenium(II) dimer as starting materials. These scaffolds were screened for their anticancer activity against breast cancer (MCF7) and human Epitheloid Cervix Carcinoma (HeLa) cell line. It was established that compounds [(η(6)-pcymene)RuCl(κ(2)-N,N-(3,5-dinitro-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4o), [(η(6)-pcymene)RuCl(κ(2)-N,N-N-(3,5-dibromo-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4c), [(η(6)-pcymene)RuCl(κ(2)-N,N-(3,5-dibromo-6-methylpyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4j) and [(η(6)-pcymene)RuCl(κ(2)-N,N-3(3-bromo-5-methyl-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4b) were significantly active against both the cell lines. Show less

The ligands 2-pyridin-2-yl-1H-benzimidazole (HL(1)), 1-methyl-2-pyridin-2-ylbenzimidazole (HL(2)), and 2-(1H-imidazol-2-yl)pyridine (HL(3)) and the proligand 2-phenyl-1H-benzimidazole (HL(4)) have been used to prepare five different types of new ruthenium(II) arene compounds: (i) monocationic complexes with the general formula [(η(6)-arene)RuCl(κ(2)-N,N-HL)]Y [HL = HL(1), HL(2), or HL(3); Y = Cl or BF4; arene = 2-phenoxyethanol (phoxet), benzene (bz), or p-cymene (p-cym)]; (ii) dicationic aqua complexes of the formula [(η(6)-arene)Ru(OH2)(κ(2)-N,N-HL(1))](Y)2 (Y = Cl or TfO; arene = phoxet, bz, or p-cym); (iii) the nucleobase derivative [(η(6)-arene)Ru(9-MeG)(κ(2)-N,N-HL(1))](PF6)2 (9-MeG = 9-methylguanine); (iv) neutral complexes consistent with the formulation [(η(6)-arene)RuCl(κ(2)-N,N-L(1))] (arene = bz or p-cym); (v) the neutral cyclometalated complex [(η(6)-p-cym)RuCl(κ(2)-N,C-L(4))]. The cytototoxic activity of the new ruthenium(II) arene compounds has been evaluated in several cell lines (MCR-5, MCF-7, A2780, and A2780cis) in order to establish structure-activity relationships. Three of the compounds with the general formula [(η(6)-arene)RuCl(κ(2)-N,N-HL(1))]Cl differing in the arene moiety have been studied in depth in terms of thermodynamic dissociation constants, aquation kinetic constants, and DNA binding measurements. The biologically most active compound is the p-cym derivative, which strongly destabilizes the DNA double helix, whereas those with bz and phoxet have only a small effect on the stability of the DNA double helix. Moreover, the inhibitory activity of several compounds toward CDK1 has also been evaluated. The DNA binding ability of some of the studied compounds and their CDK1 inhibitory effect suggest a multitarget mechanism for their biological activity. Show less

Deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) binding interactions for a series of ruthenium heterocyclic complexes were monitored using ultraviolet-visible (UV-Vis) spectrophotometry, fluorescence emission spectroscopy and agarose gel electrophoresis. Investigations of the DNA interactions for the metal complexes revealed that they are groove-binders with intrinsic binding constants in the order of 10⁴ - 10⁷ M^-1. Electronic spectrophotometric DNA titrations of the bis-heterocyclic metal complexes illustrated hypochromism of their intraligand electronic transitions and the presence of diffuse isosbestic points which are synonymous with homogeneous binding modes. Metal complexes with the mono-heterocyclic chelates also showed alterations in their intraligand transitions and changes in their metal-based electronic transitions which are suggestive of metal coordination to the CT-DNA structure. Using agarose gel electrophoresis assessments, Hoechst DNA binding competition studies corroborate that the metal complexes are DNA groove-binders. Optimal uptake of these metal complexes by BSA was observed based on their optimal apparent association and Stern-Volmer constants (K_app and K_SV > 10⁴ M^-1). Radical scavenging studies revealed that the metal complexes have high activities towards the neutralization of NO and DPPH radicals. Data attained from the BSA electronic spectrophotometric titrations for the majority of the metal complexes illustrated distinct hyperchromism accompanied with blue shifts which indicates unwinding of the protein strands. Predominately, the metal complexes showed moderate cytotoxicity against both triple-negative breast cancer and cervical cancer cell lines that was greater than that of 5-fluorouracil.Communicated by Ramaswamy H. Sarma. Show less

Polypyridyl ruthenium complexes as novel photosensitizers had drawn attention due to its high selectivity towards cancer cells and low toxicity to normal cells. Herein, we synthesized a lysosome-targeted polypyridyl ruthenium complex Rhein-Ru(bpy)₃ (bpy = 2,2'-bipyridine, rhein = 4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid), tethering with the Chinese medicine herb rhein. Rhein-Ru(bpy)₃ exhibited high phototoxicity with short time of irradiation against tumor cell lines with the IC₅₀ value of 2.4- 8.7 μM, and higher cytotoxicity against cisplatin-resistant A2780 cell lines, suggesting that Rhein-Ru(bpy)₃ could overcome the cisplatin resistance. Moreover, Rhein-Ru(bpy)₃ displayed low cytotoxicity towards cell lines in dark incubation, which was beneficial to reduce the toxic side effects towards normal cell lines. Besides, the confocal imaging and western blotting assay results suggested that Rhein-Ru(bpy)₃ could induce cancer cell death through the autophagy pathway. These results inspired us that lysosome-targeted photosensitizers based on ruthenium complexes showed great potential for photodynamic therapy (PDT) application in cancer treatment. Show less

The discovery of new light-triggered prodrugs based on ruthenium (II) complexes is a promising approach for photoactivated chemotherapy (PACT). The light-mediated activation of "strained" Ru(II) polypyridyl complexes resulted in ligand release and produced a ligand-deficient metal center capable of forming covalent adducts with biomolecules such as DNA. Based on the strategy of exploiting structural distortion to activate photochemistry, biologically active small molecules were coordinated to a Ru(II) scaffold to create light-triggered dual-action agents. Thirteen new Ru(II) complexes with pyridyl-pyrazol(in)e ligands were synthesized, and their photochemical reactivity and anticancer properties were investigated. Isomeric bidentate ligands were investigated, where "regular" ligands (where the coordinated nitrogens in the heterocycles are linked by C-C atoms) were compared to "inverse" isomers (where the coordinated nitrogens in the heterocycles are linked by C-N atoms). Coordination of the regular 3-(pyrid-2-yl)-pyrazol(in)es to a Ru(II) bis-dimethylphenanthroline scaffold yielded photoresponsive compounds with promising photochemical and biological properties, in contrast to the inverse 1-(pyrid-2-yl)-pyrazolines. The introduction of a phenyl ring to the 1N-pyrazoline cycle increased the distortion in complexes and improved ligand release upon light irradiation (470 nm) up to 5-fold in aqueous media. Compounds 1-8, containing pyridyl-pyrazol(in)e ligands, were at least 20-80-fold more potent than the parent pyridyl-pyrazol(in)es, and exhibited biological activity in the dark, with half-maximal inhibitory concentration (IC₅₀) values ranging from 0.2 to 7.6 μM in the HL60 cell line, with complete growth inhibition upon light irradiation. The diversification of coligands and introduction of a carboxylic acid into the Ru(II) complex resulted in compounds 9-12, with up to 146-fold improved phototoxicity indices compared with complexes 1-8. Show less

A series of mixed ligand ruthenium(II) complexes [Ru(Hdpa)2(diimine)](ClO4)2, 1-5 where Hdpa is 2,2'-dipyridylamine and diimine is 1,10-phenanthroline (phen) and a modified/extended 1,10-phenanthroline such as, 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), 5-methyldipyrido[3,2-d:2',3'-f]quinoxaline (mdpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) have been isolated and characterized by analytical and spectral methods. The complex [Ru(Hdpa)2(phen)](PF6)2 1 has been structurally characterized and the coordination geometry around Ru(II) in it is described as distorted octahedral. 1H NMR spectral data reveal that 1-5 should have a C2 symmetry lying on the diimine plane due to the rapid flapping of the coordinated Hdpa ligands. The interaction of the complexes with calf thymus (CT) DNA has been explored by using absorption and emission spectral and viscometry and electrochemical techniques and the mode of DNA binding of the complexes has been proposed. The DNA binding affinity of the complexes decreases with decrease in number of planar aromatic rings in the co-ligand supporting the intercalation of the diimine co-ligands in between the DNA base pairs. Circular dichroic spectral studies reveal that the complexes 3-5 exhibit induced circular dichroism upon binding to CT DNA. Interestingly, upon interaction with CT DNA all the complexes show an increase in anodic current in the cyclic voltammograms suggesting that they are involved in electrocatalytic guanine oxidation. Interestingly, of all the complexes, only 5 alters the DNA superhelicity upon binding with supercoiled pBR322 DNA, which is consistent with its higher DNA binding affinity. Further, the cytotoxicities of the complexes against human cervical epidermoid carcinoma cell line (ME180) have been examined. Interestingly, 5 exhibits a cytotoxicity against ME180 higher than other complexes with potency approximately 8 times more than cisplatin for 24 h incubation but 4 times lower than cisplatin for 48 h incubation. 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

Synergistic drug combinations can extend the use of poly(ADP-ribose) polymerase inhibitors (PARPi) such as Olaparib to BRCA-proficient tumors and overcome acquired or de novo drug resistance. To identify new synergistic combinations for PARPi, we screened a "micro-library" comprising a mix of commercially available drugs and DNA-binding ruthenium(II) polypyridyl complexes (RPCs) for Olaparib synergy in BRCA-proficient triple-negative breast cancer cells. This identified three hits: the natural product Curcumin and two ruthenium(II)-rhenium(I) polypyridyl metallomacrocycles. All combinations identified were effective in BRCA-proficient breast cancer cells, including an Olaparib-resistant cell line, and spheroid models. Mechanistic studies indicated that synergy was achieved via DNA-damage enhancement and resultant apoptosis. Combinations showed low cytotoxicity toward non-malignant breast epithelial cells and low acute and developmental toxicity in zebrafish embryos. This work identifies RPC metallomacrocycles as a novel class of agents for cancer combination therapy and provides a proof of concept for the inclusion of metallocompounds within drug synergy screens. Show less

Phosphrescent complexes of monodentate imidazole have been reported before, but their dissociation induced by cellular endogenous stimulus have never been explored. In our work, the dissociation of monodentate imidazole ligands from phosphorescent cyclometalated platinum (II) iridium (III) and polypyridyl ruthenium (II) complexes in GSH abundant reductive solution are firstly investigated. The release rate of ligand follows the order: Pt (II) > Ir (III) > > Ru (II) in vitro, while their corresponding bidentate complexes are barely affected. Moreover, the cellular dissociation of ligand can be monitored in time by Confocal imaging and flow cytometry. In brief, cellular penetration and nucleolus targeting ability of GSH active complexes are mainly interfered by the reductive microenvironment. Our work may help to reveal the dynamic process of coordination and release of monodentate ligands of complexes in cellular microenvironment. Show less

Three half-sandwich organometallic ruthenium(ii) complexes containing purine analogs such as triazolopyrimidines of general formula [(η⁶-p-cym)Ru(L)Cl₂], where p-cym represents p-cymene and L is 5,6,7-trimethyl-1,2,4-triazolo[1,5-a]pyrimidine (tmtp for 1), 5,7-diethyl-1,2,4-triazolo[1,5-a]pyrimidine (detp for 2) and 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one (HmtpO for 3), have been synthesized and characterized by elemental analysis, infrared, multinuclear magnetic resonance spectroscopic techniques (¹H, ¹³C, ¹⁵N), and single-crystal X-ray diffraction (for 1 and 2). All these complexes have been thoroughly screened for their in vitro cytotoxicity against MCF-7 and HeLa cell lines as well as L929 murine fibroblast cells, indicating [(η⁶-p-cym)Ru(HmtpO)Cl₂] (3) as the most active representative against the HeLa cell line and simultaneously being 64-fold less toxic to normal L929 murine fibroblast cells than cisplatin. At the same time, 3 has shown antimetastatic activity comparable to NAMI-A against HeLa cells both after 24 and 48 h of treatment in a wound healing assay. In order to better understand the mechanism of anticancer action and differences in the cytotoxic activity of 1-3, the studies were expanded to determining their lipophilicity, the kinetic stability at pH 6.5-8, the effect on reactive oxygen species (ROS) production in HeLa cells and interactions with significant biomolecules (DNA and albumin) by using molecular docking and circular dichroism (CD) experiments. Furthermore, antiparasitic studies against L. braziliensis, L. infantum and T. cruzi reveal that the newly synthesized complexes 1-3 are very promising candidates which can compete with commercial antiparasitic drugs. Complex 3 in particular, on top of exhibiting a high antiparasitic effect (IC₅₀ < 1 μM against two strains), reaches a selectivity index >1000. Show less

Title: "Half-Sandwich" Ruthenium Complexes with Alizarin as Anticancer Agents: Abstract: Upon exploration of the chemistry of the combination of ruthenium/arene with anthraquinone alizarin (L), three new complexes with the general formulas [Ru(L)Cl(η6-p-cymene)] (C1), [Ru(L)(η6-p-cymene)(PPh3)]PF6 (C2), and [Ru(L)(η6-p-cymene)(PEt3)]PF6 (C3) were synthesized and characterized using spectroscopic techniques (mass, IR, and 1D and 2D NMR), molar conductivity, elemental analysis, and X-ray diffraction. Complex C1 exhibited fluorescence, such as free alizarin, while in C2 and C3, the emission was probably quenched by monophosphines and the crystallographic data showed that hydrophobic interactions are predominant in intermolecular contacts. The cytotoxicity of the complexes was evaluated in the MDA-MB-231 (triple-negative breast cancer), MCF-7 (breast cancer), and A549 (lung) tumor cell lines and MCF-10A (breast) and MRC-5 (lung) nontumor cell lines. Complexes C1 and C2 were more selective to the breast tumor cell lines, and C2 was the most cytotoxic (IC50 = 6.5 μM for MDA-MB-231). In addition, compound C1 performs a covalent interaction with DNA, while C2 and C3 present only weak interactions; however, internalization studies by flow cytometry and confocal microscopy showed that complex C1 does not accumulate in viable MDA-MB-231 cells and is detected in the cytoplasm only after cell permeabilization. Investigations of the mechanism of action of the complexes indicate that C2 promotes cell cycle arrest in the Sub-G1 phase in MDA-MB-231, inhibits its colony formation, and has a possible antimetastatic action, impeding cell migration in the wound-healing experiment (13% of wound healing in 24 h). The in vivo toxicological experiments with zebrafish indicate that C1 and C3 exhibit the most zebrafish embryo developmental toxicity (inhibition of spontaneous movements and heartbeats), while C2, the most promising anticancer drug in the in vitro preclinical tests, revealed the lowest toxicity in in vivo preclinical screening. Show less

Phase separation of DNA is involved in chromatin packing for the regulation of gene transcription. Visualization and manipulation of DNA phase separation in living cells present great challenges. Herein, we present a Ru(II) complex (Ru1) with high DNA binding affinity and DNA "light-switch" behavior that can induce and monitor DNA phase separation both in vitro and in living cells. Molecular dynamics simulations indicate that the two phen-PPh₃ ligands with positively charged lipophilic triphenylphosphine substituents and flexible long alkyl chains in Ru1 play essential roles in the formation of multivalent binding forces between DNA molecules to induce DNA phase separation. Importantly, the unique environmental sensitive emission property of Ru1 enables direct visualization of the dynamic process of DNA phase separation in living cells by two-photon phosphorescent lifetime imaging. Moreover, Ru1 can change the gene expression pattern by modulating chromatin accessibility as demonstrated by integrating RNA-sequencing and transposase-accessible chromatin with high-throughput sequencing. In all, we present here the first small-molecule-based tracer and modulator of DNA phase separation in living cells and elucidate its impact on the chromatin state and transcriptome. 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

Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-"light switch" complexes [Ru(dppz)₂(5,5'dmb)]²⁺ and [Ru(PIP)₂(5,5'dmb)]²⁺ (dppz = dipyridophenazine, 5,5'dmb = 5,5'-dimethyl-2,2'-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor-acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)₂(5,5'dmb)]²⁺ acts to block DNA replication fork progression. Show less

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity. Show less

Looking for new metal-based anticancer treatments, in recent years many ruthenium complexes have been proposed as effective and safe potential drugs. In this context we have recently developed a novel approach for the in vivo delivery of Ru(III) complexes, preparing stable ruthenium-based nucleolipidic nanoaggregates endowed with significant antiproliferative activity. Herein we describe the cellular response to our ruthenium-containing formulations in selected models of human breast cancer. By in vitro bioscreens in the context of preclinical studies, we have focused on their ability to inhibit breast cancer cell proliferation by the activation of the intrinsic apoptotic pathway, possibly via mitochondrial perturbations involving Bcl-2 family members and predisposing to programmed cell death. In addition, the most efficient ruthenium-containing cationic nanoaggregates we have hitherto developed are able to elicit both extrinsic and intrinsic apoptosis, as well as autophagy. To limit chemoresistance and counteract uncontrolled proliferation, multiple cell death pathways activation by metal-based chemotherapeutics is a challenging, yet very promising strategy for targeted therapy development in aggressive cancer diseases, such as triple-negative breast cancer with limited treatment options. These outcomes provide valuable, original knowledge on ruthenium-based candidate drugs and new insights for future optimized cancer treatment protocols. 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 the fight against cancer, photodynamic therapy is generating great interest thanks to its ability to selectively kill cancer cells without harming healthy tissues. In this field, ruthenium(II) polypyridyl complexes, and more specifically, complexes with dipyrido[3,2-a:2',3'-c]phenazine (dppz) as a ligand are of particular interest due to their DNA-binding and photocleaving properties. However, ruthenium(II) polypyridyl complexes can sometimes suffer from low lipophilicity, which hampers cellular internalisation through passive diffusion. In this study, four new [Ru(dppz-X₂ )₃ ]²⁺ complexes (X=H, F, Cl, Br, I) were synthesized and their lipophilicity (logP), cytotoxicity and phototoxicity on cancerous and noncancerous cell lines were assessed. This study shows that, counterintuitively, the phototoxicity of these complexes decreases as their lipophilicity increases; this could be due solely to the atomic radius of the halogen substituents. Show less

Five new complexes with general formula [Ru(L_n)(PP)(bipy)]PF₆, where L_n = N,N'-dimethyl-N-Acyl thiourea, and P-P: 1,2-bis(diphenylphosphino)ethane (dppe) or 1,4-bis(diphenylphosphino)butane (dppb)) were synthesized and characterized by elemental analysis, molar conductivity, cyclic voltammetry, IR, NMR (¹H, ¹³C{¹H} and ³¹P{¹H}), and single crystal X-ray diffractometry. The cytotoxicity of compounds against lung and breast tumor cell lines was significant, where two complexes, [Ru(L₃)(bipy)(dppe)]PF₆ (3) and [Ru(L₃)(bipy)(dppb)]PF₆ (6), were selected to evaluate changes in morphology, inhibition of migration and cell death in the MDA-MB-231 lineage. The complexes caused alterations in the cell morphology and were able to inhibit cell migration at the concentrations evaluated, induce the cell cycle arrested in the Sub-G1 phase, and induced cell death by apoptosis. All the complexes presented interaction with HSA, and the interaction studies with DNA suggested weak interactions, probably by the minor groove. Show less

Several complexes of general formula [Ru(halide)(η⁶-p-cymene)(α-diimine)]⁺, in the form of nitrate, triflate and hexafluorophosphate salts, including a newly synthesized iodide compound, were investigated as potential anticancer drugs and bactericides. NMR and UV-Vis studies evidenced remarkable stability of the complexes in water and cell culture medium. In general, the complexes displayed strong cytotoxicity against A2780 and A549 cancer cell lines with IC₅₀ values in the low micromolar range, and one complex (RUCYN) emerged as the most promising one, with a significant selectivity compared to the non-cancerous HEK293 cell line. A variable affinity of the complexes for BSA and DNA binding was ascertained by spectrophotometry/fluorimetry, circular dichroism, electrophoresis and viscometry. The performance of RUCYN appears associated to enhanced cell internalization, favored by two cyclohexyl substituents, rather than to specific interaction with the evaluated biomolecules. The chloride/iodide replacement, in one case, led to increased cellular uptake and cytotoxicity at the expense of selectivity, and tuned DNA binding towards intercalation. Complexes with iodide or a valproate bioactive fragment exhibited the best antimicrobial profiles. Show less

The use of natural products as potential ligands has been explored as a strategy in the development of metal-based chemotherapy. Since ruthenium complexes are promising alternatives to traditional antitumor agents, this study evaluated the anti-melanoma potential of two ruthenium(II) complexes containing the naphthoquinone ligands lapachol (lap), [Ru(lap)(dppm)₂]PF₆, and lawsone (law), [Ru(law)(dppm)₂]PF₆, in addition to the bis(diphenylphosphino)methane (dppm) ligand, referred to as complexes (1) and (2), respectively, using a syngeneic murine melanoma model. Activation of the apoptotic pathway by the treatments was assessed by immunohistochemistry in tumor tissue. Additionally, toxicity of the treatments was evaluated by variation in body and organ weight, quantification of biochemical indicators of renal damage, and genotoxicity in bone marrow and hepatocytes. First, the antiproliferative activity of (1) and (2) was observed in B16F10 cells, with IC₅₀ values of 2.78 and 1.68 μM, respectively. The results obtained in mice showed that, unlike complex (1), (2) possesses significant anti-melanoma activity demonstrated by a reduction in tumor volume and mass (88.42%), as well as in mitosis frequency (83.86%). Additionally, complex (2) increased the levels of cleaved caspase-3, inducing tumor cell apoptosis. When compared to the metallodrug cisplatin, complex (2) exhibited similar anti-melanoma activity and lower toxicity considering all parameters evaluated. In silico studies demonstrated no difference in the binding energy of the naphthoquinone complex between complexes (1) and (2). However, the complex containing the lawsone ligand has a lower molar volume, which may be important for interactions with minor DNA grooves. The present results demonstrate the antitumor efficiency of complex (2) and a significantly lower systemic toxicity compared to cisplatin. Show less

(1) Background: Ruthenium and osmium complexes attract increasing interest as next generation anticancer drugs. Focusing on structure-activity-relationships of this class of compounds, we report on 17 different ruthenium(II) complexes and four promising osmium(II) analogues with cinnamic acid derivatives as O,S bidentate ligands. The aim of this study was to determine the anticancer activity and the ability to evade platin resistance mechanisms for these compounds. (2) Methods: Structural characterizations and stability determinations have been carried out with standard techniques, including NMR spectroscopy and X-ray crystallography. All complexes and single ligands have been tested for cytotoxic activity on two ovarian cancer cell lines (A2780, SKOV3) and their cisplatin-resistant isogenic cell cultures, a lung carcinoma cell line (A549) as well as selected compounds on three non-cancerous cell cultures in vitro. FACS analyses and histone γH2AX staining were carried out for cell cycle distribution and cell death or DNA damage analyses, respectively. (3) Results: IC50 values show promising results, specifically a high cancer selective cytotoxicity and evasion of resistance mechanisms for Ru(II) and Os(II) compounds. Histone γH2AX foci and FACS experiments validated the high cytotoxicity but revealed diminished DNA damage-inducing activity and an absence of cell cycle disturbance thus pointing to another mode of action. (4) Conclusion: Ru(II) and Os(II) compounds with O,S-bidentate ligands show high cytotoxicity without strong effects on DNA damage and cell cycle, and this seems to be the basis to circumvent resistance mechanisms and for the high cancer cell specificity. Show less

Ruthenium compounds have demonstrated promising activity in different cancer types, overcoming several limitations of platinum-based drugs, yet their global structure-activity is still under debate. We analyzed the activity of Runat-BI, a racemic Ru(III) compound, and of one of its isomers in eight tumor cell lines of breast, colon and gastric cancer as well as in a non-tumoral control. Runat-BI was prepared with 2,2'-biimidazole and dissolved in polyethylene glycol. We performed assays of time- and dose-dependent viability, migration, proliferation, and expression of pro- and antiapoptotic genes. Moreover, we studied the growth rate and cell doubling time to correlate it with the apoptotic effect of Runat-BI. As a racemic mixture, Runat-BI caused a significant reduction in the viability and migration of three cancer cell lines from colon, gastric and breast cancer, all of which displayed fast proliferation rates. This compound also demonstrated selectivity between tumor and non-tumor lines and increased proapoptotic gene expression. However, the isolated isomer did not show any effect. Racemic Runat-BI is a potential drug candidate for treatment of highly aggressive tumors. Further studies should be addressed at evaluating the role of the other isomer, for a more precise understanding of its antitumoral potential and mechanism of action. Show less

Two new ruthenium(II) complexes [Ru(dip)₂(PPβC)]PF₆ (Ru1, dip = 4,7-diphenyl-1,10-phenanthroline, PPβC = N-(1,10-phenanthrolin-5-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxamide) and [Ru(phen)₂(PPβC)]PF₆ (Ru2, phen = 1, 10-phenanthroline) with β-carboline derivative PPβC as the primary ligand, were designed and synthesized. Ru1 and Ru2 displayed higher antiproliferative activity than cisplatin against the test cancer cells, with IC₅₀ values ranging from 0.5 to 3.6 μM. Moreover, Ru1 and Ru2 preferentially accumulated in mitochondria and caused a series of changes in mitochondrial events, including the depolarization of mitochondrial membrane potential, the damage of mitochondrial DNA, the depletion of cellular ATP, and the elevation of intracellular reactive oxygen species levels. Then, it induced caspase-3/7-mediated A549 cell apoptosis. More importantly, both complexes could act as topoisomerase I catalytic inhibitors to inhibit mitochondrial DNA synthesis. Accordingly, the developed Ru(II) complexes hold great potential to be developed as novel therapeutics for cancer treatment. Show less

Title: Increasing Anticancer Activity with Phosphine Ligation in Zwitterionic Half-Sandwich Iridium(III), Rhodium(III), and Ruthenium(II) Complexes. Abstract: The synthesis and biological assessment of neutral or cationic platinum group metal-based anticancer complexes have been extremely studied, whereas there are few reports on the corresponding zwitterionic complexes. Herein, the synthesis, characterization, and bioactivity of zwitterionic half-sandwich phosphine-imine iridium(III), rhodium(III), and ruthenium(II) complexes were presented. The sulfonated phosphine-imine ligand and a group of zwitterionic half-sandwich P,N-chelating organometallic complexes were fully characterized by nuclear magnetic resonance (NMR), mass spectrum (electrospray ionization, ESI), elemental analysis, and X-ray crystallography. The solution stability of these complexes and their spectral properties were also determined. Notably, almost all of these complexes showed enhanced anticancer activity against model HeLa and A549 cancer cells than the corresponding zwitterionic pyridyl-imine N,N-chelating iridium(III) and ruthenium(II) complexes, which have exhibited inactive or low active in our previous work. The increase in the lipophilic property and intracellular uptake levels of these zwitterionic P,N-chelating complexes appeared to be associated with their superior cytotoxicity. In addition, these complexes showed biomolecular interactions with bovine serum albumin (BSA). The flow cytometry studies indicated that the representative complex Ir1 could induce early-stage apoptosis in A549 cells. Further, confocal microscopy imaging analysis displayed that Ir1 entered A549 cells through the energy-dependent pathway, targeted lysosome, and could cause lysosomal damage. In particular, these complexes could impede cell migration in A549 cells. 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

Title: Ruthenium(II)-Dithiocarbazates as Anticancer Agents: Synthesis, Solution Behavior, and Mitochondria-Targeted Apoptotic Cell Death. Abstract: The reaction of the Ru(PPh3 )3 Cl2 with HL1-3 -OH (-OH stands for the oxime hydroxyl group; HL1 -OH=diacetylmonoxime-S-benzyldithiocarbazonate; HL2 -OH=diacetylmonoxime-S-(4-methyl)benzyldithiocarbazonate; and HL3 -OH=diacetylmonoxime-S-(4-chloro)benzyl-dithiocarbazonate) gives three new ruthenium complexes [RuII (L1-3 -H)(PPh3 )2 Cl] (1-3) (-H stands for imine hydrogen) coordinated with dithiocarbazate imine as the final products. All ruthenium(II) complexes (1-3) have been characterized by elemental (CHNS) analyses, IR, UV-vis, NMR (1 H, 13 C, and 31 P) spectroscopy, HR-ESI-MS spectrometry and also, the structure of 1-2 was further confirmed by single crystal X-ray crystallography. The solution/aqueous stability, hydrophobicity, DNA interactions, and cell viability studies of 1-3 against HeLa, HT-29, and NIH-3T3 cell lines were performed. Cell viability results suggested 3 being the most cytotoxic of the series with IC50 6.9±0.2 μM against HeLa cells. Further, an apoptotic mechanism of cell death was confirmed by cell cycle analysis and Annexin V-FITC/PI double staining techniques. In this regard, the live cell confocal microscopy results revealed that compounds primarily target the mitochondria against HeLa, and HT-29 cell lines. Moreover, these ruthenium complexes elevate the ROS level by inducing mitochondria targeting apoptotic cell death. Show less