Copper(II) Complexes with Isomeric Morpholine-Substituted 2-Formylpyridine Thiosemicarbazone Hybrids as Potential Anticancer Drugs Inhibiting Both Ribonucleotide Reductase and Tubulin Polymerization: The Morpholine Position Matters.

The development of copper(II) thiosemicarbazone complexes as potential anticancer agents, possessing dual functionality as inhibitors of R2 ribonucleotide reductase (RNR) and tubulin polymerization by binding at the colchicine site, presents a promising avenue for enhancing therapeutic effectiveness. Herein, we describe the syntheses and physicochemical characterization of four isomeric proligands H2L3-H2L6, with the methylmorpholine substituent at pertinent positions of the pyridine ring, along with their corresponding Cu(II) complexes 3-6. Evidently, the position of the morpholine moiety and the copper(II) complex formation have marked effects on the in vitro antiproliferative activity in human uterine sarcoma MES-SA cells and the multidrug-resistant derivative MES-SA/Dx5 cells. Activity correlated strongly with quenching of the tyrosyl radical (Y•) of mouse R2 RNR protein, inhibition of RNR activity in the cancer cells, and inhibition of tubulin polymerization. Insights into the mechanism of antiproliferative activity, supported by experimental results and molecular modeling calculations, are presented.

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Complex formation of ML324, the histone demethylase inhibitor, with essential metal ions: Relationship between solution chemistry and anticancer activity.

N-(3-(dimethylamino)propyl-4-(8-hydroxyquinolin-6-yl)benzamide (ML324, HL) is a potent inhibitor of the iron-containing histone demethylase KDM4, a recognized potential target of cancer therapeutics. Herein, we report the proton dissociation and complex formation processes of ML324 with essential metal ions such as Fe(II), Fe(III), Cu(II) and Zn(II) using UV-visible, fluorescence, electron paramagnetic resonance and 1H NMR spectroscopic methods. The electrochemical behaviour of the copper and iron complexes was characterized by cyclic voltammetry and spectroelectrochemistry. The solid phase structure of ML324 analysed by X-ray crystallography is also provided. Based on the solution equilibrium data, ML324 is present in solution in H2L+ form with a protonated dimethylammonium moiety at pH 7.4, and this (N,O) donor bearing ligand forms mono and bis complexes with all the studied metal ions and the tris-ligand species is also observed with Fe(III). At pH 7.4 the metal binding ability of ML324 follows the order: Fe(II) < Zn(II) < Cu(II) < Fe(III). Complexation with iron resulted in a negative redox potential (E'1/2 = -145 mV vs. NHE), further suggesting that the ligand has a preference for Fe(III) over Fe(II). ML324 was tested for its anticancer activity in chemosensitive and resistant human cancer cells overexpressing the efflux pump P-glycoprotein. ML324 exerted similar activity in all tested cells (IC50 = 1.9-3.6 µM). Co-incubation and complexation of the compound with Cu(II) and Zn(II) had no impact on the cytotoxicity of ML324, whereas Fe(III) decreased the toxicity in a concentration-dependent manner, and this effect was more pronounced in the multidrug resistant cells.

DNA-dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

Effective targeting of breast cancer by the inhibition of P-glycoprotein mediated removal of toxic lipid peroxidation byproducts from drug tolerant persister cells.

Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1-/-;p53-/- mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.

ABCC1 and glutathione metabolism limit the efficacy of BCL-2 inhibitors in acute myeloid leukemia.

The BCL-2 inhibitor Venetoclax is a promising agent for the treatment of acute myeloid leukemia (AML). However, many patients are refractory to Venetoclax, and resistance develops quickly. ATP-binding cassette (ABC) transporters mediate chemotherapy resistance but their role in modulating the activity of targeted small-molecule inhibitors is unclear. Using CRISPR/Cas9 screening, we find that loss of ABCC1 strongly increases the sensitivity of AML cells to Venetoclax. Genetic and pharmacologic ABCC1 inactivation potentiates the anti-leukemic effects of BCL-2 inhibitors and efficiently re-sensitizes Venetoclax-resistant leukemia cells. Conversely, ABCC1 overexpression induces resistance to BCL-2 inhibitors by reducing intracellular drug levels, and high ABCC1 levels predicts poor response to Venetoclax therapy in patients. Consistent with ABCC1-specific export of glutathionylated substrates, inhibition of glutathione metabolism increases the potency of BCL-2 inhibitors. These results identify ABCC1 and glutathione metabolism as mechanisms limiting efficacy of BCL-2 inhibitors, which may pave the way to development of more effective therapies.

Nucleotide binding is the critical regulator of ABCG2 conformational transitions.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency.

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

Comparison of Different Clinical Chemotherapeutical Agents' Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

P-Glycoprotein Activity at Diagnosis Does Not Predict Therapy Outcome and Survival in Canine B-Cell Lymphoma.

Various mechanisms are known to be involved in the development of multidrug resistance during cancer treatment. P-glycoprotein (P-gp) decreases the intracellular concentrations of cytotoxic drugs by an energy-dependent efflux mechanism. The aim of this study was to investigate the predictive value of P-gp function based on the evaluation of P-gp activity in tumor cells obtained from canine B-cell lymphoma patients at diagnosis. P-gp function of 79 immunophenotyped canine lymphoma samples was determined by flow cytometry using the Calcein assay. Dogs were treated with either the CHOP or the L-CHOP protocol, a subset of relapsed patients received L-asparaginase and lomustine rescue treatments. Among the 79 dogs, the median overall survival time was 417 days, and the median relapse-free period was 301 days. 47 percent of the samples showed high P-gp activity, which was significantly higher in Stage IV cancer patients compared to Stage II + III and V. Whereas staging was associated with major differences in survival times, we found that the intrinsic P-gp activity of tumor cells measured at diagnosis is not predictive for therapy outcome. Further studies are needed to identify the intrinsic and acquired resistant mechanisms that shape therapy response and survival in B-cell canine lymphoma patients.

Structure-Activity Relationships of 8-Hydroxyquinoline-Derived Mannich Bases with Tertiary Amines Targeting Multidrug-Resistant Cancer.

A recently proposed strategy to overcome multidrug resistance (MDR) in cancer is to target the collateral sensitivity of otherwise resistant cells. We designed a library of 120 compounds to explore the chemical space around previously identified 8-hydroxyquinoline-derived Mannich bases with robust MDR-selective toxicity. We included compounds to study the effect of halogen and alkoxymethyl substitutions in R5 in combination with different Mannich bases in R7, a shift of the Mannich base from R7 to R5, as well as the introduction of an aromatic moiety. Cytotoxicity tests performed on a panel of parental and MDR cells highlight a strong influence of experimentally determined pKa values of the donor atom moieties, indicating that protonation and metal chelation are important factors modulating the MDR-selective anticancer activity of the studied compounds. Our results identify structural requirements increasing MDR-selective anticancer activity, providing guidelines for the development of more effective anticancer chelators targeting MDR cancer.

Cytotoxicity of cinchona alkaloid organocatalysts against MES-SA and MES-SA/Dx5 multidrug-resistant uterine sarcoma cell lines.

Since the first application of natural quinine as an anti-malarial drug, cinchona alkaloids and their derivatives have been exhaustively studied for their biological activity. In our work, we tested 13 cinchona alkaloid organocatalysts, synthesised from quinine. These derivatives were screened against MES-SA and Dx5 uterine sarcoma cell lines for in vitro anticancer activity and to investigate their potential to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR). Decorating quinine with hydrogen-bond donor units, such as thiourea and (thio)squaramide, resulted in decreased half-maximal growth inhibition values on both cell lines (1.3-21 µM) compared to quinine and other cinchona alcohols (47-111 µM). Further cytotoxicity studies conducted in the presence of the P-gp inhibitor tariquidar indicated that several analogues, especially cinchona amines and squaramides, but not thiosquaramide, were expelled from MDR cells by P-gp. Similarly to the established P-gp inhibitor quinine, 6 cinchona analogues were shown to inhibit calcein-AM efflux. Interestingly, quinine and didehydroquinine exhibited a marginally increased toxicity against the multidrug resistant Dx5 cells. Collateral sensitivity of the MDR cell line was more pronounced when the cinchona thiosquaramide was complexed with Cu(II) acetate. Based on the results, cinchona derivatives are good anticancer candidates for further drug development.

Efficient Synthesis of Acylated, Dialkyl α-Hydroxy-Benzylphosphonates and Their Anticancer Activity.

An efficient method applying acyl chlorides as reagents was developed for the acylation of the hindered hydroxy group of dialkyl α-hydroxy-benzylphosphonates. The procedure did not require any catalyst. A few acylations were also performed with the SC-enantiomer of dimethyl α-hydroxy-benzylphosphonate, and the optical purity was retained. A part of the acyloxyphosphonates was tested against eight tumor cell lines of different tissue origin at c = 50 µM concentration. The compounds elicited moderate cytostatic effect against breast, skin, prostate, colon, and lung carcinomas; a melanoma cell line; and against Kaposi's sarcoma cell lines. Then, dose-dependent cytotoxicity was assayed, and benzoylation of the α-hydroxy group was identified as a moiety that increases anticancer cytotoxicity across all cell lines. Surprisingly, a few analogues were more toxic to multidrug resistant cancer cell lines, thus evading P-glycoprotein mediated drug extrusion.

Identifying Novel Inhibitors for Hepatic Organic Anion Transporting Polypeptides by Machine Learning-Based Virtual Screening.

Integration of statistical learning methods with structure-based modeling approaches is a contemporary strategy to identify novel lead compounds in drug discovery. Hepatic organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are classical off-targets, and it is well recognized that their ability to interfere with a wide range of chemically unrelated drugs, environmental chemicals, or food additives can lead to unwanted adverse effects like liver toxicity and drug-drug or drug-food interactions. Therefore, the identification of novel (tool) compounds for hepatic OATPs by virtual screening approaches and subsequent experimental validation is a major asset for elucidating structure-function relationships of (related) transporters: they enhance our understanding about molecular determinants and structural aspects of hepatic OATPs driving ligand binding and selectivity. In the present study, we performed a consensus virtual screening approach by using different types of machine learning models (proteochemometric models, conformal prediction models, and XGBoost models for hepatic OATPs), followed by molecular docking of preselected hits using previously established structural models for hepatic OATPs. Screening the diverse REAL drug-like set (Enamine) shows a comparable hit rate for OATP1B1 (36% actives) and OATP1B3 (32% actives), while the hit rate for OATP2B1 was even higher (66% actives). Percentage inhibition values for 44 selected compounds were determined using dedicated in vitro assays and guided the prioritization of several highly potent novel hepatic OATP inhibitors: six (strong) OATP2B1 inhibitors (IC50 values ranging from 0.04 to 6 µM), three OATP1B1 inhibitors (2.69 to 10 µM), and five OATP1B3 inhibitors (1.53 to 10 µM) were identified. Strikingly, two novel OATP2B1 inhibitors were uncovered (C7 and H5) which show high affinity (IC50 values: 40 nM and 390 nM) comparable to the recently described estrone-based inhibitor (IC50 = 41 nM). A molecularly detailed explanation for the observed differences in ligand binding to the three transporters is given by means of structural comparison of the detected binding sites and docking poses.

Metal Complexes of a 5-Nitro-8-Hydroxyquinoline-Proline Hybrid with Enhanced Water Solubility Targeting Multidrug Resistant Cancer Cells.

Multidrug resistance (MDR) in cancer is one of the major obstacles of chemotherapy. We have recently identified a series of 8-hydroxyquinoline Mannich base derivatives with MDR-selective toxicity, however with limited solubility. In this work, a novel 5-nitro-8-hydroxyquinoline-proline hybrid and its Rh(η5-C5Me5) and Ru(η6-p-cymene) complexes with excellent aqueous solubility were developed, characterized, and tested against sensitive and MDR cells. Complex formation of the ligand with essential metal ions was also investigated using UV-visible, circular dichroism, 1H NMR (Zn(II)), and electron paramagnetic resonance (Cu(II)) spectroscopic methods. Formation of mono and bis complexes was found in all cases with versatile coordination modes, while tris complexes were also formed with Fe(II) and Fe(III) ions, revealing the metal binding affinity of the ligand at pH 7.4: Cu(II) > Zn(II) > Fe(II) > Fe(III). The ligand and its Rh(III) complex displayed enhanced cytotoxicity against the resistant MES-SA/Dx5 and Colo320 human cancer cell lines compared to their chemosensitive counterparts. Both organometallic complexes possess high stability in solution, however the Ru(II) complex has lower chloride ion affinity and slower ligand exchange processes, along with the readiness to lose the arene ring that is likely connected to its inactivity.

Mesenchymal-Stromal Cell-like Melanoma-Associated Fibroblasts Increase IL-10 Production by Macrophages in a Cyclooxygenase/Indoleamine 2,3-Dioxygenase-Dependent Manner.

Melanoma-associated fibroblasts (MAFs) are integral parts of melanoma, providing a protective network for melanoma cells. The phenotypical and functional similarities between MAFs and mesenchymal stromal cells (MSCs) prompted us to investigate if, similarly to MSCs, MAFs are capable of modulating macrophage functions. Using immunohistochemistry, we showed that MAFs and macrophages are in intimate contact within the tumor stroma. We then demonstrated that MAFs indeed are potent inducers of IL-10 production in various macrophage types in vitro, and this process is greatly augmented by the presence of treatment-naïve and chemotherapy-treated melanoma cells. MAFs derived from thick melanomas appear to be more immunosuppressive than those cultured from thin melanomas. The IL-10 increasing effect is mediated, at least in part, by cyclooxygenase and indoleamine 2,3-dioxygenase. Our data indicate that MAF-induced IL-10 production in macrophages may contribute to melanoma aggressiveness, and targeting the cyclooxygenase and indoleamine 2,3-dioxygenase pathways may abolish MAF-macrophage interactions.

Data-Driven Ensemble Docking to Map Molecular Interactions of Steroid Analogs with Hepatic Organic Anion Transporting Polypeptides.

Hepatic organic anion transporting polypeptides-OATP1B1, OATP1B3, and OATP2B1-are expressed at the basolateral membrane of hepatocytes, being responsible for the uptake of a wide range of natural substrates and structurally unrelated pharmaceuticals. Impaired function of hepatic OATPs has been linked to clinically relevant drug-drug interactions leading to altered pharmacokinetics of administered drugs. Therefore, understanding the commonalities and differences across the three transporters represents useful knowledge to guide the drug discovery process at an early stage. Unfortunately, such efforts remain challenging because of the lack of experimentally resolved protein structures for any member of the OATP family. In this study, we established a rigorous computational protocol to generate and validate structural models for hepatic OATPs. The multistep procedure is based on the systematic exploration of available protein structures with shared protein folding using normal-mode analysis, the calculation of multiple template backbones from elastic network models, the utilization of multiple template conformations to generate OATP structural models with various degrees of conformational flexibility, and the prioritization of models on the basis of enrichment docking. We employed the resulting OATP models of OATP1B1, OATP1B3, and OATP2B1 to elucidate binding modes of steroid analogs in the three transporters. Steroid conjugates have been recognized as endogenous substrates of these transporters. Thus, investigating this data set delivers insights into mechanisms of substrate recognition. In silico predictions were complemented with in vitro studies measuring the bioactivity of a compound set on OATP expressing cell lines. Important structural determinants conferring shared and distinct binding patterns of steroid analogs in the three transporters have been identified. Overall, this comparative study provides novel insights into hepatic OATP-ligand interactions and selectivity. Furthermore, the integrative computational workflow for structure-based modeling can be leveraged for other pharmaceutical targets of interest.

[Mathematical and computational methods to model chemotherapy- induced resistance]. / Matematikai és számítógépes módszerek a kemoterápiás kezelések során kialakuló rezisztencia modellezésére.

Chemotherapy plays an important role in the treatment of cancer. While clinical chemotherapy protocols can lead to remission in some patients, in many cases tumor progression occurs despite continued treatment. In the present study we summarize mathematical approaches to model tumor growth and response to treatment, focusing on anticancer therapy resistance. We present results obtained at the recently founded Cybermedical Competence Center at Óbuda University, focusing on the development of a new therapy optimization concept that aims to optimize traditional chemotherapy.

[Development of novel treatment strategies for drug resistant cancer]. / Új stratégiák fejlesztése a gyógyszerrezisztens daganatok kezeléséhez.

There are about 14 million new cancer cases and 8 million deaths every year. Every second man and one in every three women will get cancer during their lifetimes. Following decades of steady increase, death rates have stabilized due to increased awareness and prevention, early detection, and the emergence of more effective therapy. Yet despite all the advances cancer remains a major killer. Despite improved therapies, nearly all current treatments face the same problem: for many patients, they ultimately stop working. Therapy resistance is the ultimate challenge facing cancer researchers and patients today. In this review we present an overview of the most important resistance mechanisms, discussing progress in therapies designed to prevent or overcome anticancer therapy resistance. Finally, we present recent findings from our own laboratory on the development of new experimental models and new therapeutic approaches to combat multidrug resistant cancer.

Half-sandwich organometallic Ru and Rh complexes of (N,N) donor compounds: effect of ligand methylation on solution speciation and anticancer activity.

A series of half-sandwich polypyridyl complexes was synthesized and compared focusing on structural, cytotoxic and aqueous solution behaviour. The formula of the synthesized complexes is [M(arene)(N,N)Cl]Cl, where M: Ru or Rh, arene: p-cymene, toluene or C5Me5-, (N,N): 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 1,10-phenanthroline (phen) or 2,9-dimethyl-1,10-phenanthroline (neo). The structures of five half-sandwich complexes were determined by X-ray crystallography. It was found that introducing methyl groups next to the coordinating nitrogen atoms of the bidentate ligand causes steric congestion around the metal centre which changes the angle between ligand planes. The ligands and the Rh complexes showed significant cytotoxicity in A2780 and MES-SA cancer cell lines (IC50 = 0.1-56 µM) and in the cisplatin-resistant A2780cis cells. Paradoxically, phen and dmb as well as their half-sandwich Rh complexes showed increased toxicity against multidrug resistant MES-SA/Dx5 cells. In contrast, coordination to Ru caused loss of toxicity. Solution equilibrium constants showed that the studied metal complexes have high stability, and no dissociation was found for Ru and Rh complexes even at micromolar concentrations in a wide pH range. However, in the case of Ru complexes a slow and irreversible decomposition, namely arene loss, was also observed, which was more pronounced in light exposure in aqueous solution. In the case of neo, the methyl groups next to the nitrogen atoms significantly decrease the stability of complexes. For Rh complexes, the order of the stability constants corrected with ligand basicity (log K*): 9.78 (phen) > 9.01 (dmb) > 8.89 (bpy) > 3.93 (neo). The coordinated neo resulted in an enormous decrease in the chloride ion affinity of Ru compounds. Based on the results, a universal model was introduced for the prediction of chloride ion capability of half-sandwich Rh and Ru complexes. It combines the effects of the bidentate ligand and the M(arene) part using only two terms, performing multilinear regression procedure.