The Copper Reduction Potential Determines the Reductive Cytotoxicity: Relevance to the Design of Metal-Organic Antitumor Drugs.

Copper-organic compounds have gained momentum as potent antitumor drug candidates largely due to their ability to generate an oxidative burst upon the transition of Cu2+ to Cu1+ triggered by the exogenous-reducing agents. We have reported the differential potencies of a series of Cu(II)-organic complexes that produce reactive oxygen species (ROS) and cell death after incubation with N-acetylcysteine (NAC). To get insight into the structural prerequisites for optimization of the organic ligands, we herein investigated the electrochemical properties and the cytotoxicity of Cu(II) complexes with pyridylmethylenethiohydantoins, pyridylbenzothiazole, pyridylbenzimidazole, thiosemicarbazones and porphyrins. We demonstrate that the ability of the complexes to kill cells in combination with NAC is determined by the potential of the Cu+2 → Cu+1 redox transition rather than by the spatial structure of the organic ligand. For cell sensitization to the copper-organic complex, the electrochemical potential of the metal reduction should be lower than the oxidation potential of the reducing agent. Generally, the structural optimization of copper-organic complexes for combinations with the reducing agents should include uncharged organic ligands that carry hard electronegative inorganic moieties.

Liposomal form of 2-alkylthioimidazolone-based copper complexes for combined cancer therapy.

Aim: To develop an optimized approach for encapsulating a 2-alkylthioimidazolone-based copper coordination compound within liposomes, which could offer treatment of cancer and bacterial infections by reactive oxygen species generation toxicity mechanisms. Materials & methods: For drug-loaded liposome preparation, lipids and drug mixture in organic solvents was injected into copper salt solution, forming a coordination compound simultaneously embedded in the lipid bilayer. In vitro tests were performed on MCF7 and MDA-MB-231 breast cancer cells. Results: Liposomes had a loading capacity of up to 1.75% (molar drug-to-lipid ratio). In vitro tests showed increased viability and accumulation of the liposomal formulation compared with free drug as well as lack of cytotoxicity in hepatocytes. Conclusion: This optimized technique for encapsulating large copper complexes in liposomes could be used to improve their delivery and better treat cancer and bacterial infections.

This work introduces a new technique for copper-containing drugs encapsulation in a drug-delivery system. The drug, a promising copper compound, is embedded in lipid nanovesicles ­ tiny fat particles ­ for intravenous injection. In addition to chemical characterization of the obtained drug form, tests on cancer cells showed a noticeable effect, whereas healthy cell types were not harmed. Copper possesses not only anticancer effects but also antimicrobial properties, which are also shown by the drug form, and a test of combined suppression of cancer cell lines and bacteria was successful. Hence, the obtained drug form has the potential for dual treatment of cancer and bacterial infections.

New Titanocene (IV) Dicarboxylates with Potential Cytotoxicity: Synthesis, Structure, Stability and Electrochemistry.

The search for new anticancer drugs based on biogenic metals, which have weaker side effects compared to platinum-based drugs, remains an urgent task in medicinal chemistry. Titanocene dichloride, a coordination compound of fully biocompatible titanium, has failed in pre-clinical trials but continues to attract the attention of researchers as a structural framework for the development of new cytotoxic compounds. In this study, a series of titanocene (IV) carboxylate complexes, both new and those known from the literature, was synthesized, and their structures were confirmed by a complex of physicochemical methods and X-ray diffraction analysis (including one previously unknown structure based on perfluorinated benzoic acid). The comprehensive comparison of three approaches for the synthesis of titanocene derivatives known from the literature (the nucleophilic substitution of chloride anions of titanocene dichloride with sodium and silver salts of carboxylic acids as well as the reaction of dimethyltitanocene with carboxylic acids themselves) made it possible to optimize these methods to obtain higher yields of individual target compounds, generalize the advantages and disadvantages of these techniques, and determine the substrate frames of each method. The redox potentials of all obtained titanocene derivatives were determined by cyclic voltammetry. The relationship between the structure of ligands, the reduction potentials of titanocene (IV), and their relative stability in redox processes, as obtained in this work, can be used for the design and synthesis of new effective cytotoxic titanocene complexes. The study of the stability of the carboxylate-containing derivatives of titanocene obtained in the work in aqueous media showed that they were more resistant to hydrolysis than titanocene dichloride. Preliminary tests of the cytotoxicity of the synthesised titanocene dicarboxilates on MCF7 and MCF7-10A cell lines demonstrated an IC50 ≥ 100 µM for all the obtained compounds.

Copper coordination compounds with (5Z,5Z')-2,2'-(alkane-α,ω-diyldiselenyl)-bis-5-(2-pyridylmethylene)-3,5-dihydro-4H-imidazol-4-ones. Comparison with sulfur analogue.

A series of new organic ligands (5Z,5Z')-2,2'-(alkane-α,ω-diyldiselenyl)-bis-5-(2-pyridylmethylene)-3,5-dihydro-4H-imidazol-4-ones (L) consisting of two 5-(2-pyridylmethylene)-3,5-dihydro-4H-imidazol-4-one units linked with polymethylene chains of various lengths (n = 2-10, where n is the number of CH2 units) have been synthesized. The reactions of these ligands with CuCl2·2H2O and CuClO4·6H2O gave Cu2+ or Cu1+ containing mono- and binuclear complexes with Cu2LCl x (x = 2-4) or CuL(ClO4) y (y = 1, 2) composition. It was shown that the agents reducing Cu2+ to Cu1+ in the course of complex formation can be both a ligand and an organic solvent in which the reaction is carried out. This fundamentally distinguishes the selenium-containing ligands L from their previously described sulfur analogs, which by themselves are not capable of reducing Cu2+ during complexation under the same conditions. A higher cytotoxicity and reasonable selectivity to cancer cell lines for synthesized complexes of selenium-containing ligands was shown; unlike sulfur analogs, ligands L themselves demonstrate a high cytotoxicity, comparable in some cases to the toxicity of copper-containing complexes.

Copper-Containing Nanoparticles and Organic Complexes: Metal Reduction Triggers Rapid Cell Death via Oxidative Burst.

Copper-containing agents are promising antitumor pharmaceuticals due to the ability of the metal ion to react with biomolecules. In the current study, we demonstrate that inorganic Cu2+ in the form of oxide nanoparticles (NPs) or salts, as well as Cu ions in the context of organic complexes (oxidation states +1, +1.5 and +2), acquire significant cytotoxic potency (2-3 orders of magnitude determined by IC50 values) in combinations with N-acetylcysteine (NAC), cysteine, or ascorbate. In contrast, other divalent cations (Zn, Fe, Mo, and Co) evoked no cytotoxicity with these combinations. CuO NPs (0.1-1 µg/mL) together with 1 mM NAC triggered the formation of reactive oxygen species (ROS) within 2-6 h concomitantly with perturbation of the plasma membrane and caspase-independent cell death. Furthermore, NAC potently sensitized HCT116 colon carcinoma cells to Cu-organic complexes in which the metal ion coordinated with 5-(2-pyridylmethylene)-2-methylthio-imidazol-4-one or was present in the coordination sphere of the porphyrin macrocycle. The sensitization effect was detectable in a panel of mammalian tumor cell lines including the sublines with the determinants of chemotherapeutic drug resistance. The components of the combination were non-toxic if added separately. Electrochemical studies revealed that Cu cations underwent a stepwise reduction in the presence of NAC or ascorbate. This mechanism explains differential efficacy of individual Cu-organic compounds in cell sensitization depending on the availability of Cu ions for reduction. In the presence of oxygen, Cu+1 complexes can generate a superoxide anion in a Fenton-like reaction Cu+1L + O2 → O2-. + Cu+2L, where L is the organic ligand. Studies on artificial lipid membranes showed that NAC interacted with negatively charged phospholipids, an effect that can facilitate the penetration of CuO NPs across the membranes. Thus, electrochemical modification of Cu ions and subsequent ROS generation, as well as direct interaction with membranes, represent the mechanisms of irreversible membrane damage and cell death in response to metal reduction in inorganic and organic Cu-containing compounds.

Novel Copper-Containing Cytotoxic Agents Based on 2-Thioxoimidazolones.

A series of 73 ligands and 73 of their Cu+2 and Cu+1 copper complexes with different geometries, oxidation states of the metal, and redox activities were synthesized and characterized. The aim of the study was to establish the structure-activity relationship within a series of analogues with different substituents at the N(3) position, which govern the redox potentials of the Cu+2/Cu+1 redox couples, ROS generation ability, and intracellular accumulation. Possible cytotoxicity mechanisms, such as DNA damage, DNA intercalation, telomerase inhibition, and apoptosis induction, have been investigated. ROS formation in MCF-7 cells and three-dimensional (3D) spheroids was proven using the Pt-nanoelectrode. Drug accumulation and ROS formation at 40-60 µm spheroid depths were found to be the key factors for the drug efficacy in the 3D tumor model, governed by the Cu+2/Cu+1 redox potential. A nontoxic in vivo single-dose evaluation for two binuclear mixed-valence Cu+1/Cu+2 redox-active coordination compounds, 72k and 61k, was conducted.