Casiopeinas of Third Generations: Synthesis, Characterization, Cytotoxic Activity and Structure-Activity Relationships of Mixed Chelate Compounds with Bioactive Secondary Ligands.

Casiopeinas are a family of copper(II) coordination compounds that have shown an important antineoplastic effect and low toxicity in normal cells. These compounds induce death cells by apoptosis through a catalytic redox process with endogenous reducing agents. Further studies included a structural variation, improving the activity and selectivity in cancer cells or other targets. In the present work we report the third generation, which contains a bioactive monocharged secondary ligand, as well as the design, synthesis, characterization and antiproliferative activity, of sixteen new copper(II) coordination compounds with curcumin or dimethoxycurcumin as secondary ligands. All compounds were characterized by elemental analysis, FTIR, UV-Vis, magnetic susceptibility, mass spectra with MALDI-flight time, cyclic voltammetry, electron paramagnetic resonance (EPR) spectroscopy and X-ray diffraction. Crystallization of two complexes was achieved in dimethylsulfoxide (DMSO) with polar solvent, and crystal data demonstrated that a square-based or square-base pyramid geometry are possible. A 1:1:1 stoichiometry (diimine: copper: curcuminoid) ratio and the possibility of a nitrate ion as a counterion were supported. 1H, 13C NMR spectra were used for the ligands. A sulforhodamine B assay was used to evaluate the cytotoxicity effect against two human cancer cell lines, SKLU-1 and HeLa. Electronic descriptors and redox potential were obtained by DFT calculations. Structure-activity relationships are strongly determined by the redox potential (E1/2) of copper(II) and molar volume (V) of the complexes. These compounds can be used as a template to open a wide field of research both experimentally and theoretically.

The mitochondrial apoptotic pathway is induced by Cu(II) antineoplastic compounds (Casiopeínas®) in SK-N-SH neuroblastoma cells after short exposure times.

The family of Copper(II) coordination compounds Casiopeínas® (Cas) has shown antiproliferative activity in several tumour lines by oxidative cellular damage and mitochondrial dysfunction that lead to cell death through apoptotic pathways. The goal of this work is looking for the functional mechanism of CasIIgly, CasIIIia and CasIIIEa in neuroblastoma metastatic cell line SK-N-SH, a paediatric extra-cranial tumour which is refractory to several anti-carcinogenic agents. All Cas have shown higher antiproliferative activity than cisplatin (IC50 = 123 µM) with IC50 values of 18, 22 and 63 µM for CasIIgly, CasIIIEa and CasIIIia, respectively. At low concentrations and early times (4 h), these compounds cause a disruption of the mitochondrial transmembrane potential (Δψm). Concomitantly, an important depletion of intracellular glutathione and an increase of reactive oxygen species (ROS) hydrogen peroxide and radical superoxide were observed. On the other side, the lower cytotoxic effect of Casiopeínas on cultures of human peripheral blood lymphocytes (IC50CasIIgly  = 1720 µM, IC50 CasIIIEa  = 3860 µM and IC50 CasIIIia  = 4700 µM) show the selectivity of these compounds over the tumour cells compared with the non-transformed cells. Chemically, glutathione (GSH) interacts with Casiopeínas® through the coordination of sulphur atom to the metal centre, process which facilitates the electron transfer to get Cu(I), GSSG and the posterior production of ROS. Additionally, the molecular structure of CasIIIia as nitrate is reported. These results have shown that the anticarcinogenic activity of Casiopeínas® on neuroblastoma SK-N-SH is through mitochondrial apoptosis due to the enhanced pro-oxidant environment promoted by the presence of the coordination copper compounds.

Antineoplastic copper coordinated complexes (Casiopeinas) uncouple oxidative phosphorylation and induce mitochondrial permeability transition in cardiac mitochondria and cardiomyocytes.

Copper-based drugs, Casiopeinas (Cas), exhibit antiproliferative and antineoplastic activities in vitro and in vivo, respectively. Unfortunately, the clinical use of these novel chemotherapeutics could be limited by the development of dose-dependent cardiotoxicity. In addition, the molecular mechanisms underlying Cas cardiotoxicity and anticancer activity are not completely understood. Here, we explore the potential impact of Cas on the cardiac mitochondria energetics as the molecular mechanisms underlying Cas-induced cardiotoxicity. To explore the properties on mitochondrial metabolism, we determined Cas effects on respiration, membrane potential, membrane permeability, and redox state in isolated cardiac mitochondria. The effect of Cas on the mitochondrial membrane potential (Δψm) was also evaluated in isolated cardiomyocytes by confocal microscopy and flow cytometry. Cas IIIEa, IIgly, and IIIia predominately inhibited maximal NADH- and succinate-linked mitochondrial respiration, increased the state-4 respiration rate and reduced membrane potential, suggesting that Cas also act as mitochondrial uncouplers. Interestingly, cyclosporine A inhibited Cas-induced mitochondrial depolarization, suggesting the involvement of mitochondrial permeability transition pore (mPTP). Similarly to isolated mitochondria, in isolated cardiomyocytes, Cas treatment decreased the Δψm and cyclosporine A treatment prevented mitochondrial depolarization. The production of H2O2 increased in Cas-treated mitochondria, which might also increase the oxidation of mitochondrial proteins such as adenine nucleotide translocase. In accordance, an antioxidant scavenger (Tiron) significantly diminished Cas IIIia mitochondrial depolarization. Cas induces a prominent loss of membrane potential, associated with alterations in redox state, which increases mPTP opening, potentially due to thiol-dependent modifications of the pore, suggesting that direct or indirect inhibition of mPTP opening might reduce Cas-induced cardiotoxicity.

Doble synergetic anticancer activity through a combined chemo-photodynamic therapy and bioimaging of a novel Cas-ZnONPs all-in-one system.

A strategy for cancer treatment was implemented, based on chemo-photodynamic therapy, utilizing a novel formulation, low-cost system called Cas-ZnONPs. This system consisted of the incorporation of Casiopeina III-ia (CasIII-ia), a hydrophilic copper coordination compound with well-documented anti-neoplastic activity, on Zinc oxide nanoparticles (ZnONPs) with apoptotic activity and lipophilicity, allowing them to permeate biological barriers. Additionally, ZnONPs exhibited fluorescence, with emission at different wavelengths depending on their agglomeration and enabling real-time tracking biodistribution. Also, ZnONPs served as a sensitizer, generating reactive oxygen species (ROS) in situ. In in vitro studies on HeLa and MDA-MB-231 cell lines, a synergistic effect was observed with the impregnated CasIII-ia on ZnONPs. The anticancer activity had an increase in cellular inhibition, depending on the dose of exposure to UV-vis irradiation. In in vivo studies utilized zebrafish models for xenotransplanting stained MDA-MB-231 cells and testing the effectiveness of Cas-ZnONPs treatment. The treatment successfully eliminated cancer cells, both when combined with Photodynamic Therapy (PDT) and when used alone. However, a significantly higher concentration (50 times) of Cas-ZnONPs was required in the absence of PDT. This demonstrates the potential of Cas-ZnONPs in cancer treatment, especially when combined with PDT.

(Acetylacetonato-κ2O,O')(2,2'-bipyridine-κ2N,N')iodidocopper(II): a compound with two molecules in the asymmetric unit due to different π-π interactions.

The crystal structure of the title complex, [Cu(C5H7O2)I(C10H8N2)], in the space group P1 with Z = 4, is stabilized by π-π interactions and weak C-H···I interactions. The presence of two molecules in the asymmetric unit is associated with different intermolecular π-π interactions between two symmetry-related molecules of each type.

Antimicrobial effect of Casiopeinas® copper- and ruthenium-based compounds on Aggregatibacter actinomycetemcomitans and in vitro cell viability onto osteoblasts cells.

OBJECTIVES: The present study aims to evaluate the antimicrobial property of Casiopeinas® copper- and ruthenium-based compounds against Aggregatibacter actinomycetemcomitans serotype b (ATCC® 43,718™), as well as the cytotoxicity on an osteoblasts cell line of both compounds. MATERIAL AND METHODS: The antibacterial effect of the copper-based compounds (CasII-gly, CasIII-ia) and the ruthenium-based compound (RuN-6) at four different concentrations was evaluated as the inhibition ratio of the bacterial growth after 48 h under anaerobic conditions, and the cell viability was measured through resazurin assay. RESULTS: The copper- and ruthenium-based compounds used for this assay were (CasII-gly, CasIII-ia, and RuN-6), showing inhibitory activity between 39 and 62% compared to the antibiotic employed as control 66%. Cell viability was established between 61 and 96%. CONCLUSIONS: Casiopeinas® and ruthenium showed dose and time dependent, inhibitory activity on A. actinomycetemcomitans, and low toxicity on cells (osteoblast) underexposure. The compound CasII-gly showed the best antimicrobial effect, and it could be considered a possible antimicrobial agent in periodontal therapy.

DNA, a target of mixed chelate copper(II) compounds (Casiopeinas®) studied by electrophoresis, UV-vis and circular dichroism techniques.

Casiopeinas are a family of mixed chelate copper(II) complexes with antiproliferative and antineoplastic activities, results that have positioned them as an alternative for cancer treatment. Because DNA is one of their principal targets, it is of our interest to find out the effect of substituents on the diiamine ligands over mode of interaction. Therefore, we studied 21 Casiopeinas upon DNA by gel electrophoresis, UV-vis and circular dichroism (CD) spectroscopic techniques, previously studied by DFT calculations and Quantitative Structure-Activity Relationship (QSAR). According to electrophoresis results, the interaction modes between Casiopeinas with DNA may be through the intercalation or in the minor groove. UV-vis spectroscopy showed a hypochromic or hyperchromic effect as a consequence of each interaction. The analysis suggests the binding along the minor groove and intercalation are both influenced by the substituents in the diimine ligands and depend on the nature of the secondary moiety (acetylacetonate or glycinate). Additionally, a new band in electrophoresis and CD spectra suggests adducts formation. In general, we prove that molecules with the highest molecular weight, electron donating substituents and glycinate as secondary ligand are intercalating agents; unlike molecules with electron withdrawing substituents as chloride or nitro and acetylacetonate as secondary ligand which interact in the minor groove.

1H -NMR Metabolomics Study of the Effect of Cisplatin and Casiopeina IIgly on MDA-MB-231 Breast Tumor Cells.

The knowledge of the metabolic processes of designed metallodrugs for cancer treatment is an area that has been not profoundly studied. Casiopeina IIgly (CasIIgly), which belongs to the Casiopeínas® family, is a copper (II) coordination compound that has shown good biological activity against several cancer cells, low toxicity in normal cells, and antineoplastic activity in in vivo murine and xenografted models. In this work we employed a triple-negative highly metastatic breast carcinoma line (MDA-MB-231), which is one of the cancer types with a great mortality index, for 1H-NMR metabolomic analysis using cisplatin and CasIIgly, in order to quantify the effect of metallodrugs in the metabolic profile of this cell tumor line as a consequence of treatment at different times. Our findings indicate that cisplatin mainly contributes to phospholipid biosynthesis while CasIIgly affects processes such as carbohydrates and nucleotides metabolism. Also, we observed that CasIIgly treatment has an important and fast effect over MDA-MB-231 cell metabolism, which makes it a good alternative for treatment in this type of cancer.

Autophagy Promotes Survival of CHP-212 Neuroblastoma Cells Treated With Casiopeínas®.

BACKGROUND: Neuroblastoma is the main solid extracranial tumor of childhood. The amplification of N-myc oncogene (MYCN) and 1p deletion are the main molecular alterations. These features are what make treatment impossible, especially in high-risk patients with metastases. MATERIALS AND METHODS: Our study investigated the processes undergone by CHP-212 neuroblastoma cells, after being treated with Casiopeínas® (Cas) IIgly, IIIEa, and IIIia for 2, 10, and 24 h. RESULTS: At 2 h, all the treatments Ied to apoptosis [defined by the presence of B-cell lymphoma 2 (BCL2), BCL2-associated X protein, cytochrome c, and caspase-3]. In addition, autophagy with specific molecules beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I (ratio >1). Later at 10 h, autophagy-associated proteins were observed, and at 24 h, only survival proteins nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), and extracellular signal-regulated kinases (ERK)2/ERK1>1 were found. Another relevant finding was the presence of caspase-10 throughout the study, especially in cells treated with CasIIgly and CasIIIEa. CONCLUSION: These relationships indicate a possible mechanism of action of Casiopeínas on neuroblastoma.

Genotoxic assessment of the copper chelated compounds Casiopeinas: Clues about their mechanisms of action.

Casiopeinas is the generic name of a group of copper chelated complexes designed to be used as antineoplastic. Some of these compounds have shown promising results, and in fact, one of them named Casiopeina III-ia has completed preclinical trials and is ready to start clinical phase I in Mexico. As part of the tests that have to be done to every molecule intended to be used in humans, bacterial assays are required because of their sensitivity, speed and reproducibility and among them, Ames test and the SOS Chromotest are widely used to evaluate DNA damage. With the aim to contribute to complete safety information related to genotoxicity and support the hypothesis about their mode of action, four different Casiopeinas (Cas II-gly, Cas III-Ea, Cas III-ia and Cas III-Ha) were tested for genotoxicity with these assays, as well as differential cytotoxicity upon Escherichia coli mutants defectives in some DNA repair mechanisms. However, although it is well known that these molecules produce DNA breakage, the results of the Chromotest and Ames test were negative. Despite this is controversial, a possible explanation is that there is a direct interaction between DNA and the Casiopeinas tested.

Casiopeina III-Ea, a copper-containing small molecule, inhibits the in vitro growth of primitive hematopoietic cells from chronic myeloid leukemia.

Several novel compounds have been developed for the treatment of different types of leukemia. In the present study, we have assessed the in vitro effects of Casiopeina III-Ea, a copper-containing small molecule, on cells from patients with Chronic Myeloid Leukemia (CML). We included primary CD34+ Lineage-negative (Lin-) cells selected from CML bone marrow, as well as the K562 and MEG01 cell lines. Bone marrow cells obtained from normal individuals - both total mononuclear cells as well as CD34+ Lin- cells- were used as controls. IC50 corresponded to 0.5µM for K562 cells, 0.63µM for MEG01 cells, 0.38µM for CML CD34+ lin- cells, and 1.0µM for normal CD34+ lin- cells. Proliferation and expansion were also inhibited to significantly higher extents in cultures of CML cells as compared to their normal counterparts. All these effects seemed to occur via a bcr-abl transcription-independent mechanism that involved a delay in cell division, an increase in cell death, generation of Reactive Oxygen Species and changes in cell cycle. Our results demonstrate that Casiopeina III-Ea possesses strong antileukemic activity in vitro, and warrant further preclinical (animal) studies to assess such effects in vivo.

Pharmacokinetics and hematotoxicity of a novel copper-based anticancer agent: casiopeina III-Ea, after a single intravenous dose in rats.

Casiopeina III-Ea is a mixed chelate copper (II) complex that has shown cytotoxic and antitumor activity in vitro and in vivo. The aim of this study was to investigate the pharmacokinetics of total copper derived from casiopeina III-Ea administered by intravenous bolus injection to Wistar rats. Other objective was to evaluate the hematotoxicity produced by this compound in those animals. Wistar rats received a single intravenous dose of 4 mg/kg of casiopeina III-Ea. Blood samples were taken and pharmacokinetics evaluated. Furthermore, erythrocyte copper levels were determined to identify a potential target and Zn levels were analyzed to determine a possible change. For the evaluation of hematotoxicity, both blood and urine samples were collected for hematological and biochemical analyses; moreover, Fe determination was performed. Blood copper and zinc levels, red blood cell copper levels as well as copper, zinc, and iron amounts excreted into urine were analyzed by ICP-MS. The blood concentration-time profile of copper derived from casiopeina III-Ea was fitted to a two-compartment model with a zero-order input. Cumulative amounts of Cu, Zn, and Fe excreted into rat urine after administration of casiopeina III-Ea were different with respect to control. Hematological and biochemical data indicated a hemolytic toxicity. Pharmacokinetic analysis of total copper derived from casiopeina III-Ea provided a general knowledge about distribution and elimination process of this compound. Additionally, the systemic exposure of the copper derived from casiopeina III-Ea accounts for the hematotoxicity of this complex at test dose.

Copper(II) mixed chelate compounds induce apoptosis through reactive oxygen species in neuroblastoma cell line CHP-212.

In the present work we report the antiproliferative activity of Cu(II) coordination compounds, CasIIgly ([Cu(4,7-dimethyl-1,10-phenanthroline) (glycinato) (H2O)]NO3), CasIIIia ([Cu(4,4'-dimethyl-2,2'-bipyridine) (glycinato) (H2O)]NO3), and CasIIIEa ([Cu(4,7-dimethyl-1,10-phenanthroline) (acetylacetonato) (H2O)]NO3), against human tumoral cell line CHP-212 (estromal neuroblastoma). Additionally, the molecular structure of CasIIIEa was reported. The IC50 values obtained for the evaluated compounds are in the range 18 to 47 µM, representing an inhibition potency increase of 5 to 12 times compared with cisplatin (IC50=226.7 µM). After 2h of incubation with the evaluated compounds, cells showed high levels of reactive oxygen species and a considerable GSH depletion, besides an important disruption of the mitochondrial membrane with release of cytochrome C and besides the presence of caspase-3, an effector caspase that is activated in the last step of apoptosis cascade. The results confirm that cell death in neuroblastoma CHP-212 treated with Casiopeínas occurs via apoptosis. Due to the lack of expression of caspase-8, cell death is principally by the mitochondrial pathway. Thus, one of the most interesting findings of this work is the identification of a very important damage in neuroblastoma cells induced by Cu(II) coordination compounds in a very short exposition times.