The potential of phenothiazinium dyes as cytotoxicity markers in cisplatin-treated cells.

Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications.

Gold(III) complexes with thiosemicarbazonate ligands as potential anticancer agents: Cytotoxicity and interactions with biomolecular targets.

Gold(III) complexes have been studied for the past years due to their anticancer properties and great affinity to biotargets, such as enzymes and proteins, which support their pharmacological applications. Within this scope, in this work the antiproliferative activities of two Au(III)-thiosemicarbazonate complexes, [AuClL1] (1, L1: (E,Z)-N-ethyl-N'-(3-nitroso-kN)butan-2-ylidene)carbamohydrazonothioato-k2N2,S) and [Au(Hdamp)L2]Cl (2, L2: N-(N'',N''-diethylaminothiocarbonyl)-N'(N''', N'''-dimethylcarbothioamide)benzamidineto-kN,k2S and Hdamp: 2-(N,N-dimethylaminomethyl)-phenyl-C1), and their affinities to possible biological targets were investigated. Three different tumor cell lines were used to perform the cytotoxicity assays, including one cisplatin-resistant model, and the results showed lower EC50 for 1 over 2 in every case: B16F10 (4.1 µM and 15.6 µM), A431 (4.0 µM and >50 µM) and OVCAR3 (4.2 µM and 24.5 µM). However, a lower toxicity to fibroblast 3T3 cell line was observed for 2 (30.58 µM) when compared to 1 (7.17 µM), resulting in comparable therapeutic indexes. Both complexes presented strong affinity to HSA: they distorted the secondary structure of the protein, as verified by circular dichroism, but 1 additionally presented the apparent fluorescence quenching constant (Kapp) ten times greater than 2, which was probably due to the fact of 1 being able to denature HSA. The ethidium bromide displacement assay showed that neither 1 nor 2 are strong DNA intercalators, which is in agreement with what was observed through the UV-vis titration. In both cases, the 260 nm band presented hyperchromism, which can indicate ionic interactions or DNA damage. In fact, 1 was able to damage the pGEM plasmid, similarly to cisplatin, as verified by agarose gel electrophoresis and Atomic Force Microscopy. Biophysical studies in cancer cells model membranes were also performed in order to investigate the interaction of the gold complexes to lipid bilayers and revealed that the compounds interact with the membranes by exhibiting partition coefficients of 103 order of magnitude. Overall, both complexes were found to be promising candidates for the development of a future anticancer drug against low sensitive or cisplatin resistant tumors.

Anticancer and antitrypanosomal activities of trinuclear ruthenium compounds with orthometalated phenazine ligands.

Trinuclear ruthenium complexes with orthometalated phenazines of general formula [Ru3(µ3-O)(µ2-OAc)5(L)(py)2]PF6 (L = dppn, benzo[i]dipyrido[3,2-a:2',3'-c]phenazine, 1; dppz, dipyrido[3,2-a:2',3'-c]phenazine, 2; CH3-dppz, 7-methyldipyrido[3,2-a:2',3'-c]phenazine, 3; Cl-dppz, 7-chlorodipyrido[3,2-a:2',3'-c]phenazine, 4) were investigated for their cytotoxic activity toward the B16F10 murine melanoma and the L929 non-cancer cell lines and against Trypanosoma cruzi (2-4). This study also reports a multi-technique investigation into how complexes 1-4 interact with DNA and human serum albumin, HSA. At concentrations ranging from 2 to 50 µM, all the complexes reduced B16F10 murine melanoma cell viability by over 50%. Complex 4 had the highest cytotoxic effect in the series, diminishing B16F10 cell viability to 38% at 2 µM, with an overall order for anticancer activity of 4 > 2 > 3 > 1. Complexes 2-4 showed remarkable activity in inhibiting epimastigote and amastigote forms of T. cruzi. Complex 2 showed better antitrypanosomal activity than the reference drug (IC50 = 1.19 µM and IC50 = 0.25 µM for epimastigote and amastigotes forms, respectivily). Ethidium bromide (EB) displacement assays showed that DNA intercalation progressively increases with the extension of the π-conjugation of the cyclometalating ligand and the presence of substituents in the phenazinic portion (1 > 4-3 > 2), showing that complex 1 is a stronger intercalator than EB itself (Kapp > 107 M-1). Viscosity measurements followed the same trend. Cytotoxicity against cancer cells and antitrypanosomal activity follow the same order, which is different to the tendency of DNA intercalation, suggesting DNA is not the main target of these complexes. Compound 1-4 showed very high affinity with HSA (Kb ∼109 M-1). Circular dichroism results also showed that the complexes alter significantly the secondary structure of the HSA, lowering the α-helix % from 86.2 (pure protein) to less than 5% for compounds 1, 2 and 4 at 2.8 µM. These findings demonstrated the important role of phenazines for the biological activity of triruthenium compounds.

Organometallic Gold(III) Complex [Au(Hdamp)(L14)]+ (L1 = SNS-Donating Thiosemicarbazone) as a Candidate to New Formulations against Chagas Disease.

Chagas disease remains a serious public health concern with unsatisfactory treatment outcomes due to strain-specific drug resistance and various side effects. To identify new therapeutic drugs against Trypanosoma cruzi, we evaluated both the in vitro and in vivo activity of the organometallic gold(III) complex [Au(III)(Hdamp)(L14)]Cl (L1 = SNS-donating thiosemicarbazone), henceforth denoted 4-Cl. Our results demonstrated that 4-Cl was more effective than benznidazole (Bz) in eliminating both the extracellular trypomastigote and intracellular amastigote forms of the parasite without cytotoxic effects on mammalian cells. In in vivo assays, 4-Cl in PBS solution loses the protonation and becomes the 4-neutral. 4-Neutral reduced parasitaemia and tissue parasitism in addition to protecting the liver and heart from tissue damage at 2.8 mg/kg/day. All these changes resulted in the survival of 100% of the mice treated with the gold complex during the acute phase. Analyzing the surviving animals of the acute infection, the parasite load after 150 days of infection was equivalent to those treated with the standard dose of Bz without demonstrating the hepatotoxicity of the latter. In addition, we identified a modulation of interferon gamma (IFN-γ) levels that may be targeting the disease's positive outcome. To the best of our knowledge, this is the first gold organometallic study that shows promise in an in vivo experimental model against Chagas disease.

The role of ancillary ligand substituents in the biological activity of triruthenium-NO complexes.

Two novel triruthenium clusters, [Ru3(µ3-O)(µ-OOCCH3)6(NO)L2]PF6 (L = 4­acetylpyridine, 1, or 4­tert­butylpyridine, 2) release NO. Their spectroscopic and electrochemical characterization confirmed their structure. These complexes efficiently deliver NO in solution under irradiation at λirrad = 377 nm and/or through chemical reduction with ascorbic acid. Clusters 1 and 2 elicit vasodilation and, at concentrations of 10-5 M, can relax up to 100% of pre-contracted rat aorta. Complex 2 is more cytotoxic to murine melanoma B16F10 cells than complex 1: at 50 times lower concentration than complex 1, complex 2 decreases cell viability to 50% in the dark or under irradiation with visible light (λirrad = 527 nm). The higher cytotoxicity of complex 2 can be assigned to its larger hydrophobicity, promoted by the methylated tert­butylpyridine ancillary ligand in its structure. Investigation into human serum albumin (HSA) fluorescence quenching by clusters 1 or 2 revealed that complex 2 quenches HSA luminescence with a very high Stern-Vomer constant (KSV = 9.49 × 107 M-1 at T = 298 K) and suggested that the nature of the interaction between complex 2 and HSA is hydrophobic (ΔH = 80.81 kJ/mol and ΔS = 334.71 J/K mol). HSA lifetime and circular dichroism data pointed to a static quenching mechanism for both complexes. Together, our results show that a hydrophobic substituent in the cluster ancillary ligand improves NO release ability, cytotoxicity, and interaction with a bio-target.

New uses for old complexes: The very first report on the trypanocidal activity of symmetric trinuclear ruthenium complexes.

This work reports on the trypanocidal activity of a series of symmetric triruthenium complexes combined with azanaphthalene ligands of general formula [Ru3O(CH3COO)6(L)3]PF6 (L=(1) quinazoline (qui), (2) 5-nitroisoquinoline (5-nitroiq), (3) 5-bromoisoquinoline (5-briq), (4) isoquinoline (iq), (5) 5-aminoisoquinoline (5-amiq), and (6) 5,6,7,8-tetrahydroisoquinoline (thiq)). All complexes within the series presented in vitro trypanocidal activity against both the trypomastigote and amastigote forms of T. cruzi. The IC50 values obtained for complexes 1-6 ranged from 1.39 to 165.9µM for the trypomastigote form and from 1.06 to 53.16µM for the amastigote form. These values were lower than the values observed for the metallic core [Ru3O(CH3COO)6(CH3OH)3]+ itself and for the free ligands in all cases. Remarkably, complex 6 displayed lower IC50 values than the reference drug (benznidazole) for the acute (trypomastigote form) and chronic (amastigote form) phases of Chagas disease. These findings, combined with the low toxicity against healthy cells (LLK-MK2 strain) and a high SI value (Selectivity Index >10) make complex 6 an excellent candidate for in vivo tests.

An extended π-system and enhanced electronic delocalization on symmetric [Ru3O(CH3COO)6(L)3]n complexes combined with azanaphthalene ligands.

We report on the investigation of a new series of symmetric trinuclear ruthenium complexes combined with azanaphthalene ligands: [Ru3O(CH3COO)6(L)3]PF6 where L = (1) quinazoline (qui), (2) 5-nitroisoquinoline (5-nitroiq), (3) 5-bromoisoquinoline (5-briq), (4) isoquinoline (iq), (5) 5-aminoisoquinoline (5-amiq), and (6) 5,6,7,8-tetrahydroisoquinoline (thiq). The crystal structure of complex 1, [Ru3O(CH3COO)6(qui)3]PF6, was determined by X-ray diffraction analysis, showing a high degree of co-planarity between the [Ru3O] plane and the azanaphthalene ligands. Spectroscopic (UV-visible, NMR and infra-red) and electrochemical (cyclic voltammetry and spectroelectrochemistry) data showed correlation with the pKa values of the azanaphthalene ligands and this dependence was rationalized in terms of the molecular orbital of the [Ru3O] unit and the structure of the ligands. By analysing the spectroscopic and electrochemical correlations, the ability of the azanaphthalene ligands to extend the electronic π-system of the [Ru3O] unit to the periphery of the compounds was demonstrated. This electronic effect accounts for the planarity of the structure of complex 1. It was also shown through molecular modeling results that, to explain the spectroscopic and electrochemical behaviour of these species, it is not possible to neglect the electronic mixing between the metallic and the acetate orbitals. Finally, this work revealed that electronic coupling is more pronounced in the azanaphthalene series of complexes than in pyridinic analogues and it is this coupling that determines the spectroscopic and electrochemical behaviour of the new species.

Precursor Ion Scan Mode-Based Strategy for Fast Screening of Polyether Ionophores by Copper-Induced Gas-Phase Radical Fragmentation Reactions.

The potential of copper(II) to induce gas-phase fragmentation reactions in macrotetrolides, a class of polyether ionophores produced by Streptomyces species, was investigated by accurate-mass electrospray tandem mass spectrometry (ESI-MS/MS). Copper(II)/copper(I) transition directly induced production of diagnostic acylium ions with m/z 199, 185, 181, and 167 from α-cleavages of [macrotetrolides + Cu]2+. A UPLC-ESI-MS/MS methodology based on the precursor ion scan of these acylium ions was developed and successfully used to identify isodinactin (1), trinactin (2), and tetranactin (3) in a crude extract of Streptomyces sp. AMC 23 in the precursor ion scan mode. In addition, copper(II) was also used to induce radical fragmentation reactions in the carboxylic acid polyether ionophore nigericin. The resulting product ions with m/z 755 and 585 helped to identify nigericin in a crude extract of Streptomyces sp. Eucal-26 by means of precursor ion scan experiments, demonstrating that copper-induced fragmentation reactions can potentially identify different classes of polyether ionophores rapidly and selectively.