Development, validation and application of a GC-MS method for the simultaneous detection and quantification of neutral lipid species in Trypanosoma cruzi

The development and validation of an analytical method for the simultaneous analysis of five neutral lipids in Trypanosoma cruzi epimastigotes by GC-MS is presented in this study. The validated method meets all validation parameters for all components and the chromatographic conditions have been optimized during its development. This analytical method has demonstrated good selectivity, accuracy, within-day precision, recovery and linearity in each of the established ranges. In addition, detection and quantification limits for squalene, cholesterol, ergosterol and lanosterol have been improved and it is worth highlighting the fact that this is the first time that squalene-2,3-epoxide validation data have been reported. The new validated method has been applied to epimastigotes treated with compounds with in vitro anti- T.cruzi activity. This new methodology is straightforward and constitutes a tool for screening possible sterol biosynthesis pathway inhibitors in Trypanosoma cruzi, one of the most studied targets in Chagas disease treatment. Therefore, it is an interesting and useful contribution to medicinal chemistry research

Phenazine N,N′-dioxide scaffold as selective hypoxic cytotoxin pharmacophore. Structural modifications looking for further DNA topoisomerase II-inhibition activity

Phenazine-5,10-dioxides have been identified as prodrugs for antitumour therapy that undergo hypoxic-selective bioreduction, in the solid tumour cells, to form cytotoxic species. We investigated structural modifications of the phenazine-5,10-dioxide scaffold attempting to find new selective hypoxic cytotoxins with additional ability to inhibit DNA topoisomerase II. Four series of new phenazine-5,10-dioxides aryl-substituted connected by different linkers were prepared. The clonogenic survivals of V79 cells on aerobic and anaerobic conditions were determined, and studies of oxic DNA-interaction and hypoxic DNA topoisomerase II-inhibition, for the most relevant derivatives, were performed. Four new hypoxic-selective cytotoxins were identified at the assayed doses. In some of them were operative the DNA-interaction and/or the inhibition of DNA topoisomerase II. For one of the unselective cytotoxin biotransformation studies were performed on aerobic and anaerobic conditions, explaining the lack of selectivity

Synthesis and biological evaluation of quinoxaline di-N-oxide derivatives with in vitro trypanocidal activity

Abstract: We report the synthesis and in vitro activity against T. cruzi epimastigotes of 15 novel quinoxaline derivatives. Ten of the derivatives presented IC50 values lower than the reference drugs Nfx and Bzn; four of them standed out with IC50 values lower than 1.5 M. Moreover, unspecific cytotoxicity and genotoxicity studies are also reported. Compound 14 showed a SI higher than 24, whereas compound 10 was the only one that was negative in the genotoxicity screening

New copper-based complexes with quinoxaline 'N POT.1', 'N POT.4'-dioxide derivatives, potential antitumoral agents

Taking into account our previous studies on cytotoxic metal compounds, new copper complexes with 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives as ligands were synthesized and characterized by different spectroscopic methods. The hypoxic selective cytotoxicity towards V79 cells and the superoxide dismutase-like activity of the complexes were determined and related to physicochemical properties of the compounds. In particular, the copper(II) complex with 3-amino-6-chloro-7-fluoroquinoxaline-2-carbonitrile N1,N4-dioxide showed cytotoxic selectivity in hypoxia being the most lipophilic compound of the series. On the contrary, the complex with 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide was cytotoxic but not selective and that with 3-amino-7-chloro-6-methoxy-quinoxaline-2-carbonitrile N1,N4-dioxide was not cytotoxic towards V79 cells neither in oxia nor in hypoxia in the assayed conditions. The σm Hammett substituent electronic descriptor was related to the effect in hypoxic conditions and the SOD-like activity was correlated to the effect in normoxia.Comisión Honoraria de Lucha Contra el Cáncer (CHLCC)Comisión Sectorial de Investigación Científica (CSIC)PEDECIB

Novel quinoxaline 1,4-di-N-oxide derivatives as new potential antichagasic agents

As a continuation of our research and with the aim of obtaining new agents against Chagas disease, an extremely neglected disease which threatens 100 million people, eighteen new quinoxaline 1,4-di-Noxide derivatives have been synthesized following the Beirut reaction. The synthesis of the new derivatives was optimized through the use of a new and more efficient microwave-assisted organic synthetic method. The new derivatives showed excellent in vitro biological activity against Trypanosoma cruzi. Compound 17, which was substituted with fluoro groups at the 6- and 7-positions of the quinoxaline ring, was the most active and selective in the cytotoxicity assay. The electrochemical study showed that the most active compounds, which were substituted by electron-withdrawing groups,possessed a greater ease of reduction of the N-oxide group

Identification of Thioredoxin Glutathione Reductase Inhibitors That Kill Cestode and Trematode Parasites

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites

N1-aryl-2-(trifluoromethyl)benzo[b][1,8]naphthyridin-4(1H)-one as convenient platform to design high photostable and long-lived dyad fluorophore with potential application in live-cell imaging

The design of long-lived and high photostable fluorescent probes with high quantum yields (QYs) is of great significance for biological applications. It favors the monitoring of individual molecular events for extended period of time without side-effect consequences into cell. From a series of N1-aryl-2-(trifluoromethyl)benzo[b][1,8]naphthyridin-4(1H)-ones, we found that their photophysical properties were successfully tuned through the modulation of the intramolecular charge-transfer (ICT), generating the largest lifetimes, highest QYs and photostability for those fluorophores with a high CT character. The ICT was primarily controlled by incorporation of methoxy group at 7-position and secondarily by electron-deficient N1-arene at 1-position of benzo[b] [1,8] naphthyridin-4(1H)-one core. The trifluoromethyl at 2-position seems to strength the ICT because it reduced gap energies and increase dipole moments in their derivatives compared to 2-methyl or -hydrogen analogues. Importantly, from the 7-methoxy derivatives, two high photo-stable (15- to 17-fold more than fluorescein) and long-lived (9.75–14.20 ns) fluorophores with acceptable QYs and HOMO-LUMOs were identified. In particular, the fluorophore 3k showed a low cytotoxicity in J774.1A macrophage, pH insensibility and appropriate lipophilicity, which in combination with their good photophysical properties favored the application of the 7-methoxy dyes for bioimaging, giving blue-fluorescent macrophages in relatively short time. Then, the N1-aryl-2-(trifluoromethyl)benzo[b] [1,8] naphthyridin-4(1H)-one by its molecular extension and planarity emerges as an interesting platform to generate long-lived and highly photostable dyes and, the key point is to favor the strength of the internal ICT mechanism through the incorporation of appropriate donor (D) and acceptor (A) groups along the D-A chain.Fil: Romero, Angel H.. Universidad de la República; UruguayFil: Romero Cordero, Ivan Exehomio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Aguilera, Elena. Universidad de la República; UruguayFil: Cerecetto, Hugo. Universidad de la República; Urugua

A photobasic D-A fluorophore with a high intramolecular charge-transfer as a model strategy for designing organic proton-coupled electron-transfer modulators: An analysis based on steady-state fluorescence, isotopic effect and theoretical study

Modulation of the photoinduced proton-coupled electron transfer (PCET) mechanism is a topic of great interest because of its pivotal role in energy-transfer processes, the development of fuel cells and activation of small molecules. Herein we show that the N1-(4-chlorophenyl)-2-(trifluoromethyl)benzo[b][1,8]naphthyridin-4(1H)-one fluorophore is a convenient platform to promote selectively diverse PCET mechanisms upon phenol binding. The key points for the PCET-probe are: (i) the inclusion of an internal basic moiety into the donor (D)-acceptor (A) chain and (ii) the occurrence of intramolecular charge transfer (ICT) upon excitation. Weak fluorescence quenching and detrimental KIEs were observed when a low-CT fluorophoric analogue was used as a PCET probe. The diverse PCET mechanisms upon phenol binding were recognized from a steady-state spectroscopic study, UV-vis spectroscopy, isotopic effect experiments and theoretical calculations. A combined photoinduced electron transfer and PT-ET was identified for nitrophenols, whereas carbonylphenols promoted an apparent combined PT-ET/CPET mechanism with a quenching response dependent on the carbonyl position in the aryl ring. Alkyl-, methoxyphenols, phenol and some aminophenols involved combined CPET and ET-PT mechanisms and halophenols a CPET mechanism, whereas strongly electron-donating hydroxyl- or N-acetamidophenols and 4-aminophenol involved a weak PCET mechanism. The present investigation opens a new perspective for the rational design of selective and effective photobasic organic modulators of PCET mechanisms based on the same principles as typical metallic complexes: (i) a weak base as a hydrogen catcher and (ii) an internal ICT to enhance the basicity of the PCET modulator.Fil: Romero, Angel H.. Universidad de la Republica; UruguayFil: Romero Cordero, Ivan Exehomio. Universidad Central de Venezuela; Venezuela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Gotopo, Lourdes. Universidad Central de Venezuela; VenezuelaFil: Cabrera, Gustavo. Universidad Central de Venezuela; VenezuelaFil: Cerecetto, Hugo. Universidad de la República; Urugua

Development, validation and application of a GC-MS method for the simultaneous detection and quantification of neutral lipid species in Trypanosoma cruzi

The development and validation of an analytical method for the simultaneous analysis of five neutral lipids in Trypanosoma cruzi epimastigotes by GC-MS is presented in this study. The validated method meets all validation parameters for all components and the chromatographic conditions have been optimized during its development. This analytical method has demonstrated good selectivity, accuracy, within-day precision, recovery and linearity in each of the established ranges. In addition, detection and quantification limits for squalene, cholesterol, ergosterol and lanosterol have been improved and it is worth highlighting the fact that this is the first time that squalene-2,3-epoxide validation data have been reported. The new validated method has been applied to epimastigotes treated with compounds with in vitro anti- T.cruzi activity. This new methodology is straightforward and constitutes a tool for screening possible sterol biosynthesis pathway inhibitors in Trypanosoma cruzi, one of the most studied targets in Chagas disease treatment. Therefore, it is an interesting and useful contribution to medicinal chemistry research