This review summarizes the evolution of our research on anticancer agents, from early efforts on hypoxia-selective cytotoxins to more recent developments in chemoprevention, molecular targeting, and radiopharmacy. Initial studies focused on the design of N-oxide-containing heterocycles as bioreductive-prodrugs activated under tumor hypoxia. While early triazine N-oxides and furoxans showed limited selectivity, these studies underscored the importance of redox-properties in biological activities. This led to the identification of phenazine 5,10-dioxides as a more suitable pharmacophore, affording compounds with improved potency- and hypoxia-selectivity, supported by mechanistic-, physicochemical-, and in vivo studies. Parallel efforts explored metal-based complexes and formulation strategies enhancing bioavailability and therapeutic performance. Alongside these efforts, cancer chemopreventive-agents were investigated, particularly chalcone-derived scaffolds and related hybrids capable of modulating phase I/II enzymes through Nrf2 activation. Additionally, attention has shifted toward targeted- and diagnostic-approaches, including radiopharmaceuticals for hypoxia-imaging and the use of biomolecular recognition systems, such as aptamers, polypeptides, and antibodies, to selectively address tumor-associated biomarkers. These strategies include aptamer-based biotherapeutics for drug delivery and imaging, as well as approaches combining tyrosine kinase receptor targeting with BNCT. Furthermore, bioorthogonal-methodologies have been explored enabling selective in situ activation and targeting. Together, these studies illustrate a multidisciplinary approach integrating chemistry, biology, and pharmacology toward more selective anticancer strategies.

Neurodegenerative diseases affect millions worldwide, posing significant challenges to public health. Oxidative stress and increased caspase-3 activity are key features in the development and progression of neurological disorders leading to neuronal damage and cell loss. As part of our ongoing efforts to identify new molecules with enhanced neuroprotective potential, we have synthesized six novel thiazole-nitrone hybrid compounds, designed as dual-action antiapoptotic and antioxidant agents. The nitrone moiety is known for its spin-trapping ability, which confers antioxidant and neuroprotective properties, while thiazole-containing compounds have been described as antioxidants and potent caspase-3 inhibitors. Our findings demonstrate that these hybrid compounds effectively reduce intracellular reactive oxygen species (ROS) levels and mitigate apoptosis rates in a hippocampal cell line by decreasing active caspase-3 activity, confirming their antioxidant and antiapoptotic properties. Notably, two of the six evaluated compounds exhibited the most promising neuroprotective profiles, including favorable blood-brain barrier permeability. Altogether, these results position the identified molecules as excellent candidates for further structure-based optimization, contributing to the development of more efficient therapeutic strategies against neurodegenerative disorders.

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy -performed in a large and diverse chemolibrary- complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 μM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12-20 μM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7-45 μM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known "garbage in, garbage out" machine learning principle.

Management and disposal of domestic waste in rural locations often face difficulties, posing a potential environmental problem. Solving it implies valuing the relationship between the human being and the environment, to plan specific environmental education actions. The identification of both beliefs and behaviors of the population of Paso Hospital, (rural town in Rivera, Uruguay) regarding environment, is taken as a case study. 184 inhabitants were surveyed, the data was analyzed using the R program, performing chi-square tests, and calculating association coefficients (Cramer's V). It is observed that the assessment of local environmental problems regarding the use of natural resources varies according to the educational level. There is a clear need to carry out deeper research to better understand the relationship between beliefs and behaviors concerning the environment, involving local people, analysis and communication of environmental impacts in the territories.

Implementar procesos participativos para involucrar a diferentes actores sociales vinculados a problemas ambientales locales puede contribuir a mejorar la comprensión de las causas de estos y empoderar a los ciudadanos frente a la toma de decisiones colectivas. La importancia de esta participación ciudadana se refleja en la experiencia llevada a cabo en Tranqueras (Rivera, Uruguay), donde se convocó a distintos actores para efectuar un monitoreo participativo de la calidad de agua del río Tacuarembó. El proceso implicó la realización de talleres para la visualización de problemáticas ambientales y el diseño de un muestreo, colecta participativa y análisis de la calidad del agua. Esta experiencia generó un espacio de intercambio de saberes entre la comunidad y la Universidad que conduce a la reflexión sobre la relevancia de la dimensión epistemológica de los problemas ambientales, la forma en que se llega a su conocimiento y las distintas perspectivas con las que se abordan.

The current investigation comes from concerns expressed by local social actors in the municipality of Vichadero, Rivera, Uruguay and the lack of data on the conditions of the bodies of water in the area. The objective based on the perception of the community, was to identify the main environmental problems of the area, from a socio-environmental diagnosis using participatory methodologies, empowering through the collective production of knowledge from the people involved. This approach was put in dialogue with the results of physical-chemical and mutagenic analysis of the quality of surface water in the micro watershed of the area. This research provides evidence that similar processes could promote the involvement of local actors in monitoring and research processes associated with the environmental management of a locality.

We determined the chemosensitizer effect of phenazine dioxide derivatives to cisplatin and the possible mechanism of action on bladder cancer cells. Anti-proliferative activity of nine phenazine dioxide derivatives in presence or absence of cisplatin was evaluated in two bladder tumor human cells T24 and 253 J and one non tumor cell line V79-4. The sensitizer effect of the combined treatment was determined by chromosomal aberrations and micronucleus test. A possible mechanism of action of the sensitizer compounds as HDACi was also investigated.The phenazine dioxide 2c combined with cisplatin induced a cell cycle arrest on bladder cancer cells and resensitize the invasive and cisplatin resistant 253 J cell line. The HDAC inhibitory activity appears as one of the mechanism of action of the compound. The low toxicity levels against normal cells point out the phenazine dioxide derivative 2c as a very good scaffold for further design of HDACi sensitizer agents.

A new family of imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide with antiproliferative Trypanosoma cruzi properties was identified from a neural network model published by our group. The synthesis and evaluation of this new class of trypanocidal agents are described. These compounds inhibit the growth of Trypanosoma cruzi, comparable with benznidazole or nifurtimox. In vitro assays were performed to study their effects on the growth of the epimastigote form of the Tulahuen 2 strain, as well as the epimastigote and amastigote forms of CL clone B5 of Trypanosoma cruzi. To verify selectivity towards parasite cells, the non-specific cytotoxicity of the most relevant compounds was studied in mammalian cells, i.e. J774 murine macrophages and NCTC clone 929 fibroblasts. Furthermore, these compounds were assayed regarding the inhibition of cruzipain. In vivo studies revealed that one of the compounds, 19, showed interesting trypanocidal activity, and could be a very promising candidate for the treatment of Chagas disease.

The aim of this study was to investigate the capability of phenazine dioxides, recognized bioreductive antitumour agents, as carriers for (99m)Tc in order to generate potential theranostic radiopharmaceuticals towards hypoxic solid tumours. Two different phenazine dioxides were used as ligands for the (99m)Tc-tricarbonyl core in order to prepare the potential radiopharmaceuticals. The main physicochemical and biological properties were evaluated. Biodistribution of the two radiotracers was studied at different time points after intravenous injection in tumour bearing animals. Both compounds were obtained in high yield and radiochemichal purity. They were stable in labelling milieu, in human plasma and in the presence of histidine. Biodistribution studies in mice were characterized by slow blood clearance and persistent liver uptake, results that correlate with the values of lipophilicities and protein binding. Both the complexes showed good tumour uptake, which remained constant during the studied period. Tumour/muscle ratios proved very favourable, comparable to those of FMISO in the same animal model. On the other hand, tumour/blood ratios were low due to high blood uptake. The use of phenazine dioxides as ligands for the preparation of potential (99m)Tc-radiopharmaceuticals towards solid tumours is possible since tumour uptake and retention are promising although high blood and liver uptake are drawbacks worth consideration.

Metabolism of three different agro-pesticides widely used in Uruguay, the insecticides imidacloprid and thiamethoxam and the antiparasite nitroxinil, by bovine ruminal fluid, as supply of anaerobic microorganims, was studied. Complete ruminal fluid was incubated with each of the agrochemicals in different conditions, varying time, nutrients, and nitroethane supplementation as methanogenesis modificator. Only biotransformation was detected for nitroxinil in some of the studied variables. In the optimized condition only one product was generated and the chemical structure of this main metabolite was elucidated using combined spectroscopies evidencing a structural motive unrelated with the products of the corresponding mammal biotransformation results of reduction, and substitution processes. The ruminal generation of the metabolite was confirmed. In order to employ this anaerobic microbial system as potential bioremediator of agrochemical-contaminated soils, the toxicity, against mammal cells, and the mutagenicity, using Ames test, of the product of biotransformation were studied. The lack of toxic effects encouraged us to propose the ruminal system as a plausible system for agrochemicals bioremediation.

In the development of new drugs, it is very important to know the effects these may bring to those who consume them. Drugs which act upon certain diseases must not cause toxic side effects on healthy organs. These toxic side effects can be quite varied, i.e. mutagenicity, clastogenicity, teratogenicity, etc., but undoubtedly the mutagenicity officiate in the selection process, during preclinical testing, to advance in clinical trials. Mutagenic compounds are removed and cannot continue its development. There are preclinical studies of mutagenicity and genotoxicity, ranging from in vitro to in vivo studies. Particularly, Ames test is recommended by ICH as the first input in these studies. Herein, we investigated the mutagenicity of an in-house chemical library of eighty five N-oxide containing heterocycles using Ames test in Salmonella thyphimurium TA 98 with and without S9 activation and the use of neural networks in order to predict this nondesired activity. N-oxide containing heterocycles are especially relevant regarding its pharmacological activities as antitrypanosoma, anti-leishmania, anti-tuberculosis, anti-cancer, chemopreventive, anti-inflammatory, anti-atherogenic, and analgesic agents. In some cases, a relationship was found between the presence of N-oxide and mutagenicity. Specifically, benzofuroxan system seems to be responsible for the mutagenicity of certain agents against Chagas disease and certain anti-inflammatory agents. However other N-oxides, such as furoxans with anti-inflammatory and anti-atherosclerosis activities, seem to lack mutagenicity. In other cases, such as quinoxaline dioxides with anti-parasitic activity, mutagenicity shows to be substituent dependent. Applying CODES neural network two models were defined, one without metabolism and other with metabolism. These models predict the mutagenicity with and without metabolism in an excellent manner.

As a contribution to the identification of the relevant species for biological activity and the understanding of structure–activity relationships of [VIVO(L-2H)(NN)] antitrypanosomal complexes (NN is a bidentate polypyridyl DNA intercalator; L is a tridentate salicylaldehyde semicarbazone derivative), new [VVO2(L-2H)] complexes and [VIVO(L-2H)(NN)] complexes including bipy or dppz (dipyrido[3,2-a: 2′,3′-c]phenazine) co-ligands are prepared and characterized in the solid state and in solution. Their activity is evaluated on Trypanosoma cruzi. The lipophilicity, as structural descriptor related to bioactivity, of the whole [VIVO(L-2H)(NN)] series is determined. Furthermore, the antiproliferative effect of those new compounds showing activity against T. cruzi is evaluated on the genetically related parasite T. brucei with the aim to develop broad spectrum agents. The new [VIVO(L-2H)(dppz)] complexes are about ten to fifteen times more toxic to T. cruzi than the bipy analogues and show quite good in vitro activity on T. brucei brucei. They are shown to interact with DNA, suggesting that this biomolecule may be the parasite target. The stability of the VIVO-complexes in solution is accessed by several techniques. Globally the data suggest that the relevant species for biological activity are the [VIVO(L-2H)(NN)] compounds, their order of activity being dependent on the NN nature, but not much on the substitution on the salicylaldehyde semicarbazone moiety. A parabolic relationship between biological response and lipophilicity (determined as RM=log [(1/Rf)−1] by a TLC method) is obtained. From this correlation an optimum RM value, close to 1.44, was found, which may be used as design guide for future development of antitrypanosomal compounds.

Microorganisms of bovine rumen fluid have been studied as biocatalysts for the reduction of different N-oxide containing aromatic heterocycles, i .e. quinoxaline N 1 ,N 4 -dioxide, phenazine N 5 ,N 10 -dioxide, indazole N 1 -oxide, benzofuroxan and furoxan. In anaerobiosis, the microorganisms biocatalyzed the reduction of some N-oxides, quinoxaline and phenazine dioxides, to yield the corresponding heterocycle while in the case of benzofuroxans the heterocycle-opening, o-nitroaniline was generated as the unique product. Contrarily, the furoxan was not biotransformed in the studied conditions. The indazole N 1 oxide was biotransformed very slowly and incompletely in the corresponding indazole. Only one of the studied indazole N 1 -oxide derivative was completely converted to a new compound, different to the expected reduced indazole. For that, a semi-preparative scale biotransformation was performed and the new product was spectroscopically identified.

Phenazine 5,10 dioxides N- and O-substituted were synthesized using microwave irradiation from the corresponding amino-and hydroxy-derivatives. Attempts to obtain phenazine 5,10-dioxides N- and O-substituted in conventional heating conditions, when it occurred, conducted to the desired products in extremely low yields with very long reaction times. In order to obtain phenazine 5,10-dioxide N- and O-substituted in good yields and lower reaction times, microwave irradiation was studied. In these experiments, we obtained new 2-benzyloxy, 2-benzylamino and 2-sulphonamino phenazine 5,10-dioxide derivatives successfully, in low yields, however, significantly higher than those obtained with conventional heating. Theoretical studies suggest that low nucleophilicity of 2-amino and 2-hydroxy phenazines studied is related to the presence of the moieties N,N-dioxide.

Fenazina 5,10 dioxidos N-e O- substituidos foram sintetizadas utilizando a irradiacao de micro-ondas a partir dos correspondentes amino e hidroxi-derivados. As tentativas de N - ou O -substituicao em condicoes de aquecimento convencionais, quando ocorreu, conduziu a formacao dos produtos desejados em rendimentos extremamente baixos e em tempos de reacao muito longos. A fim de obter as fenazinas 5,10-dioxidos N -ou- O -substituidas em melhores rendimentos e em menor tempo, foi estudada a reacao em condicoes de irradiacao de micro-ondas. Nestes estudos, obteve-se com exito novos derivados 2-benziloxi, benzilamino e 2-sulfonamino phenazine 5,10-dioxidos, em baixos rendimentos, porem, significativamente maiores que aqueles obtidos atraves de aquecimento convencional. Estudos teoricos sugerem que a baixa nucleofilicidade das 2-amino and 2-hidroxifenazinas estudas esteja relacionada a presenca da subunidade N,N -dioxido. DOI: 10.5935/1984-6835.20130083

Micro-organismos do fluido ruminal de bovinos foram estudados como biocatalizadores para a reducao de diferentes heterociclos aromaticos contendo N- oxido, isto e, quinoxalina N 1 , N 4 -dioxido, fenazina N 5 , N 10 -dioxido, indazol N 1 -oxido, benzofuroxano e furoxano. Em anaerobiose, os micro-organismos biocatalisam a reducao de alguns N -oxidos, dioxidos de quinoxalina e fenazina para se obter os heterociclos correspondentes, enquanto no caso do heterociclo benzofuroxano, ocorre abertura do anel, gerando a o- nitroanilina como unico produto. Ao contrario, o furoxano nao foi biotransformado nas condicoes estudadas. O indazol N 1 -oxido foi biotransformado lenta e incompletamente ao indazol correspondente. Excecao foi observada apenas para um dos N 1 -oxidos de indazol, que foi completamente convertido a um composto novo, diferente do indazol reduzido esperado. A biotransformacao em escala semi-preparativa foi realizada e o novo produto obtido e identificado por metodos espectroscopicos. DOI: 10.5935/1984-6835.20130082

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.

The goal of this work was to include an interdisciplinary research-oriented laboratory experiment taken from medicinal chemistry into the undergraduate biochemistry or organic studies. Phenazine 5,10-dioxides with selective cytotoxicity under hypoxia and with relevance in solid tumor treatment were selected to simulate different metabolism states. In this laboratory experiment, metabolism of 7-bromo-2-hydroxyphenazine-5,10-dioxide under simulated hypoxia and normoxia was studied using rat liver cytosol. Differential hypoxic and normoxic metabolism were monitored by thin-layer chromatography. Specific enzyme inhibitors, dicoumarol, ketoconazole, and menadione, were employed to identify the enzymatic system involved. This experiment introduced students to the concept of drug metabolism, activation, and mechanism of action. Furthermore, basic biochemical techniques of enzyme isolation and study were also discussed.

A bioactive-guided investigation of the hydro-ethanolic extract of aerial parts of Aristeguietia glutinosa Lam. resulted in the isolation of two diterpenoids, (+)-15-hydroxy-labd-7-en-17-al (1) and (+)-13,14,15,16-tetranor-labd-7-en-l7,12-olide (2), as the anti- Trypanosoma cruzi active principles. The structures of 1 and 2 were determined by spectroscopic analysis. The hydro-ethanolic extract showed anti- T. cruzi activity (IC 50 = 19.6 μg/mL) whereas the isolated compounds 1 and 2 were near to seven- and one and a half-fold (IC 50 = 3.0 and 15.6 μg/mL), respectively more active than the original extract. Labdene 1, equipotent as the reference compound (Nifurtimox), displayed low hemolytic activity, low toxicity against murine macrophages, and absence of mutagenicity. These results support the vernacular medicinal use of this plant as an anti- T. cruzi agent.