Trypanosoma Cruzi Amastigotes Research Articles

Structural design, synthesis, and anti-Trypanosomatidae profile of new Pyridyl-thiazolidinones

The present work reports the synthesis of a novel series of pyridine-thiazolidinones with anti-Trypanosoma cruzi and leishmanicidal activities (compounds 10–27), derived from 2 or 4-pyridine thiosemicarbazones (1–9). The in vitro assays were performed with Trypanosoma cruzi trypomastigotes and amastigotes, as well as with Leishmania amazonensis promastigotes and amastigotes. The cytotoxicity profile was evaluated using the cell line RAW 264.7. From the 18 pyridine-thiazolidinones, 5 were able to inhibit trypomastigotes. Overall, all compounds inhibited amastigotes, highlighting compounds 15 (0.60 μM), 18 (0.64 μM), 17 (0.81 μM), and 27 (0.89 μM). Compounds 15 and 18 were able to induce parasite cell death through necrosis induction. Analysis by scanning electron microscopy showed that T. cruzi trypomastigotes treated with compounds 15 and 18 induced morphological changes such as shortening, retraction and curvature of the parasite body and leakage of internal content. Regarding the antiparasitic evaluation against Leishmania amazonensis, only compound 27 had a higher selectivity compared to Miltefosine against the amastigote form (IC50 = 5.70 μM). Our results showed that compound 27 presented an antiparasitic activity for both Trypanosoma cruzi and Leishmania amazonensis. After in silico evaluation, it was suggested that the new pyridine-thiazolidinones had an appropriate drug-likeness profile. Our results pointed out a new chemical frame with an anti-Trypanosomatidae profile. The pyridine-thiazolidinones presented here for the first time could be used as a starting point for the development of new antiparasitic agents.

Enzymatic synthesis of amlodipine amides and evaluation of their anti-Trypanosoma cruzi activity.

Advancing with our project about the development of new antiparasitic agents, we have enzymatically synthesized a series of amides derived from amlodipine, a calcium channel blocker used as an antihypertensive drug. Through lipase-catalyzed acylation with different carboxylic acids, nineteen amlodipine derivatives were obtained, eighteen of which were new compounds. To optimize the reaction conditions, the influence of several reaction parameters was analyzed, finding different requisites for aliphatic carboxylic acids and phenylacetic acids. All synthesized compounds were evaluated as antiproliferative agents against Trypanosoma cruzi, the etiological agent of American trypanosomiasis (Chagas' disease). Some of them showed significant activity against the amastigote form of T. cruzi, the clinically relevant form of the parasite. Among synthesized compounds, the derivatives of myristic and linolenic acids showed higher efficacy and lower cytotoxicity. These results added to the advantages shown by the enzymatic methodology, such as mild reaction conditions and low environmental impact, making this approach a valuable way to synthesize these amlodipine derivatives with an application as promising antiparasitic agents.

A Novel Protocol for the Synthesis of 1,2,4-Oxadiazoles Active against Trypanosomatids and Drug-Resistant Leukemia Cell Lines.

Cancer and parasitic diseases, such as leishmaniasis and Chagas disease, share similarities that allow the co-development of new antiproliferative agents as a strategy to quickly track the discovery of new drugs. This strategy is especially interesting regarding tropical neglected diseases, for which chemotherapeutic alternatives are extremely outdated. We designed a series of (E)-3-aryl-5-(2-aryl-vinyl)-1,2,4-oxadiazoles based on the reported antiparasitic and anticancer activities of structurally related compounds. The synthesis of such compounds led to the development of a new, fast, and efficient strategy for the construction of a 1,2,4-oxadiazole ring on a silica-supported system under microwave irradiation. One hit compound (23) was identified during the in vitro evaluation against drug-sensitive and drug-resistant chronic myeloid leukemia cell lines (EC50 values ranging from 5.5 to 13.2 µM), Trypanosoma cruzi amastigotes (EC50 = 2.9 µM) and Leishmania amazonensis promastigotes (EC50 = 12.2 µM) and amastigotes (EC50 = 13.5 µM). In silico studies indicate a correlation between the in vitro activity and the interaction with tubulin at the colchicine binding site. Furthermore, ADMET in silico predictions indicate that the compounds possess a high druggability potential due to their physicochemical, pharmacokinetic, and toxicity profiles, and for hit 23, it was identified by multiple spectroscopic approaches that this compound binds with human serum albumin (HSA) via a spontaneous ground-state association with a moderate affinity driven by entropically and enthalpically energies into subdomain IIA (site I) without significantly perturbing the secondary content of the protein.

Induction of Autophagy by Ursolic Acid Promotes the Elimination of Trypanosoma cruzi Amastigotes From Macrophages and Cardiac Cells.

Chagas disease, caused by the parasite Trypanosoma cruzi, is an infectious illness endemic to Latin America and still lacks an effective treatment for the chronic stage. In a previous study in our laboratory, we established the protective role of host autophagy in vivo during T. cruzi infection in mice and proposed this process as one of the mechanisms involved in the innate immune response against this parasite. In the search for an autophagy inducer that increases the anti-T. cruzi response in the host, we found ursolic acid (UA), a natural pentacyclic triterpene with many biological actions including autophagy induction. The aim of this work was to study the effect of UA on T. cruzi infection in vitro in the late infection stage, when the nests of intracellular parasites are forming, in both macrophages and cardiac cells. To test this effect, the cells were infected with T. cruzi for 24 h and then treated with UA (5–10 µM). The data showed that UA significantly decreased the number of amastigotes found in infected cells in comparison with non-treated cells. UA also induced the autophagy response in both macrophages and cardiac cells under the studied conditions, and the inhibition of this pathway during UA treatment restored the level of infection. Interestingly, LC3 protein, the main marker of autophagy, was recruited around amastigotes and the acidic probe LysoTracker localized with them, two key features of xenophagy. A direct cytotoxic effect of UA was also found on trypomastigotes of T. cruzi, whereas epimastigotes and amastigotes displayed more resistance to this drug at the studied concentrations. Taken together, these data showed that this natural compound reduces T. cruzi infection in the later stages by promoting parasite damage through the induction of autophagy. This action, in addition to the effect of this compound on trypomastigotes, points to UA as an interesting lead for Chagas disease treatment in the future.

Intramolecular Interactions Enhance the Potency of Gallinamide A Analogues against Trypanosoma cruzi.

Gallinamide A, a metabolite of the marine cyanobacterium Schizothrix sp., selectively inhibits cathepsin L-like cysteine proteases. We evaluated the potency of gallinamide A and 23 synthetic analogues against intracellular Trypanosoma cruzi amastigotes and the cysteine protease, cruzain. We determined the co-crystal structures of cruzain with gallinamide A and two synthetic analogues at ∼2 Å. SAR data revealed that the N-terminal end of gallinamide A is loosely bound and weakly contributes in drug-target interactions. At the C-terminus, the intramolecular π-π stacking interactions between the aromatic substituents at P1' and P1 restrict the bioactive conformation of the inhibitors, thus minimizing the entropic loss associated with target binding. Molecular dynamics simulations showed that in the absence of an aromatic group at P1, the substituent at P1' interacts with tryptophan-184. The P1-P1' interactions had no effect on anti-cruzain activity, whereas anti-T. cruzi potency increased by ∼fivefold, likely due to an increase in solubility/permeability of the analogues.

Front Cover: Trypanocidal Activity of Dysphania ambrosioides, Lippia alba, and Tetradenia riparia Essential Oils against Trypanosoma cruzi (Chem. Biodiversity 12/2021)

Front Cover. The essential oils from Dysphania ambrosioides (L.) Mosyakin & Clemants (Amaranthaceae), and Lippia alba (Mill.) N.E. Brown (Verbenaceae) leaves, grown in Brazil Southeast showed high activities against Trypanosoma cruzi trypomastigote and amastigote forms. These essential oils displayed moderate toxicity against LLCMK2 cells, but the essential oil of D. ambrosioides showed a high selectivity index (SI) for trypomastigote and amastigote forms. On the other hand, the essential oil of Tetradenia riparia (Hochst.) Codd (Lamiaceae) grown in Brazil Southeast displayed the best in vivo trypanocidal effects by reducing the parasitemia after intraperitoneal administration as reported by Pagotti et al. in their full paper at 10.1002/cbdv.202100678.

Cytotoxicity, antiprotozoal, and anti-inflammatory activities of eight curry powders and comparison of their UPLC-ESI-QTOF-MS chemical profiles.

Curry powder is a blend of spices that is extensively consumed worldwide and mainly in Central Asia. Its preparation is strictly related to each locality and, because of the health benefits of its constituents, eight commercial forms of this condiment were biologically and chemically investigated. This study aimed to compare their chemical profile as well as their anti-inflammatory, cytotoxic, and antiparasitic activities. Curry samples 1 and 7 inhibited leukocyte influx and myeloperoxidase activity, while only 7 was active on protein exudate and NOx species. 2, 6, and 8 displayed trypanocidal effect against Trypanosoma cruzi amastigote, whereas 6 showed antileishmanial activity on Leishmania amazonensis amastigote. 2, 6, and 8 also inhibited the growth of THP-1 cells used as the parasite's host. Among the cytotoxic samples (4 and 6), curry sample 6 induced apoptosis in MDA-MB-231 cells. Nevertheless, 4 and 6 were unselectively cytotoxic to non-tumoral and tumoral cells. The anti-inflammatory, cytotoxicity, and antiparasitic assays were respectively performed by carrageenan-induced pleurisy test, Alamar blue assay, and intracellular parasite-host cell model. Ultra-performance liquid chromatographic-electrospray ionization mass spectrometric data from the spices revealed both similar and different metabolites in their composition. The results obtained indicate that different formulations can contribute different health benefits as a result of their chemical composition. © 2018 Society of Chemical Industry.

Synthesis and SAR of new isoxazole-triazole bis-heterocyclic compounds as analogues of natural lignans with antiparasitic activity

Despite the impressive scientific and technological advances of recent decades, no effective treatment is currently available for Chagas disease. Our research group has been studying the design and synthesis of analogues of natural lignans aiming to identify compounds with antiparasitic activity. This article reports the synthesis of 42 novel bis-heterocyclic derivatives and the structure-activity relationship study conducted based on results of biological assays against Trypanosoma cruzi amastigotes. Thirty-seven compounds were active, and eight of them had GI50 values lower than 100 μM (GI50 88.4–12.2 μM). A qualitative structure activity relationship study using three dimensional descriptors was carried out and showed a correlation between growth inhibitory potency and the presence of bulky hydrophobic groups located at rings A and D of the compounds. Compound 3-(3,4-dimethoxyphenyl)-5-((4-(4-pentylphenyl)-1H-1,2,3-triazol-1-yl)methyl)isoxazole (31) was the most active in the series (GI50 12.2 μM), showing, in vitro, low toxicity and potency similar to benznidazole (GI50 10.2 μM). These results suggest that this compound can be a promising scaffold for the design of new trypanocidal compounds.

Lipids From Trypanosoma cruzi Amastigotes of RA and K98 Strains Generate a Pro-inflammatory Response via TLR2/6.

Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-α and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.

Synthesis and biological evaluation of potential inhibitors of the cysteine proteases cruzain and rhodesain designed by molecular simplification

Analogues of 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]indol-4-amine 1, a known cruzain inhibitor, were synthesized using a molecular simplification strategy. Five series of analogues were obtained: indole, pyrimidine, quinoline, aniline and pyrrole derivatives. The activity of the compounds was evaluated against the enzymes cruzain and rhodesain as well as against Trypanosoma cruzi amastigote and trypomastigote forms. The 4-aminoquinoline derivatives showed promising activity against both enzymes, with IC50 values ranging from 15 to 125µM. These derivatives were selective inhibitors for the parasitic proteases, being unable to inhibit mammalian cathepsins B and S. The most active compound against cruzain (compound 5a; IC50=15µM) is considerably more synthetically accessible than 1, while retaining its ligand efficiency. As observed for the original lead, compound 5a was shown to be a competitive enzyme inhibitor. In addition, it was also active against T. cruzi (IC50=67.7µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50=3.1µM) with remarkable selectivity index (SI=128) compared to uninfected fibroblasts. Both 5a and 4b exhibit drug-like physicochemical properties and are predicted to have a favorable ADME profile, therefore having great potential as candidates for lead optimization in the search for new drugs to treat Chagas disease.

Assessing anti-T. cruzi candidates in vitro for sterile cidality

Total clearance of the T. cruzi infection – referred to herein as “sterile cure” – seems to be a critical prerequisite for new drug candidates for Chagas disease, ensuring long-term beneficial effects for patients in the chronic indeterminate stage. This requirement is notably supported by the recent findings of clinical studies involving posaconazole and fosravuconazole, where the majority of patients treated eventually relapsed after an apparent clearance of parasitaemia at the end of treatment. We have adapted an in vitro system to predict the ability of a compound to deliver sterile cure. It relies on mouse peritoneal macrophages as host cells for Trypanosoma cruzi amastigotes. The macrophages do not proliferate, allowing for long-term testing and wash-out experiments. Giemsa staining followed by microscopy provides a highly sensitive and specific tool to quantify the numbers of infected host cells. Combining macrophages as host cells and Giemsa staining as the read-out, we demonstrate that posaconazole and other CYP51 inhibitors are unable to achieve complete clearance of an established T. cruzi infection in vitro in spite of the fact that these compounds are active at significantly lower concentrations than the reference drugs benznidazole and nifurtimox. Indeed, a few macrophages remained infected after 96 h of drug incubation in the presence of CYP51 inhibitors–albeit at a very low parasite load. These residual T. cruzi amastigotes were shown to be viable and infective, as demonstrated by wash-out experiments. We advocate characterizing any new anti-T. cruzi early stage candidates for sterile cidality early in the discovery cascade, as a surrogate for delivery of sterile cure in vivo.

1,3,4-Thiadiazole derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones cause death in Trypanosoma cruzi through oxidative stress

This work evaluated the in vitro and in vivo activity of TDZ 2 on Trypanosoma cruzi amastigotes and determined the possible mechanism of action of this compound on T. cruzi death. TDZ 2 inhibited T. cruzi proliferation in vitro and had low haemolytic potential. It also induced morphological and ultrastructural alterations. We observed a reduction of cell volume, the depolarization of the mitochondrial membrane, an increase in ROS production, lipoperoxidation of the cell membrane, lipid bodies formation and production of nitric oxide, a decrease in reduced thiols levels and, presence of autophagic vacuoles. The in vivo study found a reduction of parasitemia in animals treated with TDZ 2 alone or combined with benznidazole. Altogether, the alterations induced by TDZ 2 point to an oxidative stress condition that lead to T. cruzi cell death.

Antitrypanosomal activity of 5-nitro-2-aminothiazole-based compounds

A small series of 5-nitro-2-aminothiazole-based amides containing arylpiperazine-, biphenyl- or aryloxyphenyl groups in their core were synthesized and evaluated as antitrypanosomatid agents. All tested compounds were active or moderately active against Trypanosoma cruzi amastigotes in infected L6 cells and Trypanosoma brucei brucei, four of eleven compounds were moderately active against Leishmania donovani axenic parasites while none were deemed active against T. brucei rhodesiense. For the most active/moderately active compounds a moderate selectivity against each parasite was observed. There was good correlation between lipophilicity (clogP value) and antileishmanial activity or toxicity against L6 cells. Similarly, good correlation existed between clogP values and IC50 values against T. cruzi in structurally related subgroups of compounds. Three compounds were more potent as antichagasic agents than benznidazole but were not activated by the type I nitrorectusase (NTR).

Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed.

Antigen targeting to dendritic cells allows the identification of a CD4 T-cell epitope within an immunodominant Trypanosoma cruzi antigen.

Targeting antigens to dendritic cells (DCs) by using hybrid monoclonal antibodies (mAbs) directed against DC receptors is known to improve activation and support long-lasting T cell responses. In the present work, we used the mAb αDEC205 fused to the Trypanosoma cruzi amastigote surface protein 2 (ASP-2) to identify a region of this protein recognized by specific T cells. The hybrid αDEC-ASP2 mAb was successfully generated and preserved its ability to bind the DEC205 receptor. Immunization of BALB/c mice with the recombinant mAb in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)) specifically enhanced the number of IFN-γ producing cells and CD4+ T cell proliferation when compared to mice immunized with a mAb without receptor affinity or with the non-targeted ASP-2 protein. The strong immune response induced in mice immunized with the hybrid αDEC-ASP2 mAb allowed us to identify an ASP-2-specific CD4+ T cell epitope recognized by the BALB/c MHCII haplotype. We conclude that targeting parasite antigens to DCs is a useful strategy to enhance T cell mediated immune responses facilitating the identification of new T-cell epitopes.

Psilostachyin C: a natural compound with trypanocidal activity

In this study, the antiprotozoal activity of the sesquiterpene lactone psilostachyin C was investigated. This natural compound was isolated from Ambrosia scabra by bioassay-guided fractionation and was identified by spectroscopic techniques. Psilostachyin C exerted in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes, trypomastigotes and amastigotes, with 50% inhibitory concentration (IC 50) values of 0.6, 3.5 and 0.9 μg/mL, respectively, and displayed less cytotoxicity against mammalian cells, with a 50% cytotoxic concentration (CC 50) of 87.5 μg/mL. Interestingly, this compound induced ultrastructural alterations, as seen by transmission electron microscopy, in which vacuolisation and a structural appearance resembling multivesicular bodies were observed even at a concentration as low as 0.2 μg/mL. In an in vivo assay, a significant reduction in the number of circulating parasites was found in T. cruzi-infected mice treated with psilostachyin C for 5 days compared with untreated mice (7.4 ± 1.2 × 10 5 parasites/mL vs. 12.8 ± 2.0 × 10 5 parasites/mL) at the peak of parasitaemia. According to these results, psilostachyin C may be considered a promising template for the design of novel trypanocidal agents. In addition, psilostachyin C inhibited the growth of Leishmania mexicana and Leishmania amazonensis promastigotes (IC 50 = 1.2 μg/mL and 1.5 μg/mL, respectively).

Isoquinoline-based analogs of the cancer drug clinical candidate tipifarnib as anti- Trypanosoma cruzi agents

We developed a synthetic route to prepare isoquinoline analogs of the cancer drug clinical candidate tipifarnib. We show that these compounds kill Trypanosoma cruzi amastigotes grown in mammalian host cells at concentrations in the low nanomolar range. These isoquinolines represent new leads for the development of drugs to treat Chagas disease.

CDNA cloning and partial characterization of amastigote specific surface protein from Trypanosoma cruzi

Trypanosoma cruzi amastigote surface proteins are the target of both humoral and cell-mediated immune responses; however, few such molecules have been thoroughly studied. In order to study a T. cruzi amastigote-specific protein (SSP4), we used antibodies against the deglycosylated form of this molecule to clone cDNA. The selected cDNA clone (2070 bp) encodes for a 64 kDa protein product whose sequence analysis revealed no N-glycosylation signal. The DNA sequence showed high homology with a member of a previously reported dispersed repetitive gene family of T. cruzi. Antibodies against the recombinant protein reacted strongly with a 66 kDa protein and weakly with an 84 kDa protein in amastigote extracts. Immunoelectron microscopy studies showed that intracellular amastigotes express the native protein on their surfaces and flagellar pockets. The antibody label was also associated with an amorphous material present in the parasitic cavity and in direct contact with the parasite surface, which suggest that amastigotes are releasing this material. On cell-free amastigotes, the antibody showed strong decoration of the cell surface and labeling of intracellular vesicles. Immunofluorescence analysis showed that the superficial protein is expressed shortly after trypomastigotes begin to transform into amastigotes. Anti-recombinant protein antibodies recognized proteins of 100 kDa and 50–60 kDa in protein extracts of rat heart and skeletal muscle, respectively.

Trypanocidal activity of coumarins and styryl-2-pyrones from Polygala sabulosa A.W. Bennett (Polygalaceae)

Bioactivity of fractions and compounds obtained from Polygala sabulosa against Trypanosoma cruzi epimastigote, blood trypomastigote and amastigote forms were evaluated in vitro. Dichloromethane and ethyl acetate fractions showed a strong trypanocidal activity on epimastigotes (IC50 < 10.4 µg/mL). Chromatographic analysis by TLC of these fractions confirmed the presence of previously described compounds (dihydrostyryl-2-pyrones, styryl-2-pyrones and 6-methoxy-7-prenyloxycoumarin). The dichloromethane fraction was fractioned by silica gel column chromatography to afford the compound α-spinasterol and the ethyl acetate fraction yielded apigenin, quercetin and a quercetin-3-O-glucoside, being the first description for the Polygala genus. 4-Methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone, 4-methoxy-6-(11,12-dimethoxystyryl)-2-pyrone, 6-methoxy-7-prenyloxycoumarin and quercetin-3-O-glucoside showed a weak activity against blood trypomastigotes (IC50 < 1008.6 µg/mL). The prenylated coumarin was the most active compound against both epimastigote and trypomastigote forms, IC50 10.5 and 88.2 µg/mL, respectively. The hemolytic activity and cell toxicity of each active compound was also assessed. Furthermore, 4-methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone and 6-methoxy-7-prenyloxycoumarin reduced 4 times the T. cruzi infection rate for Vero cells at 100 and 50 µg/mL, respectively. These results show for the first time active compounds against T. cruzi in P. sabulosa.

1,4-Bis(alkylamino)benzo[ g]phthalazines able to form dinuclear complexes of Cu(II) which as free ligands behave as SOD inhibitors and show efficient in vitro activity against Trypanosoma cruzi

The synthesis of a new series of 1,4-bis(alkylamino)benzo[ g]phthalazines 1– 3 is reported, and their ability to form dinuclear complexes with Cu(II) assayed. The geometry of the complexes is dependent on the nature of the electron-donor sites at the sidechains. Compounds 1 and 2, that contain sp 3 or sp 2 nitrogens at the end of the alkylamino groups, originate monopodal dinuclear complexes which seem to include endogenous OH bridges, and the sidechains seem to actively participate in complexation. However, the substitution of nitrogen by oxygen in 3 leads to a tripodal dinuclear complex in which the sidechains are not involved. The in vitro antiparasitic activity on Trypanosoma cruzi epimastigotes and amastigotes and the SOD activity inhibition have been evaluated for compounds 1– 3, and, as expected, 1 and 2 show in all cases relevant results, whereas 3 is always the less active among the three substrates tested.