Biological Evaluation and X‐ray Co‐crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase,an Enzyme from the Redox Metabolism of Trypanosoma

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure‐based design. The best inhibitors were freely soluble and showed competitive inhibition constants (K_i) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half‐maximal inhibitory concentration, IC₅₀) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X‐ray co‐crystal structures confirmed the binding of the ligands to the hydrophobic wall of the “mepacrine binding site” with the new, solubility‐providing vectors oriented toward the surface of the large active site.     

Activity of Fluorine‐Containing Analogues of WC‐9 and Structurally Related Analogues against Two Intracellular Parasites: Trypanosoma cruzi and Toxoplasma gondii

Two obligate intracellular parasites, Trypanosoma cruzi, the agent of Chagas disease, and Toxoplasma gondii, an agent of toxoplasmosis, upregulate the mevalonate pathway of their host cells upon infection, which suggests that this host pathway could be a potential drug target. In this work, a number of compounds structurally related to WC‐9 (4‐phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, were designed, synthesized, and evaluated for their effect on T. cruzi and T. gondii growth in tissue culture cells. Two fluorine‐containing derivatives, the 3‐(3‐fluorophenoxy)‐ and 3‐(4‐fluorophenoxy)phenoxyethyl thiocyanates, exhibited half‐maximal effective concentration (EC₅₀) values of 1.6 and 4.9 μm , respectively, against tachyzoites of T. gondii, whereas they showed similar potency to WC‐9 against intracellular T. cruzi (EC₅₀ values of 5.4 and 5.7 μm , respectively). In addition, 2‐[3‐ (phenoxy)phenoxyethylthio]ethyl‐1,1‐bisphosphonate, which is a hybrid inhibitor containing 3‐phenoxyphenoxy and bisphosphonate groups, has activity against T. gondii proliferation at sub‐micromolar levels (EC₅₀=0.7 μm ), which suggests a combined inhibitory effect of the two functional groups.     

Antichagasic,Leishmanicidal, and Trichomonacidal Activity of 2‐Benzyl‐5‐nitroindazole‐Derived Amines

Three different series of new 5‐nitroindazole derivatives—1‐(ω‐aminoalkyl)‐2‐benzylindazolin‐3‐ones (series A ; ten compounds), 3‐(ω‐aminoalkoxy)‐2‐benzylindazoles (series B ; four compounds) and 3‐alkylamino‐2‐benzylindazoles (series C ; five compounds)—have been synthesized and evaluated against the protozoan parasites Trypanosoma cruzi, Leishmania amazonensis, and Trichomonas vaginalis: etiological agents of Chagas disease, cutaneous leishmaniasis, and trichomoniasis, respectively. Many indazoles of series A , B , and C were efficient against T. cruzi. Some compounds in series A , after successfully passing the preliminary screening for epimastigotes, exhibited activity values against amastigotes of several T. cruzi strains that were better than or similar to those shown by the reference drug benznidazole and displayed low nonspecific toxicity against mammalian cells. On the other hand, preliminary studies against promastigotes of L. amazonensis showed high leishmanicidal activity for some derivatives of series A and C . With regard to activity against T. vaginalis, some indazoles of series B and C were rather efficient against trophozoites of a metronidazole‐sensitive isolate and showed low nonspecific toxicities toward Vero cell cultures. Additionally, some of these compounds displayed similar activity against metronidazole‐sensitive and resistant isolates, showing the absence of cross‐resistance between these derivatives and the reference drug.     

Binding to Large Enzyme Pockets: Small‐Molecule Inhibitors of Trypanothione Reductase

The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure‐based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small‐molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so‐called “mepacrine binding site”. The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.     

Toward the Development of Dual‐Targeted Glyceraldehyde‐3‐phosphate Dehydrogenase/Trypanothione Reductase Inhibitors against Trypanosoma brucei and Trypanosoma cruzi

A significant improvement in the treatment of trypanosomiases has been achieved with the recent development of nifurtimox–eflornithine combination therapy (NECT). As an alternative to drug combinations and as a means to overcome most of the antitrypanosomatid drug discovery challenges, a multitarget drug design strategy has been envisaged. To begin testing this hypothesis, we designed and developed a series of quinone–coumarin hybrids against glyceraldehyde‐3‐phosphate dehydrogenase/trypanothione reductase (GAPDH/TR). These enzymes belong to metabolic pathways that are vital to Trypanosoma brucei and Trypanosoma cruzi, and have thus been considered promising drug targets. The synthesized molecules were characterized for their dual‐target antitrypanosomal profile, both in enzyme assays and in in vitro parasite cultures. The merged derivative 2‐{[3‐(3‐dimethylaminopropoxy)‐2‐oxo‐2H‐chromen‐7‐yl]oxy}anthracene‐1,4‐dione ( 10 ) showed an IC₅₀ value of 5.4 μM against TbGAPDH and a concomitant K_i value of 2.32 μM against TcTR. Notably, 2‐{4‐[6‐(2‐dimethylaminoethoxy)‐2‐oxo‐2H‐chromen‐3‐yl]phenoxy}anthracene‐1,4‐dione (compound 6 ) displayed a remarkable EC₅₀ value for T. brucei parasites (0.026 μM ) combined with a very low cytotoxicity toward mammalian L6 cells (7.95 μM ). This promising low toxicity of compound 6 might be at least partially due to the fact that it does not interfere with human glutathione reductase.     

Improved Model of Lanosterol 14α‐Demethylase by Ligand‐Supported Homology Modeling: Validation by Virtual Screening and Azole Optimization

Lanosterol 14α‐demethylase (CYP51) is an important target for antifungal drugs. An improved three‐dimensional model of CYP51 from Candida albicans (CACYP51) was constructed by ligand‐supported homology modeling and molecular dynamics simulations. The accuracy of the constructed model was evaluated by its performance in a small‐scale virtual screen. The results show that known CYP51 inhibitors were efficiently discriminated by the model, and it performed better than our previous CACYP51 model. The active site of CACYP51 was characterized by multiple copy simultaneous search (MCSS) calculations. On the basis of the MCSS results, a series of novel azoles were designed and synthesized, and they showed good in vitro antifungal activity with a broad spectrum. The MIC₈₀ value of four of these compounds against C. albicans is 0.001 μg mL^?1, indicating that they are promising leads for the discovery of novel antifungal agents.     

Aryloxyethyl Thiocyanates Are Potent Growth Inhibitors of Trypanosoma cruzi and Toxoplasma gondii

As a part of our project aimed at searching for new safe chemotherapeutic agents against parasitic diseases, several compounds structurally related to the antiparasitic agent WC‐9 (4‐phenoxyphenoxyethyl thiocyanate), which were modified at the terminal phenyl ring, were designed, synthesized, and evaluated as growth inhibitors against Trypanosoma cruzi, the etiological agent of Chagas disease, and Toxoplasma gondii, the parasite responsible of toxoplasmosis. Most of the synthetic analogues exhibited similar antiparasitic activity and were slightly more potent than our lead WC‐9. For example, two trifluoromethylated derivatives exhibited ED₅₀ values of 10.0 and 9.2 μM against intracellular T. cruzi, whereas they showed potent action against tachyzoites of T. gondii (ED₅₀ values of 1.6 and 1.9 μM against T. gondii). In addition, analogues of WC‐9 in which the terminal aryl group is in the meta position with respect to the alkyl chain bearing the thiocyanate group showed potent inhibitory action against both T. cruzi and T. gondii at the very low micromolar range, which suggests that a para‐phenyl substitution pattern is not necessary for biological activity.     

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas’ disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Using a homogenate of S. cerevisiae cells expressing T. cruzi OSC, we have tested 19 inhibitors: aza, methylidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, selected as representative of different classes of substrate analog inhibitors of OSC. The IC₅₀ values of inhibition (the compound concentration at which the enzyme is inhibited by 50%) are compared with the values obtained using OSC of pig liver and S. cerevisiae. Many inhibitors of pig liver and S. cerevisiae OSC show comparable IC₅₀ for T. cruzi OSC, but some phenylthiovinyl derivatives are 10–100 times more effective on the T. cruzi enzyme than on the pig or S. cerevisiae enzymes. The expression of proteins of pathogenic organisms in yeast seems very promising for preliminary screening of compounds that have potential therapeutic activity.     

Structural Design,Synthesis and Structure–Activity Relationships of Thiazolidinones with Enhanced Anti‐Trypanosoma cruzi Activity

Pharmacological treatment of Chagas disease is based on benznidazole, which displays poor efficacy when administered during the chronic phase of infection. Therefore, the development of new therapeutic options is needed. This study reports on the structural design and synthesis of a new class of anti‐Trypanosoma cruzi thiazolidinones ( 4 a – p ). (2‐[2‐Phenoxy‐1‐(4‐bromophenyl)ethylidene)hydrazono]‐5‐ethylthiazolidin‐4‐one ( 4 h ) and (2‐[2‐phenoxy‐1‐(4‐phenylphenyl)ethylidene)hydrazono]‐5‐ethylthiazolidin‐4‐one ( 4 l ) were the most potent compounds, resulting in reduced epimastigote proliferation and were toxic for trypomastigotes at concentrations below 10 μM , while they did not display host cell toxicity up to 200 μM . Thiazolidinone 4 h was able to reduce the in vitro parasite burden and the blood parasitemia in mice with similar potency to benznidazole. More importantly, T. cruzi infection reduction was achieved without exhibiting mouse toxicity. Regarding the molecular mechanism of action, these thiazolidinones did not inhibit cruzain activity, which is the major trypanosomal protease. However, investigating the cellular mechanism of action, thiazolidinones altered Golgi complex and endoplasmic reticulum (ER) morphology, produced atypical cytosolic vacuoles, as well as induced necrotic parasite death. This structural design employed for the new anti‐T. cruzi thiazolidinones ( 4 a – p ) led to the identification of compounds with enhanced potency and selectivity compared to first‐generation thiazolidinones. These compounds did not inhibit cruzain activity, but exhibited strong antiparasitic activity by acting as parasiticidal agents and inducing a necrotic parasite cell death.     

α‐Ketoheterocycles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas disease, and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinylsulfone‐based cysteine protease inhibitors, a series of α‐ketoheterocycles were developed as reversible inhibitors of a recombinant L. mexicana cysteine protease, CPB2.8. Three isoxazoles and especially one oxadiazole compound are potent reversible inhibitors of CPB2.8; however, in vitro whole‐organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease.     

Dual Parasiticidal Activities of Phthalimides: Synthesis and Biological Profile against Trypanosoma cruzi and Plasmodium falciparum

Chagas disease and malaria are two neglected tropical diseases (NTDs) that prevail in tropical and subtropical regions in 149 countries. Chagas is also present in Europe, the US and Australia due to immigration of asymptomatic infected individuals. In the absence of an effective vaccine, the control of both diseases relies on chemotherapy. However, the emergence of parasite drug resistance is rendering currently available drugs obsolete. Hence, it is crucial to develop new molecules. Phthalimides, thiosemicarbazones, and 1,3-thiazoles have been used as scaffolds to obtain antiplasmodial and anti-Trypanosoma cruzi agents. Herein we present the synthesis of 24 phthalimido-thiosemicarbazones ( 3 a – x ) and 14 phthalimido-thiazoles ( 4 a – n ) and the corresponding biological activity against T. cruzi, Plasmodium falciparum, and cytotoxicity against mammalian cell lines. Some of these compounds showed potent inhibition of T. cruzi at low cytotoxic concentrations in RAW 264.7 cells. The most active compounds, 3 t (IC₅₀=3.60 μM), 3 h (IC₅₀=3.75 μM), and 4 j (IC₅₀=4.48 μM), were more active than the control drug benznidazole (IC₅₀=14.6 μM). Overall, the phthalimido-thiosemicarbazone derivatives were more potent than phthalimido-thiazole derivatives against T. cruzi. Flow cytometry assay data showed that compound 4 j was able to induce necrosis and apoptosis in trypomastigotes. Analysis by scanning electron microscopy showed that T. cruzi trypomastigote cells treated with compounds 3 h , 3 t , and 4 j at IC₅₀ concentrations promoted changes in the shape, flagella, and surface of the parasite body similar to those observed in benznidazole-treated cells. The compounds with the highest antimalarial activity were the phthalimido-thiazoles 4 l (IC₅₀=1.2 μM), 4 m (IC₅₀=1.7 μM), and 4 n (IC₅₀=2.4 μM). Together, these data revealed that phthalimido derivatives possess a dual antiparasitic profile with potential effects against T. cruzi and lead-like characteristics.     

Therapeutic miR-21 Silencing Reduces Cardiac Fibrosis and Modulates Inflammatory Response in Chronic Chagas Disease

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), remains a serious public health problem for which there is no effective treatment in the chronic stage. Intense cardiac fibrosis and inflammation are hallmarks of chronic Chagas disease cardiomyopathy (CCC). Previously, we identified upregulation of circulating and cardiac miR-21, a pro-fibrotic microRNA (miRNA), in subjects with CCC. Here, we explored the potential role of miR-21 as a therapeutic target in a model of chronic Chagas disease. PCR array-based 88 microRNA screening was performed in heart samples obtained from C57Bl/6 mice chronically infected with T. cruzi and serum samples collected from CCC patients. MiR-21 was found upregulated in both human and mouse samples, which was corroborated by an in silico analysis of miRNA-mRNA target prediction. In vitro miR-21 functional assays (gain-and loss-of-function) were performed in cardiac fibroblasts, showing upregulation of miR-21 and collagen expression upon transforming growth factor beta 1 (TGFβ1) and T. cruzi stimulation, while miR-21 blockage reduced collagen expression. Finally, treatment of T. cruzi-infected mice with locked nucleic acid (LNA)-anti-miR-21 inhibitor promoted a significant reduction in cardiac fibrosis. Our data suggest that miR-21 is a mediator involved in the pathogenesis of cardiac fibrosis and indicates the pharmacological silencing of miR-21 as a potential therapeutic approach for CCC.     

Synthesis and Evaluation of 1‐(1‐(Benzo[b]thiophen‐2‐yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase

Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP ( 1 , 1‐(1‐benzo[b]thiophen‐2‐yl‐cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (K_i=1 μM ) and biologically active against bloodstream T. brucei (EC₅₀=10 μM ), but with poor selectivity against mammalian MRC5 cells (EC₅₀=29 μM ). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.     

Development of Small‐Molecule Trypanosoma brucei N‐Myristoyltransferase Inhibitors: Discovery and Optimisation of a Novel Binding Mode

The enzyme N‐myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high‐throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X‐ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X‐ray crystallography/structure‐based design approach, the benzomorpholinone series was further optimised, increasing activity against T. brucei NMT by >1000‐fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T. brucei.     

X‐ray Structural Analysis of Tau‐Tubulin Kinase 1 and Its Interactions with Small Molecular Inhibitors

Tau‐tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer’s disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14–313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co‐expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high‐affinity ATP‐competitive inhibitors, 3‐[(6,7‐dimethoxyquinazolin‐4‐yl)amino]phenol ( 1 ) and methyl 2‐bromo‐5‐(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamino)benzoate ( 2 ), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation‐primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199–D200 peptide backbone conformation together with altered hydrogen bonding with compound 2 . These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer’s disease.     

Monoamine Oxidase (MAO) Inhibitory Activity: 3‐Phenylcoumarins versus 4‐Hydroxy‐3‐phenylcoumarins

Monoamine oxidase (MAO) is a useful target in the treatment of neurodegenerative diseases and depressive disorders. Both isoforms, MAO‐A and MAO‐B, are known to play critical roles in disease progression, and as such, the identification of novel, potent and selective inhibitors is an important research goal. Here, two series of 3‐phenylcoumarin derivatives were synthesized and evaluated against MAO‐A and MAO‐B. Most of the compounds tested acted preferentially on MAO‐B, with IC₅₀ values in the micromolar to nanomolar range. Only 6‐chloro‐4‐hydroxy‐3‐(2’‐hydroxyphenyl)coumarin exhibited activity against the MAO‐A isoform, while still retaining good selectivity for MAO‐B. 6‐Chloro‐3‐phenylcoumarins unsubstituted at the 4 position were found to be more active as MAO‐B inhibitors than the corresponding 4‐hydroxylated coumarins. For 4‐unsubstituted coumarins, meta and para positions on the 3‐phenyl ring seem to be the most favorable for substitution. Molecular docking simulations were used to explain the observed hMAO‐B structure–activity relationships for this type of compound. 6‐Chloro‐3‐(3’‐methoxyphenyl)coumarin was the most active compound identified (IC₅₀=0.001 μM ) and is several times more potent and selective than the reference compound, R‐(?)‐deprenyl hydrochloride. This compound represents a novel tool for the further investigation of the therapeutic potential of MAO‐B inhibitors.     

Structure‐Based Design of a Monosaccharide Ligand Targeting Galectin‐8

Galectin‐8 is a β‐galactoside‐recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3‐O‐[1‐carboxyethyl]‐β‐d ‐galactopyranoside (compound 6 ) as a ligand to target the N‐terminal domain of galectin‐8 (galectin‐8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3′‐O‐sialylated lactose (3′‐SiaLac), with galectin‐8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin‐8N for 6 was 32.8 μm , whereas no significant affinity was detected for the C‐terminal domain of galectin‐8 (galectin‐8C). The crystal structure of the galectin‐8N– 6 complex validated the predicted binding conformation and revealed the exact protein–ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin‐8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide‐based scaffold as a binder of galectin‐8.     

Regulation of Phosphatidic Acid Levels in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.     

Natural Products from Annonaceae as Potential Antichagasic Agents

Chagas disease, a neglected tropical disease, is endemic in 21 Latin American countries and particularly prevalent in Brazil. Chagas disease has drawn more attention in recent years due to its expansion into non-endemic areas. The aim of this work was to computationally identify and experimentally validate the natural products from an Annonaceae family as antichagasic agents. Through the ligand-based virtual screening, we identified 57 molecules with potential activity against the epimastigote form of T. cruzi. Then, 16 molecules were analyzed in the in vitro study, of which, six molecules displayed previously unknown antiepimastigote activity. We also evaluated these six molecules for trypanocidal activity. We observed that all six molecules have potential activity against the amastigote form, but no molecules were active against the trypomastigote form. 13-Epicupressic acid seems to be the most promising, as it was predicted as an active compound in the in silico study against the amastigote form of T. cruzi, in addition to having in vitro activity against the epimastigote form.