In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

Design, synthesis and identification of novel molecular hybrids based on naphthoquinone aromatic hydrazides as potential trypanocide and leishmanicidal agents.

In pursuit of potential agents to treat Chagas disease and leishmaniasis, we report the design, synthesis, and identification novel naphthoquinone hydrazide-based molecular hybrids. The compounds were subjected to in vitro trypanocide and leishmanicidal activities. N'-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)-3,5-dimethoxybenzohydrazide (13) showed the best performance against Trypanosoma cruzi (IC50 1.83 µM) and Leishmania amazonensis (IC50 9.65 µM). 4-Bromo-N'-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzohydrazide (16) exhibited leishmanicidal activity (IC50 12.16 µM). Regarding trypanocide activity, compound 13 was low cytotoxic to LLC-MK2 cells (SI = 95.28). Furthermore, through molecular modeling studies, the cysteine proteases cruzain, rhodesain and CPB2.8 were identified as the potential biological targets.

Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites.

BACKGROUND: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.

Structural insights into drug transport by an aquaglyceroporin.

Pentamidine and melarsoprol are primary drugs used to treat the lethal human sleeping sickness caused by the parasite Trypanosoma brucei. Cross-resistance to these two drugs has recently been linked to aquaglyceroporin 2 of the trypanosome (TbAQP2). TbAQP2 is the first member of the aquaporin family described as capable of drug transport; however, the underlying mechanism remains unclear. Here, we present cryo-electron microscopy structures of TbAQP2 bound to pentamidine or melarsoprol. Our structural studies, together with the molecular dynamic simulations, reveal the mechanisms shaping substrate specificity and drug permeation. Multiple amino acids in TbAQP2, near the extracellular entrance and inside the pore, create an expanded conducting tunnel, sterically and energetically allowing the permeation of pentamidine and melarsoprol. Our study elucidates the mechanism of drug transport by TbAQP2, providing valuable insights to inform the design of drugs against trypanosomiasis.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

Synthesis and Anti-Chagas Activity Profile of a Redox-Active Lead 3-Benzylmenadione Revealed by High-Content Imaging.

Chagas disease, or American trypanosomiasis, is a neglected tropical disease which is a top priority target of the World Health Organization. The disease, endemic mainly in Latin America, is caused by the protozoan Trypanosoma cruzi and has spread around the globe due to human migration. There are multiple transmission routes, including vectorial, congenital, oral, and iatrogenic. Less than 1% of patients have access to treatment, relying on two old redox-active drugs that show poor pharmacokinetics and severe adverse effects. Hence, the priorities for the next steps of R&D include (i) the discovery of novel drugs/chemical classes, (ii) filling the pipeline with drug candidates that have new mechanisms of action, and (iii) the pressing need for more research and access to new chemical entities. In the present work, we first identified a hit (4a) with a potent anti-T. cruzi activity from a library of 3-benzylmenadiones. We then designed a synthetic strategy to build a library of 49 3-(4-monoamino)benzylmenadione derivatives via reductive amination to obtain diazacyclic benz(o)ylmenadiones. Among them, we identified by high content imaging an anti-amastigote "early lead" 11b (henceforth called cruzidione) revealing optimized pharmacokinetic properties and enhanced specificity. Studies in a yeast model revealed that a cruzidione metabolite, the 3-benzoylmenadione (cruzidione oxide), enters redox cycling with the NADH-dehydrogenase, generating reactive oxygen species, as hypothesized for the early hit (4a).

Anti-trypanosomal Lignans Isolated from Salvadoran Peperomia pseudopereskiifolia.

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Discovery of Strong 3-Nitro-2-Phenyl-2H-Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Trypanosoma cruzi Glucokinase.

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

Optimization of benzenesulfonyl derivatives as anti-Trypanosomatidae agents: Structural design, synthesis, and pharmacological assessment against Trypanosoma cruzi and Leishmania infantum.

Leishmaniasis and Chagas disease are neglected tropical diseases caused by Trypanosomatidae parasites. Given the numerous limitations associated with current treatments, such as extended treatment duration, variable efficacy, and severe side effects, there is an urgent imperative to explore novel therapeutic options. This study details the early stages of hit-to-lead optimization for a benzenesulfonyl derivative, denoted as initial hit, against Trypanossoma cruzi (T. cruzi), Leishmania infantum (L. infantum) and Leishmania braziliensis (L. braziliensis). We investigated structure - activity relationships using a series of 26 newly designed derivatives, ultimately yielding potential lead candidates with potent low-micromolar and sub-micromolar activities against T. cruzi and Leishmania spp, respectively, and low in vitro cytotoxicity against mammalian cells. These discoveries emphasize the significant promise of this chemical class in the fight against Chagas disease and leishmaniasis.

Lignans Isolated from Piper truncatum Act as New Potent Antitrypanosomal Compounds.

The hexane extract from twigs of Piper truncatum Vell (Piperaceae) displayed activity against Trypanosoma cruzi and was subjected to chromatographic steps to afford six dibenzylbutyrolactolic lignans, being four knowns: cubebin (1), (-)-9α-O-methylcubebin (2), (+)-9ß-O-methylcubebinin (3) and 3,4-dimethoxy-3,4-demethylenedioxycubebin (4) as well as two new, named truncatin A (5) and B (6). Initially, in vitro activity against trypomastigotes was evaluated and compounds 1, 4 and 6 exhibited EC50 values of 41.6, 21.0 and 39.6 µM, respectively. However, when tested against amastigotes, the relevant clinical form in the chronic phase of Chagas disease, compounds 1-6 displayed activities with EC50 values ranging from 1.6 to 13.7 µM. In addition, the mammalian cytotoxicity of compounds 1-6 was evaluated against murine fibroblasts (NCTC). Compounds 2, 3 and 4 exhibited reduced toxicity against NCTC cells (CC50>200 µM), resulting in SI values of>21.9,>14.5 and>121.9, respectively. Compound 4 showed the highest potency with an SI value twice superior to that determined by the standard drug benznidazole (SI>54.6) against the intracellular amastigotes. These data suggest that lignan 4 can be considered a possible scaffold for designing a new drug candidate for Chagas disease.

Immunomodulatory potential of Sarcophaga argyostoma larval hemolymph as a natural alternative to berenil in treating Trypanosoma evansi in vivo.

This study compared effects of diminazene aceturate (berenil), commonly used to treat domestic animals infected with Trypanosoma evansi, with the hemolymph of Sarcophaga argyostoma larva. The hemolymph may be acting as a possible natural alternative to berenil, based on immunomodulation mediated inflammatory response. Inflammatory mediators and histopathological changes in liver, kidney, and spleen of albino mice experimentally infected with T. evansi were studied. Mice were divided into five groups: G1, uninfected, untreated (negative control); G2, T. evansi infected (positive control); G3, infected and treated with berenil; G4, infected and treated with hemolymph; G5, infected and treated with hemolymph 3 days before infection (prophylactic group). Animals in (G4) and (G5) exhibited a significant overall reduction in serum levels of IFN-γ. However, the reduction in TNF-α and IL-6 levels was more limited compared to (G2) and (G3). Notably, an elevation in IL-10 levels was observed compared to animals in other groups. Furthermore, the groups treated with hemolymph demonstrated an alleviation of T. evansi infection in contrast to the other groups. This study highlights that the administration of Sarcophaga argyostoma larval hemolymph at a dosage of 0.5 ml/kg significantly inhibited T. evansi organisms in vivo, showcasing a pronounced trypanocidal effect.

Ascofuranone antibiotic is a promising trypanocidal drug for nagana.

Trypanosomosis is a disease complex which affects both humans and animals in sub-Saharan Africa, transmitted by the tsetse fly and distributed within the tsetse belt of Africa. But some trypanosome species, for example, Trypanosoma brucei evansi, T. vivax, T. theileri and T. b. equiperdum are endemic outside the tsetse belt of Africa transmitted by biting flies, for example, Tabanus and Stomoxys, or venereal transmission, respectively. Trypanocidal drugs remain the principal method of animal trypanosomosis control in most African countries. However, there is a growing concern that their effectiveness may be severely curtailed by widespread drug resistance. A minimum number of six male cattle calves were recruited for the study. They were randomly grouped into two (T. vivax and T. congolense groups) of three calves each. One calf per group served as a control while two calves were treatment group. They were inoculated with 105 cells/mL parasites in phosphate buffered solution (PBS) in 2 mL. When parasitaemia reached 1 × 107.8 cells/mL trypanosomes per mL in calves, treatment was instituted with 20 mL (25 mg/kg in 100 kg calf) ascofuranone (AF) for treatment calves, while the control ones were administered a placebo (20 mL PBS) intramuscularly. This study revealed that T. vivax was successfully cleared by AF but the T. congolense group was not cleared effectively.Contribution: There is an urgent need to develop new drugs which this study sought to address. It is suggested that the AF compound can be developed further to be a sanative drug for T. vivax in non-tsetse infested areas like South Americas.

Selenosugars targeting the infective stage of Trypanosoma brucei with high selectivity.

Earlier evidences showed that diglycosyl diselenides are active against the infective stage of African trypanosomes (top hits IC50 0.5 and 1.5 µM) but poorly selective (selectivity index <10). Here we extended the study to 33 new seleno-glycoconjugates with the aim to improve potency and selectivity. Three selenoglycosides and three glycosyl selenenylsulfides displayed IC50 against bloodstream Trypanosoma brucei in the sub-µM range (IC50 0.35-0.77 µM) and four of them showed an improved selectivity (selectivity index >38-folds vs. murine and human macrohages). For the glycosyl selenylsulfides, the anti-trypanosomal activity was not significantly influenced by the nature of the moiety attached to the sulfur atom. Except for a quinoline-, and to a minor extent a nitro-derivative, the most selective hits induced a rapid (within 60 min) and marked perturbation of the LMWT-redox homeostasis. The formation of selenenylsulfide glycoconjugates with free thiols has been identified as a potential mechanism involved in this process.

Lead Optimization of the 5-Phenylpyrazolopyrimidinone NPD-2975 toward Compounds with Improved Antitrypanosomal Efficacy.

Human African trypanosomiasis (HAT) still faces few therapeutic options and emerging drug resistance, stressing an urgency for novel antitrypanosomal drug discovery. Here, we describe lead optimization efforts aiming at improving antitrypanosomal efficacy and better physicochemical properties based on our previously reported optimized hit NPD-2975 (pIC50 7.2). Systematic modification of the 5-phenylpyrazolopyrimidinone NPD-2975 led to the discovery of a R4-substituted analogue 31c (NPD-3519), showing higher in vitro potency (pIC50 7.8) against Trypanosoma brucei and significantly better metabolic stability. Further, in vivo pharmacokinetic evaluation of 31c and experiments in an acute T. brucei mouse model confirmed improved oral bioavailability and antitrypanosomal efficacy at 50 mg/kg with no apparent toxicity. With good physicochemical properties, low toxicity, improved pharmacokinetic features, and in vivo efficacy, 31c may serve as a promising candidate for future drug development for HAT.

Tractable Quinolone Hydrazides Exhibiting Sub-Micromolar and Broad Spectrum Antitrypanosomal Activities.

Nagana and Human African Trypanosomiasis (HAT), caused by (sub)species of Trypanosoma, are diseases that impede human and animal health, and economic growth in Africa. The few drugs available have drawbacks including suboptimal efficacy, adverse effects, drug resistance, and difficult routes of administration. New drugs are needed. A series of 20 novel quinolone compounds with affordable synthetic routes was made and evaluated in vitro against Trypanosoma brucei and HEK293 cells. Of the 20 compounds, 12 had sub-micromolar potencies against the parasite (EC50 values=0.051-0.57 µM), and most were non-toxic to HEK293 cells (CC50 values>5 µM). Two of the most potent compounds presented sub-micromolar activities against other trypanosome (sub)species (T. cruzi and T. b. rhodesiense). Although aqueous solubility is poor, both compounds possess good logD values (2-3), and either robust or poor microsomal stability profiles. These varying attributes will be addressed in future reports.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

PURPOSE: This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. METHODS: A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. RESULTS: The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. CONCLUSION: It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.

In vitro and in vivo antitrypanosomal activity of the fresh leaves of Ranunculus Multifidus Forsk and its major compound anemonin against Trypanosoma congolense field isolate.

BACKGROUND: Animal trypanosomiasis is a major livestock problem due to its socioeconomic impacts in tropical countries. Currently used trypanocides are toxic, expensive, and the parasites have developed resistance to the existing drugs, which calls for an urgent need of new effective and safe chemotherapeutic agents from alternative sources such as medicinal plants. In Ethiopian traditional medicine fresh leaves of Ranunculus multifidus Forsk, are used for the treatment of animal trypanosomiasis. The present study aimed to evaluate the antitrypanosomal activity of the fresh leaves of R. multifidus and its major compound anemonin against Trypanosoma congolense field isolate. METHODS: Fresh leaves of R. multifidus were extracted by maceration with 80% methanol and hydro-distillation to obtain the corresponding extracts. Anemonin was isolated from the hydro-distilled extract by preparative TLC. For the in vitro assay, 0.1, 0.4, 2 and 4 mg/ml of the test substances were incubated with parasites and cessation or drop in motility of the parasites was monitored for a total duration of 1 h. In the in vivo assay, the test substances were administered intraperitoneally daily for 7 days to mice infected with Trypanosoma congolense. Diminazene aceturate and 1% dimethylsulfoxide (DMSO) were used as positive and negative controls, respectively. RESULTS: Both extracts showed antitrypanosomal activity although the hydro-distilled extract demonstrated superior activity compared to the hydroalcoholic extract. At a concentration of 4 mg/ml, the hydro-distilled extract drastically reduced motility of trypanosomes within 20 min. Similarly, anemonin at the same concentration completely immobilized trypanosomes within 5 min of incubation, while diminazene aceturate (28.00 mg/kg/day) immobilized the parasites within 10 min. In the in vivo antitrypanosomal assay, anemonin eliminates parasites at all the tested doses (8.75, 17.00 and 35.00 mg/kg/day) and prevented relapse, while in diminazene aceturate-treated mice the parasites reappeared on days 12 to 14. CONCLUSIONS: The current study demonstrated that the fresh leaves of R. multifidus possess genuine antitrypanosomal activity supporting the use of the plant for the treatment of animal trypanosomiasis in traditional medicine. Furthermore, anemonin appears to be responsible for the activity suggesting its potential as a scaffold for the development of safe and cost effective antitrypanosomal agent.

Structure-activity relationships of novel N-imidazoylpiperazines with potent anti-Trypanosoma cruzi activity.

Background: Chagas disease is caused by the parasite Trypanosoma cruzi, and the lack of effective and safe treatments makes identifying new classes of compounds with anti-T. cruzi activity of paramount importance. Methods: Hit-to-lead exploration of a metabolically stable N-imidazoylpiperazine was performed. Results: Compound 2, a piperazine derivative active against T. cruzi, was selected to perform the hit-to-lead exploration, which involved the design, synthesis and biological evaluation of 39 new derivatives. Conclusion: Compounds 6e and 10a were identified as optimized compounds with low micromolar in vitro activity, low cytotoxicity and suitable preliminary absorption, distribution, metabolism and excretion and physicochemical properties. Both compounds reduced parasitemia in mouse models of Chagas disease, providing a promising opportunity for further exploration of new antichagasic compounds.