Discovery of Novel Inhibitors of Cruzain Cysteine Protease of Trypanosoma cruz.

Chagas disease (CD) is a parasitic disease endemic in several developing countries. According to the World Health Organization, approximately 6-8 million people worldwide are inflicted by CD. The scarcity of new drugs, mainly for the chronic phase, is the main reason for treatment limitation in CD. Therefore, there is an urgent need to discover new targets for which new therapeutical agents could be developed. Cruzain cysteine protease (CCP) is a promising alternative because this enzyme exhibits pleiotropic effects by acting as a virulence factor, modulating host immune cells, and interacting with host cells. This systematic review was conducted to discover new compounds that act as cruzain inhibitors, and their effects in vitro were studied through enzymatic assays and molecular docking. Additionally, the advances and perspectives of these inhibitors are discussed. These findings are expected to contribute to medicinal chemistry in view of the design of new, safe, and efficacious inhibitors against Trypanosoma cruzi CCP detected in the last decade (2013-2022) to provide scaffolds for further optimization, aiming toward the discovery of new drugs.

Exploring the dark side of tertiary and quaternary structure dynamics in MtbFBPaseII.

Tuberculosis (TB) is a major global cause of mortality, primarily stemming from latent tuberculosis infection (LTBI). Failure to fully treat LTBI can result in drug-resistant forms of TB. Therefore, it is essential to develop novel drugs with unique mechanisms of action to combat TB effectively. One crucial metabolic pathway in Mycobacterium tuberculosis (Mtb), which contributes to TB infection and persistence, is gluconeogenesis. Within this pathway, the enzyme fructose bisphosphatase (FBPase) plays a significant role and is considered a promising target for drug development. By targeting MtbFBPaseII, a specific class of FBPase, researchers have employed molecular dynamics simulations to identify regions capable of binding new drugs, thereby inhibiting the enzyme's activity and potentially paving the way for the development of effective treatments.Communicated by Ramaswamy H. Sarma.

Exploring the conformational dynamics of the CRD4: a model for receptor lysosomal activity shifts in search of 'sweet' and 'sour' states.

The macrophage mannose receptor (RMM) is a crucial component of the immune system involved in immune responses, inflammation resolution, and tissue remodeling. When RMM is activated by a specific ligand, it undergoes internalization, forming an endosome that matures into a lysosome. Within the lysosome, structural changes in RMM facilitate the dissociation of ligands for further processing. However, the precise details of these structural changes are not well understood. In this study, we used molecular dynamics simulations to investigate the conformational dynamics of a specific region called CRD4 in RMM. Our simulations explored different conditions, including pH variations and the presence of Ca2+ ions. By analyzing the simulation data, we found that conformational changes primarily occur in loop regions, while the secondary structure remains stable. The binding site of CRD4, essential for ligand interaction, is located on the protein surface between two specific loop regions. Ligand binding is stabilized by three important amino acids. Interestingly, the interaction patterns differ between monosaccharide and disaccharide ligands. These findings improve our understanding of CRD4's dynamics and how it recognizes ligands. They provide insights into the structure of CRD4 and its role in ligand dissociation within lysosomes. The study also highlights the significance of loop regions in functional dynamics and interactions. Further research is needed to fully uncover the complete structure of CRD4, understand ligand binding modes, and explore the influence of environmental factors. This study lays the foundation for future investigations targeting carbohydrate-protein interactions and the development of therapeutics based on RMM's unique properties.Communicated by Ramaswamy H. Sarma.

Covalent Inhibitors for Neglected Diseases: An Exploration of Novel Therapeutic Options.

Neglected diseases, primarily found in tropical regions of the world, present a significant challenge for impoverished populations. Currently, there are 20 diseases considered neglected, which greatly impact the health of affected populations and result in difficult-to-control social and economic consequences. Unfortunately, for the majority of these diseases, there are few or no drugs available for patient treatment, and the few drugs that do exist often lack adequate safety and efficacy. As a result, there is a pressing need to discover and design new drugs to address these neglected diseases. This requires the identification of different targets and interactions to be studied. In recent years, there has been a growing focus on studying enzyme covalent inhibitors as a potential treatment for neglected diseases. In this review, we will explore examples of how these inhibitors have been used to target Human African Trypanosomiasis, Chagas disease, and Malaria, highlighting some of the most promising results so far. Ultimately, this review aims to inspire medicinal chemists to pursue the development of new drug candidates for these neglected diseases, and to encourage greater investment in research in this area.

The Role of Flavanones as Scaffolds for the Development of New Treatments against Malaria and African and American Trypanosomiases.

Parasitic infections are diseases transmitted by parasites usually found in contaminated food, water, or insect bites. Generally classified as neglected tropical diseases, malaria and trypanosomiases are some of the most prominent parasitic diseases that cause significant loss of life annually. In 2020, an estimated 241 million malaria cases were reported, with 627,000 deaths worldwide. An estimated 6 to 7 million people are infected with Trypanosoma cruzi worldwide, whereas an estimated 1000 global cases of African human trypanosomiasis were reported in 2020. Flavanones are a group of compounds that belong to the flavonoid family and are chemically obtained by direct cyclization of chalcones. Recent pharmacological studies have demonstrated the effectiveness of plant flavanones in inhibiting the growth of the parasites responsible for malaria and trypanosomiases. The present work aims to summarize up-to-date and comprehensive literature information on plant flavanones with antimalarial and antitrypanosomal activities. The mechanisms of action of the antiparasitic flavanones are also discussed. A literature search was performed for naturally occurring flavanones and antimalarial and antitrypanosomal activities by referencing textbooks and scientific databases (SciFinder, Wiley, American Chemical Society, Science Direct, National Library of Medicine, Scientific Electronic Library Online, Web of Science, etc.) from their inception until April 2022. Based on in vitro experiments, more than sixty flavanones were reported to exhibit antimalarial, anti-T. cruzi, and anti-T. brucei activities. Previous studies demonstrated that these compounds bind to PGP-like transporters of P. falciparum to reverse the parasite's resistance. Other reports pinpointed the direct effect of these compounds on the mitochondria of the malaria parasite. Moreover, flavanones have shown strong docking to several validated T. cruzi and T. brucei protein targets, including adenosine kinase, pteridine reductase 1, dihydrofolate reductase, and trypanothione reductase, among others. Flavanones, isolated and characterized from diverse plant parts, were reported to exhibit moderate to high activity against P. falciparum, T. cruzi, and T. brucei in in vitro studies. These potentially active flavanones can be used as scaffolds for the development of new antiparasitic agents. However, more studies on the cytotoxicity, pharmacokinetics, and mechanisms of action of potent flavanones should be performed.

Neglected tropical diseases and systemic racism especially in Brazil: from socio-economic aspects to the development of new drugs.

Neglected tropical diseases (NTDs) are highly prevalent communicable diseases in tropical and subtropical countries, generally not economically attractive for drug development and related to poverty. In Brazil, more specifically, socioeconomic inequalities and health indicators are strongly influenced by skin color, race, and ethnicity, due to the historical process of slavery. In this context, it is important to understand the concept of systemic racism: a form of indirect racial discrimination present in many institutions, which determines the process of illness and death of the black population, the ethnic group most affected by these diseases. The main objective of this paper was to carry out a literature review on the socioeconomic aspects of these diseases, relating them to institutional racism, and to encourage reflection on the influence of this type of racism in the NTDs context. Therefore, we present a paper that brings a evident correlation between racism versus neglected populations, which are affected by equally neglected diseases. A more humane and comprehensive view is needed to realize that these illnesses affect neglected and vulnerable populations, who require decent living conditions, health, and social justice. We hope to provide, with this paper, enough, but not exhaust, knowledge to initiate the discussion about neglected diseases, their socioeconomic aspects and institutional racism.

Resveratrol and Curcumin for Chagas Disease Treatment-A Systematic Review.

Chagas disease (CD) is a neglected protozoan infection caused by Trypanosoma cruzi, which affects about 7 million people worldwide. There are two available drugs in therapeutics, however, they lack effectiveness for the chronic stage-characterized mainly by cardiac (i.e., cardiomyopathy) and digestive manifestations (i.e., megaesophagus, megacolon). Due to the involvement of the immuno-inflammatory pathways in the disease's progress, compounds exhibiting antioxidant and anti-inflammatory activity seem to be effective for controlling some clinical manifestations, mainly in the chronic phase. Resveratrol (RVT) and curcumin (CUR) are natural compounds with potent antioxidant and anti-inflammatory properties and their cardioprotective effect have been proposed to have benefits to treat CD. Such effects could decrease or block the progression of the disease's severity. The purpose of this systematic review is to analyze the effectiveness of RVT and CUR in animal and clinical research for the treatment of CD. The study was performed according to PRISMA guidelines and it was registered on PROSPERO (CDR42021293495). The results did not find any clinical study, and the animal research was analyzed according to the SYRCLES risk of bias tools and ARRIVE 2.0 guidelines. We found 9 eligible reports in this study. We also discuss the potential RVT and CUR derivatives for the treatment of CD as well.

Therapeutic Potential of Flavonoid Derivatives for Certain Neglected Tropical Diseases.

BACKGROUND: Neglected tropical diseases (NTDs) are infectious diseases that mostly affect people living in tropical and subtropical regions, especially in impoverished areas. Ubiquitously found in plants, flavonoids are a group of compounds that have been reported to exhibit a wide range of biological activities against parasites (Leishmania sp., Trypanosoma cruzi, Trypanosoma brucei, Brugia malayi, etc.) that cause certain NTDs. AIMS: The present study aims to highlight and discuss our recent reports on the implication of flavonoids in drug development for NTDs, such as leishmaniasis, Chagas disease, African trypanosomiasis, filariasis, among others. RESULTS: Today, studies show that flavonoids exhibit in vitro antileishmanial, anti-trypanosomiasis, antifilarial activities, among others. Furthermore, the molecular hybridization of flavonoids with the triazole groups has led to the development of compounds with improved biological activity. The incorporation of chemical groups, such as NO2, F, and Cl groups, during the process of design and synthesis, leads to the enhancement of pharmacological activity. CONCLUSION: Flavonoids are useful metabolites that can be prospected as potential leads for the development of new agents against certain NTDs. However, research opportunities, including cytotoxicity and in vivo studies, mechanisms of action, bioavailability of these compounds, remain to be investigated in the future.

Triazole-containing Heterocycles: Privileged Scaffolds in Anti-Trypanosoma Cruzi Drug Development.

BACKGROUND: Chagas disease is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi and is transmitted to humans through the excreta of infected blood-sucking triatomine bugs. According to the World Health Organization, 6 to 7 million people are infected with T. cruzi worldwide, mainly in Latin America, with more than 10000 deaths annually. AIM OF THE STUDY: The present study aims to provide comprehensive literature information on the importance of triazole-containing heterocycles in developing anti-Chagas disease agents. METHODOLOGY: The embodied information was acquired without date limitation by December 2020 using various electronic databases including, SciFinder, PubMed (National Library of Medicine), Science Direct, Wiley, ACS (American Chemical Society), SciELO (Scientific Electronic Library Online), Google Scholar, Springer, Scopus, and Web of Science. RESULTS: Upon in vitro studies, more than 100 triazole-containing heterocycles have been predicted as active compounds against the pathogen responsible for the American trypanosomiasis. However, less is known about their in vivo activity in animal models and their clinical studies in humans. Moreover, the pharmacokinetic studies of these bioactive compounds are still pending. Despite the variety of mechanisms of action attributed to most of these molecules, the exact mechanism involved is still controversial. Thus, in vivo experiments, followed by pharmacokinetics, and the mechanism of action of the most active compounds, should be the subject of future investigation. CONCLUSION: All in all, recent studies have demonstrated the importance of triazole-containing heterocycles in search of potential candidates for drug development against Chagas disease. Nonetheless, the use of new catalysts and chemical transformations is expected to provide avenues for the synthesis of unexplored triazole derivatives, leading to the development of triazole-containing compounds with new properties and trypanocidal activity.

Image-Based In Vitro Screening Reveals the Trypanostatic Activity of Hydroxymethylnitrofurazone against Trypanosoma cruzi.

Hydroxymethylnitrofurazone (NFOH) is a therapeutic candidate for Chagas disease (CD). It has negligible hepatotoxicity in a murine model compared to the front-line drug benznidazole (BZN). Here, using Trypanosoma cruzi strains that express bioluminescent and/or fluorescent reporter proteins, we further investigated the in vitro and in vivo activity of NFOH to define whether the compound is trypanocidal or trypanostatic. The in vitro activity was assessed by exploiting the fluorescent reporter strain using wash-out assays and real-time microscopy. For animal experimentation, BALB/c mice were inoculated with the bioluminescent reporter strain and assessed by highly sensitive in vivo and ex vivo imaging. Cyclophosphamide treatment was used to promote parasite relapse in the chronic stage of infection. Our data show that NFOH acts by a trypanostatic mechanism, and that it is more active than BZN in vitro against the infectious trypomastigote form of Trypanosoma cruzi. We also found that it is more effective at curing experimental infections in the chronic stage, compared with the acute stage, a feature that it shares with BZN. Therefore, given its reduced toxicity, enhanced anti-trypomastigote activity, and curative properties, NFOH can be considered as a potential therapeutic option for Chagas disease, perhaps in combination with other trypanocidal agents.

Malaria and tuberculosis as diseases of neglected populations: state of the art in chemotherapy and advances in the search for new drugs.

Malaria and tuberculosis are no longer considered to be neglected diseases by the World Health Organization. However, both are huge challenges and public health problems in the world, which affect poor people, today referred to as neglected populations. In addition, malaria and tuberculosis present the same difficulties regarding the treatment, such as toxicity and the microbial resistance. The increase of Plasmodium resistance to the available drugs along with the insurgence of multidrug- and particularly tuberculosis drug-resistant strains are enough to justify efforts towards the development of novel medicines for both diseases. This literature review provides an overview of the state of the art of antimalarial and antituberculosis chemotherapies, emphasising novel drugs introduced in the pharmaceutical market and the advances in research of new candidates for these diseases, and including some aspects of their mechanism/sites of action.

Perspectives About Self-Immolative Drug Delivery Systems.

Self-immolative drug delivery system is one of the delivery systems, which have drawn attention, in recent research, highlighting the improvement they generate in drug selectivity and efficacy. Self-immolative linkers, or spacers, are covalent groups, which have the role of cleavaging two bonds between a protector group and a drug, in the case of drug delivery systems, after a stimuli.The cascade of reactions allows to control the release of the drug. The choice of the adequate self-immolative linker is essential and depend on many variables and goals as well. Many approaches can be explored when designing a system adequate for achieving these goals, especially prodrugs. Some of the most used stimuli-responses for self-immolative drugs - enzyme triggers, chemical triggers, as pH, redox system, 1,4-, 1,6-, 1,8-eliminations, photodegradable triggers, multiple triggers, among others - are described in this ten-year review, along with their application as theranostic agents. We intend that the examples presented in this review inspire researchers working on drug delivery systems to further explore their application.

Searching for drugs for Chagas disease, leishmaniasis and schistosomiasis: a review.

Chagas disease, leishmaniasis and schistosomiasis are neglected diseases (NDs) and are a considerable global challenge. Despite the huge number of people infected, NDs do not create interest from pharmaceutical companies because the associated revenue is generally low. Most of the research on these diseases has been conducted in academic institutions. The chemotherapeutic armamentarium for NDs is scarce and inefficient and better drugs are needed. Researchers have found some promising potential drug candidates using medicinal chemistry and computational approaches. Most of these compounds are synthetic but some are from natural sources or are semi-synthetic. Drug repurposing or repositioning has also been greatly stimulated for NDs. This review considers some potential drug candidates and provides details of their design, discovery and activity.

Flavonoids as efficient scaffolds: Recent trends for malaria, leishmaniasis, Chagas disease, and dengue.

Endemic in 149 tropical and subtropical countries, neglected tropical diseases (NTDs) affect more than 1 billion people annually with over 500,000 deaths. Among the NTDs, some of the most severe consist of leishmaniasis, Chagas disease, and dengue. The impact of the combined NTDs closely rivals that of malaria. According to the World Health Organization, 216 million cases of malaria were reported in 2016 with 445,000 deaths. Current treatment options are associated with various limitations including widespread drug resistance, severe adverse effects, lengthy treatment duration, unfavorable toxicity profiles, and complicated drug administration procedures. Flavonoids are a class of compounds that has been the subject of considerable scientific interest. New developments of flavonoids have made promising advances for the potential treatment of malaria, leishmaniasis, Chagas disease, and dengue, with less toxicity, high efficacy, and improved bioavailability. This review summarizes the current standings of the use of flavonoids to treat malaria and neglected diseases such as leishmaniasis, Chagas disease, and dengue. Natural and synthetic flavonoids are leading compounds that can be used for developing antiprotozoal and antiviral agents. However, detailed studies on toxicity, pharmacokinetics, and mechanisms of action of these compounds are required to confirm the in vitro pharmacological claims of flavonoids for pharmaceutical applications. HIGHLIGHTS: In the current review, we have tried to compile recent discoveries on natural and synthetic flavonoids as well as their implication in the treatment of malaria, leishmaniasis, Chagas disease, and dengue. A total of 373 (220 natural and 153 synthetic) flavonoids have been evaluated for antimalarial, antileishmanial, antichagasic, and antidengue activities. Most of these flavonoids showed promising results against the above diseases. Reports on molecular modeling of flavonoid compounds to the disease target indicated encouraging results. Flavonoids can be prospected as potential leads for drug development; however, more rigorously designed studies on toxicity and pharmacokinetics, as well as the quantitative structure-activity relationship studies of these compounds, need to be addressed.

Targeting Leishmania amazonensis amastigotes through macrophage internalisation of a hydroxymethylnitrofurazone nanostructured polymeric system.

Dextran-coated poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared and were evaluated for enhanced delivery of a promising anti-Leishmania drug candidate, hydroxymethylnitrofurazone (NFOH), to phagocytic cells. Currently available chemotherapy for leishmaniasis, such as pentavalent antimonials, presents low safety and efficacy. Furthermore, widespread drug resistance in leishmaniasis is rapidly emerging. To overcome these drawbacks, the use of nanosized delivery systems can reduce systemic drug toxicity and increase the drug concentration in infected macrophages, therefore improving treatment of leishmaniasis. PBCA-NPs containing NFOH (PBCA-NFOH-NPs) were prepared by an anionic emulsion polymerisation method. The z-average and polydispersity index (PDI) were determined by photon correlation spectroscopy, the zeta potential by microelectrophoresis and the entrapment efficiency by HPLC. Cytotoxicity was determined using macrophages from BALB/c mice. Efficacy tests were performed using Leishmania amazonensis promastigotes and amastigotes. The z-average of PBCA-NFOH-NPs was 151.5 ± 61.97 nm, with a PDI of 0.104 ± 0.01, a zeta potential of -10.1 ± 6.49 mV and an entrapment efficiency of 64.47 ± 0.43%. Efficacy in amastigotes revealed IC50 values of 0.33 µM and 31.2 µM for the nanostructured and free NFOH, respectively (95-fold increase). The cytotoxicity study indicated low toxicity of the PBCA-NFOH-NPs to macrophages. The selectivity index was 370.6, which is 49-fold higher than free NFOH (7.6). Such findings indicated that improved efficacy could be due to NP internalisation following site-specific drug delivery and reactivation of immune protective reactions by the NP components. Thus, PBCA-NFOH-NPs have the potential to significantly improve the treatment of leishmaniasis, with reduced systemic side effects.

Drug design for neglected disease in Brazil.

Neglected tropical diseases (NTD) are a group of 17 diseases transmitted by virus, protozoa, helminthes and bacteria. These illnesses are responsible for millions of deaths per year, affecting mainly the poorest populations in the world. The therapeutic drugs available are obsolete, toxic, have questionable efficacy and there are reports of resistance. Therefore, the discovery of new, safe, effective and affordable active molecules is urgently needed. Considering that, the main purpose of this mini-review is to show the current scenario concerning drug design for neglected disease in Brazil. NTD are a very broad topic. Thus, we selected four infections for discussion: Chagas disease, leishmaniasis, malaria and tuberculosis. According to CNPq (National Counsel of Technological and Scientific Development) directories, there are many Brazilian groups studying these respective diseases. The papers published possess high quality and some of them, the most recent, are briefly discussed here. Medicinal chemistry approaches such as synthesis of novel series of molecules and biological activity evaluation, studies of structure-activity relationships (qualitative and quantitative), molecular modeling calculations and electrochemistry are some of the tools applied in the design of the compounds.

Medicinal Chemistry of New Compounds to Treat Tuberculosis.

Tuberculosis (TB), a 19th century disease, is still present in the beginning of the Third Millennium. It has been considered pandemic, since around two billion people are infected with M. tuberculosis. Multi-drug resistant TB has been the biggest challenge for chemotherapy. In order to face this severe health problem, many institutions, private and public ones, have been investing in the search for new and better drug candidates. The pipeline of potential anti-TB drugs presents new molecules and formulations that have been submitted to pre-clinical and clinical assays. Medicinal Chemistry has an important role towards the objective of finding new leads through classic and modern processes. This paper reviews some aspects of this search, emphasizing the features of the main compounds under investigation and those that are in preliminary and final clinical trials and includes the contribution of our laboratory (LAPEN) in the area of designing new anti-TB drug candidates.

Reverse phase high-performance liquid chromatography for quantification of hydroxymethylnitrofurazone in polymeric nanoparticles

Hydroxymethylnitrofurazone (NFOH) is a new compound with potential leishmanicidal and trypanocidal activity. Despite its effectiveness, the formulators have to overcome its poor aqueous solubility. Recently, polymeric nano-scale drug delivery systems have proposed for the treatment of neglected diseases. As several studies have confirmed the advantages of such formulations, and this approach provides new analytical challenges, including the need to detect trace amounts of the drug. A suitable method was developed and validated for NFOH determination bound to poly (n-butylcyanoacrylate) (PBCA) nanoparticles. The chromatographic separation was achieved using a C18 column maintained at 25 ºC and an isocratic mobile phase consisting of water and acetonitrile: 80:20 (v/v) at a flow rate of 1.2 mL min-1 and UV-detection at 265 nm. Investigated validation parameters included selectivity, linearity, accuracy, precision and robustness (changes in column temperature, mobile phase composition and flow). The method was specific, the peak of NFOH had no interference with any nanoparticle excipients and no co-elution with main degradation product (nitrofurazone). Linearity was over the range of 0.94 13.11 μg mL-1 (r2=0.999). The method was accurate and precise, recovery of 100.7%, RSD of 0.4%; intra-day and inter-day RSD range 9.98-9.99 μg mL-1 and 0.3% to 0.5%, respectively. Robustness confirmed that method could resist the applied changes. Application of the optimized method revealed an encapsulation efficiency of 64.4% (n=3). Therefore, the method was successfully developed and validated for the determination of the encapsulation efficiency of NFOH-PBCA nanoparticles.

Hidroximetilnitrofural (NFOH) é um novo composto que possui atividade leishmanicida e tripanomicida potencial. Um método apropriado foi desenvolvido e validado para a determinação de NFOH em nanopartículas de poli(n-butil cianoacrilato) (PBCA). A separação cromatográfica foi obtida usando uma coluna C18 (5 µm de tamanho de partícula, 4,6 mm de diâmetro e 150 mm de comprimento), mantida a 25 °C, fase móvel composta de água e acetonitrila 80:20 (v/v), fluxo de 1,2 mL min- 1 e detecção por UV a 265 nm. Investigaram-se os seguintes parâmetros de validação: seletividade, linearidade, exatidão, precisão e robustez (mudanças na temperatura de coluna, proporção da fase móvel e fluxo). O método mostrou-se específico, o pico de NFOH não apresentou interferência dos picos provenientes dos excipientes das nanopartículas e separado do principal produto de degradação (nitrofural). A linearidade foi obtida na faixa de 0,94-13,11 μg mL- 1 (r2=0,999). O método mostrou exatidão (recuperação de 100,7%, DPR de 0,4 %) e precisão (intra-dia e inter-dia, 9,98-9,99 μg mL- 1 e DPR 0,3% a 0,5%, respectivamente). A robustez provou que o método pode resistir às mudanças propostas. Aplicação do método otimizado revelou eficiência de encapsulação de 64,4% (n=3). Portanto, o método foi desenvolvido e validado com sucesso para a determinação da eficiência de encapsulação de nanopartículas de NFOH-PBCA.

Design, synthesis and biological evaluation of hybrid bioisoster derivatives of N-acylhydrazone and furoxan groups with potential and selective anti-Trypanosoma cruzi activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.