Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic inhibitor with an IC50 value of 86 nm without inhibition effects on the mammalian enzyme. These molecules also affected cruzipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these derivatives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an important feature in the research and development of future drugs for neglected diseases.

Nematode parasites have a profound impact on humankind, infecting nearly one-quarter of the world’s population, as well as livestock. There is a pressing need for discovering nematicides due to the spread of resistance to currently used drugs. The free-living nematode Caenorhabditis elegans is a formidable experimentally tractable model organism that offers key advantages in accelerating nematicide discovery. We report the screening of drug-like libraries using an overnight high-throughput C. elegans assay, based on an automated infrared motility reader. As a proof of concept, we screened the “Pathogen Box” library, and identical results to a previous screen using Haemonchus contortus were obtained. We then screened an in-house library containing a diversity of compound families. Most active compounds had a conjugation of an unsaturation with an electronegative atom (N, O, or S) and an aromatic ring. Importantly, we identified symmetric arylidene ketones and aryl hydrazine derivatives as novel nematicides. Furthermore, one of these compounds, (1E,2E)-1,2-bis(thiophen-3-ylmethylene)hydrazine, was active as a nematicide at 25 µm, but innocuous to the vertebrate model zebrafish at 50 µm. Our results identified novel nematicidal scaffolds and illustrate the value of C. elegans in accelerating nematicide discovery using a nonlabor-intensive automated assay that provides a simple overnight readout.

A series of fifty arylideneketones and thiazolidenehydrazines was evaluated against Leishmania infantum and Leishmania braziliensis. Furthermore, new simplified thiazolidenehydrazine derivatives were evaluated against Trypanosoma cruzi. The cytotoxicity of the active compounds on non-infected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their anti-parasitic effects. Seven thiazolidenehydrazine derivatives and ten arylideneketones had good activity against the three parasites. The IC50 values for T. cruzi and Leishmania spp. ranged from 90 nM–25 µM. Eight compounds had multi-trypanocidal activity against T. cruzi and Leishmania spp. (the etiological agents of cutaneous and visceral forms). The selectivity of these active compounds was better than the three reference drugs: benznidazole, glucantime and miltefosine. They also had low toxicity when tested in vivo on zebrafish. Trying to understand the mechanism of action of these compounds, two possible molecular targets were investigated: triosephosphate isomerase and cruzipain. We also used a molecular stripping approach to elucidate the minimal structural requirements for their anti-T. cruzi activity.

Nitric oxide (NO) represents a valuable target to design antitrypanosomal agents by its high toxicity against trypanosomatids and minimal side effects on host macrophages. The progress of NO-donors as antitrypanosomal has been restricted by the high toxicity of their agents, which usually is based on NOheterocycles and metallic NO-complexes. Herein, we carried out the design of a new class of NO-donors based on the susceptibility of the hydrazine moiety connected to an electron-deficient ring to be reduced to the amine moiety with release of NO. Then, a series of novel 2-arylquinazolin-4-hydrazine, with the potential ability to disrupt the parasite folate metabolism, were synthesized. Their in vitro evaluation against Leishmania and Trypanosoma cruzi parasites and mechanistic aspects were investigated. The compounds displayed significant leishmanicidal activity, identifying three potential candidates, that is, 3b, 3c, and 3f, for further assays by their good antiamastigote activities against Leishmania braziliensis, low toxicity, non-mutagenicity, and good ADME profile. Against T. cruzi parasites, derivatives 3b, 3c, and 3e displayed interesting levels of activities and selectivities. Mechanistic studies revealed that the 2arylquinazolin-4-hydrazines act as either antifolate or NO-donor agents. NMR, fluorescence, and theoretical studies supported the fact that the quinazolin-hydrazine decomposed easily in an oxidative environment via cleavage of the N−N bond to release the corresponding heterocyclic-amine and NO. Generation of NO from axenic parasites was confirmed by the Griess test. All the evidence showed the potential of hydrazine connected to the electron-deficient ring to design effective and safe NO-donors against trypanosomatids.

Chagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as public health problems by the World Health Organization. These diseases affect millions of people around the world however, efficient and low-cost treatments are not available. Different steroid molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks, plants, fungi). These molecules have complex structures that make de novo synthesis extremely difficult. In this work, we designed new and simpler compounds with antiparasitic potential inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound 8 (ID_1260) was active in vitro (IC50 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD50 >2000 mg/kg) and no genotoxic effects, being a promising compound for anti-trypanosomatid drug development.