Despite the fact that Carlos Chagas completely described, near to a century ago [1] the vector, microorganism and clinical signs, American trypanosomiasis, remains the largest parasitic disease burden on the American continent. This illness, also known as Chagas’ disease, is caused by the trypanosomatid parasite Trypanosoma cruzi (T. cruzi). Like other neglected diseases, it is an important health problem due to inadequate therapy and the lack of an effective vaccine [2]. It has not received much attention by the pharmaceutical industry mainly due to economic considerations. Current clinical treatment of Chagas’ disease relies on two drugs (Figure 1): nifurtimox (Nfx, 3-methyl-4-(5-nitrofurfurylidene-amino)tetrahydro-4H-1,4-thiazine-1,1-dioxide, Lampit®, recently discontinued by Bayer) and benznidazole (Bnz, N-benzyl-2-(2-nitroimidazolyl)acetamide, Rochagan®, Roche, now produced by LAFEPE, Brazil) discovered empirically more than three decades ago [3]. These drugs are nitroheterocycles that present significant side effects, e.g., Nfx may cause weight loss, skin rash, psychosis, leucopenia, neurotoxicity, peripheral neuropathy, tissue abnormalities, nausea and vomiting; while Bnz may trigger edema, fever, skin rash, peripheral neuropathy, lymphadenopathy, agranulocytosis, thrombocytopenic purpura, articular and muscular pain [4]. The use of these drugs during the acute phase is widely accepted, but their efficacy in the chronic phase is still controversial. Currently, the treatment plan of chronically infected patients is based on the hypothesis that Chagasic cardiomyopathy may be triggered by persistent parasitic infection [5]. Since 2004, the BENEFIT (BENznidazole Evaluation For Interrupting Trypanosomiasis Program) multicenter study involves a randomized, double-blind, placebo-controlled, Bnz clinical trial of 3,000 patients with Chagasic cardiomyopathy from 35 centers of Latin America [6]. Similarly, Nfx, combined with eflornithine, is involved in a multicentre, randomized, open-label, active control phase III trial for treatment of patients with African Trypanosoma brucei gambiense trypanosomiasis [7].

Apart from their arguable efficacies in all the disease stages, Nfx y Bnz have showed different efficacies according to both endemic geographical areas and T. cruzi strains [8]. However the most relevant problems are their toxic and genotoxic behaviors that convert them into inappropriate drugs for treatment of any kind of disease [4,9,10,11]. Given the unsatisfactory pharmaceutical performance of the currently available drugs, new approaches to specific chemotherapy of Chagas’ disease have been advanced in the last three decades. They will be discussed in the following sections focusing in the synthetic medicinal chemistry and on those compounds at the final stage of the “hit-to-lead” phase and with possibilities of entering the clinical phase.

Medicinal chemistry, as an interdisciplinary science, has combined all its tools in the discovery of anti-Chagas drugs. Accordingly, efforts have come from biochemistry/molecular biology, computational chemistry, pharmacognosy, pharmacology, drug repositioning, and organic and inorganic chemistry areas. Studies have been done in the different stages of the drug discovery process – hit selection, synthetic development to lead identification, synthetic modifications to lead optimization, and preclinical steps – contributing in a synergistic manner allowing the identification of potential drug candidates.

The information of the complete genome sequences of T. cruzi revealed that its genome contains nearly 10,000 protein-coding genes [12]. This vast amount of new information allows the identification of targets in an accurate manner [13,14,15,16,17]. From a medicinal chemistry point of view, several potential biological targets for drugs development have been identified, e.g., geranyltransferase type I, farnesyltransferase, farnesyl pyrophosphate synthase, trans-sialidase, cAMP-specific phosphor-diesterases, polyamine and trypanothione synthetic pathways, cystein proteases, glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, membrane sterols synthetic pathways, or α-hydroxyacid dehydrogenase, but in some cases their validity and druggability as anti-Chagas targets have been placed in doubt.

Computational chemistry has reinforced the generated T. cruzi genomic/proteomic information using tools at the hit selection stage, e.g., virtual screening to identify inhibitors of specific parasite biomolecules [18,19,20], or at the lead optimization step, e.g., developing theoretical models that explain activities [21,22,23]. Figure 2 shows some examples of selected hits with specific enzymatic inhibitory activities.

Latin America vegetation has supplied a great number of active compounds where the pharmacognosts have identified relevant hits to treat Chagas’ disease. Significant leads have come from Argentine, Brazil, Bolivia, Chile, Paraguay and Peru (Figure 2) [24,25,26,27,28,29]. However, scarce examples where medicinal chemistry involve in chemical modifications to attempt improvement of the hits’ activities [29,30,31,32,33].

A great deal of work in the pharmacology/toxicology areas has been published by Argentinean, Brazilian and Chilean research teams. Castro’s group in Argentine has worked on the toxicological profile of the current anti-Chagas drugs, Nfx and Bnz [4], while the Chilean team of Morello has driven aspects related to Nfx’s mechanism of action and improvement of its activity by drug-combination [34,35]. On the other hand, the Brazilian group of de Castro has generated relevant information on experimental chemotherapies for Chagas’ disease working in vitro but also in vivo (Figure 2c, see below, Section 3) [36,37].

Drug repositioning, or drug profiling, is a medicinal chemistry tool that has also been employed in the lead identification stage for Chagas’ disease drugs. The concept of drug profiling, concerning in the research of either discontinued-, off-patent, or another-application-drug for novel indications, has been developed by Urbina from Venezuela [38]. The concept of the biological redundancy has been successfully applied by Urbina employing well-known antifungal drugs as anti-Chagas agents (Figure 2) [39]. The idea that these drugs have undergone extensive toxicological and pharmacokinetic studies support that their indication as anti-Chagas drugs would involve less risk, cost and time than conventional discovery. Based on previous reports on amiodarone’s (14, Figure 2) in vitro antifungal activity [40], Urbina found that this drug, used as an antiarrhythmic in Chagasic cardiomyopathy, also possess a synergic anti-T. cruzi effect when it is co-administered together with the antifungal posaconazole (compound 11, Figure 2) [41].

Organic and inorganic medicinal chemistry, mainly from academic centers and collaborative networks, has contributed with relevant information from the design, the synthesis, the structural modifications optimizing identified-hits, and the structure-activity relationships. Some of these results and approaches will be discussed in the following section describing those agents emerge from the “active-to-hit” stage.

A significant number of candidates, from the “active-to-hit” phase, have been submitted to some of the subsequent studies corresponding to the “hit-to-lead” stage. The compounds will be described according to the laboratory and country origins, and aspects related to their drug-like properties –e.g., oral bioavailability, toxicity, mutagenicity- will be analyzed and discussed.

From the Laboratório de Biologia Celular-Instituto Oswaldo Cruz in Brazil, the ergosterol inhibitor compound 54 (as a geometric isomers-mixture, Figure 4) has been studied in a model of infected animals (oral administration of 5 mg/kg b.w. for nine consecutive days, starting on the day of inoculation). This compound produced a consistent suppression of parasitemia combined with full protection against death, 100% of animals’ survival at the end of the assay vs 75% for Bnz-treated animals (at 100 mg/kg b. w.) [107,108]. On the other hand, the diamidines 55 and 56 (Figure 4), active in vitro against the different forms of the parasite, have been studied in vivo using different drug-administration schedules by i.p. route [109,110,111]. Compound 55 reduced cardiac parasite load and down-modulated the expression of CD8⁺ T cells in the heart tissues that revert the electrocardiography alterations in acute and chronic infection, leading to an increase in the animals’ survival rates. The best results with diamidine 56 were obtained when the animals received 25 mg/kg b.w. in two intervals, one at the onset and the second at the parasitemia peak, with 100% of animals’ survival, reduction of the cardiac parasitism although only a reduction of 40% in the parasitemia levels. These classes of compounds deserve further investigation, for example pharmacokinetic properties and metabolic stability, based on the adequate anti-T. cruzi in vivo results.

From University of São Paulo, Brazil, Franco’s team has worked in the inorganic medicinal chemistry field evaluating in vivo ruthenium complexes 57, 58 and 59 (Figure 4) [112,113]. Complexes 57 and 58 have been developed as NO-releasing compounds and they probed to be active in vitro against the parasite while in vivo results, i.p. administration of 100 nmol of each compound/kg b.w. daily for 15 consecutive days, showed that they were discrete in both the decreasing of the parasitemia and the animals’ survival rates with decreasing the occurrence of myocarditis (mainly for 58). Complex 59, synthesized using imidazole-nitrogen from Bnz as coordinating atom, has been evaluated in vivo for its toxicity -LD₅₀ using up-and-down test protocol- and for its trypanonosomicidal activity in a murine model of acute Chagas’ disease. The antiparasitic studies were done using different dosage’ schedules, e.g., oral and i.p. administration and doses of 100 nmol/kg b.w./day or 385 nmol/kg b.w./day, at least 1,000-fold lower than LD₅₀, for 15 consecutive days or for only three days, the fifth, sixth, and seventh days that precede the parasitaemic peak. Compound 59 proved to protect the infected animals eliminating, according to the micrographs, the hearts and skeletal muscles amastigotes. This compound is hydrosoluble but no information about pharmacokinetic properties and metabolic stability was mentioned.

Maldonado’s laboratory, from The University of Texas (United States) has evaluated in vivo the competitive inhibitor of T. cruzi lipoamide dehydrogenase 60 (Figure 4) [114,115]. The naphthoquinone was administered 24 hours post-infection -10 mg/kg b.w./ initially one daily i.p. dose for 3 consecutive days, and then 2 more doses, with an interval of 1 day between the doses- observing depletion in parasitemia and animals’ survival up to 60% at 70 days post-infection without apparent signs of toxicity on the uninfected treated animals.

Compounds 20 and 21 (K777 and WRR483, respectively, Figure 3), from Sandler Center (United States) have been analyzed in different models of acute Chagas’ disease, i.e. mice and dogs [116,117,118]. For example, when 20 was administered orally at 50 mg/kg b. w. twice daily for 14 days, to infected dogs the myocardial damage was ameliorated according to serum cardiac troponin I levels and histopathology findings. This compound, which is water soluble, orally bioavailable (20%), neither toxic nor mutagenic [119] and produces an additive effect on parasite killing when used in combination with Bnz [54], has now entered formal preclinical drug development investigations [54]. On the other hand, the vinyl-sulfone 21, an analog of the parent 20 contains a protonizable arginine side-chain that turn it into a better cruzipain inhibitor with activity against the parasite in culture and in an animal model of disease [54].

Compound 26 (Figure 3), from the synthetic medicinal chemistry projects at the University of Washington (United States) had the best in vivo profile when it was administered orally, at 20 or 50 mg/kg b.w. twice per day for 21 days, to mice in an acute model of Chagas’ disease [65]. From the pharmacokinetic studies the authors have not observed significant 26 losses out to 5 h, showing its mouse plasma stability. The animals tolerated the drug treatments without apparent side effects however studies of toxicity/mutagenicity were not done. Although the in vivo activity of 26 was lower than that of posaconazole (11, Figure 2), its synthesis is much simpler and its potential cost of goods lower.

Urbina, initially from Instituto Venezolano de Investigaciones Científicas (Venezuela) and now at the University of Cincinnati in the United States, has worked in vivo with well-known ergosterol biosynthesis inhibitors. The antifungals terbinafine, ketoconazole or itraconazole displayed suppressive, but not curative, effects against T. cruzi infections in animal models of Chagas’ disease [120]. However, triazoles (11–13, Figure 2; 61 and 62, Figure 4) selective inhibitors of fungal and protozoan cytochrome P-450-dependent CYP51 were potent inducing radical parasitological cure in murine models of acute and chronic Chagas’ disease [121,122,123,124,125,126,127]. The remarkable in vivo antiparasitic activities of some of them result from a combination of their potent and selective intrinsic anti-T. cruzi activity and special pharmacokinetic properties (long terminal half-life and large volumes of distribution). For example, posaconazole, 11, has terminal phase half-life of 7–9 h in mice and rats [128]. On the other hand, azole 61 has very short half-life in mice but working in a canine model demonstrated curative activity against established infections of the virulent Y strain of T. cruzi with very low toxicity but drug resistance was encountered with the Berenice-78 strain [39]. Another example of inadequate pharmacokinetic properties in mice was evidenced in the treatment with 62 however these results do not necessarily rule out the potential usefulness of this compound in the treatment of human T. cruzi infections since its activity is very high. According to Urbina, triazoles 11 and 62 would be entering into the clinical studies for the treatment of human chronic Chagas disease in the next 12 months [39] (http://www.dndi.org/press-releases/532-eisai-and-dndi-enter-into-a-collaboration.html) moreover taking in account that 11 has been demonstrated to be active in a case of a Chagas’ disease patient compromised with systemic lupus erythematosus [129].

Another interesting group of compounds that have been studied in vivo by Urbina are the quinuclidine derivatives (e.g., 63 and 64, Figure 4) [130] under development for cholesterol control in humans. The in vivo behavior of compound 63 resulted better than that of 64 producing animal survival rates of 100% and arresting development of parasitemia in the studied murine model of acute disease and in one of the assayed condition, oral treatment at a concentration of 50 mg/kg of b. w./day for 30 consecutive days. The better antiparasitic properties of 63 could be due to its better pharmacokinetic properties, with lower clearance rate in rats than 64 and wide tissue distribution, such as heart, spleen, liver, and instestines [130].

Others examples that should be mentioned for their in vivo relevance are the approaches with products isolated from natural resources. One example comes from Paraguay [25] where canthin-6-one (4, Figure 2), isolated from Zanthoxylum chiloperone as well as the total alkaloids extract from stem bark led parasitological clearance in the experimental conditions, oral or subcutaneal administration at 5mg/kg b. w./day for 2 weeks for the pure product and 50mg/kg b. w./day for 2 weeks for alkaloidal extract. Another example is the sesquiterpene lactone 5 (Figure 2) isolated from Ambrosia tenuifolia Sprengel (Asteraceae) that decreased the parasitemia levels during the experiment and promoted an animals’ survival rate of 100% when it was administered i.p. at 1 mg/kg b. w./day for five consecutive days [26].

Our group at Universidad de la República (Uruguay), in collaboration with Argentinean and Paraguayan teams has assessed nineteen nitroheterocyclic derivatives and ten N-oxide derivatives in different murine models of acute Chagas’ disease [131,132,133,134,135,136]. In our first approach, we found that each 5-nitrofuran derivative was more in vivo active than the corresponding 5-nitrothiophene analogue, e.g., comparing animals’ survival rates of derivatives 65, 66, 67 and 68 (Figure 5) where they were administered orally during 10 days at 66 mg/kg b. w./day. After that, a series of ten 5-nitrofurans were tested oral and intraperitoneally. Based on the toxicity on murine models investigated with a single administration of 7.5- and 6-fold the therapeutic dose (60 mg/kg b. w.) we selected three among the new derivatives (71, 74, and 75 Figure 5) for testing in vivo anti-T. cruzi activities. A clear relationship between chemical structure and both acute toxicity and in vivo anti-T. cruzi activity was observed, showing that derivatives 65, 67, 69, and 70 promoted higher toxicity, in healthy animals, than derivatives 71–73 and carbazate 74 (Figure 5) was lesser in vivo anti-trypomastigote agent than the semicarbazones 65, 67, and 71, or the amide 75 (Figure 5). Derivative 71 showed an excellent anti-T. cruzi profile for the smooth muscle with adequate profile in the cardiac one. Unfortunately, derivatives 71, 74, and 75 have demonstrated mutagenicity in the Ames test (against S. Typhimurium TA 98) with and without S9 fraction (unpublished data). The 5-nitroindazoles 76 and 77 were selected, for their lack of in vitro unspecific cytotoxicity, to be evaluated in a murine model of acute Chagas’ disease, oral administration at 30 mg/kg b. w./day for 10 days (using Bnz as positive control at 50 mg/kg b. w./day for 10 days). Taking into account that the doses of the tested compounds were very different, 78 mmol/kg b. w./day for 76 and 77 and 192 mmol/ kg b. w./day for Bnz, compound 76 showed better in vivo performance than the reference drug, Bnz, in animals’ survival rates and anti-T. cruzi antibody levels. On the other hand, according to parasitemia, animals’ survival rates and antibodies levels compound 77 have an in vivo behavior as good as Bnz. Although further structural modifications and deeper pharmaceutical studies have been done [137,138], this class of compounds deserves further investigation based on the adequate anti-T. cruzi in vivo results. The same experimental protocol has been used to evaluate the benzimidazole di-N-oxides 78 and 79 (Figure 5) where both compounds were able to rescue mice from death and lowered anti-T. cruzi antibodies levels, especially 78, with only 10 doses in a short-term scheme. Regrettably, compound 78 has demonstrated mutagenicity in the Ames test (against S. Typhimurium TA 98) in absence of metabolic activation (unpublished data). Pharmacokinetic and metabolic-stability studies for compounds 65–77 were not performed.

Benzofuroxans 80–82 (as geometric isomer mixtures, Figure 5), developed as T. cruzi cruzipain inhibitors, have been evaluated in an acute model of Chagas’ disease, oral administration at 60 mg/kg b. w./day for 30 days (6 day treatment, 1 day rest, until 30 doses completed). The mixture of isomers 81 has the best in vivo profile among the three tested benzofuroxans and better than Bnz considering the antibody levels. Furthermore, no signs of toxicity were observed in the studied animals, during the treatment, and none of the animals treated with 80–82 died during the treatment while in the control group the survival rate was 75%. Following the same experimental protocol, and as DNDi project, benzofuroxans 83–88 (Figure 5), and the geometric isomers-mixtures, have been studied in vivo against different T. cruzi strains, e.g., Tulahuen 2 and Colombiana (resistant to Nifurtimox and Benznidazole) strains, and two wild strains, one isolated from the wild reservoir Didelphis marsupialis and another one from a Uruguayan patient. The results showed that 83 and the equimolecular mixture 83:84 were able to reduce the parasite loads of animals with fully established T. cruzi infections with, in some cases, comparable results to that obtained with both Nfx and Bnz. The results of histological studies indicated that they could be adequate agents in preventing inflammatory infiltrates and tissue damage, particularly in heart of infected animals. The main problem found in these studies was the low compound solubility in the chosen vehicles. Furthermore, according to the acute-toxicity study administration of compound 83, at high doses, did not produce remarkable damage on the animals. As part of the research project supported by DNDi organization others preclinical studies with 83–88, e.g., scaling-up and analytical procedures, in vitro metabolism and toxicity, have been performed [139,140,141,142,143]. The mutagenic studies have indicated that benzofuroxans 81, 82 (data not published), 83, 85–88 are mutagenic, in some cases with metabolic activation, like Nfx and Bnz. Derivatives 80, 84 and the equimolecular mixture 83:84 were not-mutagenic in both conditions, unfortunately the isomer 84 is metabolized in vitro, and probably in vivo, to 83 by hepatocyte microsomal and cytosolic fractions. In deep studies of the metabolites responsible of mutagenicity for derivative 83, one of the main metabolite, 89, and not the other, 90, is mutagenic with S9 activation.

In conclusion, there is currently a number of candidates for preclinical and clinical trials for the identification of new effective anti-Chagas’ drugs, guaranteeing tolerability, safety profile, and selectivity. Another relevant aspect that should be studied and considered is the cost of production of the new agents that could guarantee patient access to these drugs.