Design, synthesis and biological evaluation of new oxazines with potential antiparasitic activity

A series of new oxazines with potential antiparasitic activity was prepared using Diels-Alder reactions, based on terpenes derived from eucarvone as dienes and nitrosoarenes with different electronic characteristics as dienophiles. The biological activity was evaluated with in vitro assays against Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei rhodesiense. Some of these oxazines have activities in the 20-50 μM range, and may be leaders for the development of novel antiparasitic drugs with improved pharmacological properties.     

Design,Synthesis and Biological Evaluation of Potent Azadipeptide Nitrile Inhibitors and Activity‐Based Probes as Promising Anti‐Trypanosoma brucei Agents

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small‐molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain‐like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile‐containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target‐based screening) and trypanocidal activity (i.e., whole‐organism‐based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity‐based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease‐relevant bloodstream form (BSF) and the insect‐residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti‐trypanosome agents, which possess better activities than existing drugs. The activity‐based probes generated from this study could also serve as valuable tools for parasite‐based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile‐containing compounds as potential anti‐parasitic agents.     

Structural Optimization and Biological Activity of Pyrazole Derivatives: Virtual Computational Analysis,Recovery Assay and 3D Culture Model as Potential Predictive Tools of Effectiveness against Trypanosoma cruzi

Chagas disease, a chronic and silent disease caused by Trypanosoma cruzi, is currently a global public health problem. The treatment of this neglected disease relies on benznidazole and nifurtimox, two nitroheterocyclic drugs that show limited efficacy and severe side effects. The failure of potential drug candidates in Chagas disease clinical trials highlighted the urgent need to identify new effective chemical entities and more predictive tools to improve translational success in the drug development pipeline. In this study, we designed a small library of pyrazole derivatives (44 analogs) based on a hit compound, previously identified as a T. cruzi cysteine protease inhibitor. The in vitro phenotypic screening revealed compounds 3g, 3j, and 3m as promising candidates, with IC₅₀ values of 6.09 ± 0.52, 2.75 ± 0.62, and 3.58 ± 0.25 µM, respectively, against intracellular amastigotes. All pyrazole derivatives have good oral bioavailability prediction. The structure–activity relationship (SAR) analysis revealed increased potency of 1-aryl-1H-pyrazole-imidazoline derivatives with the Br, Cl, and methyl substituents in the para-position. The 3m compound stands out for its trypanocidal efficacy in 3D microtissue, which mimics tissue microarchitecture and physiology, and abolishment of parasite recrudescence in vitro. Our findings encourage the progression of the promising candidate for preclinical in vivo studies.     

In silico identification and evaluation of new Trypanosoma cruzi trypanothione reductase (TcTR) inhibitors obtained from natural products database of the Bahia semi-arid region (NatProDB)

Trypanosoma cruzi Trypanothione Reductase (TcTR) is one of the therapeutic targets studied in the development of new drugs against Chagas' disease. Due to its biodiversity, Brazil has several compounds of natural origin that were not yet properly explored in drug discovery. Therefore, we employed the Virtual Screening against TcTR aiming to discover new inhibitors from the Natural Products Database of the Bahia Semi-Arid region (NatProDB). This database has a wide chemical diversity favoring the discovery of new chemical entities. Subsequently, we analyzed the best docking conformations using self-organizing maps (AuPosSOM) aiming to verify their interaction sites at TcTR. Finally, the Pred-hERG, the Aggregator Advisor, the FAF-DRUGS and the pkCSM results allowed us to evaluate, respectively, the cardiotoxicity, aggregation capacity, presence of false positives (PAINS) and its toxicity. Thus, we selected three molecules that could be tested in in vitro assays in the hope that the computational results reported here would favor the development of new anti-chagasic drugs.     

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.     

Synthesis and Evaluation of Trypanocidal Activity of Chromane-Type Compounds and Acetophenones

American trypanosomiasis (Chagas disease) caused by the Trypanosoma cruzi parasite, is a severe health problem in different regions of Latin America and is currently reported to be spreading to Europe, North America, Japan, and Australia, due to the migration of populations from South and Central America. At present, there is no vaccine available and chemotherapeutic options are reduced to nifurtimox and benznidazole. Therefore, the discovery of new molecules is urgently needed to initiate the drug development process. Some acetophenones and chalcones, as well as chromane-type substances, such as chromones and flavones, are natural products that have been studied as trypanocides, but the relationships between structure and activity are not yet fully understood. In this work, 26 compounds were synthesized to determine the effect of hydroxyl and isoprenyl substituents on trypanocide activity. One of the compounds showed interesting activity against a resistant strain of T. cruzi, with a half effective concentration of 18.3 µM ± 1.1 and an index of selectivity > 10.9.     

Protein–ligand interaction of T. cruzi trans-sialidase inhibitors: a docking and QM/MM MD study

The trans-Sialidase from Trypanosoma cruzi (TcTS) might be a key enzyme in search of new and more effective anti-Trypanosoma cruzi agents. In this report, molecular docking and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations were employed to determine the binding mode of the three TcTS inhibitors, two quinolinones derivatives (DHQ and THQ) and DANA. The results show that the sulfone group from THQ plays an important role in the protein-inhibitor interactions. In addition, a detailed analysis of the interactions of these inhibitors with key residues inside the binding pocket of TcTS has been carried out using AM1/MM. The residues Asp59, Asp247, Arg35, Arg245, and Arg314 are appointed as key residues to affinity energy of the complexes studied. Finally, the B3LYP/MM and AM1/MM methods were used to calculate global interaction energy, in order to understand the potential of these inhibitors. Among the inhibitors studied, THQ is confirmed as the most efficient one for inhibiting TcTS.     

Solid-phase microextraction in bioanalysis: New devices and directions

The primary objective of this review is to discuss recent technological developments in the field of solid-phase microextraction that have enhanced the utility of this sample preparation technique in the field of bioanalysis. These developments include introduction of various new biocompatible coating phases suitable for bioanalysis, such as commercial prototype in vivo SPME devices, as well as the development of sampling interfaces that extend the use of this methodology to small animals such as mice. These new devices permit application of in vivo SPME to a variety of analyses, including pharmacokinetics, bioaccumulation and metabolomics studies, with good temporal and spatial resolution. New calibration approaches have also been introduced to facilitate in vivo studies and provide fast and quantitative results without the need to achieve equilibrium. In combination with the drastic improvement in the analytical sensitivity of modern liquid chromatography–tandem mass spectrometry instrumentation, full potential of in vivo SPME as a sample preparation tool in life sciences can finally be explored. From the instrumentation perspective, SPME was successfully automated in 96-well format for the first time. This opens up new opportunities for high-throughput applications (>1000 samples/day) such as for the determination of unbound and total drug concentrations in complex matrices such as whole blood with no need for sample pretreatment, studies of distribution of drugs in various compartments and/or determination of plasma protein binding and other ligand–receptor binding studies, and this review will summarize the progress in this research area to date.     

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH⁺ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding β-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit μM EC₅₀ against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH⁺-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.     

Synthesis of oligosaccharide fragments of the glycosylinositolphospholipid of Trypanosoma cruzi: a new selenoglycoside glycosyl donor for the preparation of 4-thiogalactofuranosyl analogues

A new selenoglycoside, phenyl 2,3,5,6-tetra-O-acetyl-4-thio-1-selenogalactofuranose, has been synthesized. This 4-thiogalactofuranosyl donor was used in the syntheses of heteroatom analogues of the di-, tri-, and tetrasaccharides corresponding to the oligosaccharide β-d-Galf-(1→3)-α-d-Manp-(1→2)-(β-d-Galf-(1→3))-α-d-Manp. These compounds represent fragments of the terminal end of the glycosylinositolphospholipid oligosaccharide found in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, and are intended for use as inhibitors of the enzymes that construct the native oligosaccharides. The syntheses employed the selective activation of a phenyl 4-thio-1-selenogalactofuranoside glycosyl donor over ethyl 1-thioglycoside glycosyl acceptors with NIS/TfOH.     

SARs for the Antiparasitic Plant Metabolite Pulchrol. 3. Combinations of New Substituents in A/B-Rings and A/C-Rings

The natural products pulchrol and pulchral, isolated from the roots of the Mexican plant Bourreria pulchra, have previously been shown to possess antiparasitic activity towards Trypanosoma cruzi, Leishmania braziliensis and L. amazonensis, which are protozoa responsible for Chagas disease and leishmaniasis. These infections have been classified as neglected diseases, and still require the development of safer and more efficient alternatives to their current treatments. Recent SARs studies, based on the pulchrol scaffold, showed which effects exchanges of its substituents have on the antileishmanial and antitrypanosomal activity. Many of the analogues prepared were shown to be more potent than pulchrol and the current drugs used to treat leishmaniasis and Chagas disease (miltefosine and benznidazole, respectively), in vitro. Moreover, indications of some of the possible interactions that may take place in the binding sites were also identified. In this study, 12 analogues with modifications at two or three different positions in two of the three rings were prepared by synthetic and semi-synthetic procedures. The molecules were assayed in vitro towards T. cruzi epimastigotes, L. braziliensis promastigotes, and L. amazonensis promastigotes. Some compounds had higher antiparasitic activity than the parental compound pulchrol, and in some cases even benznidazole and miltefosine. The best combinations in this subset are with carbonyl functionalities in the A-ring and isopropyl groups in the C-ring, as well as with alkyl substituents in both the A- and C-rings combined with a hydroxyl group in position 1 (C-ring). The latter corresponds to cannabinol, which indeed was shown to be potent towards all the parasites.     

Synthesis, characterization and anti-Trypanosoma cruzi evaluation of ferrocenyl and cyrhetrenyl imines derived from 5-nitrofurane

A series of new cyrhetrenyl and ferrocenylimines complexes derived from 5-nitrofurane were designed, synthesized and characterized. The ¹H and ¹³C NMR spectra indicate that these compounds adopt an anti-(E) conformation in solution, and confirmed for 1b by the X-ray crystal crystallography. The electronic effects of cyrhetrenyl (1b) and ferrocenyl (1a) bound directly to nitrogen have been correlated with the chemical shift of the iminic carbon. The trypanocidal activity (Tulahuen strain of Trypanosoma cruzi) of these compounds has been studied with respect to the substituent on the nitrogen atom of the 5-nitrofurfurylideneamino pharmacophore. Even though all the resulting derivatives were less active than Nifurtimox, the cyrhetrenyl complex (1b), compared with ferrocenyl (1a) or purely organic (4a and 4b) analogues, was more efficient as antichagasic agent. This result is likely due to the enhanced lipophilic character of the molecules or from a possible synergy between the cyrhetrenyl and 5-nitrofurane groups.     

New techniques and strategies in drug discovery

Great success has been witnessed in last decades, some new techniques and strategies have been widely used in drug discovery. In this roadmap, several representative techniques and strategies are highlighted to show recent advances in this filed. (A) A DOX protocol has been developed for accurate protein-ligand binding structure prediction, in which first principle method was used to rank the binding poses. Validation against crystal structures have found that DOX prediction achieved an impressive success rate of 99%, indicating significant improvement over molecular docking method. (B) Virtual target profiling is a compound-centric strategy enabling a parallel implementation of interrogating compounds against various targets in a single screen, which has been used in hit/lead identification, drug repositioning, and mechanism-of-action studies. Current and emerging methods for virtual target profiling are briefly summarized herein. (C) Research on targeted autophagy to treat diseases has received encouraging progress. However, due to the complexity of autophagy and disease, experimental and in silico methods should be performed synergistically for the entire process. This part focuses on in silico methods in autophagy research to promote their use in medicinal research. (D) Histone deacetylases (HDACs) play important roles in various biological functions through the deacetylation of lysine residues. Recent studies demonstrated that HDACs, which possess low deacetylase activities, exhibited more efficient defatty-acylase activities. Here, we review the defatty-acylase activity of HDACs and describe examples for the design of isoform selective HDAC inhibitor. (E) The FDA approval of three kinase allosteric inhibitors and some others entering clinical study has spurred considerable interests in this targeted drug discovery area. (F) Recent advances are reviewed in structure-based design of novel antiviral agents to combat drug resistance. (G) Since nitric oxide (NO) exerts anticancer activity depending on its concentration, optimal levels of NO in cancer cells is desirable. In this minireview, we briefly describe recent advances in the research of NO-based anticancer agents by our group and present some opinions on the future development of these agents. (H) The field of photoactivation strategies have been extensively developed for controlling chemical and biological processes with light. This review will summarize and provide insight into recent research advances in the understanding of photoactivatable molecules including photoactivatable caged prodrugs and photoswitchable molecules.     

Electrochromic conjugated polyheterocycles and derivatives--highlights from the last decade towards realization of long lived aspirations

Polymer electrochromism has been considered one of the liveliest branches of conducting polymer research, a tradition continued in the last decade. We have witnessed numerous significant advances, making commercial applications closer than ever. This feature article highlights these advances by separating them into 3 sections. The material design section emphasizes the new molecular structures that have been utilized as electrochromic materials and their promising results. The color control of polymeric electrochromics section focuses on the recent achievements towards realization of full color electrochromic display devices, lastly the advances en route commercial applications section demonstrates how some of the major drawbacks towards commercialization have been successfully addressed.     

Manganese(II) complexes with N-methyl-4-nitrobenzaldehyde, N-methyl-4-nitroacetofenone,and N-methyl-4-nitrobenzophenone thiosemicarbazone: Investigation of in vitro activity against Trypanosoma cruzi

Thiosemicarbazones are known to be active against different pathogenic microorganisms including Trypanosoma cruzi, the etiological agent of Chagas disease. In the search for new therapeutic drugs against this illness, the complexes [Mn(H4NO₂Fo4M)₂Cl₂] (1), [Mn(H4NO₂Ac4M)₂Cl₂] (2) and [Mn(H4NO₂Bz4M)₂Cl₂] (3) of N⁴-methyl-4-nitrobenzaldehyde thiosemicarbazone (H4NO₂Fo4M), N⁴-methyl-4-nitroacetophenone thiosemicarbazone (H4NO₂Ac4M) and N⁴-methyl-4-nitrobenzophenone thiosemicarbazone (H4NO₂Bz4M) were obtained and screened in vitro against bloodstream and intracellular forms of T. cruzi. H4NO₂Fo4M, H4NO₂Ac4M and their Mn(II) complexes displayed poor effect on bloodstream trypomastigotes, with IC₅₀ values ranging from 68 to >200 μM. However, although H4NO₂Bz4M was also not active, its corresponding Mn(II) complex presented high effect on this T. cruzi form, with an IC₅₀ value of 19 μM. The effect of complex (3), against trypomastigotes of T. cruzi supports further in vitro as well as in vivo studies.     

Synthesis and Anti-Trypanosoma cruzi Biological Evaluation of Novel 2-Nitropyrrole Derivatives

Human American trypanosomiasis, called Chagas disease, caused by T. cruzi protozoan infection, represents a major public health problem, with about 7000 annual deaths in Latin America. As part of the search for new and safe anti-Trypanosoma cruzi derivatives involving nitroheterocycles, we report herein the synthesis of ten 1-substituted 2-nitropyrrole compounds and their biological evaluation. After an optimization phase, a convergent synthesis methodology was used to obtain these new final compounds in two steps from the 2-nitropyrrole starting product. All the designed derivatives follow Lipinski’s rule of five. The cytotoxicity evaluation on CHO cells showed no significant cytotoxicity, except for compound 3 (CC₅₀ = 24.3 µM). Compound 18 appeared to show activity against T. cruzi intracellular amastigotes form (EC₅₀ = 3.6 ± 1.8 µM) and good selectivity over the vero host cells. Unfortunately, this compound 18 showed an insufficient maximum effect compared to the reference drug (nifurtimox). Whether longer duration treatments may eliminate all parasites remains to be explored.     

Isolation and Structural Elucidation of Compounds from Pleiocarpa bicarpellata and Their In Vitro Antiprotozoal Activity

Species of the genus Pleiocarpa are used in traditional medicine against fever and malaria. The present study focuses on the isolation and identification of bioactive compounds from P. bicarpellata extracts, and the evaluation of their antiprotozoal activity. Fractionation and isolation combined to LC-HRMS/MS-based dereplication provided 16 compounds: seven indole alkaloids, four indoline alkaloids, two secoiridoid glycosides, two iridoid glycosides, and one phenolic glucoside. One of the quaternary indole alkaloids (7) and one indoline alkaloid (15) have never been reported before. Their structures were elucidated by analysis of spectroscopic data, including 1D and 2D NMR experiments, UV, IR, and HRESIMS data. The absolute configurations were determined by comparison of the experimental and calculated ECD data. The extracts and isolated compounds were evaluated for their antiprotozoal activity towards Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum, as well as for their cytotoxicity against rat skeletal myoblast L6 cells. The dichloromethane/methanol (1:1) root extract showed strong activity against P. falciparum (IC₅₀ value of 3.5 µg/mL). Among the compounds isolated, tubotaiwine (13) displayed the most significant antiplasmodial activity with an IC₅₀ value of 8.5 µM and a selectivity index of 23.4. Therefore, P. bicarpallata extract can be considered as a source of indole alkaloids with antiplasmodial activity.     

Coibanoles, a new class of meroterpenoids produced by Pycnoporus sanguineus

Three new terpenoids of mixed biosynthetic origin were isolated from the culture filtrate of the endophytic fungus Pycnoporus sanguineus. Their structures were determined by extensive spectroscopic analyses. We have named these tricyclic and tetracyclic metabolites ‘coibanoles A-C’ in reference to Coiba Island and Coiba National Park, Panamá, from which the plant and endophyte were collected. The extract was inactive to the human parasites Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum at a test concentration of 10 μg/mL.     

Heteroleptic Oxidovanadium(V) Complexes with Activity against Infective and Non-Infective Stages of Trypanosoma cruzi

Five heteroleptic compounds, [V^VO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC₅₀ values of 0.29–3.02 μM. IN ligand and the new [V^VO₂(IN-H)] complex showed negligible activity. The most active compound [V^VO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [V^VO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC₅₀ values of [V^VO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 μM concentration of [V^VO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.     

Antimony(III) complexes with pyridine-derived thiosemicarbazones: Structural studies and investigation on the antitrypanosomal activity

The antimony(III) complexes [Sb(2Fo4Ph)Cl₂] (1), [Sb(2Ac4Ph)Cl₂] (2) and [Sb(2Bz4Ph)Cl₂] (3) were prepared with N(4)-phenyl-2-formyl- (H2Fo4Ph), 2-acetyl- (H2Ac4Ph) and 2-benzoylpyridine (H2Bz4Ph) thiosemicarbazones. The antimony(III) complexes presented antitrypanosomal activity against the epimastigote and trypomastigote forms of Trypanosoma cruzi. Complexes (1) and (2) exhibited higher activity than the reference drugs benznidazole and nifurtimox.