Promising hit compounds against resistant trichomoniasis: Synthesis and antiparasitic activity of 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles.

A series of 11 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles (2-12) has been prepared starting from 1-benzyl-5-nitroindazol-3-ol 13, and evaluated against sensitive and resistant isolates of the sexually transmitted protozoan Trichomonas vaginalis. Compounds 2, 3, 6, 9, 10 and 11 showed trichomonacidal profiles with IC50 < 20 µM against the metronidazole-sensitive isolate. Moreover, all these compounds submitted to cytotoxicity assays against mammalian cells exhibited low non-specific cytotoxic effects, except compounds 3 and 9 which displayed moderate cytotoxicity (CC50 = 74.7 and 59.1 µM, respectively). Those compounds with trichomonacidal effect were also evaluated against a metronidazole-resistant culture. Special mention deserve compounds 6 and 10, which displayed better IC50 values (1.3 and 0.5 µM respectively) than that of the reference drug (IC50 MTZ = 3.0 µM). The high activity of these compounds against the resistant isolate reinforces the absence of cross-resistance with the reference drug. The remarkable trichomonacidal results against resistant T. vaginalis isolates suggest the interest of 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles to be considered as good prototypes to continue in the development of new drugs with enhanced trichomonacidal activity, aiming to increase the non-existent drugs to face clinical resistance efficiently for those patients in whom therapy with 5-nitroimidazoles is contraindicated.

Activity profile of two 5-nitroindazole derivatives over the moderately drug-resistant Trypanosoma cruzi Y strain (DTU TcII): in vitro and in vivo studies.

In previous studies, we have identified several families of 5-nitroindazole derivatives as promising antichagasic prototypes. Among them, 1-(2-aminoethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one, (hydrochloride) and 1-(2-acetoxyethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one (compounds 16 and 24, respectively) have recently shown outstanding activity in vitro over the drug-sensitive Trypanosoma cruzi CL strain (DTU TcVI). Here, we explored the activity of these derivatives against the moderately drug-resistant Y strain (DTU TcII), in vitro and in vivo. The outcomes confirmed their activity over replicative forms, showing IC50 values of 0.49 (16) and 5.75 µm (24) towards epimastigotes, 0.41 (16) and 1.17 µm (24) against intracellular amastigotes. These results, supported by the lack of toxicity on cardiac cells, led to better selectivities than benznidazole (BZ). Otherwise, they were not as active as BZ in vitro against the non-replicative form of the parasite, i.e. bloodstream trypomastigotes. In vivo, acute toxicity assays revealed the absence of toxic events when administered to mice. Moreover, different therapeutic schemes pointed to their capability for decreasing the parasitaemia of T. cruzi Y acute infected mice, reaching up to 60% of reduction at the peak day as monotherapy (16), 79.24 and 91.11% when 16 and 24 were co-administered with BZ. These combined therapies had also a positive impact over the mortality, yielding survivals of 83.33 and 66.67%, respectively, while untreated animals reached a cumulative mortality of 100%. These findings confirm the 5-nitroindazole scaffold as a putative prototype for developing novel drugs potentially applicable to the treatment of Chagas disease and introduce their suitability to act in combination with the reference drug.

In vitro assessment of 3-alkoxy-5-nitroindazole-derived ethylamines and related compounds as potential antileishmanial drugs.

Leishmaniasis is a widespread neglected tropical disease complex that is responsible of one million new cases per year. Current treatments are outdated and pose many problems that new drugs need to overcome. With the goal of developing new, safe, and affordable drugs, we have studied the in vitro activity of 12 different 5-nitroindazole derivatives that showed previous activity against different strains of Trypanosoma cruzi in a previous work. T. cruzi belongs to the same family as Leishmania spp., and treatments for the disease it produces also needs renewal. Among the derivatives tested, compounds 1, 2, 9, 10, 11, and 12 showed low J774.2 macrophage toxicity, while their effect against both intracellular and extracellular forms of the studied parasites was higher than the ones found for the reference drug Meglumine Antimoniate (Glucantime®). In addition, their Fe-SOD inhibitory effect, the infection rates, metabolite alteration, and mitochondrial membrane potential of the parasites treated with the selected drugs were studied in order to gain insights into the action mechanism, and the results of these tests were more promising than those found with glucantime, as the leishmanicidal effect of these new drug candidates was higher. The promising results are encouraging to test these derivatives in more complex studies, such as in vivo studies and other experiments that could find out the exact mechanism of action.

Experimental models in Chagas disease: a review of the methodologies applied for screening compounds against Trypanosoma cruzi.

One of the main problems of Chagas disease (CD), the parasitic infection caused by Trypanosoma cruzi, is the lack of a completely satisfactory treatment, which is currently based on two old nitroheterocyclic drugs (i.e., nifurtimox and benznidazole) that show important limitations for treating patients. In this context, many laboratories look for alternative therapies potentially applicable to the treatment, and therefore, research in CD chemotherapy works in the design of experimental protocols for detecting molecules with activity against T. cruzi. Phenotypic assays are considered the most valuable strategy for screening these antiparasitic compounds. Among them, in vitro experiments are the first step to test potential anti-T. cruzi drugs directly on the different parasite forms (i.e., epimastigotes, trypomastigotes, and amastigotes) and to detect cytotoxicity. Once the putative trypanocidal drug has been identified in vitro, it must be moved to in vivo models of T. cruzi infection, to explore (i) acute toxicity, (ii) efficacy during the acute infection, and (iii) efficacy in the chronic disease. Moreover, in silico approaches for predicting activity have emerged as a supporting tool for drug screening procedures. Accordingly, this work reviews those in vitro, in vivo, and in silico methods that have been routinely applied during the last decades, aiming to discover trypanocidal compounds that contribute to developing more effective CD treatments.

Simple dialkyl pyrazole-3,5-dicarboxylates show in vitro and in vivo activity against disease-causing trypanosomatids.

The synthesis and antiprotozoal activity of some simple dialkyl pyrazole-3,5-dicarboxylates (compounds 2-6) and their sodium salts (pyrazolates) (compounds 7-9) against Trypanosoma cruzi, Leishmania infantum and Leishmania braziliensis are reported. In most cases the studied compounds showed, especially against the clinically significant amastigote forms, in vitro activities higher than those of the reference drugs (benznidazole for T. cruzi and glucantime for Leishmania spp.); furthermore, the low non-specific cytotoxicities against Vero cells and macrophages shown by these compounds led to good selectivity indexes, which are 8-72 times higher for T. cruzi amastigotes and 15-113 times higher for Leishmania spp. amastigotes than those of the respective reference drugs. The high efficiency of diethyl ester 3 and its sodium salt 8 against the mentioned protozoa was confirmed by further in vitro assays on infection rates and by an additional in vivo study in a murine model of acute and chronic Chagas disease. The inhibitory capacity of compounds 3 and 8 on the essential iron superoxide dismutase of the aforementioned parasites may be related to the observed anti-trypanosomatid activity. The low acute toxicity of compounds 3 and 8 in mice is also reported in this article.

In vitro trichomonacidal activity and preliminary in silico chemometric studies of 5-nitroindazolin-3-one and 3-alkoxy-5-nitroindazole derivatives.

A selection of 1,2-disubstituted 5-nitroindazolin-3-ones (1-19) and 3-alkoxy-5-nitroindazoles substituted at positions 1 (20-24) or 2 (25-39) from our in-house compound library were screened in vitro against the most common curable sexually transmitted pathogen, Trichomonas vaginalis. A total of 41% of the studied molecules (16/39) achieved a significant activity of more than 85% growth inhibition at the highest concentration assayed (100 µg mL(-1)). Among these compounds, 3-alkoxy-5-nitroindazole derivatives 23, 24, 25 and 27 inhibited parasite growth by more than 50% at 10 µg mL(-1). In addition, the first two compounds (23, 24) still showed remarkable activity at the lowest dose tested (1 µg mL(-1)), inhibiting parasite growth by nearly 40%. Their specific activity towards the parasite was corroborated by the determination of their non-specific cytotoxicity against mammalian cells. The four mentioned compounds exhibited non-cytotoxic profiles at all of the concentrations assayed, showing a fair antiparasitic selectivity index (SI > 7·5). In silico studies were performed to predict pharmacokinetic properties, toxicity and drug-score using Molinspiration and OSIRIS computational tools. The current in vitro results supported by the virtual screening suggest 2-substituted and, especially, 1-substituted 3-alkoxy-5-nitroindazoles as promising starting scaffolds for further development of novel chemical compounds with the main aim of promoting highly selective trichomonacidal lead-like drugs with adequate pharmacokinetic and toxicological profiles.

Biological approaches to characterize the mode of action of two 5-nitroindazolinone prototypes on Trypanosoma cruzi bloodstream trypomastigotes.

The phenotypic activity of two 5-nitroindazolinones, i.e. 2-benzyl-1-propyl (22) and 2-benzyl-1-butyl (24) derivatives, previously proposed as anti-Trypanosoma cruzi prototypes, was presently assayed on bloodstream trypomastigotes (BT) of the moderately drug-resistant Y strain. Further exploration of putative targets and cellular mechanisms involved in their activity was also carried out. Therefore, transmission electron microscopy, high-resolution respirometry and flow cytometry procedures were performed on BT treated for up to 24 h with the respective EC50 value of each derivative. Results demonstrated that although 22 and 24 were not as active as benznidazole in this in vitro assay on BT, both compounds triggered important damages in T. cruzi that lead to the parasite death. Ultrastructural alterations included shedding events, detachment of plasma membrane and nuclear envelope, loss of mitochondrial integrity, besides the occurrence of a large number of intracellular vesicles and profiles of endoplasmic reticulum surrounding cytoplasmic organelles such as mitochondrion. Moreover, both derivatives affected mitochondrion leading to this organelle dysfunction, as reflected by the inhibition in oxygen consumption and the loss of mitochondrial membrane potential. Altogether, the findings exposed in the present study propose autophagic processes and mitochondrial machinery as part of the mode of action of both 5-nitroindazolinones 22 and 24 on T. cruzi trypomastigotes.

A new type of quinoxalinone derivatives affects viability, invasion, and intracellular growth of Toxoplasma gondii tachyzoites in vitro.

Quinoxalinone derivatives, identified as VAM2 compounds (7-nitroquinoxalin-2-ones), were evaluated against Toxoplasma gondii tachyzoites of the RH strain. The VAM2 compounds were previously synthesized based on the design obtained from an in silico prediction with the software TOMOCOMD-CARDD. From the ten VAM2 drugs tested, several showed a deleterious effect on tachyzoites. However, VAM2-2 showed the highest toxoplasmicidal activity generating a remarkable decrease in tachyzoite viability (in about 91 %) and a minimal alteration in the host cell. An evident inhibition of host cell invasion by tachyzoites previously treated with VAM2-2 was observed in a dose-dependent manner. In addition, remarkable alterations were observed in the pellicle parasite, such as swelling, roughness, and blebbing. Toxoplasma motility was inhibited, and subpellicular cytoskeleton integrity was altered, inducing a release of its components to the soluble fraction. VAM2-2 showed a clear and specific deleterious effect on tachyzoites viability, structural integrity, and invasive capabilities with limited effects in host cells morphology and viability. VAM2-2 minimum inhibitory concentration (MIC50) was determined as 3.3 µM ± 1.8. Effects of quinoxalinone derivatives on T. gondii provide the basis for a future therapeutical alternative in the treatment of toxoplasmosis.

Novel solid dispersions of benznidazole: preparation, dissolution profile and biological evaluation as alternative antichagasic drug delivery system.

Solid dispersions (SD) of benznidazole (BNZ) in sodium deoxycholate (NaDC) or low-substituted hydroxypropylcellulose (L-HPC) were developed by freeze-drying process to improve the solubility of this low water-soluble drug and consequently, its trypanocidal activity. Although the dissolution studies showed a progressive decrease in the release rate of BNZ when formulated in the presence of NaDC, the increase in the surfactant concentration resulted in a better trypanocidal profile on epimastigotes, as well as in an enhancement of the unspecific cytotoxicity. However, such an effect was not so evident on amastigotes and in vivo (blood-trypomastigotes), where high concentrations of surfactant (BNZ:NaDC ≥ 1:6) experimented a loss of activity, correlating this fact with the minor cession of BNZ these formulations accomplished in acidic locations (i.e., dissolution test medium). According to the in vitro results, we reformulated the promising SD-1:3 (IC50 epimastigotes = 33.92 ± 6.41 µM, IC50 amastigotes = 0.40 ± 0.05 µM and LC50 = 183.87 ± 12.30 µM) replacing NaDC by L-HPC, which achieved the fastest dissolution profile. This fact, together with the safety this carrier ensures (LC50 > 256 µM), prompted us to evaluate the cellulose SD in vivo, improving the effectiveness of its NaDC equivalent (%AUPC = 96.65% and 91.93%, respectively). The results compiled in the present work suggest these solid dispersions as alternative drug delivery systems to improve the limited chemotherapy of Chagas disease.

Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

Protozoan parasites have been one of the most significant public health problems for centuries and several human infections caused by them have massive global impact. Most of the current drugs used to treat these illnesses have been used for decades and have many limitations such as the emergence of drug resistance, severe side-effects, low-to-medium drug efficacy, administration routes, cost, etc. These drugs have been largely neglected as models for drug development because they are majorly used in countries with limited resources and as a consequence with scarce marketing possibilities. Nowadays, there is a pressing need to identify and develop new drug-based antiprotozoan therapies. In an effort to overcome this problem, the main purpose of this study is to develop a QSARs-based ensemble classifier for antiprotozoan drug-like entities from a heterogeneous compounds collection. Here, we use some of the TOMOCOMD-CARDD molecular descriptors and linear discriminant analysis (LDA) to derive individual linear classification functions in order to discriminate between antiprotozoan and non-antiprotozoan compounds as a way to enable the computational screening of virtual combinatorial datasets and/or drugs already approved. Firstly, we construct a wide-spectrum benchmark database comprising of 680 organic chemicals with great structural variability (254 of them antiprotozoan agents and 426 to drugs having other clinical uses). This series of compounds was processed by a k-means cluster analysis in order to design training and predicting sets. In total, seven discriminant functions were obtained, by using the whole set of atom-based linear indices. All the LDA-based QSAR models show accuracies above 85% in the training set and values of Matthews correlation coefficients (C) vary from 0.70 to 0.86. The external validation set shows rather-good global classifications of around 80% (92.05% for best equation). Later, we developed a multi-agent QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses.

Further insights into biological evaluation of new anti-Trypanosoma cruzi 5-nitroindazoles.

Twelve molecules from a series of 35 new 5-nitroindazole derivatives, selected from a successful primary screening on Trypanosoma cruzi epimastigotes, have been evaluated against intracellular amastigotes according to the previous results of their trypanocidal activity and unspecific cytotoxicity. 2-Benzyl-1-propyl (22), 2-benzyl-1-isopropyl (23), and 2-benzyl-1-butyl (24) 5-nitroindazolin-3-ones have inhibited the growth of amastigotes similarly to the reference drugs benznidazole and nifurtimox, inducing complete growth inhibition at concentrations lower than 8 µM (IC50 < 5 µM) and accomplishing great selectivity indexes on the intracellular form of the parasite (SI > 30). Further in vivo assays were developed only for two of the most active molecules (22 and 24), reaching significant reductions in parasitemia levels (52 % and 77%, respectively) after their oral administration to infected mice. In addition, none of the mice in experimental and benznidazole groups died, unlike in the control group which is only treated with the vehicle. The trypanocidal properties found in some of the 5-nitroindazole derivatives assayed in the present work represent an interesting contribution to the urgent need for searching new antichagasic drugs.

In vivo genotoxicity and cytotoxicity assessment of a novel quinoxalinone with trichomonacide activity.

The compound VAM2-6 (1-methyl-7-nitro-4-(5-(piperidin-1-yl)pentyl)-3,4-dihydroquinoxalin-2(1H)-one) has previously been shown to have an in vitro efficacy of 100% at a concentration of 100 µg ml(-1) against Trichomonas vaginalis, a protozoon parasite that causes the sexually transmitted disease trichomoniasis. Because VAM2-6 is a quinoxaline derivative and given the lack of studies on the genotoxic activity of this compound, the present study was undertaken to evaluate its ability to induce DNA damage in the peripheral blood of mice using single-cell gel electrophoresis (SCGE or comet assay) and the micronucleus (MN) assay. Cell viability was assessed using a fluorochrome-mediated viability test. The compound was tested on CD1 mice at 60, 40 and 10 mg kg(-1) body weight administrated intraperitoneal (i.p.) in a single dose. Peripheral blood samples were collected 24 and 48 h after treatment. N-Ethyl-N-nitrosourea (ENU) was used as a positive control for the comet and micronucleus assays. The results showed that i.p. VAM2-6 induced single-strand DNA breaks and increased the average number of micronuclei in the treated mice in a dose-dependent manner at 60, 40 and 10 mg kg(-1). Cell viability decreased at 24 h but recovered at 48 h for all three evaluated doses. Therefore, the chemical structure of VAM2-6 should be modified to reduce its genotoxic potential.

Biological assay of a novel quinoxalinone with antimalarial efficacy on Plasmodium yoelii yoelii.

Compound 1-methyl-7-nitro-4-(5-(piperidin-1-yl)pentyl)-3,4-dihydroquinoxalin-2(1H)-one (VAM2-6) was evaluated against a blood-induced infection with chloroquine-sensitive Plasmodium yoelii yoelii lethal strain in CD1 mice in a 4-day test scheme. LD50 of the compound was 56.51 mg/kg and LD10 was 20.58 mg/kg (taken as the highest dose). Animals were treated by oral gavage of 20, 10, and 5 mg/kg. Mice in the untreated control group showed a progressively increasing parasitemia leading to mouse death on 6 days post-infection; in this group, all mice showed parasites in the blood on the fifth day of sampling; the mean parasitemia on that day was 19.4%. A 4-day dosage of 20 mg/kg of VAM2-6 showed a 97% chemosuppression of total parasitemia on the fifth day, a 28 days survival time, and 20% of cured animals. A 4-day dosage of 10 and 5 mg/kg showed 85 and 37%, respectively, chemosuppression of total parasitemia on the fifth day; but all mice died from days 6 to 9 post-infection with increasing parasitemia. Mice treated with chloroquine at 5 mg/kg survived during the experiment. The results obtained in this study showed that the infection outcome of P. yoelii yoelii-infected mice is affected by VAM2-6 compound by slowing down the parasite replication, retarding the patency time, and increasing their survival time. Although compound VAM2-6 was active at higher doses than chloroquine, these results leaves a door open to the study of its structure in order to improve its antimalarial activity.

Discovery of nitroheterocycles active against African trypanosomes. In vitro screening and preliminary SAR studies.

A selection of 76 nitroheterocycles and related compounds from our in-house compound library was screened in vitro against the parasite Trypanosoma brucei rhodesiense, causative agent of human African trypanosomiasis (HAT). The unspecific cytotoxicity of the compounds was also evaluated against rat myoblast L6-cells to measure the selectivity of the compounds towards the parasite. This screening revealed some preliminary structure-activity relationships (SAR) among the series, and six hit compounds showing interesting activity (IC(50)≤10µM) and fair selectivity (SI>17). The 7-nitroquinoxalin-2-one and 5-nitroindazole scaffold derivatives 58 and 35, respectively, are particularly interesting because of their established oral bioavailability in mice. These hits represent interesting starting points for a medicinal project aimed at identifying the SAR behind this class of compounds.

Antileishmanial, antitrypanosomal, and cytotoxic screening of ethnopharmacologically selected Peruvian plants.

Extracts (34) from eight plant species of the Peruvian Amazonia currently used in traditional Peruvian medicine, mostly as antileishmanial remedies and also as painkiller, antiseptic, antipyretic, anti-inflamatory, antiflu, astringent, diuretic, antipoison, anticancerous, antiparasitic, insecticidal, or healing agents, have been tested for their antileishmanial, antitrypanosomal, and cytotoxic activity. Plant species were selected based on interviews conducted with residents of rural areas. The different plant parts were dried, powdered, and extracted by maceration with different solvents (hexane, chloroform, and 70% ethanol-water). These extracts were tested on promastigote forms of Leishmania infantum strain PB75, epimastigote forms of Trypanosoma cruzi strain Y, and the mammalian CHO cell line. Parasite viability and nonspecific cytotoxicity were analyzed by a modified MTT colorimetric assay method. The isolation and identification of pure compounds from selected extracts were performed by column chromatography, gas chromatography mass spectrometry (GC-MS; mixtures), spectroscopic techniques [MS, infrared (IR), ultraviolet (UV)], and mono and two-dimensional (1)H and (13)C nuclear magnetic resonance (NMR; COSY, HSQC, NOESY) experiments. Chondodendron tomentosum bark and Cedrela odorata were the most active extracts against Leishmania, while C. odorata and Aristoloquia pilosa were the most active against Trypanosoma, followed by Tabebuia serratifolia, Tradescantia zebrina, and Zamia ulei. Six compounds and two mixtures were isolated from Z. ulei [cycasin (1)], T. serratifolia {mixtures 1-2, and naphthoquinones 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione (2) and 2-(1-hydroxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione (3)}, and C. tomentosum [chondrocurine (4); (S,S')-12-O-methyl(+)-curine (5); and cycleanine (6)]. Four compounds and the two mixtures exhibited significant activity.

5-Nitroindazole derivatives as potential therapeutic alternatives against Acanthamoeba castellanii.

Amoebas of the genus Acanthamoeba are distributed worldwide, including species with a high pathogenic capacity for humans. In a similar way to what occurs with other parasitic protozoa, the available treatments show variable effectiveness in addition to high toxicity, which demands the development of new treatments. Positive results of 5-nitroindazole derivatives against several protozoa parasites suggest that these compounds may be a promising tool for the development of efficient antiparasitic drugs. In the present work we have evaluated the in vitro activity of ten 5-nitroindazole derivatives against Acanthamoeba castellanii trophozoites and cysts. To that end, AlamarBlue Assay Reagent® was used to determine the activity against trophozoites compared to the reference drug chlorhexidine digluconate. Cytotoxicity of the compounds was evaluated using Vero cells. The activity on cysts was evaluated by light microscopy and using a Neubauer chamber to quantifying cysts and presence of trophozoites, as an indication of cyst. Our results showed the effectiveness of the 5-nitroindazole derivatives tested against both trophozoites and cysts of A. castellani highlighting 5-nitroindazole derivative 8 which showed a 80% activity on cysts, which is higher than that of the reference drug. Moreover, 5-nitroindazole derivatives 8, 9 and 10 were more effective on trophozoites than the reference drug showing IC50 values lower than 5 µM. Taking together these results, these 5-nitroindazole derivatives specially compound 8, might be a promising alternative for the development of more efficient treatments against A. castellani infection.

5-Nitroindazole-based compounds: further studies for activity optimization as anti-Trypanosoma cruzi agents.

In this study, a new series of eleven 5-nitroindazole derivatives (10-20) and a related 6-nitroquinazoline (21) was synthesized and tested in vitro against different forms of the kinetoplastid parasite Trypanosoma cruzi, etiological agent of Chagas disease. Among these compounds, derivatives 11-14 and 17 showed trypanocidal profiles on epimastigotes (IC50 = 1.00-8.75 µM) considerably better than that of the reference drug benznidazole, BZ (IC50 = 25.22 µM). Furthermore, the lack of cytotoxicity observed for compounds 11, 12, 14, 17 and 18 over L929 fibroblasts, led to a notable selectivity (SI) on the extracellular replicative form of the parasite: SIEPI > 12.41 to > 256 µM. Since these five derivatives overpassed the cut-off value established by BZ (SIEPI ≥ 10), they were moved to a more specific assay against the intracellular and replicative form of T. cruzi, i.e, amastigotes. These molecules were not as active as BZ (IC50 = 0.57 µM) against this parasite form; however, all of them showed remarkable IC50 values lower than 7 µM. Special mention deserve compounds 12 and 17, whose SIAMA were > 246.15 and > 188.23, respectively. The results compiled in the present work, point to a positive impact over the trypanocidal activity of the electron withdrawing substituents introduced at position 2 of the N-2 benzyl moiety of these compounds, especially fluorine, i.e., derivatives 12 and 17. These outcomes, supported by the in silico prediction of good oral bioavailability and suitable risk profile, reinforce the 5-nitroindazole scaffold as an adequate template for preparing potential antichagasic agents.

Hydrogen-bond-mediated self-assembly of 26-membered diaza tetraester crowns of 3,5-disubstituted 1H-pyrazole. Dimerization study in the solid state and in CDCl3 solution.

By using an improved synthetic method reported earlier, the cyclic stannoxanes obtained from RN-diethanolamine (R = Me, Bu) and dibutyltin oxide have been reacted with 1H-pyrazole-3,5-dicarbonyl dichloride to afford 26-membered diaza tetraester crowns (1, R = Me; 3, R = Bu) and 39-membered triaza hexaester crowns (2, R = Me; 4, R = Bu). The new structures were identified from their analytical and spectroscopic ((1)H and (13)C NMR, FAB-MS, and/or ESI-MS) data. Both diaza tetraester crowns (1 and 3), containing two 1H-pyrazole units, self-assemble into dimeric species through the formation of four hydrogen bonds involving the two NH pyrazole groups and the two tertiary amine groups of both crowns, as proved by X-ray crystallography and NMR analysis. Preliminary NMR, ESI-MS, MALDI-TOF-MS, and molecular modeling studies suggest that, in CDCl(3) solution, 1 interacts with ethyleneurea (ETU), affording 1:1, 2:1, and 2:2 1-ETU complexes.

X-ray diffraction, solution structure, and computational studies on derivatives of (3-sec-butyl-2,3-dihydro-1H-isoquinolin-4-ylidene)acetic acid: compounds with activity as calpain inhibitors.

A thorough experimental and computational study of derivatives of (3-sec-butyl-2,3-dihydroisoquinolin-4-ylidene)acetic acid was performed. Some of these compounds are calpain inhibitors and could be useful as therapeutic agents, since this enzyme is a Ca(2+)-dependent cysteine protease involved in a wide variety of metabolic and physiological processes, whose over-activation is associated to several pathological conditions. To gain a better understanding of the structure-activity relationships, a structural analysis was carried out with (1)H and (13)C NMR spectroscopy and DFT calculations together with the X-ray diffraction data of three compounds. The solid state structures showed that the crystal packing as well as the intermolecular interactions depend on the substituent nature of the COOR group. Also, the reactivity of the exocyclic double bond was theoretically evaluated, finding that the more reactive compound is the most potent inhibitor of calpain (IC(50) = 25 nM).

Selective electrochemical discrimination between dopamine and phenethylamine-derived psychotropic drugs using electrodes modified with an acyclic receptor containing two terminal 3-alkoxy-5-nitroindazole rings.

Electrochemical discrimination between dopamine and psychotropic drugs which have in common a skeletal structure of phenethylamine, can be obtained using acyclic receptors L(1) and L(2), containing two terminal 3-alkoxy-5-nitroindazole rings. Upon attachment to graphite electrodes, L(1) and L(2) exhibit a well-defined, essentially reversible solid state electrochemistry in contact with aqueous media, based on electrolyte-assisted reduction processes involving successive cation and anion insertion/binding. As a result, a distinctive, essentially Nernstian electrochemical response is obtained for phenethylammonium ions of methamphetamine (METH), p-methoxyamphetamine (PMA), amphetamine (AMPH), mescaline (MES), homoveratrylamine (HOM), phenethylamine (PEA) and dopamine (DA) in aqueous media.