Evaluation of phthalazinone phosphodiesterase inhibitors with improved activity and selectivity against Trypanosoma cruzi.

BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. OBJECTIVES: As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. METHODS: In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. RESULTS: Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. CONCLUSIONS: The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

New 1,2,3-triazole-based analogues of benznidazole for use against Trypanosoma cruzi infection: In vitro and in vivo evaluations.

Chagas disease has spread throughout the world mainly because of the migration of infected individuals. In Brazil, only benznidazole (Bnz) is used; however, it is toxic and not active in the chronic phase, and cases of resistance are described. This work aimed at the synthesis and the trypanocidal evaluation in vitro and in vivo of six new Bnz analogues (3-8). They were designed by exploring the bioisosteric substitution between the amide group contained in Bnz and the 1,2,3-triazole ring. All the compounds were synthesized in good yields. With the exception of compound 7, the in vitro biological evaluation shows that all Bnz analogues were active against the amastigote form, whereas only compounds 3, 4, 5, and 8 were active against trypomastigote. Compounds 4 and 5 showed the most promising activities in vitro against the form of trypomastigote, being more active than Bnz. In vivo evaluation of compounds, 3-8 showed lower potency and higher toxicity than Bnz. Although the 1,2,3-triazole ring has been described in the literature as an amide bioisostere, its substitution here has reduced the activity of the compounds and made them more toxic. Thus, further molecular optimization could provide novel therapeutic agents for Chagas' disease.

Antichagasic, Leishmanicidal, and Trichomonacidal Activity of 2-Benzyl-5-nitroindazole-Derived Amines.

Three different series of new 5-nitroindazole derivatives-1-(ω-aminoalkyl)-2-benzylindazolin-3-ones (series A; ten compounds), 3-(ω-aminoalkoxy)-2-benzylindazoles (series B; four compounds) and 3-alkylamino-2-benzylindazoles (series C; five compounds)-have been synthesized and evaluated against the protozoan parasites Trypanosoma cruzi, Leishmania amazonensis, and Trichomonas vaginalis: etiological agents of Chagas disease, cutaneous leishmaniasis, and trichomoniasis, respectively. Many indazoles of series A, B, and C were efficient against T. cruzi. Some compounds in series A, after successfully passing the preliminary screening for epimastigotes, exhibited activity values against amastigotes of several T. cruzi strains that were better than or similar to those shown by the reference drug benznidazole and displayed low nonspecific toxicity against mammalian cells. On the other hand, preliminary studies against promastigotes of L. amazonensis showed high leishmanicidal activity for some derivatives of series A and C. With regard to activity against T. vaginalis, some indazoles of series B and C were rather efficient against trophozoites of a metronidazole-sensitive isolate and showed low nonspecific toxicities toward Vero cell cultures. Additionally, some of these compounds displayed similar activity against metronidazole-sensitive and resistant isolates, showing the absence of cross-resistance between these derivatives and the reference drug.

Identification of Pyrazolo[3,4-e][1,4]thiazepin based CYP51 inhibitors as potential Chagas disease therapeutic alternative: In vitro and in vivo evaluation, binding mode prediction and SAR exploration.

American trypanosomiasis or Chagas disease (CD) is a vector borne pathology caused by the parasite Trypanosoma cruzi (T. cruzi), which remains a serious global health problem. The current available treatment for CD is limited to two nitroderivatives with limited efficacy and several side effects. The rational design of ergosterol synthetic route inhibitors (e.g. CYP51 inhibitors) represents a promising strategy for fungi and trypanosomatids, exhibiting excellent anti-T.cruzi activity in pre-clinical assays. In the present work, we evaluate through different approaches (molecular docking, structure activity relationships, CYP51 inhibitory assay, and phenotypic screenings in vitro and in vivo) the potency and selectivity of a novel CYP51 inhibitor (compound 1) and its analogues against T.cruzi infection. Regarding anti-parasitic effect, compound 1 was active in vitro with EC50 3.86 and 4.00 µM upon intracellular (Tulahuen strain) and bloodstream forms (Y strain), respectively. In vivo assays showed that compound 1 reduced in 43% the parasitemia peak but, unfortunately failed to promote animal survival. In order to promote an enhancement at the potency and pharmacological properties, 17 new analogues were purchased and screened in vitro. Our findings demonstrated that five compounds were active against intracellular forms, highlighting compounds 1e and 1f, with EC50 2.20 and 2.70 µM, respectively, and selectivity indices (SI) = 50 and 36, respectively. Against bloodstream trypomastigotes, compound 1f reached an EC50 value of 20.62 µM, in a similar range to Benznidazole, but with low SI (3). Although improved the solubility of compound 1, the analogue 1f did not enhance the potency in vitro neither promote better in vivo efficacy against mouse model of acute T.cruzi infection arguing for the synthesis of novel pyrazolo[3,4-e][1,4]thiazepin derivatives aiming to contribute for alternative therapies for CD.

Antichagasic and trichomonacidal activity of 1-substituted 2-benzyl-5-nitroindazolin-3-ones and 3-alkoxy-2-benzyl-5-nitro-2H-indazoles.

Two series of new 5-nitroindazole derivatives, 1-substituted 2-benzylindazolin-3-ones (6-29, series A) and 3-alkoxy-2-benzyl-2H-indazoles (30-37, series B), containing differently functionalized chains at position 1 and 3, respectively, have been synthesized starting from 2-benzyl-5-nitroindazolin-3-one 5, and evaluated against the protozoan parasites Trypanosoma cruzi and Trichomonas vaginalis, etiological agents of Chagas disease and trichomonosis, respectively. Many indazolinones of series A were efficient against different morphological forms of T. cruzi CL Brener strain (compounds 6, 7, 9, 10 and 19-21: IC50 = 1.58-4.19 µM for epimastigotes; compounds 6, 19-21 and 24: IC50 = 0.22-0.54 µM for amastigotes) being as potent as the reference drug benznidazole. SAR analysis suggests that electron-donating groups at position 1 of indazolinone ring are associated with an improved antichagasic activity. Moreover, compounds of series A displayed low unspecific toxicities against an in vitro model of mammalian cells (fibroblasts), which were reflected in high values of the selectivity indexes (SI). Compound 20 was also very efficient against amastigotes from Tulahuen and Y strains of T. cruzi (IC50 = 0.81 and 0.60 µM, respectively), showing low toxicity towards cardiac cells (LC50 > 100 µM). In what concerns compounds of series B, some of them displayed moderate activity against trophozoites of a metronidazole-sensitive isolate of T. vaginalis (35 and 36: IC50 = 9.82 and 7.25 µM, respectively), with low unspecific toxicity towards Vero cells. Compound 36 was also active against a metronidazole-resistant isolate (IC50 = 9.11 µM) and can thus be considered a good prototype for the development of drugs directed to T. vaginalis resistant to 5-nitroimidazoles.

Activities of psilostachyin A and cynaropicrin against Trypanosoma cruzi in vitro and in vivo.

In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection.

In vitro and in vivo studies of the antiparasitic activity of sterol 14α-demethylase (CYP51) inhibitor VNI against drug-resistant strains of Trypanosoma cruzi.

Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 µM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.

Copper(II)-fluoroquinolone complexes with anti-Trypanosoma cruzi activity and DNA binding ability.

Copper(II) complexes of fluoroquinolone antibacterial agents levofloxacin (LEV) and sparfloxacin (SPAR), containing or not a nitrogen donor heterocyclic ligand, 2,2'-bipyridine (bipy) or 1,10-phenathroline (phen), were prepared and characterized. The complexes are of the type [CuCl(2)(H(2)O)(L)], [CuCl(bipy)(L)]Cl and [CuCl(2)(phen)(L)], where L = LEV or SPAR. The data suggest that LEV and SPAR act as zwitterionic bidentade ligands coordinated to Cu(II) through the carboxylate and ketone oxygen atoms. The electron paramagnetic resonance spectra of the [CuCl(bipy)(L)]Cl and [CuCl(2)(phen)(L)] complexes (L = LEV and SPAR) in aqueous and DMSO solutions indicate mixture of mononuclear and binuclear forms. The Cu(II) complexes, together with the corresponding ligands, were evaluated for their trypanocidal activity in vitro against Trypanosoma cruzi, the causative agent of Chagas disease. The assays performed against bloodstream trypomastigotes showed that all complexes were more active than their corresponding ligands. Complexes [CuCl(2)(phen)(LEV)] and [CuCl(2)(phen)(SPAR)] were revealed, among all studied compounds, to be the most active with IC(50) = 1.6 and 4.7 µM, respectively, both presenting a superior effect than benznidazole. The interactions of fluoroquinolones and their Cu(II) complexes with calf-thymus DNA were investigated. These compounds showed binding properties towards DNA, with moderated binding constants values, suggesting that this structure may represent a parasite target.

In vitro and in vivo investigation of the efficacy of arylimidamide DB1831 and its mesylated salt form--DB1965--against Trypanosoma cruzi infection.

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA--DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T. cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC(50) value/48 h of 5-40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T. cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T. cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole.

The efficacy of novel arylimidamides against Trypanosoma cruzi in vitro.

The present study aimed to determine the in vitro biological efficacy and selectivity of 7 novel AIAs upon bloodstream trypomastigotes and intracellular amastigotes of Trypanosoma cruzi. The biological activity of these aromatic compounds was assayed for 48 and 24 h against intracellular parasites and bloodstream forms of T. cruzi (Y strain), respectively. Additional assays were also performed to determine their potential use in blood banks by treating the bloodstream parasites with the compounds diluted in mouse blood for 24 h at 4°C. Toxicity against mammalian cells was evaluated using primary cultures of cardiac cells incubated for 24 and 48 h with the AIAs and then cellular death rates were determined by MTT colorimetric assays. Our data demonstrated the outstanding trypanocidal effect of AIAs against T. cruzi, especially DB1853, DB1862, DB1867 and DB1868, giving IC50 values ranging between 16 and 70 nanomolar against both parasite forms. All AIAs presented superior efficacy to benznidazole and some, such as DB1868, also demonstrated promising activity as a candidate agent for blood prophylaxis. The excellent anti-trypanosomal efficacy of these novel AIAs against T. cruzi stimulates further in vivo studies and justifies the screening of new analogues with the goal of establishing a useful alternative therapy for Chagas disease.

Trypanosoma cruzi: activity of heterocyclic cationic molecules in vitro.

Chagas disease remains a serious public health problem in several Latin American countries. New chemotherapy is urgently needed since current drugs are limited in efficacy and exhibit undesirable side effects. Aromatic diamidines and analogs are well known anti-parasitic agents and in this study, we have evaluated the in vitro trypanocidal effect of several different heterocyclic cationic compounds, including diamidines (DB1195, DB1196 and DB1345), a monoamidine (DB824), an arylimidamide (DB613A) and a guanylhydrazone (DB1080) against amastigotes and bloodstream trypomastigotes of Trypanosoma cruzi, the etiological agent of Chagas disease. Our present findings showed that all compounds exerted, at low-micromolar doses, a trypanocidal effect upon both intracellular parasites and bloodstream trypomastigotes of T. cruzi. The activity of DB1195, DB1345, DB824 and DB1080 against bloodstream forms was reduced when these compounds were assayed in the presence of mouse blood possibly due to their association with plasma constituents and/or due to metabolic instability of the compounds. However, trypanocidal effects of DB613A and DB1196 were not affected by plasma constituents, suggesting their potential application in the prophylaxis of banked blood. In addition, potency and selectivity of DB613A, towards intracellular parasites, corroborate previous results that demonstrated the highly promising activity of arylimidamides against this parasite, which justify further studies in experimental models of T. cruzi infection.

In vitro and in vivo studies of the trypanocidal activity of a diarylthiophene diamidine against Trypanosoma cruzi.

Aromatic diamidines are DNA minor groove-binding ligands that display excellent antimicrobial activity against fungi, bacteria, and protozoa. Due to the currently unsatisfactory chemotherapy for Chagas' disease and in view of our previous reports regarding the effect of diamidines and analogues against both in vitro and in vivo Trypanosoma cruzi infection, this study evaluated the effects of a diarylthiophene diamidine (DB1362) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas' disease. The data show the potent in vitro activity of DB1362 against both parasite forms that are relevant for mammalian infection at doses which do not exhibit cytotoxicity. Ultrastructural analysis and flow cytometry studies show striking alterations in the nuclei and mitochondria of the bloodstream parasites. In vivo studies were performed at two different drug concentrations (25 and 50 mg/kg/day) using a 2-day or a 10-day regimen. The best results were obtained when acutely infected mice were treated with two doses at the lower concentration, resulting in 100% survival, compared to the infected and untreated mice. Although it did not display higher efficacy than benznidazole, DB1362 reduced both cardiac parasitism and inflammation, and in addition, it protected against the cardiac alterations (determined by measurements) common in T. cruzi infection. These results support further investigation of diamidines and related compounds as potential agents against Chagas' disease.