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Indazoles have previously been identified as molecules with antiprotozoal activity. In this study, we evaluate the in vitro activity of thirteen 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) against L. amazonensis, L. infantum, and L. mexicana. In vitro, cytotoxicity against mouse peritoneal macrophages and growth inhibitory activity in promastigotes were evaluated for all compounds, and those showing adequate activity and selectivity were tested against intracellular amastigotes. Transmission and scanning electron microscopy were employed to study the effects of 3-alkoxy-1-benzyl-5-nitroindazole and 2-benzyl-5-nitroindazolin-3-one derivatives on promastigotes of L. amazonensis. Compounds NV6 and NV8 were active in the two life stages of the three species, with the latter showing the best indicators of activity and selectivity. 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) showed in vitro activity comparable to that of amphotericin B against the promastigote stage of Leishmania spp. Two compounds were also found to be active the amastigote stage. Electron microscopy studies confirmed the antileishmanial activity of the indazole derivatives studied and support future research on this family of compounds as antileishmanial agents. [ABSTRACT FROM AUTHOR]

Drug repositioning is an efficient strategy to search for new treatment alternatives that is especially valuable for neglected parasitic diseases such as leishmaniasis. Tamoxifen and raloxifene are selective estrogen receptor modulators (SERMs) that have shown antileishmanial activity. Clomiphene is a SERM structurally similar to tamoxifen, whose antileishmanial potential is unknown. That is why the objective of the present work was to evaluate its antileishmanial activity in vitro and in vivo in comparison with tamoxifen. The inhibitory effect against promastigotes of L. amazonensis, L. major, and L. mexicana was evaluated for both compounds, as well as the cytotoxicity against mouse peritoneal macrophages, the growth inhibitory activity in intracellular amastigotes of L. mexicana, and the in vivo activity in mice experimentally infected with L. mexicana. Clomiphene was about twice as active as tamoxifen against both promastigotes and intracellular amastigotes, with IC50 values of 1.7–3.3 µM for clomiphene and 2.9–6.4 µM for tamoxifen against all three species of promastigotes and 2.8 ± 0.2 µM and 3.7 ± 0.3 µM, respectively, against L. mexicana amastigotes. Clomiphene structurally affected several parasite organelles in a concentration-dependent fashion, leading to the death of both promastigotes and intracellular amastigotes. Interestingly, the macrophage host cell did not appear damaged by any of the clomiphene concentrations tested. With oral administration at 20 mg/kg for 14 days, both compounds showed similar effects in terms of reducing the growth of the lesions, as well as the weight of the lesions and the parasite load at the end of the follow-up period. The results showed the potential of SERMs as antileishmanial drugs and support further testing of clomiphene and other compounds of this pharmacological group. [ABSTRACT FROM AUTHOR]

Leishmaniasis is an infectious disease caused by protozoan species in the genera Leishmania and Endotrypanum. Current antileishmanial drugs are limited due to adverse effects, variable efficacy, the development of resistant parasites, high cost, parenteral administration and lack of availability in endemic areas. Therefore, active searching for new antileishmanial drugs has been done for years, mainly by academia. Drug screening techniques have been a challenge since the intracellular localization of Leishmania amastigotes implies that the host cell may interfere with the quantification of the parasites and the final estimation of the effect. One of the procedures to avoid host cell interference is based on its detergent-mediated lysis and subsequent transformation of viable amastigotes into promastigotes, their proliferation and eventual quantification as an axenic culture of promastigotes. However, the use of detergent involves additional handling of cultures and variability. In the present work, cultures of intracellular amastigotes were incubated for 72 h at 26 °C after exposure to the test compounds and the transformation and proliferation of parasites took place without need of adding any detergent. The assay demonstrated clear differentiation of negative and positive controls (average Z´ = 0.75) and 50% inhibitory concentrations of compounds tested by this method and by the gold standard enumeration of Giemsa-stained cultures were similar (p = 0.5002) and highly correlated (r = 0.9707). This simplified procedure is less labor intensive, the probability of contamination and the experimental error are reduced, and it is appropriate for the automated high throughput screening of compounds. • Intracellular L. mexicana amastigotes transform in vitro into promastigotes at 26 °C. • Detergents are not needed for the transformation of amastigotes into promastigotes. • High Z' values are obtained with the transformation-proliferation assay (TPA). • Highly correlated IC 50 values determined by the TPA and the gold-standard technique. [ABSTRACT FROM AUTHOR]