Thio- and selenosemicarbazones as antiprotozoal agents against Trypanosoma cruzi and Trichomonas vaginalis

Abstract Herein, we report the preparation of a panel of Schiff bases analogues as antiprotozoal agents by modification of the stereoelectronic effects of the substituents on N-1 and N-4 and the nature of the chalcogen atom (S, Se). These compounds were evaluated towards Trypanosoma cruzi and Trichomonas vaginalis. Thiosemicarbazide 31 showed the best trypanocidal profile (epimastigotes), similar to benznidazole (BZ): IC50 (31)=28.72 μM (CL-B5 strain) and 33.65 μM (Y strain), IC50 (BZ)=25.31 μM (CL-B5) and 22.73 μM (Y); it lacked toxicity over mammalian cells (CC50 > 256 µM). Thiosemicarbazones 49, 51 and 63 showed remarkable trichomonacidal effects (IC50 =16.39, 14.84 and 14.89 µM) and no unspecific cytotoxicity towards Vero cells (CC50 ≥ 275 µM). Selenoisosters 74 and 75 presented a slightly enhanced activity (IC50=11.10 and 11.02 µM, respectively). Hydrogenosome membrane potential and structural changes were analysed to get more insight into the trichomonacidal mechanism.


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CL-B5 lacZ epimastigote susceptibility assays. Trypanocidal activity of the compounds was firstly evaluated over this DTU TcVI lacZ transfected strain in presence of the chromogenic substrate chlorophenol red β-D-galactopyranoside (CPRG) 4,9 . Cultures of log-phase epimastigotes in LIT medium were seeded in 96-well microplates (200 µL/well) at a density of 2.5 × 10 5 parasites/mL and incubated within serial solutions of compounds for 72 h at 28 °C. Then, 50 µL of CPRG solution in 0.9% Triton X-100 (final concentration 200 µM, pH 7.4) was added per well and after 3 h of incubation at 37 °C, absorbance read at 595 nm (ELx808 ELISA reader, Biotek Instruments Inc). Percentages of epimastigote growth inhibition (%EGI) and concentration that inhibits 50% of epimastigote growth (IC50) were calculated as previously reported 4 .
Y strain epimastigote susceptibility assays. The most promising compounds of these series, were also tested against the moderately-drug resistant Y strain (DTU TcII) in presence of the redox indicator resazurin, following the methodology standardized by Rolón et al. 10 . Accordingly, 3 × 10 6  Unspecific cytotoxicity assays on L929 fibroblasts. The cytotoxic profile of compounds was simultaneously explored over cultures of L929 cells. Accordingly, 100 µL/well of a suspension in MEM containing 10 × 10 4 cells were seeded in 96-well microplates and maintained for 3 h at 37 °C with 5% CO2. After cell attachment, culture medium was replaced by 200 µL of compounds serially diluted in fresh MEM and plates incubated for 72 h at the aforementioned conditions of temperature and humidity. Then, 20 µL of a resazurin solution in PBS (2 mM, pH 7.0) was added and plates incubated for 3 h (37 °C, 5% CO2). Fluorescence intensity was read at 535 (excitation) and 590 (emission) nm in a Infinite 200 multifunctional microplate reader (Tecan). Percentages of cytotoxicity (%CL929) and concentration that produces 50% of cell death (CC50) were calculated as reported by Fonseca-Berzal et al. 4 .

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Unspecific cytotoxicity assays on J774 macrophages. For the most promising compounds, the potential toxic effects were also explored on J774 macrophages 8 . Briefly, a suspension of 50 × 10 4 cells in RPMI medium was distributed in 96-well microplates by adding 100 µL/well. After 3 h of incubation at 37 °C with 5% CO2, culture medium was discarded and 200 µL of compounds serially diluted in RPMI added. Cells were treated for 48 h (37 °C, 5% CO2) and then, 20 µL of a resazurin solution in PBS (1 mM, pH 7.0) added to each well. Fluorescence intensity was read at 535 (excitation) and 590 (emission) nm in a Infinite 200 multifunctional microplate reader (Tecan). Finally, percentages of cytotoxicity (%CJ774), CC50 and SI on Y epimastigotes were calculated 4,11 .
CL-B5 lacZ intracellular amastigote susceptibility assays. Those compounds with trypanocidal profile over epimastigotes similar to that of BZ, were moved to a more specific assay against intracellular amastigotes. These assays were carried out by applying the CPRG method reported by Fonseca-Berzal et al. 4 . Accordingly, 10,000 L929 cells were seeded in 48-well plates and infected with CL-B5 lacZ tissue culture-derived trypomastigotes at 1:6 ratio (cell:parasite). After 24 h of incubation at 33 °C with 5% CO2, non-penetrated parasites were rinsed with PBS and then, infected cultures treated with compounds diluted in fresh MEM for 7 days in similar conditions of temperature and humidity. Finally, 50 µL of CPRG solution in 3% Triton X-100 (final concentration 400 µM, pH 7.4) was added and after 3 h of incubation at 37 °C, absorbance read at 595 nm in a Infinite 200 multifunctional microplate reader (Tecan). Percentages of amastigote growth inhibition (%AGI), IC50 and SI on CL lacZ amastigotes were also calculated.
For each experiment, the assays were run at the same conditions three times separately (n=3) and therefore, both activity and cytotoxicity results are expressed as the mean value of the respective IC50 or CC50 ± standard deviation (SD). To a solution of the corresponding aniline 1-10 (3.0 g) in a 1:1 CH2Cl2-H2O mixture (24 mL) were added triphosgene (1.5 equiv.) and CaCO3 (2.0 equiv.); the corresponding mixture was vigorously stirred for 2 h, at rt. After that, it was filtrated, and the organic layer was separated, dried over Na2SO4, filtrated and concentrated under vacuum.

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Isothiocyanates 11-20 were directly used for the next step, without any further purifications.
General procedure for the preparation of thiosemicarbazides 22-30.
To a solution of isothiocyanates 11-20 (1.0 equiv.) in CH2Cl2 was dropwise added hydrazine (1.2 equiv.). After the addition, the solution was kept at rt for 3 h, yielding precipitation of the target compounds. The solid was filtrated and washed with CH2Cl2 to give derivatives 22-30.

General procedure for the preparation of thiosemicarbazones 36-65.
A solution of thiosemicarbazides 21-30 (500 mg) and the corresponding aromatic aldehyde