In vitro and in vivo activities of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol against Trypanosoma cruzi

Abstract

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

FIG. 1.

Chemical structures of the 1,3,4-thiadiazole-2-arylhydrazone derivatives.

FIG. 2.

Effects on parasitemia curves and cumulative mortality of oral treatment of T. cruzi-infected mice with a single dose on dpi 5 of 200 mg/kg of the compound indicated in the group name. (a) Results for the TcS1-200 and TcMg-200 groups. (b) Results for the TcS2-200, TcS3-200, TcS4-200, and TcS5-200 groups. Asterisks indicate P values of ≤0.05 for comparison of the results for each treated group with those for group Tc.

FIG. 3.

Transmission electron microscopy images of T. cruzi trypomastigotes treated with S1, S3, or megazol for 24 h. (a) Control parasite showing the typical elongated body and normal morphology of the mitochondrion (M) and kinetoplast (K). N, nucleus. (b) A concentration of 5.3 μM S1 led to expansion of the flagellar membrane (thick arrows), dilation of the nuclear membrane (white arrowhead), and autophagosome-like structures (asterisks). (c) A concentration of 17 μM S3 induced increased vesiculation near the flagellar pocket (star) and formation of autophagosome-like structures (asterisk). (d and e) A concentration of 9.9 μM megazol induced alterations in the kDNA network (arrows) and in the mitochondrion (arrowhead) and also an intense vacuolization (white stars). Bars: 0.5 μm.

FIG. 4.

Scanning electron microscopy images of T. cruzi trypomastigotes treated with S1, S2, S3, or megazol for 24 h. (a) Untreated parasites display normal morphology and surface topology. (b) S1 at 5.3 μM led to rounding of the parasite's body (arrows). (c) S2 at 11.6 μM induced shrinkage of the cell surface (arrow) and body (thick arrow) and shortening of the flagellum (arrowhead). (d) S3 at 17 μM caused rounding of parasites (arrows). (e) Megazol at 9.9 μM led to torsion (asterisk) and rounding (arrow) of the body of the parasites and shortening of the flagellum (arrowhead). Bars: 4 μm.

FIG. 5.

Effects of various concentrations of S1 (a and b) and megazol (c and d) on T. cruzi-infected HMCs. The percent infection and the endocytic index were quantified; mean results ± standard deviations are shown. After 24 h of plating HMCs (10⁵ cells/well) onto gelatin-coated glass coverslips, the cultures were infected at a 10:1 ratio (parasites/HMCs), and after 24 h of incubation, S1 or megazol was added as described in Materials and Methods.

FIG. 6.

Effects on parasitemia curves and cumulative mortality of oral treatment with 10 consecutive doses of 100 mg/kg of S2 (TcS2-100) and S3 (TcS3-100) from dpi 6 to 15. Asterisks indicate P values of ≤0.05 for comparison of the results for each treated group with those for the Tc group.