The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P<0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R = 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously.

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Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene) (WHO 1993), the causative agent of this disease. Lately, great advances have been made in the control of vectorial and transfusion transmission of the disease, through spraying programs to eliminate the triatomine vectors and screening of blood banks (TDR 2000). Nevertheless, since the discovery of this disease more than 90 years ago, there is still no efficient treatment. A large number of different compounds have been assayed in a variety of ways, for instance cysteine-protease inhibitors that recognize cruzipain active site are effective in acute and chronic murine models (Engel et al. 1998): phenothiazines inhibit trypanothione reductase, acting in this way as promising trypanocidal agents (Chan et al. 1998). In spite of the amount of work conducted in studying trypanothione reductase and proteases in particular, still the drugs available for clinical use are restricted to benznidazole and nifurtimox (de Castro 1993). Both can reduce symptoms and mortality in the acute phase of the illness, but are not effective in achieving parasitologic cure or preventing the chronic phase. In addition, both Previous works reported the antichagasic properties of 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine-1,1-dioxides ). Attempts to synthesize new compounds with this structure failed and only the cyanoacetamide derivatives were obtained by partial hydrolysis of one of the nitrile groups from 5-nitro-arylidenemalononitriles (di Maio et al. 1999). The anti-epimastigote activity of these compounds was then evaluated and it was noteworthy that the activity of 5-nitro-thienyl-malononitrile (5NO 2 TM) was higher than nifurtimox activity (Muelas-Serrano et al. 2001a, b).The interesting activity of these new nitro-derivatives without the tetrahydrothiazine moiety of nifurtimox encouraged us to carry out new experiments to study in depth the properties of these compounds. MATERIALS AND METHODSCell culture -Murine J774 macrophages were grown in plastic 25 µl flasks in RPMI 1640 medium (Sigma) suplemented with 20% heat inactivated (30 min, 56 o C) foetal calf serum (FCS) and 100 IU penicillin/ml + 100 µg/ml streptomycin, in a humidified 5% CO 2 /95% air atmosphere at 37 o C and subpassaged once a week.Parasites -Trypanosoma cruzi Chagas, 1909 (Y strain) was grown at 28 o C in liver infusion tryptose (LIT) supplemented with 10% FCS and antibiotics. Epimastigote forms were harvested on day 14 of culture (stationary phase) and washed three times in Grace medium. To induce metacyclogenesis, parasites were then cultured in fresh Grace medium supplemented with 10% FCS and haemin (25 µg/ml). Nine days after cultivation at 28 o C, metacyclic forms were counted. The proportion of metacyclic forms was around 30% at this stage. These metacyclic forms were used to infect J774 macrophages, for anti-amastigote assays.Cell infection -J774 macrophages were d...

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A study of some antiparasitic properties of several homoallylamines and related tetrahydroquinolines and quinolines, previously described, was carried out using in vitro activity assays against the epimastigote form of Trypanosoma cruzi and against Trichomonas vaginalis. Unspecific cytotoxicity against murine macrophages was also studied. Although the antichagasic and trichomonacidal activities are not comparable to those of the standard drugs, nifurtimox and metronidazole, some of the compounds exhibit an interesting specific antiparasitic activity.