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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