In Vitro and in Vivo Assays of 3,5-Disubstituted-Tetrahydro-2H-1,3,5-Thiadiazin-2-Thione Derivatives against Trypanosoma cruzi

Abstract

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione Key words: antichagasic drugs -amastigotes -J774 macrophages -cytotoxicity assays -antitrypanosomal activity assaysthiadiazine thione derivativesTrypanosoma cruzi is the etiological agent of Chagas disease. It is estimated that 16-18 million people are chronically infected (WHO 1993). The current treatment is dependent on two nitroheterocyclic drugs, the nitrofuran nifurtimox (Lampit ®), whose production has now been discontinued, and the 2-nitroimidazole benznidazole (Rochagan ®) (Croft et al. 1997). Both drugs are effective in reducing the severity of acute and congenital Chagas disease, but have no role in the therapy of chronic infections. Both must be administrated for extended periods, often cause severe side effects and achieve parasitological cure in only about 50% of treated patients (Kirshhoff 1994). There is a considerable need for the development of new compounds to approach the chemotherapy for this disease.In this context new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives have been synthesized. In a previous work it was reported the antibacterial, antifungal, antiviral, anthelmintic, and tuberculostatic activities of tetrahydro-2H-1,3,5-thiadiazin-2-thione as prodrugs. The isothiocyanates formed by hydrolysis of the thiadiazine ring interact with and inactivate cysteine proteinases (Ertan et al.1992 The anti-epimastigote activity of these new thiadizines was previously evaluated (Ochoa et al. 1999). All of them were effective at 100 µg/ml, and some of them even at 1 µg/ml (1m, S-1n, RS-2b, 2c, S-2d, RS-2f, S-2g and S-2j). Once these first in vitro studies were accomplished, new biological assays have been performed in the present work, to study the non-specific toxicity and anti-amastigote activity of these compounds. MATERIALS AND METHODSCell culture -Murine J774 macrophages is a cell line that was kindly provided by the National Centre for Sanitary Microbiology, Virology and Immunology of Instituto Carlos III (Spain). It was grown in plastic 25 ml flasks in RPMI 1640 medium (Sigma) supplemented with 20% heat inactivated (30 min 56ºC) foetal calf serum (FCS) and 100 IU penicillin/ml + 100 µg/ml streptomycin, in a humidified 5% CO 2 /95% air atmosphere at 37ºC and subpassaged once a week.Parasites -T. cruzi (Y strain) was grown at 28ºC 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 into fresh Grace medium supplemented with 10% FCS and haemin (25 µg/ml). Nine days after cultivation at 28ºC, metacyclic forms were counted in order to infect macrophages. The proportion of metacyclic forms was around 30% at this stage.Cell infection -J774 macrophages were detached by EDTA-PBS (ethylendiamine tetraacetic acid-phosphate-

Evaluation of nitric oxide scavenging activity, In Vitro and Ex Vivo, of selected medicinal plants traditionally used in inflammatory diseases

“…The extent of the reduction was estimated spectrophotometrically (570 nm) after dissolving the formazan dye with DMSO. Thus, the viability (IC 50 ) of the cells after treatment with plant extracts was determined in comparison with 'untreated' control cells (Muelas et al, 2002). macrophage cells (2 × 10 6 cells/mL), treated with LPS (10 µg/mL) in the presence or absence of different doses of the plant extracts, were incubated at a temperature of 37°C in a humidified 5% CO 2 incubator for 48 h. The cells were scraped out from the culture plates, washed twice in cold PBS, and boiled for 5 min with lysis buffer [20 mM Tris-HCl, pH 7.5, 1 mM EDTA and 0.1% (v/v) β-mercaptoethanol] mixed with SDS-PAGE Laemmli buffer (4 ×) [20 mM Tris-HCl, pH 6.8, 4% SDS, 20% β-mercaptoethanol, 2 mM EDTA, 0.004% bromophenol blue, 40% glycerol].…”

Genotoxicidad del nifurtimox en deferentes líneas celulares utilizando el ensayo cometa

“…Estas actividades dependieron de la concentración del medicamento y del tipo de célula utilizado. La variabilidad en la toxicidad del nifurtimox ha sido demostrada en ensayos in vitro utilizando varios tipos de células de mamífero hospederas del T. cruzi ya que este medicamento es utilizado como medicamentos de referencia en estudios de screening de nuevos compuesto anti-tripanosomas (26)(27)(28)(29). Igualmente y dado que el nifurtimox está siendo evaluado como medicamento en el tratamiento de algunos canceres como neuro y meduloblastoma, su actividad antitumoral ha sido demostrada en diferentes tipos de células cancerosas (30).…”

Liquid phase organic synthesis of 3,5-disubstituted 1,3,5-thiadiazinane-2-thione derivatives on polyethylene glycol (PEG) support

“…14 The synthesized molecules were evaluated in vitro and in vivo against Trypanosoma cruzi and Trichomonas vaginalis showing significant antiprotozoal activity that, in some cases, was further correlated to a non-specific toxicity effect. 15 In another study the remarkable cytotoxicity properties of these derivatives against HeLa and HT-29 cells were also tested, 16 and we have developed a theoretical model to predict the influence of structure on the antitumor activity for this type of compounds. 17 The antiparasitic properties of some 5-carboxyalkyl-3-furfuryl-1,3,5-thiadiazinane-2-thione against both extracellular promastigotes and intracellular amastigotes of Leishmania amazonensis have also been reported.…”

“…21 Taking to account the interaction mechanism suggested for this heterocycle, the incorporation of two THTT moiety in the same structure must increase the activity of these derivatives as antiparasitic agents. 22 We have previously demonstrated the feasibility of the synthesis of thiadiazinane-2-thiones in solution [14][15][16][18][19][20][21] by reaction of the appropriate amine with carbon disulfide and potassium hydroxide, to give the dithiocarbamate potassium salt, which is not isolated, followed by cyclocondensation with formaldehyde and the selected amino acids or pseudopeptides able to provide the nitrogen atom at the N-5 position of the thiadiazinane ring. Based on experimental evidence and DFT studies, a probable cyclization route to 1,3,5-thiadiazinane-2-thiones in aqueous medium was proposed.…”