In a search for antiprotozoan compounds, 34 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazine-2-thione derivatives were synthesized and tested in vitro against Trypanosoma cruzi and Trichomonas vaginalis. Some of them showed important antiprotozoan activity. In vivo assays of compounds which showed remarkable in vitro activity against T. vaginalis were carried out.

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-

Abstract. The cytotoxicity of 18 new 1,2,6-thiadiazin-3,5-dione 1,1-dioxides was evaluated. This group of products was previously assayed against epimastigotes of Trypanosoma cruzi and some of them showed a high antiprotozoal activity. Thereafter 13 compounds with a high anti-epimastigote activity and low cytotoxicity were selected to be assayed against amastigotes. Some of the products showed the same or even lower cytotoxicity than nifurtimox and benznidazole, but most of them were very toxic for macrophages at 100 µg/ml. Only one of the compounds had an anti-amastigote activity similar to that of reference drugs at 10 µg/ml, but unfortunately this disappeared at lower concentrations.Chagas' disease is a major health problem in South and Central America, affecting 16-18 million people (WHO 1991). In spite of such high prevalence, only two synthetic compounds, nitroheterocycles nifurtimox (Lampit ® ) and benznidazole (Rochagan ® ), are in use (Croft et al. 1997). Both are effective in the early stages of trypanosomiasis, but are practically useless in the chronic disease. Only 50% of patients are parasitologically healed after treatment (Kirchhoff 1994). The limited efficacy as well as their toxic side effects justify the continued research for trypanocidal substances.In this way, new 1,2,6-thiadiazine-3,5-dione 1,1-dioxides have been synthesised (Di Maio et al. 1999). In previous works were reported the anti-Trypanosoma cruzi properties of 3,5-diamino-4-(5'-nitro-2-furfurylidene) 4H-thiadiazine 1,1-dioxide and those of some 4-heteroarylidene-1,2,6-thiadiazine-3,5-dione 1,1-dioxide . The presence of nitro substituents in the pentaheterocyclic moiety, as a source of free radicals, seems to be a factor that increases anti-T. cruzi activity (Di Maio et al. 1999).Previous anti-T. cruzi activity of these new thiadiazines was evaluated on epimastigotes. Almost all of them were effective at 100 µg/ml. Some of them remained active at 10 µg/ml (3a, 3b, 3c, 3d, 4a, 4b, 4c and 4d), but none at 1 µg/ml (Di Maio et al. 1999). After the first screening, new in vitro studies have been performed to analyse the nonspecific toxicity and antiamastigote activity of the compounds. MATERIALS AND METHODSCell culture. Murine J774 macrophages were grown in plastic 25ml 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. Trypanosoma cruzi Chagas, 1909 (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 in 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...

As a contribution to the search of antiparasitic activity in plants from Panama, a total of 37 extracts obtained from 14 species were assessed against Trypanosoma cruzi (Trypanosomatidae), Trichomonas vaginalis (Trichomonadidae), and Heligmosomoides polygyrus (Heligmosomidae). Also, the cytotoxicity to macrophages was determined, in order to discard, among the active extracts, those cytotoxic to mammalian cells. Tripanocidal activity and cytotoxicity to macrophages were performed by a colorimetric method using the tetrazolium salt MTT, assays on T. vaginalis by counting viable protozoa, and the anthelmintic activity was determined by enzymatic assays of acetylcholinesterase and acid phosphatase activities. Extracts from Bixa orellana L. (Bixaceae) and Clusia rosea Jacy. (Clusiaceae) were the most interesting both as antiprotozoan and nematocides.

In a search for antichagasic drugs, 14 new 4-(nitroarylidene)-1,2,6-thiadiazin-3,5-dione 1,1-dioxide derivatives were synthesized and tested in vitro against the epimastigote form of Trypanosoma cruzi and some of them showed important antiprotozoan activity. Attempts to synthesize new 4-(nitroarylidene)-3,5-diamino-1,2,6-thiadiazine 1,1-dioxides were unsuccessful. The antichagasic properties of nitroarylidene-malononitrile and nitroarylidene-cyanoacetamide derivatives, thus obtained, were also tested. The cytotoxic properties against Vero cells of compounds which showed remarkable in vitro antichagasic activity were evaluated. All compounds tested exhibited high toxicity percentages at 100 micrograms/ml. However, compound 3c showed in vitro antichagasic and cytotoxic properties such as nifurtimox at the dose of 10 micrograms/ml.