Solid-phase and solution-phase protocols for the synthesis of furo[3,4-d]pyrimidines, pyrrolo[3,4-d]pyrimidines, and pyrimido[4,5-d]pyridazines are reported. The multistep solid-phase sequence involves the initial high-speed, microwave-promoted acetoacetylation of hydroxymethylpolystyrene resin with methyl 4-chloroacetoacetate. The immobilized 4-chloroacetoacetate precursor was subsequently subjected to three-component Biginelli-type condensations employing urea and a variety of aromatic aldehydes. The resulting 6-chloromethyl-functionalized resin-bound dihydropyrimidones served as common chemical platforms for the generation of the desired heterobicyclic scaffolds using three different traceless cyclative cleavage strategies. The corresponding furo[3,4-d]pyrimidines were obtained by microwave flash heating in a rapid, thermally triggered, cyclative release. Treatment of the chloromethyl dihydropyrimidone intermediates with a variety of primary amines followed by high-temperature microwave heating furnished the anticipated pyrrolo[3,4-d]pyrimidine scaffolds via nucleophilic cyclative cleavage. In a similar way, reaction with monosubstituted hydrazines resulted in the formation of pyrimido[4,5-d]pyridazines. All compounds were obtained in moderate to good overall yields and purities.

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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-