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A series of new phthalazine derivatives 1-4 containing imidazole rings were prepared. The monoalkylamino substituted derivatives 2 and 4 were more active in vitro against T. cruzi and less toxic against Vero cells than both their disubstituted analogues and the reference drug benznidazole. Compounds 2 and 4 highly inhibited the antioxidant parasite enzyme Fe-SOD, and molecular modeling suggested that they interact with the H-bonding system of the iron atom moiety. In vivo tests on the acute phase of Chagas disease gave parasitemia inhibition values twice those of benznidazole, and a remarkable decrease in the reactivation of parasitemia was found in the chronic phase for immunodeficient mice. Glucose metabolism studies showed that compounds 1-4 did not affect the succinate pathway but originated important changes in the excretion of pyruvate metabolites. The morphological alterations found in epimastigotes treated with 1-4 confirmed extensive cytoplasm damage and a high mortality rate of parasites.

The in vivo trypanosomicidal activity of the imidazole-based benzo[g]phthalazine derivatives 1-4 and of the new related pyrazole-based compounds 5 and 6 has been studied in both the acute and chronic phases of Chagas disease. As a rule, compounds 1-6 were more active and less toxic than benznidazole in the two stages of the disease, and the monosubstituted derivatives 2, 4, and 6 were more effective than their disubstituted analogs. Feasible mechanisms of action of compounds 1-6 against the parasite have been explored by considering their inhibitory effect on the Fe-SOD enzyme, the nature of the excreted metabolites and the ultrastructural alterations produced. A complementary histopathological analysis has confirmed that the monosubstituted derivatives are less toxic than the reference drug, with the behavior of the imidazole-based compound 4 being especially noteworthy.

The synthesis and trypanosomatic behavior of a new series of 1,4-bis(alkylamino)benzo[g]phthalazines 1- 4 containing the biologically significant imidazole ring are reported. In vitro antiparasitic activity against Trypanosoma cruzi epimastigotes is remarkable, especially for compound 2, whereas toxicity against Vero cells is very low. Conversion of epimastigotes to metacyclic forms in the presence of the tested compounds causes significant decreases in the amastigote and trypomastigote numbers. Fe-SOD inhibition is noteworthy, whereas effect on human Cu/Zn-SOD is negligible.

A convenient synthesis of the proton-ionizable crown 3 is reported that uses dibutyltin oxide. In acetonitrile, the reaction of 3 (LH(2)) with phenethylamine and homoveratrylamine (molar ratio 1:2) affords solid dinuclear complexes [LH(2)]2RNH(2) (4a,b), which spectroscopic (FAB-MS, IR, (1)H and (13)C NMR) data point toward a strong participation of the pyrazole nitrogens in the amine complexation. In DMSO-d(6) solution, a (13)C NMR study demonstrates the formation in situ of analogous neutral 4a-d[LH(2)]2RNH(2) or charged 5a-d[L(2)(-)]2RNH(3)(+) dinuclear complexes by reaction of 3 [LH(2)] or 3'[L(2)(-)]2Na(+) with RNH(2) (phenethylamine, homoveratrylamine, dopamine, and norepinephrine) or their RNH(3)(+)Cl(-) salts, respectively. Differences between the structure of complexes 4 and 5 have been evaluated by taking the homoveratrylamine derivatives 4b and 5bas models. An (1)H and (13)C NMR study (by raising the temperature) and measurements of intermolecular NOE effects (from NOESY and ROESY spectra) demonstrate that both complexes behave as prototropic isomers showing different conformations. By increasing the ionic strength, the 4b isomer structure becomes similar to that of 5b. The molecular modeling (GenMol software) of 4a-d and 5a-d shows that the assemblage in which both amine molecules are on the same side of the crown is the more stable. Lipophilic amines afford more stable complexes than hydrophilic ones and charged species are much more stable than the neutral ones.

The tricyclic heteroaromatic nucleus of 1,4-bis(alkylamino)benzo[g]phthalazine can be protonated at physiological pH, depending on the nature of the side chains. The interaction of the 3-methoxypropyl derivative with calf thymus and closed, circular DNA has been studied with UV-vis spectroscopy and NMR. The effect of drug binding on the topology of closed, circular DNA was determined by topoisomerase-I catalyzed relaxation of the complex followed by gel electrophoresis. The results strongly support intercalative binding and suggest that this series of compounds are promising targets for anticancer activity evaluation.