Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti- Agents.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles () and 2-guanidino benzimidazoles (), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of DHFR and PTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against and and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole resulted to be the most potent ( = 9 nM) and highly selective DHFR inhibitor, 6000-fold over PTR1 and 394-fold over DHFR. The 5,6-dimethyl tricyclic analogue , despite showing a lower potency and selectivity profile than , shared a comparable antiparasitic activity against in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles and revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of and parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.