Pyrazoline- and hydrazone-linked thiazole-triazole hybrids as potent dual-stage antileishmanial agents with antifolate activity

Abstract

Leishmaniasis remains a neglected tropical disease with limited and often toxic therapeutic options, underscoring the need for new and safer antileishmanial agents. In this work, two series of thiazolyl-triazole hybrids were designed using scaffold hybridization and cyclic-to-acyclic structural remodeling strategies. Series 6a-i incorporated a ring-closed pyrazoline moiety within the hybrid framework, whereas series 8a-i represented the corresponding ring-opened acyclic hydrazone analogues derived from structural opening of the pyrazoline core. In vitro evaluation against Leishmania major revealed that the ring-opened hydrazone derivatives showed markedly improved activity compared with their pyrazoline counterparts, indicating the favorable contribution of the acyclic hydrazone-based scaffold organization to antileishmanial potency. In particular, compounds 8b , 8f and 8i emerged as the most promising antileishmanial candidates, exhibiting potent activity against both promastigote and axenic amastigote forms. Against promastigotes, these compounds displayed half-maximal inhibitory concentration (IC 50 ) values of 2.92 ± 0.18, 3.63 ± 0.24 and 2.10 ± 0.12 μM, respectively, outperforming miltefosine (IC 50 = 7.84 ± 0.36 μM). They also retained strong activity against axenic amastigotes, with IC 50 values of 5.02 ± 0.26, 4.89 ± 0.22 and 4.24 ± 0.18 μM, respectively, compared with miltefosine (IC 50 = 8.08 ± 0.22 μM). Mechanistic investigation using a folate-reversal assay showed concentration-dependent recovery of parasite viability upon co-treatment with folic or folinic acid, suggesting involvement of the folate pathway, likely through inhibition of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and/or pteridine reductase 1 (PTR1). The selected compounds also showed low cytotoxicity toward Vero cells, with half-maximal cytotoxic concentration (CC 50 ) values of 154.5-281.9 μM and selectivity index (SI) values of 53-125 against promastigotes and 31-62 against axenic amastigotes. Molecular docking within the PTR1 active site provided qualitative support for the proposed antifolate mechanism. Overall, these findings identify the ring-opened hydrazone-containing thiazolyl-triazole scaffold as a promising and selective platform for further development of antileishmanial agents.