Ring-Opening Simplification of Thiazolyl-Pyrazoline-Triazole Hybrids: Synthesis, SAR and Antifolate Mechanism Against Leishmania major

Abstract

Leishmaniasis, a neglected tropical disease with a severely limited and toxicity-prone therapeutic arsenal, urgently demands structurally innovative drug candidates. Capitalizing on the well-established antileishmanial potential of thiazole, triazole, and pyrazole pharmacophores, we describe a rational scaffold-hybridization strategy and ring-opening approach employing two distinct molecular architectures: a rigid pyrazoline ring as a cyclic linker connecting the thiazole and triazole units in series 6a-r, and a simplified acyclic hydrazone linker replacing the pyrazoline core in series 8a-h. In vitro evaluation against Leishmania major revealed that open hydrazone-linked derivatives (8a-h) significantly outperformed their rigid pyrazoline counterparts (6a-r). In particular, compounds 8d and 8h, carrying a para-fluorophenyl thiazole moiety, emerged as outstanding dual-stage lead compounds, exhibiting sub-micromolar anti-promastigote activity (IC50 = 0.94 ± 0.12 and 0.89 ± 0.14 µM, respectively), representing an ~8- to 9-fold potency gain over miltefosine (IC50 = 7.84 µM), along with robust intracellular anti-amastigote inhibition (IC50 = 3.29 and 2.68 µM, respectively). SAR analysis established a clear substituent rank order for the thiazole aryl moiety (R₁): F >> CH₃ ≈ Cl ≈ H, identifying fluorine as the critical potency determinant. Mechanistic investigation through a folate-reversal assay suggested that the antileishmanial activity is mediated via an antifolate mechanism, potentially involving the parasite-specific enzymes DHFR-TS and PTR1. Parasite survival was restored in a dose-dependent manner upon co-treatment with folic or folinic acid, reaching 82-88% at 20 µM and 87-96% at 100 µM folic acid, depending on the compound tested. Furthermore, 8d and 8h demonstrated excellent safety profiles in VERO cells (CC50 = 305.6 and 585.6 µM; SI = 323 and 655, respectively), validating this open hydrazone-linked thiazole–triazole scaffold as a promising platform for antileishmanial drug development. Finally, molecular docking studies of compounds 8d and 8h within the PTR1 active site were performed to provide a qualitative structural hypothesis for potential binding modes and to support, in a general manner, the proposed antifolate mechanism of action.