Design, Synthesis, and Biological Studies of C-5-Substituted Diazenyl Derivatives of Uracil as Potent and Selective Antileishmanial Agents Targeting Uridine Biosynthesis Pathway Enzymes.

Herein, we describe the design and synthesis of a series of C-5-substituted diazenyl derivatives of uracil, exhibiting selective and potent antileishmanial but not antibacterial or antifungal activity. The formation of the substituted derivatives was confirmed by using FTIR, ¹H, ¹³C NMR, and HRMS analysis. Among all of the sets of tested compounds, only three [4a, 6b, and 8b] showed the highest activity against Leishmania donovani (LD) promastigote and amastigote models of LD infections. Further, the cytotoxicity assays performed using three different cell lines, Vero cells, J774 cells, and THP1 cells, along with erythrocyte hemolysis assay showed the highest biocompatibility for the 4a, making it a lead compound for further biological assays. The LD cell death associated with 4a was not linked with ergosterol depletion, a common mechanism of action of antileishmanial drugs like amphotericin B (AmB). However, the LD cell death in the presence of 4a was reversed significantly through supplementation of uridine monophosphate (UMP), indicating the specific role of uridine biosynthesis pathway as the target of 4a. Furthermore, the in silico studies predicted ornithine monophosphate decarboxylase enzyme (OMPDCase) from LD as the plausible target for 4a. The proteomics analysis showed stronger downregulation of the aforementioned OMPDCase and also for a few other enzymes that are involved in the UMP biosynthesis pathway. This indicates that OMPDCase and other enzymes that regulate the UMP biosynthesis may be the target of 4a. Overall, the C-5-substituted diazenyl derivatives of uracil are presented here as novel and potent antileishmanial agents that can be used for treating visceral leishmaniasis (VL) wherein at present drug resistance and side effects of existing drugs demand a look for safer alternatives.