Design, synthesis, and biological evaluation of (E)-3-amino-N′-substituted benzylidene-6-chloropyrazine-2-carbohydrazide derivatives as anti-mycobacterial agents

Abstract

Pyrazinamide is a powerful sterilizing agent that reduces the treatment duration required to cure tuberculosis and works synergistically with both new and existing anti-tuberculosis drugs. Thirty-one derivatives of (E)-3-amino-N′-substituted benzylidene-6-chloropyrazine-2-carbohydrazide (20a-20ae) were designed and synthesized. The structures of these compounds were confirmed through various analytical methods, such as 1H NMR, 13C NMR, and mass spectrometry. To better understand the arrangement of atoms and confirm the structures, single crystals of 20 m and 20aa were grown and analyzed. The final derivatives, 20a-20ae, were evaluated for their anti-mycobacterial activity against the Mycobacterium tuberculosis (M.tb) H37Ra strain using the Microplate Alamar Blue Assay (MABA). Among all the synthesized compounds tested, 20 m and 20s showed potent activity with a minimum inhibitory concentration (MIC) of 3.13 μg/mL (8.66 μM and 11.37 μM, respectively). 20q and 20r also displayed significant anti-TB activity with an MIC of 6.25 μg/mL (23.66 μM and 21.47 μM, respectively). The MIC values of the remaining compounds ranged from 12.5 to >50 μg/mL (34.62 μM to 172.96 μM). To further evaluate the binding interaction within the active site of the enzyme aspartate decarboxylase (PanD) from M. tb (PDB: 6P02), a molecular docking analysis of compound 20s was performed. Finally, 100 ns molecular dynamics simulations were carried out to comprehend the stability, conformation, and intermolecular interactions of the co-crystal ligand and the highly active compound 20s with the selected target protein. Further, in order to better understand bacterial resistance and pathogenesis and to create efficient treatments against significant drug-resistant pathogens, in vitro anti-mycobacterial activity of the compounds with MIC ≤12.5 μg/mL (43.24 μM) was assessed for their effectiveness against the ESKAPE group of pathogens using the MABA method. Results indicate that 20e exhibited the most promising activity with an MIC of 50 μg/mL (172.9 μM) against Staphylococcus aureus among the ESKAPE group of pathogens.