Investigating the Mechanism of Antimycobacterial and Antiproliferative Activity of (<i>E</i>)‐<i>N</i>’‐Benzylidenepyrazine‐2‐Carbohydrazides and their Derivatives

A series of 33 (E)-N'-benzylidenepyrazine-2-carbohydrazides and their derivatives were synthesized and tested for biological activity. Benzylidene derivatives with 2-OH substitution on the phenyl ring (18: R = 2-OH, 21: R = 2,3-diOH, and 22: R = 2,4-diOH) exhibit various biological activities. Compounds 18 and 21 demonstrate antimycobacterial activity against Mycobacterium tuberculosis H37Ra, M. tuberculosis H37Rv, and M. aurum, with minimum inhibitory concentration values ranging from 15.625 to 62.5 μg mL^-1. Compounds 18, 21, and 22 show mild cytotoxicity on several human cell lines (IC₅₀ ranging from 70.2 to 500 μM). Crystallographic studies confirm the (E)-configuration of compound 18 and a nearly planar molecular conformation. Due to their structural similarity with salicylaldehyde isonicotinoyl hydrazone (SIH), a known iron chelator, selected compounds were tested for iron-chelating properties, revealing comparable or superior activity. Mechanistic assays targeting enoyl-[acyl carrier protein] reductase (InhA), isocitrate lyase (ICL), and lipid/mycolic acid biosynthesis show no significant inhibition, suggesting a nonspecific mechanism potentially linked to iron chelation. A correlation is observed between chelating activity and cytotoxicity, while antimycobacterial activity appears to involve additional mechanisms. Pharmacokinetic studies with compound 18 reveal no specific plasma metabolites, and no significant metabolites are detected after incubation with human liver microsomes.