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Benzylidene derivatives with 2\u2010OH substitution on the phenyl ring (<jats:bold>18<\/jats:bold>: <jats:italic>R<\/jats:italic>\u2009=\u20092\u2010OH, <jats:bold>21<\/jats:bold>: <jats:italic>R<\/jats:italic>\u2009=\u20092,3\u2010diOH, and <jats:bold>22<\/jats:bold>: <jats:italic>R<\/jats:italic>\u2009=\u20092,4\u2010diOH) exhibit various biological activities. Compounds <jats:bold>18<\/jats:bold> and <jats:bold>21<\/jats:bold> demonstrate antimycobacterial activity against <jats:italic>Mycobacterium tuberculosis<\/jats:italic> H37Ra, <jats:italic>M. tuberculosis<\/jats:italic> H37Rv, and <jats:italic>M. aurum,<\/jats:italic> with minimum inhibitory concentration values ranging from 15.625 to 62.5\u2009\u03bcg\u2009mL<jats:sup>\u22121<\/jats:sup>. Compounds <jats:bold>18<\/jats:bold>, <jats:bold>21<\/jats:bold>, and <jats:bold>22<\/jats:bold> show mild cytotoxicity on several human cell lines (IC<jats:sub>50<\/jats:sub> ranging from 70.2 to 500\u2009\u03bcM). Crystallographic studies confirm the (<jats:italic>E<\/jats:italic>)\u2010configuration of compound <jats:bold>18<\/jats:bold> 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\u2010chelating properties, revealing comparable or superior activity. Mechanistic assays targeting enoyl\u2010[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 <jats:bold>18<\/jats:bold> reveal no specific plasma metabolites, and no significant metabolites are detected after incubation with human liver microsomes.<\/jats:p>","DOI":"10.1002\/cmdc.202500085","type":"journal-article","created":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T12:59:06Z","timestamp":1756472346000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Investigating the Mechanism of Antimycobacterial and Antiproliferative Activity of (<i>E<\/i>)\u2010<i>N<\/i>\u2019\u2010Benzylidenepyrazine\u20102\u2010Carbohydrazides and their Derivatives"],"prefix":"10.1002","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7011-4102","authenticated-orcid":false,"given":"Priam\u2010Amedeo","family":"Houngbedji","sequence":"first","affiliation":[{"name":"Faculty of Pharmacy in Hradec Kr\u00e1lov\u00e9 Charles University  Ak. 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