Pyridine Regioisomerism and Aryl Substitution Govern the Divergent Cyclization of Isoniazid-Derived Thiosemicarbazides: Mechanistic DFT Analysis and Antimycobacterial Evaluation

Isoniazid is an established antitubercular pharmacophore widely used as a structural platform for developing compounds with potential activity against Mycobacterium tuberculosis. This study investigated isoniazid-derived thiosemicarbazides as precursors for the synthesis of thiazolidin-4-one derivatives, focusing on the influence of pyridine regioisomerism and aryl substitution on cyclization outcomes. Thiosemicarbazides containing 2-, 3-, or 4-pyridyl moieties and fluoro- or chloro-substituted phenyl groups were synthesized and subjected to cyclization with ethyl bromoacetate. Density functional theory (DFT) calculations were performed to compare the competing pathways leading to thiazolidin-4-one and 1,3,4-oxadiazole formation, and the resulting heterocyclic compounds were evaluated against four M. tuberculosis strains. An initial control experiment using a 2-pyridyl/2-fluorophenyl thiosemicarbazide yielded the target thiazolidin-4-one as the sole product. However, investigation of the broader substrate series revealed pronounced substrate-dependent differences in cyclization selectivity. Most substrates afforded 1,3,4-oxadiazoles, whereas 2-pyridyl thiosemicarbazides bearing ortho-fluoro or ortho-chloro substituents selectively formed thiazolidin-4-ones. DFT calculations provided an energetic rationale for the observed divergence in cyclization pathways. Despite systematic structural diversification, none of the investigated 1,3,4-oxadiazole or thiazolidin-4-one derivatives, including ester-functionalized analogues, exhibited appreciable antimycobacterial activity (MIC ≥ 512 µg/mL). These findings demonstrate that pyridine regioisomerism combined with aryl substitution strongly influences the cyclization pathways of isoniazid-derived thiosemicarbazides. However, the pronounced structure–reactivity relationship governing cyclization was not accompanied by a meaningful structure–activity relationship against Mycobacterium tuberculosis. The combined experimental and computational results provide insight into the factors governing cyclization selectivity and inform the design of future INH-derived analogues.