New Hydrazone Derivatives Featuring Isatin and Piperazine Moieties: Synthesis, Cytotoxicity Evaluation, and Molecular...

In this study, ten novel compounds (IPH1-IPH10) were designed by integrating three pharmacophores (isatin, piperazine, and hydrazone) commonly found in the molecular structures of anticancer agents. The target molecules were obtained by the reaction of in-house prepared 4-(4-(pyridin/pyrimidin-2-yl)piperazin-1-yl)benzohydrazide with various isatin derivatives. After confirming their proposed chemical structures with different spectral techniques, the final compounds were tested for their cytotoxicity against colorectal cancer (CRC) cell lines (HT-29, SW480, and DLD-1). Among several derivatives exhibiting remarkable antiproliferative activity against CRC cell lines while sparing healthy fibroblasts (L929), IPH9 stood out for its exceptional anticancer potency (IC₅₀ = 1.13 μM) with high selectivity (SI = 69.50). Structure-activity relationships analysis suggested that electron-withdrawing substituents on the isatin core played a key role in enhancing cytotoxic potential. Cell cycle and immunocytochemical analyses on the selected compounds showed that, unlike IPH8, IPH9 induced G1 phase cell cycle arrest by regulating Bax and Caspase-3 expression and significantly increased apoptosis. Moreover, molecular docking studies were performed to investigate the potential interactions of IPH9 with Bax and Caspase-3. In silico calculations confirmed the favorable drug-likeness and oral bioavailability of these molecules, supporting their potential as anticancer agents with preferable selectivity profiles. Overall, supported by biological and computational data, our study introduces a new approach to designing molecules as effective cytotoxic agents targeting CRC, based on isatin and piperazine pharmacophores.