Multi-Kinase Inhibition by New Quinazoline–Isatin Hybrids: Design, Synthesis, Biological Evaluation and Mechanistic Studies

<b>Background/Objectives:</b> Cancer is a worldwide health concern and is the second leading cause of death, responsible for nearly one in six deaths. Discovery of new anticancer agents is still a challenge for medicinal chemists and further research will improve patients' chances of survival. Protein kinases are among the most popular and successful biological targets for developing anticancer drugs. In this context, protein kinases were selected as targets, and a series of isatin-quinazoline hybrids were synthesized. <b>Methods:</b> Their antiproliferative activity was evaluated against four cancer cell lines (HepG2, MCF-7, MDA-MB-231, and HeLa) and one normal fibroblast cell line (WI38) using MTT assays. <b>Results:</b> The tested compounds showed variable cytotoxic effects on the four cancer cell lines. Compound <b>6c</b> exhibited the most potent anticancer activity against all cancer cells. In addition, this compound was tested for the effect on the expression of anti-apoptotic Bcl-2 protein and pro-apoptotic proteins Bax, caspase-3, and caspase-9, which revealed induction of apoptosis similar to staurosporine. Furthermore, an annexin V-FITC/PI dual staining assay confirmed that compound <b>6c</b> induced cell death by apoptosis. Flow cytometric analysis revealed that compound <b>6c</b> induced cell cycle arrest at the sub-G1 and S phases in the HepG2 cell line. Moreover, compound <b>6c</b> was found to be a multi-kinase inhibitor with potent inhibitory activity on CDK2, EGFR, VEGFR-2, and HER2, with IC₅₀ values of 0.183 ± 0.01, 0.083 ± 0.005, 0.076 ± 0.004, and 0.138 ± 0.07 μM, respectively. Finally, a molecular docking simulation was conducted to predict possible binding interactions with the active site of CDK2. <b>Conclusions:</b> These findings suggest that compound <b>6c</b> is a promising multi-kinase inhibitor with potent anticancer activity, warranting further investigation as a potential therapeutic agent.