ABSTRACT A total of 48 tetrahydrooxazolo‐[5′,4′:4,5]pyrimido[1,2‐ a ]azepinones were designed and synthesized from a scaffold hopping approach. All compounds were confirmed by analysis using 1 H NMR, 13 C NMR, and HRMS techniques. The synthesized compounds were evaluated against the human cancer cell lines (HeLa, MCF‐7, A549) in vitro, and the structure–activity relationships were summarized . The compound E43 exhibited the best inhibitory activity against HeLa, MCF‐7, A549, displaying IC 50 values of 1.48 ± 0.13, 3.01 ± 0.09, and 5.11 ± 0.13 μM. Molecular docking indicated that compound E43 may bind to protein (PDB:6FEX) via hydrogen bond and π stacking. Further, molecular dynamics simulations indicated a relatively low binding free energy (−40.06 kJ·mol −1 ) of compound E43 with protein. In conclusion, these findings suggested that E43 is promising as a potential novel anticancer drug candidate worthy of further investigation.