Synthesis, Computational Evaluation, and Anticancer Assessment of Benzofuran-Based Pyrano[2,3- c ]pyrazole Derivatives

Abstract A new series of benzofuran-based pyrano[2,3-c]pyrazole derivatives 4a–e was synthesized and evaluated through integrated synthetic, computational, and biological studies. The target compounds were prepared via a multistep route involving Sonogashira coupling, iodine-mediated cyclization, Suzuki–Miyaura coupling, and final K2CO3-mediated one-pot cyclocondensation under mild reaction conditions. The optimized protocol afforded the desired derivatives in high to excellent yields (88–98%), and their structures were confirmed by 1H NMR, 13C NMR, and HRMS analyses. ADMET prediction indicated acceptable drug-likeness profiles, favorable intestinal permeability, and low predicted toxicity risks for the synthesized derivatives. Molecular docking studies against CDK2, the androgen receptor, VEGFR2, and Bcl-2 yielded favorable docking scores and predicted ligand-residue interactions within the selected binding pockets. Among the synthesized compounds, 4c showed the most favorable docking scores toward CDK2 and the androgen receptor, whereas 4a showed the most favorable scores toward VEGFR2 and Bcl-2. The most favorable complexes obtained from molecular docking were further examined by 100 ns molecular dynamics simulations, where RMSD, RMSF, and Rg analyses supported their dynamic stability and structural compactness during the simulation period. Biological evaluation demonstrated moderate, cell line-dependent antiproliferative activity, with 4a showing the lowest EC50 values against A549 and LNCaP cells (75.58 and 86.40 μM, respectively). Mechanistic assays indicated that the active derivatives promoted apoptosis, regulated apoptosis-related gene expression, suppressed cell migration and colony formation, enhanced intracellular ROS generation, disrupted mitochondrial membrane potential, and affected cell-cycle progression. These findings suggest that benzofuran-based pyrano[2,3-c]pyrazoles represent biologically relevant heterocyclic scaffolds with measurable anticancer activity supported by computational and cellular evidence.