Novel benzofuran-furan-pyrano[2,3-c]pyrazole hybrids as potential anticancer agents: design, synthesis, in silico profiling, and multi-mechanistic biological evaluation
The development of anticancer agents capable of affecting multiple cancer-related cellular processes remains an important strategy in cancer drug discovery. In this study, a novel series of benzofuran-furan-pyrano[2,3-c]pyrazole hybrids was synthesized and comprehensively investigated through in vitro biological assays and in silico analyses. The synthesized compounds exhibited concentration-dependent cytotoxicity against LNCaP, MDA-MB-231, A549, and Caco-2 cancer cells, with EC50 values ranging from 20.49 to 274.37 µM while displaying lower toxicity toward non-malignant HUVEC cells. Among the synthesized derivatives, compounds 4j, 4h, and 4d showed the strongest antiproliferative activities and significantly suppressed cancer cell migration, invasion, and colony formation. Mechanistic studies demonstrated that the active hybrids induced G1/S phase cell cycle arrest, increased intracellular reactive oxygen species (ROS) production, disrupted mitochondrial membrane potential, and promoted apoptotic cell death. These findings were further supported by quantitative real-time PCR, which revealed downregulation of BCL2 together with upregulation of BAX, CASP3, CASP8, CASP9, and TP53, indicating activation of both intrinsic and extrinsic apoptotic pathways. In silico analysis of absorption, distribution, metabolism, excretion, and toxicity (ADMET) predicted favorable drug-likeness and pharmacokinetic properties for the synthesized hybrids. Molecular docking suggested potential interactions with CDK2, the androgen receptor, VEGFR-2, and Bcl-2, with compound 4f exhibiting the most favorable overall docking profile. Collectively, these findings demonstrate that benzofuran-furan-pyrano[2,3-c]pyrazole hybrids possess promising multifaceted in vitro anticancer activity by inhibiting cancer cell proliferation, migration, invasion, and clonogenicity while inducing ROS-mediated mitochondrial apoptosis, highlighting these hybrid scaffolds as attractive candidates for further anticancer drug development.