Targeting <i>P</i> -glycoprotein with novel halogenated 4 <i>H</i> -pyran-coumarin hybrids: a combined experimental, computational approach to tumor-sensitive and -resistant (breast) cancer cell lines

The over expression of <i>P</i>-glycoprotein (<i>P</i>-gp) is a primary mechanism of multidrug resistance (MDR) in cancer, urgently necessitating the development of novel and effective inhibitors. This work presents a new series of halogenated 4<i>H</i>-pyran-coumarin hybrids designed to overcome this resistance. Six novel hybrids (<b>4a-f</b>) were synthesized and characterized using IR, MS, and 1D/2D NMR techniques. Their antitumor potential was evaluated against sensitive (MCF-7, Caco-2) and resistant (MCF-7/ADR) cancer cell lines, alongside two normal lung cell lines (HFL-1, WI-38), using MTT assays. The ability to inhibit <i>P</i>-gp expression was assessed <i>via</i> ELISA, and efflux pump inhibition was tested with a Rhodamine 123 accumulation assay. Molecular docking and dynamics simulations were employed to investigate interactions with the <i>P</i>-gp binding site. Compounds <b>4a</b> (2-F), <b>4c</b> (2-Cl), and <b>4d</b> (3-Cl) emerged as the most potent leads. They exhibited significant cytotoxicity against MCF-7 (IC₅₀ = 8.14-11.86 µM) and Caco-2 (IC₅₀ = 9.04-13.61 µM) cells and, crucially, effectively reversed <i>P</i>-gp-mediated MDR in MCF-7/ADR cells (IC₅₀ = 15.09-22.79 µM), outperforming Doxorubicin (IC₅₀ = 50.9 µM). These compounds also demonstrated selectivity over normal cells. Mechanistic studies revealed they significantly down regulated <i>P</i>-gp expression. Docking studies confirmed strong binding interactions within the <i>P</i>-gp drug-binding pocket, which were stabilized by key residues like Gln721, as further validated by molecular dynamics simulations. We have successfully developed a novel class of 4<i>H</i>-pyran-coumarin hybrids with potent dual antitumor and MDR-reversal activity. The most promising compounds, particularly the 3-chloro derivative <b>4d</b>, act through down regulation of <i>P</i>-gp expression and represent highly promising leads for further development as therapeutic agents to combat chemotherapy-resistant cancers.