Quinoxaline-based cyanoacrylate hybrids as multi-target anti-proliferative and anti-inflammatory agents with potent activity against hepatocellular carcinoma: induction of apoptosis and modulation of oxidative stress signaling.

A set of quinoxaline-based derivatives incorporating a cyanoacrylate pharmacophore were designed, synthesized, and biologically evaluated for their dual anticancer and anti-inflammatory potential. Cytotoxic activity was assessed against HepG-2, HCT-116, MCF-7, and Panc-1 cancer cell lines using the MTT assay. Among the synthesized compounds, compound 9 exhibited the highest potency, particularly against HepG-2 cells (IC50 = 7.81 ± 1.1 µM), surpassing doxorubicin (IC50 = 15.96 ± 0.61 µM), and demonstrated improved selectivity toward WI-38 normal fibroblasts (IC50 = 67.21 µM; SI ≈ 7.81). Mechanistic investigations in HepG-2 cells revealed that compound 9 induced pronounced G0/G1 cell cycle arrest, increasing the cell population from 54.39% to 86.21% (∼1.6-fold), with a concomitant reduction in S-phase cells (∼3.1-fold decrease). Apoptosis analysis showed a significant increase in total apoptotic cells from 3.12% to 35.14% (∼11.3-fold), predominantly driven by late apoptosis (∼129-fold increase). These findings were supported by gene expression analysis, where compound 9 upregulated p53 (∼5.9-fold), BAX (∼3.7-fold), cytochrome c (∼3.9-fold), and caspase-7 (∼2.6-fold), while downregulating Bcl-2 (∼0.64-fold), indicating activation of the intrinsic mitochondrial apoptotic pathway. In LPS-stimulated RAW264.7 macrophages, compound 9 exhibited potent anti-inflammatory activity without significant cytotoxicity. The compound markedly reduced intracellular reactive oxygen species (ROS) levels in a dose-dependent manner (from 591.52 to 60.62 pg mL-1; ∼9.8-fold reduction) and significantly suppressed nitric oxide production. Furthermore, it downregulated key pro-inflammatory cytokines, including TNF-α (∼0.36-fold), IL-1β (∼0.47-fold), and IL-6 (∼0.51-fold), with effects comparable to celecoxib. Molecular docking studies indicated favorable binding of compound 9 within the active sites of IL-1β, TNF-α, and IL-6Rα, with the highest affinity toward IL-6Rα (-7.93 kcal mol-1). These interactions were further validated by 100 ns molecular dynamics simulations, which supported stable protein-ligand complexes, consistent RMSD profiles, preserved structural compactness, and persistent key interactions throughout the trajectories. Collectively, these findings identify compound 9 as a promising multi-target quinoxaline-based agent with dual antiproliferative and anti-inflammatory activities, mediated through induction of mitochondrial apoptosis and modulation of oxidative stress and cytokine signaling pathways.