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

Abstract

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.