Design, synthesis, and evaluation of artesunate-benzofuran hybrids as dual anticancer and anti-inflammatory candidates: In silico profiling and in vitro validation in Caco-2, HepG2, Ishikawa, and RAW 264.7 cell lines

Abstract

Colorectal, liver, and uterine cancers remain major clinical challenges due to their high prevalence and mortality. In this study, a series of artesunate-benzofuran hybrid compounds (5A-5E) were designed and synthesized through a multistep approach and structurally characterized by ¹H/¹³C NMR, FTIR, and LC-MS/MS analyses. Their anticancer and anti-inflammatory potential was evaluated using complementary in silico and in vitro methodologies. Drug-likeness and ADMET predictions indicated favorable pharmacokinetic behavior and low toxicity. Molecular docking studies demonstrated strong binding affinities toward key cancer-related targets, DNA polymerase β (POLB), caspase-8 (CASP8), and protein kinase A (PKA), with binding energies ranging from -91.66 to -170.97 a.u., exceeding those of tamoxifen (TAM) (-88.40 to -114.88 a.u.) and 5-fluorouracil (5FU) (-52.27 to -53.79 a.u.). Molecular dynamics simulations further confirmed the stability of the ligand-protein complexes. In vitro cytotoxicity assays against Caco-2, HepG2, and Ishikawa cell lines revealed selective anticancer activity with minimal toxicity. Compounds 5A and 5C displayed the most pronounced effects, with half maximal inhibitory concentration (IC₅₀) values of 51.48 µM (Caco-2) and 35.16 µM (HepG2) for 5A, and 58.32 µM (Caco-2) and 50.98 µM (HepG2) for 5C. Mechanistic investigations showed apoptosis induction via caspase activation, inhibition of cell migration, G0/G1 cell-cycle arrest, and suppression of colony formation. In addition, compounds 5A-5C significantly reduced lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages. These results support artesunate-benzofuran hybrids as promising multitarget candidates for the development of anticancer agents with concurrent anti-inflammatory activity.