Conformationally adaptive benzimidazolium-chalcone hybrid salts as selective TGF-β1 inhibitors

The transforming growth factor-β (TGF-β) signaling pathway is crucial in promoting tumor growth, enabling tumors to evade immune responses, and contributing to resistance against therapies. As a result, it is a significant target for Cancer treatment. However, its full potential remains untapped because selectively inhibiting it without affecting normal cells is challenging. This study reports the design, synthesis, and comprehensive evaluation of novel benzimidazolium-chalcone hybrid salts (3a-3e) that strategically combine two privileged scaffolds with complementary Anticancer mechanisms. Following complete structural characterization by Elemental Analysis, FT-IR, and NMR spectroscopy, an integrated experimental and computational workflow supports a three-strategy hypothesis rather than a single lead compound. Compound 3e emerged as the most selective derivative, showing moderate anti-proliferative activity in U87 cells while maintaining reduced toxicity toward non-cancerous cells. Although direct pathway-level validation was beyond the scope of the present study, the slight reduction observed in extracellular TGF-β1, together with anti-migratory and anti-migratory and anti-clonogenic effects, along with in silico interaction patterns, supports 3e as a promising lead scaffold for further mechanistic investigation. In vitro studies using the glioblastoma cell line, U87, displayed promising Anticancer activity of benzimidazolium-chalcone hybrid salts. Compounds 3a, 3b, and 3c demonstrated limited selectivity, with IC₅₀ ratios between Cancer and normal cells ranging from 1.5- to 1.7-fold. Compound 3d showed a 1.6-fold and 2.0-fold selectivity advantage over BEAS-2B and HUVEC cells, respectively. In contrast, compound 3e demonstrated the most favorable selectivity profile, with an IC₅₀ of 41.09 μM in U87 cells and IC₅₀ values of ≥96.60 μM in normal cell lines. Molecular docking predicted binding affinities ranging from -9.91 to -11.67 kcal/mol. However, no significant correlation was observed between docking scores and biological activity (R² = 0.068, p = 0.671). Molecular dynamics simulations (3 × 100 ns) confirmed stable ligand binding for all compounds (protein-ligand minimum distance: ∼0.20 nm), with per-residue energy decomposition revealing that compound 3a binds mainly through extensive hydrophobic contacts (92% van der Waals), while compound 3e forms unique polar interactions with His283 and Tyr282. Principal component analysis revealed distinct conformational profiles (variance: 3a = 0.715, 3d = 1.364, 3e = 0.531), suggesting a possible connection between conformational restriction and cellular safety. ADMET profiling confirmed drug-like properties for compound 3e with no PAINS alerts or CYP3A4 inhibition. These findings support a preliminary hypothesis that links physicochemical properties and interaction quality, rather than static binding affinity, to therapeutic selectivity in TGF-β1 modulation design.