Design, synthesis, and evaluation of 3-fluoro-2-hydroxybenzaldehyde derivatives as TLR2 small molecule antagonists

Aberrant activation of Toll-like Receptor 2 (TLR2) is closely associated with inflammatory diseases, autoimmune disorders, and certain cancers, underscoring the importance of developing potent and selective TLR2 antagonists. Guided by the structure activity relationship (SAR) of the lead compound C29, along with a constructed 4D-QSAR model (Q² = 0.505, R² = 0.895) and molecular docking studies, a series of novel 3-fluoro-2-hydroxybenzaldehyde derivatives (Series A-C) were rationally designed, synthesized, and systematically evaluated. Specifically, Series A aimed to strengthen hydrogen bond interactions, Series B introduced quinoline moieties to better occupy the S1 hydrophobic pocket, and Series C optimized the linker structures. In vitro biological assessment using the HEK-Blue™ hTLR2 reporter gene assay demonstrated that the synthesized compounds exhibited significantly enhanced inhibitory effects (IC₅₀ = 0.94-17.33 μM) compared to the C29 (IC₅₀ = 47.35 μM). Notably, compound B1 emerged as the most potent candidate, achieving sub-micromolar TLR2 inhibitory activity (IC₅₀ = 0.94 μM). Selectivity analysis confirmed its excellent specificity toward TLR2 over Other TLR subtypes. Furthermore, pharmacokinetic evaluations revealed that B1 possessed acceptable metabolic stability but limited oral absorption. Molecular dynamics (MD) simulations (200 ns) of representative compound-TLR2 complexes provided deep insights into their binding modes. Comprehensive evaluations, including root mean square deviation (RMSD), MM/PBSA binding free energies, principal component analysis (PCA), and free energy landscape (FEL), supported the improved conformational stability and optimized binding modes of the novel antagonists compared to the C29 complex. Collectively, these findings establish a solid theoretical and experimental foundation for the further rational design and optimization of TLR2 antagonists.