Design and optimization of N-(3-((4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)amide derivatives as potent anti-inflammatory agents against LPS-induced acute lung injury.

Efficient treatment of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) can be achieved by inhibiting the inflammatory cascade and reducing pulmonary inflammation. A series of novel N-(3-((4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)amide derivatives were rationally designed and synthesized as cathepsin L (CTSL) inhibitors to reduce the expression of pro-inflammatory cytokines. Among these compounds, B5 exhibited dose-dependent inhibition of pro-inflammatory cytokine production in HBE cells, with IC50 values of 2.51 μM for IL-6 and 1.15 μM for IL-8, without inducing significant cytotoxicity in vitro. In addition, B5 effectively inhibited CTSL enzymatic activity, with an IC50 value of 5.52 μM. In LPS-induced ALI mouse model, treatment with B5 (20 mg/kg) significantly reduced inflammatory cell infiltration into lung tissue, thereby markedly alleviating lung injury. Mechanistic studies revealed that B5 suppresses CTSL maturation, which in turn attenuates activation of the downstream NF-κB and p38 MAPK signaling pathways. Molecular docking further demonstrated that B5 establishes stable binding in the active site of CTSL through multiple noncovalent interactions with key residues Asp162, Cys25, and Glu63. Specifically, the NH linker, the pyrimidine ring, and the amino group participate in hydrogen bonding, π-sulfur interactions, and electrostatic interactions, respectively. These findings indicate that B5 exhibits potent anti-inflammatory activity both in vitro and in vivo and represents a promising lead compound for the treatment of ALI.