Design, synthesis, and biological evaluation of glycyrrhetinic acid-cinnamamide derivatives as HMGB1/TLR4 pathway inhibitors for sepsis-induced liver injury.

Sepsis-associated systemic inflammation represents a predominant etiology of acute liver injury, with the high-mobility group box 1(HMGB1)-mediated late-phase inflammatory response playing a central pathogenic role. As a key damage-associated molecular pattern molecule, HMGB1 activates downstream pro-inflammatory cascades through binding to Toll-like receptor 4(TLR4), triggering early-phase cytokine release (TNF-α, IL-1β) and upregulating inflammatory effectors(iNOS, COX-2), thereby establishing a self-amplifying cycle of inflammatory signaling and oxidative stress. Guided by this pathological framework, we developed a series of novel 3-aminoglycyrrhetinic acid derivatives through rational drug design targeting the HMGB1/TLR4 axis. Structural optimization of glycyrrhetinic acid involved C-30 benzyl esterification, C-3 amination, and subsequent conjugation with differentially substituted cinnamic acids, yielding 18 novel analogs. Through systematic pharmacological screening, compound 18 (featuring a 3',4'-dihydroxycinnamamide pharmacophore) emerged as a superior multi-target therapeutic candidate in LPS-challenged RAW264.7 and HepG2 models. It demonstrated concentration-dependent suppression of the upstream mediators TNF-α, IL-1β, HMGB1, and TLR4, while concurrently inhibiting downstream effectors iNOS and COX-2, achieving potent NO reduction (IC50 = 1.13 µM). Notably, compound 18 exhibited dual mechanistic activity by significantly attenuating intracellular ROS generation and restoring mitochondrial membrane integrity, indicating synchronized control of inflammatory and oxidative pathways. Molecular docking analysis indicates that the 3',4'-dihydroxy structure of compound 18 exhibits significantly higher binding affinity with HMGB1 compared to the monohydroxy-substituted compounds 17 and GA. In a murine sepsis model, treatment with compound 18 resulted in remarkable amelioration of hepatic histopathological damage, showing significantly enhanced efficacy compared to the parent compound GA. Mechanistic investigation confirmed that its hepatoprotective effects are mediated through suppression of the hepatic HMGB1/TLR4 signaling axis and subsequent modulation of iNOS/COX-2 expression. Pharmacokinetic studies demonstrated that compound 18 exhibited an oral bioavailability of 39.47%, significantly superior to GA, and showed enriched distribution in liver tissue (accounting for 42.0%). These findings establish compound 18 as a promising lead candidate for developing targeted therapeutics against HMGB1/TLR4-driven acute liver injury in sepsis, providing new strategic insights for inflammatory liver pathology intervention.