Discovery and Characterization of Neuroactive Secondary Metabolites from Acacia sieberiana with Potential Analgesic Properties.

Acacia sieberiana (syn. Vachellia sieberiana), widely used in African traditional medicine for pain, was investigated using an ethnopharmacology-guided workflow integrating LC-MS/MS-based metabolomics and molecular networking to prioritize bioactive secondary metabolites. Fractionation of the ethanolic stem bark extract enabled targeted isolation and identification of piperine (1) and moupinamide (3) by HRMS and NMR. Moupinamide and its positional isomer (tamgermanetin) were synthesized via HATU-mediated coupling to support preliminary structure-activity studies. In a capsaicin-induced mouse model, the extract significantly attenuated acute nociceptive behavior without affecting the secondary mechanical hypersensitivity. Functional screening using human iPSC-derived nociceptors in a multielectrode array (MEA) platform showed that moupinamide potently reduced neuronal firing, with reduced efficacy under high-heat conditions, suggesting a non-TRPV1-mediated mechanism. GPCR profiling of moupinamide revealed minimal receptor engagement, and further pharmacological studies demonstrated retained activity under potassium channel blockade and suppression of Nav1.7-induced hyperexcitability, consistent with voltage-gated sodium channel modulation. These findings highlight A. sieberiana-derived alkaloid amides as promising nonopioid analgesic leads and underscore the utility of metabolomics-guided discovery strategies.