Anacyphrethines A and B as potent analgesics: Multiple ion channel inhibitors with an unprecedented chemical architecture.

Multi-target analgesics with minimal side effects and high efficacy are a key research focus in addressing the global pain crisis. Using a molecular networking approach, five pairs of potent analgesic alkaloid enantiomers were isolated from the roots of (. ). Their structures were elucidated by comprehensive spectroscopic data analysis, including LR-HSQMBC and H-N HMBC, quantum C NMR DP4+ and ECD calculations, and single-crystal X-ray diffraction analysis. Anacyphrethines A () and B () are highly conjugated and polymethylated 6/6/6/6/5/7/5/5-fused octacyclic tetraazabic alkaloids possessing an unprecedented 8,14,18,24-tetraaza-octacyclo[16.8.2.1.0.0.0.0.0] nonacosane motif. Their biosynthetic pathways are proposed involving key aldol, hydroamination, and Schiff base reactions. All isolates showed potent analgesic effects . Even at a lower dose of 0.2 mg/kg, (±)- and (+)- still exhibited more potent analgesic activities than morphine. Interestingly, the racemic mixture (±)- showed stronger analgesic effect than either pure enantiomer alone at higher doses of 5 and 1 mg/kg; while, (±)- showed significant analgesic activities comparable to (+)- at lower doses of 0.2 and 0.04 mg/kg. (+)- had stronger analgesic effect than (-)- at five tested does. Further tests on 44 analgesic-related targets demonstrated that (+)- showed significant inhibitory effects against many ion channels such as TRPM8, Kv1.2, Kv1.3, and Ca2.1 with IC values of 1.10 ± 0.26, 4.20 ± 0.07, 2.20 ± 0.24, and 10.40 ± 0.69 μmol/L, respectively, while (-)- primarily inhibited TRPC6, Kv1.2, and Kv1.3 ion channels with IC values of 0.81 ± 0.05, 0.91 ± 0.04, and 1.50 ± 0.13 μmol/L, respectively, without affecting the opioid receptors, suggesting their non-opioid analgesic potentials. The molecular dockings provided structural guidance to develop potent non-opioid analgesics.