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 <i>Anacyclus pyrethrum</i> (<i>A</i>. <i>pyrethrum</i>). Their structures were elucidated by comprehensive spectroscopic data analysis, including LR-HSQMBC and ¹H-¹⁵N HMBC, quantum ¹³C NMR DP4+ and ECD calculations, and single-crystal X-ray diffraction analysis. Anacyphrethines A (<b>1</b>) and B (<b>2</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[16.8.2.1^1,23.0^4,28.0^5,17.0^9,16.0^11,15.0^21,27] nonacosane motif. Their biosynthetic pathways are proposed involving key aldol, hydroamination, and Schiff base reactions. All isolates showed potent analgesic effects <i>in vivo</i>. Even at a lower dose of 0.2 mg/kg, (±)-<b>1</b> and (+)-<b>1</b> still exhibited more potent analgesic activities than morphine. Interestingly, the racemic mixture (±)-<b>1</b> showed stronger analgesic effect than either pure enantiomer alone at higher doses of 5 and 1 mg/kg; while, (±)-<b>1</b> showed significant analgesic activities comparable to (+)-<b>1</b> at lower doses of 0.2 and 0.04 mg/kg. (+)-<b>1</b> had stronger analgesic effect than (-)-<b>1</b> at five tested does. Further tests on 44 analgesic-related targets demonstrated that (+)-<b>1</b> showed significant inhibitory effects against many ion channels such as TRPM8, Kv1.2, Kv1.3, and Ca_v2.1 with IC₅₀ values of 1.10 ± 0.26, 4.20 ± 0.07, 2.20 ± 0.24, and 10.40 ± 0.69 μmol/L, respectively, while (-)-<b>1</b> primarily inhibited TRPC6, Kv1.2, and Kv1.3 ion channels with IC₅₀ 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.