摘要Synthetic cannabinoids(SC)target the human cannabinoid receptor 1(hCB1)and are extensively metabolized,but the metabolite activity on the hCB1 receptor after a SC intake is largely unknown.In this study we compared the in vitro hCB1 receptor activity of 26 metabolites of the synthetic cannabinoid receptor agonists(SCRA)JWH-018,AM-2201,THJ-018 and THJ-2201 as a model system for SC metabolite activity to elucidate their structure-activity relationships.The efficacy and potency of metabolites were assessed using an AequoScreen hCB1 receptor assay in triplicates and 7-8 concentration points(20 μg/mL-9.5 ng/mL)were used to construct dose-response curves and to determine EC50 and Emax.In silico docking and molecular dynamics were performed using a model of the active form of the hCB1 receptor with all the metabolites.Final poses were simulated to assess stability under physiological conditions.We showed that carboxylic acid metabolites and 2-hydroxyindole biotransformational products were inactive,while 5-hydroxypentyl SCRA metabolites decreased efficacy to＜70％,qualifying them as partial agonists.Eighteen metabolites retained＞70％efficacy of their parent compound.Metabolite potencies ranged from 13-3500 nM where the most potent were the 4-hydroxypentyl derivatives of THJ-2201 and THJ-018 and the 4-hydroxyindole derivatives of AM-2201 and JWH-018,also known to be prevalent in vivo metabolites.The efficacy data from in silico experiments were correlated with the in vitro results demonstrating a linear trend(R2=0.9457),significant(P＜0.0001)at the 95％confident interval between the binding energies and efficacies of the compounds investigated.In silico analysis with docking and molecular dynamics simulations showed that active metabolites maintained a minimum of six amino acid interactions involving all substructures.The in silico molecular dynamics simulations revealed that the efficacy and potency seemed to be driven by a complex network of hydrophobic weak amino acid-ligand interactions.Most prevalent were CH-π interactions and π-π stackings.This study demonstrates the clear structure-activity relationships well correlated to the molecular dynamics simulations,suggesting that metabolites,especially the 4-hydroxy pentyl metabolites,may contribute to the overall effect of SCs in vivo.