Design, Synthesis And Activity Study Of GABA_A Receptor Agonist

Insomnia is one of the most prevalent sleep disorders,which is a variety of physical,mental and behavioral clinical manifestation.Patients with insomnia tend to have an altered quality of life,impaired daytime functioning and an increased risk of work accidents.With the pace of life speeds up,insomnia has become a killer to human health.GABAA receptors are the major inhibitory neurotransmitter receptors in the central nervous system and can be modulated by a variety of clinically important drugs.Previous studies have shown that,as therapeutic targets,the GABAA receptors are of critical physiological importance and their malfunctions are related to a number of known diseases including anxiety,schizophrenia and epilepsy.Traditionally,therapy for insomnia has focused on the regulation of the GABAA receptors.Zopiclone is a kind of non-benzodiazepine drugs,which exerts a depressant action on the CNS and produces the hypnotic activity by selectively binding at GABAA receptor.Zopiclone can significantly reduce the sleep latency,increase the sleep duration and improve the quality of sleep.In the present study,in exploring new types of sedative-hypnotic drugs,we synthesized four series of compounds(XA,XB,XC and XD),which were designed from the structure of zopiclone.Totally,83 compounds were synthesized and confirmed by 1H-NMR and LC-MS,and 80 compounds were not reported before.Chiral resolution of XB-42,XB-43 and XB-44 were achieved using L(+)tartaric acid as a chiral selector,and the ee%of products are both higher than 90%.To determine the absolute configuration of XB derivatives,we prepared the sing crystal of(-)XB-42 using solvent evaporation method,and the structure of(-)XB-42 was confirmed as the S configuration with X-Ray Single Crystal Diffractometer.The target compounds were assayed in vitro using[3H]flumazenil as radiolabeled ligand for the GABAA receptor to evaluate their biological activities.The effects of the compounds,XA,XB,XC and XD on radio ligand binding to GABAA receptor competitive inhibition were examined in comparison with flumazenil and zopiclone.The results of binding tests showed that XA and XB were the more potent GABAA receptor ligands.XA-01,XA-03,XA-04 and XA-05 displayed IC50 in the 0.1 μM range.Moreover,XB-02 and XB-09 displayed IC50 in the 0.1 nM range,which are worthy to be further investigated.XC and XD derivatives did not show high competitive inhibition to GABAA receptor,but inhibition ratio of XC-01 and XD-10 are both larger than 80%,which is useful to further drug design.The biological results of chiral compounds(XB-42,XB-43 and XB-44)showed that the activity for GABAA receptor of the dextrorotatory form of XB derivatives is higher than that of the levorotatory form.The IC50 value of(+)XB-42 is 10-14M,which is lower than eszopiclone.And(+)XB-42 has the prospect of becoming a potential GABAA receptor agonist.In the present study,the open-state and closed-state models of the GABAA receptor were built using homology modelling.Molecular dynamics simulation using the closed-state and open-state receptors as initial models were carried out,and the simulation time was 15 ns each for the two systems.Then the changes of the electrostatic potential of the two conformations,the pore radius of the channel and the root-mean-square fluctuation values were calculated.We found that the entrance of the ECD and the end of the TMD were highly positively charged in GABAA receptor.This structural feature might play an important role in ion selectivity.Then,the free-energy of chloride ion permeations through the closed-state and open-state GABAA receptor has been estimated using ABF(adaptive biasing force)simulation.We observed two major energy barriers in the closed-state GABAA receptor,locating at 20’ and L9’,and one in the open-state GABAA receptor,locating at T6’.By using ABF simulation,the overall free-energy profile is obtained for Cl-transporting through GABAA receptor,which gives a complete map of the ion channel of Cl-permeation.Our studies pave the way for the further studies of the physiology function of the GABAA receptor.