Design, Synthesis And Anti-fatigue Activity Of AMPA Receptor Modulators

Objective The present study was designed to develop new anti-fatigue drug candidateswith AMPA receptor as target. A series of novel benzamide analogues were synthesized asAMPA receptor modulators. Physicochemical properties and docking studies of targetcompounds were performed. Further pharmacological studies were carried out to screenpotential compounds with significant anti-fatigue effect and proper pharmacokineticproperties.Methods Based on benzamide as parent structure, a series of novel benzamideanalogues were designed as AMPA receptor modulators using the classical drug design theory.Particular attention was paid to the influence of the3,4-methylenedioxy and methoxy groupsin the benzene ring as well as to the cycloalkyl amines in the benzamides N-heterocycle chain.The target compounds were synthesized employing a twostep approach starting fromcorresponding methoxy or3,4-methylenedioxy substituted benzoic acid. The esterficationreaction of N-succinimidyl-trifluoroacetate with methoxy or3,4-methylenedioxy substitutedbenzoic acid gave the corresponding substituted benzoic acid-N-succinimidyl ester. Theseintermediates were successively converted into target compounds by the condensationreaction with cycloalkyl amines. Crystals were grown by vapor diffusion. Docking ofcompound A2and D2was performed using AutoDock4.0software, in order to assess possible binding abilities and modes of them with AMPA receptor. The purity and Log P dataof target compounds were determined by HPLC method.The anti-fatigue activities of these compounds were investigated by weight-loadedforced swimming test and sleep deprivation test. Modafinil and caffeine were chosen aspositive control. KunMing mice were orally administered with corresponding compounds forseven days, then the weight-loaded forced swimming test and sleep deprivation test wereperformed. The forced swimming time and sleep deprivation time were recorded. After thelast experiment, blood samples and corresponding tissues were collected to determine thelevels of liver glycogen, muscle glycogen, lactic acid, blood urea nitrogen, superoxidedismutase, glutathione peroxidase, catalase, lactate dehydrogenase and creatine kinase. Thebinding affinity test was determined by radioligand binding method.Pharmacokinetic properties of candidate compound A2were investigated andcorresponding parameters were calculated such as half-life period and bioavailability. Theacute toxicity test was performed to predict the toxicity and calculate the LD50data of A2.Results The target compounds were successfully synthesized by the designed syntheticroute, which demonstrated that the synthetic route was reliable. In this study, a total of24compounds including19target compounds,4intermediates and modafinil were synthesized.Their structures were confirmed by nuclear magnetic resonance (1H NMR), massspectrometry (MS), infrared spectrum (IR) and elemental analysis. CA online (SciFinder)database confirmed that all of the compounds were novel except A1and modafinil. Crystalsof A, A1, A2and D2were gained, and their structural analysis results were obtained by X-raysingle crystal diffraction. Judging by the docking results, it can be assumed that compound A2and D2possess potent binding abilities with AMPA receptor.The results of HPLC determination showed that the purity of target compounds were allabove98%, which was suitable for the pharmacological research. The Log P data was0.912.24, which was suitable for the physicochemical properties.In the present study, all of the compounds had no significant effect on the body weightcompared to the control group. The results of weight-loaded forced swimming test showedthat compound A2, C2, A1and D1significantly increased the swimming time compared withthe control group, indicating that they possess an anti-fatigue effect. Meanwhile, the results of sleep deprivation test showed that compound A2, A1, C3, C1and D1significantly increasedthe sleep deprivation duration time compared with the control group. Results from this studyshowed that mice in A2, C2and A1groups significantly increased their liver glycogen andmuscle glycogen levels, compared with control group, indicating they enhance the exerciseendurance may owing to increased the storage of liver glycogen. Lactic acid and blood ureanitrogen levels in A2, A1and C2groups were significantly lower than the control group,which indicated that these compounds facilitated mice recovery from fatigue due to reduce theaccumulation of fatigue-related metabolites. Among all of the target compounds, A2exhibitedthe most significant anti-fatigue effect. The binding affinity test showed that compound A2represented a strong binding affinity with AMPA receptor.The half-life period was81.34min (i.g.) and82.57min (i.v.), and the bioavailability was30.8%. The results of acute toxicity test showed that it had no obvious toxic effects and noexcessive central excited in the dose range0.2mmol/kg1.0mmol/kg both by i.g. and i.v.route. The LD50data was1.3306g/kg (i.g.) and0.6066g/kg (i.v.).Conclusion The present study was designed to choose AMPA receptor as the target,and a series of novel benzamide analogues were synthesized as AMPA receptor modulators. Inthis study, a total of24compounds including19target compounds,4intermediates andmodafinil were synthesized. Their structures were confirmed by1H NMR, MS, IR andelemental analysis. CA online (SciFinder) database confirmed that22compounds were noveland firstly synthesized. The purity of target compounds was all above98%, which wassuitable for the pharmacological research. The Log P data was0.912.24, which was suitablefor the physicochemical properties. Compound A2, C2, A1and D1showed significantanti-fatigue effect. The biological results obtained with the new compounds unveiled that A2appeared to possess the most potent anti-fatigue activity together with proper pharmacokineticparameters and LD50data. Thus, these studies may provide as experiment basis for novelanti-fatigue durg development.