Synthesis,Anti-Depression Activity Evaluation,and Molecular Docking Studies On Glycyrrhetinic Acid Derivatives As High-Mobility Group Box-1 Inhibitors

Depression is a mental disorder characterized by depressed mood,loss of interest,aversion to activity,cognitive impairment and inattention.Globally,over 300 million people of all ages suffer from depression,which makes it one of the leading causes of disability.Although conventional antidepressant therapies are prevalent,about one-third of all patients are nonresponsive to those treatments and the mechanisms underlying the development of depression remain abstruse.Recently,abundant data has shown that immune system,especially the inflammatory system,becomes an important contributor to the pathogenesis of depression.In 2015,Chun-Lei Jiang's research group of the Second Military Medical University reported the depression-inducible effect of High mobility group box-1(HMGB1)for the first time.HMGB1,acted as a late-phase inflammation mediator,is released actively post LPS administration and is involved in LPS induced depressive-like behavior.In addition,the inhibition of HMGB1 has therapeutic effect on depressive disorder.HMGB1,a highly conserved nuclear protein,is released or secreted from the cell in response to damage or stress.It is named for its rapid migration in polyacrylamide gel electrophoresis(PAGE).Extracellular HMGB1 has a variety of activities by binding to multiple receptors.Receptor for advanced glycation endproducts(RAGE),Toll-like receptor(TLR)2 and TLR4 are three major receptors of HMGB1.When HMGB1 binds to RAGE,it activates the nuclear factor?B(NF-?B)pathway,subsequently induces the production of many cytokines and promotes the inflammatory response.When HMGB1 binds to TLR2and TLR4,it activates the MyD88 dependent NF-?B pathway,promotes the expression and release of inflammatory cytokines such as TNF-?,IL-6 and IL-1?.HMGB1 has three major structural domains:two DNA binding motifs,called the Box A and Box B,and an acidic carboxyl terminus.Natural products play a major role in drug discovery,and nearly half of the new drugs introduced into the market over the past two decades are natural products or their derivatives.Glycyrrhizic acid(GL)is a triterpene glycoside of licorice root extract and possesses a wide range of pharmacological properties,especially anti-inflammatory properties.In 2007,Mollica et al.firstly reported that GL inhibited HMGB1 by directly binding(K_d is about 150?M)to each of the two HMG boxes and thus prohibiting its chemoattractant and mitogenic activities.Research objective:Based on the results of the docking between glycyrrhizic acid and HMGB1,and the development of HMGB1 and depressive behavior,the aim of this study is to select glycyrrhetinic acid(the glycoside part of glycyrrhizic acid)which is the core skeleton of interaction with protein HMGB1 as the lead structure to optimize the modification and to investigate the antidepressant activity of the derivatives.Under the condition of preserving the mother nucleus structure of pentacyclic triterpenoids of glycyrrhetinic acid and the main effector groups which are 11 carbonyl groups and 30 carboxyl groups,the structural modification of A ring of glycyrrhetinic acid is carried out.Furthermore,some of the triterpenic acids with heterocyclic group attached to ring“A”of the skeleton have been reported to exhibit potent anti-inflammatory activity.Therefore,we determine the modification of the fused heterocyclic ring in the A ring of glycyrrhetinic acid.It is expected that the introduction of A ring specific fused heterocyclic structure and side chain structure can enhance the interaction between the target compound and HMGB1.At the same time,by adjusting the lipids and water partition coefficients of the target compounds,the ability of the target compounds to pass through the blood-brain barrier(BBB)is improved,which is beneficial to their antidepressant activities.The purpose of our research is to provide new evidence for finding new antidepressant lead compounds.Research contents:1.Design and synthesis of glycyrrhetinic acid derivatives.Through esterification,oxidation,nucleophilic substitution,cyclization and hydrolysis reaction,we introduced pyrazole ring,substitute pyrazole ring,indole ring,substituted indole ring and isooxazole ring into A ring of glycyrrhetinic acid,and obtained 16 derivatives of glycyrrhetinic acid.Using glycyrrhizic acid,glycyrrhetinic acid and carbenoxolone as positive control,the ability of the target compound to inhibit the release of inflammatory cytokines TNF-?,IL-6and IL-1?was detected by enzyme-linked immunosorbent assay(ELISA)in RAW 264.7cells,and the effect of the target compound on the release of NO was also detected.The results showed that the N-Phenyl substituted pyrazole-fused derivatives exhibit good anti-inflammatory activity at the concentration of 30 and 10?M.According to this result,we further selected"N-phenyl substituted pyrazole"as the preferred structure for modification.By introducing different substituents on the phenyl group,21 new N-phenyl substituted pyrazole a ring fused glycyrrhetinic acid derivatives were synthesized.The structures of all key intermediates and target compounds had been confirmed by nuclear magnetic resonance spectroscopy(NMR)and high resolution mass spectrometry.2.In vitro activity studies of glycyrrhetinic acid derivatives.A total of 39 compounds were synthesized and tested for anti-inflammatory activity in RAW264.7 cells and BV2 cells.The ability of the target compound to inhibit the release of inflammatory cytokines TNF-?and IL-6 in RAW264.7 cells was detected by flow cytometry.The results showed that 20 N-phenyl-substituted pyrazole derivatives at the concentrations of 30?M and 10?M reflect obvious anti-inflammatory activity,and the derivative GA-21T and GA-51T could still exhibit anti-inflammatory activity below 10?M.Derivatives with better anti-inflammatory activity were further detected by ELISA in BV2 cells to detect the ability to inhibit the release of inflammatory cytokines TNF-?and IL-6.The results showed that the derivative GA-25T exhibites excellent anti-inflammatory activity in BV2 cells,especially at the concentration of 10?M,its anti-inflammatory effect is equivalent to that of fluoxetine.Finally,GA-21T,GA-25T and GA-51T were obtained as the optimal selection compounds.The experiment of combining three preferred compounds with HMGB1 is under way.3.Evaluation of antidepressant models of selected compounds in vivo.The antidepressant activity of the selected compounds in vivo was evaluated by two methods including direct intracerebral administration after LPS modeling and intraperitoneal administration after rHMGB1 modeling.Experimental results of direct intracerebral administration after LPS modeling showed that the derivative GA-21T could significantly improve the depressive behavior induced by LPS.Experimental results of intraperitoneal administration after rHMGB1 modeling showed that the derivative GA-21T could significantly improve the depressive behavior induced by rHMGB1.Further in vivo experiments are under way.4.Molecular docking of selected compounds.The selective compounds GA-21T was subjected to a docking study with HMGB1 by computer simulation of molecular docking techniques.From molecular docking point of view,the binding mode of selected compounds with HMGB1 is described.The results showed that the selective compound GA-21T has hydrogen bond with HMGB1.The binding mode was similar with glycyrrhizic acid and glycyrrhetinic acid.Further docking research is ongoing.ConclusionIn summary,36 glycyrrhetinic acid derivatives were synthesized from glycyrrhetinic acid as a leading compound by attaching heterocyclic group to its A ring and introducing side chain structure.The anti-inflammatory activity test in vitro showed that some of the target compounds could significantly inhibit the release of inflammatory cytokines TNF-?and IL-6.Further evaluation of antidepressant activity in vivo showed that GA-21T could significantly improve depressive behavior induced by LPS and rHMGB1.The results provide new evidence for finding new antidepressant lead compounds based on active natural product HMGB1 inhibitors.Further in vivo testing of antidepressant activity and the binding activity of selected compounds to HMGB1 are ongoing.