Design,synthesis And Biological Evaluation Of The Novel Agonists For The A_2AAR

One of the main reasons for the failure of the research and development in the treatment of central nervous system diseases is the obstruction of the blood-brain barrier?BBB?,which prevents the drug from being delivered to the central nervous system and accumulating in the brain to achieve effective concentration and produce a corresponding therapeutic effect.Therefore,a key factor in the successful development of medicines targeted brain is the need to overcome the blood-brain barrier,and the study of open blood-brain barriers has become a hot spot for intracerebral administration.Trans-blood-brain barrier administration has been a challenging area of research for the past several decades,and researchers have put considerable effort into developing various drug delivery systems.A series of strategies have revealed that it is very difficult to transport drugs and contrast agents across the blood-brain barrier.However,central nervous system diseases,such as brain tumors,strokes,trauma and neurodegenerative diseases,as well as the treatment of neurotoxic agents,require effective therapeutic drugs for treatment.It is particularly important to design drugs that can penetrate the blood-brain barrier.Therefore,it is necessary to develop a highly effective blood-brain barrier disruption?BBBD?strategy with minimal neurological damage and capable of delivering larger molecular weight drugs with better pharmacokinetic properties.BBB limits the entry of molecules into the brain through two main structural features.First,tight junctions?TJs?structures that block endothelial cells cause low permeability of molecules in the blood through the BBB.On the other hand,compared with peripheral vascular endothelial cells,there are few pathways of vascular endothelial cell transport on brain capillary endothelial cells?BCECs?,and the level of active efflux transporter is high,such as P-glycoprotein?P-gp?.Considering the critical role of TJs in limiting the entry of molecules into the brain,reversibly changing the tightness of TJs may be a viable way to up-regulate BBB permeability.Temporary opening of TJs is a viable method of brain drug delivery because of the high throughput efficiency and no molecular weight limitations of therapeutic drugs.Bynoe et al.have recently demonstrated that specific activation of A_2A adenosine receptors(A_2AAR)in mouse BCECs can promote brain drug absorption.Further studies have shown that activation of A_2A adenosine receptors can up-regulate BBB permeability and temporarily increase the intercellular space of brain capillary endothelial cells.The A_2AAR signal pathway alters cytoskeletal elements by modulating intracellular actin,resulting in cell morphogenesis,disruption of TJs integrity,and increased barrier permeability.Therefore,these studies have greatly expanded the potential application areas and development space of A_2AAR agonists,and the development of highly effective A_2AAR agonists is of great significance for the study of the strategy of opening the blood-brain barrier.To obtain potent A_2A receptor selective agonists,A_2AAR agonists were designed using a fragment-based computer-aided design approach.The distribution characteristics of residues at the active site of the receptor were analyzed,and a fragment library was constructed by splitting drug molecules in MDDR?MDL Drug data report?database.Comb Lig,which is a design strategy of small molecule derivative based on fragments,was proposed.Using the high-quality fragment design library,the fragment growth algorithm was used to design the agonists for the A_2A receptor.Three types of A_2AAR agonists were designed at the 2-and 6-position substitutions of the A_2AAR agonist adenosine;In order to investigate the effect of the purine skeleton on agonists,we designed a class of tricyclic skeleton compounds.Using Comb Lig and self-built molecular fragment library,the efficiency and diversity of our design of new compounds were improved.Finally,based on the comprehensive analysis,the target compounds for synthesis and activity testing were determined.In the synthesis of the target compound,this study was repeatedly explored through synthetic route optimization design and reaction conditions.The synthesis of 35two-class 2-substituted compounds,22 one-class 6-substituted compounds and 13one-class tricyclic skeleton compounds was completed.According to the position and structure of the linked side chains,there are four main synthetic routes involved in the synthesis of target compounds.The reactions include substitution,cyclization,aminolysis,suzuki and other reaction steps,and the important steps in the synthesis route are optimized and improved.A total of 70 target compounds were synthesized in this study,and their structures were confirmed by MS,¹H-NMR and ¹³C-NMR.Preliminary in vitro bioactivity evaluations were performed on all synthesized compounds.1.In the A_2AAR affinity activity test,all four class of compounds showed strong affinity activity.There are 9 compounds with affinity less than 10 n M in the2-substituted target compound,6 compounds with affinity less than 10 n M in the6-substituted target compound,and 6 compounds with less than 10 n M affinity in the target compound of the new tricyclic skeleton.2.In the A₁AR affinity activity test,15target compounds were highly selective for A_2AAR with affinity more than 10?M for A₁AR.3.Four class of compounds were selected for c AMP agonistic activity test.The results showed that both the 2-substituted target compounds and the 6-substituted target compounds have agonistic activity and are A_2AAR agonists;and the novel tricyclic skeleton target compounds do not have agonistic activity and are A_2AAR inhibitors.From the preliminary bioactivity evaluation,we found 5 agonists?ZM0409,ZM1017,ZM0335,ZM0643 and ZM0909?with A_2AAR strong affinity(K_i h A_2AAR<10 n M)and high selectivity?K_i h A₁AR>10?M?.These compounds have follow-up research and development value,and further evaluation studies are underway.In this study,by studying the interaction between known agonists and the receptor,the active A_2AAR crystal structure was selected as the target,and novel A_2AAR agonists were designed based on the structure-based molecular design method.By the synthesis and biological activity evaluation of the target compound,some small molecules with high affinity for the A_2AAR was found,and 46 target compounds have nanomolar affinity with the A_2AAR,and 21 compounds have affinity less than 10 n M.Based on the fragment-based molecular design strategy,we designed the compound ZM0409(K_iindicates that ZM0409 is one of the strongest affinity and highly selective A_2AAR agonists discovered to date.In addition,through the docking study and QSAR analysis of the target compound,it provides a guidance on structure-activity relationship for the further design of new structural agonists.Overall,this study is a successful application of fragment-based drug design methods in A_2AAR agonist design.