Design And Synthesis Of Molecular Probes For The Determination Of The Target Of The Anti-HBV Lead PA-XY1

Despite the existence of an effective vaccine,hepatitis B virus(HBV)infection is still a major publica health problem worldwide,which continues to cause nearly 1million deaths each year.HBV infection can lead to liver failure,fibrosis,cirrhosis,and hepatocellular carcinoma(HCC).There will be approximately 63 million newly-added person of chronic HBV infection and 17 million HBV-related deaths between 2015 and 2030.IFN-? is clinically useful anti-HBV drug but has substantial side effects.Nucleos(t)ide analogues(lamivudine,adefovir,dipivoxil,entecavir,telbivudine,tenofovir and so on)acting as competitive inhibitors of the viral DNA polymerase have been approved to treat chronic HBV infection.However,the nucleos(t)ide analogues is no inhibitory effect on HBe Ag or HBs Ag expression being associated with the occurrence of HCC.Moreover,rapid development of drug-resistant virus becomes a big problem.Hence,there is an urgent need for new drugs with novel target and mechanism.Phyllanthusol A(PA)is an anti-HBV natural sesquiterpenoid isolated from Phyllanthus acidus.PA inhibited the secretion of hepatitis B virus e antigen(HBe Ag),but has high toxicity and low activity.Therefore,the structural design was carried out,which led to synthesize an anti-HBV lead,PA-XY1.PA-XY1 showed potential anti-HBV in vivo and in vitro and significantly reduced the expression of hepatocellular nucleo-factor-4?(HNF-4?).The result suggested that PA-XY1 showed different mechanism of action from clinical anti-HBV drugs.However,target protein of PA-XY1,a new anti-HBV lead,is unavailable.Hence,there is an urgent need for finding targets of PA-XY1.Therefore,this thesis used active protein spectrum technology(activity-based protein profiling,ABPP)to identify target proteins of PA-XY1.First of all,the thesis summarized the research progress of ABPP in the discovery of targets of active natural product in order to provide ideas for the follow-up research work.Secondly,the thesis used PA-XY1 as the active unit to synthesis of more than 20 molecules.We successfully synthesized four generations of small molecular probes.Among them,ABPP small molecule probe(I)labeled biotin and ABPP small molecule probe(IV)labeled rodamine.At the same time,considering that PA-XY1 may either take a covalent combination or non-covalent binding with the target protein,we designed and synthesized PAL-ABPP small molecule probe(II)to fish non-covalent binding proteins.However,taking into account that the light affinity and label volume of PAL-ABPP small molecule probes affects the marking efficiency of the probe molecules,we have synthesized the PAL-CC-ABPP small molecule probe(III)with a view to increasing the marking efficiency by using the acetylene base as a potential reporting group to introduce the reporting group through click-chemical(Cilck-Chemistry)after binding the probe molecule to the protein.By optimization of the optical and connectiong affinity groups,we successfully synthesized four generation probes showed potential anti-HBV activity same with that of the active unit(PA-XY1).The aforementioned rearch results gave supportting for identification target proteins of PA-XY1.Finally,this thesis synthesized two pairs of configuration isomers(4R,5R),(4S,5S),(4S,5R),(4S,5R),(4R,5S)in order to determine the absolute configuration of the new compound 4,5-dihydroxyaxaphline acting on AD isolated from Piper retrofractum.It is note that only 4R,5S-dihydroxyaxyaline showed potential anti-AD activity,while other configurations showed no anti-AD activity.The result suggested that chiral sites of 4,5-dihydroxyaxaxaphonite may be a key active center for anti-AD activity.Further studies showed that 4R,5S-dihydroxyaxaphline acting on AD should be due to new mechanisms or targets,not inhibition of cholinesterase.The above results provide new active molecules for the discovery of AD drugs or study on the AD targets.