Study On The Deuterated Drug And Structural Optimization Based On Targets And Mechanisms Of Action Of HBV Capsid Protein Inhibitor GLS4

Hepatitis B virus(HB V)is a hepatotropic DNA virus that relies on blood,mother-to-child and sexual transmission.Long-term HBV infection can lead to chronic hepatitis B,cirrhosis,liver failure,and hepatocellular carcinoma.According to statistics from the World Health Organization,there are about 257 million people with HBV in the worldwide,accounting for about 4%of the world’s population.China is a high endemic area of HBV,and there are about 90 million HBV carriers.At present,HBV therapies mainly include interferon and nucleos(t)ide analogues,but neither of these two categories can completely cure HBV.The tolerance of interferon is poor,while nucleos(t)ide analogues are easy to rebound and develop resistance after drug withdrawal.Wait.Therefore,the research of new HBV inhibitors is imminent.HBV inhibitors currently in clinical and pre-clinical research target proteins through the entire life cycle of HBV,and then capsid(core)protein allosteric modulators(CpAMs)have attracted much attention due to the versatility of the targets.Among the many kinds of CpAMs,Heteroaryldihydropyrimidine(HAP)derivative GLS4 is currently in clinical phase Ⅱ with favorable activity,but it is easily metabolized in the body with low bioavailability and poor water solubility.The combined use of ritonavir limits the clinical application of GLS4 to a certain extent.Therefore,the second chapter of this paper aims at its shortcomings of being easily metabolized in the body,replacing the 6-position morpholine ring of dihydropyrimidine parent ring with deuterated morpholine,where is the metabolizable site,in order to use the isotope kinetic effect of deuterated drugs.The deuterated drug of GLS4(GLS4D)was synthesized.It is hoped that the antiviral effect of GLS4 can be maintained while improving its metabolic properties.The activity test found that in the Hep38.tet.7 cell line,GLS4D has a slightly better inhibitory effect on HBV DNA replication(EC50=0.038±0.004 μM)than GLS4(EC50=0.082±0.018 μM),with low cytotoxicity(CC50>1 μM).Therefore,the metabolic properties of GLS4D were further studied.In the human liver microsomal stability experiment,the metabolism rate of GLS4D is 232.1 mL/min/kg,about ten times faster than that of GLS4(20 mL/min/kg);the inhibition experiment on CYP450 enzyme subtypes shows that GLS4D is a metabolic substrate of a variety of CYP450 enzymes,including 2C9,2C19,2D6 and 3A4.Finally,a pharmacokinetic study of GLS4D in SD rats was carried out,and it was found that GLS4D has lower absorption,wider distribution,faster clearance,and lower bioavailability.In general,the deuterium strategy has not improved the rapid metabolism of GLS4,and more in-depth research on the metabolic mechanism of GLS4 is needed.The third chapter of this thesis aims at the shortcomings of GLS4’s easy metabolism and poor water solubility.On the basis of retaining the dominant skeleton of GLS4,utilizing the medicinal chemistry strategy of skeleton transition,the spiro ring structure is introduced at the 6 position of the dihydropyrimidine parent ring.Due to the spiro ring’s relatively rigid and three-dimensional structure,it can change the water solubility,lipid solubility,and absorption,distribution,metabolism and excretion(ADME)properties of drug molecules to a certain extent,and can improve activity,reduce toxicity;increase half-life,and break through patents.Therefore,a total of 29 HAP-spiro compounds were designed and synthesized by introdcing the spiro ring segment to the solvent opening region at the 6 position.Among them,compound 4r has the best activity(EC50=0.2 μM),which is better than the positive control lamivudine,and a quater of GLS4 potency,with low cytotoxicity(CC50>87.03 μM).And predicted cLogP of 4r is 4.5,which indicated a better true value than GLS4(cLogP=4.747).The PROteolysis TArgeting Chimeria(PROTAC)technology shines in the application of multiple targets due to its multiple advantages.The antiviral mechanism of GLS4 is to induce the misassembly of the HBV capsid,and PROTAC can directly degrade the targeted protein.If the PROTAC strategy is used for the HBV capsid,can it directly degrade the viral capsid?Can we use the versatility of viral proteins to exert greater antiviral potential?In order to solve this confusion,the fourth chapter of this paper utilized the PROTAC strategy on the HBV capsid protein based on the GLS4 backbone to test its antiviral activity and cytotoxicity at the cellular level,and conduct a preliminary investigation of the antiviral mechanism.After investigating the target protein structure,binding pocket characteristics and tissue specificity of hepatocytes,a total of 15 GLS4-PROTAC analogs were designed and synthesized.They were divided into three series:only GLS4 plus linker without E3 ligase ligand(A series),GLS4 plus linker and E3 ligase ligand(B series)and GLS4 plus linker and blocked E3 ligase ligand(C series).A and C series are used as negative controls.All GLS4-PROTAC analogs showed a strong inhibition of HBV DNA replication in the initial screening at the concentration levels of 20 μM and 5 μM;the re-screening results showed that the EC50 values of all GLS4-PROTAC analogs were at low micromolar levels,and the three compounds’ activity is close to the positive drug 3TC and about 10 times lower than GLS4,Ⅱ-8c(EC50=0.48±0.08 μM),Ⅱ-8i(EC50=0.46±0.06 μM)and Ⅱ-8j(EC50=0.43±0.05 μM).The antiviral activity of A series is lower than B and C series,while the cytotoxicity of A series is generally higher than B and C series.This shows that the introduction of E3 ligase ligand into GLS4-linker will increase the antiviral activity and reduce the cytotoxicity,which preliminarily verifies the rationality of the design of GLS4-PROTAC.The effect of HBV capsid assembly and core protein expression was tested:the capsid blot results showed that GLS4 has no much difference than the control on the capsid,which was the consistent with the capsid misassembly induced by GLS4 at low concentrations.But all the GLS4-PROTACs,except Ⅱ-7b,significantly reduce the amount of capsid.This indicates that the antiviral mechanism of GLS4-PROTACs is different from GLS4.GLS4 induces capsid misassembly at low concentrations,while GLS4-PROTACs significantly reduce capsids.From the core protein blots,there is a very good agreement between the expression of core protein and the formation of capsids.GLS4-PROTACs significantly reduced the total amount of core protein,and multiple compounds directly reduced the signal intensity of the core protein to less than 10%,indicating that GLS4-PROTAC analogs are causing the core protein degradation.In general,GLS4-PROTACs show better antiviral activity and lower cytotoxicity,and their antiviral mechanism has been initially verified.GLS4-PROTACs can reduce core protein accumulation and reduce capsid formation to perform antiviral effect.