Design,Synthesis And Evaluation Of Novel Dihydropyrimidine Derivatives As Hepatitis B Virus Inhibitors

Chronic hepatitis B(CHB)is a contagious chronic liver disease caused by continuous infection of the hepatitis B virus.Nearly 400 million people worldwide test positive for hepatitis B surface antigen,which seriously affects human life and health as a public health challenge.HBV is highly infectious and is mainly transmitted through unprotected sex,mother-to-child,blood,and damaged skin.Without effective treatment,the disease will further worsen malignant diseases such as liver cancer.At present,although the marketed drugs can control the deterioration of the patients’ condition,the existing medicines still cannot completely cure CHB.Therefore,it is of great significance to develop novel anti-HBV drugs.The life cycle of HBV includes adsorption,invasion,gene repair,transcription,translation,nucleocapsid assembly,DNA replication,envelope formation and release Among them,the nucleocapsid not only serves as a safe site for the generation of the HBV genome,but also participates in important life cycles such as secretion,envelope and maturation.Therefore,it is reasonable and feasible to regard HBV nucleocapsid as a novel target for drug design.At present,multiple HBV nucleocapsid crystal-ligands have been discovered,which is conducive to the anti-HBV drug design.Hetero-aryldihydro-pyrimidine HBV nucleocapsid assembly regulators have been thoroughly studied.Among them,GLS4 exhibited great activity with EC50 value of 12 nM in HepG2.2.15 cell line and its phase Ⅱ clinical study was conducted by the HEC Company in China.However,GLS4 has poor water solubility and unstable metabolism in vivo(cLogP=4.7,t1/2=1.78 h)which limits its clinical application.Therefore,the structure of GLS4 needs to be optimized to improve its properties.In order to develop novel HBV nucleocapsid inhibitors,we used a covalent strategy targeting serine residue at the protein-solvent interface in HBV nucleocapsid in the second chapter.Thus we designed and synthesized 9 compounds with boronic acid as covalent warheads and evaluated their biological activities,aiming to increase the activity and increase water solubility of HAPs.The anti-HBV activity results showed that the EC50 values of P-9 and P-8 were 0.38 μM and 0.4 μM respectively in HepG2.2.15 cells.However,they demonstrated cytotoxicity that CC50 values were 29.2 μM and 32.5 μM respectively and the SI values were 77.63 and 81.37 respectively.The molecular simulation results showed that the covalent interaction between the boronic acid group of P-8 and the Ser141 of the nucleocapsid.This simulation results may be the reason for maintaining antiviral activity of HAPs which validates the design idea as well.In the third chapter,we used the molecular hybridization strategy based on crystallographic overlay to design and synthesize a series of amide compounds in order to improve the metabolic stability of GLS4.The activity results showed that Y-7 and Y-9 had a good inhibitory activity on HBsAg(99.1%;94.6%)and HBeAg(83.6%;82.1%)secretion at 10 μM which greatly exceeded the lead compound GLS4 and marketed drug 3TC.The different substituent on the benzene ring has a great effect on the activity.The nitro-substituted compounds(the inhibition rates are greater than 40%at 10 μM)have a good effect on the inhibition of HBV DNA proliferation.Among them,Y-11 exhibited better activity whose EC50 value is 1.24 μM.However,its activity is still far from the lead compound GLS4(EC50=0.02 μM).In summary,we designed and synthesized two types of novel active compounds based on the SARs of HAPs,then conducted in vitro biological activity evaluation and did molecular simulation experiments.In order to develop novel covalent HBV nucleocapsid inhibitors,we introduced boronic acid into HAPs to seek nucleocapsid inhibitors for the first time and afforded promising lead compounds with value of further research(exemplified by P-8:EC50=0.4 μM;CC50=32.5 μM).Overall,our research further enriched the SARs of HAPs and lay a solid foundation for the in-depth study of dihydro-pyrimidine-based HBV nucleocapsid inhibitors by targeting protein-solevent interface.