Discovery,Structural Modification And Biological Evaluation Of Novel Benzoamide Derivatives As HBV Capsid Assembly Modulators

Currently,available clinical HBV therapeutic agents are viral reverse transcription inhibitors and interferons.However,many of these medicines demonstrated obvious side effects in clinic,including renal toxicity,bone toxicity,and drug resistance caused by long-term treatments.In addition,none of current therapies can realize functional cure for HBV infections.HBV capsid protects the viral genome and creates the environment for reverse transcription of pg RNA to DNA.The disruption of HBV capsid assembly led to blocking the replication of viral particles and the amplification of covalently-closed circular DNA（ccc DNA）.Therefore,capsid assembly modulator is expected to achieve functional cure for hepatitis B,and targeting capsid assembly is regarded as a hotspot in the development of novel anti-HBV drug.Several assembly modulator-HBV capsid co-crystal structures were evaluated by re-docking and cross-docking and 5E0I,5WRE were picked up as templates for further molecular docking and virtual screening.The SPECS small-molecule compound library was screened using Glide SP and Prime MM-GBSA and 90 candidates were selected.Among them,10 compounds were chosen for biological evaluation with Hep G2.2.15cells.The results showed that arylamine acetaminobenzamide derivative XS-5 exhibited a moderate anti-HBV activity with an IC₅₀of 3.03μM,which is worthy of further structural modification and optimization.Then,based on the molecular docking result of XS-5,the acetaminobenzamide skeleton was remained and the systematically structural optimization were carried out for methanesulfonamide in the solvent zone and phenethylamine in a hydrophobic pocket,which result in the design and synthesis of 55 substituted benzamide derivatives.In vitro HBV DNA replication evaluation showed that several of them are potent capsid modulators with submolecular IC₅₀ values against HBV-DNA replication,especially,four compounds A-21,B-13,B-23,and B-24 demonstrated the most promising activities with IC₅₀ value range from 0.1μM to 0.4μM.Then,compound B-13 was chosen for metabolic stability evaluztion and it demonstrated favorable stability in rat whole blood,simulated gastric fluid,simulated intestinal fluid,and human liver microsomes.The in vivo pharmacokinetics and mechanism study of B-13 is ongoing.In summary,a series of novel substituted benzamide derivatives were discovered as HBV capsid assembly modulators by virtual screening and strutural modification.Through the optimization of the structure of hit XS-5,several target compounds exhibited submicromolar IC₅₀ values against HBV DNA replication and this result served as a promising starting point for further research on novel HBV capsid assembly modulators.