HBV-IN-54 is a selective and orally active HBV inhibitor. It demonstrates anti-HBV activity in HepAD38 cells (EC50 = 0.020 μM, CC50 > 100 μM) and HLCZ01 cells (EC50 = 0.024 μM, CC50 > 100 μM), and effectively inhibits viral replication in vivo. HBV-IN-54 exhibits favorable physicochemical properties and high plasma stability, making it suitable for research related to hepatitis B (HBV).

HBV-IN-54 (compound 35a) effectively binds to the HBV core protein, maintaining hydrogen bond interactions with Trp-102, Thr-128, and Leu-140, and interacts with Ser-106, Thr-142, and Asn-136. The compound exhibits an EC₅₀ of 0.039 μM and a CC₅₀ of 53.11 μM in HepAD38 cells, with a selectivity index (SI) of 1361.8. In the HLCZ01 cell line, HBV-IN-54 demonstrates anti-HBV activity with an EC₅₀ of approximately 0.024 μM when used at concentrations between 0.3-100 μM over six days. Regarding metabolic stability, HBV-IN-54 (1 mg/mL) shows a half-life (T₁/₂) of 30.8 minutes in human liver microsomes, with parameters C = 1 mg/mL, CL_mint = 22.5 μL/min/mg protein, MPPGL = 48.8 mg protein/g tissue, OW = 25.7 g tissue/kg body weight, and CL_int,sce = 28.2 mL/min/kg.

HBV-IN-54 (compound 35a), administered orally at a dose of 25 mg/kg once daily for 21 consecutive days, demonstrates in vivo antiviral activity in a pAAV-HBV1.2 plasmid-induced HBV mouse model.