Multisite Occupancy and Multidimensional Optimization: Design and Evaluation of Piperazine-Thioureidobenzamide Derivatives as Potent HBV Capsid Assembly Modulators

Hepatitis B virus (HBV) capsid assembly modulators are a promising class of agents for chronic HBV infection. Thioureidobenzamide compound <b>17i</b> exhibits potent anti-HBV activity but suffers from poor water solubility, high cytotoxicity, and limited drug-like properties. Herein, we report the design of piperazine-thioureidobenzamide derivatives via multisite occupancy and multidimensional optimization strategies. The representative compound <b>35a</b> exhibited potent anti-HBV activity and low cytotoxicity in HepAD38 cells (EC₅₀ = 0.020 μM, CC₅₀ > 100 μM) and HLCZ01 cells (EC₅₀ = 0.024 μM, CC₅₀ > 100 μM). Molecular dynamics simulations revealed that <b>35a</b> retained critical hydrogen bonds with Trp-102, Thr-128, and Leu-140, while forming new hydrogen bonds with Ser-106, Thr-142, and Asn-136. Multidimensional optimization endowed <b>35a</b> with improved solubility, ideal LogP and high plasma stability. In HBV carrier mouse models, <b>35a</b> inhibited viral replication more effectively than <b>17i</b>, highlighting its potential as a promising candidate drug for further development.