Pyrazolo[3,4- d]pyrimidine-based neplanocin analogues identified as potential de novo pharmacophores for dual-target HBV inhibition

The discovery of selective and potent inhibitors through de novo pathways is essential to combat drug resistance in chronic hepatitis B (CHB) infections. Recent studies have highlighted that neplanocin A (NepA) derivatives are biologically selective inhibitors of the hepatitis B virus (HBV). In this study, we designed, synthesized, and evaluated various pyrazolo[3,4-d]pyrimidine-based NepA analogues (4a-h) for their anti-HBV activity. Notably, analogue 4g demonstrated significant activity against HBV replication, with EC₅₀ (HBV DNA) = 0.96 μM, CC₅₀ > 100 μM and EC₅₀ (HBsAg) = 0.82 μM, showing selective inhibition of HBsAg secretion. The SAR analysis concluded that replacing the polar 4-NH₂ group with -CH₃ also acted as a weak H-bonding donor, and the presence of 3-iodo was found to be desirable for the activity/toxicity profile. The nucleoside analogues exhibited a distinct mechanism of action compared to existing nucleoside analogues for the selective inhibition of HBsAg secretion. Based on these findings, compound 4g represents a promising lead molecule for the development of new anti-HBV agents with unique mechanisms of action.