Synthesis of Nitazoxanide Derivatives as Potent and Orally Available HBx-DDB1 Inhibitors against Hepatitis B Virus.

Nitazoxanide-based analogues were synthesized and evaluated for HBx-DDB1 interaction inhibition using a split-luciferase assay. Compared to the nitazoxanide (NTZ, IC50 = 1.2 μM) the compound 49a exhibited potent inhibition of the HBx-DDB1 interaction with an IC50 of 0.083 μM. Consistent with its HBx-DDB1 inhibitory effect, compound 49a showed antiviral activity in HBV-infected cells. Compound 49a displayed excellent metabolic stability in human liver microsomes (82% remaining after 30 min), minimal hERG inhibition, and limited CYP interference, showing only moderate inhibition of CYP2C9. In vivo pharmacokinetic studies in mice and rats revealed rapid absorption (T max = 0.5-3.5 h), low systemic clearance (0.09-0.10 L/h/kg), and high oral bioavailability (>90%). These findings establish compound 49a as a promising lead for the development of direct-acting antivirals targeting chronic HBV infection.