Synthesis of a novel L-nucleoside , β-L-D4A and its inhibition on the replication of hepatitis B virus in vitro

AimNucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, β-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome. Methodsβ-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, ¹H NMR and MS. 2.2.15 Cells were placed at a density of 5×10⁴ per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a ³²P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC₅₀ was calculated. 2.2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC₅₀ was calculated. ResultsThe synthesized compound structure conformed with β-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC₅₀ 0.2 μmol·L^-1. The experiment of cytotoxicity gained IC₅₀ 200 μmol·L^-1. Conclusionβ-L-D4A has been synthesized successfully. β-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.