Anti-hepatitis B virus activities of purine derivatives of oxetanocin A.

X, which respectively have 2-amino-adenine, guanine, hypoxanthine, xanthine as the base moiety5). HB611 cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (General Scientific Laboratories), 100 ^g/ml of streptomycin, 100IU/ml of benzylpenicillin (Gibco) and 200 jug/ml of geneticin (Gibco) at 37°C in 5% CO2 -95% air. The cells were seeded in 35-mrn CORNING wells at a density of 1 x105 cells/well, using 1.2 ml of the medium. After 2 days of incubation, the mediumwas replaced with the same medium containing the test compound. The cells were incubated for a further 15 days, during which time the mediumcontaining the drug was exchanged every 3 days. The cells were then harvested and cellular DNA was prepared2), and digested with restriction enzyme Hind III (Takara Shuzo Co., Ltd.). An aliquot (3 fig) was electrophoresed in 1.5% agarose gel, followed by blotting onto a nylon membrane GeneScreen Plus according to Southern6). The filter was hybridized to a random primed 32P labeled HBVDNAprobe, and washed twice with 2xstandard saline citrate containing 1 % SDS at 65°C for 30 minutes. It was then autoradiographed, and the results were analyzed using a densitometric analyzer (Shimadzu, Chromatoscana S93O). As can be seen from Fig. 1, because HindIII does not cleave the HBVDNAsequencer), the slow-migrating band (I) represents chromosomally integrated HBVDNA, and the fast-migrating bands (S, Dl and D2) are the extrachromosomal, replicative intermediates of HBVDNA.Among