Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N ⁴ -Hydroxy- and 5-Methyl-β- l -Deoxycytidine Analogues

Abstract

, and ␤-L-5-methyl-2-deoxycytidine (EC 50 , 0.9 M). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the ␤-L-cytidine derivatives was also assessed. In accordance with the cell culture data, ␤-L-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 M. The cytotoxicities of some of the 4-NHOH-modified ␤-L-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH 2 group. The 50% cytotoxic concentrations for ␤-L-Hyd4C in HepG2 and HL-60 cells were 2,500 M and 3,500 M, respectively. In summary, our results demonstrate that at least ␤-L-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.Complete and sustained suppression of viral replication remains the most important goal in the treatment of patients with chronic hepatitis B virus (HBV) infection. Nucleoside and nucleotide analogues have greatly improved the disease outcome for these patients and have also prevented hepatic decompensation or the development of hepatocellular carcinoma. While ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine (3TC; lamivudine) has been approved for the treatment of HBV infections, a series of further ␤-Lnucleosides are under clinical investigation as inhibitors of hepadnavirus infections. These include ␤-L-2Ј,3Ј-dideoxy-3Ј-The most important advantage associated with some, but not all, of these ␤-L-nucleosides seems to be a much higher selectivity for the viral target than for the cellular targets, resulting in reduced cytotoxicity. This quality of ␤-L-nucleosides stimulated the search for new ␤-L-nucleosides but also the synthesis of enantiomeric isomers of effective ␤-D-nucleosides (7).Some of the unmodified pyrimidine and purine ␤-L-nucleosides were synthesized for the first time more than 35 years ago (12, 28, 34) but were considered to be metabolized very poorly in mice (14). A systematic biochemical investigation of the ␤-L-enantiomers was initiated later and demonstrated the antihuman immunodeficiency virus (HIV) activity of ␤-L-2Ј,3Ј-dideoxycytidine (␤-L-ddC) in a cellular system (18). In addition, it was shown that ␤-L-dT might be phosphorylated by herpes simplex virus type 1 (HSV-1) thymidine kinase and further, by cellular enzymes, to ␤-L-dTTP (35).As a consequence, the effects of ␤-L-dTTP on various cellular DNA polymerases, TdT, the Klenow fragment of Escherichia coli DNA polymerase I, and viral DNA polymerases such as HSV-1 DNA polymerase and HIV reverse transcriptase were investigated (6,32,38). We showed that ␤-L-TTP does not influence the activity of any of the cellular DNA polymerases (␣, ␤, ␥, ␦, or ε) or of HIV reverse transcriptase, but we demonstrated for the first time a strong inhibition of human and duck HBV DNA polymerases by ␤-L-dTTP (concentrations resulting in 50% inhibition of HBV DNA polymerase activity [IC 50 ], 0.46 M and 1.0 M, respectively) (37). Investigations on the cellular level and in vivo experim...

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β- l -2′,3′-Didehydro-2′,3′-Dideoxy-N 4 -Hydroxycytidine: Relevance for Activation in HepG2 Cells

“…HepG2 cells were assessed by reversed-phase HPLC. Figure 2A shows the HPLC radiochromatogram of extracts from HepG2 cells exposed to 2 M L-[ 3 H]Hyd4C for 24 h. The retention times of the parent nucleoside and 5Ј-triphosphate were identical to those of unlabeled standards synthesized in our laboratory (12).…”

“…Between them, a series of strong inhibitors of HBV replication could be found. L-Hyd4C emerged as the most effective one, suppressing HBV replication in HepG2.2.15 cells with an ED 50 of 0.03 M and surprisingly displaying an extremely low cytotoxicity (CD 50 in HepG2 cells ϭ 2,500 M) (12).…”

“…A reduction of L-Hyd4C should produce L-d4C, a compound which is known to display both a relatively high cytotoxicity (CD 50 ϭ 20 M) and strong antiviral activity against HBV and HIV replication (9). The extremely low cytotoxicity of L-Hyd4C and the lack of any effect on HIV replication let us assume that this modification is unlikely (12;Matthes,unpublished). This view is further supported by our radiochromatograms from extracts of HepG2 cells incubated with L-[ 3 H]Hyd4C (Fig.…”

“…Fractions of 1 ml (each) were collected, and the radioactivity was measured with a high-salt-capacity scintillation fluid. The L-Hyd4C metabolites were identified by use of the UV spectra ( max ϭ 235, 271 nm; min ϭ 207, 257 nm), using the 5Ј-triphosphate of L-Hyd4C as a standard (12), and by enzyme digestion of the dried methanol extracts with 1.0 U of alkaline phosphatase (60 l; 10 mM Tris-HCl, pH 10.8, at 37°C for 12 h).…”

“…Between them, ß-L-2Ј,3Ј-didehydro-2Ј,3Ј-dideoxy-N 4 -hydroxycytidine (L-Hyd4C) ( Fig. 1) emerged as the most effective in suppression of virus production in HepG2.2.15 cells (50% effective dose [ED 50 ] ϭ 0.03 M), displaying an extremely low cytotoxicity (50% cytotoxic dose [CD 50 ] for HepG2 cells ϭ 2,500 M) (12).…”