A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3-OH group of the ␤-L-2-deoxyribose of the ␤-L-2-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides ␤-L-2-deoxycytidine, ␤-L-thymidine, and ␤-L-2-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (␣, ␤, and ␥) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10 8 genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.Infection with hepatitis B virus (HBV) is a major world health problem, affecting 5% of the population. More than 2 billion people have been infected with the virus, and 350 million of them are chronic carriers at risk of death from cirrhosis and liver cancer (49).Several strategies have been evaluated for the treatment of chronic HBV infection with the goal of eliminating persistent viral replication and preventing progression to chronic active hepatitis and liver failure. Currently, the only approved treatment options are alpha interferon (IFN) and lamivudine (␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine [3TC]). Unfortunately, the rate of response to IFN is low, and drug-associated side effects are significant (24,55). Individuals who are immunosuppressed (e.g., transplant recipients or those coinfected with the human immunodeficiency virus [HIV]) rarely respond to IFN therapy (13). Lamivudine is a well-known example of the class of ␤-Lnucleoside analogs that has recently drawn attention as antiviral and anticancer agents (52). As with IFN, however, a complete antiviral response, as assessed by HBe seroconversion, is seen in only a minority of patients after 1 year of treatment (27). In addition, cessation of lamivudine therapy or development of viral resistance may lead to a marked rebound in viral replication which can be life threatening (hepatitis flare) in HIV-HBV-coinfected patients (2, 30). Lamivudine resistance is now recognized in 16 to 32% of HBV-infected patients after 1 year of treatment and in as many as 58% after 2 to 3 years (14,27,30).Since the Food and Drug Administration approved lamivudine for the treatment of HIV infection in the United States in 1996 and for HBV in 1998, intensive studies on "unnatural" L-nucleosides as agents against HIV, HBV, and herpesviruses (including Epstein-Barr virus [EBV]) and as anticancer agents have been conducted (23). Now, through an extensive structure-activity analysis, we have found that the 3Ј-OH group of the ␤-L-2Ј-deoxyribose of the ␤-L-2Ј-deoxynucleoside series confers unique specificity for anti-HBV activity. In this ...

Hepatitis B virus (HBV) is the major cause of acute and chronic hepatitis, leading to progressive development of necroinflammatory changes in the liver, which can result in cirrhosis and hepatocellular carcinoma (1, 7). Approximately 350 million people (5% of the world's population) are chronically infected with HBV, and 1 million of these patients die every year as a result of this infection (11). Although the development of an effective vaccine to prevent HBV infection has shown promising results and should lead to its eventual eradication, antiviral chemotherapy remains the only effective method to prevent the progression of the disease in chronic carriers (8). Initially, alpha interferon was used as therapy for chronic HBV infection; however, the majority of patients did not benefit, and side effects were significant in some patients (15). At present, ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine (lamivudine) is the only nucleoside analogue approved for use for the treatment of chronic hepatitis B; however, upon the cessation of treatment serum HBV DNA levels return to pretreatment levels. This rebound is also associated with the appearance of drug-resistant virus that is mutated at the active site of the viral reverse transcriptase (9). Therefore, the development of new antiretroviral agents active against HBV is needed.Recently, ␤-L-thymidine (L-dT) and ␤-L-2Ј-deoxycytidine (LdC) were shown to be potent and specific inhibitors of HBV replication both in vivo and in vitro (50% effective concentrations [EC 50 s], 0.19 to 0.24 M in human hepatoma 2.2.15 cells) (2). In a phase I-II clinical trial, treatment with L-dT has also been demonstrated to cause marked reductions in HBV DNA levels in chronically infected patients

Variable temperature-and pH-dependent 1 H NMR conformational analyses of 3 J HH coupling constants and NOE enhancements in the 4Ј-thionucleosides 1-10 in D 2 O, complemented by ab initio calculations, have given insight into the interplay of anomeric and other stereoelectronic effects that are modulated by the substitution of ring oxygen by sulfur in natural nucleosides. The N S pseudorotational equilibrium of the 2Ј-deoxy-4Ј-thionucleosides 1-4 is slightly shifted towards S-type conformers, while their ribo analogues 5-8 exhibit ca. 50 : 50 ratio at 278 K and neutral pH. α-4Ј-Thionucleosides 9 and 10 display a strong preference for N-type conformers. The S-C-N anomeric effect in 1-4 is stronger in purine than in pyrimidine 4Ј-thionucleosides, which is opposite to natural 4Ј-oxonucleosides, and increases in the following order: thymine < cytosine < guanine < adenine. The S-C-N anomeric effect in 1-4 is weaker than the O-C-N anomeric effect in their 4Ј-oxo counterparts. We have observed considerable population of up to 40% of γ Ϫ rotamers across the C4Ј-C5Ј bond, which has been attributed to the preference of the [S4Ј-C4Ј-C5Ј-O5Ј] fragments in 1-10 for trans over gauche conformation. Similarly, the 3Ј-OH group drives the N S equilibrium in 1-10 towards N where the [S4Ј-C4Ј-C3Ј-O3Ј] fragment adopts trans conformation. The 2Ј-OH group has been found to preferentially stabilise N-type sugar conformation in the 4Ј-thioribonucleosides 5-8 where it occupies a pseudoaxial orientation. The pK a values in 1-10 are almost identical to the pK a values of their natural counterparts, which shows that the acid-base character of the constituent heterocyclic moieties does not change upon substitution of oxygen with sulfur atom. The shift of the N S pseudorotational equilibrium in 1-8 towards N upon protonation and towards S upon deprotonation of the nucleobase is smaller by up to 10 percentage points in comparison to their 4Ј-oxo counterparts. This can be attributed to less efficient tuning of the S-C-N anomeric effect in 1-8 by protonation and deprotonation of the nucleobase. 1D difference NOE experiments indicated predominant anti orientation of the nucleobase in 2-10. Ab initio calculations at up to MP2/6-31G**//6-31G** level have shown two energy minima in the North and South regions of conformational space with the energy barriers between 17.5 and 28.5 kJ mol Ϫ1 in the East region. Interestingly, the energy barrier in the West region is comparable or even lower than the barrier in the East region of conformational space in 1-10, which is in contrast to natural nucleosides.

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