In order to study the structure-activity relationships, several dioxolane pyrimidine nucleosides have been synthesized and their anti-HBV activities have been evaluated in 2.2.15 cells. From the study it was found that 5-fluoro-cytosine derivatives exhibited the most potent anti-HBV activity. Recently, several nucleosides have been reported as potent antiviral agents against human hepatitis B virus (HBV). These include β-L-(2-hydroxymethyl-1,3-oxathiolan-4-yl)cytosine (3TC), β-L-(2-hydroxymethyl-1,3-oxathiolan-4-yl)-5-fluorocytosine (FTC), β-L-2',3'-dideoxy-5-fluoro-cytidine (L-FddC), 2'-fluoro-5-methyl-β-L-arabinofuranosyluracil (L-FMAU), and β-D-9-(2-hydroxymethyl-1,3-dioxolan-4-yl)-2,6-diaminopurine (DAPD). These nucleosides are currently undergoing preclinical and clinical studies as anti-HBV agents (Figure 1). Previously, we have found that (-)-β-L-dioxolane-cytosine [(-)-OddC] exhibited extremely potent anti-HBV activity in 2.2.15 cells. However, its in vitro cellular cytotoxicity precluded its use as a useful anti-HBV agent. We have also synthesized and evaluated the corresponding D-isomer as a potential anti-HBV agent. The D-isomer was found to be significantly less toxic than the L-isomer, however, its anti-HBV potency was also significantly reduced, comparing to the potency of the L-isomer. Thus, it was of interest to study the structure-activity relationships (SAR) of the D-dioxolane nucleosides to search for more potent anti-HBV agents while maintaining low cellular toxicity. Particularly, of interest was the study of the SAR of cytosine derivatives due to the finding that the cytosine derivatives exhibit, in general, more potent anti-HBV activity than the other analogues. Therefore, herein we report the synthesis and anti-HBV activity of 5-substituted D-dioxolane cytosine nucleosides (5-F, Cl, Br, and I) along with other previously unreported pyrimidine derivatives. The dioxolane intermediate 1, which was prepared from D-mannose according to the method previously reported by our laboratory, was condensed with silylated 5-substituted N-benzoylcytosine derivatives (5-F, Cl, Br, and I) to anomeric mixtures of the 5-substituted cytosine-nucleosides 2-9 (Scheme 1). The individual isomers were separated by silica gel column chromatography, and were subsequently treated with n-Bu4NF to remove the 5'-silyl protecting group followed by the treatment with methanolic ammonia to give the final products 10-17. Some uracil derivatives have been also prepared by condensation of the appropriate silylated heterocyclic bases with the dioxolane acetate 1 followed by a routine work-up procedure. The structural assignments of the synthesized derivatives were made on the basis of the 1H NMR studies. Cis- and trans- arrangements of the 5'-CH2 group with the cytosine ring were established by the nuclear Overhauser effect. The anti-HBV activity of the newly synthesized D-dioxolane cytosine nucleosides indicated that the 5-fluoro-cytosine derivative exhibited the most potent anti-HBV activity while the 5-chloro and 5-bromo derivatives exhibited significantly less potent anti-HBV activity in a human hepatoma cell line carrying the HBV (2.2.15 cells). The 5-iodo derivative was also found to be less potent than the 5-fluoro derivative. Previously, we have found that the β-D-cytosine derivative (EC50 0.01 μM) exhibited potent anti-HBV activity. Several uracil (uracil, 5-bromo, and 5-fluoro) and cytosine (5-methyl-cytosine) derivatives have also been evaluated as potential anti-HBV agents. However, these compounds did not exhibit any significant antiviral activity. In order to compare the anti-HBV potency, several other cytosine nucleosides are also included in Table 1. In summary, we have studied the structure-activity relationships of D-dioxolane-cytosine nucleosides as anti-HBV agents and have discovered that the cytosine and 5-fluoro-cytosine derivatives are the most potent anti-HBV agents with less cellular toxicity than the β-L-5-fluoro-cytosine derivative. Therefore, any modification other than the 5-fluoro group at C5 position of the pyrimidine ring significantly reduces the anti-HBV activity. In view of the high selectivity (>1500) exhibited by the cytosine and 5-fluoro-cytosine derivative, further virological and biochemical studies are warranted.