N4-Acyl-Modified D-2′,3′-Dideoxy-5-Fluorocytidine Nucleoside Analogues with Improved Antiviral Activity

Abstract

A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.

Development of a Novel Mouse Model to Evaluate Drug Candidates against Hepatitis B Virus

“…In the presence of tet, undetectable levels of virus were observed in cell culture supernatant, whereas in the absence of tet high titres of HBV were observed (Ladner et al, 1997). Subsequent studies have shown the utility of HepAD38 cells for drug discovery and development against HBV (Bijsterbosch et al, 2001;Shi et al, 2003;Ying et al, 2003). In this report, HepAD38 cells transplanted subcutaneously into nude mice resulted in the development of high virus titres in blood.…”

Evaluation of Single and Combination Therapies with Tenofovir Disoproxil Fumarate and Emtricitabine In Vitro and in a Robust Mouse Model Supporting High Levels of Hepatitis B Virus Replication

“…In the presence of Tet, virus replication was suppressed, while in the absence of Tet, very high titers of HBV were observed (29). Subsequent studies have shown the utility of this cell line for drug discovery and the development of lead compounds active against HBV (41). Previously, we demonstrated that HepAD38 cells transplanted subcutaneously into nude mice resulted in the development of viremia, and treatment of these mice with drugs active against HBV demonstrated significant antiviral activity in vivo, suggesting that this simple small-animal model could be used to assess new therapeutic approaches and combination therapies against wild-type (wt) HBV (17).…”