G. Gosselin

Nucleoside Analogues as Chemotherapeutic Agents: A Review

Abstract The importance of nucleoside analogues in chemotherapy and in other potential therapeutic approaches as immunomodulation or regulation of gene expression, is reviewed.

Nm 283, an efficient prodrug of the potent anti-HCV agent 2'-C-methylcytidine.

In order to improve the oral bioavailability of 2′-C-methylcytidine, a potent anti-HCV agent, the corresponding 3′-O-L-valinyl ester derivative (NM 283) has been synthesized. Based on its ease of synthesis and its physicochemical properties, NM 283 has emerged as a promising antiviral drug for treatment of chronic HCV infection.

3′-Substituted Thymine α-L-nucleoside Derivatives as Potential Antiviral Agents: Synthesis and Biological Evaluation

Hitherto unknown 1-(3-deoxy-3-substituted-α-L-lyxofuranosyl)thymines and their 2-deoxy derivatives related to azidothymidine (AZT) and its congeners have been synthesized and their antiviral properties examined. They were prepared by nucleophilic substitution with inversion of configuration from 3′-O-trifluoromethanesulphonate α-L-arabinofuranonucleosides and their 2′-deoxy derivatives. All the prepared compounds were tested for their activity against a variety of RNA and DNA viruses, but they did not show significant antiviral activity.

Synthesis and Antiviral Evaluation of SATE-Foscarnet Prodrugs and New Foscarnet—AZT Conjugates

The synthesis of a range of di- and triester derivatives of phosphonoformate (PFA; foscarnet) as potential lipophilic, membrane-soluble prodrugs is described. In addition to normal alkyl esters in the carboxylate and phosphonate residues of PFA, the bioreversible S-(pivaloyl)thioethyl (t-butyl-SATE) group was introduced in an attempt to deliver PFA after bioactivation inside the cells. Furthermore, PFA—AZT conjugates were prepared in order to develop combinational drugs. The key synthetic step was in all cases the formation of the P—C bond to build up the different PFA esters. In contrast to the diester derivatives, the triesters of PFA showed high hydrolytic instability during chromatographic purification. The compounds were evaluated in vitro for their ability to inhibit viruses in several tissue culture systems. All PFA alkyl di- and triesters proved poorly active or inactive against human immunodeficiency virus (HIV) and inactive against hepatitis B virus. In contrast, the PFA—AZT conjugates exhibited significant anti-HIV activity. However, this activity was nearly completely lost in thymidine kinase-deficient cells, suggesting a fast unselective chemical hydrolysis of the conjugates to yield the nucleoside analogue AZT in the cell culture medium. Furthermore, no synergistic effect of PFA and AZT was observed.

NEW UNNATURAL L-NUCLEOSIDE ENANTIOMERS : FROM THEIR STEREOSPECIFIC SYNTHESIS TO THEIR BIOLOGICAL ACTIVITIES

Abstract Several purine and pyrimidine β-L-dideoxynucleosides were stereospecifically synthesized and their antiviral properties examined. Two of them, namely β-L-2′,3′-dideoxyadenosine (β-L-ddA) and its 2′,3′-didehydro derivative (β-L-d4A) were found to have significant anti-human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) activities in cell culture.

Comparison of cytotoxicity of mononucleoside phosphotriester derivatives bearing biolabile phosphate protecting groups in normal human bone marrow progenitor cells

The effects of three mononucleoside phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) which incorporate biolabile phosphate protecting groups, namely S-acetyl-2-thioethyl (MeSATE), S-(2-hydroxyethylsulfidyl)-2-thioethyl (DTE), and pivaloyloxymethyl (POM) were studied and compared to their nucleoside parent in human myeloid colony-forming cells. Moreover, the relative antiviral potency of these three pronucleotides were determined in primary human peripheral blood mononuclear cells infected with human immunodeficiency virus type 1. The results indicate that the SATE and DTE pro-moieties, as well as their degradation products, do not induce additional toxicity. The bis(MeSATE) phosphotriester derivative of AZT emerged as the most selective inhibitor with an in-vitro therapeutic index of the same order of magnitude as observed for AZT. This study has been extended to the corresponding bis(MeSATE) and bis(DTE) phosphotriester derivatives of 2′,3′-dideoxyuridine (ddU).

Synthetic Approaches to a Mononucleotide Prodrug of Cytarabine

Synthetic pathways to a mononucleotide prodrug of cytarabine (Ara-C) bearing S-pivaloyl-2-thioethyl (tBuSATE) groups, as biolabile phosphate protections, are reported. Using a common phosphoramidite approach, two different kinds of nucleoside protecting groups have been investigated. During this study, we observed an intermolecular migration of the Boc protecting group in the course of the tert-butyldimethylsilyl ether cleavage using tetrabutyl ammonium fluoride.

Antiviral Activity and Intracellular Metabolism of Bis(tButylSATE) Phosphotriester of β-L-2′,3'Dideoxyadenosine, a Potent Inhibitor of HIV and HBV Replication

The β-L-nucleoside analogue β-L-2′,3′-dideoxy adenosine (β-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide β-L-2′,3′-dideoxyadenosine-5′-monophosphate (β-L-ddAMP) (Placidi et al., 2000). However, the nucleotide β-L-2′,3′-dideoxyadenosine-5′-triphosphate (β-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 μM without inhibiting human DNA polymerases α, β, or γ up to a concentration of 100 μM. These results suggested that prodrugs of β-L-ddAMP may bypass the poor metabolic activation of β-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of β-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, β-L-ddAMP-bis(tButylSATE) was synthesized. β-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of β-L-ddAMP-bis(tButylSATE) demonstrated that β-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of β-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCS, respectively. The intracellular concentrations of β-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.

Human Immunodeficiency Virus Resistance to AZT in MOLT4/8 Cells is Associated with a Lack of AZT Phosphorylation and is Bypassed by AZT-Monophosphate SATE Prodrugs

Human T lymphoid MOLT4/8 cells were grown continuously for more than 2 years in a medium containing 3′-azido-2′,3′-dideoxythymidine (zidovudine; AZT) at a concentration of 250 μM. These cells, designated MOLT-4/8rAZT250, were used to test the cytotoxic and antiviral activity of AZT. Intracellular accumulation of AZT, expression of the multidrug resistance 1 (MDR-1) gene, thymidine kinase (TK) gene and activity of the TK enzyme in cellular extracts were measured. The results showed that both the cytotoxic and antiviral activity of AZT were significantly lower in MOLT4/8rAZT250 than in MOLT4/8 cells; concentrations required to inhibit 50% production of the p24 human immunodeficiency virus type 1 (HIV-1) antigen of two laboratory strains were at least 100-fold higher in resistant cells. The MDR-1 gene was not expressed in the resistant cells. TK mRNA expression was significantly lower in the resistant than in the sensitive cells. TK enzymatic activity for deoxythymidine phosphorylation was impaired in MOLT4/8rAZT250 cells compared to the sensitive cells. AZT was phosphorylated only in the sensitive cells whereas no phosphorylation of AZT was found in the resistant cells. We tested whether several AZT-monophosphate triesters, which bypass cellular TK, could overcome resistance to the cytotoxic and antiviral activity of AZT. The bis(t-butylSATE) phosphotriester derivative of AZT showed comparable cytotoxic and antiviral activity in sensitive and resistant cells. The results demonstrated that MOLT4/8rAZT250 cells exert resistance to the anti-HIV activity of the drug mainly owing to the lack of AZT phosphorylation and that resistance may be bypassed by using AZT-monophosphate SATE prodrugs.

Inhibition of Hepatitis B Virus Replication by Nucleoside Enantiomers of β-2′,3′-Dideoxypurine Analogues

Various purine β-L-2′,3′-dideoxynucleoside analogues with both sugar and base modifications including β-L-ddG, β-L-ddl, β-L-ddA, 2′-azido-β-L-araddA, 2′-amino-β-L-araddA, 2′,5′-anhydro-β-L-araddA, 2′-azido-β-L-ddA, 2′-amino-β-L-ddA, 2′-fluoro-β-L-ddA, 3′-azido-β-L-ddA, 3′-amino-β-L-ddA, 3′-fluoro-β-L-ddA, 2,6-diamino-β-L-2′,3′-dideoxyfuranosylpurine, 6-cyclopropylamino-β-L-ddA, 2′-azido-6-N-triphenylphosphine-β-L-araddA, 2-amino-6-methylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2-amino-6-cyclopropylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2-amino-6-cyclopentylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2′,3′-didehydro-β-L-ddA and 2′,3′-didehydro-6-N-triphenyl phosphine-β-L-ddA were synthesized and evaluated as potential inhibitors of hepatitis B virus (HBV) replication in HBV DNA-transfected human hepatoblastoma-derived Hep-G2 cells (2.2.15 cells). β-L-ddA, 2′-azido-β-L-ddA, 3′-azido-β-L-ddA, 2″,3′-didehydro-β-L-ddA (β-L-D4A) and a modified base of β-L-D4A, inhibited HBV replication in vitro. β-L-D4A was the more potent and selective antiHBV agent with a 50% effective concentration value of 0.1 μM and a selectivity index of 1800. On the basis of this finding, studies are in progress to synthesize new purine derivatives with the β-L unnatural configuration which hopefully will lead to identifying additional potent and highly selective anti-HBV agents.