Challenges resulting from novel viruses or new strains of known viruses call for new antiviral agents. Nucleoside analogs that act as inhibitors of viral polymerases are an attractive class of antivirals. For nucleosides containing thymine, base pairing is weak, making it desirable to identify nucleobase analogs that pair more strongly with adenine, in order to compete successfully with the natural substrate. We have recently described a new class of strongly binding thymidine analogs that contain an ethynylmethylpyridone as base and a Cnucleosidic linkage to the deoxyribose. Here we report the synthesis of the 3'-azido-2',3'-deoxyribose derivative of this compound, dubbed AZW, both as free nucleoside and as ProTide phosphoramidate. As a proof of principle, we studied the activity against Herpes simplex virus type 1 (HSV1). Whereas the ProTide phosphoramidate suffered from low solubility, the free nucleoside showed a stronger inhibitory effect than that of AZT in a plaque reduction assay. This suggests that strongly pairing C-nucleoside analogs of pyrimidines have the potential to become active pharmaceutical ingredients with antiviral activity.

High fidelity base pairing is important for the transmission of genetic information. Weak base pairs can lower fidelity, complicating sequencing, amplification and replication of DNA. Thymidine 5'-monophosphate (TMP) is the most weakly pairing nucleotide among the canonical deoxynucleotides, causing high errors rates in enzyme-free primer extension. Here we report the synthesis of an ethynylpyridone C-nucleoside analog of 3'-amino-2',3'dideoxythymidine monophosphate and its incorporation in a growing strand by enzyme-free primer extension. The ethynylpyridone C-nucleotide accelerates extension more than five-fold, reduces misincorporation and readily displaces TMP in competition experiments. The results bode well for the use of the C-nucleoside as replacements for thymidine in practical applications.

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Strongly pairing ethynylpyridone C-nucleosides are attractive surrogates for thymidine in oligonucleotides. Exploratory work on the antiviral activity of 3'-azidothymidine (AZT) derivatives with ethynylpyridone as base had identified strong lipophilicity as a limiting factor. Two strategies are being pursued to overcome this issue. In order to make the base more polar, the ethynyl group has been replaced with a cyano group, leading to a cyanopyridone C-nucleoside, whose eleven-step synthesis is reported here, together with the synthesis of a 3'-azido-2',3'-dideoxynucleoside derivative. The base pairing with adenine in a DNA duplex was studied by UV melting analysis of a self-complementary hexamer containing the 6-cyano-2'-deoxynucleoside instead of thymidine. A melting point increase of 2 °C compared to the unmodified control was found. The other strategy employs a phosphoramidate prodrug design with less lipophilic amino acid esters. Here, anti-HIV test of the alaninyl and prolinyl methyl esters of AZT gave promising results in cell culture experiments, increasing the selectivity index up to 5.8-fold for the III B strain and up to 5-fold for the ROD strain of the virus, as compared to the parent nucleoside. These findings help to design the next generation of pyridone C-nucleosides with potential applications as antivirals.