Synthesis of Nucleoside Analogues and Elucidation of their Antiviral Mechanisms

Viral infections continue to challenge global healthcare systems, necessitating the development of effective antiviral therapies. This thesis attempts to address this need through the exploration of nucleoside analogues as potential antiviral drugs, presented in three chapters.

Following viral infection, the enzyme viperin catalyses the formation of 3ʹ-deoxy-3ʹ,4ʹdidehydrocytidine triphosphate (ddhCTP) as part of the innate immune response. Incorporation of ddhCTP by viral polymerases into nascent RNA has been shown to inhibit the replication of several viruses through an obligate chain termination mechanism. However, as a triphosphate species, exogenously synthesised ddhCTP has limited cellular permeability. As a result, ddhCTP is non-viable as a therapeutic intervention. The non-phosphorylated analogue, ddhC, has been shown to traverse cell membranes, where subsequent phosphorylation by host kinases resulted in elevated levels of intracellular ddhCTP. Chapter one of this thesis sought to improve the cellular uptake of ddhC through prodrug strategies. Following intracellular localisation, enzymatic cleavage of the prodrug moiety of the nucleoside analogue and metabolism to their respective triphosphate forms was envisioned to attenuate viral replication. In this research, the 5ʹ position was derivatised with ester groups and a phosphoramidite (ProTide) moiety. In addition, the enantiomeric form and a 5-fluoro analogue of ddhC (as their prodrug forms) were also synthesised to investigate the effects of these nucleoside modifications.

While some 5ʹ ester derivatives showed improved antiviral activity against Epstein-Barr Virus (EBV) compared to native ddhC, the inhibition remained moderate, and some modifications led to reduced activity. An EBV polymerase inhibition assay was conducted with collaborators to determine whether the antiviral activities of ddhC and its analogues were linked to EBV polymerase inhibition by ddhCTP. The assay results revealed that although not as efficient as the tenofovir diphosphate control, there was evidence of inhibition by ddhCTP, implying higher intracellular concentrations of ddhCTP could inhibit EBV polymerase.

Chapter two investigated the synthesis of 5ʹ prodrugs of cyclopropanated nucleosides featuring a 2-oxabicyclo[3.1.0]hexane scaffold. These compounds were envisioned to be isosteric analogues of ddhC, where the 3ʹ,4ʹ-double bond was appended with a cyclopropane group. In addition, the presence of the cyclopropane ring results in conformational restriction which may confer altered biological activity. Although the synthesis of this class of compounds has been reported, their prodrug forms have not been evaluated in antiviral assays. Anticipating intracellular uptake issues like ddhC, the prodrug strategy was envisioned to improve their activity as antiviral compounds. To this end, cyclopropanated ddhC and ddhA analogues were prepared using literature methodology, which were then functionalised to their corresponding 5ʹ prodrug forms. Simultaneously, a novel methodology was developed to access this class of compounds by direct cyclopropanation of a ddh-type scaffold. Preliminary results are promising; however, this methodology requires further development. Antiviral testing of the cyclopropanated nucleosides revealed limited antiviral activity of the cyclopropanated cytidine analogues. On the other hand, cyclopropanated adenosine analogues displayed high cytotoxicity profiles, suggesting off-target effects.

Finally, chapter three details the design and synthesis of a Galidesivir phosphoramidite which will be used towards the synthesis of modified RNA sequences. Galidesivir is an imino-C-nucleoside that demonstrates broad-spectrum antiviral activity against viruses of different families. Initially reported as a delayed chain terminator, recent research suggests this is not the case and that an alternative mechanism of viral inhibition exists. The preparation of a Galidesivir phosphoramidite would enable synthesis of modified oligonucleotides whose analysis by biophysical methods would help elucidate Galidesivir’s antiviral mechanism. In this chapter, various protecting group strategies were investigated, culminating in the successful preparation of Galidesivir phosphoramidite. The protecting group strategy developed in this chapter should be adaptable towards the synthesis of other types of imino-C-nucleoside phosphoramidites for future oligonucleotide synthesis endeavours