Dinucleoside phosphorothioates represent first-in-class antiviral agents that act by novel mechanisms involving the activation of cellular innate immune receptors RIG-I and NOD2 and induction of the production of intracellular interferon for antiviral defense. A lead compound SB 9200 has been evaluated in Phase I clinical trials in chronically infected HCV patients and is also being advanced into Phase II clinical trials for HBV, and RSV. As part of clinical development, we carried out thermal stability studies of several dinucleotide compounds. Interestingly, it was observed that upon heating, the dinucleotide analogs underwent fragmentation resulting in the cleavage of the internucleotidic linkage. To fully characterize this novel reaction, we evaluated a series of analogs with different substitution patterns at the ribose, deoxyribose, nucleobase and the phosphorothioate group. The requisite dinucleoside phosphorothioates were assembled using the appropriate protected nucleosides via standard phosphoramidite chemistry to generate dinucleoside phosphorothioates, which upon chemo-selective S-alkylation using the iodo-alkyl derivatives gave the corresponding S-alkyl phosphorothioates (A). The S-alkylated derivatives were subjected to heating at 70-90 oC for 72 h. The resulting fragmentation products were identified as the cyclonucleosides B and the phosphorothioate diesters C (Fig.1) which were further confirmed by total synthesis. The mechanism of the fragmentation reaction can be rationalized as shown (Fig. 1). Interestingly, the rate of the reaction is influenced by the nature of the S-alkyl groups, substitutions on the sugar ring and the nucleosidic bases. Furthermore, the P-chirality had significantly more impact on the rate of fragmentation of the Sp-diastereomer, which was faster compared to that of the Rp-diastereomer. Molecular dynamics and quantum mechanics studies were conducted to further elucidate the mechanism and to provide a rational basis for the structure- and stereochemistry-related effects on this novel fragmentation reaction. Support of this research from the National Institutes of Health with grants (UO1 AI058270 and RO1 AI094469) through NIAID are gratefully acknowledged.

Thermal degradation of the antiviral dinucleoside phosphorothioate analogs: Novel fragmentation pathway results in the formation of cyclonucleosides

251st National Meeting (2016)

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