Synthesis and Biological Evaluation of Phosphoester and Phosphorothioate Prodrugs of STING Agonist 3′,3′-c-Di(2′F,2′dAMP)

Cyclic dinucleotides (CDNs) are second
messengers that bind to
the stimulator of interferon genes (STING) and trigger the expression
of type I interferons and proinflammatory cytokines. Here we evaluate
the activity of 3′,3′-c-di­(2′F,2′dAMP)
and its phosphorothioate analogues against five STING allelic forms
in reporter-cell-based assays and rationalize our findings with X-ray
crystallography and quantum mechanics/molecular mechanics calculations.
We show that the presence of fluorine in the 2′ position of
3′,3′-c-di­(2′F,2′dAMP) improves its activity
not only against the wild type (WT) but also against REF and Q STING.
Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl
phosphoester prodrugs of CDNs. Masking the negative charges of the
CDNs results in an up to a 1000-fold improvement of the activities
of the prodrugs relative to those of their parent CDNs. Finally, the
uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to
the parent CDN is rapid, reaching a peak intracellular concentration
within 2 h.