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

Abstract

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.

Enzymatic Synthesis of 3′–5′, 3′–5′ Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations

“…As seen in Figure , there are three outliers, namely, CDN1 , CDN23 , and 3′3′c-diGMP, which are discussed below. For the remaining compounds, a reasonable correlation between the computed values and experimental “proxies” to true thermodynamic data has been obtained ( R 2 = 0.7 after exclusion of three clear outliers discussed below and the inclusion of five ligands with an identical macrocyclic skeleton reported previously) …”

“…CDN8 and CDN9 contain two 2′-fluoro-2′-deoxyAMP or 2′-deoxy-2,2′-difluoroAMP subunits. A biological characterization of CDN8 (3′,3′c-di­(2′F,2′dAMP)) was described by us and others previously, and it was shown that this 3′3′CDN had improved binding to the STING in both DSF and cell-based assays relatively to 3′3′c-diAMP. Clearly, the enzymatic synthesis of 3′,3′c-di­(2′F,2′dAMP) is possible and relatively easy compared to the tedious organic synthesis.…”

“…Interestingly, some of the prepared 3′3′CDNs ( CDN8 , CDN13 , CDN19 , and CDN23 ) activated the REF STING only about 10-fold less efficiently than the WT STING (Tables –). We have recently shown that the mono- and bisphosphorothioate substitutions of CDN8 with an Rp or RpRp configuration can further decrease the difference in REF and WT STING EC 50 values to the levels seen for 2′3′cGAMP . Thus, further modifications of CDN13 , CDN19 , and CDN23 could result in their better binding to the REF STING.…”

“…We have recently shown that the mono-and bisphosphorothioate substitutions of CDN8 with an Rp or RpRp configuration can further decrease the difference in REF and WT STING EC 50 values to the levels seen for 2′3′cGAMP. 73 Thus, further modifications of CDN13, CDN19, and CDN23 could result in their better binding to the REF STING.…”

“…For the remaining compounds, a reasonable correlation between the computed values and experimental "proxies" to true thermodynamic data has been obtained (R 2 = 0.7 after exclusion of three clear outliers discussed below and the inclusion of five ligands with an identical macrocyclic skeleton reported previously). 73 With respect to the aforementioned structural assumption of the closed form of the STING protein, contradictory reports exist in the literature concerning the closed vs open conformation of the STING:3′3′c-diGMP complex. 20,48,78,79 This can perhaps explain the larger than expected difference from the correlation line for the 3′3′c-diGMP (∼15 kcal mol −1 in the calculated ΔG′ int_rel ).…”