Cyclic diguanylate analogues: Facile synthesis, STING binding mode and anti-tumor immunity delivered by cytidinyl/cationic lipid

Herein 2-cyanoethoxy-N,N,N′,N′-tetraisopropyl-phosphorodiamidite(10, PIII, 3.5 eq.) could synergistically react with 3′,5′-dihydroxyl groups in a dinucleotide(PV) at the cyclization step for the synthesis of cyclic dinucleotides (CDNs) (c-di-GMP, cGAMP etc.) and their phosphorothioated analogues. A dynamic PIII-PV coordination mechanism has been proposed for the cyclization procedure which is confirmed by the variant 31P NMR data and molecular simulation. Among the mono-phosphorothioated CDNs, two stereoisomers showed different capacity for STING activation and the reason was predicted by molecular modeling. While compound 12b1 showed most potent ability to elicit cytokines (IFNβ, IL-6, Cxcl9 and Cxcl10) induction compared to another stereoisomer. Also, 12b1 significantly inhibited the tumor growth in the EO771 model with both 0.1 μg (i.t.) and 2 μg (i.v.) administration through the aid of a Mix delivery system developed by our group, and achieved a 31% long-term survival rate of tumor-bearing mice. 12b1/Mix significantly improved the percentage of CD8+ or CD4+ effector memory T (Tem, CD44highCD62Llow) cells and CD8+ central memory T (Tcm, CD44highCD62Lhigh) cells in the blood of EO771 mice, inducing the immune memory against EO771 tumor cells. Relatively lower dose regimens of 12b1(0.1 μg)/Mix displayed better tumor suppression by more potent STING pathway activation and higher levels of cytokines induction in the tumor. Outline: c-di-GMP analogues were synthesized by phosphoramidite approach via PIII-PV coordination in the cyclization step. Compound 12b1 delivered with DNCA/CLD potently inhibited the EO771 tumor growth and induced anti-tumor memory. [Display omitted] •Phosphorothioated CDNs analogues were prepared using phosphoramidite method.•PIII-PV coordinating cyclization mechanism was proposed and confirmed basically.•12b1,12d2 and 12e2 significantly improved the cytokine levels.•12b1 potently inhibited the EO771 tumor growth and induced anti-tumor memory.