PDE-stable 2'3'-cGAMP analogues, containing 5'-S-phosphorothioester linkage, as STING agonists

The stimulator of interferon genes (STING) has emerged as a promising target for cancer immunotherapy. 2'3'-cGAMP, a natural agonist of STING, shows anticancer activity via stimulation of immune cells but it is susceptible to degradation in vivo by hydrolytic enzymes. Consequently, the cyclic dinucleotide analogues that are being evaluated in the clinic as immunotherapies contain the hydrolytically stable phosphorothioate moiety, whereby the sulfur moiety is exo to the phosphate containing ring. The synthesis of these phosphorothioates however produces diastereomers, which presents separation challenges. An alternative phosphorothioate (referred to as endo-S-phosphorothioate) whereby the sulfur atom is endo to the cyclic phosphate ring (i.e. 5'-S-phosphorothioester linkage) would not have chirality at phosphorus and hence not pose diastereomer separation problems. Herein, we report the design and synthesis of novel 5'-endo-phosphorothioate substituted 2'3'cGAMP analogues that are hydrolytically stable towards both ectonucleotide phosphodiesterase I (ENPP1, a mammalian phosphodiesterase) and poxvirus immune nucleases (poxin, a phosphodiesterase in Poxvirus) but retains STING-TBK1-IRF activation, comparable to clinical candidate, ADU-S100 in THP1 monocytes.