Design, Synthesis, and Biochemical and Biological Evaluation of Novel 7-Deazapurine Cyclic Dinucleotide Analogues as STING Receptor Agonists

Abstract

Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.

Scheme 1. Naturally Occurring CDNs (1a–d) and 7-Deaza Variants (1e,f) Reported in Our Previous Study

Scheme 2. 7-Deazapurine NTPs for Enzymatic Synthesis

Scheme 3. Enzymatic Synthesis of 2′3′-cGA^RMPs

Reagents and conditions: (i) GTP, Tris–HCl [pH 8.0], MgCl₂, dsDNA, mcGAS, 37 °C 16 h.

Scheme 4. Enzymatic Synthesis of 7-Deazaguanine Containing CDNs

Reagents and conditions: (i) ATP, Tris–HCl [pH 8.0], MgCl₂, dsDNA, mcGAS, 37 °C 16 h; (ii) Tris–HCl [pH 8.0], MgCl₂, dsDNA, mcGAS, 37 °C 16 h.

Scheme 5. Enzymatic Synthesis with Bacterial Enzymes

Reagents and conditions: (i) GTP, HEPES [pH 8.0], MgCl₂, NaCl, DTT, DncV, 37 °C 16 h; (ii) HEPES [pH 8.0], MgCl₂, NaCl, DTT, DisA, 50 °C 16 h.

Scheme 6

Reagents and conditions: (i) TMSCl, BzCl/py, 0 °C—rt, 16 h; (ii) DMTrCl/py, rt, 16 h; (iii) TBDMSCl, AgNO₃, py/THF, rt 16 h; (iv) (1) PO(OPh)₂/py, rt, 1 h, (2) H₂O, rt, 5 min; (v) (1) DCA/DCM, rt, 15 min, (2) TES, rt, 30 min.

Scheme 7

Reagents and conditions: (i) DCA/DCM, rt, 10 min; (ii) (1) 13/MeCN, rt, 10 min, (2) tBuOOH, rt, 30 min; (iii) (1) DMOCP/py, rt, 110 min, (2) I₂, H₂O, rt, 10 min; (iv) CH₃NH₂/EtOH, rt, 3 h; (v) Et₃N·3HF/py, Et₃N, 50 °C, 3.5 h.

Figure 1

Open isomerized side product of a cross-coupling reaction.

Scheme 8

Reagents and conditions: (i) RB(OH)₂, Cs₂CO₃, TPPTS, Pd(OAc)₂/H₂O–MeCN (2:1), 100 °C, 30 min. ^bRBpin was used instead of RB(OH)₂. ^cOpen isomer 16 (13%) was also isolated.

Scheme 9

Reagents and conditions: (i) PhB(OH)₂, Cs₂CO₃, TPPTS, Pd(OAc)₂/H₂O–MeCN (2:1), 100 °C, 30 min.

Figure 2

Crystal structures of human STING in complex with 2′3′-cGA^RMPs. (A) Binding poses of compounds are shown with 2Fo–Fc maps contoured at 1σ. Compounds are distinguished by carbon colors (5f in green, PDB 8A2H; 5k in purple, PDB 8A2J; 5l in yellow, PDB 8A2I; and 5m in blue, PDB 8A2K), while nitrogen, oxygen, and phosphorus atoms are colored blue, red, and orange. (B) AMP side view of 5f, 5k, 5l, and 5m binding poses superposed with 2′3′-cGAMP (PDB 4KSY), showing the trend in positions of 7-deazaadenosine for all of our compounds, which differs from the position of adenosine in 2′3′-cGAMP (in white). (C) Side view superposition of ligands showing similarity of localization of nucleobases in this orientation. (D) Human STING dimer shown as a biological unit with differently colored monomers with 2′3′-cGAMP located in the binding site (PDB 4KSY). (E) Bottom view and (F) GMP side view of superposed ligands.

Figure 3

Interaction scheme of 5f (A; PDB 8A2H), 5k (B; PDB 8A2J), 5l (C; PDB 8A2I), and 5m (D; PDB 8A2K) in STING ligand-binding site, as seen in our structures.