Structure-activity relationship study of amidobenzimidazole derivatives as stimulator of interferon genes (STING) agonists

Highlights

• 88 compounds were designed and synthesized as amidobenzimidazole monomer STING agonists.

• Compound 72 potently activated the STING Signaling in PBMCs.

• Compound 72 showed high selectivity for human STING.

Abstract

Stimulator of interferon genes (STING) is a crucial adaptor protein that can regulate the innate immune response by inducing the secretion of type Ι interferons and other cytokines after recognizing endogenous or exogenous DNA. Due to the key role of STING in the innate immune system, the activation of STING pathway is expected to be an efficacious immunotherapeutic tactic to treat cancer. In this study, we performed a structure-activity relationship study of amidobenzimidazole monomer, led to a series of ABZI STING agonist derivatives with potent STING-activating effects. Among them, compound 72, as a representative compound, markedly activated the STING-TBK1-IRF3 signaling pathway and significantly increased the mRNA and protein levels of IFN-β, CXCL10 and IL-6 in both WT THP-1 cells and human peripheral blood mononuclear cells (hPBMCs). In addition, it was confirmed that compound 72 was highly selective for human STING, specifically targeting human STING signaling and showing no activation of m-STING.

Introduction

Stimulator of interferon gene (STING, also known as MITA, ERIS, or MPYS) is a stimulatory molecule residing on the endoplasmic reticulum (ER) [1]. As an adaptor protein, STING can regulate the innate immune response by linking the upstream DNA sensor cyclic GMP-AMP synthase (cGAS) with downstream recruitment of tank-binding kinase 1 (TBK1), leading to the activation of the IRF and NF-κB pathways and the expression of type I interferons (IFNs) and other inflammatory cytokines [[2], [3], [4]], such as CXCL10, TNF-α and IL-6. The research value of STING inhibitors in immunotherapy was presented [5]. In addition, activation of the STING pathway was found to be crucial for priming tumor-specific CD8⁺ T cells to eliminate tumor cells, and the release of IFN-β can promote dendritic cell (DC) maturation, making tumor antigen presentation for the cross-priming of CD8⁺ T cells possible [[6], [7]]. Therefore, STING agonists have received increasing attention as antitumor agents.

First-generation STING agonists are cyclic dinucleotide (CDN) derivatives, mimetics of the endogenous STING activator 2′,3′-cGAMP (1, Fig. 1A), and several have already entered clinical trials [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]]. However, because of the poor biochemical properties of CDN derivatives, most are limited to intratumoral administration. Some groups are working on developing non-CDN small-molecule agonists. DMXAA (2, Fig. 1B) [20], one of the first reported non-CDN small-molecule STING agonists, has high efficacy. However, it is a mouse-specific STING agonist that cannot bind to human STING [[21], [22], [23]]. More recently, other non-CDN STING agonists have been reported by several groups, including GSK (3, Fig. 1C) [[24], [25], [26]], Scripps (4, Fig. 1D) [27], Merck (5, Fig. 1E) [[28], [29], [30]], and others [31].

Our previous study reported a series of amidobenzimidazole monomer STING agonists and identified lead compound 6 with improved biochemical and cellular potency (with EC₅₀ = 0.287 μM in the THP1-Dual cell line), but the in vivo antitumor potency was modest [32]. Additionally, compound 6 had poor stability and was easily metabolized (with a residual of 36.4% of the initial concentration after incubation with rat plasma for 1 h). Because of the significant antitumor efficacy of compound 6 in vitro and the limited structure-activity relationship (SAR) study reported, compound 6 was considered amenable to structural modifications, which we believe to be potentially capable of improving its biological activity in vivo. In this regard, structural optimization was performed to discover more potent amidobenzimidazole monomer STING agonists. Herein, we describe the design, synthesis, and biological evaluation of these new STING agonists.