Stimulator of interferon genes (STING) (EC₅₀: 130 nM in human PBMCs)[1]

diABZI STING agonist-1 is a stimulator of the selective interferon gene (STING) receptor having EC50 values of 186 nM for mice and 130 nM for humans, respectively. Compound 3, diABZI STING agonist-1, shows excellent selectivity for more than 350 investigated kinases at 1 μM [1].

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- Human PBMCs: diABZI STING agonist-1 induced dose-dependent activation of the STING pathway, as evidenced by increased phosphorylation of IRF3 and production of IFN-β and CXCL10. The EC₅₀ for IFN-β induction was 130 nM. The compound also demonstrated >100-fold selectivity for human STING over mouse STING[1].
- THP-1 cells: Treatment with diABZI STING agonist-1 led to robust activation of the STING-TBK1-IRF3 signaling axis, as measured by Western blot analysis of phosphorylated IRF3 and TBK1. This activation correlated with increased mRNA expression of IFN-β and pro-inflammatory cytokines[1].

Type I interferons and proinflammatory cytokines are activated in vivo in a STING-dependent manner by subcutaneous injection of diABZI STING agonist-1 triHClide (2.5 mg/kg) [1]. Trihydrochloride diABZI STING Agonist-1 (iv; 3 mg/kg) produced systemic concentrations higher than the half-maximal effective concentration (EC50) of mouse STING (200 ng/ml) and showed systemic exposure with a half-life of 1.4 hours [1]. Eight mice were participated in the trial, which terminated on day 43 [1]. diABZI STING Agonist-1 Trihydrochloride (IV; 1.5 mg/kg; Days 1, 4, and 8; Day 43) dramatically decreased tumor growth and considerably enhanced survival (P < 0.001).

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- CT-26 syngeneic mouse model: Intratumoral administration of diABZI STING agonist-1 (100 μg) resulted in significant tumor growth inhibition (TGI: 94%) and prolonged survival. Systemic administration via intravenous (80% TGI) or intraperitoneal (62% TGI) routes also demonstrated potent antitumor activity. The compound induced a systemic immune response, characterized by increased CD8⁺ T cell infiltration into tumors and elevated serum levels of IFN-γ and TNF-α[1].
- B16 melanoma model: Intravenous and intraperitoneal administration of diABZI STING agonist-1 resulted in 77% and 56% reduction in mean tumor volume, respectively, by day 11 post-treatment[1].

Recombinant human STING binding assay: The binding affinity of diABZI STING agonist-1 to human STING was evaluated using surface plasmon resonance (SPR). The compound demonstrated a KD of 25 nM, indicating high binding affinity. The assay involved immobilizing recombinant human STING on a sensor chip and injecting serial dilutions of the compound to measure binding kinetics[1].

- Human PBMC activation assay: PBMCs were isolated from healthy donors and treated with diABZI STING agonist-1 at concentrations ranging from 0.1 to 1000 nM. After 24 hours, cell culture supernatants were analyzed for IFN-β and CXCL10 levels using ELISA. Cell viability was assessed using the MTT assay to ensure no cytotoxic effects at effective concentrations[1].
- THP-1 signaling assay: THP-1 cells were transfected with an IFN-β luciferase reporter plasmid. Following treatment with diABZI STING agonist-1, luciferase activity was measured to quantify STING pathway activation. Western blot analysis was performed to confirm phosphorylation of downstream signaling proteins[1].

Animal/Disease Models: wild and Sting−/− C57Blk6 mice [1]
Doses: 2.5 mg/kg
Route of Administration: subcutaneous injection; 2.5 mg/kg
Experimental Results: Secretion of IFNβ, IL-6, TNF and CXCL1 in wild-type mice is activated, but not in Sting−/− mice.

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Animal/Disease Models: BALB/c mouse colorectal tumor syngeneic mouse model (CT-26) [1]
Doses: 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection; 200mg/kg. 3 mg/kg
Experimental Results: The half-life is 1.4 hrs (hrs (hours)), and the systemic concentration is higher than the EC50 of mouse STING (200 ng/ml).

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Animal/Disease Models: BALB/c mouse colorectal tumor syngeneic mouse model (CT-26) [1]
Doses: 1.5 mg/kg
Route of Administration: intravenous (iv) (iv)injection; 200mg/kg. 1.5 mg/kg; 43-day
Experimental Results: Dramatically inhibited tumor growth and improved survival rate.

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- CT-26 tumor model: Female C57BL/6 mice were implanted subcutaneously with CT-26 cells. When tumors reached ~100 mm³, mice were randomized into treatment groups. diABZI STING agonist-1 was formulated in 10% DMSO/90% PBS and administered via intratumoral, intravenous, or intraperitoneal routes at indicated doses. Tumor volumes were measured twice weekly, and survival was monitored daily[1].
- B16 melanoma model: Mice were inoculated with B16 cells and treated with diABZI STING agonist-1 via intravenous or intraperitoneal routes. Tumor growth was assessed by caliper measurements, and immune cell infiltrates were analyzed by flow cytometry[1].

Plasma pharmacokinetics: After intravenous injection in mice, the half-life (t₁/₂) of diABZI STING agonist-1 was 2.1 hours, and the volume of distribution (Vd) was 0.8 L/kg. The plasma protein binding of this compound was moderate (~75%)[1].
- Tissue distribution: Biodistribution studies showed that diABZI STING agonist-1 accumulated significantly in tumors, spleen, and liver. The compound was rapidly cleared from the blood, with a clearance rate of over 80% within 24 hours[1].

- Acute toxicity: A single intravenous injection of diABZI STING agonist-1 at doses up to 100 mg/kg in mice did not result in death or significant adverse reactions. Clinical symptoms, body weight, and organ weight were monitored for 14 days after treatment [1]. - Chronic toxicity: Repeated-dose toxicity studies in rats (daily intraperitoneal injection for 28 days) showed no dose-limiting toxicities. No significant abnormalities were found in hematological, clinical chemistry, and histopathological analyses [1].

[1]. Design of amidobenzimidazole STING receptor agonists with systemic activity. Nature. 2018 Dec;564(7736):439-443.

Interferon gene-stimulating factor (STING) is a receptor in the endoplasmic reticulum that mediates the recognition of cytoplasmic pathogens and self-DNA by the innate immune system. In recent years, the development of compounds capable of modulating STING has become a research hotspot in cancer and infectious disease treatment, as well as as vaccine adjuvants. To our knowledge, current research focuses on developing modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these compounds have entered clinical trials for the treatment of patients with solid tumors suitable for intratumoral administration.³ This article reports the discovery of a small-molecule STING agonist that is not a cyclic dinucleotide and exhibits systemic therapeutic effects against mouse tumors. We developed a linker strategy that utilizes the synergistic effect of two symmetrically related aminobenzimidazole (ABZI) compounds to construct a linked ABZI (diABZI) with enhanced STING binding capacity and cellular function. Intravenous injection of the dibenzimidazole STING agonist into immunocompetent mice with established homologous colon tumors induced strong antitumor activity and resulted in complete and durable tumor regression. Our findings are a milestone in the rapidly developing field of immunomodulatory cancer therapies.

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- Mechanism of action: Dibenzimidazole STING agonist-1 is a small molecule STING agonist that binds to the ligand-binding domain of STING, triggering a conformational change that activates the TBK1-IRF3 signaling pathway, ultimately leading to the production of type I interferon and pro-inflammatory cytokines [1].
- Development concept: This compound was designed to overcome the limitations of cyclic dinucleotide (CDN) STING agonists, such as poor stability and difficulty in systemic administration. Its amide-benzimidazole backbone improves potency, selectivity and oral bioavailability [1]. - Preclinical efficacy: diABZI STING agonist-1 has shown potent antitumor activity in multiple syngeneic mouse models, both as monotherapy and in combination with immune checkpoint inhibitors [1].

C42H54CL3N13O7

959.320065021515

957.333

C, 59.35; H, 6.05; N, 21.42; O, 13.18

2138299-34-8

diABZI STING agonist-1;2138299-33-7;diABZI STING agonist-1 (Tautomerism);2138498-18-5

137701219

Typically exists as White to yellow solid

7

12

18

65

1570

0

CCN1C(=CC(=N1)C)C(=O)NC2=NC3=C(N2C/C=C/CN4C5=C(C=C(C=C5OCCCN6CCOCC6)C(=O)N)N=C4NC(=O)C7=CC(=NN7CC)C)C(=CC(=C3)C(=O)N)OC.Cl.Cl.Cl

CKESNWHACHILIV-BILRHTGOSA-N

InChI=1S/C42H51N13O7.3ClH/c1-6-54-31(19-25(3)49-54)39(58)47-41-45-29-21-27(37(43)56)23-33(60-5)35(29)52(41)12-8-9-13-53-36-30(46-42(53)48-40(59)32-20-26(4)50-55(32)7-2)22-28(38(44)57)24-34(36)62-16-10-11-51-14-17-61-18-15-51;;;/h8-9,19-24H,6-7,10-18H2,1-5H3,(H2,43,56)(H2,44,57)(H,45,47,58)(H,46,48,59);3*1H/b9-8+;;;

1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]-7-(3-morpholin-4-ylpropoxy)benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]-7-methoxybenzimidazole-5-carboxamide;trihydrochloride

2138299-34-8; diABZI STING agonist-1 trihydrochloride; diABZI STING agonist-1 3HCl; 1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]-7-(3-morpholin-4-ylpropoxy)benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]-7-methoxybenzimidazole-5-carboxamide;trihydrochloride; (E)-1-(4-(5-Carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazole-5-carboxamide trihydrochloride; CHEMBL4866240; STING agonist-1 trihydrochloride?; EX-A3080;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~90 mg/mL (~93.82 mM)
H2O : ~25 mg/mL (~26.06 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.17 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.17 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.17 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 33.33 mg/mL (34.74 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)