TLR7/8 agonist 14 is a dual-target agonist of TLR7 and TLR8, with EC50 values of 75 μM for TLR7 and 96 μM for TLR8. This compound enhances the secretion of pro-inflammatory cytokines TNF-α, IL-1β, IL-8, and IFN-γ, while also increasing cytokine secretion and CD86 expression levels. TLR7/8 agonist 14 is applicable in colorectal cancer research.

TLR7:0.53 μM (EC50)

TLR7/8 agonist 14 (compound 69) demonstrates immune-regulatory properties in vitro at concentrations of 1-5 μM over 24 hours. At a concentration of 5 μM for 24 hours, it functions as an adjuvant candidate to promote adaptive anti-tumor immunity in immature dendritic cells. At 20 μM, it activates the downstream MyD88-dependent signaling pathways, resulting in AP-1 activation and the phosphorylation of MEK-1, p38, IκB, and ERK1/2 in THP-1 cells, thus initiating further downstream signaling. TLR7/8 agonist 14 has a half-life (t ₁/₂) of 5.6 minutes and a microsomal intrinsic clearance rate (CL int (mic)) of 246.9 μL/min/mg, with a liver intrinsic clearance rate (CL int (liver)) of 222.2 ml/min/kg in human liver microsomes at 10 mM for 1 hour.

Compound 69, a TLR7/8 agonist (2.5-10 mg/kg, administered subcutaneously once or intratumorally on days 1, 2, and 4), exhibits promising antitumor activity without systemic toxicity in a CT26 colorectal cancer syngeneic mouse model.