Design, Synthesis, and Biological Evaluation of Aryl Pyrazolopyrimidines as Toll-Like Receptor 7 Agonists

Abstract

Compounds containing pyrazolopyrimidine scaffolds were designed and synthesized as toll-like receptor 7 (TLR7) agonists. Thirty-three compounds, including 22 novel compounds, were prepared, and their structures were identified using nuclear magnetic resonance spectroscopy and mass spectrometry. TLR7 agonist activity was determined in HEK-Blue hTLR7 reporter cells. Among the compounds tested, 2-((4-methoxyphenyl)amino)-7-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile showed the highest activity, and further in vitro biological experiments were performed using this compound. Treatment with the title compound activated the TLR7-mediated NF-κB pathway, triggering the IRAK4-IKKα/β-IκBα-p65 NF-κB signaling cascade, which led to an increase in the expression of NF-κB-regulated innate cytokines such as TNFα and IL-1β in RAW264.7 macrophages. These findings suggest that the title compound acts as a TLR7 agonist and enhances the innate immune response.