Design, synthesis, and anti-inflammatory effects of novel pyrazolopyrimidine compounds inhibitors

The JAK-STAT signaling pathway is a critical therapeutic target for inflammatory diseases like rheumatoid arthritis (RA). This study designed and synthesized 28 novel pyrazolo[1,2- b ]pyrimidine derivatives, using tofacitinib as a lead compound. Their anti-inflammatory activity was evaluated in LPS-induced RAW264.7 macrophages and a zebrafish inflammation model. Among the synthesized compounds, D1 and D9 demonstrated potent anti-inflammatory effects, significantly inhibiting neutrophil migration in zebrafish and reducing the production of nitric oxide (NO), TNF-α, and IL-6 in vitro and in vivo. In vitro, the inhibition effect on JAK kinases were performed using HTRF (Homogenous Time-Resolved Fluorescence) detection technology, which is more convenient and stable than traditional methods. D9 exhibits a certain degree of inhibitory effect on TYK2 (IC 50 = 110.6 μM). Molecular docking studies revealed stable binding interactions, including hydrogen bonds, with key residues in JAK proteins. ADME predictions indicated favorable drug-like properties for both candidates. These findings identify D1 and D9 are promising lead compounds for developing novel anti-inflammatory activities. • We designed and synthesized 28 novel pyrazolopyrimidine derivatives as potential JAK inhibitors. • Compounds D1 and D9 potently inhibited NO, TNF-α, and IL-6 production in LPS-induced RAW264.7 cells. • D1 and D9 significantly suppressed neutrophil migration in an LPS-induced zebrafish inflammation model. • Molecular docking revealed stable binding interactions with key amino acids in the JAK protein active site. • Promising in silico ADME profiles suggest D1 and D9 possess favorable drug-like properties.