ITK-IN-6 is a potent and selective ITK inhibitor (Kd = 387 nM). It binds directly to the ITK kinase domain. This compound prevents the release of pro-inflammatory cytokines and the activation and differentiation of Th2 and Th17 cells. ITK-IN-6 improves asthma progression by reducing inflammatory cell infiltration, mucus production, and IgE generation. It significantly inhibits airway inflammation and is used in asthma research.

ITK-IN-6 (Compound C-161) demonstrates a 62.27% inhibition of ITK downstream transcriptional activity after 24 hours in the Jurkat-NFAT-Luc stable cell line. At concentrations of 0.07-5 μM for 12 hours, ITK-IN-6 shows good safety in CD4+ T cells and reduces Th2 and Th17 differentiation in vitro. With exposure of 0.15-2.5 μM for 12 hours, it blocks key T cell activation factors and inhibits T cell surface activation markers in Jurkat cells. ITK-IN-6 (0.625-2.5 μM, 12 h) specifically inhibits ITK and hinders proximal T cell receptor signaling, significantly suppressing CD69 expression in Jurkat T cells. It also suppresses IL-2 expression in a dose-dependent manner at both transcriptional and protein levels in Jurkat cells, with an IC50 of 494 nM. Furthermore, treatment with 0.625-2.5 μM ITK-IN-6 for 30 minutes significantly reduces intracellular Ca2+ flux induced by anti-CD3 in Jurkat cells.

ITK-IN-6 (Compound C-161) administered intraperitoneally at 10 mg/kg once daily from days 7 to 11, effectively alleviates airway inflammation in C57BL/6J mice induced by house dust mites. It achieves this by inhibiting Th2/Th17 differentiation and cytokine production, while demonstrating a good safety profile.