RIPK1 plays a key role in the necroptosis pathway that regulates inflammatory signaling and cell death in various diseases, including inflammatory and neurodegenerative diseases. Herein, we report a series of potent RIPK1 inhibitors, represented by compound 70. Compound 70 efficiently blocks necroptosis induced by TNFα in both human and mouse cells (EC₅₀ = 17–30 nM). Biophysical assay demonstrates that compound 70 potently binds to RIPK1 (K_d = 9.2 nM), but not RIPK3 (K_d > 10,000 nM). Importantly, compound 70 exhibits greatly improved metabolic stability in human and rat liver microsomes compared to compound 6 (PK68), a RIPK1 inhibitor reported in our previous work. In addition, compound 70 displays high permeability in Caco-2 cells and excellent in vitro safety profiles in hERG and CYP assays. Moreover, pre-treatment of 70 significantly ameliorates hypothermia and lethal shock in SIRS mice model. Lastly, compound 70 possesses favorable pharmacokinetic parameters with moderate clearance and good oral bioavailability in SD rat. Taken together, our work supports 70 as a potent RIPK1 inhibitor and highlights its potential as a prototypical lead for further development in necroptosis-associated inflammatory disorders.

RIPK1 在坏死性凋亡通路中发挥关键作用，该通路调节各种疾病中的炎症信号传导和细胞死亡，包括炎症和神经退行性疾病。在此，我们报告了一系列有效的 RIPK1 抑制剂，以化合物70为代表。Compound 70可有效阻断人和小鼠细胞中由 TNFα 诱导的坏死性凋亡 (EC ₅₀  = 17–30 nM)。生物物理测定表明，化合物70与 RIPK1 (K _d  = 9.2 nM) 有效结合，但不与 RIPK3 (K _d  > 10,000 nM) 结合。重要的是，与化合物6相比，化合物70在人和大鼠肝微粒体中的代谢稳定性显着提高(PK68)，一种 RIPK1 抑制剂，在我们之前的工作中报道过。此外，化合物70在 Caco-2 细胞中显示出高渗透性，在 hERG 和 CYP 测定中具有出色的体外安全性。此外， 70的预处理显着改善了 SIRS 小鼠模型中的低温和致死性休克。最后，化合物70在 SD 大鼠中具有良好的药代动力学参数，具有中等清除率和良好的口服生物利用度。总之，我们的工作支持70作为一种有效的 RIPK1 抑制剂，并强调其作为进一步发展坏死性凋亡相关炎症性疾病的原型线索的潜力。