Receptor-interacting protein kinase-1 (RIPK1) is involved in the necroptosis pathway, which regulates inflammatory signaling and cell death in a variety of diseases, including inflammatory and neurodegenerative disorders. We identified a novel hit compound 36 by a cell-based screening assay (anti-necroptosis EC₅₀ = 58 nM). Starting from compound 36, we designed a series of scaffolds to improve anti-necroptosis activity, physicochemical properties and metabolic stability. The isothiazolo[5,4-b]pyridine backbone proved to be a promising scaffold which provided a number of potent necroptosis inhibitors. Compound 56, for example, effectively blocked necroptosis in both human and mouse cells (EC₅₀ = 1–5 nM). A binding assay showed that compound 56 potently binds to RIPK1 (K_d = 13 nM), but not RIPK3 (K_d > 10,000 nM). Kinase functional assay (ADP-Glo) confirmed that compound 56 inhibits RIPK1 phosphorylation with an IC₅₀ at 5.8 nM. Importantly, compound 56 displayed excellent cross-species liver microsomal metabolic stability (t1/2 > 90 min). Furthermore, compound 56 exhibited favorable in vitro safety profiles in hERG and CYP assays. Finally, pre-treatment with 56 significantly reduced hypothermia and lethal shock in the systemic inflammatory response syndrome mice model. Taken together, compound 56 represented a promising prototype for the development of therapeutic agent to treat inflammation-related diseases.

受体相互作用蛋白激酶 1 (RIPK1) 参与坏死性凋亡通路，该通路调节多种疾病（包括炎症和神经退行性疾病）中的炎症信号和细胞死亡。我们通过基于细胞的筛选试验（抗坏死性凋亡 EC ₅₀  = 58 nM）鉴定了一种新的命中化合物36 。从化合物36开始，我们设计了一系列支架来提高抗坏死性凋亡活性、理化性质和代谢稳定性。异噻唑并 [5,4- b ] 吡啶主链被证明是一种很有前途的支架，它提供了许多有效的坏死性凋亡抑制剂。例如，化合物56可有效阻断人和小鼠细胞的坏死性凋亡 (EC ₅₀ = 1–5 纳米）。结合测定显示化合物56有效结合 RIPK1 (K _d  = 13 nM)，但不结合 RIPK3 (K _d  > 10,000 nM)。激酶功能测定 (ADP-Glo​​) 证实化合物56抑制 RIPK1 磷酸化，IC ₅₀为 5.8 nM。重要的是，化合物56显示出出色的跨物种肝微粒体代谢稳定性（t1/2 > 90 分钟）。此外，化合物56在 hERG 和 CYP 测定中表现出良好的体外安全性。最后，用56预处理可显着降低全身炎症反应综合征小鼠模型的低温和致死性休克。合起来，化合物56代表了开发治疗炎症相关疾病的治疗剂的有前途的原型。