Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4‑<i>d</i>]pyrrolo[2,3‑<i>b</i>]pyridine Scaffold

Janus kinases are major drivers of immune signaling and have been the focus of anti-inflammatory drug discovery for more than a decade. Because of the invariable colocalization of JAK1 and JAK3 at cytokine receptors, the question if selective JAK3 inhibition is sufficient to effectively block downstream signaling has been highly controversial. Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381 (23) featuring high isoform and kinome selectivity. Crystallography revealed that this inhibitor induces an unprecedented binding pocket by interactions of a nitrile substituent with arginine residues in JAK3. Herein, we describe detailed structure–activity relationships necessary for induction of the arginine pocket and the impact of this structural change on potency, isoform selectivity, and efficacy in cellular models. Furthermore, we evaluated the stability of this novel inhibitor class in in vitro metabolic assays and were able to demonstrate an adequate stability of key compound 23 for in vivo use.贾努斯激酶（Janus kinases）是免疫信号传导的关键驱动因子，十余年来一直是抗炎药物研发的核心靶点。由于JAK1与JAK3始终在细胞因子受体处共定位，选择性JAK3抑制是否足以有效阻断下游信号传导这一问题，长期以来颇具争议。近期，本团队发现了共价可逆型JAK3抑制剂FM-381（编号23），其对激酶亚型及激酶组具备优异的选择性。晶体学研究显示，该抑制剂通过其腈基取代基与JAK3内精氨酸残基的相互作用，诱导出了前所未有的结合口袋。本文详述了诱导该精氨酸结合口袋所需的构效关系，以及这一结构变化对细胞模型中化合物效价、亚型选择性及药效的影响。此外，我们通过体外代谢实验评估了这一新型抑制剂类别的稳定性，并证实关键化合物23具备适用于体内实验的足够稳定性。