AbstractJanus tyrosine kinase 3 (JAK3) is expressed in lymphoid cells and is involved in the signalling of T cell functions. The development of a selective JAK3 inhibitor has been shown to have a potential benefit in the treatment of autoimmune disorders. In this article, we developed the 4-aminopiperidine-based compound RB1, which was highly selective for JAK3 inhibition, with an IC₅₀ of value of 40 nM, but did not inhibit JAK1, JAK2 or tyrosine kinase 2 (TYK2) at concentrations up to 5 µM. Furthermore, RB1 also exhibited favourable selectivity against a panel of representative kinases. In a battery of cytokine-stimulated cell-based assays, this potent inhibitor of JAK3 activity with good selectivity against other kinases could potently inhibit JAK3 activity over the activity of JAK1 or JAK2 (over at least 100-fold). A combination of liquid chromatography-mass spectrometry (LC-MS) experiments validated that RB1 covalently modified the unique cysteine 909 residue in JAK3. In vivo, RB1 exerted significantly improved pathology in the joints of a collagen-induced arthritis mouse model. The reasonable pharmacokinetics properties (F = 72.52%, T1/2 = 14.6 h) and favourable results of toxicology experiments (LD₅₀ > 2 g/kg) suggest that RB1 has the potential to be an efficacious treatment for RA.