Several small-mol. Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacol. characterization of BTK inhibitor acalabrutinib [compound 1, ACP-196 (4-[8- amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin1-yl]-N-(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochem. and cellular selectivity than ibrutinib and spebrutinib (compounds 2 and 3, resp.). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Determination of the inhibitory potential of anti-IgM-induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclin. and clin. studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approx. 99% median target coverage at 3 and 12 h and around 90% at 24 h in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacol. differentiators vs. ibrutinib and spebrutinib and is currently being evaluated in clin. trials.