Based on our results, 40 represents a potent and selective PIKfyve inhibitor with sufficient in vivo stability and oral bioavailability to be used in animal studies and which does not elicit adverse side effects when mice are treated. Our previously disclosed negative control compound can be utilized alongside this second-generation PIKfyve chemical probe, 40, in efficacy studies. In summary, we provide an orthogonal scaffold to those that have been used to interrogate PIKfyve inhibition in vivo and which lacks a structural liability of the most advanced PIKfyve inhibitor, apilimod. The half-life of 40 (>5 hours) suggests that it may have optimal pharmacokinetic properties compared to apilimod, but more extensive studies are required to confirm this hypothesis. As a promising second-generation PIKfyve probe, compound 40 can be used to interrogate the effect of PIKfyve inhibition in vivo with the goal of identifying diseases that are responsive to this therapeutic approach.