Optimizing macrocycle ring size can also affect the potency of irreversible covalent kinase inhibitors. Shen et al. have recently reported that decreasing the ring size of macrocycle 34 by 3 atoms, by reducing the length of a flexible polymethylene linker to give 35, resulted in up to a ~40fold increase in potency towards the EGFR resistance mutant D746-750/T790M in vitro (Figure ). We note, however, that the IC50 values used for these determinations were based on a single time-point, rather than a full kinetic analysis of the irreversible labeling by these compounds. Consequently, it impossible to say how much of this effect is due to altered binding to the target versus differential rates of covalent reaction once bound, limiting our ability to interpret how the change in macrocycle structure affected interaction with the target kinase.