A striking effect of macrocycle ring size on cellular activity that was attributed to a difference in membrane permeability is provided by the macrocyclic c-Met inhibitors recently reported by Wang et al.. The authors report that shortening a flexible polymethylene linker so as to reduce the macrocycle ring size by one atom, from 24 to 23 atoms (42, 43), resulted in a 230-fold increase in potency in a cellular antiproliferation assay while giving no measurable difference in inhibitory potency against c-Met in in vitro assays (Figure ). Only a two-fold difference in permeability was found between these compounds in Caco-2 assays, however, casting doubt on whether differential membrane permeability is the explanation for this large effect of ring size on cellular potency. An alternative explanation is that 43 does indeed bind more strongly to the c-Met in vitro, compared to 42, but this difference is not reflected as a difference in potency in vitro because both compounds are potent enough to be at the 'floor' of the assay. That is, if the assay contained ~6 nM kinase protein, then at least 3 nM inhibitor would be required to occupy 50% of the target regardless of how strongly it binds, such that even an infinitely potent compound could not give an IC50 value lower than 3 nM. The authors do not report the exact concentration of c-Met protein used in the assay, but the available information 50 supports a concentration in the range 1-15 nM, consistent with the notion that this assay will not distinguish between compounds with potencies below the low nM range. Other potential explanations exist for the disconnect between the SAR seen in vitro versus in cells. For example, it is possible that the antiproliferative activity of 43 was not due to inhibiting c-Met, but rather to interaction with some off-target against which 43 is more potent than 42. However, we consider this and other alternative possibilities to be less likely.