The acyclic precursor, 6, was derived from a lead compound developed to target the Aurora A kinase. Upon cyclization, the resulting compound 5 showed poor activity against Aurora A (IC50 = 1 µM), but good antiproliferative activity in cancer cell lines. The results from a kinase selectivity panel led the authors to attribute this cellular activity to the inhibition of CDK2, FLT3, and JAK2, against which 5 showed IC50 values of 13-73 nM (Figure ). Based on computational modeling, the authors hypothesized that the different selectivity seen for 5 was due to formation of a salt bridge between the basic tertiary amine nitrogen in 5 and an active site aspartate residue that is present in CDK2 and FLT3 but not in Aurora A. Since a pan-CDK/FLT3/JAK2 inhibitor was considered desirable as a potential treatment for cancer, the compound series was further optimized to exploit these adventitiously discovered activities. As noted above in our discussion of potency, the ability to form this salt bridge is likely not a major contributor to the potency gain seen for 5 versus the acyclic precursor 6 against CDK2 or FLT3. However, this feature does appear to contribute substantially to selectivity; macrocyclization preorganizes the ligand conformation so as to stabilize the salt bridge, and possibly other interactions, thereby bringing them into play as selectivity determinants. The developers of the JAK2/JAK2 V617F /FLT3 inhibitor pacitrinib (27) similarly report that macrocyclization of an acyclic inhibitor led to substantially improved selectivity against JAK1 and JAK3, which they attribute to stabilizing certain interactions with the protein, though the detailed selectivity profile of the acyclic lead was not reported. The modulation of selectivity for PI3Ka versus mTOR achievable with different macrocyclized variants of the acyclic dual inhibitor GSK-2126458, reported by Alvarez et al., provides another example. Macrocyclization per se is not always a route towards better kinase selectivity. As discussed above, kinase inhibitor selectivity depends on often subtle differences in active site geometry, as well as on how a particular ligand chemotype engages with the binding site and whether binding to the protein induces differential strain in the bound ligand. It is unsurprising, therefore, that examples exist in which cyclizing a kinase ligand did not greatly affect selectivity.