For example, in the MerTK inhibitors discussed above, the acyclic precursor 12 inhibited four kinases, MerTK, AXL, Tyro3, and FLT3, with IC50 values of 0.74, 14, 17, and 0.8 nM, respectively (Figure ). Macrocyclizing this compound to generate 13 changed the affinities for binding to each of these kinases to a similar degree, such that selectivity for these four targets did not change by more than 2.5-fold in any case. Similarly, in the PI3K inhibitors reported by Spencer at al., described above, macrocyclization of 36 to give 37 gave only a moderate (2-to 6-fold) increase in selectivity for PI3Kd versus PI3Ka, b and g. In the case of the pyrazolopyrimidine inhibitors of casein kinase reported by Krämer et al. (e.g. 24, Figure ), too, macrocyclization had remarkably little effect on selectivity across a panel of 56 kinases, as assessed using differential scanning fluorimetry. Achieving the desired selectivity profile for a macrocyclic kinase inhibitor can require careful optimization of the geometry and/or flexibility of the macrocycle ring. For example, in the case of zotiraciclib, 5 (Figure ) modification of the linker length, geometry, and flexibility resulted in significant changes in selectivity toward CDK2, JAK2 and FLT3, and considerable optimization was required to find a compound that inhibited all three of these target kinases with high affinity. A striking example of the effect that linker length can have on kinase selectivity is provided by the work that led to the JAC2/FLT3 inhibitor pacritinib, reported by William et al.. In this case, decreasing the macrocycle ring size from 18 to 17 atoms (compounds 29 and 28) had little effect on potency towards FLT3, but caused a 60-fold increase in selectivity for this target against TYK2 and a >20-fold increased selectivity versus CDK2, while causing smaller but still substantial improvements in selectivity for JAK2 against these off-targets (Figure ). In other kinase-inhibitor systems, modification of the linker length and geometry did not substantially affect selectivity. For example, extensive exploration of linker geometry and flexibility had little effect on the selectivity of the MerTK inhibitor 14 with respect to AXL, Tyro3 and FLT3 (Figure ), or on the selectivity of Amgen's 4 for inhibiting Pim1 versus Pim2 (Figure ). It might be expected that the ability of substituents to modulate selectivity will be increased by their limited conformational flexibility when appended to a macrocyclic ring. However, for macrocyclic kinase inhibitors we were unable to find much data that would allow us to probe this concept directly because authors rarely report extensive parallel SAR data for the cyclic and corresponding acyclic compounds. One exception is the Cephalon ALK inhibitor, 46, described above (Figure ). In the acyclic precursor, 47, addition of a methoxy group to an aromatic substituent to give 44 caused only a 3-fold increase in affinity for ALK, and enhanced selectivity against IR to a similar extent. In contrast, the same substitution in the context of the corresponding macrocyclic compound (48 versus 45) resulted in a 30-fold increase in potency against ALK, once again with little effect on activity towards IR, thereby giving a 24-fold increase in ALK/IR selectivity. Interestingly, the sharp SAR seen for this substitution was not observed in all macrocyclic variants; in a macrocycle identical to 46 except for the introduction of a carbon-carbon double bond to rigidify the linker, the same -H to -OCH3 substitution had no significant effect on potency (49 versus 50). This example establishes that macrocyclization can cause the substituent SAR to differ considerably from that seen for the analogous acyclic molecule, and that SAR of macrocycles can be nonlinear in the sense that a structural change in one part of the molecule can substantially affect the SAR observed for changes at other locations. Importantly, this complexity of SAR does not prevent the medchem optimization of macrocyclic compounds. For example, Alvarez et al. reported an extensive exploration of linker plus substituent SAR that led to a ~200-fold increase in the selectivity of the macrocyclic for PI3Ka kinase inhibitors versus mTOR. The above examples illustrate that macrocyclization per se can, at times, significantly affect kinase selectivity, but the effects are not currently predictable and must therefore be established empirically. Within a given macrocyclic chemotype, optimizing the ring size, linker geometry, and linker flexibility can sometimes be used to tune selectivity, but in other cases does not significantly affect this property. Thus, the linker SAR landscape, with respect to selectivity, can be either rugged or smooth. Even in the latter case, however, where selectivity is relatively insensitive to linker structure, achieving a desired selectivity profile can still be facilitated by macrocyclization by exploiting the sharpened substituent SAR that results from the ensuing constraints on how the substituents are positioned in three-dimensional space. Operationally, when working to optimize the selectivity of a macrocyclic inhibitor, a functional group substitution that originally gives no improvement may be beneficial when applied to a later compound with a different linker structure, or vice versa. Thus, it can be useful to re-explore key aspects of substituent SAR after a change in ring size or linker geometry. Notwithstanding the somewhat unpredictable effects that can be observed, it is clear that macrocyclization frequently introduces additional levers for modulating selectivity, thereby adding to the toolbox of features available to optimize the pharmacological properties of a compound.