(Figure ). Cyclization of 15 with flexible linkers, to create macrocycles with ring sizes from 19-22 atoms, resulted in binding for TRKA and TRKC that ranged from similar to that of the acyclic precursor to ~20-fold weaker. Among these, the highest affinity was associated with the smallest macrocycle ring size (16), and this was only ~2-fold greater than the affinity seen for the acyclic 15. This finding implies that the larger ring sizes suffer from strain in the bound conformation arising from steric congestion in the flexible linker, and even the best linker tested failed to achieve a substantially beneficial trade-off of pre-organization over strain. Macrocyclization also had no discernible effect on affinity in a series of dual PI3 kinase-d/g inhibitors recently reported by AstraZeneca. Comparison of the activity of the acyclic compound 17 with macrocyclic congeners 18-21, containing a variety of flexible linkers, had no very great effect on potency towards either PI3Kg or PI3Kd in vitro or in cells (Figure ). Macrocyclization apparently failed to yield any other major benefits aside from affinity, as the compound eventually advanced into the clinic was the acyclic AZD8154 (22). Macrocyclization similarly had no measurable effect on the potency of the acyclic inhibitor GSK-2126458 towards PI3Ka, though substantially improved selectivity against off-target kinases was achieved. Macrocyclization of the acyclic pyrazolopyrimidine casein kinase-2 inhibitor 23 by Krämer et al. to give the macrocyclic 24 (Figure ) also resulted in a slight decrease in binding affinity, from 12 to 82 nM, as measured by isothermal titration calorimetry. Several of the above examples illustrate that optimizing linker length can have a significant impact on the binding affinity of macrocyclic kinase inhibitors. The MerTK inhibitor in Figure was one example where increasing the ring size by one atom, among other changes, improved the affinity of the initial macrocycle 13 from 65 nM down to low nM levels (e.g. 14), with potency being relatively insensitive to further expansion of the ring. Similarly, in the discovery of the CDK/JAK2/FLT3 inhibitor zotiraciclib, increasing the ring size of compound 25 from 17 to 18 atoms, to give compound 26, resulted in a 7-to 26-fold increase in in vitro potency against its three main kinase targets (Figure ). In contrast, in the work that led to the JAK2/FLT3