In contrast with the above examples, in other reported cases macrocyclization did not lead to improved affinity. For instance, in the work of Wang et al. on inhibitors of Mer tyrosine kinase and FLT3, the authors started with the acyclic lead compound UNC2025 (12) which inhibits MerTK and FLT3 with IC50 values of 0.74 and 0.8 nM, respectively (Figure ). The X-ray crystal structure of a related compound bound to MerTK indicated that the hydroxycyclohexyl and N-butyl groups of 12 lie close together when bound to the protein target, suggesting that a macrocyclic compound might be devised that would lock the compound in the bound conformation. As proof of concept they prepared a cyclic variant, 13, in which these two substituents were modified to connect via an amide bond to make a 14-membered ring (Figure ). In fact, cyclization decreased potency for MerTK by 85-fold to 65 nM relative to the acyclic precursor 12, corresponding to a loss of binding energy of ~2.5 kcal/mol. Based on a crystal structure of 14 bound to MerTK, the authors concluded that the constrained geometry of the ring holds the amide NH group in 13 in a position where it is unable to form the same hydrogen bonds with the protein that were inferred to exist for the hydroxyl group of 12. It is also possible that adopting the bound conformation involves some conformational strain within the relatively small ring present in 13, which could contribute to the weakened binding compared to the acyclic precursor. The involvement of strain is supported by the observation that making the ring more flexible, by eliminating the amide and/or increasing the ring size by one or more atoms, for example as in compound 14, increased potency to low nanomolar levels, provided that a hydrogen bond donor group (hydroxyl or amine) was appended to the ring. However, it is also possible that this binding improvement results, in whole or in part, from better positioning of the hydrogen bond donor.