The PI3Kd inhibitors described by Spencer et al. provide an interesting example of the complex interplay that can exist between the effects on affinity of macrocycle ring size versus other aspects of ring structure. These authors described a series of N-pyridinyl 3,4dihydrobenzooxazines, exemplified by 36, that were macrocyclized by connecting the terminal sulfonamide and amine substituents via polymethylene linkers of various lengths (e.g. 37) (Figure ). In addition to varying the ring size, the investigators also tested the effects of inverting the sulfonamide and of substituting the ring amine with an amide. In the most favorable case, closing the ring with an ethylene linker, for example going from 36 to 37, resulted in a 30-fold increase in potency against PI3Kd in an in vitro enzymatic assay. The affinity increase due to cyclization was similar whether the macrocycle contained an amine or an amide, although both amines (i.e. 36/37) showed ~10-fold greater potency than the corresponding lactams (38/39). However, in a variant of the chemotype in which the sulfonamide was reversed (40), macrocyclization via an ethylene (41) resulted in an 8-fold decrease in binding affinity compared to the acyclic counterpart, illustrating that the effect on affinity of a ring closure can be highly sensitive to the structure elsewhere in the macrocycle ring. In this reversed sulfonamide series it was necessary to increase the linker length to four methylenes to achieve any increase in affinity compared to the acyclic precursor 41; in contrast, for the original series the shortest linker, ethylene, gave the best binding. Interestingly, in this chemotype overall, the macrocycles containing a three-carbon linker tended to be less potent than those in which the ring was closed via either a 2-carbon ethylene or a 4carbon polymethylene chain. The above examples illustrate several important points about the effect of macrocyclization on binding affinity. It is clear that, while substantial affinity gains can be achieved through macrocyclization, a higher affinity is not guaranteed even with acyclic precursor ligands that appear to bind in a geometry that readily lends itself to macrocycle ring closure. In practice, it can be necessary to undertake substantial work to vary the length, geometry, and rigidity of the linker that closes the macrocycle ring to maximize conformational preorganization while minimizing strain. Although it is unwise to attempt to draw general conclusions from so few examples, we note that in none of the ligand-target combinations discussed above can macrocyclization per se be shown to increase affinity by more than ~500-fold (3.7 kcal/mol increase in free energy of binding), even after extensive optimization of the ring geometry; an increase of 10-to 50-fold (1.4-2.4 kcal/mol) is more representative of what is typically seen (Table ). This observation suggests that it would be optimistic to expect macrocyclization alone to improve, for example, a micromolar binder to low nanomolar levels of affinity. In all likelihood, none of the compounds described above reflects a perfect degree of conformational preorganization, because achieving this may not be possible within the constraints of the bond lengths and angles available to organic compounds, and perhaps also because doing so was not the primary goal of the investigators. Currently, there is no rigorous and validated theoretical value for the maximum affinity increase that might be achieved solely from conformational preorganization, though estimates in the classic literature have proposed values of the order of ~0.4 kcal/mol per bond rotation that is frozen, at 37 °C, corresponding to roughly a 2-fold increase in binding affinity for each rotatable bond that becomes fully frozen upon macrocyclization. However, this estimate assumes that only a single or a very narrow range of dihedral angles for any given bond can be accommodated in the bound ligand, and that in the macrocyclic each bond is constrained at precisely this preferred dihedral angle. In practice, the entropic benefit that is observed will be reduced by any vibration about the preferred dihedral angle that can occur in the bound ligand. How the experimentally observed affinity increases upon cyclization, in the literature cases described above, compares to this theoretical expectation is shown in Table . These results suggest that an affinity enhancement of up to 3-fold per rotatable bond that becomes constrained due to macrocyclization can be achieved in practice, and that this enhancement can be propagated for as many as 5 or 6 bonds.