Lorlatinib (51), developed by Pfizer, represents an exquisite example of how ADME was improved through macrocyclization (Figure ). A previously approved (acyclic) oral drug, Crizotinib (52), inhibits ALK plus the hepatocyte growth factor receptor c-Met and the protooncogenic receptor tyrosine kinase ROS1. Crizotinib has robust efficacy in ALK-positive nonsmall cell lung carcinoma. However, some patients develop resistance to the drug, attributed to development of an L1196M mutation in ALK, leading to brain metastases that cannot be treated with crizotinib. To treat these metastases, there was a need for a pan-ALK inhibitor that could inhibit ALK L1196M and that could cross the blood brain barrier (BBB). X-ray crystal structures of crizotinib complexed with ALK showed that the compound binds in a U-shaped conformation. This observation suggested that a macrocyclic chemotype might be derived that would allow exploration of different property space, potentially enabling the selectivity profile and ADME properties they were seeking and particularly BBB penetrance. As was seen in some of the other cases discussed above, macrocyclization of 52 resulted in a substantial increase in target binding affinity. This is illustrated by comparing the activity of 53, one of the macrocyclic compounds they studied en route to lorlatinib, with its close acyclic analogue 54. The macrocyclic compound shows a >10-fold increased affinity for wild-type ALK and ~100-fold improved affinity towards the ALK L1196M resistance mutant (Figure ), with a >100-fold improved potency against both forms in cell-based assays. The main focus of the optimization efforts, however, was to limit the overall size and lipophilicity of the compounds, based in part on in-house data showing that molecular weight and logD positively correlate with the BA/AB efflux ratio associated with the action of efflux receptors such as the multi-drug resistance proteins (MRP), and thus tend to reduce BBB permeability. To monitor lipophilicity during lead optimization, the investigators devised a numerical index, LipE, to quantify binding effectiveness per unit of lipophilicity: LipE = pKI (or pIC50) -logD. Their analysis showed that, among the inhibitor chemotypes they studied, only the macrocyclic compounds had LipE values >6, thereby outperforming the acyclic compounds with respect to the combination of potency and lipophilicity. They additionally aimed for a logD value in the range 2 to 3, and found that many of the macrocycles also satisfied this criterion. Specifically, compared with the acyclic 54, the macrocycle 53 showed a LipE value that was 2.3 units higher, while maintaining the logD value in the target range (Figure ). As hoped, these changes resulted in a substantial reduction in the BA/AB MDR efflux ratio, from 17 for 54 to 4 for the macrocycle 53. Compound 53 also showed increased metabolic stability, compared to 54, in a human liver microsome assay, with the resulting calculated total intrinsic clearance reduced from the relatively high value of 28 mL/min/kg to below the lower limit of detection of the assay. The final macrocyclic drug, lorlatinib (51), differs from 53 only in the substitution of the pyrazole ring. Compared to the acyclic drug crozitinib that was the starting point for the project, lorlatinib has a 60-fold increased potency against ALK, a 40-fold better potency against the resistance mutant ALK L1196M , and a 30-fold improved BA/AB efflux ratio of 1.5, predictive of good BBB penetration. It has been shown that some macrocycles can have higher membrane permeability and oral absorption than acyclic compounds of comparable size and composition, with the classic example of this effect being cyclosporine A. This advantage has been attributed to the ability of certain macrocycles to undergo a so-called "chameleonic" conformational change.