Macrocycle was synthesized as described
Macrocycle was synthesized as described in , . Under SAr conditions, 2-chloronicotinonitrile was heated with -butyl 5-aminopentanoate in refluxing ethanol (). The resulting aminopyridine was treated with mesyl chloride and the nitrile was subsequently reduced to afford amine . Chloroaminopyrimidine intermediate was heated with under basic conditions to generate diaminopyrimidine ()., Following -Boc deprotection and -butyl ester hydrolysis, crude amino c-Myc Peptide was subjected to a HATU-mediated macrolactamization under dilute conditions to provide in 56% yield over two steps. In contrast to , exhibited moderate selectivity for Pyk2 over FAK (Pyk2 IC=3.1nM, FAK IC=17nM, ) demonstrating the validity of the macrocyclization strategy. The excellent cellular permeability of was also notable (Caco-2: A to B=30×10cm/s and B to A=32×10cm/s) and displayed a moderate improvement over acyclic inhibitor in the absorptive direction (Caco-2: A to B=11×10cm/s and B to A=29×10cm/s). Interestingly, a small molecule crystal structure of revealed the presence of atropisomers and in the solid state (a). For , the linker alkyl chain appeared disordered but was successfully modeled as 80/20 occupancy. Co-crystallization of macrocycle with Pyk2 confirmed preservation of the binding mode, inhibitor conformation, protein-inhibitor interactions and revealed the kinase adopting a DFG-in conformation (b). An overlay of the Pyk2-bound conformation of with the solid state conformation of indicated that the latter was the relevant atropisomer for inhibition (c). Attempts to separate the atropisomers in chiral HPLC and SFC screens were unsuccessful, suggesting that rapid interconversion was occurring on the HPLC timescale., On the other hand, chiral HPLC analysis of the -phenylmethanesulfonamide macrocycle (Pyk2 IC=0.9nM) suggested a 1:1 ratio of atropisomers, however baseline resolution of the split peak could not be achieved, presumably due to on-column interconversion. To increase the energy barrier for atropisomer interconversion, we focused our attention on -phenylsulfonamides, prepared the bulkier ethyl sulfonamide , and also explored the effect of alkylation of the secondary benzylic amine in the form of the tertiary analogue . 1-Iodobenzylamine hydrochloride proved useful as a starting material for both compounds, with copper mediated -arylation of the sulfonamide and selective alkylation of the sulfonamide over the carbamate under basic conditions as key steps (). Chiral HPLC analysis of and were qualitatively similar and showed two peaks separated by a plateau, which suggested the atropisomers may be separable on preparative scale., Unfortunately, however, peak resolution for could not be achieved due to on-column interconversion. Nonetheless, the rapidly interconverting class 1 atropisomers constituting both and showed >100-fold improvement in Pyk2 IC (1.5nM and 1.9nM respectively) when compared to their corresponding acyclic diaminopyrimidines and , together with 5-fold selectivity over the FAK enzyme (). The effect of various macrocycle linkers on Pyk2 and FAK biochemical potencies was also investigated (). Shortening the 5-carbon linker of to four carbons yielded as an 18-membered macrocyclic dual Pyk2/FAK inhibitor with a 10-fold loss in Pyk2 potency (IC=30nM). Replacing the tertiary methyl sulfonamide moiety in with a secondary sulfonamide within the linker afforded 19-membered macrocycle , an equipotent Pyk2 inhibitor (IC=3.5nM) with a slight erosion in FAK selectivity compared to . Rigidifying macrocycle with a phenyl-containing linker did not improve selectivity. -Phenylsulfonamide macrocycles – suffered from poor solubility (<1.6μM at pH 7), and short half-lives were observed for – in mouse, dog and/or human liver microsomes (<60min). For the next generation of macrocyclic Pyk2 inhibitors, we employed an intramolecular Heck reaction for the ring closure step. Macrocycles with morpholine as R and/or 3-pyridyl regioisomers were prepared as a tactic to enhance solubility (). Intermediate , used as a precursor for the synthesis of macrocycle , was prepared via a copper-catalyzed -arylation of methanesulfonamide. Heck-mediated macrocyclizations of – under dilute conditions provided inhibitors – bearing 3-carbon linkers, thus decreasing the number of sites for potential metabolism (). In some cases, the alkene was reduced with Pd/C under an atmosphere of hydrogen to yield macrocycles and . Assessment of the activities of the acyclic precursors – relative to the macrocycles – provided further evidence of the potency switch toward Pyk2 and selectivity improvement in the latter. Strikingly, the acyclic tertiary amine was markedly less active against both enzymes but macrocyclization to provided ∼2500-fold and 500-fold increases in potency against Pyk2 and FAK respectively. Saturation of the macrocyclic linker in led to a modest improvement in Pyk2 potency, albeit with a relative loss in selectivity. Incremental improvements in kinetic solubility at physiological pH were noticed between , and (0.3, 1 and 3μM respectively). Macrocycle , bearing an unsaturated 3-carbon linker, exemplified the most metabolically stable analogue tested in the human microsomal stability assay (=263min).