Hitting a Moving Target: How Does an N-Methyl Group Impact Biological Activity?

Macrocycles have several advantages over small‐molecule drugs when it comes to addressing specific protein–protein interactions as therapeutic targets. Herein we report the synthesis of seven new cyclic peptide molecules and their biological activity. These macrocycles were designed to understand how moving an N‐methyl moiety around the peptide backbone impacts biological activity. Because the lead non‐methylated structure inhibits the oncogenic regulator heat‐shock protein 90 (Hsp90), two of the most potent analogues were evaluated for their Hsp90 inhibitory activity. We show that incorporating an N‐methyl moiety controls the conformation of the macrocycle, which dramatically impacts cytotoxicity and binding affinity for Hsp90. Thus, the placement of an N‐methylated amino acid within a macrocycle generates an unpredictable change to the compound's conformation and hence biological activity. Seven cyclic peptides were synthesized and tested for biological activity. These new macrocycles were designed to understand how moving an N‐methyl group around the peptide backbone impacts activity. The lead non‐methylated structure was found to inhibit heat‐shock protein 90 (Hsp90), which prompted us to test two of the most potent analogues for their Hsp90‐inhibitory activity. Our data show that the N‐methyl group controls the conformation of the macrocycle, which has a dramatic impact on cytotoxicity and binding affinity.