Passive Membrane Permeability of Macrocycles Can Be Controlled by Exocyclic Amide Bonds Jennifer L. Hickey, †,‡ Serge Zaretsky, † Megan A. St. Denis, †,‡ Sai Kumar Chakka, †,‡ M. Monzur Morshed, †,‡ Conor C. G. Scully, † Andrew L. Roughton, ‡ and Andrei K. Yudin* ,† † Davenport Research Laboratories, Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada ‡ Encycle Therapeutics Inc., 101 College Street, Suite 314, Toronto, Ontario M5G 1L7, Canada * S Supporting Information ABSTRACT: We have developed a strategy for synthesizing passively permeable peptidomimetic macrocycles. The cyclization chemistry centers on using aziridine aldehydes in a multicomponent reaction with peptides and isocyanides. The linker region in the resulting product contains an exocyclic amide positioned α to the peptide backbone, an arrangement that is not found among natural amino acids. This amide provides structural rigidity within the cyclic peptidomimetic and promotes the creation of a stabilizing intra- molecular hydrogen bonding network. This exocyclic control element also contributes to the increased membrane permeability exhibited by multi- component-derived macrocycles with respect to their homodetic counterparts. The exocyclic control element is employed along with a strategic placement of N-methyl and D-amino acids to produce passively permeable peptides, which contain multiple polar residues. This strategy should be applicable in the pursuit of synthesizing therapeutically relevant macrocycles. ■ INTRODUCTION The discovery of new biologically active molecules is complicated by the need to consider not only the binding and specificity toward the intended biomolecular target but also the properties required for pharmacological activity in vivo. Peptide ligands often exhibit high binding affinity toward their target receptors; however, they have insufficient stability, resulting in limited therapeutic application. 1,2 Linear peptides typically undergo enzymatic degradation prior to executing their pharmaceutical objective, which has made cyclization an attractive modification. 3 It has been shown that peptide cyclization can have a beneficial impact on essential druglike properties such as membrane permeability, 4-6 metabolic stability, 7 and overall pharmacokinetics. 8,9 Evidence suggests that limited flexibility within the backbone allows the formation of intramolecular hydrogen bonds, contributing to increased passive permeability and improving overall oral bioavailabil- ity. 8,10-12 Moreover, the structural preorganization may also reduce the entropy cost of receptor binding by eliminating unproductive conformations, subsequently increasing binding affinity compared to those of linear analogues. 8, 13-16 Consequently, macrocyclic peptides have been the subject of sustained interest as therapeutically relevant molecules. Macrocycles typically have high molecular masses (>500 Da), moving them outside of what has traditionally been considered orally bioavailable drug space. The conventional metrics for “druglikeness” are of limited value in addressing macrocycles as the guidelines have been constructed exclusively through the evaluation of small molecule drug candidates. 17-20 Therefore, understanding the druglike properties required for oral bioavailability when it comes to beyond the “Rule of Five” molecules continues to be a work in progress. Modified sets of desired property ranges for the design of oral macrocyclic drugs have recently been proposed. 8,21-23 Although the data sets are small, it is apparent that macrocycles display consistent properties that are clearly distinct from those observed for conventional drugs. Aside from a few rare instances, cell permeable macrocycles do not contain more than one polar side chain. 21,24,25 In fact, a large proportion of passively permeable peptide macrocycles consist solely of lipophilic amino acids, such as leucine or alanine, limiting their therapeutic relevance. 10,11,26-29 These compounds are often poorly water-soluble and require complex formulation strategies prior to administration. As approximately 50% of drug candidates fail because of poor druglike properties and/or pharmacokinetics, mostly attributable to poor water solubility, 30-34 the significant challenge for peptide macrocycle drug development is to maintain membrane permeability while increasing water solubility. We recently reported the discovery of a structural control element, an unnatural exocyclic amide motif, 35 that is integrated into peptide macrocycles via aziridine aldehyde-mediated macrocyclization (Scheme 1). 36-38 This exocyclic control Received: February 11, 2016 Article pubs.acs.org/jmc © XXXX American Chemical Society A DOI: 10.1021/acs.jmedchem.6b00222 J. Med. Chem. XXXX, XXX, XXX-XXX