Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.

An aldol-based ‘build/couple/pair’ (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti- aldol reactions were performed to produce four stereoisomers of a Boc protected γ-amino acid. In addition both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes, namely: nucleophilic aromatic substitution (SNAr), Huisgen [3+2] cycloaddition and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on solid-phase to yield a 14,400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships (SSAR) was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.