Fine-Tuning Side Chain Substitutions: Impacts on the Lipophilicity–Solubility–Permeability Interplay in Macrocyclic Peptides

Macrocyclic drugs are promising for targeting undruggable proteins, including those in cancer. Our prior work identified BE-43547A₂ (BE) as a selective inhibitor of pancreatic cancer stem cells in PANC-1 cultures, but its high lipophilicity limits clinical application. To address this, we designed derivatives retaining BE's backbone while modifying tail groups to improve its properties. A concise total synthesis enabled a versatile late-stage intermediate (compound <b>17</b>), serving as a platform for efficient diversification of BE analogs via modular click chemistry. This approach introduced a central triazole ring connected by flexible alkyl spacers. Key properties, including lipophilicity, solubility, and Caco-2 permeability, were experimentally determined. These derivatives exhibited reduced lipophilicity and improved solubility but unexpectedly lost cellular activity. Direct target engagement studies using MicroScale Thermophoresis (MST) revealed compound-dependent deactivation mechanisms: certain derivatives retained binding to eEF1A1 with only modestly reduced affinity (e.g., compound <b>29</b>), while others showed no detectable binding (e.g., compound <b>31</b>). Microsecond-scale molecular dynamics simulations and free-energy calculations showed that, for derivatives retaining target affinity, tail modifications disrupted the delicate balance of drug-membrane and drug-solvent interactions, resulting in substantially higher transmembrane free-energy penalties (>5 kcal/mol) compared to active compounds (<2 kcal/mol). These insights emphasize the need to simultaneously preserve both target engagement and optimal permeability when modifying side chains in cell-permeable macrocyclic peptides, positioning compound <b>17</b> as a robust scaffold for future lead optimization. This work furnishes a blueprint for balancing drug-like properties with therapeutic potency in macrocyclic therapeutics.