C13 Side-Chain Modification of Nannocystin A Analogues: Balancing Cytotoxicity, Cellular Distribution, and eEF1A Binding

Nannocystin A is a macrocyclic natural product with potent cytotoxic activity and a reported mechanism involving eukaryotic elongation factor 1A (eEF1A). Here, we investigated the structure–activity relationships of the nannocystin-derived macrocyclic framework by integrating chemical synthesis, cytotoxicity assays, molecular docking, and in silico prediction of cellular distribution. Evaluation of analogues 1–6, bearing modifications at distinct regions of the macrocycle, revealed differential effects on cellular activity, predicted cellular distribution, and compatibility with the proposed eEF1A-binding site, identifying the C13 side chain as a tractable position for further modification. A focused series of C13-modified analogues 7–12 was therefore synthesized and evaluated. Among these, the homoleucine analogue 10 displayed the most favorable activity profile, retaining sub-nanomolar cytotoxicity toward SMMC7721 and Hep3B cells (IC50 = 0.52 and 0.53 nM, respectively). Computational analysis suggested that the favorable profile of 10 may reflect an appropriate balance between predicted cellular access and compatibility with the proposed eEF1A-binding site, whereas bulkier or more hydrophobic C13 substituents were associated with increased predicted membrane retention and/or altered binding orientations. These findings identify C13 as a tunable region of the nannocystin-derived macrocyclic framework and provide a structure–activity framework for further optimization of eEF1A-targeting nannocystin analogues.