Design, Synthesis, and Cytotoxic Evaluation of New Structurally Simplified and Highly Potent Third-Generation Tubulysin Derivatives

Tubulysins belong to a class of natural products originally isolated from myxobacteria culture and are known to induce cell apoptosis through inhibition of microtubule assembly. Herein, we report the computationally designed, structurally simplified, and first solid-phase peptide synthesis of novel third-generation tubulin inhibitors in high yields. These inhibitors are devoid of tubuvaline and tubuphenylalanine fragments previously considered essential for tubulin inhibition activity. The most potent inhibitor contains four fragments arranged from the N terminal to the C terminal as N-methyl pipecolic acid, isoleucine, valine-thiazole, and asparagine. The hydrophilic tubulin inhibitors demonstrated significant anticancer activity, with IC₅₀ values in the low nanomolar range (IC₅₀ = 13–53 nM) within a 48 hours incubation period across prostate, lung, breast, skin, and cervical cancer cell lines. The synthetic strategy incorporates a simplified valine-thiazole ring structure, retaining both biological activity and chiral integrity of the molecules. The method enables the synthesis of potent tubulin inhibitors by avoiding multistep synthetic and purification procedures, supporting the inhibitor's applicability for large-scale synthesis and potential therapeutic development. The structural modifications at the N-terminal result in the loss of activity from nM to µM range, whereas the C-terminal modification had minimal impact on the potency.