Design, Synthesis, Development, and Biological Evaluation of Third-Generation Tubulin Inhibitors for Treatment of Cancer

Abstract

Tubulysins, a family of natural products originally isolated from myxobacteria culture, inhibit tubulin polymerization, leading to apoptosis, thereby making them potential candidates for anticancer drug development. In this article, we report an advancement in the synthesis of third-generation tubulin inhibitors developed in our laboratory by leveraging the first application of solid-phase peptide synthesis (SPPS) to circumvent the limitations of conventional multistep solution-phase synthesis. The tubulin inhibitors have been synthesized in high yields without incorporating tubuvaline and tubuphenylalanine fragments present in the natural tubulysins, which are known to be essential for their anticancer activity. The most active inhibitor contains 4 fragments, i.e., N-methyl-d-pipecolic acid (N-Mep), l-isoleucine (l-Ile), valine-thiazole, and asparagine (Asn), arranged sequentially from the N-terminus to the C-terminus. The protocols enumerate an efficient synthesis of valine thiazole and N-methyl-d-pipecolic acid fragments, which are tethered using SPPS with a single purification step, resulting in high yields and enhancing their potential for scalable synthesis and therapeutic applications. The newly synthesized tubulin inhibitors exhibit anticancer activities with IC50 values in the low nanomolar range. The incorporation of a simplified thiazole ring structure preserves the biological activity and chiral integrity of the molecules. The structure-activity relationship studies highlight that small changes at the N-terminal fragment with various heterocyclic groups significantly reduce potency from nM to µM. In contrast, aliphatic C-terminal changes have little effect, though aromatic C-terminal modifications significantly impact the activity. This article describes an efficient method to synthesize simplified tubulin inhibitors, supporting further development of potent anticancer drugs. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Fmoc-protected valine thiazole and N-methyl-d-pipecolic acid fragments Basic Protocol 2: Synthesis and purification of tubulin inhibitors (11a to 11l) using SPPS Basic Protocol 3: In vitro cytotoxicity assay of tubulin inhibitors (11a to 11l) against cancer cells derived from prostate, cervical, breast, lung, and skin using MTT assay. This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine