Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline

Synthetic tubulysins 24a-m, having non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multi-step synthesis. Key step was the formation of differently N-substituted Ile-Tuv fragments 10 via aza-Michael reaction of azido-Ile derivatives 8 with the α,β-unsaturated oxo-thiazole 5. A SAR study using a panel of human tumor cell lines showed strong anti-proliferative activity for all compounds 24a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of Tubulysin A, vinorelbine, and paclitaxel. Furthermore, 24a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumor cell lines with acquired resistance to doxorubicin. Compounds 24e and 24g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24e and 24g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, whose growth was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.