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

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,ß-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g 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 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which 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.

Replacement of isobutyl by trifluoromethyl in pepstatin A selectively affects inhibition of aspartic proteinases.

Two bis-trifluoromethyl pepstatin A analogues, carboxylic acid 1 and its methyl ester 2, have been synthesised in order to probe the properties and size of the trifluoromethyl (Tfm) group and compare it to the "bigger" isobutyl that is present in pepstatin A. The results demonstrate that Tfm can effectively replace the isobutyl chain as far as inhibitory activity against plasmepsin II (PM II), an aspartic proteinase from Plasmodium falciparum, is concerned. On the other hand, replacement of isobutyl by Tfm selectively affected activity against other aspartic proteinases tested. Two lines of evidence led to these conclusions. Firstly, compounds 1 and 2 retained single-digit nanomolar inhibitory activity against PM II, but were markedly less active against PM IV, cathepsin D and cathepsin E. Secondly, the X-ray crystal structures of the three complexes of PM II with 1, 2 and pepstatin A were obtained at 2.8, 2.4 and 1.7 A resolution, respectively. High overall similarity among the three complexes indicated that the central Tfm was well accommodated in the lipophilic S1 pocket of PM II, where it was involved in tight hydrophobic contacts. The interaction of PM II with Phe111 appeared to be crucial. Comparison of the crystal structures presented here, with X-ray structures or structural models of PM IV and cathepsin D, allowed an interpretation of the inhibition profiles of pepstatin A and its Tfm variants against these three enzymes. Interactions of the P1 side chain with amino acids that point into the S1 pocket appear to be critical for inhibitory activity. In summary, Tfm can be used to replace an isobutyl group and can affect the selectivity profile of a compound. These findings have implications for the design of novel bioactive molecules and synthetic mimics of natural compounds.