Naturally occurring antimitotic pentapeptide dolastatin 10 (1, Figure 1) [1] and its synthetic analogue auristatin E (AE, 2) [2] possess subnanomolar cytotoxicity against many human cancer cell lines and are over a hundred-to a thousand-times more potent than many pharmaceuticals, including taxol and doxorubicin, respectively, which are currently used in clinic. These pentapeptides have mechanism of actions similar to that of taxol, but through processes that appear opposing to each other; that is, dolastatins and auristatins inhibit tubulin polymerization, whereas taxol promotes it and stabilizes the microtubules. [3] In the end, both dolastatins or auristatins and taxol inhibit cell proliferation. Additionally, auristatins function as the vascular disrupting agents and damage the established tumor vessels, thereby likely causing a more pronounced effect than taxol in vivo. [4] Nonetheless, the therapeutic efficacies of the auristatins and dolastatins are poor as they also cause nonselective toxicity to normal cells. [5] Seattle Genetics has pioneered the selective delivery of auristatins to tumor cells by further modifying them to suitable analogues, including di-desmethylauristatin E (DDAE, 3) and monomethylauristatin E (MMAE, 4), and conjugating them to tumor-targeting antibodies (Ab) in the Fc region through a linker. [6] Upon uptake of the conjugates in tumor cells, free drugs are released by lysosomal protease-catalyzed hydrolysis of the linker. Many such auristatin-Ab conjugates have shown very high efficacy in tumor models, and several conjugates are in clinical trials for the treatment of various cancers. [7] We are interested in this class of molecules as they could also feed our ongoing program in the development of prodrugs [8] and their conjugates to the programmed Abs (progAbs). [9] First, we prepared and evaluated a series of AE (2) analogues and compared their cytotoxicity to the previously described analogues, DDAE and MMAE. An examination of the results revealed that new auristatin analogues were highly potent, but they were not as cytotoxic as compounds 1-4. Therefore, we focused on MMAE and DDAE, and prepared and evaluated their prodrugs, which undergo tumor-associated protease-catalyzed activation. Herein, we report the results of our preliminary study comparing their in vitro and in vivo efficacy and selectivity to the parent cytotoxins MMAE and DDAE.
Tumor-associated proteases have been implicated in tumor angiogenesis, growth and metastasis formation, and found as both the positive and negative regulators of the disease. [10] Often, these proteases are overexpressed both intracellularly and extracellularly in cancer cells. Of particular interest to us in this study is legumain, [11] which is a cysteine protease and the Figure 1. Structure of dolastatin 10, auristatins and auristatin prodrugs. Cleavage sites are indicated and labeled as follows: a) legumain-catalyzed proteolysis; b) autocleavage through 1,6-elimination; c) autocleavage through CO 2 loss.