Bis-arylidenecycloalkanones structurally related to the nonselective isopeptidase inhibitor G5 were synthesized
and tested for cytotoxic activity against glioblastoma cells. Cytotoxicities correlate well with Hammett σ constants for substituted
arylidene groups, confirming the proposed inhibition mechanism. A new inhibitor (2c) based on the 4-hydroxycyclohexanone
scaffold, which favors apoptosis over necrosis, was selected for further development. 2c inhibited representative deubiquitinases
with micromolar IC50, and its proapoptotic activity was studied on several cancer cell lines. Inhibitor 2c was conjugated to PEG
via dicarbamate and diester linkers. While the dicarbamate was inactive, the diester (2cPE) behaves like a prodrug and is
converted into the active species 2c by secreted esterase activities. Finally, 2cPE was also tested in vivo on A549 lung carcinoma
xenografts generated in mice. Intravenous treatment with 2cPE led to a significant reduction in primary tumor growth, without
appreciable toxicity to mice.
