Design, synthesis and biological evaluation of novel inhibitors of human and protozoan proteases

Proteases play many important roles in several key pathways, thus representing potential drug targets for the treatment of various diseases. Many protease inhibitors are already commercially available and many others are currently in the early stage of the clinical trials for the treatment of several diseases. The general approach to develop a specific inhibitor is to create a special motif to block the active site; generally a small molecule. An irreversible inhibition is normally desirable in the case of parasitic proteases, on the contrary in the case of human proteases it is more convenient to develop a reversible inhibitor to avoid undesidered or off-targets effects. The main research goal of my PhD work was focused on the design, synthesis, and biological evaluation of a series of amide derivatives acting as immunoproteasome inhibitors. The compounds were designed to act as noncovalent inhibitors, thus being a promising therapeutic strategy because of the lack of all the drawbacks associated to the irreversible inhibition. In this scenario, the research group with whom I worked during my PhD has been actively involved in the development of novel 20S proteasome inhibitors; in particular, a series of amides were identified, some of which inhibited the ChT-L activity of 20S proteasome with Ki values in the submicromolar range. Docking studies allowed us to show the noncovalent binding mode of the most active inhibitors by simulations into the yeast 20S proteasome crystal structure. Noteworthy, the designed compounds lack of the electrophilic warhead and act as non-covalent proteasome inhibitors that, with respect to covalent inhibitors, might be a promising alternative to use in therapy, because of the lack of all drawbacks and side effects related to irreversible inhibition. In order to design novel immunoproteasome inhibitors, we decided to firstly screen several amide derivatives, already synthesized in our laboratories, against the three immuno-subunits, to identify active compounds. Among the tested compounds, N-benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide showed a relevant result, selectively inhibiting the β1i subunit with a Ki of 2.23 μM. Therefore, this compound was selected as hit compound to design a panel of derivatives characterized by structural variations at the N-substituent and at the methylene linker between the pyridone scaffold and the amide function. During the last period of my PhD, I focused my research activity on the optimization of a high reactive vinyl ketone which was previously identified as a potent rhodesain inhibitor of T. b. rhodesiense, with a k2nd value of 67000·x 103 M-1 min-1 coupled with a potent binding affinity (Ki = 38 pM). Based on the structure of the lead compound, we designed a new series of Michael acceptors. Herein, the highly reactive vinyl ketone warhead and the HPhe at the P1 site were kept unchanged. The HPhe moiety at the P1 position assures resistance towards endopeptidases, leading to a greater stability in vivo with respect to the corresponding analogs bearing a natural amino acid side chain at P1. We then decided to replace the Cbz group with some benzo-fused rings, one of which is the 2,3-dihydrobenzo[b][1,4]dioxine moiety, present in potent rhodesain inhibitors. A panel of aromatic moieties, variously decorated with halogen atoms, were also introduced at the P3 site, in agreement to the structure of peptidic inhibitors bearing at the P3 site 3,5-difluoro- or 4-CF3-phenyl moieties or taking into consideration non peptide rhodesain inhibitors based on a triazine nucleus variously decorated with halogen–substituted aromatic nuclei. This approach could also allow us to investigate the size of S3 pocket and to concurrently allow for the formation of halogen bonds and/or hydrophobic interactions. We also investigated the relevance of the L-Phe residue at the P2 site, by synthesizing the L-cyclohexylalanine, 4-F-L-phenylalanine and 4-methyl-L-phenylalanine derivatives.