Ubiquitin-proteasome system in eukaryotic cells is the main system for degradation of intracellular proteins and consequently for maintaining homeostasis and cell activity. In addition to the constitutive proteasome 26S, which is present in all cell types, the immunoproteasome is found mainly in cells of immune origin. Expression of immunoproteasome is induced when inflammation and other stressful situations occur. It is used as a therapeutic target for treatment of inflammatory, cancerous, autoimmune and neurodegenerative diseases. Most inhibitors used for treatment, or undergoing a clinical trial, are non-selective, which is the main reason they cause many side effect. Accordingly, development is oriented in the direction of finding inhibitors selective for the β5i subunit, which would reduce the occurrence of side effect. This was also our main goal of the work for the master's thesis.
Benzyl carbamate protection was attached to the backbone of the substituted piperidine so that the t-butyloxycarbonyl protecting group could be removed from the secondary amine group in the second step. The amide was then formed on the free amine group using coupling reagents. The other compounds were then synthesized differently, because the losses in the removal of the benzyl carbamate protecting group by catalytic hydrogenation were excessive. Instead of attaching acryloyl chloride to the piperidine amine group in the last step, we did so first. The t-butyloxycarbonyl protection was then removed. The synthesis of amide or amine with the help of coupling reagents followed. A carboxylic acid was attached to the free amine group, which was activated by coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We also added Hydroxybenzotriazole (HOBT) to reduce the likelihood of by-product formation and racemization. Thus we obtained amide derivatives. A reductive amination reaction was used for the synthesis of amine derivatives. The free amine group was converted to the imine and reduced with sodium acetoxyborohydrate (NaBH(OAc)3). The reaction took place in an inert argon atmosphere. Secondary amine derivatives were stained with ninhydrin on TLC (amides were not).
Nine final compounds were successfully synthesized and biochemically evaluated. We measured residual activity of enzyme in the presence of inhibitor and calculated IC50. Two of them are micromolar immunoproteasome inhibitors. Unfortunately they are non-selective – in addition to the β5i subunit, they probably reversibly inhibit β5, β1i and β1 subunits.
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