The ubiquintin-proteasome system is central to protein homeostasis in cells, and is responsible for the degradation of misfolded and damaged proteins. Research has shown that in autoimmune diseases, cancer, and neurodegenerative disorders, immunoproteasome expression is increased under the influence of inflammatory cytokines. As a result, the scope of research in the field of selective immunoproteasome inhibitors, which have fewer adverse side effects compared to non-selective inhibitors, has increased in the last decade.
In the master thesis, we focused on the synthesis of 2-cyanobenzimidazoles and 2-cyanobenzoxazoles, which are heterobicycles with an electrophilic warhead – nitrile. The nitrile group can form a covalent bond with Cys48 of immunoproteasome subunit 5i, which allows selective inhibition of the immunoproteasome, since the constitutive subunit 5c lacks a cysteine amino acid at this sub-site. Substituted ortho-phenylenediamines or 2-aminophenols were used for the first step – the cyclization reaction of dinucleophile and Appel's salt. The synthesized 2-cyanobenzimidazoles were then methylated with methyl iodide to give two positional isomers of 2-cyano-N-methylbenzimidazole, which were separated and purified by column chromatography. The structures of the positional isomers were confirmed by 2-D nuclear magnetic resonance experiments. Methyl carboxylates of 2-cyanobenzoxazoles were converted to carboxylic acids using the Lewis acid aluminum triiodide (AlI3) generated in situ under anhydrous conditions to avoid hydrolysis of the activated nitrile.
The results of the biochemical assay on the 5i subunit of immunoproteasome showed that compounds 1, 2, 4B, 7–10 inhibited the immunoproteasome, with compound 7 beeing the most potent inhibitor with an IC50 value of 12 ± 1 µM. 2-Cyanobenzoxazoles were generally more potent inhibitors than 2-cyanobenzimidazoles, but the latter may be a better starting point for further optimization of small molecule inhibitors of the immunoproteasome 5i subunit due to their better stability and lower reactivity.
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