Ubiquitin-proteasome system in eukaryotic cells is the main system responsible for the degradation of intracellular proteins and maintaining their homeostasis. In addition to the constitutive proteasome, which is responsible for regulation of cell processes with selective degradation of proteins, immunoproteasome has also been characterized. It is mainly located in cells of immune origin however, its expression can be induced when inflammation, stressful or autoimmune situations occur. Immunoproteasome has important role in degradation of proteins, which have been modified because of oxidative stress and inflammatory responses, and therefore represents an important therapeutic target for the treatment of inflammatory, cancerous, autoimmune, and neurodegenerative diseases.
There are three proteasome inhibitors present on the market and few additional inhibitors are undergoing clinical trial procedures. Most of them do not distinguish between catalytically active subunits of both proteasomes and this lack of selectivity is the main reason for many side effects of currently available proteasome targeting drugs. Development of immunoproteasome-selective inhibitors is oriented to preparation of non-peptide inhibitors of β5i subunit of immunoproteasome, as this would reduce the incidence of side effects, increase metabolic stability of compounds, increase their bioavailability and improve physicochemical properties.
In the master´s thesis, we designed and synthesized N-(4-morpholinobenzyl)-1-(piperidin-4-yl)methanamine-based inhibitors, which possessed an electrophilic »warhead« in their structure, aimed to form a covalent bond with the catalytic threonine β5i subunit and thus to contribute to inhibition of immunoproteasome significantly.
After reductive amination of the appropriately-substituted amine and aldehyde we obtained the main secondary amine intermediate, which was further N-acylated by attaching various fragments that differed in size and terminal functional group. Finally, tert-butylcarbamate protecting group was removed under acidic conditions. The final compounds were characterized by means of 1H NMR, HRMS, IR and 13C NMR methods. To assess inhibitory activities, we biochemically evaluated six successfully synthesized compounds by measuring the residual β5i activity of the enzyme in the presence of inhibitors. None of the synthesized compound inhibited the immunoproteasome however, the results will contribute to the understanding of structure-activity relationship of this compound class immunoproteasome inhibitors.
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