Trends in treating complex diseases are shifting towards the use of targeted and specific medicines for a more effective response to therapy and a lower likelihood of adverse events. Targets for treatment are different and specific, depending on the indication. Some types of cancer and autoimmune diseases have upregulated proteasome and immunoproteasome expression and activity. These two proteasome types differ in three ß subunits, which represent the possibility of subtype-specific inhibition.
Currently there are three non-selective peptide proteasome inhibitors available for cancer therapy. In this thesis, we wanted to synthesize several compounds of covalent nature that were designed to bind into the active site of ß5i subunit of the immunoproteasome. The covalent interaction should occur between the threonine residue in ß5i subunit of the immunoproteasome and the warhead, the electrophilic part in the compound. Our goal was to synthesize compounds with good water solubility, low IC50 and high selectivity compared to other subunits of immunoproteasome and constitutive proteasome.
Structures of covalent inhibitors were designed using the program Open-Eye BROOD and then adjusted to our synthetic options in the lab. To the core structure, consisting of 4-BOC protected 4-morpholinobenzaldehyde and 4-amino(methyl)morpholine, after the BOC group deprotection, a warhead was attached via amide bond. We added coupling reagents EDC and HOBt to weak electrophiles such as cyanoacetic acid and potassium oxirane-2-carboxylate. No coupling reagents were added to anhydrides and acid chlorides. After BOC deprotection, we used a base to deprotonate NH3+ group; for pH modifications glacial acetic acid was used. Reactions were performed under argon atmosphere or equipped with a calcium trap; and monitored by TLC in a suitable mobile phase. Purification of compounds was carried out by evaporation of solvents, use of diethyl ether, extractions, and by column chromatography.
Synthesized and purified compounds were assayed for inhibition of the immunoproteasome and for the most potent compound IC50 was determined. All final compounds have good water solubility. The most potent derivative was the 4-aminomethyl-based compound and an acrylamide warhead. The IC50 of this compound was in the micromolar range, which is less potent compared to the active substances on the market, because they have an IC50 in the nanomolar range. The selectivity assays showed equipotent inhibition of the ß5 subunit of the constitutive proteasome. Nevertheless, we managed to synthesize a set of compounds which potency and selectivity could be improved with additional optimizations.
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