The Ubiquitin-Proteasome pathway is of key value for the controlled degradation of proteins in mammalian cells with 26S proteasome (cCP), being responsible for over 80% of the protein degradation. Another member of the proteasome family, the immunoproteasome (iCP), has an indirect influence on the immune response of the body since it contributes to the formation of peptides optimised for binding to MHC class I molecules. It is also involved in cytokine modulation and T cell differentiation. Therefore, iCP is a promising target for the treatment of autoimmune diseases and cancer.
This master's thesis' main aim was to synthesise biphenylpiperidine covalent inhibitors of threonine proteases, a group of enzymes to which cCP and iCP also belong with a main goal to achieve selective binding of compounds into the active site of the β5i subunit of the immunoproteasome. The general structure of the synthesised compounds was a biphenylpiperidine scaffold to which we attached acrylamide and nitrile »warheads«. We expected that our compounds would non-covalently bind in the proximity of the nucleophilic catalytic threonine (Thr1) in the active site of the β5i subunit of human immunoproteasome and subsequently »warheads« would covalently interact with Thr1. We have also synthesised six analogous compounds with carboxyl groups instead of »warheads« to evaluate the non-covalent inhibition potential of synthesised derivatives.
Our initial starting materials were tert-butyloxycarbonyl (Boc) protected piperidine-3-ilmethyl amine and piperidine-4-ilmethyl amine. We then attached lipophilic fragments to the piperidine nitrogen through amine or amide bonds. Amines were formed through the reaction of reductive amination with corresponding aldehydes. Amides were formed by the use of carboxylic acids with coupling reagents or with acid chlorides. After the removal of the Boc-protecting group with acidolysis, acrylic and cyanoacetic acids were attached via amide bond. We successfully synthesized eight potential covalent inhibitors of the immunoproteasome. The starting point for the carboxylic acid derivatives were nipecotic and isonipecotic acid. After initial protection with methyl ester, the attachment of lipophilic fragments was achieved as stated above. The methyl ester protection was removed through alkaline hydrolysis.
The biochemical assays performed at the Faculty of Pharmacy, University of Ljubljana have reported that none of the end compounds exhibited substantial effect on the target enzyme, therefore further optimization is needed.
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