Design,Synthesis And Biological Evaluation Of Peptidyl Enoxyketone Immunoproteasome Inhibitors

Proteasome is of outstanding importance for cellular homeostasis,participating in many physiological processes.Proteasome in vertebrate can be divided into three types,termed the constitutive proteasome（cCP）,the immunoproteasome（iCP）,and the thymoproteasome（tCP）.CCP is present in all tissues,whereas iCP is constitutive in hematopoetic cells,especially lymphocytes and monocytes.Inhibition of proteasome leads to an accumulation of proteins relating to cell growth and proliferation,resulting in cell cycle arrest and apoptosis,and ultimately produces anti-cancer effects.Systemic side effects of bortezomib and carfilzomib may result from the combined inhibition of cCP and iCP.Immunoproteasome has tissue-specific distribution,and a preferential expression of iCP was detected in MM cells.By selectively targeting iCP,it may be possible to keep the anticancer activity unchanged and,at the same time,to circumvent the dose-limiting toxicities associated with pan-proteasome inhibitors.In addition,iCP is responsible for the generation of major histocompatibility complex（MHC）class I ligands.Given the role of iCP in the immune response,targeting iCP is a rational approach to treat hematologic malignancies,autoimmune and inflammatory diseases.Only one clinical trial for immunoproteasome inhibitor has been launched so far.In order to develop novel iCP selective inhibitors and overcome the shortcomings such as poor stability,which most peptide compounds have,the following work had been carried out.Immunoproteasome-selective inhibitors with D-amino acid:We chose E83 previously reported by our group as lead compound and introduced different R₁,R₂ and R₃ side chains to E83 molecular scaffold while keeping the N-cap unchanged.We synthesized eight tripeptidyl epoxyketone derivatives（Series One）,focusing on investigating the influence of D-amino acid substitution in R₃ residue on stability.This series of compounds exhibited moderate acitivity and selectivity.Modeling of compounds within proteasome catalytic binding sites suggested that the N-cap was too large.Based on these findings,we shortened the N-cap and introduced different N-cap moieties.Using D-amino acid substitution strategy in R₃ residue,we synthesized 30 analogues（Series Two）.SARs indicated that the chirality（D）of the alanine in R₃ residue was crucial for increasing the selectivity forβ5i overβ5c while maintaining or even enhancingβ5i-affinity at the same time.Notably,the analogues B25(β5i IC₅₀=26.0 nM,25-fold selectivity)and B30(β5i IC₅₀=25.1 nM,24-fold selectivity)with D stereochemistry at R₃ residue proved to be as potent as ONX-0914(β5i IC₅₀=28.2 nM,7-fold selectivity).Compounds B25 and B30 were much more higher in selectivity than ONX-0914.Besides,B25（remaining 50%at 30 min）and B30（remaining 55%at 30 min）were better in metabolic stability than ONX0914（remaining 40%at 30 min）which are promising compounds for further development.Inhibitors（A5,B12,B25,B27 and B30）displayed moderate antiproliferative activity against RPMI-8226 and MM.1S cell lines（0.11¹.92μM）.The effects of these compounds on cancer cell proliferation and cytokine expression are still under investigation.Immunoproteasome-selective inhibitors withβ-amino acid:In an effort to expand the structural diversity of our designed proteasome inhibitors and develop compounds with increased stability,we directed our work to the exploration of tripeptidyl inhibitor containingβ-amino acids,and synthesized twelve analogues.Among them,compound C2(β5i IC₅₀=33.5 nM,3.5-fold selectivity)was the most potent one which was as potent as ONX-0914(β5i IC₅₀=31.2 nM,4.2-fold selectivity),and itsβ5i selectivity was similar to ONX-0914.Compound C2（remaining 60%at 30 min）containingβ-amino acids were more stable in mouse liver microsomal than B30（remaining 55%at 30 min）withα-amino acids,indicating that the introduction ofβ-amino acid into compounds resulted in an increased enzymatic stability.Compound C2 is worth further studying.My work was also involved in studying the synthetic technology of E83.1-（tert-butoxycarbonyl）piperidine-4-carboxylic acid was used as the starting material and the target compound was obtained after a series of chemical reactions including condensation reaction,deprotection reaction,nucleophilic substitution reaction and hydrolysis reaction.The overall yield was about 33%and the purity of the target compound determined by HPLC was 98.1%.This optimized synthesis process has the advantages of easy,low-cost and high purity of the final product which was of great value.