Sinteza in vrednotenje substituiranih 1H-benzo[d]imidazol-2-karbonitrilov in 2-cianobenzo[d]oksazolkarboksilatov kot kovalentnih zaviralcev imunoproteasoma

Krajnc, Tina (author), Knez, Damijan (mentor), Gobec, Martina (co-mentor)

Ubikvintin-proteasomski sistem je pomemben za zagotavljanje homeostaze proteinov v celicah, saj je odgovoren za razgradnjo napačno zvitih in poškodovanih proteinov. Raziskave so pokazale, da se pri avtoimunskih boleznih, raku in nevrodegenerativnih spremembah pod vplivom vnetnih citokinov poveča izražanje imunoproteasoma, zaradi česar se je v zadnjem desetletju povečal obseg raziskav na področju selektivnih zaviralcev imunoproteasoma, ki imajo, v primerjavi z neselektivnimi zaviralci, manj neželenih učinkov. V okviru magistrske naloge smo se osredotočili na sintezo 2-cianobenzimidazolov in 2-cianobenzoksazolov, ki so heterobicikli z elektrofilno bojno glavo – nitrilom. Ciano skupina lahko tvori kovalentno vez s Cys48 v 5i podenoti imunoproteasoma, kar omogoča selektivno zaviranje 5i podenote imunoproteasoma, saj 5c konstitutivna podenota proteasoma na tem mestu nima cisteinskega aminokislinskega preostanka. Pri sintezi smo izhajali iz substituiranih orto-fenilendiaminov oziroma 2-aminofenolov, kjer je v prvi stopnji potekala ciklizacija med dinukleofilom in Appelovo soljo. Sintetizirane 2-cianobenzimidazole smo v drugem koraku z uporabo metil jodida pretvorili do dveh položajnih izomerov 2-ciano-N-metilbenzimidazola, ki smo ju očistiti in ločiti s kolonsko kromatografijo, strukturo posameznega položajnega izomera pa smo potrdil z 2-dimenzionalnimi eksperimenti jedrske magnetne resonance. Metil karboksilate 2-cianobenzoksazolov smo pretvorili do karboksilnih kislin z in situ pripravljeno Lewisovo kislino aluminijev trijodid (AlI3) v brezvodnih pogojih, da smo se izognili hidrolizi aktiviranega nitrila. Rezultati biokemijskega testiranja spojin na 5i podenoti imunoproteasoma so razkrili, da spojine 1, 2, 4B, 7–10 zavirajo imunoproteasom, najmočnejši zaviralec pa je bila spojina 7 z IC50 vrednostjo 12 ± 1 µM. 2-Cianobenzoksazoli so v splošnem močnejši zaviralci kot 2-cianobenzimidazoli, vendar so slednji zaradi boljše stabilnosti in manjše reaktivnosti lahko boljša osnova za nadaljnjo optimizacijo nizkomolekularnih zaviralcev 5i podenote imunoproteasoma.

• Language: Slovenian
• Keywords: benzimidazol, benzoksazol, imunoproteasom, kovalentni zaviralci, nitril
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-140580
• Publication date in RUL: 16.09.2022

Secondary language

• Language: English
• Title: Synthesis and evaluation of 1H-benzo[d]imidazole-2-carbonitriles and 2-cyanobenzo[d]oxazolecarboxylates as covalent inhibitors of immunoproteasome

Abstract

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.

• Keywords: benzimidazole, benzoxazole, covalent inhibitors, immunoproteasome, nitrile