Sinteza N-(piperidin-3-ilmetil)acilamidnih zaviralcev imunoproteasoma

Levačič, Luka (author), Sosič, Izidor (mentor), Obreza, Aleš (co-mentor)

Razgradnja znotrajceličnih proteinov v sesalskih celicah poteka po ubikvitin-proteasomskem sistemu (UPS). Ta je zelo dobro reguliran, a se lahko ob nekaterih stresnih dejavnikih začne inducirati nastanek imunoproteasoma, ki predstavlja izoformo konstitutivnega proteasoma. Njegova čezmerna ekspresija je povezana s številnimi imunskimi, rakavimi, nevrodegenerativnimi in vnetnimi bolezenskimi stanji. Zato je imunoproteasom v zadnjih letih postal pomembna terapevtska tarča. Kljub precejšnjemu znanstvenemu vložku pa je večina trenutno znanih peptidnih zaviralcev dokaj neselektivna, kar se lahko odraža v neželenih učinkih. Zato je precej raziskav usmerjenih v odkrivanje nepeptidnih zaviralcev imunoproteasoma, ki bi imeli selektivno delovanje in hkrati tudi ustrezne fizikalno-kemijske lastnosti. V magistrski nalogi smo se osredotočili na načrtovanje in sintezo N-(piperidin-3-ilmetil)acilamidov, ki so v strukturi imeli še t. i. elektrofilno bojno glavo. Namen uvedbe te glave je bil doseči tvorbo kovalentne vezi s katalitičnim treoninom v aktivnem mestu podenote β5i imunoproteasoma, s čimer bi se protein inaktiviral. Izhajali smo iz 3-aminometil substituiranega piperidina, zaščitenega z zaščitno skupino Boc na piperidinskem dušiku. V prvi stopnji smo z reduktivnim aminiranjem na izhodno spojino vezali derivat morfolina. V naslednji stopnji smo na novonastalo sekundarno aminsko skupino pripenjali različne substituente, ki so se razlikovali po velikosti in terminalni funkcionalni skupini. Pripenjanje je potekalo bodisi s kislinskimi kloridi bodisi s karboksilnimi kislinami ob dodatku ustreznih sklopitvenih reagentov. V zadnji stopnji smo odstranili zaščitno skupino Boc s 4 M HCl v dioksanu. Sintetizirali smo šest načrtovanih spojin ter jih biokemijsko vrednotili, pri čemer smo preverili, ali zavirajo katalitično aktivnost podenote β5i imunoproteasoma. Kljub temu da noben izmed pripravljenih derivatov ni zaviral encima pri 50 µM, so pridobljeni podatki dodatna informacija o odnosu med strukturo in delovanjem spojin tega kemijskega razreda.

• Language: Slovenian
• Keywords: Imunoproteasom, UPS, zaviralci, podenota β5i, elektrofilna bojna glava
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-133644
• Publication date in RUL: 07.12.2021

Secondary language

• Language: English
• Title: Synthesis of N-(piperidine-3-ylmethyl)acylamide immunoproteasome inhibitors

Abstract

Degradation of intracellular proteins in mammalian cells is catalyzed with a ubiquitin-proteasome system (UPS), which is well regulated. In certain cases the formation of immunoproteasome is induced and it represents the isoform of constitutive immunoproteasome. Its excessive expression is linked with numerous immune, cancer, neurodegenerative and inflammatory diseases. Therefore, the immunoproteasome has become an important therapeutic target in the recent years. However, despite the considerable scientific input, the majority of known peptidic inhibitors are currently quite non-selective, which may lead to side effects. Because of this, there is quite a lot of research directed in discovering non-peptidic immunoproteasome inhibitors that would have selective action and at the same time appropriate physico-chemical properties. In this master's thesis we focused on the design and synthesis of N-(piperidine-3-ylmethyl)acylamides with additional »electrophilic warhead« in their structure. The purpose of the latter was to enable the formation of covalent bond with catalytic threonine in the active site of β5i subunit in immunoproteasome that would result in inactivation of the protein. We started from a 3-aminomethyl substituted piperidine, protected with a Boc protecting group on a piperidine nitrogen atom. In the first step, we linked the mentioned substance with a morpholine derivative via reductive amination. In the next step, we attached various substituents on the newly formed secondary amine, which were different in size and terminal functional group. The coupling took place with acid chloride or carboxylic acid and coupling reagents. In the final step, we removed Boc protecting group using 4 M HCl in dioxane. We synthesized six final compounds and evaluated them biochemically, where we checked whether they inhibit the catalytic activity of β5i subunit of the immunoproteasome. Despite the fact that none of the prepared derivatives showed the inhibition at 50 µM, the obtained data serve us as an additional information about the relationship between the structure and mechanism of action of substances of this chemical class.

• Keywords: Immunoproteasome, UPS, inhibitors, β5i subunit, electrophilic warhead