Design and synthesis of amide derivates of 3-amino- and 3-aminomethylpiperidines as immunoproteasome inhibitors

Kopičenje poškodovanih ali odsluženih proteinov v celici poruši homeostatsko ravnovesje in povzroči celično smrt. Eden od mehanizmov, ki zagotavljajo razgradnjo takih proteinov, je ubikvitin-proteasomski sistem. Pri človeku poznamo tri oblike proteasomov. Konstitutivni proteasom je prisoten v vseh celicah našega organizma, timoproteasom je v kortikalnih epitelijskih celicah timusa, imunoproteasom najdemo večinoma v hematopoetskih celicah, ob prisotnosti INFγ, TNFα ter oksidativnega stresa pa tudi drugje. Slednji ima pomembno fiziološko vlogo v imunskem sistemu, vnetnem odgovoru ter zaščiti celic pred oksidativnim stresom. Dokazano je bilo, da sta prekomerno izražanje in aktivacija imunoproteasoma povezana z vnetnimi, avtoimunskimi, nevrodegenerativnimi in rakavimi obolenji. Zato predstavlja imunoproteasom obetavno tarčo za zdravljenje teh bolezni. Do zdaj so dovoljenje za promet pridobili trije neselektivni zaviralci proteasomov. V magistrski nalogi smo želeli sintetizirati selektivne, nepeptidne zaviralce podenote β5i imunoproteasoma z ustreznimi fizikalno-kemijskimi lastnostmi in z manj neželenih učinkov, ki so značilni za neselektivne zaviralce. Najprej smo po različnih sinteznih poteh sintetizirali 3-aminometilpiperidinski in 3-aminopiperidinski skelet. Najbolj uspešna, časovno in glede izkoristkov, je bila reakcija reduktivnega aminiranja. Po odstranjevanju Boc zaščitne skupine, kjer smo prav tako preizkusili in med sabo primerjali reakciji s trifluorocetno kislino in klorovodikovo kislino v dioksanu, smo na osnovni skelet pripenjali elektrofilne bojne glave. Elektrofilne bojne glave so ob ustreznem pozicioniranju v aktivnem mestu imunoproteasoma odgovorne za tvorbo kovalentne vezi s katalitičnim treoninom (Thr). Kot reagente smo uporabili akriloil klorid, kloroacetil klorid, bromoacetil bromid, 2-cianoocetno kislino in kalijev oksiran-2-karboksilat. Pri reakciji z 2-cianoocetno kislino in kalijevim oksiran-2-karboksilatom smo uporabili sklopitvena reagenta EDC in HOBt, da smo aktivirali karboksilno kislino. Sintetizirali smo tudi oksatiazolonski derivat. Tukaj je bil osnovni skelet malo drugačen. Na (4-morfolinofenil)metanol, ki smo ga predhodno aktivirali z metansulfonil kloridom, smo pripeli piperidin-3-karboksamid, nato pa smo izvedli ciklizacijo z uporabo klorokarbonilsulfenil klorida. Uspelo nam je sintetizirati sedem končnih spojin (reakciji z bromoacetil bromidom in kalijevim oksiran-2-karboksilatom nista bili uspešni), ki smo jih biokemijsko vrednotili. Akrilamidna derivata zavirata imunoproteasom v mikromolarni koncentraciji, vendar sočasno zavirata tudi katalitično aktivnost konstitutivnega proteasoma. Selektivnosti nismo uspeli doseči, kljub temu pa naše delo predstavlja dobro izhodišče za nadaljnje raziskave tega strukturnega razreda.The accumulation of damaged or non-functional proteins in cells disturbs homeostasis and may lead to cell death. One of the mechanisms by which such proteins are degraded is called ubiquitin proteasome system. In humans, three different isoforms of proteasomes are found. Constitutive proteasome is present in all cells of our organism, thymoproteasome is found in cortical epithelium cells of thymus, and immunoproteasome in hematopoietic cells. In the presence of INFγ, TNFα, and oxidative stress the expression of immunoproteasome can be induced in other tissues as well. The immunoproteasome plays an important role in immune system and protection against oxidative stress. However, its elevated expression and hyperactivity are associated with inflammation, autoimmune and neurodegenerative diseases, as well as cancer. That is why the immunoproteasome represents a promising target for these diseases. Three nonselective proteasome inhibitors received marketing authorization up to now. In master’s thesis, we wanted to synthesize selective, nonpeptidic inhibitors of the β5i subunit of immunoproteasome with good physicochemical properties and minimal side effects that are characteristic for nonselective proteasome inhibitors. First, 3-aminomethylpiperidine and 3-aminopiperidine scaffolds were synthesized using different reaction pathways. Reductive amination was the most successful as far as the time consumption and yields are concerned. After removal of Boc protective group, where two different synthetic pathways were used as well (reaction with trifluoroacetic acid and reaction with 4 M HCl in dioxane), electrophilic warheads were attached to the scaffold. When properly positioned, electrophilic warheads form a covalent bond with threonine in the active site of immunoproteasome. Acryloyl chloride, chloroacetyl chloride, bromoacetyl bromide, 2-cyanoacetic acid and potassium oxirane-2-carboxylate were used as reagents. To activate the acetic part of 2-cyanoacetic acid and potassium oxirane-2-carboxylate coupling reagents EDC and HOBt were used. In addition, the oxathiazolone derivate was synthesized, starting from different scaffold. Namely, (4-morpholinophenyl)methanol was first activated with methanesulfonyl chloride, followed by the addition of piperidine-3-carboxamide. Then, cyclization was preformed using chlorocarbonylsulfenyl chloride. Seven final compounds were successfully synthesized (reactions with bromoacetyl bromide and potassium oxirane-2-carboxylate were not successful) and evaluated biochemically. Among these, the acrylamide-based inhibitors proved to be the most promising since they showed β5i inhibition with IC50 values in the low micromolar range. We did not manage to achieve selectivity, because compounds almost equipotently inhibited subunits of the immunoproteasome and the constitutive proteasome. Despite not achieving envisaged goals in their entirety, our work represents a solid starting point for further investigations