Synthesis of 4\u27-cyclohexyl-substitued inhibitors of immunoproteasome with psoralene scaffold

Podvrsta encima proteasoma, ki ima vlogo pri vzdrževanju homeostaze celice z uravnavanjem razgradnje poškodovanih, nepravilno zvitih ali denaturiranih proteionov, se imenuje imunoproteasom. Ta varuje celico pred poškodbami, nastalimi zaradi vnetja ali infekcije z virusi. Večinoma se izraža v celicah imunskega sistema, še zlasti v antigen predstavitvenih celicah. Ugotovljeno je bilo tudi, da sta količina in aktivnost imunoproteasoma v celicah povečani pri nekaterih boleznih, kot so rakava, nevrodegenerativna ali avtoimunska obolenja. To dejstvo je pripeljalo do razvoja novih sinteznih učinkovin, ki imajo za svojo tarčo imunoproteasom in s tem prispevajo k izboljšanju omenjenih bolezenskih stanj. V magistrski nalogi smo se ukvarjali s sintezo šestih novih spojin, katerih namen je zaviranje imunoproteasoma. Po končani sintezi smo izvedli še farmakodinamsko vrednotenje sintetiziranih spojin. Pri sintezi spojin smo najprej izvedli Pechmannovo kondenzacijo med resorcinolom in 2-acetil dietilsukcinatom ter resorcinolom in dietil-2-acetilpentandioatom. Pechmannovi kondenzaciji je sledila nukleofilna substitucija z 2-bromo-cikloheksiletanonom, ki smo ga predhodno sintetizirali z bromiranjem acetilcikloheksana. Nato smo izvedli kondenzacijo spojine, pri čemer je nastal psoralenski obroč, ki je bil na četrtem mestu substituiran z metilno skupino. Kondenzaciji je sledila funkcionalizacija karboksilne kisline, pri čemer smo na sintetizirane spojine pripenjali N-hidroksisukcinimid, 2-(metilamino)acetonitril in 2-aminoacetonitril. Vse sintetizirane spojine so na 4\u27 mestu psoralenskega skeleta imele pripet cikloheksil, ki naj bi izboljšal fizikalne in kemijske lastnosti spojin ter s tem omogočil boljšo zaviralno aktivnost na kimotripsinski podenoti imunoproteasoma. Farmakodinamsko smo spojine ovrednotilli tako, da smo določili rezidualno aktivnost kimotripsinske podenote imunoproteasoma. Najboljšo zaviralno aktivnost sta pokazali spojini 7 in 10, ki sta bili strukturno gledano aktivirana estra z N-hidroksisukcinimidom. Na podlagi pridobljenih rezultatov lahko sklepamo tudi, da je zamenjava fenilnega obroča z nasičenim obročem neugodna, saj pride pri teh spojinah v primerjavi s tistimi, ki imajo na tem mestu fenilno skupino, do zmanjšanja zaviralnega delovanja spojin. Prav tako smo ugotovili, da skrajšanje stranske verige na tretjem mestu psoralenskega skeleta za eno CH2 skupino pri aktiviranih estrih privede do poslabšanja kovalentne interakcije s treoninom v aktivnem mestu, saj je zaviralna aktivnost teh spojin šibkejša.Proteasome has a significant role in the maintenance of cell homeostasis by regulating degradation of damaged, misfolded or denatured proteins. Immunoproteasome is an isoform of proteasome and it protects the cell from the damage caused by inflammation or virus infection. The inhibition of the immunoproteasome can lead to accumulation of damaged or misfolded proteins which in turn leads to cell death. Nevertheless, studies have shown that the increased expression and activity of the immunoproteasome in cells correlates with cancer, neurodegenerative and autoimmune illnesses. This reason led to the development of new synthetic substances, which selectively target the immunoproteasome and lead to the improvement of earlier mentioned diseases. In this thesis, we managed to synthesize six new substances, which inhibit the immunoproteasome. All synthesized compounds were evaluated biochemically in vitro. The first step in the preparation of final compounds was the Pechmann condensation involving resorcinol and 2-acetyldiethylsuccinate or resorcinol and diethyl-2-acetylpentandioate. These condensations were followed by the nucleofilic substitution with previously synthesized 2-bromo-cyclohexyletanone, which was synthesized by reacting bromine with acetylcyclohexane. After that, we performed a condensation reaction which resulted in the formation of a psoralene ring with a methyl substituent at position 4. In the last step, carboxylic acid was derivatized by forming an activated ester with N-hydroxysuccinimide or by forming amides with 2-(methylamino)acetonitrile and 2-aminoacetonitrile, respectively. All of the synthesized substances had a cyclohexyl substituent at position 4\u27 place of the psoralene ringthe aim of this substitution pattern was to improve the physico-chemical properties in comparison with phenyl-bearing compounds, and concomitantly retain or improve inhibitory activity on the chymotrypsin-like subunit of immunoproteasome. In the biochemical evaluation of synthesized molecules, we determined the residual activity of the chymotrypsin-like subunit of immunoproteasome. The best ability to suppress the activity of the chyomtrypsin-like subunit was shown by compounds 7 and 10, which were chemically the succinimide esters. Based on the results, we can conclude that replacing the phenyl ring with its saturated counterpart leads to a reduction in inhibitory activity. In addition shortening of the side chain of succinimide esters by one CH2 group decreases the covalent interaction with threonine in the active site