Sinteza derivatov 2-(4-okso-3-fenilizoksazolo[5,4-d]pirimidin-5(4H)-il)-N-fenilacetamida iz različno substituiranih aldehidov in anilinov kot zaviralcev indolamin 2,3-dioksigenaze 1

Bombek, Domen (author), Sova, Matej (mentor)

Z imunoterapijo raka želimo ponovno vzpostaviti ravnovesje imunskega sistema, katerega delovanje rakaste celice s procesom imunskega preurejanja uporabijo za izboljšanje preživetja in pospešeno rast tumorja. Povečano izražanje inhibitornih receptorjev imenovanih kontrolne točke, ki v organizmu skrbijo za periferno toleranco in nadzorujejo jakost imunskega odziva, je eden izmed mehanizmov, kjer tumorske celice izkoristijo imunski sistem za zagotovitev imunskega pobega. Indolamin 2,3-dioksigenaza 1 (IDO1) je znotrajcelična hemvsebujoča oksidoreduktaza, ki sodeluje pri kinureninski poti razgradnje triptofana do kinurenina ter ostalih metabolitov. Znižana koncentracija triptofana in povišana koncentracija kinurenina in ostalih metabolitov v organizmu sproži zaviranje imunskega odziva in širjenje tumorja. IDO1 zato predstavlja ustrezno tarčo za iskanje novih protirakavih učinkovin, ki v organizmu ponovno vzpostavijo imunsko ravnovesje. V sklopu magistrske naloge smo želeli sintetizirati nove potencialne zaviralce IDO1 na osnovi strukture 2-(4-okso-3-fenilizoksazolo[5,4-d]pirimidin-5(4H)-il)-N-fenilacetamida. Osnovna sintezna pot do končnih produktov je obsegala pet stopenj, alkilante za reakcijo N-alkiliranja pa smo pripravili z enostopenjsko sintezo. Intermediatom in končnim produktom smo z različnimi analitskimi metodami določili njihove fizikalno-kemijske lastnosti in potrdili njihovo istovetnost in čistost. Končnim spojinam smo z uporabo biokemijskega testiranja na osnovi spremljanja fluorescence ovrednotili njihovo zaviralno aktivnost na IDO1. Vsi pridobljeni produkti, razen N-(4-nitrofenil)-2-(4-okso-3-(p-tolil)izoksazolo[5,4-d]pirimidin-5(4H)-ilacetamida (14), so se izkazali za slabe zaviralce encima. Slednji je izkazal 45 % zaviranje pri 100 μM, zaradi česar je bil edini ustrezen produkt, ki mu je bilo smiselno določiti vrednost IC50, ki je znašala 145 μM. Uspelo nam je torej sintetizirati en zaviralec IDO1 z aktivnostjo v mikromolarnem območju, hkrati pa smo pridobili nove podatke o vplivu substitucij na fenilnem obroču vezanim na izoksazolno in acetamidno skupino, ki jih bomo lahko uporabili za nadaljnje delo pri razvoju novih zaviralcev encima IDO1.

• Language: Slovenian
• Keywords: indolamin 2, 3-dioksigenaza 1, rak, imunoterapija, zaviralec, kinureninska pot, imunsko preurejanje
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-136916
• Publication date in RUL: 25.05.2022

Secondary language

• Language: English
• Title: The synthesis of 2-(4-oxo-3-phenylisoxazolo[5,4-d]pyrimidin-5(4H)-yl)-N-phenylacetamide derivatives from substituted aldehydes and anilines as indoleamine 2,3-dioxygenase 1 inhibitors

Abstract

The goal of cancer immunotherapy is to restore the balance of immune system that is being used by cancer cells in order to improve their survival and cancer growth in a process called immunoediting. One of mechanisms of immunoediting is increased expression of inhibitory receptors named checkpoint inhibitors, which control peripheral tolerance and regulate intensity of immune response. Their increased expression allows tumor cells to achieve immune escape. Indolamine 2,3-dioxygenase 1 (IDO1) is a cytosolic heme containing oxidoreductase, which is involved in kynurenine pathway catabolism of tryptophane into kynurenine and other metabolites. Lowered tryptophane levels and increased kynurenine levels induce immunotolerance and tumor growth. That makes IDO1 a suitable target for cancer therapy research, that aims to restore the balance of immune system. The aim of this thesis was to synthesize new potential IDO1 inhibitors, based on 2-(4-oxo-3-phenylisoxazolo[5,4-d]pyrimidin-5(4H)-yl)-N-phenylacetamide structure. The main synthetic pathway consisted of five steps and the synthesis of alkylating agents used for N-alkylation reaction was performed in only one step. Various analytical methods were used to determine the physicochemical properties of intermediates and final products and confirm their identity and purity. Fluorescence-based biochemical assay was used to determine the inhibitory activity of final products on IDO1. All final products, apart from N-(4-nitrophenyl)-2-(4-oxo-3-(p-tolyl)isoxazolo[5,4-d] pyrimidin-5(4H)-yl)-N-phenylacetamide (14), were found as weak inhibitors of enzyme activity. The latter showed 45 % inhibition at 100 μM concentration, therefore being the only suitable product for further determination of IC50 value of 145 μM. We were able to synthesize one micromolar IDO1 inhibitor and gain new data about the effects of substitution on the phenyl ring attached to isoxazolo and acetamide moiety, that can be used for future work and development of IDO1 inhibitors.

• Keywords: indoleamine 2, 3-dioxygenase 1, cancer, immunotherapy, inhibitor, kynurenine pathway, immunoediting