Optimizacija sinteze in vrednotenje zaviralcev indolamin 2,3-dioksigenaze 1 s substituiranimi 3-fenilizoksazolo[5,4-d]pirimidin-4(5H)-oni

Mlinarič, Larisa (author), Sova, Matej (mentor)

Indolamin 2,3-dioksigenaza 1 (IDO1) je hem vsebujoči citosolni encim, ki katalizira prvo stopnjo pretvorbe triptofana, pri tem kot glavni toksični metabolit nastaja kinurenin. Pomanjkanje triptofana in povečana koncentracija kinurenina v tumorskem mikrookolju vpliva na tri signalne poti v celici, kar vodi do prekritja tumorskih celic imunskemu sistemu in s tem do napredovanja bolezni. K napredovanju bolezni prav tako prispeva z IDO1 povzročena neovaskularizacija. Iz teh razlogov predstavlja IDO1 zanimivo potencialno tarčo v imunoterapiji raka. Trenutno je v kliničnih študijah kar nekaj zaviralcev IDO1. Študije pa kažejo predvsem na sinergistično delovanje v kombinaciji z uveljavljenimi načini zdravljenja raka. Namen naše naloge je bil na osnovi 3-fenilizoksazolo[5,4-d]pirimidin-4(5H)-ona sintetizirati in biokemijsko ovrednotiti nove zaviralce IDO1, od katerih bi vsaj eden zaviral delovanje encima v nizkem mikromolarnem območju. Prav tako pa smo želeli v sklopu magistrske naloge optimizirati s stališča izkoristkov in čistosti intermediatov najbolj problematičen korak splošne sintezne poti, ki ga predstavlja sinteza izoksazol-4-karboksamida. Sinteza novih zaviralcev IDO1 je temeljila na večstopenjski sintezni poti, s katero smo pripravili devet končnih spojin, ki smo jim z različnimi analitskimi tehnikami potrdili istovetnost in čistost. Z biokemijskim testiranjem na osnovi merjenja fluorescence pa smo ovrednotili njihovo zaviralno delovanje na IDO1. V nizkem mikromolarnem območju so svoje vrednosti IC50 imele tri spojine, in sicer 31, 32 in 33. Kot najmočnejši zaviralec IDO1 pa se je izkazal 2-(3-(3,4-difluorofenil)-4-oksoizoksazolo[5,4-d]pirimidin-5(4H)-il)-N-(4-nitrofenil)acetamid (31) z vrednostjo IC50 68,48 µM. Na osnovi analize molekulskega sidranja spojine 31 v aktivno mesto encima smo poizkušali napovedati potencialne interakcije, ki jih spojina 31 tvori z aminokislinskimi ostanki v aktivnem mestu. Na osnovi naših rezultatov lahko zaključimo, da spojina 31 predstavlja pomembno izhodišče za nadaljnje raziskave in razvoj močnejših novih zaviralcev IDO1 kot potencialnih protitumornih učinkovin.

• Language: Slovenian
• Keywords: indolamin 2, 3-dioksigenaza 1 zaviralci, imunoterapija raka, metabolizem triptofana, imunski pobeg
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-117401
• Publication date in RUL: 09.07.2020

Secondary language

• Language: English
• Title: Synthesis optimization and evaluation of substituted 3-phenylisoxazolo[5,4-d]pyrimidin-4(5H)-one indoleamine 2,3-dioxygenase 1 inhibitors

Abstract

Indolamine 2,3-dioxygenase 1 (IDO1) is a heme-containing cytosolic enzyme that catalyzes the first step of tryptophan metabolism, which leads to the formation of kynurenine as the major toxic metabolite. Lack of tryptophan and increased concentration of kynurenine in the tumor microenvironment affects three signaling pathways in the cell, leading to the immune escape of tumor cells and disease progression. IDO1-induced neovascularization also contributes to disease progression. For these reasons, IDO1 represents an interesting potential target in cancer immunotherapy. There are currently quite a few IDO1 inhibitors in clinical trials. So far the findings show a synergistic effect in combination with established treatments for cancer. The aim of our thesis was to synthesize and biochemically evaluate novel IDO1 inhibitors on the basis of 3-phenylisoxazolo[5,4-d]pyrimidin-4(5H)-one in order to obtain at least one inhibitor in the micromolar range. Furthermore, we also wanted to optimize the yields and purity of intermediates of the most problematic step of the general synthetic pathway, which is the synthesis of isoxazole-4-carboxamide. The synthesis of new IDO1 inhibitors was based on the multi-step synthetic route, which was used to prepare nine final compounds. Their identity and purity was confirmed by various analytical techniques. Biochemical evaluation based on fluorescence measurement was used to determine their inhibitory activity on IDO1. Three compounds (31, 32 and 33) had their IC50 values in low micromolar range. The most potent inhibitor of IDO1 was 2-(3-(3,4-difluorophenyl)-4-oxoisoxazolo[5,4-d]pyrimidin-5(4H)-yl)-N-(4-nitrophenyl)acetamide (31) with an IC50 value of 68,48 μM. Based on the molecular docking of compound 31 into the enzyme's active site we predicted potential interactions of 31 with amino acid residues. According to results obtained for 31 it represents an important starting point for further research and development of more potent new IDO1 inhibitors as potential anticancer agents.

• Keywords: indolamine 2, 3-dioxygenase 1 inhibitors, cancer immunotherapy, tryptophan metabolism, immune escape