Načrtovanje, sinteza in vrednotenje derivatov 3-(4-fluorofenil)izoksazolo[5,4-d]pirimidin-4(5H)-ona kot zaviralcev indolamin 2,3-dioksigenaze 1

Šmon, Špela (author), Sova, Matej (mentor)

Za fazo pobega, ki je del širšega procesa, imenovanega imunsko urejanje, je značilno progresivno razmnoževanje tumorskih celic do pojava vidnega tumorja in kliničnih simptomov. K njenemu razvoju pomembno doprinese razvoj tumorskega mikrookolja, ki nastane tudi kot posledica delovanja citoplazemskega encima indolamin 2,3-dioksigenaza 1 (IDO1). IDO1 je hem-vsebujoča dioksigenaza, ki katalizira začetno stopnjo (in hkrati določa tudi hitrost) presnove L-triptofana do L-kinurenina, kar privede do nastanka toksičnih kinureninskih presnovkov. Rezultat je zaviranje imunskega odgovora in nadalje širjenje malignega tumorskega tkiva. Iz tega razloga predstavlja IDO1 zanimivo tarčo v imunoterapiji raka. Žal pa so razvoju zaviralcev tega encima pridruženi mnogi zapleti, kot je na primer nespecifično zaviralno delovanje na novo odkritih spojin. Navkljub omenjenim težavam pa je zadnja leta kar nekaj zaviralcev IDO1 v kliničnih študijah in nekatere izmed spojin tudi kažejo obetavne klinične izide. V sklopu magistrske naloge smo na osnovi znanega zaviralca IDO1 3-(4-fluorofenil)izoksazolo[5,4-d]pirimidin-4(5H)-ona, ki je bil predhodno odkrit z virtualnim rešetanjem, v okviru petstopenjske sinteze pripravili devet končnih spojin (zaviralcev), katerim smo z različnimi analitskimi tehnikami potrdili istovetnost in čistost ter z biokemijskim testiranjem na osnovi merjenja fluorescence ovrednotili njihovo zaviralno delovanje na encimu. V sklopu sinteze smo se v zadnjih stopnjah spoprijemali z zelo nizkimi izkoristki poteka reakcij, kar je otežilo analitiko in biokemijsko vrednotenje končnih spojin. Kljub temu da so si vse končne spojine strukturno med seboj zelo podobne, pa se je izkazalo, da tri spojine (23, 28 in 29) dovolj močno zavirajo encim, zato smo jim določili še njihove vrednosti IC50. Najmočnejše zaviralno delovanje z vrednostjo IC50 10,9 µM smo določili za metil 4-(2-(4-okso-3-(tiofen-2-il)izoksazolo[5,4-d]pirimidin-5(4H)il)acetamido)benzoat (29), ki smo ga tudi sidrali v aktivno mesto encima in predpostavili potencialne interakcije. Spojina 29 predstavlja nov strukturni razred zaviralcev in pomembno izhodišče za nadaljnje raziskave in razvoj močnejših novih zaviralcev IDO1 kot potencialnih protitumornih učinkovin.

• Language: Slovenian
• Keywords: indolamin 2, 3-dioksigenaza 1 (IDO1), kinurenin, imunoterapija, tumor, zaviralci IDO1
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-108335
• Publication date in RUL: 28.06.2019

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of 3-(4-fluorophenyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one derivatives as indoleamine 2,3-dioxygenase 1 inhibitors

Abstract

The escape phase, which is a part of a more general process of cancer immunoediting, is known for progressive growth of tumor cells, which contributes to tumor visibility and manifestation of clinical symptoms. The tumor microenvironment, which among other things is generated as a consequence of cytoplasmatic enzyme indolamine 2,3-dioxygenase 1 (IDO1), significantly contributes to the development of the escape phase. IDO1 is a heme-containing dioxygenase and catalyzes the first (and at the same time rate-limiting step) of metabolism of L-tryptophane to L-kynurenine, which leads to the formation of toxic kynurenine metabolites. As a result, the immune response is inhibited and further on the malignant tumor tissue is spread. Accordingly, IDO1 is an interesting target in cancer immunotherapy. Unfortunately, the development of IDO1 inhibitors is associated with many complications, e.g. novel compounds show inhibitory activity through unspecific mechanisms. Despite these problems, a large number of IDO1 inhibitors are undergoing clinical trials in the last years and some of them have shown promising clinical outcomes. As a part of the Master's thesis, using five-step synthetic route, we synthesized nine final compounds (inhibitors) on the basis of a known IDO1 inhibitor 3-(4-fluorophenyl)isoxazolo[5,4-d]pyrimidin-4(5H)-on, which was previously discovered by virtual screening. Their identity and purity was confirmed by analytic techniques and their inhibitory activity on the enzyme was evaluated by a biochemical assay. In the last steps of the synthesis we had to cope with very low yields obtained after the reactions, which made the analytic and biochemical evalution of final compounds more difficult. Even though all final compounds have very similar structures, it was proved that three of them (23, 28 and 29) are sufficiently potent inhibitors to determine their IC50 values. The most potent inhibitory activity with an IC50 value of 10,9 µM was determined for methyl-4-(2-oxo-3-(tiophene-2-yl)isoxazolo[5,4-d]pyrimidin-5(4H)-il)acetamido)benzoate (29), which was also docked into the enzyme's active site for prediction of potential interactions. Compound 29 represents a new chemical class of inhibitors and an important starting point for further research and development of more potent novel IDO1 inhibitors as potential anticancer drugs.

• Keywords: indolamine 2, 3-dioxygenase 1 (IDO1), kynurenine, immunotherapy, tumor, IDO1 inhibitors