Alkiliranje 3-(4-fluorofenil)izoksazolo[5,4-d]pirimidin-4(5H)-ona in vrednotenje zaviralnega delovanja produktov na indolamin 2,3-dioksigenazo 1

Lazić, Valerija (author), Sova, Matej (mentor), Dolšak, Ana (co-mentor)

Rak je bolezen, ki nastane zaradi nenadzorovane in nenormalne rasti celic. Zanj obstaja kar nekaj oblik zdravljenja, med novejše spada tudi imunoterapija, katere temelj je ponovna vzpostavitev ravnovesja imunskega sistema. Indolamin 2,3-dioksigenaza 1 (IDO1) je hem vsebujoča oksidoreduktaza, ki katalizira razgradnjo L-triptofana do L-kinurenina in toksičnih kinureninskih derivatov, kar lahko vodi do zaviranja imunskega odgovora in širjenja tumorja. Z novimi odkritji imunosupresivnih funkcij in vloge IDO1 pri imunskem pobegu rakavih celic, so prepoznali aktivnost encima v korist raku. Iz tega razloga predstavlja IDO1 zanimivo tarčo za razvoj protirakavih učinkovin. Zaviranje encima predstavlja inovativno in obetavno strategijo za zdravljenje številnih vrst raka. Kar nekaj zaviralcev, ki so trenutno v kliničnih študijah, se bo v prihodnosti morda lahko uporabljalo kot podpora pri zdravljenju raka. V magistrski nalogi smo na osnovi že znanega zaviralca IDO1 5-(2-(3,4-dihidrokinolin-1(2H)-il)-2-oksooetil)-3-(4-fluorofenil)isoksazolo[5,4-d]pirimidin-4(5H)-ona, ki je bil predhodno odkrit z virtualnim rešetanjem, v okviru petstopenjske sinteze pripravili sedem končnih spojin. Vse končne spojine so si med seboj strukturno podobne, saj temeljijo na enakem osnovnem skeletu 3-(4-fluorofenil)izoksazolo[5,4-d]pirimidin-4(5H)-onu, na katerega smo preko amidnega dušika pripenjali različne substituente. Končnim spojinam smo s pomočjo analitskih metod potrdili istovetnost in čistost, z biokemijskim testiranjem pa smo preverili zaviralno aktivnost le-teh na encimih IDO1, indolamin 2,3-dioksigenazi 2 (IDO2) in triptofan 2,3-dioksigenazi (TDO). Nobena od končnih spojin, ki smo jih testirali, ni izkazovala tolikšne zaviralne aktivnosti, da bi ji bilo smiselno določiti IC50. Najboljši rezultat smo dobili pri spojini 3-(4-fluorofenil)-5-(4-nitrobenzil)izoksazolo[5,4-d]pirimidin-4(5H)-onu (spojina 8), ki je pri koncentraciji 100 µM dosegla 24 % zaviranje encima IDO1. Z našo študijo smo dokazali, da je za zaviralno delovanje na IDO1 nujno potrebna prisotnost amida pri substituentih, ki jih pripenjamo na osnovni 3-(4-fluorofenil)izoksazolo[5,4-d]pirimidin-4(5H)-onski skelet.

• Language: Slovenian
• Keywords: indolamin 2, 3-dioksigenaza 1, imunoterapija, rak, zaviralci IDO1
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-124069
• Publication date in RUL: 24.12.2020

Secondary language

• Language: English
• Title: Alkylation of 3-(4-fluorophenyl)isoxazolo[5,4-d]pyrimidine-4(5H)-one and products’ inhibitory activity evaluation on indoleamine 2,3-dioxygenase 1

Abstract

Cancer is a disease caused by uncontrolled and abnormal cell growth. There are quite a few ways of treatment with immunotherapy being the most recent one, which is based on the restoration of the immune system's balance. Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing oxidoreductase that catalyzes the breakdown of L-tryptophan to L-kynurenine and later toxic kynurenine derivates, which can lead to the suppresion of the immune response and tumor spread. With new discoveries of immunosuppressive functions and the role of IDO1 in the immune escape of cancer cells, the scientists identified the activity of the enzyme in favor of cancer. For this reason, IDO1 represents an interesting target for anticancer drug development. IDO1 inhibition is thus an innovative and promising strategy for the treatment of many types of cancer. There are quite a few inhibitors in clinical studies that could be used in the future to support cancer treatment. In this Master's thesis, we synthesized seven final compounds using five-step synthetic procedure, on the basis of a known IDO1 inhibitor 5-(2-(3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-3-(4-fluorophenyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one, previously discovered by virtual screening. All final compounds are structurally similar to each other as they are based on the same basic scaffold 3-(4-fluorophenyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one, to which various substituents were attached via the amide nitrogen. The identity and purity of the final compounds were confirmed by analytical methods, and their inhibitory activity on the IDO1, indolamine 2,3-dioxygenase 2 (IDO2) and tryptophan 2,3-dioxygenase (TDO) enzymes was evaluated by the biochemical assay. None of the final compounds exhibited a sufficient inhibitory activity that it would make sense to determine IC50 values. The compound 3-(4-fluorophenyl)-5-(4-nitrobenzyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one compound (8) showed the most potent inhibitory effect on IDO1 with 24 % inhibiton at the concentration of 100 µM. With our study we proved that the presence of an amide moiety in substituents attached to isoxazolo[5,4-d]pyrimidin-4(5H)-one is essential for potent IDO1 inhibitory activity.

• Keywords: indoleamine 2, 3-dioxygenase 1, immunotherapy, tumor, IDO1 inhibitors