Sinteza tiofenskih analogov 3-fenilizoksazolo[5,4-d]pirimidin-4(5H)-ona z zaviralnim delovanjem na indolamin 2,3-dioksigenazo 1

Novak, Teja (author), Sova, Matej (mentor)

Indolamin 2,3-dioksigenaza 1 je eden izmed treh encimov, ki sodelujejo pri razgradnji aminokisline triptofan po kinureninski poti. Gre za inducibilen, citosolen, hem-vsebujoči encim. Njegovo izražanje je povišano v tumorskih celicah ter v mikrookolju tumorja, kar pa predstavlja slabo prognozo za pacienta. Znižane koncentracije triptofana in povišane koncentracije njegovega metabolita kinurenina, namreč privedejo do določenih imunosupresivnih pojavov, kot so: zorenje limfocitov T v smeri regulatornih celic, aktivacija mieloidnih supresorskih celic, hkrati pa pride tudi do zmanjšanja količin efektorskih limfocitov T in naravnih celic ubijalk. Povišane koncentracije kinurenina naj bi prispevale celo k rasti žilja in invadiranju tumorja. IDO 1 zato predstavlja potencialno tarčo za razvoj zaviralcev v boju proti rakavim obolenjem. Kar nekaj potencialnih učinkovin je trenutno prisotnih v kliničnih študijah, obetavni pa so predvsem rezultati pri uporabi zaviralcev IDO v kombinaciji z že uveljavljenimi kemoterapevtiki. Namen magistrske naloge je bil sinteza potencialnih zaviralcev IDO1 na osnovi strukture 3-fenilizoksazolo[5,4-d]pirimidin-4(5H)-ona (rezultat virtualnega rešetanja). Osnovna sintezna pot je obsegala 5 stopenj. Intermediatom in končnim produktom smo z analitskimi metodami določili fizikalno-kemijske lastnosti ter potrdili njihovo istovetnost. Z biokemijskimi testi pa smo končnim produktom določili še zaviralno aktivnost in jakost delovanja. Naš cilj dela je bil pridobiti zaviralec IDO1 s čim nižjo vrednostjo IC50 (mikromolarna ali nižja). Vsi sintetizirani končni produkti so pokazali zaviralno aktivnost napram encimu IDO1, vendar je samo spojina N-(4-nitrofenil)-2-(4-okso-3-(tiofen-3-il)izoksazolo[5,4-d]pirimidin-5(4H)-il)acetamid (18) povzročila več kot 50 % zaviranje pri 100 µM, in sicer 67,8 %. Omenjeno zmanjšanje delovanja encima je bilo zadostno za določitev vrednosti IC50, ki je za to spojino znašala 39,6 µM. Dobili smo torej zaviralec IDO1 v mikromolarnem območju, katerega struktura ponuja dodatne informacije in dobro izhodišče za optimizacijo in izboljšave na področju razvoja zaviralcev encima IDO1.

• Language: Slovenian
• Keywords: Indolamin 2, 3-dioksigenaza 1, rak, zaviralec, imunski sistem, toleranca
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-125957
• Publication date in RUL: 10.04.2021

Secondary language

• Language: English
• Title: Synthesis of thiophene analogs of 3-phenylisoxazolo[5,4-d]pyrimidin-4(5H)-one with inhibitory activity against indoleamine 2,3-dioxygenase 1

Abstract

Indolamine 2,3-dioxygenase 1 is one of three enzymes that catabolize amino acid tryptophan via the kynurenine pathway. It is an inducible, cytosolic and heme-containing enzyme. Its expression is elevated in the tumor cells and tumor microenvironment. That parameter indicates a bad prognosis for the patient. Depleted concentrations of tryptophan and increased concentrations of its metabolite kynurenine, lead to many immunosuppressive actions, such as: differentiation of lymphocytes to regulatory T cells, activation of myeloid-derived suppressor cells and suppression of cytotoxic T cells and natural killer cells. Furthermore, high concentrations of kynurenine promote vascularization and invasion of tumor. Therefore, IDO 1 presents a great potential target for the development of inhibitors to fight against cancer. There is a number of potential candidates in clinical trials. Up to date, the most promising results have been obtained when an inhibitor was administered in combinations with already well-established chemotherapeutic drugs. The aim of this thesis was to synthesize potential inhibitors of IDO1, based on structure of 3-phenylisoxazolo[5,4-d]pirimidin-4(5H)-one, obtained by virtual screening. The synthetic pathway consisted of 5 steps. The intermediates and final products were characterized by analytical methods to determine their physicochemical properties and confirm their identity. Final products were then used in biochemical assay to determine their inhibitory potency. Our main goal was to obtain IDO1 inhibitor with low IC50 value (in micromolar range or less). All synthesized final compounds showed inhibitory activity against the enzyme IDO1. However, only one compound N-(4-nitrophenyl)-2-(4-oxo-3-(thiophen-3-yl)isoxazolo[5,4-d]pyrimidin-5(4H)-yl)acetamide (18) exhibited higher inhibition than 50 %, i.e. 67,8 %. The inhibitory potency was sufficient to further determine the IC50 value of 39,6 µM. We synthesized a micromolar IDO1 inhibitor, providing new information about structure-activity relationship for additional optimization and improvements towards more potent IDO1 inhibitors.

• Keywords: Indolamine 2, 3-dioxygenase, cancer, inhibitor, immune system, tolerance