Sinteza pirolidinskih, piperidinskih in piperazinskih derivatov 6-(trifluorometil)izoksazolo[5,4-d]pirimidina kot agonistov Tollu podobnega receptorja 7

Smajić, Benjamin (author), Sova, Matej (mentor)

Imunski sistem je osnovni obrambni sistem našega telesa in se deli na prirojeni in pridobljeni. Celice prirojene imunosti zagotavljajo prvo in osnovno linijo obrambe našega telesa pred patogeni. Slednje zaznajo z receptorji za prepoznavanje molekulskih vzorcev (PRR), ti pa prepoznajo z patogeni in poškodbami povezane molekulske vzorce (PAMP in DAMP). Ena izmed najbolj opisanih skupin PRR so Tollu podobni receptorji (TLR). TLR so membranski glikoproteini tipa 1 in do danes so pri vretenčarjih odkrili 13 različnih tipov TLR-jev, od tega jih 10 najdemo pri človeku. Eden izmed teh je tudi TLR7, na katerega smo se osredotočili v sklopu magistrske naloge. Nahaja se na endosomalnih membranah znotraj celic in njegovo naloga je, da prepoznava enoverižne RNA virusov. Terapevtsko bi lahko bili agonisti TLR7 uporabni pri zdravljenju astme, rakavih obolenj in virusnih okužb. Edini registrirani agonist TLR7 je imikvimod. Namen naše naloge je bil sintetizirati in biološko ovrednotiti potencialne selektivne agoniste TLR7, ki so strukturni analogi spojine, ki je izkazovala agonistično delovanje na TLR7; sinteza le – te pa je temeljila na povezavi strukturnih lastnosti spojin, ki so bile agonisti TLR7 ali pa zaviralci encima indolamin-2,3-dioksigenaze. Končne spojine smo sintetizirali po šeststopenjskem postopku z dvema različnima izhodnima spojinama. V zadnji, šesti stopnji, smo na biciklični sistem pripenjali različne pirolidinske, piperidinske ter piperazinske derivate in tako sintetizirali 10 končnih spojin. Sintetiziranim spojinam smo preverili topnost v celičnem gojišču in potencialno citotoksičnost ter tudi ovrednotili agonistično delovanje na TLR7 in TLR8. Sintetiziranim spojinam, ki so izkazovale preliminarno agonistično delovanje na TLR7 (spojine 6, 7 in 20), smo nato še določili vrednost EC50. Kot najmočnejši agonist TLR7 se je izkazal 3-(3-bromo-4-fluorofenil)-4-(3-metilpiperidin-1-il)-6-(trifluorometil)izoksazolo[5,4-d]pirimidin (spojina 20), z izračunano EC50 = 16,4 ± 0,1 µM. Nobena izmed spojin ni izkazovala agonističnega delovanja na TLR8. Na podlagi teh rezultatov lahko pridemo do zaključka, da spojine 6, 7 in 20 tvorijo dobro izhodišče za nadaljnje raziskave novih selektivnih agonistov TLR7 kot potencialnih imunomodulatornih učinkovin.

• Language: Slovenian
• Keywords: imunski sistem, Tollu podobni receptor, TLR7, agonist, pirimidin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-141635
• Publication date in RUL: 03.10.2022

Secondary language

• Language: English
• Title: Synthesis of pyrrolidine, piperidine and piperazine derivatives of 6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine as Toll-like receptor 7 agonists

Abstract

The immune system is the primary defense system of our body and it is divided into innate and acquired immunity. Cells of innate immunity provide our body with the first and primary line of defense against pathogens. The latter are detected by pattern recognition receptors (PPRs), which recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs). One of the most characterised groups of PPRs are Toll-like receptors (TLRs). TLRs are type 1 membrane glycoproteins and to date 13 different types of TLRs have been identified in vertebrates, of which 10 are found in humans. One of them is TLR7, on which we focused as part of our master's thesis. It is located inside the cells on endosomal membranes and its function is to recognise single-stranded RNA viruses. Therapeutically, TLR7 agonists could be useful in the treatment of asthma, cancer and viral infections. The only registered TLR7 agonist is imiquimod. The aim of our work was to synthesize and biologically evaluate potential selective TLR7 agonists, which are structural analogues of a compound that exhibited agonist activity on TLR7. The synthesis of that compound was based on the combination of structural properties of compounds that were either TLR7 agonists or inhibitors of the enzyme inoleamine-2,3-dioxygenase. The final compounds were synthesized using a six-step procedure with two different starting compounds. In the last, sixth step, various pyrrolidine, piperidine and piperazine derivatives were attached to the bicyclic system, resulting in the synthesis of 10 final compounds. The synthesised compounds were screened for solubility in cell culture medium and potential cytotoxicity, and also evaluated for agonist activity on TLR7 and TLR8. The synthesised compounds that showed preliminary agonist activity on TLR7 (compounds 6, 7 and 20) were then subjected to further EC50 determination. The most potent TLR7 agonist was 3-(3-bromo-4-fluorophenyl)-4-(3-methylpiperidin-1-yl)-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (compound 20), with a calculated EC50 of 16.4 ± 0,1 µM. None of the compounds showed agonist activity on TLR8. Based on these results, it can be concluded that compounds 6, 7 and 20 form a good starting point for further research investigation of new selective TLR7 agonists as potential immunomodulatory agents.

• Keywords: immune system, Toll-like receptor, TLR7, agonist, pyrimidine