Načrtovanje in sinteza derivatov 3,5-dimetilizoksazola z antagonističnim delovanjem na Tollu podobni receptor 8

Gojkovič, Jan (author), Sova, Matej (mentor)

Prirojen imunski sistem predstavlja prvo obrambno linijo pred okužbami, pri čemer ima ključno vlogo prepoznavanje molekulskih vzorcev, povezanih s patogeni (PAMP), ter molekulskih vzorcev, povezanih s poškodbo (DAMP). Za to prepoznavo skrbijo receptorji za prepoznavanje molekulskih vzorcev (PRR), med katerimi so Tollu podobni receptorji (TLR) najpomembnejši in najtemeljiteje raziskani. Med njimi izstopa TLR8, ki je lokaliziran na membrani endosoma in se aktivira predvsem ob prisotnosti enoverižne RNA. Zaradi svoje povezave z razvojem številnih vnetnih in avtoimunskih bolezni predstavlja TLR8 privlačno tarčo za razvoj selektivnih antagonistov, ki bi lahko uravnavali pretiran vnetni odziv. V okviru magistrske naloge smo sintetizirali 15 končnih spojin s potencialnim antagonističnim delovanjem na TLR8, pri čem smo izhajali iz spojine zadetka 10 (lit.), ki so jo z virtualnim rešetanjem odkrili Matziol in sod. Na podlagi te spojine smo načrtovali nove derivate 3,5-dimetilizoksazola, pri čemer smo v prvi stopnji 3,5-dimetilizoksazol pripeli na metilni ester 3-nitro- in 3-kloro-4-hidroksibenzojske kisline. V drugi stopnji je sledila bazično katalizirana hidroliza estra, nato pa smo po optimizaciji sklopitvenega postopka v tretji stopnji sintetizirali različne amide iz derivatov 4-aminopiperidina. Spojinam, na katere smo uvajali 4-aminopiperidin z zaščitno skupino Boc, smo v četrti stopnji odstranili zaščito, v peti stopnji pa smo na sekundarno aminsko skupino pripenjali alil bromid. Končne spojine smo okarakterizirali z različnimi analitskimi tehnikami ter jih biokemijsko ovrednotili na celični liniji HEK-Blue™ hTLR8, s čimer smo določili antagonistično aktivnost na TLR8 in citotoksičnost. Ugotovili smo, da uvedba kloro skupine ohrani antagonistično delovanje na TLR8, medtem ko so nitro derivati aktivnost izgubili. Rezultati testiranj so pokazali, da imajo klorirani derivati 6, 8, 10 in 21 IC50 v nanomolarnem območju in so močni antagonisti TLR8, pri čemer je bila najboljša spojina 10 z vrednostjo IC50 0,68 µM. Test citotoksičnosti je pokazal, da je bila končna viabilnost celic pri najmočnejših antagonistih z izjemo spojine 21 ustrezna. Z našimi rezultati smo razširili odnos med strukturo in delovanjem tega tipa spojin, pridobljeni podatki pa predstavljajo odlično izhodišče za nadaljnje racionalno načrtovanje antagonistov TLR8.

• Language: Slovenian
• Keywords: imunski sistem, Tollu podobni receptorji, TLR8, antagonist
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-174229
• Publication date in RUL: 30.09.2025

Secondary language

• Language: English
• Title: Design and synthesis of 3,5-dimethylisoxazole derivatives with antagonistic activity on Toll-like receptor 8

Abstract

The innate immune system represents the first line of defence against infections, playing a key role in the recognition of pathogen-associated molecular patterns (PAMP-) and damage-associated molecular patterns (DAMP-). This recognition is mediated by pattern recognition receptors (PRR-), among which the Toll-like receptors (TLR) are the most important and most extensively studied. TLR8 is localized on the endosomal membrane and is primarily activated by single-stranded RNA. Due to its association with the development of various inflammatory and autoimmune diseases, TLR8 represents an attractive target for the development of selective antagonists that could regulate excessive inflammatory responses. As part of this master's thesis, we synthesized 15 compounds with potential antagonistic activity against TLR8, based on a hit compound 10 (lit.), which was identified by Matziol et al. through virtual screening. Based on this compound, we designed new derivatives of 3,5-dimethylisoxazole. In the first step, the 3,5-dimethylisoxazole moiety was attached to the methyl ester of 3-nitro- and 3-chloro-4-hydroxybenzoic acid. In the second step, base-catalyzed hydrolysis of the ester was performed, followed by optimization of the coupling procedure in the third step to synthesize various amides from 4-aminopiperidine derivatives. For compounds where 4-aminopiperidine with a Boc protecting group was introduced, deprotection was carried out in the fourth step, and in the fifth step, allyl bromide was attached to the secondary amino group. The final compounds were characterized using various analytical techniques and biochemically evaluated on the HEK-Blue™ hTLR8 cell line to determine their TLR8 antagonist activity and cytotoxicity. The introduction of a chloro substituent preserved antagonistic activity, whereas the introduction of a nitro group resulted in a complete loss of activity. The test results showed that four chloro-substituted compounds (6, 8, 10, 21) exhibited IC50 values in the nanomolar range and are potent TLR8 antagonists, with compound 10 being the most active with IC50 value of 0.68 µM. The cytotoxicity test showed that the final cell viability for the most potent compounds was appropriate except for compound 21. With our results, we expanded the structure-activity relationships of this class of compounds, and the data obtained provide an excellent starting point for further rational design of TLR8 antagonists.

• Keywords: immune system, Toll-like receptors, TLR8, antagonist