Sinteza derivatov 4-hidroksi-3-metoksibenzamida z antagonističnim delovanjem na Tollu podobni receptor 8

Tratar, Katjuša (author), Sova, Matej (mentor)

Imunski sistem je ključen za varovanje organizma pred vdorom mikroorganizmov in drugih škodljivih dejavnikov. Delimo ga na prirojenega in pridobljenega. Prirojeni imunski sistem predstavlja prvo linijo obrambe in se odziva hitro, a nespecifično, medtem ko pridobljena imunost omogoča specifičen in dolgotrajen odziv. Del prirojene imunosti so Tollu podobni receptorji (TLR), ki so ključni za prepoznavo s patogeni povezanih molekulskih vzorcev in s poškodbo povezanih molekulskih vzorcev. Aktivacija teh receptorjev preko celičnih signalnih poti vodi do povečanega izražanja genov za provnetne citokine in s tem povezano aktivacijo imunskega sistema. V okviru magistrske naloge smo se osredotočili na TLR8. Gre za endosomalni receptor, ki prepoznava enoverižno RNA in ima tako ključno vlogo pri protivirusnem in protitumornem odzivu. Pretirana aktivacija TLR8 in prepoznava telesu lastnih RNA pa je povezana z razvojem avtoimunskih bolezni. Zaradi tega antagonisti TLR8 predstavljajo velik potencial za zdravljenje bolezni, kot so sistemski lupus eritematozus, revmatoidni artritis, dermatomiozitis in psoriaza. V okviru magistrske naloge smo na osnovi zadetka virtualnega rešetanja z izkazanim antagonističnim delovanjem na TLR8 načrtovali in sintetizirali 8 končnih spojin. Sinteza je potekala iz metil 4-hidroksi-3-metoksibenzoata, na katerega smo preko hidroksilne skupine pripeli 3-klorometil-5-metilizoksazol in 4-klorometil-3,5-dimetilizoksazol. Temu je sledila hidroliza estra in sinteza različnih amidov. Identiteto in čistost končnih spojin smo potrdili s tekočinsko kromatografijo visoke ločljivosti, z masno spektrometrijo visoke ločljivosti in jedrsko magnetno resonanco. Antagonistično delovanje in citotoksičnost vseh končnih spojin smo preverili na celični liniji HEK-Blue hTLR8. Antagonistično delovanje so pokazale samo spojine, ki so imele na osnovni 4-hidroksi-3-metoksibenzamidni skelet pripet dimetilizoksazolni obroč in N-alkiliran piperidin. Med njimi se je spojina 12 izkazala za citotoksično. Spojini 11 in 13 sta z IC50 vrednostjo 1,57 μM oziroma 1,38 μM izkazali najmočnejše antagonistično delovanje in tako predstavljata dobro izhodišče za nadaljnji razvoj in optimizacijo antagonistov TLR8.

• Language: Slovenian
• Keywords: imunski sistem, Tollu podobni receptorji, TLR8, avtoimunske bolezni, antagonisti
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-168591
• Publication date in RUL: 18.04.2025

Secondary language

• Language: English
• Title: Synthesis of 4-hydroxy-3-methoxybenzamide derivatives with antagonistic activity on Toll-like receptor 8

Abstract

The immune system is essential for protecting the body from invading microorganisms and other harmful agents. It is divided into the innate and the adaptive immune system. The innate immune system serves as the first line of defense and responds quickly but non-specifically, whereas adaptive immunity provides a specific and long-lasting response. An essential component of innate immunity are the Toll-like receptors (TLR), which play a key role in recognizing pathogen-associated molecular patterns and damage-associated molecular patterns. Activation of these receptors triggers signalling pathways, leading to the increased expression of pro-inflammatory cytokine genes and consequently, to activation of the immune system. In this thesis, we focused on TLR8, an endosomal receptor that recognizes single-stranded RNA and plays a crucial role in antiviral and antitumor responses. However, excessive activation of TLR8 and recognition of self-RNA are associated with the development of autoimmune diseases. For this reason, TLR8 antagonists hold great potential for the treatment of diseases such as systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, and psoriasis. In this master's thesis, we designed and synthesized eight final compounds based on a virtual screening hit that demonstrated antagonistic activity against TLR8. The synthesis was carried out using methyl 4-hydroxy-3-methoxybenzoate, to which 3-chloromethyl-5-methylisoxazole and 4-chloromethyl-3,5-dimethylisoxazole were attached via the hydroxyl group. This was followed by ester hydrolysis and the synthesis of various amides. The identity and purity of final compounds were confirmed by high-resolution liquid chromatography, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. The antagonistic activity and cytotoxicity of all final compounds were tested on the HEK-Blue hTLR8 cell line. Antagonistic activity was shown only in compounds containing a dimethylisoxazole ring and N-alkylated piperidine attached to the main 4-hydroxy-3-methoxybenzamide scaffold. Among them, compound 12 exhibited cytotoxicity. Compounds 11 and 13 showed the strongest antagonistic activity, with IC50 values of 1.57 μM and 1.38 μM, respectively. Therefore, they represent a good starting point for further development and optimization of TLR8 antagonists.

• Keywords: immune system, Toll-like receptors, TLR8, autoimmune diseases, antagonists