Sinteza agonistov Tollu podobnega receptorja 7 s 6-(trifluorometil)izoksazolo[5,4-d]pirimidin-4-aminskim skeletom

Pavlin Gregorčič, Laura (avtor), Sova, Matej (mentor)

Terapija bolezenskih stanj, katerih vzrok je pomanjkljivo ali prekomerno delovanje imunskega sistema, vključuje predvsem uporabo učinkovin, ki delujejo na tarče pridobljene, specifične imunosti. Obetavno področje pa je v zadnjih letih postal razvoj učinkovin, katerih tarča so receptorji prirojene imunosti. Tollu podobni receptorji (TLR) so pomemben del prirojene imunosti, ki predstavlja prvo linijo obrambe pred vdorom tujkov. TLR specifično prepoznavajo različne, za patogene in poškodbe značilne molekulske vzorce in preko kompleksne signalne poti sprožijo hiter vnetni odziv, ki se kaže v povečani proizvodnji citokinov in drugih vnetnih dejavnikov. Od desetih znanih TLR, ki jih najdemo pri človeku, smo se v okviru magistrske naloge osredotočili na TLR7, endosomski receptor, ki s svojo zunanjo domeno specifično prepoznava enoverižno RNA virusov in je pomembna tarča učinkovin – agonistov TLR7 za zdravljenje virusnih okužb, rakavih obolenj in astme. Trenutno edini agonist TLR7 na tržišču je imikvimod. Sinteza potencialnih agonistov TLR7 je temeljila na strukturnih značilnostih spojine, ki je združevala elemente znanih agonistov TLR7 in zaviralcev encima indolamin-2,3-dioksigenaze ter je izkazovala agonistično delovanje na TLR7. Šeststopenjska sinteza je zajemala sintezo oksimov iz komercialno dostopnih aromatskih aldehidov, njihovo pretvorbo do imidoil kloridov, nastanek izoksazolnega obroča in nato bicikličnega sistema izoksazolo[5,4-d]pirimidina, pretvorbo do kloridov in v zadnji stopnji pripenjanje različnih aminov na biciklični sistem. Z izbiro topil in reakcijskih pogojev smo poskušali doseči čim boljše izkoristke in višjo čistost spojin. Sintetiziranim spojinam smo ovrednotili topnost v celičnem mediju, preverili morebitno citotoksičnost na celični liniji HEK 293 in agonistično delovanje na TLR7. Ugotovili smo, da so sintetizirane spojine zaradi svoje lipofilnosti zelo slabo topne v celičnem mediju, zato je testiranje agonizma potekalo pri nizkih koncentracijah, pri katerih pa spojine ne izkazujejo agonističnega delovanja. Kljub neuspelemu poskusu pridobitve novih agonistov TLR7 lahko zaključimo, da smo postavili izhodišče za nadaljnje raziskave na tem področju in optimizirali sintezni postopek do biciklične spojine izoksazolo[5,4-d]pirimidina.

• Jezik: Slovenski jezik
• Ključne besede: Tollu podobni receptorji, TLR7, agonisti, izoksazolo[5, 4-d]pirimidini, rak, virusna okužba.
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-131606
• Datum objave v RUL: 30.09.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthesis of Toll-like receptor 7 agonists with 6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine scaffold

Izvleček

The therapy of diseases due to immunodeficiency or the excessive immune response includes primarily the use of drugs which act on targets of adaptive immunity. However, the development of active substances that target the receptors of innate immunity represents the promising field in recent years. Toll-like receptors (TLRs) represent a significant part of the innate immunity and the front-line defence system against foreign pathogens. TLRs recognise different pathogen- and damage-associated molecular patterns triggering a rapid inflammatory response via complex signalling pathways resulting in increased production of cytokines and other inflammatory agents as a part of adaptive immunity. Out of ten known TLRs found in humans, we focused in this thesis on TLR7, an endosomal receptor, which with its ectodomain specifically recognises single-stranded viral RNA thus being an important target for potential drugs – i.e., TLR7 agonists for treating virus infections, cancer, and asthma. Currently, the only TLR7 agonist on the market is imiquimod. The synthesis of TLR7 agonists was based on structural features of the compound which combined elements of known TLR7 agonists and inhibitors of indoleamine 2,3-dioxygenase and which demonstrated agonist activity on TLR7. The six-step synthetic route included the synthesis of oximes from commercially available aromatic aldehydes, their transformation to imidoyl chlorides, leading to the formation of isoxazolopyrimidine rings and then bicyclic system of isoxazolo[5,4-d]pyrimidine, the transformation into chlorides and in the final stage linking different amines onto the bicyclic system. By the selection of solvents and reaction conditions, we tried to achieve the highest yields and purity of the compounds. We assessed the solubility of synthesized compounds in the cell media, evaluated potential cytotoxicity in the cell line HEK 293 and agonist activity on TLR7. It was found that synthetised compounds, due to their lipophilicity, dissolve poorly in cell media; therefore, agonism tests were carried out at low concentrations, where no agonist activity of compounds was observed. Despite the unsuccessful attempt in producing a new TLR7 agonist, the foundations for further research in this field have been laid and the process of synthesis has been optimised up to bicyclic isoxazolo[5,4-d]pyrimidine compound.

• Ključne besede: Toll-like receptors, TLR7, agonists, isoxazolo[5, 4-d]pyrimidine, cancer, viral infection.