20.500.12556/RUL-189329 Vrednotenje reaktivnosti karbamoil fluoridov kot bojnih glav pri načrtovanju kovalentnih zaviralcev Reactivity evaluation of carbamoyl fluorides as warheads in the design of covalent inhibitors Razvoj kovalentnih zaviralcev predstavlja pomemben pristop pri iskanju novih učinkovin za zdravljenje različnih bolezni, pri čemer sta ključna izziva ustrezna selektivnost in optimalna reaktivnost. V zadnjem času se veliko pozornosti namenja karbamoil fluoridom, ki zaradi svojih kemijskih lastnosti predstavljajo obetavne kovalentne bojne glave za različne tarčne proteine, vključno s kaspazami. Kaspaze so cisteinske proteaze z osrednjo vlogo pri uravnavanju apoptoze in nevrovnetja. Ena najbolje raziskanih je kaspaza-1, ki sproži vnetni odziv z aktivacijo provnetnih citokinov IL-1β in IL-18 ter cepitvijo gasdermina D, kar vodi v piroptotično celično smrt. Zaviralci kaspaze-1 imajo zato velik potencial za razvoj novih učinkovin za zdravljenje nevrodegenerativnih bolezni. V okviru magistrske naloge smo sintetizirali dve modelni sondi, karbamoil fluorida 14 in 15. Ključni sintezni korak je bila z bakrom(I) katalizirana azid-alkin cikloadicija (CuAAC), t. i. »klik« reakcija, pri kateri je nastal 1,4-disubstituiran 1,2,3-triazol. Poleg sintetiziranih sond smo proučevali tudi reaktivnost treh manjših karbamoil fluoridov 1–3 v prisotnosti različnih nukleofilov. Rezultati kažejo, da je reaktivnost karbamoil fluoridov odvisna od njihovega strukturnega ogrodja. Najmanj reaktivna je bila spojina 1, derivat izoindolina, sledila je spojina 2, derivat benzil piperidina. Spojina 2 je v prisotnosti tiolnih nukleofilov, zlasti ditiotreitola razpadla hitreje in je bila bolj reaktivna tudi v prisotnosti drugih aminokislin. Najbolj reaktiven je bil indolin 3, pri katerem mezomerni vpliv aromatskega obroča poveča dovzetnost karbamoil fluoridne skupine za napad nukleofilov. V prisotnosti aminokislin s tiolno skupino je popolnoma razpadla že v nekaj urah. Vse tri spojine so bile zelo stabilne v etanolu. Modelna sonda 15 je bila manj reaktivna kot sonda 14. Spojini se razlikujeta le v distančniku: pri 14 je ta alifatska veriga, pri 15 pa tetraetilenglikol. Sonda 15 je poleg tiolnih nukleofilov hitro reagirala tudi z L- lizinom, kar kaže, da lahko pri uporabljenih pogojih reagira tudi z amino skupinami in da njena reaktivnost ni omejena le na tiole. Rezultati prispevajo k boljšemu razumevanju vpliva strukture karbamoil fluoridov na njihovo reaktivnost ter predstavljajo izhodišče za načrtovanje novih kovalentnih sond z ustrezno stabilnostjo in reaktivnostjo. The development of covalent inhibitors represents an important strategy in the search for new therapeutic agents for various diseases, with achieving adequate selectivity and optimal reactivity remaining key challenges. In recent years, considerable attention has been devoted to carbamoyl fluorides, whose chemical properties makes them promising covalent warheads for targeting various proteins, including caspases. Caspases are cysteine proteases that play a central role in the regulation of apoptosis and neuroinflammation. One of the best-studied members is caspase-1, which triggers inflammatory responses by activating the pro-inflammatory cytokines IL-1β and IL-18 and cleaving gasdermin D, thereby inducing pyroptotic cell death. Caspase-1 inhibitors hence hold considerable potential for the development of novel therapeutic agents for neurodegenerative diseases. As part of this master’s thesis, two model probes, carbamoyl fluorides 14 and 15., were synthesized. The key synthetic step was a copper(I)-catalysed azide–alkyne cycloaddition (CuAAC), a so-called click reaction that afforded a 1,4-disubstituted 1,2,3-triazole. In addition to the synthesised probes, we investigated the reactivity of three smaller carbamoyl fluorides 1– 3, in the presence of various nucleophiles. The results showed that the reactivity of carbamoyl fluorides depends on their structural scaffold. Compound 1, an isoindoline derivative, was the least reactive, followed by compound 2, a benzylpiperidine derivative. Compound 2 underwent rapid degradation in the presence of thiol nucleophiles, particularly dithiothreitol, and was also more reactive in the presence of other amino acids. Indoline derivative 3, was the most reactive, as the resonance effect of the aromatic ring increases the susceptibility of the carbamoyl fluoride group to nucleophilic attack. In the presence of thiol-containing amino acids, it decomposed completely within only a few hours. All three compounds were highly stable in ethanol. Model probe 15 was less reactive than probe 14. They differ only in their linker: 14 contains an aliphatic chain, whereas 15 contains tetraethylene glycol linker. In addition to thiol nucleophiles, 15 reacted rapidly with L-lysine, indicating that under the conditions employed it can also react with amino groups and that its reactivity is not restricted to thiols. These findings contribute to a better understanding of how the structure of carbamoyl fluorides affects their reactivity and provide a basis for designing new covalent probes with appropriate stability and reactivity. karbamoil fluorid reaktivnost kaspaza-1 nevrovnetje klik reakcija carbamoyl fluoride reactivity caspase-1 neuroinflammation click reaction true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-10-03 08:45:41 2026-10-03 08:45:49 2026-10-04 04:15:35 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 133324 34634.pdf 34634.pdf 1 8785CA99344D4B531E7839762E8F6D19 58784414cb117a6a884916329e660d837c44422c2676091867d2b0fa38127f18 f24d19c7-bef5-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=249801 Fakulteta za farmacijo 0 0 0