Sinteza azastilbenskih zaviralcev ligaz Mur

Kramarič, Sabina (author), Hrast, Martina (mentor)

Boj proti bakterijskim infekcijam predstavlja enega pomembnejših dosežkov medicine 20. stoletja. Vendar pa je prekomerna in tudi napačna uporaba protimikrobnih učinkovin povzročila pojav odpornosti patogenih bakterij na antibiotike in s tem postavila resno grožnjo javnemu zdravju, obenem pa tudi potrebo po stalnem razvoju novih učinkovin. Medtem ko številni antibiotiki v klinični uporabi delujejo preko inhibicije kasnejših stopenj sinteze peptidoglikana, so začetni koraki biosinteze citoplazemskih prekurzorjev peptidoglikana kot protibakterijske tarče slabo izkoriščeni. Mednje spadajo tudi Mur encimi, ki kot ključen sestavni del bakterij omogočajo načrtovanje zaviralcev s potencialnim baktericidnim učinkom ter širokim spektrom delovanja. V okviru magistrske naloge smo sintetizirali analoge predhodno odkrite spojine z azastilbenskim skeletom, ki je zavirala encime MurC-F v mikromolarnem območju. Furan s kisikom na orto mestu smo preko Suzukijevega pripajanja nadomestili z različnimi heterocikli, na mestu 5-substituiranega tetrazola smo sintetizirali 2-oksazolin, 2-imidazolin in 2-tiazolin ter reducirali dvojno vez azastilbena. Sintetizirane spojine smo ovrednotili z biokemijskim testiranjem na encimih MurC, D, E in F bakterije E. coli. Vsem smo določili rezidualno aktivnost [%], najobetavnejšim spojinam pa tudi IC50 vrednost [µM]. Primerjava spojin 3a in 4a prikazuje pomen prisotnosti tetrazola pri doseganju zaviralnega delovanja na MurD-F, medtem ko pri zaviranju MurC tetrazol ni optimalna izbira. Ključno komponento pri doseganju aktivnosti na vseh štirih Mur ligazah predstavlja furan s kisikom na orto položaju, saj ostali analogi z drugimi heterocikli na tem mestu niso pokazali zaviralnega učinka na nobenem od encimov. Pri tem ima pomembno vlogo položaj kisika na orto mestu in ne zgolj njegova prisotnost v heterociklu, kar potrjuje primerjava med spojinama 4b in 4f. Najmočnejše zaviralno delovanje je dosegla spojina 13, ki z aktivnostjo na MurC, D in F spada med multiple inhibitorje, ki zmanjšujejo možnost razvoja rezistence bakterij na protibakterijske učinkovine. Dobro protibakterijsko delovanje na sev S. aureus smo izmerili le pri spojini 12 z MIK vrednostjo 10,65 µg/mL. V sklopu našega dela smo torej uspeli preučiti vpliv različnih substituentov na inhibicijo encimov MurC-F, pri čemer predvsem spojina 13 predstavlja osnovo za morebitno nadgradnjo multiplih azastilbenskih zaviralcev.

• Language: Slovenian
• Keywords: Mur ligaze, multipli inhibitorji, protibakterijske učinkovine, azastilbeni
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-110660
• Publication date in RUL: 18.09.2019

Secondary language

• Language: English
• Title: Synthesis of azastilbene inhibitors of Mur ligases

Abstract

The fight against bacterial infections has been one of the great success stories of 20th century medicine. However, extensive use, and also misuse of antibiotics have caused emergence of antimicrobal-resistant pathogenic bacteria, which now present a serious threat to public health and a need for a constant development of new antibacterial agents. While a large number of antibiotics in clinical use act by inhibiting later steps in peptidoglycan synthesis, the earlier steps of the biosynthesis of cytoplasmic peptidoglycan precursor are poorly exploited as antibacterial targets. Among them, Mur enzymes, as essential part of bacteria, enable design of inhibitors with potencial bactericidal and wide spectrum activity. In this master’s thesis we synthetized analogues of previously discovered azastilbene compound, that inhibited MurC-F in micromolar range. Via Suzuki coupling, we replaced furan, containing oxygen in ortho position, with various heterocycles; as well we synthetized 2-oxazoline, 2-imidazoline and 2-thiazoline instead of 5-substituted terazole and reduced azastilbene’s double bond. Synthesized compounds were tested for their inhibitory effect on enzymes MurC, D, E and F from E. coli. Residual activity [%] was measured for each of them, and for the most potential coumpounds, IC50 values [μM] were determined. Comparison of compounds 3a and 4a shows the importance of tetrazole at reaching inhibitory activities on MurD-F, while for MurC inhibition, tetrazole is not the best option. Furan with oxygen in ortho position seems to be essential, as the rest of analogues with other heterocycles in this place, does not show inhibitory activity on any Mur enzyme. Beside the presence of oxygen in furan, its ortho position is also significant, as we are able to see from comparison of compounds 4b and 4f. The best inhibitor was compound 13 that shows inhibitory activity on MurC, D and F, therefore it is classified as multiple inhibitor, which minimize the possibility of resistance development. Only compound 12 achieved significant antibacterial activity on S. aureus with values 10,65 μg/mL. To conclude, we manage to study the effect of different substituents on MurC-F inhibition, wherein compound 13 represents the basis for further studies of multiple azastilbene inhibitors.

• Keywords: Mur ligase, multiple inhibitors, antibacterial agents, azastilbenes