Sinteza in vrednotenje novih N-(2-(3-karbamoilbenziloksi))fenilpirolamidov kot potencialnih zaviralcev človeške DNA-topoizomeraze IIα

Frankovič, Monika (author), Zidar, Nace (mentor)

Rak je kompleksna bolezen, ki predstavlja enega najpomembnejših javnozdravstvenih problemov razvitega sveta. Vsako leto se zaradi vse bolj nezdravega načina življenja in staranja prebivalstva število obolelih za rakom in posledično umrlih za posledicami te bolezni veča. Raziskovalci po celem svetu težijo k odkrivanju novih učinkovitih protirakavih zdravilnih učinkovin, med katerimi so se kot pomembni izkazali zaviralci encima DNA-topoizomeraza IIα. Na podlagi predhodnega dela in ugotovitev raziskovalcev s Katedre za farmacevtsko kemijo Fakultete za farmacijo Univerze v Ljubljani smo v sklopu magistrske naloge pripravili manjšo serijo novih N-(2-(3-karbamoilbenziloksi))fenilpirolamidnih katalitičnih zaviralcev človeške DNA-topoizomeraze IIα, ki se vežejo na ATP-vezavno mesto N-terminalnega dela encima. Vse pripravljene spojine smo ustrezno ovrednotili s kromatografskimi in spektroskopskimi metodami ter jim določili fizikalno-kemijske lastnosti. Zaviralno aktivnost na encimu DNA-topoizomeraza IIα smo določili trem končnim spojinam, 12, 16 in 20, z uporabo komercialno dostopnega relaksacijskega testa. Ker so vse tri spojine izkazovale zaviralno aktivnost na encimu v nizkih mikromolarnih koncentracijah, smo vsem določili tudi vrednosti IC50. Najboljšo zaviralno aktivnost je izkazovala spojina 20 s srednjo zaviralno koncentracijo 6,3 μM. Sledili sta ji spojini 16 in 12 z IC50 7,7 μM in 27 μM. Zaviralno delovanje spojin smo preverili tudi na celični liniji jetrnega karcinoma HepG2, pri čemer smo dobili izredno vzpodbudne rezultate. Vse tri testirane spojine so izkazale zelo dobro citotoksično delovanje. Kot najbolj aktivna se je izkazala spojina 16, z vrednostjo IC50 0,13 µM. Sledili sta ji spojini 20 in 12, z vrednostima IC50 1,6 µM in 1,9 µM. Na podlagi teh rezultatov smo potrdili domnevo, da je za vezavo v aktivno mesto encima pomemben pirolamidni strukturni element, ki oponaša adeninski fragment molekule ATP in tvori ključne vodikove vezi z aminokislinskim ostankom Asn120. Poleg tega pa smo potrdili ključno vlogo bazičnega centra za citotoksično delovanje, ki je v našem primeru prisoten na aminopiperidinskem oziroma aminopirolidinskem fragmentu molekule.

• Language: Slovenian
• Keywords: rak, človeška DNA-topoizomeraza IIα, ATP-kompetitivni zaviralec, protirakava učinkovina, N-fenilpirolamid
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-127687
• Publication date in RUL: 19.06.2021

Secondary language

• Language: English
• Title: Synthesis and evaluation of novel N-(2-(3-carbamoylbenzyloxy))phenylpyrrolamides as potential inhibitors of human DNA topoisomerase IIα

Abstract

Cancer is a complex disease that represents one of the biggest public health problems of the developed world. Each year, due to the increasingly unhealthy lifestyle and population aging, the number of people suffering from cancer and consequently deaths due to this disease are increasing. Researchers around the world are striving to discover new effective anti-cancer drugs, among which DNA topoisomerase IIα inhibitors have proven to be very important. Based on the previous work and the discoveries of researchers from the Department of Pharmaceutical Chemistry at the Faculty of Pharmacy, University of Ljubljana, we prepared a small series of new N-(2-(3-carbamoylbenzyloxy))phenylpyrrolamide catalytic inhibitors of human DNA topoisomerase IIα, which bind to ATP-binding domain on the N-terminal part of the enzyme. All prepared compounds were evaluated by chromatographic and spectroscopic methods and their physicochemical properties were determined. Inhibitory activity on the DNA topoisomerase IIα enzyme was determined for the three final compounds, 12, 16 and 20, using a commercially available in vitro relaxation assay. All three compounds showed inhibitory activities on the enzyme at low micromolar concentrations. The best inhibitory activity was shown by compound 20 with a mean inhibitory concentration of 6.3 μM, following by compounds 16 and 12, with IC50 values of 7.7 μM and 27 μM. The inhibitory activity of compounds was also tested on the human liver cancer cell line HepG2, and extremely encouraging results were obtained. All tested compounds showed very good cytotoxic activity. Compound 16 proved to be the most potent, with an IC50 value of 0.13 µM. It was followed by compounds 20 and 12, with IC50 values of 1.6 µM and 1.9 µM. Based on these results, we can confirm our assumption that a pyrrolamide structural element that mimics the adenine fragment of the ATP molecule and forms key hydrogen bonds with the amino acid residue Asn120 is important for binding to the active site of the enzyme. In addition, we confirmed the key role of the basic center for cytotoxic activity, which in our case is present on the aminopiperidine or aminopyrrolidine moiety.

• Keywords: cancer, human DNA topoisomerase IIα, ATP-competitive inhibitor, anticancer drug, N-phenylpyrrolamide