Sinteza derivatov N-(2-izopropoksifenil)-pirolamida kot zaviralcev človeške DNA-topoizomeraze IIα

Rozman, Sebastjan (author), Zidar, Nace (mentor)

Trend naraščanja števila rakavih obolenj je glavna gonilna sila številnih raziskovalnih projektov povezanih z odkrivanjem novih načinov zdravljenja raka. Glede na veliko hitrost replikacije rakavih celic, so se encimi iz družine topoizomeraz, ki modulirajo topologijo molekule DNA in s tem omogočajo podvajanje in prepisovanje DNA, uveljavili kot logična in obetavna tarča za zdravljenje raka. Na podlagi ugotovitev obsežnega raziskovalnega dela Katedre za farmacevtsko kemijo na Fakulteti za farmacijo Univerze v Ljubljani smo zasnovali in sintetizirali novo serijo zaviralcev človeške DNA-topoizomeraze IIα z N-(2-izopropoksifenil)-pirolamidno splošno strukturo. Predvidevali smo, da bo zamenjava kislega strukturnega elementa na desni strani molekule z bolj bazičnim strukturnim elementom izboljšala citotoksične lastnosti naših spojin, hkrati pa da bo ohranitev pirolamidnega strukturnega fragmenta vezanega na 2-izopropoksi substituiran benzenski skelet zagotovila dober zaviralni učinek na encim. Vse sintetizirane spojine smo ustrezno ovrednotili s spektroskopskimi in kromatografskimi metodami in določili njihove fizikalno-kemijske lastnosti. Zaviralne lastnosti petih končnih in treh predkončnih spojin na človeško DNA-topoizomerazo IIα smo ovrednotili s komercialno dostopnim relaksacijskim testom. Vsem petim končnim spojinam, ki so izkazovale dobro zaviralno aktivnost na encimu, smo preko testiranja na celičnih linijah MCF-7 in HepG2 določili zaviralno aktivnost na proliferacijo rakavih celic. Vseh pet končnih spojin je izkazovalo zelo dobre zaviralne lastnosti na encimskem testu z IC50 vrednostmi v razponu med 1,4 µM in 37 µM, kjer je bila najmočnejša spojina 22, ki je izkazovala IC50 vrednost 1,4 µM. Tudi citotoksično testiranje je razkrilo obetavne rezultate, saj so imele štiri od petih končnih spojin odlične zaviralne lastnosti na obeh celičnih linijah z vrednostmi IC50, ki se gibale med 1,5 µM in 9,3 µM. Najmočnejši zaviralec je bila spojina 22, ki je izkazovala vrednost IC50 4,2 µM na celično linijo MCF-7 in 1,5 µM na celično linijo HepG2. Z dobrimi rezultati encimskega relaksacijskega testiranja smo potrdili pomembnost pirolamidnega fragmenta vezanega na 2-izopropoksi substituiran benzenski skelet kot najmanjšega strukturnega motiva, ki omogoča dobro ATP kompetitivno inhibicijo človeške DNA-topoizomeraze IIα. Poleg tega so odlične inhibitorne lastnosti štirih končnih spojin (19, 20, 21 in 22) na obeh rakavih celičnih linijah potrdile pozitivni vpliv uvedbe bazičnega centra v obliki amidopiperidinskega strukturnega elementa za doseganje dobre citotoksične aktivnosti.

• Language: Slovenian
• Keywords: rak, človeška DNA-topoizomeraza IIα, ATP kompetitivni zaviralec, N-(2-izopropoksifenil)-pirolamid, citotoksičnost
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-122952
• Publication date in RUL: 18.12.2020

Secondary language

• Language: English
• Title: Synthesis of N-(2-isopropoxyphenyl)-pyrrolamide derivatives as human DNA topoisomerase IIα inhibitors

Abstract

The increasing incidence of cancer has been the driving force behind numerous research projects dealing with new cancer treatments. Based on the rapid replication of cancer cells, enzymes from the family of topoisomerases have established themselves as logical and promising targets for cancer treatment due to their ability to modulate DNA topology needed for DNA replication and transcription. Based on the extensive research work performed at the Department of pharmaceutical chemistry on the Faculty of Pharmacy, University of Ljubljana, we designed and synthesised a new series of human DNA topoisomerase IIα inhibitors with N-(2-isopropoxyphenyl)-pyrrolamide basic structure. We proposed that a replacement of the acidic structural part on the right-hand side of the molecule with a more basic moiety would improve cytotoxic properties of our compounds, while the pyrrolamide fragment bound to the 2-isopropoxy substituted benzene backbone will preserve the inhibitory effect on the enzyme. All the synthesised compounds were evaluated using spectroscopic and chromatographic methods and their physicochemical properties were determined. The inhibitory properties of five final and three pre-final compounds were evaluated using an in vitro DNA topoisomerase IIα relaxation assay to confirm their inhibitory activities on the enzyme. Lastly, all five final compunds that showed adequate inhibitory activity in the enzyme relaxation test, were subjected to cytotoxic testing using MCF-7 and HepG2 cancer cell lines. All five final compounds exerted very good inhibitory properties on the enzyme test with an IC50 value ranging between 1,4 µM and 37 µM. The most potent was compound 22, which showed an IC50 of 1,4 µM. Cytotoxic testing also revealed promising results, with 4 out of the 5 final compounds having excellent inhibitory properties on both cell lines with an IC50 ranging from 1,5 – 9,3 µM. The most potent compound was once again compound 22, which showed an IC50 of 4,2 µM and 1,5 µM on the MCF-7 and HepG2 cell lines respectively. Through the good results from the relaxation assay testing, we confirmed the importance of the pyrrolamide fragment bound to the 2-isopropoxy substituted benzene backbone as the smallest structural motif that allows good ATP competitive inhibition of human DNA topoisomerase IIα. Furthermore, the excellent inhibitory properties of four final compounds (19, 20, 21 and 22) on both cancer cell lines confirmed the importance of introducing a basic centre in the form of an amidopiperidine structural element to achieve good cytotoxic activity.

• Keywords: cancer, human DNA-topoisomerase IIα, ATP competitive inhibitor, N-(2-isopropoxyphenyl)-pyrrolamide, cytotoxicity