Synthesis of N-methylated N-phenylpyrrolamide derivatives as human DNA topoisomerase II inhibitors

Rak je skupek bolezni, pri katerih pride do nenadzorovane rasti celic tkiva, ne glede na potrebe telesa. Že desetletja rak globalno predstavlja velik zdravstveni izziv. Z boljšim razumevanjem bolezni so razvili nove pristope zdravljenja, kar je omogočilo boljše rezultate zdravljenja. Med uveljavljene pristope zdravljenja prištevamo tudi učinkovine, ki delujejo na človeško DNA-topoizomerazo II (topo II). To je encim, ki sodeluje pri sproščanju in uvajanju topoloških sprememb v molekulo DNA. Pri ljudeh obstajata dve izoobliki encima – IIα in IIβ. Prva se v visokih koncentracijah nahaja v hitro delečih se celicah, med katere spadajo tudi rakave celice, druga pa je vseskozi prisotna v jedru celic. Pri razvoju protirakavih učinkovin zato stremimo k selektivnosti na izoobliko IIα. Učinkovine, ki zavirajo topoizomerazo II povzročijo nepravilno podvajanje DNA, to pa vodi do apoptoze celice. V praksi se trenutno uporablja skupina učinkovin imenovana topoizomerazni strupi, ki so učinkoviti pri zdravljenju več oblik raka npr. levkemija, limfom, sarkom, itd. Pri njihovi uporabi pa lahko pride do neželenih učinkov, med katere prištevamo kardiotoksičnost in nastanek sekundarnih oblik raka. Druga skupina učinkovin, ki delujejo na ta encim, so katalitični zaviralci. Ti lahko delujejo na več različnih prijemališč na encimu. Najbolj obetavni so tisti, ki delujejo na ATP-vezavno mesto. Naš cilj je bil sintetizirati tri analoge znanih zaviralcev topoizomeraze II, ki delujejo na ATP-vezavno mesto. Znani zaviralci v strukturi vsebujejo N-fenilpirolamidni del. Na dušikov atom N-fenilpirolamida smo dodatno vezali metilno skupino in preverili, kako to vpliva na delovanje encima in kakšna je aktivnost v primerjavi z izhodnimi spojinami. Sintetiziranim spojinam smo potrdili strukturo, jim določili fizikalno-kemijske lastnosti s spektroskopskimi metodami (jedrska magnetna resonanca, infrardeča spektroskopija, masna spektrometrija) in jih ustrezno okarakterizirali ter določili njihovo čistost s kromatografskimi metodami (tankoplastna in kolonska kromatografija, tekočinska kromatografija visoke ločljivosti). Zaviralno delovanje smo ovrednotili na bakterijski topoizomerazi – DNA-girazi bakterije E. coli. Ta ima podobno strukturo ATP-vezavne domene kot človeška DNA-topoizomeraza IIα. Določili smo srednjo zaviralno koncentracijo (IC50). Končne sintetizirane spojine (8, 18 in 27) so v primerjavi z nemetiliranimi analogi izkazale slabše zaviralno delovanje. Metilna skupina lahko ovira nastanek vodikovih vezi med amidno skupino in encimom.Cancer is a group of diseases in which there is uncontrolled growth of tissue cells, regardless of the needs of the body. Cancer has been a major global health challenge for decades. With a better understanding of the disease, new treatment approaches have been developed to achieve better treatment outcomes. Established treatment approaches include agents that target human DNA topoisomerase II (topo II). Topo II is an enzyme involved in releasing and introducing topological changes in the DNA molecule. In humans, there are two isoforms of the enzyme – IIα and IIβ. The first is found in high concentrations in rapidly dividing cells, including cancer cells, while the other is found in all cells in the nucleus. Therefore, in development of anticancer agents, we aim for selectivity for the IIα isoform. Agents that inhibit topoisomerase II cause improper DNA replication, which in turn leads to cell apoptosis. In practice, a group of agents known as topoisomerase poisons are currently used for this purpose. Topoisomerase poisons are effective in the treatment of several forms of cancer, e.g. leukaemia, lymphoma, sarcoma, etc. Side effects may occur with their use including cardiotoxicity and the formation of secondary forms of cancer. Another group of agents that act on this enzyme are catalytic inhibitors. These can target several different receptors on the enzyme. The most promising are those that act at the ATP binding site. Our goal was to synthesize three analogues of known topoisomerase II inhibitors that act at the ATP binding site. Known inhibitors contain N-phenylpyrrolamide in their structure. We attached an additional methyl group to the nitrogen atom of N-phenylpyrrolamide and investigated how this affects the activity of the compounds and how high the activity is compared to the original compounds. We confirmed the structure of the synthesized compounds, determined their physicochemical properties by spectroscopic methods (nuclear magnetic resonance, infrared spectroscopy, mass spectrometry) and characterized them accordingly, and determined their purity using chromatographic methods (thin-layer and column chromatography, high-resolution liquid chromatography). The inhibitory effect was evaluated on bacterial topoisomerase - DNA gyrase of the E. coli bacterium. This enzyme has a similar ATP binding domain structure as human DNA topoisomerase IIα. The mean inhibitory concentration (IC50) was determined. The final synthesized compounds (8, 18 and 27) showed lower inhibitory activity compared to the unmethylated analogues. The methyl group may hinder the formation of hydrogen bonds between the amide group and the enzyme