Synthesis and evaluation of novel derivatives of 4,6-substitued-1,3,5-triazin-2(1H)-ones as catalytic inhibitors of human DNA topoisomerase IIα

Rak je bolezen, za katero je značilna nekontrolirana rast celic v različnih organih. Encim človeška DNA topoizomeraza IIα (topo IIα) ima ključno vlogo pri uspešnem podvajanju celic, saj omogoča topološke spremembe molekule DNA in je uveljavljena protirakava tarča. Z zaviranjem encima dosežemo ustavitev procesa celične delitve, ki ima za posledico celično smrt. Zaradi poznanih neželenih stranskih učinkov klinično že uveljavljenih topoizomeraznih strupov, kot so kardiotoksičnost in nastanek sekundarnih oblik raka, poteka intenziven razvoj katalitičnih zaviralcev, ki bi encim zavrli preko alternativnih mehanizmov ter tako zmanjšali pojavnost stranskih učinkov. V magistrski nalogi smo sintetizirali in ovrednotili nove predstavnike kemijskega razreda 4,6-substituiranih-1,3,5-triazin-2(1H)-onov, ki delujejo kot katalitični zaviralci človeške DNA topo IIα in ciljajo na vezavno mesto za ATP ter tako razširili poznavanje odnosa med strukturo in delovanjem (SAR). Pri razvoju smo izhajali iz predhodno sintetiziranih spojin in na substitucijsko mesto 6 triazinonskega obroča uvedli nove biciklične substituente, ki smo jih izbrali z analizo dostopne serije sorodnih spojin farmacevtske družbe Novartis. S kondenzacijo strukturno novih bicikličnih derivatov amidin izotiouree in etoksikarbonil izotiocianata smo najprej pripravili monosubstituirane 1,3,5-triazin-2(1H)-onske obroče. Na mesto 4 smo nato pripeli predhodno identificirane najugodnejše benzilne substituente. V nekaterih primerih smo na koncu reakcije izolirali zmes dveh spojin, ki je še nismo uspeli ločiti, smo pa z različnimi spektroskopskimi in analitičnimi metodami kot so TLC, NMR in HRMS uspeli potrditi identiteto obeh nastalih triazinonov. Zaviralno delovanje končnih spojin in dobljenih zmesi smo ovrednotili z in vitro testom razpletanja kinetoplastne DNA, ki ga katalizira topo IIα. Vse sintetizirane spojine in zmesi spojin so izkazovale boljšo zaviralno aktivnost kot referenca etopozid. Predvsem so visoko topo IIα zaviralno aktivnost izkazovale spojine z benzotiofenskim obročem na mestu 6, med katerimi so izstopale spojine z alkil bromidno skupino na mestu 2 benzotiofenskega obroča. Molekulsko sidranje v tarčno vezavno mesto za ATP na N-terminalni domeni topo IIα je pokazalo, da triazinonski obroč sintetiziranih spojin tvori vodikovo vez s karbonilnim kisikom aminokisline Asn120, ki je ključna za uspešno medmolekulsko prepoznavanje. Naši rezultati so potrdili, da uvedba bicikličnih derivatov na mesto 6 v kemijski seriji 4,6-substituiranih-1,3,5-triazin-2(1H)onov vodi do spojin z dobro topo IIα zaviralno aktivnostjo. Derivati, ki vsebujejo benzotiofenski biciklični substituent na tem mestu, so še posebej dobro izhodišče za nadaljnji razvoj in optimizacijo tega kemijskega razreda katalitičnih zaviralcev kot potencialnih novih protirakavih učinkovin.Cancer is a common disease characterized by the uncontrolled growth of cells in various organs. The enzyme human DNA topoisomerase IIα (topo IIα) plays a key role in the successful cell replication, as it allows topological changes of the DNA molecule, and is an established anti-cancer target. By inhibiting this enzyme the process of cell division is halted, which results in cell death. Due to known negative side effects of clinically established topoisomerase poisons, such as cardiotoxicity and induction of secondary malignancies, there is an intense on-going development of catalytic inhibitors that would inhibit the enzyme via alternative mechanisms thus reducing the incidence of side effects. In this master\u27s thesis we successfully synthesized and evaluated new representatives of the chemical class of 4,6-substituted-1,3,5-triazin-2(1H)-ones, which act as catalytic inhibitors of the human DNA topo IIα targeting its ATP binding site. This expanded the structure-activity relationships (SARs) of this chemical class. In the development, we started from previously synthesized compounds and introduced new bicyclic substituents at the substitution site 6 of the triazinone ring, which were selected by analyzing an accessible series of related compounds developed by pharmaceutical company Novartis. Initially, the 1,3,5-triazin-2(1H)-one ring was prepared by a condensation of the corresponding structurally new bicyclic derivatives of the amidine isothiourea and ethoxycarbonyl isothiocyanate. Subsequently, we attached the preidentified most advantageous benzyl substituents to the scaffold position 4 to yield the final compounds. In some cases, at the end of the reaction, a mixture of two compounds was isolated. These mixtures have not yet been separated, but the identity of the two resulting triazinones was confirmed by various spectroscopic and analytical methods such as TLC, NMR and HRMS. The inhibitory activity of the obtained compounds and mixtures was evaluated by the in vitro kinetoplast DNA decantentation assay catalyzed by topo IIα. All synthesized compounds and their mixtures exhibited improved inhibitory activity compared to the reference etoposide. Particularly favorable topo IIα inhibitory activity was demonstrated by compounds with incorporated benzothiophene ring present at the triazinone position 6, of which compounds with the alkyl bromide group at position 2 of the benzothiophene ring additionally stood out. Molecular docking into the targeted ATP binding site located on the N-terminal domain of human topo IIα displayed that the triazinone ring of the newly obtained compounds forms a hydrogen bond with the carbonyl oxygen of the amino acid Asn120, which can be considered a crucial interaction for successful intermolecular recognition. Our results confirmed that the introduction of new bicyclic substituents at the position 6 of the 1,3,5-triazin-2(1H)-one scaffold yields compounds with promising topo IIα inhibitory activity. Furthermore, derivatives with incorporated benzothiophene substituent at this position comprise a particularly good starting point for further development and optimization of this chemical class of catalytic inhibitors as potential anticancer drugs