Structural, theoretical and biological investigation of ruthenium(II) complexes with imidazole and isothiazole derivatives

U okviru ove doktorske disertacije sintetisani su i okarakterisani kompleksi rutenijuma(II) sa imidazolima ([Ru(η 6 -p-cymene)(N-MeIm)3]Cl2·2H2O i [Ru(η 6 -pcymene)(N-PrIm)Cl2]) i izotiazolima ([Ru(η 6 -p-cymene)Cl2(5-MA-3-PyCN-ITZ)], [Ru(η 6 -pcymene)Cl2(5-MA-4-MPipCN-ITZ)], [Ru(η 6 -p-cymene)Cl2(5-MA-3-MorphCN-ITZ)], [Ru(η 6 -pcymene)Cl2(5-PhA-3-PyCN-ITZ)], [Ru(η 6 -p-cymene)Cl2(3-Morph-5-PhACN-ITZ)]) kao ligandima. Karakterizacija novosintetisanih jedinjenja izvršena je primenom savremenih spektroskopskih metoda analize (1H i 13C NMR, IR i UV-Vis), kao i standardnim metodama (elementalna mikroanaliza i tačka topljenja). Rendgenskom strukturnom analizom potvrđene su strukture kompleksa, kao i izotiazolovih liganada 5- MA-3-PyCN-ITZ, 5-MA-4-MPipCN-ITZ, 5-MA-3-MorphCN-ITZ, 5-PhA-3-PyCN-ITZ i 3- Morph-5-PhACN-ITZ. Snimanjem emisionih fluorescentnih i apsorpcionih spektara ispitivane su interakcije liganada i kompleksa sa DNK koji je izolovan iz grudne žlezde teleta (CTDNA) i sa albuminom humanog seruma (HSA). Cilj ispitivanja interakcija sa biološkim makromolekulima se ogleda u određivanju mogućih načina vezivanja i konstanti vezivanja. Nakon ispitivanja, potvrđeno je da se interakcija odvija preko statičkog mehanizma gašenja. Primenom MTT testa citotoksičnosti, kao i metoda za detekciju apoptoze i autofagije, ispitivana je in vitro biološka aktivnost liganada i kompleksa. Kompleks [Ru(η 6 -p-cymene)(N-MeIm)3]Cl2·2H2O pokazuje najveću citotoksičnost, a najsenzitivnija je HeLa ćelijska linija. Vestern blot analiza i protočna citometrija ukazuju na apoptozu kao primaran način ćelijske smrti. Posmatrajući ćelijski ciklus, u S fazi uočava se sličnost u mehanizmu delovanja kompleksa rutenijuma(II) sa agensima iz platinske grupe metala. Teorijska ispitivanja liganada i kompleksa su odrađena primenom savremenih metoda kompjuterske hemije. Strukturne karakteristike kompleksa (veze, uglovi i torzioni uglovi) koji su optimizovani primenom teorijskih modela B3LYP/def2-TZVP, B3LYP/SDD i M06 u kombinaciji sa SDD baznim setom, u skladu su sa eksperimentalnim podacima dobijenim rendgenskom strukturnom analizom. Kompjuterska molekulska simulacija – doking, je korišćena za ispitivanje interakcija između liganada i kompleksa sa biološkim makromolekulima DNK i HSA. Modeliranje i simulacija je bazirana na akva vrstama novosintetisanih kompleksa rutenijuma(II), jer su hloro kompleksi slično cisplatini, manje reaktivni. Doking liganada i kompleksa čije su strukture određene rendgenskom strukturnom analizom, ispitan je korišćenjem AutoDock, AutoDock Vina i Gold softvera. Uočeno je slaganje predviđenih i eksperimentalnih rezultata. Najbolje vezana konformaciona vrsta [Ru(p-cymene)L(H2O)H]2+ je proverena primenom MOPAC metode PM6-MOZYME, pri čemu je potvrđena kovalentna veza između rutenijuma(II) i N(7) koji potiče od dG7 nukleotida. Interakcije između DNK i liganada ili kompleksa su utvrđene pomoću Discovery Studio Visualizer, pri čemu su sva ispitivana jedinjenja u energetski najpovoljnijoj konformaciji pokazala interakcije sa DT5 iz A lanca i DC18 iz B lanca DNK. Korišćenjem GOLD softverskog paketa, proučavano je vezivanje liganada i kompleksa za HSA. Dokovanje je izvršeno na svakom aktivnom mestu HSA, a rezultati ukazuju da je glutamin-292 uobičajno mesto vezivanja u asocijativnom mehanizmu vezivanja proteina.SUMMARY Within this PhD dissertation new complex compounds of ruthenium(II) were synthetized and characterized. Complexes contain coordinated imidazole type ligands ([Ru(η 6 -p-cymene)(NMeIm)3]Cl2·2H2O and [Ru(η 6 -p-cymene)(N-PrIm)Cl2]) or isothiazole type ligands ([Ru(η 6 -pcymene)Cl2(5-MA-3-PyCN-ITZ)], [Ru(η 6 -p- cymene)Cl2(5-MA-4-MpipCN-ITZ)], [Ru(η 6 -pcymene)Cl2(5-MA-3-MorphCN-ITZ)], [Ru(η 6 -p-cymene)Cl2(5-PhA-3-PyCN-ITZ)] and [Ru(η 6 - p-cymene)Cl2(3-Morph-5-PhACN-ITZ)]). Modern spectroscopic analytic methods (1H NMR, 13C NMR, IR and UV-Vis) and standard methods (elemental analysis and melting point) were employed in characterization of novel compounds. X-ray structural analysis was used to confirm structures of complexes and isolated ligands. Fluorescent emission spectroscopy and absorption spectroscopy were utilized in examination of interactions of both ligands and complexes with calf thymus DNA (CT-DNA) and with human serum albumin (HSA). The purpose of looking into interactions with biological macromolecules is determining possible binding modes and binding constants. It was found that interactions happen via static quenching mechanism. MTT cytotoxicity test and method for apoptosis and autophagy detection were used to examine in vitro biological activity of novel compounds. Complex [Ru(η 6 -p-cymene)(NMeIm)3]Cl2·2H2O shows the highest cytotoxicity and the most sensitive was HeLa cell line. Western blot and flow cytometry analisys both point towards apoptosis as primary cell death mechanism. During observations of cell cycle in the S phaze similarity was noticed between acting of ruthenium(II) complexes and acting of complexes that contain elements from platinum group of metals. For theoretical examination of ligands and complexes, modern computational chemistry methods were applied. Structural properties of complexes (bonds, angles and torsions), which were optimized with theoretical models B3LYP/def2-TZVP, B3LYP/SDD and M06 in combination with SDD basis set, are in agreement with experimentally determined data gained through X-ray structural analisys. Molecular simulation – docking, was utilized for examination of interactions of ligands and complexes with DNA and HSA. Modelling and simulation were based on aqua species of novel ruthenium(II) complexes, because chloro complexes are less reactive, similar to the behavior of cisplatin. For the docking of ligands and complexes programs AutoDock, AutoDock Vina, and GOLD were used. Conformation of [Ru(p-cymene)L(H2O)H]2+ species that showed the highest binding score was validated using MOPAC method PM6-MOZYME and it was determined that covalent bonding between ruthenium(II) and N(7) atom of dG7 nucleotide may occur. Program Discovery Studio Visualizer was used to visualize and inspect possible interactions and it was noted that in the most energetically favorable conformation interactions that form are with DT5 from A chain and DC18 from B chain of DNA. Program GOLD was used to study interactions of novel compounds with HSA molecule. Docking experiments were repeated for every possible binding place on the target molecule and results point to glutamine-292 as most probable binding residue in associative protein binding mechanism