Sinteza, karakterizacija i biološka aktivnost kompleksa srebra(i) i zlata(iii) sa aromatičnim heterocikličnim jedinjenjima koja sadrže azot u prstenu

Abstract

U okviru ove doktorske disertacije opisana je sinteza, strukturna karakterizacija i rezultati ispitivanja antimikrobne i citotoksične aktivnosti kompleksa srebra(I) i zlata(III) sa aromatičnim heterocikličnim jedinjenjima koja sadrže azot u prstenu. U cilju definisanja mehanizma delovanja novosintetisanih kompleksa ispitivane su njihove interakcije sa biomolekulima (DNK i BSA), hemolitički potencijal i mogućnost formiranja reaktivnih kiseoničnih vrsta (eng. "Reactive oxygen species, ROS"). U prvom delu disertacije prikazana je sinteza, spektroskopska karakterizacija i biološka aktivnost kompleksa srebra(I) koji nastaju u reakcijama: 1) AgNO3 sa piridazinom (pydz), pirimidinom (pm), pirazinom (pz), hinoksalinom (qx) i fenazinom (phz). Ovi kompleksi su polinuklearni, pri čemu njihova geometrija zavisi od položaja azota u prstenu odgovarajućeg liganda (RSC Advances, 6 (2016) 13193-13206); 2) AgNO3 sa hinazolinom (qz) i AgBF4 sa ftalazinom (phtz). U reakciji između AgNO3 i qz nastaje polinuklearni [Ag(NO3)(qz)]n kompleks, dok u reakciji između AgBF4 i phtz nastaje dinuklearni [{Ag(CH3CN)}2(µ-phtz)2](BF4)2 kompleks (MedChemComm, 7 (2016) 282-291); 3) različitih AgX soli sa 1,7-fenantrolinom (1,7-phen), pri čemu nastaju mononuklearni [Ag(NO3-O,O’)(1,7-phen-N7)2] i [Ag(1,7-phen-N7)2]X kompleksi (X = ClO4-, CF3SO3-, BF4- i SbF6-). U ovim kompleksima, 1,7-phen je monodentatno koordinovan za srebro(I) jon preko sterno manje zaklonjenog N7 atoma azota (European Journal of Medicinal Chemistry, 156 (2018) 760-773); 4) različitih AgX soli (X = NO3-, ClO4-, BF4-, CF3COO-, SbF6-) sa 4,7- fenantrolinom (4,7-phen), pri čemu nastaju polinuklearni [Ag(NO3-O)(4,7-phen-µ- N4,N7)]n, [Ag(ClO4-O)(4,7-phen-µ-N4,N7)]n, {[Ag(4,7-phen)][Ag(4,7- phen)(H2O)](BF4)2}n, [Ag(CF3COO-O)(4,7-phen-µ-N4,N7)]n i dinuklearni [Ag2(H2O)(4,7-phen)3](SbF6)2 kompleksi. Kompleksi su okarakterisani primenom spektroskopskih metoda (1H i 13C NMR, IR, UV-vis), masene spektrometrije i elektrohemijskih metoda (ciklična voltametrija). Strukture sintetisanih kompleksa su određene primenom rendgenske strukturne analize. Ispitivana je stabilnost kompleksa u rastvoru, pri čemu su kompleksi veoma stabilni 24 i 48 h nakon rastvaranja. Pored toga, praćena je stabilnost kompleksa na svetlosti i vazduhu, pri čemu je uočena njihova spora fotodegradacija. Sintetisani kompleksi srebra(I) su ispitivani prema različitim sojevima Gram-pozitivnih (Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis) i Gram-negativnih (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Klebsiella pneumoniae) bakterija i prema različitim Candida sojevima (C. albicans, C. parapsilosis, C. glabrata, C. krusei). Ovi testirani sojevi mogu biti uzročnici različitih infekcija kože, mekih tkiva i rana, kao i respiratorne i nozokomijalne (intrahospitalne) infekcije. Antimikrobna aktivnost sintetisanih kompleksa srebra(I) je izražena kao vrednost minimalne inhibitorske koncentracije (eng. "Minimum inhibitory concentration, MIC"). U cilju određivanja terapeutskog potencijala ovih kompleksa, ispitivana je njihova antiproliferativna aktivnost (citotoksičnost) prema normalnoj ćelijskoj liniji fibroblasta pluća (MRC5). Ispitivane su interakcije sintetisanih kompleksa sa DNK, pri čemu su kod većine kompleksa uočene elektrostatičke interakcije,najverovatnije preko malog žleba. Pored toga, u cilju definisanja mehanizma delovanja, ispitivan je hemolitički potencijal, to jest sposobnost da utiču na ćelijsku membranu, kao primarni ciljni molekul, većine sintetisanih kompleksa, pri čemu je uočeno da oni uzrokuju lizu eritrocita, čime oštećuju ćelijsku membranu i omogućavaju isticanje ćelijskih komponenata iz unutrašnjosti ćelije u međućelijski prostor. Ispitivana je mogućnost inhibicije filamentnog rasta C. albicans sintetisanih kompleksa koji pokazuju inhibiciju rasta gljiva, pri čemu većina kompleksa u potpunosti inhibira i razvoj hifa. Za [Ag(NO3-O,O’)(1,7-phenN7)2] kompleks, koji pokazuje najveću antifungalnu aktivnost, ispitivan je uticaj na formiranje reaktivnih kiseoničnih vrsta, što se smatra jednim od mehanizama delovanja kompleksa srebra(I). Pored toga, za [{Ag(NO3)}2(µ-pydz)2]n kompleks koji je pokazao najveću aktivnost prema Pseudomonas aeruginosa bakteriji, kao i za [Ag(NO3-O,O’)(1,7-phen-N7)2], prikazani su rezultati ispitivanja in vivo embriotoksičnosti na modelu zebrica (Danio rerio). U drugom delu disertacije prikazana je sinteza, spektroskopska karakterizacija i biološka aktivnost različitih kompleksa zlata(III) sa aromatičnim heterocikličnim ligandima koji sadrže azot u prstenu i to: 1) mononuklearnih kompleksa zlata(III) sa diazinima, piridazinom (pydz), pirimidinom (pm) i pirazinom (pz), diazanaftalenom, hinoksalinom (qx) i tricikličnim aromatima, fenazinom (phz), 1,7- i 4,7- fenantrolinom (1,7-phen i 4,7-phen) (RSC Advances, 6 (2016) 13193-13206; Journal of Inorganic Biochemistry, 174 (2017) 156-168); 2) dinuklearnih kompleksa zlata(III) sa 4,4’-bipiridinom (4,4’-bipy) i 1,2- bis(4-piridil)etanom (bpe) (MedChemComm, 7 (2016) 1356-1366). Svi kompleksi su okarakterisani primenom spektroskopskih metoda (1H i 13C NMR, IR, FIR, UV-vis). Strukture sintetisanih kompleksa su određene primenom rendgenske strukturne analize. Ispitivana je stabilnost kompleksa u rastvoru, pri čemu su oni veoma stabilni 24 i 48 h nakon rastvaranja. Pored toga, ispitivana je stabilnost kompleksa na svetlosti i vazduhu, pri čemu nije uočena fotodegradacija. Primenom teorije funkcionala gustine (DFT) izvršena je optimizacija geometrije sintetisanih kompleksa u vakuumu i odgovarajućem rastvaraču. Kompleksi zlata(III) su ispitivani prema različitim sojevima Grampozitivnih (S. aureus, L. monocytogenes, Micrococcus luteus) i Gram-negativnih (E. coli, P. aeruginosa, S. typhimurium) bakterija i prema gljivi C. albicans. U cilju određivanja indeksa selektivnosti, odnosno terapeutskog potencijala ispitivanih kompleksa, minimalne inhibitorske koncentracije su poređene sa njihovom antiproliferativnom aktivnošću na normalnim ćelijskim linijama fibroblasta pluća MRC5. Ispitivane su interakcije kompleksa zlata(III) sa pydz, pm, pz, qx i phz sa DNK primenom gel elektroforeze, pri čemu se većina ispitivanih kompleksa interkalira između dvostrukog lanca DNK. Primenom fluorimetrijskih metoda ispitivane su interakcije dinuklearnih kompleksa zlata(III) sa 4,4’-bipy i bpe sa BSA. Na osnovu vrednosti Stern-Volmerovih konstanti, može se zaključiti da oba kompleksa imaju identičnu tendenciju vezivanja za BSA. Za komplekse zlata(III) sa 1,7- i 4,7-phen određene su IC50 vrednosti (μM) na normalnim ćelijskim linijama fibroblasta pluća (MRC5) i ćelijama tumora grlića materice (HeLa) i adenokarcinoma pluća (A549) i poređene su sa odgovarajućim vrednostima za auranofin (zlato(I) kompleks koji se koristi u kliničkoj praksi). Ispitivana jedinjenja nisu posebno aktivna prema tumorskim ćelijama, ali i ne pokazuju toksičnost prema normalnim ćelijama fibroblasta pluća, dok je auranofin izuzetno citotoksičan prema ispitivanim ćelijskim linijama. Pored toga, ispitivana je in vivo embriotoksičnosti na modelu zebrica (Danio rerio). Zbog uočene pojave redukovane repne cirkulacije kod zebrica, ispitivan je antiangiogeni potencijal kompleksa zlata(III) sa 1,7- i 4,7-phen, pri čemu su oni inhibirali angiogenezu i pokazali znatno manju toksičnost od auranofina i metastatskog agensa sunitiniba (Journal of Inorganic Biochemistry, 174 (2017) 156- 168). Dobijeni rezultati u okviru ove disertacije predstavljaju značajan doprinos sintezi novih kompleksa srebra(I) i zlata(III) kao potencijalnih terapeutskih agenasa, koji bi se primenjivali za lečenje multirezistentnih bakterijskih i gljivičnih infekcija i različitih vrsta tumora.

In this PhD thesis, synthesis, structural characterization and evaluation of the antimicrobial and cytotoxic activities of the silver(I) and gold(III) complexes with aromatic nitrogen-containing heterocycles have been reported. In order to define the mechanism of action of novel complexes, their interactions with biomolecules (DNA and BSA) have been investigated, as well as their hemolytic potential and ability to produce reactive oxygen species (ROS). In the first part of this thesis, synthesis, structural characterization and biological evaluation of the silver(I) complexes are reported. These silver(I) complexes were obtained in the reactions of: 1) AgNO3 with pyridazine (pydz), pyrimidine (pm), pyrazine (pz), quinoxaline (qx) and phenazine (phz). The obtained complexes are polynuclear, and their geometry depends on the nitrogen position in the aromatic ring of the corresponding ligand (RSC Advances, 6 (2016) 13193-13206); 2) AgNO3 with quinazoline (qz) and AgBF4 with phthalazine (phtz). In the reaction between AgNO3 and qz, polynuclear [Ag(NO3)(qz)]n complex was formed, while the reaction between AgBF4 and phtz led to the formation of dinuclear [{Ag(CH3CN)}2(µ- phtz)2](BF4)2 complex (MedChemComm, 7 (2016) 282-291); 3) different AgX salts and 1,7-phenanthroline (1,7-phen). In these reactions, the mononuclear [Ag(NO3-O,O’)(1,7-phen-N7)2] and [Ag(1,7-phen-N7)2]X (X = ClO4-, CF3SO3-, BF4- and SbF6-) complexes were formed. In these complexes, 1,7-phen is monodentately coordinated to the Ag(I) ion through the sterically less hindered N7 nitrogen (European Journal of Medicinal Chemistry, 156 (2018) 760-773); 4) different AgX salts (X = NO3-, ClO4-, BF4-, CF3COO-, SbF6-) and 4,7- phenanthroline (4,7-phen). In these reactions, polynuclear [Ag(NO3-O)(4,7-phen-µ- N4,N7)]n, [Ag(ClO4-О)(4,7-phen-µ-N4,N7)]n, {[Ag(4,7-phen)][Ag(4,7- phen)(H2O)](BF4)2}n, [Ag(CF3COO-O)(4,7-phen-µ-N4,N7)]n and dinuclear [Ag2(H2O)(4,7-phen)3](SbF6)2 complexes were formed. The synthesized silver(I) complexes were characterized by spectroscopic techniques (1H and 13C NMR, IR, UV-vis), mass spectrometry and electrochemical methods (cyclic voltammetry). Their structures were determined by single-crystal X-ray diffraction analysis. The stability of the complexes was investigated in solution. It was found that these complexes were very stable during 24 and 48 h after their dissolution. Moreover, the air/light stability of the complexes was monitored, showing a slow photodegradation of the complexes during time. The synthesized silver(I) complexes were evaluated against different Gram-positive (Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis) and Gramnegative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Klebsiella pneumoniae) bacteria and various Candida strains (C. albicans, C. parapsilosis, C. glabrata, C. krusei). All these pathogens can lead to many skin and soft tissue, respiratory, wound and nosocomial infections. The antimicrobial activity of the complexes was expressed as the minimum inhibitory concentration (MIC) value. In order to determine the therapeutic potential of the complexes, their antiproliferative effect on the normal human lung fibroblast cell line MRC5 was evaluated. The interactions of the synthesized silver(I) complexes with DNA were studied. It was found that some of the complexes bind to DNA through the electrostatic interactions, most likely through a minor groove. In order to define the mechanism of action of silver(I) complexes, their hemolytic potential was investigated. It was found that these complexes can cause lysis of the red blood cells by disrupting permeability of the cell membrane and leakage of cellular constituents. Most of the silver(I) complexes completely inhibit development of hyphae of C. albicans. The [Ag(NO3-O,O’)(1,7-phen-N7)2] complex showing the highest antifungal activity can induce the formation of reactive oxygen species. This is very important because one of the possible mechanism of action of silver(I) complex is related to the ROS formation leading to the cell death. Additionally, in vivo embryotoxicity of [{Ag(NO3)}2(μ-pydz)2]n complex showing the highest activity towards P. aeruginosa and [Ag(NO3-O,O’)(1,7-phen-N7)2] was evaluated on the zebrafish model (Danio rerio). In the second part of the thesis, synthesis, structural characterization and biological evaluation of gold(III) complexes with aromatic N-heterocycles have been reported. The synthesized series of gold(III) complexes includes: 1) mononuclear gold(III) complexes with diazines, pyridazine (pydz), pyrimidine (pm) and pyrazine (pz), diazanaphthalene, quinoxaline (qx) and tricyclic aromatic compounds, phenazine (phz), 1,7- and 4,7- phenanthroline (1,7-phen and 4,7-phen) (RSC Advances, 6 (2016) 13193-13206; Journal of Inorganic Biochemistry, 174 (2017) 156- 168); 2) dinuclear gold(III) complexes with 4,4’-bipyridine (4,4’-bipy) and 1,2-bis(4- pyridyl) ethane (bpe) (MedChemComm, 7 (2016) 1356-1366). The synthesized gold(III) complexes were characterized by different spectroscopic techniques (1H and 13C NMR, IR, FIR, UV-vis). Their structures were determined by single-crystal X-ray diffraction analysis. The stability of the complexes was investigated in the solution. The obtained results showed that these complexes are very stable during 24 and 48 h after their dissolution. Additionally, the air/light stability of the complexes was investigated, indicating that no photodegradation process was observed. Density functional theory (DFT) was used for the optimization of the complexes’ geometry in vacuo and in the corresponding solvent. Gold(III) complexes with aromatic N-heterocycles were evaluated against different Gram-positive (S. aureus, L. monocytogenes, Micrococcus luteus) and Gram-negative (E. coli, P. aeruginosa, S. typhimurium) bacteria and the fungus C. albicans. In order to determine the selectivity index and therapeutic potential of the investigated gold(III) complexes, the MIC values were compared with their antiproliferative activity on the normal MRC5 cell line. The DNA interactions of the gold(III) complexes with pydz, pm, pz, qx and phz were investigated by gel electrophoresis. Most of these complexes could be intercalated between the double stranded DNA. The interactions of dinuclear gold(III) complexes with 4,4’-bipy and bpe and BSA were investigated by fluorimetry. Based on the values of the Stern-Volmer constants, it can be concluded that both of complexes have identical binding tendency to BSA. The IC50 values (μM) on the MRC5 cell line and two cancer cell lines (cervical cancer cell line (HeLa) and adenocarcinoma cell line (A549)) were determined for the gold(III) complexes with 1,7- and 4,7-phenanthroline and compared with those for auranofin. These complexes were not active on cancer cell lines, but also do not show toxicity on the normal lung fibroblast cell line, while auranofin was very cytotoxic. Additionally, in vivo embryotoxicity of the gold(III) complexes with 1,7- and 4,7-phenanthroline was studied on the zebrafish model (Danio rerio). Prompted by the observed reduced caudal circulation during the embryotoxicity assessment, the potential of gold(III) complexes with 1,7- and 4,7-phenanthroline to affect angiogenesis in vivo was evaluated on the zebrafish model. The obtained results showed that both of these complexes inhibited angiogenesis with significantly lower toxicity in comparison to auranofin and sunitinib, clinically used agents (Journal of Inorganic Biochemistry, 174 (2017) 156-168). The results of this thesis can contribute to the development of new silver(I) and gold(III) complexes as potential therapeutic agents that could be used for the treatment of multiresistant bacterial and fungal infections and cancer.