Sinteza i biološka aktivnost Rh(III) kompleksa sa ligandima poliaminopolikarboksilatnog tipa

Abstract

U okviru ove doktorske disertacije sintetisani su i okarakterisani kompleksi rodijuma(III) sa helatnim poliaminopolikarboksilatnim ligandima edta tipa, među kojima su dva neutralna: cis-equatorial-[Rh(ed3a)H2O] i cis-polar-[Rh(1,3-pd3a)H2O], kao i tri anjonska cis-equatorial-[Rh(ed3a)Cl]-, cis-polar-[Rh(1,3-pd3a)Cl]- i trans(O5O6)- [Rh(eddadp)]-. Sva dobijena jedinjenja okarakterisana su primenom standardnih metoda (elementalna mikroanaliza, tačka topljenja), kao i savremenim spektroskopskim metodama analize (1H i 13C NMR, IR i UV-Vis). Strukture sintetisanih kompleksa su određene primenom rendgenske strukturne analize, osim u slučaju cis-polar-[Rh(1,3-pd3a)H2O]∙2H2O kompleksa gde nije izolovan kristal pogodan za difraktometrijsku analizu. Njegova geometrija je pretpostavljena na osnovu spektralne analize, DFT proračuna, kao i poređenjem sa sličnim kompleksima koji su strukturno okarakterisani. Konstante protonovanja liganada H3ed3a i H31,3-pd3a, konstante stabilnosti kompleksa i stehiometrija kompleksa sa ovim ligandima proučavane su u vodenom rastvoru, pri čemu je potvrđeno je postojanje samo mononuklearnih kompleksa kao što su [MHL] ([Rh(Hed3a)]+, [Rh(H1,3-pd3a)]+), [ML] ([Rh(ed3a)], [Rh(1,3-pd3a)]) i [MLH-1] ([RhOH(ed3a)]-, [RhOH(1,3-pd3a)]-). Biološka aktivnost ovih jedinjenja in vitro je ispitana primenom MTT testa citotoksičnosti, kao i metodama za detekciju apoptoze. Najveću citotoksičnost je pokazao kompleks [Rh(ed3a)(H2O)]·H2O, dok je najneaktivniji [Rh(1,3-pd3a)(H2O)]·2H2O kompleks, a HeLa ćelijska linija je najsenzitivnija. Rezultati protočne citometrije i Vestern blot analize ukazuju na apoptozu kao primaran način ćelijske smrti i to na kaspaza-zavistan način. Na kraju, kompjuterske metode su korišćene za interpretaciju strukturnih parametara eksperimentalno dobijenih kompleksa Rh(III), kao i njihovih mogućih izomera. Utvrđeno je da je B3LYP metoda i brojčano i vizuelno najpribližnija eksperimentalnim rezultatima dobijenim iz X-ray struktura. Detaljna analiza energetske zavisnosti geometrijskih izomera i uobičajenih parametara deformacije ((Oh), (M-O-C), (N)) ukazuje na nemogućnost formiranja trans(O6) izomera u slučaju [M(eddadp)]- sistema. U slučaju [M(1,3-pddadp)]- sistema nizak energetski profil omogućava formiranje trans(O6) izomera u slučaju kompleksa rodijuma(III), dok visoka energija u slučaju trans(O5)- [Co(1,3-pddadp)]- isključuje mogućnost formiranja ovog kompleksa. Granična energija, iza koje je nemoguće očekivati formiranje geometrijskih izomera, mogla bi imati vrednost oko 6 kcal mol- za [Rh(eddadp)]- i [Rh(1,3-pddadp)]- sisteme. NBO i NRT analize sistema [M(eddadp)]- i [M(1,3-pddadp)]- (M = Rh(III), Co(III)) pokazuju da svi izomeri predstavljaju snažno delokalizovane strukture, a sve dobijene rezonantne strukture pokazuju da je jon metala tri-koordinovan preko N2O ili NO2 hromofore, osim Co(III) u trans(O6)-[Co(eddadp)]- kompleksu koji je di-koordinovan. Takođe, kovalentnost je više izražena u rodijum(III) kompleksima nego u slučaju Co(III) kompleksa. Rezultati kompjuterski simuliranog dokinga između HSA i cis-equatorial- [Rh(ed3a)(H2O)] i cis-polar-[Rh(1,3-pd3a)(H2O)] kompleksa ukazuju da četiri vodonične veze čine rodijumov kompleks čvrsto vezanim za HSA i sasvim bezbednim za njegov transport do ciljnih ćelija.

Within this PhD dissertation, the complexes of rhodium(III) with chelate polyaminopolycarboxylate edta-type of ligands have been synthesized and characterized, among which are two neutral cis-equatorial-[Rh(ed3a)(H2O)] and cis-polar-[Rh(1,3-pd3a)(H2O)], as well as three anionic cis-equatorial-[Rh(ed3a)Cl]-, cis-polar-[Rh(1,3-pd3a)Cl]- and trans(O5O6)- [Rh(еddadp)]-. The synthesized rhodium(III) complexes were characterized by the use of standard methods (elemental microanalysis, melting point), as well as by modern spectroscopic analysis techniques (1H and 13C NMR, IR and UV-Vis). Structures of the synthesized complexes were determined by X-ray structural analysis, except in the case of cis-polar-[Rh(1,3-pd3a)(H2O)]∙2H2O complex. Its geometry is assumed based on spectral analysis, DFT calculations, as well as by comparison with similar complexes that are structurally characterized. The protonation constants of H3ed3a и H31,3-pd3a ligands, the stability constants of the complexes and the stoichiometry of the complexes with these ligands were studied in aqueous solution, whereby only mononuclear complexes have been confirmed: [MHL] ([Rh(Hed3a)]+, [Rh(H1,3-pd3a)]+), [ML] ([Rh(ed3a)], [Rh(1,3-pd3a)]) и [MLH-1] ([RhOH(ed3a)]-, [RhOH(1,3- pd3a)]-). The biological activity of these compounds in vitro was investigated using the MTT cytotoxicity test, as well as methods for the detection of apoptosis. The greatest cytotoxicity was demonstrated by the complex [Rh(ed3a)(H2O)]·H2O, while the most inactive was [Rh(1,3- pd3a)(H2O)]·2H2O, and the HeLa cell line is the most sensitive. The results of flow cytometry and Western blot analysis suggest apoptosis as the primary mode of cell death, in a caspase-dependent manner. Finally, computer methods were used to interpret the structural parameters of the experimentally obtained Rh(III) complexes, as well as their possible isomers. It was found that the B3LYP method is both numerically and visually most similar to experimental results obtained from X-ray structures. A detailed analysis of the energy dependence of geometric isomers on the usual deformation parameters ((Oh), (M-O-C), (N)) indicates the inability of the formation of trans(O6) isomer in the case of [M(eddadp)]- system. In the case of [М(1,3- pddadp)]- system low energy profile allows the formation of trans(O6) isomer for rhodium(III) complexes, while high energy of trans(O5)-[Co(1,3-pddadp)]- exclude the possibility for these complex to be formed. The boundary energy, beyond which it is impossible to expect the formation of geometric isomers, could have a value of about 6 kcal mol-1 for [Rh(eddadp)]- and [Rh (1,3-pddadp)]- systems. NBO and NRT analyzes of the [М(eddadp)]- and [М(1,3- pddadp)]- (M = Rh(III), Co(III)) systems show that all isomers are strongly delocalized structures, and all resulting resonant structures show that the metal ions are tri-coordinated through N2O or NO2 chromophores, except Co(III) in trans(O6)-[Co(eddadp)]- complex ion which adopts di-coordination in an octahedral surrounding. Also, covalency is more pronounced in the complexes of rhodium(III) than in the case of cobalt(III) complexes. The results of a computer simulated docking between the HSA and cis-equatorial- [Rh(ed3a)(H2O)] and cis-polar-[Rh(1,3-pd3a)(H2O)] complexes indicate that four hydrogen bonds make rhodium complex tightly bounded to HSA and quite safe for his transport to the target cells.