Sinteza i antitumorna aktivnost paladijum(II) sa derivatima oksalne i malonske kiseline

U okviru ove disertacije sintetisani su paladijum(II) kompleksi sa helatnim ligandima amidnog tipa. Sintetisani su derivati diamida malonske i oksalne kiseline sa aminokiselinskim ostacima, ostacima antranilne kiseline ili propandiaminskim ostacima vezanim za obe strane amidne jedinice. Derivati oksalne kiseline: (3,3'-[1,2-dioksoetan-1,2diil)diimino] dipropionska kiselina) (H4obp); (N,N'-bis(3-aminopropil) etandiamid) (H2apox); (2-({[(3-aminopropil)amino] (okso)acetil}amino) benzoeva kiselina) (H3obap); (2,2'-[(1,2-dioksoetan-1,2- diil)diimino] (H4obbz) i malonske kiseline: (2,2'-[1,3-dioksopropan- 1,3diil)diimino] disirćetna kiselina) (H4mda) i 3,3'- [(1,3-dioksopropan-1,3- diil)diimino] dipropanska kiselina (H4mdp) su korišćeni za sintezu i ispitivanje biološke aktivnosti odgovarajućih paladijum(II) kompleksa. Prva faza disertacije podrazumevala je primenu potenciometrijskih metoda u cilju ispitivanja stabilnosti vodenih rastvora samih liganada, smeše paladijum(II) jona i odgovarajućih liganada u različitim koncentracionim odnosima, kao i Pd-L-A sistema da bi se ispitao uticaj binarnih kompleksa [PdL] na nukleozide koji ulaze u sastav DNK. Ispitivanje interakcija sintetisanih jedinjenja i molekula HSA i DNK vršeno je primenom fluorescentne i apsorpcione spektroskopije. Dobijeni rezultati su pokazali da postoji dobro vezivanje naših sistema sa biološki značajnim molekulima, što nas je dovelo do sledeće faze koja je podrazumevala in vitro eksperimente. Biološka aktivnost ovih jedinjenja in vitro ispitana je primenom MTT testa citotoksičnosti i metoda za detekciju apoptoze. Eksperimentalno dobijeni rezultati su u saglasnosti sa teorijskim i ukazuju da biološka aktivnost kompleksa paladijuma(II) veoma zavisi od strukture azot-kiseonik donorskih liganada. Najveću osetljivost je pokazala HeLa ćelijska linija, a posebno kada su u pitanju K2[Pd(mdp)] · H2O i H4obbz jedinjenja. Nedavne studije su pokazale da se kod nekih paladijum(II) kompleksa sa helat O- donorskim ligandima može očekivati da indukuju ER stres i jedro nezavisnu signalizaciju apoptoze. U skladu sa tim ispitivan je (teorijski i eksperimentalno) potencijalni mehanizam delovanja leka koji podrazumeva interakcije HSP proteina (protein toplotnog šoka) i šaperona (Grp78 iz endoplazmatskog retikuluma (ER) i Hsc70 iz citosola) i [Pd(L)]n- jedinice ispitivanih sistema. Između odabranih makromolekula, gde su naši kompleksi računski simulirani, grupa šaperon proteina (Grp78 iz ER i Hsc70 iz citosola) su se pokazali kao bolji domaćini od oligonukleotida (DNK). Takođe, Vestern Blot analiza pronalazi da je ekspresija Bcl-2 proteina i cepanje PARP u skladu sa našim pretpostavkama. Analiza ćelijskog ciklusa vršena je primenom elektroforetske Vestern blot analize i protočne citometrije. Rezultati su pokazali da antitumorna aktivnost paladijum(II) kompleksa sa različitim vrstama liganada na HeLa humanim ćelijama izazivaju apoptozu na kaspaza-zavisan način. Takođe, ispitivan je potencijalni mehanizam dejstva K2[Pd(mdp)] · H2O i H4obbz jedinjenja na malignoj HeLa ćelijskoj liniji. Rezultati su pokazali da testirana jedinjenja indukuju apoptozu u velikom procentu ispitivanih ćelija. Pored toga, progresija ćelijskog ciklusa HeLa ćelija tretiranih ovim jedinjenjima pokazuju zaustavljanje sinteze DNK i povećanje ćelijske populacije u G0/G1 fazi kao mogući mehanizam koji pokreće apoptozu. Na kraju, primenom klasične i QM/MM molekulske dinamike istražene su konformacione promene, slobodne energije vezivanja liganada-receptora i njihove kovalentne interakcije i predložen mehanizam dejstva ispitivanih sistema. Utvrđena je primetna korelacija između IC50 (toksičnost) i ΔG (slobodne vezivne energije) vrednosti kod sintetisanih jedinjenja. Na osnovu dobijenih rezultata, pre svega kada su u pitanju K2[Pd(mdp)] · H2O i K2[Pd(mda)] · H2O kompleksi, u planu je sledeća faza ispitivanja farmakoloških svojstava paladijumovih lekova.As part of this thesis palladium(II) complexes were synthesized with chelate amide type ligands. There were synthesized the derivatives of malonic and oxalic acid from the amino acid residues, residues of an anthranilic acid or propandiamine residues bounded to both sides of the amide units. Oxalic acid derivatives: (3,3'-[1,2-dioksoethane-1,2diyl)diamines] dipropionic acid) (H4obp); (N,N'-bis(3-aminopropyl)ethanediamide) H2apox); (2-({[(3- aminopropyl)amino] (oxo)acetyl} amino)benzoic acid) (H3obap), (2,2'-[(1,2-dioksoetan-1,2- diyl)diamines] (H4obbz) and malonic acid: (2,2'-[1,3-dioksopropan-1,3diyl)diamines] diacetic acid) (H4mda) and 3,3'-[(1,3-dioksopropan-1,3-diyl)diamines] dipropionic acid (H4mdp), the ligands were prepared and used for synthesis and biology tests of the corresponding palladium(II) complexes. The first phase of this thesis, involved the application of potentiometric methods in order to investigate the stability of aqueous solutions of ligands, a mixture of palladium(II) ions and the corresponding ligands in different relations of concentration, and Pd-L-A system in order to investigate the effect of binary complexes [PdL] to nucleosides entering in DNA. The fluorescent and absorption spectroscopy were used to see interactions of the synthesized compounds and HSA and DNA molecules. The results showed that there is a good binding of our system with biologically significant molecules, which led us to the next phase, involving the in vitro experiments. The in vitro biological activity of these compounds was examined by the MTT cytotoxicity assay and methods for detecting apoptosis. Experimental results correspond with theoretical and indicate that the biological activity of the palladium(II) compounds is highly dependent on the structure of the nitrogen-oxygen donor ligands. HeLa cell line has been shown the highest sensitivity, especially in the case of K2[Pd(mdp)] · H2O and H4obbz compounds. Recent studies have shown that some palladium(II) complexes with chelating O-donor ligands may be expected to induce an ER stress and core independent apoptosis signaling. Accordingly, it was investigated (theoretically and experimentally) a potential mechanism of drug action which involves the interaction of proteins HSP (heat shock protein) and the chaperone (Grp78 from the endoplasmic reticulum (ER) and HSC70 from the cytosol ) and [Pd(L)]n- units. Between chosen macromolecules, where our compounds were computationally simulated, the group of chaperon proteins (Grp78 from ER and Hsc70 from cytosol) have evinced as better hosts than oligonucleotides (DNA). Also, Western Blot analysis found that the expression of Bcl-2 protein and the cleavage of PARP are in accordance with our assumptions. Analysis of cell cycle was performed by method of electrophoretic analysis of the Western blot and flow cytometry. The results showed that the antitumor activity of palladium(II) complexes with different types of ligands on the HeLa human cells induce apoptosis in caspase-dependent manner. Also, it was examined a potential mechanism of action of K2[Pd(mdp)] · H2O and H4obbz compounds to malignant HeLa cell line. The results showed that the tested compounds induce apoptosis in a large percentage of the examined cells. In addition, the progression of the cell cycle of HeLa cells treated with these compounds, show the stopping of DNA synthesis and an increase of the cell population in the G0/G1 phase as a possible mechanism by which trigger apoptosis. Finally, classical and QM/MM molecular dynamics were used for studied conformational change, free energy of binding ligands and their receptor-covalent interactions, and proposed mechanism of action of the tested system. There was a noticeable correlation between the IC50 (toxicity) and ΔG (free binding energies) values of the synthesized compounds. The results, especially when it comes to K2[Pd(mdp)] · H2O and K2[Pd(mda)] · H2O complexes, indicate the next phase of pharmacological properties of our drugs