Sinteza, karakterizacija i citotoksičnost kompleksa zlata (III) sa estrima R2edda-tipa : doktorska disertacija
Pantelić, Nebojša Đ., 1982-
Sabo, Tibor, 1958-
Zmejkovski, Bojana
Kaluđerović, Goran
Gruden-Pavlović, Maja, 1970-
Stanojković, Tatjana
U ovom radu opisane su sinteze, karakterizacija i antiproliferativna aktivnost kompleksa zlata(III) sa dialkil estrima (S,S)-etilendiamin-N,N’-di-2-propanske kiseline, (S,S)-H2eddip, (S,S)-etilendiamin-N,N’-di-2-(4-metil)-pentanske kiseline, (S,S)-H2eddl, (S,S)-etilendiamin-N,N’-di-2-(3-cikloheksil)-propanske kiseline, (S,S)-H2eddch. Sintetisan je novi diizoamil estar sa (S,S)-H2eddip. Ovaj ligand prekursor je dobijen refluktovanjem suspenzije kiseline i apsolutnog izoamil alkohola, kome je prethodno ukapan tionil-hlorid. Estar je dobijen u obliku dihidrohlorida, [(S,S)-H2iAm2eddip]Cl2. Okarakterisan je elementalnom analizom, infracrvenom i NMR spektroskopijom, masenom spektrometrijom i polarimetrijskom analizom. Kompleksi zlata(III) dobijeni su u reakciji natrijum-tetrahloridoaurata(III) dihidrata, Na[AuCl4]∙2H2O, sa O,O’-dialkil-(S,S)-etilendiamin-N,N’-di-2-propanoatom, (R = nBu, nPe, iBu, iAm, cPe) ili O,O’-dialkil-(S,S)-etilendiamin-N,N’-di-2-(4-metil)-pentanoatom, (R = nPr, nBu, nPe, iBu) ili O,O’-dialkil-(S,S)-etilendiamin-N,N’-di-2-(3-cikloheksil)-propanoatom, (R = Me, Et, nPr, nBu, iBu, iAm) u metanolu i litijum-hidroksida u molskom odnosu 1:1:2, a kompleksi su dobijeni nakon dodavanja čvrstog amonijum-heksafluorofosfata. Kompleksi su opšte formule [AuCl2{(S,S)-R2eddip}]PF6 (R = nBu, nPe, iBu, iAm, cPe) i [AuCl2{(S,S)-R2eddl}]PF6 (R = nPr, nBu, nPe, iBu), [AuCl2{(S,S)-R2eddch}]PF6 (R = Me, Et, nPr, nBu, iBu, iAm). Okarakterisani su elementalnom analizom, UV/Vis, infracrvenom i NMR spektroskopijom i masenom spektrometrijom. Za sve sintetisane komplekse urađeni su DFT proračuni. Da bi se bolje razumeo mehanizam delovanja kompleksa zlata(III) kao antitumorskih agenasa, urađeno je elektrohemijsko ispitivanje svih kompleksa iz serije [AuCl2{(S,S)-R2eddip}]PF6 i [AuCl2{(S,S)-R2eddch}]PF6. Cikličnom i diferencijalnom pulsnom voltametrijom utvrđeno je da se redukcija zlato(III) kompleksa vrši do zlato(I) vrste, u vidu dva ireverzibilna elektronska koraka, praćena gubitkom hlorido liganda. Pojava redukcionog koraka AuIII/Au0 se isključuje zbog izostanka elementalnog zlata na platinskoj elektrodi što je potvrđeno nakon redukcije pri konstantnom potencijalu od −0,8 V (vs. Ag/AgCl) u trajanju od 15 minuta. Antiproliferativna aktivnost sintetisanih kompleksa određena je prema tumorskim ćelijama: humanog adenokarcinoma materice (HeLa), humanog malignog melanoma (Fem-x), humane mijeloidne leukemije (K562), kao i na zdravim humanim mononuklearnim ćelijama, izolovanim iz periferne krvi (PBMC), kao i na stimulisanim na proliferaciju PBMC ćelijama (PBMC + PHA) ili ćelijama fetalnog plućnog fibroblasta (MRC-5). Svi kompleksi iz serije [AuCl2{(S,S)-R2eddip}]PF6 pokazuju visoku citotoksičnu aktivnost prema svim ćelijskim linijama, a najveću prema Fem-x ćelijama. Najnižu IC50 vrednost prema Fem-x ćelijama pokazuje kompleks [AuCl2{(S,S)-iAm2eddip}]PF6, ali istovremeno i najvišu prema HeLa i K562 ćelijskim linijama. Indeks selektivnosti ovih kompleksa pokazuje da su manje toksični i znatno selektivniji od cisplatine. Posebno treba istaći da je kompleks [AuCl2{(S,S)-iAm2eddip}]PF6 4 puta aktivniji u 28 puta selektivniji od cisplatine. Kompleksi iz serije [AuCl2{(S,S)-R2eddl}]PF6 pokazuju najveću aktivnost prema K562 ćelijama koja je uporediva sa referentnom supstancom, cisplatinom. Iz serije [AuCl2{(S,S)-R2eddch}]PF6, najaktivniji je kompleks kada je R = iAm prema K562 ćelijama koji je aktivniji i od cisplatine, ali je umereno aktivan prema HeLa ćelijskoj liniji. Ovaj kompleks je ujedno i najselektivniji. Svi ispitivani kompleksi indukuju apoptozu. Ispitivanje stabilnosti kompleksa [AuCl2{(S,S)-iBu2eddip}]PF6 u DMSO-u i fiziološkom medijumu (PBS, pH 7,4) praćeno je pomoću UV/Vis i 13C NMR spektroskopije. Ispitivani kompleks je stabilan u DMSO-u tokom 24-časovnog praćenja UV/Vis spektroskopijom. Snimljeni 13C NMR spektri kompleksa u PBS-u tokom vremena (0, 2, 24 i 48 h) pokazuju koordinacione promene u kompleksu tako da verovatno dolazi do supstitucije hlorido liganada molekulima vode pri čemu nastaju [AuCl(H[AuCl(H[AuCl(H[AuCl(H[AuCl(H[AuCl(H[AuCl(H2O){(O){(O){(O){(S,S )-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]2+ ili [Au(H[Au(H[Au(H[Au(H[Au(H2O) 2{( S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]3+ . U cilju ispitivanja mogućnosti da se kompleksi zlata(III) redukuju u ćeliji sa biološki relevantnim reducentom, praćena je reakcija kompleksa [AuCl2{(S,S)-iBu2eddip}]PF6 sa askorbinskom kiselinom, snimanjem 13C NMR spektara. Ispitivanja su pokazala da askorbinska kiselina trenutno redukuje kompleks, što ukazuje na visoku mogućnost istog ishoda u živim ćelijama. Takođe, praćena je interakcija kompleksa [AuCl2{(S,S)-iBu2eddip}]PF6 sa goveđim serum albuminom (BSA) pomoću UV/Vis spektroskopije. Pretpostavlja se da se kompleks zlata(III), redukuje cisteinom iz albumina do zlato(I) vrste, što se može videti na spektrima nakon 2 sata reakcije. Nakon 24 i 48 h, UV/Vis spektri ukazuju da dolazi do disproporcionisanja zlata(I) do odgovarajućih zlato(III) jedinjenja i elementalnog zlata.
Hemija - Opšta i neorganska hemija / Chemistry - General and Inorganic Chemistry Datum odbrane: 18.05.2015.
This thesis describes synthesis, characterization and antiproliferative activity of gold(III) complexes with dialkyl esters of (S,S)-ethylenediamine-N,N’-di-2-propanoic acid, (S,S)-H2eddip, (S,S)-ethylenediamine-N,N’-di-2-(4-methyl)-pentanoic acid, (S,S)-H2eddl and (S,S)-ethylenediamine-N,N’-di-2-(3-cyclohexyl)-propanoic acid, (S,S)-H2eddch. A novel diisoamyl ester of (S,S)-H2eddip is synthesized. Thionyl chloride was introduced into a flask containing absolute isoamyl alcohol. (S,S)-H2eddip∙HCl was added forming a suspension which was reflucted. The ester was obtained as a dihydrochloride, [(S,S)-H2iAm2eddip]Cl2. This compound was characterized by elemental analysis, IR and NMR spectroscopy, mass spectrometry and polarimeter analysis. Gold(III) complexes are synthesized in a reaction of sodium-tetrachloroaurate(III) dihydrate, Na[AuCl4]∙2H2O, with O,O’-dialkyl-(S,S)-ethylenediamine-N,N’-di-2-propanoate, (R = nBu, nPe, iBu, iAm, cPe) or O,O’-dialkyl-(S,S)-ethylenediamine-N,N’-di-2-(4-methyl)-pentanoate, (R = nPr, nBu, nPe, iBu) or O,O’-dialkyl-(S,S)-ethylenediamine-N,N’-di-2-(3-cyclohexyl)-propanoate, (R = Me, Et, nPr, nBu, iBu, iAm) in methanol and lithium hydroxide in molar ratio 1:1:2. Desired complexes were obtained after addition of amonium hexafluorphosphate to the reaction mixture. Complexes general formulae are: [AuCl2{(S,S)-R2eddip}]PF6 (R = nBu, nPe, iBu, iAm, cPe), [AuCl2{(S,S)-R2eddl}]PF6 (R = nPr, nBu, nPe, iBu) and [AuCl2{(S,S)-R2eddch}]PF6 (R = Me, Et, nPr, nBu, iBu, iAm). These compounds are characterized by elemental analysis, UV/Vis, IR and NMR spectroscopy and mass spectrometry. DFT calculations were done for all synthesized complexes. In order to explain the mechanism of action of gold(III) complexes as antitumor agents, redox chemistry was studied by cyclic and differential pulse voltammetry of [AuCl2{(S,S)-R2eddip}]PF6 and [AuCl2{(S,S)-R2eddch}]PF6 complexes. The investigation confirmed two successive irreversible reduction steps followed by loss of chlorido ligands where AuI species were the final reduction product. The occurrence of the AuIII/Au0 reduction is rejected due to the lack of metalic gold at platinum electrode. This observation was also confirmed by potentiostatic reduction at –0.8 V (vs. Ag/AgCl) electrode for 15 min. In vitro antiproliferative activity of gold(III) complexes was determined against several tumor cell lines: human adenocarcinoma (HeLa), human myelogenous leukemia (K562), human malignant melanoma (Fem-x) as well as against normal and stimulated for proliferation human peripheral blood mononuclear cells (PBMC, PBMC + PHA) or human embryonic lung fibroblast (MRC-5). All complexes from series [AuCl2{(S,S)-R2eddip}]PF6 exhibit high activity against all three cancer lines, the highest against Fem-x cells. The lowest IC50 value is observed against Fem-x cells by complex [AuCl2{(S,S)-iAm2eddip}]PF6 and at the same time the highest against HeLa and K562. This complex is 4 times more active i 28 times more selective than cisplatin. Generally, selectivities of these complexes are significantly greater than cisplatin. Complexes from series [AuCl2{(S,S)-R2eddl}]PF6 show the highest activity against K562 cells comparable to the reference compound cisplatin. Complex [AuCl2{(S,S)-iAm2eddch}]PF6 was found to be the most effective against K562 cell line as well as to have higher activity in relation to cisplatin, but it was found moderately active against HeLa cell line. This complex also expressed the highest selectivity. All tested complexes induce apoptosis. The stability of [AuCl2{(S,S)-iBu2eddip}]PF6 was investigated in DMSO and physiological medium (PBS, pH 7,4) and experiments have been monitored by UV/Vis and 13C spectroscopy. Complex [AuCl2{(S,S)-iBu2eddip}]PF6 is stable in DMSO during 24 h monitoring by UV/Vis spectra. Stability study of [AuCl2{(S,S)-iBu2eddip}]PF6 in PBS, examined by 13C NMR spectroscopy at different time intervals (0, 2, 24 and 48 h), immediately showed coordination changes in the complex which presumably indicates instant coordination of water by displacement of the chlorido ligands to provide [AuCl(H[AuCl(H[AuCl(H[AuCl(H[AuCl(H[AuCl(H[AuCl(H2O){(O){(O){(O){(S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]2+ or [Au(H[Au(H[Au(H[Au(H[Au(H2O) 2{( S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]3+ species.species.species.species.species.species.species.species. In order to investigate the possibility of [AuCl[AuCl[AuCl[AuCl[AuCl2{( S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]PF6 being reduced in cells with a biologically relevant reductant, time-depending 13C NMR spectroscopy was performed for the reaction of [AuCl[AuCl[AuCl[AuCl[AuCl2{( S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]PF6 with ascorbic acid. It was found that ascorbic acid reduces the complex readily and instantly, indicating a high possibility of the same outcome in living cells. Also, interaction of [AuCl[AuCl[AuCl[AuCl[AuCl2{( S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]PF6 with bovine serum albumin (BSA) was examined by UV/Vis spectrometry. It is assumed that [AuCl[AuCl[AuCl[AuCl[AuCl2{( S,SS,SS,S)-iBu 2eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]eddip}]+ might be reduced with cysteine leading to gold(I) species which can be seen in spectra after 2 h of reaction. After 24 and 48 h, UV/Vis spectra indicate that gold(I) species disproportionate to corresponding gold(III) species and elemental gold.
srpski
2015
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OSNO - Opšta sistematizacija naučnih oblasti, Metali
zlato(III) kompleksi, ligandi R2edda-tipa, oksido-redukciona svojstva, DFT proračuni, antiproliferativna aktivnost, apoptoza, ćelijski ciklus
OSNO - Opšta sistematizacija naučnih oblasti, Metali
gold(III) complexes, R2edda-type ligands, oxidoreductive properties, DFT calculations, antiproliferative activity, apoptosis, cell cycle