Anticancer Activity of Palladium & Ruthenium Complexes: Effect of Steric Hindrance on Cytotoxicity and GSH Resistance

Purkait, Kallol (2019) Anticancer Activity of Palladium & Ruthenium Complexes: Effect of Steric Hindrance on Cytotoxicity and GSH Resistance. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

The research work is based upon designing of metal complexes targeting to inhibit thiol deactivation and for the purpose of controlling fast hydrolysis and anticancer activity. Small molecules, especially metal complexes have achieved great success in diagnostics and chemotherapy. The platinum drugs are used in almost 50% of chemotherapy against cancer. Gallium nitrate is of immense use as a calcium resorption inhibitor. Gadolinium complexes are very useful, as a contrast agent, in magnetic resonance imaging. However, there is an increasing demand for more potential molecules in this regard, including those that can exhibit potential against various resistant forms of cancer. This thesis work involves the use of steric hindrance to control the reactivity of metal complexes against cellular thiols. The target metals in this regard are Palladium and Ruthenium. Chapter I of the thesis starts with a brief introduction of cancer and provides relevant discussion on the action of selected metal complexes that have shown promises against cancer, in clinical or pre-clinical trials. The discussion encompasses the use of biologically relevant molecules as part of the ligand systems, particular correlation of hydrolysis vs. cytotoxicity and coordinating atoms vs. cytotoxicity. There is emphasis upon steric hindrance and rate of hydrolysis in Palladium(II) and Ruthenium(II) complexes and how they are correlated to the cytotoxicity and the mechanistic aspects. Later in this chapter, the scope of the thesis is briefed for a better understand of the work flow in the following chapters. Platinum drugs are used worldwide to treat several types of cancer and most importantly ca. 50 % of total used drugs used are platinum based, in spite of the fact that they show thiol mediated deactivation. The primary reason for this lies in the tuning of the leaving groups, viz., cisplatin, carboplatin and oxaliplatin, which decreases reactivity towards thiols. The activation step of these complexes involve hydrolysis followed by binding with DNA bases. However, low solution lifetime due to quick hydrolysis leads to side effects. Palladium(II) complexes are isostructural with platinum(II) complexes but are kinetically more labile in solution. For the purpose of correlating the effect of steric bulk in palladium complexes with hydrolysis and GSH binding we designed a series of cis-dichloro palladium(II) complexes, altering steric hindrance. In this attempt, the synthesized cis-dichloro palladium(II) complexes have been evaluated for their anticancer activity against prostate (DU145), pancreatic (MIA PaCa-2), liver (HepG2) and triple negative breast (MDA-MB-231) cancer under normoxic and hypoxic conditions. The solution stability studies in DMF:phosphate buffer (pH 7.4) mixture (4:6 v/v) showed hydrolysis and degradation depending upon the complex studied. Complex II-5 appeared to be the most stable. Their selectivity towards the cancer cells were compared with their cytotoxicity against normal human foreskin fibroblasts (HFF-1). II-1 and II-2 showed ca. 20-30 times more toxicity (IC50 5.4 and 7.2 μM) against cisplatin resistant DU145 cells (cisplatin IC50, >150 μM). Complex II-5 showed 6 to 7 times more toxicity (IC50, 23.8 μM), compared to cisplatin but was 2.5 times less toxic (IC50, 66.3 μM) to the non-cancerous HFF- 1, unlike cisplatin (IC50, 9.7 μM). Complex II-5 showed an increase in toxicity under hypoxic conditions against pancreatic cancer (MIA PaCa-2). In presence of glutathione, both the complexes II-3 and II-4 undergo degradation which was further confirmed by their toxicity studies against glutathione induced cells. Complexes II-1, II-2 and II-5 showed cell cycle arrest at G0/G1 phase and followed the apoptotic pathway of cell killing. The Pd-complexes in chapter-II showed potent activity against DU145 cells along with hypoxia activation. But all of them were poorly soluble in aqueous solutions. This became a problem and hindered their studies and further development. In addition, steric bulk in the vicinity of the metal centre was not much successful to control glutathione deactivation. Hence, to probe if the change of metal ion can improve the properties, we used RuII which is probably another alternative for Pt as an anticancer chemotherapeutic agent due to its kinetic inertness. Three ligands with increased steric hindrance, two of which were used in Chapter-II were used to synthesize tetrahedral piano-stool type RuII-p-cymene complexes. The gradual increase in steric bulk of the ligands was achieved from aniline, 2,6-dimethyl aniline and 2,6-diisopropyl aniline. The solution stability studies showed that the steric bulk increases solution lifetime. Most sterically hindered complex showed higher stability and in presence of 110 mM chloride concentration, all the three complexes showed stability. Similarly, when complexes were incubated with 25 equiv. of glutathione, complexes III-1 and III-2 showed binding whereas III-3, with the highest steric hindrance, showed the highest stability. Cytotoxicity studies showed that the toxicity of the complexes is comparable with cisplatin. Hypoxia activation was observed for III-3. Moreover, III-3 showed resistivity against GSH inhibition as found from toxicity studies. Faster hydrolysis does lead to higher cytotoxicity, but it appears that the pathway of action also changes as evident from the cell cycle arrest, MMP change and caspase activation data of the complexes. Complex III-3 followed the intrinsic apoptosis pathway for cell killing. In addition, complexes III-1 – III-3 may exhibit potential anti-metastatic and antiangiogenic activities. Hence, by applying steric bulk in the vicinity of metal centre, hydrolysis rate and resistivity against GSH binding can be controlled. Recent literature showed that the increase in cellular toxicity, cellular uptake and alteration of mechanism of cell killing may also be dependent on the coordinated halide in RuII-p-cymene complexes. Hence, to probe the effect of the labile halide atom upon solution stability, GSH resistivity and cytotoxicity, the bromo and iodo analogues of complexes III-1 to III-3 have been synthesized. Solution stability studies in buffer solution showed faster chloride exchange in presence of extracellular chloride concentration (110 mM) at pH 7.4 for the most sterically hindered IV-5 and IV-6, whereas it is minimum for least sterically hindered IV-1 and IV-2. Interestingly, the least sterically hindered complex, IV-2, having a Ru-I bond instead of a Ru- Cl bond, showed higher stability in presence of GSH whereas the most sterically hindered ligand bearing complex (IV-6) with a Ru-I bond showed degradation. Compared to the bromo complexes, iodo complexes are more stable and show higher kinetic inertness, which was evident from the chloride exchange studies and GSH reactivity. Complexes III-1, IV-1 and III-3 showed maximum HSA binding constant. All the complexes showed better activity against cancer cells than cisplatin. The effect of HSA binding on cytotoxicity was studied. The cytotoxic efficacy of complex IV-5, which displayed the least binding constant with HSA, increased when it was pre-incubated with HSA. The cell cycle studies with the representative complexes IV-5 and IV-6 showed that similar to III-1, these complexes also arrested the cell cycle at G2/M phase and induced apoptosis. In this chapter, we investigated the anticancer activity of hydrolytically stable complexes and probed their activities in GSH induced cells. Among the five complexes, three were redox innocent and two were having ligands with redox non-innocence. The redox non-innocent ligands (hydroquinone derivative, V-L1 and planer ring-based system, V-L2) were used for the purpose of achieving an increase in activity in hypoxia. The solution stability of the RuIIp- cymene complexes were compared, where V-2 showed stability up to 24 h. The quinone containing ligand system V-L1 showed quick reaction with thiols and was converted to the hydroquinone species in presence of NADH. The cytotoxicity against a panel of two cancer cell lines showed that V-1 is four times more toxic towards cancer cells than normal fibroblasts. V-1 and V-3 showed resistivity against GSH inhibition. During complexation with RuII, the quinone ligand V-L1 was reduced to hydroquinone, thereby attaining stability. Which diminished the cytotoxicity of RuII complex. However, the quinone ligand system (V-L1) itself was hypoxia active without complexation and so was complex V-5. But both of them were highly deactivated by GSH and showed more toxicity towards normal cells. V-L1 could generate ROS for cell killing and disrupt cellular redox balance by oxidation of NADH quantitatively. V-L1 also showed antimigration effects. The complexes mediated apoptosisbased cell killing as exhibited by V-2 and V-3.

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