Nitrogen Based Heterocycles and their Metal Complexes: From Anti-Cancer to Anti-Dyslipidemic Agents

Bhattacharyya, Sudipta (2017) Nitrogen Based Heterocycles and their Metal Complexes: From Anti-Cancer to Anti-Dyslipidemic Agents. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

The thesis starts with a brief discussion on anticancer chemotherapeutic agents of relevance to the type of work described in the latter chapters in the thesis, is provided. The examples cited are mostly clinical drugs or compounds/complexes in clinical trials with selective discussion on their properties. The discussion starts with the legendary platinum drugs in clinic or in clinical trial followed by emphasis on copper, zinc and ruthenium based metal complexes. A handful examples of copper, zinc and ruthenium based complexes that have showed promises have been presented. In some cases their unique mechanism of action has been described. It must be noted here that there are more complexes of Cu, Zn and Ru which have potential but the discussion is limited to a selected few. Special emphasise has been given on bis(pyrazole)methane based metal complexes of copper and zinc to understand the effect of such tight binding ligands upon coordination with the metal centre. Among reported complexes, benzimidazole-p-cymene based ruthenium compounds are discussed as they are related to the scope of the thesis. Their different mechanism of action has been compared. During the work progress especially when targeting the VEGFR2 in vitro using a Ru(II) benzimidazole complex (chapter 3) it was realized that cholesterol lowering agents may also be able to kill cancer cells targeting cancer. The role of cholesterol and cholesterol lowering agents in clinic hence is described with discussion on how various dedicated groups have increased the potential of this class of compounds. Here also different modification on those molecules has been depicted and classified. The discussion is followed by the reactivity of nitrogen mustard which is interesting ligand to tune for control of activity, has been discussed for its interaction with DNA based on different examples of clinical drugs of this category. Broad discussions are presented on various types of modification of the nitrogen mustard based prodrug cyclophosphamide, since the last chapter deals with tuning the activity and rendering fluorescence based tracking ability to phosphoramide analogues. The tight binding ability of the bis(pyrazole)methane based complexes led us to work on complexes of bio-essential metal ions copper(II) and zinc(II) which is reported in Chapter 2. The project was initiated to compare the role of redox active and inactive metal using similar ligand framework. In addition the effect of glucosamine conjugation on the zinc(II) complex of a ligand framework whose Cu(II) analogue has been reported was performed. Copper(II) complex (1 & 2) were found to exhibit poor activity against MCF7 cells but they were found to act as a chemical nuclease agent in presence of known cellular reducing agent ascorbic acid (5 eq) through involvement of hydrogen peroxide production in vitro. On the other hand the zinc(II) complex of same ligand (L1 in Chapter 2) did not show any hydrolytic DNA cleavage even at higher concentration (170 μM) but the toxicity was poor for both 1 and 3. However, the Zn(II)complex of the acetylated glucosamine analogue of bis(pyrazole) carboxylic acid derivative (L2 in Chapter 2) (5) was found to drastically increase the cellular internalisation and cytotoxicity through Caspase3/7 mediated apoptosis pathways. Inspired by the effect of internalization but mostly poor activity of the complexes it was realized that if a ligand could be designed that would be also active after dissociation from the complex than there is scope of activation of multiple pathways. In chapter 3, such a ligand has been designed that has the potential to inhibit the kinase domain of VEGFR2. The Ru(II)-arene complexes of this ligand have been synthesised with change in their labile halide coordination from Cl (6), Br(7) to I(8). A detailed solution stability studies on the complexes (6-8) showed that their potential to exchange halide in chloride solution (4mM NaCl) affected their hydrolysis and solution half-life. The stability order is as follows 7> 6 >8. The complexes were found to be vulnerable to the known cellular detoxifying agent glutathione (GSH) as per NMR studies, where they liberated the free ligand (HL3 in Chapter 3). The toxicity profile and cellular internalisation of complexes follows the order or 6 >7 >8. In spite of their instability there is small effect of excess GSH on their cytotoxicity. The complexes follow apoptotic pathways of cell killing through mitochondria mediated pathways. All the complexes turned out to be efficient in inhibition of cell migration and vascularisation in vitro/in ovo conditions. Western blot studies suggested that they have the ability to decrease expression of vascular endothelial growth factor receptor 2 (VEGFR2), Ras and Akt. While studying the literature on the VEGFR2 pathway of action it was found that the cell membrane needs cholesterol and so does certain other pathways in cells. It was found that there were evidences of higher cholesterol in certain forms of cancer. The bis(pyrazole) ligand happened to have carboxylic acid group and an alpha hydrogen with the carbon bridging the pyrazole rings this compound was good precursor to generate β-lactams some of which are good cholesterol lowering agents by inhibiting uptake of cholesterol. This made the grounds for the work in chapter 4. Here Staudinger syntheses was used to generate β-lactams having bispyrazole and nitrogen mustard the age old alkylating moiety which is still exploited and is present in more than seven anticancer chemotherapeutic drugs in clinic even today. The mustard conjugated β-lactam (11) was synthesised along with their non mustard analogues (9, 10) to check the effect of alkylating agent. Though mustard analogue did not turn out to give the expected cytotoxicity profile but the hypotheses of targeting cholesterol depletion to kill cancer cells did work. The most active cholesterol depleting compound also happened to be toxic to the liver cancer cell HepG2. Then inspired from the single β-lactam compound ezetimibe in market that inhibits cholesterol absorption, all the compounds were checked for their potential as cholesterol inhibitor. From in vitro studies it was found that the p-chlorobenzene containing 10 is the most potential candidate in the series to inhibit total cellular cholesterol. The poor alkylating ability of the aromatic mustard in chapter 4 was intriguing and led to get deeper in controlling the reactivity using the popular main group element phosphorous in its pentavalent state. There are many compounds reported with phosphorous and mustard and the class was called phosphoramide. One popular molecule of this class in clinic was cyclophosphamide which needed activation and suffers from huge dose loss during activation in liver. In chapter 5, two fluorescent cyclic phosphoramide analogues (12 & 13) were designed and their studies reported. Among them, 12 did not show any dissociation but the cytotoxicity profile showed that it was active unlike cyclophosphamide which could be activated only through liver. Another compound 13 produced only ca. 25% of dissociated/solvated product as observed in the respective NMR spectra. Both of them exerted enhanced in vitro cytotoxicity in presence of ascorbic acid. 12 could not be deactivated by GSH the most common cellular reducing agent that deactivates the activated cyclophosphamide and similar molecules. Cellular localisation of 13 was probed using its red fluorescence inside cell. 13 was found to localise more in endoplasmic reticulum and mitochondria as per fluorescence microscopic analysis. Both of the compounds follow apoptotic pathways of cell killing with properties to inhibit cell migration and inhibition of cancer stem cells at sub IC₁₀ dose.

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