Date of Award

Winter 2016

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Celine J. Marmion

Funder/Sponsor

Science Foundation Ireland under Grants No. [08/RFP/CHE1675] and [11/RFP.1/CHS/3094]. We also gratefully acknowledge the Programme for Research in Third Level Institutions (PRTLI), administered by the HEA for funding. We also thank colleagues in COST CM1105 for fruitful discussions and collaborations.

Keywords

Anti-Cancer, anti-Bacterial, Ruthenium, Platinum, Copper, Histone Deacetylase Inhibitors, Ciprofloxacin

Abstract

Despite nearly 50% of all anti-cancer treatments being Pt-based, there is an urgent need to develop novel therapeutics beyond those currently in use. The first Pt-based anti-cancer chemotherapeutic, cisplatin, was granted clinical approval in 1978. Only two further Pt drugs have gained full global approval namely carboplatin and oxaliplatin. Although hugely successful, the widespread application and efficacy of Pt drugs are hindered by their toxic side effects, their limited activity against many human cancers and their susceptibility to acquired drug resistance. As a consequence, many investigations have been conducted into trying to develop novel chemotherapeutics that would (i) selectively target cancerous cells and thus have a more favourable toxicity profile as compared to PtII drugs and/or (ii) have a mechanism of action different to classical Pt drugs and thus potentially overcome resistance issues.

Herein, we describe our attempts to develop a new class of drug - rationally designed to overcome the above mentioned shortcomings of classical Pt drugs.

First, we took into consideration immunosuppressed patients undergoing chemotherapy. We successfully synthesised a dual-functioning anti-bacterial and anti-cancer RuII arene complex incorporating a derivative of the antibiotic ciprofloxacin (CipA), namely [Ru(h6-p-cym)(CipA-H)Cl]. The X-ray structures of the CipA derivative and this novel RuII complex were determined. This complex showed potent cytotoxicity towards a range of human cancer cells (both cisplatin and oxaliplatin resistant and p53 knockout) and is as potent as the clinically approved cisplatin. To get some further insight into its mechanism of action, cell cycle analysis and induction of apoptosis were carried out. These studies indicate that [Ru(h6-p-cym)(CipA-H)Cl] begins to induce apoptosis after 24 h of drug exposure, but most importantly that this may not be the only cell death process occurring. To examine its toxicity in healthy cell lines, a simple in vivo Galleria mellonella larvae model was utilised. The complex was well tolerated across the concentration range tested. The anti-bacterial activity of the CipA ligand and [Ru(h6-p-cym)(CipA-H)Cl] was also evaluated in nine different bacterial strains. While the ligand alone showed excellent anti-bacterial activity across all nine strains, the complex only showed moderate activity on a selected strain of Escherichia coli and its clinical isolate and on a strain of Pseudomonas aeruginosa. Despite being highly cytotoxic, we concluded that the difference between the concentrations that show anti-cancer (average IC50 between all cell lines ca. 2 mM) and anti-microbial activity (ca. 50 mM) was too great to warrant further investigations given the likelihood of a build up of resistance.

In the literature are numerous examples of Cu complexes possessing anti-microbial activity. Cu complexes have also been developed as anti-cancer agents with two CuII complexes of the Casiopeínas® family in clinical trials, namely Cas II-gly and Cas III-ia. Cu-phen derivatives have received much attention due to their ability to intercalate DNA as well as their ability to act as chemical nuclease agents. We thus sought to combine into one drug molecule the anti-microbial and anti-cancer properties of CipA with the anti-cancer properties of the Cu-phen framework. We successfully synthesised and fully characterised a library of novel dual-targeting Cu-N,N’-CipA complexes in which N,N’ is 1,10-phen or a designer ligand, DPQ or DPPZ. The X-ray structure of the novel CuII-phen-CipA complex, [Cu(1,10-phen)(CipA-H)Cl], where the Cu ion is coordinated by two CipA oxygen atoms and two 1,10-phen nitrogen atoms in the equatorial plane, and by a Cl ion in the axial position, has been determined. All complexes possessed potent anti-cancer activity towards two human cancer cells (MCF-7 and DU145) and are as potent as the clinically approved doxorubicin. In a similar manner as for the Ru work, we also wanted to determine the toxicity of these chemotypes towards healthy cells. The same simple in vivo model was utilised. The least toxic complex was found to be the DPQ analogue. To understand the mechanism of action of these novel complexes, DNA binding properties and interactions with nucleic acids were explored. These studies suggested that all tested complexes could be considered as DNA intercalators. All complexes were also found to exhibit excellent nuclease activity. The mechanism of action of quinolones is not yet fully understood. It is envisaged that the quinolones bind to DNA, inhibiting bacterial topoisomerases thus preventing the bacteria from replicating. Keeping this in mind, inhibition of topoisomerase I was carried out. The complexes were found to inhibit topoisomerase I, where, from the series of chemotypes investigated, Cu-DPPZ-CipA showed highest topoisomerase I inhibitory activity with the formation of fully relaxed DNA appearing at 2.5 mM.

Secondly, in recent years, many investigations into new molecular targets which may present unique opportunities for therapeutic exploitation have been carried out. Histone deacetylase (HDAC) enzymes have been identified as novel cancer targets, the inhibition of which suppresses tumour cell proliferation. one such example is the clinically approved HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), which not only possesses potent anti-cancer activity but also demonstrates selectivity towards tumour cells over normal cells. SAHA is well tolerated by patients at doses which induce a potent anti-cancer effect. Herein, we report a novel synthetic route to derivatise SAHA in such way so as to introduce a carboxy linker into the capping group of SAHA generating mono-carboxySAHA in excellent purity and good yield. Our plan was to develop a library of PtIV–HDAC inhibitor prodrugs, which would feature a cisplatin core and two axial mono-carboxySAHA ligands. Preliminary molecular modelling studies indicated that incorporation of a carboxy linker would not compromise the HDAC inhibitory activity of these derivatives. We also included a second derivative in our studies, mono-carboxy-CH2-SAHA developed a little later by a colleague. We describe in detail our attempts to synthesise these novel PtIV-HDACi prodrugs, including the introducion of a carboplatin core to increase solubility as well as exploring the possibility of utilisation of PEG chemistry. We have preliminary evidence that we successfully synthesised cis,cis,trans-[PtIV(NH3)2(CBDA-2H)(monocarboxy-CH2-SAHA-H)2] (where CBDA is cyclobutanedicarboxylate) in greater than 90% purity.

We also wanted to gain a deeper insight into the mechanism of action of a lead candidate cis-[PtII(NH3)2(malSAHA-2H)] (Pt-malSAHA) developed by our Group. This complex was found to be highly stable in solution. Studies involving reaction of Pt-malSAHA with S-containing nucleophiles in acetonitrile/water mixture, resulted in displacement of one of the arms of the malonato linker indicating that acetonitrile competed with the nucleophiles for complexation to Pt. Nevertheless, this did suggest that our complex may be activated in a similar way to that of carboplatin although further studies involving a different solvent system would be requied to validate our findings.

In conclusion, we have developed a series of novel Ru, Cu and Pt complexes as potential anti-cancer agents. Of those developed, the Cu complexes demonstrased greatest promise and warrant further investigation.

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2016.

Available for download on Sunday, November 03, 2019

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