The investigations into cellular targets for 41 and 42 looked at number of potential targets, including secondary DNA structures. Both strongly interacted with G-Quadruplexes and i- motifs. These are formed from single stranded DNA rich in guanine and cytosine respectively. The potential for other nucleotides to form secondary structures has been investigated. Thymine can also from quartets however they are less stable the G-quartets because they are unable to form a stabilising interaction with alkali metal cations.34
5.5.1 G-quadruplexes
G-quadruplex forming sequences are found in the telomeric region of DNA, at the ends of eukaryotic chromosomes.35 Telomeres are made up of many repeating guanine units and
correspondingly cytosine in the other strand. They are supposed to protect or buffer the coding region of the chromosome from degradation. These single stranded guanine rich sequences can assemble into four stranded DNA structures called G-quadruplexes.34 A G-quadruplex is formed from two or more π stacked guanine tetrads (G-quartet) linked in a stable planar arrangement by hydrogen bonded Hoogsteen pairings (Figure 5.11). In addition, the presence of alkali metal cations (represented as an orange circle, Figure 5.11) are important as they coordinate to oxygen atoms and can influence the stability of the structure. The stability of the G-quadruplexes is also affected by variable length of the loops on the exterior of the G- quadruplexes, which are made of DNA bases not participating in the formation of a quartet.34 The loops also allow the formation of cavities on the exterior of the G-quadruplexes which could be potentially interact with drug molecules.
112
Figure 5.11. left) A G-quadruplex; middle) a representation of quartet; right) a
representation of the stacked system showing stacked quartet and loops of different sizes.34
Telomerase is a normally inactive enzyme that allows telomere extension and is activated in many human cancers.36 Extending telomeres, extends the lifetime of a replicating cell, further
removing cancer cells from normal cellular regulation. Telomerase recognizes only the linear form of DNA, so stabilizing the G-quadruplex structure in the telomeres could inhibit telomerase activity leading to the inhibition of cancer cells’ immortality.37
A G-quadruplex sequence is also found in the promotor region of the MYC proto-oncogene, its formation can repress the transcription of this gene.38 The MYC proto-oncogene is
overexpressed in almost 80% of solid tumours, therefore it is an interesting target to allow potential control of oncogene expression.
The ability of gold(I) complexes to stabilise G-quadruplexes has been investigated in the case of the caffeine-based bis(NHC)gold(I) cation (Figure 5.12). Crystallographic data showed three gold complexes per G-quadruplex interacting through π stacking into accessible tetrads.39 Mass spectrometry confirmed the occurrence of aggregates of the complex with G-
quadruplexes with ratios from 1:1 to 1:3 in solution.
113 The gold(III) complexes, shown in Figure 5.13, interact with G-quadruplexes but not with double-stranded DNA.40 The strongest binding affinity value was observed for the binuclear complex on the left. This affinity for G-quadruplexes was comparable to other metal-based complexes.41
Figure 5.13. Gold(III) complexes used to stabilise G-quadruplexes.
5.5.2 iMotifs
DNA sequences rich in guanine have a complementary sequence rich in cytosine, these can also form secondary structures which are known as i-motifs (Figure 5.14).42 There is considerably less know about i-motifs compared to G-quadruplexes. I-motifs were first observed in acidic conditions and are formed of two parallel duplexes, arranged in an antiparallel fashion, which intercalate through hemiprotonated cytosine-cytosine base pairs (left, Figure 5.14).43 The ability to trigger folding depending on the pH of the media has been investigated, allowing reversible i-motif formation.42
Figure 5.14. a) Hemiprotonated cytosine- cytosine base pair; b) Schematic view of an i-
motif.42
There are examples of interactions between other nucleotides in the loop regions contributing to stabilisation of i-motifs, such as the formation of an A-T base pair.44 The nature of cations
114 present in solution is also thought to affect the conformations formed.45 Furthermore,
development of sequences of i-motifs that are stable at physiological pH instead of acidic pH have been investigated.46 Again, stabilization of i-motifs is an interesting potential target for anticancer drugs because they have been shown to alter both telomerase activity and oncogene expression in similar manor to G-quadruplexes.42
These secondary structures are of interest, but their formation requires single cytosine-rich strands of DNA to be freed from their complement. In order to understanding their formation DNA transcription has been examined, a process during which the double helix of duplex DNA is unwound releasing single stranded DNA.47
5.6 Objectives
This work follows the previous research by the Bochmann and Che groups into gold(III) NHC complexes stabilised by pincer ligands that have shown promising anticancer properties. As discussed, alteration of the NHCs with biologically relevant molecules has been probed, but simple NHC complexes such as N and 41 are more potent anticancer agents. Extending the series of complexes in a systematic manner using three pincer ligands and three NHC ligands with minor changes could help to identify factors that influence anticancer properties.
The C^N^C ligand systems chosen to be investigated were 1,5-diphenyl substituted pyrazine, pyridine or p-methoxy substituted pyridine. Both the pyridine and pyrazine based C^N^C gold(III) have been used anticancer studies, however the p-methoxy substituted pyridine had previously only been used in the synthesis of gold(III) hydrides.48 Previous work has shown gold(III) NHCs based on 1,3-dimethylbenzimidazolium, 1,3-dimethylcaffeine and 1,3- dimethylimidazolium to have anticancer properties. To minimise the parameters being varied, the dimethyl NHC analogues were used for all complexes.