Triangulación de instrumentos

Since DNA is considered a major target of metal-based anticancer compounds, reactivity towards 9-EtG was examined as a model for guanine binding in DNA. All complexes formed the 9-EtG adduct13G, with complex13exhibiting 100% binding within 15 min. This is consistent with previous reports of similar complexes.19 The pyridyl complexes 18-20 show a decrease in the ability to bind to 9-EtG, with only 22-38% formation of 13G after 24 h. This may indicate that pyridyl complexes18- 20are less likely to bind to DNA in the cell compared to complex13due to the more stable Ir-N bond.18,31

The electron donating -NMe2 group on the pyridine ligand in complex 19 stabilises

the Ir-N(pyridyl) bond, reducing the reactivity of the complex with 9-EtG. The opposite effect was observed for complex20which bears the electron-withdrawing – CF3group, resulting in a greater extent of binding to 9-EtG. The observed reactivity

towards 9-EtG parallels the expected reactivity of each complex 13 >20 > 18 > 19 showing that modulation of activity towards DNA nucleobases can be achieved by careful selection of the monodentate ligand.

4.4.3.2 GSH

The concentration of GSH in cells is high, ranging from 0.5 – 10 mM in mammalian cells.33 The interaction of metallodrugs with GSH can lead to inactivation as in the case with cisplatin where binding of GSH results in the drug being pumped out of the cell by specific membrane transport systems,34 while thiolate adducts of cisplatin exhibit reduced reactivity towards biological targets such as DNA.35 It has also been

detoxification of cisplatin by GSH.36 In some cases, reaction of metal complexes with GSH may be integral to their antiproliferative activity, as in the case for some octahedral Pt(IV)37 and Ru(III)38 complexes. The half-sandwich ruthenium(II) complex [(η6-pcym)Ru(azpy)I]PF6(azpy = N,N-dimethylphenyl- or hydroxyphenyl-

azopyridine) undergoes ligand-centred catalytic oxidation of GSH to GSSG resulting in enhanced levels of ROS, where coordination of the azpy chelating ligand to the ruthenium activates its catalytic activity.39 It is believed that adducts of GSH with iridium complexes such as [(η5-Cpx)Ir(2-PhPy)SG] may also be less reactive, resulting in the decreased attack on potential biological targets.17 This may be modulated by the type of monodentate ligand present on the complex.

Within the pyridyl series, the order of stability in the presence of GSH followed from most to least stable19>18~20(Figures 4.6 and 4.8). Complexes18and20exhibit a similar degree of reactivity towards GSH compared to chlorido complex 13. Modulation of reactivity by pyridyl ligands is observed only in the case of 19 indicating that the electron-donating –NMe2group is essential for enhanced stability

in the presence of GSH. In contrast, the iridium complex [(η5-CpxBiPh)Ir(2- PhPy)Py]PF6 shows markedly enhanced stability in the presence of GSH compared

to its chlorido analogue [(η5-CpxBiPh)Ir(2-PhPy)Cl].17 This may indicate that the presence of the extended cyclopentadienyl capping ligand also plays a role in the reactivity of the pyridine complex towards GSH. This could be due to steric hindrance caused by the biphenyl rings compared with the Cp* ligand studied in this work. It has been previously demonstrated that the use of CpxPhand CpxBiPhcapping ligands in some half-sandwich iridium(III) complexes leads to slower rates of hydrolysis for chlorido complexes.29 This may also result in more stable pyridine monodentate for CpxBiPhcomplexes compared with Cp* complexes.

The reaction of complexes 13 and 18-20 with GSH resulted in HPLC traces with peaks having almost identical retention times (Figure 4.6), indicating that the reaction products are independent from the nature of the monodentate ligand. LC- MS analysis of the reaction between complex 13 and GSH resulted in the identification of several reaction products (Figure 4.7 and Table 4.5). The identification of the glutathione bridged Ir dimer [(η5-Cp*)Ir(µ-SG)3Ir(η5-Cp*)]2-

was supported by a peak corresponding to free chelating ligand 2-(2ˈ-

methylphenyl)pyridine. Analysis of the formation of products over time suggests that the bridged species forms quickly and its concentration stays constant over the 24 h period. This may suggest that [(η5-Cp*)Ir(µ-SG)3Ir(η5-Cp*)]2-is a kinetic product of

the reaction.

In the presence of air, the Ir-GSH adducts [(η5-Cp*)Ir(2ˈ-methylphenyl)pyridine)(S-

O)G]- (sulfenate) and [(η5-Cp*)Ir(2ˈ-methylphenyl)pyridine)(S-O2)G]- (sulfinate)

were observed, but no [(η5-Cp*)Ir(2ˈ-methylphenyl)pyridine)SG]-was found. The Ir- (S-O)G and Ir-(S-O2)G adducts appear to increase over time. As the extinction

coefficient for each species is unknown, the definitive concentration of each species in solution cannot be determined; however, it can be deduced which species increase/decrease in concentration during the course of the reaction in relation to the other species present.

An unidentified Ir-containing species was also observed, which appears to be an intermediate species, as exemplified in Figure 4.8 for the reaction between 19 and GSH. Its concentration in solution increases during the first 8 h of reaction, but then decreases after 24 h. The concentration of this species also decreases over time for the reaction between 13 and GSH, but initially forms quickly due to the faster reaction of13compared to19.

Intriguingly, binding of GSH to the iridium may result in oxidation of the sulfur atom by dioxygen. This was further examined by performing the reaction under argon (Figure 4.9 and Table 4.6). LC-MS analysis identified two new peaks that could correspond to the adduct [(η5-Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)SG]-. The peaks contained m/z species including [(η5-Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)]+

and free GSH, which may be dissociating during the ionisation process. An m/z of 801.13 was detected which is the exact mass of [(η5-Cp*)Ir(2-(2ˈ-

methylphenyl)pyridine)SG]-. As the LC-MS was run in positive mode, this species is 2 Daltons lower than the detectable species {[(η5-Cp*)Ir(2-(2ˈ-

methylphenyl)pyridine)SG]+2H}+(calculated m/z of 803.25). Therefore, it cannot be determined with certainty that the unoxidised Ir-SG adduct has been detected. The Ir-(S-O)G adduct was detected as a minor species and no Ir-(S-O2)G was observed,

indicating that the presence of O2 gas is likely to promote the oxidation of the bound

sulfur atom. The small presence of Ir-(S-O)G may be a consequence of the exposure of the reaction mixture to air upon injection into the LC-MS. These oxidised species have not been previously reported for half-sandwich Ir(III) complexes unless coupled with another reaction. Such an oxidation was demonstrated for the iridium complex [(η5-CpxBiPh)Ir(2-PhPy)Py]PF6, but only when coupled to the catalytic

oxidation of NADH to NAD+to produce H2O2 where oxidation of the sulfur atom

then occurs.40 Further addition of H2O2 was required to produce the glutathione-

sulfinate adduct.

The Ru(II) arene complex [(η6-biphenyl)Ru(ethylenediamine)Cl]PF6was shown to

bind to GSH through the thiolate sulfur atom and was oxidised to the sulfenate complex by dioxygen, although further oxidation to the sulfinate was not observed.28 This resulted in a Ru-(S-O)G adduct that then facilitated the binding to guanine,

while the Ru-SG adduct did not. Performing the same reaction under argon supressed the formation of the sulfenate complex. Therefore, it can be postulated that if complexes13and18-20are capable of forming these oxidised GSH adducts, their reactivity may not be severely decreased as the adducts formed may be more labile than the Ir-SG adduct.

4.4.3.3 Catalytic Oxidation of NADH

The use of platinum-group chelated metal half-sandwich complexes for the catalytic oxidation of NADH to NAD+22,41,42 and NAD+ to NADH43-45 has been well documented, where the catalytic activity appears to take place at the monodentate site. The catalytic oxidation of NADH to NAD+may be an important feature of half- sandwich iridium(III) complexes,46 and could potentially occur in cells.22 The catalytic activity of [(η5-Cp*)Ir(2-PhPy)Cl] complexes was evaluated in Chapter 3. They can oxidise NADH, potentially contributing to the mechanism of action responsible for the antiproliferative activity.

The chlorido complex 13 and the less reactive Py-NMe2 complex 19 were

investigated for their catalytic activity towards the oxidation of NADH to NAD+. Intriguingly, both 13and19exhibited similar catalytic activity, with a TON after 24 h of 16 and TOF of 1.4 h-1at an NADH concentration of 127 µM (Figure 4.10). This implies that under the conditions used, the monodentate ligand has little effect on the catalytic activity of the complex, despite these complexes exhibiting different reactivities towards aquation, 9-EtG binding and GSH stability. The use of pyridyl monodentate ligands in place of chloride may protect the complex from possible deactivation by other reactions that can occur inside the cell (e.g. with GSH,

proteins), whilst potentially allowing the complex to modulate the ratio of NADH/NAD+.17,18