E ↵ ect of Functional Groups on Aquation

The azpyNMe2 complex 1 is the most potent complex against A2780 ovar- ian carcinoma of the nineteen complexes studied (Table 3.10). However all azpyNMe2 complexes tested have almost no oxidant activity towards NADH (Figure 3.15), indicating that they possess a MoA that does not involve NADH oxidation. The remainder of this chapter details further investigations into possible MoAs of the azpyNMe2 complexes.

To elucidate the MoA of a complex, it is necessary to know what form it takes when in solution. The activation step of CDDP involves the hydrolysis of the metal-Cl bond. Many anticancer complexes of other transition metals share this activation step, in many cases the rate of hydrolysis is dependent on the bound halide.114 _{To investigate whether changing the halide bound} to the iridium centre a↵ects hydrolysis, solutions of complexes 1 and 4 were monitored over 24 h by1_{H-NMR in 10% d6-DMSO:D2O, 0.1% 1,4-dioxane at} 310 K (Figure 3.16 and 3.17) and the extent of hydrolysis calculated by the peak integrals (Table 3.11). The presence of DMSO ensured the solubility of the complexes and 1,4-dioxane was used as the reference peak to calibrate the spectra. Further experiments were carried out under the same conditions using 120 mM NaCl to assess whether the presence of excess chloride would suppress hydrolysis of the Cl bond. This concentration of chloride matches that of the cell medium in which antiproliferative screenings were carried out, therefore spectra under these conditions provide an indication of what form the complexes would likely take in solution under biological conditions.

N N N Ir Cl PF6 N Complex1 10 min. 0 mM [Cl] 24 h. 0 mM [Cl] 24 h. 120 mM [Cl] NMe2 Cpx methyls Acetone DMSO in D2O Ether Cpx _methyls NMe2 Emergent Peak Emergent Irreversible Peak

Figure 3.16: Hydrolysis of complex 1 studied by 600 MHz 1_{H-NMR spectra} of a 100 µM solution of complex 1 in 10% d6-DMSO:D2O, 0.1% 1,4-dioxane (v/v) at 310 K, unbu↵ered at pD 8. Aliphatic region shown above, aromatic region shown below. Spectra shown 10 min after sample preparation, 24 h after sample preparation, and 24 h after sample preparation in 120 mM NaCl solution. Emergent peaks not corresponding to original complex denoted by

N N N Ir I PF6 N [ [ Complex4 10 min. 0 mM [Cl] 24 h. 0 mM [Cl] 24 h. 120 mM [Cl] Cp* methyls Cp* methyls Emergent Peak 10 min. 0 mM [Cl] 24 h. 0 mM [Cl] 24 h. 120 mM [Cl]

Azo pyridine ring

Azo phenyl ring Azo phenyl ring

Azo pyridine ring

Emergent Peak

Figure 3.17: Hydrolysis of complex4 studied by 600 MHz1_{H-NMR spectra of} a 100µM solution of complex1in 10% d6-DMSO:D2O, 0.1% 1,4-dioxane (v/v) at 310 K, unbu↵ered at pD 8. Spectra shown 10 min after sample preparation, 24 h after sample preparation, and 24 h after sample preparation in 120 mM NaCl solution. Emergent peaks not corresponding to original complex denoted by red arrows.

Table 3.11: Hydrolysis data for complexes 1 and 4 monitored over 24 h by 1_{H-NMR in 10% d}

6-DMSO:D2O, 0.1% 1,4-dioxane at 310 K. Aliphatic region shown above, aromatic region shown below. Experiments were repeated with the addition of 120 mM NaCl before or after 24 h to assess suppression and reversibility of hydrolysis by chloride.

Complex Structure % Extent of

Hydrolysis (0 mM [Cl]) % Extent of Hydrolysis (120 mM [Cl]) 1 [Cpxbiph_Ir(azpyNMe 2)Cl]PF6 79% 0% 4 [Cp*_Ir(azpyNMe 2)I]PF6 0% 40%*

*Complex 4 did not hydrolyse in the presence on 120 mM [Cl], instead 40% of the complex exchanged its iodido ligand for a chlorido one.

To determine whether the hydrolysis of complex 1 was reversible by post- hydrolysis addition of chloride, the experiment was repeated without chloride then 120 mM chloride was subsequently added after 24 h incubation as solid NaCl, and another spectrum was taken 10 min thereafter.

N N N Ir Cl PF6 N Complex1 Acetone Cpx _methyls

Azo phenyl ring Azo phenyl ring

Azo pyridine ring

Cp biphenyl rings

Emergent Peak Emergent Irreversible Peak

Figure 3.18: Aliphatic (top) and aromatic (bottom) regions of a 600 MHz 1_{H-NMR spectrum of a 100µM solution of complex1 in 10% d6-DMSO:D2O,} 0.1% 1,4-dioxane (v/v) at 310 K, unbu↵ered at pD 8. Spectrum taken 10 min after addition of 120 mM [Cl] to a previously chloride-free solution that had been incubated for 24 h. Emergent peaks not corresponding to original complex denoted by pink arrows. Traces of acetone/ether from the NMR tube are visible.

Complex1has been shown to be stable in 100% DMSO, spectra in Appendix Figure S21, pg. 292. Therefore the appearance of new peaks in the spectra of chlorido complex 1 over 24 h in addition to concomitant reduction in the

peaks corresponding to the original complex can be attributed to the hydrolysis of complex 1 in the presence of H2O. The spectrum of Cp* iodido complex 4, however, does not change over 24 h, indicating that complex 4 does not hydrolyse in the presence of H2O. The spectrum of chlorido complex 1 under the same conditions with 120 mM NaCl shows no change after 24 h. However, some of the new peaks that appear upon hydrolysis do not disappear upon subsequent addition of 120 mM NaCl. Conversely, the spectrum of Cp* iodido complex 4 with 120 mM NaCl contains new peaks after 24 h. The chemical shifts of these new peaks match exactly that of Cp* chlorido complex3 under the same conditions, therefore the most likely explanation is that there is some I_! Cl exchange occurring when complex4 is in solution with 120 mM NaCl. A peak at m/z = 709.2 was observed in the ESI-MS spectrum of complex 1 after 24 h in salt-free aqueous solution which matches the theoretical m/z of an analogue of complex 1 in which the metal-bound chloride is replaced by OH.

3.3.7

E↵ect of Chelating Ligands on Chiral Enantiomer