Ligand-centred redox activation of inert organoiridium anticancer catalysts

Organometallic complexes with novel activation mechanisms are attractive anticancer drug candidates. Here, we show that half-sandwich iodido cyclopentadienyl iridium(iii) azopyridine complexes exhibit potent antiproliferative activity towards cancer cells, in most cases more potent than cisplatin. Despite their inertness towards aquation, these iodido complexes can undergo redox activation by attack of the abundant intracellular tripeptide glutathione (GSH) on the chelated azopyridine ligand to generate paramagnetic intermediates, and hydroxyl radicals, together with thiolate-bridged dinuclear iridium complexes, and liberate reduced hydrazopyridine ligand. DFT calculations provided insight into the mechanism of this activation. GS− attack on the azo bond facilitates the substitution of iodide by GS−, and leads to formation of GSSG and superoxide if O2 is present as an electron-acceptor, in a largely exergonic pathway. Reactions of these iodido complexes with GSH generate Ir-SG complexes, which are catalysts for GSH oxidation. The complexes promoted elevated levels of reactive oxygen species (ROS) in human lung cancer cells. This remarkable ligand-centred activation mechanism coupled to redox reactions adds a new dimension to the design of organoiridium anticancer prodrugs.


S2.1 X-ray crystallography
Suitable crystals were selected and mounted on a glass fiber with Fomblin oil and placed on a Rigaku Oxford Diffraction SuperNova diffractometer with a dual source (Cu at zero) equipped with an AtlasS2 CCD area detector. The crystals were kept at 150 ± 2 K, except the crystal of complex 5·MeOH kept at 296 ± 2 K, during data collection. Using Olex2, 1 all the structures were solved with the ShelXT 2 structure solution program using direct methods and refined with the ShelXL 3 refinement package using least squares minimization. X-ray crystallographic data for complexes 1'·MeOH, 2, 3·MeOH, 5·MeOH, 7', 8

S2.3 Electrospray mass spectrometry
Electrospray ionization mass spectra (ESI-MS) were obtained for samples in methanol on a Bruker Esquire 2000 spectrometer. The mass spectra were recorded with a scan range of either m/z 50-500 or 400-1000 in positive ion mode.

S2.4 Elemental analysis
CHN elemental analyses were carried out on a CE-440 elemental analyzer by Warwick Analytical (UK) Ltd.

S2.5 pH measurements
pH or pH* (pH meter reading without correction for effect of deuterium on the sensor) values for samples in H 2 O or D 2 O were measured at ca. 298 K, using a HATCH minilab pocket pH meter with ISFET silicon chip pH sensor, calibrated with buffer solutions of pH 4, 7, and 10.

S2.6 UV-Vis Spectroscopy
A Cary 300 UV-vis recording spectrophotometer was used with 1 cm pathlength quartz cuvettes (3.0 mL) and a PTP1 Peltier temperature controller. Spectra were recorded using UV Winlab software and plotted using Origin 2018. Experiments were carried out at 298 K from 800 to 200 nm with 0.5 nm intervals.

S2.7 Determination of pK a
Changes in the UV-vis absorption spectra of 20-30 µM solutions of complexes 1-6 and 9-10 in 10% methanol/90% Milli-Q water in 1 cm path-length quartz cuvettes (3.0 mL) from pH 2-12, were monitored by UV-Vis spectroscopy, after addition of μL amounts of either dilute perchloric acid in acetic acid, or dilute KOH. Changes in absorbance maxima at different pH values were plotted and fitted to the Henderson-Hasselbalch equation to determine the pK a .

S2.8 Determination of molar extinction coefficients
The UV-vis spectra of the iridium complexes 1-6 were recorded at 3 different concentrations from 800 nm to 200 nm, and the concentrations were subsequently determined by ICP-OES protocol. Linear plots of absorbance versus concentrations for each complex gave the molar extinction coefficient as the gradient, according to the Beer-Lambert law.

S2.9 Determination of the hydrolysis kinetics for complex 9
HPLC analysis with the detection wavelength at 254 nm relevant to 610 nm was conducted for complex 9 (50 µM) dissolved in MeOH/H 2 O (5/95, v/v) and incubated at 310 K, at different time interval over 24 h. The normalized peak integral of complex 9 was calculated based on the aqua complexes. The curve of the integral versus time (min) was were plotted and fitted to the pseudo firstorder kinetics to determine the hydrolysis rate and half-life time.

S2.10 Cyclic voltammetry
All the electrochemical measurements used a three-electrode configuration: the reference electrode was Ag/AgNO 3 (0.1 M in acetonitrile); the working electrode was a polished platinum disc and the counter electrode was a large surface area platinum wire. The electrolyte solution was 0.1 M [Bu 4 N]PF 6 in HPLC grade acetonitrile solution. The concentration of the metal in all the samples, including the ferrocene reference, was 1 mM in 5 ml electrolyte which had been scanned as blank in advance after bubbling with N 2 at least for 15 min. The scan rate was 100 mV/s. Between each sample interval, all the electrodes were washed with acetone three times and dried under an air flow.

S2.12 Catalysis of GSH oxidation
The catalysis of GSH oxidation to GSSG by iodido iridium complexes 1, 3, 7 and 8, free ligand phenol-azopyridine (HO-azpy), and the Ir-SG complexes 7-SG and 3-SG was studied by 1 H NMR spectroscopy. The tested compound and GSH were in 5% d 6  Then the NMR tube was sealed with a cap and parafilm during the reaction.
The turnover number (TON) for the reactions was calculated as follows:

S2.14 Partition coefficient determination
The lipophilicity of complexes 3 and 7 is compared by determining their octanolphosphate buffer (2 mM, pH 7.4) partition coefficients using the "shake-tube" method. 4 On account of the deprotonation of the phenolic group at physiological pH, which would affect the partition of the complex, phosphate buffer (pH 7.4) was used to mimic the physiological environment.

OES)
ICP-OES analyses were carried out on a PerkinElmer Optima 5300 DV series Optical Emission Spectrophotometer. The iridium plasma standard was diluted with 3.6% HNO 3 in type-1 Milli-Q water to give freshly-prepared calibrants at concentrations of 700, 600, 500, 400, 300, 200, 100, 50, and 0 ppb, which were adjusted to match the sample matrix by standard addition of sodium chloride.
Total dissolved solids did not exceed 0.2% w/v. Data were acquired and processed using WinLab32 V3.4.1 for Windows.

S2.17 Cell culture
Cancer cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% (v/v) of fetal calf serum, 1% (v/v) of 2 mM glutamine, and 1% (v/v) penicillin/streptomycin. Cells were grown as adherent monolayers at 310 K in a 5% CO 2 humidified atmosphere and passaged at ca.
80% confluency using trypsin-EDTA. A2780cis cisplatin-resistant cells were exposed to cisplatin for 24 h upon reaching 80-90% confluence. After this time, the cells were washed with PBS and supplied with fresh medium.

S2.18 In vitro cell growth inhibition
Briefly

S2.19 Toxicity in zebrafish
Zebrafish experiments using Singapore wild-type zebrafish embryos were S12 carried out at School of Life Sciences (University of Warwick Committee. Zebrafish were housed in 3.5 L tanks and fed 2 -4 times a day.
Fish were mated once a week starting at two pairs per 1 L breeding tank.

S2.20 Ir accumulation in cancer cells
The accumulation of iridium in A549 human lung carcinoma cells was determined by ICP-MS. 1x10 6 cells were seeded onto a 100 mm Petri dish and incubated in compound-free media for 24 h at 310 K in a 5% CO 2 humidified atmosphere. For a further 24 h the cells were incubated in media containing a range (0.25 , 0.5 , 1 and 2 IC 50 ) of compound concentrations × × × × (determined by ICP-OES). Following this, cells were counted and cell pellets were collected and digested as ICP-MS protocol.

S2.21 ROS determination
Flow cytometry analysis of ROS/superoxide generation in A549 cells caused by exposure to complex 3 and 7 was carried out using the Total were carried out to establish statistical significance of the variations.

S2.22 Computational details
All calculations were performed in the framework of Density Functional Theory employing the hybrid Becke three-parameter exchange functional 6 and the Lee-Yang-Parr correlation functional, B3LYP. 7 To carry out such calculations the Gaussian 09 suite of program 8 was used. Grimme approach was adopted to include dispersion corrections for nonbonding interaction using atom pairwise additive schemes, 9 denoted as DFT-D3 method.
The LANL2DZ effective core potential was used for the Ir atom, 10 along with the split valence basis set. The standard triple-ζ quality 6-311+G** basis sets of Pople and coworkers were used for the atoms directly participating in the process, whereas in order to reduce the computational effort, the 6-31G* basis sets were employed for peripheral atoms.
In order to establish both the nature of intercepted stationary points as minima and transition states and calculate zero-point energy (ZPE) and Gibbs free energy corrections, vibrational frequencies at the same level of theory were calculated. The intercepted transition states are first order saddle points on a S14 potential energy surface (PES) and their vibrational spectrum is characterized by one imaginary frequency, corresponding to a negative force constant, which means that in one direction, in the nuclear configuration space, the energy has a maximum, while in all the other directions the energy has a minimum.
Furthermore, IRC (intrinsic reaction coordinate) analysis was employed to carefully check transition state structures to be properly connected to the correct minima. 11,12 The effects due to the presence of the solvent were included using the S15

S3.2 Synthesis of iridium complexes
All the iridium iodido complexes were synthesized following a general procedure: the ligand (0.10 mmol, 2.0 mol equiv.) was added to 10 mL           Table S5). Raising the pH to 11.0 led to a dramatic red-shift of this MLCT band to 558-586 nm with a much higher extinction coefficient (26900-35800 M -1 ·cm -1 , Table S5).