Cytotoxic activity and protein binding through an unusual oxidative mechanism by an iridium ( I ) – NHC complex †

A new NHC iridium(I) complex (1) showing significant antiproliferative properties in vitro is described here. Its crystal structure, solution behaviour and interactions with the model proteins cytochrome c (cyt c) and lysozyme were investigated. High resolution ESI-MS measurements suggest that this iridium(I) complex acts as a prodrug and binds cyt c tightly through an unusual ‘‘oxidative’’ mechanism. Eventually, an iridium(III)–NHC fragment is found associated to the protein.

General: Reactions were carried out under N 2 by using standard Schlenk techniques.All solvents were of analytical grade.Chemicals were obtained from commercial sources and used without further purification. 1H and 13 C NMR spectra were recorded at room temperature on a Bruker DRX 400 in deuterated solvents which were also used as internal reference.The chemical shifts are reported in ppm (parts per million) relative to TMS.Coupling constants, J, are reported in Hz, multiplicities being marked as: singlet (s), doublet (d), triplet (t) or multiplet (m).ESI mass spectra were measured on a Bruker Esquire 6000 mass spectrometer.HR-ESI mass spectra were recorded with an LTQ Orbitrap high-resolution mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with a conventional ESI source.FAB mass spectrometry was performed with a Fisons VG Instruments Autospec spectrometer.The mass to charge relation (m/z) is given as a dimensionless number.Elemental analyses carried out on a Vario EL (Elementar Analysensysteme GmbH, Hanau, D) in C, H, N mode.

Cell Culture and Cytotoxicity
Dulbecco's Modified Eagle's Medium (DMEM), containing 10% fetal calf serum, 1% penicillin and streptomycin, was used as growth medium.MCF-7, HT-29 and HEK-293T cells were detached from the wells with trypsin and EDTA, harvested by centrifugation and resuspended again in cell culture medium.The assays have been carried out on 96 well plates with 6000 (10000) cells per well for MCF-7 and HT-29 (HEK-293T).After 24 h of incubation at 37°C and 10% CO 2 , the cells were treated with the compounds (with DMSO concentrations of 0.5%) with a final volume of 200 µl per well.For a negative control, one series of cells was left untreated.The cells were incubated for 48 h followed by adding 50 µl MTT (2.5 mg/ml).After an incubation time of 2 h, the medium was removed and 200 µl DMSO were added.The formazan crystals were dissolved and the absorption was measured at 550 nm, using a reference wavelength of 620 nm.Each test was repeated in quadruplicates in three independent experiments for each cell line.Time dependent cell growth studies.Cell growth was monitored continuously using the xCELLigence RTCA (Real Time Cell Analyser) system (Roche).The background of the Eplates was determined in 50 µL of medium.Subsequently, 50 µl of a suspension of the HEK-293T cells (10000 cells per well) were added and the cells were incubated for 24 h at 37°C and 10% CO 2 .The cells were then treated with the compound (10 µM, 20 µM and 30 µM with DMSO concentrations of 0.5%) with a final volume of 200 µl per well.For a negative control, one series of cells was treated with media alone (0.5% DMSO).Each treatment was performed in triplicates.The impedance was monitored every 15 minutes for additional 72 h.

Figure
Figure S1. 1 H NMR spectrum of 1 recorded in CD 2 Cl 2 at 400 MHz.

Figure S2 .
Figure S2.UV/Vis spectrum of 1 (200 µM) in DMSO.Spectrum was recorded in 10 min intervals during the first 60 min and hourly for additional 47h.

Figure S4 .
Figure S4.UV/Vis spectrum of 1 (100 µM) in ammonium acetate buffer (50% DMSO, pH 6.8).After the first run, 10 eq. of H 2 O 2 were added.The spectrum was recorded in 10 min intervals during the first 60 min and hourly for additional 47h.

Figure S6 .
Figure S6.Example of a fitted dose-response curve for the determination of the IC 50 value of 1 on MCF-7 cells.

Figure S7 .
Figure S7.Example of a fitted dose-response curve for the determination of the IC 50 value of 1 on HEK-293T cells.

Figure S8 .
Figure S8.Example of a fitted dose-response curve for the determination of the IC 50 value of 1 on HT-29 cells.