124I Radiolabeling of a AuIII‐NHC Complex for In Vivo Biodistribution Studies†

Abstract AuIII complexes with N‐heterocyclic carbene (NHC) ligands have shown remarkable potential as anticancer agents, yet their fate in vivo has not been thoroughly examined and understood. Reported herein is the synthesis of new AuIII‐NHC complexes by direct oxidation with radioactive [124I]I2 as a valuable strategy to monitor the in vivo biodistribution of this class of compounds using positron emission tomography (PET). While in vitro analyses provide direct evidence for the importance of AuIII‐to‐AuI reduction to achieve full anticancer activity, in vivo studies reveal that a fraction of the AuIII‐NHC prodrug is not immediately reduced after administration but able to reach the major organs before metabolic activation.


Supplementary Figures
X-ray structural analysis. X-ray quality crystals of complexes 3 and 4 were successfully obtained by liquid-liquid diffusion. Diffraction data were collected at the X-ray diffraction beamline (XRD1) of the Elettra Synchrotron (Trieste, Italy), at 100 K with a monochromatic wavelength of 0.700 Å The structural analysis revealed that complex 3 comprises an Au(III) metal center oriented in a rigorously square-planar environment with two trans-carbene ligands and two trans-iodide ions, counterbalanced by a PF 6 − anion. The two imidazole rings are almost coplanar (dihedral angle between their mean planes = 4.6°), while they are twisted by 84.69° relative to the C 2 I 2 coordination plane. The neutral complex 4 shows a structure comparable to that of 3, having one carbene ligand replaced by a chloride. Bond distances and angles of both compounds (see Table S2) are comparable to those measured for structurally related Au(III) complexes. [1][2][3] CCDC 1989306-1989307 contain the supplementary crystallographic data for this paper.         [4] Figure S14. Impact of the investigated Au compounds on cell cycle distribution. The indicated cell models were exposed to increasing concentrations of auranofin (left), 1 (middle), and 3 (right), for 24 h and cell cycle distribution was analysed by FACS sorting of PI-stained cells.
One representative experiment is shown.

General aspects
If not stated otherwise solvents and reagents were used in commercial grade without purification. Potassium iodide was purchased from Fisher Scientific and used as received.
Other chemicals were purchased from Sigma Aldrich, deuterated solvents from Cortecnet SI18 while other HPLC grade solvents were purchased from Scharlab (Sentmenat, Barcelona, Spain). Au(I) carbenes 1 and 2 were synthesized according to procedures already reported by some of us. [5] 1 H and 13 C NMR spectra were recorded on a Bruker Fourier TM 300 instrument equipped with Sample Xpress system or on a Bruker 500 Avance equipped with a z gradient BBOF probe.
Spectra were analysed using Mestrenova 12 (MestreLab Research S.L). Mass spectrometric analysis of the compounds was carried out on an Aquity Ultra Performance Liquid Chromatography (UPLC) separation module, coupled with a LCT-TOF Premier XE mass spectrometer (Waters, Manchester, UK). Reverse phase C18 column (50×2.1 mm, 1.7 μm particle size, Waters) was used as the stationary phase. Elemental analyses have been performed on a LECO ® microanalyzer.

X-ray crystallography
Data collections of the compounds reported were performed at the X-ray diffraction beamline (XRD1) of the Elettra Synchrotron (Trieste, Italy), with a Pilatus 2M image plate detector.
Complete datasets were collected at 100 K (nitrogen stream supplied through an Oxford Cryostream 700) with a monochromatic wavelength of 0.700 Å with the rotating crystal method.
The diffraction data were indexed, integrated and scaled using XDS. [7] The structures were solved by direct methods using SIR2014. [8] Fourier analysis and refinement were performed by the full-matrix least-squares methods based on F 2 implemented in SHELXL-2014. [9] The Coot program was used for modeling. [10] Anisotropic thermal motion was allowed for all nonhydrogen atoms. Hydrogen atoms were included at calculated positions with isotropic factors U = 1.2 Ueq, Ueq being the equivalent isotropic thermal factor of the bonded non hydrogen atom. Crystallographic and refinement data are reported in Table S1.

Stability of the Au(III) complexes in solution
Milli-Q (MQ) water was purified with a Millipore Direct-Q® 3 UV apparatus. Buffers were prepared by dissolving the suitable salts in MQ water and adjusting the pH.

DFT calculations
Calculations were performed using Gaussian16 package. [11] Geometry optimization was carried out using pbe1 algorithm [12] and CEP-31G functional basis sets. [13] Solvent was considered by means of the conductor like polarized continuum model (CPCM) with water as the implicit solvent. [14] Geometry was optimized and frequency calculations were carried out to avoid the presence of imaginary modes. Results have been compared with RX structures to check the suitability of the model. Louis, Missouri, USA).

Cell viability assays (MTT and ATP assays)
These assays were performed as published. [16] Shortly, 2-4x10 4 cells/ml were seeded into 96well plates, left to adhere overnight, then treated with the indicated concentrations of auranofin,

Hoechst 33258/propidium iodide (HoePI) staining.
Hoechst 33342/propidium iodide (Hoe/PI) double staining for detection of (early as well as late) apoptotic cells was performed as described in detail by Grusch et al. [4] A2780 cells were seeded in duplicates (1.25x10 4 cells per well) into 24 well plates, allowed to recover overnight, and treated with the indicated concentrations of gold compounds for 24h. Hoechst (1 µg/ml) / PI (2.5 µg/ml) was added for 1 h and pictures were taken on a Nikon eclipse Ti-e fluorescence microscope with a sCMOS pco.edge camera (100x magnification). Data evaluation was SI22 performed by counting the number of all cells as well as early and late apoptotic cells using the ImageJ software.

Cell cycle analysis by flow cytometry
3-5x10 5 cells per well were seeded into 6 well plates, allowed to recover for 24h, and treated with the indicated concentrations of auranofin, 1, or 3 for 24 h. Afterwards, propidiumiodidebased DNA content analysis (PI-staining) was performed as published. [17] PI fluorescence intensity measurements were carried out on a LSRFortessa flow cytometer (BD Biosciences, East Rutherford, NJ,USA) and data were analysed by Flowing Software 2.5.1 (Perttu Terho, Turku, Finland).

Clone formation assay
5x10 4 A2780 cells were incubated for 2h in PBS supplemented with calcium and magnesium (HyClone, GE Healthcare) with the indicated concentrations of 1 or 3 with/without 50µM ascorbic acid. Then, cells were centrifuged, the supernantant was removed, and the cell pellet was resuspended in 700 µL growth medium containing 10% FBS. Either 100 µL or 200 cells/well (7x10 3 or 14x10 3 cells/well) were seeded in duplicates into 24 well plates and further incubated to test for clone formation ability. Cell clones were visualized after 10 days by crystal violet staining as described [16] and scanned on a Typhoon scanner (Typhoon TRIO Variable Mode Imager, GE Healthcare Life Sciences). Afterwards, crystal violet was dissolved again by 2% SDS (in H 2 O), and absorbance (corresponding to the number of adherent cell clones) was measured at 560 nm on a Tecan infinite M200 pro spectrophotometer.

Western blot analysis
Western blot experiments were performed as published. In short, the indicated cell models were exposed in 6-well plates to increasing concentrations of the indicated gold compounds for 24 h before prepartation of total protein extracts for immunoblotting. [17] Radiochemical synthesis of compound 3

General aspects
Na Agilent 1200 series HPLC system equipped with a UV-Vis and a radioactivity detector (Gabi, Raytest) was used. A Mediterranea Sea18 column (4.6x150 mm, 5 μm particle size, Teknokroma, Spain) was used as the stationary phase and MQ water with 1% trifluoroacetic acid (TFA); B: acetonitrile; flow rate = 1mL/min were used as the mobile phase.

Pharmacokinetic Study
Anesthesia was induced to rats with 5% isoflurane and maintained by 2% of isoflurane in 100% minutes. The liquid phase was separated from the precipitate by decantation and was injected into the HPLC system, using the same conditions as for quality control.

In vivo PET Studies
PET studies were carried out in rats (n=2 per compound) using an eXplore Vista-CT small animal PET-CT system (GE Healthcare). Anaesthesia was induced with 5% isoflurane and maintained by 1. 5  All the scans were recorded in the 400-700 KeV energetic window. CT acquisitions were also performed at the end of each PET scan, providing anatomical information for unambiguous localization of the radioactive signal. After the imaging session, animals were sacrificed and organs were harvested for further processing (only for the case of [ 124 I]3).
PET images were reconstructed (decay and CT-based attenuation corrected) with filtered back projection (FBP) using a Ramp filter with a cut off frequency of 1 Hz. Images were analyzed using PMOD image analysis software (PMOD Technologies Ltd, Zürich, Switzerland). With that aim, frames acquired in the tame ranges corresponding to 0-1 min, 1-5 min, 5-15 min, 15-35 min and 35-60 min were averaged and volumes of interest (VOIs) were manually drawn in different organs (liver, kidneys, bladder, brain, lungs and stomach) using the CT images as anatomical reference. VOIs were then transferred to the PET images and time activity curves (decay corrected) were obtained for each organ as cps/cm 3 . Curves were transformed into real activity (Bq/cm 3 ) curves. Injected dose normalization was finally applied to data to get time activity curves as percentage of injected dose per cm 3 of tissue.

ICP Analysis.
After complete decay of the radioactivity, the extracted organs were digested in aqua regia overnight at 70°C. The samples were filtered through cotton and diluted with 2% HNO 3 to