I-radiolabeling of a Au(III)-NHC complex for in vivo biodistribution studies
§ contributed equally Au(III) complexes with N-Heterocyclic Carbenes (NHCs) ligands have shown remarkable potential as anticancer agents, yet their fate in vivo has not been thoroughly examined and understood. Herein we report on the synthesis of new Au(III)-NHC complexes via direct oxidation with radioactive [ 124 I]I2 as a valuable strategy to monitor the in vivo biodistribution of this class of compounds using positron emission tomography (PET). In combination with in vitro analyses, this radiolabeling strategy provides direct evidence of the importance of Au(III)-to-Au(I) reduction for achieving full anticancer activity.In modern medicine Au compounds are recognized for their beneficial activity against rheumatoid arthritis, being the clinically-approved auranofin the most representative drug of this type. [1][2][3] Nevertheless, the discovery that auranofin has also potent anticancer effects, [4,5]
<!>Supplementary Figures<!>General aspects<!>Synthesis of bis(1-butyl-3-methyl-imidazol-2-ylidene)-gold-diiodide hexafluorophosphate (3)<!>SI19<!>X-ray crystallography<!>Stability of the Au(III) complexes in solution<!>DFT calculations<!>Cell culture<!>Cell viability assays (MTT and ATP assays)<!>Cell cycle analysis by flow cytometry<!>General aspects
<p>together with the unceasing quest for metal-based drugs alternatives to cisplatin and its analogues, has stimulated the development of Au complexes as novel chemotherapeutic agents against cancer. [6][7][8] The pioneering work of Che, Ott, Messori and Casini, among others, demonstrated that Au(I) and Au(III) complexes bearing N-heterocyclic carbene ligands (NHCs) display unique anticancer activity profiles in vitro [9][10][11] and reduce tumor size in treated mice xenografts. [12][13][14][15][16] While the great majority of studies has so far focused on Au(I)-NHCs, reports on Au(III) analogues emerged more recently. [17][18][19][20] Under careful examination, these studies highlight that Au(III) complexes frequently display antiproliferative effects comparable to their Au(I) counterparts. [21,22] The reason for such resemblance likely lies in the limited stability of Au(III)-NHCs in biological environments and their rapid reduction to Au(I) metabolites. Surprisingly, this fundamental aspect has been overlooked in a significant portion of the studies reported on Au(III)-NHCs and their mode of action, generating some confusion on whether or not Au(III)-NHCs should solely be considered, and hence designed, as prodrugs of active Au(I) complexes. Among the exceptions in this panorama is, for example, the work of Che and coworkers, who prepared a series of Au(III)-NHCs for intracellular reductive activation by thiols [23] and demonstrated that the increased stability of pincer cyclometalated Au(III)-NHCs in biological settings led to remarkable in vitro and in vivo anticancer activity. [13,24] Furthermore, knowledge concerning the fate of Au(III)-NHCs after their administration in vivo is still largely insufficient. The study of the biodistribution of Au(III) compounds in vivo and the evaluation of their metabolization to Au(I) species is a very challenging task that has not been thoroughly addressed so far, albeit this is a key step in the development of new Au drugs. [25] Two studies used nuclear imaging and 198 Au radiolabelling to examine the biodistribution of Au(III) complexes bearing (bis)thiosemicarbazone-based and bis(pyrrolide-imine) Schiff-base ligands. [26,27] Nevertheless, such a radiolabelling strategy is not ideal to investigate the Au(III)to-Au(I) reduction processes of drug (or prodrug) candidates in animal models, since the radioactive signal of 198 Au does not report on the changes of the metal coordination sphere and does not provide information on ligand exchange or loss.</p><p>In this context, we describe herein a new strategy that exploits Au(I)/Au(III) coordination chemistry for labeling Au(III)-NHCs complexes with radioactive ligands and studying their organ accumulation in vivo. In our approach, Au(III) compounds are labelled with iodine-124 ( 124 I), a radionuclide used for positron emission tomography (PET), [28] so that they can be unequivocally discerned from their Au(I) counterparts and tracked by PET. By combining imaging studies with ex vivo analysis, direct evidence of the metabolization and biodistribution of Au(III)-NHCs overtime can be obtained. Of note, Messori and collaborators recently found that coordination of iodide enhanced the anticancer potency of Au derivatives. [29] To this aim, we selected and prepared Au(I/III) monocarbenic and biscarbenic complexes of the 1-butyl-3-methyl-imidazol-2-ylidene NHC ligand (1-4, Figure 1a). The Au(I) complexes 1 and 2 were previously reported by some of us and have potent in vitro antiproliferative activity against cisplatin-resistant cancer cell lines. [30,31] Furthermore, the homoleptic bis-carbene 2 showed promising properties both as thioredoxin reductase inhibitor [32] and as G-quadruplex binder. [33] The novel Au(III) complexes 3 and 4 were obtained by direct oxidation of 1 and 2, respectively, with elemental iodine. These reactions were carried out in CH3CN at room temperature, adapting a procedure reported earlier by Baron et al. for other Au derivatives. [34] We pursued and achieved a relatively easy-to-perform synthesis (ESI), with mild experimental conditions and high yield. These are fundamental prerequisites for translating synthetic preparations to the radiochemistry laboratory, where the use of elaborate procedures needs to be minimized or better avoided.  In order to understand the behavior of Au(III) carbenes in biological systems, we initially evaluated their stability in solution by UV-Vis spectroscopy and 1 H-NMR. Au(III)-to-Au(I) reduction was conveniently monitored by the decrease of the characteristic Au(III) absorption band between 300-450 nm (Figure S3) or by the shift of the 1 H-NMR peak relative to NHC ligands at around 7 ppm (Figure S4). The biscarbenic derivative 3 showed a double set of 1 H-NMR signals for the alkyl chains of the NHC ligands. 1 H-NMR experiments at variable temperature demonstrated that such resonances are due to the presence of syn/anti isomers in solution (Figure S5). Accordingly, DFT calculations determined an energy difference between the two isomers of only 0.3 kJ/mol, being the syn structure more stable than the anti.</p><p>UV-Vis and 1 H-NMR showed that 3 was stable in phosphate buffer (PB) and phosphate buffer saline (PBS) over 24 h (Figure S6 and S7). Instead, the UV-Vis spectra of 4 changed significantly in the same time window (Figure S8) and 1 H-NMR in PBS indicated that the monocarbene evolved to form the biscarbene 3 (Figure S9). Furthermore, 3 was stable for approximately 30-60 min in cell culture medium containing fetal bovine serum (Figure S10 and S11).</p><p>DFT electronic structure analysis of 3 and 4 revealed that these Au(III) compounds had LUMO orbitals with σ-antibonding character toward the Au-I bonds, in agreement with their tendency to undergo reductive elimination reactions. Conversely, 1 and 2 displayed bonding LUMOs toward Au-NHC bonds (Figure 1c), possibly accounting for the good stability of Au(I)-NHCs in biological environments. [31] Similar results were found for the anti isomers of 3 and 1 (Figure S12). Overall, experiments and theory confirmed that 3 had superior stability than its monocarbenic analogue 4. For this reason, we selected this complex for the radiochemistry work and subsequent in vivo studies.</p><p>Before starting in vivo biodistribution experiments, we conducted in vitro studies comparing 1 and 3 to examine the impact of the Au(III)/Au(I) reduction process on the anticancer effects. A viability screening on a panel of cancer cell lines, comprising models with acquired resistance against cisplatin and oxaliplatin, was performed. The activity of 1 and 3 was also compared to the well-established Au compound auranofin (Table 1).</p><p>Overall, while both Au-NHC compounds showed strong antiproliferative activities, with IC50 values falling in the low micromolar range for most of the cancer cell lines examined, only moderate differences between 1 and 3 were observed. For instance, 3 was more active than 1 in some of the p53 wild-type models (HCT116-p53wt, A2780). Moreover, knock-out of p53 significantly sensitized cells against 1 but not against 3, confirming an impact of p53 signaling on either uptake or activation of the two Au-NHC complexes, as recently reported for 1. [30] Interestingly, an analysis of the platinum resistance cell models revealed a clear dissimilarity in the mode-of-action and/or resistance behavior of the two Au-NHCs compared to auranofin.</p><p>For example, the cisplatin-resistant subline of A2780 exhibited hypersensitivity against 1 and 3 as compared to the parental cell line, while, in sharp contrast, resistance against auranofin was observed. The situation was reversed in the HCT116-p53ko background with acquired oxaliplatin resistance, which induced only a moderate insensitivity against auranofin but a marked one against both 1 and 3. When analyzing the impact of the Au compounds on cell viability and proliferation using Hoechst/PI staining, it became immediately evident that auranofin predominantly induced cell death (PI-positive red cells), while 1 and 3 were predominantly cytostatic (Figure S13).</p><p>Accordingly, cell cycle distribution analysis revealed that a 24 h incubation with both 1 and 3 induced a significant and dose-dependent cell accumulation in the G1 phase of different models, with exception of the A2780 parental cells (Figure S14). Furthermore, an ATP-based assay indicated for both Au-NHCs (but to a much lesser extent for auranofin) a higher anticancer activity compared to the MTT-based results (Figure S15), suggesting a strong effect of Au-NHC compounds on mitochondrial ATP production. Viability assays in the A2780 and HCT116 models were also performed in the presence of N-acetyl cysteine (NAC), a known ROS scavenger and reducing agent. While the anticancer activities of 1 and 3 remained widely unaffected, we observed a clear protective effect for auranofin in all drug-sensitive parental models (Figure S16).</p><p>The sum of these data suggested a significantly different mode-of-action for the Au-NHC agents as compared to auranofin. However, an overall similar toxicity profile was observed for 1 and 3, advocating that the reduction of 3 to yield 1 took place extremely rapidly in cell culture medium. This could occur even before addition to the cell cultures, considering that viability experiments required initial dilutions of the compounds in cell culture medium supplemented with FBS (in which 3 is poorly stable as shown in Figure S11).</p><p>Therefore, we performed the MTT cell viability assay in the absence of serum or under hypoxic conditions (Figure 2a). Serum reduction to 0.1% resulted in an enhanced activity for all Au complexes, whereas the reducing hypoxic environment sensitized against auranofin but protected against Au-NHCs. The comparable response of 1 and 3 to these specific experimental conditions highlighted that the Au(III) reduction process was not critical and, obviously, other factors played a role (e.g.</p><p>inactivation of the Au complexes by a serum component). For this reason, we performed another experiment exposing A2780 cells to the Au complexes for 2 h in PBS with and without ascorbic acid (AA) as reducing agent, followed by a colony formation assay without drugs in 24-well plates. The densitometric evaluation of these colony formation experiments revealed an enhanced activity of 1 as compared to 3 over the entire concentration range, as well as a significant chemosensitization of 3 by AA at the different concentrations analyzed (Figure 2b).</p><p>On the contrary, 1 tended to be even less active in the presence of AA, ultimately proving that 3 needs reduction for exhibiting optimal anticancer activity in vitro.</p><p>To mimick the in vivo sample administration conditions for biodistribution studies (vide infra), we used, in addition to the standard DMSO stock solution, also a PEG-400-based formulation (30% PEG, 5% DMSO, 65% PB) of the two Au-NHC complexes in vitro. Mostly, the IC50 values for the PEG-400 formulation remained in a comparable concentration range, but the activity of 1 tended to be increased whereas 3 tended to be less active in several cell models (violin blot in Figure S17a). As illustrative example, selecetd dose-respose curves for MCF-7 breast cancer cells are shown in Figure S17b. These data suggest that the reduction process of 3 to 1 might be delayed by the presence of PEG-400, depending on the cellular background.</p><p>Additionally, the PEG-400 formulation at concentrations above the IC50 value was distinctly more active as compared to the DMSO counterpart, suggesting upregulation of the cytotoxic activities of both 1 and 3 (HCT116 cell models shown representatively in Figure S17c).</p><p>Interestingly, this effect was able to overcome the resistance against the Au-NHC compounds observed in the oxaliplatin-resistant HCT116-p53ko/OxR model (compare Table 1). In order to prove whether 3 is protected against immediate reduction to 1 in the presence of PEG-400, combination experiments with AA and NAC were performed. Indeed, a sensitizing effect of coincubation with the two reducing agents was primarily observed in the case of 3 while the effects were variable and minor for 1 (Figure 2c). Together, the PEG-400 formulation had superior performance in terms of anticancer activity, resistance circumvention, and, especially, delayed prodrug activation essential for in vivo biodistribution studies.</p><p>Radiolabeling of 3 for in vivo work was carried out following the scheme of The solid was then isolated by centrifugation, washed with water and reacted with 1 in CH3CN for 1 h. As determined by high performance liquid chromatography (HPLC) connected in series with a radioactivity detector, we obtained a 95% purity of the product (Figure S18). The radiochemical yield (35%) was relatively high considering a maximum theoretical yield of 40% (ESI). Due to its modest water solubility (< 0.4 mg/ml with 15% DMSO) and the toxicity of DMSO at high concetrations, radiolabeled 3 was solubilized in a PEG-400 based formulation (30% PEG, 5% DMSO, 65% PB) for subsequent intravenous injection in rodents. We selected such a formulation on the basis of tolerability and solvent dose limits suggested by Thackaberry et al. [35] Indeed, [ 124 I]KI dissolved in this mixture and used as control proved to be well tolerated by intravenously administered rats. HPLC experiments confirmed that the radiolabeled 3 was stable over 24 h in the PEG-400 formulation (Figure S19). Crucially, time dependent UV-Vis spectra indicated that formulated 3 was more stable than the complex alone in PBS against reduction by stoichiometric amounts of AA (Figure S20). In buffer, 3 was completely reduced by AA in less than 20 min, whereas in the PEG-based formulation the Au(III)-associated UV-Vis band at 300-450 nm could be observed even after 2 h (~ 70% reduction).</p><p>In a first in vivo experiment, we investigated the biological half-life of compound 3 measuring its time-activity curve in blood (that is, the concentration of radioactivity in blood as a function of time). To such aim, the arterial input function was determined by catheterization and online blood sampling using a previously reported procedure with minor modifications. [36] In brief, a 640 µM solution of [ 131 I]3 dissolved in the PEG-400 formulation (1.11 MBq) was intravenously administered to a wild type rat bearing a bypass between the femoral artery and the femoral vein, in order to achieve an extracorporeal circulation of the blood through a radioactivity detector. Blood concentration of the radioactive signal versus time was measured and the resulting curve was analyzed using a nonlinear regression method (Figure 4a). Subsequently, we explored the accumulation of radioactivity in the whole body after intravenous administration of [ 124 I]3 (1.2 MBq, 640 μM) using PET imaging (Figure 4b). The amount of compound injected per animal was ca. 0.45 mg/kg, which is in the range of pharmacological doses used in therapeutic studies. Hence, no further dilution with nonradioactive compound was carried out. [ 124 I]KI (1.9 MBq, 1 mM) was also administered in a separate animal (Figure S21a) using the PEG-400 formulation as a control. Visual inspection of PET images (Figure 4b) showed high accumulation of radioactivity in the heart at short times (ID: injected dose) at the last time point. Such accumulation was expected since the stomach is the natural site of iodine metabolism. [37] Additionally, we determined the body distribution of Au by ICP-MS harvesting the organs of the rats treated with the radiolabeled compound immediately after termination of the imaging sessions. Comparing such data with the PET results in terms of % ID per gram of tissue (Figure 4d), we observed a good correlation in the liver, kidneys, and lungs confirming the idea of a rapid accumulation of the Au complex 3 in such organs. At the same time, the differences between ICP-MS and PET quantification were visible in the bladder, the brain and were particularly significant in the stomach. This clearly indicates that Au concentration is low and that free [ contrary to what is reported in the literature for 1 and other similar derivatives, [30,38] the modeof-action profile of 3 and 1 strikingly differs from that of auranofin, the reference Au compound to date.</p><!><p>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 PF6 − 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 C2I2 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.</p><!><p>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.</p><p>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.</p><p>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.</p><!><p>Compound 3 was obtained by adapting a procedure reported by Baron et al. for dinuclear Au(III) NHC complexes. [6] The gold(I) precursor 1 (50 mg, 0.08 mmol) was dissolved in 7 ml of acetonitrile. 2.5 equivalents of I2 (51 mg, 0.2 mmol) were added from a freshly prepared 0.5 M stock solution in acetonitrile. The reaction was protected by light. After 1 h solvent was removed under reduced pressure, the resulting orange solid was washed with hexane to remove the excess of I2 and dried under vacuum. (60 mg, 86% yield). Single crystals suitable for x-ray diffraction analysis were obtained by stratification of diethyl ether onto a concentrated solution of 3 in acetonitrile.</p><!><p>The synthetic procedure was the same followed for 3. Complex 2 (50 mg, 0.135 mmol), I2 (85 mg, 0.338 mmol). After 1 h stirring at room temperature, the reaction mixture was filtered on celite to remove what seemed to be Au(0) (black powder). Solvent removal under reduced pressure gave an oily solid residue. The residue was dissolved in a little amount of DCM and precipitated with hexane to obtain a purple solid. (41 mg, 48% yield). Single crystals suitable for x-ray diffraction analysis were obtained by stratification of hexane onto a concentrated solution of 4 in dichloromethane.</p><!><p>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.</p><p>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.</p><p>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.</p><!><p>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. Concentrated stock solutions of the compounds (5 mM or 10 mM) were freshly prepared in DMSO, in deuterated or non-deuterated acetonitrile and then diluted in the suitable buffers. UV-Vis absorption spectra were recorded on a Jasco V-730 spectrophotometer. All spectra were recorded using quartz cuvettes (Hellma Analytics) of 1000 µL or 2000 µL volume and an optical path of 1 cm.</p><!><p>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.</p><!><p>The human cancer cell lines MCF-7, A375, and N87 were purchased from American Type Culture Collection (Manassas, VA, USA). A2780 and A2780cis were purchased from Sigma-Aldrich (MO, USA). HCT116-p53wt and HCT116-p53ko were kindly donated by Dr. B.</p><p>Vogelstein (John Hopkins University, Baltimore, USA, and the respective oxaliplatin-resistant HCT116 sublines were generated in the lab of Walter Berger at the Institute of Cancer Research (Vienna, Austria). [15] All cell lines were cultured in growth media (MCF- Louis, Missouri, USA).</p><!><p>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, 1, 3, or oxaliplatin for 72 h. Gold drugs were standardly dissolved in DMSO (10mM stock solutions for 1, 3, and auranofin). Oxaliplatin was dissolved in H2O (10mM stock solution). For analysis of the activity of 1 and 3 dissolved in the PEG-400-based formulation, stock solutions were prepared using a mixture of 30% PEG-400, 5% DMSO, and 65% PB instead of DMSO.</p><p>For testing the effect of N-acetylcysteine (NAC) and ascorbic acid (AA) on the anticancer activity of the gold compounds, cells were pre-incubated with 1 mM NAC or 50 µM AA for 1h before the respective gold drugs were added for 72 h. For serum reduction experiments, cells treated with the indicated gold drugs were incubated in growth medium containing 0.1% instead of 10% FBS. For analysis of the influence of hypoxic conditions, cells treated with the indicated gold drugs were incubated under 1% oxygen in a Heracell 150i incubator (Thermo Scientific). Cell viability was either determined using the 3-( 4 Hoechst 33258/propidium iodide (HoePI) staining.</p><p>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. [17] 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.</p><!><p>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. [18] 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).</p><p>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 H2O), and absorbance (corresponding to the number of adherent cell clones) was measured at 560 nm on a Tecan infinite M200 pro spectrophotometer.</p><!><p>Na</p><p>Animals were maintained and handled in accordance with the Guidelines for Accommodation For in vivo studies, compound 3 was dissolved in 1000 µL mixture of 5% DMSO, 30% PEG400 in PBS to a final concentration of 640 µM. Stability in this formulation over 24 h was evaluated through UV-Vis absorption spectroscopy and HPLC analysis. For UV-Vis experiments solutions of 150 µM 3 where prepared in the formulation alone or with addition of 10% FBS, protected from light and analysed over 24 h or 1 h respectively. For HPLC analysis, 10 µL of 124 I-radiolabelled 3 in formulation were taken and diluted in 20 µL of a 1:1 mixture of CH3CN/H2O. 20 µL of the obtained solution were injected into the HPLC. For the analysis, an 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.</p>