Morwen R M Williams

Benoît Bertrand
email: benoit.bertrand@upmc.fr

David L Hughes

Zoë A E Waller

Claudia Schmidt

Ingo Ott

Maria O'connell

Mark Searcey
email: m.searcey@uea.ac.uk

Manfred Bochmann
email: m.bochmann@uea.ac.uk

Cyclometallated Au(III) dithiocarbamate complexes: synthesis, anticancer evaluation and mechanistic studies

A series of cationic mixed cyclometallated (C^N)Au(III) dithiocarbamate complexes has been synthesized in good yields [HC^N = 2-(p-t-butylphenyl)pyridine]. The crystal structure of [(C^N)AuS2CNEt2]PF6 (3) has been determined. The cytotoxic properties of the new complexes have been evaluated in vitro against a panel of human cancer cell lines and healthy cells and compared with a neutral mixed (C^C)Au(III) dithiocarbamate complex (C^C = 4,4′-di-t-butylbiphenyl-2,2′-diyl). The complexes appeared to be susceptible to reduction by glutathione but were stable in the presence of Nacetyl cysteine. The potential mechanism of action of this class of compounds has been investigated by measuring the intracellular uptake of some selected complexes, by determining their interactions with higher order DNA structures, and by assessing the ability to inhibit thioredoxin reductase. The complexes proved unable to induce the formation of reactive oxygen species. The investigations add to the picture of the possible mode of action of this class of complexes.

Introduction

Since the approval in the late 70's of cis-diamminodichloroplatinum(II) (cisplatin) for the treatment of various cancers including testicular, ovarian, prostate or lung cancers, 1 the field of metal-based anticancer drugs has grown rapidly. However, platinum drugs have several limitations, including resistances and heavy side effects affecting fastly growing tissues. 2 This has led to a search for the possible replacement of platinum by other transition metals, such as iron, 3 copper, 4 ruthenium 5 or gold, 6 and promising results have been obtained. Organometallic complexes of gold in oxidation states +I and +III have attracted particular attention for anti-cancer purposes due to their high stability in physiological media and the ease of their derivatization. The evolution of the field has been regularly reviewed. 7 Cyclometallated Au(III) with (C^N), (N^N^C) or (C^N^C) ligands have been tested and show activities up to low-micromolar range. 8,[START_REF] Bertrand | Pincer Compounds -Chemistry and Applications[END_REF] Mono-cyclometallated (C^N)Au(III) complexes have appeared particularly interesting due to the large palette of (C^N) ligands available, including derivatives of N,Ndimethylbenzylamine, [START_REF] Parish | [END_REF] phenylpyridine, 11 benzylpyridine, 12 or iminophophorane. 13 A broad range of ancillary ligands is tolerated as well. Some representative examples are collected in Figure 1. 14 Although the mechanism of action of these complexes could not yet be fully elucidated, 6a investigations have shown that cyclometallated (C^N)Au(III) complexes inhibit several enzymes such as cathepsin B and K, 12 thioredoxin reductase (TrxR) 8c, 15 or poly(ADP)-ribose polymerase 1 (PARP-1). 16 Complex B bearing a highly hydrophilic biguanide ligand (Figure 1) has been demonstrated to induce reticulum endoplasmic stress and to present anti-angiogenic effects at sub-cytotoxic concentrations. 17 Dithiocarbamate (DTC) ligands are also well-known to efficiently stabilize Au(III) cations and have been widely used for making anticancer drug candidates (see for example Figure 1 structures C -E). 7c Complexes based on the scaffold C were highly active in vitro against several human cancer cell lines, induced the formation of reactive oxygen species (ROS), and triggered apoptosis via mitochondria-related pathways. 18,19 Inhibition of proteasomes by Au(DTC) complexes has also been demonstrated both in vitro and in vivo. 20 The association of (C^N) cyclometallated ligands with dithiocarbamates ligands has been explored in Au(III)-based drug candidates using either iminophosphorane or phenylpyridine ligands (D and E in Figure 1) and gave compounds with in vitro IC50 values in the low to submicromolar range against human cancer cells. 21,22 From a mechanistic point of view, (C^N)Au(DTC) complexes present several features in common with their pure DTC analogues, including cell death through both apoptosis and necrosis 21 and inhibition of proteasomes. 22 Furthermore, complex E has been demonstrated to have anti-angiogenic activity, similar to B which bears the same (C^N) ligand, and to inhibit deubiquitinases, enzymes regulating the ubiquitination of proteins. 22 However, the complete mechanism of action of that class of organogold complexes is far from fully elucidated.

Here we report the synthesis of a series of gold(III) compounds that combine the stabilisation by a cyclometallated (C^N) framework with dithiocarbamate (DTC) ligands, in an effort to probe their effect on a panel of human cancer cell lines in vitro, including lung cancer (A549), breast cancer (MCF-7 and MDA-MB-231), colon cancer (HCT-116) and leukaemia (HL60) cells and the normal human umbilical vein endothelial cells (HUVEC). We also explore their activity against different cellular targets associated with each class of compounds, namely the induction of ROS (dithiocarbamates), the interaction with non-canonical DNA structures (C^N chelates), and the inhibition of TrxR (typical of dithiocarbamates and some cyclometallated compounds).

Results and discussion

Synthesis and characterization

The (C^N) cyclometalated gold dichloride precursor 1 and the (C^C)Au(DTC) complex 7 were synthesized by previously reported methods. 22,24 An overnight reaction with sodium dialkyldithiocarbamate hydrate in methanol followed by the addition of aqueous potassium hexafluorophosphate or silver hexafluoroantimonate gave the cyclometallated dialkyl-dithiocarbamate complexes 2-6 in good yields (71-94 %), as depicted in Scheme 1.

Scheme 1: Synthesis of the [(C^N)Au(DTC)] + complexes 2-6, including the numbering scheme used for NMR signal assignments.

The 1 H NMR spectra of complexes 2-6 showed a significant downfield shift in the characteristic H 2 doublet signal from δ 9.71 to 8.40-8.78 ppm, as well as a downfield shift in the signal for H 11 from δ 8.03 to 7.03-7.15 ppm, confirming the formation of the different complexes. The coordination of the dithiocarbamate ligand was indicated by the observation of two inequivalent alkyl groups. The C 13 { 1 H} NMR resonance for the NCS2 carbon appeared between δC 190.4 and 198.7 ppm. Bands in the IR spectra between 1550 and 1579 cm -1 are attributed to the delocalised dithiocarbamate NCS2 system. In the particular case of complex 6 bearing a non-symmetrically substituted DTC ligand based on sarcosine ethyl ester, the two possible isomers (Figure 2) were observed in the 1 H NMR spectrum in a ratio of 1:0.8.

In vitro antiproliferative activity

Although the dithiocarbamate complexes 2 -7 were poorly soluble in an aqueous cell culture medium, they were soluble enough in DMSO not to precipitate when diluted up to 100 µM in the aqueous culture medium with 1% DMSO. IC50 values for the six complexes were then determined on a panel of human cancer cell lines. These included solid tumour cell lines; lung adenocarcinoma cells (A549), breast adenocarcinoma (MCF-7 and MDA-MB-231) and human colon cancer (HCT-116) and suspension cells (promyelocytic leukaemia, HL60), as well as healthy Human Umbilical Vein Endothelial Cells (HUVEC) for comparison. Results were determined using a colorimetric MTS assay after 72 h of incubation in comparison to cisplatin (see Experimental part). The results are reported in Table 1. Complexes 2 -4 all showed similar cytotoxicity profiles, with high levels of cytotoxicity towards all the tested cell lines. The compounds were more active than the dichloro precursor 1 on all comparable cell lines. The two complexes presenting the shortest alkyl substituents on the dithiocarbamate ligand (2 and 3) showed an activity that was, repectively, 7 and 12 times higher thanthat of cisplatin towards A549 lung cancer and more than 15 times more active towards MCF-7 breast cancer and HL60 leukaemia cells. The complexes also showed more than twice the activity of cisplatin towards the colon cancer HCT-116 cells lines, while complex 3 was15 times more active than cisplatin against the MDA-MB-231 metastatic breast cancer cells.

It was thought possible that an increase in lipophilicity of the complex could induce an increase in the cellular uptake. However, the more lipophilic pyrrolidine and dibutyl variants 4 and 5 were slightly less cytotoxic compared to 2 and 3, although they still showed extremely promising levels of cytotoxicity in comparison to cisplatin. These results are in line with data reported on similar [(C^N)Au(DTC)] + complexes. 22 However, despite their promising anticancer activities all four complexes showed poor selectivity towards the Human Umbilical Vein Endothelial Cells (HUVEC), with IC50 values in the submicromolar range. Further tests on other healthy cell lines are needed for a more accurate conclusion as HUVEC are perhaps not the most reliable comparison,since they typically show a reduced sensitivity towards cisplatin. 29 The sarcosine ethyl ester dithiocarbamate, complex 6 showed surprisingly low activities across the panel of cells, with IC50 values of more than 100 µM for A549, MCF-7, MDA-MB-231 and HCT-116. This could be due to poor cellular uptake as the complex is highly hydrophilic and therefore might struggle to cross the cell membrane. The complex also showed poor activity towards HL60, leukaemia cells, with an IC50 of 17.8 µM. In that case, the replacement of the two chlorido ligands by the sarcosine ester-based DTC ligand lead to the reduction in the antiproliferative activity of the Au(III) complex.

However, this complex also showed the best selectivity profile of the new dithiocarbamates towards this cell line, (selectivity factor SHL60/HUVEC = 1.9).

The neutral (C^C) complex 7 also showed reduced cytotoxicity compared to its cationic (C^N) analogue 3, particularly towards the HCT-116, MDA-MB-231 and A549 cells, although it was reasonably active towards MCF-7, breast cancer and HL60 leukaemia cells, (IC50 values of 8.8 and 1.9 µM respectively). Once again, the reduced activity could be due to reduced cellular uptake as the (C^C) cyclometalated ligand and the absence of acharge makes this complex very lipophilic.

Reaction with glutathione and N-acetyl cysteine

Many gold(III) complexes show only weak and reversible binding to DNA, which is the primary target of platinum-based drugs. 30 However, cytotoxic gold compounds have shown high reactivity towards model proteins. Consequently gold-protein interactions are generally thought to be responsible for the cytotoxic effects of these complexes and are regarded as their primary targets. 6c Metal-protein binding sites generally involve the side chain residues of amino acids such as the thioether and thiolate sulphur atoms in L-methionine and L-cysteine, respectively. 31 Complex 2 as a representative for cationic [(C^N)Au(DTC)] + compounds was reacted with glutathione (GSH) and N-acetyl cysteine (NAC), which were selected as reliable models of target biomolecules. Although both GSH and NAC present the same thiol group, they have very different redox potentials, with the potential of NAC being 63 mV more positive than that of GSH. This makes NAC a weaker reductant compared to GSH. 32 On treating 2 at room temperature with GSH in a 1:1 mixture of DMSO-d6 and D2O, 1 H NMR spectroscopy showed the immediate splitting of the signal of the CH2S group (multiplet at 2.75 ppm for GSH) into two signals (doublets of doublets at 2.8 and 3.1 ppm) characteristic of the formation of oxidized glutathione (GSSG) (Figure 4A). 33 Although no signals for the complex could be seen due to its poor solubility in these solvents, the formation of a pale-yellow product was observed, which suggested reduction to a gold(I)

species. This reduction of 2 by GSH could also be followed by UV/Vis spectroscopy (Figure 4B). Upon mixing a 1 mM solution of 2 in DMSO with 1 mM of GSH in H2O, the characteristic ligand-to-metal charge-transfer (LMCT) band 34 of 2 at 350 nm disappeared, confirming that fast reduction occurred, as already observed for gold(III) complexes with bipyridyl ligands. 35 The appearance of a broad UV/vis band at 400-450 nm could also be tentatively attributed to a gold(I) species. 36 The same features could also be seen when other dithiocarbamate derivatives were reacted with GSH (Figure S1-5).

Cellular uptake

Cellular uptake of drugs can have a significant influence on the cytotoxicity of prospective chemotherapeutic agents. 37 Inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantify the amount of intracellular gold and determine whether the uptake of the dithiocarbamate complexes correlated with their cytotoxicity. Complexes 2 and 5 (the dimethyl and dibutyl variants)

were selected as these represented, respectively, the shortest and longest alkyl substituents R in the [(C^N)Au III (S2CNR2)] + series. The sarcosine ethyl ester variant 6 was selected because it showed extremely poor cytotoxicity towards all tested cell lines, together with 7, the (C^C) diethyl dithiocarbamate, as it was charge-neutral and the only complex with a (C^C) ligand. MCF-7 cells were incubated for 6 h with 10 µM concentrations of each of the complexes in 1% DMSO. The results of three independent experiments are depicted in Figure 6. As we previously observed for other organometallic complexes including cyclometallated Au(III) complexes, 26,27 there was a correlation between the trends in the cellular uptake study (uptake of 2 >> 5

> 7 > 6) and in the in vitro cytotoxicity study of the complexes (IC50 2 ≈ 5 < 7 << 6) as depicted in Figure 6B. The most cytotoxic complexes 2 and 5, which have IC50 values of 1.4 and 1.7 µM respectively, both show high levels of uptake, particularly the dimethyl variant, 2 which displays more than three times the cellular uptake of complex 5. Complex 7, the neutral (C^C) dithiocarbamate, is moderately cytotoxic towards MCF-7 cells with an IC50 of 8.8 µM and also displays quite a low cellular uptake. Both 2 and 5 present significantly higher levels of cellular uptake than 7 (28 and 8 times respectively). Complex 6, the most hydrophilic complex bearing a sarcosine ethyl ester substituent, has both very low in vitro cytotoxicity (IC50 value of > 100 µM) and also poor cellular uptake. It seems therefore that there is a delicate balance between lipophilicity, charge and solubility under physiological conditions which must be met for compounds to be able to cross the phospholipid bilayer cell membrane. This behaviour is consistent with our previous observations on the cytotoxicity of pincer-stabilised complexes of the type [(C^N pz ^C)Au(NHC)] + (NHC = N-heterocyclic carbene). 27

Quantification of reactive oxygen species

Induction of reactive oxygen species (ROS) is a recognized mechanism of action for metal-based drugs, including Au III (DTC) complexes. 18b The cyclometallated dithiocarbamate complexes investigated here were therefore tested for the production of ROS. The amount of intracellular ROS was measured after treatment of MCF-7 cells with 100 µM, 50 µM and 10 µM concentrations of each complex. The results are summarized in Figure 7. Contrary to what has been observed in the case of AuCl2(S2CNMe2), 18b none of the tested complexes appeared to increase the production of ROS and therefore this mode of action can be ruled out for these cyclometallated gold dithiocarbamates. However, this is in line with reports for other cyclometallated Au(III) complexes, 28,38 suggesting that in our case of mixed cyclometallated/DTC complexes, the inability to generate ROS is related to the cyclometallated ligand structure.

Figure 7: ROS measurements in MCF-7 cells after 24 h incubation with complexes 2 -7.

Interactions with non-canonical DNA structures

Higher-order DNA structures like G-quadruplexes and i-motifs are emerging as promising targets for the development of new anticancer agents. 39 G-quadruplexes are four-stranded DNA secondary structures that form in guanine-rich DNA sequences. They are formed by the stacking of tetrads of guanine residues linked via hydrogen-bonding and stabilized by the presence of typically monovalent cations in the centre of the tetrad. 40 The selective stabilization of G-quadruplex structures has been investigated as a method of controlling key cellular events such as telomerase activity or oncogene expression making them potentially interestinganticancer targets. Cyclometallated Au(III) complexes have been reported to selectively stabilize the non-canonical DNA G-quadruplex and i-motif structures. 27,41b We thus investigated the influence of the DTC ligands on the interaction between the cyclometallated Au(III) moiety and G-quadruplexes and i-motifs.

The established Fӧrster resonance energy transfer (FRET) based DNA melting method measures the changes in fluorescence as the spatial distance between two fluorophores at the ends of DNA is changed as they are brought closer together or moved further apart. Comparison of the melting temperature of DNA in the absence and presence of compounds can reveal any compound-induced DNA stabilization or denaturation. 43 Here, FRET results are expressed as the changes in DNA melting temperature (ΔTm) when DNA (0.2 µM) is dosed with 50 µM concentrations of each drug. Thus the higher ΔTm is in the presence of a compound, the higher is the DNA stabilization and thus the stronger is the compound-DNA interaction. The DNA melting assay was used to give a broad indication of the DNA binding capabilities of the dithiocarbamate complexes 2 -7 towards different DNA targets. We used a G-quadruplex forming sequence from the human telomere (hTeloG), the human telomeric i-motif sequence (hTeloC), and i-motif forming sequences from the promoter regions of HIF-1-α (hif-1-α) and c-Myc (c-MycC), as well as double-stranded DNA.

The human telomeric i-motif forming sequence, hTeloC is most stable at acidic pH. The binding capabilities of the complexes were therefore assessed at a transitional pH of 6.0 (the pH where the structure is 50 % folded). 44 The c-MYC i-motif, is also more stable at acidic pH, was measured at the respective transitional pH of 6.6. 39d The remaining sequences were all tested at the transitional pH of hif-1-α, pH 7.2. 45 Results of the FRET-melting assay with complexes 2 -7 are depicted in Figure 8. Complexes 2, 3, 4 and 5 showed high levels of DNA stabilization towards all higher-order DNA structures tested. These four complexes showed particularly high levels of stabilization towards two of the i-motif forming sequences, the human telomerici-motif sequence, (hTeloC) and the i-motif forming sequence from the promoter region of c-Myc (c-MycC). For these two sequences, ΔTm values were between 47-51 °C for complexes 2, 3 and 4 and > 60 °C for complex 5. For the G-quadruplex forming sequence, (hTeloG) and the i-motif forming sequence, (hif-1-α), ΔTm values were somewhat lower, between 26-37 °C for all four complexes, indicating lower levels of complex-DNA stabilization and thus weaker DNA binding.

In good agreement with the cytotoxicity data, both 6, the least cytotoxic of the [(C^N)Au(DTC)] + complexes, and 7, the neutral (C^C)Au(DTC), showed only low levels of DNA interaction towards all tested structures. For both types of complexes, ΔTm values were below 10 °C for all of the DNA structures tested. These values could be a result of the lack of a cationic charge as this is consistent with our previous work, which highlights the importance of a cationic charge on the ability of gold complexes to interact with higher-order nucleic acid structures. 27 This suggests that mixed cyclometallated/dithiocarbamate Au(III) complexes behave similarly to pure cyclometallated Au(III)

complexes, with the nature of the DTC ligand acting only to modulate the strength of the gold complex/DNA interaction. The results of the FRET-melting assay are generally in good agreement with the cytotoxicity data, with the most cytotoxic complexes displaying the highest levels of stabilization of the higher-order DNA structures. This suggests that the stabilization of these structures could be a possible mechanism action of these complexes.

Inhibition of thioredoxin reductase

Thioredoxin reductase (TrxR) is a homodimeric enzyme whose role consists of reducing thioredoxin values dropping to sub-micromolar levels. The most hydrophilic complex, 6, the sarcosine ethyl ester derivative, was non-toxic across all of the cell lines. The most lipophilic complex, the (C^C) derivative 7, also showed only low cytotoxicity. Cellular uptake studies in MCF-7 breast cancer cells indicated that there was a correlation between the in vitro cytotoxicity of the complexes and their ability to enter the cell. Complex 2 and 5 both showed high levels of cellular uptake in comparison to 6 and 7. However, this correlation is non-linear, and therefore cell uptake is only one of the preconditions for cytotoxicity.

Redox stability and mechanistic studies on targets have been carried out, including ROS production for the (DTC)Au(III) complexes and G-quadruplex and i-motif DNA structures for the cyclometallated Au(III) compounds, as well as TrxR inhibition studies as a common target to both classes of compounds. This has enabled us to investigate the influence of each structural element on the pharmacological properties of the other. We observed that cyclometallated gold(III) dithiocarbamate complexes presented an intermediate redox stability between the pure cyclometallated and pure dithiocarbamate Au(III) complexes. The presence of a cyclometallated ligand completely inhibited the ability of the (DTC)Au(III) to generate ROS, while the interaction of the mixed complexes with Gquadruplexes and i-motifs followed the same trend as for non-DTC cyclometallated Au(III), with only a small influence of the nature of the DTC ligand. Moreover, complex 3 presented an intermediate inhibitory activity against TrxR between those of the pure cyclometallated and dithiocarbamate Au(III) complexes, suggesting that cyclometallated/dithiocarbamate Au(III) complexes should be considered as an independent class of compounds in some cases. Alltogether these data highlight the potential of fine tuning the reactivity of this class of organogold complexes.

For a better understanding of the full mechanism of action, the activity of cyclometallated/dithiocarbamate Au(III) complexes against other reported targets of each single classes, such as proteasome, PARP-1 or cathepsins, should be investigated. Moreover, beyond those reported targets, specific targets of this peculiar class of compound might emerge.

Experimental

When required, manipulations were performed using standard Schlenk techniques under dry nitrogen or in an MBraun glove box. Nitrogen was purified by passing through columns of supported P2O5 with moisture indicator, and activated 4 Å molecular sieves. Anhydrous solvents were freshly distilled from appropriate drying agents. 1 H and 13 C{ 1 H} spectra were recorded using a Bruker Avance DPX-300 spectrometer. 1 H NMR spectra (300.13 MHz) were referenced to the residual protons of the deuterated solvent used. 13

Synthesis of complex 2

A solution of sodium dimethyldithiocarbamate hydrate (0.020 g, 0.140 mmol) in methanol (10 mL) was added dropwide to a suspension of (C^N)AuCl2 (0.060 g, 0.125 mmol) in methanol (15mL) and stirred at room temperature overnight. A colour change from white to pale yellow was observed during the addition. An excess of a saturated aqueous solution of KPF6 was then added, which caused an immediate fluffy white precipitation. The solid was filtered off and purified by dissolving in a minimal amount of acetonitrile (2 mL) and precipitating with excess diethyl ether (20 mL). The product was filtered and dried under vacuum (0.069 g, 0.103 mmol, 82%

. IR: νmax (neat)/cm -1 : 1579 (dithiocarbamate).

Synthesis of complex 3

Following the procedure described for 2, complex 3 was made from (C^N)AuCl2 (0.060 g, 0.125 mmol) and NaS2CNEt2•H2O (0.028 g, 0.125 mmol) as a white microcrystalline solid (0.083 g, 0.118 mmol, 94%). Anal.Calcd. for C20H26AuF6N2PS2 ). IR: νmax (neat)/cm -1 : 1559.

Synthesis of complex 4

Following the procedure described for 2, complex 4 was made from (C^N)AuCl2 (0.060 g, 0.125 mmol)

and sodium pyrrolidinedithiocarbamate hydrate (0.085 g, 0.502 mmol). A colour change from white to pale yellow was observed during the addition. This solid product was filtered and dried under vacuum ). IR: νmax (neat)/cm -1 : 1556.

(

Synthesis of complex 5

Following the procedure described for 2, complex 5 was made from (C^N)AuCl2 (0.060 g, 0.125 mmol) and NaS2CN n Bu2 (0.105 g, 0.379 mmol). A colour change from white to pale yellow was observed during the addition. The solvent was removed under vacuum and the dark yellow residue was dissolved in dichloromethane (15 mL). Excess AgSbF6 in dichloromethane (5mL) was then added and the solvent was removed under vacuum. The solid was purified by dissolving in minimal acetonitrile (2 mL) and precipitating the product with an excess of diethyl ether (20 mL). ). IR: νmax (neat)/cm -1 : 1550.

Synthesis of complex 6

Following the procedure described for 2, complex 6 was made from (C^N)AuCl2 (0.060 g, 0.125 mmol)

and sarcosine ethyl ester dithiocarbamate hydrate (0.100 g, 0517 mmol). The solid was filtered and purified by dissolving in minimal acetonitrile (2 mL) and precipitating the product with an excess of diethyl ether (20 mL). The product was filtered and dried under vacuum (0.073 g, 0.098 mmol, 78 %).

Anal Data were processed using the CrysAlisPro-CCD and -RED 48 programs. The structure was determined by the intrinsic phasing routines in the SHELXT program 49 and refined by full-matrix least-squares methods, on F 2 's, in SHELXL. 50 The non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were included in idealised positions and their Uiso values were set to ride on the Ueq values of the parent carbon atoms. At the conclusion of the refinement, wR2 = 0.049 and R1 = 0.029 (47) for all 11,737 reflections weighted w = [σ 2 (Fo 2 ) + (0.0117P) 2 + 10.284P] -1 with P = (Fo 2 + 2Fc 2 )/3; for the 'observed' data only, R1 = 0.024.

X-Ray Diffraction

In the final difference map, the highest peaks (to ca 1.0 eÅ -3 ) were close to the gold atoms.

Scattering factors for neutral atoms were taken from reference 51. Computer programs used in this analysis have been noted above, and were run through WinGX 52 on a Dell Precision 370 PC at the University of East Anglia.

Antiproliferation assay

The human A549 and HL60 cancer cell lines (from ECACC) were cultured in RPMI 1640 medium with 10% fetal calf serum, 2 mM L-glutamine, 100 U mL -1 penicillin and 100 µg mL -1 streptomycin (Invitrogen). The cells were maintained under a humidified atmosphere at 37 °C and 5% CO2.

Uptake study

MCF-7 cells were grown in 75 cm 2 flasks up to 70% of confluence in 10 mL of culture medium.

Compounds 2, 5, 6 and 7 were added to the flasks (100 µL of 1 mM solution in DMSO) and incubated for 6 h at 37 °C with 5% CO2. Negative controls were used by incubating cells with DMSO alone under the same conditions. After removal of the medium and washing of the cells with PBS pH 7.4, the cells were detached using a trypsin solution. After quenching of trypsin with fresh medium, centrifugation and removal of the supernatant, the cell pellet was resuspended into 1 mL of PBS pH 7.4 and split into twice 500 µL for metal and protein quantification. The number of cells (expressed per million cells) of each sample was determined by measuring the protein content of the treated samples using a BCA assay (ThermoFischer Scientific) corrected by the amount of protein/10 6 cells determined for each cell type by measuring the protein content of an untreated sample and dividing by the corresponding number of cells measured with a hematocytometer following a reported procedure. 53 Microwave digestion was used to solvate the samples to liquid form. Nitric acid and hydrogen peroxide were used in a Milestone Ethos 1 microwave system using SK-10 10 place carousel. The digest was ramped to 200C in 15 min., holding at 200C for 15 min. The sample was weighed into a microwave vessel before digestion, and decanted and rinsed into a pre-weighed PFA bottle after digestion. ICP-MS samples were spiked with rhodium internal standard and run on a Thermo X series 1 ICP-MS. The isotopes selected were 63 Cu, 65 Cu, 107 Ag, 109 Ag and 197 Au. Certified standards and independent reference were used for accuracy.

Acid blanks were run through the system and subtracted from sample measurements before corrections for dilution.

ROS assay

100 μL of MCF-7 cells were seeded at a density of 1 × 10 5 cells per mL in a 96-well black plate with a transparent bottom. The cells were incubated at 37 °C for 24 h. The medium was removed, and replaced Control samples for each run were prepared with the same quantity of DMSO with the DNA in buffer.

Fluorescence melting curves were determined in a QIAgen Rotor-Gene Q-series PCR machine, using a total reaction volume of 20 μL. Measurements were made with excitation at 470 nm and detection at 510 nm. Final analysis of the data was carried out using QIAgen Rotor-Gene Q-series software and Origin or Excel.

Inhibition of mammalian TrxR

To determine the inhibition of mammalian TrxR, an established microplate-reader-based assay was performed. 54 For this purpose, commercially available rat liver TrxR (Sigma Aldrich) was used and diluted with distilled water to achieve a concentration of 3.58 U/mL. The compound was freshly dissolved as stock solutions in DMSO. Aliquots (25 mL) of the enzyme solution and either potassium phosphate buffer (25 µL; pH 7.0) containing the compound in graded concentrations or buffer (25 µL) without the compound, but DMSO (positive control) were added. A blank solution (DMSO in buffer; 50µL) was also prepared (final concentrations of DMSO: 0.5% v/v). The resulting solutions were

Figure 1 :

 1 Figure 1: Exemples of (C^N) cyclometallated and dithiocarbamate Au(III) complexes investigated for anticancer purposes.

Figure 2 :

 2 Figure 2: Representation of the two structural isomers of 6.

Figure 3 :

 3 Figure 3: Structure of one of the two cations 3 + . Hydrogen atoms, PF6 -anions and the solvent molecule are omitted for clarity. Ellipsoids are shown at 50% probability. Selected bond distances [Å] and angles [°]: Au1-N1 2.061(3), Au1-C10 2.038(3), Au1-S21 2.3984(8), Au1-S22 2.2821(8), C10-Au1-N1 80.64(11), C10-Au1-S22 98.36(9), N1-Au1-S21 105.30(7), S22-Au1-S21 75.54(3), N1-Au1-S22 177.22(7), C10-Au1-S21 173.08(9), S21-C23-S22 111.47(17).

Figure 4 :

 4 Figure 4: a) 1 H NMR spectra of a 1:1 mixture of 2 with GSH at room temperature, in comparison with the starting materials, GSH and GSSG (DMSO-d6/D2O 1:1).b) UV/Vis spectra of complex 2 (1 mM), GSH (1 mM) and a mixture of complex 2 and GSH (1mM), (DMSO-H2O 1:1).

Figure 5 :

 5 Figure 5: 1 H NMR spectra of a 1:1 mixture of 2 with NAC at different reaction times at room temperature, in comparison with the starting materials 2 and NAC (CD3CN).

Figure 6 :

 6 Figure 6: a) Cellular uptake of complexes 2, 5, 6 and 7 and a DMSO control in MCF-7 cells after 6 h of treatment at 10 µM in 1% DMSO. Data represent the average ± standard deviation of three experiments. The significance of the results was analysed by t-test. *p value < 0.05. b) Correlation between IC50 and cell uptake of complexes 2, 5, 6 and 7 in MCF-7 cells.

  39a,b Recently, there have been several examples of potential chemotherapeutic gold(I, III) complexes that target G-quadruplexes.27,41 A caffeine-based [Au(NHC)2] + cation has been co-crystallized with a G-quadruplex, showing that DNAligand interaction occurred via π-stacking on the accessible tetrads.42 i-Motifs are higher-order DNA structures that form in cytosine-rich DNA sequences via hydrogen bonding between hemiprotonated pairs of cytosines. As i-motifs require cytosine-rich sequences, they are likely to form in the complementary strands opposing G-quadruplexes in the genome. 39c,d Stabilization of i-motif structures has been shown to alter gene expression of the oncogene bcl-2 and disrupt telomerase activity, and therefore compounds that stabilize these structures also have potential as anticancer drugs. 39d

Figure 8 :

 8 Figure 8: Stabilization of different DNA structures (0.2 µM) in 10 mM sodium cacodylate, 100 mM NaCl when dosed with 50 µM concentrations of dithiocarbamate complexes 2 -7. Data represents the average and standard deviations of two experiments.

(

  Trx) in an NADPH-dependent manner. Trx is responsible for the subsequent reduction of different proteins including ribonucleotide reductase and peroxiredoxin. Such inhibition of TrxR leads to apoptosis through mitochondrial membrane permeabilization and release of cytochrome c.46 Gold complexes are well-known to selectively inhibit TrxR over the closely related enzyme glutathion reductase via the direct coordination of the gold cation to the selenocysteine residue present in the active site of TrxR.47 Considering that dithiocarbamate Au(III) complexes 18b and, in some cases depending on the ancillary ligand, cyclometallated Au(III) complexes15 have been reported to inhibit TrxR, and taking into account the ability of our [(C^N)Au(DTC)] + complexes to react with the thiol containing tripeptide GSH, we tested complex 3, as a representative of that class of compounds, in vitro as a potential TrxR inhibitor using mammalian TrxR (see Experimental part for details). The IC50 of complex 3 was 0.31 ± 0.03 µM, demonstrating TrxR as another potential intracellular target of that class of compounds. It is worth noting that this value is intermediate between those of pure cyclometallated complexes (tenth to low micromolar range)15 and those of pure dithiocarbamate complexes (low nanomolar range). 18b This suggests the possibility to modulate the inhibitory activity of Au(III) complexes through the association of cyclometallated and dithiocarbamate ligands.ConclusionsCyclometallated gold(III) dithiocarbamate complexes of the type [(C^N)Au III (S2CNR2)] + show cytotoxicities that vary with the length and polarity of the N-alkyl substituents R and decrease in the order R = Me ≈ Et > pyrrolidine ≈ n Bu >> sarcosine ethyl ester, with cationic complexes being much more active than neutral (C^C)-chelated analogues. The complexes were tested for their in vitro cytotoxicity towards a panel of human cancer cell lines including lung adenocarcinoma cells (A549), breast adenocarcinoma (MCF-7 and MDA-MB-231), human colon cancer (HCT-116) and promyelocytic leukaemia (HL60), as well as healthy Human Umbilical Vein Endothelial Cells (HUVEC) for comparison. Complexes 2, 3, 4 and 5 showed the highest levels of cytotoxicity with IC50

Crystal data: 2 (

 2 C20H26AuN2S2), 2(PF6), MeCN, M = 1442.02; CCDC Code: 1854835. Monoclinic, space group P21/c (no. 14), a = 11.9382(2), b = 26.6092(3), c = 16.1416(3) Å, β = 93.060(2) , V = 5120.33(14) Å 3 . Z = 4, Dc = 1.871 g cm -3 , F(000) = 2808, T = 140(1) K, μ(Mo-Kα) = 60.3 cm -1 , λ(Mo-Kα) = 0.71073 Å. Crystals are large, pale yellow-green prisms. From a sample under oil, one, a block (fragment) ca 0.10 x 0.21 x 0.33 mm, was mounted on a glass fibre and fixed in acold nitrogen stream on an Oxford Diffraction Xcalibur-3/Sapphire3-CCD diffractometer equipped with Mo-Kα radiation and graphite monochromator. Intensity data were measured by thin-slice ω-and φ-scans. Total no. of reflections recorded, to θmax = 27.5, was 87,240 of which 11,737 were unique (Rint = 0.036); 10,802 were 'observed' with I> 2σI.

  with 50 μM H2DCFDA (from Life Technologies) solution in PBS for 40 min. H2DCFDA was removed and replaced with fresh medium. The cells were left for recovery for 20 min at 37 °C. Basal fluorescence was measured at 485/520 nm on a POLARstar Optima. The cells were incubated with 10 μM, 50 μM, or 100 μM of compounds, 1% DMSO (negative control) and 100 μM of H2O2 (positive control) for 24 h. Fluorescence was read at 485/520 nm. Basal fluorescence was subtracted from the fluorescence in the treated cells to calculate the amount of fluorescence caused by the compounds. FRET melting assay. Assessment of compound-induced stabilization of DNA was performed using a fluorescence resonance energy transfer (FRET) DNA melting based assay. The sequences used were hTeloCFRET (5′-FAMd[TAA-CCC-TAA-CCC-TAA-CCC-TAA-CCC]-TAMRA-3′); hif-1-αFRET (5′-d[CGC-GCT-CCC-GCC-CCC-TCT-CCC-CTC-CCC-GCG-C]-TAMRA-3′), hTeloGFRET (5′-FAM-d[GGG-TTA-GGG-TTA-GGG-TTA-GGG]-TAMRA-3′), cMycCFRET (5'-FAM-d[CCC-CAC-CTT-CCC-CAC-CCT-CCC-CAC-CCT-CCC-C]-TAMRA-3') and DSFRET FAM-d(TAT-AGC-TAT-A-HEG(18)-TAT-AGC-TAT-A)-TAMRA-3′). The labelled oligonucleotides (donor fluorophore FAM is 6-carboxyfluorescein;acceptor fluorophore TAMRA is 6-carboxytetramethyl-rhodamine) were prepared as a 220 nM solution in 10 mM sodium cacodylate buffer at the indicated pH with 100 mM sodium chloride and then thermally annealed by heating to 95°C in a heat block and cooling overnight. Strip-tubes (QIAgen) were prepared by aliquoting 20 μL of the annealed DNA, followed by 0.5 μL of the compound solutions.

Table 1 :

 1 IC50 values for complexes 1 -7 in comparison to cisplatin against different human cancer cell lines and healthy HUVEC cells after 72 h of incubation.

				IC50 ± SD (µM) a
	Complex	A549	MCF-7	HL60	HCT-116	MDA-MB-231	HUVEC
	1 b	43.6 ± 4.1	10.8 ± 3.5	6.0 ± 0.5	n. d.	n. d.	n. d.
	2	4.6 ± 0.7	1.4 ± 0.3	0.2 ± 0.05	2.5 ± 0.3	8.6 ± 1.0	0.8 ± 0.04
	3	2.9 ± 0.6	1.2 ± 0.2	0.2 ± 0.03	2.4 ± 0.4	1.9 ± 0.1	0.7 ± 0.02
	4	10.8 ± 0.3	1.1 ± 0.03	1.4 ± 0.07	6.0 ± 0.4	3.0 ± 0.2	0.7 ± 0.05
	5	5.3 ± 0.3	1.7 ± 0.04	0.8 ± 0.01	3.5 ± 0.3	3.1 ± 0.1	1.2 ± 0.2
	6	>100	>100	17.8 ± 2.1	>100	>100	33.9 ± 5.4
	7	>100	8.8 ± 0.2	1.9 ± 0.3	>100	>100	3.3 ± 0.03
	Cisplatin	33.7 ± 3.7 c 21.2 ± 3.9 c	3.7 ± 0.3 c	5.3 ± 0.2 d	28.4 ± 0.1 d	>100
	a Mean ± the standard error of at least three independent experiments. b Values from ref. 26. c Values
	from ref. 27. d Values from ref. 28.

  1H, H 2 ), 8.26 (dt,3 JH-H = 8.1 Hz,4 JH-H = 1.5 Hz, 1H, H 4 ), 8.07(d, 3 JH-H = 8.1 Hz, 1H, H 5 ), 7.72(d, 3 JH-H = 8.3 Hz, 1H, H 8 ), 7.59-7.50 (m, 2H, H 3+9 ), 7.13(d, 4 JH-H = 1.6 Hz, 1H, H 11 ), 3.98 (t,3 JH-H = 6.6 Hz, 4H, H 14+14' ), 2.24 (m, 4H, H 15+15' ), 1.36 (s, 9H, t Bu).13 C{ 1 H} NMR (CD2Cl2, 75 MHz): δ 190.4 (s, C 13 ), 164.4 (s, C 12 ), 157.1 (s, C 6/7 ), 151.8 (s, C 6/7 ), 148.4 (s, C 2 ), 143.6 (s, C 4 ), 140.5 (s, C 10 ), 126.6 (s, C 8 ),

	126.5 (s, C 9 ), 125.3 (s, C 3 ), 125.3 (s, C 11 ), 122.0 (s, C 5 ), 52.0 (s, C 14/14' ), 35.8 (s, C(CH3)3), 30.7 (s,
	C(CH3)3), 24.6 (s, C 15/15' ), 24.2 (s, C 15/15'

0.062 g, 0.089 mmol, 71 %). Anal.Calcd. for C20H24AuF6N2PS2.4H2O (770.54): C, 31.18; H, 4.19; N, 3.64. Found C, 30.85; H, 3.78; N, 3.25. 1 H NMR (CD2Cl2, 300 MHz, 298 K): δ 8.48 (d, 3 JH-H = 5.6 Hz,

  C 2' ), 150.0 (s, C 2 ), 144.1 (s, C 4' ), 144.1 (s, C 4 ), 141.6 (s, C 10' ), 141.4 (s, C 10 ), 127.4 (s, C 8' ), 127.4 (s, C 8 ), 126.8 (s, C 9' ), 126.7 (s, C 9 ), 126.1 (s, C 3 ), 126.0 (s, C 3' ), 124.7 (s, C 11' ), 124.7 (s, C 11 ), 122.5 (s, C 5' ), 122.5 (s, C 5 ), 62.6 (s, C 14' ), 62.6 (s, C 14 ), 55.3 (s, C 15' ), 54.1 (s, C 15 ), 35.9 (s, C(CH3)3), 31.4 (s, C 17' ), 31.1 (s, C(CH3)3), 30.0 (s, C 17 ), 14.3 (s, C 18 ), 14.3 (s, C 18' ). IR: νmax (neat)/cm -1 : 1736 (C=O), 1558 (dithiocarbamate).

	.Calcd. for C21H26AuF6N2PS2.8H2O (906.64): C, 27.82; H, 4.89; N, 3.09. Found C, 27.34; H, 4.42;
	N, 3.29. 1 H NMR ((CD3)2SO, 300 MHz, 298 K): δ 8.77 (d, 3 JH-H = 5.6 Hz, 0.8H, H 2' ), 8.71 (d, 3 JH-H =
	5.6 Hz, 1H, H 2 ), 8.40-8.27 (m, 3.6H, H 4+4'+5+5' ), 7.94 (m, 1.8H, H 9+9' ), 7.63-7.49 (m, 3.6H, H 3+3'+8+8' ),
	7.15 (d, 4 JH-H = 1.4 Hz, 1H, H 11 ), 7.01 (d, 4 JH-H = 1.4 Hz, 0.8H, H 11' ), 4.82 (s, 1.6H, H 15' ), 4.78 (s, 2H,
	H 15 ), 4.21 (m, 3.6H, H 17+17' ), 3.54 (s, 3H, H 14 ), 3.45 (s, 2.4H, H 14' ), 1.29 (s, 9H, t Bu), 1.27 (s, 7.2H, t Bu ' ),
	1.23 (m, 5.4H, H 18+18' ). 13 C{ 1 H} NMR ((CD3)2SO, 75 MHz): δ 198.7 (s, C 13 ), 166.3 (s, C 16' ), 166.2 (s,
	C 16 ), 163.5 (s, C 12' ), 163.3 (s, C 12 ), 156.0 (s, C 6'/7' ), 156.0 (s, C 6/7 ), 151.5 (s, C 6'/7' ), 151.2 (s, C 6/7 ), 150.1
	(s,

  The human MCF-7, HCT116 and MDA-MB-231 cancer cell lines (from ECACC) were cultured in DMEM medium with 10% fetal calf serum, 2 mM L-glutamine, 100 U mL -1 penicillin and 100 µg mL -1 streptomycin (Invitrogen). The HUVEC cells were cultured in Endothelial Cell Growth Medium with growth and antibiotic supplement. The cells were maintained under a humidified atmosphere at 37 °C

	and 5% CO2. Inhibition of cancer cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)5-
	(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using the CellTiter 96
	Aqueous One Solution Cell Proliferation Assay (Promega) and following the manufacturer's
	instructions. Briefly, the cells (3 × 10 4 per 100 µL for HL60, 8 × 10 3 per 100 µL for A549, MCF-7,
	HCT116, MDA-MB-231 and HUVEC) were seeded in 96-well plates and left untreated or treated with
	1 µL of DMSO (vehicle control) or 1 µL of complexes diluted in DMSO at different concentrations, in
	triplicate for 72 h at 37 °C with 5% CO2. Following this, MTS assay reagent was added for 4 h and
	absorbance measured at 490 nm using a Polarstar Optima microplate reader (BMG Labtech). IC50 values
	were calculated using GraphPad Prism Version 5.0 software.

Acknowledgements

This work was supported by the European Research Council. M.B. is an ERC Advanced Investigator Award holder (grant no. 338944-GOCAT).

incubated with moderate shaking for 75 min at 37°C in a 96-well plate. A portion (225 µL) of the reaction mixture (1 mL of reaction mixture consists of: 500 µL potassium phosphate buffer pH 7.0, 80 µL EDTA solution (100 mM, pH 7.5), 20 µL BSA solution (0.2 %), 100 µL of NADPH solution (20 mM), and 300 µL distilled water) was added to each well and the reaction was immediately initiated by the addition of 20 mM DTNB solution in ethanol (25 µL). After thorough mixing, the formation of 5-TNB was monitored with a microplate reader at 405 nm ten times in 35 second intervals for about 6 min. The increase in 5-TNB concentration over time followed a linear trend (r 2 = 0.990), and the enzymatic activities were calculated as the slopes thereof (increase in absorbance per second). For each tested compound, the non-interference with the assay components was confirmed by a negative control experiment using an enzyme-free test solution. IC50 values were calculated as the concentration of the compounds decreasing the enzymatic activity of the untreated control by 50%, and are given as the means and error of three repeated experiments.

Conflicts of interest

There are no conflicts of interest to declare.


Morwen R M Williams

Benoît Bertrand
email: benoit.bertrand@upmc.fr

Julio Fernandez-Cestau

Zoë A E Waller

Maria A O'connell

Mark Searcey
email: m.searcey@uea.ac.uk

Manfred Bochmann
email: m.bochmann@uea.ac.uk

Acridine-decorated cyclometallated gold(III) complexes: synthesis and antitumour investigations

C^N) and (C^N^C) cyclometalated Au(III) represent a highly promising class of potential anticancer agents. We report here the synthesis of seven new cyclometalated Au(III) complexes with five of them bearing an acridine moiety attached via (N^O) or (N^N) chelates, acyclic amino carbenes (AAC) and Nheterocyclic carbenes (NHC). The antiproliferative properties of the different complexes were evaluated in vitro on a panel of cancer cells including leukaemia, lung and breast cancer cells. We observed a trend between the cytotoxicity and the intracellular gold uptake of some representative compounds of the series. Some of the acridine-decorated complexes were demonstrated to interact with ds-DNA using FRETmelting techniques.

Introduction

The worldwide approval of cisplatin for the treatment of several cancers in the late 1970s was the starting point of the modern use of metal complexes in medicine. 1 However, the poor selectivity of cisplatin for cancer cells over healthy fast growing cells induces severe side effects which limits the dose of chemotherapy given to patients, 2 and both intrinsic and acquired resistance reduces the effectiveness of platinum-based drugs. 3 For these reasons there has been an increasing interest in the development of alternatives to platinum-based compounds for therapeutic purposes, including iron, copper, ruthenium, titanium and goldcomplexes. [4][5][6][7][8] In particular, cyclometalated gold(III) complexes have been attracting significant interest. 9,10 Both bidentate, (C^N) or tridentate, (C^N^C), (C^N^N) or (N^C^N) cyclometalated ligands have been used to stabilize the gold(III) centre, enabling the study of fundamental organometallic reactions, the characterization of catalytic intermediates, and the development of photoluminescent materials and of anticancer drug candidates. [11][12][13] Henderson and coworkers introduced a series of neutral cyclometalated (C^N)Au(N^N) chelate complexes containing phenylenediamine and ethylenediamine moieties, with the nitrogen atoms bearing electron-withdrawing moieties. The derivatives were tested for their in vitro cytotoxicity against mouse leukaemia cells (P388) and some showed very promising results in comparison to cisplatin. Several different (C^N) cyclometalated ligands were used, including the 6-membered ring based on 2-benzyl pyridine, and more rigid 2-phenyl pyridine complexes, combined with (N^O) and (O^O) chelate ligands (Figure 1, structures A -C). 14,15 Other examples include (C^N)-(N^N) derivatives reported by Zhang et al., such as the stable and water soluble cationic gold(III) complex, [Au(butyl-C^N)biguanidine]Cl, (Figure 1, structure D) which combined the lipophilic, cyclometalated (C^N) ligand and the hydrophilic, H-bonding NH2 groups of the biguanidine ligand. The complex showed high cytotoxicity towards cancer cell lines, with IC50 values in the low micromolar range, as well as a high selectivity profile towards cancer cells over healthy CCD-19-Lu cells. 16 Figure 1. Examples of previously reported cyclometallated gold(III) complexes tested for anticancer purposes. [14][15][16][17][18] We have recently described the synthesis and anticancer activity of novel cyclometalated (C^N pz ^C) gold III complexes with acyclic amino carbene (AAC) ligands enabling the introduction of amines and amino ester derivatives in the [(C^N pz ^C)Au(carbene)] + scaffold (Figure 1, structures E and F). The antiproliferative screening of these complexes identified them as promising potential chemotherapeutic agents. 17,18 Gold(III) N-heterocyclic carbene (NHC) complexes appear to be more stable towards reducing agents like GSH and are highly cytotoxic towards cancer cell lines; for example, complex F showed IC50 values in the micromolar and sub-micromolar range against a panel of cancer cell lines. 18 In spite of the structural and electronic similarity between gold(III) and platinum(II) compounds, the two metals appear to have very different intracellular targets and the binding of gold(III) complexes to DNA is often weak and reversible. 19 However, it is possible to enhance interactions of gold(III) complexes with doublestranded DNA by optimizing the choice of the ligand toward that application. 20 Acridines are known to bind to DNA by intercalation; by the direct insertion of the three planar aromatic rings in between the base pairs of DNA which causes unwinding of the double helix. 21,22 For example, Janočková et al. synthesized a series of acridine analogues, (figure 2) and demonstrated their ability to inhibit cancer cell growth in acute promyelocytic leukaemia cells (HL60) with IC50 values in the low micromolar range. DNA binding studies towards calf thymus DNA showed a strong interaction through DNA intercalation resulting in the inhibition of certain enzymes such as topoisomerase 1 and 2. 21 Howell et al. also studied the DNA binding and in-vitro cytotoxicity of 9-aminoacridinecarboxamides (Figure 2), towards various types of tertiary DNA structures. The derivatives were shown to target Gquadruplex structures with some selectivity. 23 There are also examples of acridine motifs being used to enhance the cytotoxicity of metal-based chemotherapeutic agents with platinum, iron, iridium or rhodium metal centres. In all cases, the presence of the acridine increased the interactions of the complexes with DNA. 24,25 Although examples of cytotoxic gold(I)-acridine hybrids have been reported with thiourea and NHC ligands (Figure 2, structures K and L), 26 to the best of our knowledge, no gold(III)-acridine conjugates have been described so far. In the present article, we report on the synthesis and anticancer activity of cyclometalated gold(III) complexes decorated with acridinyl substituents, based on the starting materials 1 -3 (Figure 2). We focus on (i) neutral (C^N)-(N^O) and (C^N)-(N^N) derivatives, and (ii) (C^N pz ^C) pincer complexes where the acridine functionality is joined by either an AAC or an NHC ligand. The complexes were tested for their cytotoxicity towards three cancer cell lines, namely lung adenocarcinoma (A549), breast cancer (MCF-7) and leukaemia (HL60). The DNA binding abilities of the complexes towards dsDNA was also investigated using DNA FRET-melting assays. The reaction of the (C^N)gold(III) dichlorides 1 and 2 with 2-acetamidophenol in refluxing methanol in the presence of excess trimethylamine afforded high yields of the products 4 and 5, respectively (Scheme 2), as previously reported. 15 Complex 6 was synthesized by reacting 1 with 1,2-diacetamidobenzene and silver oxide in refluxing dichloromethane. The reaction of 2 with 1,2-diacetamidobenzene/Ag2O was also attempted but caused opening of the less rigid 6-membered ring.

Results and discussion

Synthesis and characterization

Complex 6 was poorly soluble in the deuterated NMR solvents and therefore signals in both the 1 H and particularly the 13 C{ 1 H} NMR were poorly resolved. Scheme 2. Synthesis of the (C^N) cyclometalated gold(III) complexes 4-9.

Complex 7 was obtained in high yield by reacting 1 with L1 in refluxing methanol in the presence of an excess of trimethylamine, followed by precipitation with water. The reaction of 2 and L1 under the same conditions was also successful and gave 8 in moderate yield. The corresponding (C^N)-(N^N) complex, complex 9 was obtained in good yield by the reaction of 1 with L2 under the same conditions. Once again, the reaction was also attempted with the 2-benzyl pyridine gold dichloride starting material but caused ring opening.

At 25 °C solutions of 8in CD2Cl2 gave broadened NMR spectra indicative of hindered rotation.

These sharpen on cooling to-10C and show well-resolved signals (see ESI, Figure S10) of the chemically inequivalent acridine protons H [22][23][24][25] . The characteristic AB system of two doublets for the methylene bridge at 4.43 ppm and 3.91 ppm was observed, indicating the diastereotopic nature of these protons. For the three acridine-based complexes 7-9, only one set of signals was observed for each proton, demonstrating the formation of only one isomer in each case, as was also seen for the simpler (N^O) and (N^N) complexes 4-6.

Slow diffusion of light petroleum into dichloromethane solutions of complexes 7 and 9 yielded crystals suitable for X-ray diffraction (Figure 3). In both compounds the square planar geometry of the gold(III) centres is slightly distorted due to the small bite angles of the cyclometalated ligand [respective C11-Au-N1 angles 81.1° (7) and 80.9° (9)]. In 7, the acridine-substituted nitrogen atom is in trans position to the nitrogen of the cyclometalated ligand, placing it on the same side as the tert-butyl substituent, while the O atom, as the weakest trans influence ligand, is trans to the stronger C-donor. The adopted conformation is therefore a reflection of the electronic preferences. The acridine moiety is twisted out of the coordination plane, with an Au1-N2-C22-C23torsion angle of 110 °, presumably to avoid steric hindrance with the tert-butyl substituent. As suggested by the NMR spectra, only one isomer of the complex was obtained.

In complex 9, on the other hand, the second ligand is an N^N chelate, with both donor atoms having similar electronic properties. Therefore, in this case a conformation is adopted which places the acridine substituent distal from the t-butyl group, to minimise steric interactions. Here, too, the acridine moiety is twisted out of the coordination plane (torsion angle Au1-N3-C22-C23 67.3°). Selected bond distances (Å) and angles (º). Au1-N1 2.063 (7), Au1-C11 2.015(6), Au1-N2 1.956(8), Au1-N3 2.081(6), N1-Au1-C11 80.9(3), C11-Au1-N2 95.2(3), N2-Au1-N3 80.3(3), N3-Au1-N1 103.9(3), Au1-N2-H100 119(6), N1-Au1-N2 175.4(3), C11-Au1-N3 173.0(3), torsion Au1-N3-C22-C23 67.3 (9). The pyrazine-based pincer complex (C^N pz ^C)AuCl (3) was synthesized as reported previously. 27 Conversion of 3 into the cationic xylylisocyanide complex [(C^N pz ^C)Au(CNxyl)] + , followed by reaction with 9-aminoacridine afforded the functionalized acyclic amino carbene complexes 10 (Scheme 3). The yield was reduced due to the competitive hydrolysis of the highly electrophilic isocyanide adduct to the gold(III) formamide byproduct, as previously observed. 17 Upon formation of 10, a broad NH singlet appears in the 1 H spectrum at 10.34 ppm and the upfield H 2 shift to 9.13 ppm confirms that a reaction had occurred and suggests that the acyclic amino carbene has been formed. The signal for H 8 at 9.96 ppm is also in good agreement with H 8 signals observed for the AAC complexes synthesized previously. 17 The IR spectrum of 10 showed a carbene vibration at 1586 cm -1 and two NH stretches at 3388 cm -1 and 3270 cm -1 .

Complex 11 was synthesized in good yield by reacting an acridine functionalized imidazolium salt with silver oxide, followed by the addition of 3 and potassium hexafluorophosphate by adapting a known procedure (Scheme 4). 28 The slight downfield shift in the signal for H 2 and a larger downfield shift in H 8 from 7.88 to 7.21 ppm in the 1 H NMR spectrum and the appearance of a signal at δC 154.9 ppm for the Au-bound NHC suggested carbene formation.

Biological Activity

Although insoluble in aqueous cell culture medium, all the complexes and their ligand precursors were soluble enough in DMSO not to precipitate when diluted in 100 µM aqueous cell medium with 1% of DMSO. Complexes 1-11 and the acridine ligand precursors L1 and L2 were screened for their antiproliferative properties in vitro on a panel of human cancer cell lines, including lung adenocarcinoma cells (A549), breast adenocarcinoma (MCF-7), and promyelocytic leukaemia (HL60). The IC50, (the concentration at which cell viability is reduced to 50%), was then determined using a colorimetric MTS assay after 72 h of incubation, with the cisplatin included for comparison (see Experimental part for details). The results are summarized in Table 1.

L1 >100 >100 >100 L2
23.9 ± 3.5 14.4 ± 0.9 16.9 ± 0.9 Complexes 4-11 showed greater or comparable activity to cisplatin under the conditions used in this assay and were overall more toxic than the gold dichloride precursors, complexes 1 and 2. The free ligands, L1 and L2, showed reduced toxicity particularly L1 which was inactive against all of the cell lines tested, with an IC50 value of >100 µM against all three. L2 also showed reduced toxicity in comparison to the gold complexes, which indicates that the gold III centre contributes significantly to the cytotoxicity.

Overall, the mono-cyclometalated (C^N)-(N^X) complexes, 4-9 showed greater activity than cisplatin against both the MCF-7 and the A549 human cancer cell lines and comparable activity against the HL60 cell line. Complexes 4-9 all showed similar activities against the MCF-7 cell lines, with IC50values in the 2-10 µM range. Complexes 5, 7 and 8 showed particularly high toxicity with values between 2-4 µM. IC50 values for the A549 cell line were slightly higher, between 10-20 µM for all of the complexes. Here, the simpler structures, complexes 4 and 5 showed the greatest overall toxicity.

Changing the (C^N) cyclometalated ligand from the rigid 5-ring (phenyl pyridine)to the flexible 6-ring ligand (2-benzylpyridine) appeared to have no significant effect on the cytotoxicity of the complexes. For example, for the HL60 leukaemia cell line, complexes 4 and 5 have IC50values of 2.9 and 2.7 µM, respectively. The same trend was found for complexes 7(1.4 µM) and 8(1.5 µM). This is different to results obtained by Kilpin et al., who found that complexes containing a six-membered cyclometalated ring displayed higher cytotoxicity than those with a five-membered cyclometalated ring. 14 For all three cell lines, the (N^O) chelates appeared to be slightly more cytotoxic than the corresponding N^N chelates, which is in contrast to the values obtained for the free acridine ligands, L1 and L2. For example, for the A549 lung cancer cell line the (N^O) chelate, complex 4, has an IC50 of 12.7 µM, while the corresponding (N^N) chelate 6 has an IC50 of 22.7 µM. The same pattern can be seen for the acridine-functionalized derivatives: 7, the (N^O) chelate, is more toxic than its (N^N) analogue 9

against the all three cancer cell lines. Here the effect is most notable against the leukaemia HL60 cell line, (IC50 of 1.4 and 6.3 µM for 7 and 9, respectively).

As a comparison of complexes 4 versus 7 and 5 versus 8 shows, adding an acridine functionality appeared not to increase the overall cytotoxicity of the mono-cyclometalated (C^N)-(N^X) complexes.

This also holds for the (N^N) chelates 6 compared to 9, where the toxicity differences due to acridine are not significant.

The [(C^N pz ^C)Au(III)(AAC)] + complex 10 showed a higher cytotoxicity than cisplatin against both the MCF-7 and the A549 cancer cell lines, although it showed reduced activity against the HL60 cells (20.7 µM). These values are in good agreement with data on AAC complexes we reported earlier, particularly when considering the amino ester derivatives (e.g. GlyOEt -IC50 values of 6.4, 13.0 and 16.7

µM for MCF-7, A549 and HL60 respectively). 17 The other (C^N pz ^C) pincer complex, 11 with acridine functionality bound to the NHC ligand, showed the highest overall toxicity, with values in the low µM regions for all three cell lines. It was more than ten times more toxic than cisplatin against MCF-7 cells and 3-4 times more toxic against the A549 and HL60 cell lines. Although our incubation time is longer (72 h instead of 24 h), it is interesting to note that 11 is active against A549 cells while the related Au I complex [Au(MeImAcr)2] + is not. 26b The higher cytotoxicity of the [(C^N pz ^C)Au(NHC)] + complexes with respect to the [(C^N pz ^C)Au(AAC)] + complexes is in line with our previous results. 17

Cellular uptake into MCF-7 cells

The cellular uptake and the accumulation of drugs inside the cell are both major factors that influence the cytotoxicity of prospective anticancer agents. 29 Inductively coupled plasma-mass spectrometry, (ICP-MS) was used to quantify the amount of intracellular gold and thus measure the amount of compound taken up by the cell. In order to see whether the cytotoxicity of the complexes was influenced by their ability to be taken up by the cell, complexes 2, 6, 7 and 11 were chosen for uptake studies, since they show a range of antiproliferative activities against MCF-7 cells ranging from poor (IC50(2) of 38. There was a clear correlation between cellular uptake and the in vitro cytotoxicity of the complexes, with the most cytotoxic complexes showing the highest levels of cell uptake (see Figure 6 and ESI, Figure S1). This suggests that the primary limitation for the poorly toxic complexes is their very low uptake into the cell. Such a correlation between cellular uptake and antiproliferative activity has also been observed for other gold complexes, including [(C^N pz ^C)Au(NHC] + complexes. 18,30 The increase in cell uptake seems to follow the increasing lipophilicity of the complexes, which might suggest an internalization via passive diffusion, reminiscent of the process recently reported by Casini et al. for a phosphine analogue of 2. 31

DNA binding assays

Acridine derivatives are known to interact with duplex DNA as well as higher order DNA structures and this can influence the cytotoxic properties of the compounds. 22,23 DNA intercalators are of interest as probes in the study of ligand-DNA interactions and also as potential therapeutic agents. 32 to measure the interaction of a particular drug molecule with the DNA. 33 Results are expressed as the changes in DNA melting temperature (ΔTm) when double stranded DNA (0.2 µM) is dosed with 50 µM concentrations of each drug (Figure 5). Complex 8, the acridine functionalized 2-benzylpyridine derivative, showed high levels of DNA stabilization. This suggests that it is interacting with the ds DNA in some manner, potentially due to the hemi-lability of (C^N) ligand which could favour intercalation by decreasing steric hindrance.

Interestingly, the [(C^N pz ^C)Au(NHC)] + complex 11 appeared to show significant interaction with ds-DNA, while the closely related complex F (Figure 1) lacking the acridine moiety interacted with ds-DNA rather poorly. 18 However, considering the poor DNA intercalation behaviour of the acridine ligands L1

and L2 and the lack of interaction of most of the acridine-decorated complexes, it seems likely that the interactions observed for 8 and 11 are due to mechanisms other than the simple intercalation of the acridine moiety. Overall, considering that 7 and 8 present very similar cytotoxicities but drastically different interactions with DNA, these data suggest that acridine-DNA interactions have little bearing on the cytotoxicity mechanism of this series of compounds.

Reaction with Glutathione

Reduction of gold(III) complexes by glutathione (GSH) to gold(I) and gold(0) products is a typical feature of this class of 34 The stability of 11 and 7 towards reduction by GSH was therefore explored by 1 H NMR spectroscopy. Each complex was mixed at room temperature with reduced GSH in a 1:1 mixture of DMSO-d6 and D2O and monitored by over time. We had previously shown that a benzimidazole-based NHC complex of (C^N pz ^C)Au(III) (Figure 1, structure F) is stable towards reduction by GSH. 18 By contrast, the acridine substituted [(C^N pz ^C)Au(NHC)] + complex 11 appeared less stable, and over a 24 hour period the gradual formation of oxidized glutathione (GSSG) was observed, along with the disappearance of signals for reduced GSH (Figure 6). One can hypothesize that the higher steric hindrance around gold due to the acridine substituent on the NHC could facilitate the protolytic Au-C bond cleavage leading to S-coordination of GSH and subsequent reduction of the gold(III) ion, following a reaction pathway similar to the one recently found for the reaction of adamantylthiol with (C^N^C)Au pincer compounds. 35 The same pattern was seen for the acridine substituted (C^N)-(N^O) chelate complex, 7 (Figure S2).

Conclusion

A new series of mono cyclometalated (C^N)(N^X)Au(III) complexes as well as two bis-cyclometalated (C^N pz ^C) pincer complexes were synthesized, with and without acridine functionality. These complexes were tested for their cytotoxicity towards three cancer cell lines. These studies identified complex 11, the NHC derivative, as the most promising candidate, with IC50 values in the low µM range for all three cell lines. However, the introduction of an acridine functionality did not appear to increase the cytotoxicity of the complexes. Cell uptake studies demonstrated a correlation between cellular uptake and the in vitro cytotoxicity of the complexes and indicated that cellular uptake is the primary limitation for non-toxic gold(III) complexes. DNA binding studies showed that two of the derivatives, complex 8 and the NHC derivative 11, demonstrate high levels of DNA stabilization suggestive of DNA intercalation. The lack of a correlation between the cytotoxicity data and the interaction with ds-DNA may suggest that the addition of the acridine moiety onto these scaffolds did not result in improved DNA targeting. Taken together, those data suggest that DNA is probably not the main target of that class of compounds and that more experiments are required to fully elucidate its involvement in the mechanism of action of these complexes.

Moreover, both the pincer NHC complex [(C^N pz ^C)Au(NHC)] + 11 and the mono-cyclometalated (C^N)Au(N^O) derivative 7 were prone to reduction by glutathione at a time scale commensurate with incubation, which suggests that these compounds are reduced to gold(I) species in the cellular environment. Further work is required to investigate the localization and accumulation of those complexes within the cells and other possible modes of action of these complexes. 15,27,36,37

Materials and methods

General

Synthesis of complex 4

A mixture of 2-(4-t-butyl)phenyl pyridine gold dichloride, ( 1), (0.200 g, 0.418 mmol), 2-acetamidophenol (0.100 g, 0.661 mmol) and aqueous trimethylamine (2 mL) was refluxed in methanol (30 mL) for 30 min.

The reaction was then cooled and water (40 mL) was added causing an immediate orange precipitation.

The flask was then placed in the freezer for two days. The orange solid was filtered and purified by dissolving in minimal dichloromethane (2 mL) and precipitating the product with an excess of light petroleum (bp. 40-60 °C) (20 mL). This was filtered and dried under vacuum (0.181 g, 0.325 mmol, 78%

Synthesis of complex 7

A mixture of 2-(4-t-butyl)phenyl pyridine gold dichloride, (1), (0.235 g, 0.491 mmol), ligand L1 (0.300 g, 1.048 mmol) and aqueous trimethylamine (2 mL) was refluxed in methanol (30 mL) for 30 min. The reaction was then cooled and water (40 mL) was added causing an immediate dark brown precipitation.

The flask was placed in the freezer for two days. The brown solid was filtered off and purified by dissolving in dichloromethane (2 mL), followed by precipitation an excess of light petroleum (bp. 40-60 °C) (20 mL). This product was filtered off and dried under vacuum (0.273 g, 0.394 mmol, 80%). Anal.

Calcd. for C34H28AuN3O (691.19): C, 59.05; H, 4.08; N, 6.08. Found: C, 58.84; H 4.13; N 6.02. 1 H NMR (CD2Cl2, 300 MHz, 298 K): 9.61 (dd, 3 JH-H = 5.9 Hz, 4 JH-H = 1.0 Hz, 1H, H 2 ), 8.49 (dd, 3 JH-H = 8.7 Hz, 4 JH-H = 0.7 Hz, 2H, H 21/24 ), 8.30 (d, 3 JH-H = 8.7 Hz, 2H, H 21/24 ), 8.08 (dt, 3 JH-H = 8.1 Hz, 4 JH-H = 1.0 Hz, 1H, H 4 ), 7.82 (d, 3 JH-H = 8.

, C(CH3)3), 29.9 (s, C(CH3)3).

Synthesis of complex 8

A mixture of 2-benzylpyridine gold dichloride, (2), (0.200 g, 0.459 mmol), ligand L1 (0.178 g, 0.622 mmol) and aqueous trimethylamine (2 mL) was refluxed in methanol (30 mL) for 30 min. The reaction was then cooled and water (40 mL) was added causing an immediate dark brown precipitation. The flask was then placed in the freezer for two days. The brown solid was then filtered and purified by dissolving in minimal dichloromethane and acetone (3 mL). the product was precipitated with an excess of light petroleum (bp. 40-60 °C) (20 mL), filtered off and dried under vacuum (0.201 g, 0.309 mmol, 67%

Synthesis of complex 9

A mixture of 2-(4-t-butyl)phenyl pyridine gold dichloride, (1), (0.200 g, 0.418 mmol), ligand L2 (0.177 g, 0.620 mmol) and aqueous trimethylamine (2 mL) was refluxed in methanol (30 mL) for 30 min. The reaction was then cooled and water (40 mL) was added causing an immediate light brown precipitation.

The flask was then placed in the freezer for two days. The brown solid was then filtered and purified by dissolving in minimal dichloromethane (2 mL). The product was precipitated with an excess of light

Synthesis of complex 10

A mixture of (C^N pz ^C)AuCl, (3), (0.060 g, 0.104 mmol), 2,6-dimethylphenyl isocyanide (0.016 g, 0.125 mmol), AgSbF6 (0.043 g, 0.125 mmol) and a few pellets of 4A molecular sieves were combined in a flame-dried Schlenk flask under a nitrogen atmosphere with dry dichloromethane (15 mL). The mixture was left to stir at room temperature for 4 h. A white precipitate of AgCl was removed by filtration through celite, and the filtrate collected under an N2 atmosphere. The solvent was evaporated to a minimum and the product precipitated with an excess of light petroleum (bp. 40-60 °C). The supernatant was removed and the residue dried under vacuum to yield a yellow solid. 9-amino acridine (0.028 g, 0.146 mmol) was added to a flame-dried Schlenk flask. The solution was sonicated for 30 min and transferred to a separate flame-dried Schlenk flask containing the [(C^N pz ^C)Au(2,6-dimethylphenyl isocyanide)]SbF6. The reaction was then stirred for 16 h at room temperature and the precipitate removed from solution via filtration through a celite plug. Next, the solvent was removed under vacuum and the solid residue redissolved in dichloromethane (2 mL). The product was precipitated using a 2:1 mixture of light petroleum (bp. 40-60 °C) / diethyl ether (5 mL). After removal of the solvent, the residue was dried under vacuum to yield a dark yellow solid. Proton NMR showed a mixture of the desired product and the formamide hydrolysis product. The solid was washed twice with dry dichloromethane (40 mL) followed by light petroleum (10 mL) and dried under vacuum (0.053 g, 0.048 mmol, 46% .

X-ray crystallography

Description of the crystals: The crystals were mounted on a MiTeGen MicroMesh and fixed in a cold nitrogen stream. Diffraction intensities were recorded at 298, 173 or 140 K on a Rigaku HG Saturn724+ (2×2 bin mode) diffractometer or an Oxford Diffraction Xcalibur-3 instrument, both equipped with Mo-Kα radiation. Data collection, refinement and reduction were performed using the CrystalClear-SM Expert 3.1 b27 or CrysAlisPro software and the absorption correction was applied at this stage. 38 All structures were solved using SHELXS/T and refined by full-matrix least-squares methods on F 2 with SHELXL. 39 Non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were included in idealised positions. No missed symmetry was reported by PLATON. 40 Computer programs used in this analysis were run through WinGX. 41 Scattering factors for neutral atoms were taken from reference. [START_REF]International Tables for X-ray Crystallography[END_REF] standard and run on a Thermo X series 1 ICP-MS. The isotopes selected were 63 Cu, 65 Cu, 107 Ag, 109 Ag and 197 Au. Certified standards and independent reference were used for accuracy. Acid blanks were run through the system and subtracted from sample measurements before corrections for dilution.

FRET assay.

The initial FRET melting screen was performed using a fluorescence resonance energy transfer (FRET)

DNA melting based assay. The sequence used was DSFRET FAM-d(TAT-AGC-TAT-A-HEG(18)-TAT-AGC-TAT-A)-TAMRA-3′). The labelled oligonucleotide (donor fluorophore FAM is 6carboxyfluorescein; acceptor fluorophore TAMRA is 6-carboxytetramethyl-rhodamine) were prepared as a 220 nM solution in 10 mM sodium cacodylate buffer at the indicated pH with 100 mM sodium chloride and then thermally annealed. Strip-tubes (QIAgen) were prepared by aliquoting 20 μL of the annealed DNA, followed by 0.5 μL of the compound solutions. Control samples for each run were prepared with the same quantity of DMSO with the DNA in buffer. Fluorescence melting curves were determined in a QIAgen Rotor-Gene Q-series PCR machine, using a total reaction volume of 20.5 μL. Measurements were made with excitation at 470 nm and detection at 510 nm. Final analysis of the data was carried out using QIAgen Rotor-Gene Q-series software and Origin or Excel.

Electronic Supporting Information

Correlation between IC50 and cell uptake, NMR spectra.CCDC numbers 1850050 (7•CH2Cl2) and 1850051 (9•CH2Cl2) contain the crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic

Figure 2 .

 2 Figure 2. Examples of acridine derivatives and acridine-based metal complexes G -L tested for anticancer applications, and the structures of starting materials 1 -3 showing the numbering scheme used for the NMR assignments.

FollowingScheme 1 .

 1 Scheme 1. Synthesis of the substituted acridine ligands L1 and L2.

Figure 3 .

 3 Figure 3. Top: crystal structure of complex 7•CH2Cl2 (dichloromethane and hydrogen atoms omitted for clarity). Selected bond distances (Å) and angles (º). Au1-N1 2.028(3), Au1-C11 2.034(4), Au1-O1 2.049(3), Au1-N2 1.990(3), C11-Au1-N1 81.1(1), C11-Au1-N2 103.8(1), N2-Au1-O1 82.5(1), O1-Au1-N1 92.7(1), N1-Au1-N2 174.3(1), C11-Au1-O1 173.1(1), torsion Au1-N2-C22-C23 110.4(3). Bottom: Crystal structure of complex 9•CH2Cl2 (dichloromethane and hydrogen atoms omitted for clarity).

Scheme 3 .

 3 Scheme 3. Synthesis of the acridine functionalized [(C^N pz ^C)Au III (AAC)] + 10.

Scheme 4 .

 4 Scheme 4. Synthesis of the acridine-functionalized [(C^N pz ^C)Au III (NHC)] + 11.

a

  Mean ± the standard error of at least three independent experiments. b Values from ref.18

  7 µM) to high (IC50(11) of 1.5 µM) via intermediate activities (IC50(6) and IC50(7) of 9.9 µM2.7 µM respectively). Moreover, that selection of compounds includes a dichloro complex (2), a simple (N^X) chelate (6) and examples of acridine derivatives with (C^N) and (C^N pz ^C) cyclometalated ligands (7 and 11 respectively). MCF-7 cells were incubated for 6 h with 10 µM concentrations of each of the complexes in 1% DMSO. The results of three independent experiments are depicted in Figure4.

Figure 4 .

 4 Figure 4. Cellular uptake of complexes 2, 6, 7 and 11 and a DMSO control in MCF-7 cells after 6 h of treatment at 10 µM in 1% DMSO. Data represent the average ± standard deviation of three experiments. The significance of the results was analysed by t-test. **p value < 0.01.

  DNA intercalation is a reversible mode of DNA-ligand binding which consists of the direct insertion of planar aromatic moieties in between the base pairs of DNA, which causes unwinding to the double helix. There are several examples of gold(III) complexes acting as DNA intercalators, including both aminoquinoline, 20a and terpyridine 20b derivatives, which have been shown to bind to intracellular DNA by intercalation of the planar gold(III) coordination plane into the DNA base pairs. To determine whether or not the acridine-functionalized complexes interact with DNA in the present cases, a Fӧrster resonance energy transfer (FRET) DNA melting assay was used to measure the ability of 4-11, L1 and L2 to stabilize double stranded DNA. FRET is used as a tool for detecting spatial relationships between two fluorophores and involves measuring the changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved further apart as a result of DNA stabilization or denaturation. As a result of the large differences in fluorescence between folded and unfolded DNA structures it is possible

Figure 5 .

 5 Figure 5. Stabilization of double-stranded DNA (0.2 µM) by complexes 4-11 and L1/L2 at 50 µM concentrations measured by FRET DNA melting assay. Data represents the average and standard deviation of two separate experiments.

Figure 6 . 1 H

 61 Figure 6. 1 H NMR spectra of a 1:1 mixture of 11 with GSH at room temperature, in comparison with the starting materials, 11, GSH and GSSG (DMSO-d6/D2O 1:1).

Table 1 :

 1 Cytotoxic effects of complexes 1-11 and ligands L1 and L2 in comparison to cisplatin towards different human cancer cell lines after 72 h of incubation.

	Complex	A549	IC50 ± SD (µM) a MCF-7	HL60
	1	43.6 ± 4.1	10.8 ± 3.5	6.0 ± 0.5
	2	>100	38.7 ± 4.7	12.8 ± 0.3
	3 b	> 50	8.6 ± 1.1	3.7 ± 0.3
	4	12.7 ± 2.5	5.2 ± 1.0	2.9 ± 0.7
	5	10.8 ± 2.7	3.6 ± 1.4	2.7 ± 0.4
	6	22.7 ± 2.5	9.9 ± 2.1	2.5 ± 0.1
	7	16.6 ± 2.8	2.7 ± 0.9	1.4 ± 0.4
	8	19.9 ± 3.9	4.3 ± 0.4	1.5 ± 0.1
	9	17.2 ± 4.4	6.7 ± 1.6	6.3 ± 0.1
	10	15.0 ± 2.0	7.5 ± 0.6	20.7 ± 0.4
	11	7.6 ± 0.6	1.5 ± 0.1	1.1 ± 0.1

  remarks. When required, manipulations were performed using standard Schlenk techniques under dry nitrogen or in a MBraun glove box. Nitrogen was purified by passing through columns of supported

	P2O5 with moisture indicator, and activated 4 Å molecular sieves. Anhydrous solvents were freshly
	distilled from appropriate drying agents. 1 H and 13 C[ 1 H] spectra were recorded using a Bruker
	AvanceDPX-300 spectrometer. 1 H NMR spectra (300.13 MHz) were referenced to the residual protons of
	the deuterated solvent used. 13 C[ 1 H] NMR spectra (75.47 MHz) were referenced internally to the D-
	coupled 13 C resonances of the NMR solvent. Elemental analyses were carried out at London Metropolitan

University. Complexes 1, 2, 3 and 5 were synthesized following reported procedures.

  ).

	Anal. Calcd. for C23H23AuN4O2.2H2O (592.45): C, 46.63; H, 4.59; N, 4.73. Found: C, 46.23; H 4.21; N
	4.76. 1 H NMR (CD2Cl2, 300 MHz, 298 K): 8.97 (d, 3 JH-H = 5.1 Hz, 1H, H 2 ), 8.07 (t, 3 JH-H = 8.1 Hz, 1H,
	H 4 ), 7.83 (d, 3 JH-H = 8.1 Hz, 1H, H 5 ), 7.75 (d, 4 JH-H = 1.5 Hz, 1H, H 11 ), 7.51-7.40 (m, 2H, H 3+8 ), 7.37 (dd,
	3 JH-H = 8.3 Hz, 4 JH-H = 1.5 Hz, 1H, H 9 ), 7.22 (dd, 3 JH-H = 7.7 Hz, 4 JH-H = 1.3 Hz, 1H, H 14/17 ), 6.84 (m, 1H,
	H 15/16 ), 6.66 (dd, 3 JH-H = 7.7 Hz, 4 JH-H = 1.3 Hz, 1H, H 14/17 ), 6.49 (m, 1H, H 15/16 ), 2.32 (s, 3H, H 20 ), 1.35 (s,
	9H, Synthesis of complex 6
	A mixture of 2-(4-t-butyl)phenyl pyridine gold dichloride, (2) (0.200 g, 0.418 mmol), 1,2-
	diacetamidobenzene (0.081 g, 0.421 mmol) and silver I oxide (200 mg, 0.863 mmol) was added to
	dichloromethane (30 mL) and refluxed for 24 h. The mixture was then cooled, filtered through celite and
	the filtrate was concentrated under vacuum. A large excess of diethyl ether (20 mL) was added to
	precipitate the product as a pale-yellow, fluffy, solid. This was filtered and dried under vacuum (0.224 g,
	0.375 mmol, 89%). Anal. Calcd. for C25H26AuN3O2 (597.47): C, 50.26; H, 4.39; N, 7.03. Found: C, 50.12;
	H 4.38; N 6.97. 1 H NMR (CD2Cl2, 300 MHz, 298 K): 9.30 (s, 1H, H 2 ), 8.02 (t, 3 JH-H = 7.8 Hz, 1H, H 4 ),
	7.79 (d, 3 JH-H = 7.8 Hz, 1H, H 5 ), 7.55 (d, 4 JH-H = 1.4 Hz, 1H, H 11 ), 7.48 (d, 3 JH-H = 8.0 Hz, 1H, H 8 ), 7.41-
	7.28 (m, 3H, H 9+3+14/17 ), 7.09 (m, 1H, H 14/17 ), 6.99 (dt, 3 JH-H = 7.3 Hz, 4 JH-H = 1.4 Hz, 1H, H 15/16 ), 6.91 (dt,
	3 JH-H = 7.3 Hz, 4 JH-H = 1.4 Hz, 1H, H 15/16 ), 2.27 (s, 3H, H 20/22 ), 2.15 (s, 3H, H 20/22 ), 1.34 (s, 9H, t Bu). 13 C[H]
	NMR (CD2Cl2, 75 MHz): 174.1 (s, C 19/21 ), 173.1 (s, C 19/21 ), 164.6 (s, C 12 ), 154.8 (s, C 2 ), 153.3 (s, C 6/7 ),
	145.5 (s, C 10 ), 142.2 (s, C 4 ), 140.5 (s, C 13/18 ), 130.8 (s, C 11 ), 125.5 (s, C 3/9 ), 124.6 (s, C 15/16 ), 124.2 (s, C 8 ),
	124.0 (s, C 14/17 ), 123.0 (s, C 3/9 ), 122.7 (s, C 15/16 ), 119.7 (s, C 5 ), 35.3 (s, C(CH3)3), 30.8 (s, C(CH3)3).

t Bu). 13 C[H] NMR (CD2Cl2, 75 MHz): 173.7 (s, C 19 ), 164.2 (s, C 12 ), 161.3 (s, C 6/7 ), 155.5 (s, C 6/7 ), 147.0 (s, C 2 ), 144.7 (s, C 10 ), 144.3 (s, C 13/18 ), 142.7 (s, C 4 ), 139.4 (s, C 13/18 ), 133.0 (s, C 11 ), 125.4 (s, C 3 ), 124.8 (s, C 8 ), 124.2 (s, C 9 ), 123.8 (s, C 14/17 ), 123.4 (s, C 14/17 ), 120.2 (s, C 5 ), 115.6 (s, C 15/16 ), 115.3 (s, C 15/16 ), 35.3 (s, C(CH3)3), 30.8 (s, C(CH3)3), 25.2 (s, C 20 ).

  1 Hz, 1H, H 5 ),7.75 (m, 2H, H22/23 ),7.54 (m, 1H, H 3 ), 7.48-7.36 (m, 3H, H 8+22/23 ),7.01 (dd,3 JH-H = 8.2 Hz,4 JH-H = 1.7 Hz, 1H, H 9 ), 6.85 (dd,3 JH-H = 7.7 Hz,4 JH-H = 1.2 Hz, 1H, H 14/17 ), 6.48 (dt,3 JH-H = 7.7 Hz,4 JH-H = 1.2 Hz, 1H, H15/16 ), 6.12 (dt,3 JH-H = 7.7 Hz,4 JH-H = 1.2 Hz, 1H, H15/16 ), 5.39 (dd,3 JH-H = 7.7 Hz,4 JH-H = 1.2 Hz, 1H, H 14/17 ), 5.23 (d, 4 JH-H = 1.7 Hz, 1H, H 11 ), 0.35 (s, 9H, t Bu). 13 C[H] NMR (CD2Cl2, 75 MHz): 163.3 (s, C 2 ), 156.1 (s, C 12 ), 155.2 (s, C 6/7 ), 153.3 (s, C 6/7 ), 151.0 (s, C 19 ), 150.9 (s, C 20 ), 147.5 (s, C 10 ), 142.5 (s, C 13/18 ), 141.9 (s, C 4 ), 140.2 (s, C 13/18 ), 130.3 (s, C 21 ), 130.2 (s, C 22 ), 127.4 (s, C 9 ), 126.2 (s, C 23 ), 125.4 (s, C 8 ), 124.5 (s, C 25 ), 124.3 (s, C 24 ), 124.0 (s, C 11 ), 123.5 (s, C 3 ), 120.2 (s, C

5

), 118.4 (s, C

15/16

), 117.0 (s, C

15/16

), 115.1 (s, C 14/17 ), 113.7 (s, C 14/17 ), 34.

  ). C 6/8 ), 151.5 (s, C14/19 ), 150.7 (s, C 2 ), 141.7 (s, C 4 ), 138.5 (s, C 14/19 ), 132.3 (s, C 20/21/26 ), 130.2 (s, C 22/25 ), 130.1 (s, C 9/12 ), 129.8 (s, C 22/25 ), 129.5 (s, C 23/24 ), 126.4 (s, C 9/12 ), 126.4 (s, C 10/11 ), 126.4 (s, C 10/11 ), 126.1 (s, C 5 ), 125.6 (s, C 23/24 ), 124.7 (s, C 3 ), 118.3 (s, C 16/17 ), 116.7 (s, C 16/17 ), 115.4 (s, C 15/18 ), 113.4 (s, C 15/18 ), 47.8 (s, C 7/7' ).

	Anal.Calcd.for C31H22AuN3O.5H2O (739.58): C, 50.35; H, 4.36; N, 5.68. Found: C, 50.63; H 3.36; N 7.45.
	1 H NMR (CD2Cl2, 300 MHz, 263 K): 9.23 (d, 3 JH-H = 5.6 Hz, 1H, H 2 ), 8.51 (d, 3 JH-H = 8.6 Hz, 1H, H 22/25 ),
	8.46 (d, 3 JH-H = 8.6 Hz, 1H, H 22/25 ), 8.21 (d, 3 JH-H = 8.6 Hz, 1H, H 22/25 ), 8.03 (dt, 3 JH-H = 7.7 Hz, 4 JH-H = 1.4
	Hz, 1H, H 4 ), 7.95 (d, 3 JH-H = 8.6 Hz, 1H, H 22/25 ), 7.80 (m, 1H, H 23/24 ), 7.67 (d, 3 JH-H = 7.7 Hz, 1H, H 5 ),
	7.62-7.46 (m, 3H, H 3+23/24 ), 7.25 (t, 3 JH-H = 8.6 Hz, 1H, H 23/24 ), 6.78-6.71 (m, 2H, H 15/18+9/12 ), 6.55 (t, 3 JH-H
	= 7.4 Hz, 1H, H 16/17 ), 6.47 (t, 3 JH-H = 7.4 Hz, 1H, H 10/11 ), 6.16 (t, 3 JH-H = 7.4 Hz, 1H, H 16/17 ), 5.99-5.84 (m,
	2H, H 9/12+10/11 ), 5.69 (dd, 3 JH-H = 7.4 Hz, 4 JH-H = 1.0 Hz, 1H, H 15/18 ), 4.43 (d, 2 JH-H = 15.3 Hz, 1H, H 7/7' ),
	3.91 (d, 2 JH-H = 15.3 Hz, 1H, H 7/7' ). 13 C[H] NMR (CD2Cl2, 75 MHz): 157.2 (s, C 13 ), 157.1 (s, C 6/8 ), 151.7
	(s,

  JH-H = 7.7 Hz,4 JH-H = 1.1 Hz, 1H, H14/17 ), 6.44 (t,3 JH-H = 7.7 Hz, 1H, H15/16 ), 6.36-6.18 (m, 3H, H 8+11+15/16 ), 5.62 (dd,3 JH-H = 7.7 Hz,4 JH-H = 1.1 Hz, 1H, H 14/17 ), 1.46 (s, 9H, t Bu). 13 C[H] NMR (CD2Cl2, 75 MHz): 164.6 (s, C 2 ), 155.3 (s, C 12 ), 154.5 (s, C 6/7 ), 150.5 (s, C 6/7 ), 149.6 (s, C 19 ), 148.0 (s, C 20 ), 145.6 (s, C 10 ), 145.0 (s, C 13/18 ), 140.4 (s, C 4 ), 139.7 (s, C 13/18 ), 130.2 (s,

	petroleum (bp. 40-60 °C) (20 mL), filtered off and dried under vacuum (0.214 g, 0.310 mmol, 74%). Anal.
	Calcd. for C34H29AuN4.3H2O (744.65): C, 54.84; H, 4.74; N, 7.52. Found: C, 54.25; H; 4.20 N; 7.41. 1 H
	NMR (CD2Cl2, 300 MHz, 298 K): 8.40 (dd, 3 JH-H = 8.7 Hz, 4 JH-H = 0.7 Hz, 2H, H 21/24 ), 8.24 (d, 3 JH-H = 8.7
	Hz, 2H, H 21/24 ), 7.78-7.68 (m, 4H, H 22/23+2+4/5 ), 7.64-7.55 (m, 2H, H 3+4/5 ), 7.41 (dd, 3 JH-H = 8.2 Hz, 4 JH-H =
	1.7 Hz, 1H, H 9 ), 7.35 (m, 2H, H 22/23 ), 6.97 (dd, 3 C 21/24 ), 130.0 (s, C 21/24 ), 126.5 (s, C 25 ), 126.0 (s, C 9 ), 125.5 (s, C 22/23 ), 124.9 (s, C 8 ), 124.8 (s, C 22/23 ), 124.6
	(s, C 11 ), 122.3 (s, C 3 ), 120.1 (s, C 5 ), 118.2 (s, C 15/16 ), 116.3 (s, C 15/16 ), 113.3 (s, C 14/17 ), 112.6 (s, C 14/17 ),
	35.6 (s, C(CH3)3), 31.0 (s, C(CH3)3).

  reaction was stirred for a further 15 min before being heated to reflux overnight. After cooling to room temperature, the orange solid was filtered and washed twice with light petroleum (bp. 40-60 °C) before being dried under vacuum (0.551 g, 1.925 mmol, 92%).1 H NMR ((CD2)SO, 300 MHz, 298 K): 10.18 (s, 1H, H 8 ), 8.26 (d,3 JH-H = 8.8 Hz, 1H, H 3/6 ), 8.18 (d,3 JH-H = 7.9 Hz, 1H, H 3/6 ), 8.08-7.88 (m, 5H, Acridin-9-ylamino)-benzoic acid (1.500 g, 4.772 mmol) and N,N'-carbonyldiimidazol (CDI) (0.774 g, 4.773 mmol) were placed in a flame dried flask, fitted with a condenser, and stirred in dry tetrahydrofuran (50 mL) under a nitrogen atmosphere for 15 min. O-phenylenediamine (0.675 g, 6.242 mmol) was then added and the reaction was stirred for a further 15 min before being heated to reflux overnight. After cooling to room temperature, the orange solid was filtered and washed twice with light petroleum (bp. 40-60 °C) before being dried under vacuum (1.102 g, 3.862 mmol, 81%).1 H NMR ((CD2)SO, 300 MHz, 298 K): 8.24 (d,3 JH-H = 8.1 Hz, 2H, H11/14 ), 8.02 (d,3 JH-H = 8.1 Hz, 2H, H 11/14 ), 7.90 (t,3 JH-H = 8.1 Hz, 2H, H 12/13 ), 7.33 (t,3 JH-H = 8.1 Hz, 2H, H 12/13 ), 7.18 (t,3 JH-H = 7.7 Hz, 1H, H 4/5 ), 7.05 (d,3 JH-H = 7.7 Hz, 1H, H 3/6 ), 6.89 (d,3 JH-H = 7.6 Hz, 1H, H 3/6 ), 6.60 (t,3 JH-H = 7.6 Hz, 1H, H 4/5 ).13 C[H] NMR ((CD2)SO, 75 MHz): 156.5 (s, C 2/7/9 ),145.1 (s, C 2/7/9 ), 140.3 (s, C 2/7/9 ), 135.3 (s, C 12/13 ), 129.9 (s, C 4/5 ), 127.2 (s, C 3/6 ), 126.2 (s, C 11/14 ), 125.0 (s, C 10/15 ), 123.7 (s, C 12/13 ), 119.2 (s, C 11/14 ), 116.9 (s, C 4/5 ), 116.3 (s, C 3/6 ),113.6 (s, C 10/15

	). Anal. Calcd. for
	C46H45AuF6N5Sb (1100.62): C, 50.20; H, 4.12; N, 6.36. Found: C, 49.97; H 4.03; N 6.35. 1 H NMR H 4/5+11+14 ) 8.28-7.50 (m, 4H, H 12+13 ), 7.03 (t, 3 JH-H = 7.9 Hz, 1H, H 4/5 ). 13 C[H] NMR ((CD2)SO, 75 MHz):
	((CD3)2SO, 300 MHz, 298 K): 10.34 (s, 1H, NH), 9.13 (s, 2H, H 2 ), 8.26 (d, 3 JH-H = 8.2 Hz, 2H, H 20/23 ), 156.8 (s, C 2/7/9 ), 152.9 (s, C 2/7/9 ), 140.5 (s, C 2/7/9 ), 136.1 (s, C 11/14 ), 131.9 (s, C 4/5 ), 130.5 (s, C 10/15 ), 128.1 (s,
	7.96 (d, 4 JH-H = 1.5 Hz, 2H, H 8 ), 7.85 (d, 3 JH-H = 8.4 Hz, 2H, H 5 ), 7.74-7.57 (m, 4H, H 20/23+21/22 ), 7.30 (dd, C 3/6 ), 128.0 (s, C 12/13 ), 125.9 (s, C 3/6 ), 124.6 (s, C 12/13 ), 121.0 (s, C 10/15 ), 119.9 (s, C 11/14 ), 117.8 (s, C 4/5 ),
	3 JH-H = 8.4 Hz, 4 JH-H = 1.5 Hz,2H, H 6 ), 7.25-7.14 (m, 3H, H 14+15 ), 6.96 (t, 2H, 3 JH-H 7.5 Hz, 2H, H 21/22 ), 114.1 (s, C q ).
	2.56 (s, 6H, H 16 ), 1.20 (s, 18H, t Bu). 13 C[H] NMR (CD3CN, 75 MHz): 167.6 (s, C 9 ), 156.0 (s, C 4 ), 155.6
	(s, C 3 ), 144.7 (s, C 7 ), 139.9 (s, C 2 ), 139.6 (s, C 13 ), 135.1 (s, C 8 ), 134.0 (s, C 20/23 ), 128.6 (s, C 15 ), 127.7 (s, Synthesis of L2
	C 14 ), 127.6 (s, C 20/23 ), 127.6 (s, C 12 ), 127.0 (s, C 5 ), 124.5 (s, C 6 ), 123.6 (s, C 21/22 ), 123.5 (s, C 21/22 ), 117.8 (s, 4-(
	C 18 ), 117.8 (s, C 19 ), 117.8 (s, C 24 ), 35.7 (s, C(CH3)3), 31.4 (s, C(CH3)3), 19.3 (s, C 16 ). νmax (neat)/cm -1 : 3388
	(NH), 3270 (NH) 2957 ( t Bu), 2867 (Ar), 1586 (carbene).
	Synthesis of complex 11
	[(MeImAcr)H]Cl (0.070 g, 0.232 mmol) and silver oxide (0.043 g, 0.186 mmol) and acetonitrile (15 mL)
	were combined in a Schlenk and stirred for 56 h. (C^N pz ^C)AuCl, (3), (0.133 g, 0.232 mmol) and KPF6
	(0.128 g, 0.695 mmol) were then added to the flask and the reaction was stirred for a further 16 h before
	being filtered through a celite plug. The filtrate was concentrated under vacuum and the product was
	precipitated using a 2:1 mixture of light petroleum (bp. 40-60 °C) / diethyl ether (5 mL), and after
	removing the solvent, dried under vacuum to yield a dark yellow solid (0.170 g, 0.180 mmol, 78%). Anal.
	Calcd. for C41H39AuF6N5P.5MeCN (1148.99): C, 53.31; H, 4.74; N, 12.19. Found: C, 52.92; H, 5.10; N,
	11.80. 1 H NMR (CD3CN, 300 MHz, 298 K): 8.68 (s, 2H, H 2 ), 8.12-8.02 (m, 3H, H 11+19 ), 7.96 (d, 1H, 3 JH-H
	= 1.9 Hz, H 12 ), 7.88-7.77 (m, 4H, H 16+18 ), 7.55 (d, 2H, 3 JH-H = 8.3 Hz, H 5 ), 7.45 (t, 2H, 3 JH-H = 7.8 Hz, H 17 ),
	7.33 (dd, 2H, 3 JH-H = 8.3 Hz, 4 JH-H = 1.7 Hz, H 6 ), 7.21 (d, 2H, 4 JH-H = 1.7 Hz, H 8 ), 4.15 (s, 3H, H 13 ), 1.30
	(s, 18H, t Bu). 13 C[H] NMR (CD3CN, 75 MHz): 166.1 (s, C 9 ), 156.8 (s, C 4 ), 156.3 (s, C 3 ), 154.9 (s, C 10 ),
	146.6 (s, C 20 ), 144.6 (s, C 7 ), 140.5 (s, C 14 ), 139.3 (s, C 2 ), 133.3 (s, C 18 ), 133.0 (s, C 8 ), 128.9 (s, C 17 ), 127.0
	(s, C 12 ), 126.9 (s, C 19 ), 126.7 (s, C 5 ), 126.3 (s, C 11 ), 125.2 (s, C 6 ), 123.5 (s, C 16 ), 122.2 (s, C 15 ), 39.1 (s,
	C 13 ), 35.1 (s, C(CH3)3), 30.3 (s, C(CH3)3).
	Synthesis of L1

2-(Acridin-9-ylamino)-benzoic acid (0.600 g, 2.100 mmol) and N,N'-carbonyldiimidazol(CDI) (0.341 g, 2.103 mmol) were placed in a flame dried flask, fitted with a condenser and stirred in dry tetrahydrofuran (20 mL) under a nitrogen atmosphere, for 15 min. 2-amino phenol (0.301 g, 2.758 mmol) was then added and the

Acknowledgements

This work was supported by the European Research Council. M.B. is an ERC Advanced Investigator Award holder (grant no. 338944-GOCAT). We are grateful to the EPSRC National Crystallographic Service, Southampton, UK, for the collection of crystallographic data sets for samples of 7 and 9. 43

Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Antiproliferation assay

The human A549 and HL60 cancer cell lines (from ECACC) were cultured in RPMI 1640 medium with 10% fetal calf serum, 2 mM L-glutamine, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin (Invitrogen). The cells were maintained under a humidified atmosphere at 37 °C and 5% CO2. The human MCF-7 cancer cell line (from ECACC) was cultured in DMEM medium with 10% fetal calf serum, 2 mM L-glutamine, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin (Invitrogen). The cells were maintained under a humidified atmosphere at 37 °C and 5% CO2. Inhibition of cancer cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) assay using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) and following the manufacturer's instructions. Briefly, the cells (3 × 10 4 per 100 µL for HL60, 8 × 10 3 per 100 µL for A549 and MCF-7) were seeded in 96-well plates and left untreated or treated with 1 µL of DMSO (vehicle control) or 1 µL of complexes diluted in DMSO at different concentrations, in triplicate for 72 h at 37 °C with 5% CO2. Following this, MTS assay reagent was added for 4 h and absorbance measured at 490 nm using a Polarstar Optima microplate reader (BMG Labtech). IC50 values were calculated using GraphPad Prism Version 5.0 software.

Uptake study

MCF-7 cells were grown in 75 cm 2 flasks up to 70% of confluence in 10 mL of culture medium.

Compounds 2, 5, 6 and 11 were added to the flasks (100 µL of 1 mM solution in DMSO) and incubated for 6 h at 37 °C with 5% CO2. Negative controls were used by incubating cells with DMSO alone under the same conditions. After removal of the medium and washing of the cells with PBS pH 7.4, the cells were detached using a trypsin solution. After quenching of trypsin with fresh medium, centrifugation and removal of the supernatant, the cell pellet was resuspended into 1 mL of PBS pH 7.4 and split into twice 500 µL for metal and protein quantification. The number of cells (expressed per million cells) of each sample was determined by measuring the protein content of the treated samples using a BCA assay (ThermoFischer Scientific) corrected by the amount of protein/10 6 cells determined for each cell type by measuring the protein content of an untreated sample and dividing by the corresponding number of cells measured with a hematocytometer following a reported procedure. Microwave digestion was used to solvate the samples to liquid form. Nitric acid and hydrogen peroxide were used in a Milestone Ethos 1 microwave system using SK-10 10 place carousel. The digest was ramped to 200 °C in 15 minutes holding at 200 °C for 15 min. The sample was weighed into a microwave vessel before digestion, and decanted and rinsed into a pre-weighed PFA bottle after digestion. ICP-MS samples were spiked with rhodium internal

Conflicts of interest

There are no conflicts of interest to declare.