Benoît Bertrand

Alexander S Romanov

Mark Brooks

Josh Davis

Claudia Schmidt

Ingo Ott

Maria O'connell

Manfred Bochmann

Synthesis, Structure and Cytotoxicity of Cyclic (Alkyl)(Amino) Carbene and Acyclic Carbene Complexes of Group 11 metals

A series of complexes of cyclic (alkyl)(amino)carbene (CAAC) complexes of copper, silver and gold have been investigated for their antiproliferative properties. A second series of acyclic carbene (ACC) complexes of gold(I) were prepared by nucleophilic attack on isocyanide complexes by amines and amino esters, to give (ACC)AuCl, [(ACC)Au(PTA)] + (PTA = triazaphosphaadamantane), as well as mixed-carbene compounds [(CAAC)Au(ACC)] + . Representative complexes were characterised by X-ray diffraction which confirmed the mononuclear linear structures without close intermolecular contacts or aurophilic interactions. The redox properties of these complexes have been determined. The compounds were tested against a panel of human cancer cell lines including leukemia (HL 60), breast adenocarcinoma cells (MCF-7) and human lung adenocarcinoma epithelial cell lines (A549), which show varying degrees of cisplatin resistance. The pro-ligand iminium salts and the PTA complexes were non-toxic. By contrast, the CAAC complexes show high cytotoxicity, with IC50 values in the sub-micromolar to ~100 nanomolar range, even against cisplatin-insensitive MCF-7 and A549 cells. Cationic bis-carbene complexes [( Me2 CAAC)2M] + (6 -8, M = Cu, Ag and Au) proved particularly effective. The mechanism of cell growth control by these complexes remains to be established, although possible modes of action such as inhibition of thioredoxin reductase (TrxR), which is a common pathway for gold NHC compounds, or the formation of reactive oxygen species (ROS) through redox processes, could be ruled out as primary pathways.

Introduction

Carbene complexes of copper(I), silver(I) and gold(I) have been shown to display promising activities as antiproliferative agents towards a large number of cancer cell lines. Gold complexes have been extensively studied, and their structures and cytotoxicity profiles have been the subject of a number of reviews. [1][2][3][4][5][START_REF]CH3 CNxylyl) ppm. IR νmax (neat)/cm -1 : 2215 (CNxylyl). Synthesis of [( Ad CAAC)Au{C(NHxylyl)(pyrrolidine)}]SbF6 (14a) The solution of 13a (50 mg, 0.053 mmol) and pyrrolidine (9 µL, 0.106 mmol) in 6 mL of MeCN was stirred overnight in the dark, filtered through a thin layer of Celite, washed with a saturated ammonium chloride solution, and dried with sodium sulphate. The solution was filtered and volatiles were removed under vacuum. The residue was taken up in dichloromethane and the product was precipitated with hexane as a white powder (41 mg, 0.040 mmol, 75%)[END_REF] Copper is an essential trace element and participates in a wide variety of catalytic functions in the body. Its homeostasis is guaranteed by different active transporters and chaperones. Copper complexes are being discussed in connection with developing treatments against a range of diseases, including neurodegenerative diseases, Alzheimer's and Parkinson's disease, as well as cancers. 7 Most of the compounds studied for anti-cancer activity are based on copper(II), and its cytotoxic activity is thought to be possibly related to its redox behaviour and generation of reactive oxygen species (ROS), to mitochondrial toxicity, to binding to intracellular thiols and interference in respiratory processes, as well as DNA cleavage and activation of apoptotic pathways in cancer cells. [8][9][10][11] Carbene complexes of silver-too, have become attractive for their cytotoxic activity, which in some cases exceeds that of gold analogues. [12][13][14][15][16] The ligands used in these compounds are N-heterocyclic carbenes (NHCs) based on the imidazole framework and may be mono-or bi-dentate. NHC complexes generally fall into two categories: (i) mono-carbene adducts of metals salts that also carry a labile ligand X, such as in (NHC)MX, usually X = halide, where the X ligand can be expected to be readily substituted under physiological conditions and is susceptible to substitution by intracellular thiols; (ii) ionic bis-carbene complexes [M(NHC)2] + X -, which may be redox active but can only bind to thiols after carbene dissociation. In any case, silver NHC complexes are known for their lability and have long been used as NHC transfer reagents for the synthesis of NHC complexes of other metals. 17,[START_REF] Canal | A carbene transfer agent[END_REF] This implies that the structural integrity of (NHC)Ag compounds under physiological conditions cannot be taken for granted, so that establishing quantitative structure-activity relationships may not be straightforward.

In the present study we have chosen cyclic (alkyl)(amino)carbene (CAAC) ligands [19][START_REF] Bertrand | [END_REF][21] coordinated to coinage metals to investigate the potential of these complexes as antiproliferative agents. CAAC ligands have the advantage over NHCs that they bind significantly more strongly and are not prone to dissociation. [START_REF] Romanov | For photochemical applications of CAAC complexes[END_REF] For example, CAAC ligands have been found capable of forming stable complexes even with zero-valent gold and copper. 23 For these reasons CAAC silver complexes are rather poor carbene transfer agents; for example they failed to give the expected carbene transfer to rhodium and ruthenium targets. 24 These characteristics are however beneficial for biological applications, as further supported by recent results on polycyclic CAAC complexes reported by Waldmann and coworkers. 25 We also report the extension of carbene anti-cancer complexes to acyclic carbenes, in combination with CAAC and other ligands. Acyclic carbenes (ACC) offer the advantage of facile modification and functionalization via a common precursor and provide a potentially very versatile method for improving solubility, cell uptake and selectivity of cytotoxicity.

Although acyclic carbene adducts of gold were among the first examples of carbene complexes to be reported, 26 their biological activity has hardly been explored. 27

Results and Discussion

Synthesis and Structure. The compounds (CAAC)MCl 1 -5 (M = Cu, Ag, Au, Chart I) and of the bis-carbene compounds [( Me2 CAAC)2M]X (6, M = Cu, X = I; 7, M = Ag, X = OTf; 8, M = Au, X = Cl) were prepared following previously reported procedures. 19c,21,22a,24 Due to steric hindrance bis-CAAC complexes can be obtained only for methyl-and ethyl substituted CAAC ligands but not for adamantyl-substituted CAAC. The iminium salt ligand precursors Me LH, Et LH and Ad LH were also included in the toxicity testing programme for comparison.

All the metal complexes are mononuclear in solution and in the solid state.

Chart I: Structure of the CAAC-M(I) complexes 1-8 and the corresponding pro-ligands.

Complexes with a combination of CAAC and acyclic carbene ligands were obtained by nucleophilic addition of amines and amino esters to gold(I) isocyanide complexes, 26 as outlined in Scheme 1. Both neutral complexes (ACC)AuCl and ionic complexes [(L)Au(ACC)] + X -were prepared to evaluate the influence of charge and solubility (ACC = acyclic carbene; L = Ad CAAC or triazaphosphaadamantane, PTA). The reaction of gold isocyanide complexes (RNC)AuCl (R = xylyl, t Bu) with pyrrolidine in dichloromethane at room temperature in the dark gave compounds 9a and 9b as white powders. Similarly, the reaction of sarcosine hydrochloride in acetonitrile in the presence of potassium carbonate afforded the amino acid derivatives 10a and 10b as orange to purple powders in good yields.

The chloride ligands in 9 and 10 can be readily be substituted by PTA, chosen because of its water solubility, to give the ionic complexes 11a,b and 12a,b, respectively (Scheme 1). The structures of 11a and 11b were confirmed by X-ray crystallography (vide infra). In solution 12a appears as a mixture of the two possible orientations of the sarcosine substituent of the ACC ligand in a ratio of approximately 0.75:0.25, while complex 12b exists in solution as a mixture of all four possible stereoisomers of the ACC ligand; these lead to distinct 1 H NMR resonances for the N-H hydrogen with an integration ratio of approximately 0.1:0.2:0.2:0.5.

The reaction of ( Ad CAAC)AuCl with xylyl isocyanide and silver hexafluoroantimonate in dichloromethane under anhydrous conditions afforded the isocyanide complex 13a, which reacted with pyrrollidine in acetonitrile gave the mixed-carbene complex 14a as a white powder in 76% yield.

Scheme 1. Synthesis of neutral and ionic gold(I) complexes of acyclic carbenes

X-ray diffraction studies. The molecular structures of representative neutral and ionic ACC complexes with and without amino acid substituents were determined by X-ray diffraction (Figure 1). The structures of the CAAC complexes 13a and 14a are shown in Figure 2. All compounds show an almost linear coordination geometry, with the largest deviation observed for the bulky mixed-carbene complex 14a (C1-Au-C28 173.4(1)°). The Au-C (ca. 2.00 Å) and Au-Cl (ca. 2.29 Å) bond lengths deviate within the error of the experiment for the complexes 9a, 10a and 10b while being almost equidistant with other (ACC)AuCl reported in the literature. 28 The Au-C bond length for complex 11a is elongated by 0.06 Å compared to complexes 9-10 due to moderate structural trans-influence of the phosphine (PTA) ligand vs. chloride anion, in accordance with the general trend. 29 The mixed-carbene complex 14a shows that the Au-CCAAC bond length is slightly shorter than Au-CACC. The open chain carbene ligands possess an essentially planar geometry with N-C-N angles greater than 116°, similar to the values previously observed for acyclic carbene ligands. 28 1). The mono(carbene) silver chloride 4 in MeCN undergoes an irreversible reduction process with an anodic shift compared to the analogous copper (1, 240 mV) and gold compounds (5, 160 mV). A reoxidation peak from the reduction process could not be detected at all scan rates from 0.05 to 2 Vs -1 in MeCN, likely due to reaction with the electrolyte or solvent. To solve this problem we have performed cyclic voltammetry in THF with the less nucleophilic supporting electrolyte [ n Bu4N][B(C6F5)4]. In this solvent the reduction process shows quasi-reversible character but at a significantly lower potential, with E1/2 at -1.62 V (Figure 3). This indicates the feasibility of chemical reduction of the silver complex 4. Its oxidation behaviour is very similar to the copper and gold analogues and at all scan rates shows one irreversible process without a back-peak, regardless of the solvent or supporting electrolyte. The ionic homoleptic bis(carbene) complexes [( Me2 CAAC)2M]X (M = Ag, X = OTf; M = Au, X = BF4) show quasi-reversible one-electron reduction processes (Figure 4), with a significant anodic shift of the reduction potential E1/2 by 200 mV compared to mono(carbene) metal chloride complexes (see Table 1). The quasi-reversibility of the reduction peak is witnessed by the negligible shift of 10 mV in the peak position Ep on increasing the scan rate and the peak-to-peak separation ΔEp of 66-94 mV (at 100 mV/s), which is close to the ideal value of 59 mV for a one-electron reversible couple. Both silver (7) and gold (8) complexes show ipa/ipc values approaching unity, which is the ideal value for a reversible couple. For instance, the ipa/ipc ratio increases from 0.33 (at 50 mVs -1 ) to 0.54 (at 2 Vs -1 ) for silver [( Me2 CAAC)2Ag]OTf and from 0.49 (at 50 mVs -1 ) to 0.80 (at 2 Vs -1 ) for gold complex [( Me2 CAAC)2Au]BF4 (Figure 4). The reversibility of the reduction process for silver (7) and gold (8) complexes is markedly different from the copper analogue [( Me2 CAAC)2Cu]I (6), which shows quasi-reversible reduction only at high scan rates (0.5 to 2 Vs -1 ). By contrast, at all scan rates the heteroleptic bis-carbene gold complex 14a showed irreversible reduction (Figure 4), which was shifted cathodically by 220 mV compared to the homoleptic gold complex 8.

Unlike the bis-carbene copper complex 6, the irreversible oxidation of silver (7) and gold complexes 8 and 14a is observed at high anodic potential as expected (1.33 V for silver and over 1.63 V for gold complexes) (Table 1). The oxidation potential for 8 was completely masked by solvent discharge (THF or MeCN), while silver (7) and the heteroleptic gold complex 14a showed only partial overlap (Figure 4). We have previously reported that chemical oxidation of mono-carbene and bis-carbene gold complexes with PhICl2 produced photochemically unstable Au(III) complexes or decomposition products. 21 Table 1. Formal electrode potentials (peak position Ep for irreversible and E1/2 for quasireversible processes (*), V, vs. FeCp2), onset potentials (E, V, vs. FeCp2), peak-to-peak separation in parentheses for quasi-reversible processes (ΔEp in mV), EHOMO/ELUMO (eV) and band gap values (ΔE, eV) for the redox changes exhibited by copper, silver, and gold complexes. Antiproliferative properties on cancer cells in vitro. Although the complexes appeared to be poorly soluble in aqueous media, all compounds were soluble enough in DMSO not to precipitate when diluted with aqueous buffer solution up to 100 µM with 1% DMSO. Stock solutions of cisplatin were prepared in pure water. For an initial screening, the CAAC complexes 1 -8 were selected for the determination of the IC50 on a panel of human cancer cell lines including leukemia (HL 60), breast adenocarcinoma cells (MCF-7) and human lung adenocarcinoma epithelial cells (A549) (Table 2). is about an order of magnitude lower. The exception here is the surprisingly low activity of ( Ad CAAC)CuCl 1 against A549, which contrasts with the low IC50 value of only 0.53 ±0.06 μM for the ethyl analogue, ( Et CAAC)CuCl 2. The silver complex ( Ad CAAC)AgCl 4 is slightly more toxic than its copper analogue 1 on the MCF-7 cells. Overall 4 shows a similar trend: sub-micromolar activity against HL-60 and MCF-7 and low micromolar values against the more resistant A549 lines. These copper and silver CAAC complexes are an order of magnitude more potent than the gold complex 5, which is less effective than copper and silver against HL-60 and MCF-7 and, surprisingly, non-toxic against A549. In the series of adamantyl-substituted CAAC complexes, against HL-60, MCF-7 and A549 silver shows the highest and gold the lowest cytotoxicity: IC50(5) > IC50(1) ≥ IC50(4). On the other hand, such a metal dependence has been not observed in the cationic series, for which very similar, and remarkably low, IC50 values were determined for all metals against each of the tested cancer cell lines. Indeed, all [(CAAC)2M] + complexes 6 -8 demonstrated very strong antiproliferative effects, with IC50 values down up to sub-micromolar levels, including against A549 cells which are particularly difficult to treat with cisplatin.

Varying the substituents on the CAAC ligand in the copper series leads to only small differences in activity against HL 60 and MCF-7 cell lines, while against A549 cells a dramatic substituent dependence of the cytotoxic properties was observed (IC50 = 17.4 ± 0.8; 0.53 ± 0.06 and 2.0 ± 0.5 µM for 1, 2 and 3 respectively), indicating that Et2 CAAC complexes are the most toxic of this series.

The acyclic carbene complexes 9 -12 and 14a were screened against lung (A549) and breast (MCF-7) cancer cells at concentrations of 10 and 100 μM. The cell viability was measured after 72 hours of incubation using the standard MTS assay (see experimental part).

The results are reported in Figure 5. proliferative activity against MCF-7 at the lower concentration of 10 μM (Fig. 5B).

Moreover, while complexes 9a, 9b, 10b, 11a, 11b and 12b completely inhibited cell growth at the higher concentration of 100 μM, the two complexes presenting the ACC ligand with the xylyl/sarcosine combination, 10a and 12a, appeared to have a reduced activity even at that high concentration (cell viability of 20.2 ± 7.7 and 71 ± 24 % for 10a and 12a respectively). Combined with the data against A549 cells, this suggests a deleterious effect of that particular substituent combination on the antiproliferative properties of these compounds.

By contrast, the mixed-carbene complex [( Ad CAAC)Au(ACC)] + 14a fully inhibited both A549 and MCF-7 cells growth at both high and low concentrations, making it the most promising complex of the ACC series. For that reason it is the only one that was used for the determination of IC50 against our human cancer cell lines panel (Table 2). Like all [(CAAC)2M] + complexes, 14a demonstrated IC50 values in the low to sub-micromolar range against the panel of tested cell lines (IC50 = 0.10 ± 0.01; 2.3 ± 0.9 and 6.1 ± 0.3 μM against HL60, MCF-7 and A549 cells respectively). This demonstrates the potential of using a mixed [(CAAC)Au(ACC)] + scaffold for the introduction of functionalities, for example as vectors to improve cell uptake selectivity or as imaging probes.

Mechanistic investigations: inhibition of TrxR Thioredoxin reductase (TrxR) is a homodimeric enzyme whose role consists of reducing thioredoxin in an NADPH-dependent manner. Thioredoxin (Trx) in turn reduces different proteins including peroxiredoxin (involved in the regulation of cellular level of hydrogen peroxide). 30 TrxR has been reported as a potential intracellular target for complexes of various metals, including Ru, Fe and Au. [31][32][33] Among those, Au(I)-NHC complexes were found to be particularly potent TrxR inhibitors. 3,4,34 Based on those finding we have tested our most toxic complexes 1-8 as potential TrxR inhibitors (see Experimental for details). We first performed a screening of all complexes at 10 μM against mammalian TrxR. The results are reported in Table 3. Although none of the copper containing compounds (1 -3 and 6) showed any strong inhibitory effects on TrxR up to a concentration of 10 μM (less than 50% inhibition), we could notice a trend between the bulkiness of the CAAC ligand and the inhibitory activity of the corresponding Cu-Cl complexes. Indeed, the adamantyl complex 1 was much less potent than the ethyl and methyl complexes 2 and 3. Moreover, for the same substituent (methyl) the coordinatively saturated bis-CAAC complex 6 was a much weaker TrxR inhibitor than its mono-CAAC analogue 3. Those data are in good agreement with the reported mechanism of action of Au(I) complexes which involves the direct coordination of the metal centre to the selenocysteine residue of the active site of TrxR. 34 The silver and gold complexes, both in the mono-and bis-CAAC series, inhibited TrxR at the concentration of 10 μM. Those compounds where thus selected for the determination of their IC50 against mammalian TrxR.

The IC50 values follow the trend (CAAC)MCl < [(CAAC)2M] + , as already noticed in the copper series. The best inhibitor was the mono-CAAC gold complex 5. However the TrxR inhibition activity of 5 (IC50 = 2.6 ± 0.8 μM) is almost 10 times lower than that of reported NHC-Au-Cl complexes, 35 while 8 showed an inhibiting effect similar to the previously reported [(NHC)2Au] + complexes. 36 In particular regarding gold complexes, the higher activity of monocarbene complexes versus biscarbene analogues has been observed in several recent studies. 36a,b,37 The decreased inhibitory activity of 5 compared to other monocarbene gold NHC complexes might be explained by the steric protection of the gold centre, which is expected to inhibit the ability of the gold centre to bind to the selenocyteine residue of the active site of TrxR.

Altogether, the data show that although inhibition of TrxR might account partially for the observed antiproliferative properties of some the CAAC compounds investigated here (complexes 4, 5, 7 and 8), the IC50 values are an order of magnitude higher than the IC50 values derived from cell growth inhibition. This leads to the conclusion that TrxR inhibition is most probably not the main mode of action of the CAAC class of compounds.

Mechanistic investigations: enhancement of ROS formation. Reactive oxygen species (ROS) are naturally produced by mitochondria and are involved in different intracellular processes. However, their production, location and quantity is tightly controlled. ROS overproduction can induce cellular stress leading to cell death. 38 We measured the intracellular ROS amount of A549 cells after incubation with the most toxic complexes 1 -8

and 14a for 24 hours (Figure 6). In the CuCl series there is a clear correlation between the substituent on the CAAC ligand and the ability of the compounds to induce ROS production, with the least bulky Me2 CAAC complex 3 inducing about the same amount of ROS production as the positive control H2O2, whereas the most bulky adamantly-substituted complex 1 presented the same amount of ROS as the negative control samples. The Ad CAAC complexes 1, 4 and 5 did not trigger the formation of ROS. None of the cationic bis-CAAC complexes 6 -8 led to ROS formation either, despite the fact that they contain the sterically least demanding Me2 CAAC ligand, and despite the differences in their oxidation potentials. Of course, these bis-carbene complexes lack readily available coordination sites, and overall the results suggest that the accessibility of the metal for further complexation is of particular importance for the generation of ROS. Moreover, while (NHC)AuCl and [(NHC)2Au] + complexes have been reported to enhance the formation of intracellular ROS, 35,36a we found that neither neutral

Experimental

General Remarks. Unless stated otherwise all reactions were carried out in air. Solvents were distilled and dried as required. The carbene ligands Me2 CAAC, Et2 CAAC, Ad CAAC, 19 and complexes [( Et2 CAAC)2Cu]I, 22b and (CAAC)MCl (M = Cu, Ag, Au; X = Cl, Br, I), [21][START_REF] Romanov | For photochemical applications of CAAC complexes[END_REF][23][24] were obtained according to literature procedures. 1 H, 13 C{ 1 H} and 19 F NMR spectra were recorded using a Bruker Avance DPX-300 MHz NMR spectrometer. 1 H NMR spectra (300.13 MHz)

and 13 C{ 1 H} (75.47 MHz) were referenced to CD2Cl2 at δ 5.32 ( 13 C, δ 53.84), C6D6 at δ 7.16

( 13 C, δ 128.4), CDCl3 at δ 7.26 ( 13 C, δ 77. 16) or C6D5Br at δ 7.30 for the most downfield signal ( 13 C, δ 122.5) ppm. 19 F NMR spectra (282.4 MHz) were referenced externally to CFCl3 and internally to C6F6 (δF -164.9). IR spectra were recorded using a Perkin-Elmer Spectrum One FT-IR spectrometer equipped with a diamond ATR attachment.

Electrochemical experiments were performed using an Autolab PGSTAT 302N computercontrolled potentiostat. Cyclic voltammetry (CV) was performed using a three-electrode configuration consisting of either a glassy carbon macrodisk working electrode (GCE) (diameter of 3 mm; BASi, Indiana, USA) combined with a Pt wire counter electrode (99.99 %; GoodFellow, Cambridge, UK) and an Ag wire pseudoreference electrode (99.99 %;

GoodFellow, Cambridge, UK). The GCE was polished between experiments using alumina slurry (0.3 μm), rinsed in distilled water and subjected to brief sonication to remove any adhering alumina microparticles. The metal electrodes were then dried in an oven at 100 °C to remove residual traces of water, the GCE was left to air dry and residual traces of water were removed under vacuum. The Ag wire pseudoreference electrodes were calibrated to the ferrocene/ferrocenium couple in MeCN at the end of each run to allow for any drift in potential, following IUPAC recommendations. 39 All electrochemical measurements were performed at ambient temperatures under an inert Ar atmosphere in MeCN containing complex under study (0.

Inhibition of mammalian Thioredoxin Reductase

To determine the inhibition of mammalian 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 (increase in absorbance per second) thereof. 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 compound decreasing the enzymatic activity of the untreated control by 50%, and are given as the means and error of three repeated experiments.

ROS assay. 100 μL of A549 cells were seeded at a density of 1 x 10 5 cells/mL in a 96-well black plate with a transparent bottom. The cells were incubated at 37 C for 24h. The The principal crystallographic data and refinement parameters are listed in Table 4. The structures were solved by direct methods and refined by the full-matrix least-squares against F 2 in an anisotropic (for non-hydrogen atoms) approximation. All hydrogen atom positions were refined in isotropic approximation in "riding" model with the Uiso(H) parameters equal to 1.2 Ueq(Ci), for methyl groups equal to 1.5 Ueq(Cii), where U(Ci) and U(Cii) are respectively the equivalent thermal parameters of the carbon atoms to which the corresponding H atoms are bonded. All calculations were performed using the SHELXTL software. 41

  None of complexes show aurophilic metal-metal interactions. The molecules are arranged into a three-dimensional network of weak interactions, based on N1-H1A•••X (X = N, O, or F) hydrogen bonds between neighbouring molecules.

Figure 1 .

 1 Figure 1. Molecular structures of 9a (top left), 10a (top right), 10b (bottom left) and the cation in 11a (bottom right). Ellipsoids are shown at the 50% level. Except for N-H, hydrogen atoms are omitted for clarity. Selected bond distances [Å] and angles [°]: 9a: Au1-C1 1.999(3), Au1-Cl1 2.2891(8), C1-N1 1.342(4), C1-N2 1.323(4); C1-Au1-Cl1 178.68(9), N1-C1-N2 117.6(3). 10a: Au1-C1 2.006(5), Au1-Cl1 2.2977(12), C1-N1 1.325(6), C1-N2 1.342(6); C1-Au1-Cl1 176.37(15), N1-C1-N2 117.3(4). 10b: Au1-C1 2.011(2), Au1-Cl1 2.2929(5), C1-N1 1.324(2), C1-N2 1.340(2); C1-Au1-Cl1 176.78(5), N1-C1-N2 117.27(17). 11a: Au1-C1 2.065(3), Au1-P1 2.2725(7), C1-N1 1.339(3), C1-N2 1.308(4); C1-Au1-P1 178.38(8), N1-C1-N2 118.6(2).

Figure 2 .

 2 Figure 2. Molecular structures of the complex cations in 13a (left), and 14a (right). Ellipsoids are shown at the 50% level. Except for N-H, hydrogen atoms are omitted for clarity. Selected bond distances [Å] and angles [°]: 13a: Au1-C1 2.034(3), Au1-C28 1.971(3), C1-C2 1.520(3), C1-N1 1.294(3), N2-C28 1.153(4); C1-Au-C28 174.51(11), Au-C28-N2 173.5(3). 14a: Au1-C1 2.037(3), Au1-C28 2.043(3), C1-C2 1.530(4), C1-N1 1.302(4), N2-C28 1.326(4), C28-N3 1.337(4); C1-Au-C28 173.4(1), N2-C28-N3 116.4(3).

Electrochemistry.

  Cyclic voltammetry (CV) was used to analyse the redox behaviour of the CAAC copper, silver and gold complexes in MeCN and THF solutions using [ n Bu4N]PF6 or [ n Bu4N][B(C6F5)4] as the supporting electrolytes (Table

Figure 3 .

 3 Figure 3. Full range cyclic voltammogram of ( Ad CAAC)AgCl (4), recorded using a glassy carbon electrode in THF solution (0.7 mM) with [ n Bu4N][B(C6F5)4] as supporting electrolyte (0.13 M).

Figure 4 .

 4 Figure 4. Top Left: full range cyclic voltammogram of homoleptic complex [( Me2 CAAC)2Au]BF4 (8). Top right: Varied scan rate behaviour recorded using a glassy carbon electrode in MeCN solution (1.4 mM) with [ n Bu4N][PF6] as supporting electrolyte (0.13 M). Bottom left: full range cyclic voltammogram of the open-chain carbene complex 14a. Bottom right: Varied scan rate behaviour recorded using a glassy carbon electrode in MeCN solution (2.0 mM) with [ n Bu4N][PF6] as supporting electrolyte (0.13 M).

Figure 5 : 9 -

 59 Figure 5: Inhibition of A) A549 cells and B) MCF-7 cells proliferation by ACC complexes 9 -12 and 14a; data represent the average ± standard error of three experiments.

Figure 6 :

 6 Figure 6: ROS measurement in A549 cells after 24 h incubation with complexes 1 -8 and 14a. Data represent the average ± standard error of three experiments.

(CAAC)AuCl 5

 5 nor the homo-and heteroleptic bis-carbene cations [(CAAC)2Au] + 8 and [(CAAC)Au(ACC)] 14a are able to trigger an increase in intracellular ROS. These data, taken together with the relatively low inhibitory effect on TrxR, suggest that the mode of action of the CAAC-based complexes is different from that of their analogues with more labile NHC ligands. Evidently, the primary inhibitory mechanism of the new CAAC complexes remains to be established.ConclusionAcyclic carbene complexes of gold(I) are readily accessible from the reaction of the corresponding isocyanide complexes with amines, including amino acid esters. This method was explored for its potential for attaching bio-relevant substituents to non-labile carbene ligands. These ACC complexes, together with eight neutral and cationic CAAC-based complexes of Cu, Ag and Au, were screened for their antiproliferative properties on a panel of human cancer cells. Compared to the more conventional imidazolylidene-type NHC complexes, CAAC compounds offer the advantage that they are not prone to ligand exchange and retain their structural integrity in aqueous media and under physiological conditions.While none of the chloride or PTA derivatives demonstrated particularly promising cytotoxic effects, the CAAC-containing complexes showed interesting IC50 values down to submicromolar levels. In particular, the cationic [( Me2 CAAC)2M] + complexes (6 -8, M = Cu, Ag and Au) and the mixed-carbene complex [( Ad CAAC)Au(ACC)] + 14a proved to be the most potent of the series. The CAAC containing complexes appeared to only poorly inhibit TrxR, suggesting the TrxR inhibition by binding to the selenium site is unlikely to be the primary intracellular target. While Cu complexes with sterically less-hindered CAAC ligands appeared to enhance the formation ROS, none of the other CAAC-containing complexes seemed to do so up to a concentration of 100 μM. Overall, our study documents that CAACbased complexes show promise for anticancer applications. The use of mixed CAAC/ACC combinations is of particular interest since it opens the way to further functionalization of this scaffold. Studies to understand the possible mode of action of those compounds as well as their conjugation to bio-vectors are currently ongoing.

  medium was removed, and replaced with 50 μM H2DCFDA in phosphate buffer solution for 40 min. H2DCFDA was removed and replaced with fresh medium and the cells were left for recovery for 20 min at 37 C. Basal fluorescence was measured at 485/520 nm on a POLARstar Optima. Cells were incubated with 10 μM, 50μM, or 100 μM of compounds, 1% DMSO (negative control) and 100 μM of H2O2 (positive control) for 24h. Fluorescence was read at 485/520 nm. Basal fluorescence was subtracted from the fluorescence in treated cells to calculate the amount of fluorescence caused by the compounds.X-Ray Crystallography.Crystals suitable for X-ray diffraction were obtained by the slow evaporation of dichloromethane/hexane solutions for 9a, 10a, 10b and 14a, acetone/toluene solutions for 11a and 11b, or by layering a CH2Cl2 solution with hexanes for 13a. Complex 11a crystallizes as a solvate with one disordered molecule of CH2Cl2. Two positions for CH2Cl2 molecule were successfully resolved upon cooling the crystal of 11a to 140 K leading to occupancies 0.6 and 0.4. The disordered CH2Cl2 molecule was refined in the geometry of isosceles triangle and DFIX restraints on C1S-Cl1 bond length (1.72(1) Å) and Cl1•••Cl2 distance (2.90(1) Å). The atom C3 was disordered over two half-populated positions for complex 14a. Crystals were mounted in oil on glass fibres and fixed in the cold nitrogen stream on a diffractometer. Data were collected using an Oxford Diffraction Xcalibur-3/Sapphire3-CCD diffractometer, using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 140 K. Data were processed using CrystAlisPro-CCD and -RED software.40

Table 2 .

 2 Effect

	Complex	HL 60	MCF-7	A549
	1	0.14 ± 0.06	0.49 ± 0.02	17.4 ± 0.8
	2	0.12 ± 0.01	0.32 ± 0.02	0.53 ±0.06
	3	0.32 ± 0.04	0.57 ± 0.07	2.0 ± 0.5
	4	0.18 ± 0.06	0.32 ± 0.10	5.7 ± 0.7
	5	2.2 ± 0.2	4.1 ± 0.8	43.8 ± 0.4
	6	0.13 ± 0.03	1.4 ± 0.6	0.31 ± 0.08
	7	0.10 ± 0.01	1.2 ± 0.3	0.39 ± 0.06
	8	0.09 ± 0.01	1.6 ± 0.6	0.5 ± 0.1
	14a	0.10 ± 0.01	2.3 ± 0.9	6.1 ± 0.3
	Cisplatin	3.7 ± 0.3	21.2 ± 3.9	33.7 ± 3.7
	Ad LH	8.0 ± 1.3	9.03 ± 0.09	> 50
	Me LH	> 50	> 50	> 50
	Et LH	> 50	19.5 ± 4.9	> 50
	All three ligand precursors

of compounds 1-8, 14a, the pro-ligand iminium salts Ad LH, Et LH and Me LH and cisplatin on viability of cancer cell lines (IC50, μM) after 72 h incubation. R LH are non-toxic. The metal complexes, irrespective of whether they are neutral halides or of the ionic bis-carbene type, are particularly active on HL 60, and MCF-7 cell lines, with IC50 values from the low-micromolar to the ~100 nanomolar range. These complexes are substantially more active than cisplatin. The copper complexes 1 -3 show high activity against HL-60 and MCF-7, with IC50 values in the low submicromolar range. Their activity against A549, a cell line which typically shows high cisplatin resistance,

Table 3 .

 3 Effects

	Complex	Inhibition at 10 µM (% control) a	IC50 ± SD (µM) b
	1	17.6	n.d.
	2	52.3	n.d.
	3	43.0	n.d.
	4	69.0	4.6 ± 0.3
	5	91.6	2.6 ± 0.8
	6	27.1	n.d.
	7	56.8	6.8 ± 0.1

of compounds 1 -8 and 14a and of the pro-ligand salts Ad LH, Et LH and Me LH on mammalian TrxR activity. a Results of only one experiment. b Data represent the average ± standard error of three experiments.

Synthesis of [(1,3,5-triaza-7-phosphaadamantane)Au{C(NH t Bu)(pyrrolidine)}]PF6

  , 0.50 mmol) were added. The mixture was stirred overnight at room temperature, filtered and the acetone removed under vacuum. The product was taken up in dichloromethane and precipitated with hexane to give a white powder (60 mg, 0.07 mmol, 70%).1 H NMR (DMSO, 300 MHz): δ 9.41 (s, 1H, NH), 7.21 (m, 3H, Harom.), 4.45(d,

	NMR (CDCl3, 300 MHz): δ 5.95 (s, 1H, NH), 4.85 (s, 2H, NCH2), 4.22 (q, 3 JH-H = 7 Hz, 2H, (sept, J = 6.7 Hz, 2H, CH(CH3)2), 2.59 (br d, J = 10.2 Hz, 2H, CH2), 2.48 (t, J = 6.7 Hz, 2H,
	OCH2), 2.96 (s, 3H, NCH3), 1.65 (s, 9H, t Bu), 1.30 (t, 3 JH-H = 7 Hz, 3H, CH2CH3). 13 C{ 1 H} NCH2 (C1) pyrrolidine), 2.25-1.53 (m, 14H, adamantyl CH and CH2) overlapping with 2.12
	NMR (CDCl3, 75 MHz): 193.6 (s, Ccarbene), 168.9 (s, C=O), 62.7 (s, OCH2), 62.0 (s, NCH2), (s, 6H, 2CH3, CNxylyl) and 2.00 (m, J = 7.1 Hz, 2H, CH2 (C3) pyrrolidine) and 1.58 (m, 2H,
	55.1 (s, C(CH3)3), 34.6 (s, NCH3), 31.8 (s, C(CH3)3), 14.27 (s, CH2CH3). IR νmax (neat)/cm -1 : CH2 (C2) pyrrolidine), 1.32 (s, 6H, 2CH3) overlapping with 1.33 (d, J = 6.7 Hz, 6H,
	3364 (NH), 1734 (C=O), 1548 (carbene). CH(CH3)2) and 1.31 (d, J = 6.7 Hz, 6H, CH(CH3)2) ppm. 13 C{ 1 H} NMR (75 MHz, CD2Cl2):
	δ 255.6 (C carbene CAAC), 201.6 (C open chain carbene), 145.6 (o-C aniline), 137.5 (NCipso
	Synthesis of [(1,3,5-triaza-7-phosphaadamantane)Au{C(NHxylyl)(pyrrolidine)}]PF6 CNxylyl), 136.9 (o-C, CNxylyl), 136.4 (Cipso aniline), 130.3 (p-CH, aniline), 129.1 (p-CH
	(11a) overlapping with m-CH, CNxylyl), 125.7 (m-CH aniline), 79.0 (Cq), 65.5 (Cq), 53.86 (NCH2,
	ClAu{C(NHxylyl)(pyrrolidine)} (42 mg, 0.10 mmol) was dissolved in acetone and 1,3,5-C1 pyrrolidine overlaps with residual CHDCl2), 48.7 (CH2), 46.0 (NCH2 C4 pyrrolidine),
	triaza-7-phosphaadamantane (PTA, 16 mg, 0.10 mmol) and potassium hexafluorophosphate 38.8 (CH2), 37.3, 35.2, 34.3 (CH2), 29.5, 29.4, 28.0, 27.2, 26.5, 25.1 (CH2, C3 pyrrolidine),
	24.7 (CH2, C2 pyrrolidine), 23.2 (CH3), 18.7 (CH3 CNxylyl) ppm. IR νmax (neat)/cm -1 : 3331 (NH), 1546 (carbene). Biological testing Antiproliferation assay. Human HL 60 and A549 cancer cell lines (from ECACC) were cultured in RPMI 1640 medium with 10% fetal calf serum, 2mM L-glutamine, 100U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). Cells were maintained in a humidified (92 mg), 127.9 (s, CHarom.), 71.7 atmosphere at 37 °C and 5% CO2. The human MCF-7 cancer cell line (from ECACC) were
	(s, NCH2N), 54.8 (s, CH2 1/4 ), 47.0 (s, CH2 1/4 ), 51.1 (broad s, PCH2N), 30.8 (s, CH2 2/3 ), 24.6 cultured in DMEM medium with 10% fetal calf serum, 2mM L-glutamine, 100U/mL
	(s, CH2 2/3 ), 18.6 (s, CH3 xylyl). 31 P{ 1 H} NMR (DMSO, 121 MHz): -51.2 (broad s, 1P, PTA), -penicillin and 100 µg/mL streptomycin (Invitrogen). Cells were maintained in a humidified
	144.2 (h, 1 JP-F = 708 Hz, 1P, PF6). IR νmax (neat)/cm -1 : 3160 (NH), 1557 (carbene). 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
	(11b) (MTS) assay using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay
	The complex was prepared as described for 11a, from ClAuC(NH t Bu)(pyrrolidine) (27 mg, (Promega) and following the manufacturer's instructions. Briefly, cells (3 × 10 4 /100 µL for
	0.07 mmol), 1,3,5-triaza-7-phosphaadamantane (11 mg, 0.07 mmol) and potassium HL 60 and 8 × 10 3 /100 µL for both A549 and MCF-7) were seeded in 96-well plates and left
	hexafluorophosphate (64 mg, 0.35 mmol) as a white powder (35 mg, 0.05 mmol, 77%). Anal. untreated or treated with 1 µL of DMSO (vehicle control) or 1 µL of complexes diluted in
	Calcd for C15H30N5PAuPF6 (671.9): C, 28.60; H, 4.88; N, 10.42. Found: C, 28.62; H, 5.21; DMSO at different concentrations in triplicate for 72 h at 37 °C with 5% CO2. Following this,
	N, 10.20. 1 H NMR (DMSO, 300 MHz): δ 7.19 (s, 1H, NH), 4.57 (d, 3 JH-H = 12 Hz, 3H, MTS assay reagent was added for 4 h and absorbance measured at 490 nm using a Polarstar 14 mM) and supporting electrolyte [ n Bu4N][PF6] (0.13 mM). Data NCH2N), 4.36 (d + s, 3 JH-H = 12 Hz, 3H + 6H, NCH2N + PCH2N), 3.82 (broad s, 2H, CH2 1/4 ), Optima microplate reader (BMG Labtech). IC50 values were calculated using GraphPad
	were recorded with Autolab NOVA software (v. 1.11). Elemental analyses were performed 3.28 (broad s, 2H, CH2 1/4 ), 1.93 (broad s, 2H, CH2 2/3 ), 1.81 (broad s, 2H, CH2 2/3 ), 1.49 (s, 9H, Prism Version 5.0 software.
	by London Metropolitan University. Compound 9b has been synthesized according to t Bu). 13 C{ 1 H} NMR (DMSO, 75 MHz): 202.3 (s, Ccarbene), 71.8 (d, 3 JP-C = 8 Hz, NCH2N), reported procedure. 28 55.9 (s, CH2 1/4 ), 53.5 (s, C(CH3)3), 50.9 (broad s, PCH2N), 46.1 (s, CH2 1/4 ), 31.8 (s, C(CH3)3),
	24.4 (s, CH2 2/3 ), 24.3 (s, CH2 2/3 ). 31 P{ 1 H} NMR (DMSO, 121 MHz): -52.0 (broad s, 1P,
	Synthesis of ClAu{C(NHxylyl)(pyrrolidine)} (9a) PTA), -144.21 (h, 1 JP-F = 708 Hz, 1P, PF6). IR νmax (neat)/cm -1 : 3360 (NH), 1545 (carbene).

3

JH-H =

15

Hz, 3H, NCH2N), 4.28

(d, 3

JH-H = 15 Hz, 3H, NCH2N), 4.02 (s, 6H, PCH2N), 3.78 (t, 3 JH-H = 6 Hz, 2H, H 1 or H 4 ), 3.40 (t, 3 JH-H = 6 Hz, 2H, H 1 or H 4 ), 2.19 (s, 6H, CH3 xylyl), 2.05 (m, 2H, H 2 or H 3 ), 1.89 (m, 2H, H 2 or H 3 ). 13 C{ 1 H} NMR (DMSO, 75 MHz): (Ccarbene not visible), 138.2 (s, Carom. ipso), 136.2 (s, Carom. ortho), 128.2 (s, CHarom.

  commercially available rat liver TrxR (Sigma Aldrich) was used and diluted with distilled water to achieve a concentration of 3.58 U/mL. The compounds were 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 compounds in graded concentrations or
	buffer (25 µL) without the compounds, 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 incubated with moderate shaking for 75 min at 37°C in a
	96-well plate. A portion (225 µL) of 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
	ethanolic DTNB solution (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

TrxR, an established microplate-reader-based assay was performed. (Ref: Chem. Eur. J. 2017, 23, 1869 -1880) For this purpose,

Table 4 .

 4 Summary of crystallographic data and structure refinement = 2 (Fo 2 ) + (aP) 2 + bP, where P = 1/3(Fo 2 + 2Fc 2 )

	9a	10a	10b	11a	13a	14a

Xylylisocyanide gold chloride (50 mg, 0.18 mmol, 1 eq.) was dissolved in dichloromethane.

Pyrrolidine (49 mg, 0.90 mmol, 5 eq.) was added to this solution. The mixture was left overnight at room temperature in the dark, filtered through celite and then washed with saturated aqueous ammonium chloride solution. The clear solution was dried with sodium sulphate for 1h, filtered and the volatiles were removed under vacuum. The product was precipitated with a dichloromethane/hexane mixture to form a white powder which was dried under vacuum (47 mg, 0.14 mmol, 79%

Synthesis of ClAuC(NHxylyl)(sarcosine) (10a)

Sarcosine hydrochloride (210 mg, 1.50 mmol) was dissolved in acetonitrile and stirred with potassium carbonate (207 mg, 1.5 mmol) for 1h. The resulting mixture was paper-filtered and (xylylNC)AuCl (110 mg, 0.30 mmol) was added and left to mix for an hour. The solution was purified by filtration through Celite, washing with ammonium chloride and drying over anhydrous Na2SO4 for 1h. This mixture was then paper-filtered and the volatiles were removed under vacuum. The product was precipitated with hexane and diethyl ether to give an orange powder (95 mg, 0.09 mmol, 66% 128.9 (s, CHarom.), 128.8 (s, CHarom.), 62.1 (s, OCH2), 61.2 (s, NCH2), 35.9 (s, NCH3), 18.9 (s, CH3 xylyl), 14.3 (s, CH2CH3). IR νmax (neat)/cm -1 : 3219 (NH), 1745 (C=O), 1547 (carbene).

Synthesis of ClAuC(NH t Bu)(Sarcosine) (10b)

The compound was prepared as described for 10a, from sarcosine hydrochloride (44 mg, 0.32 mmol) and t-butyl isocyanide gold chloride (50 mg, 16 mmol)

Synthesis of [(1,3,5-triaza-7-phosphaadamantane)Au{C(NHXylyl)(sarcosine)}]PF6 (12a)

The complex was prepared as described for 11a, from ClAu{C(NHxylyl)(sarcosine)} (50 mg, 0.104 mmol), 1,3,5-triaza-7-phosphaadamantane (16 mg, 0.104 mmol) and potassium hexafluorophosphate (96 mg, 0.52 mmol). The product was isolated as a pink powder as a mixture of two stereoisomers in a ratio 3/1 (66 mg, 0.09 mmol, 85% 3 JH-H = 13 Hz, 3H, NCH2N), 4.34 (d, 3 JH-H = 13 Hz, 3H, NCH2N), 4.26 (s, 0.5H, NCH2 isomer 2), 4.23 (q, 3 JH-H = 7 Hz, 2H, OCH2), 3.96 (s, 6H, PCH2N), 3.45 (s, 0.7H, NCH3 isomer 2), 3.13 (s, 2.3H, NCH3 isomer 1), 2.24 (s, 6H, CH3 xylyl), 1.27 (q, 3 JH-H = 7 Hz, 3H, CH2CH3). 13 The complex was prepared as described for 11a, from ClAu{C(NH t Bu)(sarcosine)} (50 mg, 0.12 mmol), 1,3,5-triaza-7-phosphaadamantane (19 mg, 0.12 mmol) and potassium hexafluorophosphate (110 mg, 0.60 mmol) as a red powder which was a mixture of four stereoisomers in a ratio 46/24/21/9. ( (d, 3 JH-H = 13 Hz, 3H, NCH2N), 4.45 (s, 0.5H, NCH2 isomer 2), 3.45 (m, 9.4H, NCH2N + PCH2N + NCH2 isomer 3 and 4), 4.17 (q, 3 JH-H = 7 Hz, 2H, OCH2), 3.45 (s, 0.6H, NCH3 isomer 3 and 4), 3.04 (s, 0.7H, NCH3 isomer 2), 2.97 (s, 1.5H, NCH3 isomer 1), 1.52 (s, 4.3H, t Bu isomer 1), 1.49 (s, 2.4H, t Bu isomer 2), 1.32 (s, 2.9H, t Bu isomer 3 and 4), 1.24 (t, 3 JH-H = 7 Hz, 3H, CH2CH3). 13