domain: chim sdv

text:
Morwen Williams

Adam I Green

Julio Fernandez-Cestau

David L Hughes

Maria A O'connell

Mark Searcey

Benoît Bertrand
email: b.bertrand@uea.ac.uk

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

(C^N pz ^C)Au III complexes of acyclic carbene ligands: synthesis and anticancer properties †

A series of cyclometallated gold(III) complexes supported by pyrazine-based (C^N pz ^C)-type pincer ligands were synthesized via two different pathways. Nucleophilic attack on the isocyanide complex [(C^N pz ^C)Au(C≡NC6H3Me2-2,6)]SbF6 (2) gave [(C^N pz ^C)Au(ACC)]SbF6 complexes with aniline (4•SbF6), adamantylamine (5), glycine ethyl ester (6), alanine methyl ester (7), valine methyl ester (8), phenylglycine methyl ester (9) and methionine methyl ester (10) substituents (ACC = acyclic carbene). The pathway via isocyanide insertion into gold-amide bonds was also investigated; e.g. the reaction of xylyl isocyanide with (C^N pz ^C)AuNHPh followed by protonation with HBF4•OEt2 gave the acyclic carbene complex 4•BF4. To the best of our knowledge compounds 6 -10 represent the first examples of gold(III) acyclic carbene complexes bearing amino acid functions. The compounds provide a versatile platform for the study of anti-proliferative properties of gold(III) complexes. Tests against human adenoma-type lung cancer cells identified 5, 6, 7 and 10 as particularly promising and demonstrate the synthetic flexibility of acyclic carbene complexes and the potential of that class of compounds for anticancer applications.

Compared to cisplatin, amino ester-containing ACC complexes showed improved selectivity for MCF-7 breast cancer cells over healthy fibroblasts.

Introduction

Since the late 1970s cis-diamminedichloroplatinum(II) (cisplatin) 1 has become one of the most widely used compounds in the clinic for the treatment of various type of cancer including ovarian, testicular, bladder cancer, melanoma, non-small cell lung cancer, small cell lung cancer, lymphomas and myelomas. 2 Since the introduction of cisplatin only two other platinum-based compounds (carboplatin and oxaliplatin) have received worldwide approval as anticancer drugs. 3 However, despite their clinical success, platinum-based metallodrugs present major drawbacks such as a limited spectrum of action, acquired resistance to treatment, as well as severe side effects limiting administrable doses to patients. 4 For these reasons, alternatives are being sought, and organometallic gold(I/III) complexes have demonstrated promising anticancer properties. 5 Indeed, complexes presenting Nheterocyclic carbene (NHC) ligands, alkynyl ligands and cyclometallated arylpyridine ligands give rise to Au(I/III) complexes which are very stable in physiological environments. Special interest has focused on cyclometallated complexes in which the Au(III) centre is stabilized by (C^N), (C^N^N) or (C^N^C) pincer ligands. 5b,6 These complexes tolerate a broad range of ancillary ligands such as phosphines, 7,8 NHCs, 9 or N-donor ligands, 10,11 which enables optimization of the biological properties of these complexes. The cytotoxicity of some of these gold complexes has been demonstrated to be due to the inhibition of various enzymes such as thioredoxin reductase (TrxR), poly-(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP-1) and aquaporins. 12 Gold(I/III) complexes have also been shown to stabilize the G-quadruplex structure of DNA. 13 Post-metallation modifications have appeared as a very efficient and high-yielding method of introducing elaborated scaffolds into metal complexes, for example for derivatising metal gold complexes with peptides, 14 aptamers 15 or other metal complexes. 16 The first gold(III) acyclic carbenes were reported in the 1970s by Balch et al. and Minghetti and co-workers. 17,18 The compounds were described to behave as oxidising agents.

Further early work on gold(III) open-carbene systems by Usón and Villacampa et al.

describes the synthesis of mononuclear, mono-carbene gold(III) complexes. 19 The authors utilised existing synthetic methodology, reacting primary amines or diamines with gold(III) isocyanide complexes to produce acyclic carbene complexes. Since then few investigations on these systems have been reported, except for a small number of papers describing cyclometallated gold(III) carbenes produced either by insertion of an isocyanide into an Au-N bond 20,21 or by nucleophilic attack of a gold-CNR complex by an amine. 22 We became interested in acyclic carbene complexes since their formation promised to present a simple way of functionalizing gold complexes with bio-inspired moieties bearing a amine functions such as amino acid derivatives.

Fig. 1 Structures of previously reported Au(III) conjugates with amino acid derivatives and of the benzimidazolylidene complex AuNHC.

As we have recently reported, pyrazine-based pincer complexes 23 with benzimidazolylidene ligands [(C^N pz ^C)Au(NHC)] + (AuNHC, Fig. 1) possess promising anticancer properties against human leukemia, breast cancer and lung cancer cells. 24 However, although the compounds showed lower toxicity against healthy lung fibroblasts, the selectivity of the compound for cancer cells needs to be improved. Considering the high stability of the [(C^N pz ^C)Au(carbene)] + scaffold under physiological conditions, including their tolerance to increased concentrations of glutathione (GSH), the attachment of a potential vector via the carbene ligand seemed attractive in the search for improving target selectivity.

In recent years, coupling of organometallics to peptides has appeared as a promising way to increase their selectivity to cancer cells. 25 This concept has been applied predominantly to gold(I) complexes using mono-or polypeptide vectors. 26 Although some examples of Au(III) complexes conjugated to amino acid derivatives have been reported (Fig. 1), 26a,c,f,27 to the best of our knowledge no examples of cyclometallated Au(III) complexes bearing amino acid moieties have been synthesized so far. Within this context, we report here the synthesis and characterization of the first cyclometallated Au(III) complexes decorated with amino ester-based acyclic diamino carbene (ACC) ligands. The compounds were screened for their antiproliferative properties against a human lung cancer cell line (A549).

The most promising ones were then tested against a broader panel of human cancer cells, including lung adenocarcinoma (A549), leukemia (HL60), colon cancer cells (HCT116), breast cancer cells (MCF-7 and MDA-MB-231), as well as healthy fibroblasts (MRC5) for comparison.

Results and Discussion

Synthesis and characterization. Following reported procedures, [17][18][19] we started by exploring the amine attack on cationic gold(III) isocyanide complexes, which seemed the most promising route to the expected amino-acid derivatised acyclic carbene complexes.

Treatment of (C^N pz ^C)AuCl ( 1) with 2,6-Me2C6H3N≡C and AgSbF6 under strictly anhydrous conditions gave the isocyanide complex 2 in good yield (Scheme 1). The synthesis of 2 was confirmed by NMR and IR spectroscopy and elemental analysis. The 1 H and 13 C NMR signals were assigned and confirmed with HMQC experiments. The IR spectrum showed a weak ν(C≡N) signal at 2267 cm -1 . This value is in good agreement with previously reported [(C^N^C)AuCNR] + complexes. 28 Complex 2 appeared to be very sensitive to hydrolysis and in the presence of moisture readily formed the formamide complex 3, with characteristic IR bands of ν(NH) = 3296 cm -1 and ν(C=O) = 1643 cm -1 .

Complex 2 was reacted with dry, distilled aniline at room temperature to give the corresponding acyclic diamino carbene complex 4•SbF6 in good yield (Scheme 1). The complex displays two NH peaks in the 1 H NMR spectrum, at δ 9.7 and 9.4 ppm. The ν(C=N) stretching frequency of 4•SbF6 is found at 1586 cm -1 . The reaction of 2 with adamantylamine gave compound 5, which shows an up-field shift of the characteristic H 2 peak to 8.9 ppm and the appearance of broad 1 H NMR singlets for the NH moieties at 8.3 and 6.6 ppm. The chemical shift difference is much larger than in 4•SbF6, illustrating the difference between the aryl and alkyl amine substituents of the carbene. The IR spectrum of 5 shows the characteristic νC=N(carbene) band at 1585 cm -1 , together with two distinct ν(NH) bands of the carbene at 3312 cm -1 and 3210 cm -1 . The [(C^N pz ^C)Au(ACC)] + complexes are very stable even in solution, which contrasts with recent reports on (ACC)AuCl3 complexes. 29 The reaction of the amino esters Gly-OEt, Ala-OMe, Val-OMe, PhGly-OMe and Met-OMe with 2 afforded the corresponding amino ester complexes 6-10 in good yields. All complexes feature the ν(carbene) band at 1585 cm -1 in their IR spectra, along with a ν(C=O) band at ~1740 cm -1 . While the 1 H and 13 C{ 1 H} NMR spectra of 6, which contains a nonchiral glycine moiety, reflected the mirror symmetry of the (C^N pz ^C) pincer ligand, the 1 H and 13 C{ 1 H} NMR spectra of 7-10 showed the loss of lateral symmetry of the (C^N pz ^C) framework, indicated by a doubling of the tert-butyl, H 5 , H 8 and CH3(xylyl) signals. The effect is most prominent in the spectra of 8 and 9, which contain the sterically most demanding side chains. The NMR spectra were fully assigned using HMQC and HMBC NMR experiments. Scheme 1 Synthesis of (C^N pz ^C)Au complexes 2-10.

The alternative pathway, the insertion of isocyanides into the gold-amide bond, was also briefly explored. The reaction of 1 with anilines in the presence of base afforded 11a, b in high yields (Scheme 2). Both complexes 11a and 11b were reacted with 2,6dimethylphenylisocyanide at room temperature overnight. 11a gave the corresponding insertion product 12 in good yield, whereas 11b, with the more bulky 2,6-diisopropylaniline ligand, did not react and returned the unreacted starting material. NMR analysis of compound 12 revealed the presence of a NH proton at 6.71 ppm in the 1 H NMR spectrum and a signal at 124.7 ppm in the { 1 H} 13 C corresponding to the inserted carbon. The formation of 12 was confirmed by the IR spectrum which showed the NH vibration at 3340 cm -1 and the C=N stretch at 1607 cm -1 . Insertion of tert-butyl isocyanide and cyclohexyl isocyanide into 11a was attempted in a variety of reaction conditions. Tert-butyl isocyanide inserted as expected but promptly rearranged to produce the cyanide complex, as was previously noticed for pyridine-based complexes. 27 Cyclohexyl isocyanide generated a mixture of products which proved impossible to separate and purify. Protonation of 12 using HBF4OEt2 produced the air-stable carbene complex 4•BF4, with the same spectroscopic characteristics as its SbF6 The distances and angles in the C^N pz ^CAu moiety are comparable to those described previously. 23,29,[START_REF] Fernandez-Cestau | Organimetallics[END_REF] In the neutral amidate complexes 3 and 12 the Au-C7/C25 distances of 2.0251(18) and 2.031(4) Å, respectively, are comparable to the Au-C σ-bond distances in gold alkyls, typically in the range 2.00-2.15 Å. 23,[START_REF] Fernandez-Cestau | Organimetallics[END_REF][START_REF] Roşca | [END_REF] In the cationic Au III -carbene complexes the Au-C7/C25 distances (2.037(11) Å 5, 2.010(3) Å 6) are in the upper range of distances observed for complexes of cyclic carbenes [(C^N^C)Au(NHC)] + (1.967 to 2.017 Å), 23,32 possibly due to a combination of steric hindrance and lower donor strength of the acyclic carbenes. The carbene adopts an almost perfectly perpendicular orientation relative to the (C^N pz ^C)Au plane [torsion angles C22-Au-C7-N8 90.7 (8) in 5, and C6-Au-C25-N3 90.9(3) in 6], in contrast with the value of 115-120º found in [(C^N pz ^N)Au(1,3-dimethylbenzimidazol-2-ylidene)]PF6. 24 This conformation of the carbene allows the formation of supramolecular interactions.

As we showed recently, pyrazine-based pincer complexes have a tendency to aggregate through the formation of π•••π interactions that involve the pyrazine rings. 33 In the present complexes, however, the presence of N-H protons in the ancillary ligand and the formation of intermolecular H•••N pz interactions are the main driving force for aggregation. Fig. 3 shows illustrative examples (see ESI for further details of intermolecular interactions of 2, 3, 5, 11a and 12 in the solid state).

As can be seen in Figs. 3a and3b for complex 6 and Fig. S3 for Rather than forming a π-stack of the arene rings, the two planar neighbouring (C^N^C)Au moieties show a head-to-head alignment of the pyrazine ring of one molecule with the Au-N bond of another, with an offset of about 1.15 Å. The formimidate complex 12•C7H8 also forms dimers through xylylNH•••N pz interaction (2.407 Å). Here, too, the pyrazine ring of one molecule is aligned with the Au-N bond of the second, although in this case the off-set is minimal (Fig. 3c).

The neutral complexes 3 and 11a form polymeric zig-zag chains. In the case of complex 3 (Fig. 3d) the intermolecular interactions involve again NH•••N pz contacts (2.280 Å), as well as additional O•••H-C interactions between the oxygen atoms of the formamido ligand and protons of the pyrazine ring of a neighbouring molecule. In the case of 11a, additional interactions contributing to chain formation involve the ortho protons of the phenyl ring of the aniline (see ESI, Fig. S4b).

In the absence of NH functional groups leading to NH•••N(pz) interactions, π-stacking of the pyrazine rings seems to be preferred, as illustrated by the structure of the isocyanide complex 2 (see ESI. Fig. S1b). The presence or absence of such hydrogen bonding patterns is likely to be of importance not just for crystal packing arrangements but also for the interactions of pyrazine-based functionalised complexes with biomolecules and cells. In vitro antiproliferative activity. Although the complexes proved to be only poorly soluble in purely aqueous media, all compounds are soluble enough in DMSO not to precipitate when diluted with aqueous buffer solution up to 100 µM with 1% DMSO. An initial screening of compounds 1, 3 -10 and 12 was carried out on human A549 lung adenocarcinoma cells. This cell line was chosen for its ability to discriminate efficiently between structurally very similar (C^N pz ^C)Au III carbene complexes. 24 The inhibition of the proliferation of A549 cells was determined using the established MTS assay (see ESI) after 72 h of incubation with the starting chloride complex 1 and compounds 3 -10 and 12 at concentrations of 100 µM and 10 µM (Fig. 4). Both neutral complexes 3 and 12 did not show any antiproliferative effects against A549 cells at both concentrations. This is consistent with our previous results using stable neutral (C^N^C)Au complexes bearing phenylacetylide and thiophenolate ligands. 24 Complexes 5 -10 all inhibited A549 cell proliferation at a concentration of 100 µM. However, of those only 5 -7 and 10 showed a cell viability of A549 cells below 50 % at 10 µM (cell viability of 9.6 %, 38.4 %, 14.9 % and 23.5 % for 5, 6, 7 and 10, respectively). The two complexes with the most lipophilic side-chains (isopropyl for 8, and phenyl for 9) turned out to have a reduced activity compared to the other amino ester decorated

[(C^N pz ^C)Au(ACC)]X complexes.
The four complexes which inhibited cell proliferation by more than 50 % at 10 μM concentration were selected for the determination of the IC50 values (i.e. the concentration required to inhibit 50 % cell growth) on a panel of human cell lines, including (i) solid tumour cells, that is human lung (A549), breast (MCF-7) and colon (HCT-116) adenocarcinomas; (ii) breast adenocarcinoma (MDA-MB-231) as an example of highly metastatic cancer cells; (iii) leukemia (HL60) as an example of suspension cells; and (iv) healthy fibroblasts (MRC-5). The results are reported in Table 1.

Complexes 5 -7 and 10 all showed significantly higher cytotoxicity than cisplatin against A549, MCF-7 and MDA-MB-231 cells, while only the adamantyl derivative 5 was more toxic than cisplatin against HL60 and HCT-116 cells. In particular, the new compounds show promising antiproliferative effects against A549, MCF-7 and MDA-MB-231 cells, which are cell lines with the least sensitivity towards cisplatin, and which are the most difficult to treat. Overall the activity is similar to that of our benzimidazole-based compound AuNHC. 24 The adamantyl compound 5 was over ten times more potent than AuNHC against HCT-116 and MBA-MB-231 cells. Among the series of amino ester derivatives, the glycinebased complex 6 appeared as the least toxic in the series. Overall, the low micromolar activities of the [(C^N pz ^C)Au(ACC)]X complexes, including against A549, MCF-7 and MDA-MB-231 cell lines which show only poor sensitivity towards cisplatin, render them promising for the development of anticancer drugs. Complexes 6, 7 and 10 appeared to have a reduced activity against the healthy fibroblasts compared to the heterocyclic complex AuNHC. Indeed, all amino ester-containing complexes compounds 6, 7, 10 and AuNHC show improved selectivity for MCF-7 cells versus MRC-5 fibroblasts (selectivity factor SMCF7/MRC5 >1) and proved significantly more selective than cisplatin (SMCF7/MRC5 = 0.5). Only the adamantyl compound 5 is less selective).

Reactive oxygen species quantification. Inducing the formation of reactive oxygen species (ROS) is a well-known mechanism of action of metal-based drugs and especially of Au(I)-NHC complexes. 34 Our panel of acyclic carbene complexes was therefore tested in this respect. We measured the amount of intracellular ROS after treatment of A549 cells with 100 µM, 50 µM and 10 µM concentrations of the four toxic complexes 5 -7 and 10 and the nontoxic complex 4•SbF6 as negative control (see Experimental for details). The results are summarized in Fig. 5. We observed no particular increase in ROS formation for any of the tested complexes, including the cytotoxic ones. These data suggest therefore that induction of ROS is unlikely to be the cause of cytotoxicity, and this mode of action can therefore be ruled out for this class of compounds. Reaction with glutathione. Glutathione (GSH) is a tripeptide present at millimolar levels inside cells and is found overexpressed in most cancer cells. GSH is involved in many different cellular functions such as xenobiotic detoxification and reactive oxygen species (ROS) scavenging. 35 Moreover, GSH has been related to resistance mechanism to cisplatin. 36 Although GSH is known to reduce Au(III) complexes bearing (N^N) or (N^N^N) chelating ligands, 37 we demonstrated the stability of bis-cyclometallated Au(III)-NHC complex toward reduction and substitution with GSH. 24 We have therefore explored the reactivity of GSH

Experimental Section

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 [C^N pz ^C]Au(2,6-dimethylphenyl formamide) (3)

A mixture of 2 (0.055 g, 0.061 mmol) and K2CO3 (0.025 g, 0.182 mmol) dissolved in a mixture of THF (5 mL) and water (2 mL) was stirred at room temperature for 3 h. The THF was removed under vacuum and water (10 mL) was added. The product was extracted using dichloromethane (3 × 10 mL) and the organic layer washed with water (3 × 10 mL) and dried with anhydrous Na2SO4. The solution was evaporated and the solid residue washed twice with light petroleum (bp. 40-60 °C) and dried, to yield a bright yellow solid (0.038 g, 68 %).

Anal IR: νmax (neat)/cm -1 : 3300 (NH), 3189 (NH), 2965 (CH), 1742 (C=O), 1582 (carbene).

Synthesis of [(C^N pz ^C)Au{C(NHCH(Ph)CO2Me)(NHC6H3Me2-2,6)}]SbF6 (9)

D-phenylglycine methyl ester HCl (0.030 g, 0.146 mmol) was added to a flame-dried Schlenk flask charged with K2CO3 (0.012 g, 0.087 mmol), molecular sieves and acetonitrile (5 mL).

The solution was sonicated for 30 min and transferred to a separate flame-dried Schlenk flask containing 2 (0.066 g, 0.073 mmol). The reaction was stirred for 16 h at room temperature and the precipitate removed from solution via filtration through a celite plug. The solvent was removed under vacuum and the solid residue re-dissolved in dichloromethane (2 mL). The product was crashed out of solution using a 2:

1
L-methionine methyl ester HCl (0.035 g, 0.175 mmol) was added to a flame-dried Schlenk flask charged with K2CO3 (0.012 g, 0.087 mmol), molecular sieves and acetonitrile (5 mL).

The solution was sonicated for 30 min and transferred to a separate flame-dried Schlenk flask containing 2 (0.079 g, 0.087 mmol). 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 re-dissolved in dichloromethane (2 mL). The product was crashed out of solution 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 an orange solid (0.069 g, 74%

X-ray crystallography

Pale-yellow square plates of 2 were grown by slow evaporation of a saturated solution of the crude product in CH2Cl2. 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. 42

Antiproliferation assay

The human A549 and HL60 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 atmosphere at 37°C and 5%

CO2. The human MCF-7, HCT116 and MDA-MB-231 cancer cell lines (from ECACC) and the healthy fibroblasts MRC-5 cells were cultured in DMEM medium with 10% fetal calf serum, 2mM L-glutamine, 100U/mL penicillin and 100 µg/mL streptomycin (Invitrogen).

Cells were maintained in a humidified atmosphere at 37 °C and 5% CO2. Inhibition of cancer

analogue 4 Scheme 2

 42 Scheme 2 Synthesis of acyclic carbene 4•BF4 from gold(III) anilide.

Fig. 2 6 .

 26 Fig. 2 (a) Crystal structure of complex 5•1.5 C7H8. Ellipsoids set at 50% probability. Toluene molecules are omitted for clarity. Selected bond distances [Å] and angles [°]: Au-N1 2.01(1), Au-C7 2.03(1), Au-C22 2.10(1), Au-C62 2.09(1), N1-Au-C7 175.8(4), N1-Au-C22 80.4(4), N1-Au-C62 80.5(4), C22-Au-C7 101.6(5), C62-Au-C7 97.4(4), C22-Au-C62 160.9(4).(b)

  5•1.5 C7H8, the carbene complexes crystallize as dimers held together by xylylNH•••N pz interactions (2.187 Å in 5•1.5 C7H8; 2.443 Å in 6). There are also C(aryl)H•••F interactions with the SbF6 anions.

Fig. 3

 3 Fig. 3 (a) Side view of the crystal packing of complex 6, showing the carbene NH•••N(pz) and CH•••F interactions. (b) Top view of the dimer found in the crystal packing of complex 6. (c) Top view of the aggregation in Complex 12•C7H8. (d) Side view of the aggregation in complex 3.

  The acyclic diamino carbene complexes 4•SbF6 and 4•BF4 (featuring an aniline substituent) demonstrated only poor antiproliferative effects on the tested cell line. Although the cell viability when treated with 4•SbF6 was noticeably lower than when treated with 4•BF4 at 100 µM concentration (cell viability of 38 % and 65 % for 4•SbF6 and 4•BF4, respectively), at a concentration of 10 µM no difference was observed, indicating that the SbF6 anion has only a marginal effect on the antiproliferative properties of the compounds at low concentrations.

Fig. 4

 4 Fig. 4 Inhibition of A549 cells growth by complexes 1, 3 -10 and 12 in DMSO; data represent the average ± standard error of three experiments.

Fig. 5

 5 Fig. 5 ROS measurement in A549 cells after 24 h incubation with complexes 4•SbF6, 5 -7 and 10.

towards two representative complexes, 6 and 7 ,

 7 by 1 H NMR spectroscopy. Both 6 and 7 were mixed at room temperature with GSH in DMSO-d6/D2O 1/1 over a period of time of 24 h (Fig.6 and S6). Although neither 6 nor 7 showed any reaction with GSH over the first hour, over 24 h we observed the formation of oxidized glutathione (GSSG), the disappearance of the signals of the [(C^N^C)Au (III) (ACC)] + cations and the formation of a pale yellow product, which we tentatively attribute to the reduction product (C^N^CH)Au (I) (ACC). This leads to the conclusion that acyclic carbene complexes are more sensitive to GSH than benzimidazole-type Au(III) carbene adducts.24

Figure 6 : 1 H

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

  Complex 3 was crystallized by slow diffusion of light petroleum in a saturated solution of the complex in CH2Cl2. Yellow square plates with stoichiometry 5•1.5 toluene were grown by diffusion of petroleum into a saturated solution of the complex in toluene. Crystals of 6 and 11a were grown by the slow evaporation of saturated dichloromethane solutions. Slow diffusion of light petroleum into a saturated solution of 12 produced yellow needles with stoichiometry 12•toluene. 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

  4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-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, cells (3 × 10 4 /100 µL for HL-60, 8 × 10 3 /100 µL for A549, MCF-7, HCT116, MDA-MB-231 and 2 × 10 3 /100 µL for MRC-5) were seeded in 96well 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 490nm using a Polarstar Optima microplate reader (BMG Labtech). IC50 values were calculated using GraphPad Prism Version 5.0 software.ROS assay100 μL of A549 cells were seeded at a density of 1 × 10 5 cells/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 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. Cells were incubated with 10 μM, 50μM, 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 treated cells to calculate the amount of fluorescence caused by the compounds.

Table 1 :

 1 Effects of compounds 5-7, 10, AuNHC and cisplatin on cell viability of a panel of human cancer cell lines and of healthy fibroblast MRC-5 cells after 72 h incubation.

				IC50 ± SD (µM) a
	Complex	A549	MCF-7	HL60	HCT-116	MDA-MB-231	MRC-5	SMCF7/MRC5
	5	6.1 ± 1.1 5.2 ± 0.2	0.8 ± 0.1	0.6 ± 0.1	0.3 ± 0.1	0.37 ± 0.03	0.07
	6	13.0 ± 3.6 6.4 ± 1.6	16.7 ± 2.5	8.4 ± 0.5	5.1 ± 2.0 8.8 ± 1.0	1.4
	7	7.9 ± 1.5 3.9 ± 0.6	8.1 ± 0.9	8.1 ± 0.4	4.7 ± 0.7 6.9 ± 1.1	1.8
	10	7.7 ± 1.6 3.4 ± 0.2	6.9 ± 0.6	7.3 ± 0.4	11.1 ± 2.8	6.4 ± 0.7	1.9
	AuNHC	7.8 ± 1.3 b	0.56 ± 0.02 b	0.3 ± 0.1 b	11.2 ± 1.5	5.7 ± 0.4	1.4 ± 0.4 b	2.5
	Cisplatin	33.7 ± 3.7 b	21.2 ± 3.9 b	3.7 ± 0.3 b	5.3 ± 0.2	28.4 ± 0.1	10.7 ± 3.0 b	0.5

a Mean ± standard error of at least three independent experiments.

b

Values from ref.

24.

  2.58 (s, 3H, CH3 xylyl ), 2.44, (s, 3H, CH3 xylyl ), 1.39(d, 3 JH-H = 7.2 Hz, 3H, CH3 ala. ), 1.32 (s, 9H, t Bu), 1.32 (s, 9H, t Bu).13 C{H} NMR (CD3CN, 75 MHz): δ 176.2 (s, C 10 ), 170.9 (s, C=O), 165.2 (s, C 9 ), 164.9 (s, C 9' ), 157.0 (s, C 7 ), 156.8 (s, C 7' ), 156.4 (s, C 4 ), 156.3 (s, C 4' ), 145.0 (s, C 3 ), 144.9 (s, C 3' ), 139.8 (s, C 2 ), 139.7 (s, C 2' ), 135.9 (s, C ortho ), 135.7 (s, C ortho' ), 133.9 (s,

	Synthesis of [(C^N pz ^C)Au{C(NHPh)(NHC6H3Me2-2,6)}]SbF6 (4•SbF6)
	Complex 2 (0.066 g, 0.073 mmol) and aniline (0.014 g, 0.146 mmol) were combined in a
	flame-dried Schlenk flask, with molecular sieves, under N2 atmosphere, dissolved in
	dichloromethane and filtered through a celite plug. The product was precipitated by addition
	C 8 ), 132.9 (s, C 8' ), 132.4 (s, C ipso ), 129.9 (s, CH xylyl ), 129.5 (s, CH xylyl ), 129.4 (s, CH xylyl ), of a 2/1 mixture of light petroleum (bp. 40-60 °C) and diethyl ether. The product was finally dried under vacuum to yield a bright yellow solid (0.060 g, 0.050 mmol, 58%). Calcd. for 127.1 (s, C 5 ), 126.8 (s, C 5' ), 125.3 (s, C 6 ), 125.1 (s, C 6' ), 58.6 (s, NCH), 52.6 (s, OCH3), 35.3
	(s, C(CH3)3), 30.4 (s, C(CH3)3), 30.4 (s, C(CH3)3), 18.6 (s, CH3 xylyl ), 18.5 (s, CH3 xylyl ), 17.4 (s, C39H42AuF6N4Sb•3H2O (1053.6): C, 44.46; H, 4.59; N, 5.32. Found: C, 44.02; H 4.94; N 5.25. 1 H NMR (CD3CN, 300 MHz, 298 K): δ 9.68 (s, 1H, NH), 9.40 (s, 1H, NH), 8.96 (s, 2H, CH3 ala. ). IR: νmax (neat)/cm -1 : 3319 (NH), 3062 (NH), 2961 (CH), 1750 (C=O), 1582
	H 2 ), 7.80-7.71 (m, 4H, CH Ar ), 7.46-7.32 (m, 7H, CH Ar ), 7.24-7.14 (m, 3H, CH Ar ), 2.53 (s, 6H, (carbene).
	CH xylyl ), 1.34 (s, 18H, t Bu). IR: νmax (neat)/cm -1 : 3311 (NH) , 2958 (CH), 1586 (carbene).
	The product was
	C43H52AuN4SbF62H2O (1093.66): C, 47.22; H, 5.16; N, 5.12. Found: C, 47.16; H, 4.71; N, crashed out of solution using a 2:1 mixture of light petroleum (bp. 40-60 °C) / diethyl ether (5
	5.30. 1 H NMR (CD2Cl2, 300 MHz, 298 K): δ 8.89 (s, 2H, H 2 ), 8.31 (s, 1H, NH), 7.73 -7.70 mL), and after removing the solvent, dried under vacuum to yield a bright yellow solid (0.068
	(m, 4H, H 5/8 ), 7.46 (dd, 3 J = 1.8 Hz, 4 J = 8.30 Hz, 2H, H 6 ), 7.40 -7.34 (m, 3H, CH xylyl ), 6.61 g, 75%). Anal. Calcd. for C39H48AuN4O2SbF6 (1037.56): C, 45.15; H, 4.66; N, 5.40. Found:
	(s, 1H, NH), 2.51 (s, 6H, CH xylyl ), 2.04 (s, 9H, CH ad. + CH2 ad. ), 1.54 -1.51 (m, 6H, CH2 ad ), C, 44.86; H, 4.52; N, 5.52. 1 H NMR (CD3CN, 300 MHz, 298 K): δ 9.26 (s, 1H, NH), 8.80 (s,
	2H, H 2 ), 7.62 (2d, 3 J = 9.0 Hz, 2H, H 5+5' ), 7.80 (d, 4 J = 1.89 Hz, 1H, H 8 ), 7.53 (s, 1H, H 8 ), 1.36 (s, 18H, t Bu). 13 C{ 1 H} NMR (CD2Cl2, 75 MHz): δ 172.4 (s, C 10 ), 166.5 (s, C 9 ), 157.9 (s, C 7 ), 157.3 (s, C 4 ), 144.5 (s, C 3 ), 139.5 (s, C 2 ), 135.9 (s, C ortho ), 134.6 (s, C 8 ), 130.9 (s, 7.25-7.28 (m, 4H, NH + H 6+6'+8' ), 7.13-7.24 (m, 3H, CH xylyl ), 4.26 (m, 1H, NCH), 3.15 (s,
	. Calcd. for C33H36AuN3O (687.6): C, 57.64; H, 5.28; N, 6.11. Found: C, 57.42; H, 5.18; CH xylyl ), 130.5 (s, CH xylyl ), 127.0 (s, C 5 ), 125.6 (s, C 6 ), 57.5 (s, C ad. ), 44.6 (s, CH2 ad. ), 36.0 (s, 3H, OCH3), 2.33 (s, 3H, CH3 xylyl ), 2.30 (s, 3H, CH3 xylyl ), 1.88 (m, 1H, CH iPr ), 1.09 (s, 9H,
	N, 6.19. 1 H NMR (CD2Cl2, 300 MHz, 298 K): δ 8.80 (s, 2H, H 2 ), 8.00 (d, 3 J = 2.01 Hz, 2H, C(CH3)3), 35.6 (s, CH ad ), 31.3 (s, C(CH3)3), 29.8 (s, CH2 ad. ), 19.5 (s, CH3 xylyl ). IR: νmax t Bu), 1.08 (s, 9H, t Bu), 0.61 (d, 3 JH-H = 6.9 Hz, 6H, CH3 iPr ). 13 C{ 1 H} NMR (CD3CN, 75
	H 8 ), 7.60 (d, 3 J = 8.16 Hz, 2H, H 5 ), 7.33 (dd, 3 J = 2.07 Hz, 4 J = 8.16 Hz, 2H, H 6 ), 7.16 (s, 3H, (neat)/cm -1 : 3311 (NH) , 2958 (CH), 1586 (carbene). MHz): δ 177.7 (s, C 10 ), 171.7 (s, C=O), 166.5 (s, C 9 ), 165.6 (s, C 9' ), 157.9 (s, C 7 ), 157.8 (s,
	CH xylyl ), 7.07 (s, 1H, NH), 2.50 (s, 6H, CH xylyl ), 1.34 (s, 18H, t Bu). 13 C{ 1 H} NMR (CD2Cl2, C 7' ), 157.4 (s, C 4 ), 157.3 (s, C 4' ), 145.9 (s, C 3 ), 145.8 (s, C 3' ), 140.7 (s, C 2 ), 136.7 (s, C xylyl ),
	75 MHz): δ 168.8 (s, C 10 ), 167.5 (s, C 9 ), 156.6 (s, C 7 ), 156.4 (s, C 4 ), 144.8 (s, C 3 ), 138.4 (s, 136.6 (s, C xylyl ), 134.6 (s, C 8 ), 134.0 (s, C 8' ), 133.1 (s, CH xylyl ), 130.7 (s, CH xylyl ), 130.6 (s,
	C 2 ), 136.7 (s, C ipso ), 135.8 (s, C ortho ), 134.9 (s, C 8 ), 128.5 (s, CH xylyl ), 127.2 (s, CH xylyl ), 126.0 [(C^N pz ^C)Au{C(NHCH2CO2Et)(NHC6H3Me2-2,6)}]SbF6 (6) CH xylyl ), 127.9 (s, C 5 ), 127.8 (s, C 5' ), 126.5 (s, C 6 ), 126.2 (s, C 6' ), 69.5 (s, NCH), 53.2 (s,
	(s, C 5 ), 124.3 (s, C 6 ), 35.7 (s, C(CH3)3), 31.4 (s, C(CH3)3), 19.7 (s, CH3 xylyl ). IR: νmax OCH3), 36.3 (s, C(CH3)3), 36.3 (s C(CH3)3), 32.3 (s, CH iPr ), 31.4 (s, C(CH3)3), 31.3 (s,

(neat)/cm -1 : 3296 (NH), 2962 (CH), 1643 (C=O).

Synthesis of [(C^N pz ^C)Au{C(NH-1-Ad)(NHC6H3Me2-2,6)}]SbF6 (5)

Complex 2 (0.050 g, 0.055 mmol) and 1-adamantylamine (0.017 g, 0.110 mmol) were combined in a flame-dried Schlenk flask, with molecular sieves, under N2 atmosphere and dissolved in dichloromethane (5 mL). The solution was stirred at room temperature for 16 h.

The product was precipitated with an excess of light petroleum (bp. 40-60 °C), filtered off and dried under vacuum to yield a bright yellow solid (0.031 g, 53%). Anal. Cald. for Glycine ethyl ester HCl (0.020 g, 0.146 mmol) was added to a flame-dried Schlenk flask charged with K2CO3 (0.012 g, 0.088 mmol) and acetonitrile (10 mL). The solution was sonicated for 30 min and transferred to a separate flame-dried Schlenk flask containing 2

Synthesis of [(C^N pz ^C)Au{C(NHCH( i Pr)CO2Me)(NHC6H3Me2-2,6)}]SbF6 (8)

L-valine methyl ester HCl (0.029 g, 0.175 mmol) was added to a flame-dried Schlenk flask charged with K2CO3 (0.012 g, 0.088 mmol) and acetonitrile (5 mL). The solution was sonicated for 30 min and transferred to a separate flame-dried Schlenk flask containing 2 (0.079 g, 0.087 mmol). The mixture was stirred for 16 h at room temperature and the precipitate removed by filtration through a celite plug. Next, the solvent was removed under vacuum and the solid residue re-dissolved in dichloromethane (2 mL). C(CH3)3), 19.9 (s, CH3 xylyl ), 19.7 (s, CH3 xylyl ), 19.5 (s, CH3 iPr ), 19.4 (s, CH3 iPr ), 0.9 (s, CH iPr ).

Synthesis of (C^N pz ^C)Au{C(NPh)(NHC6H3Me2-2,6)} (12)

  CH Ar. ), 128.9 (s, C Ar. ), 127.0 (s, C 5 ), 124.8 (s, C 6 ), 124.7 (s, C 10 ), 123.4 (s, CH Ar. ),

	124.7 (s, C 6 ), 120.0 (s, C meta,para ), 117.1 (s, C ortho ), 36.6 (s, C(CH3)3), 31.3 (s, C(CH3)3). IR:
	). Anal. Calcd. for C39H48AuN4O2SSbF6 (1069.6): C, 43.79; H, 4.52; N, 5.24. Found: C, 43.58; H, 4.37; N, 5.08. 1 H NMR (CD2Cl2, 300 MHz, 298 K): δ 8.85 (s, 2H, H 2 ), 8.71 (s, 1H, NH), 8.29 (d, 3 JH-H = 9.0 Hz, 1H, NH), 7.74 -7.62 (m, 4H, H 5/8 ), 7.48 -7.35 (m, 5H, H 6 + H xylyl ), 5.00 (m, 1H, NCH), 3.60 (s, 3H, OCH3), 2.63 (s, 3H, CH3 xylyl ), 2.52 (s, 3H, CH3 xylyl ), 2.14 (m, 2H, SCH2), 1.66 (s, 3H, SCH3), 1.36 (s, 9H, t Bu), 1.33 (s, 9H, t Bu), 1.14 (s, 2H, CHCH2). 13 C{ 1 H} NMR (CD2Cl2, 75 MHz): δ 178.2 (s, C 10 ), 170.6 (s, C=O), 165.8 (s, C 9 ), 165.1 (s, C 9' ), 158.2 (s, C 7 ), 158.0 (s, C 7' ), 157.0 (s, C 4 ), 156.9 (s, C 4' ), 144.7 (s, C 3 ), 144.6 (s, C 3' ), 139.5 (s, C 2 ), 139.4 (s, C 2' ), 136.3 (s, C Ar. ), 135.7 (s, C Ar. ), 134.0 (s, C 8 ), 132.9 (s, C 8' ), 131.7 (s, CH Ar. ), 130.9 (s, CH Ar. ), 130.5 (s, CH Ar. ), 130.2 (s, C Ar. ), 127.3 (s, C 5 ), 127.1 (s, C 5' ), 125.9 (s, C 6 ), 125.8 (s, C 6' ), 61.6 (s, OCH3), 36.0 (s, NCH), 31.9 (s, CHCH2), 31.3 (s, C(CH3)3), 31.2 (s, C(CH3)3),30.4 (s, C(CH3)3), 30.1 (s, C(CH3)3), 19.4 (s, CH3 xylyl ), 19.3 (s, CH3 xylyl ), 18.9 (s, SCH2), 14.8 (s, SCH3). IR: νmax (neat)/cm -1 : 3304 (NH), 3169 (NH), 2960 (CH), 1744 (C=O), 1585 (carbene). Synthesis of (C^N pz ^C)Au(NHPh) (11a) A mixture of 1 (0.050 g, 0.087 mmol) and KO t Bu (0.012 g, 0.104 mmol) in a flame-dried Schlenk flask with molecular sieves under N2 atmosphere was dissolved in toluene (5 mL) and stirred at room temperature for 3 h. Distilled aniline (8.26 µL, 0.087 mmol) was added and the mixture stirred for a further 3 h. The solvent was removed under vacuum and the product dissolved in dichloromethane. The solution was filtered through celite and the solvent removed under vacuum. The solid residue was washed twice with light petroleum and dried under vacuum to yield a black solid (0.050 g, 91 %). Anal. Calcd. for C30H32AuN3 (631.6): C, 57.05; H, 5.11; N, 6.65. Found: C, 57.17; H, 5.26; N, 6.55. 1 H NMR (CD2Cl2, 300 MHz, 298 K): δ 8.69 (s, 2H, H 2 ), 7.60 (d, 3 JH-H = 8.1 Hz, 2H, H 5 ), 7.41 (d, 3 JH-H = 1.8 Hz, 2H, H 8 ), 7.30 (dd, 3 JH-H = 8.4 Hz, 4 JH-H = 2.1 Hz, 2H, H 6 ), 7.01 (dd, 3 JH-H = 6.75, 2H, CH meta ), 6.80 (dd, 3 JH-H = 7.8 Hz, 4 JH-H = 1.2 Hz, 2H, CH ortho ), 6.58 (t, 3 JH-H = 7.2, 1H, CH para ), 3.84 (s, 1H, NH), 1.23 (s, 18H, t Bu). 13 C{ 1 H} NMR (CD2Cl2, 75 MHz): δ 170.7 (s, C 9 ), 156.3 (s, C 7 + νmax (neat)/cm -1 : 3315 (NH), 2951 (CH). Synthesis of (C^N pz ^C)Au(NHC6H3 i Pr2-2,6)}] (11b) The procedure for 11a was followed to make 11b from 1 (0.050 g, 0.087 mmol), KO t Bu (0.024 g, 0.218 mmol) and distilled 2,6-diisopropylaniline (0.015 g, 0.087 mmol). The solvent was removed under vacuum and the product dissolved in dichloromethane, filtered through celite and the solvent removed under vacuum to give an oily precipitate which was recrystallized from dichloromethane/ light petroleum at -20 C to yield a brown solid (0.046 g, 74 %). Anal. Calcd. for C36H44AuN3 (715.7): C, 60.41; H, 6.20; N, 5.87. Found: C, 60.29; H, 6.27; N, 5.74. 1 H NMR (CD2Cl2, 300 MHz, 298 K): δ 8.81 (s, 2H, H 2 ), 7.65 (d, 3 JH-H = 8.1 Hz, 2H, H 5 ), 7.28 (dd, 3 JH-H = 8.1 Hz, J = 2.0 Hz, 2H, H 6 ), 7.13 (d, J = 7.6 Hz 2H, CH meta ), 7.02 (m, 3H, H 8 CH para ), 4.12 (s, 1H, NH), 3.69 (h, 3 JH-H = 6.9 Hz, 2H, CH iPr ) 1.23 (s, 18H, 31.1 (s, C(CH3)3), 28.2 (s, CH iPr ), 23.0 (s, CH3 iPr ). IR: νmax (neat)/cm -1 : 3322 (NH), 2958 (CH). Product 11a (0.174 mmol) was made in-situ as described and, without prior purification, was treated with 2,6-dimethylphenyl isocyanide (0.023 g, 0.174 mmol). The mixture was stirred for 16 h. The solvent was removed under vacuum and the product dissolved in dichloromethane and filtered through Celite. The solvent removed again under vacuum to give a yellow oil which was washed with ice-cold hexane. The residue was dried under vacuum to yield a yellow solid (0.108 g, 83 %). Anal. Calcd. for C39H41AuN4 (762.8): C, 61.41; H, 5.42; N, 7.35. Found: C, 61.44; H, 5.36; N, 7.25. 1 H NMR (CD3CN, 300 MHz, 298 K): δ 8.87 (s, 2H, H 2 ), 8.05 (s, 2H, H 8 ), 7.67 (d, 3 JH-H = 8.4 Hz, 2H, H 5 ), 7.34 (dd, 3 JH-H = 8.1 (CH), 1607 (C=N). Synthesis of [(C^N pz ^C)Au{C(NHPh)(NHC6H3Me2-2,6)}]BF4 (4•BF4) A solution of 12 (0.050 g, 0.067 mmol) in diethyl ether at -78 ºC was treated with HBF4•OEt2 (9 µL, 0.067 mmol). There was an immediate colour change from yellow to orange. The mixture was allowed to warm to room temperature and left to stir for 30 min. The solvent was removed under vacuum to yield an orange powder (0.057 g, 99 %). Anal. Calcd. for C39H42AuN4BF43H2O (904.6): C, 51.78; H, 5.35; N, 6.19. Found: C, 51.23; H, 4.05; N, 6.25. 1 H NMR (CD3CN, 300 MHz, 298 K): δ 9.71 (s, 1H, NH), 9.47 (s, 1H, NH), 8.98 (s, 2H, H 2 ), 7.82-7.79 (m, 4H, CH Ar. ), 7.48-7.37 (m, 7H, CH Ar. ), 7.24-7.17 (m, 3H, CH Ar. ), 2.55 (s, 6H, Hz, 36.0 (s, C(CH3)3), 31.5 (s, C(CH3)3), 20.0 (s, CH3 xylyl ). IR: νmax (neat)/cm -1 : 3340 (NH), 2957 CH3 xylyl ), 1.36 (s, 18H,
	C 4 ), 144.2 (s, C 3 ), 138.4 (s, C 2 ), 133.9 (s, C 10 ), 131.5 (s, C 8 ) 129.2 (s, C ipso ), 125.6 (s, C 5 ),

t Bu), 1.15

(d, 3

JH-H = 6.9 Hz, 12H, CH3 iPr ).

13

C{

1

H} NMR (CD2Cl2, 75 MHz): δ 169.6 (s, C 9 ), 156.4 (s, C 7 ), 155.8 (s, C 4 ), 148.4 (s, C ipso ), 144.4 (s, C 3 ), 141.8 (s, C ortho ), 138.4 (s, C 2 ), 129.3 (s, CH meta ), 125.3 (s, C 5 ), 124.2 (s, C 8 ), 123.7 (s, C 6 ), 121.9 (s, CH para ), 35.5 (s, C(CH3)3),

4

JH-H = 2.1 Hz, 2H, H 6 ), 7.14-7.12 (m, 4H, CH Ar. ), 6.96-6.94 (m, 4H, CH Ar. ),

6

.71 (s, 1H, NH), 2.52 (s, 6H, CH3 xylyl ), 1.36 (s, 18H, t Bu). 13 C{ 1 H} NMR (CD3CN, 75 MHz): δ 167.3 (s, C 9 ), 156.7 (s, C 7 ), 156.5 (s, C 4 ), 146.0 (s, C 3 ), 139.8 (s, C 2 ), 135.9 (s, C 8 ), 130.7 (s, C Ar. ), 129.4 (s, t Bu). 13 C{ 1 H} NMR (CD3CN, 75 MHz): δ 176.2 (s, C 10 ), 165.8 (s, C 9 ), 157.8 (s, C 7 ), 157.4 (s, C 4 ), 145.7 (s, C 3 ), 140.6 (s, C 2 ), 139.9 (s, C Ar. ), 136.6 (s, C Ar. ), 134.1 (s, C 8 ), 133.5 (s, C Ar. ), 130.9 (s, CH Ar. ), 130.6 (s, CH Ar. ), 130.5 (s, CH Ar. ), 128.8 (s, CH Ar. ), 127.7 (s, C 5 ), 126.1 (s, C 6 ), 124.9 (s, CH Ar. ), 36.3 (s, C(CH3)3), 31.4 (s, C(CH3)3), 19.4 (s, CH3 xylyl ). IR: νmax (neat)/cm -1 : 3215 (NH), 2956 (CH), 1586 (carbene).

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 6, 11a and 12•toluene. 43 †Electronic supplementary information (ESI) available: Crystal structure diagrams, NMR spectra. See DOI: 10.1039/xxxxxx. CCDC 1561647 (2), 1561651 (3), 1561652 (5•1.5C7H8), 1561648 (6), 1561649 (11a) and 1561650 (12•C7H8) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.


P Lautridou
email: pascal.lautridou@subatech.in2p3.fr

Results of the radio-detection experiment CODALEMA

Keywords: Radio detection, Ultra-High-Energy Cosmic Rays PACS: 95.55.Jz, 95.85.Ry, 96.40 ---

published or not. The documents may come

Results of the radio-detection experiment CODALEMA

Introduction

Since many years, the origin of non-thermal cosmic radiation has become a very stimulating field of researches. Recent results [1] have maybe opened a new window in the domain of UHECR: the arrival directions of the charged cosmic particles (above 10 19 eV) can be correlated with the locations of active galactic nuclei. These objects could be the source of the radiation, but definite conclusions will be made only when more data and better performances in detection will be obtained. Similarly, Ultra High Energy Cosmic Neutrinos have not yet been discovered but, at the ground level, the detection of inclined Extensive Air Showers induced by ν τ interactions in rock targets could be envisaged [2]. The produced τ could propagate in the rocks, emerges into the atmosphere and decays, giving rise to a shower of particles. Detection of very inclined EAS could then constitute a timely objective. To meet these challenges, one of the prospects perhaps lies in the development of the radio-detection of EAS. This runway was initiated first in the late 60' [3,[START_REF] Allan | Progress in elementary particle and cosmic ray physics[END_REF] but despite its general advantage -the radio waves propagate in the atmosphere without much attenuation -work ceased quickly in the mid 70'. Since 2003, some experiments, of relatively small scales, like LOPES [START_REF] Badea | Proceedings of CRIS2004[END_REF] and CODALEMA [6] in Europe, Radio-AUGER at PAO in Argentina [7,8] revisit this topic. Several comprehensive theoretical frameworks have also been completed or initiated in the last five years [9,10,11,12].

The present article reports on the contributions of the experiment CODALEMA to this effort. It will also present some new preliminary results and it will try to point out some possible next studies in the domain.

Developments of the detection

Although CODALEMA has greatly evolved over 5 years, the main idea that has driven our efforts has concerned the evolution of the detection method itself, by moving from a frequency analysis (as performed in the 70's) to a study in terms of waveform (Fig. 1) of the radio signal (available in the years 2000). This small progress has led to the basic and innovative idea that the transient approach can give, in a natural way, a triggering capability to this detection method.

The gained experience has enabled to enlarge step by step our prospective and since 2006, additional questions are currently addressed taking advantage of several other sites: at PAO, the radio detection in the range of 10 18 eV is studied using first prototypes of autonomous radio stations [7]; some limited measurements have been conducted at ARRAGAT (Armenia) for the detection of gammas at lower energy (< 10 16 eV), but without success due to the noise induced by the particle detectors; the site of the 21CMA detector is tested as one of the most interesting site for the detection of horizontal showers and neutrinos around 10 17 eV [2].

At the beginning of the experiment, few of the 144 conic logarithmic antennas, taken away from the Nançay Decameter Array [13], were used to successfully detect radio signals [14,15] using LeCroy digital oscilloscopes (8 bits, 500 MHz frequency sampling, 10 µs recording time). In 2006, emphasis on simplicity, size, cost and performance was used as guidelines to develop a new broadband antenna based on a short, fat active dipole concept [16]. It is made of two 0.6 m long, 0.1 m wide and 1 mm thick aluminum slats, separated by a 10 mm gap, hold horizontally by a plastic mast at 1 m above the ground (Fig. 2). The wires are loaded by a dedicated high input impedance low noise 34 dB amplifier (1 nV/√Hz) with a 3 dB bandwidth in 0.1-220 MHz. This design makes the antenna resonating around 115 MHz with a slowly varying gain over a wide band from 1 to 100 MHz. Validation of the dipole sensitivity has been obtained by observing its correlation pattern with the Decameter Array [START_REF] Charrier | IEEE Antennas and propagation Society International Symposium[END_REF] during the transit of the radio source Cas.A. This new sensor collects data since the end of 2006, linked to VME waveform digitizer boards [START_REF] Breton | 10th Workshop on Electronics for LHC and future Experiments[END_REF] (12 bits, 300 MHz analog bandwidth, 1GS/s of sampling, 2.5 µs recording time) and it is very robust. At Nançay, the only downside has been the detection below 10 MHz, which is not efficient enough without specific captors (this situation is better at PAO), and the detection efficiency in the range 100-200 MHz which is fluctuating due to intermittent transmitters (this domain of frequency will be relevant in clean RF sites). Hence, at the first stage of the off line analysis, the antenna waveforms are usually numerically filtered from the recorded 1-200 MHz bandwidth to a 24-82 MHz window. Presently, the radio array consists in 21 dipole antennas, put in E-W polarization, with a spacing of around 90 m and forming a cross with 600 meter long arms (see Fig. 3). It is completed by 3 dipole antennas setup in a N-S polarization [START_REF] Ardouin | summited to Astropart[END_REF]. Since 2007, a new and more sophisticated particle detector array, made of 17 scintillator stations located on a grid of approximately 85 m of pitch has been installed [START_REF] Lebrun | the CODALEMA collaboration, 30 th Int. Cosmic Ray Conf. ICRC'07[END_REF]. It covers a 340×340 m 2 area whose center roughly matches the radio array center. This upgrade enables now to extract the energy of the shower and to study the energy correlation with the radio-signal. EAS arrival direction is obtained using the time of flight between different stations. Shower size and core position are calculated from the measured particle densities in the detectors. The deduced lateral distribution is fitted with a NKG lateral distribution function via a minimization algorithm. For shower cores located inside the particle detector array (so called "internal events"), an estimation of the energy is relevant. A Constant Intensity Cut (CIC) method is used to define a vertical equivalent shower size N 0 . From Aires simulations for proton primaries, the energy is related to N 0 by the formula E = 2.14 10 10 N 0 0.9 , with a resolution of the order of ∆E/E ~ 30 % at E = 10 17 eV.

One of the starting points of the CODALEMA experiment has been perhaps to realize that it was possible to develop a method of analysis of radio waveform analogous to those already used in particle and nuclear physics (tagging of transients, TOF method are extensively described in Ref. [14,15]). In the initial phase, the tagging of a radio transient was obtained using leading edge discrimination in a bandwidth free of emitters. Such a method has the advantage of being simple and very efficient provided the chosen bandwidth remains free from occasional emitters. In 2007, a new approach to pulse detection was implemented using a linear prediction [START_REF] Press | Numerical Recipes in C[END_REF][START_REF] Ardouin | summited to Astropart[END_REF] that helps dealing with the opposite situation. It computes an estimate for a point in a time series, as a linear combination of the preceding values of the recorded radio waveform. With this new algorithm, the number of pulse candidates increases by 30 % with respect to the results of the leading edge discrimination technique.

As mentioned earlier, another important point for the future of this detection method was to convince oneself of the possibility to build an effective online radio trigger. Preliminary tests were conducted in 2004 at Nançay using a simple narrow band filter (33-65 MHz) inserted on the antenna signal which was sent to an amplitude threshold discriminator [14]. At the time of these results, the conclusion was that the trigger rate varied between 0.02 Hz and 2 Hz depending of the weather conditions and the daynight cycle, making feasible a method of an autonomous triggering of the antennas based on the radio transient alone. As demonstrated by the tests at PAO in 2007 [START_REF] Acounis | Auger Internal Note[END_REF], this method has to be tuned site per site. However, to avoid misunderstandings in trigger responses, the extreme simplicity of this triggering method has been kept for the new selfcontained radio stations in development.

To have confidence in the interpretation of the data, one unavoidable point has consisted also to understand and qualify the detection performances [START_REF] Lamblin | 30 th Int. Cosmic Ray Conf. ICRC'07[END_REF]. This has been possible taking advantage of two natural phenomena. On one hand, the accuracy in the arrival direction has been extracted using a radio emission from the sun. The distribution of the residues, between the reconstructed arrival directions and the true sun locations in the sky, has provided an estimation of the standard deviation of 0.7°. On the other hand, calibration of gains and sensitivities of the antennas have been deduced from the analysis of the antenna noise. It was found that versus the time, the behavior of the noise follows the galactic signal modulation. This has enable to cross calibrate the antenna responses in electric field.

Results

The showers are first identified through a selection in the time distribution of coincidence between the arrival of the particle pancake and the radio front, and another one in the angular direction difference between the pancake and the radio front [15]. The energy distributions [START_REF] Ardouin | summited to Astropart[END_REF] measured in coincidence by the ground particle array (for internal events) and by the dipole array are superimposed on the Fig. 4. A threshold behavior of the radio detector is clearly visible below 10 17 eV but, for a similar set up, the energy threshold of the ground particle array is far below, around 10 15 eV. Both distributions converge at the highest energies. This reflects the increase of the radio-detection efficiency, which is regularly increasing starting at 3 10 16 eV and reaches roughly 50 % at 2 10 17 eV. This indicates the present domain of detection of this method but technical improvements, should enable to cover the knee region, and especially for inclined showers.

After tagging of the EAS, it becomes possible to extract the distribution of the radio signal at ground and to enlighten one of its most singular features: the distribution of the EAS signal follows a bell shape, when a far source exhibits a flat distribution. This distinguishes the cosmic shower from a man made or from an atmospheric radio wave and suggests that it becomes possible to use only the radio signal with a self-trigger to identify an EAS. As for particle detector arrays, it is possible to estimate the shower core locations using the radio signals. The first way is to determine the center of the bell shaped distribution when the shower falls inside the antenna array. The alternative is to fit the lateral electric field profile, translated in the shower frame, by an exponential law. Indeed, the lateral dependence of the electric field profile seems to follow a decreasing exponential (as suggested 30 years ago by H.R. Allan [START_REF] Allan | Progress in elementary particle and cosmic ray physics[END_REF]) E (d) = E 0 exp (-d/d 0 ) where d is the distance between the shower axis and each fired antenna and d 0 is the slope parameter for the considered event (see Fig. 5). Present results indicate also that lateral profiles of the electric fields, arrival directions and core locations do not depend strongly on the frequency below 200 MHz. This suggests that the most relevant median value of frequency window is not firmly established; the minimum width in frequency is not well defined (wide, narrow?). The results obtained so far indicate then that the antenna designs are not crucial except for mechanical considerations and detection capabilities of very inclined showers. From the viewpoint of detection, another striking feature is that this radio signal can be measured at more than 300 m for energies around 10 17 eV.

The figure 6 represents the sky maps in local coordinates (zenith, azimuth) of the radio events arrival directions seen by the East-West antennas. A striking feature is the shape of the azimuth distribution and the large asymmetry in counting rate between the North and South sectors. This pattern has been interpreted as an effect of geomagnetic field on the electric field generation [START_REF] Ardouin | summited to Astropart[END_REF]. Because the Lorentz force acting on the charged particles is at the origin of the different emission mechanisms of the electric field involving the geomagnetic field [10,12], the electric field magnitude should depend on the values of the vector cross product v∧B. Near the detection threshold, the number of detected radio events should be proportional to the absolute value of the electric field. Indeed, calculations for the recovery of the sky map of the events made under these assumptions looks very similar to the observed ones. This simulation has taken into account that, in the experiment, the signal amplitude given by the E-W dipole should be proportional to |(v∧B) EW |, which is the magnitude of the projection of the vector cross product on the East-West axis (orientation of the antennas). These results suggest that the behavior of the measured electric field can be fairly reproduced by simply considering the vector cross product v∧B of the Lorentz force, and that the radio signal induced by the geomagnetic field dominates with the current CODALEMA observation conditions.

New issues

To complete this review, it could be interesting to flash some other results, which are far from finding, at the moment, complete explanations but one has to point out that these analyses are still regarded as very preliminary. These issues could be part of the next challenges in the development of the radio.

Calibration in energy of the radio signal is one of the keys for the future of this new technique. Our first studies of the correlation of the radio signal with the particle energy have been initiated in 2008 and these analyses are far to be conducted at end. In the simplest picture, the maximum value of the electric field at the shower core (E 0 ) could be considered as an energy estimator for the radio signal. This quantity can be extracted, on an event-by-event basis, from the fit of the electric field profiles assuming the exponential dependency E 0 exp (-d/d 0 ). The resulting scatter plot of E 0 (in a.u.) as a function of the energy E p deduced from the particle analysis is presented Fig. 7. It is computed using all the detected "internal events" without any other cuts. It exhibits a satisfactory correlation pattern. A linear fit of the correlation gives Log (E 0 sinα cosθ) = 1.1 Log E p -14.3 which leads to E 0 ~ N p 0.99 with the assumption that N = E p 0.9 . The scatter on E 0 indicates that ∆ E 0 / E 0 = 26 % when ∆ E p / E p = 30 %. At first glance, the radio emission seems coherent but it is found also that about 50% of internal events (especially when the Signal / Noise declines) do not exhibit clean exponential dependences at small impact parameters (see Fig. 8). Although many sources might be at the origin of this behavior (such as possible superposition of several mechanisms in the electric field generation or restrictions on the use of exponential model), current analysis suggest that the near environment of the sensors would be more the main cause (disturbances of the radio signal by trees, buildings) .To pass over this difficulty, a "minimum" estimator E mean , which represents the mean of the recorded amplitude, has been tried. Unlike the previous observable, E mean doesn't include any explicit dependence of the distance to the shower axis. Indeed, the computed scatter plot is very similar to the previous one. A linear fit of the correlation gives Log (E mean sinα cosθ) = 0.84 Log E p -9.9 . At this stage of our understanding, the radio estimators E 0 or E mean could be questionable (furthermore both are correlated to the detector geometry and both depend strongly from the measured electric field profiles) but they seem still very robust and the main trend already obtained by LOPES is observed [START_REF] Falcke | [END_REF].

Curvature of the particle pancake is usually linked to the nature of the primary. It was tempting to look at the counterpart in radio. What is observed with CODALEMA is very promising for the interpretations of this parameter. The distribution of the curvatures deduced from the event-by-event analysis of the arrival times of the radio fronts is shown on Fig. 9. It is made using all the detected events without selection. An interpretation of the result, in term of distance to the main emitting source, suggests that the time of the maximum amplitude of the filtered radio signal is well linked to the maximum of development of the shower (in opposition with the first observations of LOPES [START_REF] Falcke | [END_REF]). Such a discrepancy can hardly be attributed to the time accuracy of the detection (~8ns), which could basically acts as a limiting factor. In addition, due to the filtering, the radio-signals are characterized by a fairly constant duration, quasi-independent in a few nanoseconds of the distance. Accordingly, the time accuracy is not expected to change greatly with the distance to the core. This situation seems also to differ in many aspects from ground particle measurements and needs further investigations (for instance, each antenna shows all the shower longitudinal development).

Following recent calculations of T. Huege [START_REF] Huege | 30 th Int. Cosmic Ray Conf. ICRC'07[END_REF], the information contained in the lateral profile of the radio signal can be correlated to the X max of the shower, to the energy and to the nature of the primary. Indeed, the ratio of the field strength (E 275 /E 725 ) measured at 2 distances (275 m and 725 m) from the shower core and for zenith angles around 60°, is proposed to distinguish between primary particle types. In the 32-64 MHz band, the predictions lead to the following trend: for 4 < E 275 /E 725 < 8, the nature of the primary evolves from Fe to γ. With CODALEMA, the event-by-event estimation of the parameter d 0 of the electric field profile (interpreted with the exponential formula), makes possible to compute the ratio E 275 /E 725 without detecting at 725 m. The resulting distribution is presented Fig. 10. Part of the distribution locates in the expected range but around 20% of the events show a ratio greater than expected by the simulations. This discrepancy could come from our distribution of zenith angles, which is centered around 35°. Again to solve this apparent puzzle, additional investigations are needed.

Conclusion

Various antennas (log-spiral teepee, short dipole) linked to various electronics (LNA, VME ADC or Scope, Filters) have been tested successfully in CODALEMA. It appears that the detection method is robust, the signal is firm: pretty independent of the antenna and of the electronics.

Much progress has been made in the interpretation of the radio signals. The analysis and the detection methods are in stage of stabilization (see also the results of LOPES) but this effort still demands inputs from the experiments and the theory.

For instance, a deeper understanding of the electric field profile is mandatory in order to deduce a satisfactory energy calibration. A firm confirmation of the polarization state of the electric field is essential to improve the detection and to distinguish between different theoretical approaches such as geosynchrotron or transverse current models.

Beside the continuation of the R&D of CODALEMA and of LOPES, which are aimed at clearing the ground, a super hybrid detector covering a large area at PAO is envisaged. This new perspective has already been taken into account by the CODALEMA collaboration, which develops a self-contained radio station. It will be operational for the end of 2008.

The radio-detection has entered a phase of detailed studies of the mechanism of emission and it is not far from being ready for intensive use. The potential of this detection method can be reasonably considered but the true interest of the radio-detection might yet to be invented.

Fig. 1 :

 1 Fig. 1: Radio waveforms of an EAS observed in the 23-130 MHz band at Nançay (scales in µs and V). Each plot corresponds to an antenna located along one of the two 600 m long arms.

Fig. 2 :

 2 Fig. 2: View of the new broadband antenna with its front-end amplifier box used since 2006.

Fig. 3 :

 3 Fig. 3 : Schematic view of the CODALEMA experimental setup on January, 2008 superimposed on an aerial view of the Nançay observatory centered on the Decameter array. Plastic scintillators are depicted as red squares. Yellow and green "T" represent dipole antenna oriented respectively in the E-W and N-S directions.

Fig. 4 :

 4 Fig.4: Energy distribution of "internal" events measured by the ground particle detector array (black circles) and seen in coincidence with the antenna array (blue squares).

Fig. 5 :

 5 Fig. 5: Electric field profile, footprint at ground, arrival direction and core position reconstructed by using radio detection.

Fig. 6 :

 6 Fig. 6: Sky map of the observed events in the East-West polarization. The direction of the geomagnetic field at Nançay is indicated by the dot.

Fig. 7 :

 7 Fig. 7: Scatter plot of the radio energy estimator E 0 in function of the energy E p deduced from the particle analysis (α is the geomagnetic angle and θ is the zenith angle).

Fig. 8 :

 8 Fig. 8: Problematic profile of electric field, footprint at ground, direction of arrival and core position of a reconstructed EAS.

Fig. 9 :

 9 Fig. 9: Histogram of the number of events as a function of the curvature radius.

Fig. 10 :

 10 Fig. 10: Event distribution versus the ratio E 275 /E 725 deduced with the exponential model of the electric field profile.

Acknowledgments

The authors acknowledge the support of the French Agence Nationale de la Recherche, under grant ANR-NT05-2_42808.


Dr Florent Laroche

Advanced Industrial Archaeology and TechnoMuseology A new virtual life for industrial heritage

All through history humans have invented and created so as to improve their standard of living, many machines have been built; simple ones but also very complex others. Enterprise continuously adjusts its operating modes and its production tools to optimise its added value creation. Machines which are obsolete as soon as they don't meet the demand are stopped, stored and very often dismantled. Industrial sites disappear and the workers leave the industrial world taking their know-how with them. To anticipate and avoid this loss to our scientific and technological knowledge an idea was pioneered by Professor Michel Cotte a few years ago [M. Cotte, S. Deniaud (2005), "CAO et patrimoine : perspectives innovantes", revue Archéologie industrielle en France,n°46,Paris,. Our team has since then formalised the methodology and tested it over more than ten case studies. Moreover, we take note that the rescue and proper maintenance of physical objects is very costly for museums. Moreover, sometimes, dismantling of the equipment is impossible due to the deteriorated condition of the machine. So, avoid those problems, we propose to preserve it as a numerical object. We focus on the mechanical and technical points of view. For instance, in a factory, there is the building itself but also all the equipment it contains. Taking into account the technical point of view can provide a better understanding of the past. Consequently, engineers and industrial engineering tools and methods can give answers for capitalising knowledge, conserving and popularising old machines. Even so, the work of historians is not discarded. Researches for a better understanding of the socio-ethno-historical context of the object are necessary for formalising and validating technical hypotheses. It is a new way of thinking about heritage projects: social sciences and engineering sciences must merge together. Tools and methods from both domains have to fit and to interact as shown by the puzzle figure below.

Our proposition consists in overturning the time axis of the design process generally used for developing contemporary technical products. That means that we begin at the end of machine lifetime and come back to the initial need that define why the technical object had been created. First step is the digitalisation of the physical object and the capitalisation of the know-how learnt by studding the machine. Next, thanks to virtual reality technologies, we can valorise this amount of knowledge. This global process is what we call Advanced Industrial Archaeology. First of all, if physical data exists it must be captured. The basic tools are decametres, slide callipers, micrometer callipers. However, 3D digitalisation can be employed for measuring complex machines or inaccessible components inside the object: TMM (Tridimensionnal Measuring Machines), laser radar, X-ray, 3D scanner laser with topographical reconstruction in real-time. Those contemporary industrial tools must be chosen carefully as the gait is not the same when digitalising ancient objects and modern industrial products. For example, to avoid damaging a machine, contact can sometimes be impossible or even forbidden: technology used must be without contact based on a passive system. Main difference between passive and active systems is due to the technology that emits a light beam or not. Simultaneously, Knowledge Management methods which are commonly used by businesses must be coupled to historical studies. This step allows capitalising external knowledge, identifying know-how of workers, giving information about suppliers or customers, etc. Next, the technical object and its environment are re-designed thanks to Computer Aided Design (CAD) software. Those programmes are commonly used by modern industries for designing complex products such as airplanes or cars. Considered as experimental researches or didactic applications, our teams use Catia V5 by Dassault Systèmes (the same software used for developing the Airbus A380 aircraft). Moreover, even though old objects are mainly inert, they were animated by mechanisms. Consequently, they have to be virtually restored and simulated in order to validate the operating of the machine. At this step, we prefer to use CAD programmes instead of Computer Graphics programmes (CG). These are employed for creating animated pictures, movies, etc. With CG programmes, simulations and dynamics are not realistic. An entire "virtual world" is created in which objects move. But this world does not have the properties of the terrestrial physical laws such as the fundamental principles of mechanics (for example gravity, stress, speed, acceleration). Then this heritage engineering phase allows obtaining what businesses call Digital Mock-Up (DMU). Nowadays, the digital model of a product is the principal element for industrial production. DMU is the converging point for organising departments of an enterprise: Research & Development, design, manufacturing, marketing, sales, etc. In the same way, the virtual representation of an old technical object can be associated, linked and enriched thanks to external cultural knowledge and anthropological knowhow: it is what we call the Digital Heritage Reference Model (DHRM). This Technical Heritage File allows capitalising knowledge from the past into a digital media and a virtual simulating state. Thus, the DMU can become a new museological tool and a reference model for museum curators. Obviously we must insist that digital files will never replace physical objects. The DMU is only another way to represent reality: it widened the definition of the Artefact. Obviously, the value of using digital representation is intended to experts or museum curators. But it has other applications… Old machines do not usually function or cannot be exhibited in a museum due to problems of cost and security (component wear, machine driver requirement, etc.). Nowadays, thanks to virtual reality technologies, the DHRM can be used to present the artefact. Unlike videos and thanks to interactivity, it is easier to understand how they work: the visitor is no longer a spectator but an actor. Virtual Reality is a new mediation tool that allows visitors to immerse themselves in a virtual environment: the machine can be tested virtually to its extreme limits; the level of detail can be adapted by the museum according to the targeted public, etc. Indeed, it lets visitors investigating themselves the technical system from their own point of view (global immersion, technical analyses, local study of components, social integration of the technical environment, etc.). Understanding an old technological object can be easy for former workers but it can be difficult for curators or visitors of their museums. Virtual Reality tools can become a springboard for ensuring that the know-how of our scientific and technical industrial heritage will be apprehensible and comprehensible by everyone. This is the next step after Advanced Industrial Archaeology: we call it Techno-Museology, a new kind of museology for this 3 rd millennium.

The images below show two of the experiences we have performed in France. The input data of the study was one drawing of a steamboat designed in 1861 by DCNS (latitude 47°11'47 North and longitude 1°41'00 West). The other example is a salt washing machine (c.1914-1963) belonging to the Musée des Marais Salants in Batz-sur-Mer in Brittany (latitude 47°16'44 North and longitude 2°28'41 West).


Guo Xian Tan

Christian Viard-Gaudin
email: christian.viard-gaudin@univ-nantes.fr

Alex C Kot
email: eackot@ntu.edu.sg

A Stochastic Nearest Neighbor Character Prototype Approach for Online Writer Identification

 by making use of a stochastic nearest neighbor algorithm to estimate the character prototype distribution. The proposed method is text independent and relies on the automatic segmentation of the handwritten text at the character level. Our results show that this approach attained a writer identification rate of 99.2% when a reference database of 120 writers is used. Experiments related to the effect of the length of text of the document on the performance of the writer identification system are also reported.

Introduction

With the advent of pen-input devices such as Tablet PC and personal handheld devices, entering data into computers is now not just being limited to using the keyboard. Numerous initiatives have thus been funded to develop algorithms and computing platforms to handle the surge in demand for this seamless and natural interaction between human users and the machine [START_REF] Oviatt | Designing the User Interface for Multimodal Speech and Pen-Based Gesture Applications: State-of-the-Art Systems and Future Research Directions[END_REF]. All these led to the emergence and proliferation of handwritten on-line documents. Online handwritten digital documents are defined as those digital documents that not only provide information obtainable from offline digital documents, but also 1 Allographs are different shapes and forms of the same alphabet. contain temporal information of the handwriting process [START_REF] Jain | Indexing and Retrieval of On-line Handwritten Documents[END_REF]. Such additional information provides vital clues as to the identities of the writer. Writer identification has ubiquitous applications in digital rights management and forensic analysis in the prevention of fraud and identity theft cases. In addition, we can also perform writer-adaptation in tablet PCs and create, store or retrieve a profile of handwriting styles of the writers if we are able to automatically determine their identities.

Various techniques currently exist for writer identification. They can be generally classified into text-dependent or text-independent techniques, which can make use of both global as well as local features. Yashushi et al. proposed a HMM-based approach [START_REF] Yasushi | Textindicated Writer Verification Using Hidden Markov Models[END_REF][START_REF] He | Writer identification of Chinese handwriting documents using hidden Markov tree model[END_REF][START_REF] Caillault | Mixed Discriminant Training of Hybrid ANN/HMM Systems for Online Handwriting Word Recognition[END_REF] that is robust to noise and small shape changes. However, this model is text-dependent. Jain et al. proposed to use dynamic time warping [START_REF] Jain | Indexing and Retrieval of On-line Handwritten Documents[END_REF][START_REF] Niels | Automatic Allograph Matching in Forensic Writer Identification[END_REF] on the stroke direction, curvature and the height as features to do word matching. Such a method is language independent, but at the expense of a high computational complexity. Schomaker et al. [START_REF] Schomaker | Automatic Writer Identification Using Connected-Component Contours and Edge-Based Features of Uppercase Western Script[END_REF][START_REF] Bulacu | Text-Independent Writer Identification and Verification Using Textural and Allographic Features[END_REF] used textural information such as the slant and curvature to transform them into probability density function as well as allographic information such as the graphemes to cluster into codebooks. This approach achieved significant improvements when compared to traditional techniques.

One newly-established technique by Chan et al. [START_REF] Chan | Online Text Independent Writer Identification Using Character Prototypes Distribution[END_REF] makes use of character prototyping, where statistical information about the writer is being matched based on the prototype of that character. A top1 accuracy of 95% was achieved based on this text-independent approach. The advantage of this method is that it is more consistent at the character level as opposed to using the grapheme or word level. Our proposed work further improves upon the work done by Chan et al. by 978-1-4244-2175-6/08/$25.00 ©2008 IEEE adopting a fuzzy-algorithmic approach, resulting in significant improvements in the performance.

The remainder of this paper is organized as follows: Section 2 describes the proposed methodology and experimental setup. Following that, section 3 then presents the experimental results. Finally, discussions and future areas to explore are described in section 4.

System Architecture

The writer identification system can be broadly divided into three main stages as shown in figure 1. The purpose of the prototype training stage is to build a set of character prototypes to model the different allographs of the 26 Latin alphabets ('a' to 'z'). The character prototypes are built and trained using the IRONOFF database [START_REF] Viard-Gaudin | The IRESTE On/Off (IRONOFF) Dual Handwriting Database[END_REF] of 16585 isolated French words written by 373 subjects. The segmentation and extraction of the characters to build the set of character prototypes is done by an industrial text recognition engine, MyScript [START_REF]Vision Objects Industrial Text Recogniser SDK[END_REF]. Thereafter, in the document labeling stage, the same recognition engine is used to segment and label the online handwritings of a writer document at the character level. Each of the extracted characters is then mapped to the set of character prototypes corresponding to its alphabet and transformed into a distribution of frequency vectors. Finally, in the last classification stage, frequency vectors are then classified to identify the writers.

In this paper, we propose a stochastic nearest neighbor algorithm at the document labeling stage. After an automatic segmentation and preprocessing process, the extracted characters are then assigned using a Mahalanobis distance to the respective character prototypes built in the previous prototype training stage. This distance is used to take care of the specificities of the feature space and the resulting shape of the clusters in this space. Hence, the variances among the different feature vectors are factored into the proposed methodology. The assignment to the respective prototypes, termed as stochastic nearest neighbor assignment, is derived from a fuzzy c-means algorithm [START_REF] Han | Data Mining: Concepts and Techniques[END_REF][START_REF] Pedrycz | Collaborative Clustering with the use of Fuzzy C-means and its Quantification[END_REF] which uses an exponential kernel function as described in Eq. 1 to determine a partial membership to the respective character prototypes.

( ) ( , ) k dist p x α represents the Mahalanobis distance. In Eq. 1, β is a tuning parameter which is set to be 0.01 and N is the number of prototypes used, which is set to 10 based on our past results.

( ) ' ' 1 exp ( , ) ( | ) exp ( , ) k k N k k dist p x P p x dist p x α α α β β = -× = -× ∑ (1)
1 1 ( | ) M k k x tf P p x M α = = ∑ (2) ', ' 1 1 , 1 log N R k i k i k R k i i tf idf tf ε ε = = = + = + ∑ ∑ ∑ (3)
( | ) k P p x α is then used to calculate the distribution of frequency vectors, the term frequency (tf), as shown in Eq. 2. Each component of the term frequency vector will be weighted with the inverse document frequency (idf), as shown in Eq.3. The usage of tf and idf is commonly employed in information theory for document retrieval [START_REF] Salton | Term-weighting approaches in automatic text retrieval[END_REF] and will be used for classification later. In Eq. 2, M is the number of characters corresponding to the alphabet α. In Eq. 3, R is the number of reference writers and ε is a small value to prevent any numerical problems.

Experimental Results

We have collected online handwritten documents from 120 writers. Each writer wrote 2 documents, with a digital pen and paper technology: one is considered as a reference document and the other is taken as a test document. As presented in table 1, using Euclidean distance between the frequency vectors during classification resulted in our stochastic nearest neighbor approach attaining a high accuracy of 98.3% for writers that are ranked correctly in the top 1 position. This translates into a misclassification error of 2 misclassified writers, with both of them being misclassified in the top 2 position. This indicates that the writer identification system has confused the misclassified documents with only 1 other document from a different writer out of 120 writers. Comparisons with the method by Chan et al. [START_REF] Chan | Online Text Independent Writer Identification Using Character Prototypes Distribution[END_REF] show a significant improvement. An accuracy of 96.7% (4 misclassified writers out of 120) was achieved using their technique. Their methodology hinges on the concept that each character can only be assigned to one particular prototype. This is flawed in reality because there often exist overlapping handwriting styles for different writers. Our observations reveal that there are numerous instances when the characters are close to more than one prototype in the vector space. For such instances, the discrete allocation of prototypes used by Chan et al. does not yield good results. A writer whose dominant handwriting style does not fit well into an existing prototype will be weakly modeled using their approach. Therefore, in our proposed methodology, each character is assigned a certain degree of all the prototypes depending on how close they are to that prototype, allowing us to realize a higher accuracy.

Classification using different distance measure

Experiments were also conducted using different distance measures for classification. It is observed that the Chi 2 distance measure, as described in Eq. 4, outperforms the Euclidean measure in our writer identification system, achieving a top 1 writer 2 1-Nearest Neighbor algorithm adopted by [START_REF] Chan | Online Text Independent Writer Identification Using Character Prototypes Distribution[END_REF] identification rate of 99.2%. This is equivalent to a misclassification error of only 1 misclassified writer, with the misclassified writer being in the top 2 position. The rationale, which can explain the better result obtained with the Chi 2 distance, is that the Chi 2 distance considers a relative difference between the two components of the distributions instead of an absolute difference as with the Euclidean distance and this is more meaningful with respect to the style of writings that we would like to distinguish.

Dist (writer

i , writer T ) = ( ) 2 , , 1 , ,
N k k i k T k k i k T i d f t f t f t f t f = - + ∑ (4)

Effect of length of text in test documents

A series of experiments have been conducted to investigate the amount of text required for sufficient accuracy of the writer identification system. Figure 2 shows the achievable of the identification rate by varying the number of characters in the test documents, while keeping the reference documents unchanged. A random reduction of characters from all the alphabets in the original test document is carried out until the required number of characters per test document is attained. As illustrated in figure 2, the misclassification error remains almost constant when at least 160 characters are present in each test document and then suffers a drastic loss in performance once it falls below this threshold. This can be explained by the fact that the handwriting styles of different writers cannot be effectively modeled without sufficient allographic information. A minimum length of text is necessary to perform a reasonably accurate statistical representation of the handwriting styles. Figure 2 also suggests that beyond this minimum threshold, any further increase in the amount of allographic information does not serve to improve the accuracy of the system. Hence, our methodology requires a minimum threshold of 160 characters in each test document for sufficient performance to be achieved. This is equivalent to approximately 30 words, or 3 lines.

Discussions

From the experimental results, the proposed methodology is able to generate high accuracies of 99.2% (one misclassified writer out of 120) with the misclassified writer being identified correctly in the rank 2 nd position. This is a remarkable improvement over Chan et al.'s results [START_REF] Chan | Online Text Independent Writer Identification Using Character Prototypes Distribution[END_REF], where they reported an accuracy of 95.12% (four misclassified writers out of 82), with all the misclassified writers only being correctly identified in the rank 11 th position. Furthermore, we have also determined that the length of text for each document has to be at least 160 characters in order to achieve sufficient performance when using this stochastic nearest neighbor character prototype approach for writer identification.

In this paper, the segmentation of the characters from the handwritten documents was done automatically using a text recognition engine based on the French language, where a corresponding French lexicon was attached to increase segmentation accuracies. This tool attained an average character recognition accuracy of 91% on the document datasets used in our experiments. With such a level of accuracy, characters which are not correctly recognized or segmented do not play a pivotal role on the identification writer rate. Hence, it will be interesting to investigate the performance of the writer identification system where the language domain is unknown or in an environment which contains a mixture of multi-lingual documents. Our current work focuses on this aspect of developing a general framework for writer identification based on multiple languages.

Figure 1 .

 1 Figure 1. Block diagram of character prototype methodFor each writer i,

Figure 2 .

 2 Figure 2. Number of misclassifications against the number of characters in each test document.

Table 1 .

 1 Performance of writer identification using different distance measures for classification

	1NN 2	Stochastic Nearest Neighbor Approach Euclidean distance Chi 2 distance
	96.7%	98.3%	99.2%
	This improvement over Chan et al.'s results can be
	explained as follows.

Acknowledgements

This research is jointly supported by Nanyang Technological University of Singapore, the French Merlion Scholarship, and the ANR grant CIEL 06-TLOG-009.