Margaux Elie

Umuhire Gilbert

Eric Mahoro

Jean-Luc Duverger

Richard Renaud

Sylvain Daniellou

Gaillard

Gilbert Umuhire Mahoro

Eric Duverger

Jean-Luc Renaud

Richard Daniellou

Sylvain Gaillard
email: sylvain.gaillard@ensicaen.fr

Cytotoxicity of cationic NHC copper(I) complexes coordinated to 2,2'-bis-pyridyl ligands

INTRODUCTION

Cancer therapy research was strongly impacted by the discovery of the cytotoxicity of cis-platin against different cancer cell lines, [START_REF] Rosenberg | Platinum Compounds: a New Class of Potent Antitumour Agents[END_REF] and the development of its congeners. [START_REF] Dasari | Cisplatin in cancer therapy: molecular mechanisms of action[END_REF][START_REF] Johnstone | The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs[END_REF] Transition metal complexes have been then extensively studied for anticancer applications. [START_REF] Michael | Novel Metals and Metal Complexes as Platforms for Cancer Therapy[END_REF][START_REF] Zhang | Recent Progress and Future Potential for Metal Complexes as Anticancer Drugs Targeting G-quadruplex DNA[END_REF][START_REF] Romero-Canelon | Next-generation metal anticancer complexes: multitargeting via redox modulation[END_REF][START_REF] Baile | Metal Ion Complex -Potential Anticancer Drug-A Review[END_REF][START_REF] Bergamo | Linking the future of anticancer metal-complexes to the therapy of tumour metastases[END_REF][START_REF]overview on metal anticancer compounds, see also the special issue 48 of Dalton Trans[END_REF] An important attention was focused on gold complexes which were considered as alternative to cis-platin because of their different biological mechanisms. Indeed, many research groups have demonstrated that the cytotoxicity of gold(I) and gold(III) complexes [START_REF] Cisnetti | Metal/N-heterocyclic carbene complexes: opportunities for the development of anticancer metallodrugs[END_REF][START_REF] Bertrand | A golden future in medicinal inorganic chemistry: the promise of anticancer gold organometallic compounds[END_REF][START_REF] Zou | Chemical biology of anticancer gold(III) and gold(I) complexes[END_REF] is mainly driven by the metal center which binds cysteine or selenocysteine protein fragments, inhibits thioredoxin reductase (TrxR) or leads to mitochondria swelling. [START_REF] Hickey | Mitochondria-Targeted Chemotherapeutics: The Rational Design of Gold(I) N-Heterocyclic Carbene Complexes That Are Selectively Toxic to Cancer Cells and Target Protein Selenols in Preference to Thiols[END_REF][START_REF] Hindi | The Medicinal Applications of Imidazolium Carbene-Metal Complexes[END_REF][START_REF] Rubbiani | Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties[END_REF][START_REF] Messori | The mode of action of anticancer gold-based drugs: a structural perspective[END_REF][START_REF] Rana | Novel Gold(I)-and Gold(III)-N-Heterocyclic Carbene Complexes: Synthesis and Evaluation of Their Anticancer Properties[END_REF][START_REF] Porchia | New insights in Au-NHCs complexes as anticancer agents[END_REF] These effects on cancer cells would allow to overcome some cell-line resistance to cisplatin. The NHC complexes based on isoelectronic less expensive and more abundant transition metals like silver(I) and copper(I) have been also investigated because they might behave similarly from an organometallic point of view. [START_REF] Teyssot | Metal-NHC complexes: a survey of anti-cancer properties[END_REF][START_REF] Mercs | Beyond catalysis: N-heterocyclic carbene complexes as components for medicinal, luminescent, and functional materials applications[END_REF][START_REF] Gautier | Advances in metal-carbene complexes as potent anti-cancer agents[END_REF][START_REF] Liu | Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs[END_REF][START_REF] Oehninger | N-Heterocyclic carbene metal complexes in medicinal chemistry[END_REF][START_REF] Aher | Recent developments of metal N-heterocyclic carbenes as anticancer agents[END_REF] Indeed, gold(I) and copper(I) complexes present similar affinities toward selenium and could be considered to have similar biological mechanism. [START_REF] Wang | Interactions of Cu(I) with selenium-containing amino acids determined by NMR, XAS, and DFT studies[END_REF] However, regarding the biological mechanisms proposed in the literature for different organometallics congeners of the column 11, they seems to act differently and the simple previous assumption appears to be an incorrect hypothesis. Indeed, as example, the group of Hecht has reported that the cleavage of DNA by bleomycin depends on the presence of copper(I) as metal cofactor. [START_REF] Ehrenfeld | Copper-Dependent Cleavage of DNA by Bleomycin[END_REF] Gautier et al. assumed that NHC copper complexes could generate reactive oxygen species (ROS) leading to a DNA strand break, responsible of the observed cytotoxicity. [START_REF] Lazreg | Synthesis of Homoleptic and Heteroleptic Bis-N-heterocylic Carbene Group 11 Complexes[END_REF] Consequently, the development of new cytotoxic transition metal complexes and more specifically Earth-abundant metal based complexes, might increase the panel of anticancer agents and furnish more drugs to overcome some cancer cell line resistance.

In the literature, several copper(I) complexes have been reported to exhibit IC 50 micromolar (µM) range against different cancer cell lines. These copper(I) complexes are generally coordinated to phosphane and/or nitrogen ligands. [START_REF] Marzano | Copper Complexes as Anticancer Agents, Anti-Cancer Agents in[END_REF][START_REF] Gandin | Novel mixed-ligand copper(I) complexes: role of diimine ligands on cytotoxicity and genotoxicity[END_REF][START_REF] Santini | Advances in copper complexes as anticancer agents[END_REF][START_REF] Komarnicka | Copper(I) complexes with phosphine derived from sparfloxacin. Part II: a first insight into the cytotoxic action mode[END_REF][START_REF] Lopes | New copper(I) and heteronuclear copper(I)-ruthenium(II) complexes: Synthesis, structural characterization and cytotoxicity[END_REF][START_REF] Morais | Important cytotoxic and cytostatic effects of new copper(i)phosphane compounds with N,N, N,O and N,S bidentate ligands[END_REF] Unlike its group 11 congeners, (NHC) copper(I) complexes are scarcely reported and are based on two families of general formula [CuX(NHC)] and [Cu(NHC) 2 ][X]. [START_REF] Teyssot | Metal-NHC complexes: a survey of anti-cancer properties[END_REF][START_REF] Mercs | Beyond catalysis: N-heterocyclic carbene complexes as components for medicinal, luminescent, and functional materials applications[END_REF][START_REF] Gautier | Advances in metal-carbene complexes as potent anti-cancer agents[END_REF][START_REF] Liu | Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs[END_REF][START_REF] Oehninger | N-Heterocyclic carbene metal complexes in medicinal chemistry[END_REF][START_REF] Aher | Recent developments of metal N-heterocyclic carbenes as anticancer agents[END_REF] Gautier et al. have firstly selected the [CuX(NHC)] complexes and compared their activities on different cancer cell lines such as MCF-7, MCF-7R, LNCaP, HL60 and KB. They demonstrated that the copper complexes stopped the cell cycle progression at the G1 phase. [START_REF] Lazreg | Synthesis of Homoleptic and Heteroleptic Bis-N-heterocylic Carbene Group 11 Complexes[END_REF] Then, the group of Tacke has synthesized copper complexes bearing benzyl-substituted NHC ligands and studied their cytotoxicity against CAKI-1. [START_REF] Streciwilk | Synthesis and cytotoxicity studies of p-benzyl substituted NHC-copper(I) bromide derivatives[END_REF][START_REF] Walther | In Vitro and In Vivo Investigations into The Carbene Copper Bromide Anticancer Drug Candidate WBC4[END_REF][START_REF] Tacke | Benzyl-substituted carbene-metal complexes: Potential for novel antibiotics and anticancer drugs[END_REF] In-vivo studies on nude mice with these aforementioned complexes showed encouraging results as strong inhibition growth of the tumor was noticed. [START_REF] Walther | In Vitro and In Vivo Investigations into The Carbene Copper Bromide Anticancer Drug Candidate WBC4[END_REF][START_REF] Tacke | Benzyl-substituted carbene-metal complexes: Potential for novel antibiotics and anticancer drugs[END_REF] We have recently reported a series of cationic (NHC)copper(I) complexes bearing 2,2'-dipyridylamine ligands (dpa) and exhibiting highly blue emissive properties. [START_REF] Marion | NHC copper(I) complexes bearing dipyridylamine ligands: synthesis, structural, and photoluminescent studies[END_REF][START_REF] Elie | Designing NHC-Copper(I) Dipyridylamine Complexes for Blue Light-Emitting Electrochemical Cells[END_REF][START_REF] Elie | Role of the Bridging Group in Bis-Pyridyl Ligands: Enhancing Both the Photo-and Electroluminescent Features of Cationic (IPr)Cu(I) Complexes[END_REF] We anticipated that such complexes would be of interest as cytotoxic agents. Indeed, their straightforward synthesis [START_REF] Gaillard | Highly efficient and economic synthesis of new substituted amino-bispyridyl derivatives via copper and palladium catalysis[END_REF] allows rapid modulation of the electronic and steric properties of the dpa ligands. Moreover, the functionalization via alkylation of the central nitrogen atom of the dpa ligand may open a route to their potential vectorization. [START_REF] Rauterkus | Cisplatin analogues with 2,2′dipyridylamine ligands and their reactions with DNA model nucleobases[END_REF][START_REF] Wang | Efficient Iridium Catalysts for Base-Free Hydrogenation of Levulinic Acid[END_REF][START_REF] Sauvageot | Antipseudomonal activity enhancement of luminescent iridium(iii) dipyridylamine complexes under visible blue light[END_REF] Adding a recognized biological framework to cytotoxic complexes would also allow their preferential accumulation in the targeted cells, and consequently, reduce the quantity of prodrug absorbed by the patient. [START_REF] Ren | Recent development of biotin conjugation in biological imaging, sensing, and target delivery[END_REF] Before going further with the development of such sophisticated ligands, the influence of different functionalized ligands (NHC and dpa) and anions on the cytotoxicity against cancer cells had to be evaluated. This study aimed to demonstrate (i) the influence of the substitution on dpa ligand or the nature of the anion on the anticancer activity and (ii) whether a wellfunctionalized dpa moiety could open a route to vectorization. [START_REF] Sauvageot | Antipseudomonal activity enhancement of luminescent iridium(iii) dipyridylamine complexes under visible blue light[END_REF] Moreover, the emissive properties of our cationic copper(I) complexes may also be interesting as potential fluorescent probes to detect the copper accumulation in the cell as their photophysical design is now understood. [START_REF] Ma | Bioactive luminescent transition-metal complexes for biomedical applications[END_REF][START_REF] Hemmert | Luminescent bioactive NHC-metal complexes to bring light into cells[END_REF] Herein, we present a comparative study of the cytotoxicity of various cationic (NHC) (dpa) copper(I) complexes and neutral (NHC) copper complexes having different anionic ligands such as chloride, iodide, hydroxide and cyanide (Fig. 1).

Figure 1. NHC copper complexes used in this study

MATERIAL AND METHODS

Synthesis of the copper(I) complexes.

General Considerations. All reactions were carried out using standard Schlenk technique under an atmosphere of dry argon. Solvents were purchased from Carlo Erba and degassed prior to use by bubbling argon gas directly in the solvent. Solvents for NMR spectroscopy were dried over molecular sieves. NMR spectra were recorded on 400 MHz and 500 MHz Bruker spectrometers. Proton ( 1 H) NMR information is given in the following format: multiplicity, coupling constant(s) (J) in Hertz (Hz), number of protons. Carbon ( 13 C) NMR spectra are reported in ppm (δ) relative to residual CHCl 3 (δ 77.0) unless noted otherwise. HRMS were performed by LCMT analytical services. NMR solvent was passed through a pad of basic alumina before uses. 2,2'-dipyridylamine (dpa) was purchased from Sigma-Aldrich and used without prior purification. The complexes [CuCl(SIPr)] ( 9), [CuCl(IMes)] [START_REF] Cisnetti | Metal/N-heterocyclic carbene complexes: opportunities for the development of anticancer metallodrugs[END_REF] and [CuCl(SIMes)] (11) were purchased from Strem and used without prior purification. The imidazolium salts used for the preparation of the N-Heterocyclic Carbenes [START_REF] Arduengo | Imidazolylidenes, Imidazolinylidenes and Imidazolidines[END_REF][START_REF] Nolan | Synthesis of 1,3 disubstituted Imidazolium Salts[END_REF][START_REF] Arduengo | Fused polycyclic nucleophilic carbenes -synthesis, structure, and function[END_REF][START_REF] Lane | Iron-catalyzed formation of 2-aminopyridines from diynes and cyanamides[END_REF] and the 2,2'dipyridylamine derivatives [START_REF] Elie | Designing NHC-Copper(I) Dipyridylamine Complexes for Blue Light-Emitting Electrochemical Cells[END_REF][START_REF] Gaillard | Highly efficient and economic synthesis of new substituted amino-bispyridyl derivatives via copper and palladium catalysis[END_REF] were synthesized as previously reported. Complexes [CuCl(NHC)]

(1, 5-8) where synthesized according to reported procedures. [START_REF] Citadelle | Simple and versatile synthesis of copper and silver N-heterocyclic carbene complexes in water or organic solvents[END_REF][START_REF] Santoro | A general synthetic route to [Cu(X)(NHC)] (NHC = N-heterocyclic carbene, X = Cl, Br, I) complexes[END_REF] Complexes [CuI(IPr)] (2) [START_REF] Zheng | A novel and efficient (NHC)CuI (NHC=N-heterocyclic carbene) catalyst for the oxidative carbonylation of amino compounds[END_REF] [CuOH(IPr)] (3) [START_REF] Fortman | A Versatile Cuprous Synthon: [Cu(IPr)(OH)] (IPr = 1,3 bis(diisopropylphenyl)imidazol-2-ylidene)[END_REF] were prepared by the reported methods. Complexes [Cu(NHC)(dpa)][PF 6 ] 12, 18-26 were prepared following our previously reported procedure. [START_REF] Elie | Designing NHC-Copper(I) Dipyridylamine Complexes for Blue Light-Emitting Electrochemical Cells[END_REF] Complex [CuCN(IPr)] (4). In a flame-dried Schlenk tube under argon atmosphere, [CuCl(IPr)] (1) (0.3 mmol, 150 mg, 1 equiv.) and KCN (0.3 mmol, 19 mg, 1 equiv.) were introduced in degassed MeOH (5 mL) and the reaction mixture was stirred under reflux (50 °C) for 4 hours. After returning to room temperature, the reaction mixture was concentrated to dryness under vacuum. The complex was then dissolved in dichloromethane and filtered through a pad of Celite® and concentrated again under vacuum. A purification by recrystallization by slow diffusion of pentane in a THF solution of the complex led to the pure complex (4) as a white powder (143 mg, 97 % yield). 1 H-NMR (CDCl 3 , 400 MHz): δ 1.22 (d, J = 6.9 Hz, 12H), 1.27 (d, J = 6.9 Hz, 12H), 2.50 (sept, J = 6.9 Hz, 4H), 7.14 (s, 2H), 7.30 (d, J = 7.8 Hz, 4H), 7.50 (t, J = 7.8 Hz, 4H) ppm. (spectroscopic data in good agreement with the literature). [START_REF] Fortman | A Versatile Cuprous Synthon: [Cu(IPr)(OH)] (IPr = 1,3 bis(diisopropylphenyl)imidazol-2-ylidene)[END_REF]

General procedure for the synthesis of complexes having general formula of [Cu(IPr)(dpa)][X] 13-17.

Following the general procedure described previously for the synthesis of complexes 12, [START_REF] Elie | Designing NHC-Copper(I) Dipyridylamine Complexes for Blue Light-Emitting Electrochemical Cells[END_REF] the KPF 6 salt of the aqueous treatment at the end of the reaction was replaced by the appropriate salt to give complexes 13-17.

[Cu(IPr)(dpa)][BF 4 ] complex 13. Following the general procedure with copper complex 1 (162.5 mg, 0.33 mmol), dpa ligand (59.9 mg, 0.35 mmol) and NaBF 4 as salt for the final aqueous solution, complex 6.9 Hz, 12H), 1.22 (d, J = 6.9 Hz, 12H), 2.65 (sept, J = 6.9 Hz, 4H), 6.13 (dd, J = 5.5 and 1.5 Hz, 2H), 6.28 (t, J = 6.3 Hz, 2H), 7.20-7.23 (m, 4H), 7.33 (d, J = 7.8 Hz, 4H), 7.44 (dt, J = 1.8 and 6.9 Hz, 2H), 7.58 (t, J = 7.8 Hz, 2H), 8.81 (s, 1H) ppm. 13 C-NMR (CDCl 3 , 100 MHz): δ 24.0 (4xCH 3 ), 24.2 (4xCH 3 ), 28.3 (4xCH) [Cu(IPr)(dpa)][BPh 4 ] complex 14. Following the general procedure with copper complex 1 (162.5 mg, 0.33 mmol), dpa ligand (59.9 mg, 0.35 mmol) and NaBPh 4 as salt for the final aqueous solution, complex 14 was obtained as a white powder (293 mg, 94% yield). [Cu(IPr)(dpa)][NTf 2 ] complex 15. Following the general procedure with copper complex 1 (146.3 mg, 0.30 mmol), dpa ligand (53.6 mg, 0.32 mmol) and LiNTf 2 as salt for the final aqueous solution, complex 15 was obtained as a white powder (269 mg, 99% yield). 1 H-NMR (CDCl 3 , 500 MHz): δ 1.08 (d, J = 6.7 Hz, 12H), 1.23 (d, J = 6.7 Hz, 12H), 2.65 (sept, J = 6.7 Hz, 4H), 6.17 (d, J = 4.4 Hz, 2H), 6.33 (t, J = 6.3 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 7.22 (s, 2H), 7.33 (d, J = 7.8 Hz, 4H), 7.48 (t, J = 7.2 Hz, 2H), 7.58 (t, J = 7.8 Hz, 2H), 8.49 (br s, 1H) ppm. 13 C-NMR (CDCl 3 , 100 MHz): δ 23.9 (s, 4xCH 3 ), 24.1 (s, 4xCH 3 ), 28.7 (s, 4xCH), 114.9 (s, 2xCH), 116.3 (s, 2xCH), 119.9 (q, J = 321 Hz, 2xC, 2xCF 3 from NTf 2 anion), 123.5 (s, 2xCH), 124.7 (s, 4xCH), 130.6 (s, 2xCH), 135.9 (s, 2xC), 139.0 (s, 2xCH), 146.0 (s, 4xC), 147.5 (s, 2xCH), 153.0 (s, 2xC), 183.0 (s, C) ppm.

[Cu(IPr)(dpa)][B(3,5-(CF 3 ) 2 Ph

) 4 ] complex 16. Following the general procedure with copper complex 1 (87.8 mg, 0.18 mmol), dpa ligand (32.5 mg, 0.19 mmol) and NaB(3,5-(CF 3 ) 2 Ph) 4 as salt for the final aqueous solution, complex 16 was obtained as a white powder (208 mg, 78% yield). 1 H-NMR (CDCl 3 , 400 MHz): δ 1.05 (d, J = 6.9 Hz, 12H), 1.22 (d, J = 6.9 Hz, 12H), 2.63 (sept, J = 6.9 Hz, 4H), 6.19 (dd, J = 5.5 and 1.3 Hz, 2H), 6.39 (t, J = 6.9 Hz, 2H), 6.49 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 7.24 (s, 2H),

Biological study details

The growth level of four cancer cell lines was determined using a colorimetric MTT (thiazolyl blue tetrazolium bromide, Sigma) assay. Cancer cell lines and growth medium were obtained from CLS Cell Line Service GmbH, Eppelheim, Germany). Human skin melanoma SK-Mel-28 and human brain glioma HS-683 were grown in DMEM supplemented with 4.5 g/l glucose, L-glutamine and 10% FBS.

The human lung carcinoma cell line A549 was grown in DMEM:Ham's F12 (1:1) supplemented with Lglutamine and 5% FBS and human breast adenoma carcinoma MCF-7 in EMEM supplemented with Lglutamine, sodium pyruvate, NEAA and 10 % FBS. MTT assay is based on the reduction of the yellow product thiazolyl blue tetrazolium bromide (MTT) to purple-blue formazan by mitochondrial dehydrogenase of metabolically active cells. The number of living cells after incubation in the presence (or absence, control) of the tested molecule is directly proportional to the blue color which was measured by spectrophotometry. Briefly, cells were seeded (200 µl of a 5.10 4 cells/ml suspension) in 96-well culture plates (TPP, Trasadingen, Switzerland) and incubated for 24h. Each compound (starting from DMSO solutions, stable for months) was assessed in serial dilution (four concentrations in 0.5% DMSO at the highest concentration) in six replicates (n=6) and incubated for 72 h. Thereafter, MTT (5 mg/ml solution in PBS) was added to each well (10% v/v) and cells were further incubated for 4 hours.

Then, after removing the culture medium, the blue crystals were dissolved in 100 µL SDS-acidicisopropanol solution (0.5% SDS; 80 mM HCl) and absorbance measured at 540 nm using a 620 nm reference. Absorbance of the serial dilution of each cell line treated under the same conditions but without the tested compounds was measured to generate a standard curve allowing IC 50 determination (IC 50 is defined as the concentration reducing cell growth by 50%).

RESULTS AND DISCUSSION

Synthesis and characterization

The imidazolium salts [START_REF] Arduengo | Imidazolylidenes, Imidazolinylidenes and Imidazolidines[END_REF][START_REF] Nolan | Synthesis of 1,3 disubstituted Imidazolium Salts[END_REF][START_REF] Arduengo | Fused polycyclic nucleophilic carbenes -synthesis, structure, and function[END_REF][START_REF] Lane | Iron-catalyzed formation of 2-aminopyridines from diynes and cyanamides[END_REF] and 2,2'-dipyridylamine derivatives [START_REF] Elie | Designing NHC-Copper(I) Dipyridylamine Complexes for Blue Light-Emitting Electrochemical Cells[END_REF][START_REF] Gaillard | Highly efficient and economic synthesis of new substituted amino-bispyridyl derivatives via copper and palladium catalysis[END_REF] were prepared according to reported procedures.

The [CuCl(NHC)] precursors 1 and 5-8 were synthesized following the Nolan-Cazin procedure using Cu 2 O as metal precursor and the appropriate imidazolium salt and complexes 1, 5-8 were isolated in 40 to 94% yield (Scheme 1, i). [START_REF] Citadelle | Simple and versatile synthesis of copper and silver N-heterocyclic carbene complexes in water or organic solvents[END_REF][START_REF] Santoro | A general synthetic route to [Cu(X)(NHC)] (NHC = N-heterocyclic carbene, X = Cl, Br, I) complexes[END_REF] Then, complex 2 was prepared in 53% isolated yield by reacting 1,3-bis(-2,6-di-iso-propylphenyl)-4,5-dichloroimidazolium chloride in the presence of KOtBu and CuI (Scheme 1, ii). [START_REF] Zheng | A novel and efficient (NHC)CuI (NHC=N-heterocyclic carbene) catalyst for the oxidative carbonylation of amino compounds[END_REF] The other NHC copper(I) complexes 9-11 were commercially available and were introduced in order to evaluate other N-aromatic substituent on the NHC ligand. The (NHC)copper hydroxide complex 3 was synthesized via a known procedure [START_REF] Fortman | A Versatile Cuprous Synthon: [Cu(IPr)(OH)] (IPr = 1,3 bis(diisopropylphenyl)imidazol-2-ylidene)[END_REF] and was also used as precursor for the synthesis of some cationic complexes described below (Scheme 1, iii). The exchange of the chlorine ligand by a cyano ligand in degassed methanol furnished the complex 4 in 97% isolated yield (Scheme 1, iv). Scheme 1. General scheme for the synthesis of neutral complexes 1-8.

Then, complexes 1, 3 and 5-8 were used as precursors for the synthesis of cationic copper complexes bearing 2,2'-dipyridylamine (dpa) ligands. Complexes 12-21, 23, 24 and 26 were obtained from 1, or 5-8 via the exchange of the chloride atom by a dpa ligand in refluxed methanol, followed by an anion metathesis (Scheme 2, i). [START_REF] Elie | Designing NHC-Copper(I) Dipyridylamine Complexes for Blue Light-Emitting Electrochemical Cells[END_REF] The expected cationic (NHC)copper(I) complexes were isolated in 76 to 99% yields. In details, the synthesis of complexes 12 to 17 were carried out with KPF 6 , NaBF

Cytotoxicity studies.

Half growth inhibition concentration (IC 50 ) of all the copper(I) complexes were measured on four cancer lines, namely MCF-7 (breast cancer), A-549 (lung carcimona), SK-Mel-28 (melanoma) and HS-683 (glioma) in order to establish structure/cytotoxicity relationships toward each cancer cell lines independently. The choice for the three first cancer lines was directed by the previous studies with gold complexes. [START_REF] Teyssot | Metal-NHC complexes: a survey of anti-cancer properties[END_REF][START_REF] Mercs | Beyond catalysis: N-heterocyclic carbene complexes as components for medicinal, luminescent, and functional materials applications[END_REF][START_REF] Gautier | Advances in metal-carbene complexes as potent anti-cancer agents[END_REF][START_REF] Liu | Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs[END_REF][START_REF] Oehninger | N-Heterocyclic carbene metal complexes in medicinal chemistry[END_REF][START_REF] Aher | Recent developments of metal N-heterocyclic carbenes as anticancer agents[END_REF] In addition, the cytotoxicity of [(NHC)CuCl] complexes against MCF-7 was also reported by Gautier and this cell line was used as reference for this work. [START_REF] Lazreg | Synthesis of Homoleptic and Heteroleptic Bis-N-heterocylic Carbene Group 11 Complexes[END_REF] The growth level of the four cancer cell lines was determined using a colorimetric MTT (thiazolyl blue tetrazolium bromide, Sigma) assay. MTT assay is based on the reduction of the yellow product thiazolyl blue tetrazolium bromide (MTT) to purple-blue formazan by mitochondrial dehydrogenase of metabolically active cells. The number of living cells after incubation in the presence (or absence, control) of the tested molecule is directly proportional to the blue color, which was measured by spectrophotometry. Absorbance spectra were measured to generate a standard curve allowing IC 50 determination (IC 50 is defined as the concentration reducing cell growth by 50 %). All active complexes were then subjected to their IC 50 evaluation on a 10 000 cells population.

Results on the MCF-7 are presented in table 1 and chart 1. Etoposide and 5-fluorouracil, two commercially available generic drugs classically used to treat cancer, were used as positive controls. We were pleased to observe cytotoxicity toward the breast cancer MCF-7 cell lines for all copper complexes with IC 50 around 100 to 1000 fold lower than the two reference compounds i.e. Etoposide and 5fluorouracil (Table 1, Chart 1). Concerning the neutral copper complexes 1-11, the IC 50 were found between 0.015 ± 0.020 µM (for complex 4 and 9, entries 4 and 9, Table 1) and 0.30 ± 0.05 µM (for complexes 2 and 7, entries 2 and 7, Table 1) except for complex 11 bearing SIMes ligand with a much higher IC 50 of 1.45 ± 0.10 than other neutral (NHC)copper(I) complexes. This surprising IC 50 for complex 11 is 20 times higher than the IC 50 previously reported by Gautier et al. (0.075 ± 0.002 µM on a 3 000 cells population). [START_REF] Lazreg | Synthesis of Homoleptic and Heteroleptic Bis-N-heterocylic Carbene Group 11 Complexes[END_REF] Nevertheless, the IC 50 value of complex 11 is still lower than the IC 50 of cis-platin in similar conditions. [START_REF] Johnson | Recent Developments in the Medicinal Applications of Silver-NHC Complexes and Imidazolium Salts[END_REF] Then, when neutral complexes 1-4 are now compared, the best X ligand appeared to be the cyanide present in complex 4. Indeed, 4 exhibited a IC 50 of at least 10 fold lower than the complexes 1-3 (Entries 1-4, Table 1). Gratefully, cationic copper(I) complexes 12-26 also presented a cytotoxic activity against MCF-7 cell-line. To the best of our knowledge, no cytotoxicity against breast cancer MCF-7 cell lines has ever been reported for cationic (NHC)copper(I) complexes. Most of the IC 50 ranged from 0.03 ± 0.01 to 0. 1). The comparison of these IC 50 gave us some trends on the structure/cytotoxicity property relationships, (i) the presence of electron donor or withdrawing substituent on the pyridine ring of the dpa skeleton in these cationic copper(I) complexes led to higher IC 50 and unsubstituted dpa should be favoured, and (ii) large fluorinated tetraarylborate anions B(3,5-F 3 C-C 6 H 3 ) 4 -and B(C 6 F 5 ) 4 -have a negative effect on the cytotoxicity against MCF-7. The latter might be due to solubility or lipophilicity issues. Very interestingly, the N-alkylated dpa complex 26 presents a higher IC 50 than the most active cationic copper(I) complex but still below 1 µM (0.19 ± 0.17 µM, Entry 30, Table 1). Nevertheless, this complex appears more active than Etoposide, 5F-uracil and the cis-platin. In the second survey, all the copper complexes were studied toward lung carcimona A-549. All complexes were found active, the IC 50 were determined and reported in table 2 and chart 2. In the neutral complexes series, complexes 1-3, 5-6 and 8-10 exhibited low IC 50 between 0.04 ± 0.01 µM (complex 1) and 0.15 ± 0.03 µM (complex 10) (Entries 1-3, 5-6, 8-10, Table 2). Then, complexes 4 and 7 presented a higher IC 50 of 0.6 ± 0.2 and 0.30 ± 0.06 µM respectively, but still lower than Etoposide and 5-fluorouracil (Entries 4 and 7 vs. 12 and 13). Noteworthy, because of solubility issues above 3 µM of complex 11 its IC 50 was not determined (Entry 11, Table 2). In details, the copper complexes of general formula [CuX(NHC)], 1 and 2 appeared to be more active than other inner sphere anion such as HO -(complex 3) and NC -(complex 4) (Entries 1-4, Table 2). In sharp contrast with the results on the MCF-7 cell line, the complex 4 bearing a cyanide ligand showed the lowest activity with a IC 50 of 0.6 ± 0.2 µM (Entry 4, Table 2). Cationic copper(I) complexes presented much higher IC 50 than neutral (NHC)copper complexes, except for complexes 13-15 having unsubstituted dpa and IPr ligands (Entries 15-17, Table 2). However, these copper complexes remained more active than the reference drugs (Entries 12-13, Table 2). In this cationic copper(I) complexes series, the non-coordinating anion appeared to be structurally important as complexes 13-15 with BF 4 -, BPh 4 -and NTf 2 -anions exhibited IC 50 (at maximum 0.06 ± 0.02 µM) similar to the most active neutral copper complexes 1 and 8 (Entries 1, 8, 15-18, Table 2). Then, adding substituents on the IPr motif decreased the IC 50 (Complex 12 vs.

complexes 18-21, entries 14 and 20-23, Table 2) and, when the dpa substitution is considered, the Nsubstituted dpa complex presented the lowest IC 50 (complex 26 vs. complexes 12, 22-25), with a value of 0.78 ± 0.10 µM for complex 26 (Entry 28, Table 2). The cytotoxicity of the copper(I) complexes toward the Melanoma SK-Mel-28 cancer cells was then evaluated (Table 3 and Chart 3). For neutral complexes 1-11, all the IC 50 were in a smaller range (0.04 ± 0.002 for 5 to 0.42 ± 0.04 for 11) compared to the two previous cancer cell lines. In addition, complexes 1-11 have IC 50 at least 15 fold lower than etoposide (IC 50 of 6.5 ± 0.8), the most active commercially available reference used in this study (Entries 1-13, Table 3). Concerning the cationic copper complexes, the range of IC 50 was found between 0.09 ± 0.02 for complex 20 and 1.80 ± 0.60 µM for complex 23 (Entries 14-28, Table 3). The role of the anion seemed to have a little impact on the activity and its nature was not as important as observed with the MCF-7

and A-549 cell lines (Entries 14-19, Table 3). Then, it appeared that electron donating substituent on the IPr moiety decreased the IC 50 (Complex 12 vs complexes 18-20, Entries 14 and 20-22, Table 3), while, in sharp contrast, the electron withdrawing chloride substituent on the C4 and C5 positions of the IPr ligand led to an increase of the IC 50 (Complex 12 vs complex 21, Entries 14 and 23, Table 3). The fourth cancer cell line we studied in this work was Glioma HS-683. The IC 50 were presented in table 4 and chart 4. Fortunately, all the copper(I) complexes presented a higher cytotoxicity toward Glioma HS-683 than the commercially available drugs etoposide and 5-fluorouracil, which exhibited IC 50 of 4.00 ± 0.52 and 16.7 ± 12.2, respectively (Entries 12 and 13, Table 4). And even more interesting was the micromolar range of the IC 50 of some (NHC) copper(I) complexes (Entries 1-3, 5-6, 8-11, 16-17, 20, 22 and 24, Table 4). As example, in the neutral copper complex family, the most active (NHC) copper(I) complexes were 5 and 10 with IC 50 of 0.21 ± 0.01 and 0.20 ± 0.05, respectively (Entries 5 and 10, Table 4). Cationic copper(I) complexes exhibited a lower cytotoxicity than neutral copper(I) complexes 1-11 except complex 14, for which an IC 50 of 0.22 ± 0.01 µM was determined, similarly to complexes 5 and 10 (Entry 16 vs entries 5 and 10, Table 4). In the cationic complex series, IC 50 were not determined for 16 and 17 due to solubility issues above 3 µM (Entries 16 and 17, Table 4). Complexes 23 and 25 presented the lowest cytotoxicity with IC 50 of 3.60 ± 0.40 and 3.50 ± 0.70, respectively (Entries 25 and 27, Table 4). Concerning complex 26, having the N-alkylated dpa ligand, its IC 50 was found at 1.8 ± 0,1 µM representing a cytotoxicity 10 fold lower than the one of 14 (Entries 16 and 28, Table 4). Even if BPh 4 -anion is very hydrophobic, this anion appeared to bring the highest cytotoxicity to cationic NHC copper complexes as 14 presented the lowest IC 50 compared to complexes 12-17 (Entry 16, Table 4). Therefore, an eventual anion metathesis on complex 26 might lead to more active complexes and balance the negative effect of the N-alkyl substitution on the dpa ligand. Finally, the evolution and stability of complexes 1 and 12 in DMSO-d6 solution was evaluated by 1 H NMR spectroscopy (Figure S42 and S43 in supplementary information).

After 14 h, no evolution of both complexes was noticed. Then, the stability study was assessed for complex 12 in biological medium (1%DMSO/DMEM+GlutaMAX-ITM solution) in absence of cells and was monitored by UV-visible absorption spectroscopy for 25 hours (Figure 2). As control, the same experiment was performed in the biological medium.

Surprisingly, the absorption spectra of the medium presented important changes in both UV (274 nm) and visible region (560 nm) (See Supporting Information, Figure S1). As the ligands (dpa and NHC) and complex 12 have only their maximal absorption wavelength in the UV region, our analysis of the evolution, in function of time, of the absorption spectra of Encouragingly, the UV-visible spectra in the 1%DMSO/DMEM+GlutaMAX-I TM solution did not dramatically change in the 260 and 315 nm region within 4 to 5 h. After this period of time, a slow decrease of the absorbance was observed: 13 and 26% at 260 nm, 17 and 39% at 312 nm after 8 and 25 h, respectively. In a few words, the integrity of the cationic complex 12 seems to be maintained during the first 4 to 5 h in the biological medium and such stability might open a door to vectorizable copper complexes bearing a dpa ligand.

CONCLUSION

We reported the synthesis of several new neutral and cationic (NHC)copper(I) complexes.

The cytotoxicity of all these copper(I) complexes was studied on three cancer lines, breast cancer MCF-7, lung carcimona A-549 and Melanoma SK-Mel-28 which were previously reported to be sensitive to (NHC)gold(I) complexes. In addition, we also reported cytotoxicity of these (NHC)copper(I) complexes toward Glioma HS-683. This work further demonstrates that copper complexes can act as anticancer agents like their group 11 Finally, complexes bearing substituted dpa present higher IC 50 , whatever the electron donating or electron withdrawing properties of the substituent. Such results might be due to steric hindrance issues. Finally, the complex 26, which could be seen as a model of a future vectorizable complex if the dpa is substituted by a biologically recognized framework, has higher IC 50 than its non-substituted congeners. However, its cytotoxicity against the four cell lines is higher than commercially available anticancer agents. All these observations might give some guidelines for the future design of more efficient cationic copper(I) complexes.

  1 H-NMR (CDCl 3 , 400 MHz): δ 1.06 (d, J = 6.8 Hz, 12H), 1.23 (d, J = 6.8 Hz, 12H), 2.62 (sept, J = 6.8 Hz, 4H), 5.67 (d, J = 8.4 Hz, 2H), 5.91 (s, 1H), 6.07 (d, J = 4.4 Hz, 2H), 6.22 (t, J = 6.2 Hz, 2H), 6.79 (t, J = 6.8 Hz, 4H, BPh 4 ), 6.97 (t, J = 7.3 Hz, 8H, BPh 4 ), 7.13 (s, 2H), 7.16 (dd, J = 11.4 and 4.4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 4H), 7.49 (br s, 8H, BPh 4 ), 7.56 (t, J = 7.8 Hz, 2H) ppm. 13 C-NMR (CDCl 3 , 125MHz): δ 24.0 (4xCH 3 ), 24.2 (4xCH 3 ), 28.7 (4xCH), 115.2 (2xCH), 116.3 (2xCH), 123.4 (4xCH, BPh 4 ), 123.4 (2xCH), 124.7(4xCH), 125.8 (q, 2 J 13C-11B = 2.8 Hz, 8xCH, BPh 4 ), 130.6 (2xCH), 135.9 (2xC), 136.2 (8xCH, BPh 4 ), 138.7 (2xCH), 146.0 (4xC), 147.3 (2xCH), 152.0 (2xC), 163.1 (q, 1 J 13C-11B = 49.3 Hz, 4xC, BPh 4 ), 181.9 (C) ppm. 11 B-NMR (CDCl 3 , 100MHz): δ -6.40 ppm. IR (neat): ν 3339, 2961, 1619, 1578, 1466, 1157, 731, 705 cm -1 . HRMS (ESI): m/z calcd for C 37 H 45 CuN 5 [M-BPh 4 ] + : 622.2971; found: 622.2989. Elemental Anal. Calcd for C 61 H 65 BCuN 5 : C, 77.73; H, 6.95; N, 7.43; found: C, 77.53; H, 7.12; N, 7.49.

7. 33

 33 (d, J = 7.8 Hz, 4H), 7.42 (dt, J = 1.8 and 6.9 Hz, 2H), 7.49 (br s, 4H, B(3,5-(CF 3 ) 2 Ph) 4 ), 7.57 (t, J = 7.8 Hz, 2H), 7.70 (br s, 8H, B(3,5-(CF 3 ) 2 Ph) 4 ) ppm. 13 C-NMR (CDCl 3 , 100 MHz): δ 23.8 (4xCH 3 ), 24.1 (4xCH 3 ),28.7 (2xCH), 113.4 (2xCH), 117.3 (2xCH), 117.4-117.5 (m, 4xCH, B(3,5-(CF 3 ) 2 Ph) 4 ), 123.5 (2xCH), 124.5 (q, 1 J 13C-19F = 272.5 Hz, 8xCF 3 , B(3,5-(CF 3 ) 2 Ph) 4 ), 124.7 (4xCH), 128.8 (qq, 2 J 13C- 19F = 31.4 Hz and 4 J 13C-19F = 2.8 Hz, 8xC, B(3,5-(CF 3 ) 2 Ph) 4 ), 130.7 (2xCH), 134.8 (8xCH, B(3,5-(CF 3 ) 2 Ph) 4 ), 135.8 (2xC), 139.5 (2xCH), 146.0 (4xC), 148.4 (2xCH), 152.0 (2xC), 161.7 (q, 1 J 13C-11B = 49.8 Hz, 4xC, B(3,5-(CF 3 ) 2 Ph) 4 ), 182.5 (C).

4 ,Scheme 2 . 4 .

 424 Scheme 2. General scheme for the synthesis of cationic complexes 12-26.

Chart 1 .

 1 IC 50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on MCF-7.

Chart 2 .

 2 IC 50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on A-549.

Chart 3 .

 3 IC 50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on SK-Mel-28.

Chart 4 .

 4 IC 50 of neutral (NHC)copper(I) complexes 1-11 (red) and [Cu(NHC)(dpa)][X] 12-26 (blue) on HS-683.

complex 12 ,Figure 2 .

 122 Figure 2. Stability of 12 followed by UV-vis spectroscopy over 25 h in 1%DMSO/ DMEM+GlutaMAX-I™ solution (c = 50 µM) and UV-visible spectra of 12 in dichloromethane solution (c = 100 µM) (red line, inset) and in 1%DMSO/ DMEM+GlutaMAX-I™ solution (c = 50 µM) in absence of cell line (dark line, inset).

  congeners and are more active than commercially available anti-cancer agents. Some of the cationic (NHC)copper(I) complexes 12-26, bearing NHC and dpa ligands exhibit similar submicromolar IC 50 than neutral [CuX(NHC)] complexes 1-11 and, in all cases, lower than the commercial Etoposide and 5-fluorouracil drugs.Chart 5 depicts the whole IC 50 , on the four cell lines determined in this study. This overview highlights that neutral (NHC) copper(I) complexes are in general more active than the cationic (NHC) copper(I) analogs bearing dpa ligands. Nevertheless, in few cases, the cytotoxicity of some cationic copper(I) complexes is comparable to the one observed with the neutral copper(I) complexes and depends on the nature of the anion, the electronic property or the steric hindrance of their ligands. Complexes 13-15 having a small anion, present similar cytotoxicity to neutral copper(I) complexes toward MCF-7 and lung carcimona A-549. Complexes 18-20, having electron rich substituent on the NHC moiety, present also IC 50 in the range of the most active neutral (NHC)copper(I) complexes toward MCF-7 and SK-Mel-28.

Chart 5 .

 5 IC 50 of complexes 1-26 on the four cancer cell lines (MCF-7, A-549, SK-Mel-28 and HS-68)

19 F-NMR (CDCl 3 , 377 MHz): δ 151.27 ppm. 11 B-NMR (CDCl 3 , 100 MHz): δ

	, 115.3 (2xCH), 116.1 (2xCH), 123.4 (2xCH), 124.7 (4xCH), 130.61 (2xCH),
	136.0 (2xC), 138.9 (2xCH), 146.0 (4xC), 147.4 (2xCH), 153.2 (2xC), 183.2 (C) ppm. -0.76 ppm. IR (neat): ν 2964,
	1634, 1582, 1478, 1228, 1072, 761 cm -1 . HRMS (ESI): m/z calcd for C 37 H 45 CuN 5 [M-BF 4 ] + : 622.2971;
	found: 622.2989. Elemental Anal. Calcd for C 37 H 45 BCuF 4 N 5 : C, 62.58; H, 6.39; N, 9.86; found: C,
	62.31; H, 6.52; N, 9.89.

19 F-NMR (CDCl 3 , 400 MHz): δ

  -78.80 (s, 2xF, 2xCF 3 from NTf 2 anion) ppm. IR (neat) ν 3357, 1626, 1581, 1469, 1356, 1197, 1139, 1050, 806, 762, 740, 696 cm -1 . HRMS (ESI): m/z calcd for C 37 H 45 CuN 5 [M-NTf 2 ] + : 622.2971; found: 622.2973. Elemental Anal. Calcd for C 39 H 45 CuF 6 N 6 O 2 S 2 : C, 51.85; H, 5.02; N, 9.30; found: C, 51.67; H, 5.21; N, 9.33.

11 B-NMR (CDCl 3 , 100 MHz): δ

  19 F-NMR (CDCl 3 , 377 MHz): δ -60.41 ppm. -6.60 ppm. IR (neat): ν 2967, 1625, 1585, 1473, 1352, 1272, 1156, 1124, 887, 681, 668 cm -1 . HRMS (ESI): m/z calcd for C 37 H 45 CuN 5 [M-B(3,5-(CF 3 ) 2 Ph) 4 ] + : 622.2971; found: 622.2959. Elemental Anal. Calcd for C 69 H 57 BCuF 24 N

5 : C, 55.75; H, 3.87; N, 4.71; found: C, 55.62; H, 4.02; N, 4.72. [Cu

(IPr)(dpa)][B(F 5 Ph) 4 ] complex 17.

  Following the general procedure with copper complex 1 (97.5 mg, 0.20 mmol), dpa ligand (36 mg, 0.21 mmol) and NaB(F 5 Ph) 4 as salt of the final aqueous solution, complex 17 was obtained as a white powder (198 mg, 76% yield).1

H-NMR (CDCl 3 , 400 MHz): δ

	1.07
	(d, J = 6.7 Hz, 12H), 1.23 (d, J = 6.7 Hz, 12H), 2.65 (sept, J = 6.7 Hz, 4H), 6.21 (d, J = 4.8 Hz, 2H),
	6.42 (t, J = 6.3 Hz, 2H), 6.61 (d, J = 8.3 Hz, 2H), 6.89 (br s, 1H), 7.24 (s, 2H), 7.35 (d, J = 7.8 Hz, 4H),
	7.50 (t, J = 7.9 Hz, 2H), 7.59 (t, J = 7.8 Hz, 2H) ppm. 13 C-NMR (CDCl 3 , 100 MHz): δ 23.8 (s, 4xCH 3 ),
	24.1 (s, 4xCH 3 ), 28.7 (s, 4xCH), 113.62 (s, 2xCH), 117.2 (s, 2xCH), 123.5 (s, 2xCH), 124.8 (s, 4xCH),
	130.7 (s, 2xCH), 135.0 (m, 4xC, B(F 5 Ph) 4 ), 135.9 (s, 2xC), 137.5 (m, 8xC, B(F 5 Ph) 4 ), 139.5 (s, 2xCH),
	146.0 (s, 4xC), 147.0 (m, 4xC, B(F 5 Ph) 4 ), 148.3 (s, 2xCH), 149.3 (m, 8xC, B(F 5 Ph) 4 ), 152.2 (s, 2xC),
	182.7 (s, C) ppm.

19 F-NMR (CDCl 3 , 400 MHz): δ

  -166.8 (t, J = 17.5 Hz, 8xF, B(F 5 Ph) 4 ), -163.0 (t, J = 20.5 Hz, 8xF, B(F 5 Ph) 4 ), -132.7 (br s, 4xF, B(F 5 Ph) 4 ) ppm.

11 B-NMR (CDCl 3 , 100 MHz): δ

  -16.67 (s, B(F 5 Ph) 4 ) ppm. IR (neat): ν 2966, 1638, 1515, 1462, 1088, 979, 773 cm -1 . HRMS (ESI): m/z calcd for C 37 H 45 CuN 5 [M-B(F 5 Ph) 4 ] + : 622.2971; found: 622.2974. Elemental Anal. Calcd for C 61 H 45 BCuF 20 N 5 :

	C, 56.26; H, 3.48; N, 5.38; found: C, 56.02; H, 3.65; N, 5.41.

  28 ± 0.05 excepted for complexes 16, 17 and 25 (Entries 20, 21 and 29, Table1) and were generally slightly higher than those determined for neutral copper(I) complexes 1-10. These values seem

to indicate that large anions (complexes 16 and 17) and electron deficient substituent at the 5 position of the pyridine ring in dpa ligand (complex 25) are not suitable for a high cytotoxicity. The best activities were found for complexes 15 and 20, both having an unsubstituted dpa, small anion and electron rich NHC (complex 20), with a IC 50 of 0.03 ± 0.01 and 0.04 ± 0.03 µM, respectively (Entries 19 and 24, Table

Table 1 .

 1 In vitro IC 50 growth inhibitory concentrations (µM) for breast cancer MCF-7.

	Entry	Neutral complex	IC50 (µM) a	Entry	Cationic complex	IC50 (µM) a
	1	1	0.15 ± 0.03	16	12	0.16 ± 0.10
	2	2	0.30 ± 0.05	17	13	0.08 ± 0.02
	3	3	0.22 ± 0.05	18	14	0.10 ± 0.01
	4	4	0.015 ± 0.020 19	15	0.03 ± 0.01
	5	5	0.14 ± 0.02	20	16	1.6 ± 0.4
	6	6	0.03 ± 0.03	21	17	0.6 ± 0.1
	7	7	0.30 ± 0.05	22	18	0.22 ± 0.09
	8	8	0.03 ± 0.04	23	19	0.15 ± 0.90
	9	9	0.015 ± 0.020 24	20	0.04 ± 0.03
	10	10	0.10 ± 0.01	25	21	0.28 ± 0.05
	11	11	1.45 ± 0.10	26	22	0.20 ± 0.10
	12	11 b	0.075 ± 0.002 27	23	0.15 ± 0.07
	13	Cis-platin[55] 8.8 ± 1.3	28	24	0.22 ± 0.12
	14	Etoposide	25.0 ± 0.8	29	25	1.00 ± 0.80
	15	5F-Uracil	19.5 ± 2.0	30	26	0.19 ± 0.17

a ± values, means standard error of at least 6 determinations. b IC 50 of complex 11 previously reported by Gautier et al. on 3000 cells populations.

[START_REF] Lazreg | Synthesis of Homoleptic and Heteroleptic Bis-N-heterocylic Carbene Group 11 Complexes[END_REF]

Table 2 .

 2 In vitro IC 50 growth inhibitory concentrations (µM) for lung carcinoma A-549.

	Entry	Neutral complex	IC 50 (µM) a	Entry	Cationic complex IC 50 (µM) a
	1	1	0.04 ± 0.01	14	12	1.60 ± 0.10
	2	2	0.07 ± 0.03	15	13	0.05 ± 0.02
	3	3	0.10 ± 0.02	16	14	0.06 ± 0.02
	4	4	0.6 ± 0.2	17	15	0.06 ± 0.01
	5	5	± 0.01	18	16	2.0 ± 0.2
	6	6	0.050 ± 0.005 19	17	0.60 ± 0.10
	7	7	0.30 ± 0.06	20	18	0.56 ± 0.17
	8	8	0.050 ± 0.005 21	19	0.66 ± 0.13
	9	9	0.08 ± 0.02	22	20	0.63 ± 0.20
	10	10	0.15 ± 0.03	23	21	0.70 ± 0.10
	11	11	> 3 b	24	22	1.70 ± 0.40
	12	Etoposide	3.0 ± 0.2	25	23	2.90 ± 0.40
	13	5F-Uracil	3.5 ± 0.3	26	24	1.47 ± 0.50
				27	25	1.30 ± 0.10
				28	26	0.78 ± 0.10
	a ± values, means standard error of at least 6 determinations. b higher concentrations were not considered due to
	solubility	issue	and	IC 50	was	not	determined.

Table 3 .

 3 In vitro IC 50 growth inhibitory concentrations (µM) for melanoma SK-Mel-28.

	Entry		Neutral complex		IC 50 (µM) a	Entry		Cationic complex	IC 50 (µM) a
	1		1		0.045 ± 0.005	14		12		0.47 ± 0.10
	2		2		0.16 ± 0.03	15		13		0.29 ± 0.03
	3		3		0.100 ± 0.025 16		14		0.22 ± 0.12
	4		4		0.16 ± 0.02	17		15		0.24 ± 0.02
	5		5		0.040 ± 0.002	18		16		0.95 ± 0.05
	6		6		0.18 ± 0.40	19		17		0.52 ± 0.03
	7		7		0.13 ± 0.01	20		18		0.13 ± 0.05
	8		8		0.25 ± 0.04	21		19		0.12 ± 0.02
	9		9		0.21 ± 0.01	22		20		0.09 ± 0.02
	10		10		0.35 ± 0.25	23		21		1.30 ± 0.30
	11		11		0.42 ± 0.04	24		22		0.90 ± 0.40
	12		Etoposide		6.5 ± 0.8	25		23		1.80 ± 0.60
	13		5F-Uracil		13.4 ± 2.0	26		24		0.50 ± 0.20
						27		25		1.20 ± 0.40
						28		26		0.60 ± 0.20
	a	±	values,	means	standard	error	of	at	least	6	determinations.

Table 4 .

 4 In vitro IC 50 growth inhibitory concentrations (µM) for Glioma HS-683.

	Entry	Neutral complex	IC 50 (µM)	Entry	Cationic complex	IC 50 (µM)
	1	1	0.65 ± 0.01	14	12	1.30 ± 0.10
	2	2	0.58 ± 0.03	15	13	1.07 ± 0.70
	3	3	0.77 ± 0.01	16	14	0.22 ± 0.01
	4	4	1.5 ± 0.2	17	15	0.89 ± 0.18
	5	5	0.21 ± 0.01	18	16	> 3 b
	6	6	0.39 ± 0.12	19	17	> 3 b
	7	7	1.1 ± 0.2	20	18	0.63 ± 0.20
	8	8	0.39 ± 0.01	21	19	1.10 ± 0.10
	9	9	0.81 ± 0.13	22	20	0.80 ± 0.20
	10	10	0.20 ± 0.05	23	21	1.40 ± 0.20
	11	11	0.41 ± 0.02	24	22	0.70 ± 0.10
	12	Etoposide	4.00 ± 0.52	25	23	3.60 ± 0.40
	13	5F-Uracil	16.7 ± 12.2	26	24	2.10 ± 0.10
				27	25	3.50 ± 0.70
				28	26	1.80 ± 0.10

a ± values, means standard error of at least 6 determinations. b higher concentrations was not considered due to solubility issue and IC 50 was not determined.

Table of Abbreviation

 of

dpa 2,2'-dipyridylamine derivative. IC 50 half growth inhibitory concentration NHC N-Heterocyclic Carbene

Acknowledgements

This work was supported by the "Ministère de la Recherche et des Nouvelles Technologies", CNRS (Centre National de la Recherche Scientifique) and the LABEX SynOrg (ANR-11-LABX-0029). We thank the "Agence Nationale de la Recherche", within the CSOSG program (ANR-12-SECU-0002-02), the ANR program (ANR-15-CE39-0006), the "Région Basse-Normandie" (M.E.) and Normandie University (G.U.M.) for funding. SG thanks Johnson Matthey for the gift of metals.

Supporting Information.

Supporting information available: experimental procedures for the synthesis of the iridium (III) complexes, their characterization, and the biological evaluation of the inhibition. See DOI: 10.1039/c000000x/

Highlights

We evaluated cytotoxicity of cationic NHC copper(I) complexes bearing dipyridylamine ligands We compared cytotoxicity of cationic NHC copper(I) complexes bearing dipyridylamine ligands with their neutral NHC copper(I) complexes congeners


Paul Raterron
email: praterro@nsf.gov

Caroline Bollinger

Sébastien Merkel

Olivine intergranular plasticity at mantle pressures and temperatures

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The plasticity of olivine-rich rocks constraints that of Earth's upper mantle.

Consequently, there has been considerable effort to quantify olivine aggregate rheology in terms of flow laws which can be implemented in geodynamical models for mantle thermal convection. Experimental studies investigated the effects of temperature and stress [a review [START_REF] Hirth | Rheology of the upper mantle and the mantle wedge: a view from the experimentalists, in: Inside the Subduction Factory[END_REF], pressure [e.g., [START_REF] Durham | New measurement of activation volume in olivine under anhydrous conditions[END_REF][START_REF] Hilairet | Deformation of olivine under mantle conditions: An in situ high-pressure, high-temperature study using monochromatic synchrotron radiation[END_REF][START_REF] Bollinger | Olivine dislocation creep: revisiting experimental data to 8 GPa pressure[END_REF], fluid fugacities [e.g., [START_REF] Kohlstedt | The role of water in high-temperature rock deformation[END_REF][START_REF] Keefner | Dependence of dislocation creep of dunite on oxygen fugacity: implications for viscosity variations in Earth's mantle[END_REF][START_REF] Ohuchi | Flow behavior and microstructures of hydrous olivine aggregates at upper mantle pressures and temperatures[END_REF], grain size [START_REF] Warren | Grain size sensitive deformation mechanisms in naturally deformed peridotites[END_REF], lattice preferred orientations [e.g., [START_REF] Hansen | Laboratory measurements of viscous anisotropy of olivine aggregates[END_REF] and melt fractions [e.g., [START_REF] Hirth | Experimental constraints on the dynamics of partially molten upper mantle 2: deformation in the dislocation creep regime[END_REF]. The traditional view is to assign one dominant deformation mechanism to given deformation conditions [START_REF] Frost | Deformation Mechanisms Maps: The Plasticity and Creep of Metals and Ceramics[END_REF] and implement the flow law with specific dependences on temperature, stress or grain size. At the microscopic scale, however, olivine aggregate plasticity involves numerous mechanisms operating concurrently, within the grains and at grain boundary (Fig. 1). To this day, the fundamental question of the amount of strain accommodated in the mantle through grain-to-grain interactions versus that accommodated within the grain remains unanswered.

Strain accommodation at grain boundary has been attributed to several distinct mechanisms. A model for the deformation of aggregates by grain-boundary diffusion was introduced by [START_REF] Coble | A model for boundary diffusion controlled creep in polycrystalline materials[END_REF] to explain the high-temperature plasticity of alumina. Coble creep requires the rearrangement of grain interfaces by grain-boundary sliding (GBS). The corresponding flow law exhibits a linear dependence on stress and a strong inverse dependence on grain size (d), theoretically to the power p = -3. For persistently small grain sizes -when grain growth is for example impeded by Zener pinning -Coble creep may contribute to superplastic flow, which has been characterized at room pressure in olivine rich aggregates [e.g., [START_REF] Hiraga | Mantle superplasticity and it selfmade demise[END_REF]. Grain-boundary sliding can also be assisted by dislocation motions within grains, which contribute to relax stress concentration at triple junctions. This mechanism, called dislocation-assisted grain boundary sliding (disGBS), has been observed at low pressure in olivine [START_REF] Hirth | Rheology of the upper mantle and the mantle wedge: a view from the experimentalists, in: Inside the Subduction Factory[END_REF][START_REF] Hansen | Grain boundary sliding in San Carlos olivine: Flow law parameters and crystallographic-preferred orientation[END_REF]. It is characterized by a strain rate depending strongly on stress, typically to the power n ~ 3, with an inverse dependence on grain size to a power p within [-2, -0.6]. Other deformation mechanisms, which do not exist in single crystals, accommodate strain in olivine aggregates.

Motions of disclinations -defects identified along grain boundaries in olivine [START_REF] Cordier | Disclinations provide the missing mechanism for deforming olivine-rich rocks in the mantle[END_REF] -can accommodate strain. Furthermore, interactions between grains, in materials with limited number of intracrystalline deformation mechanisms, generate locally high stress concentration [e.g., [START_REF] Castelnau | Micromechanical modeling of viscoplastic behavior of olivine[END_REF]. In materials with anisotropic elastic and plastic properties such as olivine this may promote high stress and strain transmission patterns percolating throughout the aggregates [START_REF] Burnley | The importance of stress percolation patterns in rocks and other polycrystalline materials[END_REF]. Conversely, the stress field associated with single-crystal deformation can only be relaxed by intracrystalline deformation mechanisms, such as dislocation motions (glide, climb and cross slip) and intracrystalline diffusion (e.g., Nabarro-Herring diffusion).

Recently, [START_REF] Tielke | Observation of grain size sensitive power law creep of olivine aggregates over a large range of lattice-preferred orientation strength[END_REF] compared olivine single-crystal and aggregate hightemperature rheology at low pressure, and quantified the contribution of both intracrystalline and intergranular mechanisms to the aggregate strain. They determine that olivine aggregates deform up to 4.6 times faster than what would be expected assuming only intracrystalline plasticity; the latter's contribution to strain rate was calculated from micromechanical modeling of dislocations activity. Following a similar approach, we here compare olivine single-crystal and aggregate high-temperature plasticity, as measured experimentally at the high pressures representative of mantle conditions. We demonstrate that grain-to-grain interactions significantly contribute to accommodating strain in experiments. Extrapolation to mantle stress conditions along geotherms suggests that intergranular plasticity may also dominate upper mantle plasticity.

Methods: intracrystalline vs intergranular plasticity

Comparing aggregates and single-crystal deformation data allows quantifying the strain rate contributions of intergranular deformation mechanisms. Assuming that intracrystalline (IC) and intergranular (IG) mechanisms operate concurrently, we have:

ε = ε + ε (1),
where ε is the strain rate of the aggregate, ε is the contribution of intracrystalline processes and ε is due to grain-to-grain interactions. The plasticity of aggregates can thus be quantified by introducing the following ratio:

ε /ε = 1 + ε /ε (2),
It should range from 1, when all the aggregate strain is accommodated within the grains, to +∞, when the strain is fully accommodated through grain-to-grain deformation processes.

Values for ε and ε (and their ratio ε /ε ) can be directly extracted from previously published rheological data (Figure 2).

We used ε values for San Carlos olivine aggregates deformed in axisymmetric compression at mantle pressure and temperature in the Deformation-DIA (D-DIA) apparatus, as reported by [START_REF] Durham | New measurement of activation volume in olivine under anhydrous conditions[END_REF], [START_REF] Hilairet | Deformation of olivine under mantle conditions: An in situ high-pressure, high-temperature study using monochromatic synchrotron radiation[END_REF] and [START_REF] Bollinger | Olivine dislocation creep: revisiting experimental data to 8 GPa pressure[END_REF] (supplementary Table S1). For each ε reported value, the corresponding ε can be calculated at identical pressure (P), temperature (T) and differential stress (σ) by combining experimental flow laws for San Carlos olivine single crystals [START_REF] Mackwell | The role of water in the deformation of olivine single crystals[END_REF][START_REF] Bai | High-temperature creep of olivine single crystals.1. Mechanical results for buffered samples[END_REF][START_REF] Raterron | Experimental deformation of olivine single crystals at mantle pressure and temperature[END_REF], 2012;[START_REF] Girard | Hydrolytic weakening of olivine at mantle pressure: evidence of [100](010) slip system softening from single crystal deformation experiments[END_REF] (supplementary materials, Table S2). Indeed, assuming homogeneous stress throughout the aggregate (lower bound approach, i.e. Sachs' bound) -one of the simplest end-member assumption when analytically addressing aggregate strain -and random grain orientations in the aggregate, ε reads: account for pressure, temperature, water content, and oxygen fugacity (fO2) and are based on data from the literature (Supplementary information). For the dry experiments reported by [START_REF] Durham | New measurement of activation volume in olivine under anhydrous conditions[END_REF] (squares in Fig. 2), we assumed a dry-crystal rheology when calculating ε ; for the wet experiments reported by [START_REF] Hilairet | Deformation of olivine under mantle conditions: An in situ high-pressure, high-temperature study using monochromatic synchrotron radiation[END_REF] and [START_REF] Bollinger | Olivine dislocation creep: revisiting experimental data to 8 GPa pressure[END_REF] (triangles and circles in Fig. 2), we assumed wet-crystal rheology. ε was also calculated assuming oxygen fugacity conditions comparable to those of the experiments. [START_REF] Durham | New measurement of activation volume in olivine under anhydrous conditions[END_REF] experiments, where the Ni/NiO buffer controlled fO2, we assumed the Ni/NiO buffer oxygen fugacity [START_REF] Frost | Introduction to oxygen fugacity and its petrologic importance[END_REF]. For the unbuffered experiments [START_REF] Hilairet | Deformation of olivine under mantle conditions: An in situ high-pressure, high-temperature study using monochromatic synchrotron radiation[END_REF][START_REF] Bollinger | Olivine dislocation creep: revisiting experimental data to 8 GPa pressure[END_REF] -where oxygen fugacity was not controlled but specimens were placed within a boron nitride sleeve which promotes low fO2 conditions [Wendland et al., 1985] -we assumed the oxygen fugacity of the iron-wüstite buffer (IW) which borders the olivine stability field.

ε = 0.123 (ε [ ] + ε [ ] + ε [ ] ) (3)

Results: aggregate strain rate ε versus intracrystalline strain rate ε

Figure 2.a shows the aggregate strain rate ε , as measured experimentally (Table S1), versus the intracrystalline strain rate ε (Eq. 3) calculated at identical P, T and σ.

Because of our initial assumption (Sachs' bound), Eq. 3 tends to overestimate the intracrystalline strain rate. This may occasionally lead to ε < ε when strain is fully accommodated by intracrystalline processes. Remarkably, we have ε ≥ ε (within uncertainty) for all but one experimental point, by factors reaching ~20 at 1673 K and ~2000 at 1373 K. This shows that, in high-pressure deformation experiments, i) a significant fraction of aggregate strain is accommodated by mechanisms involving grain-to-grain interactions, even in regimes where dislocation creep was observed. It also shows that ii) this fraction tends to increase with decreasing temperature. Also show on Figure 2.a are the data reported by Tielke el al. (2016) obtained at low pressure (open diamonds, green is for 1523 K). For these data, shear strain rates and stresses were converted into compressional strain rates and stresses before plotting. Tielke et al. report that the aggregate strain rate is up to 4.6 times higher than the intracrystalline strain rate as calculated using a micromechanical modeling; Figure 2.a shows that these data fall, indeed, in the vicinity of the line corresponding to a ratio of 4.6 between the measured strain rate and the intracrystalline strain rates calculated here using the analytical approach described above. It is remarkable that using two different approaches, Tielke et al. and we obtained similar results. This give us confidence in the analytical approach used here. is achieved, i.e. strain is fully accommodated within the grains (ε = ε ). A further increase of stress will have no effect on Eq. 2 ratios. This result is in agreement with the conventional interpretation that increasing stress and strain rate at given temperature favors dislocation creep [START_REF] Frost | Deformation Mechanisms Maps: The Plasticity and Creep of Metals and Ceramics[END_REF]] -a grain-size insensitive creep involving mostly intracrystalline plasticity -with respect to grain-size sensitive creep which involves intergranular plasticity.

The contribution of grain-to-grain deformation processes to the aggregate strain also decreases with temperature (Figs. 2a and 2.b). This effect may result from a combination of factors, such as: an increasing aggregate grain size with temperature, i.e., a decreasing grainboundary surface/bulk volume ratio favoring intracrystalline deformation mechanisms; an increasing activity of disclinations with decreasing T favoring intergranular plasticity; higher stress and strain concentrations near grain boundaries at lower T, or a more effective stress percolation at moderate T [Burnley, 2012] promoting high-strain networks throughout the aggregates, accounted here for as intergranular strain.

A linear fit through log(ε /ε ) in Figure 2.b leads to the empirical equation:

log(ε /ε ) = 20.4 -0.0115 ) -0.0045 * (4),
where T is in K, and the differential stress σ is in MPa. ε /ε must remain ≥ 1. Therefore, ε /ε = 1 is here imposed when Eq. 4 gives values < 1. Note that, although empirical, Eq. 4 accounts for all dry and wet data with good consistency between studies. Combining Eqs. 2 and 4, a first-order estimate of olivine aggregate strain rate can be calculated from the intracrystalline strain rate ε (Eq. 3) through the following composite flow law:

ε = Max[1; 10^(20.4 -0.0115 ) -0.0045 *)] × ε (5),
where Max[i; j] is the maximum of i and j , T is in K and σ is in MPa. Note that the effects of pressure, temperature, stress, oxygen fugacity, or hydrous conditions are accounted for in the intracrystalline flow laws (Eq. 3, supplementary materials, Table S2). In the following, we explore the implications for the upper mantle of the effect of pressure and temperature on intracrystalline vs intergranular plasticity.

Discussion: Extrapolation to mantle conditions

Figure 3.a shows log"ε Agg /ε IC # calculated along two oceanic geotherms (20-Ma and 80-Ma) for a differential stress σ = 1 MPa -i.e., a shear stress μ = 1/√3 MPa which is a reasonable value for the mid to deep upper mantle [START_REF] Bürgmann | Rheology of the lower crust and upper mantle: evidence from rock mechanics, geodesy and field observations[END_REF] -and along a classic continental geotherm (supplementary Fig. S1, see also [START_REF] Turcotte | Geodynamics[END_REF])

for σ = 1 and 50 MPa. The latter stress value is representative of shear zone in the coldest part of the lithosphere, and of experimental conditions. For this calculation, pressure was calculated with an upper-mantle average density of 3.35 g/cm 3 (i.e. a 32.9 MPa/km vertical pressure gradient), and oxygen fugacity set at FMQ-2 which is reasonable for the upper mantle [START_REF] Herd | Basalts as probes of planetary interior redox state[END_REF]. We assumed wet condition for this plot (supplementary materials). and continental (blue curves) geotherms, as calculated using the composite flow law of Eq. 5

for a differential stress of 1 MPa. Wet conditions are assumed when using Eq. 5, as well as for plotting the Hirth and [START_REF] Hirth | Rheology of the upper mantle and the mantle wedge: a view from the experimentalists, in: Inside the Subduction Factory[END_REF] dislocation creep flow law with hydroxyl content COH = 300 ppm H/Si. The intracrystalline strain rate calculated from Eq.3 is shown for comparison, together with two low-temperature flow laws [START_REF] Raterron | Low temperature olivine rheology at high pressure[END_REF][START_REF] Demouchy | Low strength of Earth's uppermost mantle inferred from tri-axial deformation experiments on dry olivine crystals[END_REF]. In both contexts, the composite flow law in Eq. 5 leads to viscosities about two orders of magnitude lower that the Hirth and Kohlstedt's dislocation creep flow law.

Interestingly, in the shallow (cold) upper mantle, the composite flow law of Equation 5 is in relatively good agreement with the low-temperature flow laws reported for olivine, thus captures the change in rheology between high-temperature and low-temperature plasticity.

Changing the differential stress to 50 MPa does not significantly affect these results

(Supplemental Figure S2). At deeper depths (i.e. 400 km), the difference between the predictions of Eq. 5 and the dislocation creep law of Hirth and [START_REF] Hirth | Rheology of the upper mantle and the mantle wedge: a view from the experimentalists, in: Inside the Subduction Factory[END_REF] is due to the pressure-induced change of dominant slip system in olivine [START_REF] Raterron | Activities of olivine slip systems in the upper mantle[END_REF].

It should be emphasized here that polycristalline specimens in high-pressure experiments have small grain sizes, typically ranging from 1 to 50 µm, which increases significantly their surface versus volume ratio when compared to that of mantle rocks with estimated grain sizes ranging from tenths of millimetre to centimetres. This enhances grain-to-grain interactions, hence intergranular plasticity, in laboratory specimens and may artificially lower their strenght with respect to that of mantle rocks. The results reported here (Eq. 5 and Fig. 2) may, thus, significantly overestimate how much strain can be accomodated by grain-to-grain interactions in the coarse-grain mantle. However, our results may apply more directly in the context of mantle shear zones, where grain-size reduction weakens sheared peridotites [START_REF] Warren | Grain size sensitive deformation mechanisms in naturally deformed peridotites[END_REF][START_REF] Skemer | Torsion experiments on coarsegrained dunite: implications for microstructural evolution when diffusion creep is suppressed[END_REF].

Concluding remarks

According to our results and extrapolation, we conclude that olivine strain is mostly accommodated by deformation mechanisms involving grain-to-grain interactions at mantle pressures and temperatures, which results in a much weaker strength as that obtained when combining single-crystal dislocation creep flow laws. Such a phenomenon was recently observed at low pressure [START_REF] Tielke | Observation of grain size sensitive power law creep of olivine aggregates over a large range of lattice-preferred orientation strength[END_REF], but is much more marked at high pressure where intergranular plasticity largely dominate deformation.

Uncertainties remain regarding the additional deformation mechanisms, present in aggregates and absent in single crystals, responsible for the measured low strength of aggregates with respect to that of single crystals. Several candidate mechanisms are mentioned in the introduction, such as disclinations, grain boundary sliding, stress/strain percolation, etc., but our analysis does not allow to favor one over another.

Furthermore, the empirical model presented here is extracted from deformation experiments carried out at high differential stresses on aggregates with small grain sizes compared to mantle conditions where stresses are much lower and grain sizes larger -.

Further investigation is necessary to quantify the effects of increasing grain size and decreasing stress on Eq.4 parameters. As mentioned above, one may speculate that, due to the larger grain sizes, intracrystalline mechanisms may accommodate more strain in the Earth's mantle than in experiments and, hence, reduce the effect of grain-to-grain interactions highlighted here. Another source of discrepancy when extrapolating the present results to mantle processes is the presence of secondary phases such as pyroxenes, garnet and possibly partial melts in mantle peridotites which are absent in the present laboratory specimens.

Keeping in mind the above reservations, let us however emphasize that olivine classical flow laws, whether assuming dislocation or diffusion creep, fail to explain the fast surface displacement observed by GPS after large earthquakes [e.g., [START_REF] Freed | Steady-state laboratory flow laws alone fail to explain postseismic observations[END_REF], S1) versus predictions based on intracrystalline strain rate ε as calculated from Eq. 3 and Table S2 parameters. Rate04 for [START_REF] Raterron | Low temperature olivine rheology at high pressure[END_REF]. See text for further explanation. The effect of intergranular strain relaxation mechanism is apparent through the reduction of viscosity by a factor of ~100 (solid lines) at shallow depths relative to that obtained from the classical Hirth and Kohlstedt's flow laws or laws based on intracrystalline deformation (dashed lines). Do note, however, that such effect is probably reduced in the mantle because of larger grain sizes than in experiments. At deeper depths, the difference between the present law and that of [START_REF] Hirth | Rheology of the upper mantle and the mantle wedge: a view from the experimentalists, in: Inside the Subduction Factory[END_REF] is due to the pressure-induced change of dominant slip system in olivine.

  , where ε [ ] , ε [ ] , and ε [ ] are the strain rates of oriented single crystals and the 0.123 geometrical factor arises from averaging over crystal orientations[Raterron et al., 2011]. The indexes indicate the crystallographic orientation of the compression directione.g.,[110]c direction is at 45° angle from [100] and [010] directions. [110]c, [011]c, [101]c promote respectively [100](010) dislocation slip, [001](010) dislocation slip, and [100](001) and [001](100) slips together. The single-crystal strain rates ε [ ] , ε [ ] , and ε [ ]

Figure 2 .

 2 Figure 2.b shows log"ε /ε # versus stress; the color code indicate approximatetemperatures. The ratio ε /ε varies over orders of magnitude, from ~ 1 to ~2×10 3 . When ε /ε ~ 1 the aggregate strain is fully accommodated within the grains, while ε /ε ≫ 2 indicates that strain is mostly accommodated by grain-to-grain interactions (intergranular mechanisms). Within our model, one should always satisfy ε /ε ≥ 1 (Eq. 2) which,

Figure 3 .

 3 Figure 3.a suggests that, like in experiments, deformation in the upper mantle is largely

Figure 1 :

 1 Figure 1: Schematics of mechanisms accommodating strain in olivine aggregates:

Figure 2 :

 2 Figure 2: A) Aggregate strain rate εas measured in experiments (TableS1) versus

Figure 3 :

 3 Figure 3: A) ratio of the aggregate strain rate by the intracrystalline strain rate (ε /ε )

Figure 1 20Figure

 1 Figure 1

which requires a much weaker strength for the lithosphere, as the one we propose here. Also, the particularly deep weakening predicted here along a continental geotherm may provide an explanation for the elusiveness of the lithosphere-asthenosphere boundary beneath cratons [e.g., Eaton et al., 2009], since it should reduce the lithosphere-asthenosphere viscosity contrast. We thus conclude that grain-to-grain interactions are an important component of olivine plasticity at mantle pressures, and may likely contribute to the weakening of the Earth's upper mantle with respect to that calculated from classical flow laws for olivine.

Acknowledgements

This research was supported by the Agence Nationale de la Recherche (ANR) Grant BLAN08-2_343541 "Mantle Rheology". We thank two anonymous reviewers for their thoughtful insights which helped improving the original manuscript. Part of the work was carried out while PR was serving at the National Science Foundation.


Guillaume Fiquet
email: guillaume.fiquet@sorbonne-universite.fr

Chandrabhas Narayana

Christophe Bellin

Abhay Shukla

Imène Esteve

Art L Ruoff

Gaston Garbarino

Mohamed Mezouar

I Estève

Structural phase transitions in aluminium above 320 GPa

Keywords: aluminium, structural transition, x-ray diffraction, multi-megabar

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The extreme pressure phase diagram of materials is important not only for the understanding of the interiors of planets or stars, but also for the fundamental understanding of the relation between the crystal and electronic structures. Structural transitions induced by extreme pressures are governed by the deformation of the charge density of the valence electrons which bears the brunt of the increasing compression while the relative volume occupied by the nearly incompressible ionic core electrons increases. This fact is said to hold not only in the few hundred GPa range but also beyond the terapascal regime where compression pushes ion cores together.

Experimental limitations will however keep the TPa regime in the realm of predictions, at least as far as static pressure is concerned.

Early first principles calculations (1) for aluminium (Al) predicted an fcc -> hcp -> bcc structural transition trend over a pressure range of roughly 0-500 GPa and these have since been repeatedly confirmed (2)(3)(4) and refined (5)(6)(7). This non-intuitive generic transition from a compact (fcc or hcp) to a more open (bcc) structure with increasing pressure is said to be driven by the increasingly smaller and restricted volume available for the valence electrons. This tends to reduce electronic bandwidth by the occupation of an s-d band at a few hundred GPa (as opposed to the dispersive s-p band at low pressures). Ultimately, theoretical calculations indicate that valence electrons can be localized in "interstitial" spaces in an open-packed incommensurate host-guest structure similar to that predicted at 3.2 TPa (8). That d electrons play a role in these transitions is strongly suggested by the fact that the structure sequence with increasing pressure is mirrored in transition metals as the number of d electrons increases (9) (the analogy has obvious limitations since the underlying magnetism intervenes in transition metals (10)). As the unit cell volume is reduced to fractions approaching half or less, the initially unoccupied d bands in simple metals and in particular in aluminium approach, narrow, and descend below the Fermi level triggering structural changes which can be intuitively understood since the bcc structure is more compatible with a bonding interaction between second nearest neighbour atoms than the fcc structure (1,2). Such a transition has earlier been reported in Mg (11) and Pb (12) where it takes place at lower pressures.

A simple system like Al is not only important as a benchmark for theory, but can also be used as a standard for pressures in the TPa range and beyond, which are targeted at dynamic compression facilities such as the National Ignition Facility (NIF) at the Lawrence Livermore National Laboratory in the US or Laser Mégajoule (LMJ) in Bordeaux in France. Confirming predictions of aluminium structure at extremely high densities is thus paramount. According to a recent first principle calculation (7), aluminium should undergo a phase transition from fcc to hcp structure around 200 GPa and another transition from hcp to bcc with further compression beyond 300 GPa. The hcp phase around 217 GPa was reported in an earlier room-temperature static highpressure experiment where a maximum pressure of 330 GPa was achieved (13). An earlier classical shock-compression study found no evidence of the predicted fcc to hcp transition (14) but it is expected that fcc aluminium melts at 125-150 GPa along principal Hugoniot (see (16)). More recently, bcc aluminium has been synthesized in the non-equilibrium conditions of an ultra-fast laser-induced micro-explosion confined inside a sapphire (α-Al 2 O 3 ) rod (15). This recent report of the bcc super-dense phase of Al is interpreted as a complex route of synthesis via a spatial separation of Al and O ions in short-lived hot non-equilibrium plasma of solid-state density. The microexplosion confined inside a sapphire capsule leads to this bcc-Al phase, which survives in a compressed state after fast quenching. However, if confined micro-explosions provide an interesting route to create and recover high-density polymorphs, such an experiment does not allow the determination of a transition pressure nor does it show any evidence of the presence of a quenched hcp phase. Very recently, an experiment succeeded to combine nanosecond in situ x-ray diffraction and simultaneous velocimetry measurements to determine the crystal structure and pressure of ramp-compressed aluminium at stress states between 111 and 475 GPa (16). The solid-solid Al phase transformations, fcc-hcp and hcp-bcc, are reported at 216 ± 9 and 321 ± 12 GPa, respectively. In this article, we confirm this observation with a synchrotron-based X-ray diffraction experiment carried out within a diamond-anvil cell and report indications of the onset of the transition towards a bcc structure at pressures beyond 320 GPa.

Experimental

Sample preparation

Central to our experiment was the establishment of a protocol for reaching pressures exceeding 350 GPa in a diamond-anvil cell with a pure aluminium sample (Fig. 1) confined inside a conventional gasket (Fig. 2), which limits the deviatory stress component to moderate values. Very fine-grained aluminium powder is very difficult to handle and is pyrophoric in nature. It can thus easily oxidize. We used instead an aluminium foil (99.999 % purity, 15 µm thick, Goodfellow) as starting material.

Samples were pre-cut from this foil with a focused ion beam (FIB) so as to obtain cylinders (Fig. 1) with dimensions fitting the holes prepared in pre-indented rhenium gaskets.

High-pressure cells preparation and sample loading

Our experiments were conducted in symmetrical diamond anvil cells equipped with single bevelled diamonds (8° bevel) mounted on X-ray transparent cubic boron nitride seats. Two separate experimental runs were carried out at ambient temperature with diamonds having centre flat culets of 35 and 20 µm diameters respectively. The first experiment with the 35 µm culet anvil reached 257 GPa whereas the second one reached pressures of about 370 GPa. In our experiments, no pressure-transmitting medium was used. As noted by Akahama et al. (13), Al has a relatively low shear modulus and is expected to keep low uniaxial stresses in the diamond-anvil cell.

Success of experiments in this pressure range are highly dependent on handling samples inside a hole of less than 10 µm, which has to be perfectly centred on a culet of 20 µm made on a gasket pre-indented to less than 10 µm. Manipulating a sample of such small dimensions inside a highly contoured gasket terrain to place it perfectly into the hole is yet another challenge. In the present experiment, we have used the focused ion beam for drilling such tiny holes (see 17) in rhenium with a centring precision better than 1 μm and causing very little defects to the gaskets. Rhenium gaskets were pre-indented to reach an initial thickness of about 12 μm, and then drilled with the focused ion beam (FIB). Preparation of gaskets and sample loading were carried out in the FIB chamber, so as to have a perfect sample loading in the pressure chambers (see Fig. 2). Above 300 GPa, pressure was estimated according to the equation of state of rhenium (18). In the fcc stability field, pressure was measured with available equations of state reported for aluminium (13,19). Both methods yield pressure measurements in very good agreement (i.e. within error bars) up to 300 GPa. Alternatively, the equation of state for rhenium proposed by Dubrovinsky et al. (20) could be used but the latter yields significant pressure overestimate when compared to other measurements (as large as 70 GPa at 300 GPa).

X-ray diffraction experiments

In situ X-ray diffraction high-pressure experiments were conducted at the high-pressure beamline ID27 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). X-ray diffraction pattern were collected using an angle dispersive monochromatic set-up with a wavelength of 0.3738 Å (iodine K-edge at 33.3 keV) focused with KB mirrors down to a spot of 2.5 x 3 μm FWHM at sample location. Such a spot size explains why rhenium diffraction lines are completely absent in the first run conducted with bevelled diamonds with a central culet of 35 µm in diameter and a sample chamber of 14 µm in diameter which reached a pressure of 240 GPa (see Fig. 3).

Rhenium lines are visible in the second run designed to reach a higher pressure (Fig. 5) with a smaller sample having a diameter of about 8 µm mounted on 20 µm culets.

Exposure time varied from 60 s below 100 GPa to 240 s at pressures exceeding 370 GPa, thus compensating for the thickness reduction as pressure was increased. We used a two-dimensional MAR CCD detector located at a distance of 207 mm from the sample. Images were then integrated using the Fit2D software (21) in order to obtain a conventional diffraction pattern. Data analysis was then carried out using the GSAS package (22,23). Cell parameters were refined using LeBail method for the extraction of reflection intensity. Preferred orientations were not refined. Graphics were realized with the Datlab software (courtesy K. Syassen, MPI Stuttgart). All X-ray diffraction patterns are background subtracted.

Results and discussion

X-ray pattern analysis

The first run presented in Fig. 3 show a smooth evolution of the fcc structure at lower pressures. The pattern at 213 GPa can be unambiguously assigned to a fcc lattice with a lattice parameter a= 3.246 (1) Å and a unit cell volume of 34.190 (31) Å 3 . With four atoms per unit cell in the fcc structure, the atomic volume is 8.548 Å 3 . As pressure was increased, new peaks appeared around 220 ± 5 GPa. At the pressure of 235 GPa, as shown in Fig. 4, these new lines can be unambiguously assigned to a hcp structure with lattice parameters a= 2.266 (1) Å and c=3.720 (2) Å and a unit cell volume of 16.542 (23) Å 3 , coexisting with an fcc lattice with a=3.211 (1) Å and a unit cell volume of 33.097 (30) Å 3 . We find, within error, a similar atomic volume, V A = 8.274 (7) Å 3 for the fcc structure and V A = 8.269 (11) Å 3 for the hcp structure and a large coexistence domain which can be explained by the small enthalpy difference between the two phases (6,7,13). These observations are in perfect agreement with the transition pressure of 215-222 GPa reported in the previous X-ray diffraction work cited above (13) or in the dynamic compression experiment (16). Alternatively, a slightly lower pressure of 210 GPa is obtained when using another room temperature high-pressure equation of state for aluminium (19). This fcc to hcp transition takes place at a compression of 0.5, in general agreement with theoretical predictions (e.g. ref 7) or experimental measurements (13).

In the second run, we explored pressures above 300 GPa. Patterns are cut at a maximum angle of 20 degrees 2-theta because of the use of higher diamonds. With such a configuration, diamond seats and cell mechanical opening prevented us to collect diffraction at higher angles. In this second run (see Fig. 5), we also observe without ambiguity the progressive growth of the hcp phase at the expense of the fcc phase, with the complete disappearance of the fcc peaks at a pressure which can be estimated of about 280 GPa according to the equation of state of aluminium (19) or that of rhenium (18). In this run, the presence of rhenium reflections could not be avoided with a sample chamber typically smaller than 8 µm as soon as pressure has been increased. At 235 GPa, lattice parameters a= 2.266 (1) Å and c=3.720 (2) Å for the hcp structure yield a c/a ratio of 1.642 (2). At 370 GPa, with a= 2.182 (2) Å and c=3.557 (5), c/a ratio is 1.630 (4). It thus seems that the trend followed by c/a ratio is a decrease when pressure is increased. Between 320 GPa and 350 GPa, the most interesting feature is the observation of a splitting of the 002 reflection of the hcp structure (see Fig. 6). The remaining peaks corresponding to the hcp structure (100 and 101) do not show any significant broadening nor splitting. It is indeed shown in Fig. 6B and 6C that a new line here interpreted as the 110 bcc grows as a shoulder of the hcp 002 line. Some diffracted intensity detected around a 2-theta value of 17° close to the aluminium 102 hcp reflection (see Fig. 5) can tentatively be interpreted as the 200 bcc reflection.

Discussion

The hcp <-> bcc transformations with pressure are martensitic transitions which result from small relative movements of atoms. The hcp -> bcc transition has been studied in some detail from the theoretical point of view for the case of Mg (24,25). It is thought to involve a distortion of the regular hexagonal atomic arrangement in the (001) hcp plane as well as a shear between adjacent (001) planes (see Figure 7). The distortion accounts for the transformation of the (001) hcp planes into the (110) bcc planes while the shear transforms the ABAB stacking along the [001] hcp direction to one compatible with the bcc structure. This mechanism principally involves the (001) hcp planes and would thus manifest itself by changes in peaks with 00l hcp character. This is exactly what we observe in our experiments as shown in Fig. 6 with a clear splitting of the 002 hcp line when pressure exceeds 320 GPa. It is expected the 002 hcp reflection should totally disappear with the appearance of a single 110 bcc peak when the transition is completed. However, it is likely the martensitic nature of such a transition makes the phase transition sluggish at ambient temperature where the transformation is kinetically inhibited, as observed in other system at room temperature (see 26). Both low-pressure and high-pressure structures coexist on a large pressure domain and hcp reflections can still be observed at the pressure of 370 GPa as shown shown in Fig. 5 and6, although the bcc features are less marked at this maximum pressure because of a diminution of the quality of our diffraction images. Though, our observations are compatible with the observations reported in the ramp-compressed aluminium experiment (16) where coexistence of the two high-pressure structures is no longer observed above 380 GPa. It is however likely kinetic barriers can be more easily overcome in shock experiments whereas it was impossible to heat up our sample kept under such pressure conditions in our experiment. We propose therefore that the peak splitting observed above 320 GPa correspond to the onset of the hcp to bcc structure transition, with compression along the [001] axis. In a rigid atom model, the bcc->hcp transition can be explained simply by using the relationships between atomic radius r and lattice parameters a 0 and c 0 with a 0 =2r, c 0 ≈1.633a 0 for a hcp structure and a 0 = 4r/√3 for a bcc structure, since the atomic packing fraction is higher in the hcp phase. In addition, the relations a hcp =√3a bcc /2 and c hcp =√2a bcc can also be written for such a transition (see 27 for instance) and the cell parameters we could deduce from our experiments satisfy these relations. In our experiment, the unit cell volume fitted for the hcp structure at 320 GPa is V hcp =15.655 (41) Å 3 while that calculated assuming that the split peak corresponds to 002 bcc yields V bcc =15.719 (56), which are indiscernible within error bars at the onset of the transition. Observed pattern and reflections are shown for the two phases at 320 GPa in Fig. 7. At this pressure, aluminium d-spacing for bcc 110 reflection is 1.771, which is comparable to values reported at the same pressure in (16).

We note that the transition to the bcc phase is predicted in the region between 290 and account for zero-point thermal vibrations or neglect these. These pressures compare well with the onset of the transition that we place at 320 GPa. We also have a perfect agreement with the 321 ± 12 GPa measured by velocimetry in the dynamic compression experiments (16). According to theoretical studies, the calculated enthalpy difference between these phases is only a few mRy (6,7,11) and one would experimentally expect these phases to co-exist over a large pressure range as in the case of the fcc to hcp transition. We can thus anticipate a very sluggish hcp to bcc transition. Our observations correspond to the first step of this transition, built here on a distortion of the hcp lattice.

Our experiment thus confirms the measured and predicted fcc-hcp-bcc phase transitions for Al. It also supports the predicted mechanism for this martensitic transition via a distortion and shear of the (001) hcp planes. Experiments permitting higher pressures than those reached here will be needed to detect a pure bcc phase unequivocally.

Though a recent static pressure experiment has reached a pressure exceeding 1 TPa (29), our experiment performed on a sample of physical interest in conventional static pressure geometry, still opens new perspectives. It provides a large pressure window (up to 4 Mbar) for the study of a wide variety of materials and phenomena in conventional diamond-anvil cell geometry. These range from structural phase transitions or the detection of novel physical properties (such as superconductivity) in elemental or more complicated materials of physical or geophysical interest (30,31).

Figure 1 .

 1 Figure 1. Secondary electron SEM images: series of sample cylinders shaped with focused ion beam (FIB) on the edge of the pure aluminium sample foil (top). Individual bulk aluminium sample piece during lift-out procedure. The small bridge holding the sample can easily be cut once micro-manipulator is attached. A platinum deposition (2 x 2 μm light square shape on the top) is visible (bottom). This pressure marker was unfortunately not detected during X-ray diffraction experiments.

Figure 2 .

 2 Figure 2. Secondary electron SEM images: 10 μm hole drilled with FIB at the center of the 20 μm inner culet print on the rhenium gasket. Outer culet is 300 μm in diameter (top). Sample cylinder loaded with micro-manipulator in sample chamber (bottom).

Figure 3 .

 3 Figure 3. Diffraction pattern of an fcc aluminium sample compressed in a rhenium gasket at room temperature to 256 GPa in a diamond-anvil cell. Reflections corresponding to the hcp structure are detected at 213 GPa and pressures above.

Figure 4 .

 4 Figure 4. Analysis of X-ray diffraction pattern of aluminium collected at a pressure of 235 GPa, showing coexisting fcc and hcp structures. Cell parameters are a= 3.211 (1) Å and volume is V=33.097 (30) Å 3 for the fcc phase. Cell parameters are a=2.266 (1) Å and c=3.720 (2) Å with volume V=16.539 (23) Å 3 for the hcp phase.

Figure 5 .

 5 Figure 5. Series of full diffraction pattern collected between 180 to 370 GPa from bottom to top of the figure. Green lines (fcc), blue lines (hcp) and red lines (bcc) are guide to the eye for the different aluminium phases. The fcc phase is no longer observed at pressures exceeding 280 GPa. With the reduced size of the rhenium gasket pressure chamber, rhenium diffraction lines cannot be avoided at these pressures.

Figure 6 .

 6 Figure 6. (A) Detailed view of the peak splitting observed at pressures above 320 GPa. Pattern collected between 280 to 370 GPa from bottom to top of the figure. Blue lines (hcp) and red lines (bcc) are guide to the eye for the different aluminium phases. (B) Cake image of the pattern recorded at 310 GPa, showing hcp 002 spotty reflections (C) Cake image of the pattern recorded at 330 GPa, showing the splitting of hcp 002 and bcc 110 reflections. A thin black line serves as a reference guide for the 002 hcp reflection.

Figure 7 .

 7 Figure 7. Observed diffraction at 320 GPa at the onset of the hcp -bcc transition. Fitted cell parameters are a= 2.230 (2) Å and c=3.635 (3) Å for a unit cell volume of 15.655 (41) Å 3 for the hcp structure, coexisting with a bcc lattice with a=2.505 (3) Å and a unit cell volume of 15.719 (56) Å 3 .

Figure 8 .

 8 Figure 8. Left panel: View of adjacent (001) planes with ABAB stacking in the hcp structure, seen along the [001] axis. Right panel: View of adjacent (110) planes in the bcc structure derived from the (001) hcp planes by distortion and shear, with underlying bcc unit cells.

Acknowledgements

The focused ion beam (FIB) and scanning electron microscopy (SEM) facility of the Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie is supported by Région Ile de France grant SESAME 2006 N°I-07-593/R, INSU-CNRS, INP-CNRS, University Pierre et Marie Curie-Paris 6, and by the French National Research Agency (ANR) grant ANR-07-BLAN-0124-01. G. Fiquet acknowledges the program MATISSE that contributed to the visit of Pr. Narayana at IMPMC-Sorbonne Université. MATISSE is a Cluster of Excellence led by Sorbonne Universit s and managed by ANR within the Investissements d'Avenir program under reference ANR-11-IDEX-0004-02.