Dr Benjamin Rousselle

Aurélie Massot

Dr Malorie Privat

Lucile Dondaine

Dr Audrey Trommenschlager

Dr Florence Bouyer

Dr Jérôme Bayardon

François Ghiringhelli

Ali Bettaieb

Dr Christine Goze

Prof François Ghiringhelli

Prof Ali Bettaieb

Prof Catherine Paul

Dr Raluca Malacea-Kabbara

Prof Ewen Bodio
email: ewen.bodio@u-bourgogne.fr

] B Rousselle

E Bodio

C Paul

Conception and Evaluation of Fluorescent Phosphine-Gold Complexes: From Synthesis to in vivo Investigations

Keywords: theranostic, gold complex, fluorescence, coumarin, phosphine

Introduction

The research of new antineoplastic agents has been giving pride of place to metal-based drugs for years and more particularly since the discovery of the anticancer properties of cisplatin by Rosenberg. [1,2] While many researches have focused on platinum derivatives, [3][4][5] the anti-cancer properties of many other metals such as titanium, [6] ruthenium [7] ... have been investigated. [8][START_REF] Zhao | Advances in Inorganic Chemistry[END_REF][START_REF] Tong | Advances in Inorganic Chemistry[END_REF][START_REF] Murray | [END_REF][12][13][14][15][16][17][18][19][20] In our case, we are interested in gold salts. [21][22][23][24] They have been used since ancient times and are still under investigation today. Indeed, gold complexes have been exploited in modern medicine to fight against parasitic and bacterial infection, or inflammation phenomena. [25][26][27][28] Additionally, the structural analogy of certain gold (III) complexes with cisplatin has inspired some researchers to investigate their potential in fighting cancer. [29][30][31][32] Various studies have opened up new avenues by stabilizing these complexes and showing that in a biological environment, they could be reduced to gold (I), which initiated research on gold (I) complexes. [31,33,34] However, it was the repositioning of auranofin, the drug used to treat acute rheumatoid arthritis, as an anti-cancer drug, that really kick-started the development of anti-cancer gold complexes. [35] Indeed, this phosphine-gold complex with a peracetylated thioglucose as "X" ligand is now being studied in several clinical trials for the treatment of different types of cancer (e.g. Epithelial Ovarian, Primary Peritoneal, Fallopian Tube Cancer [NCT01747798, NCT03456700], Chronic Lymphocytic Leukemia [NCT01419691], Non-Small Cell Lung Cancer [NCT01737502]…). [36][37][38][39][40] Thus, for the past ten years, we have been developing various gold complexes with a preference for phosphine-gold derivative, [41][42][43][44][45][46] and, if possible, complexes, which can be tracked by optical imaging. [18,21,23,[47][48][49][50][51][52] The development of such trackable therapeutics enabled us to get crucial information on the behavior of such new therapeutics in vitro. In particular, we developed a smart fluorescent "ON / OFF" probe. [49,53] It allowed us to prove that the P-Au bond was stable in vitro and in vivo (in zebrafish larvae), in contrast to commonly received ideas that this bond degrades in a biological environment. More precisely, the fluorescence of our system is quenched when the P-Au bond is broken (Figure 1, upper left part). Interestingly, this coumarinbased probe associated with gold (I) exhibited interesting antiproliferative properties on cancer cells, while not showing significant toxicity in zebrafish. The choice of coumarin as a fluorophore was not made at random. Indeed, this fluorophore is perfect for in vitro imaging: it is stable, has a good brightness and responds even in two-photon imaging. Moreover, it also presents interesting biological properties, either as an antibacterial or antiinflammatory agent. [54][55][56][57][58] This last property is interesting because gold(I) complexes often also have anti-inflammatory properties. Knowing that some anti-cancer treatments induce inflammation phenomena which seem to be the cause of side effects, [59,60] the development of a drug with an anti-inflammatory effect should make it possible to limit such issues.

In addition, we have shown in a recent study that combining a phosphonium moiety with a phosphine-gold complex increases dramatically the anti-proliferative properties of the resulting molecule with regards to corresponding gold complex (Figure 1, upper right part). [61] Indeed, the phosphonium group allows to improve the crossing of cell membranes thanks to the combination of the water-solubilizing character of its positive charge and the lypophilicity due to the delocalization of this charge, when phosphonium is substituted by aromatic groups. [62] Moreover, phosphoniums are known to target mitochondria and have intrinsic anticancer activity. [63,64] In the present study (Figure 1, bottom part), we therefore decided to combine these two strategies in order to optimize the previous systems and to benefit from their respective advantages: i.e. to have a trackable gold complex (green part), which has a strong antiproliferative activity (orange part). Thus, in a first step, the target compound was synthesized, characterized and its photophysical, antiproliferative, and fluorescence properties in vitro were compared to those of previously reported compounds. In a second step, an in vivo study was performed to determine which is the most promising theranostic. [49,53] and on phosphine-phosphonium-gold complexes (orange part) [61] Results and Discussion Synthesis Obtaining phosphine-phosphonium complexes involves an alkylation reaction of a phosphine of interest with a (2bromomethylphenyl)phosphine derivative. In our case, the phosphine of interest is the coumarin phosphine that we used previously to synthesize the smart theranostic. It is synthesized in two steps according to the strategy reported by Prat and coll (Scheme 1). [65] The first step consists in a cyclization reaction between 4-iodophenylacetonitrile and 2-hydroxy-4methoxybenzaldehyde in presence of piperidine, which results in the coumarin core III. Then, an organometallic coupling enables the introduction of the chosen phosphine. On the other side, [(2bromomethyl)phenyl] diphenylphosphine borane IX is obtained in three steps (Scheme 1): [61] reaction of 2-bromobenzyl alcohol with chlorodiphenyl phosphine and DIPEA, followed by boration with BH3, then a halogen-metal exchange is carried out in the presence of tBuLi allowing a phospha-Fries rearrangement to give [(2-hydroxymethyl)phenyl] phosphine borane VIII (these different changes are easily monitored by 31 P-NMR), and the desired intermediate is finally obtained thanks to the Appel's reaction, which allows the conversion of the alcohol function to the bromide derivative. Once these two intermediates are in hands, the phosphonium-phosphine ligand 1 has been obtained in 43% yield by reacting them together.

The 31 P-NMR spectrum shows two characteristic signals at -15.1 ppm for the phosphine moiety and at 22.7 ppm for the phosphonium group. The final gold complexes are obtained by reacting chloro(tetrahydrothiophene)gold (I) precursor with IV for the synthesis of X and bromo(tetrahydrothiophene)gold(I) precursor with 1 for the synthesis of 2. In the latter case, we used the brominated version of the gold salt to avoid obtaining a chlorido and bromido-gold mixture induced by the bromide counter-ion of 1. This last reaction is carried out in dichloromethane at room temperature and affords the corresponding gold(I)-phosphonium-phosphine complex 2 in 94% yield (Scheme 1). Its 31 Scheme 1. Synthesis of phosphine-gold(I) complex X, phosphonium XI, and phosphonium-phosphine gold(I) complex 2.

Photophysical properties

One of the interests of introducing a coumarin moiety, beyond its contribution from a biological activity point of view, is that it enables the tracking of the therapeutic agent by fluorescence imaging. We have therefore characterized the photophysical properties of the new gold complex 2 and its corresponding ligand 1 (Figure 2). For comparison, we have indicated the properties of IV, V, X, and XI previously measured. [49,53] It is worth noting that the maximum absorption and emission wavelengths are little affected by the various modifications: no impact of complexation with gold and bathochromic shift of 10 nm in the case of the phosphonium derivatives. Likewise, the molar absorption coefficients are similar, although they are higher for XI.

Conversely, quantum yields are strongly modified by the presence of a lone pair on the phosphorus. This free lone pair generates photo-induced electron transfer (PET) (e.g. ligands IV and V and 1, even if in this last case the PET is lower, due to an increased distance between the fluorophore and the phosphorus atom). When the latter is engaged in the formation of a "P-C" bond in the case of phosphonium (e.g. XI) or of a "P-Au" bond for gold complexes (e.g. X), fluorescence is restored (X and XI 30 to 40 times brighter than IV respectively, and 2 is almost 70% of the brighter than 1) (Figure 3).

N°

λabs [a] (nm)

λem [b] (nm)

ε [c] (M -1 .cm -1 )
Φf [d] (%) V, X, XI, [49] 1, and 2 in DMSO at 298 K. [a] λabs = wavelength of maximal absorption. [b] λem = wavelength of maximal emission. [c] ε = molar absorption coefficient. [d] Φf = fluorescence quantum yield, reference: diphenylanthracene (Φf = 0.97, λexc = 355 nm, in cyclohexane [66] ).

IV

In vitro investigations

While coumarin derivatives exhibit good signal of fluorescence, their photophysical properties are not ideal for in vitro imaging. Indeed, their maximum absorption wavelength, which is between 350 and 360 nm is too low to be compatible with many microscopes. In addition, a low wavelength is also synonymous with high energy, thus, such excitation wavelength can degrade the tissues observed. Fortunately, we previously noticed that coumarins could respond to two-photon imaging. The advantage of this technique is that it allows the use of longer wavelength photons -in our case 700-720 nm -which will not damage the tissues.

As expected, both X and 2 gave good response in two-photon imaging, and thus, can be easily tracked in vitro (Figure 4). By looking at the images obtained by irradiation at 720 nm of CT26 cancer cells, previously incubated for 1 h at 37°C with 50 μM of the gold complex 2, we can see that it enters the cytoplasm, even if it also appears to accumulate in vesicles. Interestingly, the power of the laser needed for obtaining an equivalent signal for 2 is three times lower than for X. Furthermore, no drop in fluorescence was observed during the experiments. This would tend to confirm that the "P-Au" bond is stable in a cell medium, even if in this case, we must remain cautious because the difference in brightness between 2 and its corresponding ligand 1 is by a factor of approximately 2 and not of 30 as in the case of X and IV. As compound 2 can be easily tracked in vitro by two-photon imaging, its anti-proliferative properties were evaluated on three human cell lines of lung (A549), breast (MDA-MD-231), and colon (SW480) cancers (Table 1). On the three cell lines, after 48 h, the gold complex 2 appears strongly active. It exhibits activities of the range of paclitaxel, it is 4 to 18 times more active than oxaliplatin and more than 20 times more efficient than 5-FU. Moreover, if we take the IC50 that we have obtained for coumarin-gold X on MDA-MB-231 and SW480, [49,53] 2 is 17 to 26 times more active than X.

It appears that the modification of the structure of the complex very significantly improved its anti-proliferative activity. In addition, it would come in second position, if we compare it to the seven phosphonium-phosphine-gold (I) complexes that we had synthesized and studied previously. [61] This confirms our observations by highlighting that promoting the delocalization of the phosphonium charge over a maximum of the aromatic cycles improves the anti-proliferative properties of the compound. Before proceeding with the in vivo experiments, the antiproliferative properties of X and 2 were investigated onto lung (LLC1), breast (4T1), and colon (CT26) murine cancer cell lines (Table 2). Indeed, given the importance of the immune system in the tumor response, we wanted to preserve it and therefore use syngeneic models. Again, complex 2 exhibits anti-proliferative properties far superior to X.

In vivo studies

Proliferation tests have shown that compound 2 is about as active on the three murine cancer cell lines, while X displays its best activity on the CT26 cancer cell line. The anti-cancer effect of X and 2 was therefore evaluated in Balb/c mice bearing subcutaneous CT26 tumor (Figure 5). When the tumor size reaches a volume of about 50 mm 3 , the mice were injected intraperitoneally twice a week at a dose of 0.1 mg / kg of X or of 2. Tumors have been measured regularly and it appears that, as might be expected, complex X exhibits limited anti-cancer activity at this concentration. On the other hand, phosphoniumphosphine-gold 2 has a very significant anti-cancer effect with a strong decrease of tumor growth. A similar anticancer effect was obtained by Milovanovic and coll. using a mass concentration of oxaliplatin 20 times higher than the one we used for compound 2 under conditions similar to ours. [67] Furthermore, in our case, no sign of toxicity was observed in any of the mice.

Conclusion

During this study, the synthesis of a novel optical theranostic agent was described as well as its characterization. It appears that the phosphonium-phosphine unit not only increases the solubility in water but also dramatically improves the antiproliferative properties of an average factor of 20 of the complex compared to its phosphine-gold analogue. We also noticed that it had a better signal on two-photon imaging so that it could be tracked in the cells. Finally, it exhibits very significant anti-cancer activities in vivo without any sign of toxicity, whilst its phosphinegold analogue does not exhibit significant anti-cancer activity. These very promising results prompt us, in a future study, to explore in more details the mechanisms of action involved, to know whether it may be interesting to modify the ligand "X" carried by the gold to further improve the biological activity of the complex and, in parallel, to determine the maximum tolerated dose to further optimize its activity in vivo.

Experimental Section Material and methods

All synthetic manipulations were carried out using standard Schlenk techniques. All reagents were purchased either from Acros Organics, Sigma-Aldrich, Fischer Scientific, Alfa Aesar, TCI or ABCR and used without further purification. THF and dichloromethane were dried over alumina cartridges using a solvent purification system MB-SPS-800 model from M. BRAUN. Column chromatography were conducted on silica gel 60-200 μm or 40-63 μm purchased from Sigma-Aldrich. NMR spectra ( 1 H, 13 C, 31 P) were recorded with Brucker 300 MHz, 500 MHz or 600 MHz apparatus, using tetramethylsilane as internal reference for 1 H, 13 C NMR, phosphoric acid (85%) as external reference for 31 P NMR. Abbreviations used to describe the multiplicity of the signal are: s (singlet), d (doublet), t (triplet), q (quartet), bs (broad singlet), m (multiplet). Mass spectra were performed under electrospray ionization conditions (ESI) with a Thermo LTQ Orbitrap XP. Infrared spectra were recorded on a FT-IR instrument (Bruker Alpha), and the data are given in cm -1 . To a solution of [(2-bromomethyl)phenyl]diphenylphosphine borane IX (185 mg, 0.5 mmol) in DCM (2 mL) was added the tertiary phosphine V (545 mg, 1.25 mmol, 2.5 equivalents) and the reaction was stirred 24h. After removing most of the solvent, Et2O (5 mL) was added and a precipitate appeared which was filtered and purified on silica gel using MeOH/DCM 1:10 as eluent. Compound 1 was obtained as a yellow solid in 43% yield (170 mg, 0.21 mmol).

Mp: 162°C (dec). To a solution of phosphine phosphonium 1 (80 mg, 0.1 mmol) in DCM (4 mL), bromo(tetrahydrothiophene) Au (I) (37 mg, 0.1 mmol) was added under argon at RT and the reaction was stirred for 1h. The resulting mixture was concentrated and the product was precipitated using pentane and washed twice with a mixture of pentane/DCM 10:1. Complex 2 was obtained as a white solid in 94% yield (100 mg, 0.094 mmol).

Mp : 188 °C (dec).

Drug solutions:

The newly phosphonium-phosphine gold(I) complex 2 was diluted into dimethylsulfoxide (DMSO) (Sigma, France). Reference molecules, approved in clinic for colon, breast or lung cancer, were acquired from commercial solutions: oxaliplatin (Oxaliplatin Dakota Pharm® 5 mg/ml), 5fluorouracil (Fluorouracil Accord® 50 mg/ml) and paclitaxel (Paclitaxel Kabi® 6 mg/ml). In cell culture, maximum concentration of DMSO did not exceed 3 % in the medium.

Determination of cytotoxicity:

The crystal violet staining test and MTS assay were chosen to determine the cytotoxicity of different compounds on respectively human and murine cancer cell lines. 10 5 cells were seeded in 96-well plates and incubated for 24h to allow for cell adherence. Twenty four hours later, cells were treated for 48 hours by increasing concentrations (from 0 to 500 μM or from 0 to 25 μM according to the drug) of the newly synthetized or reference molecules. After treatment, cytotoxicity on human cancer cell lines was assessed by crystal violet staining, at λ = 570 nm (spectrophotometer UVM 340, Bioserv). The cytotoxic activity of compounds and drug references on murine cancer cell lines was determined using the MTS assay (Promega®). In order to test the potential anti-tumor effect of 2 and X compounds, Balb/c mice (8 weeks old, Charles River, France) were grafted by subcutaneous injection of murine colon cancer CT26 cells (5.10 5 cells). Seven days after tumor implantation (tumor volume around 50 mm 3 ), tumor-bearing mice were randomized into three groups, one received 0.1 mg/kg of 2 or X compounds (in DMSO) and the other received the same quantity of DMSO in saline (vehicle) by intraperitoneal injection (IP). These IP injections were performed two times a week and tumor volume assessed three times per week. Mice whose tumor reached 1,500 mm 3 were euthanized due to ethical reasons.

Entry for the Table of Contents

A promising gold-based optical theranostic agent was conceived. This phosphine-phosphonium gold(I) complex can be tracked by two-photon imaging thanks to a coumarin moiety. It displayed strong anti-proliferative properties against cancer cell lines and induced an anti-cancer effect in vivo.

Institute and/or researcher Twitter usernames: ((optional)) @ICMUB

Figure 1 .

 1 Figure 1. Illustration of the strategy developed in this work: combination of the work we have done on smart theranostics (coumarin-phosphine-gold complexes, green part)[49,53] and on phosphine-phosphonium-gold complexes (orange part)[61]

  P NMR spectrum shows two signals at 23.Ph or p-Tol) [Au(tht)Cl] DCM, r.t. = p-Tol) -V (Ar = Ph)

Figure 2 .

 2 Figure 2. (top) normalized absorption (blue) and emission (orange) spectra of compound 2 in DMSO at 298 K.(bottom) Photophysical data of compounds IV, V, X, XI,[49] 1, and 2 in DMSO at 298 K.[a] λabs = wavelength of maximal absorption.[b] λem = wavelength of maximal emission.[c] ε = molar absorption coefficient.[d] Φf = fluorescence quantum yield, reference: diphenylanthracene (Φf = 0.97, λexc = 355 nm, in cyclohexane[66] ).

Figure 3 .

 3 Figure 3. Explanation of the difference of quench phenomenon intensity for the different compounds (top) and its impact on the brightness (brightness, B = εΦf) of the corresponding compounds (bottom).

Figure 4 .

 4 Figure 4. Biphoton images of the compounds X and 2 on CT26 cell line. Cells are incubated, or not (Ctrl with DMSO), with 50 μM of the compounds for 1 h at 37°C, then fixed and permeabilized with 2% paraformaldhehyde and mounted with Prolong. Two-photon images are recorded upon 720 nm excitation (Chameleon IR laser from Coherent) and fluorescence emission collected through channel 1 ("DAPI channel") 400/492 nm.

Figure 5 .

 5 Figure 5. Anti-tumor effect of X and 2 compounds. CT26 Tumor-bearing BALB/c mice (n=6/group) were treated intraperitoneally twice per week with 0.1 mg/kg of compounds X or 2 (in DMSO) or with the same quantity of DMSO (vehicle). Tumor growth was determined 3 times per week using a caliper. Results are expressed as means ± SEM, Two-way Anova:*p<0.05, **p≤0.01, ***≤0.001.

Synthesis 3 -

 3 {4-{[(2diphenylphosphanyl)phenyl]methyldiphenylphosphonium}phenyl}-7methoxy-2H-chromen-2-one bromide 1 :

1 H

 1 NMR (500 MHz, Methylene Chloride-d2) δ 8.19 (s, 1H, Haro), 8.07 (dd, J = 8.5, 3.1 Hz, 2H, Haro), 7.92 -7.87 (m, 2H, Haro), 7.71 -7.64 (m, 5H, Haro), 7.62 -7.54 (m, 6H, Haro), 7.39 -7.23 (m, 9H, Haro), 6.98 -6.88 (m, 6H, Haro), 6.88 -6.82 (m, 1H, Haro), 5.04 (d, J = 13.8 Hz, 2H, CH2), 3.92 (s, 3H, OCH3).{ 1 H} 13 C NMR (151 MHz, Methylene Chloride-d2) δ 164.68 (Caro), 160.88 (Caro), 156.78 (Caro), 143.72 (Caro), 143.33 (d, J = 3.3 Hz, Caro), 139.40 (dd, J = 12.8, 6.1 Hz, Caro), 136.25 (d, J = 3.1 Hz, Caro), 135.57 (d, J = 3.4 Hz, Caro), 135.25 (d, J = 10.1 Hz, Caro), 135.22 (d, J = 9.8 Hz, Caro), 134.81 (d, J = 7.7 Hz, Caro), 134.54 (d, J = 19.8 Hz, Caro), 132.58 (dd, J = 24.0, 8.9 Hz, Caro), 131.80 (t, J = 4.5 Hz, Caro), 131.16 (d, J = 12.6 Hz, Caro), 130.95 (d, J = 3.6 Hz, Caro), 130.88 (Caro), 130.67 (d, J = 12.8 Hz, Caro), 130.50 (d, J = 3.9 Hz, Caro), 130.28 (Caro), 129.70 (d, J = 7.2 Hz, Caro), 122.21 (Caro), 118.19 (d, J = 85.8 Hz, Caro), 116.93 (d, J = 87.4 Hz, Caro), 113.97 (Caro), 113.80 (Caro), 101.24 (Caro), 56.83 (OCH3), 30.85 (dd, J = 48.3, 24.2 Hz, CH2). { 1 H} 31 P NMR (202.5 MHz, Methylene Chloride-d2) δ 22.67 (s), -15.09 (s). IR (cm -1 ): 3052, 1719, 1590, 1506, 1435, 1360, 1270, 1161, 1112, 1021, 984, 939, 824, 781, 740, 718, 690, 637, 623, 595, 540, 512. HR-MS (ESI): m/z calcd for C47H37O3P2 [M-Br] + : 711.22124 Da; Found : 711.22307 Da. {3-{4-{[(2-diphenylphosphanyl)phenyl]methyldiphenylphosphonium} phenyl}-7-methoxy-2H-chromen-2-one} AuBr 2 :

  dimethylthiazol-2-yl)-5-(3carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega, Charbonnières, France) was added in each well and absorbance at 490 nm was measured after 3h incubation at 37 °C. The resulting IC50 values were calculated using GraphPad Prism 5.0 software. Each treatment was performed in three independent experiments.Determination of the anti-tumor effect:All experiments followed the guidelines of the Federation of European Animal Science Associations and were all approved by the Ministry of agriculture and food (France, Apafis number: #15181) and by the Ethics Committee of Burgundy University (Dijon, France).

Table 1 .

 1 Determination of the IC50 values (μM) of complexes X and 2, compared those of oxaliplatin and 5-FU on A549, MDA-MD-231, and SW480 cancer cell lines by crystal violet assay at 48 h (values are presented as the mean ± SEM of at least three independent experiments with three replicates).

	Compound	A549	IC50 (µM) MDA-MB-231	SW480
	X	N.T.

33.1  14.8* 42.0  11.7* 2 0.42  0.01 1.28  0.44 2.47  0.97 Oxaliplatin 7.5  0.40 14.4  1.90 9.2  1.40 5-FU 9.1  0.90 22.3  0.60 52.5  8.40 Paclitaxel 0.33  0.04 0.39  0.02 2.0  0.74

Table 2 .

 2 Determination of the IC50 values (μM) of X and 2 compounds on LLC1, 4T1 and CT26 cancer cell lines by MTS assay at 48 h (values are presented as the mean ± SEM of at least three independent experiments with three replicates).

	Compound	LLC1	IC50 (µM) 4T1	CT26
	X	94.9  16.0	40.2  0.23	25.4  0.4
	2	4.81  0.34	4.72  0.10	4.67  0.09

  NMR (500 MHz, Chloroform-d) δ 8.26 (s, 1H, Haro), 8.11 (d, J = 7.1 Hz, 2H, Haro), 7.85-7.78 (m, 2H, Haro), 7.72 -7.60 (m, 11H, Haro), 7.60 -7.43 (m, 8H, Haro), 7.43-7.34 (m, 5H, Haro), 6.93 (dd, J = 8.6, 2.4 Hz, 1H, Haro), 6.87 (d, J = 2.4 Hz, 1H, Haro), 6.80 (dd, J = 12.6, 7.8 Hz, 1H, Haro), 5.66 (d, J = 14.9 Hz, 2H, CH2), 3.92 (s, 3H, OCH3).

	{ 1 H} 13 C NMR (151 MHz, Methylene Chloride-d2) δ 164.64 (Caro), 160.86
	(Caro), 156.76 (Caro), 143.80 (Caro), 143.45 (d, J = 3.3 Hz, Caro), 136.48 (d,
	J = 3.2 Hz, Caro), 136.26 (d, J = 7.5 Hz, Caro), 135.48 (d, J = 11.2 Hz, Caro),
	135.47 (d, J = 10.1 Hz, Caro), 135.26 (d, J = 14.3 Hz, Caro), 133.6 (Caro),
	133.56 (d, J = 2.3 Hz, Caro), 133.27 (m, Caro), 132.68 (dd, J = 10.6, 7.7 Hz,
	Caro), 132.51 (d, J = 9.7 Hz, Caro), 131.46 (d, J = 12.7 Hz, Caro), 131.04 (d,
	J = 12.9 Hz, Caro), 130.85 (Caro), 130.70 (d, J = 12.2 Hz, Caro), 129.82 (dd,
	J = 57.5, 6.7 Hz, Caro), 127.87 (d, J = 62.9 Hz, Caro), 122.31 (Caro), 117.70
	(d, J = 86.4 Hz, Caro), 116.43 (d, J = 87.4 Hz, Caro), 113.94 (Caro), 113.82
	(Caro), 101.24 (Caro), 56.82 (OCH3), 30.36 (dd, J = 50.1, 12.8 Hz, CH2).
	{ 1 H} 31 P NMR (202.5 MHz, Chloroform-d) δ 26.76 (s), 23.12 (s).
	IR (cm -1 ): 2926, 1719, 1589, 1506, 1435, 1403, 1360, 1270, 1216, 1161,
	1100, 1024, 996, 939, 822, 774, 748, 717, 688, 622, 596, 543, 495.
	HR-MS (ESI): m/z calcd for C47H37AuBrO3P2 [M-Br] + : 987.10613 Da;
	Found: 987.10788 Da.
	Cell lines and culture conditions:
	Human colon cancer (SW 480), breast cancer (MDA-MB-231), and lung
	cancer (A-549) and murine colon cancer (CT26), breast cancer (4T1), and
	lung cancer (LLC1)cell lines were obtained from the American Type
	Culture Collections (Manassas, VA, United States). They were cultured in
	RPMI 1640 (SW480, MDA-MB-231, A-549 and CT26) or DMEM (4T1 and
	LLC1) medium (Biowhittaker, France) supplemented with 10% fetal bovine
	serum (Biowhittaker, France) at 37°C under a humidified atmosphere
	containing 5% CO2. All cell lines were maintained as exponentially growing
	monolayers in mycoplasma free culture condition checked by polymerase
	chain reaction (PCR) analysis (PCR Mycoplasma Test Kit I/C, PromoKine,
	PromoCell France).

1 H

Acknowledgements

The Ministère de l'Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), the Conseil Régional de Bourgogne (PhD JCE grant # 2018Y-08158), and the French Research National Agency (ANR) via project JCJC ''SPID" ANR-16-CE07-0020 are gratefully acknowledged. This work is part of the projects ''Pharmacoimagerie et agents théranostiques" et ''Chimie durable, environnement et agroalimentaire" supported by the Université de Bourgogne and the Conseil Régional de Bourgogne through the Plan d'Actions Régional pour l'Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. We thank the Conseil Régional de Bourgogne Franche-Comté for its support via the project ANER "AZTHEC". This work was also supported by grants from Ligue contre le Cancer, Comité de Côte d'Or. FrenchBIC, and GDR AIM are acknowledged for fruitful discussion. Ms M.-J. Penouilh and Mr. Q. Bonnin are gratefully acknowledged for HR-MS analysis, and Dr M. Picquet for NMR analyses. The authors would like to thank Dr V. Comte-Candas, Ms. P. Girard, and Ms. M. Guillemin for their technical support, Ms. V. Saint-Giorgio (zootechny center, UBFC), Ms. A. Legrand (Cytometry Plateform, UMR1231 UBFC), and Ms. E. Noirot (Plateforme DImaCell, AgroSup Dijon INRAE, Université Bourgogne Franche-Comté).


Dr Tarek Khelfa
email: tarekkhelfa@yahoo.fr

Rabeb Lachhab

Hiba Azzeddine

Zhiguo Chen

Jairo Alberto Muñoz

José María Cabrera-Marrero

François Brisset

Anne-Laure Helbert

Thierry Baudin

Mohamed Khitouni

Effect of ECAP and Subsequent Annealing on Microstructure, Texture, and Microhardness of anAA6060 Aluminum Alloy

Keywords: Aluminum alloy, ECAP, Microstructure, Texture, Hardness, Activation energy

AA6060 aluminum alloy was subjected to severe plastic deformation (SPD) through Equal Channel Angular Pressing (ECAP) up to 8 passes via route BC. ECAPed samples isochronally annealed for 1 hour at a temperature range of 150-450 °C. The microstructure and texture of the studied material were evaluated by Electron Backscatter Diffraction (EBSD), and the microhardness was characterized by Vickers microhardness testing. It was found that shearing texture is typically enhanced after ECAP processing. Grain size and grain growth kinetics were also studied. ECAP led to a substantial rise in hardness, with stability following 4 passes. Microstructures and material properties were relatively stable up to annealing temperatures of 150 °C. Some sub-micrometer grains were kept 2 in the 8 passes sample to annealing temperatures of 300 °C. Annealing at elevated temperature resulted in a reduction in hardness leading to a rise in grain size and a decrease in dislocation density. After annealing temperature up to 450 °C, the texture index reveals a tendency to the texture weakening and randomization. The activation energy required for the grain growth of the AA6060 alloy was exceptionally low above 300 °C.

Introduction

Equal-channel angular pressing (ECAP) as among the most efficient and attainable processes of severe plastic deformation (SPD) has extensive applications thanks to the manufacture of nanostructured (NS) and UFG metals and alloys [START_REF] Valiev | Bulk nanostructured materials from severe plastic deformation[END_REF][START_REF] Valiev | Plastic deformation of alloys with submicron-grained structure[END_REF][START_REF] Valiev | Structure and properties of ultrafine-grained materials produced by severe plastic deformation[END_REF][START_REF] Valiev | Principles of equal-channel angular pressing as a processing tool for grain refinement[END_REF][START_REF] Segal | Kopylov Plastic working of metals by simple shear[END_REF]. On the other hand, it is well established that using SPD processes, the physical and mechanical properties of metallic materials can be significantly improved [START_REF] Inoue | Improvement of strength, toughness and ductility in ultrafine-grained low-carbon steel processed by warm bi-axial rolling[END_REF]. Additionally, SPD processes may also cause important changes in the evolution of microstructure and crystallographic texture relative to traditional processing methods [START_REF] Naseri | Effect of strain path on microstructure, deformation texture and mechanical properties of nano/ultrafine grained AA1050 processed by accumulative roll bonding (ARB)[END_REF]. AA6000 series aluminum alloys have drawn interest in the aviation, automotive, electrical engineering, and building industries thanks to their lightweight, excellent strength, high conductivity, and good corrosion resistance [START_REF] Bobruk | Enhanced strengthening in ultrafinegrained Al-Mg-Si alloys produced via ECAP with parallel channels[END_REF][START_REF] Yuan | Effects of La addition on the mechanical properties and thermal-resistant properties of Al-Mg-Si-Zr alloys based on AA 6201[END_REF][START_REF] Ji | Microstructures and properties of Al-Mg-SI alloy overhead conductor by horizontal continuous casting and continuous extrusion forming process[END_REF][START_REF] Murashkin | Enhanced mechanical properties and electrical conductivity in ultrafine-grained Al alloy processed via ECAP-PC[END_REF]. Several studies have concentrated on the evaluation of AA6000 series alloys after ECAP processing [START_REF] Kim | Optimization of strength and ductility of 2024 Al by equal channel angular pressing (ECAP) and post-ECAP aging[END_REF][START_REF] Horita | Equal channel angular pressing of commercial aluminum alloys: grain refinement, thermal stability and tensile properties[END_REF][START_REF] Khelfa | Structure and microstructure evolution of Al-Mg-Si alloy processed by equal-channel angular pressing[END_REF][START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF]. For example in [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF], AA6060 samples were severely ECAPed at ambient temperature up to 12 ECAP passes via route BC. In overall behavior, samples exhibited an equiaxed array of UFG microstructure and a rapid increase in high-angle grain boundaries (HAGB) fraction after 5 passes while a greater number of passes demonstrate a limited effect on strength and no significant change in the average grain size. In spite of the fact that UFG alloys can have remarkable mechanical properties, they can not be utilized at high temperatures except the microstructures are steady. In fact, the annealing temperature can strongly affect the microstructure evolution which has a significant impact on the physical, chemical, and mechanical characteristics.

Then, developing a better comprehension of the thermal stability behavior of UFG structures produced by ECAP claims interest from the scientific community. Recently, extensive research work has been performed to examine the impact of annealing on microstructure development of highly deformed materials (Cu, Al, Fe, Mg) [START_REF] Liang | Influence of microstructure on thermal stability of ultrafine-grained Cu processed by equal channel angular pressing[END_REF][START_REF] Rangaraju | Effect of cryo-rolling and annealing on microstructure and properties of commercially pure aluminium[END_REF][START_REF] Muñoz | Thermal stability of ARMCO iron processed by ECAP[END_REF][START_REF] Hanna | An investigation of the thermal stability of a Mg-Dy alloy after processing by high-pressure torsion[END_REF][START_REF] Lugo | Thermal stability of ultrafine grains size of pure copper obtained by equal-channel angular pressing[END_REF] and Al alloys [START_REF] Mao | Grain refinement, thermal stability and tensile properties of 2024 aluminum alloy after equal-channel angular pressing[END_REF][START_REF] Nikulin | The Effect of Temperature on Microstructure Evolution in a 7055 Aluminum Alloy Subjected to ECAP[END_REF][START_REF] Dhal | Precipitation phenomena, thermal stability and grain growth kinetics in an ultra-fine grained Al 2014 alloy after annealing treatment[END_REF][START_REF] Prangnell | Continuous recrystallisation of lamellar deformation structures produced by severe deformation[END_REF]. It was reported by Liang et al. [START_REF] Liang | Influence of microstructure on thermal stability of ultrafine-grained Cu processed by equal channel angular pressing[END_REF] that a lower number of ECAP passes results in retarded recrystallization process to longer annealing duration or elevated annealing temperature. With approximately the same strength properties and improved thermal stability, the microstructure got after 2 ECAP passes has some benefit in terms of the cost of production as opposed to 16 ECAP passes. Studying the annealing behavior of Al-0.13% and Al-3%Mg treated by the ECAP process, Prangnell et al. [START_REF] Prangnell | Continuous recrystallisation of lamellar deformation structures produced by severe deformation[END_REF] mentioned that the early stages of annealing were considered to be particularly susceptible to the misorientation and distribution of the low angle boundaries maintained inside the structure of deformation. According to Dhal et al. [START_REF] Dhal | Precipitation phenomena, thermal stability and grain growth kinetics in an ultra-fine grained Al 2014 alloy after annealing treatment[END_REF], after annealing treatment, the precipitation of Cu2Al and AlCuMgSi particles in UFG Al 2014 alloy resulted in better thermal stability. In contrast, Han and Langdon [START_REF] Han | Significance of microstructural thermal stability in an Al-2219 alloy processed by severe plastic deformation, Ultrafine Grained Materials II[END_REF] gave proof that despite the fact that precipitates of θ′ or θ are present in the AA 2219 aluminum alloy microstructure, they were not deemed powerful enough to inhibit the substantial growth of grain at high temperatures. In this context, the main challenge in the thermal stability of Al alloys is to increase the upper service limit at high temperatures [START_REF] Czerwinski | Thermal Stability of Aluminum Alloys[END_REF].

The current research aims to investigate the impact of annealing temperature on the microstructure morphology, texture and microhardness evolution of AA6060 aluminum alloy ECAPed up to eight ECAP passes.

Experimental procedure

The material studied is AA6060 aluminum alloy in the aged condition T6 as an extruded rod with a 10 mm diameter and with the chemical composition listed in Table 1. Rods were machined in about 60 mm long cylinders. The initial grain size was ~52 μm. ECAP was done using die with two channels that intersect at an angle of = 90° and an outer curvature angle of Ψ= 37°. With this configuration, a total strain of 1 is impaired in each pass [START_REF] Iwahashi | Principle of equal-channel angular pressing for the processing of ultra-fine-grained materials[END_REF]. The ECAP processing was carried out with a 0.02 m/s pressing velocity at room temperature. The lubricant used in the experiments was molybdenum disulfide (MoS2). Specimens were deformed up to eight ECAP passes through route BC, i.e., the billet is rotated in the same sense by 90° about its longitudinal axis between each pass [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF].

Annealing treatments were conducted at various temperatures after ECAP: 150, 200, 250, 300, 350, 400 and 450 °C for 1 hour under high vacuum, with a heating rate of 20 °C min -1 .The microstructures of the specimens were examined through electron backscatter diffraction (EBSD) observations in the Scanning Electron Microscope (SEM) with a Field-Emission Gun (FEG) SUPRA 55 VP operating at 20 kV. For EBSD observations, all specimens were cut in the transverse direction (TD) plane from the core area and then mechanically polished from 1200 grit silicon carbide paper till 0.02µm colloidal silica, followed by electro-polishing utilizing Struers A2 electrolyte at 22 V. The examined regions were 100 x100 µm 2 with a step size of 0.1 µm for specimens annealed at 150 °C, and 200 x 200 μm 2 with a step size of 0.2 μm for specimens annealed at 250 °C, 300 °C, 350 °C and 450 °C. TSL Orientation Imaging Microscopy (OIM™) software was used to perform the EBSD data collection.

Quantitative texture analysis was performed using the MTex software to calculate the pole figure (111) and the orientation distribution function (ODF). The ODF was measured using the harmonic approach (L equal to 22) with every orientation modeled with a Gaussian function possessing a 5 ° half-width. [START_REF] Bachmann | Texture analysis with MTEX[END_REF]. The linear intercept approach was used to calculate the mean grain size. The grain size values were determined with a 5 ° grain tolerance angle.

Vickers micro hardness of the specimens was characterized by SHIMADZU type HMV-2 tester using 100 g load (penetration time of 10 s) in the Extrusion Direction (ED)-Transversal Direction (TD) plane. At least 5 hardness indentations in the central regions of each specimen performed randomly. As shown in Figure 1a, the microstructure of the as-received alloy displays equiaxed grain morphology with an average grain size of ~ 52.6 μm. With increasing ECAP passes at room temperature , as shown in (Figures 1b,1c, 1d and 1e), the microstructure is evolved from a banded structure to a UFG one with elongated and smaller equiaxed grains mostly oriented along the shear direction. At lower temperatures and strain (Figures 2c, 3c, and4c), OIM maps display duplex microstructures characteristic of discontinuous recrystallization mechanism, clearly distinguishing deformed areas from recrystallized grains. At more advanced stages of recrystallization, i.e. larger ECAP passes (Figures 5d and5e), the deformed structure's directionality is gradually lost. The microstructures arising from annealing 1 ECAP specimen show that the total recrystallization mechanism begins at a later stage compared to higher ECAP passes. Indeed, annealed at 150 and 250°C, the one and two pass samples (Figures 2a-2b andFigures 3a-3b) have retained almost the same microstructure as the deformed state. However, small recrystallized grains can be noticed in the large deformed grains and gathered in islands at grain boundaries (see the yellow square in Figure 3b) after annealing at 250°C. A similar observation can be found in the sample ECAPed four times at 250°C in a more advanced stage. According to Humphreys et al. [29], discontinuous recrystallization usually takes place on subsequent annealing for the greater original grain sizes and lower strains. Comparable retard in the recrystallization temperature has also been found by several authors [START_REF] Ferrasse | Texture evolution during equal channel angular extrusion (ECAE): part II. An effect of post-deformation annealing[END_REF][START_REF] Jazaeri | The transition from discontinuous to continuous recrystallization in some aluminium alloys: I -the deformed state[END_REF].

Results and discussion

Microstructural evolution during annealing

As previously stated [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF], annealing in the range of 100-500 °C of AA6060 alloy processed by ECAP leads to the formation of solute rich clusters/Guinier-Preston (GP) zones and to an important reduction in the peak temperature correlated with the 50% of recrystallization with increasing ECAP passes. It was suggested that in the annealed sample after one ECAP pass, precipitated clusters may exert a pinning effect (Smith-Zener pinning) on grain boundaries, which inhibits and/or delays the mechanism of recrystallization. Studying the recrystallization mechanism at particles in rolled aluminum, Humphreys [START_REF] Humphreys | The nucleation of recrystallization at second phase particles in deformed aluminium[END_REF] has deduced that the recrystallization arises with a random orientation at a pre-existing sub-grain inside the deformation region. Then, the nucleation takes place by rapid subboundary migration. Finally, once the deformation zone is consumed, the grain growth may stop.

Complete recrystallization seems to occur after annealing at temperatures of 450, 300, 300, and 350 °C for samples ECAPed once, twice, four and eight times, respectively as displayed at Figures 2e,3c,4c, and 5d. These results indicate that the kinetics of recrystallization significantly based on the strain imposed. It is well established that a rise in strain contributes to a rise in stored energy, which is the major driving energy for recrystallization. Consequently, the mechanism of recrystallization is accelerated. Inhomogeneous grain growth is obtained and some coarse grains can be observed in the EBSD maps at higher annealing temperatures (see Figures 2e, 3e, 4e, and5e) due to the intense grain growth. However, some small regions with fine grains are still noted. As reported in the literature [START_REF] Humphreys | chapter 1-Introduction[END_REF], the smaller grains are removed during annealing, while the larger grains grow, and the grain boundaries adopt a lower energy configuration. In certain conditions, this normal grain growth may give way to the selective growth of a few large grains, by the process of abnormal grain growth or secondary recrystallization (Figure 4e, for example).

During annealing, changes in the relative proportions of HAGB were found to provide a valid way to determine the nature of the recrystallization mechanism [START_REF] Jazaeri | Quantifying recrystallization by electron backscatter diffraction[END_REF]. As discontinuous recrystallization occurs, the HAGB fraction rises fairly rapidly, while the recrystallizing grains consume the deformed substructure. On the other hand, there is a slight shift in the HAGB fraction during continuous recrystallization [START_REF] Jazaeri | Quantifying recrystallization by electron backscatter diffraction[END_REF]. The evolution of HAGB fraction during isochronal annealing is illustrated in Figure 6a. HAGB fraction increases sharply and continually for the one pass specimen, at temperatures range between 250-300 °C in the 2 and 4 ECAP specimens and between 250 and 350 °C for the 8 ECAP passes specimen, indicating discontinuous recrystallization.

The average grain size of the AA6060 alloy processed by ECAP as a function of the annealing temperature is listed in Table 2. A large reduction can be observed from 52.60 µm in the as-received sample to ~ 0.57 µm after eight ECAP passes. In addition, it can be found that for the 1 and 2 ECAP specimens, the average grain size increases rapidly to 6.25 and 5.85 μm after annealing at 250 °C, respectively, and after that remains nearly stable in the 300-450 °C temperature range. On the other hand, the average grain sizes of the 4 and 8 ECAP passes specimens remain almost stable after annealing at 150 °C. However, annealing at 450 °C increases the grain sizes of the 4 and 8 ECAP passes materials to 8.16 and 6.77 μm, respectively, as shown in Figures 4e and5e.

Nevertheless, despite a rise in the annealing temperature, it can be found that for the 4 ECAP specimen, the HAGB fraction remains nearly stable in the 300-450 °C temperature range and above 350 °C for the 2 and 8 ECAP passes specimens. This observation cannot indicate the occurrence of continuous recrystallization since larger grains have been developed during recrystallization. Earlier observations [29,[START_REF] Jazaeri | The transition from discontinuous to continuous recrystallization in some aluminium alloys: I -the deformed state[END_REF], have suggested that continuous recrystallization occurs in specimens where a crucial HAGB fraction was formed during deformation (~64%). Thus, in the case of sufficiently large strains, the HAGB fraction rises to a reasonable value to maintain the microstructure against discontinuous recrystallization. In the present work, the AA6060 alloy has exhibited its maximum value of ~55% in HAGB fraction after deformation up to 8 ECAP passes [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF] which seems to be insufficient to achieve continuous recrystallization.

Figures 6b-6c summarize the grain size evolution and the recrystallized grains, respectively.

Based on the grain orientation spread approach (GOS) available in OIM-software, the recrystallized grain fraction was determined. GOS represents the average deviation between every point orientation in the grain and the average grain orientation [START_REF] Wouters | Effects of crystal structure and grain orientation on the roughness of deformed polycrystalline metals[END_REF]. The recrystallized grains are distinct from the deformed ones by a threshold GOS value of 1° [START_REF] Cho | Determination of a mean orientation in electron backscatter diffraction measurements[END_REF]. In the present study, GOS ≤1° are considered to be fully recrystallized.

As plotted in Figure 6b, the average recrystallized grains is gradually increasing with rising annealing temperature. Initially, the measured grain sizes are marked by a stage of relatively slow evolution until 300 °C. Further annealing up to 450 °C shows a faster evolution, and the smallest grain size is obtained after 8 passes, which is inconsistent with several previous findings [START_REF] Ferrasse | Texture evolution during equal channel angular extrusion (ECAE): part II. An effect of post-deformation annealing[END_REF][START_REF] Abib | Thermal stability of Cu-Cr-Zr alloy processed by equal-channel angular pressing[END_REF]. From this standpoint, it can be considered that the control of grain growth is more efficient after larger strain.

According to Humphreys predictions [START_REF] Humphreys | A unified theory of recovery, recrystallization and grain growth, based on the stability and growth of cellular microstructures-I. The basic model[END_REF], as the HAGB fraction produced on deformation increased, the microstructure gradually becomes stable against the catastrophic grains growth that bounded by HAGBs because of the energies and mobilities of HAGBs are not sensitive to boundary misorientation comparing to low angle boundaries. The average grain sizes after 1, 2, 4 and 8 ECAP passes after annealing at 450 °C are approximately identical to 18 μm. This grain size is well below than the one of the as-received alloy (52 µm) [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF] and could enhance its superplasticity ability.

The recrystallized grains fraction evolution in Figure 6c shows curves with a sigmoidal shape composing mainly of three distinct stages. The recrystallization fraction rate in the first stage ranges from 150 °C to 250 °C, and it grows slowly due to the incubation period. The second stage highlights a roughly considerable increase of the recrystallized grain fraction up to 350 °C. After that, a flat region with quite constant evolution takes place since the recrystallization is complete.

Texture evolution

Texture of the initial state

Figure 7 presents the texture of the as-received AA6060 alloy in terms of recalculated pole figure {111} and ODF section at φ2 = 0 and 45°, respectively. The as-received alloy exhibits a typical extrusion texture, where a typical <001> fiber is formed. In this texture, the directions <001> are preferably aligned along the extrusion direction [START_REF] Hirsch | Texture and Anisotropy in Industrial Applications of Aluminum Alloys[END_REF]. It also becomes apparent that the texture is dominated by the presence of components like Cube and Goss.

Texture evolution after ECAP and subsequent annealing

The ideal ECAP orientations for face-centered cubic (FCC) alloys utilizing a channel angle  = 90° are presented and their characteristic positions in Euler space (1,, 2) are given in Table 3. Copper and Al alloys using route Bc [START_REF] Higuera | Texture Analysis in Ultrafine Grained Coppers Processed by Equal Channel Angular Pressing[END_REF][START_REF] Reyes-Ruiz | Texture and Lattice Distortion Study of an Al-6061-T6 Alloy Produced by ECAP[END_REF].

These texture components seem to be stable until the 4 ECAP pass. Additionally, the ODF section of the sample extruded twice (Figure 10a) shows a weak A component. The intensity of the present orientations rises with rising the ECAP passes number up to 4 passes with a deviation from their ideal positions about 5-10° toward 1. This deviation is attributed to the effect of friction and dies geometry [START_REF] Beyerlein | Texture evolution in equal-channel angular extrusion[END_REF].

The presentation of ODF sections in the range of 0-360° in 1 revealed that the monoclinic symmetry is not evident. The monoclinic symmetry loss is frequently reported in route Bc due to a rotation around the ED axis between each pass [START_REF] Shaeri | Microstructure and texture evolution of Al-7075 alloy processed by equal channel angular pressing[END_REF]. After 8 ECAP passes, Figure 11 allows seeing significant texture changes. The texture intensity decreases and A component appears after annealing (Figure 11). This behavior leads to the formation of B fiber {hkl}<110> (containing the A, A , B and B components) [START_REF] Beyerlein | Texture evolution in equal-channel angular extrusion[END_REF]. Conversely, the A fiber {111}<uvw> (containing A, A , * 1 A and * 2 A components) [START_REF] Beyerlein | Texture evolution in equal-channel angular extrusion[END_REF] is not complete due to the absence of * 2 A component. Molodova et al. [START_REF] Molodova | Thermal stability of ECAP processed pure copper[END_REF] also reported the strengthening of C and B components with a rising ECAP passes number for UFG pure copper via route Bc. In the meantime, the domination of * 1

A was reported in copper, and Al-7075 alloy with increasing the number of ECAP passes using route Bc [START_REF] Shaeri | Microstructure and texture evolution of Al-7075 alloy processed by equal channel angular pressing[END_REF][START_REF] Li | Texture evolution during multi-pass equal channel angular extrusion of copper: Neutron diffraction characterization and polycrystal modeling[END_REF]. Several authors [START_REF] Beyerlein | Texture evolution in equal-channel angular extrusion[END_REF][START_REF] Li | Texture evolution during multi-pass equal channel angular extrusion of copper: Neutron diffraction characterization and polycrystal modeling[END_REF] reported that the texture components in route Bc are not stable (both in position and intensity) during the first 4 passes because of the complexity in the imposed deformation, which makes the texture characterization relatively tricky compared to route A. Accordingly, the development of the different shear texture components in the first ECAP passes strongly depends on the initial texture [START_REF] Beyerlein | Texture evolution in equal-channel angular extrusion[END_REF][START_REF] Li | Texture evolution during equal channel angular extrusion: Effect of initial texture from experiment and simulation[END_REF][START_REF] Suwas | Evolution of crystallographic texture during equal channel angular extrusion of copper: The role of material variables[END_REF]. Thus, the ECAP processing with route Bc of material with strong <100> fiber leads to the development of a strong C component in the first passes [START_REF] Suwas | Evolution of crystallographic texture during equal channel angular extrusion of copper: The role of material variables[END_REF]. This observation is in good agreement with the present finding, where the initial texture of the alloy developed a welldefined <100> fiber (Figure 8). The texture weakening with increasing ECAP passes was reported in various FCC alloys processed by ECAP via route Bc [START_REF] Reyes-Ruiz | Texture and Lattice Distortion Study of an Al-6061-T6 Alloy Produced by ECAP[END_REF][START_REF] Shaeri | Microstructure and texture evolution of Al-7075 alloy processed by equal channel angular pressing[END_REF][START_REF] Abib | On the microstructure and texture of Cu-Cr-Zr alloy after severeplastic deformation by ECAP[END_REF]. According to some researchers [START_REF] Reyes-Ruiz | Texture and Lattice Distortion Study of an Al-6061-T6 Alloy Produced by ECAP[END_REF], this texture evolution is attributed to the formation of new grains with different or random orientations.

The texture after 1 ECAP pass seems to be stable up to annealing at 350 °C for 1 h. As the annealing temperature rises, the C component remains the dominant texture although their intensities decrease. Interestingly, annealing at 450 °C leads to some changes in the texture of the specimen with one pass where C component tends to disappear and B to appear. The A-fiber and B-fiber are partially present due to the absence of A and B component. It is important to note that this typical ECAP texture was evaluated upon annealing at 300 °C in the 2 ECAP passes specimen and rapidly after annealing at 250 °C in case of the 4 ECAP passes specimen. The development of shear texture with decreasing annealing temperature could be attributed to the increase of stored energy introduced by high strain, thereby the acceleration of static recrystallization.

A significant shear texture weakening can be noticed in all annealed specimens. The texture weakening and randomization were also reported after annealing of deformed pure copper via route Bc with increasing ECAP passes [START_REF] Molodova | Thermal stability of ECAP processed pure copper[END_REF]. Besides, intensity differences of A/ A and B/ B components are observed in the annealed samples implying the absence of the monoclinic symmetry.

The orientation changes during different annealing temperatures could be an indication of the discontinuous recrystallization occurrence. Indeed, it is well established that the evolution of the recrystallization texture is strongly related to the recrystallization mechanism [START_REF] Jazaeri | The transition from discontinuous to continuous recrystallization in some aluminium alloys: I -the deformed state[END_REF][START_REF] Jazaeri | The transition from discontinuous to continuous recrystallization in some aluminium alloys: IIannealing behavior[END_REF]. The deformation texture was found retained during continuous recrystallization [START_REF] Abib | Thermal stability of Cu-Cr-Zr alloy processed by equal-channel angular pressing[END_REF], while discontinuous recrystallization could lead to new orientations [START_REF] Tirsatine | On the recrystallization and texture of Fe-36%Ni alloy after accumulative roll bonding and annealing at 600[END_REF]. Indeed, the weakening of the recrystallization texture was associated with the appearance of large grains due to the occurrence of discontinuous recrystallization [START_REF] Molodova | Thermal stability of ECAP processed pure copper[END_REF]. The texture strengthening can be measured using the following equation [START_REF] Bunge | Texture Analysis in Materials Science: Mathematical Methods[END_REF]:

I = 1 8𝜋 2 ∫G 𝑓 2 (𝑔). 𝑑𝑔 (1)
where f(g) is the ODF intensity at g orientation and G is the Euler space.

Figure 12 displays the texture index evolution against the annealing temperature of AA6060 alloy after ECAP processing and subsequent annealing.

This figure demonstrates that the texture indexes is almost stable during annealing up to 250 °C for all ECAPed samples, except for the 4 ECAP pass one where the texture index decreases rapidly from 8.5 to 3.2 upon annealing at 150 °C. The decrease in the texture strength can be noticed after annealing at 250 °C followed by stabilization until annealing at 450 °C where the texture index value is similar for almost all the ECAPed samples (~ 1.4).

Microhardness and Geometrically Necessary Dislocations (GND) density evolution after ECAP processing and annealing

Figure 13a shows the microhardness variation after annealing treatments. Before annealing, the microhardness values rise steadily with rising the ECAP passes number. The tendencies of microhardness-temperature relation for all specimens are similar, i.e., the microhardness values vary slightly till the temperature of 150 °C. Then, the microhardness sharply drops during annealing temperatures between 150 °C and 300 °C, and finally, it stabilizes for temperatures higher than 350 °C.

As shown in Figure 13a, the microhardness of specimens after 1 and 2 passes undergo a gradual increase during annealing up to 150 °C. This evolution is related to the creation and the precipitation of solute rich clusters which create a pinning effect of precipitates on dislocations causing the strengthening of the material. An analysis of differential scanning calorimetry (DSC) carried out on the identical material and under the identical ECAP conditions [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF] demonstrated the presence of an exothermic peak in the range of 150-225 °C. This peak was associated with the GP zones precipitation.

A comparison with materials ECAPed 4 and 8 times revealed that the softening of these latter began at slightly lower annealing temperatures than the samples processed once and twice. In the range of temperature between 150-300 °C, the lowest microhardness value has been recorded for the four passes sample. In keeping with the rule of Johnson-Mehl-Avrami-Kolmogorov (JMAK) [START_REF] Humphreys | Recrystallisation and Related Annealing Phenomena[END_REF], this decrease becomes faster as the level of deformation rises. After registering almost the same microhardness values before annealing, the 8 ECAP pass sample seems to be more stable against annealing up to 300 °C having higher microhardness value than the 4 ECAP pass sample. The character of the curve indicates a shift in the process that governs the mechanical properties' deterioration. According to Hall-Petch relation [START_REF] Hall | The deformation and ageing of mild steel: III discussion of results[END_REF][START_REF] Petch | The cleavage strength of polycrystals[END_REF], the strength of severely deformed UFGed material is affected mainly by the grain size and the dislocation densities [START_REF] Estrin | A dislocation-based model for all hardening stages in large strain deformation[END_REF]. Therefore, it is expected that the coarsening of grain and the elimination of dislocations during annealing would regulate the hardness and strength of the material. Figure 6b, displaying the evolution of grain size, and Figure 13b, displaying the evolution of dislocation density with isochronal annealing, need to be checked at the same time.

The Kernel Average misorientation (KAM) method characterizing the local misorientation is used to calculate the Geometrically Necessary Dislocations (GND) density in all the microstructure. Its principle is well explained in the literature [START_REF] Betanda | Measurement of stored energy in Fe-48%Ni alloys strongly cold-rolled using three approaches: Neutron diffraction, Dillamore and KAM approaches[END_REF]. From misorientation measurements, the density of dislocation (ρGND) can be calculated using the next equation [START_REF] Takayama | Stored energy and Taylor factor relation in an Al-Mg-Mn alloy sheet worked by continuous cyclic bending[END_REF][START_REF] Liu | Effect of grain orientation on deformation structure in coldrolled polycrystalline aluminium[END_REF][START_REF] Calcagnotto | Orientation gradients and geometrically necessary dislocations in ultrafine grained dual-phase steels studied by 2D and 3D EBSD[END_REF]:

𝜌 𝐺𝑁𝐷 = 𝛼 𝜃 𝐾𝐴𝑀 𝑋𝑏 ( 2
)
where α is the parameter depending on the type of grain boundary and it is is equivalent to two for the tilting boundaries, four for twist ones and three for the mixture of the two types, X = nd is the kernel size (n the closest neighbors to be identified (n=3)) and d is the scan step (d=0.1 µm for specimens annealed at 150 °C and d= 0.2 µm for specimens annealed at 250 to 450 °C ) and b is the Burgers vector (b= 0.286 nm in aluminum).

The grain growth is negligible in the low temperature region (i.e.,150 °C-250 °C), while the dislocation density exhibits a dramatic collapse suggesting regeneration of the dislocation structure associated with UFG structure creation. In this temperature region, the most likely re-arrangement and mutual annihilation of dislocations with opposite signs occur during annealing. This observation ties well with earlier reports [START_REF] Stráská | Microstructure stability of ultra-fine grained magnesium alloy AZ31 processed by extrusion and equal-channel angular pressing (EX-ECAP)[END_REF][START_REF] Zi | Response of aluminum processed by extrusion preceded ECAP to isochronal annealing[END_REF]. The fine grain structure is unstable during annealing and the grain sizes of recrystallized grains increase at temperatures 250 < T < 300 °C (see Figure 6b). For all samples, the dislocation density declines abruptly in the same temperature range. Indeed, both dislocations annihilation and coarsening of grain are considered to contribute concurrently to the alloy softening. At elevated temperatures (T > 350 °C), the grain growth is likely to greatly affect the hardness of the studied alloy because no significant change occurs on dislocation density evolution.

Grain Growth Kinetics

Grain growth kinetics in a polycrystalline material is commonly described by the following relationship [START_REF] Wang | An Investigation of Microstructural Stability in An Al-Mg Alloy with Submicrometer Grain Size[END_REF]:

𝑑 2 -𝑑 0 2 = 𝑘 𝑡 (3)
where d is the mean grain size after annealing, d0 is the original grain size [START_REF] Khelfa | Microstructure and strengthening mechanisms in an Al-Mg-Si alloy processed by equal channel angular pressing (ECAP)[END_REF], t is the annealing duration (where t=1h is used in those studies) and k is a parameter that describes the mobility of the grain boundary and the interface. The grain growth rate is correlated with the factor of boundary mobility (k) and can be calculated through the use of the conventional Arrhenius equation [START_REF] Wang | An Investigation of Microstructural Stability in An Al-Mg Alloy with Submicrometer Grain Size[END_REF]:

𝑘 = 𝐴 exp( -𝑄 𝑅𝑇 ) (4)
where A is a pre-exponential constant, R is the gas constant, Q is the activation energy for boundary mobility that is correlated with the driving force needed to activate grain growth, and T is the absolute temperature of annealing.

The present data can be interpreted by constructing the plot of ln (

𝑑 2 -𝑑 0 2 𝑡
) versus l000/RT.

Figure 14 represents the annealing influence on grain growth kinetics of AA6060 alloy. This plot allows distinguishing two temperature ranges with different activation energies. The difference in the rate of grain growth from lower to higher temperatures, which reveals distinct growth regimes, has been extensively reported in the literature [START_REF] Wang | An Investigation of Microstructural Stability in An Al-Mg Alloy with Submicrometer Grain Size[END_REF][START_REF] Tighiouaret | Thermal Stability of an Mg-Nd Alloy Processed by High-Pressure Torsion[END_REF][START_REF] Park | Microstructural characteristics and thermal stability of ultrafine grained 6061 Al alloy fabricated by accumulative roll bonding process[END_REF][START_REF] Mohamed | Deformation mechanism maps based on grain size[END_REF][START_REF] Dhal | Insight into the microstructural evolution during cryosevere plastic deformation and post-deformation annealing of aluminum and its alloys[END_REF]. At low annealing temperature (T < 300 °C), Q is the energy needed for complete recrystallization of the unrecrystallized microstructure, while at the elevated annealing temperature (T > 300 °C) Q is the activation energy for coarsening of the initial UFG microstructure [START_REF] Dhal | Insight into the microstructural evolution during cryosevere plastic deformation and post-deformation annealing of aluminum and its alloys[END_REF].

One can easily observe that the activation energy continually reduces with increasing ECAP passes. A similar observation was obtained earlier [START_REF] Molodova | Thermal stability of ECAP processed pure copper[END_REF][START_REF] Higuera-Cobos | Mechanical, microstructural and electrical evolution of commercially pure copper processed by equal channel angular extrusion[END_REF]. This finding was correlated with a rise in HAGBs fraction with an increasing ECAP passes number allowing less activation energy of recrystallization.

The main activation energy (Q) determined from the slopes of the experimental results in the unrecrystallized grains at lower temperatures (≤ 300 °C), is about ~ 75 kJ mol -1 . This value is significantly smaller than the predicted value for lattice self diffusion in pure Al (~143.4 kJ mol -1 [START_REF] Mohamed | Deformation mechanism maps based on grain size[END_REF]), or the activation energy for Mg impurity diffusion in Al (~130.5 kJ mol -1 [START_REF] Rothman | Tracer Diffusion of Magnesium in Aluminum Single Crystals[END_REF]), neither that for lattice diffusion of Si in Al (~125-154 kJ mol -1 [START_REF] Huang | Subgrain growth and low angle boundary mobility in aluminium crystals of orientation {110}〈001[END_REF]). However, it is quite closer to the value expected for grain boundary diffusion in Al (~86 kJ mol -1 [START_REF] Mohamed | Deformation mechanism maps based on grain size[END_REF]) and that for diffusion across dislocation cores in Al (~82 kJ mol -1 [START_REF] Murr | Interfacial Phenomena in Metals and Alloys[END_REF]). Previous studies have claimed that low activation energy for grain boundary diffusion could be accomplished in SPD-produced UFG materials. One reason is that the nonequilibrium grain boundaries are unstable requiring a slight additional driving force to allow the grains and grain boundaries to be rearranged [START_REF] Valiev | Deformation behaviour of ultra-fine-grained copper[END_REF][START_REF] Lian | On the enhanced grain growth in ultrafine grained metals[END_REF]. Other researchers attributed the low energies to the improved atomic mobility of the nonequilibrium grain boundaries owing to the enhanced diffusivity resulting from the existence of multiple extrinsic dislocations [START_REF] Roy | Thermal Stability in Bulk Cryomilled Ultrafine-Grained 5083 Al Alloy[END_REF]. Besides, aluminum with its strong stacking fault energy favors the dislocation annihilation and offers the required driving force for the annihilation of dislocation and recrystallization [START_REF] Dhal | Precipitation phenomena, thermal stability and grain growth kinetics in an ultra-fine grained Al 2014 alloy after annealing treatment[END_REF].

The activation energy is unexpectedly low (~20 kJmol -1 ) in the completely recrystallized structure at high temperatures (> 300 °C). This value is inconsistent with numerous findings in the literature obtained after several SPD techniques of different Al alloys like Al-Mg processed by ECAP [START_REF] Wang | An Investigation of Microstructural Stability in An Al-Mg Alloy with Submicrometer Grain Size[END_REF], AA6061 subjected to ARB [START_REF] Park | Microstructural characteristics and thermal stability of ultrafine grained 6061 Al alloy fabricated by accumulative roll bonding process[END_REF], AA5083 fabricated by cryo-milling [START_REF] Dhal | Insight into the microstructural evolution during cryosevere plastic deformation and post-deformation annealing of aluminum and its alloys[END_REF] or AA2014 processed by cryo-rolling [START_REF] Dhal | Insight into the microstructural evolution during cryosevere plastic deformation and post-deformation annealing of aluminum and its alloys[END_REF]. Nevertheless, several other studies suggest a similar change to a high-temperature regime with very low activation energy [START_REF] Abbasi-Baharanchi | Thermal Stability Evaluation of Nanostructured Al6061 Alloy Produced by Cryorolling[END_REF][START_REF] Gleiter | Observations suggesting a transformation in the structure of high-angle grain boundaries in lead[END_REF][START_REF] Gondi | Grain Growth in Polycrystalline Materials[END_REF][START_REF] Pandey | Thermal and microstructural stability of nanostructured surface of the aluminium alloy 7075[END_REF]. E.g., studying the thermal stability of AA6061 aluminum alloy developed by cryo-rolling followed by optimal aging heat treatment [START_REF] Abbasi-Baharanchi | Thermal Stability Evaluation of Nanostructured Al6061 Alloy Produced by Cryorolling[END_REF], it has been recorded a decrease in the activation energy in the elevated temperature range (300-500 °C). They attributed to the dissolution of coherent and semi-coherent precipitates and to the diminution in the pinning impact that inhibits the migration of grain boundaries and then the amount of activation energy. Likewise, there have been reports of a change to quite a low activation energy in an elevated temperature regime which is near to that for boundary diffusion in high purity lead [START_REF] Abbasi-Baharanchi | Thermal Stability Evaluation of Nanostructured Al6061 Alloy Produced by Cryorolling[END_REF] and to that for liquid diffusion in zinc [START_REF] Gondi | Grain Growth in Polycrystalline Materials[END_REF]. The first study [START_REF] Gleiter | Observations suggesting a transformation in the structure of high-angle grain boundaries in lead[END_REF] has suggested the occurrence of change to various boundary structure. Nevertheless, in the second investigation [START_REF] Gondi | Grain Growth in Polycrystalline Materials[END_REF], the low energy was described as a transition in the boundary migration process from one dominated by diffusion to other requiring collaborative shuffles of atomic. Recently, a one grain growth regime was observed in the AA7075 alloy treated by ultrasonic shot peening and annealed at different temperatures up to 350°C where the activation energy for grain growth was determined to be 37.9 kJmol -1 [START_REF] Pandey | Thermal and microstructural stability of nanostructured surface of the aluminium alloy 7075[END_REF]. The latter research considered that the dominant role in grain coarsening was played by grain boundary self-diffusion.

Grain growth requires the migration of HAGBs and the kinetics are strongly influenced by many factors, including solute (impurity) drag, second phase (Zener) drag, the temperature dependence of grain boundary mobility, and grain boundary segregation [START_REF] Pandey | Thermal and microstructural stability of nanostructured surface of the aluminium alloy 7075[END_REF][START_REF] Liu | Mechanical properties and thermal stability of nanocrystallized pure aluminum produced by surface mechanical attrition treatment[END_REF]. The driving force for grain growth is typically quite low at high temperatures, particularly in comparison to the recrystallization regime [START_REF] Humphreys | chapter 11 -Grain Growth Following Recrystallization[END_REF].

According to the X-ray diffraction (XRD) analysis, Figure 15 shows the diffractogram of AA6060 samples after ECAP and annealing at 350 °C, revealed only the (111), ( 200) and (220) fundamental reflections of fcc-Al structure. This figure confirms the absence of any other phase in the XRD pattern of the studied alloy. Even the small peak recorded at 2θ ~ 40.2°, usually assigned to Mg2Si precipitates [START_REF] Khelfa | Structure and microstructure evolution of Al-Mg-Si alloy processed by equal-channel angular pressing[END_REF][START_REF] Noda | Masumoto KA Preparation and thermoelectric properties of Mg2Si1-x Gex (x=0.0~0.4) solid solution semiconductors[END_REF], cannot be considered efficient due to its negligible intensity. Therefore, the dissolution of strengthening precipitates after annealing at temperatures higher than 300 °C can contribute efficiently in decreasing the activation energy for grain growth in this range of temperature. Nevertheless, the instability of the UFG microstructure and the mechanism of grain boundary migration occurring with increasing temperature can also affect the grain growth kinetics.

Conclusion

Series of AA6060 alloy samples for thermal stability investigations were subjected through Equal Channel Angular Pressing using the Route Bc for 1, 2, 4 and 8 passes and followed by isochronal annealing at various temperatures range between 150 and 450 °C for 1 h.

The following conclusions were drawn.

• The specimens showed recrystallization and grain growth. In addition, the recrystallized microstructure typically has the randomly scattered large grains and small grains indicating the appearance of the discontinuous recrystallization process. • It was noticed from the observed transitions distinguished by sudden softening and degradation of the deformation texture components, which occurred after annealing above 150°C while the UFG microstructure of the AA6060 ECAPed material remained stable up to this temperature.

• The 8 ECAP pass sample has shown a limited grain growth and a restricted evolution after 300°C. A relatively homogeneous microstructure after annealing up to 450 °C has been obtained with reasonably small mean grain size ~ 12.5 µm compared to the 4 ECAP pass sample (~16 µm) despite their similar behavior after ECAP processing in terms of achieved grain sizes (0.57 and 0.68 µm, respectively).

• The grain growth kinetics of AA6060 alloy are followed by two stages that rely upon the range of annealing temperature. Average order values of ~75 and ~20 kJ mol -1 of activation energies were estimated in the elevated temperature 300-450 °C range and the low temperature 150-300

Figure 1 -

 1 Figure 1-5 demonstrates the TD inverse pole figure (TD-IPF) maps from EBSD illustrating the

Figures 8 -

 8  show the texture in term of recalculated pole figures (111) and ODF sections at φ2 = 45°

Figure

  Figure 8a indicates the development of a strong C component and weak B component after 1

Figure 11 2 A

 112 Figure 11 illustrates the texture during the isochronal annealing of the 8 ECAP pass specimen.

Figures captionsFigure 1 :

 1 Figures captions

Figure 2 :

 2 Figure 2: IPF maps of AA6060 after 1 ECAP passes and subsequent annealing for 1 h at: (a) 150 °C,

Figure 3 :

 3 Figure 3: IPF maps of AA6060 after 2 ECAP passes and subsequent annealing for 1 h at: (a) 150 °C,

Figure 4 :

 4 Figure 4: IPF maps of AA6060 after 4 ECAP passes and subsequent annealing for 1 h at: (a) 150 °C,

Figure 5 :

 5 Figure 5: IPF maps of AA6060 after 8 ECAP passes and subsequent annealing for 1 h at: (a) 150 °C,

Figure 6 :

 6 Figure 6: a) Fraction of HAGBs, b) Recrystallized grain size evolution and c) Evolution of the

Figure 7 :

 7 Figure 7: Recalculated pole figure (111) and ODF sections at φ2 = 0 and 45° of the as-received

Figure 8 :

 8 Figure 8 : Recalculated pole figure (111) and ODF section at φ2 = 45° of AA6060 alloy after: a) 1

Figure 9 :

 9 Figure 9 : Recalculated pole figure (111) and ODF section at φ2 = 45° of AA6060 alloy after: a) 2

Figure 10 : 29 Figure 11 :

 102911 Figure 10: Recalculated pole figure (111) and ODF section at φ2 = 45° of AA6060 alloy after: a) 4

Figure 12 :

 12 Figure 12: Evolution of the texture index before and after annealing.

Figure 13 :

 13 Figure 13: a) Hardness evolution, b) Geometrically Necessary Dislocations (GND) density evolution

Figure 14 :

 14 Figure 14 : Evolution of ln(d 2 -d0 2 /t) as a function of 1000/RT for AA6060 alloy processed by ECAP

Figure 15 :

 15 Figure 15 : XRD pattern of AA6060 alloy after ECAP process and annealing at 350°C for 1 h.

  Figure 1

•

  ECAP process through 1, 2, and 4 ECAP passes lead to the development of strong C component and weak B component. Meanwhile, the typical shear ECAP texture of FCC metals is observed after 8 ECAP passes. The texture intensity increases with increasing ECAP processing up to 4 passes and then decreases after 8 ECAP passes. The calculation of the texture index shows the propensity for texture weakening and randomization with rising annealing temperature up to 450 °C.

Table captions Table 1 :

 captions1 Chemical composition of the AA6060 alloy studied.

Table 2 :

 2 Average grain size of ECAPed samples at different temperatures

Table 3 :

 3 Ideal ECAP orientations for FCC alloys using a channel angle  of 90° 30

Acknowledgments

Tarek Khelfa and Zhiguo Chen gratefully acknowledge for the financial support from the National Natural Science Foundation of China (Grant No. 51011120052) and the double first-class discipline construction program of Hunan Province. This work also was supported by the PHC-Maghreb program N o 16MAG03. JMC thanks CONACYT (Mexico) for the economic partial funding of his sabbatical leave at UMSNH.

Funding

National Natural Science Foundation of China (Grant No. 51011120052) and the double firstclass discipline construction program of Hunan Province, and PHC-Maghreb program No 16MAG03 provided financial assistance for this research.

°C range, respectively. At elevated temperatures, the grain growth is controlled by lattice self diffusion. While at low temperatures, the grain growth is governed by high mobility of the nonequilibrium grain-boundary.

Conflict of interest

The authors declare that they have no conflict of interest.