Théo Guérin

Christophe Waterlot

Emmanuelle Lipka

Philippe Gervois

David Bulteel

Damien Betrancourt

Carla Moignard

Adrian Sorin Nica

Christophe Furman

Alina Ghinet
email: alina.ghinet@junia.com

Ecocatalysed Hurtley reaction: Synthesis of urolithin derivatives as new potential RAGE antagonists with anti-ageing properties

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

Introduction

Fruits and nuts, such as pomegranate, strawberry, raspberry, blackberry, grapes, walnuts, and chestnuts are widely consumed and recognized for their health benefits. These last have been associated with a high level of antioxidant polyphenols, particularly ellagitannins, which have presented various biological properties including antioxidant 1 , antiviral 2 , antimutagenic 3 , antimicrobial 4,5 and antitumor activities 6,7 . Ellagic acid is one of the most common hydrolysates of ellagitannins 8 , which is itself then metabolized by the action of unidentified colonic bacteria in urolithin compounds 9 , the most common found in humans and animals being urolithins A and B 9 (Figure 1). These last consist of a benzo[c]chromene ring with different phenolic hydroxylation patterns. In addition to presenting many biological activities [9][10][11][12][13][14][15] , recent studies have demonstrated their anti-aging activities 16,17 and especially their capacity to inhibit the formation of Advanced Glycation End-products 18 (AGEs). Our group is interested in organic and sustainable chemistry applied to the biology of aging by studying AGEs and the consequences of their interaction with the Receptor of Advanced Glycation End-products (RAGE), as well as the development of experimental drugs targeting RAGE. We hypothesize that RAGE is at the center of multiple inflammatory mechanisms and is involved in organ senescence. RAGE acts as a key receptor involved in "inflammation" for which we are seeking to develop an innovative therapy. Blocking RAGE via antagonist molecules will prevent and control not only acute inflammation, but also chronic inflammation and aging. Consequently, this study describes the green synthetic access to new urolithin analogues 1-24 (Figure 1) and their biological evaluation as RAGE antagonists using an ELISA assay.

Important biological activities were attributed to phloroglucinol which is the main ingredient of antispasmodic marketed Spasfon® and to phloretin which is used as flavour enhancer and is present in many cosmetic formulas. The antioxidant power of these molecules is well-documented and, interestingly, phloroglucinol was also reported as moderate inhibitor of AGEs production. 19 In the same manuscript, curcumin (Figure 1) was reported as slightly more efficient AGEs inhibitor than phloroglucinol (curcumin: IC50 = 80.87 µg/mL and phloroglucinol: IC50 = 128.9 µg/mL). 19 This suggested that bulkier polyphenolic compounds may represent an interest as AGEs inhibitors. In addition to these target anti-ageing properties, the synthesis of new polyphenolic analogues is still relevant today given the plethora of their fields of use.

Since the chemical construction of the target benzo[c]chromene ring involved the use of metal catalysts, the most common being the copper salts, our group studied the possibility to apply ecological metal-based catalyst to access this important class of urolithins. Important areas of soils of the North of France region are highly MTE-contaminated, especially with Zn, Cd, Fe but also Cu.

Consequently, the synthetic strategy was further based on the potential use of ecocatalysts to promote the benzo[c]chromene formation as an alternative to commercial copper salts.

Results and discussion

Ecocatalyst preparation and characterisation

In a context of green chemistry, the development of new ecomaterials which can be used as biosourced Lewis acids has become a research area of interest. This concept has been introduced by Pr. Grison a decade ago and is better known as « ecocatalysis » [START_REF] Grison | Ecocatalysis: A New Integrated Approach to Scientific Ecology[END_REF] . This consists of using hyperaccumulative plants on metal contaminated soils which become, after metal-uptake, biomasses enriched with metals. These last are then extracted from the biomasses in the form of Lewis acids, thus constituting plant-based catalyst, also called ecocatalyst. This technology has been applied and has been effective in a wide range of chemical transformations [START_REF] Hechelski | [END_REF] including cross-coupling transformation such as Suzuki and Heck 22 but also Ullmann reactions 23 .

If the concept of ecocatalysis was originally developed from hyperaccumulating plants, recent studies have shown that it was possible to extend the technology to so-called "tolerant" plants. Indeed, ecocatalysts have been developed by Professor Grison's team from different tree species and have been shown to be effective for Knoevenagel condensation 24 , esterification and epoxidation reaction 25 . Moreover, our group previously identified tolerant biomasses such as Lolium perenne L. 26,27,28 and Miscanthus x giganteus 29,30 that have also enabled to design new ecocatalysts that have catalyzed amidification reactions 28,30 . In this article, the new ecocatalysts obtained from Miscanthus x giganteus (Eco-MG) which has been produced in situ on heavy-metal contaminated soil were prepared and characterized.

Based on our previous studies 29,30 , it was shown that stems of miscanthus are the most appropriate organs to produce ecocatalyst since the concentration of metals was higher than in leaves.

Consequently, stems and leaves on miscanthus were sampled separately. After extracting metals from stems using HCl 2M according to the procedure described in literature 30 , the resulting yellow solution was concentrated under vacuum to provide a yellow-pale residue (Eco-MG1). The total concentration of metals (Cd, Pb, Zn, Fe, Mn, Cu, Ca, Mg, Na and K) measured in the mixture of salts was 295.37 g kg -1 . The distribution of metal was reported in Table 1 and shows that K is the predominant species (69.21%). Some other interesting catalyst precursors were measured (e.g. Fe, Zn, Mn, Mg, Ca), while the proportion of Cd and Pb were unsignificant. These preliminary data were completed using SEM-EDS experiments. These analyses allowed to understand the distribution of predominant metal species present in the Eco-MG1 and envisage the necessity of acetone wash of the raw material to concentrate interesting catalyst precursors species such as Cu or Zn and obtain Eco-MG2. Results highlighted different domains containing high amounts of K and Cl, Na and Cl, and Ca, S and O that can be related to the presence of sylvite (KCl), halite (NaCl), anhydrite (CaSO4). Surprisingly, other domains were rich in Mg, K and Cl (Figure 2). This domain could be composed of KMgCl3, a very strong Lewis acid that has been already identified in other studies 24,25 . Due to the high proportion of K in Eco-MG1, a part of it was washed with acetone in order to separate a large amount of K from the other cations. After washing, the resulting solution has been concentrated under vacuum to obtain a white powder (Eco-MG2). The total concentration of metals was 465.56 g kg -1 and the distribution of them (Table 1), confirmed an enrichment in metals of interest (Zn, Fe, Mg). The Figure 3 clearly shows the presence of metals like Zn, Fe, Mn, Mg, Ca, K and Na. Other elements appeared (e.g. P, O) and domains with P, O and Mg were highlighted suggesting the presence of magnesium phosphate (MgP4O11). Further analyses are necessary to identify the crystalline phases (e.g. KMgCl3, K2ZnCl4) in this second ecocatalyst Eco-MG2.

Ecocatalysed synthesis of urolithin B derivatives

Several synthetic pathways exist to produce urolithin compounds such as Suzuki-Coupling, 31 Kolbe oxidative cyclization, 32 intramolecular biaryl coupling of 2-halobenzoates 33 including the basic strategy of Bringmann's 'lactone concept' 34 or Pechmann reaction 35 , but the most common method is the Hurtley reaction (often seen as a sub-category of Ullmann's reaction 36 which has been discovered almost a century ago 37 by reacting 2-bromobenzoic acid with resorcinol using Cu-based Lewis acid as catalyst. In this section, the comparison of the catalytic activities of the miscanthus-based ecocatalysts with conventional commercial Cu-based catalytic species to synthesize urolithin derivatives through Hurtley reaction was realized systematically. Starting materials of natural origin (resorcinol, phloroglucinol, phloretin and resveratrol, Figure 1) were used in a major part of syntheses. The reactions were carried out in water and/or in ethanol. The reaction media resulted systematically in complete dissolution of reagents and catalyst.

The model reaction was the synthesis of urolithin B 1. First, the reaction of 2-bromobenzoic acid with resorcinol in aqueous sodium hydroxide and in the absence of catalyst only provided urolithin B 1 in very low 5% yield (entry 1, Table 2). This underlined the importance of the catalyst for this transformation. Using the initial protocol with 1.6 wt% (2 mol%) CuSO4 as catalyst 12 resulted in 61% yield of urolithin B 1 (entry 2, Table 2). The first designed eco-catalyst Eco-MG1 was tested at different ratios (10 wt% to 50 wt%) in the same Hurtley reaction and the final yield did not exceed 32% (entries 3-8, Table 2), Eco-MG1 displaying reduced catalytic efficiency compared to commercial CuSO4. Conversely, Eco-MG2 showed improved potential allowing a maximum of 70% yield of urolithin B 1, reached when using 6 wt% of ecocatalyst (entry 12, Table 2). The reduction of the catalytic charge (3 wt%, 1.5 wt% then 1 wt%) gradually reduced the yield of the reaction to 41%, 34% and 30%, respectively (entries 9-12, Table 2), while the increase in the catalytic load to 10 wt% did not permit to exceed the efficiency of 70% yield obtained using 6 wt% also (entry 13, Table 2). Next, the influence of different copper salts was studied (entries 14-16, Table 2). Interestingly, copper (II) chloride showed equipotent catalytic activity as Eco-MG2 (entry 14, Table 2). The oxidation state of copper does not seem to have a major impact on the reaction progress since the use of copper (I) chloride resulted in 62% slightly decreased yield while the copper (I) iodide was beneficial and exceeded the catalytic effectiveness of the other considered catalysts (77% yield) (entries 15 and 16, Table 2). Similar yield was obtained by using classical Ullmann conditions previously described by our group (0.5 eq. CuI, 1 eq. N,N'-DMEDA, 2 eq. Cs2CO3, dioxane, under inert atmosphere). 38 Although we have not carried out any mechanistic study, it is reasonable to postulate a mechanism similar to Ullmann-type reactions for the formation of urolithin B.

Since the ecocatalysts contain multiple metallic species (e.g. Cu, Zn, Cd, Pb, Mn and Fe), the commercial corresponding Lewis acids were next tested (2 mol% of metal) in order to learn more about active species responsible of the catalytic efficiency. The use of ZnCl2, CdCl2 and PbCl2 was detrimental for the reaction progress, inducing only 11-14% yields of urolithin B (entries 17-19, Table 2). The manganese salts (MnSO4 and MnCl2) were completely ineffective and did not show any catalytic power. The final yields were similar to the reaction realized in the absence of catalyst (compare entries 20, 21 and 1, Table 2). On the contrary, pure iron chlorides (FeCl2 and FeCl3) were more effective and allowed to obtain urolithin B in 28% and 10% yield, respectively (entries 22 and 23, Table 2). This study suggested that the interesting catalytic activity of bio-sourced Eco-MG2 was due to a synergistic effect of its metallic species and no pure Lewis acid tested was responsible of the catalytic activity alone.

Additional experiments were carried out by varying the amount of NaOH and resorcinol but these were not conclusive, they permitted to verify that, according to what was described in the presence of CuSO4, 12 2 eq. of NaOH and 2 eq. of resorcinol were necessary to obtain a good yield >60%. The reagents and the biosourced catalyst being selected (entry 12, Table 2), efforts were deployed to obtain a series of natural and synthetic urolithin analogues 1-24. Reactions were conducted in water and/or ethanol. The different bromobenzoic acids and polyphenols used as starting materials are reported in Table 3. All reactions were carried out in duplicate and by systematically comparing the efficacy of Eco-MG2 to that of classical CuSO4. Compounds 1-24 have been synthesized in variable yields (Table 3). Eco-MG2 showed superior catalytic activity in most of the transformations carried out (entries 1-5, 7, 9, 11, 13-16, 18, 19, 21 and 22, Table 3) or equivalent to CuSO4 in some cases (entries 6, 8, 10, 12, 17, 20 and 24, Table 3). The bromobenzoic acids substituted by a chloro unit were the least reactive and resulted in yields inferior to their methoxy-substituted congeners (compare entries 2 and 4, 10 and 12, 22 and 24, Table 3). The best yields were registered in the reaction of phloroglucinol with 2-bromobenzoic acids (entries 5-8, Table 3, ranging from 80 to 96%) and the most modest were observed with resveratrol as starting material (entries 21-24, Table 3, ranging from 6 to 54%). Compounds 8-24 have been synthesized for the first time and were fully characterized (see Supplementary information section). To be noted, the same reaction carried out with 3-aminophenol or 3-mercaptophenol and 2-bromobenzoic acid did not allow to obtain the corresponding products, highlighting the specificity of this reaction for polyphenols.

Biological evaluation

The inhibitory activity of the experimental drug candidates 1-24 was evaluated against AGE2-BSA/sRAGE binding using the Creative BioMart® ELISA kit according to the manufacturer's instructions. Plates were precoated with AGE2-BSA, which can bind to recombinant His-tagged sRAGE. Horse radish peroxidase (HRP)conjugated anti-His-tag monoclonal antibody specifically reacted with recombinant His-tagged sRAGE that was trapped with AGE2-BSA immobilized on the microplate well surface. Finally, an incubation with a substrate chromogenic reagent (TetraMethylBenzidine) was realized. The effective inhibition was measured spectrophotometrically and resulted in a decrease in the staining of the medium of the well compared to the well containing no experimental drug. The wells were washed and the relative amounts of the His-tagged sRAGE in the wells were evaluated spectrophotometrically using Horse radish peroxidase (HRP)-conjugated anti-His-tag monoclonal antibody. Known RAGE antagonists have been used as positive references: Azeliragon, FPS-ZM1, curcumin, urolithins A and B and papaverine, while DMSO was used as a negative control. Azeliragon is currently one of the most promising RAGE antagonists, reaching phase III clinical trials to prevent the advancement of the disease in patients with mild Alzheimer's disease and the only experimental RAGE antagonist to have been studied in clinical trials so far. 39 FPS-ZM1 was also described as a potent RAGE antagonist. 40 Curcumin and urolithins are inhibitors of the production of AGEs, 18,19 while papaverine was identified as inhibitor of HMGB1/RAGE interaction, HMGB1 being an excellent RAGE ligand and critical for acute and chronic inflammatory disorders. 41 All the starting materials resorcinol, phloroglucinol, 2,6-dihydroxyacetophenone, 2',4',6'-trihydroxypropiophenone, 2',4',6'-trihydroxy-2-phenylacetophenone, phloretin and resveratrol were also evaluated to estimate the contribution of the chemical modulations carried out on these raw materials on the biological activity of final products and conclude on the interest of these modulations. Compounds were first tested at initial concentration of 100 µM or at 10 µM for references Azeliragon and papaverine and molecules displaying more than 70% inhibition rate of AGE2-BSA/sRAGE were selected for further IC50 value determination. Results are illustrated in Table 4. To be noted, the starting materials showed no inhibition potential of AGE2-BSA/RAGE interaction at 100 µM concentration (entries 1-7, Table 4), while the newly synthesized urolithin derivatives showed excellent inhibitory activities (entries 8-31, Table 4). For the reference FPS-ZM1, we did not obtain any inhibition in this assay (entry 33, Table 4). This result corroborated well with that recently published by Mizukami et al. 42 Indeed, FPS-ZM1 was not efficient to inhibit the AGE-RAGE interaction using the ELISA Circulex AGE-RAGE in vitro Binding Assay kit (MBL, Nagoya, Japan). 42 On the contrary, Azeliragon was identified as effective inhibitor of the AGE2-BSA/sRAGE interaction with an IC50 value of approximately 10 µM (entry 32, Table 4). This value was in perfect agreement with that found by Mizukami et al.

and defined as 13.4 ± 2.8 µM. 42 Urolithin A was less active than urolithin B 1 (compare entries 34 and 8, Table 4)

showing 20% inhibition versus 76%. Urolithin B 1 was indeed identified as a promising inhibitor of AGE2-BSA/sRAGE interaction with a value of IC50 of 25.3 µM. Curcumin and papaverine showed moderate inhibitory effects with 43% inhibition at 100 µM concentration and 26% inhibition at 10 µM concentration, respectively (entries 35 and 36, Table 4). 4); iv) the OMe substituent in position 8 seemed favorable for the inhibition of AGE2-BSA/sRAGE interaction, especially when the positions 3 and 4 were substituted with a hydroxy and a small propanoyl group, respectively (e.g. compare compounds 2 (IC50=31.6 µM), 6 (IC50=14.2 µM) and 14 (IC50=17.6 µM), Table 4) while the Cl substitution was less tolerated (compound 4 (51% inhibition at 100 µM) and compound 8 (44% inhibition at 100 µM), Table 4); v) in the same line, the presence of two methoxy substituents in positions 8 and 9 was tolerated only when the position 4 was unsubstituted (e.g. compound 5 (IC50=67.3 µM) and compound 7 (IC50=12.8 µM), Table 4). Moreover, urolithin B 1 has been selected by the National Cancer Further biological studies will be realized in vitro and in vivo to explore the potential positioning of urolithins as nutraceutical and/or pharmacological anti-ageing agents.

Experimental section

Reagents and products characterization

Plant samples were collected from 5 to 6 years old Miscanthus x giganteus plantations established at a density of 20,000 plants ha -1 in contaminated agricultural plots located not far away from the former Metaleurop Nord plant (Hauts-de-France region, France). Reagents and solvents were purchased from commercials sources (TCI Europe N.V., Sigma Aldrich, Acros Organics) and were used without further purification.

Conception of biosourced catalysts

668 g of miscanthus stems powder were transformed into ashes in a muffle furnace (Nabertherm P330, Lilienthal, Germany) and treated with HCl following the procedure detailed in Guérin et al. 30 The resulting solid was dried at 80 °C for 12 h, to provide 26.7 g of a white powder. From this, 6.7 g were used as such and constituted the crude ecocatalyst (Eco-MG1) and the remaining 20 g were washed with acetone with a weight/ratio powder/acetone: 1/20 at 20 °C during 2 h in order to solubilize some metallic salts. The suspension was then filtered on sintered glass and the filtrate was evaporated to dryness to provide 0.751 g of purified ecocatalyst (Eco-MG2).

Analysis of ashes and biosourced catalysts

The concentration of heavy (Cd, Pb, Zn, Cu, Mn and Fe), alkali (Na and K) and alkaline earth (Ca and Mg) metals in the mixture of extracted Lewis acids from plants were determined by flame atomic absorption spectrometry (AA-6800, Shimadzu, Tokyo, Japan) following the recommendations described in the literature 26 . Moreover, microstructures of the two biosourced catalysts were examined using scanning electron microscope (SEM; Hitachi S-4300SE/N) fitted with a Thermo Scientific Ultra Dry energy dispersive X-ray spectrometer (EDS) in order to assess structural chemical analyses 43 .

Synthetic chemistry

Nuclear Magnetic Resonance (NMR) spectra were acquired at 400 MHz for 1 H NMR and at 100 MHz LC-MS was accomplished using an HPLC combined with a Surveyor MSQ (Thermo Electron) equipped with APCI source.

General procedure for the synthesis of uolithin-type compounds 1-24

In a 50 mL round-bottom flash equipped with a magnetic stirrer were added the bromobenzoic acid (0.5 g, 1 equiv.), sodium hydroxide pellets (2 equiv.), the 1,3-diphenol derivative (2 equiv.) and 3 mL of water (or a 1/1 mixture of water-absolute ethanol). The reaction medium was stirred at 50 °C until homogenization, and the ecocatalyst Eco-MG2 was then added (6% wt, regarding the bromobenzoic acid). The resulting mixture was stirred at 100 °C for 3 h. After cooling to room temperature, the crude solid product was isolated and purified by a simple filtration of the reaction medium followed by washing with absolute ethanol or purified by flash chromatography using n-heptane/EtOAc 100/0 to 0/100 as eluent on silica pre-packed column only when recrystallization has failed. After crystallization from EtOH or flash chromatography purification, urolithin B and derivatives have been analyzed by Electrothermal Atomic Absorption Spectroscopy (ETAAS) to highlight possible traces of metals due to the ecocatalyst. The concentrations of metals were below the limit of detection of the equipment (e.g. LDCd=0.02 µg/L; LDPb=0.06µg/L).

3-Hydroxy-6H-benzo[c]chromen-6-one (Urolithin B) (1)

. The general procedure was used with 2bromobenzoic acid (0.5 g, 2.49 mmol) and resorcinol (0.548 g, 4.98 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 1 as a beige solid in 70% yield with the same physico-chemical characteristics as previously reported; 12

3-Hydroxy-8-methoxy-6H-benzo[c]chromen-6-one (2)

. The general procedure was used with 2-bromo-5-methoxybenzoic acid (0.5 g, 2.16 mmol) and resorcinol (0.476 g, 4.32 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 2 as a beige solid in 78% yield with the same physico-chemical characteristics as previously reported; 12

3-Hydroxy-8,9-dimethoxy-6H-benzo[c]chromen-6-one (3).

The general procedure was used with 2bromo-4,5-dimethoxybenzoic acid (0.5 g, 1.92 mmol) and resorcinol (0.422 g, 3.84 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 3 as a beige solid in 80% yield with the same physico-chemical characteristics as previously reported; 12 1,3-Dihydroxy-8,9-dimethoxy-6H-benzo[c]chromen-6-one (7). The general procedure was used with 0.5g of 2-bromo-4,5-dimethoxybenzoic acid (0.5 g, 1.92 mmol) and phloroglucinol (0.484 g, 3.84 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 7 as a beige solid in 96% yield with the same physicochemical characteristics as previously reported; 12 1 H NMR (400 MHz, DMSO-d6) (ppm): 3.87 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.25 (d, J = 2.0 Hz, 1H, Ar-H), 6.39 (d, J = 2.0 Hz, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 8.58 (s, 1H, Ar-H). (8). The general procedure was used with 2bromo-5-chlorobenzoic acid (0.5 g, 2.12 mmol) and phloroglucinol (0.535 g, 4.24 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 8 as a beige solid in 81% yield; mp (EtOH): > 200 °C; (10). The general procedure was used with 2-bromo-5-methoxybenzoic acid (0. (12). The general procedure was used with 2-bromo-5-chlorobenzoic acid (0.5 g, 2.12 mmol) and 2,6-dihydroxyacetophenone (0.645 g, 4.24 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 12 as a brown solid in 51% yield; mp (EtOH): 196-197 °C; 1 H NMR (400 MHz, DMSO-d6) (ppm): 2.62 (s, 3H, CH3), 1,3-Dihydroxy-4-propanoyl-6H-benzo[c]chromen-6-one (13). The general procedure was used with 2bromobenzoic acid (0.5 g, 2.49 mmol) and 1-(2,4,6-trihydroxyphenyl)propan-1-one (0.907 g, 4.98 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 13 as a beige solid in 63% yield; mp (EtOH): 166-167 °C; 1 H NMR (400 MHz, DMSO-d6) (ppm): 1.11 (t, J = 6.9 Hz, 3H, CH3), 3.15 (q, J = 6.9 Hz, 2H, CH2), (20). The general procedure was used with 2-bromo-4,5-dimethoxybenzoic acid (0.5 g, 1.92 mmol) and phloretin (0.992 g, 3.84 mmol) in 3 mL of water/ethanol 1/1. After filtration of the reaction mixture, the crude product has been purified by flash chromatography to obtain pure compound 20 as a beige solid in 54% yield; mp (EtOAc/n-heptane): > 200 °C; 1 H NMR (400 MHz, DMSO-d6) (ppm): 2.81 (t, J = 7.2 Hz, 2H, CH2), 3.30 (t, J = 7.2 Hz, 2H, CH2), 3.83 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.19 (s, 1H, Ar-H), 6.69 (d, J = 7.9 Hz, 2H, Ar-H), 7.06 (d, J = 7.9 Hz, 2H, Ar-H), 7.42 (s, 1H, Ar-H), 8.34 (s, 1H, Ar-H). 13 (21). The general procedure was used with 2-bromobenzoic acid (0.5 g, 2.49 mmol) and (E)-5-(4-hydroxystyryl)benzene-1,3-diol (resveratrol) (1.14 g, 4.98 mmol) in 3 mL of water/ethanol 1/1. After filtration of the reaction mixture, the crude product has been purified by flash chromatography to obtain pure compound 21 as a yellow solid in 50% yield; mp (n-heptane/EtOAc): 166-168 °C; 1 (22). The general procedure was used with 2-bromo-5-methoxybenzoic acid (0.5 g, 2.16 mmol) and resveratrol (0.986 g, 4.32 mmol) in 3 mL of water/ethanol 1/1. After filtration of the reaction mixture, the crude product has been purified by flash chromatography to obtain pure compound 22 as a yellow solid in 54% yield; mp (n-heptane/EtOAc): > 200 °C; 1 (23). The general procedure was used with 2-bromo-4,5-dimethoxybenzoic acid (0.5 g, 1.92 mmol) and resveratrol (0.876 g, 3.84 mmol) in 3 mL of water/ethanol 1/1. After filtration of the reaction mixture, the crude product has been purified by flash chromatography to obtain pure compound 23 as a yellow solid in 47% yield; mp (n-heptane/EtOAc): > 200 °C; 1 H NMR (400 MHz, DMSO-d6) (ppm): 3.70 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.68 (d, J = 2.1 Hz, 1H, Ar-H), 6.79 (s, 1H, Ar-H), 6.81 (s, 1H, Ar-H), 6.87 (d, J = 16.2 Hz, 1H, CH), 7.50-7.59 (m, 4H, Ar-H + CH), 7.70 (s, 1H, Ar-H). 13 (E)-8-Chloro-3-hydroxy-1-(4-hydroxystyryl)-6H-benzo[c]chromen-6-one (24). The general procedure was used with 2-bromo-5-chlorobenzoic acid (0.5 g, 2.12 mmol) and resveratrol (0.968 g, 4.24 mmol) in 3 mL of water/ethanol 1/1. After filtration of the reaction mixture, the crude product has been purified by flash chromatography to obtain pure compound 24 as a yellow solid in 6% yield; mp (nheptane/EtOAc): > 200 °C; 1

8-Chloro-1,3-dihydroxy-6H-benzo[c]chromen-6-one

4-Acetyl-3-hydroxy-8-methoxy-6H-benzo[c]chromen-6-one

4-Acetyl-8-chloro-3-hydroxy-6H-benzo[c]chromen-6-one

(E)-3-Hydroxy-1-(4-hydroxystyryl)-6H-benzo[c]chromen-6-one

(E)-3-hydroxy-1-(4-hydroxystyryl)-8-methoxy-6H-benzo[c]chromen-6-one

(E)-3-Hydroxy-1-(4-hydroxystyryl)-8,9-dimethoxy-6H-benzo[c]chromen-6-one

Biological evaluation

ELISA assay

The in vitro inhibitors screening of AGE2 (glyceraldehyde-modified AGE)-sRAGE interaction (soluble RAGE) was performed using a 96-well AGE-BSA-coated plate (Creative BioMart® ELISA kit), according to the manufacturer's instructions. Briefly, 100 UA/mL soluble RAGE (sRAGE) was incubated with 10 or 100 µM of the tested compounds or 0.05% DMSO as a negative control on an AGE2-BSA-coated plate at room temperature for 60 minutes, shaking at 300 rpm on an orbital microplate shaker. After incubation, the wells were washed 4 times with wash buffer and horseradish peroxidase (HRP)-labeled anti-RAGE antibody was added and the plate was further incubated at room temperature for 60 minutes, shaking at 300 rpm on an orbital microplate shaker. After incubation, the wells were washed 4 times with wash buffer and the Substrate Reagent was added to each well. After an incubation at room temperature for 7 minutes, the HRP-labeled antibody-sRAGE-AGE complex

Figure 1 .

 1 Figure 1. Structures of naturally occurring ellagic acid and urolithins A and B, of curcumin and

Figure 2 .Figure 3 .

 23 Figure 2. SEM image and EDS spectrum of Eco-MG1

  Yield obtained with Ecocat-MG2 (b) Yield obtained with CuSO4

  Now, based on the results obtained with urolithin B 1 derivatives 2-24, some important structure-activity relationships could be established: i) the benzo[c]chromene derivatives are favorable to inhibit the interaction of s-RAGE with its ligand AGE2-BSA, blocking their binding and consequently, benzo[c]chromene derivatives may present an interest in blocking the inflammatory biological phenomena triggered following this binding and activation of RAGE; ii) four points of chemical modulation have been studied (positions 1, 4, 8 and 9 of the benzo[c]chromene ring); the presence of the hydroxy substituent in position 1 was tolerated and generally improved the inhibition potential (compare compounds 3 and 7); iii) the bulky substituents in position 1 or in position 4 of the benzo[c]chromene ring diminished the biological activity (compare compounds 9-11 to 15-17 and 21-24, Table

  Institute (NCI) (Germantown, USA) for biological evaluation of the cytotoxic potential on a panel of 60 cancer cell lines. No cytotoxicity was observed for urolithin B 1 at a 10 µM concentration. This result consolidates the positioning of urolithin derivatives as safe RAGE antagonists with interesting potential for further development of urolithin-inspired drug candidates (see supplementary information section for full graph of one-dose NCI assay of urolithin B 1 tested at 10 µM concentration).

[ a ]

 a Inhibition of AGE2-BSA/sRAGE interaction at a 100 µM concentration. [b] Values represent mean of three experiments. [c] Tested at 10 µM concentration. N.D. Not determined.ConclusionIn summary, two miscanthus-based ecocatalysts Eco-MG1 and Eco-MG2 have been produced and successfully investigated as green alternative catalysts for the Hurtley reaction of polyphenols with 2-bromobenzoic acids to produce urolithin derivatives 1-24. Eco-MG2 displayed excellent catalytic activity compared to classical CuSO4 catalyst used for this transformation. In a context of environmental respect, the purity of final compounds has been analysed by SFC, a green analytical technique. Natural and synthetic urolithins 1-24 have been evaluated for their inhibitory activity of the AGE2-BSA/s-RAGE interaction and displayed excellent potential, equipotent to Azeliragon, the most advanced molecule to date in drug development as a RAGE antagonist. Important SAR features have been identified.

for 13 C

 13 NMR, on a Varian 400-MR spectrometer at 25 °C with tetramethylsilane (TMS) as internal standard, DMSO-δ6. Chemical shifts (δ) are expressed in ppm relative to TMS. Splitting patterns are designed: s, singlet; d, doublet; t, triplet; q, quadruplet. Coupling constants (J) are reported in Hertz (Hz).Melting points were measured on a MPA 100 OptiMelt® apparatus and are uncorrected. IR spectra were recorded on an Agilent Technologies FT-IR Cary 630. Flash chromatography was performed with a CombiFlash Rf Companion (Teledyne-Isco System) using RediSep packed columns.The purity of final compounds has been verified by supercritical fluid chromatography (SFC), a green analytical technique using a binary mobile phase consisting of CO2 in supercritical state and a small volume of organic co-solvent (ethanol was used as co-solvent in this study) compared to classical liquid chromatography (see Supplementary Information section for SFC chromatograms of synthesized compounds). The chiral analytical column used for this study, was a C18 column, purchased from Chiral Technologies Europe (Illkirch, France). This column had dimensions 250 mm x 4.6 mm i.d. with 5 µm particle size and was coated on a silica-gel support. The chromatographic system used was an SFC-PICLAB hybrid 10-20 apparatus (PIC Solution, Avignon, France) equipped with an autosampler comprised a 48-vial plate and a 24-vial plate (model Alias, Emmen, Netherlands), three model 40P pumps: two for CO2 and a third for the modifier (Knauer, Berlin, Germany), a column oven with a Valco ten-position column selection valve, and a Valco six-position solvent switching valve. The pressure was 150 bars, the mobile phase was EtOH/CO2 30/70 and the flow 4 mL/min.

  , J = 8.3 Hz, 1H, Ar-H), 7.89 (dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 8.06 (d, J = 2.1 Hz, 1H, Ar-H), 8.24 (d, J = 8.3 Hz, 1H, Ar-H), 8.29 (d, J = 8.4 Hz, 1H, Ar-H). 13 C NMR (100 MHz, DMSO-d6) (ppm): 32.5 (CH3), 108.5 (CH), 113.6 (C), 116.1 (C), 120.2 (C), 124.2 (CH), 126.4 (C), 128.4 (CH), 132.2 (C), 133.5 (CH), 135.15 (CH), 148.4 (C), 158.2 (C), 158.7 (C), 200.4 (C). IR ν (cm -1 ): 3075 (C-OH), 1737 (C=O), 1633 (C=O), 1592 (C=C). LC/MS (APCI + ) m/z 289.38 (MH + ) (calcd for C15H9ClO4: 288.68 g/mol), tr 3.26 min.

  C NMR (100 MHz, DMSO-d6) (ppm): 29.5 (CH2), 46.5 (CH2), 55.8 (CH3), 55.9 (CH3), 99.4 (CH), 106.7 (C), 107.8 (CH), 110.1 (C), 111.6 (CH), 115.6 (2CH), 129.6 (2C), 131.7 (2CH), 148.1 (2C), 154.7 (C), 155.9 (2C), 156.7 (C), 159.8 (C), 161.7 (C), 207.0 (C). IR ν (cm -1 ): 3314 (C-OH), 1700 (C=O), 1606 (C=O), 1513 (C=C). LC/MS (APCI + ) m/z 437.03 (MH + ) (calcd for C24H20O8: 436.42 g/mol), tr 2.92 min.

  H NMR (400 MHz, DMSO-d6) (ppm): 6.73 (d, J = 2.6 Hz, 1H, Ar-H), 6.84 (d, J = 8.0 Hz, 2H, Ar-H), 6.87 (d, J = 2.6 Hz, 1H, Ar-H), 6.98 (d, J = 15.8 Hz, 1H, CH), 7.41 (d, J = 15.8 Hz, 1H, CH), 7.52 (d, J = 8.4 Hz, 2H, ArH), 7.57 (t, J = 7.3 Hz, 1H, Ar-H), 7.87 (t, J = 7.3 Hz, 1H, Ar-H), 8.15 (d, J = 7.3 Hz, 1H, Ar-H), 8.25 (d, J = 7.3 Hz, 1H, Ar-H); 13 C NMR (100 MHz, DMSO-d6) (ppm): 102.2 (CH), 107.8 (CH), 113.4 (2CH), 115.6 (CH), 119.8 (CH), 125.5 (CH), 126.0 (CH), 127.1 (C), 127.6 (C), 128.2 (C), 129.9 (2CH), 131.3 (CH), 134.7 (CH), 135.4 (C), 139.0 (C), 152.7 (C), 157.7 (C), 158.6 (C), 160.4 (C). IR ν (cm -1 ): 3165 (C-OH), 1700 (C=O), 1614 (C=C), 1588 (C=C). LC/MS (APCI + ) m/z 331.01 (MH + ) (calcd for C21H14O4: 330.34 g/mol), tr 2.68 min.

  H NMR (400 MHz, DMSO-d6) (ppm): 3.88 (s, 3H, OCH3), 6.71 (d, J = 2.1 Hz, 1H, Ar-H), 6.80-6.86 (m, 3H, Ar-H), 6.95 (d, J = 16.3, 1H, CH), 7.37 (d, J = 16.3 Hz, 1H, CH), 7.48-7.53 (d, J = 16.3 Hz, 3H, Ar-H), 7.67 (d, J = 2.1 Hz, 1H, Ar-H), 8.09 (d, J = 9.5 Hz, 1H, Ar-H). 13 C NMR (100 MHz, DMSO-d6) (ppm): 55.6 (CH3), 102.2 (CH), 107.9 (CH), 111.7 (CH), 113.4 (2CH), 115.6 (C), 121.1 (CH), 122.7 (CH), 126.0 (C), 127.3 (CH), 127.7 (C), 128.2 (2CH), 128.7 (CH), 131.2 (C), 138.1 (C), 151.7 (C), 157.6 (C), 157.7 (C), 157.9 (C), 160.3 (C). IR ν (cm -1 ): 3280 (C-OH), 1692 (C=O), 1595 (C=C), 1502 (C=C). LC/MS (APCI + ) m/z 361.06 (MH + ) (calcd for C22H16O6: 360.36 g/mol), tr 2.72 min.

  C NMR (100 MHz, DMSO-d6) (ppm): 55.0 (CH3), 55.5 (CH3), 102.1 (CH), 107.5 (CH), 107.8 (CH), 110.0 (CH), 112.5 (CH), 113.4 (2CH), 115.5 (C), 126.0 (CH), 127.4 (C), 128.0 (C), 130.5 (2CH), 131.4 (C), 138.3 (C), 147.9 (C), 152.4 (C), 153.7 (C), 157.6 (C), 157.8 (C), 160.0 (C). IR ν (cm -1 ): 3276 (C-OH), 1700 (C=O), 1606 (C=C), 1588 (C=C). LC/MS (APCI + ) m/z 391.09 (MH + ) (calcd for C23H18O6: 390.39 g/mol), tr 2.8 min.

  H NMR (400 MHz, DMSO-d6) (ppm): 6.74 (d, J = 3.0 Hz, 1H, Ar-H), 6.83 (d, J = 8.3 Hz, 2H, Ar-H), 6.88 (d, J = 3.0 Hz, 1H, Ar-H), 6.99 (d, J = 16.7 Hz, 1H, CH), 7.38 (d, J = 16.7 Hz, 1H, CH), 7.52 (d, J = 8.3 Hz, 2H, Ar-H), 7.96 (dd, J = 8.8, 2.3 Hz, 1H, Ar-H), 8.15 (d, J = 8.8 Hz, 1H, Ar-H), 8.18 (d, J = 2.3 Hz, 1H, Ar-H). 13 C NMR (100 MHz, DMSO-d6) (ppm): 102.2 (CH), 107.1 (CH), 113.6 (2CH), 115.6 (C), 121.5 (CH), 125.5 (CH), 127.5 (C), 127.6 (C), 128.4 (CH), 128.7 (CH), 131.4 (2CH), 131.8 (C), 134.2 (CH), 134.6 (C), 139.6 (C), 152.7 (C), 157.8 (C), 158.9 (C), 159.4 (C). IR ν (cm -1 ): 3168 (C-OH), 1700 (C=O), 1610 (C=C), 1588 (C=C). LC/MS (APCI + ) m/z 365.48 (MH + ) (calcd for C21H13ClO4: 364.78 g/mol), tr 2.86 min.

✓

  Miscanthus-based ecocatalysts ✓ Green ecocatalysed synthesis ✓ 24 synthesized urolithins ✓ Biological evaluation on AGE2-BSA/sRAGE interaction ✓ 4 points of pharmacomodulation ✓ First SAR as RAGE antagonistsFavorable modulation for blocking RAGE activity Disadvantageous for blocking RAGE activity

Table 1 .

 1 Distribution of metals in Eco-MG1 and Eco-MG2 MG1 a 0.05 1.16 2.62 1.66 0.27 0.61 16.26 3.93 4.21 69.21 Eco-MG2 b 0.52 13.06 28.33 16.66 2.60 7.00 6.30 22.76 1.00 1.78

	Material	Cd	Pb	Zn	Distribution of metals (%) Fe Mn Cu Ca	Mg Na	K
	Eco-

a Eco-MG1=crude Lewis acids obtained after treating ashes with 2M HCl aqueous solution b Eco-MG2=crude Lewis acids obtained after washing with acetone Eco-MG1

Table 2 .

 2 Catalytic study of the Urolithin B synthesis

	Entry Catalyst	Eq (w/w) a	Yield (%)
	1	-	-	5
	2	CuSO4	0.016 b
	3	Eco-MG1	0.010
	4	Eco-MG1	0.050
	5	Eco-MG1	0.100
	6	Eco-MG1	0.150
	7	Eco-MG1	0.200
	8	Eco-MG1	0.500
	9	Eco-MG2	0.010
	10	Eco-MG2	0.015
	11	Eco-MG2	0.030
	12	Eco-MG2	0.060
	13	Eco-MG2	0.100
	14	CuCl2	0.013 b
	15	CuCl	0.010 b
	16	CuI	0.019 b
	17	ZnCl2	0.014 b
	18	CdCl2	0.018 b
	19	PbCl2	0.028 b
	20	MnSO4	0.015 b	5
	21	MnCl2	0.012 b	6
	22	FeCl2	0.012 b
	23	FeCl3	0.016 b
	(a) Regarding bromobenzoic acid reactant
	(b) 2% mol of metals

Table 3 .

 3 Synthesis of urolithin derivatives

	Entry	Bromobenzoic acid	Polyphenol	Product	Yield (%)

Table 4 .

 4 AGE2-BSA/sRAGE inhibitory activity of urolithin derivatives 1-24 and of known positive references

	Entry	Compound	% Inh AGE2-BSA/sRAGE [a,b] IC50 (AGE2-BSA/sRAGE) µM
	1	Resorcinol	0	N.D.
	2	Phloroglucinol	0	N.D.
	3	2,6-Dihydroxyacetophenone	0	N.D.
	4	2',4',6'-Trihydroxypropiophenone	0	N.D.
	5	2',4',6'-Trihydroxy-2-phenylacetophenone	0	N.D.
	6	Phloretin	0	N.D.
	7	Resveratrol	0	N.D.
	8	1	76	25.3
	9	2	100	31.6
	10	3	100	29.7
	11	4	51	N.D.
	12	5	88	67.3
	13	6	91	14.2
	14	7	92	12.8
	15	8	44	N.D.
	16	9	100	34.8
	17	10	100	34.5
	18	11	89	65.0
	19	12	66	N.D.
	20	13	5	N.D.
	21	14	72	17.6
	22	15	53	N.D.
	23	16	69	N.D.
	24	17	58	N.D.
	25	18	62	N.D.
	26	19	58	N.D.
	27	20	62	N.D.
	28	21	38	N.D.
	29	22	63	N.D.
	30	23	66	N.D.
	31	24	26	N.D.
	32	Azeliragon	49 [c]	N.D.
	33	FPS-ZM1	14	N.D.
	34	Urolithin A	20	N.D.
	35	Curcumin	43	N.D.
	36	Papaverine	26 [c]	N.D.

  The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 4 as a brown solid in 49% yield; mp (EtOH): 170-172 °C; 1 H NMR (400 MHz, DMSO-d6) (ppm): 6.73 (s, 1H, Ar-H), 6.84 (d, J = 9.3 Hz, 1H, Ar-H), 7.90 (d, J = 9.3 Hz, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 8.13 (d, J = 9.2 Hz, 1H, Ar-H), 8.26 (d, J = 9.2 Hz, 1H, Ar-H).

	13 C NMR (100 MHz,
	DMSO-d6) (ppm): 102.9 (CH), 108.3 (CH), 113.4 (CH), 120.3 (C), 123.9 (C), 124.9 (CH), 128.4
	(CH), 131.6 (C), 134.0 (CH), 135.0 (C), 152.1 (C), 159.5 (C), 160.7 (C). IR ν (cm -1 ): 3295 (C-OH),
	1696 (C=O), 1629 (C=C), 1599 (C=C). LC/MS (APCI + ) m/z 247.35 (MH + ) (calcd for C13H7ClO3:
	246.65 g/mol), tr 3.26 min.
	1,3-Dihydroxy-6H-benzo[c]chromen-6-one (5). The general procedure was used with 2-bromobenzoic
	acid (0.5 g, 2.49 mmol) and phloroglucinol (0.628 g, 4.98 mmol) in 3 mL of water. The product was
	purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 5
	as a beige solid in 80% yield with the same physico-chemical characteristics as previously reported; 12
	1 H NMR (400 MHz, DMSO-d6) (ppm): 6.27 (d, J = 2.3 Hz, 1H, Ar-H), 6.41 (d, J = 2.3 Hz, 1H, Ar-H),
	7.46 (t, J = 7.5, 1H, Ar-H), 7.80 (t, J = 7.5 Hz, 1H, Ar-H), 8.16 (d, J = 7.5 Hz, 1H, Ar-H), 8.94 (d, J =
	7.5 Hz, 1H Ar-H).
	1 H NMR (400 MHz, DMSO-d6) (ppm): 3.87 (s, 3H, OCH3), 4.01 (s, 3H,

OCH3),

6

.72 (d, J = 2.2 Hz, 1H, Ar-H),

6

.82 (dd, J = 8.4, 1.8 Hz, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 8.16 (d, J = 8.4 Hz, 1H, Ar-H).

8-Chloro-3-hydroxy-6H-benzo[c]chromen-6-one

(4)

. The general procedure was used with 2-bromo-5-chlorobenzoic acid (0.5 g, 2.12 mmol) and resorcinol (0.467 g, 4.24 mmol) in 3 mL of water.

1,3-Dihydroxy-8-methoxy-6H-benzo[c]chromen-6-one

(6)

. The general procedure was used with 2bromo-5-methoxybenzoic acid (0.5 g, 2.16 mmol) and phloroglucinol (0.542 g, 4.32 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 6 as a beige solid in 88% yield with the same physico-chemical characteristics as previously reported;

12

1 H NMR (400 MHz, DMSO-d6) (ppm): 3.88 (s, 3H, OCH3), 6.26 (d, J = 2.2 Hz, 1H, Ar-H), 6.40 (d, J = 2.2 Hz, 1H, Ar-H), 7.46 (dd, J = 8.8, 2.8 Hz, 1H, Ar-H), 7.63 (d, J = 2.8 Hz, 1H, Ar-H), 8.92 (d, J = 8.8 Hz, 1H, Ar-H).

  5 g, 2.16 mmol) and 2,6-dihydroxyacetophenone (0.657 g, 4.32 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol washing of the precipitate to obtain pure compound 10 as a brown solid in 63% yield; mp (EtOH): 191-192 °C;

	1 H NMR (400 MHz, DMSO-d6) (ppm): 2.63 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 6.94 (d, J = 7.9 Hz,
	1H, Ar-H), 7.51 (d, J = 7.9 Hz, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 8.21 (t, J = 7.2 Hz, 2H, Ar-H); 13 C
	NMR (100 MHz, DMSO-d6) (ppm): 33.0 (CH3), 56.1 (CH3), 110.1 (CH), 111.2 (CH), 113.9 (C), 116.3
	(C), 120.3 (CH), 124.4 (C), 124.6 (CH), 126.5 (C), 128.5 (CH), 148.3 (C), 157.6 (C), 159.3 (C), 160.1
	(C), 201.3 (C). IR ν (cm -1 ): 1729 (C=O), 1629 (C=O), 1588 (C=C). LC/MS (APCI + ) m/z 284.97 (MH + )
	(calcd for C16H12O5: 284.27 g/mol), tr 2.78 min.
	4-Acetyl-3-hydroxy-8,9-dimethoxy-6H-benzo[c]chromen-6-one (11). The general procedure was used
	with 2-bromo-4,5-dimethoxybenzoic acid (0.5 g, 1.92 mmol) and 2,6-dihydroxyacetophenone (0.584
	g, 3.84 mmol) in 3 mL of water. The product was purified by simple filtration and absolute ethanol
	washing of the precipitate to obtain pure compound 11 as a brown solid in 67% yield; mp (EtOH):
	152-153 °C; 1 H NMR (400 MHz, DMSO-d6) (ppm): 2.63 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 4.02 (s,
	3H, OCH3), 6.95 (d, J = 8.4 Hz, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 8.34 (s, 1H, Ar-H);
	13 C NMR (100 MHz, DMSO-d6) (ppm): 32.5 (CH3), 55.7 (CH3), 56.2 (CH3), 103.6 (CH), 109.5 (CH),
	109.6 (CH), 111.3 (C), 113.0 (C), 115.6 (C), 126.5 (C), 130.0 (CH), 148.4 (C), 149.1 (C), 155.3 (C),
	157.3 (C), 159.4 (C), 200.9 (C). IR ν (cm -1 ): 1707 (C=O), 1606 (C=O), 1520 (C=C). LC/MS (APCI + )
	m/z 314.99 (MH + ) (calcd for C17H14O6: 314.29 g/mol), tr 3.12 min.

Acknowledgments

The authors thank the Région Hauts-de-France (Fonds européen de développement regional (FEDER))

and JUNIA Hauts-de-France for financial support of this work and technical help and facilities. The authors also acknowledge the NCI (National Cancer Institute) for the biological evaluation of urolithin B on their 60-cell panel: the testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (the URL to the Program's website: http://dtp.cancer.gov).

was then detected by measuring the absorbance at 450 nm using the microplate reader Varioskan (Thermo Fischer Scientific). These experiments were repeated three times.

CRediT authorship contribution statement


MD Jean-Charles Roy
email: jean-charles.roy@chu-rennes.fr

Jean-François Houvenaghel

Joan Duprez

Murielle Guillery

Dominique Drapier

Gabriel Robert

DYNAMICS OF COGNITIVE ACTION CONTROL IN LATE-LIFE DEPRESSION DURING ACTION SELECTION

Keywords: Apathy evaluation scale C, Congruent Condition CAC, Cognitive action control Late-life depression MoCA, Montreal cognitive assessment scale MCST, Modified Wisconsin card sorting test mR2, marginal R2

INTRODUCTION

Late-Life Depression (LLD), (>60 years old), affects 7% of the population (World Health Organization, 2017). Cognitive control deficits lie at the core of LLD being persistent during remission [START_REF] Bhalla | Persistence of neuropsychologic deficits in the remitted state of late-life depression[END_REF][START_REF] Butters | The nature and determinants of neuropsychological functioning in late-life depression[END_REF][START_REF] Diniz | Late-life depression and risk of vascular dementia and Alzheimer's disease: systematic review and meta-analysis of community-based cohort studies[END_REF], correlating with disability and predicting depressive relapses [START_REF] Alexopoulos | Comorbidity of late life depression: an opportunity for research on mechanisms and treatment[END_REF][START_REF] Alexopoulos | Executive dysfunction and long-term outcomes of geriatric depression[END_REF]. Cognitive action control (CAC) refers to "a subset of cognitive control processes involved in the requirement to coordinate one's instantaneous urges vis-à-vis actions that concord with our intentions or instructions" [START_REF] Richard Ridderinkhof | Neurocognitive mechanisms of action control: resisting the call of the Sirens[END_REF]. Such processes include cognitive inhibition, cognitive flexibility or inhibitory control. They can be altered during normal aging [START_REF] Maldonado | Age Differences in the Subcomponents of Executive Functioning[END_REF][START_REF] Rey-Mermet | Inhibition in aging: What is preserved? What declines? A meta-analysis[END_REF] but may be magnified in LLD. Indeed, CAC deficits in LLD have been associated with higher suicidality [START_REF] Mcgirr | Deterministic learning and attempted suicide among older depressed individuals: Cognitive assessment using the Wisconsin Card Sorting Task[END_REF][START_REF] Richard-Devantoy | Cognitive inhibition in older high-lethality suicide attempters[END_REF][START_REF] Hommel | The Simon effect as tool and heuristic[END_REF] possibly mediated by impulsivity [START_REF] Dombrovski | Lethal Forethought: Delayed Reward Discounting Differentiates High-and Low-Lethality Suicide Attempts in Old Age[END_REF], and apathy [START_REF] Feil | Apathy and cognitive performance in older adults with depression[END_REF][START_REF] Funes | Apathy Mediates Cognitive Difficulties in Geriatric Depression[END_REF]. However, these studies lack an integrative framework to explore in-depth CAC dysfunction. The Simon task (ST) is a tool of choice to explore CAC in LLD as it measures electively the integrity of the processes coding for perceptual and action monitoring [START_REF] Hommel | The Simon effect as tool and heuristic[END_REF] and is negligibly influenced by other domains of cognition. During this task, subjects are instructed to make a fast and accurate left or right hand response according to the color of a stimulus that is presented either to the left or right side of a central fixation point [START_REF] Hedge | The effect of irrelevant spatial correspondences on two-choice response-time[END_REF]. Conflict lies in the competition between the location (non-relevant information) and the color of the stimulus (relevant information). During non-congruent trials (i.e. when color and location do not match) participants show longer reaction time (RT) and decreased accuracy compared to congruent trials (i.e. when color and location match). These modifications between non-congruent and congruent conditions, named the congruency effect (CE), are believed to evaluate online CAC. Additionally, RT and error rates are greater when trials congruency alternates [START_REF] Gratton | Optimizing the use of information: strategic control of activation of responses[END_REF]. This so-called congruency sequence effect (CSE; [START_REF] Duthoo | The heterogeneous world of congruency sequence effects: an update[END_REF] is an adaptation of CAC to conflict [START_REF] Botvinick | Conflict monitoring and cognitive control[END_REF]. Hence, the ST provides a unifying paradigm to evaluate online (CE) and adaptive CAC (CSE). Using the ST, depressive symptoms have been associated with larger CE [START_REF] Ng | Dissociating effects of subclinical anxiety and depression on cognitive control[END_REF] interpreted as a poorer performance. However, the association between depressive symptoms and adaptive CAC has yielded mixed results: decreased CSE in depression during negative feedback [START_REF] Holmes | Task feedback effects on conflict monitoring and executive control: Relationship to subclinical measures of depression[END_REF], no association between symptoms severity and CSE [START_REF] Ng | Dissociating effects of subclinical anxiety and depression on cognitive control[END_REF] and increased CSE in depressed participants [START_REF] Van Steenbergen | Affective regulation of cognitive-control adjustments in remitted depressive patients after acute tryptophan depletion[END_REF]. This discrepancy might be due to the use of averaged RTs, thereby omitting the temporal dynamics of CAC. To resolve this, we used the activation-suppression model (Ridderinkhof, 2002a). This model incorporates the temporal dynamics of top-down suppression of impulsive action selection by distinguishing a fast and early impulsive action selection from a late and slow selective response suppression. Impulsive action selection results in increased errors for the fastest responses of the participants in conflict situations while selective response suppression of the non-relevant information takes time to buildup and is most evident at the slowest RTs. Discretizing CAC by reaction times is particularly relevant in LLD because it is suggested that abnormal cognitive control is mediated by decreased processing speed [START_REF] Nebes | Decreased working memory and processing speed mediate cognitive impairment in geriatric depression[END_REF][START_REF] Sheline | Cognitive function in late life depression: relationships to depression severity, cerebrovascular risk factors and processing speed[END_REF][START_REF] Snyder | Major Depressive Disorder is Associated with Broad Impairments on Neuropsychological Measures of Executive Function: A Meta-Analysis and Review[END_REF].

The main purpose of the present study is to show that patients with LLD have a deficit in both online and adaptive CAC. Secondly, we aim to show that LLD patients have specific patterns of impulsive action selection and selective response suppression. We hypothesized an impulsive behavior in LLD reflected by increased fast errors and lower selective suppression. Finally, we explored how impulsive action selection and selective response suppression of both online and adaptive CAC were associated to apathy and impulsivity.

MATERIALS AND METHODS

Participants

Thirty-one elderly diagnosed with DSM-5 criteria of Major Depressive Disorder and 31 healthy elderly controls (HC) participated. LLD group individuals were recruited by their psychiatrist, while HC were recruited from local retirees associations. Inclusion and exclusion criteria were assessed during a psychiatric interview by a trained old-age psychiatrist. Inclusion criteria for the LLD group were: Hamilton Depressive Rating Scale (HDRS, [START_REF] Hamilton | A rating scale for depression[END_REF]) score > 10. Exclusion criteria were: age < 60 years-old, schizophrenia, bipolar and neurological disorders, history of addiction (tobacco excluded), uncorrected vision or motor impairments, Montreal Cognitive Assessment Scale (MoCA, [START_REF] Nasreddine | The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment[END_REF] score < 20 [START_REF] Blair | Depressive Symptoms Negatively Impact Montreal Cognitive Assessment Performance: A Memory Clinic Experience[END_REF][START_REF] Dautzenberg | Clinical value of the Montreal Cognitive Assessment (MoCA) in patients suspected of cognitive impairment in old age psychiatry. Using the MoCA for triaging to a memory clinic[END_REF], cerebral modulation during the last 6 months. Written informed consents were collected. The study was approved by the local ethics committee of Rennes University Hospital in accordance with current French legislation.

Experimental design

We used the Apathy Evaluation Scale (AES, [START_REF] Marin | Apathy: a neuropsychiatric syndrome[END_REF], the Depressive Retardation Scale (DRS, [START_REF] Widlöcher | Psychomotor Retardation: Clinical, Theoretical, and Psychometric Aspects[END_REF], the Urgency Premeditation Perseverance Sensation seeking impulsive behavior scale (UPPS, [START_REF] Whiteside | The five factor model and impulsivity: Using a structural model of personality to understand impulsivity[END_REF] exploring four dimensions of impulsivity, and the State and Trait Anxiety Inventory (STAI, [START_REF] Spielberger | Manual for the State-Trait Anxiety Inventory[END_REF]. HDRS quantified depression severity (LLD patients only). General cognitive function was assessed with the MoCA. The Stroop [START_REF] Golden | A Manual for the Clinical and Experimental Use of the Stroop Color and Word Test[END_REF], Trail Making Test A and B [START_REF] Reitan | Validity of the Trail Making Test as an Indicator of Organic Brain Damage[END_REF], Modified Wisconsin Card Sorting (MCST, [START_REF] Nelson | A modified card sorting test sensitive to frontal lobe defects[END_REF] and Continuous Performance tests (CPT-3, Conners et al., 2018) assessed cognitive control, cognitive flexibility, set shifting and sustained plus selective attention, respectively.

Task and Procedure

The ST (Figure 1) was programmed under E-prime Professional version 2.0. This task comprised a familiarization phase (72 trials) followed by an experimental one (five blocks of 72 trials, with a pause between each block). All stimuli were displayed on a 60 cm white computer screen, placed 90 cm away from the participant. Each trial began with a black fixation cross displayed at the center of the screen for 1000 ms. A blue or yellow circle (target) of 2.1 cm diameter appeared on the right or left side of the cross. The distance between the center of the computer screen and the edge of the circle was of 1.1 cm. Participants were asked to press a blue or yellow button on a computer keyboard with their right or left hand, as quickly and accurately as possible, according to the color of the target while ignoring its position. The position of the target defined the congruency of the trial. The target disappeared as soon as participants pressed one of the two buttons and a new target appeared after a 1550 to 1650 ms randomly defined interval. The position of the color button was pseudo-randomized across participants, and all conditions were pseudo-randomized with an equal number of trials in each condition.

Behavioral Analysis

Only responses from the experimental phase with RT between 200 and 1500ms and less than three standard deviations from the mean individual RT were analyzed (1.66 % of the trials removed). Main analyses focused on between-group differences in RT and accuracy rates between the congruent (C) and non-congruent (NC) conditions to assess the CE, adding the interaction with preceding congruency for evaluating the CSE. The dynamics of CAC (i.e., impulsive action selection and selective response suppression) were assessed by distributional analyses (Ridderinkhof, 2002b). Impulsive action selection is represented by conditional accuracy functions (CAF) which plot correct responses as a function of the RT distribution of all responses discretized in 5 bins (62 trials per bin per participant) for each condition (C or NC) and each group (HC or LLD). An increased impulsive action selection corresponds to a lower accuracy rate at the first bin of RT (i.e. fastest responses). Selective response suppression is represented by delta plots which plot the congruency effect (difference in RT between NC and C conditions) as a function of the RT distribution of the correct responses, discretized in 5 bins. We then estimated the slopes between these bins (four slopes). According to the activation-suppression model, the steeper the fourth slope is, the more effective the selective suppression on slow responses. We performed the CAF and delta-plots considering systematically the current trial as a function of the congruency of the preceding trial (CSE). Three participants, 2 LLD and 1 HC, were excluded from the distribution analyses because 20% of their completed trials were missing.

Statistical Analysis

Statistical analyses were performed using R software (version 3.0; R core team, 2017). A significance threshold of two-sided p < 0.05 was used.

Patients characteristics

Demographic, clinical and psychological data were compared between the LLD and HC groups using χ 2 tests for qualitative variables and independent t-tests (or Wilcoxon's signedrand test, depending on visual inspection of Q-Q plots).

Behavioral data

As RTs were not normally distributed, they were first converted using a logarithmic transformation. The CSE on RT was estimated using a linear mixed model comparing the log RTs between the four conditions and both groups. The fixed factors were the congruency of the preceding trial (Pre_Cong: Pre_congruent vs. Pre_non-congruent), the congruency of the current trial (Congruency: congruent vs. non-congruent) and the group (LLD and HC). Participant was added as a random intercept. Mixed models optimize statistical power as they use all available data, account for intra-subject repeated measures correlation and easily handle unbalanced designs [START_REF] Hox | Multilevel Analysis : Techniques and Applications[END_REF]. As recommended by [START_REF] Nakagawa | A general and simple method for obtaining R2 from generalized linear mixed-effects models[END_REF],we reported the conditional (cR2) and marginal R2 (mR2) using the {MuMIn} package [START_REF] Bartoń | MuMIn: Multi-model inference[END_REF]. The linear mixed model was executed using the lme function of the {nlme} package [START_REF] Pinheiro | Nlme: Linear and Nonlinear Mixed Effects Models[END_REF]. Post-hoc Tukey-Kramer tests were performed using the {multcomp} package [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF]. The CSE on accuracy was estimated using a mixed logistic regression model comparing the correct responses between the four conditions and both groups. The fixed factors were the congruency of the preceding trial, the congruency of the current trial and the group. Participant was added as a random intercept. The model was executed with the glmer function of the {lme4} package [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF]. P-values from the logistic models were derived from the Wald Chi-square tests using the {car} package [START_REF] Fox | An R Companion to Applied Regression[END_REF]. The CE on RT and accuracy were estimated from the effect of the congruency of the current trial from the respective models. Impulsive action selection was tested using a mixed model logistic regression with accuracy at the first bin of the CAF as the independent variable. 'Group' and 'congruency of the current trial' were fixed factors while 'participant' was added as a random intercept. Selective suppression was tested with a linear regression mixed model with steepness of the fourth slope of the delta plots as independent variable and with the same factorial design as for impulsive action selection. Statistically different between-group sociodemographic variables were included as covariates.

Behavioral and Clinical Data Relationships

Associations between the ST indices and the clinical scores of LLD were tested using spearman correlations (detailed in supplemental methods). All codes are available in supplemental material. Clinical and behavioral data are available at the Mendeley data repository (10.17632/5644kyzncp.1).

RESULTS

Patient characteristics

Between-group sociodemographic, clinical and neuropsychological variables are summarized in Table 1. Twenty-seven (87%) LLD patients were taking antidepressants. LLD patients had lower educational status (p<0.001) than HC but comparable age (p=0.11) and sex ratio (p=0.57). Because of the expected effect of education status on cognitive performance [START_REF] Seblova | Education and age-related decline in cognitive performance: Systematic review and meta-analysis of longitudinal cohort studies[END_REF], all following between-group analyses were adjusted on education status.

Between group difference on the CE and CSE 3.2.1. Congruency Effect

RTs were similar between the LLD patients and HC (Group: log(RT): F(1, 59) = 3.17, p = 0.07). RTs were longer and accuracy rates lower for non-congruent than for congruent trials across all participants (Congruency: log(RT), F(1, 18362) = 2350.39, p < 0.0001, mR 2 = 0.24 , cR 2 = 0.33; accuracy, χ 2 (1, 19305) = 246.34, p < 0.0001, mR 2 = 0.13 , cR 2 = 0.49). This CE was significantly different between the groups on accuracy rates (Group × Congruency: accuracy, χ 2 (1, 19305) = 15.11, p < 0.001) but not for RT (Group × Congruency: log(RT), F(1, 18362) = 3.45, p = 0.06). Post-hoc analysis showed that LLD patients made more errors on congruent trials (Group: z = -3.91, p < 0.001) which was not found in non-congruent trials (Group: z = -1.24, p = 0.57).

Congruency sequential effect

Across groups, accuracy rates were higher for trials that followed congruent trials in comparison to trials that followed non-congruent trials, as opposed to RTs (Pre_Cong: accuracy, χ 2 (1, 19296) = 51.58, p < 0.0001; log(RT), χ 2 (1, 16888) = 0.014, p = 0.91; Figure 2). CE was associated with the congruency of the preceding trial (RT: Congruency × Pre_Cong: log(RT), χ 2 (1, 16888) = 2308.51, p < 0.0001; accuracy, χ 2 (1, 19296) = 15.11, p = 0.0001). Post-hoc comparisons indicated that the CE was larger when the preceding trial was congruent for both RT (Pre_Cong: log(RT), z = -9.06, p < 0.001) and accuracy (Pre_Cong: accuracy, z = -9.76, p < 0.001). No group differences were found on the CSE (RT: Group x Congruency × Pre_Cong: log(RT), χ 2 (1, 16888) = 0.48, p = 0.49; accuracy, χ 2 (1, 19296) = 0.58, p = 0.44).

Impulsive action selection 3.3.1. Between group differences on online impulsive action selection

Figure 3 shows the dynamics of accuracy as a function of RT for congruent and noncongruent conditions. Across groups, we found a reduced accuracy on the fastest RTs during non-congruent trials (Congruency: χ 2 (1, 3927) = 273.57, p < 0.0001; mR 2 = 0.43, cR 2 = 0.53), showing greater impulsive action selection in the non-congruent condition. The RT of the first bins were equivalent between the groups (Group: log(RT): F(1, 59) = 0.65, p = 0.42), even when only considering the congruent trials (Group: log(RT): F(1, 59) = 0.04, p = 0.27). While no main group differences across congruency was found (Group: accuracy, χ 2 (1, 59) = 0.99, p = 0.32), there was a significant interaction between group and congruency (Group x Congruency: accuracy, χ 2 (1, 3927) = 10.77, p = 0.01). Post-hoc analysis indicates that LLD patients made more errors than HC during congruent trials (Group: z = -3.44, p = 0.003) and not for non-congruent trials (Group: z = -0.86, p = 0.81). We found no effect of the group on the reaction times at the first quantile (Pre_Cong x Congruency x Group: log(RT): F(1, 59) = 0.46, p =0.50) particularly when congruent conditions were preceded by non-congruent conditions (Group: z = 0.036, p= 0.73).

Between group differences on adaptation of impulsive action selection

Figure 4 reports the CAFs of each group as a function of the congruency of the preceding trials. Across groups, the accuracy of the fastest responses was dependent of the congruency of the preceding trial (Pre_Cong: χ 2 (1, 59) = 116.95, p < 0.0001) as well as the CSE (Pre_Cong x Congruency: χ 2 (1, 59) = 40.28, p <0.0001). Hence, an adaptation effect was present on the fastest responses. There was no significant interaction between the group and CSE (Group x Pre_Cong x Congruency: χ 2 (1, 59) = 2.46, p =0.12). As the graphical reading of the CAFs suggests between-group differences in impulsive action selection when congruencies in trials change (grey plots in Figure 4), we explored an association between 'Group' and impulsive action selection for each pair of congruency sequences. Impulsive responses in LLD patients were greater when congruent trials occur after non-congruent trials (Group, z = -3.13, p = 0.03) compared to HC (Figure 4D), not found in other sequences of adaptation (all p = 0.99).

Selective response suppression 3.4.1. Between group differences on online selective response suppression

Figure 5 displays the delta plots for each group. As predicted by the activation-suppression model, the fourth slope was steeper for the slowest RT, without between group differences (Group: slope, F(1, 56) = 9.55, p = 0.008; mR 2 = 0.11 , cR 2 = 0.89).

Between group differences on adaptation of selective response suppression

Delta plots as a function of trial sequence are represented in supplementary materials (Figure S1). We did not find any significant differences in selective suppression according to the congruency of the preceding trial (Pre_Cong: slope, χ 2 (1,56) = 0.08, p = 0.78) and no significant interaction between group and preceding congruency (Group x Pre_Cong: slope, χ 2 (1,56) = 0.98, p = 0.32).

Associations between CAC metrics and symptoms severity in LLD

We did not find any significant associations between CAC metrics and apathy, impulsivity, depression or anxiety scores in LLD. These results are detailed in supplemental material.

DISCUSSION

We combined measures of online CAC (CE) and adaptive CAC (CSE) from the ST to the activation-suppression model (Ridderinkhof, 2002a) to assess impulsive action selection and response suppression. We found mixed results on the integrity of CAC in LLD: while LLD participants showed reduced cognitive control during impulsive action, they had a more effective selective response suppression compared to HC.

Influence of Depression on Global Cognitive Action Control

Regarding online CAC, we found increased errors in LLD patients on congruent conditions, unlike Ng and colleagues (2012) who found increased error rates in non-congruent trials among sub-depressive adults. However, the task they used required working memory, which may affect CAC by increasing cognitive load [START_REF] Engström | Effects of Cognitive Load on Driving Performance: The Cognitive Control Hypothesis[END_REF]. In contrast, our simpler task was specifically designed to assess the CAC. Regarding overall adaptive control abilities, we found the expected CSE on both accuracy and RTs, suggesting the task was adequately performed. We did not find any group differences, suggesting preserved overall adaptive control abilities in LLD, as shown in adult depression [START_REF] Dillon | A Computational Analysis of Flanker Interference in Depression[END_REF][START_REF] Van Steenbergen | Affective regulation of cognitive-control adjustments in remitted depressive patients after acute tryptophan depletion[END_REF]. The increased errors during congruent trials among LLD participants compared to HC suggest a subtle decreased facilitation of action selection. Indeed, congruent trials facilitate the transformation of a spatial stimulus location into a motor response [START_REF] Hasbroucq | Stimulus-response compatibility and the Simon effect: Toward a conceptual clarification[END_REF][START_REF] Kornblum | Stimulus-response compatibility with relevant and irrelevant stimulus dimensions that do and do not overlap with the response[END_REF]. Our results suggest this facilitation of the performance by the task-irrelevant dimension of the stimulus is reduced in LLD. In the ST, the CE specifically informs on response selection, as the conflict operates between the response set and the taskirrelevant dimension of the stimulus. This explains why this CE was different from the interference effect at the Stroop test, where the conflict occurs at the stage of stimulus identification [START_REF] Li | Independent Processing of Stimulus-Stimulus and Stimulus-Response Conflicts[END_REF].

Influence of depression on the dynamics of CAC 4.2.1. Influence of depression on impulsive action selection

CEs associated to LLD are underpinned by an increased impulsive online action selection in congruent condition. All participants had increased errors in non-congruent conditions for the fastest RT in accordance with the activation-suppression hypothesis. LLD participants showed a greater impulsive action selection on congruent trials suggesting a decreased facilitation by the congruent situation, hence a reduced sensitivity to the irrelevant dimension of the stimulus [START_REF] Dillon | A Computational Analysis of Flanker Interference in Depression[END_REF]. Analysis of impulsive action selection adaptation suggests that the impulsive tendency of LLD may be due to a reduced disengagement of attention from conflict. Similar to HC, LLD improved their responses when a NC trial preceded a NC trial, but showed increased impulsivity when a NC trial preceded a C trial. Thus, LLD subjects were less "helped" by the stimulus location to promote fast and accurate responses after conflict. This is consistent with altered adaptation to 'aversive' stimulus like cognitive conflict [START_REF] Botvinick | Conflict monitoring and cognitive control[END_REF][START_REF] Dreisbach | Conflicts as Aversive Signals for Control Adaptation[END_REF] in depression [START_REF] Disner | Neural mechanisms of the cognitive model of depression[END_REF][START_REF] Eshel | Reward and Punishment Processing in Depression[END_REF][START_REF] Meiran | Cognitive rigidity in unipolar depression and obsessive compulsive disorder: examination of task switching, Stroop, working memory updating and post-conflict adaptation[END_REF]. This has been related to impaired attention [START_REF] Nicoletti | Attention shifts produce spatial stimulus codes[END_REF] which LLD participants confirmed by lower CPT scores than HC.

Influence of depression on selective response suppression

LLD patients displayed a stronger selective response suppression than HC. This result aligns with studies showing increased electrophysiological markers of inhibition during Go/No-Go task in depression [START_REF] Ruchsow | Electrophysiological evidence for reduced inhibitory control in depressed patients in partial remission: a Go/Nogo study[END_REF][START_REF] Zhang | The Effect of Aging in Inhibitory Control of Major Depressive Disorder Revealed by Event-Related Potentials[END_REF]. The authors suggested that LLD participants require additional resources to activate inhibition. In our study, mild to moderate depressive subjects may have over-recruited their inhibition resources to improve their performance in the slowest responses. Indeed, HC's last quantile of RT were approximately 100ms faster than LLD. A couple of studies found similar results with depressed individuals taking more time to execute a more effective inhibitory control of pre-potent responses than HC [START_REF] Dillon | A Computational Analysis of Flanker Interference in Depression[END_REF][START_REF] Vallesi | Modulating speed-accuracy strategies in major depression[END_REF]. The absence of adaptive selective suppression across participants reproduces Ridderinkhof (2002a) as Wylie and colleagues (2010). This result is sound as the suppression of impulsive responses, maximal for the slowest RT, should be less affected by the congruency of the preceding trial.

No association between clinical features and CAC metrics

No significant correlation between CAC measures and clinical scores were found in LLD, similarly than previous results [START_REF] Austin | Cognitive function in depression: a distinct pattern of frontal impairment in melancholia?[END_REF][START_REF] Quinn | The impact of depression heterogeneity on inhibitory control[END_REF]. This might be explained by our sample wherein 80% of the patients had mild to moderate depressive symptoms [START_REF] Zimmerman | Severity classification on the Hamilton Depression Rating Scale[END_REF]. As the performance at ST would not have been specific for too severely depressed elderly (Staub et al., 2013), we may have failed to find correlations between clinical severity and CAC measures. LLD patients had higher scores in impulsive urgency and perseverance traits consistent with depressive disorder (d'Acremont and Van der Linden, 2007). The absence of association between impulsivity-trait and CAC has been found previously, reflecting poor consistency between self-reported temperament and cognitive measures in laboratory settings [START_REF] Cyders | Measurement of constructs using self-report and behavioral lab tasks: is there overlap in nomothetic span and construct representation for impulsivity?[END_REF][START_REF] Stahl | Behavioral components of impulsivity[END_REF]. Explorative results found no association between anxiety and impulsive action selection nor response suppression. They also suggest a non-linear relationship between anxiety and online CE which might explain the mixed evidence for an association between cognitive inhibition and anxiety [START_REF] Beaudreau | The Association of Anxiety and Depressive Symptoms with Cognitive Performance in Community-Dwelling Older Adults[END_REF][START_REF] Delphin-Combe | Relationship between anxiety and cognitive performance in an elderly population with a cognitive complaint[END_REF][START_REF] König | Neural Processing of Cognitive Control in an Emotionally Neutral Context in Anxiety Patients[END_REF]. We observed that anxiety and premeditation as perseverance impulsivities were correlated in LLD, which are in turn associated with shortest CE on RT, consistent with the meta-analysis from Berg and colleagues (2015). However, these analyses have to be interpreted with caution as they are insignificant after p-value correction.

Limitations

To better consider our findings, several issues might be raised.

The magnitude of the reduced facilitation after conflict found in LLD suggests that it might be a cognitive marker of the LLD. It should be outlined however that while statistically significant, the group difference is marginal and questions the translational potential of this finding. This is likely to be due to the criteria selection in terms of cognitive impairment we used in order to homogenize the population sample. By selecting participants on an estimation of their ability to complete the ST via the MoCA, we may have excluded participants with poor executive functions, thereby reducing the effect size of CAC deficit among LLD. Yet, the ST has shown to be sensitive to modifications in CAC between groups of subjects with preserved executive functioning [START_REF] Wylie | The Effect of Parkinson's Disease on the Dynamics of On-line and Proactive Cognitive Control during Action Selection[END_REF]. We cannot rule out the possibility that some members of our sample may have an underlying neurodegenerative condition that is contributing to these low scores. However, a reduced MoCA scores has been found in adult depression without mild cognitive impairment [START_REF] Blair | Depressive Symptoms Negatively Impact Montreal Cognitive Assessment Performance: A Memory Clinic Experience[END_REF][START_REF] Dautzenberg | Clinical value of the Montreal Cognitive Assessment (MoCA) in patients suspected of cognitive impairment in old age psychiatry. Using the MoCA for triaging to a memory clinic[END_REF], a score of 20 being the lowest bound of MoCA scores in this population. Hence, we chose a lower threshold of 20 for inclusion and the absence of DSM-5 criteria of mild neurocognitive impairment. Most of LLD patients were under antidepressants and anxiolytic drugs. While being difficult to disentangle effects of drugs from effects of depression, tapering medication was not feasible for ethical reasons. Other parameters of conflict adaptation, as incentives or emotion, were not considered while important [START_REF] Shenhav | The expected value of control: an integrative theory of anterior cingulate cortex function[END_REF] notably in depression [START_REF] Grahek | Motivation and cognitive control in depression[END_REF]. Further studies evaluating them may shed light on complementary mechanisms involved in CAC in LLD.

CONCLUSION

Our study suggests that LLD subjects have a modified CAC, originating from increased impulsive errors during congruent trials, which suggest that LLD individuals have marginal difficulties to disengage their attention from conflict. Also, LLD was characterized by an increased selective suppression suggesting an over-recruitment of inhibition resources. Further research should investigate how motivation and emotion might influence these alterations.

-5 5 ± 1 3 -5 Naming 3 ± 0 2 -3 3 ± 0 3 -3 Attention 6 ± 1 3 -6 6 ± 0 4 -6 Language 2.5 ± 1 2 -3 3 ± 1 2 -3 Abstraction 1 ± 1 0 -2 2 ± 0 0 -2 Delayed Recall 3 ± 2 0 -5 5 ± 1 0 -5 Orientation 6 ± 1 4 -6 6 ± 0 5 -6

Stroop test

Interference score -1.

FIGURES Figure 1

In the Simon task, subjects are instructed to make a left-or right-hand response as fast and as accurately as possible according to the color of a stimulus that is presented either to the left or right of a fixation cross. A. In congruent trials both dimensions of the stimulus (location and color) point to the desired response, facilitating the response of the subject. B. In noncongruent trials, the automatic processing of the spatial location of the stimulus (plain arrow) conflicts with the response requested by the controlled processing of color (dotted arrow). The spatial location of the stimulus has to be ignored to produce a correct response, thus leading to increased RT and errors.

Figure 2

Mean RT and accuracy rates (% correct) for congruent (C) and non-congruent (NC) trials with respect to the congruency of the preceding trial (preceding trial congruent (PreC) and non-congruent (PreNC)) in each group. Situations in which the congruency of sequential trials changes are shaded in grey. Error bars represent standard error of the mean. *: Significant between-group differences between the last slopes.

Figure 3

 3 Figure 3Conditional accuracy functions for congruent and non-congruent trial LLD and HC. Error bars represent the standard error of the mean. *: Significant between-group differences.Figure 4Conditional Accuracy Functions in individuals with LLD and HC for congruent (C) and noncongruent (NC) trials preceded by congruent trials (PreC) or by non-congruent trials (PreNC). Accuracy rates in the context of shift in congruency are reported in grey-shaded cells. Error bars represent the standard error of the means. *: Significant between-group differences.Figure 5Delta-plot of reaction times of LLD and HC participants. Error bars represent the standard error of the mean. *: Significant between-group differences between the last slopes.

AUTHORS' CONTRIBUTIONS

CONFLICTS OF INTEREST None ACKNOWLEDGMENTS

The authors are grateful to the Institute of Clinical Neurosciences of Rennes which provided financial support to the study. We are thankful to all the volunteers who participated in this study.

TABLES