Guillaume Tintori

Arona Fall

Nadhrata Assani

Yuxi Zhao

David Bergé-Lefranc

Sébastien Redon

Patrice Vanelle
email: patrice.vanelle@univ-amu.fr]

Julie Broggi
email: julie.broggi@univ-amu.fr

Generation of powerful organic electron donors by water-assisted decarboxylation of benzimidazolium carboxylate

Organic Electron Donors (OEDs) are increasingly used as efficient reducing agents in various radical reactions. However, their sensitivity to air restraints their practical use and the development of further industrial applications. To meet the community's needs, this paper reports the preparation of air-and moisture-stable carboxylate precursors that can be conveniently activated into the corresponding organic reducer. The new strategy consists in the water-assisted decarboxylation of the adduct at room temperature, providing a straightforward protocol for the single-electron transfer reduction of aryl halides and sulfonamide. The resulting aryl radicals are engaged in the first examples of OED-promoted intermolecular addition reactions. A thorough mechanistic study supports the in-situ formation of the organic reducer.

Results and discussion

14,15 the adducts were prepared in a two-step procedure involving first

Introduction

Reduction reactions promoted by electron transfer are now recognized as a powerful group-tolerant strategy for new elaborate reactions and rapid increase of molecular complexity. [START_REF]diazepine-2,12-diamine[END_REF] Long limited to toxic and highly aggressive metalbased reducing agents, reduction reactions are enjoying renewed interest since the development of metal 2 or organic readily-prepared enamine-based Organic Electron Donors (OEDs) [START_REF] For Reviews | Encyclopedia of Radicals in Chemistry, Biology and Materials[END_REF] are also attracting attention as a serious alternative as shown by the growing number of useful applications (initiators for coupling reactions or polymerizations, redox switches and flow batteries, greenhouse gas reduction).

6 They spontaneously promote single-(SET) or double electrontransfers (DET) to substrates that are thermodynamically too uphill for current SET-photoredox catalysts. At ground state, they generate radical or anionic intermediates from substrates with redox potential E 1/2 < -1.0 V. 7 UV-photoexcitation (365 nm) of OEDs further enhances their reducing power to substrates with E 1/2 up to -3.5 V. 8 Depending on their structures, the enamines are stable and can be stored and manipulated under inert atmosphere. [START_REF] For Reviews | Encyclopedia of Radicals in Chemistry, Biology and Materials[END_REF]9 In other cases, the dimeric form is elusive and decomposes after a few minutes or hours into the corresponding N-heterocyclic carbene (NHC) (Scheme 1). 9a,10 In addition to these short half-life issues, OEDs like any other strong reducing agents, are highly sensitive to oxygen.

11

To overcome these constraints, processes consisting of the activation of air-stable precursors with strong bases [START_REF] Jolly | Imidazole-derived carbenes and their elusive tetraazafulvalene dimers[END_REF]12 or under high working temperatures [START_REF] Tintori | Base-Free Generation of Organic Electron Donors from Air-Stable Precursors[END_REF] have been used to in situ generate the reducer. However, strong bases present limited group tolerance and thermal activation (150°C) impedes the use of volatile substrates or solvents. To significantly improve the practicality of OEDs, there is a need for air-stable precursors allowing the convenient in situ generation of enamine reducers under mild conditions.

Scheme 1. OED formation through air-stable precursors.

This manuscript illustrates the synthesis and the activation of carboxylate and sulfinate iminium adducts into the corresponding organic reducer. The new strategy consists in the water-assisted decarboxylation (or desulfination) of the adduct at room temperature (Scheme 1). A thorough mechanistic study supports the in-situ formation of the enamine-type reducer. Their scope will also be discussed, notably for OED-promoted intermolecular radical addition reactions.

Please do not adjust margins

Please do not adjust margins deprotonation of the iminium salt, followed by trapping of the generated species with carbon dioxide or sulfur dioxide atmosphere (Scheme 2). All the precursors were obtained in moderate to good yields (51-98%).

Scheme 2. Synthesis of carboxylate and sulfinate adducts.

While expecting easier cleavage of the SO 2 moiety than the CO 2 , 16 thermogravimetric analysis (TGA) at the solid states of these adducts surprisingly indicated that the sulfinate adducts would decompose at higher temperatures than their carboxylate counterparts (IMe- 1). However, this physical feature directly contrasted with their instability in air (leading to the formation of hydrogen sulfite salt IMe-R'.HHSO 3 ) and with their higher reactivity in solution under thermal activation (See supporting information (S.I.), Scheme S1 and Table S2).

n Bu-SO 2 , 190°C vs. IMe- n Bu- CO 2 , 141°C) (Table

17

In NHC-CO 2 series, the presence of bulkier N-groups around the carboxylate moiety facilitates the decarboxylation (Table 1, IMe-CO 2 140°C vs. I t Bu-CO 2 63°C). 18,19c At 150°C, these carboxylate adducts can be decarboxylated to generate an organic reducing species. [START_REF] Tintori | Base-Free Generation of Organic Electron Donors from Air-Stable Precursors[END_REF] Nevertheless, the steric hindrance induce by the tert-butyl groups may also impede the efficient formation of the active dimeric OED-form. To balance ease of decarboxylation and dimerization, we prepared IMen Bu-CO 2 bearing a longer N-butyl chain. However, the decarboxylation temperature of IMe-n Bu-CO 2 was not affected by this slight increase in hindrance, displaying a similar value (141°C) to that of IMe-CO 2 . Encouragingly, the 1,3dimethylbenzimidazolium adduct BzIMe-CO 2 showed a low decarboxylation temperature (52°C) and a relative stability of the generated species until its decomposition at 119°C. Despite a first decarboxylation at low temperature (50°C), the loss of the second molecule of CO 2 of the N,N'-bridged bisbenzimidazolium carboxylate adduct Bis-BzIMe-CO 2 was not clearly observed before its decomposition at 219°C (S.I., Figure S2).

The lower IR frequency of the carbonyl asymmetric stretching (1632 cm -1

) of BzIMe-CO 2 was also in line with the low frequencies usually observed for adducts more prone to decarboxylation, such as I t Bu-CO 2 (1609 cm -1 ) (Table 1). , respectively). UV-Vis spectroscopy indicated similar maximum absorption wavelengths (269-277 nm) for all imidazolium and benzimidazolium adducts (Table 1). We then evaluated the stability of our carboxylate and sulfinate adducts towards water. Indeed, Louie and Falvey showed that the decarboxylation can be facilitated by the presence of water in an organic solvent. 18,19 While stable in pure water or stored at air, N-alkyl NHC-CO 2 adducts were rapidly decarboxylated when dissolved in an water/organic solvent mixture, yielding the corresponding carbonate salt NHC.HHCO 3 upon fast protonation of the carbene intermediate (Scheme 3). 18,19 Fast decarboxylation in a 5%

H 2 O/dioxane mixture and formation of corresponding carbonate salts were indeed observed by 1 H NMR for our imidazolium and benzimidazolium NHC-CO 2 series. Sulfinate adducts NHC-SO 2 also quickly decomposed, since exposure to air moisture was enough to induce the SO 2 cleavage, leading to the corresponding hydrogen sulfite salts (S.I., Scheme S1). Like with N-aryl NHC-CO 2 , 18 water was only hydrogen-bonded to the CO 2 moiety of the aminopyridinium carboxylate adduct DMAP-Me-CO 2 , and did not trigger its decarboxylation (Scheme 3).

13 Scheme 3. Water activation of carboxylate adducts.

The same decarboxylation strategy was then performed in the presence of the substrate to reduce (1-iodo-2-(3phenoxypropylbenzene) 1a) (Table 2). 4 equivalents of precursors were used to in situ generate 2 equivalents of the dimeric OED form. While activation of IMe-CO 2 required a high This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins temperature of 150°C under previous strategy (Entries 1-2), 13 addition of 5% of degassed water (35 equiv.) led to its decarboxylation at room temperature and to the partial reduction of 1a (60%) under UV-irradiations (Entry 5).

20

As predicted above, the introduction of a longer N-alkyl chain did not ease the activation of the precursor. Compared to IMe-CO 2 , water activation of IMe-n Bu-CO 2 led to the same reduction of 1a (Entry 6, 60%). On the other hand, the higher steric hindrance of I t Bu-CO 2 facilitated the decarboxylation, providing a slight higher reduction rate (Entry 7, 69%). 21 As expected from the weakness of the longer C-SO 2 bond, the sulfinate adducts IMe-Et-SO 2 and IMe-n Bu-SO 2 performed better than their carboxylate counterparts under thermal activation (S.I., Table S2, entries 10-11, 99 and 69% respectively at 120°C versus 6% for IMe-CO 2 ) (underlying the easier loss of the SO 2 moiety). Nevertheless, the low reducing reactivity observed upon water activation (Table 2, Entries 8-9, 16 and 14%) and their poor stability at air storage clearly disqualified them as convenient OED-precursors.

As anticipated from our stability study, hydrogen-bonding of water to the aminopyridinium carboxylate DMAP-Me-CO 2 was not sufficient to induce an efficient decarboxylation, leading only to traces of reduction product (Entry 10, 6%). In line with the TGA and IR conclusions, the benzimidazolium precursor BzIMe-CO 2 offered the best reactivity, with 80% reduction at room temperature (Entry 11). The N,N'trimethylene-bridged derivative Bis-BzIMe-CO 2 might have induced faster formation of the dimeric reducing agent, N,N'bridged dibenzimidazolin-2-ylidene DiBzIMe. However, its lower reactivity (Entry 12, 27%) was consistent with the sluggish second decarboxylation process predicted by TGA.

Evaluation of the influence of water showed that a decrease or an increase over the range of 1.5-10% H 2 O slightly decreased the reduction rate (Entries 11, 14-16). As expected, absence of water was deleterious for the formation of the active reducing species (Entry 13, 61%). These fluctuations highlight the impact of the first activation step on the reaction outcome. Efficient decarboxylation of the precursor required 5% concentration of water in DMF. This 5% H 2 O concentration also provided fast decomposition rates of IMe-CO 2 in Falvey's study. 19 Likewise, a trend towards faster decomposition was observed with decreasing solvent polarity (Entries 11, 17-19). Non-polar solvents decrease the dipole moment, destabilizing the zwitterionic adduct and easing the release of CO 2 . 19 The best reduction reactivities were observed with less polar THF and 1,4-dioxane (Entries 18-19, 90 and 99%) compared to polar DMF and CH 3 CN (Entries 11 and 17, 80 and 68%). This decrease in solvent polarity did not seem to be detrimental to the second step of electron-transfer. In these optimized 5% H 2 O/1,4-dioxane conditions, the precursor BzIMe-CO 2 was thus the best candidate, giving a high yield of the reduced product 2a (Entry 19, 90%). The OED-precursor BzIMe-CO 2 was still active after several months of storage in air.

Several experiments were then performed to elucidate the mechanism behind this reduction process and confirm the formation of dimeric OEDs upon water activation of the carboxylate adduct (Table 3). First, evidence supporting an electron transfer mechanism was obtained through the addition of commonly used inhibitors. The reduction of 1a was drastically diminished in the presence of inhibitors such as the radical trap TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) (40%), oxygen (44%) or the competitive electron acceptor p-DNB (para-dinitrobenzene) (0%) (Entries 2-4). To gain insights into the active reducing species at work in this process, we first compared the reduction of 1a, directly using the active reducer di-1,3-dimethylbenzimidazolin-2ylidene DiBzIMe ( E 1/2 (DMF) = -0.88 V vs. SCE) or the precursor form BzIMe-CO 2 . With both electron sources, reduction of iodoarene 1a required UV-irradiations to proceed (Table 3, Entries 1,7 vs. 5,8).

22

This is not surprising, as dibenzimidazolinylidene-type OEDs, such as DiBzIMe, are SETreducers (E 1/2 ≈ -0.8 V vs. SCE) only able to reduce aryl iodide under high temperatures (>100°C) or UV-photoexcitation (λ = 365 nm).

5,12a

Although the contribution of water was unquestionable (Table 3, Entry 1 vs. 6), note that UV-irradiations alone could also participate to the decarboxylation step of NHC-CO 2 precursors (Table 2, Entry 3 andTable 3, Entry 6). Compared to thermal activation, 1a was reduced at drastically lower temperatures (90°C with IMe-CO 2 , 30°C with BzIMe-CO 2 ) albeit at lower conversion rates (76 and 64 %, respectively). Our UV-LED light source emits at a wavelength (λ = 365 ± 5 nm) out of the range of wavelengths absorbed by NHC-CO 2 (224-330 nm, Table 1). Hence, rather than a photoexcitation of the Please do not adjust margins Please do not adjust margins carboxylate adducts, we attribute this activation of IMe-CO 2 and BzIMe-CO 2 to localized temperature rises induced by UV radiations, assisting the elimination of CO 2 and drastically decreasing the need for external heating. The slight increase of media temperature (+5°C at r.t) under UV-irradiations plus the polarity effect of the dioxane, that destabilizes the zwitterionic adduct, might be sufficient to slowly activate the decarboxylation of BzIMe-CO 2 (T-CO2 = 52°C by TGA of the solid state, Table 1). We also compared the benzimidazolium derivatives present at the end of reduction reactions (Table 4). According to the experiment, three derivatives could be retrieved: the 1,3-dimethylbenzimidazolium salt BzIMe.HX (3), the 1,3dimethylbenzimidazolin-2-one BzIMeO (4) or the tetramethylbibenzimidazolium salt DiBzIMe 2+ (5). BzIMeO arises from the autoxidation of DiBzIMe (Entry 4) 23 and DiBzIMe 2+ is the characteristic DET-oxidized form of DiBzIMe (Entry 5). It could be argue that under UV-irradiations the carbene 1,3-dimethylbenzimidazolin-2-ylidene (BzIMe) itself could promote the single electron-transfer reduction of iodoarene 1a. Benzimidazolium salt 3 would also be the resulting oxidized species (Scheme 4). N-heterocyclic carbenes are theoretically able to donate an electron, even though they have very low reducing powers (e.g. for the 1,3-bis(2,4,6trimethylphenyl)imidazolin-2-ylidene (IMes): E 1/2 (DMF) = -0.20 V vs. SCE). [START_REF] Gorodetsky | Electrochemical reduction of an imidazolium cation: a convenient preparation of imidazol-2-ylidenes and their observation in an ionic Liquid[END_REF] In the case of the I t Bu-CO 2 precursor, the I t Bu carbene could indeed be the electron donor species considering its sterically disfavoured dimerization (Table 2, Entry 7). 21 Nonetheless, with BzIMe-CO 2 as precursor, the halflife of the BzIMe carbene ought to be very short. The inclination of N,N'-dimethylbenzimidazolium salts to rapidly and tightly dimerize upon deprotonation is well reported. NMR at 70°C in C 6 D 6 detected the formation of DiBzIMe but no trace of carbene BzIMe, even after several days of heating (Scheme 5). Moreover, when the reaction was performed with one equivalent of carboxylate BzIMe-CO 2 , only 63% reduction was observed, consistent with in situ generation of 0.5 equivalent of the reducing dimeric form DiBzIMe (the remaining 13% accounted for direct homolytic cleavage of 1a under UV-irradiations, Table 3, Entry 11). This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins Scheme 4. Candidate pathways using benzimidazolium carboxylate for the ET-reduction of iodoarenes.

The contribution of the carboxylate form as active donor species was also considered. Direct photoexcited SET from BzIMe-CO 2 to 1a would generate the corresponding aryl radical and the carboxyl radical species BzIMe-CO 2

• (Scheme 4).

BzIMe-CO 2

• would decompose into salt 3 upon decarboxylation and HAT. In fact, carboxylate derivatives are known as reducers able to promote photoinduced electron transfer to very strong acceptors (Ir* and Ru* complexes or organic dyes).

29 An electron-rich amino group near the carboxylate moiety is necessary to assist the electron transfer and the decarboxylation process. However, in our case, the electron-poor iminium moiety of BzIMe-CO 2 should suffice to strongly disfavour any electron transfer from the carboxylate group to a poor acceptor like an iodoarene. The lack of reactivity of the aminopyridinium carboxylate DMAP-Me-CO 2 (Table 3, Entry 10), presenting no sign of decarboxylation under these conditions, also makes it reasonable to conclude that the carboxylate form is not the active reducing species.

All these arguments suggest that the in situ generation of DiBzIMe proceeds according to the following mechanism (Scheme 5): Water-assisted decarboxylation of BzIMe-CO 2 leads to the formation of the 1,3-dimethylbenzimidazolin-2ylidene BzIMe, which is protonated into benzimidazolium salt 3. Falvey showed that these two steps were stepwise and not concerted. To further investigate the scope of this water-activated OED-precursor, we examined the reduction of various aryl substrates, and their subsequent intramolecular and intermolecular radical addition on alkenes. Water activation of BzIMe-CO 2 and UV-excitation of the as-formed DiBzIMe promoted the SET-reduction of various iodide and sulfonamide arenes 1a-j in moderate to excellent yields (Scheme 6). Exclusive formation of reduced products 2b and 2c (i.e. with no trace of the cyclised derivatives obtained by DET, S.I., Table S3) was in line with the SET capacities of DiBzIMe. 12a The reducing system was tolerant to different functional groups including ester (2b, 2c and 2d), ketone (2e) or cyano groups (2f), characteristic of the chemo-selectivity of OEDs. Bromoarenes with more negative reduction potentials were also partially reduced (2a, Y = Br and 2f). These highly challenging reduction reactions prove that the potency of DiBzIMe was maintained after in situ generation from BzIMe-CO 2 .

ARTICLE

Please do not adjust margins Please do not adjust margins DMF was sometimes used when the reduction was slow to enhance the electron transfer. The moderate yields obtained for 2g and 2i can be attributed to a partial loss of these volatile reduced products during the work-up. Unfortunately, as previously observed with OED, presence of an alcohol group in 2j prevented the reduction reaction.

Scheme 6. Scope of reducible and tolerated functional groups. This journal is © The Royal Society of Chemistry 20xx

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The aryl radicals obtained through reduction of the Csp 2 -I bond were further engaged in intramolecular additions on alkenes via a 5-exo-trig cyclisation reaction, leading to the 2,3dihydrobenzofuran products 7 and 10 (Scheme 7). Formation of 8 arose from a recombination between the cyclized radical and traces of iodide radical generated upon UV-induced homolytic cleavage of the C-I bonds in 6 (Table 3, Entry 11, blank reaction). The cyclized radical obtained from 9 could either abstract a hydrogen to give 10 or disproportionate to give 11. [START_REF] Curran | The Samarium Grignard Reaction. In Situ Formation and Reactions of Primary and Secondary Alkylsamarium(III) Reagents[END_REF] Starting materials 6 and 9 accounted for the mass balance.

Scheme 7. Intramolecular aryl radical additions on alkenes.

Our strategy of in situ generation of a powerful OED from an air-stable precursor also affords the first example of OEDpromoted intermolecular additions of aryl radicals to alkenes (Scheme 8). The aryl radical resulting from the complete reduction of 12 was added to the external position of the diphenylethylene 13 to obtain the 1,2-diphenylethylbenzene derivative 14 in 36% yield. The moderate yield probably accounted from the competitive formation of volatile chlorobenzene (15) upon HAT to the aryl radical. The αsubstituted pyrrole derivative 17 was obtained in excellent yield through intermolecular addition of the aryl radical of 12 on the pyrrole 16. Rearomatization of the pyrrole moiety of 17 probably occurs through radical coupling of the alkyl radical with the radical cation DiBzIMe

•+ , followed by elimination of the resulting benzimidazolium leaving group.

32 Scheme 8. Intermolecular aryl radical additions on alkenes.

Conclusions

In conclusion, we demonstrated that water can enhance the decarboxylation of air-stable imidazolium and benzimidazolium carboxylate adducts and thus allow the in situ generation of strong organic electron donors at room temperature. Photo-excited di-1,3-dimethylbenzimidazolin-2ylidene DiBzIMe then promotes the efficient single-electron transfer reduction of sulfonamide and aryl halides. The resulting aryl radicals are then engaged in intramolecular and intermolecular additions to alkenes. A thorough mechanistic study supports the activation pathway and the in-situ formation of the organic electron donor. The multiple strategies that we developed for the activation of Nheterocyclic carboxylate adducts (thermal-, water-or UVactivation) ensure their practicality and should foster their use as air-stable precursors of powerful organic electron donors in radical chemistry.

28

  Lemal briefly observed trace of BzIMe through dissociation of DiBzIMe at high temperature (140°C), but the signal rapidly disappeared upon cooling.28a This demonstrates the high stability of DiBzIMe and the reversibility of the process. In our case, both thermal decarboxylation of BzIMeCO 2 (S.I., Scheme S5) and deprotonation of BzIMe.HI salt (S.I., Scheme S2) allowed fast formation of the dimer species DiBzIMe.Monitoring of the decarboxylation of BzIMe-

  19 A second molecule of BzIMe is added to salt 3 to form the cation intermediate DiBzIMeH + . Rapid deprotonation of DiBzIMeH + with BzIMe leads to the formation of the active reducer DiBzIMe and regenerates BzIMe.HHCO 3 (3). This acidcatalysed dimerization of BzIMe, where 3 is the acid, allows a complete shift of the Wanzlick enamine/carbene equilibrium toward the dimer form DiBzIMe. 30 Small amounts of DiBzIMe could also come from the thermal decarboxylation of BzIMe-CO 2 under the UV-irradiation conditions. 20 Photo-excited DiBzIMe then transfers a single electron to the iodoarene ArI, leading to the corresponding aryl radical Ar • and the radical cation DiBzIMe •+ . The aryl radical Ar • abstracts a hydrogen from the 1,4-dioxane to generate the final reduced compound. Disproportionation or HAT of the radical cation leads to the formation of the unstable cation DiBzIMeH + which decomposes into salt 3 and carbene BzIMe. The generated carbene can in turn form 3 after protonation.

  Scheme5.Proposed mechanism.

adducts IMe-CO 2 and IMe- n Bu-CO 2 displayed

	13,18
	More thermally stable imidazolium higher values (1645 and 1657 cm	-1

Table 1 . Physical features of NHC-CO2 and NHC-SO2.

 1

	Precursor	T-CO2 or -SO2 [°C] a	COasym [cm -1 ] a	λmax [nm]
	IMe-n Bu-SO2	190	-	274
	DMAP-Me-CO2	181	1624	288
	IMe-Et-SO2	160	-	274
	IMe-n Bu-CO2	141	1657	274
	IMe-CO2	140	1645	273
	I t Bu-CO2	63, 115	1609	269
	BzIMe-CO2	52, 119	1632	270, 277
	Bis-BzIMe-CO2	50, 219	1669	272, 277
	a Data excerpted from Ref. 13 and 18 for

DMAP-Me-CO2, IMe-CO2, and I t Bu-CO2.

Table 2 .

 2 Optimization of the reduction process.

	Entry	Precursor	Temp[°C]	Solvent	%H2O a Conv(%) b
	1 c	IMe-CO2	150 d	DMF	-	>99
	2 c	IMe-CO2	120 d	DMF	-	6
	3	IMe-CO2	90	DMF	-	76
	4	IMe-CO2	90	DMF	5	>99
	5	IMe-CO2	rt	DMF	5	60
	6	IMe-n Bu-CO2	rt	DMF	5	60
	7	I t Bu-CO2	rt	DMF	5	69
	8	IMe-Et-SO2	rt	DMF	5	16
	9	IMe-n Bu-SO2	rt	DMF	5	14
	10	DMAP-Me-CO2	rt	DMF	5	6
	11	BzIMe-CO2	rt	DMF	5	80
	12	Bis-BzIMe-CO2 e	rt	DMF	5	27
	13	BzIMe-CO2	rt	DMF	-	61
	14	BzIMe-CO2	rt	DMF	1.5	77
	15	BzIMe-CO2	rt	DMF	7.5	77
	16	BzIMe-CO2	rt	DMF	10	75
	17	BzIMe-CO2	rt	CH3CN	5	68
	18	BzIMe-CO2	rt	THF	5	90
	19	BzIMe-CO2	rt	1,4-dioxane	5	99(90) f

a Degassed water. b Conversion determined by 1 H NMR analysis. c Data excerpted from Ref. 13. d Without hν. e 2 equiv. f Yield.

Table 3 .

 3 Mechanistic study.

	Entry	Reducer source	Changes to the standard conditions	Conv (%) a
	1	BzIMe-CO2	-	99
	2	BzIMe-CO2	+ TEMPO (4 equiv.)	40
	3	BzIMe-CO2	+ p-DNB (4 equiv.)	0
	4	BzIMe-CO2	+ O2 (1 atm)	44
	5	BzIMe-CO2	hν	0
	6	BzIMe-CO2	no H2O	64
	7	DiBzIMe b	-	>99
	8	DiBzIMe b	no hν	0
	9	BzIMeO (4)	-	0
	10	DMAP-Me-CO2	no H2O	12
	11	-	-	10

a Conversion determined by 1 H NMR analysis. b 2 equiv.

Table 4 .

 4 Final benzimidazolium derivatives present in the organic and aqueous phases.

	Entry	Reducer source	Oxidant	Changes to the standard conditions	Benzimidazolium derivatives a 3 4 5
	1	BzIMe-CO2	C2Cl6	-	✓	✓	x
	2	BzIMe-CO2	1a	+ hν	✓	✓	x
	3	DiBzIMe	1a	+ hν	✓	✓	x
	4	DiBzIMe	O2	no H2O	x	✓	x
	5	DiBzIMe	C2Cl6	no H2O	x	x	✓
	6	DiBzIMe	C2Cl6	no H2O, + hν	✓	x	✓

a Observed by 1 H NMR. hν (1 LED, 365 nm, 5 cm).

photocatalysts. Although used in stoichiometric amounts,

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Acknowledgements

The Agence Nationale de la Recherche is gratefully acknowledged for the JCJC ANR grant "iPOD" (17-CE07-000101). Aix Marseille Université (AMU) and the Centre National de la Recherche Scientifique (CNRS) are also acknowledged for financial support. GT thanks the Ministère de l'Enseignement supérieur et de la Recherche for his Ph.D. grant. YZ thanks the China Scholarship Council for his Ph.D. grant. We thank Dr. Vincent Remusat for NMR analyses and the Spectropole (Fédération des Sciences Chimiques de Marseille) for spectroscopic analyses.

Conflicts of interest

There are no conflicts to declare.

ARTICLE

Journal Name 8 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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Antsa Rakotozafiarison

Déborah Puyraimond-Zemmour
email: deborah.puyraimondzemmour@aphp.fr

Xavier Romand
email: xromand@chu-grenoble.fr

Matthieu Lavielle
email: matthieu.lavielle@wanadoo.fr

René-Marc Flipo

Christophe Richez
email: christophe.richez@chu-bordeaux.fr

Alain Saraux
email: alain.saraux@chu-brest.fr

Maxime Dougados
email: maxime.dougados@u-paris.fr

Laure Gossec
email: laure.gossec@aphp.fr

Anna Molto
email: anna.molto@aphp.fr

Rene-Marc Flipo
email: rene-marc.flipo@chru-lille.fr

Dr Rakotozafiarison Antsa

The adherence questionnaires in chronic inflammatory rheumatic diseases and their psychometric properties: a systematic literature review

Keywords: patient adherence, compliance, questionnaires, rheumatoid arthritis, spondyloarthritis, inflammatory arthritis

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

The adherence questionnaires in chronic inflammatory rheumatic diseases and their psychometric properties: a systematic literature review Authors: Antsa Rakotozafiarison 1* , Déborah Puyraimond-Zemmour 1,2* , Xavier Romand 3,4 , Matthieu Lavielle 5 , Anna Moltó 6,7 , Rene-Marc Flipo 8 , Christophe Richez 9,10 , Alain Saraux 11,12 , Maxime Dougados 6,7 , Laure Gossec 1,2 *The first 2 authors contributed equally. In chronic inflammatory rheumatic diseases (CIRDs) including rheumatoid arthritis (RA), spondyloarthritis (SpA), psoriatic arthritis (PsA), crystal-induced arthritis and connective tissue diseases (CTD), adherence to disease-modifying drugs is only moderate over the long term [START_REF] Van Den Bemt | Medication adherence in patients with rheumatoid arthritis: a critical appraisal of the existing literature[END_REF][START_REF] Beauvais | One-third of patients are low-adherent to methotrexate for rheumatoid arthritis at the initiation of a first biologic. A cross-sectional study investigating adherence rate and factors[END_REF]. Insufficient patient adherence to treatments may lead to complications, unnecessary treatment switches, and increased costs [START_REF] Pasma | Does non-adherence to DMARDs influence hospital-related healthcare costs for early arthritis in the first year of treatment?[END_REF]. Therefore, patient adherence to treatment should be assessed, however the optimal assessment method has not been determined [START_REF] Gossec | Recommendations for the assessment and optimization of adherence to disease-modifying drugs in chronic inflammatory rheumatic diseases: A process based on literature reviews and expert consensus[END_REF].

The objective of this systematic literature review was to assess the psychometric properties of the main drug adherence questionnaires in patients with CIRDs.

In January 2019, a systematic literature review was performed in three databases (Pubmed, Cochrane central register of clinical trials, Embase) and several websites (Clinicaltrials.gov, World Health Organisation and the French High Authority of Health) as well as congress abstracts from the American and European congresses of the previous 3 years. The scope was limited to questionnaires published in English to assess adherence to drugs such as disease-modifying drugs, in CIRDs, i.e., RA, SpA, PsA, CTD, crystal-induced arthritis, vasculitis, and auto-inflammatory diseases [START_REF] Gossec | Recommendations for the assessment and optimization of adherence to disease-modifying drugs in chronic inflammatory rheumatic diseases: A process based on literature reviews and expert consensus[END_REF][START_REF] Lavielle | Methods to improve medication adherence in patients with chronic inflammatory rheumatic diseases: a systematic literature review[END_REF]. All questionnaires used to assess adherence were collected, then a specific search using the questionnaire name was carried out to obtain data on their psychometric properties according to the OMERACT filter [START_REF] Boers | Developing core outcome measurement sets for clinical trials: OMERACT filter 2.0[END_REF]. We assessed overall validity, sensitivity (Se), specificity (Sp), internal consistency by Cronbach coefficient, reliability, and sensitivity to change. To summarise the data, and because a metaanalysis was not possible, properties are presented semi-quantitatively [START_REF] Boers | Developing core outcome measurement sets for clinical trials: OMERACT filter 2.0[END_REF].

Of 1209 publications and 194 other documents, 242 papers reported adherence. Of these, 131 articles used questionnaires: 69/154 (44.8%) in RA, 27/40 (67.5%) in systemic lupus erythematosus (SLE), 14/21 (66.6%) in SpA, 9/16 (56.2%) in PsA, 8/35 (22.8%) in crystal-induced arthritis and 4/6 (66.6%) in other CTD. Four questionnaires were used to evaluate drug adherence [START_REF] Klerk | The compliance-questionnaire-rheumatology compared with electronic medication event monitoring: a validation study[END_REF][START_REF] Thompson | Reliability and validity of a new Medication Adherence Rating Scale (MARS) for the psychoses[END_REF][9][10] (Table 1). The most used questionnaire was the MMAS (Morisky Medication Adherence Scale). The Compliance Questionnaire of Rheumatology (CQR) was validated in 85 patients with CIRD against as external standard with an electronic medication monitoring; the MASRI (Medication Adherence Self-report Inventory) was validated in 55 patients with SLE against adherence based on pharmacy refill information.

The MMAS was validated in 91 patients with gout against medication possession ratio and the MARS (Medication Adherence Report Scale) in 108 patients with RA. Reproducibility was correct; some questionnaires are subject to copyright (Table 2).

Our results indicate that four questionnaires are being used to measure medication non-adherence in CIRDs; the most used is the MMAS which is unfortunately copyrighted and not fully validated in rheumatology. The CQR and MASRI questionnaires were the mostextensively validated in rheumatology, but the CQR is long and the MASRI only used for SLE. Thus it appears that to date, a simple, reliable and valid questionnaire to assess drug adherence is lacking.

[9] Walsh JC, Mandalia S, Gazzard B G. Responses to a 1 month self-report on adherence to antiretroviral therapy are consistent with electronic data and virological treatment outcome. AIDS. 2002 Jan 25;16(2):269-77.

[10] Morisky DE, Ang A, Krousel-Wood M, Ward HJ. Predictive validity of a medication adherence measure in an outpatient setting. J Clin Hypertens (Greenwich). 2008;10(5):348-54.

Affiliations 1 :

 1 Service de Rhumatologie, Hôpital de la Pitié Salpetrière, 48-83 Bd de l'Hôpital 75013 Paris, France 2: Sorbonne Université, INSERM, Institut Pierre Louis d'Epidémiologie et de Santé Publique, 47-83 Bd Hopital, 75013 Paris, France 3: Univ. Grenoble Alpes, GREPI TIMC-IMAG, UMR 5525, 38000 Grenoble, France 4: Service de Rhumatologie, CHU Grenoble Alpes, 38000 Grenoble, France 5: Praticien privé, 419 Route du Médoc, 33520 Bruges, France 6: Service de Rhumatologie, Cochin Hospital, APHP, 75014 Paris, France 7: Université de Paris Inserm Unit 1183 (CRESS), 75014 Paris, France 8: Université de Lille, 42 Rue Paul Diez 59000 Lille, France 9: Service de Rhumatologie, Hôpital Pellegrin, 33 000 Bordeaux, France 10: Université de Bordeaux, CNRS 5164, 33000 Bordeaux, France 11: Service de Rhumatologie, CHU de la Cavale-Blanche, boulevard Tanguy-Prigent, 29200 Brest, France 12: INSERM 1227, Université de Bretagne Occidentale, 29200 Brest, France Dear Sir,

Table 1 .

 1 Questionnaires used to evaluate drug adherence in recently published studies

			RA	SpA	Lupus	PsA	Cristal induced arthritis	Vasculitis and auto-inflammatory diseases
	Total number of studies	154	21	40	16	35	6
	Number	of
	articles using adherence	69 (44.8%) 14 (66.6%) 27 (67.5%) 9 (56.2%) 8 (22.8%)	4 (66,6%)
	questionnaires
	Number questionnaires of	3	3	3	3	3	2
	Most questionnaire used	CQR	MMAS	MMAS	MMAS	MMAS	MMAS
	Other questionnaires used	MMAS, MARS	CQR, MARS	CQR, MASRI	CQR, MARS	MARS, CQR	CQR
	MMAS: Morisky Medication Adherence Scale; MASRI: Medication Adherence Self-Report
	Inventory; MARS: Medication Adherence Report Scale; CQR: Compliance Questionnaire of
	Rheumatology

Table 2 .

 2 Questionnaires performances to assess adherence in chronic inflammatory rheumatic

This study was conducted thanks to an unrestricted grant from Abbvie France thank Margaux Orange for logistic assistance during the

Disclosures of interest

Maxime DOUGADOS has received honoraria from Abbvie for his participation as the convenor of this initiative. Xavier ROMAND, Déborah PUYRAIMOND-ZEMMOUR and Matthieu LAVIELLE have received honorarium fees from

scientific discussions. AbbVie did not review the content or have influence on this letter. The authors meetings and the 104 experts for input during the meeting.


Quentin Carboué
email: quentin.carboue.qc@gmail.com

Marc Maresca
email: m.maresca@univ-amu.fr

Gaëtan Herbette
email: gaetan.herbette@univ-amu.fr

Sevastianos Roussos
email: sevastianos.roussos@imbe.fr

Rayhane Hamrouni
email: rayhan.hamrouni@gmail.com

Isabelle Bombarda
email: isabelle.bombarda@univ-amu.fr

Naphtho-Gamma-Pyrones Produced by Aspergillus tubingensis G131: New Source of Natural Nontoxic Antioxidants

Keywords: naphtho-gamma-pyrones, radical scavenging activity, hydrogen peroxide, mediated cell death assay, solid state fermentation, Aspergillus tubingensis

Introduction

Naphtho-gamma-pyrones (NγPs) are polyketide pigments that have been found to occur in higher plants belonging to the genera Cassia. They are also widely produced by fungi and, notably, Aspergillus spp. [START_REF] Nielsen | Review of secondary metabolites and mycotoxins from the Aspergillus niger group[END_REF][START_REF] Zhan | Asperpyrone D and other metabolites of the plant-associated fungal strain Aspergillus tubingensis[END_REF]. Their structures consist of a naphthalene and a γ-pyrone moiety, and there are monomeric and dimeric forms for which a diaryl bond links two NγPs together [START_REF] Lu | Bis-naphtho-γ-pyrones from Fungi and Their Bioactivities[END_REF]. The NγPs' dimerization is carried out by cytochrome P450 enzymes, which stereoselectivity depend on monomeric substrates [START_REF] Obermaier | Biaryl-Forming Enzymes from Aspergilli Exhibit Substrate-Dependent Stereoselectivity[END_REF]. This family exhibits a broad spectrum of biological activities, including antimicrobial, antiviral, insecticidal, anti-estrogenic, and antioxidant activity [START_REF] Xu | Bioactive natural products derived from mangrove-associated microbes[END_REF][START_REF] Choque | Fungal naphtho-γ-pyrones-secondary metabolites of industrial interest[END_REF][START_REF] Ma | A new pyrone derivative from an endophytic Aspergillus tubingensis of Lycium ruthenicum[END_REF]. Antioxidants are compounds that can delay, inhibit, or prevent the oxidation of oxidizable matters by scavenging free radicals and diminishing oxidative stress. Such stress corresponds to an imbalanced state, where excessive quantities of reactive oxygen species (e.g., superoxide radical anion, hydrogen peroxide, and hydroxyl radical) are present at levels more than what is required for normal cell function and overwhelm endogenous antioxidant capacity and repair [START_REF] Kim | Comprehensive Study on Vitamin C Equivalent Antioxidant Capacity (VCEAC) of Various Polyphenolics in Scavenging a Free Radical and its Structural Relationship[END_REF]. Nowadays, there is increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body, and to prevent the deterioration of fats and other constituents of foodstuffs. In both cases, there is a preference for antioxidants from natural rather than from synthetic sources [START_REF] Molyneux | The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity[END_REF]. Few studies deal with the in vitro antioxidant activity (AOA) based on the trapping of the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) and NγPs. Among the NγPs tested, some were reported to have or not antioxidant activity depending of the studies [START_REF] Cai | a new dimeric naphtho-γ-pyrone from the edible fungus Pleurotus ostreatus[END_REF][START_REF] Leutou | Induced production of 6,9-dibromoflavasperone, a new radical scavenging naphthopyranone in the marine-mudflat-derived fungus Aspergillus niger[END_REF][START_REF] Siriwardane | Chemical investigation of metabolites produced by an endophytic Aspergillus sp. isolated from Limonia acidissima[END_REF][START_REF] Zhang | Nigerasperones A~C, New Monomeric and Dimeric Naphtho-γ-pyrones from a Marine Alga-derived Endophytic Fungus Aspergillus niger EN-13[END_REF].

Among the published activities, only fonsecin shows a strong AOA compared to ascorbic acid, fonsecinone A having a moderate AOA and aurasperone A, a moderate or no significant AOA compared to pure references ascorbic acid or BHT. In the present study, we isolated various NγPs (including asperpyrone E, aurasperone A, dianhydroaurasperone C, fonsecin, fonsecinone A, fonsecin B, and ustilaginoidin A from Aspergillus tubingensis G131 and evaluated their AOA using the radical 2,2 ′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS + ) assay, commonly used in the agrifood sector and H 2 O 2 -mediated cell death assay.

Materials and Methods

Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) spectra were recorded with an Avance III Bruker NMR spectrometer operating at 600 MHz for 1 H and 150 MHz for 13 C. The chemical shifts are given in δ (ppm) and coupling constants (J) are reported in hertz (Hz). The one-(1D) and two-dimensional (2D) spectra were processed using Topspin. The NMR spectra were measured as solutions in chloroform-d or methanol-d following the solubility of the compounds in 5 mm outer diameter tubes. The structural identifications of the compounds were achieved by comparing their spectroscopic data ( 1 H, 13 C-NMR) with the values found in the literature.

Analytical HPLC Analysis

Dry samples were dissolved in ethanol and filtered. A C18 RP, Zorbax Eclipse XDB column 150 × 5 mm, 4.6 µm particle size (Agilent Technologies©, Santa Clara, CA, USA) fitted with a C18 guard column was used with a flow rate of 1 mL min -1 and an injection volume of 40 µL. Elution was performed using acidified water (H 2 O with 2% acetic acid) (solvent A) and acetonitrile (ACN) (solvent B) with a linear gradient over 55 min at a flow rate of 1 mL min -1 , starting from 30 to 100% of solvent B during 45 min, followed by 100% solvent B for 5 min. The UV absorbance of the eluent was monitored at 280 nm.

Semi-Preparative HPLC Analysis

Separation of the compounds was performed using HPLC apparatus (Agilent Technologies©, USA). The column was a C18 (250 × 10 mm, 5 µm particle size) (Ascentis©, Eden Prairie, MN, USA) with a flow rate of 3 mL min -1 and an injection volume of 100 µL, following a linear gradient starting from H 2 O/ACN (70:30, v/v) to 100% ACN during 60 min. The UV absorbance of the eluent was monitored at 280 nm.

Fungal Material

A. tubingensis G131 was provided by École Nationale Supérieure Agronomique de Toulouse (France); this fungus is non-mycotoxigenic as the synthetic genes required for the production ochratoxin A and fumonisin are absent from its genome [START_REF] Choque | Whole-genome sequencing of Aspergillus tubingensis G131 and overview of its secondary metabolism potential[END_REF]. It was conserved at 4 • C in a 15 mL bottle on potato dextrose agar (PDA). The inoculum stock was prepared by propagating the fungus in 250 mL Erlenmeyer flasks containing PDA. The cultures were incubated at 25 • C for 5-10 days. The inoculum suspension was prepared by adding 0.01% (v/v) Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) and scraping with a magnetic stirrer to recover the conidia. The quantity of conidia was counted using a Malassez cell prior to inoculation of the solid medium.

Solid State Fermentation

The SSF was performed in Raimbault columns on 50 g (dry material) of moist solid medium composed by 50% of vine shoots, 25% of wheat bran, and 25% of potato flakes. The medium was inoculated with 2.10 7 conidia g -1 (dry material) at an initial humidity of 66% and incubated at 25 • C during 7 days.

Isolation of Compounds 1-7

One kilogram of moist fermented solid matrix was extracted using 2 L of ethanol at 25 • C during 2 h. The obtained extract was filtered with Whatman No. 1 filter paper and concentrated under reduced pressure using a rotary evaporator at 40 • C. The extract (8.32 g) was suspended in 250 mL of distilled water before being partitioned with hexane (84.7 mg), dichloromethane (334.9 mg), ethyl acetate (143.52 mg), and butanol (107.5 mg). The fractions that exhibited the most powerful antioxidant activity, thus the methylene chloride and the ethyl acetate extracts, were subsequently purified by semi-preparative HPLC. Twenty fractions were obtained (F1-F20), and subsequent semi-preparative HPLC on F6, F9, F16, F17, F19, and F20 allowed to isolate compounds 1-7. Compounds 1 (16 mg) and 3-7 were isolated as yellowish powder (15, 9, 12, 7, 8, and 10 mg, respectively), whereas compound 2 (11 mg) was presented as red needles.

The spectroscopic data of the known NγPs were in accordance with literature.

Fonsecin (1). Dianhydroaurasperone C (4). Asperpyrone E (5). Fonsecinone A (6).

Radical Scavenging Capacity Assay

A colorimetric assay was adapted from Re et al. [START_REF] Re | Antioxidant activity applying an improved ABTS radical cation decolorization assay[END_REF]. It consists of the measurement of the decolorization of radical monocation ABTS •+ as it reacts with the NγPs. The decolorization was monitored in a 96-well microplate using a microplate reader Infinite 200 (Tecan©, Mennedorf, Switzerland). The reaction between the sample and the ABTS •+ solution was performed for 5 min at 25 • C and the reading was made at 734 nm. The scavenging activity is expressed in Trolox equivalent antioxidant capacity (TEAC). A calibration curve of 3,4-dihydro-6-112 hydroxy-2,5,7,8-tétramethyl-2H-1-benzopyran-2-carboxylic acid (Trolox) ranging from 15.625 to 1000 µmol L -1 was made. To compare the various TEAC, the concentrations of the sample were set to 10 µmol

L -1 . TEAC = (A 0 -A f ) sample -(A 0 -A f ) blank (A 0 -A f ) Trolox -(A 0 -A f ) blank ( 1
)
where A 0 is the initial absorbance and A f the absorbance after 5 min.

Cell Innocuity and Protective Effect Against H 2 O 2 -Mediated Cell Death

Innocuity was evaluated on normal/non-cancerous cells, i.e., Chinese hamster ovary cells (CHO, ATCC©, Manassas, VA, USA). Cells were routinely grown on 25 cm 2 flasks in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% l-glutamine, and 1% antibiotics (Invitrogen©, Carlsbad, CA, USA), and maintained in a 5% CO 2 incubator at 37 • C. For innocuity testing, CHO cells grown on flasks were trypsinized, diluted in culture media, and seeded onto 96-well plates (Greiner Bio-One©, Austria) at 20,000 cells per well [START_REF] Razafimanjato | The food-associated fungal neurotoxin ochratoxin A inhibits the absorption of glutamate by astrocytes through a decrease in cell surface expression of the excitatory amino-acid transporters GLAST and GLT-1[END_REF]. After seeding, cells were left to reach confluence for 48 h. Confluent cells were then exposed to increasing doses of compounds 1-7 or ascorbic acid for 24 h at 37 • C before evaluation of the cell viability using a resazurin-based in vitro toxicity assay kit (Sigma-Aldrich), as previously described [START_REF] Benkhaled | Elaboration of antimicrobial polymeric materials by dispersion of well-defined amphiphilic methacrylic SG1-based copolymers[END_REF][START_REF] Borie | Enediynes bearing polyfluoroaryl sulfoxide as new antiproliferative agents with dual targeting of microtubules and DNA[END_REF].

Briefly, resazurin stock solution was diluted 1:100 in sterile PBS containing calcium and magnesium (PBS++, pH 7.4). Plates were aspirated and 100 µL of the diluted solution was added per well. After 1 h incubation at 37 • C, fluorescence intensity was measured using a microplate reader (Biotek, Winnooski, VT, USA, Synergy Mx) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The fluorescence values were normalized by the controls (untreated cells) and expressed as percent viability.

Cell viability (%

of control) = A sample -A blank A control -A blank × 100 (2)
For protective effect against H 2 O 2 -mediated cell death, confluent cells on 96-well plates were exposed for 30 min to increasing doses of compounds 1-7 or ascorbic acid before treatment with 300 µmol L -1 of hydrogen peroxide for 24 h at 37 • C prior to evaluation of the cell viability using resazurin-based assay, as explained above. Results were expressed as percentage of inhibition of the hydrogen peroxide effect on the cells.

% of inhibition of the H

2 O 2 effect = 100 - A sample -A blank A control -A blank × 100 (3)

Data Analysis

All experiments were performed in triplicate. The results were fitted using R software version 3.5.1.

Results

Isolation of the NγPs

Most of the studies evaluating production of NγPs involve a microorganism growing on synthetic media [START_REF] Zhan | Asperpyrone D and other metabolites of the plant-associated fungal strain Aspergillus tubingensis[END_REF][START_REF] Leutou | Induced production of 6,9-dibromoflavasperone, a new radical scavenging naphthopyranone in the marine-mudflat-derived fungus Aspergillus niger[END_REF][START_REF] Zhang | Nigerasperones A~C, New Monomeric and Dimeric Naphtho-γ-pyrones from a Marine Alga-derived Endophytic Fungus Aspergillus niger EN-13[END_REF][START_REF] He | Fungal naphtho-γ-pyrones: Potent antibiotics for drug-resistant microbial pathogens[END_REF][START_REF] Li | Naphthoγ -pyrones from Endophyte Aspergillus niger Occurring in the Liverwort Heteroscyphus tener (Steph.) Schiffn[END_REF][START_REF] Shaaban | Seven naphtho-γ-pyrones from the marine-derived fungus Alternaria alternata: Structure elucidation and biological properties[END_REF]. In the present case, the culture was performed on an optimized solid medium containing solid agroindustrial byproducts, since these culture conditions were found to strongly increase NγP production, allowing the isolation of all produced NγPs, including the less abundant ones [START_REF] Carboué | Experimental design and solid state fermentation: A holistic approach to improve cultural medium for the production of fungal secondary metabolites[END_REF]. From the fungal ethanolic extract, seven NγPs were isolated by semi-preparative HPLC after liquid-liquid extraction (Figure 1), including: Fonsecin (1), ustilaginoidin A (2), fonsecin B (3), dianhydroaurasperone C (4), asperpyrone E (5), fonsecinone A (6), and aurasperone A (7) [START_REF] Li | Naphthoγ -pyrones from Endophyte Aspergillus niger Occurring in the Liverwort Heteroscyphus tener (Steph.) Schiffn[END_REF][START_REF] Wang | Yellow Pigments of Aspergillus niger and Aspergillus awamori: Part II. Chemical Structure of Aurasperone A[END_REF][START_REF] Priestap | New naphthopyrones from Aspergillus fonsecaeus[END_REF][START_REF] Galmarini | a Naphthopyrone Pigment from a Mutant of Aspergillus fonsecaeus[END_REF][START_REF] Shibata | Metabolic Products of Fungi. XXII. On Ustilaginoidins. (2). The Structure of Ustilaginoidin A[END_REF][START_REF] Ikeda | Aspergillus species strain m39 produces two naphtho-γ-pyrones that reverse drug resistance in human KB cells[END_REF]. The structures and carbon numbering used for NMR assignment are given in Figure 2. Compounds 1 and 3 are monomeric NγPs and compounds 4-7 are dimeric NγPs. The NγPs contain a fully conjugated system, giving rise to very characteristic UV/VIS spectra, with an absorption maximum at 280 nm.

ABTS Assay

Table 1 gives the AOA reported for NγPs in the literature. The radical scavenging activity against ABTS •+ was tested for the seven NγPs isolated at a concentration of 10 µmol L -1 . Compounds 1-7 exhibited various AOA activity, expressed in TEAC, i.e., 13.3, 14.6, 5.1, 4.6, 3.3, 2.4, to 2.4 µmol L -1 respectively (Table 1). In comparison, ascorbic acid showed a TEAC of 13.2 µmol L -1 , very close to the ones observed for foncesin (1) and ustilaginoidin A (2).

Structure-Mechanism Relationship

A linear relation between the degree of substitution with hydroxyl groups per NγP unit and the TEAC was observed for the isolated NγPs (Figure 3). The degree of substitution with hydroxyl groups per NγP unit is presented in Table 1. The proposed radical scavenging mechanism is based on a resonance stabilization of the unpaired electron through its redistribution on the aromatic cores of the NγPs (Figure 4).

CHO Cell-Based Assay

The innocuity and the protective effect of the various compounds against H 2 O 2 -mediated cell death were investigated using an in vitro model, i.e., CHO cells. With regards to the innocuity of the compounds, only compounds 1 and 2-with the highest radical scavenging potential-reduced the cells' viability at higher doses with 56.5 and 35% reduction in cell viability for 3.4 and 1.9 mmol L -1 of compounds (Figure 5). Compounds 3-7 did not influence the cell viability, even at the highest doses tested (ranging from 1 to 3 mmol L -1 ). CHO cells were exposed for 24 h to increasing concentrations of compounds 1-7 or ascorbic acid. At the end of the incubation period, cell viability was evaluated using Alamar blue assay. Error bars represent the variability associated with the measurements of triplicate samples.

About the protective effect of compounds 1-7 against H 2 O 2 -mediated cell death, and as expected, incubation of CHO cells for 24 h with 300 µmol L -1 of H 2 O 2 decreases cell viability by 75.5 ± 0.7%. Pre-exposure of CHO cells to increasing concentrations of compounds 1-7 or ascorbic acid resulted in variable degrees of protection against H 2 O 2 (Figure 6). CHO cells were exposed for 30 min to increasing concentrations of compounds 1-7 or ascorbic acid in the presence of 300 µmol L -1 of H 2 O 2 . At the end of the incubation period, cell viability was evaluated using Alamar blue assay. Error bars represent the variability associated with the measurements of triplicate samples.

Results show that the NγPs with a higher degree of substitution with hydroxyl groups exhibited a better protective effect. Indeed, a strong protective effect was measured for compounds 1, 2, and 3 with IC 50 on H 2 O 2 -mediated cell death of 3.7, 2.25, and 189.0 µmol L -1 , respectively. Compounds 4, 6, 7, and ascorbic acid exhibited a lower protective effect with IC 50 on H 2 O 2 -mediated cell death of 1.79 mmol L -1 for compound 4, and IC 50 > 1.8 mmol L -1 for compounds 5, 6, and 7.

Discussion

Compounds 1 and 3-7 have already been characterized in Aspergillus genus [START_REF] Li | Naphthoγ -pyrones from Endophyte Aspergillus niger Occurring in the Liverwort Heteroscyphus tener (Steph.) Schiffn[END_REF][START_REF] Priestap | New naphthopyrones from Aspergillus fonsecaeus[END_REF][START_REF] Akiyama | New Dimeric Naphthopyrones from Aspergillus niger[END_REF][START_REF] Huang | Cytotoxic Naphtho-γ-pyrones from the Mangrove Endophytic Fungus Aspergillus tubingensis (GX1-5E)[END_REF]. Interestingly, compound 2, which is a chaetochromin-type NγP, has been reported from the genus of Chaetomium, Fusarium, Penicillium, Metarhizium, and Ustilaginoidea, but is very scarce in Aspergillus spp., which is characteristic of asperpyrone-type NγPs and nigerone-type NγPs [START_REF] Lu | Bis-naphtho-γ-pyrones from Fungi and Their Bioactivities[END_REF][START_REF] Koyama | Absolute configurations of chaetochromin A and related bis(naphtho-gamma-pyrone) mold metabolites[END_REF][START_REF] Lu | Bioactive Bis-naphtho-γ-pyrones from Rice False Smut Pathogen Ustilaginoidea virens[END_REF][START_REF] Taniwaki | Aspergillus bertholletius sp. nov. from Brazil Nuts[END_REF]. Frisvad et al. made the hypothesis that an Aspergillus flavus strain producing ustilaginoidin C could be the consequence of horizontal transfer of gene clusters coding for ustilaginoidins from one fungus to another, while occupying the same ecological niche during evolution [START_REF] Frisvad | Safety of the fungal workhorses of industrial biotechnology: Update on the mycotoxin and secondary metabolite potential of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei[END_REF].

Concerning the AOA, fonsecin has been found to exhibit potent radical scavenging activity against DPPH radical, which is coherent with the results of Leutou et al. [START_REF] Leutou | Induced production of 6,9-dibromoflavasperone, a new radical scavenging naphthopyranone in the marine-mudflat-derived fungus Aspergillus niger[END_REF]. Zhang et al. found no significant AOA for aurasperone A, dianhydroaurasperone C, or fonsecinone A, whereas we report AOA for these compounds [START_REF] Zhang | Nigerasperones A~C, New Monomeric and Dimeric Naphtho-γ-pyrones from a Marine Alga-derived Endophytic Fungus Aspergillus niger EN-13[END_REF]. An explanation of this difference could be that these authors used concentrations of 0.088, 0.090, and 0.088 µmol L -1 respectively, which are 100 times less concentrated that the one we used for these compounds. Accordingly, Cai et al. also reported AOA for aurasperone A and fonsecinone A using a similar protocol to the one used by Zhang et al., but using concentrations 5000 time higher than them [START_REF] Cai | a new dimeric naphtho-γ-pyrone from the edible fungus Pleurotus ostreatus[END_REF][START_REF] Zhang | Nigerasperones A~C, New Monomeric and Dimeric Naphtho-γ-pyrones from a Marine Alga-derived Endophytic Fungus Aspergillus niger EN-13[END_REF]. It is, however, difficult to compare results obtained with DPPH and ABTS protocols, although both colorimetric methods use the same single electron transfer mechanism [START_REF] Dudonné | Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD, and ORAC Assays[END_REF][START_REF] Thaipong | Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts[END_REF].

Although the relationship between the structure of NγPs and their biological activities is poorly described in the literature, hypotheses have been made concerning the mechanism behind this activity, while observing the structure of other antioxidant such as naphtho-α-pyrenes or flavonoids [START_REF] Choque | Fungal naphtho-γ-pyrones-secondary metabolites of industrial interest[END_REF]. These studies suggest that the antioxidant capacity of such molecules is due to the presence of an aromatic core-undergoing a resonance structure, able to stabilize the unpaired electron through its redistribution on the aromatic core-and number of free hydroxyl groups, able to donate a hydrogen radical and thereby scavenge the free radicals. For that matter, the antioxidant capacity is a function of the number and the position of the hydroxyl groups on the molecule [START_REF] Amić | Structure-radical scavenging activity relationships of flavonoids[END_REF][START_REF] Foo | Antioxidant capacities of fucoxanthin-producing algae as influenced by their carotenoid and phenolic contents[END_REF]. This hypothesis is verified for the NγPs isolated here, as there is a linear relation between the degree of substitution with hydroxyl groups per NγP unit and the radical scavenging activity. In comparison with the results found in the literature, a graphic representation of the degree of substitution with hydroxyl groups per NγP unit and the logarithmic values of the IC 50 obtained by Leutou et al. shows that, except for aurasperone A, there is a linear relation between these variables [START_REF] Leutou | Induced production of 6,9-dibromoflavasperone, a new radical scavenging naphthopyranone in the marine-mudflat-derived fungus Aspergillus niger[END_REF]. No linear relation appears in the case of Cai et al., but the range of degree of substitution with hydroxyl groups per NγP unit they investigated was smaller (varying between 1 and 1.5) [START_REF] Cai | a new dimeric naphtho-γ-pyrone from the edible fungus Pleurotus ostreatus[END_REF]. However, the variability of values they obtained suggests that for the same number of hydroxyl groups on the molecule, the position of these groups also influences the scavenging activity. No such linear relation could have been highlighted for the results of Zhang et al., although the two highest AOA values they obtained correspond to the NγPs with the highest degree of substitution with hydroxyl groups [START_REF] Zhang | Nigerasperones A~C, New Monomeric and Dimeric Naphtho-γ-pyrones from a Marine Alga-derived Endophytic Fungus Aspergillus niger EN-13[END_REF].

With regards to the innocuity of the compounds, only the compounds with the highest radical scavenging potential reduce the cell viability at higher doses. Accordingly, Zhan et al. tested various NγPs-including the present fonsecin, fonsecin B, fonsecinone A, aurasperone A, and dianhydroaurasperone C-and none of them showed cytotoxicity against the human cancer cell lines NCI-H460 (non-small cell lung carcinoma), MIA Pa Ca-2 (pancreatic cancer), MCF-7 (breast cancer), and SF-268 (CNS cancer; glioma) [START_REF] Zhan | Asperpyrone D and other metabolites of the plant-associated fungal strain Aspergillus tubingensis[END_REF]. Following the same idea, Zhang et al. showed that none of their tested NγPs-including the present aurasperone A, dianhydroaurasperone C, and fonsecinones A-exhibited cytotoxic effect on the tumor cell lines SMMC-7721 (hepatocellular carcinoma cells) and A549 (human lung epithelial cells) at a dose ranging from 32.97 to 66.23 µmol.L -1 [START_REF] Zhang | Nigerasperones A~C, New Monomeric and Dimeric Naphtho-γ-pyrones from a Marine Alga-derived Endophytic Fungus Aspergillus niger EN-13[END_REF]. In accordance with our results concerning the protective effect of the isolated compounds on CHO against H 2 O 2 , NγP glycosides from Cassia obtusifolia have been shown to protect human liver-derived HepG2 cells against the toxicity of tert-butylhydroperoxide-an oxidative stress inducer-by significantly inhibiting the reactive oxygen species generation [START_REF] Paudel | Anthraquinone and naphthopyrone glycosides from Cassia obtusifolia seeds mediate hepatoprotection via Nrf2-mediated HO-1 activation and MAPK modulation[END_REF].

The evaluation of the radical scavenging potential of a given substance on cellular models might be a good requisite for eventual applications in cosmetics [START_REF] Rona | The cosmetic treatment of wrinkles[END_REF][START_REF] Ratz-Lyko | Methods for evaluation of cosmetic antioxidant capacity[END_REF]. It is also good complementary information in addition to the ABTS-based antioxidant measurement. Indeed, it helps to underline mechanisms in complex systems; to be effective, the antioxidants need to be absorbed, transported, distributed, and retained properly in the biological fluids, cells, and tissues [START_REF] Zhang | Evaluation of the Cellular and Animal Models for the Study of Antioxidant Activity: A Review[END_REF]. In addition, to be used in food, medicine, or cosmetics, the antioxidant molecule must be safe/non-toxic. The innocuity and the protective effect of the various compounds against H 2 O 2 -mediated cell death were investigated using an in vitro model, i.e., CHO cells. Indeed, since the Free Radical Theory of Aging, attention has been focused on the role of reactive oxygen species (ROS) in the initiation and progression of the aging process [START_REF] Beckman | The Free Radical Theory of Aging Matures[END_REF]. ROS are known to cause damage to molecules such as DNA, proteins, and lipids, and for that reason, ROS-induced damage contributes to the natural aging process-as ROS are naturally produced in the cells-but also in a variety of biological phenomena, including radiation damage, infection with xenobiotics, carcinogenesis, and neurodegenerative diseases among others [START_REF] Toyokuni | Reactive oxygen species-induced molecular damage and its application in pathology[END_REF]. Among the ROS of biological interest, the hydrogen peroxide can undergo Fenton-type reactions with metals such as Fe 2+ to produce OH • radicals. These radicals are the most noxious and interactive of the radical species and are known to initiate rounds of peroxidative damage to membrane lipids [START_REF] De Haan | Fibroblasts derived from Gpx1 knockout mice display senescent-like features and are susceptible to H 2 O 2 -mediated cell death[END_REF]. Hence, present in critical concentration, H 2 O 2 in the cytosol induces an oxidative stress that eventually results in cell death [START_REF] Zdolsek | H 2 O 2 -Mediated Damage to Lysosomal Membranes of J-774 Cells[END_REF]. Protective effects of a compound against H 2 O 2 -mediated cell death in normal/non-cancerous cells such as CHO cells is a good way to assess the efficiency of antioxidant potential of this compound in biological conditions [START_REF] Festa | Strong antioxidant activity of ellagic acid in mammalian cells in vitro revealed by the comet assay[END_REF].

Finally, although some studies have been carried out on this strain to understand its metabolism in order to optimize NγP production, further analyses are required in the case of an industrial application [START_REF] Carboué | Experimental design and solid state fermentation: A holistic approach to improve cultural medium for the production of fungal secondary metabolites[END_REF][START_REF] Lopez De Leon | Influence of the culture conditions on the production of NGPs by Aspergillus tubingensis[END_REF].

Conclusions

NγPs are fungal secondary metabolites exhibiting interesting radical scavenging properties. This effect has been documented in vitro for some members of this family. In this study, we demonstrated that seven NγPs isolated from A. tubingensis G131 have different antioxidant effect using ABTS and cell-based assays. Amongst all of the antioxidant NγPs, fonsecin and ustilaginoidin A exhibit interesting results, particularly against ROS-mediated cell death. The hypothesis behind this increased radical scavenging potential is that the higher the number of hydroxyl substitutes on the NγP skeleton is, the higher this potential will be. Such properties could be applied in the food and cosmetic industries.

  13 C-NMR (150 MHz, CDCl3/CD 3 OD, 3:7) δ: 185.4 (C-4, C-4 ′ ), 169.4 (C-2, C-2 ′ ), 164.8 (C-5, C-5 ′ ), 161.8 (C-6, C-6 ′ ), 160.8 (C-8, C-8 ′ ), 153.9 (C-10a, C-10 ′ a), 142.3 (C-9a, C-9 ′ a), 108.2 (C-5a, C-5 ′ a), 107.4 (C-3, C-3 ′ ), 104.3 (C-4a, C-4 ′ a), 101.4 (C-10, C-10 ′ ), 101.9 (C-9, C-9 ′ ), 98.2 (C-7, C-7 ′ ), 56.4 (6-CH 3 O, 6 ′ -CH 3 O), 20.7 (2-CH 3 , 2 ′ -CH 3 ). Fonsecin B (3). 1 H-NMR (600 MHz, CDCl3/CD 3 OD, 9:1) δ: 6.46 (1H, s, H-10), 6.42 (1H, d, J = 2.1 Hz, H-9), 6.23 (1H, d, J = 2.1 Hz, H-7), 3.88 (3H, s, 6-CH 3 O), 3.82 (3H, s, 8-CH 3 O), 2.93 (1H, d, J = 20.0 Hz, H-3a), 2.80 (1H, d, J = 20.0 Hz, H-3b), 1.63 (3H, s, 2-CH 3 ).

  13 C-NMR (150 MHz, CDCl3/CD 3 OD, 9:1) δ: 197.1 (C-4), 164.2 (C-5), 162.4 (C-8), 161.3 (C-6), 153.8 (C-10a), 143.4 (C-9a), 107.2 (C-5a), 103.3 (C-4a), 102.7 (C-10), 99.9 (C-2), 98.6 (C-9), 96.7 (C-7), 56.0 (6-CH 3 O), 55.4 (8-CH 3 O), 47.5 (C-3), 27.8 (2-CH 3 ).

  13 C-NMR (150 MHz, CDCl 3 ) δ: 167.1 (C-2), 166.8 (C-2 ′ ), 162.5 (C-8), 159.9 (C-10), 157.1 (C-10 ′ ), 156.5 (C-5 ′ ), 156.2 (C-8 ′ ), 116.1 (C-9 ′ ), 110.9 (C-3 ′ ), 110.7 (C-3), 109.5 (C-4 ′ a), 108.4 (C-4a), 108.4 (C-10 ′ a), 106.2 (C-7 ′ ), 105.8 (C10a), 97.6 (C-9), 96.4 (C-7), 61.8 (10 ′ -CH 3 O), 56.3 (10-CH 3 O), 55.5 (8-CH 3 O), 20.8 (2 ′ -CH 3 ), 20.8 (2-CH 3 ).

5 (C- 5 ′

 55 13 C-NMR (150 MHz, CDCl 3 ) δ: 167.1 (C-2), 166.8 (C-2 ′ ), 162.5 (C-8), 159.9 (C-10), 157.1 (C-10 ′ ), 156.), 156.2 (C-8 ′ ), 116.1 (C9 ′ ), 110.9 (C-3 ′ ), 110.7 (C-3), 109.5 (C-4 ′ a), 108.4 (C10 ′ a), 108.4 (C-4a), 106.2 (C-7 ′ ), 105.9 (C-6 ′ ), 105.8 (C10a), 97.6 (C-9), 96.4 (C-7), 61.8 (10 ′ -CH 3 O), 56.3 (10-CH 3 O), 55.5 (8-CH 3 O), 20.8 (2-CH 3 ), 20.8 (2 ′ -CH 3 ).

Figure 1 .

 1 Figure 1. Chromatogram of the ethanolic extract with molecules associated to the peaks.

Figure 1 .

 1 Figure 1. Chromatogram of the ethanolic extract with molecules associated to the peaks.

Figure 2 .

 2 Figure 2. Structures of compounds 1-7 and carbon numbering used for NMR assignment.Figure 2. Structures of compounds 1-7 and carbon numbering used for NMR assignment.

Figure 2 .

 2 Figure 2. Structures of compounds 1-7 and carbon numbering used for NMR assignment.Figure 2. Structures of compounds 1-7 and carbon numbering used for NMR assignment.

Figure 3 .

 3 Figure 3. Linear relation between the number of hydroxyl groups per N P unit and the TEAC.Figure 3. Linear relation between the number of hydroxyl groups per NγP unit and the TEAC.

Figure 3 .

 3 Figure 3. Linear relation between the number of hydroxyl groups per N P unit and the TEAC.Figure 3. Linear relation between the number of hydroxyl groups per NγP unit and the TEAC.

Figure 4 .

 4 Figure 4. Radical scavenging mechanism of the fonsecin.Figure 4. Radical scavenging mechanism of the fonsecin.

Figure 4 .

 4 Figure 4. Radical scavenging mechanism of the fonsecin.Figure 4. Radical scavenging mechanism of the fonsecin.

Figure 5 .

 5 Figure 5. Innocuity testing of compounds 1-7 on Chinese hamster ovary (CHO) cells.Figure 5. Innocuity testing of compounds 1-7 on Chinese hamster ovary (CHO) cells.

Figure 5 .

 5 Figure 5. Innocuity testing of compounds 1-7 on Chinese hamster ovary (CHO) cells.Figure 5. Innocuity testing of compounds 1-7 on Chinese hamster ovary (CHO) cells.

Figure 6 .

 6 Figure 6. Protective effect of compounds 1-7 on H O -mediated cell death in CHO cells.Figure 6. Protective effect of compounds 1-7 on H 2 O 2 -mediated cell death in CHO cells.

Figure 6 .

 6 Figure 6. Protective effect of compounds 1-7 on H O -mediated cell death in CHO cells.Figure 6. Protective effect of compounds 1-7 on H 2 O 2 -mediated cell death in CHO cells.

  1 H-NMR (600 MHz, CDCl 3 ) δ: 7.14 (1H, s, H-7), 6.99 (1H, s, H-6 ′ ), 6.48, (1H, d, J = 2.2 Hz, H-9), 6.36 (1H, brs, H-3), 6.32 (1H, brs, H-3 ′ ), 6.25, (1H, d, J = 2.2 Hz, H-7), 4.02 (3H, s, 10-CH 3 O), 3.63 (3H, s, 8-CH 3 O), 3.61 (3H, s, 10 ′ -CH 3 O), 2.56 (3H, brs, 2-CH 3 ), 2.47 (3H, brs, 2 ′ -CH3).

Table 1 .

 1 Antioxidant activity (AOA) of the NγPs reported in the literature.

	Compounds	Number of OH Per NγP Unit	ABTS TEAC (µmol L -1 ), Tested Concentration: 10 µmol L -1 (in This Work)	IC 50 (µmol L -1 ) [11]	AOA DPPH Essay Radical Scavenging (%), Tested Concentration: 50 µg L -1 [13]	[12]	Radical Scavenging (%), Tested Concentration: 250,000 µg L -1 [10]
	6,9-Dibromoflavasperone	1		21.0
	Ascorbic acid		13.23 ± 0.05 a	20.0			90.5
	Asperpyrone A	1			N
	Asperpyrones B	1					31.9
	Asperpyrone C	1			N		32.9
	Asperpyrone E	1	3.29 ± 0.11 c
	Asperpyrone F	1.5					32.8
	Aurasperone A	1	2.4 ± 0.20 d		N	N	38.6
	Aurasperone B	2		0.01	48.1
	Aurasperone E	1					33.4
	BHT				80.4
	Dianhydroaurasperone C	1.5	4.57 ± 0.36 b,c		N
	Flavasperone	1		25.0		N
	Fonsecin	3	13.32 ± 0.08 a	0.02
	Fonsecin B	2	5.12 ± 0.08 b
	Fonsecinone A	1	2.36 ± 0.07 d		N		36.9
	Fonsecinones B	1.5			13.7		38.7
	Fonsecinone C	1.5			N
	Fonsecinone D	1.5			37.5	N
	Nigerone A	2			N
	Nigerone B	2			N
	Nigerone C	2			41.6
	Rubrofusarin B	1				N
	TMC-256A1	2		0.30
	Ustilaginoidin A	3	14.59 ± 1.75 a
	TEAC: Trolox equivalent antioxidant capacity; N: No significant activity; different letters in superscript a,b,c,d indicate a significant difference, measured with a t-test at a risk α = 0.05.

Conflicts of Interest:

The authors declare no conflicts of interest.