Emeline Boukherrouba

Camille Larosa

Kim-Anh Nguyen

Jérémy Caburet

Laurent Lunven

Hugues Bonnet

Antoine Fortuné

Ahcène Boumendjel

Benjamin Boucherle

Sabine Chierici

Marine Peuchmaur
email: marine.peuchmaur@univ-grenoble-alpes.fr

Exploring the structure-activity relationship of benzylidene-2,3dihydro-1H-inden-1-one compared to benzofuran-3(2H)-one derivatives as inhibitors of amyloid fibers

Keywords: Indanones, Aurones, Alzheimer's disease, tau aggregation, AcPHF6 peptide

published or not. The documents may come     Exploring the structure-activity relationship of benzylidene-2,3-dihydro-1H-inden-1-one compared to benzofuran-3(2H)-one derivatives as inhibitors of tau amyloid fibers

Introduction

Tau, the major hydrophilic microtubule-associated protein (MAP), is involved in the normal brain function, playing a crucial role in the assembly and stability of microtubules. In Alzheimer disease (AD) and in the other related neurodegenerative diseases, known as tauopathies, tau is abnormally hyperphosphorylated. The subsequent conformational changes of this protein ultimately lead to the formation of highly disruptive and toxic intracellular aggregates, and then neurofibrillary tangles (NFTs) [START_REF] Iqbal | Tau in Alzheimer disease and related tauopathies[END_REF][START_REF] Kovacs | Neuropathology of tauopathies: principles and practice[END_REF][START_REF] Arendt | Tau and tauopathies[END_REF]. Their deposition in the brain results in neuronal dysfunction and eventually death of neurons, but the pathophysiological role of tau protein, notably in AD, is still not fully understood.

Nevertheless, the shorter critical fragment prominently involved in tau aggregation was previously identified and was referred to as PHF6 [START_REF] Bergen | Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure[END_REF]. To go further in the understanding of tau aggregation mechanism, an hexapeptide model was also developed: the AcPHF6 derivative (Ac-VQIVYK-NH2).

It is able to undergo efficient in vitro fibrillation to form cross β-sheet structures and paired helical filaments very similar to those of pathological full-length tau [START_REF] Rojas Quijano | Prediction of nucleating sequences from amyloidogenic propensities of tau-related peptides[END_REF][START_REF] Goux | The formation of straight and twisted filaments from short tau peptides[END_REF]. The inhibition of the aggregation process of tau representing a relevant therapeutic target for tauopathies, this model was mainly used for the prompt biochemical evaluation of new inhibitors [START_REF] Zheng | Macrocyclic beta-sheet peptides that inhibit the aggregation of a tau-protein-derived hexapeptide[END_REF][START_REF] Zheng | A hydrophobic surface is essential to inhibit the aggregation of a tau-protein-derived hexapeptide[END_REF][START_REF] Mohamed | Tau-derived-hexapeptide 306VQIVYK311 aggregation inhibitors: nitrocatechol moiety as a pharmacophore in drug design[END_REF][START_REF] Landau | Towards a pharmacophore for amyloid[END_REF] but it can also be useful for the search of probes that could participate in AD treatment or diagnosis [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF].

A few years ago, we explored the interaction between diverse polyhydroxylated benzylidene aurones (Fig. 1) and the tau model AcPHF6 [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF]. Firstly, this study showed that at least four groups are needed to achieve significant activity. Depending on the hydroxylation pattern, it also revealed that aurones may act as inhibitors of fiber elongation or as chemical probes of fibers. While pursuing our investigation, we focused our efforts on hydroxylated benzylidene indanones which are structurally very close to aurones.

Indanones are very useful precursors involved in the synthesis of diverse natural or synthetic heterocyclic molecules, including the probably most significant drug bearing an indanone moiety, donepezil, an acetylcholinesterase inhibitor approved for the treatment of AD (Fig. 1) [START_REF] Patil | Recent developments in biological activities of indanones[END_REF][START_REF] Menezes | Arylidene indanone scaffold: medicinal chemistry and structure-activity relationship view[END_REF].

Arylidene indanones are both closely related to aurones and chalcones (Fig. 1), incorporating the α,β-unsaturated ketone moiety of the latter in a rigid five-membered ring. Indanones are also one of the privileged structures in medicinal chemistry associated with a broad spectrum of biological activities such as anti-inflammatory, antimicrobial, antiviral or anticancer activities [START_REF] Turek | Synthesis of 1-indanones with a broad range of biological activity[END_REF][START_REF] Radhakrishnan | Inhibitory kinetics of novel 2,3-dihydro-1Hinden-1-one chalcone-like derivatives on mushroom tyrosinase[END_REF][START_REF] Kadayat | Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-inden-1one and benzofuran-3(2H)-one derivatives as a novel class of potential therapeutics for inflammatory bowel disease[END_REF]. More specifically, 2-arylidene indanone derivatives have been investigated on various biological targets related to AD, such as acetylcholinesterase inhibitors, metal-chelating agents, potential probes for β-amyloid plaques even, logically, as multi-target-directed ligands against AD [START_REF] Sheng | Design, synthesis and AChE inhibitory activity of indanone and aurone derivatives[END_REF][START_REF] Meng | Design, synthesis, and evaluation of indanone derivatives as acetylcholinesterase inhibitors and metal-chelating agents[END_REF][START_REF] Nan | 6-Methoxy-indanone derivatives as potential probes for β-amyloid plaques in Alzheimer's disease[END_REF][START_REF] Huang | Discovery of indanone derivatives as multi-targetdirected ligands against Alzheimer's disease[END_REF]. To the best of our knowledge, there is currently no evidence of interaction between tau and this family of compounds. Herein, this paper reports the design, synthesis and amyloid fibers interaction of benzylidene-2,3dihydro-1H-inden-1-one derivatives and their benzofuran-3(2H)-one analogues. The tau AcPHF6 model, using thioflavin T (ThT) fluorescence assays, circular dichroism (CD), and atomic force microscopy (AFM) were used to assess and compare the effect of the two families of compounds on amyloid fibers.

Chemistry

Indanones were prepared according to known procedures starting with the synthesis of the suitably substituted 2,3-dihydro-1H-inden-1-one derivatives 2 and 6 (scheme 1). The hydroxylation pattern of indanones were mostly chosen to obtain corresponding analogues of previously synthesized aurones [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF]. 4-Hydroxy-2,3-dihydro-1H-inden-1-one 2 was thus obtained thanks to a Friedel-Crafts acylation starting from commercially available chroman-2-one 1 in the presence of AlCl3 [START_REF] Parada | Hydrogen bonded phenolquinolines with highly controlled proton-transfer coordinate[END_REF]. Compound 6, 5,7-dimethoxy-2,3-dihydro-1H-inden-1-one, was prepared from 3,5dimethoxybenzaldehyde 3 and malonic acid via the Doebner modification of the Knoevenagel condensation [START_REF] Li | Design, synthesis, and evaluation of 2-piperidone derivatives for the inhibition of β-amyloid aggregation and inflammation mediated neurotoxicity[END_REF]. The α,β-unsaturated carboxylic acid 4 was subsequently hydrogenated to give compound 5 which was finally cyclized in the presence of methanesulfonic acid (MSA) [START_REF] Sim | Aurones as modulators of ABCG2 and ABCB1: synthesis and structure-activity relationships[END_REF]. The 2,3-dihydro-1H-inden-1-one derivatives 2 and 6 described above and the commercially available 2,3-dihydro-1H-inden-1-one 7 were then condensed with benzylaldehyde derivatives in basic conditions to give the corresponding indanones (11a-o, scheme 2 and table 1). When the condensation was carried out with methoxylated 2,3-dihydro-1H-inden-1-one and/or benzaldehyde derivatives, the hydroxyl analogues were obtained by reaction of intermediates 11 with BBr3 in dry dichloromethane to give 13a-b and 13g-o.

To complete our set of indanone and aurone analogue derivatives, diverse polyhydroxylated aurones were synthesized (12a-c, e-g, i-o and 14a-b, i, m-o, table 1) following the same procedure as described for indanones (scheme 2). The synthesis of benzofuran-3(2H)-ones 8, 9a, 9b and 10 used as starting materials were already described [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF].

Table 1

Structures of evaluated indanone and aurone derivatives. All the synthesized intermediates are not listed in this table (cf. experimental section).

Indanone (X

= CH2) Aurone (X = O) R a R b R c R a ' R b ' R c ' R d '

Results and discussion

Screening of aurones and indanones by thioflavin T fluorescence assays.

Thioflavin T (ThT) fluorescence assays were used to evaluate the potential inhibition activity of aurone and indanone derivatives toward the aggregation process of the tau AcPHF6 model [START_REF] Levine | Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides: detection of amyloid aggregation in solution[END_REF][START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF]. To avoid misleading interpretations, it was first necessary to study the spectroscopic properties of tested compounds, i.e. absorbance and fluorescence. ThT assay is based on the fluorescence of the ThT dye in presence of the characteristic β-sheet motif of amyloid fibers in formation. Thus, it may be biased by an inner filter effect of molecules which strongly absorb in the same range as the bound ThT (wavelengths of excitation and emission at 440 and 480 nm, respectively) [START_REF] Jameson | Dye-binding assays for evaluation of the effects of small molecule inhibitors on amyloid (Aβ) self-assembly[END_REF][START_REF] Hudson | The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds[END_REF]. In light of this, we recorded the UV/Vis absorption of all the molecules to check if their own spectroscopic properties would not interfere with the assays (Fig. 3). The UV/Vis spectra of indanones and their corresponding aurone derivatives are quite similar. Interestingly, the intensity and maximum absorption appeared to be lower for the indanone analogues. The highest absorption is observed for indanones 13m and 13n and their aurone analogues 14m and 14n with a maximum around 400-410 nm. It is noteworthy that these compounds along with the other ones from table 1 have a low absorption at 10 µM and 1 µM that cannot significantly quench the ThT fluorescence at the working wavelengths of the ThT assays. As for the intrinsic fluorescence of compounds, such emission was corrected by using blank wells and it remains very low at 480 nm when excited at 440 nm in comparison to the bound ThT fluorescence (Fig. S2). Furthermore, aurones and indanones do not exhibit any significant change in their emission spectra in the presence of preformed AcPHF6 fibers, except for indanones 11e and 13b (Fig. S3). Both indanones show no fluorescence on their own but a slight fluorescence emission when added to preformed fibers. The intensity is nevertheless low compared to the ThT fluorescence intensity and could only weakly undervalue the effect of both compounds. Remarkably, their corresponding aurones 12e and 14b do not show such behavior which may indicate a closer interaction with fibers of the indanone series. Interestingly, aurone and indanone analogues displayed similar inhibitory activities at 10 μM with few exceptions (compounds 11e/12e, 11f/12f and 13j/12j, Fig. 2). As expected, O-methylated indanones 11a-b and unsubstituted indanones 11c turned out to be inactive while unsubstituted aurone analogue 12c showed a very weak activity at 10 μM. Contrariwise, modest to significant activities were obtained for more polar compounds. Nevertheless, monohydroxylated indanones 11d and 11e were inactive, unlike the corresponding aurone 12e of the latter exhibited a higher, although still modest, activity. On the whole, increasing the number of hydroxyl groups on ring A and/or B appeared favourable to the inhibition properties of these classes of compounds, the most potent compounds being the tetra and pentahydroxylated ones (e.g. 13o/14o and 13b/14b). The hydroxyl group position on the B ring also influenced the potency of the inhibition, notably in the which a substitution on the R4' position seems appropriate to maximize the desired interaction.

These results are thus consistent with those obtain for polyhydroxylated aurones in our previous study [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF].

A screening was carried out with a 1 μM concentration to further assess the most potent, and some 'atypical' pair of indanone/aurone compounds (Fig. 2). While the most potent compounds were still able to inhibit the ThT signal at 1 μM, the difference observed for the 'atypical' aurone/indanone pairs (11e/12e and 11f/12f) was again clearly noticeable.

Displacement of bound ThT by fluorescence assay.

It is noteworthy that inhibition of fluorescence in ThT assays does not necessarily mean inhibition of amyloid aggregation process. Indeed, displacement of the bound ThT by the tested compounds may also occur. Thus, we recorded the emission spectrum of a solution of preformed AcPHF6 fibers in the presence of ThT right after adding an aurone or an indanone (Fig. 4). All active compounds in ThT assays induce a decrease in ThT fluorescence when added to preformed fibrils (e.g. 13a/14a, 13b/14b, 13m/14m, 13n/14n, 13o/14o, 12f). These results suggested that displacement of the bound ThT by the tested compounds in previous assay may also occur in place of or in competition with an inhibitory effect. Considering the short period of time left for this analysis, it is unlikely that the decrease of fluorescence signal was due to the disaggregation of fibers. To note, a slight increase of the fluorescence signal was observed after the addition of inactive compound 11e to fibers. This could however be explained by the fluorescence emission of 11e in presence of preformed fibers as mentioned earlier (Fig. S3).

Inhibition properties.

The inhibition values obtained from the two previous assays are quite similar for all the compounds (see for comparison Table 2 where the decrease of ThT fluorescence intensity is expressed like in ThT assay in %) except for compounds 12e, 12f, and 13a for which the values are significantly higher in the ThT fluorescence assay compared to ThT displacement assay, suggesting a greater inhibitory effect.

Table 2

% ThT fluorescence inhibition obtained by both the ThT fibrillation assay from the AcPHF6 peptide and the ThT displacement assay realized from preformed AcPHF6 fibers. The values (%) reported in the table are the mean values. For the ThT assay, these values are those reported in Fig. 2, for ThT displacement assay the values are calculated from the fluorescence emission intensities at 480 nm.

CD and AFM experiments.

The combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy and atomic force microscopy (AFM) was used to better characterize the interaction of compounds with AcPHF6 model.

CD spectroscopy provides structural information on β-sheet patterns. These β-sheets (from soluble oligomeric species or fibers) are expected to be detected during the fibrillation process, but are likely to disappear, or at least to decrease, when an inhibitor is added.

We selected a set of aurone/indanone pairs based on the results of the fluorescence experiments: the two 11e/12e and 11f/12f pairs where only the aurone analogue is active on both fluorescence assays; the 13a/14a pair where both aurone and indanone are active on ThT assay but showing a different behavior in the ThT displacement test; and finally, the 13b/14b and 13o/14o pairs where all aurones and indanones are active on both fluorescence tests.

Indanone

ThT fluorescence inhibition (%) Aurone

ThT fluorescence inhibition (%) Among all the tested compounds, the CD profiles were significantly altered only in the presence of the indanones 13a, 13b and 13o (Fig. 5). For the 11e/12e and 11f/12f pairs the CD profiles were the same for the active aurones and the corresponding inactive indanones (Fig. 5 and Fig. S4 for 11e/12e). The aurones 12e and 12f were not able to inhibit the formation of β-sheet species suggesting that their effect can be mostly attributed to a displacement of the bound ThT from fibers. The other selected pairs had all the same behavior in CD. The indanones 13a, 13b and 13o

showed a significant inhibition of the β-sheet profile in CD whereas the aurones did not. This is not in contradiction with fluorescence assays but suggests that only the indanone derivatives have an inhibitory effect on AcPHF6 fibril formation. The effect of aurones seems to be mostly due to a ThT agonist effect. Lastly, compounds 13a, 13b and 13o that have shown β-sheet inhibitory properties in DC were incubated with AcPHF6 for 24 h to obtain the AFM images depicted in figure 6. In the absence of inhibitors, AcPHF6 was able to create a tight fiber network (Fig. 6A). This fibrillation was largely inhibited in presence of 100 µM indanone 13a (Fig. 6B) since only a few aggregates remained after 24 h. Contrariwise, the indanones 13b (Fig. 6C) and 13o (Fig. 6D) at the same 100 µM concentration did not inhibit the fibrillation of AcPHF6, although the fiber network was less dense, especially for 13o. To conclude, compounds 13b and 13o were capable of reducing the amount of β-sheet fibers but are not able to stabilize aggregates, unlike compound 13a. As a comparison, AFM images of the inactive aurone 14a, the analogue of indanone 13a, are reported in figure S6.

Molecular modelling.

To better understand the difference in the way these two families of compounds interact with their target, molecular modelling was used to compare the three-dimensional structure of aurone 14a

and indanone 13a with myricetin, a known polyphenolic inhibitor of the tau fibers elongation (Fig. 7). Whereas the electronic density of the aurone and indanone analogues is quite similar, the spatial orientation of cycles A and B are substantially different. The three-dimensional shape of the indanone comes closer to that of myricetin with orthogonal cycles which can explain their common properties related to the inhibition of amyloid AcPHF6 fibers.

Conclusion

In an effort to deepen the knowledge related to the activity of polyphenol derivatives on the aggregation of a tau model, AcPHF6, we have synthesized a set of aurone and indanone derivatives. The structure-activity relationship studies highlighted the significant effect of the number and the position of hydroxyl substituents on the synthesized analogues for the close interaction with AcPHF6. Moreover, the tridimensional geometry of the molecule was shown to play a potential role in determining if it acts as inhibitors of fiber elongation or as chemical probes of fibers. In short, indanones, which possess a conformation similar to myricetin, have been identified

as relevant inhibitors when substituted at least by three hydroxyl groups. In addition, the preliminary kinetic assays carried out not on AcPHF6 model but directly on the tau protein have shown that the fibrillation inhibitory effect of indanones 13a, 13b and 13o was also found on the full length protein. Further study would be needed, but this activity similar to that of myricetin is very promising (Fig. S5). Finally, considering the large other known activities of indanones on neurodegenerative processnotably as acetylcholinesterase inhibitors or as antioxidants, it would be of particular interest to evaluate these polyhydroxylated indanones in a multi-target strategy against AD. mL/min, 30 μL injection, detection at 254 nm. Purity of all the reported compounds was ≥ 95%.

General procedure A for the synthesis of indanone compounds (11a-o)

To a solution of an indanone (1 equiv.) in EtOH (5 mL/mmol) were added a benzaldehyde derivative (1 equiv.) and then dropwise an aqueous solution of NaOH 5% (2 equiv.). After stirring for 3 to 7 h at room temperature, the reaction mixture was cooled for 30 min in an ice bath. If necessary (presence of phenolic group), the reaction mixture was acidified by an aqueous solution of HCl (1 M). If a precipitation occurred, the mixture was filtered and washed with cold water and MeOH; if no precipitation was noticed, then the mixture was extracted 3 times with EtOAc and the organic layer was dried over MgSO4, filtered and concentrated in vacuo. In the latter case, the residue was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 95:5). [START_REF] Sim | Aurones as modulators of ABCG2 and AABCB1: synthesis and structure -activity relationships[END_REF] (11n) were synthesized according to general procedure A. Pure products were obtained with a yield of 94%, 75%, 62%, 36%, 73%, 73%, 81%, 24% and 73% respectively. Spectral data are in accordance with literature.

5.1.1.1. (E)-2-(3,4,5-trimethoxybenzylidene)-2,3-dihydro-1H-inden-1-one [27] (11a), (E)-2- benzylidene-2,3-dihydro-1H-inden-1-one [28] (11c), (E)-2-(3-hydroxybenzylidene)-2,3-dihydro-1H- inden-1-one[29] (11d), (E)-4-hydroxy-2-(3-hydroxybenzylidene)-2,3-dihydro-1H-inden-1-one [29] (11f), (E)-2-(3,5-dimethoxybenzylidene)-2,3-dihydro-1H-inden-1-one [30] (11g), (E)-2-benzylidene- 5,7-dimethoxy-2,3-dihydro-1H-inden-1-one [31] (11j), (E)-5,7-dimethoxy-2-(3- methoxybenzylidene)-2,3-dihydro-1H-inden-1-one [31] (11k), (E)-2-(3,5-dimethoxybenzylidene)- 5,7-dimethoxy-2,3-dihydro-1H-inden-1-one [31] (11l) and (E)-2-(3,4-dimethoxybenzylidene)-5,7- dimethoxy-2,3-dihydro-1H-inden-1-one

(E)-4-hydroxy-2-(3,4,5-trimethoxybenzylidene)-2,3-dihydro-1H-inden-1-one (11b

General procedure B1 for the synthesis of aurone derivatives (12a-c, e-f, i, l-o)

To a solution of a benzofuran-3(2H)-one derivative in dry dichloromethane (20 mL/mmol) were added a benzaldehyde derivative (1.2 to 1.5 equiv.) and aluminium oxide (4.0 g/mmol). The suspension was stirred at room temperature under nitrogen overnight, then filtered and concentrated in vacuo. The product was recrystallized in MeOH and filtered off to afford the corresponding pure aurone derivative. products were obtained with a yield of 85%, 79%, 78%, 65%, 77%, 74%, 6%, 91%, 80% and 92%

(Z)-2-[(3,4,5-trimethoxy)phenylmethylene]benzofuran-3(2H)-one

respectively. Spectral data are in accordance with literature.

(Z)-2-(3,5-dihydroxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (12g).

The synthesis and spectral data were described in Lunven et al [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF].

General procedure B2 for the synthesis of aurone derivatives (12j-k)

4,6-dihydroxybenzofuran-3(2H)-one (1 equiv.) was dissolved in MeOH (15 mL/mmol). An aqueous solution of potassium hydroxide (50%, 1.5 mL/mmol) and a benzaldehyde derivative (1.5 equiv.)

were added to the mixture which was stirred at 70 °C in the microwave reactor for 2 to 10 h and concentrated under reduced pressure. The reaction mixture was diluted with water and acidified with 1 M HCl until pH 2-3, then extracted with EtOAc. The combined organic layers were washed with water and brine, filtered and dried over MgSO4. Filtration and concentration in vacuo gave the corresponding crude aurone derivative. This residue was purified by flash chromatography on silica gel (CH2Cl2 to CH2Cl2/MeOH 95:5). [START_REF] Haudecoeur | Investigation of binding-site homology between mushroom and bacterial tyrosinases by using aurones as effectors[END_REF] (12k) were synthesized according to general procedure B2. Pure products were obtained with a yield of 82% and 50% respectively. Spectral data are in accordance with literature.

(Z)-2-benzylidene-4,6-dihydroxybenzofuran-3(2H)-one [34] (12j) and (Z)-4,6-dihydroxy-2-(3-hydroxybenzylidene)benzofuran-3(2H)-one

General procedure C for the deprotection of the methoxylated indanone / aurone derivatives (13a-b, g-o and 14a-b, i, m-o)

To a solution of a methoxylated indanone or aurone derivatives (1 equiv.) in dry dichloromethane (10 mL/mmol) was added BBr3 (20 equiv.) at 0 °C. After stirring at room temperature and under N2 atmosphere for 24 to 72 h (until TLC showed total consumption of starting material), cold water was added and the mixture was extracted 3 times with EtOAc. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The compounds obtained were pure enough to avoid any purification.

(E)-2-(3,4,5-trihydroxybenzylidene)-2,3-dihydro-1H-inden-1-one (13a

(E)-2-(3,5-dihydroxybenzylidene)-2,3-dihydro-1H-inden-1-one (13g). The crude product

was prepared according to the general procedure C starting from 11g (100 mg, 0.36 mmol). The pure product (67 mg, 0.26 mmol, 74%) was obtained as a brown solid. Rf 0.55 for C16H11O3 251.0709, found 251.0713.

(

(E)-2-(3,5-dihydroxybenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (13h

). The crude product was prepared according to the general procedure C starting from 11h (50 mg, 0.17 mmol).

The pure product (46 mg, 0.17 mmol, 100%) was obtained as a brown solid. Rf 0.44

(

Peptide synthesis and purification

Ac-PHF6-NH2 (AcPHF6, MW=1018 g/mol) were synthesized as previously reported. [START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF] Peptides were purified with a reverse-phase high pressure liquid chromatography (RP-HPLC) on a Nucleosil C18 column (100 Ᾰ, 250 × 21 nm, 7 µM) with UV monitoring at 214 and 250 nm. During 25 min a 22 mL/min flow with a 15-35% acetonitrile in water was applied (both water and acetonitrile contained 0.1% TFA). Purified peptides were lyophilized, controlled by high resolution mass spectroscopy (HRMS, ESI+) and stored at -20 °C.

Biochemistry

Absorption spectra

Absorption spectra were acquired on a spectrophotometer SAFAS UVmc (SAFAS S.A, Monaco, France) with an optical length of 8.5 mm and a ray of 0.5 × 4 mm. Spectra were recorded using the software SAFAS SP2000 from 240 nm to 640 nm with a spectral resolution of 1 nm and an average time 0.3 s per data point from a drop of sample (5 µL). Final concentration of samples was 10 µM aurone or indanone in 50 mM phosphate buffer with a 0.5% maximum of DMSO.

Emission spectra

Emission spectra were acquired with a Perkin Elmer Luminescence Spectrometer LS50 using a quartz cell with a 0.3 cm path length and with a sample volume of 100 µL. Data were recorded using FL WinLab software from 300 to 600 nm with an excitation wavelength of 440 nm, and a scan speed of 150 nm/min. Emission spectra were recorded in 50 mM phosphate buffer with a maximum of 0.5% of DMSO (i) from 10 µM aurone or indanone, (ii) from a solution containing 10 µM aurone or indanone in presence of 100 µM of AcPHF6 fibers or (iii) from a solution of 10 µM aurone or indanone in presence of 100 µM AcPHF6 fibers prepared in presence of 10 µM ThT.

Those AcPHF6 fibers were pre-formed with AcPHF6 1mM left 24 hours in deionised water, then 10 µL of AcPHF6 fibers were added to final solutions (ii and iii). Emission spectra were collected shortly after this AcPHF6 fibers addition.

Thioflavin T fluorescence assays

The kinetic of fibrillation was performed in 96-well black U-shape polypropylene microplate (Greiner Bio-One) in a POLARstar Omega fluorescence plate reader (BMG LABTECH GmbH, Offenburg, Germany). All conditions were tested in hexaplicate whose average represents the results given in this work.

Measurements.

Fluorescence data was acquired every minute over two hours under an excitation and emission wavelengths at 440 and 480 nm, respectively. Reading (top optic, orbital averaging of 3mm diameter) was done with a 1200 gain. All results for 10 and 1 µM auronesindanones final concentrations are reported in the figure 2.

Circular dichroism

Samples (200 µL final) were prepared in low-binding tubes for a final concentration of 100 µM AcPHF6, 10 µM aurone/indanone with less than 0.2% of DMSO in 50 mM phosphate buffer, and then directly transferred into a 300 µL quartz CD cell with a 1 mm path length. CD spectra were acquired on a JASCO J-810 spectropolarimeter (Jasco corporation, Tokyo, Japan). Data was recorded at room temperature between 260 to 190 nm with a spectral resolution of 1 nm, an average time of 1 s second per data point and a bandwidth of 10 nm.

5.2.5. Atomic force microscopy. AFM samples were collected from CD's samples after 24 h of incubation in room temperature for APHF6. A 5 µL drop from each sample was absorbed onto mica surface during 10 min then it was washed two-three times by 10 µL of pure water and subsequently removing it. AFM images (40 × 40 down to 0.5 × 0.5 µm2, 512 × 512 pixel) were recorded at randomly selected surface position in peak force mode using a Dimension Icon (Bruker, Santa Barbara, CA). The cantilevers SCANASYST-Air used were triangular and had a force contact of 0.4 N/m and a resonance frequency of 70 kHz at tip scan rates of 1 Hz. AFM was processed using the flattening function of the Gwyddion microscope software.

Molecular Modeling

Compound molecular structures were built with maestro and Ligprep modules from Schrödinger suite [START_REF]Schrödinger Release 2019-2[END_REF]. Geometries where further optimized with MOPAC2016 semi-empirical package using PM7 parameterization [38,[START_REF] Stewart | Optimization of parameters for semiempirical methods VI: more modifications to the NDDO approximations and re-optimization of parameters[END_REF]. PULAY and PRECISE options where activated. Electronic densities were exported using GRAPH POTWRT keywords and mapped on respective molecular surfaces by JMol [START_REF]Jmol: an open-source Java viewer for chemical structures in 3D[END_REF].

Fig. 1 .

 1 Fig. 1. General structure of aurone, chalcone and indanone backbones (top); structures of donepezil and representative polyhydroxylated aurones previously studied [11] (bottom).

Scheme 2 .

 2 Scheme 2. Synthesis of indanone and aurone derivatives. Reagents and conditions: (i) X = CH2: NaOH aq. 5%, EtOH, rt, 24-94%; (ii) X = O: Al2O3, dry CH2Cl2, rt, 6-92%; (iii) X = O: KOH aq. 50%, MeOH, 70 °C (MW), 50-82%; (iv) HCl, EtOH, 75 °C, 17% (for 12g); (v) BBr3, dry CH2Cl2, 24-72 h, 0 °C to rt, 30-100%.

  Screening of compounds was carried out in 96-well microplates. The aggregation kinetics of the AcPHF6 peptide was recorded in phosphate buffer over 2 h in the presence of the ThT dye and of the synthesized compounds at 10 μM or 1 μM, corresponding to a peptide to compound ratio of 10:1 and 100:1, respectively. Results reported in figure 2 are expressed as the percentage of ThT fluorescence inhibition, the tau model AcPHF6 without potential inhibitors being used as control (see Fig. S1 for kinetics in presence of 11e and 12e as an illustration).

Fig. 2 .

 2 Fig. 2. ThT fluorescence inhibition (%) is obtained from ThT assay using 100 µM AcPHF6 and 10 µM ThT in 50 mM phosphate buffer pH 7.4. All assays were performed at least in triplicate and each bar value corresponds to the mean ± SD (λex and λem: wavelengths of excitation and emission).

Fig. 3 .

 3 Fig. 3. (A) UV-Vis absorption spectra of indanones (left) and aurones (right). The spectra were recorded at 10 µM of compounds in 50 mM phosphate buffer in presence of 0.5% of DMSO.

4, 6 -

 6 dihydroxy series (comparison of compounds 13l/12l,13m/14m, 13n/14n, and 13o/12o) in

Fig. 4 .

 4 Fig. 4. ThT displacement assays. The ratio compound/ThT is 1:1 and the AcPHF6 fibers were prepared from 100 µM of peptide in presence of 10 µM ThT in 50 mM phosphate buffer pH 7.4. The excitation wavelength was 440 nm.

Fig. 5 .

 5 Fig.5. CD spectra obtained after a 2 h incubation at room temperature of AcPHF6 at 100 µM in phosphate buffer 50 mM, alone or in presence of 100 µM of one of the compounds.

Fig. 6 .

 6 Fig. 6. AFM images obtained after 24 h of fibrillation of 100 µM AcPHF6 alone (A) or with 100 μM of 13a (B), 13b (C) and 13o (D).

Fig. 7 .

 7 Fig. 7. Three-dimensional structure and electronic density of aurone 14a (A), indanone 13a (B) and myricetin (C).

  Commercially available reagents and solvents were used without further purification. Reactions were monitored by thin-layer chromatography (plates coated with silica gel 60 F254 from Merck).Silica gel 60 (70-230 mesh from Macherey-Nagel) was used for flash chromatography.1 H and13 C NMR spectra were recorded at room temperature in deuterated solvents on a Brüker Avance 400 or Avance III 500 instrument (400 or 500 MHz). Chemical shifts (δ) are reported in parts per million (ppm) relative to TMS as internal standard or relative to solvent [ 1 H: δ(DMSO-d6) = 2.50 ppm, δ(CD3OD) = 3.31 ppm, δ(CDCl3) = 7.24 ppm; 13 C: δ(DMSO-d6) = 39.5 ppm, δ(CD3OD) = 49.0 ppm, δ(CDCl3) = 77.2 ppm]. Electrospray ionization ESI mass spectra were acquired on an Esquire 300 Plus Bruker Daltonis instrument with a nanospray inlet and accurate mass measurements (HRMS) were carried out on a TOF spectrometer, realized by the Analytical Department of Grenoble University. HPLC analyses were recorded on an Agilent 1100 series using a diode array detector and a C18 reversed-phase column (Nucleosil C18, Macherey-Nagel, 5 μm particle size, 125 mm × 3 mm) at 25 °C, with a mobile phase A composed of water and TFA 0.1% and a phase mobile B composed of MeOH and TFA 0.1% with a gradient 85:15 to 0:100 A:B over 10 min, 1

[ 32 ] 3 (

 323 (12a), (Z)-7-methoxy-2-(3,4,5-trimethoxybenzylidene)benzofuran-3(2H)-one[START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF] (12b), (Z)-2-benzylidenebenzofuran-3(2H)-one[START_REF] Sim | Aurones as modulators of ABCG2 and AABCB1: synthesis and structure -activity relationships[END_REF] (12c), (Z)-2-benzylidene-7-methoxybenzofuran-3(2H)-one[START_REF] Lunven | Disruption of fibers from the Tau model AcPHF6 by naturally occurring aurones and synthetic analogues[END_REF] (12e), (Z)-7-methoxy-2-(3-methoxybenzylidene)benzofuran-3(2H)-one [11] (12f), (Z)-7-methoxy-2-(2,3,4trimethoxybenzylidene)benzofuran-3(2H)-one [11] (12i), (Z)-2-(3,5-dihydroxybenzylidene)-42H)-one [33] (12n) and (Z)-2-(3,4,5-trimethoxybenzylidene)-4,6-dimethoxybenzofuran-3(2H)-one [35] (12o) were synthesized according to general procedure B1. Pure

  cyclohexane/EtOAc/MeOH 50:30:20); 1 H NMR (400 MHz, DMSO-d6) δ ppm 4.05 (bs, 2H), 6.33 (t, J = 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 2H), 7.32 (t, J = 1.9 Hz, 1H), 7.45-7.52 (m, 1H), 7.65-7.75 (m, 2H), 7.76-7.78 (m, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 32.0 (CH2), 104.5 (CH), 108.9 (2xCH), 123.5 (CH), 126.7 (CH), 127.7 (CH), 133.4 (C), 134.6 (C), 134.9 (C), 136.3 (C), 137.2 (C), 149.9 (C), 158.7 (2xC), 193.4 (C); LRMS (ESI + ) m/z (%) 253 [M+H] + (100); HRMS (ESI -) m/z calc.

9 (

 9 cyclohexane/EtOAc/MeOH 50:30:20); 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.85 (bs, 2H), 6.32 (t, J = 2.0 Hz, 1H), 6.65 (d, J = 2.0 Hz, 2H), 7.07-7.12 (m, 1H), 7.21-7.25 (m, 1H), 7.27-7.33 (m, 2H), 9.53 (s, 2H), 10.14 (s, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 29.2 (CH2), 104.4 (CH), 108.2xCH), 114.1 (CH), 120.3 (CH), 129.0 (CH), 133.4 (C), 134.6 (CH), 136,3 (C), 136.4 (C), 138.9 (C), 154.8 (C), 158.7 (2xC), 193.6 (C); LRMS (ESI -) m/z (%) 267 [M-H] -(100); HRMS (ESI -) m/z calc. for C16H11O4 267.0656, found 267.0657. 5.1.5.5. (E)-2-(2,3,4-trihydroxybenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (13i). The crude product was prepared according to the general procedure C starting from 11i (100 mg, 0.31 mmol). The pure product (72 mg, 0.25 mmol, 83%) was obtained as a brown powder. Rf 0.44 (CH2Cl2/MeOH 9:1); 1 H NMR (400 MHz, CD3OD) δ ppm 3.86 (d, J = 1.4 Hz, 2H), 6.49 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 7.6, 1.0 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.27 (dd, J = 7.6, 7.5 Hz, 1H), 7.32 (dd, J = 7.5, 1.0 Hz, 1H), 8.15 (t, J = 1.4 Hz, 1H); 13 C NMR (100 MHz, CD3OD) δ ppm 30.4 (CH2), 108.8 (CH), 115.7 (CH), 116.5 (C), 121.1 (CH), 122.3 (CH), 130.0 (CH), 131.5 (CH), 131.8 (C), 134.2 (C), 137.9 (C), 141.0 (C), 149.4 (C), 150.1 (C), 155.9 (C), 197.2 (C); LRMS (ESI -) m/z (%) 283 (100) [M-H] -, 319 (20) [M+Cl] -; HRMS (ESI -) m/z calc. for C16H11O5 283.0601, found 283.0615. 5.1.5.6. (E)-2-benzylidene-5,7-dihydroxy-2,3-dihydro-1H-inden-1-one (13j

5. 2 . 3 . 1 .

 231 AcPHF6 model. AcPHF6 peptide stock solution was dissolved, just before its addition into the plate, in water to a final concentration of 500 µM. In each well (final volume 100 µL) is added: 50 µL of 100 mM phosphate buffer, 10 µL of 100 µM ThT (10 µM final), 10 µL of pure water, 10 µL of 100 µM or 10 µM of the tested compound (10 or 1 µM final with less than 0.2% of DMSO) and 20 µL of 500 µM AcPHF6 peptide stock solution (100 µM final). Aurone-Indanone control wells were prepared under the same conditions with the exception of adding 20 µL of 100 mM phosphate buffer (50 mM final) containing 5% DMSO instead of AcPHF6 peptide.

Scheme 1 .

 1 Synthesis of the indanone cores 2 and 6. Reagents and conditions: (i) AlCl3, NaCl, 200 °C, 1 h,

	55%; (ii) malonic acid, pyridine, piperidine, 100 °C, 6 h, 94%; (iii) H2, HCO2NH4, 10% Pd/C, EtOH, rt, 16 h,
	40%; (iv) MSA, 95 °C, 6 h, 44%.

  ). The crude product was prepared according to the general procedure A starting from 2 (100 mg, 0.68

	mmol) and 2,3,4-trimethoxybenzaldehyde (265 mg, 1.35 mmol). The pure product (335 mg, 1.03
	mmol, 76%) was obtained as a grey powder. Rf 0.19 (CH2Cl2/MeOH 99:1); 1 H NMR (400 MHz,
	DMSO-d6) δ ppm 3.78 (s, 3H), 3.82-3.93 (m, 8H), 6.98 (d, J = 8.9 Hz, 1H), 7.09 (d, J = 7.5 Hz, 1H),
	7.23 (d, J = 7.5 Hz, 1H), 7.30 (dd, J = 7.5, 7.5 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.74 (s, 1H), 10.08
	(bs, 1H, OH); 13 C NMR (100 MHz, DMSO-d6) δ ppm 29.0 (CH2), 56.1 (CH3), 60.5 (CH3), 61.7
	mmol) and commercially available 3,4,5-trimethoxybenzaldehyde (133 mg, 0.68 mmol). The pure (CH3), 108.4 (CH), 114.1 (CH), 120.2 (CH), 121.4 (C), 124.9 (CH), 126.7 (CH), 129.0 (CH), 133.5
	product (78 mg, 0.24 mmol, 35%) was obtained as a yellow solid. Rf 0.41 (C), 136.2 (C), 139.1 (C), 141.9 (C), 153.7 (C), 154.8 (C), 155.3 (C), 193.5 (C); LRMS (ESI+) m/z
	(cyclohexane/EtOAc/MeOH 70:20:10); 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.74 (s, 3H), 3.89 (s, (%) 327 (100) [M+H] + , 349 (40) [M+Na] + ; HRMS (ESI+) m/z calc. for C19H16O5 327.1227, found
	6H), 3.98 (bs, 2H), 7.09-7.14 (m, 3H), 7.23-7.27 (m, 1H), 7.29-7.34 (m, 1H), 7.49-7.51 (m, 1H), 327.1220.
	10.03 (s, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 28.7 (CH2), 56.1 (2xCH3), 60.2 (CH3), 108.4
	(2xCH), 114.2 (CH), 120.5 (CH), 129.1 (CH), 130.4 (CH), 133.3 (C), 134.0 (C), 136.1 (C), 138.9 5.1.1.6. (E)-2-(2,4-dimethoxybenzylidene)-5,7-dimethoxy-2,3-dihydro-1H-inden-1-one (11m). The
	(C), 139.2 (C), 153.0 (2xC), 154.7 (C), 193.5 (C); LRMS (ESI + ) m/z (%) 327 [M+H] + (100); HRMS crude product was prepared according to the general procedure A starting from 6 (80 mg, 0.42
	(ESI + ) m/z calc. for C19H19O5 327.1232, found 327.1227. mmol) and 2,4-dimethoxybenzaldehyde (69 mg, 0.42 mmol). The purity of the product was
	checked by TLC, not by NMR due to insolubility. The pure product (65 mg, 0.19 mmol, 46%) was
	obtained as a yellowish powder. Rf 0.20 (CH2Cl2/MeOH 99:1); LRMS (ESI + ) m/z (%) 341 (100)
	[M+H] product (60 mg, 0.16 mmol, 28%) was obtained as a yellow solid. Rf 0.47
	(cyclohexane/EtOAc/MeOH 50:30:20); 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.71 (s, 3H), 3.87 (s,
	9H), 3.89 (s, 3H), 4.05 (bs, 2H), 6.51 (d, J = 1.8 Hz, 1H), 6.71 (d, J = 1.8 Hz, 1H), 7.04 (s, 2H),
	7.27-7.30 (m, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 31.7 (CH2), 55.6 (CH3), 55.9 (CH3), 56,0
	(2xCH3), 60.1 (CH3), 97.7 (CH), 102.1 (CH), 107.9 (2xCH), 119.5 (C), 130.3 (CH), 130.7 (C), 135.1
	(C), 138.6 (C), 153.0 (2xC), 154.5 (C), 159.6 (C), 166.5 (C), 189.1 (C); LRMS (ESI + ) m/z (%) 371
	[M+H]
	and commercially available 3,5-dimethoxybenzaldehyde (113 mg, 0.68 mmol). The pure product
	(80 mg, 0.27 mmol, 40%) was obtained as a light brown solid. Rf 0.38 (cyclohexane/EtOAc/MeOH
	70:20:10); 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.82 (s, 6H), 3.94 (bs, 2H), 6.62 (t, J = 2.2 Hz,
	1H), 6.95 (d, J = 2.2 Hz, 2H), 7.10 (dd, J = 7.7, 1.0 Hz, 1H), 7.25 (dd, J = 7.4, 0.8 Hz, 1H), 7.29-
	7.34 (m, 1H), 7.44-7.47 (m, 1H), 10.06 (s, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 29.0 (CH2),
	55.4 (2xCH3), 101.8 (CH), 108.7 (2xCH), 114.2 (CH), 120.6 (CH), 129.1 (CH), 132.9 (CH), 135.5
	(C), 136.2 (C), 136.7 (C), 138.8 (C), 154.8 (2xC), 160.7 (C), 193.6 (C); LRMS (ESI -) m/z (%) 295
	[M-H] 35

5.1.1.3. (E)-2-benzylidene-4-hydroxy-2,3-dihydro-1H-inden-1-one (11e

). The crude product was prepared according to the general procedure A starting from 2 (100 mg, 0.68 mmol) and commercially available benzaldehyde (72 mg, 0.68 mmol). The pure product (72 mg, 0.31 mmol, 45%) was obtained after washing with dichloromethane as a yellow solid. Rf 0.59 (cyclohexane/EtOAc/MeOH 50:30:20); 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.95 (bs, 2H), 7.11 (dd, J = 7.7, 1.0 Hz, 1H), 7.26 (dd, J = 7.5, 1.0 Hz, 1H), 7.29-7.35 (m, 1H), 7.45-7.49 (m, 1H), 7.50-7.56 (m, 3H) 7.81 (d, J = 7.3 Hz, 2H), 10.1 (s, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 29.1 (CH2), 114.1 (CH), 120.4 (CH), 129.0 (2xCH), 129.1 (CH), 129.8 (CH), 130.7 (2xCH), 132.8 (CH), 134.9 (C), 135.1 (C), 136.4 (C), 138.9 (C), 154.8 (C), 193.6 (C); LRMS (ESI -) m/z (%) 235 [M-H] - (100); HRMS (ESI -) m/z calc. for C16H11O2 235.0759, found 235.0754.

5.1.1.4. (E)-2-(3,5-dimethoxybenzylidene)-4-hydroxy-2,3-dihydro-1H-inden-1-one (11h

). The crude product was prepared according to the general procedure A starting from 2 (100 mg, 0.68 mmol) -(100); HRMS (ESI -) m/z calc. for C18H15O4 295.0970, found 295.0974.

5.1.1.5. (E)-2-(2,3,4-trimethoxybenzylidene)-4-hydroxy-2,3-dihydro-(1H)-inden-1-one (11i).

The crude product was prepared according to the general procedure A starting from 2 (200 mg, 1.+ ; HRMS (ESI + ) m/z calc. for C20H21O5 341.1389, found 341.1394.

5.1.1.7. (E)-5,7-dimethoxy-2-(3,4,5-trimethoxybenzylidene)-2,3-dihydro-1H-inden-1-one (11o).

The crude product was prepared according to the general procedure A starting from 6 (110 mg, 0.58 mmol) and commercially available 3,4,5-trimethoxybenzaldehyde (114 mg, 0.58 mmol). The pure + (100); HRMS (ESI + ) m/z calc. for C21H23O6 371.1495, found 371.1493.

Acknowledgements

This work has been partially supported by Labex Arcane and CBH-EUR-GS (ANR-17-EURE-0003) and by NeuroCoG IDEX UGA in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02). The NanoBio ICMG platforms (FR 2607) are acknowledged for providing facilities for mass spectrometry analyses (A. Durand, L. Fort, R. Gueret) and for NMR analyses (M.-C. Molina).

Appendix A. Supplementary data

Supplementary data ( 1 H and 13 C NMR spectra) related to this article can be found at http://dx.doi.org/