domain: chim sdv

text:
Amel Meguellati

Abdelhakim Ahmed-Belkacem

Wei Yi

Romain Haudecoeur

Marie Crouillère

Rozenn Brillet

Jean-Michel Pawlotsky

Ahcène Boumendjel

Marine Peuchmaur
email: marine.peuchmaur@ujf-grenoble.fr

B-ring modified aurones as promising allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase

Keywords: Aurones, Hepatitis C Virus, RNA-dependent RNA polymerase

Following our recent report showing the potential of naturally occurring aurones (2benzylidenebenzofuran-3(2H)-ones) as anti-hepatitis C virus (HCV) agents, efforts were continued in order to refine the structural requirements for the inhibitory effect on HCV RNA-dependent RNA polymerase (RdRp). In this study, we targeted the B-ring moiety of aurones with the aim to improve structural features associated with higher inhibition of the targeted polymerase. In vitro evaluation of the RdRp inhibitory activity of the 37 newly synthesized compounds pointed out that the replacement of the B-ring with a N-substituted indole moiety induced the highest inhibitory effect.

Of these, compounds 31, 40 and 41 were found to be the most active (IC50 = 2.3-2.4 µM). Docking experiments performed with the most active compounds revealed that the allosteric thumb pocket I of RdRp is the binding pocket for aurone analogues.

Introduction

Belonging to the Hepacivirus genus of the Flaviviridae family, hepatitis C virus (HCV) is responsible for a chronic infection of global health concern since it affects more than 170 million individualsapproximately 3% of the world population. The acute infection is often asymptomatic.

Nevertheless, HCV infection can become chronic and chronic hepatitis C can progress to cirrhosis, end-stage liver disease, hepatocellular carcinoma and death [START_REF] Hatzakis | The state of hepatitis B and C in Europe: report from the hepatitis B and C summit conference*[END_REF][START_REF] Gravitz | Introduction: a smouldering public-health crisis[END_REF]. To date, there is no prophylactic anti-HCV vaccine. Until the recent approval of two protease inhibitors, the standard treatment was the use of a combination of pegylated interferon α (peg-IFN) and ribavirin (RBV) [START_REF] Pawlotsky | Development of new drugs and clinical trials: promises and pitfalls[END_REF][START_REF] Zeuzem | Expert opinion on the treatment of patients with chronic hepatitis C[END_REF]. However this non-specific therapy has significant side-effects and was of limited efficiency since only a 40-50% sustained virologic response rate (SVR) was reached in patients infected with HCV genotype 1, the most prevalent genotype in Europe and North America [START_REF] Schiffer | Fighting a defiant virus[END_REF][START_REF] Fried | Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus Infection[END_REF]. The approval in 2011 of the first-generation HCV NS3 protease inhibitors telaprevir and boceprevir in combination with pegylated interferon and ribavirin improved SVR rates in patients with HCV genotype 1 infection and eventually shortened treatment duration [START_REF] Hofmann | A new standard of care for the treatment of chronic HCV infection[END_REF]. However, these therapies were poorly tolerated and resistance to the protease inhibitor emerged as an issue in patients who failed on therapy.

Therefore, the need for new direct-acting antivirals (DAAs) with a high therapeutic index is of importance [START_REF] Schlütter | Therapeutics: new drugs hit the target[END_REF][START_REF] Rice | Perspective: miles to go before we sleep[END_REF].

Increased understanding of the HCV life cycle has resulted in the discovery of several viral targets for specific antiviral therapy [START_REF] Opar | Excitement grows for potential revolution in hepatitis C virus treatment[END_REF]. HCV is a small, positive-sense, single-stranded RNA virus with a 9.6 kb-genome that is translated into a polypeptide of approximately 3000 amino acids. This polyprotein can be cleaved by a combination of host and viral proteases into ten viral proteinsstructural (core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A NS4B, NS5A and NS5B) ones [START_REF] Schaefer | Anti-hepatitis C virus drugs in development[END_REF]. Amongst HCV proteins investigated as targets for anti-HCV therapeutic development, non-structural proteins NS3 and NS5B seem to be the most promising candidates [START_REF] Wegzyn | Antiviral drug advances in the treatment of human immunodeficiency virus (HIV) and chronic hepatitis C virus (HCV)[END_REF][START_REF] Deore | NS5B RNA dependent RNA polymerase inhibitors: the promising approach to treat hepatitis C virus infections[END_REF].

The NS5B RNA-dependent RNA polymerase (RdRp) is a particularly attractive target because it is the key enzyme that catalyzes viral replication, with no close mammalian counterpart. This 65kDa enzyme shares the common structural features of other viral polymerases, shaped as a 'right hand' with characteristic thumb, finger and palm domains [START_REF] Bressanelli | Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus[END_REF][START_REF] Lesburg | Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site[END_REF][START_REF] Ago | Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus[END_REF][START_REF] Bressanelli | Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides[END_REF]. In addition to the catalytic site, at least four allosteric binding sites have been identified so far, including 'thumb' pockets I and II and 'palm' pockets I and II/III [START_REF] Barreca | Allosteric inhibition of the hepatitis C virus NS5B polymerase: in silico strategies for drug discovery and development[END_REF][START_REF] Beaulieu | Recent advances in the development of NS5B polymerase inhibitors for the treatment of hepatitis C virus infection[END_REF][START_REF] Pauwels | Binding-site identification and genotype profiling of hepatitis C virus polymerase inhibitors[END_REF][START_REF] Wang | Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase[END_REF]. A number of HCV RdRp inhibitors have been identified, including nucleoside/nucleotide analogues that target the active site, and non-nucleoside allosteric inhibitors [START_REF] Haudecoeur | Structure-activity relationships in the development of allosteric hepatitis C virus RNA-dependent RNA polymerase inhibitors: ten years of research[END_REF][START_REF] Sofia | Nucleoside, nucleotide, and nonnucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase[END_REF]. Both types of drugs have been shown to be important in new interferon-free, oral drug combination strategies against HCV.

Recently, we showed that naturally occurring aurones (2-benzylidenebenzofuran-3(2H)-ones, Fig. 1)small molecules belonging to the flavonoid familyhave potent anti-HCV properties in vitro [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF]. In this study, it was proven that aurones, represented by the naturally occurring aureusidin (3',4,4',6-tetrahydroxyaurone, 1, Fig. 1), potently inhibit HCV RdRp , with an IC50 of 5.2 µM.

Moreover, the binding site of this family of compounds was unambiguously identified to be thumb pocket I using site-directed mutagenesis experiments confirmed by molecular modeling. This first structure-activity relationship study allowed us to partially identify the key positions and substituents that are important for the inhibitory activity of HCV RdRp. Among others, the 4,6dihydroxybenzofuran-3(2H)-one moiety was found to be crucial for the inhibition activity. Based on these results, the aim of the present investigation was to maintain the 4,6-dihydroxybenzofuran-3(2H)-one moiety and to determine the aryl type that could replace the B-ring of aurones in order to enhance the inhibitory activity on HCV RdRp. The aryl ring replacing the B-phenyl of aurones was chosen among substituted phenyls, bicyclic, aryl fused moieties or small oxygen-or nitrogen-containing heterocycles. Finally, the investigation of aurones as potential anti-HCV drugs was motivated by the very low toxicity of naturally occurring aurones and derivatives. Even at high concentration (> 400 µM), aurones were not cytotoxic (MTT assay on HuH7 and HEK293) [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF].

Results and discussion

Chemistry

To synthesize the aurones of interest, we followed a previously reported general and convenient synthetic pathway [START_REF] Beney | An efficient synthesis of 4,6-dimethoxyaurones[END_REF][START_REF] Okombi | Discovery of benzylidenebenzofuran-3(2H)-one (Aurones) as inhibitors of tyrosinase derived from human melanocytes[END_REF] involving an aldol condensation between arylaldehydes and 4,6dihydroxybenzofuran-3(2H)-one under basic conditions (Scheme 1). The synthesis of aurone derivatives 4-38 was accomplished according to Scheme 1.

The prerequisite 4,6-dihydroxybenzofuran-3(2H)-one 3 was first synthesized in two steps. Reaction of phloroglucinol with chloroacetonitrile in HCl/Et2O according to Houben-Hoesch reaction afforded the chloroacetophenone 2 which was directly cyclised in acidic media to afford the 4,6dihydroxybenzofuran-3(2H)-one 3 with 84 % yield for two steps. Most of aromatic aldehydes used in the aldol condensation were commercially available except 2,4-dibutoxybenzaldehyde, employed for the synthesis of compound 7 (Table 1), and indole derivatives, used for the synthesis of compounds 20-38 (Table 2). The 2,4-dibutoxybenzaldehyde was synthesized starting from 2,4-dihydroxybenzaldehyde and using 1-bromobutane and potassium carbonate in DMF [START_REF] Pollard | Supramolecular Assemblies Formed on an Epitaxial Graphene Superstructure[END_REF] while N-substituted indolecarboxaldehydes were obtained by alkylation of the corresponding indolecarboxaldehyde with alkyl halides in the presence of sodium hydride 60% (41-99 %, Scheme 2).

i

N H N 39a Bu CHO CHO i N H 39b-p OHC R' R'' N OHC R' R'' R Scheme

In vitro evaluation of HCV-NS5B21 polymerase activity inhibition

An enzyme assay was used to assess the inhibition of the polymerase activity of a purified RdRp (NS5B protein), deleted of its 21 C-terminal amino acids in order to ensure solubility (HCV-NS5B21). The J4 genotype 1b reference strand was used. This assay measures the amount of double-stranded RNA synthesized in the presence of HCV-NS5B21, a homopolymeric RNA template and ATP, as previously described [START_REF] Ahmed-Belkacem | Silibinin and related compounds are direct inhibitors of hepatitis C virus RNAdependent RNA polymerase[END_REF]. Initial screening was performed at 20 µM concentration of aurone followed by the measurement of IC50 of those that showed higher activity at 20 µM.

Our previous study [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF] pointed out some pharmacophoric elements that were crucial for the inhibitory activity of aurones on HCV RdRp: (a) a hydroxyl group at position 4 or hydroxyl groups at positions 4 and 6 and (b) a 2',4'-or 3',4'-dihydroxylated B-ring or a hydrophobic and bulky substituent or alternative core at the B-ring. In order to refine this first study, new aurones were then synthesized. Firstly, to expand the structure-activity relationship and to improve our knowledge concerning the binding mode of this class of compounds with their target, new synthesized aurone derivatives were evaluated in which the B-ring was replaced by: (a) phenyl ring bearing weak polar and more or less bulky substituents (4-8, Table 1), (b) bis-aryl or fused aryl rings [START_REF] Rice | Perspective: miles to go before we sleep[END_REF][START_REF] Opar | Excitement grows for potential revolution in hepatitis C virus treatment[END_REF][START_REF] Schaefer | Anti-hepatitis C virus drugs in development[END_REF][START_REF] Wegzyn | Antiviral drug advances in the treatment of human immunodeficiency virus (HIV) and chronic hepatitis C virus (HCV)[END_REF]. Most of these compounds presented a fairly good activity, in the micromolar range, except for compounds 4 and 11 whose IC50 values were not determined.

During our previous study, it was shown that aurone derivatives bearing an indolyl moiety were promising NS5B inhibitors (compound 19, IC50 = 2.2 µM, Table 2) [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF]. We then naturally proceeded in our study both synthesizing new heterocyclic aurones and N-functionalized indoles.

The B-ring was subsequently exchanged with quinoleine, benzoimidazole, imidazole, pyrrole and furane cores (13-18, Table 1), but unfortunately none of these heterocycles gave a noticeable gain of activity. We can therefore conclude that the inhibition of HCV-NS5BΔ21 polymerase activity revealed that B-ring can be replaced by a variety of aryls but no smaller than phenyl, indicating that the B-ring of aurones might interact with a large pocket within the active site. except more polar substituents [START_REF] Okombi | Discovery of benzylidenebenzofuran-3(2H)-one (Aurones) as inhibitors of tyrosinase derived from human melanocytes[END_REF]. These results tend to confirm the interaction pattern of aurones with NS5B previously described for compound 19 [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF]. Indeed, the binding pocket appears to be quite large, since even bulky substituents can be accommodated (e.g. IC50 = 2.7 µM for N-phenylpropyl compound 33), and rather hydrophobic, as previously stated (the B-ring was expected to be surrounded by residues Leu392, Val494, Ile424 and Leu425). Finally, diversely substituted N-butyl indole cores were evaluated (compounds 34-38 and 41-42). Once again, these modifications were well tolerated except for the 5-hydroxy substitution of 42 which induced a great loss of activity. Nonetheless the same polar substituent at position 7 of the indole is not deleterious (IC50 = 2.3 µM for 41).

Altogether, through these results, the attachment position to the benzofuranone core and the nature of indole N-, 5-or 7-substituent revealed having far less influence. The majority of indole analogues evaluated in this study were found in a narrow range of inhibition activity (between 2.0 and 3.5 µM for at least 12 compounds). This high level of tolerance confirmed that general hydrophobic, Van der Waals interactions play a prominent role at the B-ring of aurones; therefore it might be difficult to enhance the efficiency of aurones as NS5B inhibitors by simply further modulating this position. b These yields correspond to the last step of the synthesis (hydrolysis in presence of BBr3)

Interaction of aurone derivatives with the allosteric thumb pocket I of NS5B

Using site-directed mutagenesis studies, we unambiguously identified the thumb pocket I as the binding site of aurones [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF]. This information was used to dock representatives of studied aurones into the binding pocket using Autodock tool (Fig. 2). The docking results revealed the presence of two hot spot residues, Arg503 and Gly493, interacting with 4-and 6-hydroxy groups of aurones, respectively, as already described for compound 19. While the general geometry in the binding site remains very similar for each analogue, the benzofuranone cores of indol-2-ylmethylene derivatives such as compound 21 are inverted, and subsequently build hydrogen bonds with Arg503 and Gly493 with 6-hydroxy and 4-hydroxy substituents respectively. Interestingly, compound 41, bearing a hydroxyl group at C-7 of the indole ring establishes an extra interaction with a leucine residue (Leu392). The latter interaction may explain the higher activity of 41 compared to its closed analogue 42 bearing a hydroxyl group at C-5 of the indole ring. Overall, the close IC50 values obtained for these compounds is also seen in terms of geometry and predicted free energies of binding (between -7.90 and -6.90 kcal/mol for almost all compounds).

Conclusion

In conclusion, we report here on the investigation of compounds derived from the naturally occurring aurones as HCV RdRp inhibitors. Our main objective was the pharmacomodulation of the B-ring of aurones in order to identify the optimal aryl ring. The first finding highlighted the importance of the aryl size with the indole ring being the most favorable among diverse aryl rings tested. The majority of the aurones bearing indole rings exerted a remarkable inhibition activity on HCV RdRp with IC50 values in the one digit micromolar order. Modeling studies have suggested the ability of the aurone structures to establish key interactions with three residues surrounding the thumb pocket I of HCV RdRp. The promising activity of aurones combined with their very low toxicity may place them at the center of future anti-HCV drug research.

Experimental

Chemistry

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. Melting points were measured on a Fisher micromelting point apparatus and are uncorrected.

4,6-dihydroxybenzofuran-3(2H)-one (3)

To a solution of phloroglucinol (15.0 g, 119 mmol) in diethyl ether (250 mL) at 0°C were added chloroacetonitrile (7.5 mL, 119 mmol) and dried zinc chloride (1.62 g, 11.9 mmol). The mixture was allowed to react with gaseous hydrochloric acid at 0°C for 1 h. The resulting suspension was stirred at 0°C for 1.5 h, and was allowed to react again with gaseous HCl for 30 min. The precipitate was filtered, washed with ether, dried and suspended in water (200 mL). The solution was refluxed for 5 h. After cooling, the crystals were filtered, washed with cold water and dried to

General procedure A for the synthesis of compounds (4-18 and 20-38)

To a solution of 3 in ethanol (3 mL/mmol) were added an aqueous solution of potassium hydroxide (50%, 5 mL/mmol) and a benzaldehyde derivative (1.0 to 3.5 equiv.). The solution was refluxed until TLC showed complete disappearance of the starting material (2 to 5 hours). After cooling, ethanol was removed under reduced pressure, then the residue was diluted into distilled water (50 mL/mmol) and an aqueous solution of hydrochloric acid (10%) was added to adjust the pH to 2 -3.

The mixture was then extracted with ethyl acetate or dichloromethane. The combined organic layers were washed with water and brine, dried over MgSO4, filtered off and concentrated under reduced pressure to afford the corresponding crude (Z)-2-benzylidenebenzofuran-3(2H)-one derivative.

4.1.2.1. (Z)-2-(4-butoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (4)
The crude product was prepared according to procedure A starting from

(Z)-2-(2,4-dibutoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (5)

The crude product was prepared according to procedure A starting from 3 (166 mg, 1.00 mmol) and 2,4-dibutoxybenzaldehyde [START_REF] Okombi | Discovery of benzylidenebenzofuran-3(2H)-one (Aurones) as inhibitors of tyrosinase derived from human melanocytes[END_REF] (376 mg, 1.5 mmol). After purification by column chromatography on silica gel (EtOAc/MeOH 98:2), the pure product (271 mg, 0.68 mmol, 68%)

was obtained as a red powder. m.p. = 161-162°C;

(Z)-2-(4-trifluoromethylbenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (8)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol)

(Z)-2-(4-(2-thienyl)benzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (9)

The crude product was prepared according to procedure A starting from

(Z)-2-((1-methyl-1H-pyrrol-2-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (16)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol) and

(Z)-2-((5-hydroxymethyl)furan-2-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (18)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol)

and

2-(N-butylindol-5-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (20)

The crude product was prepared according to procedure A starting from 3 (166 mg, 1.00 mmol) and aldehyde 39a (300 mg, 1.50 mmol). After purification by column chromatography on silica gel (Cyclohexane/EtOAc 2:1), the pure product (252 mg, 0.72 mmol, 72%) was obtained as a red powder. m.p. 209-210°C;

2-(N-methylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (23)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol) and 1-methylindole-3-carboxaldehyde (200 mg, 1.36 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 1:1), the pure product (144 mg, 0.47 mmol, 78%) was obtained as a red powder. m.p. > 300°C; 1

2-[N-(2-methylpropyl)indol-3-ylmethylene]-4,6-dihydroxybenzofuran-3(2H)-one (24)

The crude product was prepared according to procedure A starting from

2-[N-(3-carboxypropyl)indol-3-ylmethylene]-4,6-dihydroxybenzofuran-3(2H)-one (26)

The crude product was prepared according to procedure A starting from

2-(N-cyclohexylmethylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (27)

The crude product was prepared according to procedure A starting from 3 (207 mg, 1.

2-(N-3-methylbenzylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (29)

The crude product was prepared according to procedure A starting from 3 (200 mg, 1.20 mmol) and aldehyde 39h (200 mg, 0.80 mmol). After purification by column chromatography on silica gel (CH2Cl2/MeOH 9:1) and recrystallization in acetonitrile, the pure product (171 mg, 0.43 mmol, 54%) was obtained as orange crystals. m.p. 262-263°C;

2-(N-3-methoxybenzylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (30)

The crude product was prepared according to procedure A starting from 3 (150 mg, 0.90 mmol) and aldehyde 39i (450 mg, 1.78 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 1:1), the pure product (268 mg, 0.65 mmol, 72%) was obtained as a yellow powder. m.p. 248-249°C;

2-(N-3-bromobenzylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (31)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol)

and N-(3-bromobenzyl)indole-3-carboxaldehyde [START_REF] Wu | Analogues and derivatives of oncrasin-1, a novel inhibitor of the C-terminal domain of RNA polymerase II and their antitumor activities[END_REF] (400 mg, 1.27 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 1:1), the pure product (171 mg, 0.37 mmol, 62%) was obtained as a yellow powder. m.p. 151-152°C;

HRMS (ESI+) m/z calc. for C24H17BrNO4 462.0335, found 462.0334.

2-(N-2-phenylethylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (32)

The crude product was prepared according to procedure A starting from 3 (200 mg, 1.20 mmol) and aldehyde 39j (200 mg, 0.80 mmol). After purification by column chromatography on silica gel (CH2Cl2/MeOH 9:1) and recrystallization in acetonitrile, the pure product (145 mg, 0.36 mmol, 46%) was obtained as orange crystals. m.p. > 240°C (decomposition);

General procedure B for the synthesis of N-alkyl-indole-3-carboxaldehyde (39a-39p)

To a suspension of NaH 60% in oil (2.25 equiv.) in dry DMF (0.8 mL/mmol) was added, at 0°C and under nitrogen atmosphere, a solution of indolecarboxaldehyde (1 equiv.) in dry DMF (2.5 mL/mmol). After stirring for 30 min. at rt, alkyl halide (1.0 to 3.0 equiv.) was slowly added. After stirring overnight, the reaction was quenched by addition of water and the product was extracted with diethyl ether. The organic layer was dried over MgSO4, filtered off and concentrated under vacuum. The crude product was purified by column chromatography on silica gel.

N-butylindole-5-carboxaldehyde (39a)

The crude product was prepared according to procedure B starting from indole-5-carboxaldehyde (200 mg, 1.37 mmol) and 1-bromobutane (377 mg, 2.75 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 9:1), the pure product (134 mg, 0.67 mmol, 49%) was obtained as a yellow oil.

N-(3-methylbut-2-enyl)-indole-3-carboxaldehyde (39d)

The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde (726 mg, 5.00 mmol) and 1-bromo-

N-(cyclohexylmethyl)-indole-3-carboxaldehyde (39f)

The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde (300 mg, 2.07 mmol) and bromomethylcyclohexane (549 mg, 3.10 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (260 mg, 1.08 mmol, 52%) was obtained as a pink powder. m.p. 94-95°C; 1

N-benzyl-indole-3-carboxaldehyde (39g)

The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde

(

Fig. 1 .

 1 Fig. 1. General structure of the aurone backbone (top left), structure and activity of the naturally occurring aureusidin 1 and structures of aurone derivatives investigated in this study (the dashed line indicate the site of linkage).

1 .

 1 Scheme 1. General pathway for the synthesis of aurone derivatives. Reagents and conditions: (i) ClCH2CN, HCl, ZnCl2, Et2O, 0°C; (ii) H2O, 100°C, 5 h, 84 % (for 2 steps); (iii) ArCHO, KOH 50% in H2O, EtOH, reflux, 2-

2 .Scheme 3 .

 23 Scheme 2. Synthesis of indolecarboxaldehyde derivatives. Reagents and conditions: (i) RX (X = Br or Cl), NaH 60% in oil, DMF, rt, 18-72 h, 41-99 %.

Fig. 2 .

 2 Fig. 2. Surface views of predicted binding modes in RdRp thumb pocket I of a reported X-ray structure (PDB code 2dxs) from docking studies with Autodock. (A) Compound 2. (B) Compound 21. (C) Compound 33. (D) Compound 41.

  1 H and13 C NMR spectra were recorded at room temperature in deuterated solvents on a Brüker AC-400 instrument (400 MHz). Chemical shifts () are reported in parts per million (ppm) relative to TMS as internal standard or relative to the solvent [ 1 H: (DMSO) = 2.50 ppm, (CDCl3) = 7.24 ppm;13 C: (DMSO) = 39.51 ppm, (CDCl3) = 77.23 ppm]. Electrospray ionization ESI mass spectra were acquired by the Analytical Department of Grenoble University on an Esquire 300 Plus Bruker Daltonis instrument with a nanospray inlet. Accurate mass measurements (HRMS) were carried out on a TOF spectrometer, realized by the mass spectrometry analysis facilities of Orleans University (France).

afford 4 (

 4 16.6 g, 100 mmol, 84 %) as orange crystals, which were analytically pure and used without further purification. m.p. = 257-259°C [29]; 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.61 (s, 1H), 10.58 (s, 1H), 5.91 (s, 2H), 4.55 (s, 2H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 193.9 (C), 175.6 (C), 167.5 (C), 157.4 (C), 102.6 (C), 96.1 (CH), 90.1 (CH), 74.8 (CH2).

and 4 -

 4 trifluoromethylbenzaldehyde (200 mg, 1.15 mmol). After purification by column chromatography on silica gel (EtOAc), the pure product (138 mg, 0.43 mmol, 71%) was obtained as a yellow powder. m.p. 262-263°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.07 (bs, 2H), 8.08 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 6.69 (s, 1H), 6.23 (d, J = 1.2 Hz, 1H), 6.10 (d, J = 1.2 Hz, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 178.8 (C), 167.9 (C), 167.8 (C), 158.7 (C), 149.0 (C), 136.5 (C), 131.0 (2xCH), 128.5 (q, J = 32 Hz, CF3), 125.6 (2xCH), 122.7 (C), 106.0 (CH), 102.2 (C), 98.0 (CH), 90.7 (CH); LRMS (ESI+) m/z (%) 323 (100) [M+H] + ; HRMS (ESI+) m/z calc. for C16H10O4F3 323.0526, found 323.0527.

  7 (C), 130.0 (C), 128.2 (CH), 116.3 (CH), 115.0 (CH), 107.5 (CH), 102.4 (C), 98.0 (CH), 90.6 (CH); LRMS (ESI-) m/z (%) 293 (100) [M-H] -.4.1.2.12. (Z)-2-((1H-imidazol-5-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (15)The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol) and 1H-imidazole-5-carboxaldehyde (116 mg, 1.21 mmol), and was purified by column chromatography on silica gel (CH2Cl2/methanol 9:1) to afford 15 as a pure yellow solid (142 mg, 0.58 mmol, 97%). m.p. > 260 °C (decomposition); 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.87 (bs, 2H), 7.86 (s, 1H), 7.67 (s, 1H), 6.55 (s, 1H), 6.21 (s, 1H), 6.07 (s, 1H);13 C NMR (100 MHz, DMSO-d6) δ ppm 178.5 (C), 167.3 (C), 167.0 (C), 158.1 (C), 145.9 (C), 137.3 (CH), 130.1 (C), 125.0 (CH), 103.1 (C), 101.3 (CH), 97.6 (CH), 90.5 (CH). LRMS (ESI-) m/z (%) 243 (100) [M-H] -.

  H NMR (400 MHz, CDCl3) δ ppm 10.00 (s,1H), 8.34-8.29 (m, 1H), 7.66 (s, 1H), 7.40-7.28 (m, 3H), 3.98 (d, J = 7.2 Hz, 2H), 1.95-1.82 (m, 1H), 1.77-1.60 (m, 5H), 1.29-1.13 (m, 3H), 1,08-0,96 (m, 2H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.6 (CH), 139.1 (CH), 137.7 (C), 125.5 (C), 124.0 (CH), 122.9 (CH), 122.2 (CH), 118.0 (C), 110.5 (CH), 53.9 (CH2), 38.4 (CH), 31.1 (2xCH2), 26.3 (2xCH2), 25.7 (CH2).

Table 1

 1 Structures and inhibition activity of synthesized aurone derivatives. The dashed bound indicates the site of attachment to the methylenebenzofuranone moiety.

	Compd	Ar	Yield	Inhibition (20 M, %)	IC50 (M)
			(%)
	4	O	63	81 ± 3	n.d.
	5	O	68	90  3	4  2
	O
	6	N	68	90  1	5  3
	7	N	69	93  1	5.5  0.9
	8	CF 3	71	96.00  0.04	5.4  0.4
	9		66	97.0  0.1	4  2
		S
	10		78	93  1	4  1
	11		81	45  27	n.d.

Table 2

 2 Structures and inhibition activity of synthesized aurone indolic derivatives. The dashed bound indicates the site of attachment to the methylenebenzofuranone moiety.

	Compd	Ar	Yield	Inhibition (20 M, %)	IC50 (M)
			(%)
	19 a		65	95 ± 1	2.2 ± 0.2
	N
	20		72	94.3 ± 0.6	n.d.
		N
	21		54	98.4 ± 0.5	3.00 ± 0.04
	N
	22		66	97.8 ± 0.0	3.10 ± 0.08
	N
	23		78	90.0 ± 0.9	6 ± 1
	N
	24		81	80.70  0.07	8  3
	N
	25		80	94.7 ± 0.9	3.0 ± 0.1
	N
	26		56	70 ± 2	n.d.
		CO 2 H
	N
	27		87	93  1	3.3  0.4
	N
	28		73	96.2  0.5	2.6  0.6
	N
	29		54	95.6  0.2	2.6  0.2
	N

  3 (166 mg, 1.00 mmol) and 4-butoxybenzaldehyde (356 mg, 2.0 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 1:2), the pure product (205 mg, 0.63 mmol, 63%) was obtained as a yellow powder. m.p. = 218-219°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.88 (bs, 2H), 7.84 (d, J

= 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 6.58 (s, 1H), 6.21 (d, J = 1.6 Hz, 1H), 6.07 (d, J = 1.6 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 1.76-1.66 (m, 2H), 1.50-1.38 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 179.0 (C), 167.6 (C), 167.1 (C), 159.5 (C), 158.2 (C), 146.4 (C), 132.5 (2xCH), 124.7 (C), 114.9 (2xCH), 108.5 (CH), 102.7 (C), 97.7 (CH), 90.5 (CH), 67.3 (CH2), 30.6 (CH2), 18.7 (CH2), 13.7 (CH3); LRMS (ESI+) m/z (%) 327 (100) [M+H] + ; HRMS (ESI+) m/z calc. for C19H19O5 327.1227, found 327.1227.

  . m.p. > 280°C;1 H NMR (400 MHz, DMSO-d6) δ ppm 11.15 (s, 1H), 11.08 (s, 1H), 8.47 (d, J = 7.4 Hz, 1H), 8.25 (d, J = 7.4 Hz, 1H), 8.10-7.94 (m, 2H), 7.85-7.74 (m, 1H), 7.69-7.57 (m, 1H), 6.68 (s, 1H), 6.30 (s, 1H), 6.13 (s, 1H);13 C NMR (100 MHz, DMSO-d6) δ ppm 178.9 (C),The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol) and 1H-benzo[d]imidazole-5-carboxaldehyde (98 mg, 0.60 mmol), and was washed three times with distilled water to afford 14 as a yellow solid, which was analytically pure and used without further purification (133 mg, 0.45 mmol, 75%). m.p. > 260 °C (decomposition); 1 H NMR (400 MHz,

	4.1.2.7. (Z)-2-(4-phenylbenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (10)
	The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol)
	and 4-phenylbenzaldehyde (200 mg, 1.10 mmol). After purification by column chromatography on
	silica gel (EtOAc/MeOH 98:2), the pure product (155 mg, 0.47 mmol, 78%) was obtained as a 168.1 (C), 168.0 (C), 158.8 (C), 152.2 (C), 150.1 (C), 147.8 (C), 136.7 (CH), 130.1 (CH), 129.0
	yellow powder. m.p. = 277-278°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.96 (s, 2H), 7.99 (d, J = (CH), 127.8 (CH), 127.3 (CH), 126.8 (C), 122.4 (CH), 108.0 (CH), 102.1 (C), 98.0 (CH), 90.9 (CH);
	8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.76-7.71 (m, 2H), 7.53-7.46 (m, 2H), 7.43-7.36 (m, 1H), 6.67 LRMS (ESI+) m/z (%) 306 (100) [M+H] + ; HRMS (ESI+) m/z calc. for C18H12NO4 306.0761, found
	(s, 1H), 6.25 (d, J = 1.6 Hz, 1H), 6.10 (d, J = 1.6 Hz, 1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 306.0761.
	178.9 (C), 167.8 (C), 167.4 (C), 158.4 (C), 147.8 (C), 140.5 (C), 139.3 (C), 131.5 (C), 131.2
	(2xCH), 129.0 (2xCH), 127.9 (CH), 127.0 (2xCH), 126.7 (2xCH), 107.7 (CH), 102.5 (C), 97.8 (CH), 4.1.2.11. (Z)-2-((1H-benzo[d]imidazol-5-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (14)
	90.6 (CH); LRMS (ESI+) m/z (%) 331 (100) [M+H] + ; HRMS (ESI+) m/z calc. for C21H15O4 331.0965,
	found 331.0965.
	powder. m.p. = 276-277°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.02 (s, 2H), 8.41 (s, 1H), 8.08-
	3 (100 mg, 0.60 mmol) 7.93 (m, 4H), 7.57 (t, J = 6.4 Hz, 2H), 6.77 (s, 1H), 6.30 (s, 1H), 6.11 (s, 1H); 13 C NMR (100 MHz,
	and 4-(2-thienyl)benzaldehyde (200 mg, 1.06 mmol). After purification by column chromatography DMSO-d6) δ ppm 179.0 (C), 167.9 (C), 167.5 (C), 158.5 (C), 148.0 (C), 132.9 (C), 132.8 (C), 130.8
	on silica gel (EtOAc/MeOH 98:2), the pure product (134 mg, 0.40 mmol, 66%) was obtained as a (CH), 130.1 (C), 128.4 (2xCH), 127.6 (CH), 127.3 (CH), 127.2 (CH), 126.7 (CH), 108.2 (CH), 102.5
	red powder. m.p. 268-269°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.01 (s, 2H), 7.94 (d, J = 8.0 (C), 97.9 (CH), 90.7 (CH); LRMS (ESI+) m/z (%) 305 (100) [M+H] + ; HRMS (ESI+) m/z calc. for
	Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.63 (dd, J = 6.0, 4.0 Hz , 2H), 7.18 (dd, J = 6.0, 4.0 Hz , 1H), C19H13O4 305.0808, found 305.0808.
	6.63 (s, 1H), 6.23 (s, 1H), 6.08 (1H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 178.9 (C), 167.7 (C),

167.5 (C), 158.5 (C), 147.8 (C), 142.7 (C), 134.2 (C), 131.5 (C), 131.4 (2xCH), 128.7 (CH), 126.6 (CH), 125.7 (2xCH), 124.6 (CH), 107.6 (CH), 102.5 (C), 97.8 (CH), 90.6 (CH); LRMS (ESI+) m/z (%) 337 (100) [M+H] + ; HRMS (ESI+) m/z calc. for C19H13O4S 337.0529, found 337.0528.

4.1.2.8. (Z)-2-(1-naphthylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (11)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol) and 1-naphthaldehyde (200 mg, 1.28 mmol). After purification by column chromatography on silica gel (EtOAc/MeOH 98:2), the pure product (148 mg, 0.49 mmol, 81%) was obtained as a yellow powder. m.p. > 280°C;

1

H NMR (400 MHz, DMSO-d6) δ ppm 11.26 (bs, 2H), 8.27 (dd, J = 7.8, 7.8 Hz, 1H), 8.01 (dd, J = 7.8, 2.4 Hz, 2H), 7.68-7.58 (m, 3H), 7.26 (s, 2H), 6.31 (s, 1H), 6.28 (s, 1H);

13 C NMR (100 MHz, DMSO-d6) δ ppm 178.8 (C), 167.8 (2xC), 158.8 (C), 148.9 (C), 133.3 (C), 131.3 (C), 129.5 (CH), 128.9 (CH), 128.8 (CH), 128.2 (C), 127.1 (CH), 126.2 (CH), 125.8 (CH), 123.2 (CH), 103.1 (CH), 102.4 (C), 98.0 (CH), 90.6 (CH); LRMS (ESI+) m/z (%) 305 (100) [M+H] + ; HRMS (ESI+) m/z calc. for C19H13O4 305.0808, found 305.0808.

4.1.2.9. (Z)-2-(2-naphthylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (12)

The crude product was prepared according to procedure A starting from 3 (100 mg, 0.60 mmol) and 2-naphthaldehyde (200 mg, 1.28 mmol). After purification by column chromatography on silica gel (EtOAc/MeOH 98:2), the pure product (161 mg, 0.53 mmol, 88%) was obtained as a yellow

4.1.2.10. (Z)-2-(2-quinolinemethylene)-4,6-dihydroxybenzofuran-3(2H)-one (13)

The crude product was prepared according to procedure A starting from 3 (120 mg, 0.72 mmol) and 2-quinolinecarboxaldehyde (200 mg, 1.27 mmol). After purification by column chromatography on silica gel (EtOAc/MeOH 94:6), the pure product (186 mg, 0.59 mmol, 81%) was obtained as a yellow powderDMSO-d6) δ ppm 11.16 (bs, 2H), 9.47 (s, 1H), 8.37 (s, 1H), 8.04 J = 8.2 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 6.82 (s, 1H), 6.28 (s, 1H), 6.16 (s, 1H);

13

C NMR (100 MHz, DMSO-d6) δ ppm 178.9 (C), 167.7 (2xC), 158.6 (C), 148.0 (C), 141.9 (CH), 132.1 (C), 131.

  1 H NMR (400 MHz, DMSO-d6) δ

	ppm 10.78 (s, 1H), 10.75 (s, 1H), 8.32 (s, 1H), 7.99 (d, J = 7.3 Hz, 1H), 7.60-7.40 (m, 3H), 7.35-
	7.13 (m, 4H), 6.98 (s, 1H), 6.22 (d, J = 1.5 Hz, 1H), 6.08 (d, J = 1.5 Hz, 1H), 5.60 (s, 2H); 13 C NMR
	(100 MHz, DMSO-d6) δ ppm 178.4 (C), 167.0 (C), 166.6 (C), 158.0 (C), 145.8 (C), 136.3 (CH),

136.1 (C), 133.3 (C), 132.9 (CH), 129.7 (CH), 128.3 (C), 128.2 (CH), 127.3 (CH), 122.9 (C), 122.0 (CH), 121.1 (CH), 119.3 (CH), 110.7 (CH), 108.3 (C), 103.6 (C), 101.8 (CH), 97.6 (CH), 90.4 (CH), 49.8 (CH2); LRMS (ESI+) m/z (%) 464 (100) [M( 81 Br)+H] + , 462 (100) [M( 79 Br)+H] + , 314

  EtOAc 8:2), the pure product (809 mg, 3.81 mmol, 85%) was obtained as a light grey powder. m.p. 77-78°C; 1 H NMR (400 MHz, CDCl3) δ ppm 9.98 (s, 1H), 8.35-8.24 (m, 1H), 7.72 (s, 1H), 7.41-7.26 (m, 3H), 4.36 (t, J = 6.6 Hz, 2H), 2.38-2.13 (m, 4H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.7 (CH), 138.1 (CH), 137.0 (C), 125.7 (C), 124.6 (CH), 123.5 (CH), 122.6 (CH), 118.9 (C), 118.4 (C), 109.9 (CH), 45.4 (CH2), 25.7 (CH2), 14.8 (CH2).

	column chromatography on silica gel (cyclohexane/EtOAc 9:1), the pure product (885 mg, 4.15
	mmol, 83%) was obtained as a grey powder. m.p. 82-83°C; 1 H NMR (400 MHz, CDCl3) δ ppm 9.94
	(s, 1H), 8.32-8.24 (m, 1H), 7.69 (s, 1H), 7.37-7.25 (m, 3H), 5.43-5.33 (m, 1H), 4.68 (d, J = 7.0 Hz,
	2H), 1.81 (s, 3H), 1.80 (d, J = 0.9 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.6 (CH), 139.1
	(C), 138.0 (CH), 137.4 (C), 125.7 (C), 124.0 (CH), 123.0 (CH), 122.1 (CH), 118.1 (C), 117.9 (CH),
	110.3 (CH), 44.9 (CH2), 25.8 (CH3), 18.3 (CH3).
	4.1.3.5. N-(3-cyanopropyl)-indole-3-carboxaldehyde (39e)
	The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde
	(650 mg, 4.48 mmol) and 5-chloropentanenitrile (1.00 g, 9.66 mmol). After purification by column
	chromatography on silica gel (cyclohexane/

3-methylbut-2-ene (1.49 g, 10.00 mmol). After purification by

  610 mg, 4.20 mmol) and benzyl bromide (1.62 g, 9.46 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (0.98 g, 4.17 mmol, 99%) was obtained as a white solid. m.p. 111-112°C;1 H NMR (400 MHz, CDCl3) δ ppm 10.02 (s, 1H), The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde (610 mg, 4.20 mmol) and 3-methylbenzylbromide (1.78 g, 9.46 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (900 mg, 3.61 mmol, 86%) was obtained as a yellow solid. m.p. 78-79°C; 1 H NMR (400 MHz, CDCl3) δ ppm 10.00 (s, 1H), 8.47-8.42 (m, 1H), 7.69 (s, 1H), 7.40-7.25 (m, 4H), 7.19 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 7.02 (d, J = 7.6 Hz, 1H), 5.27 (s, 2H), 2.37 (s, 3H);13 C NMR (100 MHz, CDCl3) δ ppm 184.5 (CH), 138.8 (CH), 138.7 (C), 137.4 (C), 135.3 (C), 129.0 (CH), 128.9 (CH), 127.9 (CH), 125.4 (C), 124.3 (CH), 124.0 (CH), 122.9 (CH), 122.0 (CH), 118.2 (C), 110.5 (CH), 50.7 (CH2), 21.3 (CH3).The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde (726 mg, 5.00 mmol) and 3-methoxybenzylbromide (1.01 g, 5.00 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 9:1), the pure product (1.06 g, 4.00 mmol, 80%) was obtained as a yellow powder. m.p. 110-111°C;1 H NMR (400 MHz, CDCl3) δ ppm 9.98 (s, 1H), 8.36-8.29 (m, 1H), 7.69 (s, 1H), 7.35-7.25 (m, 4H), 6.85 (dd, J = 8.2, 2.3 Hz, 1H), 6.77-6.73 (m, 1H), 6.72-6.69 (m, 1H), 5.29 (s, 2H), 3.75 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.7 (CH), 160.3 (C), 138.7 (CH), 137.6 (C), 137.0 (C), 130.3 (CH), 125.6 (C), 124.3 (CH), 123.2 (CH), 122.3 (CH), 119.6 (CH), 118.6 (C), 113.5 (CH), 113.4 (CH), 110.5 (CH), 55.4 (CH3), 51.0 The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde (603 mg, 4.15 mmol) and 2-(bromoethyl)benzene (1.74 g, 9.35 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (420 mg, 1.68 mmol, 41%) was obtained as an orange solid. m.p. 72-73°C; 1 H NMR (400 MHz, CDCl3) δ ppm 9.90 (s, 1H), 8.45-8.41 (m, 1H), 7.42-7.37 (m, 4H), 7.33-7.26 (m, 3H), 7.10-7.04 (m, 2H), 4.37 (t, J = 7.1 Hz, 2H), 3.15 (t, J = 7.1 Hz, 2H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.4 (CH), 138.9 (CH), 137.4 (C), 136.8 (CH), 128.7 (2xCH), 128.6 (2xCH), 126.9 (CH), 125.3 (C), 123.9 (CH), 122.8 (CH), 122.1 (CH), 117.8 (C), 110.0 (C), 48.6 (CH2), 35.9 (CH2). The crude product was prepared according to procedure B starting from indole-3-carboxaldehyde (603 mg, 4.15 mmol) and 3-(bromopropyl)benzene (1.88 g, 9.35 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (902 mg, 3.43 mmol, 83%) was obtained as an orange solid. m.p. 49-50°C; 1 H NMR (400 MHz, CDCl3) δ ppm 10.03 (s, 1H), 8.49-8.46 (m, 1H), 7.62 (s, 1H), 7.39-7.26 (m, 6H), 7.21-7.17 (m, 2H), 4.08 (t, J = 7.2

	4.1.3.9. N-(3-methoxybenzyl)-indole-3-carboxaldehyde (39i)
	4.1.3.11. N-(3-phenylpropyl)-indole-3-carboxaldehyde (39k)
	8.42-8.38 (m, 1H), 7.73 (s, 1H), 7.43-7.30 (m, 6H), 7.25-7.20 (m, 2H), 5.37 (s, 2H); 13 C NMR (100
	MHz, CDCl3) δ ppm 184.7 (CH), 138.8 (CH), 137.6 (C), 135.5 (C), 129.2 (2xCH), 128.5 (CH),
	127.3 (2xCH), 125.6 (C), 124.3 (CH), 123.2 (CH), 122.2 (CH), 118.6 (C), 110.6 (CH), 51.0 (CH2).

4.1.3.8. N-(3-methylbenzyl)-indole-3-carboxaldehyde (39h) (CH2).

4.1.3.10. N-(2-phenylethyl)-indole-3-carboxaldehyde (39j)

Hz, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.21-2.11 (dt, J = 7.4, 7.2 Hz, 2H);

13

C NMR (100 MHz, CDCl3) δ ppm 184.2 (CH), 140.1 (CH), 138.5 (C), 136.9 (C), 128.4 (2xCH), 128.1 (2xCH), 126.1 (CH), 125.1 (C), 123.7 (CH), 122.6 (CH), 121.8 (CH), 117.8 (C), 110.0 (CH), 46.0 (CH2), 32.4 (CH2), 30.6 (CH2).

Acknowledgements

The authors are grateful to ANR (Agence Nationale pour la Recherche) for financial support (Labex Arcane (ANR-11-LABX-0003-01)). A.A.-B. and J.-M.P. are supported by ANRS (Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales) and FRM (Fondation pour la Recherche Médicale). The authors also thank the ICMG NMR facilities.

The crude product was prepared according to procedure B starting from 2-methylindole-3carboxaldehyde (418 mg, 2.63 mmol) and 1-bromobutane (330 mg, 2.41 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (308 mg, 1.43 mmol, 59%) was obtained as a yellow powder. m.p. 56-57°C; 1 H NMR (400 MHz, CDCl3) δ ppm 10.08 (s, 1H), 8.27-8.21 (m, 1H), 7.33-7.24 (m, 3H), 4.10 (t, J = 7.5 Hz, 2H), 2.69 (s, 3H), 1.82-1.70 (m, 2H), 1.46-1.34 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ ppm 183.9 (CH), 148.4 (C), 136.7 (C), 126.1 (C), 123.5 (CH), 123.3 (CH), 121.3 (CH), 114.3 (C), 109.9 (CH), 43.6 (CH2), 31.9 (CH2), 20.5 (CH2), 14.0 (CH3), 10.9 (CH3).

N-butyl-7-methylindole-3-carboxaldehyde (39m)

The crude product was prepared according to procedure B starting from 7-methylindole-3carboxaldehyde (400 mg, 2.51 mmol) and 1-bromobutane (685 mg, 5.00 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 9:1), the pure product (372 mg, 1.73 mmol, 69%) was obtained as a yellow powder. m.p. 90-91°C; 1 H NMR (400 MHz, CDCl3) δ ppm 9.94 (s, 1H), 8.17 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.17

N-butyl-7-methoxyindole-3-carboxaldehyde (39n)

The crude product was prepared according to procedure B starting from 7-methoxyindole-3carboxaldehyde (45 mg, 0.25 mmol) and 1-bromobutane (70 mg, 0.51 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 95:5), the pure product (54 mg, 0.23 mmol, 91%) was obtained as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ ppm 9.94 (s, 1H), 7.87 (dd, J = 7.9, 0.8 Hz, 1H), 7.56 (s, 1H), 7.17 (dd, J = 7.9, 7.9 Hz, 1H), 6.72 (dd, J = 7.9, 0.8 Hz, 1H), 4.38 (t, J = 7.2 Hz, 2H), 3.92 (s, 3H), 1.86-1.76 (m, 2H), 1.39-1.27 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.6 (CH), 147.6 (CH), 139.3 (C), 128.1 (C), 126.9 (C), 123.7 (CH), 118.0 (C), 114.6 (CH), 105.0 (CH), 55.6 (CH3), 50.5 (CH2), 33.9 (CH2), 19.9 (CH2), 13.8 (CH3).

N-butyl-5-methylindole-3-carboxaldehyde (39o)

The crude product was prepared according to procedure B starting from 5-methylindole-3carboxaldehyde (400 mg, 2.51 mmol) and 1-bromobutane (685 mg, 5.00 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 9:1), the pure product (405 mg, 1.88 mmol, 75%) was obtained as an orange powder. m.p. 55-56°C; 1 H NMR (400 MHz, CDCl3) δ ppm 9.93 (s, 1H), 8.10 (s, 1H), 7.64 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.14 (dd, J = 8.4, 1.5 Hz, 1H), 4.12 (t, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.90-1.78 (m, 2H), 1.41-1.28 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ ppm 184.6 (CH), 138.6 (C), 135.8 (C), 132.8 (CH), 125.9 (C), 125.6 (CH), 122.1 (CH), 117.8 (C), 109.9 (CH), 47.2 (CH2), 31.9 (CH2), 21.6 (CH3), 20.2 (CH2), 13.7 (CH3).

N-butyl-5-methoxyindole-3-carboxaldehyde (39p)

The crude product was prepared according to procedure B starting from 5-methoxyindole-3carboxaldehyde (200 mg, 1.14 mmol) and 1-bromobutane (312 mg, 2.28 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 9:1), the pure product (243 mg, 1.05 mmol, 92%) was obtained as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ ppm 9.91 (s, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.64 (s, 1H), 7.23 (d, J = 8.9 Hz, 1H), 6.94 (dd, J = 8.9, 2.5 Hz, 1H), 4.12 (t, J = 7.1 Hz, 2H), 3.88 (s, 3H), 1.90-1.79 (m, 2H), 1.40-1.29 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H); 13

General procedure C for the synthesis of compounds (40-42)

To a solution of derivatives 30, 36 or 38 in anhydrous CH2Cl2 (10 mL/mmol) was added BBr3 (20 equiv.) at 0°C. The solution was stirred at rt for 18 to 72 h, then EtOAc and iced water were added.

After stirring for 0.5 h, the mixture was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO4, filtered off and concentrated under reduced pressure to afford the expected crude product.

2-( N-3-hydroxybenzylindol-3-ylmethylene)-4,6-dihydroxybenzofuran-3(2H)-one (40)

The crude product was prepared according to procedure C starting from compound 30 (120 mg, 0.29 mmol). After purification by column chromatography on silica gel (CH2Cl2/MeOH 95:5), the pure product (108 mg, 0.27 mmol, 93%) was obtained as a red powder. m.p. > 300°C; 1

2-(N-butyl-7-hydroxyindol-3-ylmethylène)-4,6-dihydroxybenzofuran-3(2H)-one (41)

The crude product was prepared according to procedure C starting from compound 36 (50 mg, 0.13 mmol). After purification by column chromatography on silica gel (CH2Cl2/MeOH 9:1), the pure product (46 mg, 0.13 mmol, 95%) was obtained as a brownish red powder. m.p. 149-150°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.72 (s, 1H), 10.71 (s, 1H), 9.88 (s, 1H), 7.97 (s, 1H), 7.32 (d, J = 7.9 Hz, 1H), 6.94 (dd, J = 7.9, 7.6 Hz, 1H), 6.86 (s, 1H), 6.61 (d, J = 7.6 Hz, 1H), 6.23 (d, J = 1.2 Hz, 1H), 6.05 (d, J = 1.2 Hz, 1H), 4.48 (t, J = 6.9 Hz, 2H), 1.84-1.73 (m, 2H), 1.32-1.23 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ ppm 178.2 (C), 166.9 (C), 166.

2-(N-butyl-5-hydroxyindol-3-ylmethylène)-4,6-dihydroxybenzofuran-3(2H)-one (42)

The crude product was prepared according to procedure C starting from compound 38 (155 mg, 0.41 mmol). After purification by column chromatography on silica gel (CH2Cl2/MeOH 9:1), the pure product (143 mg, 0.39 mmol, 96%) was obtained as a red powder. m.p. 251-252°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.70 (s, 2H), 9.08 (s, 1H), 7.99 (s, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.24 (s, 1H), 6.89-6.62 (m, 2H), 6.26 (s, 1H), 6.06 (s, 1H), 4.34-4.01 (m, 2H), 1.86-1.60 (m, 2H), 1.38-1.17

Molecular Modeling

Docking studies used the reported X-ray crystallographic structure of HCV genotype 1b RdRp complexed with a known inhibitor binding to thumb pocket I of the enzyme (PDB code 2dxs) [START_REF] Ikegashira | Discovery of conformationally constrained tetracyclic compounds as potent hepatitis C virus NS5B RNA polymerase inhibitors[END_REF].

The protein structure was extracted and water molecules were deleted. The complexed ligand was used to define the binding site of docking and was successfully docked in a similar conformation in the pocket, using the same protocol as for aurones. The genetic algorithms of Autodock4 docking software performed flexible docking with small molecules while keeping the protein structure rigid.

All compounds were built and their energies minimized with Sybyl, using a conjugate gradient method and MMFF94 force field and charges. For each compound, 100 poses were generated by Autodock, clustered according to their spatial proximity and calculated free energy of binding. The pictures were built with Pymol software [32].

Biology

Expression and Purification of Recombinant HCV NS5B21. RdRp (NS5B protein) from the HCV J4 genotype 1b reference strain, truncated of its 21 C-terminal amino acids to ensure solubility (NS5B21) and carrying a hexahistidine tag (His-Tag) at its N-terminus, was expressed in Escherichia coli C41 (DE3) and purified. Chromatography was performed on Ni-NTA columns (Qiagen, Valencia, CA). The columns were washed with a buffer containing 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, and 20 mM imidazole. The bound protein was eluted in 1 mL fractions with a buffer containing 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, and 250 mM imidazole. NS5B21enriched fractions were selected with a Bradford colorimetric assay, and NS5B21 purity was determined by SDS-PAGE analysis with Coomassie staining. Purified NS5B21 fractions were pooled and dialyzed against a buffer containing 5 mM Tris (pH 7.5), 0.2 M sodium acetate, 1 mM DTT, 1 mM EDTA, and 10% glycerol. NS5B21 purity was >98%.

HCV-NS5B21 Polymerase Assay. An enzyme assay was used to measure inhibition of HCV-NS5B21 polymerase activity. The cell free HCV-NS5B21 polymerase assay is based on the amount of double-stranded RNA synthesized in the presence of HCV-NS5B21, a homopolymeric RNA template (Poly U, GE Healthcare, Chalfont St. Giles, U.K.) and ATP, measured with an intercalating agent (SYBR Green, Applied Biosystems), as previously described [START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF]. Each experiment was performed in triplicate.

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/


text:
Amel Meguellati

Abdelhakim Ahmed-Belkacem

Alessandra Nurisso

Wei Yi

Rozenn Brillet

Nawel Berqouch

Laura Chavoutier

Antoine Fortuné

Jean- Michel Pawlotsky

Ahcène Boumendjel

Marine Peuchmaur
email: marine.peuchmaur@ujf-grenoble.fr

New Pseudodimeric Aurones as Palm Pocket Inhibitors of Hepatitis C Virus RNA-dependent RNA Polymerase

Keywords: Aurones; Hepatitis C Virus, RNA-dependent RNA polymerase, 1, Introduction

The NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme for Hepatitis C Virus (HCV) replication. In addition to the catalytic site, this enzyme is characterized by the presence of at least four allosteric pockets making it an interesting target for development of inhibitors as potential anti-HCV drugs. Based on a previous study showing the potential of the naturally occurring aurones as inhibitors of NS5B, we pursued our efforts to focus on pseudodimeric aurones that have never been investigated so far. Hence, 14 original compounds characterized by the presence of a spacer between the benzofuranone moieties were synthesized and investigated as HCV RdRp inhibitors by means of an in vitro assay. The most active inhibitor, pseudodimeric aurone 4, induced high inhibition activity (IC50 = 1.3 µM). Mutagenic and molecular modelling studies reveal that the binding site for the most active derivatives probably is the palm pocket I instead of the thumb pocket I as for the monomeric derivatives.

Hepatitis C virus (HCV) infection, the major cause of liver disease worldwide, is a global health problem that affects more than 170 million people and can progress to cirrhosis, end-stage liver disease, hepatocellular carcinoma and death. [1,[START_REF] Gravitz | Introduction: a smouldering public-health crisis[END_REF] In 2014, three drugs, leading to high HCV infection cure rates, were approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), including the nucleotide NS5B polymerase inhibitor sofosbuvir, [START_REF] Gentile | A novel promising therapeutic option against hepatitis C virus: an oral nucleotide NS5B polymerase inhibitor sofosbuvir[END_REF][START_REF] Osinusi | Sofosbuvir and ribavirin for hepatitis C genotype 1 in patients with unfavorable treatment characteristics: a randomized clinical trial[END_REF] the protease inhibitor simeprevir [START_REF] You | Simeprevir for the treatment of chronic hepatitis C[END_REF][START_REF] Jacobson | Simeprevir (TMC435) with peginterferon/ribavirin for chronic HCV genotype-1 infection in treatment-naïve patients: results from quest-1, a phase III trial[END_REF] and the NS5A polymerase inhibitor daclatasvir. [START_REF] Lee | The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein[END_REF] They can be used in combination with pegylated interferon α (IFNα) and ribavirin, or as part of sofosbuvir-based IFN-free combinations. In 2015, two additional combination, including sofosbuvir/ledipasvir and ombitasvir/paritaprevir/ritonavir plus dasabuvir were approved in the US and Europe, also yielding high cure rates. However, these new treatment regimens are extremely costly and may select multidrug resistance in patients that may lead to therapy failure. Low-income countries, where therapeutic needs are high, are unlikely to benefit from these treatments in the short-to mid-term. Therefore, the need for new direct acting antivirals (DAAs) with unique mechanisms of action is still highly desired. [START_REF] Schlütter | Therapeutics: new drugs hit the target[END_REF][START_REF] Rice | Perspective: miles to go before we sleep[END_REF][START_REF] Clercq | Current race in the development of DAAs (direct-acting antivirals) against HCV[END_REF][START_REF] Cartwright | Novel drugs in the management of difficult-to-treat hepatitis C genotypes[END_REF][START_REF] Wendt | An update on the treatment of genotype-1 chronic hepatitis C infection: lessons from recent clinical trials[END_REF] In this context, the NS5B RNA-dependent RNA polymerase (RdRp), which is absent in humans and essentially required for viral replication, is a particularly attractive target as witnessed by the increasing number of rationally designed inhibitors of this enzyme. [13][14][15] At the structural level, this viral polymerase is shaped as a 'right hand' with the characteristic thumb, finger and palm domains. [16][START_REF] Lesburg | Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site[END_REF][START_REF] Ago | Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus[END_REF][START_REF] Bressanelli | Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides[END_REF] In addition to the catalytic site, at least four allosteric binding sites have been identified so far, including 'thumb' pockets I and II and 'palm' pockets I and II/III. [START_REF] Barreca | Allosteric inhibition of the hepatitis C virus NS5B polymerase: in silico strategies for drug discovery and development[END_REF][START_REF] Beaulieu | Recent advances in the development of NS5B polymerase inhibitors for the treatment of hepatitis C virus infection[END_REF][START_REF] Beaulieu | Discovery of the first thumb pocket I NS5B polymerase inhibitor (BILB 1941) with demonstrated antiviral activity in patients chronically infected with genotype 1 hepatitis C virus (HCV)[END_REF][START_REF] Wang | Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase[END_REF][START_REF] Pauwels | Binding-site identification and genotype profiling of hepatitis C virus polymerase inhibitors[END_REF] Compounds that bind to allosteric sites induce conformational changes within the enzyme, leading to loss of its catalytic activity and ultimately to a replication end of the virus.

Recently, we showed that naturally occurring aurones (2-benzylidenebenzofuran-3(2H)-ones, Figure 1)small molecules belonging to the flavonoid familyhave potent anti-HCV properties in vitro. [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF][START_REF] Meguellati | B-ring modified aurones as promising allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF] The investigation of aurones as potential anti-HCV drugs was motivated by the very low toxicity of naturally occurring aurones and their derivatives. In particular, a 4,6dihydroxyaurone derivative was previously evaluated through in vivo oral and dermal toxicity assays and no toxicity was observed even at high concentrations. [START_REF] Perrier | A method of cosmetic depigmentation care by applying at least one aurone[END_REF] The naturally occurring aureusidin (3',4,4',6-tetrahydroxyaurone, 1, Figure 1), was first identified as an inhibitor of HCV RdRp with an IC50 of 5.2 µM in an enzyme assay. Further, structure-activity relationship studies allowed us to partially identify key features that are significant for the HCV RdRp inhibitory activity of this compound. Moreover, the binding site of this family of compounds was located at the thumb pocket I using site-directed mutagenesis experiments and molecular modeling. [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF]

Results and discussion

Chemistry

In this study, we focused our efforts on two novel types of structures which differ from each other by their rigidity profile. In the first, the two benzofuranone moieties are separated by a phenyl or a biphenyl group whereas in the second one, a flexible alkyl chain was inserted between the benzofuranones as shown in figure 1.

In both cases, targeted pseudodimeric aurones were obtained following our previously reported general and convenient synthetic pathway involving aldol condensation between 4,6-dihydroxy or 4,6-dimethoxybenzofuran-3(2H)-one (2 or 3 respectively) and a bis-arylaldehyde under basic conditions (Scheme 1). [START_REF] Beney | An efficient synthesis of 4,6-dimethoxyaurones[END_REF][START_REF] Okombi | Discovery of benzylidenebenzofuran-3(2H)-one (Aurones) as inhibitors of tyrosinase derived from human melanocytes[END_REF]. It should be highlighted that the condensation of unprotected 2 with bis-arylaldehyde provided the desired compounds in satisfactory yield and no side products formation was observed.

The 4,6-dihydroxybenzofuran-3(2H)-one 2 was obtained in two steps (84%) starting from phloroglucinol and was methylated in the presence of dimethyl sulfate to give 4,6dimethoxybenzofuran-3(2H)-one 3 with 97% yield. Among the different bisaldehydes used, the benzenedicarboxaldehydes were commercially available and the biphenyl ones 18a-e (Scheme 2) were synthesized in one step by Suzukicoupling reaction (61-93%) between a bromobenzaldehyde and a formylphenylboronic acid in the presence of tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and potassium carbonate as a base (Scheme 2).

The other bisaldehydes were synthesized in one to three steps (Scheme 2). First, diethyl malonate was alkylated with a bromoalkane and sodium ethoxide in the presence of potassium hydroxybenzaldehyde in basic media following the procedure described by W. Jiang and al. [START_REF] Jiang | Chelate cooperativity and spacer length effects on the assembly thermodynamics and kinetics of divalent pseudorotaxanes[END_REF] The full series of the synthesized pseudodimeric aurones is shown in table 1.

i Br O (HO) 2 B O O O EtO O O OEt EtO O O OEt R HO OH R ii iii O R O O O R = cyclohexyl methylcyclohexyl cyclopentyl isobutyl 18a-e 19a-d

Assessment of HCV RdRp inhibition and structure-activity relationship.

An enzyme assay was used to assess the inhibition of the polymerase activity of a purified RdRp (NS5B protein), deleted of its 21 C-terminal amino acids in order to ensure solubility (HCV-NS5BΔ21). The J4 genotype 1b reference strand was used. This assay measures the amount of double-stranded RNA synthesized in the presence of HCV-NS5BΔ21, a homopolymeric RNA template and ATP, as previously described. [START_REF] Ahmed-Belkacem | Silibinin and related compounds are direct inhibitors of hepatitis C virus RNAdependent RNA polymerase[END_REF] Initial screening was performed at 20 µM concentration of the pseudodimeric aurone followed by the measurement of IC50 for those that showed the highest activity at 20 µM. Prior to that, cytotoxicity of this new family was evaluated and the model compounds 4 and 6 have been shown non-toxic even at 100 µM.

Our previous SAR studies on monomeric aurones pointed out some pharmacophoric features that were crucial for the inhibitory activity of aurones on HCV RdRp: (a) a hydroxyl group at position 4 or hydroxyl groups at positions 4 and 6, (b) a 2',4'-or 3',4'-dihydroxylated B-ring or a hydrophobic and bulky substituent or alternative core at the B-ring. [START_REF] Haudecoeur | Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase[END_REF] The importance of the hydroxylation pattern at the A-ring of benzofuranone found among monomeric aurones was also checked with these new pseudodimeric aurones: when replacing hydroxyl by methoxy (compound 6) or by hydrogen (compound 17), inhibitory activity was dramatically dropped.

In this study, we decided to explore the shape and the size of the targeted thumb pocket I while optimizing the polar/apolar interactions with the allosteric binding site. In order to probe the depth and the extent of the pocket, we first considered synthesizing pseudodimeric aurones with one or two aromatic phenyl groups between the benzofuranone moieties (4-11, Table 1).

Because of the rigidity of these compounds, different positions for the substituents were studied.

The biological evaluation of these 7 compounds revealed the importance of (a) the shape of the molecule and (b) the distance between the benzofuranone cores. The best results were obtained when the spacer between benzofuranone moieties is a simple phenyl ring with a para, para linkage (compound 4, IC50 = 1.3 µM).

Then, we investigated more flexible structures in which the two benzofuranone cores were linked via an alkyl chain included in the spacer (12-17, Table 1). We have chosen compound 12 as a derivative to begin with because the length of the alkyl chain could allow, as stated by molecular modeling studies (cf. supporting information), new π-π interactions between the aromatic linker and the second aurone moiety with histidines 428 and 34, respectively. Given the presence of an unoccupied hydrophobic sub-pocket composed by leucine 392, alanine 395, isoleucine 424 and leucine 425 residues, analogues of compound 12 bearing an alkyl hydrophobic chain in the middle of the spacer were synthesized (compounds 13-16). The biological evaluation of these new compounds showed that all substituents were globally well tolerated. Nevertheless, no significant gain in terms of inhibitory potency was observed (IC50 = 1.5 -2.0 µM).

Site directed mutagenesis studies and molecular modelling

Intrigued by the fact that the addition of the hydrophobic chain did not have a positive impact on RdRp inhibitory activity, we conducted site directed mutagenesis studies in order to check the binding site localization. Taking compounds 4 and 12-16 as representatives, mutagenesis results indicated that thumb pocket I is not the target (Figure 2). Indeed, the inhibitory activity of compounds 4 and 12-16 on the HCV-NS5BΔ21 carrying a mutation at the thumb site I (P495L) that is known to confer resistance to reported inhibitors targeting thumb site I [START_REF] Pauwels | Binding-site identification and genotype profiling of hepatitis C virus polymerase inhibitors[END_REF][START_REF] Ahmed-Belkacem | Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention[END_REF] was not affected by the amino acid substitution on NS5B (for compound 4, IC50 = 1.3 µM for HCV-NS5BΔ21-Wt versus IC50 = 1.5 µM for HCV-NS5BΔ21-P495L). The latter results reveal that thumb I is probably not the site of binding. Hence, we mapped the other allosteric binding sites (thumb II, palm I and palm II/III) and selected aurone 4 for doing it. Four mutants of HCV-NS5BΔ21 were used in this study P495L, M423T, H95Q and C316Y, corresponding respectively to mutations on thumb pocket I, thumb pocket II, palm pocket I and palm pocket II/III. [START_REF] Pauwels | Binding-site identification and genotype profiling of hepatitis C virus polymerase inhibitors[END_REF][START_REF] Ahmed-Belkacem | Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention[END_REF] The dose-response curves obtained are presented in figure 3. The amino acid substitution H95Q reduced susceptibility of compound 4 which indirectly suggests that this compound binds to (or close to) palm pocket I. Thus, molecular docking studies of compound 4

were conducted on the corresponding region of the protein, suggesting a possible favorable interaction (Figure 3A). One of the benzofuranone moieties was shown to be mainly stabilized by hydrophobic interactions in an area surrounded by apolar residues, normally occupied by palm site I inhibitors, such as Pro197, Met414, Tyr415, Ile447 and Tyr448 (Figure 3A). While the aromatic linker was further stabilized by van der Waals contacts with Phe193 and Tyr448, the second benzofuranone moiety, in contrast to the first one, occupied a more polar zone in which hydroxyl substituents may be involved in hydrogen bonds with residues such as Asn291, or with possible water molecules acting as bridges between the ligand and the protein. [START_REF] Barreca | Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study[END_REF] By following the same strategy, it was also possible to determine the interaction mode of compounds 12 and 14 to this specific region. While the position of one benzofuranone core was conserved in both cases (Figures 3B-C), the presence of a longer flexible spacer determined, for 12, the shift of the second benzofuranone moiety to a novel wide region with Ser407, Gly410, and Ile405 as possible anchoring points (Figure 3B). Concerning compound 14, characterized by a cycloalkyl chain in the middle of the spacer, the second benzofuranone ring is now involved in polar contacts with the flexible Arg158 and Arg386 residues (Figure 3C). Indeed, the presence of a long spacer in 12 and 14 determined the possibility of more contacts with the protein via the two benzofuranone extremes. Nevertheless, these moieties, being exposed to wide regions in which dynamics and solvent effects may take place, could determine entropic penalties that may explain affinities comparable to 4 (Table 1). with the MLP are reported as magenta dots.

Assessment of anti-HCV activity in the genotype 2a/2a J6/JFH1 infectious model

The anti-HCV activity of compounds 4, 12, 13, 14, 16 and 17 was assessed in the J6/JFH1

(genotype 2a/2a) infectious model using ABT-333 (Dasabuvir) as reference and compound 17

as negative control (Table 2). Indeed, compound 17 that showed no inhibition of the polymerase activity (Table 1) also does not have anti-HCV properties. Compounds 4, 12, 13, 14 and 16

showed here a low to moderate anti-HCV activity, the most active compound being only 4.5 less active than the Dasabuvir currently in the market. The fact that inhibitory effects of these 5 compounds are very close (1 to 2 μM, Table 1) whereas their anti-HCV activity are disparate could be explained either by a difference in the pharmacokinetic behavior of the compoundsit should be noted that this behavior was observed in many cases [START_REF] Newton | The discovery of 2-substiuted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity[END_REF][START_REF] Cabarcas-Montalvo | Discovery of antiviral molecules for dengue: In silico search and biological evaluation[END_REF][START_REF] Copeland | Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists[END_REF] or by a genotype specificity: whereas enzymatic assays are carried out on NS5B genotype 1, anti-HCV activity involved the genotype 2a/2a J6/JFH1 infectious model. Overall, the anti-HCV activity of the tested compounds is in relative good agreement with their respective HCV RdRp inhibitory potency. It

Conclusion

Based on the activity of naturally occurring aurones as inhibitors of NS5B RNA-dependent RNA polymerase (RdRp) and on the binding profile toward one of the allosteric sites, we investigated a series of pseudodimeric aurones using structural and ligand-based drug design. Herein, we reported the capacity of novel pseudodimeric aurones to inhibit HCV RdRp with IC50 values in the one digit micromolar order. Initially, designed to act at the thumb pocket I as the monomeric derivatives does, it was found that pseudodimeric aurones seem to target preferentially the palm pocket I as suggested by site-directed mutagenesis and molecular docking studies. The last assumption should be confirmed by co-crystallization studies. The anti-HCV activity in infectious model (genotype 2a/2a J6/JFH1) was also assessed for these new pseudodimeric aurones and results are globally correlated to their respective HCV RdRp inhibitory potency.

Experimental section

Chemistry

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.

Melting points were measured on a Fisher micromelting point apparatus and are uncorrected. H and 13

General procedure A for the synthesis of compounds (4-17)

To a solution of 2 in ethanol (7 mL/mmol) were added an aqueous solution of potassium hydroxide (50%, 5 mL/mmol) and the corresponding benzaldehyde derivative (0.45 to 0.5 equiv.). The solution was refluxed until TLC showed complete disappearance of the starting materials (2 to 5 hours). After cooling, ethanol was removed under reduced pressure, then the residue was diluted into distilled water (50 mL/mmol) and an aqueous solution of hydrochloric acid (10%) was added to adjust the pH to 2-3. The mixture was then extracted with ethyl acetate or dichloromethane. The combined organic layers were washed with water and brine, dried over MgSO4, filtered off and concentrated under reduced pressure to afford the corresponding (Z)-2benzylidenebenzofuran-3(2H)-one derivative.

4.1.3.1. (2Z,2'Z)-2,2'-(1,4-phenylenebis(methan-1-yl-1-ylidene))bis(4,6-dihydroxybenzofuran- 3(2H)-one) (4)
The crude product was prepared according to general procedure A starting from

3(2H)-one) (6)

To a solution of 4,6-dimethoxybenzofuran-3(2H)-one 3 (100 mg, 0.51 mmol, 1.0 equiv.) in 7.5 mL of methanol were added an aqueous solution of potassium hydroxide (50%, 0.75 mL) and isophthalaldehyde (35 mg, 0.25 mmol, 0.5 equiv.). After stirring for 2 h at rt, the reaction mixture was concentrated under vacuum. The residue was diluted in water (25 mL) and the mixture was extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered off and concentrated under reduced pressure. The product (70 mg, 0.

(2Z,2'Z)-2,2'-(biphenyl-3-4'-diylbis(methan-1-yl-1-ylidene))bis(4,6-dihydroxybenzofuran-3(2H)-one) (8)

The crude product was prepared according to general procedure A starting from

(2Z,2'Z)-2,2'-(biphenyl-3-3'-diylbis(methan-1-yl-1-ylidene))bis(4,6-dihydroxybenzofuran-3(2H)-one) (10)

The crude product was prepared according to general procedure A starting from 4,6dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv.) and [1,1'-Biphenyl]-3,3'dicarboxaldehyde 18d (63 mg, 0.30 mmol, 0.5 equiv.). The pure product (150 mg, 0.31 mmol, 98%) was obtained as a grey powder. m.p. > 410°C; 1

(2Z,2'Z)-2,2'-[((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1-yl-1ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (12)

The crude product was prepared according to general procedure A starting from 4,6dihydroxybenzofuran-3(2H)-one 2 (166 mg, 1.00 mmol, 1.0 equiv.) and 4,4'-[1,3propanediyl(oxy)]bisbenzaldehyde [START_REF] Jiang | Chelate cooperativity and spacer length effects on the assembly thermodynamics and kinetics of divalent pseudorotaxanes[END_REF] (142 mg, 0.50 mmol, 0.5 equiv.). After purification by column chromatography on silica gel (EtOAc/MeOH/AcOH 8:1.9:0.1), the pure product (412 mg, 0.71 mmol, 71%) was obtained as a yellow powder. m.p. 271-272°C;

(2Z,2'Z)-2,2'-[((2-cyclohexylpropane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1yl-1-ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (13)

The crude product was prepared according to general procedure A starting from

General procedure B for the synthesis of (formylphenyl)benzaldehydes (18a-e)

To a stirred solution of the corresponding commercially available formylphenyl boronic acid (1 equiv.) and bromobenzaldehyde (1 equiv.) in 1,2-dimethoxyethane (10 mL/mmol) were added Pd(PPh3)4 (0.1 equiv.) and a solution of K2CO3 (6 equiv.) in water (1mL/mmol). The reaction mixture was refluxed for 6 h and then concentrated under reduced pressure. The crude product was purified by column chromatography.

[1,1'-Biphenyl]-4,4'-dicarboxaldehyde (18a)

The crude product was prepared according to procedure B starting from 4-bromobenzaldehyde (370 mg, 2.00 mmol) and 4-formylphenylboronic acid (300 mg, 2.00 mmol). After purification by column chromatography on silica gel (toluene/EtOAc 100:0 to 92:8), the pure product (300 mg,

[1,1'-Biphenyl]-3,4'-dicarboxaldehyde (18b)

The crude product was prepared according to procedure B starting from 4-bromobenzaldehyde (370 mg, 2.00 mmol) and 3-formylphenylboronic acid (300 mg, 2.00 mmol). After purification by column chromatography on silica gel (toluene/EtOAc 100:0 to 94:6), the pure product (365 mg, 1.74 mmol, 87%) was obtained as a white powder. m.p. 82°C; 1 H NMR (400 MHz, CDCl3) δ

[1,1'-Biphenyl]-2,4'-dicarboxaldehyde (18c)

The crude product was prepared according to procedure B starting from 4-bromobenzaldehyde (370 mg, 2.00 mmol) and 2-formylphenylboronic acid (300 mg, 2.00 mmol). After purification by column chromatography on silica gel (toluene/EtOAc 100:0 to 92:8), the pure product (389 mg, 1.85 mmol, 93%) was obtained as a white powder. m.p. 99°C;

[1,1'-Biphenyl]-2,3'-dicarboxaldehyde (18e)

The crude product was prepared according to procedure B starting from 3-bromobenzaldehyde (247 mg, 1.46 mmol) and 2-formylphenylboronic acid (200 mg, 1.46 mmol). After purification by column chromatography on silica gel (toluene/EtOAc 100:0 to 95:5), the pure product (227 mg,

General procedure C for the synthesis of diethyl alkylpropanedioates (19a,b)

To a stirred suspension of NaOEt (1.5 equiv.) in ethanol (0.8 mL/mmol) were successively added diethyl malonate (1 equiv.), bromoalkane (1.5 equiv.) and KI (1.5 equiv.). The reaction mixture was refluxed for 48 h. After cooling, ethanol was removed under reduced pressure and the residue was diluted into a saturated aqueous solution of NH4Cl (2 mL/mmol). The mixture was then extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over MgSO4, filtered off and concentrated under reduced pressure to afford the corresponding crude diethyl alkylpropanedioate derivative.

Diethyl cyclohexylpropanedioate (19a)

The crude product was prepared according to procedure C starting from diethyl malonate (4.00 g, 24.97 mmol) and bromocyclohexane (6.08 g, 37.50 mmol). After purification by column chromatography on silica gel (cyclohexane/EtOAc 98:2), the pure product (1.29 g, 5.32 mmol, 21%) was obtained as a colorless liquid. RMN

General procedure D for the synthesis of 2-alkylpropane-1,3-diols (20a-d)

To a stirred suspension of LiAlH4 (3.5 equiv.) in dry THF (1.0 mL/mmol) under nitrogen atmosphere was slowly added a solution of the corresponding diethyl alkylpropanedioate (1 equiv.) in dry THF (1.0 mL/mmol) at 0°C. The reaction mixture was allowed to warm to rt and was then refluxed for 48 h before being cooled to 0°C. The reaction was quenched by careful addition of a saturated aqueous solution of NH4Cl until neutral or acidic pH. The precipitated aluminium salts were removed by filtration through a pad of Celite® and washed with diethylether or a mixture of MeOH/CH2Cl2/Et2O 1:1:1. The organic phase of the filtrate was recovered, washed with water and brine, dried over MgSO4, filtered off and concentrated under reduced pressure to afford the corresponding crude product which was pure enough to be directly used in the next step. 13 C NMR (100 MHz, CDCl3) δ ppm 22.9 (2xCH3), 25.5

(CH), 37.0 (CH2), 39.7 (CH), 66.1 (2xCH2).

General procedure E for the Mitsunobu reaction. Synthesis of bisaldehydes (21a-d)

To a solution of the corresponding 2-alkylpropane-1,3-diol 20a-d (1 equiv.) in dry THF (7 mL/mmol) were added 4-hydroxybenzaldehyde (3 equiv.), PPh3 (3 equiv.) and DIAD (3 equiv.).

The reaction mixture was stirred overnight at rt and was then concentrated under reduced pressure. The crude product was purified by column chromatography.

4,4'-[1,3-(2-cyclohexyl)propanediyl(oxy)]bisbenzaldehyde (21a)

The crude product was prepared according to procedure E starting from 2-cyclohexylpropane-

Molecular modelling

The chemical structures of pseudodimeric aurones were prepared within SYBYL X (Tripos Inc., St. Louis, MO), assigning Tripos force field atom and bond types to each ligand. The ionization state was defined as appropriate for pH 7.4. The protein structures (PDB codes: 4DRU and 3SKE) were prepared by removing co-crystallized water molecules and ligands and by adding hydrogen atoms. Automated docking was carried out with the program GOLD, version 5.2, [START_REF] Hartshorn | Diverse, high-quality test set for the validation of protein-ligand docking performance[END_REF] within the MLP strategy. [START_REF] Oberhauser | MLP tools: a PyMOL plugin for using the molecular lipophilicity potential in computer-aided drug design[END_REF][START_REF] Nurisso | Molecular docking using the molecular lipophilicity potential as hydrophobic descriptor: impact on GOLD docking performance[END_REF] Docking methodology was validated by re-docking the cocrystallized inhibitors in the corresponding pockets (RMSD < 1.5 Å, data not shown). Binding pockets were defined by considering 6 Å around the co-crystallized ligands. Preset options for the genetic search algorithm were used in calculations in which only ligand torsions were free to move. 100 solutions for each ligand-protein complex were retrieved and ranked according to the ChemPLP fitness score.

Biology

Cytotoxicity assays

The cytotoxicity assays were realized using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay on HEK293 cell lines. Cells were seeded in 96well culture plates (10 4 cells/well) and allowed to attach for 24 h at 37°C in 5% CO2. The treatment was done with a 50 µM concentration of compound in a final volume of 200 µL and allowed to grow for 72 h at 37ºC in 5% CO2. MTT (20 µL) solution (5 mg/mL) was added and incubated for 4 h at 37°C in 5% CO2. The culture medium was discarded, washed with PBS followed by addition of 100 µL of DMSO and maintained under shaking for 10 min. Absorbance was measured in a microplate reader at 570 nm. Experiments were repeated three times.

Expression and Purification of Recombinant HCV NS5BΔ21

RdRp (NS5B protein) from the HCV J4 genotype 1b reference strain, truncated of its 21 Cterminal amino acids to ensure solubility (NS5BΔ21) and carrying a hexahistidine tag (His-Tag) at its N-terminus, was expressed in Escherichia coli C41 (DE3) and purified. Chromatography was performed on Ni-NTA columns (Qiagen, Valencia, CA). The columns were washed with a buffer containing 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, and 20 mM imidazole. The bound protein was eluted in 1 mL fractions with a buffer containing 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, and 250 mM imidazole. NS5BΔ21-enriched fractions were selected with a Bradford colorimetric assay, and NS5BΔ21 purity was determined by SDS-PAGE analysis with Coomassie staining. Purified NS5BΔ21 fractions were pooled and dialyzed against a buffer containing 5 mM Tris (pH 7.5), 0.2 M sodium acetate, 1 mM DTT, 1 mM EDTA, and 10% glycerol. NS5BΔ21 purity was >98%.

HCV-NS5BΔ21 Polymerase Assay.

An enzyme assay was used to measure inhibition of HCV-NS5BΔ21 polymerase activity. The cell free HCV-NS5BΔ21 polymerase assay is based on the amount of double-stranded RNA synthesized in the presence of HCV-NS5BΔ21, a homopolymeric RNA template (Poly U, GE Healthcare, Chalfont St. Giles, U.K.) and ATP, measured with an intercalating agent (SYBR Green, Applied Biosystems), as previously described. [START_REF] Wakita | Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[END_REF] Each experiment was performed in triplicate.

J6/JFH1 infection in cell culture

Huh7.5 cells were seeded in 48-well plates at a density of 10 000 cells/well, infected 24 h later with 100 µl of HCV infectious particles (HCVcc) and incubated overnight at 37°C. After incubation, the supernatants were removed and J6/JFH1-infected cells were washed with fresh medium. Increasing concentrations of the tested compounds were added in a medium containing 1% DMSO and the cells were incubated 72 h at 37°C. Then, the cells were washed once with Dulbecco's PBS (Invitrogen), and 75 µl Renilla lysis buffer (Promega) was added to each well. Note that 20 µl of lysate was mixed with the luciferase assay substrate, as specified by the manufacturer. Luciferase activity was measured for 10 s in a Berthold luminometer.

Figure 1 .

 1 Figure 1. General structure of the aurone backbone (top left), structure and activity of the naturally occurring aureusidin 1 and structures of aurone derivatives investigated in this study (the dashed line indicate the site of linkage).

Scheme 1 .

 1 Scheme 1. General pathway for the synthesis of pseudodimeric aurones. Reagents and conditions: (i) ClCH2CN, HCl, ZnCl2, Et2O, 0°C; (ii) H2O, 100°C, 5 h, 84% (for 2 steps); (iii) Me2SO4, K2CO3, 1,2dimethoxyethane, reflux, 5 h, 97%; (iv) bis-arylaldehyde, KOH 50% in H2O, EtOH or MeOH, 2-15 h, 34-98%.

  iodide. The resulted diesters 19a,b and commercial ones 19c,d were reduced with lithium aluminium hydride in tetrahydrofuran to give the corresponding 1,3-diols 20a-d. Finally, a Mitsunobu reaction between 4-hydroxybenzaldehyde and the synthesized 1,3-diols 20a-d provided the desired bisaldehydes 21a-d with 33 to 73% yield. An analogue without alkyl chain 21e (R = H) was obtained starting from commercially available 1,3-dibromopropane and 4-

Figure 2 .Figure 3 .

 23 Figure 2. Inhibition curve of HCV-NS5BΔ21-WT r(wild-type), P495L, H95Q, C316Y and M423T mutants obtained with compound 4.

1

 1

13 C

 13 NMR (100 MHz, CDCl3) δ ppm 56.0 (CH3), 56.1 (CH3), 75.5 (C), 88.9 (CH), 93.0 (CH), 104.8 (C), 158.8 (C), 169.8 (C), 177.1 (C), 195.0 (C).

ppm 7 . 7 (

 77 67 (dd, J = 7.7, 7.6 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.89-7.95 (m, 2H), 7.99 (d, J = 8.4 Hz, 2H), 8.15 (s, 1H), 10.08 (s, 1H), 10.12 (s, 1H); 13 C NMR (100 MHz, CDCl3) δ ppm 127.2CH), 128.2 (CH), 129.8 (CH), 129.9 (CH), 130.4 (2CH), 133.2 (CH), 135.7 (C), 137.1 (C), 140.6 (C), 145.5 (C), 191.8 (2CH).

1 .

 1 08 mmol, 81%) was obtained as a white powder. m.p. 57°C, lit. 54-56°C;[START_REF] Gardinier | Cyclic versus polymeric supramolecular architectures in metal complexes of dinucleating ligands: silver(I) trifluoromethanesulfonate complexes of the isomers of bis(di(1H-pyrazolyl)methyl)-1,1'-biphenyl[END_REF] 1 H NMR (400 MHz, CDCl3) δ ppm 7.40 (dd, J = 7.7, 0.8 Hz, 1H), 7.46-7.52 (m, 1H), 7.58-7.66 (m, 3H), 7.84-7.88 (m, 1H), 7.90-7.94 (m, 1H), 7.98 (dd, J = 7.7, 1.2 Hz, 1H), 9.95 (d, J = 0.8 Hz, 1H), 10.08 (s, 1H); 13 C NMR (100 MHz, CDCl3) δ ppm 128.2 (CH), 128.4 (CH), 129.2 (CH), 129.4 (CH), 130.7 (CH), 130.8 (CH), 133.8 (C), 133.9 (CH), 135.8 (CH), 136.4 (C), 138.9 (C), 144.1 (C), 191.5 (CH), 191.8 (CH).

Table 1

 1 Structures and inhibition activity of pseudodimeric aurones. The dashed bounds indicate the sites of

	attachment to the methylenebenzofuranone moieties.

Table 2 Anti

 2

	Compound	J6/JFH1 EC50 (µM)
	Dasabuvir (ABT-333)	9.1 ± 0.2
	4	202 ± 16
	12	214 ± 21
	13	42 ± 1

-HCV activity in the genotype 2a/2a J6/JFH1 infectious model of compounds 4, 12, 13, 14, 16, 17 and ABT-133 (Dasabuvir).

  Electrosprayionization ESI mass spectra were acquired by the Analytical Department of Grenoble University on an Esquire 300 Plus Bruker Daltonis instrument with a nanospray inlet. Accurate mass measurements (HRMS) were carried out on a TOF spectrometer, realized by the mass spectrometry analysis facilities of Orleans University (France).

	4.1.1. 4,6-dihydroxybenzofuran-3(2H)-one (2)
	To a solution of phloroglucinol (15.0 g, 119 mmol) in diethyl ether (250 mL) at 0°C were added
	chloroacetonitrile (7.5 mL, 119 mmol) and dried zinc chloride (1.62 g, 11.9 mmol). The mixture
	was allowed to react with gaseous hydrochloric acid at 0°C for 1 h. The resulting suspension
	was stirred at 0°C for 1.5 h, and was allowed to react again with gaseous HCl for 30 min. The

C NMR spectra were recorded at room temperature in deuterated solvents on a Brüker AC-400 instrument (400 MHz). Chemical shifts (δ) are reported in parts per million (ppm) relative to TMS as internal standard or relative to the solvent [ 1 H: δ(DMSO) = 2.50 ppm, δ(CDCl3) = 7.24 ppm;

13

C: δ(DMSO) = 39.51 ppm, δ(CDCl3) = 77.23 ppm].

precipitate was filtered, washed with ether, dried and suspended in water (200 mL). The solution was refluxed for 5 h. After cooling, the crystals were filtered, washed with cold water and dried to afford 2 (16.6 g, 100 mmol, 84 %) as orange crystals, which were analytically pure and used without further purification. m.p. 257-259°C; 1 H NMR (400 MHz, DMSO-d6) δ ppm 4.55 (s, 2H), 5.91 (s, 2H), 10.58 (s, 1H), 10.61 (s, 1H);

13

C NMR (100 MHz, DMSO-d6) δ ppm 74.8 (CH2), 90.1 (CH), 96.1 (CH), 102.6 (C), 157.4 (C), 167.5 (C), 175.6 (C), 193.9 (C).

4.1.2. 4,6-dimethoxybenzofuran-3(2H)-one (3)

To a solution of 4,6-dihydroxybenzofuran-3(2H)-one 2 (2.00 g, 12.0 mmol) in 1,2dimethoxyethane (50 mL) were added potassium carbonate (3.70 g, 26.5 mmol) and dimethyl sulfate (2.50 mL, 26.5 mmol), and the mixture was refluxed for 5 h. After cooling, solvents were removed under reduced pressure, water (40 mL) was added, and the mixture was extracted with CH2Cl2. The combined organic layers were washed with water then brine, dried over anhydrous MgSO4, filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (eluent cyclohexane/EtOAc 40:60) to afford 3 (2.25 g, 11.6 mmol, 97 %) as an orange solid. m.p. 136°C; 1 H NMR (400 MHz, CDCl3) δ ppm 3.86 (s, 3H), 3.90 (s, 3H), 4.59 (s, 2H), 6.01 (d, J = 1.8 Hz, 1H), 6.15 (d, J = 1.8 Hz, 1H);

  1 H NMR (400 MHz, CDCl3) δ ppm 7.38 (ddd, J = 7.5, 1.2, 0.4 Hz, 1H), 7.47-7.52 (m, 3H), 7.61 (ddd, J = 7.5, 7.5, 1.4 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.98 (dd, J = 7.8, 1.2 Hz, 1H), 9.89 (s, 1H), 10.03 (s, 1H);13 C NMR

	(100 MHz, CDCl3) δ ppm 128.3 (CH), 128.6 (CH), 129.7 (2CH), 130.6 (CH), 130.7 (2CH),
	133.6 (C), 133.8 (CH), 135.8 (C), 144.1 (C), 145.2 (C), 191.5 (CH), 191.7 (CH).
	4.1.4.4. [1,1'-Biphenyl]-3,3'-dicarboxaldehyde (18d)
	The crude product was prepared according to procedure B starting from 3-bromobenzaldehyde
	(247 mg, 1.33 mmol) and 3-formylphenylboronic acid (200 mg, 1.33 mmol). After purification by
	column chromatography on silica gel (toluene/EtOAc 100:0 to 95:5), the pure product (171 mg,
	0.81 mmol, 61%) was obtained as a white powder. m.p. 94°C, lit. 90°C;[39] 1 H NMR (400 MHz,
	CDCl3) δ ppm 6.66 (dd, J = 7.7, 7.6 Hz, 2H), 7.88-7.92 (m, 4H), 8.13 (d, J = 1.6 Hz, 2H), 10.10
	(s, 2H); 13 C NMR (100 MHz, CDCl3) δ ppm 128.1 (2CH), 129.6 (2CH), 129.9 (2CH), 133.1
	(2CH), 137.2 (2C), 140.8 (2C), 192.2 (2CH).

Acknowledgements

The authors are grateful to ANR (Agence Nationale pour la Recherche) for financial support (Labex Arcane (ANR-11-LABX-0003-01)). A.A.-B. and J.-M.P. are supported by ANRS (Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales) and FRM (Fondation pour la Recherche Médicale). The authors also thank the ICMG NMR facilities.

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/