New pseudodimeric aurones as palm pocket inhibitors of Hepatitis C virus RNA-dependent RNA polymerase

Article abstract of DOI:10.1016/j.ejmech.2016.03.005

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 Combining double low line 1.3 1/4M). Mutagenic and molecular modeling 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.

Full text of DOI:10.1016/j.ejmech.2016.03.005

Accepted Manuscript
New Pseudodimeric Aurones as Palm Pocket Inhibitors of Hepatitis C Virus RNA-
dependent RNA Polymerase
Amel Meguellati, Abdelhakim Ahmed-Belkacem, Alessandra Nurisso, Wei Yi, Rozenn
Brillet, Nawel Berqouch, Laura Chavoutier, Antoine Fortuné, Jean-Michel Pawlotsky,
Ahcène Boumendjel, Marine Peuchmaur
PII:
S0223-5234(16)30179-9
10.1016/j.ejmech.2016.03.005
EJMECH 8430
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 November 2015
Revised Date: 1 March 2016
Accepted Date: 2 March 2016
Please cite this article as: A. Meguellati, A. Ahmed-Belkacem, A. Nurisso, W. Yi, R. Brillet, N. Berqouch,
L. Chavoutier, A. Fortuné, J.-M. Pawlotsky, A. Boumendjel, M. Peuchmaur, New Pseudodimeric
Aurones as Palm Pocket Inhibitors of Hepatitis C Virus RNA-dependent RNA Polymerase, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.005.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New Pseudodimeric Aurones as Palm Pocket Inhibitors of
Hepatitis C Virus RNA-dependent RNA Polymerase
Amel Meguellatia,b, Abdelhakim Ahmed-Belkacemc, Alessandra Nurissoe, Wei Yia,b,
Rozenn Brilletc, Nawel Berqoucha,b, Laura Chavoutiera,b, Antoine Fortunéa,b, Jean-
Michel Pawlotskyc,d, Ahcène Boumendjela,b and Marine Peuchmaura,b,
*
aUniv. Grenoble Alpes, Département de Pharmacochimie Moléculaire DPM UMR 5063, 38041 Grenoble, France
bCNRS, DPM UMR 5063, 38041 Grenoble, France
cINSERM U955, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France
dNational Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Hôpital Henri Mondor,
Université Paris-Est, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France
eSchool of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva, Switzerland
*Corresponding author Tel.: +33476635295; fax: +33476635298.
E-mail address: Marine.Peuchmaur@ujf-grenoble.fr
ABSTRACT
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.
Keywords: Aurones; Hepatitis C Virus, RNA-dependent RNA polymerase.
1. Introduction
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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.[1,2]
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,[3,4] the protease inhibitor simeprevir[5,6] and the NS5A
polymerase inhibitor daclatasvir.[7] They can be used in combination with pegylated interferon
(IFN ) and ribavirin, or as part of sofosbuvir-based IFN-free combinations. In 2015, two
α
α
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.[8-12]
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.[13-15] At the structural level,
this viral polymerase is shaped as a ‘right hand’ with the characteristic thumb, finger and palm
domains.[16-19] 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.[20-24]
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 family – have potent anti-HCV properties
in vitro.[25,26] 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 4,6-
dihydroxyaurone derivative was previously evaluated through in vivo oral and dermal toxicity
assays and no toxicity was observed even at high concentrations.[27] 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.[25]
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The structural analysis revealed that the targeted pocket – thumb pocket I – exhibits an
unexplored hydrophobic zone and may accommodate larger structures. Hence, we assumed
that pseudodimeric aurones would better fit in the thumb pocket I than the monomeric
derivatives, leading to higher inhibition activity. Herein, we report the design, synthesis and
activity of original pseudodimeric aurones as novel scaffolds for inhibition of NS5B polymerase.
The biological evaluation, site directed mutagenesis and molecular modelling rationalization are
reported.
OH
4'
OH
B
3'
7
HO
O
O
6
5
2'
A
C
1 (Aureusidin)
4
O
O
IC50 = 5.4 µM
OH
OR
RO
RO
O
O
Y:
Y
OR
O
O
O
O
R'
benzofuranone
benzofuranone
spacer
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).
2. Results and discussion
2.1. 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
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conditions (Scheme 1).[28,29]. 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 4,6-
dimethoxybenzofuran-3(2H)-one 3 with 97% yield.
HO
OH
HO
O
i, ii
iv
iv
O
OR
OR
OH
phloroglucinol
OH
RO
RO
2
O
O
iii
Y
MeO
O
O
O
4-17
O
OMe
3
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, 1,2-
dimethoxyethane, reflux, 5 h, 97%; (iv) bis-arylaldehyde, KOH 50% in H2O, EtOH or MeOH, 2-15 h, 34-
98%.
Among the different bisaldehydes used, the benzenedicarboxaldehydes were commercially
available and the biphenyl ones 18a-e (Scheme 2) were synthesized in one step by Suzuki-
coupling 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
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-
4

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hydroxybenzaldehyde in basic media following the procedure described by W. Jiang and al.[30]
The full series of the synthesized pseudodimeric aurones is shown in table 1.
O
O
O
Br
O
i
(HO)2B
18a-e
O
O
O
O
O
O
iv
ii
iii
HO
OH
EtO
OEt
EtO
OEt
O
O
R
R
R
19a-d
20a-d
21a-d
R = cyclohexyl
methylcyclohexyl
R = cyclohexyl
methylcyclohexyl
cyclopentyl
isobutyl
cyclopentyl
isobutyl
Scheme 2. Synthesis of bisaldehydes. Reagents and conditions: (i) Pd(PPh3)4, K2CO3, 1,2-
dimethoxyethane, reflux, 6 h, 61-93%; (ii) EtONa, RBr, KI, EtOH, reflux, 48 h; (iii) LiAlH4, THF, 0°C then
reflux for 48 h, 46-89%; (iv) 4-hydroxybenzaldehyde, PPh3, DIAD, THF, rt, 12 h, 33-73%.
2.2. 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-NS5B
∆
21). The J4 genotype 1b reference strand was used. This assay measures the
21, a
amount of double-stranded RNA synthesized in the presence of HCV-NS5B
∆
homopolymeric RNA template and ATP, as previously described.[31] 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.[25] The importance of the
5

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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).
2.3. 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-NS5B∆21 carrying a mutation at the thumb site I (P495L)
that is known to confer resistance to reported inhibitors targeting thumb site I[24,32] was not
affected by the amino acid substitution on NS5B (for compound 4, IC50 = 1.3 µM for HCV-
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NS5B∆21-Wt versus IC50 = 1.5 µM for HCV-NS5B∆21-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-
NS5B∆21 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.[24,32] 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.[33] 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).
Table 1
Structures and inhibition activity of pseudodimeric aurones. The dashed bounds indicate the sites of
attachment to the methylenebenzofuranone moieties.
7

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R2
R2
R2
O
O
Y
R2
O
O
Compd
Y group
R2
Yielda
(%)
Inhibition (20
IC50 (µM)
µM, %)
Dasabuvir (ABT-333)
95.7 ± 0.3
0.60 ± 0.03
4
OH
83
96.0 ± 0.2
1.3 ± 0.3
5
6
OH
69
55
97.0 ± 0.3
15 ± 11
3.6 ± 0.8
OMe
n.d.
7
8
OH
OH
37
72
n.d.
77 ± 4
99.2 ± 0.2
3.1 ± 0.5
9
OH
OH
OH
89
98
85
98.5 ± 0.2
95 ± 1
6 ± 1
n.d.
10
11
n.d.
91.0 ± 0.1
12
13
OH
OH
71
34
96.4 ± 0.3
95.3 ± 0.4
1.50 ± 0.07
2.0 ± 0.2
O
O
O
O
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14
15
OH
OH
78
56
94.9 ± 0.6
96.9 ± 0.1
1.8 ± 0.2
O
O
1.90 ± 0.00
O
O
16
17
OH
H
57
59
97.0 ± 0.1
0 ± 5
1.9 ± 0.1
n.d.
O
O
n.d.: not determined
aYields are given for the last step of the synthesis (condensation of bis-aldehyde and benzofuranone).
100
80
60
40
WT
P495L
H95Q
C316Y
M423T
20
0
0
2
4
6
8
10
12
14
16
Concentration of compound 4 (µM)
Figure 2. Inhibition curve of HCV-NS5B∆21-WT r(wild-type), P495L, H95Q, C316Y and M423T mutants
obtained with compound 4.
9

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A
B
C
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Figure 3. Best ranked poses of compounds 4 (A, yellow ball&sticks, ChemPLP score 62.6), 12 (B, green
ball&sticks, ChemPLP score 77.7) and 14 (C, black ball&sticks, ChemPLP score 93.8) in RdRp palm
pocket I represented as dark blue ribbons (PDB code 3SKE). RdRp residues involved in complex
stabilization are highlighted in sticks, colored and labeled whereas hydrophobic fitting points calculated
with the MLP are reported as magenta dots.
2.4. 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 compounds – it
should be noted that this behavior was observed in many cases[34-36] – 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.
Table 2
Anti-HCV activity in the genotype 2a/2a J6/JFH1 infectious model of compounds 4, 12, 13, 14, 16, 17 and
ABT-133 (Dasabuvir).
Compound
J6/JFH1 EC50 (µM)
9.1 ± 0.2
Dasabuvir (ABT-333)
4
202 ± 16
12
13
214 ± 21
42 ± 1
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14
90 ± 19
76 ± 21
> 400
16
17
3. 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.
4. Experimental section
4.1. 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.
1H and 13C NMR spectra were recorded at room temperature in deuterated solvents on a Brüker
AC-400 instrument (400 MHz). Chemical shifts (
relative to TMS as internal standard or relative to the solvent [1H:
(CDCl3) = 7.24 ppm; 13C:
(DMSO) = 39.51 ppm, (CDCl3) = 77.23 ppm]. Electrospray
δ) are reported in parts per million (ppm)
δ(DMSO) = 2.50 ppm,
δ
δ
δ
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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).
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
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; 1H NMR (400 MHz, DMSO-d6)
5.91 (s, 2H), 10.58 (s, 1H), 10.61 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ
ppm 4.55 (s, 2H),
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 1,2-
dimethoxyethane (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; 1H 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);
13C 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).
4.1.3. 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
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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)-2-
benzylidenebenzofuran-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 4,6-
dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv) and terephthalaldehyde (40
mg, 0.30 mmol, 0.5 equiv.). The pure product (108 mg, 0.25 mmol, 83%) was obtained as
apowder. m.p. > 410°C; 1H NMR (400 MHz, DMSO-d6)
2H), 7.97 (m, 4H), 11.14 (m, 4xOH); 13C NMR (100 MHz, DMSO-d6)
δ
ppm 6.20 (s, 2H), 6.30 (s, 2H), 6.62 (s,
ppm 90.7 (2xCH), 97.9
δ
(2xC), 102.3 (2xCH), 107.2 (2xCH), 130.9 (4xCH), 133.0 (2xC), 148.3 (2xC), 158.5 (2xC), 167.6
(4xC), 178.8 (2xC); LRMS (ESI−) m/z (%) 429 (100) [M−H]−; HRMS (ESI+) m/z calc. for
C24H15O8 431.0761, found 431.0762.
4.1.3.2. (2Z,2’Z)-2,2’-(1,3-phenylenebis(methan-1-yl-1-ylidene))bis(4,6-hydroxybenzofuran-
3(2H)-one) (5)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv.) and isophthalaldehyde (40,2
mg, 0.60 mmol, 0.5 equiv.). The pure product (90 mg, 0.20 mmol, 69%) was obtained as an
orange powder. m.p. > 410°C; 1H NMR (400 MHz, DMSO-d6)
6.63 (s, 2H), 7.58 (t, J = 7.6 Hz, 1H), 7.91 (d, J = 6.4 Hz, 2H), 8.35 (s, 1H), 11.02 (m, 2xOH),
11.06 (m, 2xOH); 13C NMR (100 MHz, DMSO-d6)
ppm 90.6 (2xCH), 97.9 (2xC), 102.3 (2xCH),
δ ppm 6.14 (s, 2H), 6.28 (s, 2H),
δ
107.4 (2xCH), 129.5 (CH), 131.0 (2xCH), 132.4 (CH), 132.9 (2xC), 148.1 (2xC), 158.5 (2xC),
167.6 (2xC), 167.8 (2xC), 178.9 (2xC); LRMS (ESI−) m/z (%) 429 (100) [M−H]−; HRMS (ESI+)
m/z calc. for C24H15O8 431.0761, found 431.0763.
4.1.3.3. (2Z,2’Z)-2,2’-(1,3-phenylenebis(methan-1-yl-1-ylidene))bis(4,6-dimethoxybenzofuran-
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
14

ACCEPTED MANUSCRIPT
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, 0.14 mmol, 55%) was obtained as a
pale orange powder. m.p. > 270°C; 1H NMR (400 MHz, DMSO-d6)
6H), 6.38 (d, J = 1,6 Hz, 2H), 6.74 (d, J = 2.0 Hz, 2H), 6.75 (s, 2H), 7.60 (t, J = 8.0 Hz, 1H), 7.98
(d, J = 1.6 Hz, 1H), 8.00 (d, J = 1.6 Hz, 1H), 8.40 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
ppm
δ ppm 3.91 (s, 6H), 3.93 (s,
δ
56.2 (2xCH3), 56.5 (2xCH3), 90.0 (2xCH), 94.4 (2xC), 103.9 (2xCH), 108.7 (2xCH), 129.6 (CH),
131.5 (2xCH), 132.8 (2xC), 133.0 (CH), 147.7 (2xC), 159.0 (2xC), 168.3 (2xC), 169.1 (2xC),
178.9 (2xC); LRMS (ESI+) m/z (%) 487 (100) [M+H]+; HRMS (ESI+) m/z calc. for C28H23O8
487.1387, found 487.1385.
4.1.3.4. (2Z,2’Z)-2,2’-(biphenyl-4-4’-diylbis(methan-1-yl-1-ylidene))bis(4,6-dihydroxybenzofuran-
3(2H)-one) (7)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv.) and [1,1’-Biphenyl]-4,4’-
dicarboxaldehyde 18a (63 mg, 0.30 mmol, 0.5 equiv.). The pure product (56 mg, 0.11 mmol,
37%) was obtained as a yellow powder. m.p. > 410°C; 1H NMR (400 MHz, DMSO-d6)
6.16 (s, 2H), 6.29 (s, 2H), 6.67 (s, 2H), 7.87 (d, J = 8,0 Hz , 4H), 8.01 (d, J = 8,0 Hz , 4H), 11.08
(s, 2xOH), 11.10 (s, 2xOH); 13C NMR (100 MHz, DMSO-d6)
ppm 90.7 (2xCH), 97.9 (2xC),
δ ppm
δ
102.4 (CH), 103.4 (CH), 107.6 (2xCH), 127.0 (4xCH), 128.2 (C), 128.9 (C), 131.3 (4xCH), 132.0
(2xC), 139.5 (2xC), 148.0 (2xC), 158.5 (2xC), 167.6 (2xC), 178.9 (2xC); LRMS (ESI−) m/z (%)
505 (100) [M−H]−; HRMS (ESI+) m/z calc. for C30H19O8 507.1074, found 507.1063.
4.1.3.5. (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 4,6-
dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv.) and [1,1’-Biphenyl]-3,4’-
dicarboxaldehyde 18b (63 mg, 0.30 mmol, 0.5 equiv.). The pure product (110 mg, 0.21 mmol,
72%) was obtained as a grey powder. m.p. > 410°C; 1H NMR (400 MHz, DMSO-d6)
δ ppm 6.16
(s, 2H), 6.29 (s, 2H), 6.66 (s, 1H), 6.71 (s, 1H), 7.56-7.63 (m, 1H), 7.76 (d, J = 7.2 Hz, 1H), 7.84
(d, J = 8.2 Hz, 2H), 7.93-8.04 (m, 3H), 8.17 (s, 1H), 10.98-11.34 (m, 4xOH); 13C NMR (100 MHz,
DMSO-d6)
δ ppm 90.6 (CH), 90.7 (CH), 97.8 (2xC), 102.4 (CH), 102.5 (CH), 107.6 (CH), 107.9
(CH), 127.2 (2xCH), 129.1 (2xCH), 129.7 (2xCH), 131.3 (2xCH), 131.9 (C), 133.2 (C), 139.8
15

ACCEPTED MANUSCRIPT
(C), 139.9 (C), 148.0 (C), 148.1 (C), 158.5 (2xC), 167.5 (C), 167.6 (C), 167.7 (C), 167.8 (C),
178.9 (C), 179.0 (C); LRMS (ESI−) m/z (%) 505 (100) [M−H]−; HRMS (ESI+) m/z calc. for
C30H19O8 507.1074, found 507.1073.
4.1.3.6. (2Z,2’Z)-2,2’-(biphenyl-2-4’-diylbis(methan-1-yl-1-ylidene))bis(4,6-dihydroxybenzofuran-
3(2H)-one) (9)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv.) and [1,1’-Biphenyl]-2,4’-
dicarboxaldehyde 18c (63 mg, 0.30 mmol, 0.5 equiv.). The pure product (135 mg, 0.26 mmol,
89%) was obtained as a grey powder. m.p. > 410°C; 1H NMR (400 MHz, DMSO-d6)
δ ppm 6.14
(s, 1H), 6,15 (s, 1H), 6.25 (s, 1H), 6.28 (s, 1H), 6.41 (s, 1H), 6.70 (s, 1H), 7.36-7.61 (m, 5H),
8.01 (d, J = 6.0 Hz, 2H), 8.19-8.29 (m, 1H), 10.92-11.25 (m, 4xOH); 13C NMR (100 MHz,
DMSO-d6)
δ ppm 90.5 (CH), 90.6 (CH), 97.8 (2xC), 102.3 (CH), 102.4 (CH), 105.5 (CH), 107.4
(CH), 128.1 (CH), 129.1 (CH), 129.5 (CH), 130.2 (2xCH), 130.4 (CH), 130.6 (2xCH), 131.7
(2xC), 140.4 (C), 142.1 (C), 148.1 (2xC), 158.4 (C), 158.5 (C), 167.5 (2xC), 167.7 (2xC), 178.8
(C), 178.9 (C); LRMS (ESI−) m/z (%) 505 (100) [M−H]−; HRMS (ESI+) m/z calc. for C30H19O8
507.1074, found 507.1073.
4.1.3.7. (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 4,6-
dihydroxybenzofuran-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; 1H NMR (400 MHz, DMSO-d6)
(s, 2H), 6.26 (s, 2H), 6.73 (s, 2H), 7.53-7.82 (m, 4H), 7.90-8.07 (m, 2H), 8.12-8.27 (m, 2H),
11.00 (s, 2xOH), 11.06 (s, 2xOH); 13C NMR (100 MHz, DMSO-d6)
ppm 90.7 (2xCH), 97.9
δ ppm 6.15
δ
(2xC), 102.4 (2xCH), 107.9 (2xCH), 127.5 (2xCH), 129.3 (2xCH), 129.4 (CH), 129.5 (CH), 129.6
(2xCH), 133.2 (2xC), 140.2 (2xC), 148.1 (2xC), 158.5 (2xC), 167.7 (2xC), 167.8 (2xC), 179.1
(2xC); LRMS (ESI−) m/z (%) 505 (100) [M−H]−; HRMS (ESI+) m/z calc. for C30H19O8 507.1074,
found 507.1070.
4.1.3.8. (2Z,2’Z)-2,2’-(biphenyl-2-3’-diylbis(methan-1-yl-1-ylidene))bis(4,6-dihydroxybenzofuran-
3(2H)-one) (11)
16

ACCEPTED MANUSCRIPT
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (100 mg, 0.60 mmol, 1.0 equiv.) and [1,1’-Biphenyl]-2,3’-
dicarboxaldehyde 18e (63 mg, 0.30 mmol, 0.5 equiv.). The pure product (130 mg, 0.25 mmol,
85%) was obtained as a grey powder. m.p. > 410°C; 1H NMR (400 MHz, DMSO-d6)
δ ppm 6.05-
6.20 (m, 3H), 6.25 (s, 1H), 6.40 (s, 1H), 6.68 (s, 1H), 7.32-7.69 (m, 5H), 7.45 (d, J = 6.8 Hz, 1H),
7.50 (dd, J = 7.4, 6.8 Hz, 1H), 7.61-7.56 (m, 2H), 7.83 (s, 1H), 8.03 (d, J = 6.4 Hz, 1H), 8.26 (d,
J = 7.2 Hz, 1H), 10.92-11.16 (m, 4xOH); 13C NMR (100 MHz, DMSO-d6)
δ ppm 90.6 (2xCH),
97.8 (2xC), 102.3 (2xCH), 105.5 (CH), 107.6 (CH), 128.1 (CH), 129.0 (CH), 129.1 (CH), 129.5
(CH), 129.7 (CH), 130.2 (CH), 130.3 (CH), 130.4 (CH), 131.7 (C), 132.7 (C), 140.5 (C), 142.3
(C), 148.1 (2xC), 158.4 (C), 158.5 (C), 167.6 (2xC), 167.8 (2xC), 178.8 (C), 178.9 (C); LRMS
(ESI−) m/z (%) 505 (100) [M−H]−; HRMS (ESI+) m/z calc. for C30H19O8 507.1074, found
507.1077.
4.1.3.9. (2Z,2'Z)-2,2'-[((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1-yl-1-
ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (12)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (166 mg, 1.00 mmol, 1.0 equiv.) and 4,4’-[1,3-
propanediyl(oxy)]bisbenzaldehyde[37] (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; 1H NMR (400 MHz,
DMSO-d6)
δ
ppm 10.90 (bs, 4H), 7.86 (d, J = 8.4 Hz, 4H), 7.08 (d, J = 8.4 Hz, 4H), 6.60 (s, 2H),
ppm
6.22 (s, 2H), 6.08 (s, 2H), 4.22 (s, 4H), 2.28-2.18 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
δ
28.5 (CH2), 64.4 (2xCH2), 90.5 (2xCH), 97.7 (2xCH), 102.7 (2xC), 108.4 (2xCH), 115.0 (4xCH),
125.0 (2xC), 132.5 (4xCH), 146.5 (2xC), 158.3 (2xC), 159.3 (2xC), 167.2 (2xC), 167.6 (2xC),
179.0 (2xC); LRMS (ESI+) m/z (%) 581 (100) [M+H]+, 291 (23); HRMS (ESI+) m/z calc. for
C33H25O10 581.1442, found 581.1442.
4.1.3.10. (2Z,2'Z)-2,2'-[((2-cyclohexylpropane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1-
yl-1-ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (13)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (150 mg, 0.90 mmol, 1.0 equiv.) and 4,4’-[1,3-(2-
cyclohexyl)propanediyl(oxy)]bisbenzaldehyde 21a (150 mg, 0.41 mmol, 0.45 equiv.). After
purification by column chromatography on silica gel (CH2Cl2/MeOH 9:1 to 8:2), the pure product
(92 mg, 0.14 mmol, 34%) was obtained as an orange powder. m.p. > 180°C (decomposition);
17

ACCEPTED MANUSCRIPT
RMN 1H (400 MHz, CD3OD)
δ
ppm 1.15-1.43 (m, 5H), 1.68-1.93 (m, 6H), 2.09-2.18 (m, 1H),
4.19 (dd, J = 9.7, 6.3 Hz, 2H), 4.25 (dd, J = 9.7, 5.1 Hz, 2H), 6.04 (d, J = 1.7 Hz, 2H), 6.22 (d, J
= 1.7 Hz, 2H), 6.64 (s, 2H), 7.02 (d, J = 8.9 Hz, 4H), 7.83 (d, J = 8.9 Hz, 4H); RMN 13C (100
MHz, CD3OD)
δ ppm 27.8 (CH2), 28.0 (2xCH2), 31.9 (2xCH2), 38.5 (CH), 45.3 (CH), 67.8
(2xCH2), 91.9 (2xCH), 98.8 (2xCH), 104.8 (2xC), 111.8 (2xCH), 116.2 (4xCH), 126.7 (2xC),
134.1 (4xCH), 148.5 (2xC), 160.1 (2xC), 161.8 (2xC), 169.7 (2xC), 169.8 (2xC), 183.0 (2xC);
LRMS (ESI+) m/z (%) 663 (100) [M+H]+, 359 (80), 271 (27), 193 (19); HRMS (ESI+) m/z calc.
for C39H35O10 663.2225, found 663.2216.
4.1.3.11. (2Z,2'Z)-2,2'-[((2-(methylcyclohexyl)propane-1,3-diylbis(oxy))bis(4,1-
phenylene))bis(methan-1-yl-1-ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (14)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (190 mg, 1.16 mmol, 1.0 equiv.) and 4,4’-[1,3-(2-
(methylcyclohexyl))propanediyl(oxy)]bisbenzaldehyde 21b (200 mg, 0.53 mmol, 0.45 equiv.).
After purification by recrystallization in methanol, the pure product (278 mg, 0.41 mmol, 78%)
was obtained as a yellow powder. m.p. > 200°C (decomposition); 1H NMR (400 MHz, DMSO-d6)
δ
ppm 0.80-0.97 (m, 2H), 1.04-1.48 (m, 6H), 1.53-1.78 (m, 5H), 2.29-2.41 (m, 1H), 4.09 (d, J =
4.3 Hz, 4H), 6.13 (s, 2H), 6.23 (s, 2H), 6.57 (s, 2H), 7.05 (d, J = 8.4 Hz, 4H), 7.83 (d, J = 8.4 Hz,
4H), 10.95 (s, 4H); 13C NMR (100 MHz, DMSO-d6)
ppm 25.7 (2xCH2), 26.1 (CH2), 33.0
δ
(2xCH2), 34.2 (CH), 34.9 (CH2), 35.8 (CH), 68.1 (2xCH2), 90.5 (2xCH), 97.7 (2xCH), 102.6
(2xC), 108.3 (2xCH), 115.1 (4xCH), 125.0 (2xC), 132.5 (4xCH), 146.5 (2xC), 158.3 (2xC), 159.4
(2xC), 167.2 (2xC), 167.6 (2xC), 179.0 (2xC); LRMS (ESI+) m/z (%) 677 (100) [M+H]+, 339 (23)
[M+2H]2+; HRMS (ESI+) m/z calc. for C40H37O10 677.2381, found 677.2373.
4.1.3.12. (2Z,2'Z)-2,2'-[((2-cyclopentylpropane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1-
yl-1-ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (15)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (210 mg, 1.25 mmol, 1.0 equiv.) and 4,4’-[1,3-(2-
cyclopentyl)propanediyl(oxy)]bisbenzaldehyde 21c (200 mg, 0.57 mmol, 0.45 equiv.). After
precipitation in CH2Cl2, the pure product (207 mg, 0.32 mmol, 56%) was obtained as a yellow
powder. m.p. > 180°C (decomposition); 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.21-1.38 (m, 2H),
1.44-1.69 (m, 4H), 1.76-1.89 (m, 2H), 1.95-2.13 (m, 2H), 4.12 (dd, J = 9.8, 5.9 Hz, 2H), 4.19
(dd, J = 9.8, 3.9 Hz, 2H), 6.13 (d, J = 1.7 Hz, 2H), 6.23 (d, J = 1.7 Hz, 2H), 6.57 (s, 2H), 7.05 (d,
J = 8.8 Hz, 4H), 7.83 (d, J = 8.8 Hz, 4H), 10.91 (s, 2H), 10.93 (s, 2H); 13C NMR (100 MHz,
18

ACCEPTED MANUSCRIPT
DMSO-d6)
δ
ppm 24.5 (2xCH2), 30.3 (2xCH2), 43.0 (CH), 67.2 (2xCH2), 90.5 (2xCH), 97.7
(2xCH), 102.7 (2xC), 108.4 (2xCH), 115.1 (4xCH), 125.0 (2xC), 132.5 (4xCH), 146.5 (2xC),
158.3 (2xC), 159.5 (2xC), 167.2 (2xC), 167.6 (2xC), 179.0 (2xC); LRMS (ESI+) m/z (%) 649
(100) [M+H]+, 325 (15) [M+2H]2+; HRMS (ESI+) m/z calc. for C38H33O10 649.2068, found
649.2067.
4.1.3.13. (2Z,2'Z)-2,2'-[((2-isobutylpropane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1-yl-
1-ylidene)]bis(4,6-dihydroxybenzofuran-3(2H)-one) (16)
The crude product was prepared according to general procedure A starting from 4,6-
dihydroxybenzofuran-3(2H)-one 2 (322 mg, 1.94 mmol, 1.0 equiv.) and 4,4’-[1,3-(2-
isobutyl)propanediyl(oxy)]bisbenzaldehyde 21d (300 mg, 0.88 mmol, 0.45 equiv.). After
recrystallization in methanol, the pure product (319 mg, 0.50 mmol, 57%) was obtained as an
orange powder. m.p. 184-185°C; 1H NMR (400 MHz, DMSO-d6)
δ ppm 0.91 (d, J = 6.5 Hz, 6H),
1.40 (dd, J = 7.0, 7.0 Hz, 2H), 1.68-1.80 (m, 1H), 2.27-2.37 (m, 1H), 4.10 (d, J = 5.2 Hz, 4H),
6.09 (d, J = 1.7 Hz, 2H), 6.22 (d, J = 1,7 Hz, 2H), 6.58 (s, 2H), 7.06 (d, J = 8.8 Hz, 4H), 7.84 (d,
J = 8.8 Hz, 4H), 10.88 (s, 2H), 10.92 (s, 2H); 13C NMR (100 MHz, DMSO-d6)
δ ppm 22.7
(2xCH3), 24.8 (CH), 35.7 (CH), 37.2 (CH2), 68.0 (2xCH2), 90.5 (2xCH), 97.7 (2xCH), 102.7
(2xC), 108.4 (2xCH), 115.1 (4xCH), 125.0 (2xC), 132.5 (4xCH), 146.5 (2xC), 158.2 (2xC), 159.4
(2xC), 167.2 (2xC), 167.6 (2xC), 179.0 (2xC); LRMS (ESI+) m/z (%) 637 (100) [M+H]+, 319 (18)
[M+2H]2+; HRMS (ESI+) m/z calc. for C37H33O10 637.2068, found 637.2058.
4.1.3.14. (2Z,2'Z)-2,2'-[((2-isobutylpropane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(methan-1-yl-
1-ylidene)]bis(benzofuran-3(2H)-one) (17)
To a solution of benzofuran-3(2H)-one (130 mg, 0.97 mmol, 1.0 equiv.) and 4,4’-[1,3-(2-
isobutyl)propanediyl(oxy)]bisbenzaldehyde 21d (150 mg, 0.44 mmol, 0.45 equiv.) in 15 mL of
CH2Cl2 was added 1.6 g of Al2O3. The reaction mixture was stirred overnight and was then
filtered off. The filtrate was concentrated under vacuum and the crude product was
recrystallized in methanol to afford 17 (149 mg, 0.26 mmol, 59%) as a yellow powder. m.p. 133-
134°C; 1H NMR (400 MHz, DMSO-d6)
δ ppm 0.93 (d, J = 6.5 Hz, 6H), 1.42 (dd, J = 7.0, 7.0 Hz,
2H), 1.68-1.82 (m, 1H), 2.30-2.40 (m, 1H), 4.14 (d, J = 5.3 Hz, 4H), 6.94 (s, 2H), 7.11 (d, J = 8.8
Hz, 4H), 7.31 (m, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.74-7.83 (m, 4H), 7.96 (d, J = 8.8 Hz, 4H); 13C
NMR (100 MHz, DMSO-d6)
δ ppm 22.7 (2xCH3), 24.8 (CH), 35.7 (CH), 37.2 (CH2), 68.0
(2xCH2), 112.7 (2xCH), 113.1 (2xCH), 115.3 (4xCH), 121.1 (2xC), 123.8 (2xCH), 124.1 (2xCH),
124.5 (2xC), 133.4 (4xCH), 137.3 (2xCH), 145.1 (2xC), 160.2 (2xC), 165.1 (2xC), 183.2 (2xC);
19

ACCEPTED MANUSCRIPT
LRMS (ESI+) m/z (%) 573 (100) [M+H]+, 287 (8) [M+2H]2+; HRMS (ESI+) m/z calc. for C37H33O6
573.2272, found 573.2270.
4.1.4. 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.
4.1.4.1. [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,
1.42 mmol, 71%) was obtained as a white powder. m.p. 144−146°C, lit. 146°C;[38] 1H NMR
(400 MHz, CDCl3)
δ
ppm 7.86 (d, J = 8.5 Hz, 4H), 8.06 (d, J = 8.5 Hz, 4H), 10.14 (s, 2H); 13C
NMR (100 MHz, CDCl3)
δ ppm 128.2 (4×CH), 130.6 (4×CH), 136.2 (2×C), 145.7 (2×C), 191.9
(2×CH).
4.1.4.2. [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; 1H NMR (400 MHz, CDCl3)
ppm 7.67 (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); 13C NMR (100 MHz, CDCl3)
ppm 127.7
δ
δ
(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).
4.1.4.3. [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; 1H NMR (400 MHz, CDCl3)
δ
20

ACCEPTED MANUSCRIPT
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); 13C 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] 1H 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
ppm 128.1 (2×CH), 129.6 (2×CH), 129.9 (2×CH), 133.1
(s, 2H); 13C NMR (100 MHz, CDCl3)
δ
(2×CH), 137.2 (2×C), 140.8 (2×C), 192.2 (2×CH).
4.1.4.5. [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,
1.08 mmol, 81%) was obtained as a white powder. m.p. 57°C, lit. 54−56°C;[40] 1H NMR (400
MHz, CDCl3)
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); 13C NMR (100 MHz, CDCl3)
ppm 128.2 (CH), 128.4 (CH), 129.2 (CH), 129.4 (CH),
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-
δ
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).
4.1.5. 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.
21

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4.1.5.1. 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 1H (400 MHz, CDCl3)
1.27 (t, J = 7.1 Hz, 6H), 1.26-1.36 (m, 1H), 1.58-1.77 (m, 5H), 2.01-2.16 (m, 1H), 3.14 (d, J = 9.1
Hz, 1H), 4.19 (q, J = 7.1 Hz, 4H); RMN 13C (100 MHz, CDCl3)
ppm 14.3 (2xCH3), 26.2
δ ppm 0.98-1.21 (m, 4H),
δ
(2xCH2), 26.3 (CH2), 30.9 (2xCH2), 38.1 (CH), 58.6 (CH), 61.3 (2xCH2), 169.0 (2xC).
4.1.5.2. Diethyl 2-(methylcyclohexyl)propanedioate (19b)
The crude product was prepared according to procedure C starting from diethyl malonate (4.00
g, 24.97 mmol) and (bromomethyl)cyclohexane (6.64 g, 37.50 mmol). After purification by
column chromatography on silica gel (cyclohexane/EtOAc 98:2), the pure product (1.68 g, 6.55
mmol, 26%) was obtained as a colorless liquid. 1H NMR (400 MHz, CDCl3)
2H), 1.06-1.20 (m, 4H), 1.23 (t, J = 7.1 Hz, 6H), 1.52-1.72 (m, 5H), 1.75 (dd, J = 7.4, 7.4 Hz,
2H), 3.40 (t, J = 7.7 Hz, 1H), 4.14 (q, J = 7.1 Hz, 4H); 13C NMR (100 MHz, CDCl3)
ppm 14.2
δ ppm 0.82-0.93 (m,
δ
(2xCH3), 26.2 (2xCH2), 26.5 (CH2), 33.0 (2xCH2), 35.7 (CH), 36.2 (CH2), 49.8 (CH), 61.3
(2xCH2), 169.9 (2xC).
4.1.6. 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.
4.1.6.1. 2-Cyclohexylpropane-1,3-diol (20a)
The crude product, prepared according to procedure D starting from diethyl 2-
cyclohexylpropanedioate 19a (1.20 g, 4.95 mmol), was obtained (0.36 g, 2.28 mmol, 46%) as a
22

ACCEPTED MANUSCRIPT
white solid. m.p. 96°C, lit. 93−95°C;[41] 1H NMR (400 MHz, CDCl3)
δ ppm 0.96-1.10 (m, 2H),
1.11-1.30 (m, 3H), 1.38-1.48 (m, 1H), 1.53-1.61 (m, 1H), 1.61-1.78 (m, 5H), 3.82 (dd, J = 10.5,
7.4 Hz, 2H), 3.88 (dd, J = 10.5, 3.7 Hz, 2H); 13C NMR (100 MHz, CDCl3)
δ ppm 26.7 (CH2),
26.8 (2xCH2), 30.8 (2xCH2), 36.9 (CH), 47.2 (CH), 64.9 (2xCH2).
4.1.6.2. 2-(methylcyclohexyl)propane-1,3-diol (20b)
The crude product, prepared according to procedure D starting from diethyl 2-
(methylcyclohexyl)propanedioate 19b (1.60 g, 6.24 mmol), was obtained (0.96 g, 5.57 mmol,
89%) as a white solid. m.p. 63−65°C; 1H NMR (400 MHz, CDCl3)
(dd, J = 7.0, 7.0 Hz, 2H), 1.12-1.35 (m, 4H), 1.60-1.75 (m, 5H), 1.86-1.97 (m, 1H), 3.63 (dd, J =
10.6, 7.8 Hz, 2H), 3.81 (dd, J = 10.6, 3.6 Hz, 2H); 13C NMR (100 MHz, CDCl3)
ppm 26.5
δ ppm 0.80-0.94 (m, 2H), 1.08
δ
(2xCH2), 26.8 (CH2), 33.8 (2xCH2), 35.2 (CH), 35.5 (CH2), 39.1 (CH), 67.4 (2xCH2).
4.1.6.3. 2-cyclopentylpropane-1,3-diol (20c)
The crude product, prepared according to procedure D starting from commercially available
diethyl 2-cyclopentylpropanedioate 19c (2.50 g, 10.95 mmol), was obtained (1.22 g, 8.47 mmol,
77%) as a white solid. m.p. 68°C, lit. 68−69°C;[42] 1H NMR (400 MHz, CDCl3)
δ ppm 1.10-1.22
(m, 2H), 1.47-1.76 (m, 6H), 1.76-1.85 (m, 2H), 3.74 (dd, J = 10.6, 7.5 Hz, 2H), 3.93 (dd, J =
10.6, 3.7 Hz, 2H); 13C NMR (100 MHz, CDCl3)
47. 8 (CH), 66.6 (2xCH2).
δ ppm 25.2 (2xCH2), 31.1 (2xCH2), 38.4 (CH),
4.1.6.4. 2-isobutylpropane-1,3-diol (20d)
The crude product, prepared according to procedure D starting from commercially available
diethyl 2-isobutylpropanedioate 19d (4.00 g, 18.49 mmol), was obtained (1.83 g, 13.84 mmol,
75%) as a light yellow oil. 1H NMR (400 MHz, CDCl3)
= 7.1, 7.1 Hz, 2H), 1.52-1.63 (m, 1H), 1.73-1.83 (m, 1H), 3.54 (dd, J = 10.7, 7.7 Hz, 2H), 3.70
δ ppm 0.86 (d, J = 6.6 Hz, 6H), 1.02 (dd, J
(dd, J = 10.7, 3.8 Hz, 2H), 3.84 (bs, 2H); 13C NMR (100 MHz, CDCl3)
δ ppm 22.9 (2xCH3), 25.5
(CH), 37.0 (CH2), 39.7 (CH), 66.1 (2xCH2).
4.1.7. 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.
23

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4.1.7.1. 4,4'-[1,3-(2-cyclohexyl)propanediyl(oxy)]bisbenzaldehyde (21a)
The crude product was prepared according to procedure E starting from 2-cyclohexylpropane-
1,3-diol 20a (360 mg, 2.28 mmol). After purification by column chromatography on silica gel
(cyclohexane/EtOAc 8:2), the pure product (356 mg, 0.97 mmol, 43%) was obtained as a
colorless oil. 1H NMR (400 MHz, CDCl3)
(m, 1H), 4.19 (dd, J = 9.4, 6.4 Hz, 2H), 4.25 (dd, J = 9.4, 4.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 4H),
7.83 (d, J = 8.8 Hz, 4H), 9.88 (s, 2H); 13C NMR (100 MHz, CDCl3)
ppm 26.6 (CH2), 26.7
δ ppm 1.10-1.37 (m, 6H), 1.63-1.88 (m, 5H), 2.13-2.22
δ
(2xCH2), 30.8 (2xCH2), 36.9 (CH), 43.8 (CH), 66.7 (2xCH2), 115.0 (4xCH), 130.2 (2xC), 132.2
(4xCH), 164.1 (2xC), 190.9 (2xCH); LRMS (ESI+) m/z (%) 389 (22) [M+Na]+, 367 (100) [M+H]+;
HRMS (ESI+) m/z calc. for C23H27O4 367.1909, found 367.1915.
4.1.7.2. 4,4'-[1,3-(2-(methylcyclohexyl))propanediyl(oxy)]bisbenzaldehyde (21b)
The crude product was prepared according to procedure E starting from 2-
(methylcyclohexyl)propane-1,3-diol 20b (900 mg, 5.22 mmol). After purification by column
chromatography on silica gel (cyclohexane/EtOAc 8:2), the pure product (660 mg, 1.74 mmol,
33%) was obtained as a light yellow oil. 1H NMR (400 MHz, CDCl3)
δ ppm 0.86-0.99 (m, 2H),
1.11-1.41 (m, 4H), 1.45 (dd, J = 7.0, 7.0 Hz, 2H), 1.60-1.79 (m, 5H), 2.39-2.51 (m, 1H), 4.11 (d,
J = 5.4 Hz, 4H), 7.00 (d, J = 8.7 Hz, 4H), 7.80 (d, J = 8.7 Hz, 4H), 9.85 (s, 2H); 13C NMR (100
MHz, CDCl3)
δ ppm 26.3 (2xCH2), 26.6 (CH2), 33.6 (2xCH2), 35.0 (CH), 35.6 (CH2), 36.0 (CH),
68.4 (2xCH2), 114.9 (4xCH), 130.1 (2xC), 132.0 (4xCH), 164.0 (2xC), 190.8 (2xCH); LRMS
(ESI+) m/z (%) 381 (100) [M+H]+; HRMS (ESI+) m/z calc. for C24H29O4 381.2066, found
381.2070.
4.1.7.3. 4,4'-[1,3-(2-cyclopentyl)propanediyl(oxy)]bisbenzaldehyde (21c)
The crude product was prepared according to procedure E starting from 2-cyclopentylpropane-
1,3-diol 20c (1.17 g, 8.11 mmol). After purification by column chromatography on silica gel
(cyclohexane/EtOAc 8:2), the pure product (1.50 g, 4.25 mmol, 52%) was obtained as a light
yellow oil. 1H NMR (400 MHz, CDCl3)
(m, 2H), 1.96-2.11 (m, 2H), 4.13 (dd, J = 9.3, 5.8 Hz, 2H), 4.20 (dd, J = 9.3, 3.4 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 4H), 7.75 (d, J = 8.8 Hz, 4H), 9.79 (s, 2H); 13C NMR (100 MHz, CDCl3)
ppm 24.8
δ ppm 1.15-1.29 (m, 2H), 1.45-1.68 (m, 4H), 1.78-1.89
δ
(2xCH2), 30.9 (2xCH2), 38.8 (CH), 43.9 (CH), 67.3 (2xCH2), 114.7 (4xCH), 129.9 (2xC), 131.8
(4xCH), 163.9 (2xC), 190.5 (2xCH); LRMS (ESI+) m/z (%) 375 (35) [M+Na]+, 353 (100) [M+H]+;
HRMS (ESI+) m/z calc. for C22H25O4 353.1753, found 353.1758.
24

ACCEPTED MANUSCRIPT
4.1.7.4. 4,4'-[1,3-(2-isobutyl)propanediyl(oxy)]bisbenzaldehyde (21d)
The crude product was prepared according to procedure E starting from 2-isobutylpropane-1,3-
diol 20d (1.70 g, 12.86 mmol). After purification by column chromatography on silica gel
(cyclohexane/EtOAc 9:1 to 6:4), the pure product (3.20 g, 9.40 mmol, 73%) was obtained as a
colorless oil. 1H NMR (400 MHz, CDCl3)
Hz, 2H), 1.68-1.82 (m, 1H), 2.38-2.48 (m, 1H), 4.14 (d, J = 5.6 Hz, 4H), 7.02 (d, J = 8.7 Hz, 4H),
7.83 (d, J = 8.7 Hz, 4H), 9.89 (s, 2H); 13C NMR (100 MHz, CDCl3)
ppm 22.9 (2xCH3), 25.5
δ ppm 0.97 (d, J = 6.6 Hz, 6H), 1.47 (dd, J = 7.2, 7.2
δ
(CH), 36.5 (CH), 37.6 (CH2), 68.4 (2xCH2), 115.0 (4xCH), 130.2 (2xC), 132.2 (4xCH), 164.1
(2xC), 190.9 (2xCH); LRMS (ESI+) m/z (%) 363 (25) [M+Na]+, 341 (100) [M+H]+; HRMS (ESI+)
m/z calc. for C21H25O4 341.1753, found 341.1760.
4.2. 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,[43]
within the MLP strategy.[44,45] Docking methodology was validated by re-docking the co-
crystallized 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.
4.3. Biology
4.3.1. Cytotoxicity assays
The cytotoxicity assays were realized using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) colorimetric assay on HEK293 cell lines. Cells were seeded in 96-
well culture plates (104 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
25

ACCEPTED MANUSCRIPT
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.
4.3.2. Expression and Purification of Recombinant HCV NS5B
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)
∆21
∆
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
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%.
∆21-enriched fractions were selected with a Bradford
∆
∆
∆
4.3.3. HCV-NS5B
An enzyme assay was used to measure inhibition of HCV-NS5B
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
∆21 Polymerase Assay.
∆
21 polymerase activity. The
∆
∆
Healthcare, Chalfont St. Giles, U.K.) and ATP, measured with an intercalating agent (SYBR
Green, Applied Biosystems), as previously described.[46] Each experiment was performed in
triplicate.
4.3.4. 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.
26

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