Discovery of metal ions chelator quercetin derivatives with potent anti-HCV activities

Article abstract of DOI:10.3390/molecules20046978

Analogues or isosteres of α,γ-diketoacid (DKA) 1a show potent inhibition of hepatitis C virus (HCV) NS5B polymerase through chelation of the two magnesium ions at the active site. The anti-HCV activity of the flavonoid quercetin ( 2) could partly be attri

Full text of DOI:10.3390/molecules20046978

Molecules 2015, 20, 6978-6999; doi:10.3390/molecules20046978
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Discovery of Metal Ions Chelator Quercetin Derivatives with
Potent Anti-HCV Activities
Dongwei Zhong 1,†, Mingming Liu 1,†, Yang Cao 1, Yelin Zhu 1, Shihui Bian 1, Jiayi Zhou 1,
Fengjie Wu 1, Kum-Chol Ryu 2, Lu Zhou 1,* and Deyong Ye 1,*
1
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhang-Heng Rd,
Shanghai 201203, China; E-Mails: 12211030012@fudan.edu.cn (D.Z.);
09111030009@fudan.edu.cn (M.L.); 14111030010@fudan.edu.cn (Y.C.);
09301030045@fudan.edu.cn (Y.Z.); 08301030057@fudan.edu.cn (S.B.);
09301030009@fudan.edu.cn (J.Z.); 11307120169@fudan.edu.cn (F.W.)
2
Institute of Pharmacy, HamHung Pharmaceutical University, HamHung 999093,
Democratic People’s Republic of Korea; E-Mail: yegroup@126.com
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: zhoulu@fudan.edu.cn (L.Z.);
dyye@shmu.edu.cn (D.Y.); Tel.: +86-21-5198-0117 (D.Y.); Fax: +86-21-5198-0125 (D.Y.).
Academic Editor: Derek J. McPhee
Received: 3 March 2015 / Accepted: 13 April 2015 / Published: 16 April 2015
Abstract: Analogues or isosteres of α,γ-diketoacid (DKA) 1a show potent inhibition of
hepatitis C virus (HCV) NS5B polymerase through chelation of the two magnesium ions at
the active site. The anti-HCV activity of the flavonoid quercetin (2) could partly be
attributed to it being a structural mimic of DKAs. In order to delineate the structural
features required for the inhibitory effect and improve the anti-HCV potency, two novel
types of quercetin analogues, 7-O-arylmethylquercetins and quercetin-3-O-benzoic acid
esters, were designed, synthesized and evaluated for their anti-HCV properties in
cell-based assays. Among the 38 newly synthesized compounds, 7-O-substituted derivative
3i and 3-O-substituted derivative 4f were found to be the most active in the corresponding
series (EC50 = 3.8 μM and 9.0 μΜ, respectively). Docking studies suggested that the
quercetin analogues are capable of establishing key coordination with the two magnesium
ions as well as interactions with residues at the active site of HCV NS5B.

Molecules 2015, 20
Keywords: anti-HCV activities; diketoacids mimic; quercetin derivatives; synthesis; docking
6979
1. Introduction
It was estimated that about 170 million people worldwide were chronically infected by hepatitis C
virus (HCV) and were consequently at risk of developing liver cirrhosis and/or hepatocellular
carcinoma [1]. A protective vaccine is not yet available and the previous standard of care (SOC), which is
pegylated-interferon, combined with ribavirin, is often difficult to tolerate and results in a sustained
viral response (SVR) in only 50% of patients infected with the predominant genotype 1 [2,3]. Recent
advances in the development of direct acting antivirals (DAAs) have significantly improved SVR in
patients, providing a new hope for cure in infected patients. Three NS3/4A protease inhibitors were
approved by FDA for the treatment of genotype 1 hepatitis C, and in late 2013, sofosbuvir, a
first-in-class NS5B polymerase inhibitor, was launched and became a cornerstone of recommended HCV
therapy against almost all HCV genotypes [4–7]. However, due to the potential of drug resistant
strains [8] and the high price of the new HCV drugs [9], the development of novel anti-HCV agents is
still an urgent necessity.
The RNA-dependent RNA polymerase (RdRp) of HCV, also known as protein NS5B, is a key
enzyme in the synthesis of a complementary minus-strand RNA and the subsequent synthesis of a
genomic plus-strand RNA from this minus-strand RNA template [10]. In the enzymatic reactions, two
divalent cations in the active site, such as magnesium (Mg2+) or manganese (Mn2+) play a critical role
in the ligation of the ribonucleotide triphosphate (rNTP) substrates and the promotion of a favorable
geometry of the active site. The pivotal role of the divalent cations in the active site of the HCV
polymerase supports the rational basis of the inhibition of this enzyme by metal chelating motifs.
The α,γ-diketoacid (DKA) 1a (Figure 1) has been identified as a selective and reversible inhibitor
against NS5B through high-throughput screening approaches. Subsequent structure-activity
relationship (SAR) optimization led to 1b (Figure 1), a potent HCV polymerase inhibitor [11]. The
mode of action of DKAs involves chelation of the metal ions present in the active site of the HCV
RdRp [12]. However, due to their unfavorable physicochemical and pharmacokinetic properties [13],
many DKA analogues or isosteres with NS5B inhibitory activities, including meconic acid [14] (1c,
Figure 1) and dihydroxypyrimidine carboxylic acid [12] (1d, Figure 1), have been designed and
prepared, by keeping the metal-binding moieties to achieve effective chelation of the two Mg2+ ions.
Quercetin (2, Figure 2) is a naturally occurring flavonoid with various biological activities such as
antioxidant, antiviral, anticancer, antimicrobial and anti-inflammatory properties; hence, quercetin
and its derivatives have potential as drug development leads [15]. As a part of our studies on
anti-HCV agents, quercetin was identified as an HCV inhibitor with moderate antiviral activity
(EC50 = 19.8 μM) [16]. Furthermore, it had been reported that quercetin could inhibit HCV RdRp at
low micromolar concentration (83.7% inhibition at the concentration of 20 μM in an enzyme
assay) [17]. We reasoned that the adjacent carbonyl and hydroxyl groups on A and C rings of quercetin
make it a perfect DKAs mimic to chelate the two metal ions. In consideration of the structures of 1a
and 1b (Figure 1), introduction of a 2-cyano benzyl ether group at the aromatic meta-position relative

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to the chelating moieties was shown to significantly improve the antiviral activity. Thus, we
anticipated that substituents of quercetin at the 7-O position (7-O-arylmethyl quercetins 3, Figure 2)
would mimic the meta-substituted DKA 1b and enhance the anti-HCV activities. Herein, we report the
design and synthesis of novel 7-O-arylmethyl quercetin derivatives with various aromatic substituents
and the evaluation of their anti-HCV activities. Moreover, it can be hypothesized that another
introduced carbonyl group at 3-position of quercetin, together with the adjacent carbonyl and hydroxyl
group, would be capable of chelating the two metal ions. Hence, this paper also described a series of
novel quercetin-3-O-benzoic acid esters (compounds 4, Figure 2) in an attempt to investigate whether
the introduction of another carbonyl group at 3-position would result in an improved anti-HCV
activity. In addition, several quercetin derivatives with different aliphatic groups substituted at the
3-position were also synthesized for further SAR investigations.
Figure 1. Structures and activities of α,γ-diketoacids (DKAs) and their analogues or
isosteric derivatives.
Figure 2. The design of 7-O-arylmethylquercetin derivatives 3 and quercetin-3-O-benzoic
acid ester derivatives 4.

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2. Results and Discussion
2.1. Chemistry
Synthesis of 7-O-substituted quercetin derivatives is well documented [18]. As depicted in Scheme 1,
peracetylation of quercetin (2) followed by regioselective deacetylation of 7-OAc group with
thiophenol and imidazole in N-methyl-2-pyrrolidone (NMP) at 0 °C gave the 7-O-monodeprotected
flavonol 6. Alkylation of 6 with variously substituted benzyl bromides, followed by deacetylation by
treatment with methanolic ammonia provided the desired 7-O-arylmethylquercetins 3a–3s.
R
R
R
3a
3b
3c
3d
3e
3f
H
o-F
3h
3i
m-F
m-Cl
m-Br
3n
3o
3p
p-F
p-Cl
o-Cl
o-Br
o-CH3
o-CN
o-NO2
3j
3k
3l
p-Br
m-CH3 3q
m-CN 3r
p-CH3
p-CN
p-NO2
3g
3m m-NO2 3s
Reagents and conditions: (a) Ac2O, pyridine, reflux, 6 h, 79%; (b) PhSH, imidazole, NMP, 0 °C, 1 h, 70%;
(c) R-PhCH2Br, K2CO3, acetone, rt, 3 h; (d) 2M NH3/MeOH, 0 °C, 1 h, 40%–56% (two steps).
Scheme 1. Synthesis of the 7-O-substituted quercetin derivatives.
The synthesis of 3-O-substituted quercetin derivatives is outlined in Scheme 2. In the light of the
synthetic route in the literature [19,20], tribenzylation of rutin (8) with potassium carbonate and benzyl
bromide in DMF followed by removal of the C3-rutinose with hydrochloric acid and ethanol gave
3',4',7-tri-O-benzylquercetin (9). Compound 9 was condensed with variously substituted benzoic acids,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) and a catalytic amount of 4-dimethyl-
amiopryidine (DMAP) to afford the relevant quercetin-3-O-ester derivatives 10a–10o. Alternatively,

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the etherification of compound 9 was performed in the presence of potassium carbonate with diverse
bromides or iodides to afford relevant quercetin-3-O-ether derivatives 11a–11d. Finally, the desired
final products quercetin derivatives 4a–4o and 12a–12d were obtained by hydrogenolysis and
further purification by silica gel chromatography.
R1
H
R1
3''-F
R1
4''-F
R2
Me
4a
4b
4c
4d
4e
4f
4g
4h
4i
4k
4l
12a
12b
12c
12d
2''-F
3''-NH2
3''-OCH3
3''-CN
3''-Cl
4''-NH2
4''-OCH3
4''-CN
4''-Cl
i-Pr
2''-NH2
2''-OCH3
2''-CN
4m
4n
4o
(CH2)2OH
(CH2)3CN
4j
Reagents and conditions: (a) K2CO3, BnBr, DMF, 60 °C, 3 h, then HCl con., ethanol, 70 °C, 2 h, 85%;
(b) R1-PhCOOH, EDCI, DMAP, DMF, rt, overnight; (c) H2, Pd/C, 1,4-dioxane/ethanol, rt, 6 h, 40%–79%
(two steps); (d) R2Br or R2I, K2CO3, DMF, 40 °C, 3 h.
Scheme 2. Synthesis of the quercetin-3-O-substituted benzoic acid esters and
3-O-substituted quercetin derivatives.
2.2. Biological Evaluation
The inhibition of HCV by all synthesized quercetin derivatives was evaluated following a
previously reported procedure [16]. All newly synthesized compounds were tested in authentic HCV
infection/replication system in the human hepatoma cell lines Huh7.5.1 using mycophenolic acid [21]
(MPA) as control compound. The cytotoxicity effect of the test compounds was also evaluated in the
same cell line by MTT method. Antiviral effect and cytotoxicity effect were summarized as EC50 and
CC50 respectively, as well as the selective index (SI) which is the ratio of CC50 to EC50. The stability of
quercetin-3-O-ester compounds were also tested in the same buffer as the antiviral activity assay at
37 °C and the hydrolysate was measured by HPLC. Only trace quercetin as hydrolyzate was monitored

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over 72 h which demonstrated that quercetin-3-O-ester derivatives were stable under the biological
evaluation conditions.
As clearly shown in Table 1, all 7-O-substituted quercetin derivatives 3a–3s exhibited potent
anti-HCV activities with EC50 values ranging from 3.8 μM to 8.7 μM. Among these compounds, 3''-Cl
substituted derivatives possessed the highest activities (for 3i, EC50 = 3.8 μM) and a decent selectivity
ratio (for 3i, SI = 7.2). These results tend to indicate the importance of introducing an aromatic group
at 7-O position. Substituents of quercetin at the 7-O position may mimic the meta-substituents of DKA
1b and thus enhance the anti-HCV activities. It should be noted that all the 7-O-substituted analogues
evaluated in this study were found in a narrow range of inhibition activity. This high level of tolerance
will be discussed in the following section.
Table 1. Anti-HCV activities and cytotoxic effects of quercetin-7-O-substituted derivatives.
R
Compound
EC50(μM) a,b,c CC50(μM) c,d
SI e
Position Substituent
3a
3b
3c
3d
3e
3f
-
H
F
8.7 ± 0.2
5.0 ± 0.1
4.8 ± 0.1
5.5 ± 0.2
4.9 ± 0.6
7.5 ± 0.5
7.0 ± 1.1
6.3 ± 0.2
3.8 ± 0.2
4.1 ± 0.3
4.7 ± 0.4
5.4 ± 0.5
6.6 ± 1.1
5.1 ± 0.5
3.9 ± 0.3
5.5 ± 1.5
6.7 ± 1.4
6.2 ± 0.2
4.3 ± 0.2
19.8
>50
>5.8
3.1
7.7
6.8
4.4
1.7
2.1
7.7
7.2
5.4
6.9
2.0
2.3
6.2
3.9
6.4
2.4
1.6
4.5
>2.5
2''
15.4 ± 0.1
36.7 ± 0.7
37.4 ± 0.8
21.7 ± 1.4
12.6 ± 0.1
14.6 ± 0.5
44.5 ± 3.7
27.2 ± 0.2
22.3 ± 1.2
32.5 ± 0.7
10.6 ± 0.5
15.0 ± 0.1
31.9 ± 3.4
15.1 ± 0.1
35.4 ± 1.6
15.9 ± 1.0
10.1 ± 0.1
19.6 ± 0.2
>50
Cl
Br
CH3
CN
NO2
F
3g
3h
3i
3''
4''
Cl
3j
Br
3k
3l
CH3
CN
NO2
F
3m
3n
3o
3p
3q
3r
3s
2
Cl
Br
CH3
CN
NO2
quercetin
a The anti-HCV assay was evaluated in an authentic HCV infection system in the human hepatoma cell lines,
b
c
Huh7.5.1; Concentration required to inhibit HCV RNA replication by 50%; The values obtained as the
d
average of duplicate determinations;
e Selectivity index: ratio of CC50 to EC50.
Concentration required to reduce cell proliferation by 50%;

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To examine the effects of the additional carbonyl group at the 3-position, 3-O-substituted quercetin
derivatives were designed by adding substituted aroyl groups. All the substituted benzoic acid esters
4a–4o exhibited similar or better anti-HCV activities in cell-based assays as compared to the lead
compound quercetin (Table 2).
Table 2. Anti-HCV activities and cytotoxic effects of quercetin-3-O-substituted derivatives.
12a–12d
4a–4o
R1
Compound
R2
EC50(μM) a,b,c
CC50(μM) c,d
SI e
Position
Substituent
4a
4b
4c
-
H
F
-
14.3 ± 5.1
12.9 ± 3.2
12.8 ± 2.8
11.7 ± 1.3
20.2 ± 8.6
9.0 ± 4.5
21.1 ± 1.3
15.6 ± 0.3
13.1 ± 1.0
23.0 ± 3.2
9.7 ± 3.1
23.8 ± 0.8
13.6 ± 2.1
11.4 ± 2.3
14.3 ± 2.2
11.2 ± 0.1
38.9 ± 2.5
26.3 ± 3.1
>50
>50
>50
>3.5
>3.9
>3.9
>4.3
>2.5
>5.6
>2.4
>3.2
>3.8
>3.5
3.6
2''
-
NH2
OCH3
CN
F
-
>50
4d
4e
-
>50
-
>50
4f
3''
4''
-
>50
4g
4h
4i
NH2
OCH3
CN
Cl
-
>50
-
>50
-
>50
4j
-
>50
4k
4l
F
-
35.0 ± 2.4
>50
NH2
OCH3
CN
Cl
-
>2.1
2.5
4m
4n
4o
12a
12b
12c
12d
2
-
33.9 ± 3.7
>50
-
-
>4.4
>3.5
>4.5
0.7
>50
-
-
Me
>50
-
-
i-Pr
28.8 ± 2.0
>50
-
-
(CH2)2OH
(CH2)3CN
>1.9
NA
-
-
>50
quercetin
19.8
>50
>2.5
a The anti-HCV assay was evaluated in an authentic HCV infection system in the human hepatoma cell lines,
b
c
Huh7.5.1; Concentration required to inhibit HCV RNA replication by 50%; The values obtained as the
average of duplicate determinations; d Concentration required to reduce cell proliferation by 50%; e Selectivity
index: ratio of CC50 to EC50.
Among these compounds, 3''-F substituted derivative (4f) showed more than a two-fold increase in
potency over quercetin (EC50 of 9.0 μM for 4f versus 19.7 μM for 2). In order to study the significance
of the coordination between the additional carbonyl group and the magnesium ions, we synthesized
several compounds 12a–12d in which the carbonyl group at 3-O position was replaced by aliphatic groups
of various lengths. It is not surprising that a decrease in potency was observed for these analogues,

Molecules 2015, 20
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mainly because the aliphatic substituents at the 3-position, especially when they are bulky, could not
coordinate to the magnesium ions. The maintenance of anti-HCV activities for quercetin-3-O-benzoic
acid-ester derivatives instead of the 3-O-aliphatic substituents revealed that the introduction of an
electronegative carbonyl group at 3-O position could enhance the chelation ability between the inhibitor
and metal ions, which contribute to the binding affinity in a similar way as the 3-hydroxyl group.
2.3. Molecular Docking
To rationalize the observed activities of different substituents of quercetin and investigate the
binding modes with NS5B, docking calculations were performed on the basis of the reported X-ray
crystallographic structure of HCV genotype 1b RdRp complexed with uridine triphosphate (UTP) and
divalent metal ions (PDB code: 1GX6 [22]). Initially, the lead compound quercetin 2 was docked into
NS5B active site after the removal of the original UTP. As depicted in Figure 3a, 3-, 4-, and 5-O
derivatives of quercetin chelate to the two magnesium ions (2.1 Å–2.7 Å) together with four conserved
key residues (Asp 220, Thr221, Asp318, Asp319) and two water molecules in the active site to form a
double-octahedral center. The three acidic residues (Asp220, Asp318, Asp319) were found to be
indispensable to the polymerase-catalyzed nucleotidyl transfer reaction [22]. The 3'-hydroxyl in B ring
of quercetin forms a hydrogen bond (1.6 Å) with the side chain of Asp225, the same residue that is
hydrogen bonded to the 2'-hydroxyl group of UTP. This binding mode is in accordance with our
previous results [16] that kaempferol (lacking a 3'-hydroxyl as compared to quercetin, 51.9% inhibition
at 50 μM) and isorhamnetin (with 3'-hydroxy methylated, 47.3% inhibition at 50 μM) are less potent
against HCV than quercetin (EC50 = 19.8 μM).
Figure 3. Predicted binding modes in the active site of a reported X-ray structure of HCV
RdRp (PDB code: 1GX6 [22]) from docking studies: (a) quercetin 2; (b) compound 3i;
(c) compound 4f. The two Mg2+ ions are indicated as orange spheres, carbon atoms are
shown in green, oxygen in red, chlorine in yellow, and fluorine in blue.

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Representatives of 7-O-substituted and 3-O-substituted quercetin derivatives (3i and 4f, respectively)
were also docked into the active site of NS5B. In addition to the similar chelation and hydrogen bond
found in quercetin-NS5B system, the 7-O phenyl group of 3i forms a hydrophobic interaction with
Phe193 (Figure 3b). These findings could potentially account for the much better activities of
7-O-substituent compared to quercetin (Table 1). However, due to the relatively large size of the
hydrophobic pocket around Phe193, modification of 7-O phenyl group did not give a noticeable gain
of activity. As to the 3-O-substituted analogue 4f, the carbonyl group at 3-position interacts with one
magnesium ion with a distance of 2.6 Å, the same distance as that in quercetin-NS5B system
(Figure 3a,c). Interestingly, an extra hydrogen bond (1.9 Å) was found between the carbonyl group at
3-position and the backbone NH of Phe224, which may contribute to the better activities of
quercetin-3-O-substituted derivatives compared to quercetin (Table 1).
3. Experimental Section
3.1. General Procedures
All solvents and reagents were purchased from commercial sources and used directly without
purification. Progress of all the reactions were monitored by thin-layer chromatography (TLC) with
precoated silica gel plates GF254, 10–40 µm (Huanghai, Yantai, China). Melting points (m.p.) were
determined using an X-4 microscope melting point apparatus and are uncorrected. Nuclear magnetic
resonance (NMR) spectroscopic analysis was performed on a Bruker DRX-400 spectrometer at
1
13
400 MHz for H-NMR and 100 MHz for C-NMR. Chemical shifts are reported in δ ppm (parts per
million) relative to TMS (0.00) as internal standard. Splitting parterns were assigned as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet). Mass spectra (ESI) were
performed on an Agilent 1100s mass spectrometer, and the values are reported in m/z.
3.2. Synthesis of 2-(3,4-Diacetoxyphenyl)-4-oxo-4H-chromene-3,5,7-triyl triacetate (5)
Quercetin (2, 1.0 g, 3.0 mmol), acetic anhydride (6.1 g, 60.0 mmol), and pyridine (15 mL) were
heated to reflux under argon atmosphere for 6 h. After cooling down to room temperature, the mixture
1
was poured into 50 mL of ice water and filtered to give the product 5 in 79% yield. H-NMR
(DMSO-d6): δ 7.85–7.88 (m, 2H, -ArH), 7.65 (d, J = 2.0 Hz, 1H, -ArH), 7.53 (d, J = 8.0 Hz, 1H, -ArH),
7.18 (d, J = 2.0 Hz, 1H, -ArH), 2.32–2.34 (m, 15H, -CH3-C=O); ESI-MS (m/z) 513.1 [M+H]+.
3.3. Synthesis of 4-(3,5-Diacetoxy-7-hydroxy-4-oxo-4H-chromen-2-yl)-1,2-phenylene diacetate (6)
To a solution of 5 (1.0 g, 2.13 mmol) in anhydrous NMP (30 mL) at 0 °C was added imidazole
(50 mg, 0.74 mmol) followed by thiophenol (0.22 mL, 2.13 mmol). After stirring at 25 °C until the
disappearance of the starting material as monitored by TLC, the mixture was diluted with CH2Cl2, and
then washed succesively with 1 N aq. HCl and saturated brine. The organic phase was dried over
anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 50/1, v/v) to give compound 6 as white powder in 70% yield. 1H-NMR (DMSO-d6):
δ 7.78–7.80 (m, 2H, -ArH), 7.48 (d, J = 8.2 Hz, 1H, -ArH), 6.84 (d, J = 2.3 Hz, 1H, -ArH), 6.51 (d,
J = 2.3 Hz, 1H, -ArH), 2.27–2.30 (m, 12H, -CH3-C=O); ESI-MS (m/z): 470.1 [M+H]+.

Molecules 2015, 20
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3.4. General Procedure for the Preparation of 7a–7s
K2CO3 (95 mg, 0.69 mmol) and appropriate benzyl bromide (0.35 mmol) were added to solution of
6 (200 mg, 0.23 mmol) in acetone (15 mL). The reaction mixture was stirred for 3 h at room
temperature and then filtered. The filtrate was concentrated under reduced pressure to offer the crude
product 7a–7s for the next step directly without further purification.
3.5. General Procedure for the Preparation of 3a–3s
The solution of crude compound 7a–7s in NH3/MeOH (10 mL) was stirred for 1 h at 0 °C and
concentrated under reduced pressure. This residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 50/1, v/v) to give the desired compound 3a–3s.
7-(Benzyloxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3a). Yield 50%. m.p.
1
255–257 °C; H-NMR (DMSO-d6) δ 7.69 (d, J = 2.0 Hz, 1H, -ArH), 7.54 (dd, J = 8.6, 2.0 Hz, 1H,
-ArH), 7.35–7.46 (m, 5H, -ArH) 6.88 (d, J = 8.6 Hz, 1H, -ArH), 6.78 (d, J = 2.0 Hz, 1H, -ArH), 6.41
13
(d, J = 2.0 Hz, 1H, -ArH), 5.21 (s, 2H, -CH2); C-NMR (DMSO-d6) δ 176.4, 164.3, 160.9, 156.4,
148.3, 147.8, 145.5, 136.7, 136.5, 129.0 (2C), 128.6, 128.3 (2C), 122.3, 120.5, 116.0, 115.7, 104.6,
98.5, 93.2, 70.4; ESI-MS (m/z): 392.8 [M+H]+, 412.9 [M+Na]+.
2-(3,4-Dihydroxyphenyl)-7-((2-fluorobenzyl)oxy)-3,5-dihydroxy-4H-chromen-4-one (3b). Yield 51%.
1
m.p. 254–255 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.68 (s, 1H, -OH), 9.56 (s, 1H, -OH),
9.34 (s, 1H, -OH), 7.74 (d, J = 2.0 Hz, 1H, -ArH), 7.68–7.58 (m, 1H, -ArH), 7.58 (dd, J = 8.4, 2.0 Hz,
1H, -ArH), 7.44–7.49 (m, 1H, -ArH), 7.25–7.32 (m, 2H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.86
(d, J = 2.0 Hz, 1H, -ArH), 6.55 (d, J = 2.0 Hz, 1H, -ArH), 5.27 (s, 2H, -CH2); 13C-NMR (DMSO-d6) δ
176.4, 164.1, 160.9 (d, JC-F = 245.1 Hz), 160.9, 156.4, 148.3, 147.8, 145.5, 136.6, 131.4 (d,
JC-F = 3.8 Hz), 131.2 (d, JC-F = 8.3 Hz), 125.1 (d, JC-F = 3.3 Hz), 123.5 (d, JC-F = 14.5 Hz), 122.3,
120.5, 116.0 (d, JC-F = 20.6 Hz), 116.0, 115.7, 104.7, 98.4, 93.1, 64.8 (d, JC-F = 3.6 Hz); ESI-MS (m/z):
411.1 [M+H]+, 433.0 [M+Na]+.
7-((2-Chlorobenzyl)oxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3c). Yield 49%.
1
m.p. 253–254 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.67 (brs, 1H, -OH), 9.54 (brs,
1H, -OH), 9.32 (s, 1H, -OH), 7.74 (d, J = 2.0 Hz, 1H, -ArH), 7.63–7.65 (m, 1H, -ArH), 7.58 (dd,
J = 8.4, 2.0 Hz, 1H, -ArH), 7.54–7.56 (m, 1H, -ArH), 7.40–7.46 (m, 2H, -ArH), 6.90 (d, J = 8.4 Hz,
1H, -ArH), 6.86 (d, J = 2.4 Hz, 1H, -ArH), 6.45 (d, J = 2.4 Hz, 1H, -ArH), 5.29 (s, 2H, -CH2);
13C-NMR (DMSO-d6) δ 175.8, 163.6, 160.3, 155.9, 147.8, 147.3, 145.0, 136.0, 133.4, 132.8, 130.4,
130.1, 129.4, 127.4, 121.7, 119.9, 115.5, 115.2, 104.2, 97.8, 92.6, 67.5; ESI-MS (m/z): 427.0 [M+H]+.
7-((2-Bromobenzyl)oxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3d). Yield 44%.
1
m.p. 248–249 °C; H-NMR (DMSO-d6) δ 12.53 (s, 1H, -OH), 9.67 (brs, 1H, -OH), 9.55 (brs, 1H,
-OH), 9.33 (s, 1H, -OH), 7.74 (d, J = 2.4 Hz, 1H, -ArH), 7.72 (dd, J = 8.0, 1.2 Hz, 1H, -ArH), 7.63 (dd,
J = 7.6, 1.6 Hz, 1H, -ArH), 7.58 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 7.46 (td, J = 7.6, 1.2 Hz, 1H, -ArH),
7.35 (td, J = 7.6, 1.6 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.86 (d, J = 2.0 Hz, 1H, -ArH),
6.45 (d, J = 2.0 Hz, 1H, -ArH), 5.25 (s, 2H, -CH2); 13C-NMR (DMSO-d6) δ 176.4, 164.1, 160.9, 156.5,

Molecules 2015, 20
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148.3, 147.9, 145.5, 136.6, 135.5, 133.2, 131.1, 130.0, 128.5, 123.7, 122.3, 120.5, 116.0, 115.7, 104.8,
98.4, 93.1, 70.3; ESI-MS (m/z): 471.0, 473.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-((2-methylbenzyl)oxy)-4H-chromen-4-one (3e). Yield 47%.
1
m.p. 241–242 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.66 (s, 1H, -OH), 9.53 (s, 1H, -OH),
9.33 (s, 1H, -OH) 7.74 (d, J = 2.4 Hz, 1H, -ArH), 7.58 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 7.44 (d,
J = 8.8 Hz, 1H, -ArH), 7.21–7.30 (m, 3H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.85 (d, J = 2.0 Hz,
1H, -ArH), 6.46 (d, J = 2.0 Hz, 1H, -ArH), 5.22 (s, 2H, -CH2), 2.34 (s, 3H, -CH3); 13C-NMR (DMSO-d6)
δ 175.9, 164.0, 160.4, 156.0, 147.8, 147.3, 145.1, 136.8, 134.1, 130.2, 128.6, 128.3, 125.8, 121.8,
120.0, 115.5, 115.2, 104.1, 97.9, 92.7, 68.7, 18.4; ESI-MS (m/z): 407.0 [M+H]+, 429.1 [M+Na]+.
2-(((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)methyl)benzonitrile (3f). Yield
1
40%. m.p. 248–249 °C; H-NMR (DMSO-d6) δ 12.54 (s, 1H, -OH), 9.68 (s, 1H, -OH), 9.56 (s, 1H,
-OH), 9.33 (s, 1H, -OH), 7.95 (d, J = 7.6 Hz, 1H, -ArH), 7.78–7.81 (m, 2H, -ArH), 7.74 (d, J = 2.0 Hz,
1H, -ArH), 7.57–7.63 (m, 2H, -ArH), 6.90 (d, J = 7.6 Hz, 1H, -ArH), 6.89 (d, J = 2.0 Hz, 1H, -ArH),
6.47 (d, J = 2.0 Hz, 1H, -ArH), 5.39 (s, 2H, -CH2); 13C-NMR (DMSO-d6) δ 176.4, 163.9, 160.9, 156.4,
148.4, 147.9, 145.6, 139.6, 136.6, 134.0, 133.9, 129.9, 122.3, 120.5, 117.6, 116.0, 115.7, 111.9, 104.9,
98.4, 93.3, 68.7; ESI-MS (m/z): 417.7 [M+H]+, 856.5 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-((2-nitrobenzyl)oxy)-4H-chromen-4-one (3g). Yield 43%.
m.p. 260–261 °C; 1H-NMR (DMSO-d6) δ 12.53 (s, 1H, -OH), 9.66 (s, 1H, -OH), 9.54 (brs, 1H, -OH),
9.32 (s, 1H, -OH), 8.17 (d, J = 8.4 Hz, 1H, -ArH), 7.78–7.82 (m, 2H, -ArH), 7.73 (d, J = 2.0 Hz, 1H,
-ArH), 7.63–7.67 (m, 1H, -ArH), 7.58 (dd, J = 8.8, 2.0 Hz, 1H, -ArH), 6.89 (d, J = 8.8 Hz, 1H, -ArH),
6.83 (d, J = 2.4 Hz, 1H, -ArH), 6.50 (d, J = 2.4 Hz, 1H, -ArH), 5.61 (s, 2H, -CH2); 13C-NMR
(DMSO-d6) δ 176.4, 163.8, 160.9, 154.6, 148.3, 147.9, 145.5, 136.6, 134.6, 132.2, 129.8, 129.6, 125.4,
122.3, 120.5, 116.0, 115.7, 104.9, 98.4, 93.2, 67.5; ESI-MS (m/z): 438.1 [M+H]+.
2-(3,4-Dihydroxyphenyl)-7-((3-fluorobenzyl)oxy)-3,5-dihydroxy-4H-chromen-4-one (3h). Yield 54%.
1
m.p. 243–245 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.67 (s, 1H, -OH), 9.54 (s, 1H, -OH),
9.33 (s, 1H, -OH), 7.72 (d, J = 2.4 Hz, 1H, -ArH), 7.56 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 7.45–7.48 (m,
1H, -ArH), 7.31–7.33 (m, 2H, -ArH), 7.20–7.22 (m, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.81
(d, J = 2.0 Hz, 1H, -ArH), 6.45 (d, J = 2.0 Hz, 1H, -ArH), 5.27 (s, 2H, -CH2); 13C-NMR (DMSO-d6) δ
176.4, 164.1, 162.7 (d, JC-F = 242.2Hz), 160.9, 156.4, 148.3, 147.9, 145.6, 139.6 (d, JC-F = 7.5 Hz),
136.5, 131.8 (d, JC-F = 8.3 Hz), 124.1 (d, JC-F = 2.8 Hz), 122.3, 120.5, 116.0, 115.7, 115.3 (d,
JC-F = 20.6 Hz), 114.8 (d, JC-F = 21.8 Hz), 104.7, 98.5, 93.3, 69.5; ESI-MS (m/z): 411.1 [M+H]+.
7-((3-Chlorobenzyl)oxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3i). Yield 40%.
1
m.p. 260–261 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.67 (brs, 1H, -OH), 9.54 (brs,
1H, -OH), 9.32 (s, 1H, -OH), 7.73 (d, J = 2.4 Hz, 1H, -ArH), 7.55–7.58 (m, 2H, -ArH), 7.41–7.45 (m,
3H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.81 (d, J = 2.0 Hz, 1H, -ArH), 6.46 (d, J = 2.0 Hz, 1H,
-ArH), 5.26 (s, 2H, -CH2); ESI-MS (m/z): 427.0 [M+H]+.

Molecules 2015, 20
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7-((3-Bromobenzyl)oxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3j). Yield 47%.
1
m.p. 234–236 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.68 (brs, 1H, -OH), 9.55 (brs, 1H,
-OH), 9.33 (s, 1H, -OH), 7.72 (d, J = 2.4 Hz, 1H, -ArH), 7.69 (s, 1H, -ArH), 7.56 (dd, J = 8.4, 2.0 Hz,
2H, -ArH), 7.49 (d, J = 8.0 Hz, 1H, -ArH), 7.39 (t, J = 8.0 Hz, 1H, -ArH), 6.90 (d, J = 8.8 Hz, 1H,
13
-ArH), 6.81 (d, J = 2.0 Hz, 1H, -ArH), 6.46 (d, J = 2.0 Hz, 1H, -ArH), 5.25 (s, 2H, -CH2); C-NMR
(DMSO-d6) δ 176.4, 164.1, 160.9, 156.4, 148.3, 147.8, 145.5, 139.5, 136.5, 131.4, 131.3, 130.8, 127.2,
122.3, 122.2, 120.5, 116.0, 115.7, 104.7, 98.5, 93.3, 69.4; ESI-MS (m/z): 471.0, 473.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-((3-methylbenzyl)oxy)-4H-chromen-4-one (3k). Yield 43%.
1
m.p. 224–226 °C; H-NMR (DMSO-d6) δ 12.51 (s, 1H, -OH), 9.63 (brs, 1H, -OH), 9.53 (brs, 1H,
-OH), 9.33 (brs, 1H, -OH), 7.73 (d, J = 2.0 Hz, 1H, -ArH), 7.57 (d, J = 8.8, 2.0 Hz, 1H, -ArH),
7.16–7.32 (m, 4H, -ArH), 6.90 (d, J = 8.8 Hz, 1H, -ArH), 6.79 (d, J = 2.0 Hz, 1H, -ArH), 6.43 (d,
13
J = 2.0 Hz, 1H, -ArH), 5.19 (s, 2H, -CH2), 2.33 (s, 3H, -CH3); C-NMR (DMSO-d6) δ 176.4, 164.4,
160.9, 156.4, 148.3, 147.8, 145.5, 138.2, 136.6, 136.5, 129.2, 128.9, 128.9, 125.4, 122.3, 120.5, 116.0,
115.7, 104.6, 98.5, 93.2, 70.5, 21.5; ESI-MS (m/z): 407.0 [M+H]+.
3-(((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)methyl)benzonitrile (3l). Yield
1
42%. m.p. 246–247 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.66 (s, 1H, -OH), 9.53 (s, 1H,
-OH), 9.31 (s, 1H, -OH), 7.96 (brs, 1H, -ArH), 7.82–7.86 (m, 2H, -ArH), 7.72 (d, J = 2.0 Hz, 1H,
-ArH), 7.63–7.67 (m, 1H, -ArH), 7.56 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH),
6.83 (d, J = 1.6 Hz, 1H, -ArH), 6.47 (d, J = 1.6 Hz, 1H, -ArH), 5.31 (s, 2H, -CH2); ESI-MS (m/z):
418.1 [M+H]+, 440.0 [M+Na]+.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-((3-nitrobenzyl)oxy)-4H-chromen-4-one (3m). Yield 46%.
m.p. 264–266 °C; 1H-NMR (DMSO-d6) δ 12.53 (s, 1H, -OH), 9.67 (s, 1H, -OH), 9.54 (brs, 1H, -OH),
9.31 (s, 1H, -OH), 8.35 (s, 1H, -ArH), 8.23 (dd, J = 8.4, 1.6 Hz, 1H, -ArH), 7.94 (d, J = 8.0 Hz, 1H,
-ArH), 7.72–7.76 (m, 2H, -ArH), 7.56 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH),
6.85 (d, J = 2.4 Hz, 1H, -ArH), 6.49 (d, J = 2.4 Hz, 1H, -ArH), 5.41 (s, 2H, -CH2); 13C-NMR
(DMSO-d6) δ 175.9, 163.4, 160.5, 155.9, 147.8, 147.4, 145.1, 138.6, 136.1, 134.2, 130.2, 123.0, 122.2,
121.8, 120.0, 115.5, 115.2, 104.3, 98.0, 92.9, 68.5; ESI-MS (m/z): 438.1 [M+H]+.
2-(3,4-Dihydroxyphenyl)-7-((4-fluorobenzyl)oxy)-3,5-dihydroxy-4H-chromen-4-one (3n). Yield 50%.
1
m.p.254–255 °C; H-NMR (DMSO-d6) δ 12.51 (s, 1H, -OH), 9.68 (s, 1H, -OH), 9.53 (brs, 1H, -OH),
9.33 (s, 1H, -OH) 7.72 (d, J = 2.0 Hz, 1H, -ArH), 7.52–7.58 (m, 3H, -ArH), 7.27 (d, J = 8.8 Hz, 1H, -ArH),
7.24 (d, J = 8.8 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.81 (d, J = 2.4 Hz, 1H, -ArH), 6.43
(d, J = 2.4 Hz, 1H, -ArH), 5.22 (s, 2H, -CH2); 13C-NMR (DMSO-d6) δ 175.9, 163.7, 161.8 (d,
JC-F = 244.4 Hz), 160.0, 155.9, 147.8, 147.3, 145.0, 136.0, 132.4 (d, JC-F = 2.9 Hz), 130.2 (d,
JC-F = 8.3 Hz, 2C), 121.8, 120.0, 115.5, 115.4(d, J = 21.4 Hz, 2C), 115.2, 104.1, 98.0, 92.7, 69.2;
ESI-MS (m/z): 411.1 [M+H]+.
7-((4-Chlorobenzyl)oxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3o). Yield 41%.
1
m.p. 266–267 °C; H-NMR (DMSO-d6) δ 12.51 (s, 1H, -OH), 9.67 (s, 1H, -OH), 9.53 (s, 1H, -OH),
9.32 (s, 1H, -OH), 7.71 (d, J = 2.0 Hz, 1H, -ArH), 7.56 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 7.47–7.52 (m,

Molecules 2015, 20
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4H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.80 (d, J = 2.0 Hz, 1H, -ArH), 6.44 (d, J = 2.0 Hz, 1H,
-ArH), 5.24 (s, 2H, -CH2); ESI-MS (m/z): 427.0 [M+H]+.
7-((4-Bromobenzyl)oxy)-2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-4H-chromen-4-one (3p). Yield 44%.
m.p.256–257 °C; 1H-NMR (DMSO-d6) δ 12.51 (s, 1H, -OH), 9.68 (brs, 1H, -OH), 9.54 (brs, 1H,
-OH), 9.34 (brs, 1H, -OH), 7.72 (d, J = 2.0 Hz, 1H, -ArH), 7.62 (d, J = 8.4 Hz, 2H, -ArH), 7.56 (dd,
J = 8.8, 2.0 Hz, 1H, -ArH), 7.44 (d, J = 8.4 Hz, 2H, -ArH), 6.90 (d, J = 8.8 Hz, 1H, -ArH), 6.80 (d,
13
J = 2.4 Hz, 1H, -ArH), 6.43 (d, J = 2.4 Hz, 1H, -ArH), 5.23 (s, 2H, -CH2); C-NMR (DMSO-d6) δ
176.4, 164.1, 160.9, 156.4, 148.3, 147.8, 145.5, 136.5, 136.2, 131.9 (2C), 130.4 (2C), 122.3, 121.7,
120.5, 116.0, 115.7, 104.7, 98.5, 93.3, 69.5; ESI-MS (m/z): 471.0, 473.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-((4-methylbenzyl)oxy)-4H-chromen-4-one (3q). Yield 56%.
m.p. 261–264 °C; 1H-NMR (DMSO-d6) δ 12.51 (s, 1H, -OH), 9.51–9.43 (brs, 3H, -OH), 7.73 (brs, 1H,
-ArH), 7.57 (d, J = 8.4 Hz, 1H, -ArH), 7.36 (d, J = 8.0 Hz, 2H, -ArH), 7.22 (d, J = 8.0 Hz, 2H, -ArH),
6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.78 (d, J = 1.6 Hz, 1H, -ArH), 6.41 (d, J = 1.6 Hz, 1H, -ArH), 5.18 (s,
2H, -CH2), 2.31 (s, 3H, -CH3); 13C-NMR (DMSO-d6) δ 175.9, 163.9, 160.4, 155.9, 147.9, 147.3, 145.1,
137.4, 136.0, 133.1, 129.0 (2C), 127.9 (2C), 121.8, 120.0, 115.5, 115.2, 104.1, 98.0, 92.7, 69.8, 20.8;
ESI-MS (m/z): 407.0 [M+H]+.
4-(((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)methyl)benzonitrile (3r). Yield
1
45%. m.p. 252–254 °C; H-NMR (DMSO-d6) δ 12.52 (s, 1H, -OH), 9.68 (s, 1H, -OH), 9.55 (s, 1H,
-OH), 9.32 (s, 1H, -OH), 7.90 (d, J = 8.0 Hz, 2H, -ArH), 7.72 (d, J = 1.6 Hz, 1H, -ArH), 7.67 (d,
J = 8.0 Hz, 2H, -ArH), 7.56 (dd, J = 8.4, 1.6 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.81 (d,
13
J = 1.6 Hz, 1H, -ArH), 6.46 (d, J = 1.6 Hz, 1H, -ArH), 5.37 (s, 2H, -CH2); C-NMR (DMSO-d6) δ
176.4, 163.9, 160.9, 156.4, 148.4, 147.9, 145.6, 142.5, 136.6, 133.0(2C), 128.6(2C), 122.3, 120.5,
119.2, 116.0, 115.7, 111.2, 104.8, 98.5, 93.3, 69.3; ESI-MS (m/z): 417.9 [M+H]+.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-((4-nitrobenzyl)oxy)-4H-chromen-4-one (3s). Yield 47%.
1
m.p. 253.5–254.5 °C; H-NMR (DMSO-d6) δ 12.53 (s, 1H, -OH), 9.68 (s, 1H, -OH), 9.55 (brs,
1H, -OH), 9.32 (s, 1H, -OH), 8.29 (d, J = 8.8 Hz, 2H, -ArH), 7.75 (d, J = 8.8 Hz, 2H, -ArH), 7.72 (d,
J = 2.0 Hz, 1H, -ArH), 7.56 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.83 (d,
13
J = 2.4 Hz, 1H, -ArH), 6.47 (d, J = 2.4 Hz, 1H, -ArH), 5.43 (s, 2H, -CH2); C-NMR (DMSO-d6) δ
176.4, 163.9, 161.0, 156.4, 148.4, 147.9, 147.6, 145.6, 144.6, 136.6, 128.8 (2C), 124.2 (2C), 122.3,
120.5, 116.0, 115.7, 104.8, 98.5, 93.4, 69.1, 23.0; ESI-MS (m/z): 438.1 [M+H]+.
3.6. Synthesis of 7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3,5-dihydroxy-4H-chromen-4-one (9)
A mixture of rutin (8, 5.00 g, 8.2 mmol) and potassium carbonate (2.83 g, 20.5 mmol) in dry DMF
(40 mL) was stirred under argon atmosphere for 0.5 h. Benzyl bromide (3.2 mL, 27.1 mmol) was then
added dropwise. After stirring for 3 h at 60 °C, the mixture was acidified to pH 5 with 10% aqueous
acetic acid solution, and the precipitate was collected by centrifugation. Ethanol (60 mL) was added to
the precipitate and then conc. aqueous hydrochloric acid (9 mL) of was add in portions. This reaction
mixture was stirred at 70 °C for 2 h. After cooling to room temperature, the precipitate was filtered and

Molecules 2015, 20
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washed with water. The crude product was recrystallized from CH2Cl2/EtOH to afford 9 as yellow
1
powder in 85% yield, m.p. 186–188 °C. H-NMR (DMSO-d6) δ 7.90 (d, J = 2.0 Hz, 1H, -ArH), 7.84
(dd, J = 8.8, 2.0 Hz, 1H, -ArH), 7.53–7.30 (m, 15H, -ArH), 7.25 (d, J = 8.8 Hz, 1H, -ArH), 6.86 (d,
J = 2.0 Hz, 1H, -ArH) 6.45 (d, J = 2.0 Hz, 1H, -ArH), 5.24 (s, 4H, -CH2), 5.21 (s, 2H, -CH2); ESI-MS
(m/z) 573.1 [M+H]+.
3.7. General Procedure for the Preparation of 10a–10o
A mixture of compound 9 (200 mg, 0.35 mmol), EDCI (100 mg, 0.525 mmol), DMAP (9 mg,
0.07 mmol) and appropriate benzoic acid (0.35 mmol) in DMF (20 mL) was stirred at 30 °C overnight.
The mixture was poured into 100 mL of water and filtered to give the crude products 10a–10o for the
next step directly without further purification.
3.8. General Procedure for the Preparation of 4a–4o
To a solution of the appropriate benzyl-protected quercetin-3-O-ester derivatives 10a–10o in
ethanol and 1,4-dioxane (20 mL, 3:1) was added 10% palladium on carbon (20 mg). The mixture was
stirred under hydrogen atmosphere at room temperature for 6 h. The resulting mixture was filtered,
washed with EtOH and purified by silica gel column chromatography (CH2Cl2/MeOH, 20/1, v/v) to
give the desired compounds 4a–4o.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl benzoate (4a). Yield 54%. m.p.
1
173–174 °C; H-NMR (DMSO-d6): δ 12.16 (s, 1H, -OH), 11.05 (brs, 1H, -OH), 9.90 (brs, 1H, -OH),
9.47 (brs, 1H, -OH), 8.15 (d, J = 7.9 Hz, 2H, -ArH), 7.80 (t, J = 7.9 Hz, 1H, -ArH), 7.64 (dd,
J = 7.9, 7.9 Hz, 2H, -ArH), 7.36 (d, J = 2.4 Hz, 1H, -ArH), 7.31 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 6.86
(d, J = 8.4 Hz, 1H, -ArH), 6.52 (d, J = 2.0 Hz, 1H, -ArH), 6.28 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS
(m/z) 407.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 2-fluorobenzoate (4b). Yield 54%.
1
m.p. 186–187 °C; H-NMR (DMSO-d6):δ 12.13 (s, 1H, -OH), 11.14 (brs, 1H, -OH), 10.00 (brs, 1H,
-OH), 9.54 (brs, 1H, -OH), 8.14 (ddd, J = 7.6, 7.6, 2.0 Hz, 1H, -ArH), 7.86–7.82 (m, 1H, -ArH),
7.51–7.46 (m, 2H, -ArH), 7.38 (d, J = 2.4 Hz, 1H, -ArH), 7.34 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 6.91 (d,
J = 8.4 Hz, 1H, -ArH), 6.52 (d, J = 2.0 Hz, 1H, -ArH), 6.28 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS (m/z)
425.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 2-aminobenzoate (4c). Yield 58%.
1
m.p. 198–199 °C; H-NMR (DMSO-d6): δ 12.24 (s, 1H, -OH), 11.02 (brs, 1H, -OH), 9.91 (brs, 1H,
-OH), 9.45 (brs, 1H, -OH), 7.95 (dd, J = 8.0, 2.0 Hz, 1H, -ArH), 7.38–7.34 (m, 2H, -ArH), 7.30 (dd,
J = 8.4, 2.4 Hz, 1H, -ArH), 6.85 (d, J = 8.4 Hz, 1H, -ArH), 6.84 (d, J = 7.6 Hz, 1H, -ArH), 6.74 (brs,
2H, -ArH), 6.62 (dd, J = 7.6, 7.6 Hz, 1H, -ArH), 6.51 (d, J = 2.0 Hz, 1H, -ArH), 6.26 (d, J = 2.0 Hz,
1H, -ArH); ESI-MS (m/z) 422.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 2-methoxybenzoate (4d). Yield 52%.
1
m.p. 183–184 °C; H-NMR (DMSO-d6): δ 12.23 (s, 1H, -OH), 11.11 (brs, 1H, -OH), 10.00 (brs, 1H,

Molecules 2015, 20
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-OH), 9.48 (brs, 1H, -OH), 7.98 (dd, J = 7.6, 2.0 Hz, 1H, -ArH), 7.69 (ddd, J = 8.8, 7.6, 2.0 Hz, 1H,
-ArH), 7.41 (d, J = 2.4 Hz, 1H, -ArH), 7.38 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 7.26 (d, J = 8.8 Hz, 1H,
-ArH), 7.13 (dd, J = 7.6, 7.6 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH), 6.53 (d, J = 2.0 Hz, 1H,
-ArH), 6.29 (d, J = 2.0 Hz, 1H, -ArH), 3.88 (s, 3H, -CH3); ESI-MS (m/z) 437.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 2-cyanobenzoate (4e). Yield 57%.
1
m.p. 196–197 °C; H-NMR (DMSO-d6): δ 12.24 (s, 1H, -OH), 11.02 (brs, 1H, -OH), 9.91 (brs, 1H,
-OH), 9.45 (brs, 1H, -OH), 8.45–8.43 (m, 1H, -ArH), 8.17–8.14 (m, 1H, -ArH), 8.00–7.97 (m, 2H,
-ArH), 7.38 (d, J = 2.4 Hz, 1H, -ArH), 7.35 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 6.89 (d, J = 8.4 Hz, 1H,
-ArH), 6.56 (d, J = 2.0 Hz, 1H, -ArH), 6.31 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS (m/z) 432.0 [M+H]+,
454.0 [M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 3-fluorobenzoate (4f). Yield 52%.
1
m.p. 211–213 °C; H-NMR (DMSO-d6) δ 12.11 (s, 1H, -OH), 11.07 (brs, 1H, -OH), 9.92 (brs,
1H, -OH), 9.50 (brs, 1H, -OH), 8.01 (ddd, J = 6.8, 1.6, 2.0 Hz, 1H, -ArH), 7.92 (dd, J = 9.2, 2.0 Hz,
1H, -ArH), 7.74–7.65 (m, 2H, -ArH), 7.36 (d, J = 2.0 Hz, 1H, -ArH), 7.32 (dd, J = 8.4, 2.0 Hz,
1H, -ArH), 6.87 (d, J = 8.4 Hz, 1H, -ArH), 6.53 (d, J = 2.0 Hz, 1H, -ArH), 6.28 (d, J = 2.0 Hz, 1H,
-ArH); 13C-NMR (DMSO-d6) δ 174.51, 164.73, 162.30 (d, JC-F = 3.0 Hz), 162.05 (d, JC-F = 244.3 Hz),
161.03, 156.66, 156.39, 149.37, 145.47, 131.50 (d, JC-F = 7.9 Hz), 129.98 (d, JC-F = 7.5 Hz), 129.69,
126.32 (d, JC-F = 2.5 Hz), 121.66 (d, JC-F = 21.1 Hz), 120.56, 119.41, 116.55 (d, JC-F = 23.1 Hz),
115.91, 115.09, 103.40, 99.18, 94.20; ESI-MS (m/z) 425.0 [M+H]+, 871.2 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 3-aminobenzoate (4g). Yield 50%.
1
m.p. 216–217 °C; H-NMR (DMSO-d6) δ 12.20 (s, 1H, -OH), 11.03 (brs, 1H, -OH), 9.92 (brs, 1H,
-OH), 9.45 (brs, 1H, -OH), 7.34 (d, J = 2.4 Hz, 1H, -ArH), 7.32 (d, J = 2.0 Hz, 1H, -ArH), 7.28 (dd,
J = 8.4, 2.4 Hz, 1H, -ArH), 7.25–7.21 (m, 2H, -ArH), 6.91–6.89 (m, 1H, -ArH), 6.86 (d, J = 8.4 Hz,
1H, -ArH), 6.51 (d, J = 2.0 Hz, 1H, -ArH), 6.27 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS (m/z)
422.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 3-methoxybenzoate (4h). Yield 60%.
1
m.p. 173–174 °C; H-NMR (DMSO-d6) δ 12.16 (s, 1H, -OH), 11.06 (brs, 1H, -OH), 9.92 (brs,
1H, -OH), 9.47 (brs, 1H, -OH), 7.74 (d, J = 7.6 Hz, 1H, -ArH), 7.59 (dd, J = 3.0, 2.0Hz, 1H, -ArH),
7.55 (dd, J = 7.6, 7.6 Hz, 1H, -ArH), 7.37–7.35 (m, 2H, -ArH), 7.30 (dd, J = 8.4, 2.4 Hz, 1H, -ArH),
6.86 (d, J = 8.4 Hz, 1H, -ArH), 6.52 (d, J = 2.0 Hz, 1H, -ArH), 6.27 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS
(m/z) 437.0 [M+H]+, 459.1 [M+Na]+, 895.2 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 3-cyanobenzoate (4i). Yield 49%. m.p.
1
142–143 °C; H-NMR (DMSO-d6) δ 12.08 (s, 1H, -OH), 11.08 (brs, 1H, -OH), 9.92 (brs, 1H, -OH),
9.50 (s, 1H, -OH), 8.58 (dd, J = 2.0, 2.0 Hz, 1H, -ArH), 8.45 (ddd, J = 8.0, 2.0, 2.0 Hz, 1H, -ArH),
8.27 (ddd, J = 8.0, 2.0, 2.0 Hz, 1H, -ArH), 7.86 (d, J = 8.0 Hz, 1H, -ArH), 7.37 (d, J = 2.4 Hz,
1H, -ArH), 6.83 (dd, J = 8.4, 2.4 Hz, 1H, -ArH), 6.87 (d, J = 8.4 Hz, 1H, -ArH), 6.53 (d, J = 2.0 Hz,
1H, -ArH), 6.28 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS (m/z) 432.1 [M+H]+, 454.0 [M+Na]+.

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2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 3-chlorobenzoate (4j). Purified by
1
prepared HPLC. Yield 35%. m.p. > 300 °C; H-NMR (DMSO-d6) δ 8.13 (brs, 1H, -ArH), 8.09 (d,
J = 8.0 Hz, 1H, -ArH), 7.71 (d, J = 8.0 Hz, 1H, -ArH), 7.56 (dd, J = 8.0, 8.0 Hz, 1H, -ArH), 7.33 (brs,
1H, -ArH), 7.29 (d, J = 8.0 Hz, 1H, -ArH), 6.81 (d, J = 8.0 Hz, 1H, -ArH), 6.42 (brs, 1H, -ArH), 6.21
(brs, 1H, -ArH); ESI-MS (m/z) 440.8 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 4-fluorobenzoate (4k). Yield 56%.
1
m.p. 203–205 °C; H-NMR (DMSO-d6) δ 12.14 (s, 1H, -OH), 11.06 (brs, 1H, -OH), 9.91 (brs, 1H,
-OH), 9.48 (brs, 1H, -OH), 8.24 (d, J = 8.8 Hz, 1H, -ArH), 8.23(d, J = 8.8 Hz, 1H, -ArH) 7.48 (d,
J = 8.8 Hz, 1H, -ArH), 7.46 (d, J = 8.8 Hz, 1H, -ArH), 7.36 (d, J = 2.4 Hz, 1H, -ArH), 7.31 (dd, J = 8.4, 2.4
Hz, 1H, -ArH), 6.87 (d, J = 8.4 Hz, 1H, -ArH), 6.52 (d, J = 2.0 Hz, 1H, -ArH), 6.28 (d, J = 2.0 Hz, 1H,
13
-ArH); C-NMR (DMSO-d6) 174.59, 165.72 (d, JC-F = 251.8 Hz) 164.64, 162.34, 160.98, 156.59,
156.28, 149.26, 145.40, 133.06 (d, JC-F = 9.8 Hz, 2C), 129.67, 124.36 (d, JC-F = 2.6 Hz), 120.45,
119.43, 116.33 (d, JC-F = 22.1 Hz, 2C), 115.81, 115.02, 103.35, 99.08, 94.10; ESI-MS (m/z) 425.0
[M+H]+, 871.2 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 4-aminobenzoate (4l). The product
was prepared according to procedure described above starting from 9 and 4-nitrobenzoic acid. Yield
1
55%. m.p. 233–234 °C; H-NMR (DMSO-d6) δ 12.30 (s, 1H, -OH), 11.00 (brs, 1H, -OH), 9.88 (brs,
1H, -OH), 9.42 (s, 1H, -OH), 7.78 (d, J = 8.8 Hz, 2H, -ArH), 7.35 (d, J = 2.0 Hz, 1H, -ArH), 7.28 (dd,
J = 8.4, 2.0 Hz, 1H, -ArH), 6.84 (d, J = 8.4 Hz, 1H, -ArH), 6.63 (d, J = 8.8 Hz, 2H, -ArH), 6.49 (d,
J = 2.4 Hz, 1H, -ArH), 6.26 (brs, 2H, -ArH), 6.25 (d, J = 2.4 Hz, 1H, -ArH); ESI-MS (m/z)
422.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 4-methoxybenzoate (4m). Yield 50%.
1
m.p. 194–195 °C; H-NMR (DMSO-d6) δ 12.20 (s, 1H, -OH), 11.03 (brs, 1H, -OH), 9.90 (brs, 1H,
-OH), 9.44 (s, 1H, -OH), 8.10 (d, J = 8.8 Hz, 2H, -ArH), 7.35 (d, J = 2.0 Hz, 1H, -ArH), 7.29 (dd,
J = 8.4, 2.0 Hz, 1H, -ArH), 7.14 (d, J = 8.8 Hz, 2H, -ArH), 6.84 (d, J = 8.4 Hz, 1H, -ArH), 6.51 (d,
J = 2.0 Hz, 1H, -ArH), 6.27 (d, J = 2.0 Hz, 1H, -ArH), 3.89(s, 3H, -CH3); ESI-MS (m/z) 437.0
[M+H]+, 895.2 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 4-cyanobenzoate (4n). Yield 58%.
1
m.p. 196–197 °C; H-NMR (DMSO-d6) δ 12.07 (s, 1H, -OH), 11.08 (brs, 1H, -OH), 9.92 (brs, 1H,
-OH), 9.50 (brs, 1H, -OH), 8.31 (d, J = 8.4 Hz, 2H, -ArH), 8.12 (d, J = 8.4 Hz, 2H, -ArH),7.35 (d,
J = 2.0 Hz, 1H, -ArH), 7.31 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.86 (d, J = 8.4 Hz, 1H, -ArH), 6.53 (d,
J = 2.0 Hz, 1H, -ArH), 6.28 (d, J = 2.0 Hz, 1H, -ArH); ESI-MS (m/z) 432.1 [M+H]+, 885.1 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 4-chlorobenzoate (4o). Purified by
1
preparative HPLC. Yield 40%. m.p. 194–195 °C; H-NMR (400 MHz, d6-DMSO) δ 8.14 (d,
J = 8.0 Hz, 2H, -ArH), 7.57 (d, J = 8.0 Hz, 2H, -ArH),7.34 (brs, 1H, -ArH), 7.28 (d, J = 8.0 Hz, 1H,
-ArH), 6.81 (d, J = 8.0 Hz, 1H, -ArH), 6.45 (brs, 1H, -ArH), 6.24 (brs, 1H, -ArH); ESI-MS (m/z)
440.9 [M+H]+.

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3.9. General Procedure for the Preparation of 11a–11d
To a mixture of compound 9 (200 mg, 0.35 mmol) and potassium carbonate (73 mg, 0.53 mmol) in
dry DMF (20 mL), the appropriate halide (0.53 mmol) in dry DMF (2 mL) was add, and the reaction
mixture was stirred at 40 °C for 2 h. The mixture was then poured into water (100 mL) and filtered to
give the crude product 11a–11o used or the next step directly without further purification.
3.10. General Procedure for the Preparation of 12a–12d
10% Palladium on carbon (20 mg) was added to the crude product 11a–11o in a solution of ethanol
and 1,4-dioxane (20 mL, 3:1), then the mixture was stirred under hydrogen atmosphere at room
temperature for 4 h. The resulting mixture was filtered, washed with EtOH and purified by silica gel
column chromatography (CH2Cl2/MeOH, 40/1, v/v) to give the desired compound 12a–12d.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-methoxy-4H-chromen-4-one (12a). Yield 65%. m.p. 270–271 °C;
1H-NMR (DMSO-d6) δ 12.71 (s, 1H, -OH), 10.85 (brs, 1H, -OH), 9.78 (brs, 1H, -OH), 9.41 (brs, 1H,
-OH), 7.55 (d, J = 2.0 Hz, 1H, -ArH), 7.45 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.90 (d, J = 8.4 Hz, 1H, -ArH),
6.41 (d, J = 2.0 Hz, 1H, -ArH), 6.19 (d, J = 2.0 Hz, 1H, -ArH), 3.78 (s, 3H, -CH3); ESI-MS (m/z)
317.0 [M+H]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-isopropoxy-4H-chromen-4-one (12b). Yield 70%. m.p.
1
259–262 °C; H-NMR (DMSO-d6) δ 12.79 (s, 1H, -OH), 10.82 (brs, 1H, -OH), 9.71 (brs, 1H, -OH),
9.36 (brs, 1H, -OH), 7.61 (d, J = 2.0 Hz, 1H, -ArH), 7.49 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.88 (d,
J = 8.4 Hz, 1H, -ArH), 6.40 (d, J = 2.0 Hz, 1H, -ArH), 6.19 (d, J = 2.0 Hz, 1H, -ArH), 4.51(m, 1H,
13
-CH), 1.15 (s, 3H, -CH3), 1.14 (s, 3H, -CH3); C-NMR (DMSO-d6) 178.82, 164.48, 161.77, 156.81,
148.90, 145.37, 135.77, 121.79, 121.44, 116.32, 115.88, 104.53, 98.92, 93.94, 74.74, 22.55; ESI-MS
(m/z) 345.1 [M+H]+, 711.2 [2M+Na]+.
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-(2-hydroxyethoxy)-4H-chromen-4-one (12c). Yield 61%.
1
m.p. 193–194 °C; H-NMR (DMSO-d6) δ 12.70 (s, 1H, -OH), 10.85 (s, 1H, -OH), 9.77 (s, 1H, -OH),
9.33 (s, 1H, -OH), 7.60–7.57 (m, 2H, -ArH), 6.88 (d, J = 8.0 Hz, 1H, -ArH), 6.41 (d, J = 2.0 Hz, 1H,
-ArH), 6.19 (d, J = 2.0 Hz, 1H, -ArH), 4.00 (t, J = 5.2 Hz, 2H, -CH2), 3.66 (brs, 2H, -CH2); ESI-MS
(m/z) 347.2 [M+H]+, 715.2 [2M+Na]+.
4-(2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yloxy)butanenitrile (12d). Yield 64%.
1
m.p. 169–171 °C; H-NMR (DMSO-d6) δ 12.68 (s, 1H, -OH), 10.86 (brs, 1H, -OH), 9.78 (brs, 1H,
-OH), 9.42 (brs, 1H, -OH), 7.50 (d, J = 2.0 Hz, 1H, -ArH), 7.42 (dd, J = 8.4, 2.0 Hz, 1H, -ArH), 6.91
(d, J = 8.4 Hz, 1H, -ArH), 6.41 (d, J = 2.0 Hz, 1H, -ArH), 6.20 (d, J = 2.0 Hz, 1H, -ArH), 3.97 (t,
J = 6.0 Hz, 2H, -CH2), 2.63–2.60 (m, 2H, -CH2); 1.95–1.91 (m, 2H, -CH2); ESI-MS (m/z) 370.1
[M+H]+, 761.2 [2M+Na]+.

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3.11. Biological Assays
3.11.1. Cells and Viruses Preparations
The biological assays were performed following a previously reported procedure [23]. The
Huh7.5.1 cell line and the HCV infectious virus J399EM were kindly provided by Xin-Wen Chen,
Wuhan Institute of Virology, Chinese Academy of Sciences. Huh7.5.1 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum, 0.5 mg/mL
G418, 100 IU/mL penicillin, and 100 mg/mL streptomycin.
J399EM is an infectious HCV virus derived from the JFH1 virus (genotype 2a) by insertion of
enhanced green fluorescent protein (EGFP) into the HCV NS5A region. The establishment of the
infectious J399EM HCV virus has been described previously [24]. J399EM was propagated in
Huh7.5.1 cells. Briefly, Huh7.5.1 cells were infected with J399EM at MOI = 0.1 and cultured for
4 days. Culture supernatant was collected and filtered through a 0.45 μm membrane and stored at
−80 °C as virus stock. Virus titer was tested in Huh7.5.1 cells. Stocks were serially diluted, and the
foci forming units (FFU) were determined as the number of fluorescent colonies formed in
infected Huh7.5.1.
3.11.2. Antiviral Assays in Infected Target Cells
All the compounds were first dissolved in DMSO at 50 mM, and then diluted to various
concentrations (50 μM, 25 μM, 12.5 μM, 6.3 μM and 3.1 μM). Approximately 1 × 104 Huh7.5.1 cells
per well were plated in an opaque 96-well tissue culture plate (Costar 3904). The next day, the medium
was replaced by J399EM virus at an MOI of 0.3. Four hours later, the virus inoculum was removed
and medium containing different concentrations of compounds was added. After an additional 72 h of
incubation, the culture medium was removed and the EGFP autofluorescence was measured by using a
luminometer with excitation at 488 nm and emission at 516 nm. Compounds and controls were
performed in duplicate and each experiment repeated independently at least twice.
3.11.3. Cell Viability Assays
After the EGFP signal was read, cell viability was measured in the same plate by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA]
method. Briefly, MTT (1 mg/mL) was dissolved in PBS. Cell culture supernatants were removed and
replaced with MTT and further cultured for 4 h; cells were then lysed with 10% sodium dodecyl
sulfate (SDS), 50% N,N-dimethylformamide, pH 7.2. OD570 values were read. The percentage of cell
death was calculated against the control well without compounds.
3.12. Modeling and Docking
Docking studies were carried out on quercetin and relative substituents using Schrodinger software
package [25]. The high-resolution (1.85 Å) X-ray co-crystal structure of HCV NS5B in complex with
Mn2+ and UTP (PDB code: 1GX6 [22]) was downloaded from the Protein Data Bank. Water molecules
were deleted except for those involved in coordination with the two divalent cations in the active site.

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The crystallographic Mn2+ ions were replaced with Mg2+ ions. All missing hydrogen atoms were added
by standard protein preparation protocol within Maestro followed by energy minimization using OPLS
2005 force field. Restrained minimization was later performed on heavy atoms of the protein with
root-mean-square deviation (RMSD) converged to 0.3 Å. The structures of lead compound quercetin
and the most active compound of corresponding series (3i and 4f) were built and optimized by Maestro
with default settings. Docking of the three compounds were carried out using Glide with standard
precision protocol with the Mg2+ ions defined as required constraints. The binding conformation with
the lowest score was selected to represent the predicted binding mode with HCV NS5B polymerase.
4. Conclusions
DKA analogues or isosteres show potent inhibition of HCV NS5B through chelation of the two
magnesium ions at the active site. Following our recent report which evidenced the anti-HCV potential
of the DKAs mimic quercetin (2), efforts were continued in order to delineate the structural features
required for the inhibitory effect and improve the anti-HCV potency. Accordingly, two novel quercetin
analogues, 7-O-arylmethylquercetin derivatives and quercetin-3-O-benzoic acid ester derivatives, were
synthesized and evaluated for anti-HCV activities. Experimental and modeling results highlighted the
importance of introducing an aromatic group at 7-O position, which is exemplified by compound 3i,
where a 3-chlorobenzyl substitution at quercetin 7-O position confers low micromolar inhibitory
activity (EC50 = 3.8 μM) and a decent selectivity ratio (SI = 7.2). Moreover, the electronegative
carbonyl groups at quercetin 3-O position may maintain the coordination with the magnesium ions and
more importantly, introduce other interactions with the surrounding residues and thus enhance the
anti-HCV activities, which is manifested by compound 4f (EC50 = 9.0 μM and SI > 5.6). Docking
studies suggested that the quercetin structures are capable of establishing key coordination with the
two magnesium ions as well as interactions with residues at the active site of HCV RdRp. These
results demonstrated the feasibility and potential of modifications on quercetin scaffold and provided a
promising fundamental to optimize the structures of flavonoids to obtain potent anti-HCV agents.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/04/6978/s1.
Acknowledgments
The authors acknowledge Xin-Wen Chen from Wuhan Institute of Virology, Chinese Academy of
Sciences for providing the HCV infectious virus J399EM. The authors acknowledge Jian-Ping Zuo
from Shanghai Institute of Materia Medica, Chinese Academy of Sciences for his effort on generating
bioassay data. The authors thank Zheng-Hong Yuan from Key Laboratory of Medical Molecular
Virology, Shanghai Medical College, Fudan University for his guidance. This work was supported by
the Chinese National Natural Science Foundation (Grants No. 81373330), Specialized Research Fund
for the Doctoral Program of Higher Education, Chinese Ministry of Education (No. 20130071110068)
and National Drug Innovative Program (No. 2009ZX09301- 011).

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Author Contributions
L.Z. and D.Y. conceived and designed the research; D.Z. and M.L. performed the experimental
work and wrote the manuscript; Y.C. performed the docking study; Y.Z., S.B., J.Z., F.W. and K.C.R.
participated in the experimental work, analyzed the data and discussed the results. All the authors read
and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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25. Schrodinger, L. Schrodinger Software Suite; Schrödinger, LLC: New York, NY, USA, 2011.
Sample Availability: Samples of the compounds investigated here are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).