Synthesis and antiviral evaluation of 7-O-arylmethylquercetin derivatives against SARS-associated coronavirus (SCV) and hepatitis C virus (HCV)

Article abstract of DOI:10.1007/s12272-012-0108-9

Aryl diketoacid (ADK) is well known for antiviral activity which can be enhanced by introduction of an aromatic arylmethyl substituent. A natural flavonoid quercetin has a 3,5-dihydroxychromone pharmacophore which is in bioisosteric relationship with the

Full text of DOI:10.1007/s12272-012-0108-9

Arch Pharm Res Vol 35, No 1, 77-85, 2012
DOI 10.1007/s12272-012-0108-9
Synthesis and Antiviral Evaluation of 7-O-Arylmethylquercetin Deri-
vatives against SARS-associated Coronavirus (SCV) and Hepatitis C
Virus (HCV)
Hye Ri Park1,*, Hyunjun Yoon1,*, Mi Kyoung Kim1, Sung Dae Lee2, and Youhoon Chong1
1Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701,
2
Korea and Swine Science Division, National Institute of Animal Science, RDA, Cheonan 330-801, Korea
(Received June 18, 2011/Revised July 29, 2011/Accepted August 9, 2011)
Aryl diketoacid (ADK) is well known for antiviral activity which can be enhanced by introduc-
tion of an aromatic arylmethyl substituent. A natural flavonoid quercetin has a 3,5-dihydrox-
ychromone pharmacophore which is in bioisosteric relationship with the 1,3-diketoacid moiety
of the ADK. Thus, it was of our interest to test the antiviral activity of the quercetin deriva-
tives with an arylmethyl group attached. In this study, we prepared a series of the 7-O-aryl-
methylquercetin derivatives with various aromatic substituents and evaluated their antiviral
activity against the SARS-associated coronavirus (SARS-CoV, SCV) as well as hepatitis C
virus (HCV). Single difference in the aromatic substituent fine-tuned the biological activity of
the 7-O-arylmethylquercetin derivatives to result in two different classes of derivatives selec-
tively active against SCV and HCV.
Key words: Quercetin, Arylmethyl, Hepatitis C, Severe acute respiratory syndrome (SARS)
type 1 (Amoros et al., 1992), polio-virus type 1 (Vrijsen
Selected by Editors
et al., 1988), parainfluenza virus type 3, and respira-
tory syncytial virus (RSV) (Kaul et al., 1985). Recently,
we have also shown inhibition of the helicase activity
of the SARS-associated coronavirus (SARS-CoV, SCV)
INTRODUCTION
Quercetin (1, Fig. 1) is widely distributed in the by quercetin (IC50 = 8.1 µM) (Lee et al., 2009a). More
plant kingdom and is the most abundant of the interestingly, introduction of arylmethyl substituent
flavonoid family. It is often a major component of the such as 4-ClPhCH2, 3-ClPhCH2, and 3-CNPhCH2 at
medicinal activity of the plant, and has been shown in the 7-OH position of quercetin provided the resulting
experimental studies to have numerous effects on the quercetin derivatives (
body. Quercetin appears to have many beneficial effects increased inhibitory activity against the SCV helicase
on human health, including cardiovascular protection, (IC50 = 4.1, 5.2, 2.7 M, respectively).
anticancer activity, antiulcer effects, antiallergy activity, At this point, it is worth to note that the core 3,5-
cataract prevention, and anti-inflammatory effects. In dihydroxychromone moiety of the quercetin (bold lines
addition, quercetin exerts antiviral activity against in
, Fig. 1) is in match with the 1,3-diketoacid (bold
reverse transcriptase of HIV (Harada et al., 1999) and lines in
, Fig. 1), the key pharmacophoric element of
other retroviruses, and was shown to reduce the infec- the antiviral aryl diketoacid (ADK) (
, Fig. 1), in
2, Fig. 1) with significantly
µ
1
3
3
tivity and cellular replication of herpes simplex virus atom-by-atom fashion. Also, in line with the substitut-
ed quercetins, the arylmethyloxy substituent (dotted
box in 3, Fig. 1) increased the antiviral activity of the
*These authors contributed equally to this work.
Correspondence to: Youhoon Chong, Department of Bioscience
& Biotechnology, Konkuk University, Seoul 143-701, Korea
Tel:82-2-2049-6100, Fax: 82-2-454-8217
ADKs and the substituted ADKs showed potent
inhibition of the RNA-dependent RNA polymerase
(RdRp) of hepatitis C virus (HCV) (Kim et al., 2008) as
well as the SCV NTPase/helicase (Lee et al., 2009b).
E-mail: chongy@konkuk.ac.kr
77

78
H. R. Park et al.
Fig. 1. Structures of quercetin (1), 7-O-arylmethylquercetin (2), and aryl diketoacid (3)
Prompted by these intriguing results which support tion of quercetin (5 g, 16.5 mmol) in anhydrous pyridine
the bioisosteric relationship of the 3,5-dihydroxychrom- (40 mL), and the reaction mixture was stirred at 80oC
one with 1,3-diketoacid as well as the critical role of for 4 h. After cooling to room temperature, solvent was
the arylmethyl substituents in antiviral activity, we removed under reduced pressure. The crude product
initiated structure-activity relationship study of a was purified by column chromatography on silica gel
series of 7-
1) with various aromatic substituents (R in
Also, the antiviral activity of the ADKs against both powder: H-NMR (400 MHz, CDCl3)
SCV and HCV was of another interest and thus, 8.6, 2.0 Hz, 1H), 7.70 (d, = 1.9 Hz, 1H), 7.35 (d,
antiviral screening program was extended to include Hz, 1H), 7.33 (d, = 2.2 Hz, 1H), 6.88 (d, = 2.2 Hz,
anti-HCV activity. Herein, we report preparation of a 1H), 2.43 (s, 3H), 2.34 (s, 12H).
series of the 7- -arylmethylquercetin derivatives (
O
-arylmethylquercetin derivatives (
2
, Fig. (Hexane-Acetone-CH2Cl2 = 4:1:1) to afford the desired
(4.5 g, 54% yield) as off-white
7.72 (dd,
= 8.6
2, Fig. 1). quercetin peracetate
4
1
δ
J =
J
J
J
J
O
2,
Fig. 1) with various aromatic substituents and evalua-
tion of their anti-SCV as well as anti-HCV activity.
4-(3,5-Diacetoxy-7-hydroxy-4-oxo-4
2-yl)-1,2-phenylene diacetate (5)
H-chromen-
The quercetin peracetate obtained above (4) (3 g, 5.9
mmol) and imidazole (80 mg, 1.2 mmol) were dissolv-
ed in NMP (30 mL). Thiophenol (0.5 mL, 4.7 mmol)
was slowly added to the stirred mixture at 0oC and then
MATERIALS AND METHODS
Chemicals
All chemicals were purchased from Sigma-Aldrich. the reaction mixture was stirred at room temperature.
Dulbecco’s Modified Eagle Media (DMEM), penicillin, After 2 h, the mixture was diluted with EtOAc and
streptomycin and fetal bovine serum (FBS) were pur- washed with 2 N HCl. The organic layer was dried over
chased from Invitrogen. TLC was performed on Silica MgSO4, filtered and evaporated. The crude product thus
Gel 60 F254 purchased from Merck. Column chroma- obtained was purified by column chromatography on
tography was performed using either Slica Gel 60 silica gel (Hexane-Acetone-CH2Cl2 = 2:1:1) to afford
(220-440 mesh). Melting points were measured with (2 g, 74% yield) as white powder: 1H-NMR (400 MHz,
an electrothermal melting-point apparatus (SMP10, DMSO-d6 11.33 (s, 1H), 7.83-7.80 (m, 2H), 7.51 (d, J
5
)
δ
Barloworld Scientific) in open capillary tubes and are = 8.5 Hz, 1H), 6.94 (s, 1H), 6.65 (s, 1H), 2.33 (s, 6H),
uncorrected. Nuclear magnetic resonance spectra 2.30 (s, 6H).
were recorded on a bruker 400 AMX spectrometer at
1
400 MHz for H-NMR and at 100 MHz for 13C-NMR
General procedure for preparation of the 7-
O-
with tetramethylsilane as the internal standard.
Chemical shift are reported as s (singlet), d, (doublet),
t (triplet), q (quartet), m (multiplet), or br s (broad sing- stirred solution of
arylmethylquercetins (2)
Substituted benzylbromide (1.2 eq.) was added to a
(1.0 eq.) and K2CO3 (1.2 eq.) in
5
let). Coupling constants are reported in hertz (Hz). acetone, and the reaction mixture was stirred at room
The chemical shifts are reported as parts per million temperature. The reaction was monitored by TLC.
(
δ
) relative to the solvent peak. Mass spectrometric After starting material was consumed, the inorganic
data (MS) were obtained by MALDI-TOF-TOF mass residues were removed by filtration. The filtrate was
spectrometer (Ultraflex III, Bruker Daltonik).
concentrated under reduced pressure to give a pale
yellow powder, which was used for the next step with-
out further purification. The crude product was dis-
solved in MeOH saturated with ammonia, and the
2-(3,4-Diacetoxyphenyl)-4-oxo-4H-chromene-3,5,
7-triyl triacetate (4)
Ac2O (12.5 mL, 135 mmol) was added to a stirred solu- reaction mixture was stirred at 0oC for 1 h. After con-

Antiviral Quercetin Derivatives
79
centration under reduced pressure, the residue was DMSO-d6
purified by column chromatography on silica gel 145.5, 136.5, 135.4, 133.1, 131.0, 130.8, 128.4, 123.5,
)
δ
176.3, 164.0, 160.8, 156.4, 148.3, 147.8,
(CH2Cl2-MeOH = 20:1) to give the desired product (
2
). 122.2, 120.4, 115.9, 115.6, 104.7, 98.3, 93.0, 70.2; LC/
MS (ESI) m/z Found: 471.80 (M+H)+; Calcd for
7-(Benzyloxy)-2-(3,4-dihydroxyphenyl)-3,5-di- C22H16BrO7: 471.01.
hydroxy-4 -chromen-4-one (2a)
The desired product 2a was obtained as a yellow 2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(2-iodo-
H
1
powder in 43% yield: m.p. 260-263oC (dec); H-NMR benzyloxy)-4
(500 MHz, CD3COCD3) 7.86 (s, 1H), 7.71 (d, = 8.0 The desired product 2e was obtained as a yellow
Hz, 1H), 7.52 (d, = 7.5 Hz, 2H), 7.42 (t,
= 7.5 Hz, powder in 18% yield: m.p. 252-255oC (dec); H-NMR
2H), 7.39-7.34 (m, 1H), 7.00 (d, = 8.5 Hz, 1H), 6.77 (400 MHz, CD3COCD3)
(s, 1H), 6.40 (s, 1H), 5.26 (s, 2H); 13C-NMR (100 MHz, 7.86 (d,
= 1.8 Hz, 1H), 7.73 (dd,
DMSO-d6 176.3, 164.2, 160.8, 156.3, 148.2, 147.7, 7.62 (dd, = 7.6, 1.3 Hz, 1H), 7.49 (t,
145.5, 143.9, 136.6, 136.4, 129.0, 128.3, 122.2, 116.0, 7.16 (t, = 7.6 Hz, 1H), 7.01 (d, = 8.6 Hz, 1H), 6.81
115.9, 115.6, 104.5, 98.4, 98.3, 93.2, 93.0, 70.3; LC/MS (d, = 2.1 Hz, 1H), 6.43 (d, = 2.1 Hz, 1H), 5.24 (s,
(ESI) m/z Found: 393.40 (M+H)+; Calcd for C22H17O7: 2H); 13C-NMR (125 MHz, DMSO-d6
176.4, 164.2,
H-chromen-4-one (2e)
δ
J
1
J
J
J
δ
7.97 (dd,
J
= 7.9, 0.7 Hz, 1H),
J
J
J
J = 8.5, 1.3 Hz, 1H),
)
δ
J = 7.0 Hz, 1H),
J
J
J
)
δ
393.10.
160.9, 156.5, 148.3, 147.9, 145.6, 139.8, 138.6, 136.6,
131.0, 130.7, 129.0, 122.3, 120.5, 116.1, 115.8, 104.8,
100.1, 98.4, 93.2, 74.5; LC/MS (ESI) m/z Found:
519.30 (M+H)+; Calcd for C22H16IO7: 518.99.
2-(3,4-Dihydroxyphenyl)-7-(2-fluorobenzyloxy)-3,
5-dihydroxy-4H-chromen-4-one (2b)
The desired product 2b was obtained as a yellow
1
powder in 29% yield: m.p. 255-257oC (dec); H-NMR 2-((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-
(400 MHz, CD3COCD3) 7.87 (s, 1H), 7.73 (d, = 8.3 -chromen-7-yloxy)methyl)benzo-nitrile (2f)
Hz, 1H), 7.63 (t, = 7.1 Hz, 1H), 7.46 (d, = 7.3 Hz, The desired product 2f was obtained as a yellow powder
1H), 7.29-7.20 (m, 2H), 7.00 (d,
= 8.4 Hz, 1H), 6.83 in 28% yield: m.p. 244-246oC (dec); 1H-NMR (500
(s, 1H), 6.42 (d, 7.88 (t, = 8.0 Hz, 2H), 7.83-7.77
= 1.3 Hz, 1H), 5.33 (s, 2H); 13C-NMR MHz, CD3COCD3)
(100 MHz, DMSO-d6 176.3, 164.0, 162.1, 160.8, 159.6, (m, 3H), 7.73 (d, = 8.5 Hz, 1H), 7.62 (t, = 7.3 Hz,
156.4, 148.3, 147.8, 145.5, 143.9, 136.5, 131.4, 124.9, 1H), 7.00 (d, = 8.0 Hz, 1H), 6.88 (s, 1H), 6.47 (s, 1H),
123.5, 123.3, 122.2, 120.2, 116.0, 104.6, 98.3, 93.0, 5.46 (s, 2H); 13C-NMR (100 MHz, DMSO-d6
176.3,
64.7; LC/MS (ESI) m/z Found: 411.30 (M+H)+; Calcd 163.8, 160.8, 156.3, 148.3, 147.8, 145.5, 139.5, 136.5,
δ
J
4H
J
J
J
J
δ
J
)
δ
J
J
J
)
δ
for C22H16FO7: 411.09.
134.0, 129.8, 122.2, 120.4, 117.5, 116.0, 115.9, 111.8,
104.8, 98.3, 98.2, 93.2, 93.1, 68.6; LC/MS (ESI) m/z
Found: 418.30 (M+H)+; Calcd for C23H16NO7: 418.09.
7-(2-Chlorobenzyloxy)-2-(3,4-dihydroxyphenyl)-3,
5-dihydroxy-4H-chromen-4-one (2c)
The desired product 2c was obtained as a yellow 2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(2-nitro-
1
powder in 22% yield: m.p. 252-255oC (dec); H-NMR benzyloxy)-4
(500 MHz, CD3COCD3) 7.86 (s, 1H), 7.73 (d, = 8.5 The desired product 2g was obtained as a yellow
Hz, 1H), 7.67-7.65 (m, 1H), 7.52-7.5 (m, 1H), 7.42-7.40 powder in 22% yield: m.p. 240-243oC (dec); H-NMR
(m, 2H), 7.00 (d, = 8.5 Hz, 1H), 6.82 (s, 1H), 6.43 (s, (400 MHz, CD3COCD3) 8.20 (d, = 8.1 Hz, 1H),
1H), 5.34 (s, 2H); 13C-NMR (100 MHz, DMSO-d6
7.91-7.80 (m, 3H), 7.71 (d, = 8.3 Hz, 1H), 7.66 (t,
176.3, 164.0, 160.8, 156.4, 148.3, 147.8, 145.5, 136.5, 7.6 Hz, 1H), 6.99 (d, = 8.39 Hz, 1H), 6.82 (s, 1H),
133.9, 133.3, 131.0, 130.8, 130.1, 129.7, 128.0, 127.7, 6.45 (s, 1H), 5.67 (s, 2H); 13C-NMR (100 MHz, DMSO-
122.2, 120.6, 116.0, 104.7, 93.0, 68.0; LC/MS (ESI) m/ d6 176.3, 163.7, 160.9, 156.3, 148.3, 147.8, 147.7,
Found: 427.40 (M+H)+; Calcd for C22H16ClO7: 427.06. 145.5, 136.5, 134.6, 132.1, 129.7, 129.5, 125.6, 125.1,
122.2, 115.9, 115.6, 104.8, 98.4, 93.2, 67.4; LC/MS (ESI)
7-(2-Bromobenzyloxy)-2-(3,4-dihydroxyphenyl)-3, m/z Found: 438.40 (M+H)+; Calcd for C22H16NO9:
5-dihydroxy-4 -chromen-4-one (2d) 438.08.
The desired product 2d was obtained as a yellow
H-chromen-4-one (2g)
δ
J
1
J
δ
J
)
δ
J
J =
J
)
δ
z
H
1
powder in 20% yield: m.p. 248-250oC (dec); H-NMR 2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(2-methyl-
(400 MHz, CD3COCD3) 7.86 (s, 1H), 7.74-7.64 (m, benzyloxy)-4 -chromen-4-one (2h)
3H), 7.45 (t, = 7.5 Hz, 1H), 7.33 (t, = 7.5 Hz, 1H), The desired product 2h was obtained as a yellow
7.00 (d, = 8.5 Hz, 1H), 6.79 (d,
= 1.4 Hz, 1H), 6.42 powder in 23% yield: m.p. 200-203oC (dec); H-NMR
(d, 7.86 (s, 1H), 7.72 (d, = 8.3
= 1.6 Hz, 1H), 5.29 (s, 2H); 13C-NMR (100 MHz, (400 MHz, CD3COCD3)
δ
H
J
J
1
J
J
J
δ
J

80
H. R. Park et al.
Hz, 1H), 7.47 (d,
J
= 7.0 Hz, 1H), 7.28-7.23 (m, 3H), The desired product 2l was obtained as a yellow powder
7.00 (d,
Hz, 1H), 5.26 (s, 2H), 2.40 (s, 3H); 13C-NMR (100 MHz, DMSO-d6
DMSO-d6 176.3, 171.8, 164.4, 160.8, 156.4, 148.3, (dd, = 8.5, 2.2 Hz, 1H), 7.52 (d,
147.7, 145.5, 137.2, 136.5, 134.5, 130.7, 130.5, 129.2, (t, = 7.8 Hz, 1H), 6.91 (d, = 8.5 Hz, 1H), 6.80 (d,
128.9, 128.6, 122.2, 115.9, 104.5, 98.3, 93.1, 69.1, 19.0; = 2.2 Hz, 1H), 6.45 (d,
= 2.2 Hz, 1H), 5.22 (s, 2H); 13C-
LC/MS (ESI) m/z Found: 407.40 (M+H)+; Calcd for NMR (100 MHz, DMSO-d6
176.8, 164.4, 161.3,
156.8, 148.7, 148.2, 145.9, 139.7, 137.6, 137.0, 136.9,
J = 8.4 Hz, 1H), 6.82 (s, 1H), 6.42 (d, J
= 0.7 in 14% yield: m.p. 230-233oC (dec); 1H-NMR (400 MHz,
)
δ
7.86 (s, 1H), 7.73 (d,
J
= 2.2 Hz, 2H), 7.57
)
δ
J
J = 10.2 Hz, 1H), 7.23
J
J
J
J
)
δ
C23H19O7: 407.11.
131.5, 127.9, 122.6, 120.8, 116.4, 116.1, 105.0, 98.8,
95.7, 93.6, 69.7; LC/MS (ESI) m/z Found: 519.40
(M+H)+; Calcd for C22H16IO7: 518.99.
2-(3,4-Dihydroxyphenyl)-7-(3-fluorobenzyloxy)-3,
5-dihydroxy-4H-chromen-4-one (2i)
The desired product 2i was obtained as a yellow
1
powder in 31% yield: m.p. 233-235oC (dec); H-NMR 3-((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-
(500 MHz, CD3COCD3) 7.85 (s, 1H), 7.12 (d, = 7.5 -chromen-7-yloxy)methyl)benzo-nitrile (2m)
Hz, 1H), 7.48 (q, = 8.0 Hz, 1H), 7.37 (d, = 8.0 Hz, The desired product 2m was obtained as a yellow
1H), 7.32 (d, = 10.0 Hz, 1H), 7.14 (t,
= 9.0 Hz, 1H), powder in 29% yield: m.p. 236-239oC (dec); H-NMR
7.00 (d, = 8.5 Hz, 1H), 6.81 (d, = 1.5 Hz, 1H), 6.44 (400 MHz, DMSO-d6 7.96 (s, 1H), 7.84 (t, = 8.0
(d, = 2.0 Hz, 1H), 7.65 (t,
= 2.0 Hz, 1H), 5.32 (s, 2H); 13C-NMR (100 MHz, Hz, 2H), 7.75 (d,
DMSO-d6 176.3, 164.0, 163.8, 161.4, 160.8, 156.3, 1H), 7.56 (dd, = 8.4, 2.0 Hz, 1H), 6.90 (d,
148.3, 147.8, 145.5, 139.5, 139.4, 136.5, 131.2, 124.2, 1H), 6.82 (d, = 2.0 Hz, 1H), 6.47 (d,
122.2, 120.5, 115.9, 114.8, 104.6, 98.4, 93.2, 69.4; LC/ 5.31 (s, 2H); 13C-NMR (100 MHz, CD3COCD3)
MS (ESI) m/z Found: 411.30 (M+H)+; Calcd for 165.2, 157.6, 148.3, 147.4, 145.7, 139.3, 137.0, 132.9,
δ
J
4H
J
J
1
J
J
J
J
)
δ
J
J
J
J
= 7.9 Hz,
)
δ
J
J
J = 8.4 Hz,
J
= 2.1 Hz, 1H),
176.6,
δ
C22H16FO7: 411.09.
132.6, 131.8, 130.7, 123.7, 121.5, 121.4, 119.1, 116.2,
115.8, 113.5, 105.1, 98.9, 93.7, 69.9; LC/MS (ESI) m/z
7-(3-Chlorobenzyloxy)-2-(3,4-dihydroxyphenyl)-3, Found: 418.20 (M+H)+; Calcd for C23H16NO7: 418.09.
5-dihydroxy-4H-chromen-4-one (2j)
The desired product 2j was obtained as a yellow 2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(3-nitro-
1
powder in 28% yield: m.p. 266-268oC (dec); H-NMR benzyloxy)-4
(400 MHz, DMSO-d6 7.72 (d, = 1.9 Hz, 1H), 7.57 The desired product 2n was obtained as a yellow
(d,
= 1.9 Hz, 1H), 7.55 (s, 1H), 7.45 (m, 3H), 6.90 (d, powder in 30% yield: m.p. 223-225oC (dec); H-NMR
= 8.5 Hz, 1H), 6.81 (d, = 2.0 Hz, 1H), 6.50 (d, = 2.0 (500 MHz, CD3COCD3) 8.41 (s, 1H), 8.25 (d, = 7.0
Hz, 1H), 5.26 (s, 2H); 13C-NMR (100 MHz, CD3COCD3) Hz, 1H), 8.00 (d,
= 7.5 Hz, 1H), 7.86 (s, 1H), 7.76 (t,
178.7, 165.1, 161.6, 157.4, 148.2, 147.3, 145.6, 139.7, = 8.0 Hz, 1H), 7.72 (d, = 8.5 Hz, 1H), 7.00 (d,
134.6, 130.9, 128.7, 128.1, 126.6, 123.4, 121.2, 115.9, 8.5 Hz, 1H), 6.86 (s, 1H), 6.48 (s, 1H), 5.48 (s, 2H); 13C-
115.6, 104.9, 98.7, 98.6, 70.0; LC/MS (ESI) m/z NMR (100 MHz, DMSO-d6 176.3, 171.9, 163.8,
Found: 427.10 (M+H)+; Calcd for C22H16ClO7: 427.06.
160.9, 156.3, 148.3, 148.2, 147.8, 145.5, 138.9, 136.5,
134.5, 123.1, 122.8, 122.2, 120.5, 115.9, 104.7, 98.4, 98.3,
7-(3-Bromobenzyloxy)-2-(3,4-dihydroxyphenyl)-3, 93.2, 68.9; LC/MS (ESI) m/z Found: 438.40 (M+H)+;
5-dihydroxy-4 -chromen-4-one (2k) Calcd for C22H16NO9: 438.08.
The desired product 2k was obtained as a yellow
H-chromen-4-one (2n)
)
δ
J
1
J
J
J
J
δ
J
J
δ
J
J
J =
)
δ
H
1
powder in 26% yield: m.p. 200-203oC (dec); H-NMR 2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(3-methyl-
(400 MHz, CD3COCD3) 7.86 (d, = 1.6 Hz, 1H), 7.73 benzyloxy)-4 -chromen-4-one (2o)
(s, 1H), 7.71 (d, = 1.8 Hz, 1H), 7.56-7.53 (m, 2H), The desired product 2o was obtained as a yellow powder
7.39 (t, = 7.8 Hz, 1H), 7.01 (d,
= 4.5 Hz, 1H), 6.80 in 22% yield: m.p. 206-209oC (dec); 1H-NMR (500
(d, = 2.1 Hz, 1H), 6.43 (d, = 2.0 Hz, 1H), 5.30 (s, MHz, CD3COCD3) 7.86 (s, 1H), 7.74 (s, 1H), 7.72 (dd,
2H); 13C-NMR (100 MHz, DMSO-d6
176.3, 171.9, = 8.5, 1.5 Hz, 1H), 7.31 (d,
164.0, 160.8, 156.3, 148.3, 147.8, 145.5, 139.4, 136.5, = 3.5 Hz, 1H), 7.01 (d, = 8.5 Hz, 1H), 6.80 (d,
131.5, 131.0, 127.3, 127.0, 122.3, 120.5, 120.2, 116.0, Hz, 1H), 6.41 (d,
104.6, 98.3, 93.2, 69.3; LC/MS (ESI) m/z Found: 3H); 13C-NMR (100 MHz, DMSO-d6
δ
J
H
J
J
J
J
J
δ
)
δ
J
J
= 5.0 Hz, 2H), 7.19 (t,
J
J
J
= 2.0
J
= 2.0 Hz, 1H), 5.24 (s, 2H), 2.36 (s,
176.3, 164.3,
)
δ
471.20 (M+H)+; Calcd for C22H16BrO7: 471.01.
160.8, 156.4, 148.2, 147.7, 145.5, 138.1, 136.5, 136.4,
128.9, 128.7, 125.3, 125.2, 122.2, 120.6, 120.2, 116.0,
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(3-iodo- 104.5, 98.3, 93.1, 70.4, 21.5; LC/MS (ESI) m/z Found:
benzyloxy)-4 -chromen-4-one (2l)
H
407.40 (M+H)+; Calcd for C23H19O7: 407.11.

Antiviral Quercetin Derivatives
81
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(3-meth-
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(4-iodo-
oxybenzyloxy)-4H-chromen-4-one (2p)
benzyloxy)-4H-chromen-4-one (2t)
The desired product 2p was obtained as a yellow The desired product 2t was obtained as a yellow
1
1
powder in 22% yield: m.p. 214-218oC (dec); H-NMR powder in 20% yield: m.p. 259-262oC (dec); H-NMR
(500 MHz, CD3COCD3) 7.86 (s, 1H), 7.72 (d, = 8.5 (400 MHz, DMSO-d6 7.79 (d, = 8.2 Hz, 2H), 7.73
Hz, 1H), 7.34 (t, = 8.0 Hz, 1H), 7.09 (d, = 8.0 Hz, (d, = 2.0 Hz, 1H), 7.57 (dd, = 8.5, 2.1 Hz, 1H), 7.29
1H), 7.00 (d, = 8.5 Hz, 1H), 6.93 (d, = 8.0 Hz, 1H), (d, = 8.2 Hz, 2H), 6.90 (d, = 8.5 Hz, 1H), 6.78 (d,
6.80 (d, = 2.0 Hz, 1H), 6.42 (d,
(s, 2H), 3.82 (s, 3H); 13C-NMR (100 MHz, DMSO-d6
δ
J
)
δ
J
J
J
J
J
J
J
J
J
J =
J
J
= 2.0 Hz, 1H), 5.26 2.1 Hz, 1H), 5.21 (s, 2H); 13C-NMR (100 MHz, DMSO-
)
δ
d6) δ 176.3, 164.0, 160.8, 156.3, 148.3, 147.7, 145.5,
176.3, 164.2, 160.8, 159.8, 156.3, 148.2, 147.7, 145.5, 137.7, 136.5, 136.4, 130.3, 122.2, 120.4, 115.9, 115.6,
138.1, 136.4, 130.2, 129.9, 122.2, 120.5, 120.3, 116.0, 104.6, 98.3, 94.6, 93.2, 69.6; LC/MS (ESI) m/z Found:
113.8, 113.6, 104.5, 98.4, 93.2, 70.1, 55.5; LC/MS (ESI) 519.30 (M+H)+; Calcd for C22H16IO7: 518.99.
m/z Found: 423.30 (M+H)+; Calcd for C23H19O7: 423.11.
4-((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-
2-(3,4-Dihydroxyphenyl)-7-(4-fluorobenzyloxy)-3,
4H-chromen-7-yloxy)methyl)benzo-nitrile (2u)
5-dihydroxy-4
H
-chromen-4-one (2q)
The desired product 2u was obtained as a yellow
1
The desired product 2q was obtained as a yellow powder in 29% yield: m.p. 212-215oC (dec); H-NMR
powder in 30% yield: m.p. 212-215oC (dec); H-NMR (500 MHz, CD3COCD3)
δ
7.86 (d,
= 8.0 Hz, 2H), 7.71 (d, = 8.0 Hz, 1H), 7.01 (d,
= 8.5 Hz, = 8.5 Hz, 1H), 6.82 (s, 1H), 6.45 (d, = 1.5 Hz, 1H),
= 8.5 Hz, 1H), 6.79 (s, 1H), 6.41 (s, 1H), 5.43 (s, 2H); 13C-NMR (125 MHz, CD3COCD3)
176.4,
5.27 (s, 2H); 13C-NMR (100 MHz, DMSO-d6
176.4, 163.9, 161.0, 156.4, 148.4, 147.9, 145.6, 142.5, 136.6,
J = 8.0 Hz, 3H), 7.76
1
(500 MHz, CD3COCD3)
Hz, 1H), 7.59 (t, = 8.0 Hz, 2H), 7.20 (t,
2H), 7.01 (d,
δ
7.86 (s, 1H), 7,72 (d,
J
= 8.0 (d,
J
J
J
J
J
J
J
δ
)
δ
164.2, 161.4, 160.9, 156.5, 148.3, 147.8, 145.6, 136.5, 133.0, 128.6, 122.3, 120.5, 119.2, 116.0, 115.8, 111.2,
132.9, 130.7, 130.6, 122.3, 120.5, 116.0, 115.8, 104.6, 104.8, 98.5, 93.3, 69.4; LC/MS (ESI) m/z Found:
98.5, 93.2, 69.7; LC/MS (ESI) m/z Found: 411.40 418.30 (M+H)+; Calcd for C23H16NO7: 418.09.
(M+H)+; Calcd for C22H16FO7: 411.09.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(4-nitro-
7-(4-Chlorobenzyloxy)-2-(3,4-dihydroxyphenyl)-3, benzyloxy)-4H-chromen-4-one (2v)
5-dihydroxy-4
H
-chromen-4-one (2r)
The desired product 2v was obtained as a yellow
1
The desired product 2r was obtained as a yellow powder in 24% yield: m.p. 225-227oC (dec); H-NMR
powder in 25% yield: m.p. 228-230oC (dec); H-NMR (500 MHz, CD3COCD3)
δ
8.31 (d,
= 8.5 Hz, 3H), 7.71 (d, = 8.5 Hz, 1H), 7.00 (d,
= 8.5 = 8.5 Hz, 1H), 6.83 (s, 1H), 6.46 (s, 1H), 5.48 (s, 2H);
= 1.9 Hz, 13C-NMR (100 MHz, DMSO-d6
174.5, 161.9, 159.0,
1H), 5.25 (s, 2H); 13C-NMR (100 MHz, CD3COCD3)
154.5, 146.5, 146.0, 145.7, 143.6, 142.6, 134.6, 126.8,
J = 8.5 Hz, 2H), 7.83
1
(400 MHz, DMSO-d6
(dd, = 8.4, 1.9 Hz, 1H), 7.50 (m, 4H), 6.90 (d,
Hz, 1H), 6.80 (d, = 1.8 Hz, 1H), 6.44 (d,
)
δ
7.72 (d,
J
= 1.9 Hz, 1H), 7.56 (d,
J
J
J
J
J
J
J
) δ
δ
177.1, 165.7, 162.1, 157.9, 148.9, 148.0, 146.3, 136.7, 122.2, 120.3, 118.5, 114.1, 113.8, 102.9, 96.1, 91.4,
134.7, 130.6, 129.8, 123.9, 121.8, 116.4, 116.1, 105.5, 67.1; LC/MS (ESI) m/z Found: 438.40 (M+H)+; Calcd
100.6, 99.2, 94.0, 70.0; LC/MS (ESI) m/z Found: for C22H16NO9: 438.08.
427.20 (M+H)+; Calcd for C22H16ClO7: 427.06.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(4-methyl-
7-(4-Bromobenzyloxy)-2-(3,4-dihydroxyphenyl)-3,
5-dihydroxy-4 -chromen-4-one (2s)
The desired product 2s was obtained as a yellow powder powder in 21% yield: m.p. 262-264oC (dec); H-NMR
benzyloxy)-4H-chromen-4-one (2w)
H
The desired product 2w was obtained as a yellow
1
in 22% yield: m.p. 255-258oC (dec); 1H-NMR (400 (500 MHz, CD3COCD3)
MHz, CD3COCD3) 7.85 (s, 1H), 7.71 (d, = 8.4 Hz, (dd, = 8.5, 2.5 Hz, 1H), 7.41 (d,
1H), 7.62 (d, = 8.4 Hz, 2H), 7.50 (d, = 8.4 Hz, 2H), (d, = 7.5 Hz, 2H), 7.01 (d,
7.00 (d, = 8.5 Hz, 1H), 6.79 (d, = 1.4 Hz, 1H), 6.41 = 2.0 Hz, 1H), 6.41 (d, = 2.0 Hz, 1H), 5.23 (s, 2H),
(d, 175.8,
= 1.6 Hz, 1H), 5.27 (s, 2H); 13C-NMR (100 MHz, 2.35 (s, 3H); 13C-NMR (100 MHz, DMSO-d6
DMSO-d6 174.4, 162.1, 159.0, 154.4, 146.3, 145.8, 163.8, 160.3, 155.9, 147.8, 147.2, 145.0, 137.3, 135.9,
δ
7.86 (d,
J
J
= 2.0 Hz, 1H), 7.72
= 7.5 Hz, 2H), 7.24
δ
J
J
J
J
J
J = 8.5 Hz, 1H), 6.79 (d, J
J
J
J
J
) δ
)
δ
143.6, 134.5, 134.2, 129.9, 128.4, 12.3, 119.7, 118.5, 1331, 129.0, 127.8, 121.7, 119.9, 115.5, 115.2, 104.0,
114.0, 113.7, 102.7, 96.5, 91.3, 67.5; LC/MS (ESI) m/z 97.9, 92.7, 69.8, 20.7; LC/MS (ESI) m/z Found: 423.30
Found: 471.30 (M+H)+; Calcd for C22H16BrO7: 471.01.
(M+H)+; Calcd for C23H19O8: 423.11.

82
H. R. Park et al.
2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(4-meth-
Cytostatic effect
Huh-5-2 cells were seeded at a density of 5 ×
103 per
oxybenzyloxy)-4H-chromen-4-one (2x)
The desired product 2x was obtained as a yellow well of 96-well plate in complete DMEM with the
1
powder in 19% yield: m.p. 217-219oC (dec); H-NMR appropriate concentrations of G418. Serial dilutions of
(500 MHz, CD3COCD3)
Hz, 1H), 7.45 (d, = 8.5 Hz, 2H), 7.01-6.95 (m, 3H), were added 24 h after seeding. Cells were allowed to
6.78 (d, = 2.0 Hz, 1H), 6.39 (d,
= 1.5 Hz, 1H), 5.19 proliferate for 3 days at 37oC, after which the cell
(s, 2H), 3.82 (s, 3H); 13C-NMR (100 MHz, DMSO-d6
δ 7.86 (s, 1H), 7.72 (d, J = 8.0 the test compounds in complete DMEM without G418
J
J
J
)
δ
number was determined by WST-1 assay.
176.3, 164.3, 160.7, 156.4, 156.3, 147.7, 145.4, 136.4,
130.9, 129.3, 128.4, 122.3, 122.2, 121.2, 116.7, 115.1,
113.5, 110.0, 104.4, 56.2, 54.8; LC/MS (ESI) m/z
Found: 407.30 (M+H)+; Calcd for C23H19O7: 407.11.
RESULTS AND DISCUSSION
Chemistry
Regioselective alkylation of quercetin requires selec-
tive protection of the five phenolic hydroxyl groups.
Anti-SCV assay
Carboxytetramethylrhodamine (TAMRA)-modified For preparation of the 7-O-alkylated quercetin deri-
45-base-oligomer and fluorescein-modified 25-base- vatives, we adapted a general synthetic protocol which
oligomer were purchased from Integrated DNA Tech- includes peracetylation followed by regioselective
nologies: 5'-20T25Tam (5'-TTTTTTTTTTTTTTTTTT- deacetylation (Scheme 1).
TTGAGCGGATTACTATACTACATTAGA (TAMRA)-
3') and 3'-0T25Flu (5'-(Fluorescein) TCTAATGTAGTA- dride (Ac2O) in pyridine afforded the peracetylated
TAGTAATCCGCTC-3'). The helicase substrate was pre- quercetin derivative , which underwent regioselec-
Thus, acetylation of quercetin (1) with acetic anhy-
4
pared by annealing the two oligomers, which resulted tive deacethylation with PhSH in NMP (Li et al.,
in 25 base pairs of dsDNA with single-stranded 20 dT 2003; Sabui and Venkateswaran, 2003; Lee C et al.,
of 5'-overhang. A 80
nM) in 20 mM HEPES (pH 7.4) buffer was added to quercetin 3,5,3’,4’-tetraacetates
each well of the 96-well assay plate which already tion of with variously substituted benzyl bromides
contained 1 L of various concentrations of chemical followed by deacetylation by treatment with methanolic
compounds. After 5 min incubation at rt, the FRET ammonia afforded a series of 7- -arylmethylquerce-
based dsDNA unwinding assay was started by addi- tins 2a 2x in moderate yields.
tion of 20 L 5X reaction solution [5 mM MgCl2, 45
µ
L solution of SCV helicase (150 2009a; Lee HS et al., 2010) to provide the corresponding
5
in 74% yield. Alkyla-
5
µ
O
~
µ
mM ATP, 25 mM DTT, and 100 nM dsDNA substrate
in 20 mM HEPES (pH 7.4)]. The reaction mixture was
Biological activity
All synthesized quercetin arylmethyl ethers (2a
~2x)
further incubated for 2 min at 37oC and stopped with were evaluated for their inhibitory activity against
100
M trap DNA (unmodified 25 bases 3'-0T25 oligomer) /helicase (Lee C et al., 2009a, 2009b; Lee HS et al., 2010),
in 20 mM HEPES (pH 7.4)]. The sample was excited which was summarized in Table I as IC50 values. The
at 485 nm and the fluorescence was measured at 535 anti-HCV activity of the 7- -arylmethylquercetin deri-
µL of termination solution [0.1 M EDTA and 0.4 both NTPase and helicase activity of the SCV NTPase
µ
O
nm.
vatives in the human hepatoma cell line Huh-7, carry-
ing the subgenomic HCV genotype 1 replicon with the
luc-ubi-neo fusion gene (Lohmann et al., 1999; Vroljk
Anti-HCV assay
The human hepatoma cell line Huh-7, carrying the et al., 2003) was also evaluated. INF-α was included
subgenomic HCV genotype 1 replicon with the luc-ubi- as a positive control, and the conditions of the lumi-
neo (reporter/selective) fusion gene, was kindly provid- nescence-based assay used to test the antiviral activity
ed by Dr. Ralf Bartenschlager (University of Heidelberg). of the compounds were previously described (Gozdek
Huh-5-2 cells were seeded at a density of 5
well in a tissue culture-treated white 96-well view was evaluated in the same cell line. Anti-HCV effect
×
103 per et al., 2008). The cytostatic effect of the test compounds
plate in complete DMEM supplemented with 500 µg/ and cytostatic effect are summarized as EC50 and
mL G418. After incubation for 24 h at 37oC (5% CO2), CC50, respectively, in Table I. Assays were performed
medium was refreshed (with G418) and DMSO stock in triplicate and the data in Table I are the mean of
of test compounds were added. After 4 days of incuba- three experiments.
tion at 37oC, cell culture medium was removed and
Other than the previously reported 7-
luciferase activity was determined using the Steady- quercetins such as 2j 2m, and 2r (Lee et al., 2009a)
Glo luciferase assay system (Promega). with 3''-Cl, 3''-CN, and 4''-Cl substituent, respectively,
O-arylmethyl-
,

Antiviral Quercetin Derivatives
83
Scheme 1. Synthesis of the 7-O-arylmethylquercetin derivatives (2a~2x) from quercetin (1)
no compound tested in this study showed inhibitory seems like to be originated from the cytotoxic effect of
activity against SCV NTPase/helicase. Thus, it is con- the quercetin derivatives. Considering the well known
ceivable that binding sites specific for aromatic substi- anticancer mechanisms of quercetin (Ferry et al.,
tuents might be present in the viral target enzyme. 1996; Lamson and Brignall, 2000), the cytotoxic effect
More specifically, the NTPase favors 3''-CN and 4''-Cl of the quercetin derivatives is not surprising but some
whereas the helicase has no specific preference among of them showed no cytotoxicity up to 100
the three substituents. Thus, only those five 7- -arylmethylquercetins (2a
On the other hand, anti-HCV activity of the quercetin 2g 2v 2x) showed selective anti-HCV activity with
derivatives was evaluated by the HCV replicon cell- EC50 values of 25.7, 11.1, 8.9, 23.5, and 33.9
M, respec-
µM (Table I).
O
,
,
~
µ
based assay. As this assay is based on the luciferase tively. Interestingly, the derivatives with a strongly
activity co-expressed with the viral proteins inside the electron-withdrawing nitro substituent at the ortho
Huh-7 cell line harboring the HCV replicon, estimation
of the cytotoxicity of the test compounds is an impor- the aromatic ring showed the most potent anti-HCV
tant aspect of understanding the compounds’ antiviral activity (11.1 and 8.9
M, respectively) with no cyto-
(2g, R = 2-NO2) and para (2v, R = 4-NO2) position of
µ
activity. Therefore, the cell-based antiviral assay was toxicity, which suggests a possible role of the electronic
conducted in parallel with the cytotoxicity assay. All property around the aromatic ring for bioactivity.
the quercetin derivatives synthesized in this study
showed moderate anti-HCV activity in the HCV replicon tives of 7-
cell-based assay (EC50 = 5~34
M, Table I). However, antiviral activity against SCV as well as HCV. Among
the 7- -arylmethylquercetin derivatives were generally those, three derivatives with 3''-Cl, 3''-CN, and 4''-Cl
toxic against the Huh-7 cell line harboring the HCV aromatic substituents showed selective inhibitory
replicon with CC50 values of 10~20 M (Table I) and activity against SCV NTPase/helicase. On the other
thus, the substituent-independent anti-HCV activity hand, due to the cytotoxicity associated with the test
In conclusion, in this study, we prepared 24 deriva-
O-arylmethylquercetins and evaluated their
µ
O
µ

84
H. R. Park et al.
Table I. Anti-HCV activity and inhibition of the SCV
NTPase/helicase of the 7-O-arylmethylquercetins (2a~2x)
Healthcare technology R&D Project, Ministry for
Health, Welfare & Family Affairs, Republic of Korea
(A08-4628-AA2023-08N1-00010A), by Basic Science
Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (2010-0008260),
Priority Research Centers Program through the Na-
tional Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology
(2009-0093824), by a grant from ORP 8-21-52 (NIAS),
and by a grant from 2nd Biogreen 21 program
PJ007982 (Korea Rural Development Administration).
Anti-SCV (IC50,
NTPasec Helicased EC50,
µ
M)
Anti-HCV
Ma CC50, Mb
µ
Compds
R
µ
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
2''-F
2''-Cl
2''-Br
2''-I
2''-CN
2''-NO2
2''-Me
3''-F
3''-Cl
3''-Br
3''-I
3''-CN
3''-NO2
3''-Me
3''-OMe
4''-F
4''-Cl
4''-Br
4''-I
4''-CN
4''-NO2
4''-Me
4''-OMe
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50d
>50
>50
25.4e
>50
>50
>50
>50
20.9d
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5.2d
>50
>50
2.7d
>50
>50
>50
>50
4.1d
>50
>50
>50
>50
>50
>50
25.7
10.0
9.0
>100
22.5
16.5
16.4
16.6
16.5
>100
21.3
18.9
17.8
16.8
20.0
21.1
16.7
13.2
22.4
17.5
18.2
18.6
18.9
7.0
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16.7
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